key: cord-276067-3io0xux2 authors: kanmounye, ulrick sidney; esene, ignatius n. title: covid-19 and neurosurgical education in africa: making lemonade from lemons date: 2020-05-21 journal: world neurosurg doi: 10.1016/j.wneu.2020.05.126 sha: doc_id: 276067 cord_uid: 3io0xux2 abstract never in history has the fabric of african neurosurgery been challenged as it is today with the advent of covid-19. even the most robust and resilient neurosurgical educational systems in the continent have been brought to their knees with neurosurgical trainees and young neurosurgeons bearing the brunt. in the face of this new reality, and in order to limit the impact of the current covid-19 pandemic, multiple programs have implemented physical distancing which reduces in-person interactions. in some cases, residents have been asked to stay home at least till they are instructed otherwise. this unfortunate event presents an innovative opportunity for neurosurgical education in africa. herein, we detail the framework of an online neurosurgical education initiative to advance the education of african residents and young neurosurgeons during and after the covid-19 pandemic. initiative to advance the education of african residents and young neurosurgeons during and after the covid-19 pandemic. seventeen of the 54 african countries do not have the recommended specialist neurosurgeon workforce density principally due to a lack of training programs (1) . also, few african training programs offer a wide range of neurosurgical subspecialty experience to their trainees. yet, some of the most respected neurosurgeons in the world are african neurosurgeons who have either trained on the continent or are training the future generation of neurosurgeons in their countries. these neurosurgeons are an estimable resource that we have underused. to reach greater heights, we must choose to stand on the shoulders of these giants. however, educational exchanges between our training centers are limited and pale in the face of the partnerships our training centers have with high-income country institutions. perhaps owing to the fact that it is generally cheaper and easier to travel outside of our continent than it is inside (2) . we hope therefore that the african continental free trade area will facilitate the transfer of knowledge. in the meantime, however we must find alternative ways to share the skills and experiences of our senior neurosurgeons with the younger generation. african residents and young neurosurgeons are already learning from and about world renowned neurosurgeons via online medical education platforms (3). it is clear from this that we can do a better job of integrating online medical education and our experts into our training programs. in order to limit the impact of the current covid-19 pandemic, multiple programs have implemented physical distancing which reduces in-person interactions. in some cases, residents have been asked to stay home at least till they are instructed otherwise (4). this unfortunate event presents an opportunity for neurosurgical postgraduate medical education in africa. herein, we detail the framework of an online neurosurgical education initiative to advance the education of african residents during and after the covid-19 pandemic. led by the professional societies, senior faculty from all neurosurgical subspecialties and from all the regions will be invited to host continental neurosurgery grand rounds. with permission from program directors, residents will be recruited and will be asked to register online (google some speak of the return to pre-covid "normalcy" after the pandemic but it is more likely that the post-covid period will define a new "normalcy". one in which some things that were considered impossible before will not no longer be impossible. successful organizations adapt promptly in the face of adversity. while the post-covid surprised and tested our health systems, post-covid african neurosurgical education must reinvent itself and transform its potential into achievements. resilience in this situation lies in recognizing the value of learning from those who walked the path we wish walk and using technology to our advantage. we cannot let the current crisis stifle our progression and we should not aim to return to the pre-covid "normalcy". for when life gives you lemon, make lemonade! the neurosurgical atlas: advancing neurosurgical education in the digital age impact of covid-19 on neurosurgery resident training and education the authors declare no competing interest. usk and ie contributed equally to the conceptualization, writing, review and editing of the manuscript. the authors declare no competing interest. key: cord-297103-f3jdbv47 authors: longino, kevin; kramer, holly title: racial and ethnic disparities, kidney disease, and covid-19: a call to action date: 2020-07-21 journal: kidney med doi: 10.1016/j.xkme.2020.07.001 sha: doc_id: 297103 cord_uid: f3jdbv47 nan kevin longino, mba, holly kramer, md, mph as the nation once again turns its attention to the black lives matter movement, we must ask ourselves how the movement applies to our community of kidney patients and the professionals who care for them. the fact that the call for racial justice is occurring in the midst of a public health epidemic is not a coincidence. the same factors that have spurred nationwide protests -poverty, inequality, implicit bias, and systemic racism -contribute to the disproportionate impact that kidney disease has on communities of color. while the national kidney foundation (nkf) alone cannot end the 400+ year legacy of racism that affects the day-to-day lives of so many patients, we hear our moral calling to look the challenge square in the eye. it is incumbent upon all of us -as healthcare professionals, patients, and advocates -to confront racial and ethnic disparities and work together to ensure that all people with kidney disease receive nothing but the best care our system has to offer. covid-19 is the most significant health crisis in the modern era. it has directly and indirectly resulted in overwhelming rates of illness and death, has created major disruptions to economies and everyday life both in the united states and throughout the world, and might leave survivors with chronic, permanent health complications. notably, severe covid-19 has been associated with acute kidney injury (aki), which can increase the risk of chronic kidney disease (ckd) over time. 1,2 ckd has also been associated with more severe covid-19 infection. 2, 3 covid-19 has had devastating and disproportionate consequences for communities of color. people of african american, american indian or alaska native american descent are five times more likely to be hospitalized due to covid-19 than whites, while hispanics are approximately four times more likely to be hospitalized. one in four americans dying of covid-19 are black or african american, even though members of this community represent only 13 percent of the us population. 4 the reasons for these unconscionable disparities are multifactorial but are likely closely tied to social determinants of health such as access to healthcare, socioeconomic status (ses), employment, food security, education, housing, environment, and social support. these factors in turn have been driven and exacerbated by a long history of systemic racism. some specific factors can include the higher likelihood of living in densely populated areas or in crowded housing conditions; being an essential worker who cannot work remotely; and relying on a job with a lower income or without sick leave. another significant disparity is a lack of access to healthcare, as hispanics are about three times as likely to be uninsured, and black or african americans are almost twice as likely to be uninsured. 4 many of the healthcare and socioeconomic disparities that make communities of color more vulnerable to covid-19 also make them vulnerable to kidney disease. communities of color often have higher rates of diabetes and high blood pressure, which are the first and second leading causes of kidney failure, respectively. 5 the risk of ckd increases for black or african american patients compared to white patients at every stage. early referral to nephrology is associated with improved ckd outcomes, however black or african american patients are more likely to have delayed referral or no nephrology referral at all. communities of color are also overrepresented among patients with end-stage kidney disease. for every three non-hispanics who develop kidney failure, four hispanics develop kidney failure. black or african americans are three times more likely to suffer from kidney failure than whites. 5 these disparities extend into treatment modalities as well. black and hispanic patients are less likely to receive home dialysis, and when they do begin peritoneal dialysis (pd), they are more likely to fail within the first 90 days. black or african american patients have less access to the optimal treatment of kidney transplantation. they are less likely to be waitlisted for an organ, and when they are transplanted, they wait longer, are less likely to receive a deceased or living donor organ (i.e., die on the waitlist) and have poorer graft survival at first year post-transplant. in an effort to address these challenges, nkf is advocating for access to affordable healthcare, to increase our federal investment in research, prevention, and innovations in care for people with kidney disease, and to ensure that racial and ethnic communities are not left behind. 6 the issues that underlie kidney disease, covid-19, and healthcare disparities are complex. however there are some specific strategies nkf is advocating, including calling on the federal government to provide quality, disaggregated data on all tests, hospitalizations, discharges, and deaths from covid-19 in order to fully understand the scope of the problem; and to ensure priority testing, contact tracing, access to a future vaccine, and funding for high-risk and minority communities and kidney patients. 6 nkf also supports long-term investments in public health infrastructure in historically underserved communities and increased funding for kidney disease research and awareness. the nkf and the american society of nephrology (asn) established a joint task force to reassess the inclusion of race in diagnosing kidney disease. the task force aims to evaluate the use of race in calculating egfr and ensure that gfr estimation equations provide an unbiased assessment of kidney function so that laboratories, clinicians, patients, and public health officials can make informed decisions to ensure equity and personalized care for patients with kidney disease. this work is important, but it alone cannot solve the many health and socioeconomic disparities facing black or african american and other minority communities, which are rooted in historical and ongoing systemic racism. however, this work can make progress towards addressing healthcare disparities that continue to fuel disenfranchisement among these communities. 6 the nkf is also advocating to address areas of concern for people with kidney disease in the context of the covid-19 pandemic, including prioritizing kidney patients' and clinicians' access to personal protective equipment; preserving access to essential kidney-related surgical procedures (e.g., organ transplantation, vascular access); and fighting policies that discriminate against kidney patients. 7 the nkf is also working with several partners to implement policies that accelerate patients' access to home dialysis; ensure timely implementation of kidney care payment models; ensure kidney patients and transplant patients can access greater-than-30-day supplies of critical prescriptions including immunosuppressive drugs; and ensure that vulnerable home dialysis, transplant patients, and living donors can receive needed blood draws in their homes. acute kidney injury in patients hospitalized with covid-19 kidney disease is associated with in-hospital death of patients with covid-19 chronic kidney disease is associated with severe coronavirus disease 2019 (covid 19) infection covid-19 in racial and ethnic minority groups chronic kidney disease in the united states the nkf will continue work that is tangible, results-focused, and lifesaving. the nkf will also continue long-term and focused outreach to our black or african american patients and all patients of color who develop kidney disease and the devastating complications which accompany it. key: cord-305026-t4wkv89b authors: treadwell, henrie m. title: the pandemic, racism, and health disparities among african american men date: 2020-08-07 journal: am j mens health doi: 10.1177/1557988320949379 sha: doc_id: 305026 cord_uid: t4wkv89b nan the coronavirus pandemic has amplified health disparities by race and gender, perhaps most notably among african american men. surveillance data reveal that males are disproportionately affected by covid-19, and that black populations are disproportionately affected overall (wortham et al., 2020) . it cannot and should not be a surprise that populations who are subject to poverty, fragile housing, food and fiscal insecurity, no or inadequate health insurance are disproportionately affected by . it is axiomatic that populations that have disproportionate rates of health conditions such as hypertension, diabetes mellitus, obesity, and cardiovascular disease are more susceptible to both contracting coronavirus and to adverse outcomes. the criminal justice system interjects an additional risk factor given that those captured in this matrix are predominantly african american males. the impact of covid-19 across jails, prisons, and detention centers may never be fully reported out to the community. what recent data exist are grim. in those facilities where testing has occurred, the positive test rate is 78% among the incarcerated and extremely high levels of positive tests (dolovich, 2020) . positively, the prospective heroes act (section 30110) would allow medicaid to cover health services in the last 30 days pre-release. still once released, major barriers to basic and critical health and human services still exist as three quarters of reentry service providers have scaled back or have closed their services (csg justice center staff, 2020). the lack of support services hinders opportunities for individuals to successfully reunite with families, attain employment, and address burgeoning child support and other restorative justice costs. grappling with the disparities revealed by the pandemic requires an examination and actions to ameliorate racial discrimination that has had an impact for 400 years. african american boys and men have experienced poor health and health outcomes historically, and little or nothing has changed over the years though improvements in u.s. health services and systems are reportedly the most advanced in the world. the 2019-2020 coronavirus pandemic amplifies the existing injustices. while some attribute the overall poor health and disparate rates of morbidity and mortality to individual behaviors, that assessment fails to assess the direct damage inflicted by a social and political system that has marginalized and minimized efforts to provide meaningful services even at the primary health-care level. the compendium of injuries experienced that circumscribe well-being are described as the social determinants of health. significantly, a more nuanced expression on the how the collateral damage is delivered are described as the political determinants of health. in sum, policies that do not incorporate potential for fostering racial disparities are the drivers of disease, health inequities, and the unequal burden of morbidity and mortality experienced by communities of color. examination of current health-care system policies, practices, and experiences is an inadequate starting point for a discussion of redressing health disparities. rather, historical perspectives are important to understand the depth of destruction caused by hundreds of years of exclusion from justice. it is not possible to thoroughly understand why health inequities exist without acknowledging the terrible cumulative penalty that is being paid as a result of intentionally exclusionary policy that based solely on skin color, race, ethnicity, and gender. social practices and political policies have discriminated against black men since the black codes (history.com, 2019), the man in the house rule (moffitt et al., 1994) which excluded them from the home lest funding for children be lost, and the exclusion from social security (dewitt, 2010). a racebased policy and the continuing overwhelming exclusion of boys and men over the age of 18 from medicaid (hinton & artiga, 2016) fosters exclusion that could have been mitigated if states had expanded medicaid under the affordable care act. in sum, the nation's policymakers have designed a system that discriminates and until now legislators appear loath to enact policies and programs to insure health equity for all. major systemic barriers have been erected that affect morbidity and mortality in the african american community. the lack of insurance, lack of a living wage, and employment in low-wage professions that do not provide sick leave and require that individuals go to their "essential jobs" (e.g. meat packing plants, home health care, as nurses' aides or janitorial staff, among others) despite their health symptoms or status, elevate mortality in poor communities of color. the distinction that must be made is that african american men are not disproportionately and prematurely dying simply because they are african american men. there is no evidence that the cause of the disparity is embedded in the genes. they are dying because they do not have a healthcare home as a result of not having income to pay for the visits. further and perhaps even more damaging throughout the pandemic are health messages supported by federal government leaders that insist that individuals not go to the clinic or emergency room. rather they are instructed to call their doctor. the cloak of invisibility is firmly wrapped around the population of those, mainly men of color, who have no doctor (treadwell & ro, 2008) . what are they to do and why is their plight ignored? a striking conundrum is that data are not consistently collected by race and gender making it impossible to identify population-based service deficiencies that guide the design of specific strategies, appropriate tactics, and barrier-free interventions. it is important to acknowledge that institutionalized populations are not included in population statistics unless they enter the health-care system for treatment, a subject discussed at length in the recently published manuscript, "discerning disparities: the data gap" (treadwell et al., 2019) and in the article "collecting demographic data is the first step in eliminating racism in healthcare" (eschner, 2020) . damage is perpetrated when researchers and individuals who report on morbidity, mortality, and equitable health-care access remain silent about institutionalized populations, such as african american men who are disproportionately represented in america's prisons. the silence from epidemiologists and others charged with profiling the heath care of the nation is perplexing as it allows racist and exclusionary practices to continue unabated and the public, that might respond affirmatively, to remain ignorant of the depth of health inequities linked to race and gender alone. african american men are subject to injustices on so many levels. those entities and organizations that "police" the health-care system through their incomplete data collection and reporting out strategies are failing the public, too, along with their failure to advocate for inclusive data collection. to be sure, the issues do not solely affect african american men. yet each group (e.g. hispanics, pacific islanders, and native americans) has a unique path in the united states and each group should be examined so that comparative analyses can be conducted to determine similarities that can be enacted to meet their needs, along with strategies that respond to each racial group and their heterogeneous historical and contemporary experiences. we as a nation did not come to this place by chance. race and racism have been the catalysts. the inflection point is here. the time to act to redress inequities and support health justice is now. to achieve the change that we need to see in our society, we must advocate for expanded medicaid and/or health-care coverage for all in the nation. equally important, systems must be designed to ensure that behavioral and oral health care are an integrated part of primary care. we must also address the fact that a disproportionate number of african american men are incarcerated and foster integration of data from those in the community and those incarcerated to develop a complete portrait of health status of these individuals and establish interventions that reflect the totality of need. these men do come home eventually, and their health service needs must be anticipated and accommodated, something not possible now because of parallel data systems. finally, it is important to begin the process of holding dedicated days in community clinics that invite boys and men so that their total health care and conditions, not just their prostate health, can be assessed and treated, as appropriate. and, we must expand those that research health access and analysis to include anthropologists and others in the social sciences to enable accurate delineation and enumeration of the issues, illumination of the pathways to reducing invisibility, and establishment as bedrock a rational, humane, equitable, and gender inclusive health-care system. the time to act is now. failure to act is an act that promulgates the current inequities. henrie m. treadwell https://orcid.org/0000-0003-4510-9774 survey shows reentry services halting across u the decision to exclude agricultural and domestic workers from the 1935 social security act ucla law covid-19 behind bars data project collecting missing demographic data is the first step to fighting racism in healthcare characteristics-of-remaining-uninsured-men-and-potentialstrategies-to-reach-and-enroll-them-in-health-coverage/ history state afdc rules regarding the treatment of cohabitors poverty, race, and the invisible men discerning disparities: the data gap characteristics of persons who died with covid-19 -united states key: cord-259852-skhoro95 authors: oboh, mary aigbiremo; omoleke, semeeh akinwale; imafidon, christian eseigbe; ajibola, olumide; oriero, eniyou cheryll; amambua-ngwa, alfred title: beyond sars-cov-2: lessons that african governments can apply in preparation for possible future epidemics date: 2020-08-18 journal: j prev med public health doi: 10.3961/jpmph.20.259 sha: doc_id: 259852 cord_uid: skhoro95 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has placed unprecedented pressure on healthcare systems, even in advanced economies. while the number of cases of sars-cov-2 in africa compared to other continents has so far been low, there are concerns about under-reporting, inadequate diagnostic tools, and insufficient treatment facilities. moreover, proactiveness on the part of african governments has been under scrutiny. for instance, issues have emerged regarding the responsiveness of african countries in closing international borders to limit trans-continental transmission of the virus. overdependence on imported products and outsourced services could have contributed to african governments’ hesitation to shut down international air and seaports. in this era of emerging and re-emerging pathogens, we recommend that african nations should consider self-sufficiency in the health sector as an urgent priority, as this will not be the last outbreak to occur. in addition to the regional disease surveillance systems enhancement fund (us$600 million) provided by the world bank for strengthening health systems and disease surveillance, each country should further establish an epidemic emergency fund for epidemic preparedness and response. we also recommend that epidemic surveillance units should create a secure database of previous and ongoing pandemics in terms of aetiology, spread, and treatment, as well as financial management records. strategic collection and analysis of data should also be a central focus of these units to facilitate studies of disease trends and to estimate the scale of requirements in preparation and response to any future pandemic or epidemic. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which was initially detected in wuhan, china in december 2019, has gradually snowballed into a worldwide pandemic [1] . it has placed unprecedented pressure on healthcare systems, pissn 1975 -8375 eissn 2233 even in advanced economies [1] . as of april 20, 2020, there were over 2.2 million confirmed cases globally and 152 551 recorded deaths [1] . although the numbers of cases and deaths reported in africa remain comparatively low [2] , this might be an underrepresentation of the disease burden due to the challenges of insufficient diagnostic testing kits and centres relative to the teeming high population of the continent. these inadequacies are major causes of concern for disease control efforts, given the potential for gradual but steady geometric inter-community and intra-community spread because the virus has a high reproductive number and a short serial interval [3] . given the various epidemic events that have previously oc-curred in africa, from ebola virus disease (evd) [4] to yellow fever, cholera, measles and lassa fever [5] , it would almost be safe to assume that african governments have prepared proactive measures against possible future epidemics. however, such proactive measures in africa towards pandemic situations still seem to be far-fetched, as demonstrated by the approach towards the novel sars-cov-2 pandemic. considering the high virulence of the pathogen and the rapid spread of the disease, there was a significant time lapse between the first confirmed case outside of china (thailand, on january 13, 2020) and confirmation of the first sars-cov-2 case in africa (specifically, in egypt on february 14, 2020) compared to other continents [6] . the question then arises that, beyond the current containment efforts by various african countries, could they have done better to prevent the spread of this viral infection into and within the continent based on previous experiences with other emerging and re-emerging pathogens? with the great risk of trans-continental transmission of pathogenic agents due to the high emigrational tendency of africans to western countries and vice versa (for greener pastures and/ or tourism), and dependency on imported products into the continent from countries in the global north, african leaders should have been more proactive in closing and/or reducing travel into the continent. a measure could have been applied to restrict travel even from countries with fewer than 100 confirmed sars-cov-2 cases given that the virus is highly transmissible, with a high reproductive number [3] . for example, nigeria's travel ban was implemented on march 18, 2020 (3 weeks after the first imported case) for 13 countries with more than 1000 cases at that time, despite convincing evidence regarding potential importation from other countries not listed, while a total airport ban came a week later (march 23, 2020) [7] . some proactive countries such as djibouti suspended all international flights before recording any cases of sars-cov-2 in the country. the authors are aware of the international health regulations, which frown at undue impositions of travel and trade restrictions due to a public health emergency of international concern. however, in the context of overwhelming evidence of an absent or inadequate national capacity to respond or cope with the epidemic, it is inevitable for countries to promptly wield international and local travel restrictions to slow down and reduce the peak of the epidemic. african countries, with their heavy reliance on their counterparts in the global north for medical supplies, should have purchased medical consumables, such as testing kits and per-sonal protective equipment, and should have initiated training of personnel and surveillance officers who will be needed in the event of an epidemic. it is worrisome, however, to observe different ministries of health from african countries scavenging for these supplies when they are already burdened with hundreds of active cases. apparently, borders and air spaces were left open, yet with little or no preparations made for the potential cases that were allowed into the countries. generally, the continent of africa suffers from deficiencies in human capacity and infrastructure, especially in medical and health research [8] . hence, without the availability of the necessary consumables, trained laboratory scientists, clinicians, nurses, and other health workers for effective case management and systematic contact tracing, there will still be a huge gap in the management of coronavirus disease 2019 (cov-id-19) pandemic in africa, with potentially disastrous consequences. evidence suggests that many african countries had a relatively weak epidemic preparedness and response capacity before the current outbreak [9] . these weaknesses have improved modestly, but not sufficiently to combat the covid-19 pandemic. despite the challenges mentioned above and the aftermath of the 2014 west african ebola crisis, the world bank supported the launching of the regional disease surveillance systems enhancement (redisse) with us$600 million to strengthen the health systems and disease surveillance in 16 west and central african countries. similarly, funds have been provided to support the african centers for disease prevention and control to build human capacity, provide guidelines, and coordinate epidemic prevention and response activities across the african continent. furthermore, other lessons learnt and builtup from the recent evd outbreak prompted the implementation of improved airport surveillance, temperature screening at points of entry, and training and scaling up of molecular diagnostic capacity for sars-cov-2 [10] . a few countries, such as nigeria, south africa, algeria, ghana, and egypt, initiated some of these measures somewhat earlier, but were not decisive about the implementation of travel restrictions or bans, thereby allowing the importation of sars-cov-2 virus into africa. some key lessons from the current outbreak of sars-cov-2 include the dire need to improve hospital facilities (e.g., intensive care unit upgrading and expansion through the procurement of ventilators and other life-saving equipment, upgrading emergency medical facilities, and improved diagnostic capacity), retention of highly skilled human resources, epidemic preparedness, sustained surveillance, and baseline health demographic data for chronic infectious and non-communicable diseases that could serve as potential risk factors for aggravated outcomes of new and emerging diseases. african leaders cannot afford to be complacent in responding to such issues given their previous encounters with evd, and now sars-cov-2. these experiences should serve as a wake-up call for african countries to prepare for possible future outbreaks. in conclusion, africa and the world at large will never remain the same after the covid-19 pandemic. therefore, the heads of states on the african continent should adopt preventive and precautionary measures rather than reactionary measures. in addition to the redisse fund (us$600 million) created by the world bank for strengthening health systems and disease surveillance, each country should further map out an epidemic emergency fund that will be used to address situations such as this in the future. further, we recommend that epidemic surveillance units, under the ministry of health, should be strengthened in all african countries. these units should be given the responsibility to create a secure database of previous and ongoing pandemics in terms of aetiology, spread, and treatment, as well as financial management records. strategic collection and analysis of data should also be a central focus of these unit to facilitate the analysis of disease trends and to estimate the scale of all necessary resources in preparation and response to any future pandemic or epidemic. this paper is a perspective, so it did not need ethical approval. coronavirus disease 2019 and influenza 2019-2020 africa centre for disease control and prevention. covid-19 scientific and public health policy update insight into 2019 novel coronavirus -an updated interim review and lessons from sars-cov and mers-cov ebola virus disease (evd): an unprecedented major outbreak in west africa infectious disease outbreaks in the african region: overview of events reported to the world health organization coronavirus disease 2019 (covid-19) situation report -26 update on clarification of flight restriction into nigeria due to covid-19 pandemic preparedness and vulnerability of african countries against importations of covid-19: a modelling study quagmire of epidemic disease outbreaks reporting in nigeria world health organization regional office for africa. who ramps up preparedness for novel coronavirus in the african region none. the authors have no conflicts of interest associated with the material presented in this paper. none. key: cord-305103-g0ndggwc authors: sood, lakshay; sood, vanita title: being african american and rural: a double jeopardy from covid‐19 date: 2020-05-03 journal: j rural health doi: 10.1111/jrh.12459 sha: doc_id: 305103 cord_uid: g0ndggwc nan the effect of covid-19 on african americans is better understood by analyzing the racial disparities related to previous pandemics caused by other types of coronaviruses. results from all the different studies of the 1918 spanish influenza pandemic indicate that african americans had higher mortality and case fatality rates than whites. 5 during the 2009 h1n1 influenza a pandemic, african americans had the highest overall susceptibility to complications arising from the infection, followed by whites and hispanics. 6 rural states like oklahoma documented highest hospitalization rates for african americans and lowest for whites. 6 these data are comparable to those for covid-19, where 33% of hospitalized patients nationwide (among those whose race was known) were african american, even though the latter constitute only 13% of the us population. 2 why are african americans at greater risk for covid-19, as compared to other racial/ethnic groups? there are 3 possible explanations: 1) social determinants of health; 2) comorbidities and coexposures; and 3) genetic differences. furthermore, why may rural african americans be at even greater risk than urban african americans? this health inequity is largely attributable to social determinants of health. the who defines social determinants of health as "the conditions in which people are born, grow, live, work, and age. these circumstances are shaped by the distribution of money, power, and resources at global, national and local levels. 7 8 with lower median incomes for rural than urban african american households. 9 although data for covid-19 are not known, previous analyses indicate that influenza-related hospitalization was higher for persons residing in census tracts with a greater level of poverty. 10 many african americans reside in small and/or multi-generational homes with extended families, use public transportation to commute to jobs and other places, and many of them are "essential" workers (like janitors and delivery workers), which means face-toface contact with many people (often without adequate personal protective equipment). this makes social distancing and self-isolation virtually impossible, and it increases the risk of exposure to sars-cov-2. the southeast, which has the highest concentration of african americans in the country, has more cash-poor adults without health care coverage when compared to other regions. 11 within this region, there are racial disparities in health care coverage that disproportionately affect african americans. 11 this implies that african americans in the southeast are less likely to afford covid-19 screening tests and treatments than other populations nationwide. given that about a quarter of rural hospitals may shut down due to the covid-19 pandemic, 12,13 mostly in the southeast and lower great plains, rural african americans will have difficulty accessing preventive and therapeutic services. many african americans do not have access to a primary care provider. 14 without a primary care provider, they are more likely to fall victim to misinformation about covid-19, unnecessarily use emergency rooms, or use them too late. 15 in new york city, african americans without providers could not receive covid-19-related help even from the city's 311 non-emergency line, which was overwhelmed during the outbreak. 16 african americans, particularly in the rural southeast, 17 are often suspicious of the health system, with a legacy of abuses such as the 1932-1972 tuskegee syphilis study in rural alabama, in which exclusively african american participants were allowed to die untreated. 18 unfortunately, the us health system has been shown repeatedly to offer inferior care to african americans with the same conditions and insurance as white patients, indicating a subconscious racial bias in health care. 2 as compared to whites, african americans consume less fruits and vegetables and engage in less exercise. 19 these lifestyle disparities, rooted in unequal access to economic and social resources, are even more pronounced for rural african americans. 20, 21 although dietary risk factors for covid-19 are not understood, frequent intake of fruits and vegetables protects against influenza-like illnesses, 22 and regular and moderate exercise protects against influenza-related cardiorespiratory mortality. 23 the likelihood of having obesity, diabetes, hypertension, and cardiovascular disease is higher for african americans than for whites. [24] [25] [26] [27] these diseases are also established risk factors for covid-19 prevalence and mortality. 28, 29 further, as compared to non-rural communities, rural communities may have a greater prevalence of these conditions. [30] [31] [32] for example, rural african american heart disease mortality is among the highest ever recorded anywhere in the world. 20 these comorbidities are associated with high levels of inflammatory cytokines, which may contribute to the increased morbidity and mortality in covid-19. 33 based on a 2015 national survey, 20.9% of african american men smoke, as compared to 17.3% of white men. 34 smokers have increased levels of angiotensin converting enzyme ii (ace2) receptors in their airway epithelia, which is the entry receptor for sars-cov-2, making smokers more susceptible to covid-19. 35 consistent with this information, smokers are 1.4 times more likely to have severe symptoms of covid-19 and approximately 2.4 times more likely to be admitted to an icu, need mechanical ventilation, or die, as compared to non-smokers. 36 based on a 2005-2007 national survey, marijuana use disorder is greatest among african americans compared to other races/ethnicities. 37 although not proven, it is possible that marijuana use may affect the ace2 receptor like cigarette smoke, predisposing users to census tracts with a higher percentage of african american population have higher levels of outdoor air pollution. 39 small particulate matter of less than 2.5 microns in aerodynamic diameter, also known as pm 2.5 , are particularly associated with adverse health outcomes. in a nationwide study using data from 3,000 counties in the us, an increase of only 1 g/m 3 in pm 2.5 was associated with a 15% increase in the covid-19 death rate, 40 suggesting that long-term exposure to air pollution, particularly in african american communities, increases their vulnerability to experiencing severe covid-19 outcomes. although the genetic predisposition to covid-19 has not been specifically studied, there is limited evidence to suggest that genetic differences underlie the racial susceptibility to viral infections. a recent study, mapping the expression quantitative trait loci (eqtls), demonstrates that there is extensive variation in transcriptional responses to immune challenges between individuals of african and european descent. the strongest difference in transcriptional response is observed for genes with antiviral and inflammatory-related functions. 41 in an in vitro study using macrophages, many of the genes showing european versus african ancestry-related transcriptional differences in isoform usage were in fact key regulators of innate immunity. 42 one such gene included the oas1 gene that encodes the rural african americans and this article is protected by copyright. all rights reserved. 6 isoforms with varying enzymatic activity against viral infections. 42 this limited data may help explain the genetic basis to the predisposition to viral infections in african americans. the primary goal of the pandemic containment in rural african american communities is to reduce the sars-cov-2 virus transmission. coronavirus was slow to spread to rural america. not anymore covid-19: black people and other minorities are hardest hit in us racial disparities in louisiana's covid-19 death rate reflect systemic problems the coronavirus is infecting and killing black americans at an alarmingly high rate race and 1918 influenza pandemic in the united states: a review of the literature racial disparities in exposure, susceptibility, and access to health care in the us h1n1 influenza pandemic world health organization. social determinants of health economic policy institute. the state of working america, 12th edition comparison of rural and urban america: household income and poverty influenza-related hospitalizations and poverty levels -united states the coverage gap: uninsured poor adults in states that do not expand medicaid a surge of coronavirus patients could stretch hospital resources in your area 1 in 4 rural hospitals are at risk of closure and the problem is getting worse racial disparities in geographic access to primary care in philadelphia caring for miners during the coronavirus disease-2019 (covid-19) pandemic. j rural health how to save black and hispanic lives in a pandemic perception of racial barriers to health care in the rural south african americans' views on research and the tuskegee syphilis study racial/ethnic disparities in exercise and dietary behaviors of middle-aged and older adults cardiovascular disease among women residing in rural america: epidemiology, explanations, and challenges considering intersections of race and gender in interventions that address us men's health disparities various factors associated with the manifestation of influenza-like illness effect of lifestyle factors on risk of mortality associated with influenza in elderly people disparities in diabetes: the nexus of race, poverty, and place racial disparities in cardiovascular disease risk: mechanisms of vascular dysfunction racial differences in hypertension: implications for high blood pressure management. the american journal of the medical sciences exploring racial disparity in obesity: a mediation analysis considering geo-coded environmental factors covid-19: risk factors for severe disease and death risk factors for severity and mortality in adult covid-19 inpatients in wuhan united states department of health and human services. cdc newsroom. rural americans at higher risk of death from five leading causes urban-rural differences in coronary heart disease mortality in the united states the determinants of activity and specificity in actinorhodin type ii polyketide ketoreductase obesity and its implications for covid-19 mortality. obesity (silver spring) american lung association. tobacco use in racial and ethnic populations ace-2 expression in the small airway epithelia of smokers and copd patients: implications for covid-19. the european respiratory journal covid-19 and smoking: a systematic review of the evidence race/ethnicity differences between alcohol, marijuana, and co-occurring alcohol and marijuana use disorders and their association with public health and social problems using a national sample cannabis-related illness in missouri emergency room racial, ethnic, and income disparities in air pollution: a study of excess emissions in texas exposure to air pollution and covid-19 mortality in the united states genetic adaptation and neandertal admixture shaped the immune system of human populations genetic ancestry and natural selection drive population differences in immune responses to pathogens a commentary on rural-urban disparities in covid-19 testing rates per 100,000 and risk factors why coronavirus could hit rural areas harder covid-19 and health care's digital revolution too important to ignore: leveraging digital technology to improve chronic illness management among black men key: cord-279011-arjzx85c authors: ibrahimi, sahra; yusuf, korede k.; dongarwar, deepa; maiyegun, sitratullah olawunmi; ikedionwu, chioma; salihu, hamisu m. title: covid-19 devastation of african american families: impact on mental health and the consequence of systemic racism date: 2020-09-16 journal: int j mch aids doi: 10.21106/ijma.408 sha: doc_id: 279011 cord_uid: arjzx85c african americans are bearing a disproportionate burden of morbidity and mortality due to covid-19 pandemic. to our knowledge, no previous study has delineated inequities potentially incentivized by systemic racism, and whether synergistic effects impose an abnormally high burden of social determinants of mental health on african american families in the era of covid-19 pandemic. we applied the social ecological model (sem) to portray inequities induced by systemic racism that impact the mental health of african american families. in our model, we identified systemic racism to be the primary operator of mental health disparity, which disproportionately affects african american families at all levels of the sem. programs tailored towards reducing the disproportionate detrimental effects of covid-19 on the mental health of african americans need to be culturally appropriate and consider the nuances of systemic racism, discrimination, and other institutionalized biases. covid-19 pandemic has further unveiled the distressful reality of racial disparity in the united states. african americans are bearing a disproportionate burden of morbidity and mortality due to covid-19. in many cities like chicago, although african americans are only 30% of the population, they make up more than 50% of covid-19 cases and about 70% of covid-19 deaths. 1 covid-19 health and economic consequences have a detrimental effect on mental health, 2 particularly among african americans. 3 according to kaiser health news report, compared with the rest of the population, african american adults are 20% more likely to experience mental health issues. systemic racism exacerbates the adverse impacts of social determinants of health, causing racial health disparities in african americans. 2 african americans are more susceptible to contract covid-19 due to systemic racism that historically carved out the type of work they are obliged to perform including deliberate neighborhood and school segregation policies which currently explain their increased work place and environmental exposure to covid-19 infection. as a result, a greater proportion of african americans now suffer from the stress, anxiety and depression engendered by covid-19. no previous study has delineated inequities incentivized by systemic racism, and whether synergistic effects impose an abnormally high burden of social determinants of mental health on african american families in the era of covid-19 pandemic. in this study, we hypothesized systemic racism to be the primary operator of the mental health disparity, which disproportionately affects the psychological well-being of african american families at all levels of the socio-ecological model (sem). a rapid search of the literature on covid-19 pandemic, the social determinants of mental health, systemic racism, as well as their impact on the mental health of african american families was conducted in pubmed and google scholar. next, the sem was employed to portray inequities induced by systemic racism, which aggravates the adverse effect of social determinants of mental health on african american families. the sem considers the complex interplay across individual, interpersonal, organizational, community, and public policy level factors. 4 the model emphasizes that behaviors and the social environment have a symbiotic relationship and impact each other. 4 the rapid search yielded 30 research articles and only seven of them were included in this review. we found that covid-19 disproportionately impacts african american families and their mental health due to systemic racism, which intensifies the adverse effects of the social determinants of mental health. figure 1 represents a social ecological model that delineates social determinants of mental health aggravated by systemic racism among african american families in the era of covid-19 pandemic. from the sem we found that at the individual level, african americans may experience stress from perceived knowledge of covid-19 that may be inaccurate and may stem from biased information presented by the local media and other social media networks. misinformation and more than 10 conspiracy theories have been trending since the beginning of the pandemic and have led to confusion and stress. 5 misinformation is more likely to trend among communities with limited access to quality educational resources. studies show that majority of african americans are systemically disadvantaged from proper education, distinctly afforded by asians and whites. 6 therefore, misinformation may be more likely to prey on african americans than caucasians. at the interpersonal level, heightened stress and fear may also originate from family members and relatives whose lives and jobs are affected by covid-19. because black families have been impacted disproportionally, they likely experience more stress than whites. according to the centers for disease control and prevention (cdc), african americans are less than 13% of the population but make up 33.1% of hospitalized covid-19 patients. 1 at the organizational level, african americans endure discrimination and racism at school and or work, while at the community level, african americans lack access to equitable mental healthcare, healthy food options, and recreational facilities. 6 covid-19 health problems in the african american community manifest not because african americans do not like to social distance or take care of themselves, but because resources are abominably inadequate in their neighborhood. finally, at the public policy level, african americans are disadvantaged by segregated housing and lack of access to equitable education and job opportunities. compared with caucasians, african americans are more likely to live in densely populated areas, increasing their potential contact. 7 as the number of contacts increases, so does the anxiety of being exposed to covid-19. in addition, african americans make up the majority of the "essential" workforce, including 30% of bus drivers, and 20% of food service workers. 6 these structural conditions exacerbate the impact of covid-19 on mental health as consequences of stress, fear, anxiety, and despair, which in severe cases result in posttraumatic stress disorder (ptsd) and or depression. 2 covid-19 has impacted african american lives, including their mental health, at a higher rate compared to other racial/ethnic groups. 1 as we had hypothesized, we found systemic racism to be the primary operator of the mental health disparity, which disproportionately affects the mental health of african american families at all levels of the sem. as a result of systemic racism that affects african americans at different levels, the standard mental health promotion approaches may not work in this population. studies show that public policies and health interventions that are tailored and adapted for local contexts and community characteristics are more beneficial and effective than standard approaches. 2 programs tailored towards improving the mental health of african americans should be culturally appropriate and community competent. they also need to consider the nuances of systemic racism, discrimination, and other institutionalized biases at each level of the sem in order to reduce the disproportionate detrimental effects of covid-19 on the mental health of african americans. more importantly, policymakers and program implementers need to develop laws and programs that would dismantle systemic racism. public policies have the power to mitigate the impact of covid-19 among african americans and improve their mental health outcomes or to exacerbate health disparities. interpersonal level (il): stress from family members and relaɵves whose lives and jobs may be affected by covid-19. stress from perceived knowledge that may be inaccurate and/or bias informaɵon on media and other social media networks. • african americans are bearing a disproportionate burden of morbidity and mortality due to covid-19 pandemic. • systemic racism is the primary operator of mental health disparity, which disproportionately affects african american families at all levels of the social ecological model (sem). • programs tailored towards reducing the disproportionate detrimental effects of covid-19 on the mental health of african americans need to be culturally appropriate and consider the nuances of systemic racism, discrimination, and other institutionalized biases. hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019 -covid-net, 14 states the covid-19 pandemic and mental health impacts black history month and african american mental health statistics alliance for science. covid: top 10 current conspiracy theories why are blacks dying at higher rates from covid-19? key: cord-273275-f7rbn88x authors: alkhatib, ala l.; kreniske, jonah; zifodya, jerry s.; fonseca, vivian; tahboub, mohammad; khatib, joanna; denson, joshua l.; lasky, joseph a.; lefante, john j.; bojanowski, christine m. title: bmi is associated with coronavirus disease 2019 intensive care unit admission in african americans date: 2020-08-04 journal: obesity (silver spring) doi: 10.1002/oby.22937 sha: doc_id: 273275 cord_uid: f7rbn88x objective: coronavirus disease 2019 (covid‐19) has disproportionately impacted the african american community. this study aims to identify the risk factors for severe covid‐19 disease in african american patients. methods: this was a retrospective cross‐sectional analysis of african american patients with covid‐19 treated between march 12 and april 9, 2020, at a single tertiary center. the primary outcome of interest was severe disease defined as those requiring intensive care unit (icu) admission. results: the study included 158 consecutive patients. the mean age was 57 years, and 61% were women. the mean (sd) of bmi was 33.2 (8.6) kg/m(2). overall, patients admitted to the icu were older (62 vs. 55 years, p = 0.003) and had higher bmi (36.5 kg/m(2) vs. 31.9 kg/m(2), p = 0.002). in unadjusted and adjusted analysis, the factors most associated with icu admission in this sample were age (adjusted odds ratio [aor]: 1.073; 95% ci: 1.033‐1.114), bmi (aor: 1.115; 95% ci: 1.052‐1.182), and lung disease (aor: 3.097; 95% ci: 1.137‐8.437). conclusions: this study identified risk factors for severe disease in covid‐19, specifically in an african american population. further inclusive research aimed at optimizing clinical care relevant to the african american population is critical to ensure an equitable response to covid‐19. by january 7, 2020, the novel severe acute respiratory syndrome coronavirus 2 was identified as the cause for an outbreak in china due to what is now called coronavirus disease 2019 (covid-19). on march 11, 2020 , the world health organization declared the covid-19 outbreak a global pandemic (1) . the severity and clinical presentation of covid-19 are widely variable, ranging from asymptomatic or mild disease in the majority of cases to severe respiratory failure and dysregulated inflammatory responses in a minority of patients. age and certain comorbidities, such as hypertension and diabetes, have been well identified as risk factors for the development of severe disease (1) (2) (3) . obesity is also emerging as a likely risk factor for severe disease development (4) (5) (6) . the covid-19 epidemic in the united states tracks along well-documented and historical health disparities, with early data suggesting disproportionate morbidity and ► there is a wide clinical spectrum of disease in coronavirus disease 2019 (covid-19), ranging from asymptomatic and mild cases to profound critical illness and respiratory failure. ► age and specific comorbidities such as hypertension and diabetes mellitus have been identified as independent risk factors for severe covid-19. what does this study add? ► major health disparities have been identified in the covid-19 epidemic within the united states, and this is one of the first studies to focus specifically on the risk factors within an african american population, a community that has been disproportionately impacted by this disease. ► in addition to age, obstructive lung disease, and hypertension, this study adds further evidence supporting the association between bmi and disease severity in covid-19. how might these results change the direction of research or focus of clinical practice? ► current centers for disease control and prevention recommendations describe individuals with bmi > 40 as being at "high risk" for severe illness in covid-19, whereas our results suggest that a lower threshold should be considered for this high-risk category in covid-19. ► this study calls for further mechanistic studies aimed at understanding the association observed between obesity and the severity of respiratory disease and the inflammatory response. see commentary, pg. x. mortality within the african american community (7, 8) . despite this, there is a relative dearth of analysis relevant to the african american experience. in louisiana, 55 % of covid-19-related deaths have occurred among black persons, who represent 32.7% of the state's population (9, 10) . new orleans, a predominantly african american city, has experienced among the most severe outbreaks of covid-19 in the united states. in this study, we aim to describe the baseline characteristics of laboratoryconfirmed covid-19-positive african american patients and determine the possible risk factors, including bmi, for the development of severe disease and admission to the intensive care unit (icu). this is a single-center retrospective cross-sectional study of all consecutive, self-reported african american patients confirmed to have covid-19 who presented to a tertiary academic hospital between march 12 and april 9, 2020. patients were identified through reported positive laboratory test results during this specified time period, and individual patient data were obtained through retrospective electronic medical record review by members of the investigatory team. this study was reviewed and approved by the tulane university biomedical institutional review board and was granted a waiver of consent. the primary outcome of this study was severe disease, defined here as those patients requiring icu admission for covid-19-related complications. in our study, this aligned closely with respiratory failure requiring mechanical ventilation. individuals with nonsevere disease were defined as patients who presented to the emergency department or who were admitted to the hospital and successfully discharged home without need for further escalation of care. patients with missing data or pending covid-19-confirmatory testing were excluded from the study. several covariates of interest were collected, including age, sex, bmi reported in kilograms per meter squared as calculated on admission through measurements of both weight and height, and the comorbidities diabetes mellitus (dm), hypertension, chronic kidney disease (ckd), congestive heart failure, and obstructive lung disease (including both chronic obstructive pulmonary disease and asthma). comorbidities were identified if the patient had a medical record with an established diagnosis, was on medications known to treat these comorbidities, or had previously reported laboratory values, such as an estimated glomerular filtration rate < 60 to define ckd. means and sd were calculated for age and bmi separately for non-icu and icu cases, and two-sample t tests were used to test for significant differences in these means. categorical patient characteristics were summarized with counts and percentages. pearson χ 2 tests were used to test for significant percentages among the non-icu and icu cases. multivariable logistic regression analysis was performed to examine the association between bmi and critical illness in patients with covid-19, adjusting for the potential effects of the covariates of age, sex, dm, hypertension, congestive heart failure, ckd, and obstructive lung disease (both chronic obstructive pulmonary disease and asthma). results were presented as odds ratios (or) with 95% cis and p values. data were statistically analyzed using sas software version 9.4 (sas institute inc., cary, north carolina). of the screened consecutive 183 patients with positive covid-19 test results, 158 were african american and were enrolled in our study. the mean age was 57, and 61% were women. the mean (sd) bmi was 33.2 (8.6). characteristics of the study population based on the disease severity are presented in table 1 . eighty-five percent of the severe cases admitted to the icu had respiratory failure requiring intubation and mechanical ventilation. icu mortality for severe cases was 37%; however, 21.7% of the patients were still intubated and requiring mechanical ventilation at the time of analysis, which may impact and potentially increase this percentage. patients with severe disease requiring icu admission were older (62 vs. 55 years, p = 0.003) and had higher bmi (36.5 vs. 31.9, p = 0.002). the prevalence of dm and ckd was also significantly higher in patients with severe disease (p values of 0.016 and 0.012, respectively). thirty-nine of the forty-six patients admitted to the icu during this study period were in respiratory failure requiring mechanical ventilation. an additional two patients required high-flow nasal canula, and an additional three patients required the use of a nonrebreather mask. on the basis of the available data, the average ratio of the partial pressure of oxygen to the fraction of inspired oxygen of patients admitted to the icu was 171.1 (n = 42), which is consistent with moderate acute respiratory distress syndrome; the average admission lactate dehydrogenase level was 500.5 (n = 43); the average admission ferritin level was 2,266.2 (n = 41); and the average high-sensitivity c-reactive protein level was 11.52 (n = 41). in adjusted analysis, hypertension, age, bmi, and obstructive lung disease are independent risk factors for severe disease in adjusted analysis ( in this retrospective observational cross-sectional study, we found that age, higher bmi, and obstructive lung disease were associated with severe covid-19 in an african american population. these results were unchanged after adjusting our analysis for age, sex, and underlying comorbidities. obesity www.obesityjournal.org obesity | volume 00 | number 00 | month 2020 3 brief cutting edge report in early studies of the international experience, obesity was not evaluated as an independent risk factor for critical illness; however, recent analyses regarding the covid-19 pandemic in europe and the united states suggest that obesity is highly prevalent among patients suffering from severe covid-19 (1-6, 11). as a recognized independent risk factor for severe respiratory illness in viral pneumonias such as h1n1 (12) , special attention to the needs of patients with obesity is urgently needed as the covid-19 pandemic spreads to communities with a high prevalence of obesity. current centers for disease control and prevention recommendations describe individuals with bmi > 40 as being at "high risk" for severe covid-19 illness, whereas our results suggest that a lower threshold should be considered for this high-risk category. a significant knowledge gap remains regarding the mechanisms underlying the association between viral respiratory disease severity and the cluster of illnesses that define metabolic syndrome. a severe inflammatory cytokine response appears to be the hallmark of severe covid-19. to this point, obesity is associated with low-grade inflammation associated with insulin resistance and diabetes and therefore may lead to the amplification of the inflammatory response to infection (13) . further studies looking into the mechanisms behind these observations are ongoing. a recently published report from the centers for disease control and prevention showed that a third of hospitalized patients with covid-19 in the united states were african american (14), evidencing a disproportionate impact on the african american community. further inclusive research toward optimizing clinical care relevant to the african american population is critical to ensuring an equitable response to covid-19. previous studies have identified obesity as a risk factor for severe disease in several populations (4) (5) (6) . our analysis confirmed that obesity is a significant determinant of disease severity in an african american population. these data further support the utility of bmi in the identification of patients at high risk of severe covid-19. appropriate management of obesity and the associated comorbidities of metabolic syndrome, as well as of the social determinants of health that underlie these conditions, is therefore critical to addressing the evolving covid-19 crisis.o funding agencies: this work was supported by a grant from the louisiana clinical and translational science center (u54 gm104940 to cmb). disclosure: the authors declared no conflict of interest. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study covid-19 in critically ill patients in the seattle region -case series critical care utilization for the covid-19 outbreak in lombardy, italy: early experience and forecast during an emergency response obesity in patients younger than 60 years is a risk factor for covid-19 hospital admission high prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (sars-cov-2) requiring invasive mechanical ventilation obesity is a risk factor for greater covid-19 severity covid-19 and african americans assessing differential impacts of covid-19 on black communities louisiana department of health. coronavirus (covid-19) epidemiology of covid-19 in a long-term care facility in king county, washington factors associated with death or hospitalization due to pandemic 2009 influenza a (h1n1) infection in california inflammation and emerging risk factors in diabetes and atherosclerosis hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019 -covid-net key: cord-316209-juvmabdq authors: rutayisire, erigene; nkundimana, gerard; mitonga, honore k.; boye, alex; nikwigize, solange title: what works and what does not work in response to covid-19 prevention and control in africa date: 2020-06-12 journal: int j infect dis doi: 10.1016/j.ijid.2020.06.024 sha: doc_id: 316209 cord_uid: juvmabdq abstract since the emergence of the pandemic in december 2019 in wuhan, china, as of 10:00 cest, june 6, 2020, there have been nearly 6,663,304 confirmed cases of covid-19 including 392,802 deaths worldwide. in africa, as of june 2, 2020, a total of 152,442 covid-19 cases and 4334 deaths have been reported. the five countries with the highest commutative number of cases in africa are south africa, egypt, nigeria, algeria, and ghana. africa, together with the rest of world have had to swiftly undertake measures necessary to protect the continent from irreversible effects of the covid-19 pandemic that is claiming lives and destroying livelihoods. the lower number of covid-19 cases in most african countries is attributed to inadequate health systems, low-to-absent testing capacity, poor reporting system and insufficient number of medical staff. the covid-19 pandemic poses a great threat to most african countries from cities to rural areas and has created a strong demand on already scarce resources and requires an intense mobilization of additional resources to implement established emergency contingency measures. closure of borders and movements of people restrictions within the country as measures to prevent the spread of covid-19; this has resulted in the sector being adversely affected by the loss of income. cooperative prevention and control measures are one of the promising solutions to deplete the spread of covid-19 on the continent. since its emergence in december 2019 in wuhan, china, there have been nearly 6,663,304 confirmed cases and 392,802 deaths related to coronavirus disease as reported from 215 countries and territories (who situation report-138, 2020) . on 11 march 2020, the world health organization (who) had declared covid-19 a pandemic, pointing to the over 118,000 cases of the coronavirus illnesses in over 110 countries and territories around the world and the sustained risk of further global spread. the rapidly evolving covid-19 pandemic places a heavy burden on health care systems. this burden is projected to become worse in low and middle income countries already struggling with weak health-care systems, scarce financial resources as well as protective equipment, poor testing and treatment capacities, and lack of research funding (betsch et al.,2020) . low and middle income countries need enormous global support to prepare for impending crisis (the lancet, 2020) and identify where they can allocate more resources to prevent and control covid-19. as of 9 am eat 2 june 2020, a total of 152442 covid-19 cases and 4334 deaths have been reported in 54 african countries. this is about 2.5 of all cases reported globally. since the last brief on may 26 th from africa cdc, the number of covid-19 cases has increased by 32% (37096 cases), this shows the burden covid-19 could impose on african countries. as of june 2 nd , the five countries with the highest cumulative number of cases are as of south africa (34357 cases), egypt (26384 cases), nigeria (10578 cases), algeria (9513 cases), and ghana (8070 cases) (africa cdc, outbreak brief-20, 2020) . comparatively low positive cases of covid-19 in africa is attributed to low-to-absent testing capacity, poor reporting system, and insufficient number of medical staff. evidently, covid-19 poses a great threat to most african countries from cities to rural areas (lucero-prisno et al. 2020) ; it has created a strong demand on already scarce resources and requires an intense mobilization of additional resources to support local emergency contingency measures in compliance with who and africa cdc recommendations and directives. the africa continent, as with the rest of the world, continues to confirm additional cases of covid-19. up to now, the covid-19 has no effective treatment and there are no available vaccines and can spread from both asymptomatic and symptomatic cases. covid-19 is the type of infectious disease that is highly transmissible, crosses borders and threatens countries health j o u r n a l p r e -p r o o f 5 and global economy. patients with covid-19 especially those with comorbidities may develop severe disease and experience adverse outcomes creating additional burden to healthcare systems in place. that is why, the authorities specifically in africa have the duty to respond to this pandemic with effective and appropriate interventions, policies and messages. at present in order to protect citizens' health, most of the african countries have activated their national health emergency management committees, special committees on covid-19 response that are mostly chaired by ministers of health. as new evidence become available, african countries continue to share experiences and effective strategies in order to improve covid-19 prevention and control in solidarity. africa centers for diseases control and prevention (africa cdc), world health organization and other international agencies are providing support and guidance to many african countries in response to covid-19 pandemic. since the early stage of covid-19 in africa, many african countries have received facemasks, ventilators, test kits and other medical equipment from different countries and international agencies. specifically, africa cdc is providing guidelines on contact tracing, community social distancing, africa joint continental strategy for covid-19 outbreak and weakly scientific and public health policy updates (africa cdc: outbreak brief #20). african union has established africa taskforce for novel coronavirus (aftcom) as a continental platform to better coordinate all covid-19 prevention measures across the continent. through an established task force, africa cdc supports affected countries in surveillance by proving remote technical support to the african union member states (africa cdc, feb 2020). most african countries appreciated the importance of lockdown, closure of borders and restrictions on people's movement within the country as measures to prevent the spread of covid-19. the lockdown has ranged between 14-60 days in most african countries. as cases in the continent continue to rise, member states have continued to extend imposed public health measures including total lockdown in senegal, sierra leone, and zimbabwe. mandatory wearing of masks in public is also going on in botswana and rwanda. other member states allow partial reopening of the economy and/or schools including benin, botswana, cameroon, lesotho, djibouti, nigeria and burkina faso. but, precautionary measures, such as wearing facemasks, use of hand sanitizer and gloves while maintaining social distancing remain in place. in response to africa cdc recommendation to reduce the spread of covid-19, 43 african countries have closed their borders, 7 closed international air traffic, 2 imposed travel restrictions to and from specific countries and 3 imposed entry/exit restrictions. in addition, some african to protect vulnerable citizens' health, some african governments have allocated resources to cover their basic needs. for example, zimbabwe had budgeted over $ 600 million for vulnerable households under a cost transfer program for the next 3 months; rwanda and ghana took 7 initiatives of providing food and other primary needs to needy population hit hard by covid-19 pandemic. in namibia, the government initiated the emergency income grant (eig) amounting to n$562.0 million. the grant is a once-off payment of n$750.00 in cash grant per qualifying person, on the basis of a set of eligibility criteria properly defined for vulnerable people. it is estimated to benefit up to 739,000 namibians. countries commit to leave no solution unexplored to ensure a healthy recovery from covid-19 and pursue return to work strategies once the disease is under control. unfortunately, african countries are not complying at the same level to the covid-19 prevention and control measures. disparity in responses to the pandemic across africa countries are linked to the different timing in the start of the diseases, existing lack of adequate resources for the health care, poor public health systems and community ignorance. despite reported low case-fatality of covid-19, the pandemic is likely to cause more deaths in africa if the compliance to covid-19 prevention and control measures continues to be ignored as observed in some african countries. there is a growing concern that covid-19 could spread further and heavily hit the african continent (wafaa me., & justman j.,2020), due to the existing fragile health care and public health systems, inadequate health care infrastructure, lack of access to safe water and sanitation, lack of food safety and political instability. in africa, one factor that could mitigate covid-19 related mortality is its very young population demography; in fact, more than 50% of the african population is under the age of 20 years old. however, this group of people is also surrounded by many problems including poverty, food insecurity, illiteracy and unemployment among others. lockdown policies may put them at greater risk of getting covid-19, lack of access to food may force them not to stay home, oblige them to go out for survival and thereafter get infected and eventually die of covid-19. experience from asia, europe and usa showed that people with existing health problems are most vulnerable to severe cases of covid-19. the burden of health problems in africa is proportionately higher than the rest of the world. consequently, the higher prevalence of j o u r n a l p r e -p r o o f 8 malnutrition, anemia, malaria, hiv/aid, and tuberculosis in many african countries may coincide with and worsen the ongoing covid-19 pandemic prevention and control measures in africa. taking into account the technical advice from africa cdc, and who, most countries have taken covid-19 pandemic seriously; cases are identified, tested and treated coupled with contact tracing in many african countries. however, there is always a delay in contact-tracing where some patients are still found in the general population having tested positive or becoming extremely difficult to trace along with their contacts. despite the message of self-isolation and toll free number for those with covid-19 symptoms, people in some african countries still tend to disregard the symptoms until they become severely ill. this thus increases the risk to family members in contact and the community in general. it was observed that in some african countries, people are resisting to testing over quarantine fears. for example, kenyans are resisting or simply not turning up for covid-19 testing. the main reasons fronted for this behavior are that the residents are terrified of the prospect of being found to be infected, which in turn would mean being quarantined and self incurring all quarantine costs. this resistance greatly hinders africa governments planning and interventions. considering the existing economic conditions of african citizens, the costs of quarantine are supposed to be paid by the government. african countries are struggling to increase diagnostic capacity, improve infection prevention control (ipc) as well as manage confirmed cases as need arises. if the cases continue to increase, many african countries will not be able to manage those cases; there is a need for international cooperation to reduce the burden this disease will impose on african countries. with the spread of the disease, the pandemic is dismantling gains in the social-economic fabrics of all nations and societies, and it is exposing and deepening -further emphasizing the unsustainability of previously existing weaknesses, including poverty and inequality. some people in africa have resisted staying home policies mainly due to cultural and religious beliefs. measures to impose social and physical distancing have proven to be more challenging in african countries such as senegal and tanzania (world economic forum, march 2020). it was j o u r n a l p r e -p r o o f 9 proven that during covid-19 prevention and control measures, poor people are mostly affected. for example, people demonstrated in kenya and south africa due to lack of food and sent out a clear message that they prefer to die from covid-19 instead of hunger. moreover, compliance with recommended social distancing is still a problem in some places such as public markets, banks and refugee camps. in most public markets in africa, sellers are not concerned about the risk of getting covid-19, they are neither wearing masks nor using hand washing soap, water and hand sanitizers and sellers seem only to be interested in getting money from buyers. hence, we advise africa union member states to revisit their policies by allowing a small number of people, as it is implemented in namibia, to enter in such markets while others are waiting outside of the facility and keeping the social distancing of about 1.5 meter, providing hand washing facilities and hand sanitizers. there is also a need to continue to inform the public on the importance of adherence to social distancing measure to prevent and control covid-19 (lucero-prisno et al. 2020) . in order to ease the lockdown, african governments need to make sure that the spread of covid-19 is mitigated by ensuring that people comprehend the significance of social distancing as well as wearing facemasks. african countries are battling to augment diagnostic capacities, improve ipc as well as manage confirmed cases as need arises. if new reported cases persist, numerous african countries will be unable to handle them effectively. aggressive prevention measures are one of the strategies that africa should use to prevent more covid-19 cases and deaths in coming months. cooperative prevention and control measures are one of the promising solutions to deplete the spread of covid-19 on the continent. not needed monitoring behavioural insights related to covid-19 the lancet: covid-19 will not leave behind refugees and migrants covid-19) pandemic date of issue: 2 communique by the emergency meeting of africa ministers of health on the coronavirus disease outbreak "coordinated actions to prepare and respond to covid-19 infection in africa guidance on community social distancing during covid-19 outbreak scientific and public health policy update -31 recommendations for stepwise response to covid-19 outbreak/ health department republic of south africa: corona virus (covid-19 world economic forum: why sub-saharan africa needs a unique response to covid-19 current efforts and challenges facing responses to 2019-ncov in africa africa in the path of covid-19 who coronavirus disease (covid-19) situation reports none declared key: cord-151532-mpv2wegm authors: peng, kerui; safonova, yana; shugay, mikhail; popejoy, alice; rodriguez, oscar; breden, felix; brodin, petter; burkhardt, amanda m.; bustamante, carlos; cao-lormeau, van-mai; corcoran, martin m.; duffy, darragh; guajardo, macarena fuentes; fujita, ricardo; greiff, victor; jonsson, vanessa d.; liu, xiao; quintana-murci, lluis; rossetti, maura; xie, jianming; yaari, gur; zhang, wei; lees, william d.; khatri, purvesh; alachkar, houda; scheepers, cathrine; watson, corey t.; hedestam, gunilla b. karlsson; mangul, serghei title: diversity in immunogenomics: the value and the challenge date: 2020-10-20 journal: nan doi: nan sha: doc_id: 151532 cord_uid: mpv2wegm with the recent advent of high-throughput sequencing technologies, and the associated new discoveries and developments, the fields of immunogenomics and adaptive immune receptor repertoire research are facing both opportunities and challenges. the majority of immunogenomics studies have been primarily conducted in cohorts of european ancestry, restricting the ability to detect and analyze variation in human adaptive immune responses across populations and limiting their applications. by leveraging biological and clinical heterogeneity across different populations in omics data and expanding the populations that are included in immunogenomics research, we can enhance our understanding of human adaptive immune responses, promote the development of effective diagnostics and treatments, and eventually advance precision medicine. analyses 2 . this limits the discovery of genetic diversity contributing to mendelian diseases and to explore associations between genetic variants and trait variation across populations. in recent years, awareness has been increasing about the limited generalizability of findings across populations, motivating the inclusion of diverse, multiethnic populations in large-scale genomic studies 3, 4 . for example, novel single nucleotide polymorphisms (snps) that are clinically associated with warfarin dosing were discovered in large scale genomics studies in individuals of african descent that had not been discovered in europeans 5,6 . whole-genome sequencing in individuals of african descent [7] [8] [9] and whole-exome sequencing in a southern african population 10 additional efforts have been made through international collaborations to establish reference genome datasets and recommendations for research in diverse populations; including the genomeasia 100k project, the human heredity and health in africa (h3africa) initiative, and the clinical genome resource (clingen) ancestry and diversity working group (adwg) [13] [14] [15] [16] . khatri and colleagues discovered the 3-gene signature for diagnosis of tuberculosis based on transcriptome profiles of participants from 11 countries 17 , a finding that was generalized to patient populations from every inhabited continent in a span of 3 years 18, 19 . most importantly, the 3-gene signature has been clinically translated to the point-of-care test by cepheid 20 . the inclusion of diverse populations in genomic studies has demonstrated benefits in the discovery and interpretation of gene-trait associations. similarly, greater diversity in immunogenomics research will enable the discovery of novel genetic traits associated with immune system phenotypes that are common across populations. broader inclusion of diverse populations may also enable researchers to address genetic heterogeneity in the context of translational research and clinical drug development, possibly revealing clinically relevant genomic signatures that are more prevalent in some populations than others. immunogenomics is a field in which genetic information at different levels of biological organization (epigenetics, transcriptomics, metabolomics, cells, tissues, and clinical data) has been characterized and utilized to understand the immune system and immune responses. here table 1 ). rna-seq has traditionally been mapped to study entire cellular populations instead of amplifying at the specific regions. given the complexity of the tcr and bcr genomic loci, the accurate determination of germline immune receptor genes, from bulk rna-seq or whole-genome sequencing, has proved challenging 45 . several computational methods show promise [46] [47] [48] , but the mapping rate and accuracy remain to be improved. additionally, a wide-scale comparison is needed between results obtained from methods for deriving germline receptor genes from rnaseq studies, those obtained from established methods such as, targeted pcr and sequencing of genomic dna, the sequencing and assembly of bacterial artificial chromosome (bac) and fosmid clones 49 , and those from more recent methods such as inference from airr-seq repertoires 50 . many population genetic differences have been observed in genomics studies and immunogenomics is no exception [51] [52] [53] . the current public databases of adaptive immune receptor germline genes are essential for airr-seq analysis and immunogenomics studies. however, the most widely used reference database for immunogenetics data, the international immunogenetics information system (imgt) 63 , lacks a comprehensive set of human tcr and bcr alleles representing diverse populations worldwide. the same issue exists in hla databases: over 70% of rare hla variants from oceania and west asia populations were found to be absent in the 1000 genomes project panel 64 . there is still more uncertainty due to the fact that descriptions of sample populations in databases are often self-identified based on geography or ethnicity, rather than genetic ancestry. however, progress has been made to address this issue in immunogenomics studies. for example, the airr community, an international community formed to promote high standard research in adaptive immune repertoire research, introduced the open germline receptor database (ogrdb) in september 2019 as a resource platform for germline gene discovery and validation from airr-seq data, to enrich the imgt database 65 . collaborators in our team also created vdjbase, a platform for the inferred genotypes and haplotypes from airr-seq data 66 . these efforts provide the opportunities to infer genetic ancestry. nevertheless, the majority of available germline sequences either lack population-level annotations or are biased toward samples of european descent. we argue that this shortcoming must be addressed through focused efforts that seek to include more diverse populations in immunogenomics research. as an interdisciplinary group, with expertise in biomedical and translational research, population biology, computational biology, and immunogenomics, we wish to raise awareness about the value of including diverse populations in airr-seq and immunogenetics research. in the areas of genetic disease research and cancer genomics, enhanced genetic diversity has led to demonstrable insights 67, 68 . however, the field of immunogenomics has yet to benefit from a similar growth in diversity. at the current stage of the global covid-19 pandemic, numerous vaccine trials are underway in many countries worldwide, offering opportunities to investigate genetic factors in vaccine responses. yet, this will require careful clinical study designs that can effectively address confounding factors such as environmental and socio-economic differences. hiv-1 71 , zika 72 , and sars cov-2 73 . we expect that vaccine and infection outcomes can also be shaped by genetic variability, including specific effects driven by immune-related genes 58 . here, we make several recommendations for increasing diversity in immunogenomics research. first, we propose that the community should make a greater effort to include underrepresented populations in airr-seq and immunogenomics studies. already, those that have conducted airr-seq in populations of non-european descent have uncovered evidence for extensive germline diversity. for example, in a study of south african hiv patients, scheepers and colleagues discovered 123 ighv alleles that were not represented in imgt 54 . this promoted the hiv vaccine design by understanding the immunoglobulin heavy chain variable region (ighv) profile in the south african population. in a study in the papua new guinea population, one novel ighv gene and 16 ighv allelic variants were identified from airr-seq data 55 . these discoveries of novel alleles indicate the need to generate population-based airr-seq datasets. we do not recommend generalizing airr-seq findings to populations that are underrepresented in research, due to missing variation and lack of validation, which limits our ability to leverage airr-seq datasets in biomedical applications.therefore, increasing population diversity in immunogenomics studies can lead to improvements in a wide range of applications, including drug discovery and development, vaccine design and development. promoting precision medicine for underrepresented populations and improving predictions for treatment outcomes will become more feasible in the future with broader participation and inclusion. second, we argue that there are existing genomic datasets that could potentially be leveraged to augment ig/tcr germline databases, and inform the interpretation of airr-seq and immunogenomics studies across populations. extraction of population immunogenomics information from existing genomic datasets could be an effective strategy, as well as carefully embracing non-targeted sequencing data (eg. rna-seq) to focus on genetic diversity of samples. ancestry-associated genetic markers in short-read genome sequencing may help overcome the difficulties of relying on sample metadata in airr-seq datasets. this may also be time-efficient relative to waiting for the availability of sufficiently diverse airr-seq datasets. researchers have attempted to utilize paired-end rna-seq data in the cancer genome atlas (tcga) to infer the complementarity determining region 3 (cdr3) of tumor-infiltrating t-cells 74 , and to apply a computational method to rna-seq data in the genotype-tissue expression consortium (gtex) to profile immunoglobulin repertoires 48 . similar ideas could be adapted to the direct prediction of allelic variants from short-read genomic sequence data 75, 76 . however, challenges need to be overcome, including the high levels of copy number variation and segmental duplication in the bcr and tcr loci, and the need for protocols to validate novel allelic variants gleaned from short-read sequencing data 45, 77 finally, we suggest the need for additional infrastructure and expertise in regions and countries with populations underrepresented in research, and to enhance collaborations between countries, which are critical in minimizing global health disparities. online training sessions that are customized for conducting immunogenomics research in diverse populations would be beneficial to the biomedical community, perhaps especially in those regions. the content of these trainings might include participant recruitment strategies with a commitment to outreach and education to increase participation, sample collection methods, steps to running sequence experiments onsite or in collaboration with other academic institutions or commercial companies, uploading sequencing data to appropriate repositories, and performing bioinformatics analyses. virtual learning platforms for bioinformaticians have been established by members in our group, and these could be leveraged to provide such trainings 79 . our interdisciplinary group consists of leading researchers from 13 countries, including the us, canada, norway, france, sweden, russia, the uk, israel, china, south africa, chile, peru, and french polynesia. we share concerns about the lack of diversity in immunogenomics and embrace the need for engaging our combined efforts to tackle this challenge. to spearhead the enterprise of fostering diversity in the field, we have formed this task force with the aim of developing a global consortium on diversity in immunogenomics. this consortium will seek to promote inclusive, international, and interdisciplinary research, supported by transparent and open-source learning materials and datasets, with the goal of enhancing representation of diverse populations in immunogenomics. the missing diversity in human genetic studies don't ignore genetic data from minority populations the road ahead in genetics and genomics a scientometric review of genome-wide association studies genetic variants associated with warfarin dose in african-american individuals: a genome-wide association study novel cyp2c9 and vkorc1 gene variants associated with warfarin dosage variability in the south african black population assembly of a pan-genome from deep sequencing of 910 humans of african descent the african genome variation project shapes medical genetics in africa whole-genome sequencing for an enhanced understanding of genetic variation among south africans whole-exome sequencing reveals uncaptured variation and distinct ancestry in the southern african population of botswana multi-ancestry genome-wide gene-smoking interaction study of 387,272 individuals identifies new loci associated with serum lipids research capacity. enabling the genomic revolution in africa the genomeasia 100k project enables genetic discoveries across asia h3africa: current perspectives the clinical imperative for inclusivity: race, ethnicity, and ancestry (rea) in genomics genome-wide expression for diagnosis of pulmonary tuberculosis: a multicohort analysis assessment of validity of a blood-based 3-gene signature score for progression and diagnosis of tuberculosis, disease severity, and treatment response concise whole blood transcriptional signatures for incipient tuberculosis: a systematic review and patient-level pooled meta-analysis diagnostic accuracy study of a novel blood-based assay for identification of tb in people living with hiv the science and medicine of human immunology profiling the t-cell receptor beta-chain repertoire by massively parallel sequencing high-throughput sequencing of the zebrafish antibody repertoire individual variation in the germline ig gene repertoire inferred from variable region gene rearrangements pd-1 blockade induces responses by inhibiting adaptive immune resistance linking t-cell receptor sequence to functional phenotype at the single-cell level commonality despite exceptional diversity in the baseline human antibody repertoire augmenting adaptive immunity: progress and challenges in the quantitative engineering and analysis of adaptive immune receptor repertoires ighv1-69 polymorphism modulates anti-influenza antibody repertoires, correlates with ighv utilization shifts and varies by ethnicity multi-donor longitudinal antibody repertoire sequencing reveals the existence of public antibody clonotypes in hiv-1 infection aberrant b cell repertoire selection associated with hiv neutralizing antibody breadth exploiting b cell receptor analyses to inform on hiv-1 vaccination strategies. vaccines (basel) 8 immunosequencing identifies signatures of cytomegalovirus exposure history and hla-mediated effects on the t cell repertoire b cells, plasma cells and antibody repertoires in the tumour microenvironment high-throughput immunogenetics reveals a lack of physiological t cell clusters in patients with autoimmune cytopenias analysis of the b cell receptor repertoire in six immune-mediated diseases t cell receptor β repertoires as novel diagnostic markers for systemic lupus erythematosus and rheumatoid arthritis clonally expanded cd8 t cells patrol the cerebrospinal fluid in alzheimer's disease identification of subject-specific immunoglobulin alleles from expressed repertoire sequencing data production of individualized v gene databases reveals high levels of immunoglobulin genetic diversity impre: an accurate and efficient software for prediction of t-and b-cell receptor germline genes and alleles from rearranged repertoire data de novo inference of diversity genes and analysis of non-canonical v(dd)j recombination in immunoglobulins automated analysis of immunosequencing datasets reveals novel immunoglobulin d genes across diverse species a novel framework for characterizing genomic haplotype diversity in the human immunoglobulin heavy chain locus ultrasensitive detection of tcr hypervariable-region sequences in solid-tissue rna-seq data assembly-based inference of b-cell receptor repertoires from short read rna sequencing data with v profiling immunoglobulin repertoires across multiple human tissues using rna sequencing complete haplotype sequence of the human immunoglobulin heavy-chain variable, diversity, and joining genes and characterization of allelic and copy-number variation inferred allelic variants of immunoglobulin receptor genes: a system for their evaluation, documentation, and naming variability in trbv haplotype frequency and composition in caucasian, african american, western african and chinese populations sequence diversity, natural selection and linkage disequilibrium in the human t cell receptor alpha/delta locus sequence variation in the human t-cell receptor loci ability to develop broadly neutralizing hiv-1 antibodies is not restricted by the germline ig gene repertoire genomic screening by 454 pyrosequencing identifies a new human ighv gene and sixteen other new ighv allelic variants novel substitution polymorphisms of human immunoglobulin vh genes in mexicans the immunoglobulin heavy chain locus: genetic variation, missing data, and implications for human disease the individual and population genetics of antibody immunity exploring the pre-immune landscape of antigen-specific t cells the distribution of hla dq2 and dq8 haplotypes and their association with health indicators in a general danish population kir haplotypes are associated with late-onset type 1 diabetes in european-american families imgt®, the international immunogenetics information system® 25 years on immune diversity sheds light on missing variation in worldwide genetic diversity panels ogrdb: a reference database of inferred immune receptor genes vdjbase: an adaptive immune receptor genotype and haplotype database williams-beuren syndrome in diverse populations whole-genome sequencing reveals elevated tumor mutational burden and initiating driver mutations in african men with treatment-naïve, high-risk prostate cancer & the covid-19 host genetics initiative. the covid-19 host genetics initiative, a global initiative to elucidate the role of host genetic factors in susceptibility and severity of the sars-cov-2 virus pandemic genomewide association study of severe covid-19 with respiratory failure identification of a cd4-binding-site antibody to hiv that evolved near-pan neutralization breadth recurrent potent human neutralizing antibodies to zika virus in brazil and mexico structural basis of a shared antibody response to sars-cov-2 landscape of tumor-infiltrating t cell repertoire of human cancers worldwide genetic variation of the ighv and trbv immune receptor gene families in humans correction: a database of human immune receptor alleles recovered from population sequencing data comment on 'a database of human immune receptor alleles recovered from population sequencing data living in an adaptive world: genomic dissection of the genus homo and its immune response how bioinformatics and open data can boost basic science in countries and universities with limited resources diseases of the national institutes of health under award number u01ai136677.we thank dr. nicky mulder for the valuable comments that greatly improved the manuscript. key: cord-322649-c99lszcu authors: miao, faming; zhang, jingyuan; li, nan; chen, teng; wang, lidong; zhang, fei; mi, lijuan; zhang, jinxia; wang, shuchao; wang, ying; zhou, xintao; zhang, yanyan; li, min; zhang, shoufeng; hu, rongliang title: rapid and sensitive recombinase polymerase amplification combined with lateral flow strip for detecting african swine fever virus date: 2019-05-15 journal: front microbiol doi: 10.3389/fmicb.2019.01004 sha: doc_id: 322649 cord_uid: c99lszcu african swine fever virus (asfv), the etiological agent of african swine fever (asf), a hemorrhagic fever of domestic pigs, has devastating consequences for the pig farming industry. more than 1,000,000 pigs have been slaughtered since 3 august 2018 in china. however, vaccines or drugs for asf have yet to be developed. as such, a rapid test that can accurately detect asfv on-site is important to the timely implementation of control measures. in this study, we developed a rapid test that combines recombinase polymerase amplification (rpa) of the asfv p72 gene with lateral flow detection (lfd). results showed that the sensitivity of recombinase polymerase amplification with lateral flow dipstick (rpa-lfd) for asfv was 150 copies per reaction within 10 min at 38°c. the assay was highly specific to asfv and had no cross-reactions with other porcine viruses, including classical swine fever virus (csfv). a total of 145 field samples were examined using our method, and the agreement of the positive rate between rpa-lfd (10/145) and real-time pcr (10/145) was 100%. overall, rpa-lfd provides a novel alternative for the simple, sensitive, and specific identification of asfv and showed potential for on-site asfv detection. african swine fever (asf) is a severe viral disease that manifests clinical symptoms of hemorrhagic fever caused by african swine fever virus (asfv) and can result in case fatality rates of up to 100% in domestic pigs, depending on the virus strain (galindo and alonso, 2017) . asf, which was first identified by montgomery (1921) in kenya in the 1920s, made its first incursion into europe via two successive entries into portugal (in 1957 and again in 1960) , spreading rapidly throughout western europe and then to south america and the caribbean (michaud et al., 2013) . it was eventually eradicated by the mid-1990s, with the exception of sardinia (mur et al., 2016) . however, since the second major incursion of the disease, initially into georgia in 2007, asf has spread to eastern europe and russia (rowlands et al., 2008; revilla et al., 2018) . the virus has continued to spread worldwide, including china. since the first asf case emerged in china in august 2018, more than 100 cases have been recorded in 25 provinces (zhou et al., 2018) . african swine fever normally presents with non-specific symptoms, including fever, anorexia, vomiting, and diffused hemorrhage in superficial skin. post-mortem examination shows pericardial effusion, kidney enlargement, lymphadenectasis, and darkened and enlarged spleen. all these features are indistinguishable from those seen in classical swine fever virus (csfv) infection (tauscher et al., 2015; li and tian, 2018) . at present, no effective treatment or vaccine for asfv is available, and disease control is based mainly on animal slaughtering and strict sanitary measures (cisek et al., 2016; rock, 2017) . rapid laboratory diagnosis is important for timely triage and confirmation to control and preventing this disease because of its rapid progression to death and spread. molecular tools based on the detection of the genetic information of asfv have become more widely accepted for asf diagnosis (oura et al., 2013) . polymerase chain reaction (pcr) and real-time pcr techniques have provided a supportive method to post-mortem asf diagnosis (aguero et al., 2003; zsak et al., 2005; fernandez-pinero et al., 2013) ; however, they cannot be used for field (on-site) detection in pig farms. recently, liu et al. (2019) reported that the improved realtime pcr assay using a universal probe library (upl) probe could be applied to asfv molecular diagnosis under field conditions. due to limitations of the battery-powered realtime pcr instrument, it can process only a moderate number of samples. isothermal recombinase polymerase amplification (rpa) has been successfully used to detect multiple viral pathogens, including infectious bovine rhinotracheitis virus (hou et al., 2017) , bovine coronavirus (amer et al., 2013) , ebola virus (yang et al., 2016) , bovine viral diarrhea virus, or foot-and-mouth disease virus (wang et al., 2018) . as reports of asfv detection by rpa are limited, we aimed to develop and evaluate a rapid detection tool that combines immunochromatographic strip tests, more commonly referred to as lateral flow devices (lfds), with rpa targeting the conserved asfv p72 gene. this study was conducted as part of the surveillance of the asf outbreak in china. samples were collected for asf testing and surveillance under the agreement between the ministry of agriculture and rural affairs of the chinese government and farm owners. sample collecting treatment was conducted in accordance with the protocols for viral hemorrhagic fever under the urgent interim guidance for case management established by the world organization for animal health. the protocol for this study was approved by the ethics committee of the military veterinary research, academy of military medical sciences. samples were collected by animal centers for disease control and prevention of jilin province. the viruses were inactivated in the bsl-3 lab, and the inactivated samples were transferred to the bsl-2 lab for genomic dna extraction and detection. for standard plasmids, a plasmid extraction kit was used in accordance with the manufacturer's instructions (axygen, united states). for field samples, tissue homogenates were prepared in formaldehyde, and the inactivated virus homogenate was subjected to dna extraction by using a nanomagnetic bead adsorption kit (bailing biotechnology, beijing co., ltd., china). for the whole extraction procedure, lysis, adsorption, and washing were performed, and elution with 50 µl of rnase-free water was conducted to dissolve the dna. the extracted dna was then stored at −20 • c. the p72 gene was first amplified in the rpa reaction system comprising the designed primers (labeled biotin) and a 5-carboxyfluorescein (fitc) labeled-probe, and the expected amplicons were labeled with 5 -fitc and 3 -biotin at the ends. then, the amplified products were recognized by anti-biotin and anti-fitc monoclonal antibodies on the test line, where gold nanoparticles were prefixed (figure 1) . the whole genome sequence of a p72 genotype ii virus-asfv-sy18, which was sequenced in our lab (genbank accession number mh766894)-was used as the reference sequence for rpa primer selection. rpa sense primer (5 -aagaagaaagttaatagcagatgccgataccac-3 ), rpa anti-sense primer (5 -biotin-gctcttacatacccttccacta cggaggcaatg-3 ), and rpa probe primer (5 -fam-tgg gttggtattcctcccgtggcttcaaagcaaag[thf]taa tcatcatcgcac-p-3 ) were designed by using twistdx nfo rpa kits (cambridge, united kingdom) in accordance with the manufacturer's guidelines. taqman pcr was performed to detect asfv p72 in an mx3000p multiplex quantitative pcr system (stratagene) in accordance with the manufacturer's instructions. real-time pcr primers for p72 were used as described before (king et al., 2003) . the sequences spanning the rpa amplification region were obtained through pcr and cloned into the pmd19-t plasmid to generate figure 1 | schematic of rpa-lfd assay. frontiers in microbiology | www.frontiersin.org pmd19-p72. taqman runs of the experimental samples involved at least three replicates with no-template or no-primer control and the combination of the primers. the pcr conditions were 95 • c for 2 min and 40 cycles of 95 • c for 15 s and 60 • c for 60 s. a standard curve was generated from serially diluted p72 recombinant plasmids of known copy numbers. a pair of primers and rpa probe primer was designed to specifically detect asfv circulating strains in china. the rpa assay was conducted in a 50 µl system by using a twistamp nfo kit (twistdx tm , cambridge, united kingdom). all the reagents (500 nm rpa primers, 120 nm rpa probe, 4 × rehydration buffer, and purified h 2 o) except the template or the sample dna and magnesium acetate were prepared in a master mix, which was aliquoted into each tube of a 0.2 ml tube strip containing a dried enzyme pellet. magnesium acetate (2.5 µl) was pipetted slowly into the tube lids, and subsequently, 2 µl of the sample dna was added to the tubes, the lids were closed, and magnesium acetate was centrifuged into the tubes by using a mini-spin centrifuge. the tubes were immediately used for isothermal amplification. asfv-rpa detection was combined with an lfd (yoshida, co., ltd., china). then, 2 µl of the amplified product was added to the sample pad, and the dipstick was inserted into 100 µl of the sample buffer for 3-5 min. a dilution range of 10 0 to 10 5 copies per reaction of pmd19-p72 recombinant plasmid was used to evaluate the sensitivity of recombinase polymerase amplification with lateral flow dipstick (rpa-lfd), and the amplicons were evaluated through agarose gel electrophoresis. the reactions were evaluated with prrsv/ch1r, csfv/shimen, prv/jl14, jev/sa14-14-2, pedv/cv777, and pcv2b-sd12 to examine the specificity of the developed rpa-lfd assay. the thermo-reaction procedures were optimized after different primers, probe combinations, and thermo-cycles were run in the real-time pcr system to evaluate the sensitivity of taqman asfv real-time pcr assay. the real-time pcr conditions that yielded the highest amplification efficiency were 94 • c for 5 min and 40 cycles of 94 • c for 30 s and 55 • c for 30 s by using the primer combination reported by king et al. (2003) . the realtime pcr assay was sufficiently sensitive to detect 10 2 copies per reaction (figure 2) . no cross-reactions with prrsv/ch1r, csfv/shimen, prv/jl14, jev/sa14-14-2, pedv/cv777, and pcv2b-sd12 were observed, and a positive signal was detected in asfv-sy18 and asfv-jl18. in this study, 10 5 copies of the asfv-positive pmd19-p72 plasmid were used as a template to determine the optimal rpa reaction temperature. the reaction mixture was incubated at eight different temperatures (22 • c, 26 • c, 30 • c, 34 • c, 38 • c, 42 • c, 46 • c, and 52 • c) for 15 min. the detection result showed that the test line was short at 26 • c and 52 • c, and the test line density did not enhance significantly from 30 • c to 46 • c ( figure 3a) . in this study, 10 5 copies of the asfv-positive plasmid were used as a template to determine the optimal rpa reaction time. in figure 3b , the rpa assay tested positive for asfv in 5 min. the measured dna band density revealed that the dna yield doubled after a reaction time of 10 min and increased slightly after 15 min. the intensity of the dna bands in agarose gel did not significantly change at reaction times of 15, 20, 25, and 30 min. this finding indicated that the rpa reactions during the remaining parts of this study were completed after 12 min. however, the false positive test lines appeared when the reaction time was more than 15 min (invalid result). therefore, 10 min was considered the optimum reaction time for the rpa-lfd assay, and the assay worked from 26 • c to 46 • c. in terms of convenience and safety, 38 • c was chosen as the optimum reaction temperature. the sensitivity results showed that the detection limit of the asfv rpa-lfd assay was 10 2 copies per reaction of the recombinant plasmid pmd19-p72. the amplicon in the asfv rpa-lfd assay (230 bp) was also tested through subsequent visualization with 2% agarose gel-electrophoresis after purification, and the sensitivity of rpa based on gelelectrophoresis visualization was 10 3 copies per reaction (figure 4) . in terms of the specificity of asfv rpa-lfd assay, no cross-reactions with other important viruses of pigs were observed (figure 5) , and this observation was confirmed by agarose gel electrophoresis. the 145 samples included 90 spleen samples and 55 blood samples of animals (jilin cdc). of these samples, 5 spleen tissues and 5 blood samples were positive for asfv nucleic acid as detected by rpa-lfd. taqman real-time pcr showed that 10 samples were positive. the rpa-lfd detection results were consistent with those of real-time pcr. rpa-lfd revealed that the samples showed a light test line and high cq values. of these samples, one tested sample, which showed with a short test line or no test line, was rotten, and the cq value in real-time pcr was 38, indicating weakly positive result. although the detection results between the two methods were consistent (table 1) , the rpa-lfd assay was more rapid than real-time pcr. in particular, the whole rpa-lfd assay yielded results in 30 min, whereas real-time pcr generated results approximately 2 h later. the average time from symptom onset to outcome in asf is 6-20 days; however, the pig farms were usually in rural areas or even in a mountainous area far away from the city laboratory, which required more time for sampling and transportation. once diagnosis and timely treatment of suspected asf pigs is delayed, the risk of asf exposure to other pig farms was increased. in the current outbreak, laboratory testing with taqman real-time pcr is being widely used in affected areas. however, requirements-including a sophisticated thermocycler, complex sample treatment procedures, and expensive reaction instruments-have limited its applications in point-of-care testing (king et al., 2003) . different isothermal molecule amplification assays, including loop-mediated isothermal amplification assay (lamp) (james et al., 2010) , polymerase cross-linking spiral reaction (wozniakowski et al., 2017) , cross-priming amplification method (fraczyk et al., 2016) , chimeric dna/lna-based biosensor (biagetti et al., 2018) , and droplet digital pcr (wu et al., 2018) , have been developed as a rapid, simple, and cost-effective alternative to pcr-based molecule assay. rpa has several advantages. for example, initial heating for dna denaturation is not required, and test conditions (38 • c within 20 min) are easily implemented. lamp assay for asfv detection requires a long time (60 min) and high temperature (62 • c−65 • c) (james et al., 2010) . by comparison, rpa assay takes less than 20 min at 38 • c to complete. as such, rpa assay is simpler and more easily used than lamp assay. wang et al. (2017) reported the field validation of real-time rpa for asfv, although this technique can specifically detect asfv plasmid with a detection limit of 10 2 dna copies per reaction. however, this detection assay is based on an expensive scanner device. detection methods for actual clinical samples have yet to be designed, so the application of this test to clinical samples is limited. gao et al. (2018) developed crosspriming amplification combined with immune-chromatographic strips for the rapid on-site detection of asfv, but its reaction conditions include 60 min at 59 • c and six primers for a reaction system, thereby causing non-specific amplification. the use of lateral flow assays combined with a monoclonal antibody against p72 protein of asfv can be used to detect p72 viral and recombinant protein or inactivated culture virus (sastre et al., 2016) ; however, the sensitivity of the assay is 100-fold lower than genomic amplification. recombinase polymerase amplification with lateral flow dipstick overcomes the technical difficulties posed by current amplification methods; for example, it operates at a low and constant temperature, does not require the thermal denaturation of templates, and does not rely on an expensive thermocycler (aguero et al., 2003) . in combination with a commercially magnetic nanobead-based dna extraction kit, this approach can be applied to on-site testing or rapid diagnosis under poorly equipped conditions. rpa is highly resistant to crude samples, suggesting that it can be used for on-the-spot field testing with crude nucleic acid extraction. in an rpa-nfo reaction system, nfo cleaves the primer of the probe at the thf position and effectively deblocks the probe, thereby generating a new 3 hydroxyl group that can act as a primer for polymerase extension; thus, the probe is transformed into an extension primer with an increased specificity of amplification (james and macdonald, 2015) . the potential defect of rpa-lfd assay for asfv is that it may carry over contaminants in fields because its tube must be opened after amplification is completed. therefore, precautions should be taken. for example, reaction tubes should be carefully opened and closed, gloves should frequently be changed, the progress of pre-and post-rpa amplification should be separated, and reaction time should be shortened if possible. the replacement of dttp with dutp may help prevent such carryover contamination as demonstrated in other nucleic amplified system assays. in summary, we evaluated a rapid rpa-lfd test for the rapid diagnosis of asfv. the superior performance of rpa-lfd for asfv and its consistent detection results with those of real-time pcr indicated its appropriateness for laboratory diagnosis and that it has great potential for on-site testing of asfv circulating in china. the raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. this study was conducted as part of the surveillance of the asf outbreak in china. samples were collected for asf testing and surveillance under the agreement between the ministry of agriculture and rural affairs of the chinese government and farm owners. sample collecting treatment was conducted in accordance with the protocols for viral hemorrhagic fever under the urgent interim guidance for case management established by the world organization for animal health. the protocol for this study was approved by the ethics committee of the military veterinary research institute, academy of military medical sciences. samples were collected by animals centers for disease control and prevention of jilin province. the viruses were inactivated in the bsl-3 lab, and the inactivated samples were transferred to the bsl-2 lab for genomic dna extraction and detection. highly sensitive pcr assay for routine diagnosis of african swine fever virus in clinical samples a new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay chimeric dna/lna-based biosensor for the rapid detection of african swine fever virus african swine fever virus: a new old enemy of europe molecular diagnosis of african swine fever by a new realtime pcr using universal probe library development of cross-priming amplification for direct detection of the african swine fever virus, in pig and wild boar blood and sera samples african swine fever virus: a review cross-priming amplification combined with immunochromatographic strip for rapid on-site detection of african swine fever virus rapid detection of infectious bovine rhinotracheitis virus using recombinase polymerase amplification assays recombinase polymerase amplification: emergence as a critical molecular technology for rapid, low-resource diagnostics detection of african swine fever virus by loop-mediated isothermal amplification development of a taqman pcr assay with internal amplification control for the detection of african swine fever virus african swine fever in china overcoming the challenges of pen-side molecular diagnosis of african swine fever to support outbreak investigations under field conditions comprehensive phylogenetic reconstructions of african swine fever virus: proposal for a new classification and molecular dating of the virus on a form of swine fever occurring in british east africa (kenya colony) thirty-five-year presence of african swine fever in sardinia: history, evolution and risk factors for disease maintenance virological diagnosis of african swine fever-comparative study of available tests african swine fever virus biology and vaccine approaches challenges for african swine fever vaccine developmentperhaps the end of the beginning african swine fever virus isolate, georgia development of a novel lateral flow assay for detection of african swine fever in blood on the situation of african swine fever and the biological characterization of recent virus isolates rapid detection of foot-and-mouth disease virus using reverse transcription recombinase polymerase amplification combined with a lateral flow dipstick a recombinase polymerase amplification-based assay for rapid detection of african swine fever virus polymerase cross-linking spiral reaction (pclsr) for detection of african swine fever virus (asfv) in pigs and wild boars development and application of a droplet digital polymerase chain reaction (ddpcr) for detection and investigation of african swine fever virus development and evaluation of a rapid and sensitive ebov-rpa test for rapid diagnosis of ebola virus disease emergence of african swine fever in china preclinical diagnosis of african swine fever in contact-exposed swine by a real-time pcr assay key: cord-270425-1ughypnx authors: louis-jean, james; cenat, kenney; njoku, chidinma v.; angelo, james; sanon, debbie title: coronavirus (covid-19) and racial disparities: a perspective analysis date: 2020-10-06 journal: j racial ethn health disparities doi: 10.1007/s40615-020-00879-4 sha: doc_id: 270425 cord_uid: 1ughypnx health disparity refers to systematic differences in health outcomes between groups and communities based on socioeconomic isolation. in the usa, health disparities among minority groups, especially african americans, limit their access to quality medical care and other beneficial resources and services. presently, the novel coronavirus (covid-19) highlights the extreme healthcare challenges that exist in the african american and other minority communities in the usa. african americans are dying at a rate nearly four times higher than the national average. with inadequate access to quality healthcare, viable resources, and information, covid-19 will continue to have a disastrous effect on african american communities. this communication provides a brief overview of the health inequalities resulting in african americans dying disproportionately during the covid-19 pandemic. the novel coronavirus (covid-19) emerged from wuhan, china, in late 2019 and quickly became a global pandemic that has already affected more than 115 countries and territories [1] . in symptomatic patients, this highly transmissible disease causes severe acute respiratory syndrome, which infects lower respiratory airways and results in fatal pneumonia. the effect of this pandemic is widely visible, resulting in about a 5.6% mortality rate and causing major economic and social devastations [2] . studies have shown that covid-19 is transmitted via human interactions when uninfected individuals come in contact with mucus and respiratory droplets or surfaces that contain the virus. on surfaces where it is present, the virus can remain infectious from 2 hours and up to 9 days depending on the surface materials. also, it can remain airborne for up to 3 hours post aerosolization [3] . furthermore, a small majority of carriers develop mild to no symptoms. as a result, transmission occurs rapidly and inconspicuously with both symptomatic and asymptomatic individuals unknowingly transmitting the virus, resulting in more people being infected. research data continues to show that covid-19 affects all age groups. older individuals and those with underlying health conditions are more prone to experiencing severe illnesses and death. data analysis of covid-19 from the usa highlights pre-existing health disparities among african americans as the potential cause of poor prognosis. based on reports from national health care disparities, in comparison with non-hispanic whites, african americans have a 44% greater chance of dying from stroke, 20% more likely to have asthma, 25% more likely to have heart disease, 72% more likely to have diabetes, and 23% more likely to be obese [4] . this is a major concern that government and public health officials should address as it has been shown that in 21 out of 30 states reporting data, black people accounted for a higher share of covid-19 cases than their share in the population [5] . in 19 out of 24 states, they accounted for a higher share of deaths than their share of the total population [5] . in such regard, an immediate plan of action is urgently needed to address and mitigate the effects of health disparities. this communication briefly examines the health disparities among african americans in the usa during the covid-19 pandemic. in the early phase of the outbreak in the usa, access to testing was limited to government officials, celebrities, and a selected number of healthcare workerssymptomatic or asymptomatic. it was not until 18 march 2020 when the federal government passed the families first coronavirus response act (ffcra), which allows free covid-19 testing for all individuals [6] , that large-scale testing became available nationwide. while ffcra is a great response for all citizens, the lack of testing remains, especially in minority communities. in the usa, the racial breakdown of covid-19 cases and deaths is now starting to be made available. in louisiana, where 32% of its population is african american [7] , 70% of deaths-as a result of the novel coronavirus-are among african americans. meanwhile, white americans in louisiana account for 62% of the population but recorded only 28% of covid-19 deaths [8] . of the 380 covid-19 deaths in illinois [9] , the percentage of deaths among black americans (43%) is higher compared with those among whites (36%), hispanics (8.4%), asians (3.7%), and others (6.8%). this is an area where the majority of the population is white (77%) and the black population is about 15%. the same phenomenon is occurring in michigan [10] ; the black population is about 14% but accounted for 40% of covid-19 deaths. in new york, both hispanics and african americans are dying at a relatively higher rate in comparison with other ethnic groups. they account for 29% and 22% of new york's population, respectively. however, the reported death toll [11] for hispanics and african americans is 34% and 28%, respectively. in connecticut, the black population is about 10%, but represents more than 16% of covid-19 deaths [12, 13] . in milwaukee county, the black population is about 26% but represents 77% of covid-19 deaths [14, 15] . in north carolina, 49% of deaths are among african americans, who represent 21% of the total population [16] . the trend and commonality transcend state lines. the common denominator for the high mortality rate in all these states is race and ethnic background. of note, all data presented in this communication and fig. 1 are subject to change as more data becomes available. for more up-to-date data, refer to the states department of public health and services. a recent study showed that in zip codes with high numbers of unemployed and uninsured residents, fewer test kits were available [17, 18] . most of those zip codes have disproportionate numbers of african americans. it is no surprise that underserved communities such as the african american communities would also have less access to covid-19 test kits in a time that kits are scarce. assuming that african americans are seeking medical attention for covid-19, they will most likely do so at minority-serving institutions which already have [19] : (i) lower quality care due to low budgets and lack of resources (ii) shortage of critical care physicians (iii) inadequate number of medical supplies and equipment (i.e., personal protective equipment and ventilators for critically ill patients). this is in parallel to years of ongoing racial and socioeconomic discrimination in the usa [20, 21] . while a series of landmark court cases such as simkins v moses h. cone memorial hospital (1963) and cypress v newport news hospital association (1967) litigated by the national association for the advancement of colored people (naacp) legal defense and education fund took legal actions against racial policies and discriminations in healthcare, the challenges for quality healthcare for african americans remain [22] . the pursuit of legal strategies against racist policies was an essential element in a national campaign to eliminate discrimination in healthcare delivery in the usa. as covid-19 cases and related deaths continue to rise in the usa, data demonstrates that african american communities in various cities are the most affected ( fig. 1 ). this is a challenge that the federal government and its covid-19 task force have pointed out. however, in a white house press briefing, dr. anthony fauci of the national institute of allergy and infectious diseases recently expressed, "…there is nothing we [the federal government] can do about it right now except to give them [african americans] the best possible care to avoid complications." health disparities and institutional racism [20, 21, [23] [24] [25] [26] [27] ] make the covid-19 pandemic worse for african americans. there are studies on the effects of stress and health for african americans as stress can increase vulnerability, which in turn is a factor in determinants of health disparities. perceived discrimination can add to stress, which increases vulnerability to the health effects of environmental hazards, thus adding to health disparities. harburg et al. highlighted that darkerskinned black men having racist interactions and living in neighborhoods with high rates of social instability have an increased risk and incidence of stressful experiences in daily life, which in turn increases their likelihood of high blood pressure along with other health conditions they are predisposed to from their environment [28] . conditions resulting from these conditions are listed as risk factors for more severe covid-19 cases [29] . constant streams of statistical data (fig. 1 ) about the novel coronavirus are showing that african americans are dying from covid-19-related complications at a disproportionately higher rate than other ethnic and racial groups. racial inequities in healthcare institutions, lack of access to information, higher levels of preventable chronic diseases (i.e., diabetes, asthmas, hypertension, etc.), and covid-19 testing not being widely available in minority communities are among the many factors resulting in african americans dying at disproportionate numbers during this pandemic [30] . failure to rapidly test and segregate individuals infected with covid-19 can result in major chain-of-transmission reactions and deaths. the socioeconomic discrimination has confined african americans to overpopulated housing estates (ghettos) and low-wage jobs. from the very genesis of this country, african americans have always been the essential or sacrificial workers used to ensure the continuity of this economy. from centuries of free slave labor to years of sharecropping to low-paying domestic jobs, african americans have always played a role in shaping the very essence of what america is [31, 32] . today, a large number of african americans work in retail, home healthcare, mass transit, plant factories, and prisons/jails where social distancing is almost impossible. as a result, african americans became more vulnerable to the disease combined with inadequate access to proper healthcare in their communities [13] . in many cities in the usa, stay-at-home orders are placed to mitigate the spread of the virus. this is a great effort, which has shown promising results. however, african americans and other minority groups account for 68% of the us homeless population [33] . these individuals stand no chance against the trail of devastation that covid-19 will leave behind since it is almost impossible for them to follow preventative guidelines issued by the centers for disease control and prevention (cdc) and government officials. to help flatten the curve, many doctors, nurses, and other healthcare professionals from non-profit organizations are volunteering their services to the homeless community. among other preventative measures highlighted by the cdc as well as healthcare and government officials is the use of face masks in public settings. while this effort can limit and reduce the spread of covid-19, this could pose a challenge to african americans and other minorities as they are more likely to be criminalized based on their appearances while wearing masks. of note, many individuals have failed to understand the zoonotic origin of covid-19 [34] [35] [36] [37] . as a result, significant misinformation and conspiracy theories continue to circulate on the web and social media. while the internet is an open platform to share and access information, individuals should be aware of conspiracy theories and follow credible sources for relevant information. while there is no evidence showing a correlation between covid-19 and the upcoming fifthgeneration (5g) technology, people continue to share misinformation linking the current pandemic to 5g technology. initially, there was the belief that black individuals and minorities were not susceptible to being infected with the virus. there is no available research showing that black individuals are biologically and genetically immune to the virus. without proper information, these artificial assumptions can usher individuals into disastrous directions. on a global scale, covid-19 is not only affecting people of african descent in the usa but across the world. minority groups everywhere are harassed and discriminated against during this pandemic. in china where the virus emerged, africans living in guangzhou are being evicted from their homes as the virus sparked fear and racial discrimination. of equal challenges, minorities and migrant workers from caribbean countries, such as haiti, living in the usa are being detained by the immigration and customs enforcement (ice) at detention centers where the virus is uncontrollable. these haitians-some of whom are infected with or are covid-19 carriers-are subjected to deportation to their native land of haiti, a country with a very weak public healthcare system [38] . only a few examples are highlighted here; a comprehensive analysis is underway. altogether, african americans and individuals of african descent are more vulnerable to the impact of the virus. it is no secret that the us government has misguided and often used african americans as guinea pigs for medical research. during the time of slavery, psychologists and doctors deemed it a disease for slaves to long for freedom or want to "run away." runaway slaves were diagnosed with what was called "drapetomania," which was considered, at the time, a mental illness that amplifies the desire to become a fugitive-run away [32] . furthermore, after slaves were freed, many american doctors continued to argue that former slaves were incapable of thriving as free members of society because their minds could not function beyond the established orders of slavery. among many other diseases, the syphilis outbreak in the south highlighted another dark chapter in african americans' relations with the healthcare system in america. many african americans infected with syphilis were promised treatment from the public health department. instead, those patients were denied treatment and turned into "guinea pigs" to monitor the progression of the disease. the patients were unaware and were lied to repeatedly about their conditions. many of those untreated "guinea pigs" of the syphilis experiment infected their wives and other women they had gotten in contact with. furthermore, many of them had unknowingly fathered children born with congenital syphilis. the syphilis experiment lasted for 40 years, and it was a major ethical violation. the us government had broken its laws and experimented on its citizens. the us public health service did not treat the people who were experimented on even after penicillin-the effective cure for the disease-was made available. historically, this injustice has established a certain level of distrust among african americans in the us public health system; therefore, they are more reluctant in seeking routine preventative care [39] . the time for a healthcare revolution for african americans is long overdue. this is not a surprise, as 100 years ago, the 1918 flu pandemic highlighted the racial bias and distrust that existed in the medical community [40, 41] . jim crow laws and pseudoscientific theories made the impact of the flu go unnoticed in black communities. all efforts and resources were made available to white communities as physicians believed that the virus only affected white individuals. many healthcare officials, with no reliable scientific evidence, believed that african americans were not susceptible to the flu as the linings of their "big noses" were resistant to the microorganism that affects the respiratory system. as a result, black communities were forced to battle the 1918 flu pandemic on their own, using limited resources and inadequate medical care at segregated healthcare institutions [41] . the current covid-19 pandemic reflects a mirror image of the 1918 flu pandemic for african americans and other minorities in the usa [40] . such patterns will possibly continue if the socioeconomic, health disparities, and the dividing racial lines stretch to upcoming generations. other jurisdictions have yet to publicly report their data based on race and ethnicity; regardless, the challenges remain from social and economic factors that drive health outcomes. altogether, african americans are dying at a much higher rate compared with any other ethnic group. due to various factors that affect health outcomes, a plan of action is urgently needed to respond to the challenges of this pandemic and any health threats in african american communities [42] . access to testing and proper medical treatment is necessary to ensure the safety of african americans, the most vulnerable group. the embedded racism in the healthcare system and the socioeconomic and health disparities [25, 27] continue to make the effect of the virus worst-for black americans and other minorities in the usa. overall, the lack of testing made available to the black community for covid-19, the continuous poor healthcare system for african americans, and the systematic health disparity are what makes this virus more dangerous to african americans; although covid-19 is a new pandemic, it is deeply rooted in history in the continuous plight of african americans in this country [13] . it is an unsettling reality for african americans to rely on the same systems that historically inflicted harm and damage on them to protect and serve them against this virus [31, 32, 43] . more research is needed to understand the source of exposure to covid-19 for african americans as well as a plan of care and health outcomes of african americans in various zip codes. further research can also shed light into the type of facilities a majority of african americans receive care in, such as nursing homes, and assess their preparedness looking into their access to resources such as personal protective equipment (ppe) during the covid-19 pandemic, current and past documented deficiencies from state surveys, and infection control measures taken during covid-19, and overall health outcome. more research is also needed to understand how the environment affects health outcome or the severity of covid-19 cases in terms of environmental exposures, social, and economic factors that can affect mortality, morbidity, life expectancy, healthcare expenditures, and health status amidst the covid-19 pandemic. this is relevant since a recent study found that living in areas with high air pollution is associated with an 8% increase in the covid-19 death rate [44] . black leaders in all communities, historically black colleges and universities (hbcus), and minority-serving institutions (msis) should continue to engage and educate students to respond to the needs of their communities. this will require these institutions to reinvent and alter their curriculums to provide more health and medical programs (including medical history), engineering, science, and technology. currently, these institutions are operating with low resources, many are underfunded, and others are on the verge of being permanently closed. this is very unfortunate for african americans and other minority communities especially when analyzing the impact of covid-19. as the pandemic forced all teaching to be done remotely, hbcus' and msis' students and institutions are at higher risks of not being able to satisfy their educational needs [45] . many have limited to no access to technological devices and reliable internet access to fully respond to the spontaneous remote teaching caused by the effect of the virus. the gravity of the novel coronavirus covid-19 (sars-cov-2) pandemic is yet another event showing the racialized health inequalities that exist in the usa. african americans have systematically experienced the worst health outcomes compared with any other racial and ethnic group in the usa. the burdens of covid-19 are much greater among minorities living in low-income communities where access to quality healthcare and other relevant needs is scarce. the health disparities in the usa did not start with the covid-19 pandemic. however, the virus has significantly highlighted the pre-existing racial inequalities. altogether, the socioeconomic status and the well-being of african americans require intervention and significant improvement is needed. this will require mutual inclusions of community leaders from various organizations (i.e., churches, divine nine organizations: fraternities and sororities), local government, policymakers, and researchers to mend healthcare in low-income communities. ethics approval and consent ethical approval or consent was not applicable for this paper. the authors declare that they have no conflict of interest. world heal. organ on the novel coronavirus (covid-19): a global pandemic persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents national surveillance of asthma: united states growing data underscore that communities of color are being harder hit by covid-19 louisiana state dep. heal. 2020 coronavirus disease 2019 (covid-19) in illinois test results. illinois state dep. public heal. 2020 public heal. 2020. https:// covid19tracker.health.ny.gov/views/nys-covid19-tracker/ nysdohcovid-19tracker-fatalities?%3aembed=yes&% 3atoolbar=no&%3atabs=n connect. state dep. public heal. 2020 the covid-19 pandemic: a call to action to identify and address racial and ethnic disparities covid-19: county data. wisconsin state dep covid-19 north carolina dashboard. north carolina state div. public heal the black plague. new yorker. 2020 coronavirus philadelphia: positive tests higher in poorer neighborhoods despite six times more testing in higher-income neighborhoods, researcher says. cbs philly temporal trends in critical care outcomes in u.s. minority-serving hospitals social conditions as fundamental causes of health inequalities: theory, evidence, and policy implications racial and ethnic approaches to community health (reach 2010): an overview professional and hospital discrimination and the us court of appeals fourth circuit 1956-1967 social conditions as fundamental causes of disease the weathering hypothesis and the health of african-american women and infants: evidence and speculations the us perspective: lessons learned from the racial and ethnic approaches to community health (reach) program limitations in the use of race in the study of disease causation seeking causal explanations in social epidemiology socio-ecological stress, suppressed hostility, skin color, and black-white male blood pressure: detroit preliminary estimates of the prevalence of selected underlying health conditions among patients with coronavirus disease 2019 -united states vital signs: racial disparities in age-specific mortality among blacks or african americans -united states at the dark end of the street: black women, rape, and resistance-a new history of the civil rights movement from rosa parks to the rise of black power from slavery to freedom: a history of african americans who is homeless? natl. coalit. homeless coronaviruses: a paradigm of new emerging zoonotic diseases. pathog dis novel coronavirus takes flight from bats? bats as a continuing source of emerging infections in humans dbatvir: the database of batassociated viruses covid-19) in haiti: a call for action tuskegee and the health of black men african americans, public health, and the 1918 influenza epidemic the eighteen of 1918-1919: black nurses and the great flu pandemic in the united states covid-19 and african americans why african-americans may be especially vulnerable to covid-19 exposure to air pollution and covid-19 mortality in the united states: a nationwide cross-sectional study beyond the face-to-face learning: a contextual analysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments thanks are due to miriame etienne, ruthonce stvil louis-jean, isaiah herbert, and magdonald aimé for their feedback.data availability all materials and data used in this communication are publicly available and are cited throughout the text. key: cord-256195-1hmzgwrw authors: izzy, saef; tahir, zabreen; cote, david j; al jarrah, ali; roberts, matthew blake; turbett, sarah; kadar, aran; smirnakis, stelios m; feske, steven k; zafonte, ross; fishman, jay a; el khoury, joseph title: characteristics and outcomes of latinx patients with covid-19 in comparison to other ethnic and racial groups date: 2020-09-01 journal: open forum infect dis doi: 10.1093/ofid/ofaa401 sha: doc_id: 256195 cord_uid: 1hmzgwrw background: there is limited understating of the impact of covid-19 on the latinx population. we hypothesized that latinx patients would be more likely to be hospitalized and admitted to the icu than white patients. methods: we analyzed all patients with covid-19 in 12 massachusetts hospitals between february 1 and april 14, 2020. we examined the association between race, ethnicity, age, reported comorbidities, and hospitalization and intensive care unit (icu) admission using multivariable regression. results: of 5190 covid-19 patients, 29% were hospitalized; 33% required icu and 4.3% died. 46% of patients were white, 25% latinx, 14% african american, and 3% asian american. ethnicity and race were significantly associated with hospitalization. more latinx and african american patients in the younger age groups were hospitalized than whites. latinx and african americans disproportionally required icu, with 39% of hospitalized latinx patients requiring icu compared to 33% of african americans, 24% of asian americans, and 30% of whites (p&0.007). within each ethnic and racial group, age and male gender were independently predictive of hospitalization. previously reported pre-existing comorbidities contributed to the need for hospitalization in all racial and ethnic groups (p&0.05). however, the observed disparities were less likely related to reported comorbidities, with latinx and african american patients being admitted at twice the rate of whites, regardless of such comorbidities. conclusions: latinx and african american patients with covid-19 have higher rates of hospitalization and icu admission than white patients. the etiologies of such disparities are likely multifactorial and cannot be explained only by reported comorbidities. the health, societal, and economic impacts of coronavirus disease 2019 (covid-19) have been felt worldwide, with nearly 12 million confirmed infections leading to more than 500,000 deaths by early july 2020, with numbers continuing to grow. [1] [2] [3] studies from china, italy, spain and the united states have identified several factors associated with symptomatic infection and hospitalization, with or without admission to intensive care units (icu). [4] [5] [6] [7] [8] [9] older age has been shown to significantly increase the risk for hospitalization and death. 10, 11 hypertension, heart disease, obesity, and diabetes are common comorbidities associated with hospitalization in covid-19 patients. 2, 5, 12, 13 most of these published reports either describe the disease in relatively homogenous ethnic and racial populations, 5 or they do not compare covid-19 infected patients who did and did not require hospitalization. 2 in the united states, where a racially and ethnically diverse population has been exposed to infection in the setting of known racial and ethnic health disparities, 14 several news reports have suggested that ethnic and racial minorities, especially latinx and non-latin african american individuals, may bear a higher burden of disease during the covid-19 pandemic. [15] [16] [17] [18] [19] these reports also propose that such disparities are due to higher rates of pre-existing comorbidities in latinx and non-latin african american patients. other than these limited reports, the association between ethnicity and, race, and reported pre-existing comorbidities as risk factors for hospitalization and icu admission in covid-19 patients, has yet to be examined in a large, ethnically and racially diverse population. in this paper, we hypothesized that there are ethnicity and race-related disparities in hospitalization and icu admission for covid-19 patients regardless of age and reported pre-existing comorbidities. we used medical records available from the largest not-for-profit healthcare system in massachusetts to examine the association between age, race and ethnicity, reported preexisting comorbidities, and the need for hospitalization and icu admission in a large study population of covid-19 positive patients. mass general brigham is a not-for-profit healthcare system affiliated with harvard medical school that comprises 12 m a n u s c r i p t the design of the study was approved by the mass general brigham institutional review board (irb) who deemed that the study does not include factors necessitating patient consent. we used data reporting functions available through the electronic health record (epic systems, verona, wi) shared by all mass general brigham healthcare system institutions. we collected data on all patients 18 years or older who tested positive for covid-19 during an inpatient, outpatient, or emergency room visit between february 1, 2020 and april 14, 2020. we revisited the records on april 25, 2020 to collect follow up data on mortality and other outcomes. patients who presented to mass general brigham institutions with symptoms of fever, cough, sore throat, fatigue, muscle aches, new anosmia, who were exposed to someone who tested positive for covid-19, or if referred by a healthcare provider were tested per specified testing criteria/guidelines set forth by the institution. patients were diagnosed as infected with covid-19 if sars-cov-2 rna was detected in upper or lower respiratory specimens by nucleic acid testing (nat) assays designated for emergency use authorization (eua) by the food and drug administration (fda) and in accordance with the centers for disease control and prevention (cdc) guidelines. 20,21 each assay targets at least one sars-cov-2 gene region; positive results are reported for each assay as defined by the manufacturer or reference laboratory. hospitalization at any time during the course of the illness and admission to an icu at any time during hospitalization were primary endpoints. patients who were discharged home initially but were admitted later were categorized as hospitalized patients. patients hospitalized for longer than the follow up period were censored for study outcomes. we extracted the following covariates from the electronic health records for all patients: age, gender, patient-reported race (white, african american, asian american or pacific islander, other, or unknown), patient-reported ethnicity (latin or non-latin), smoking status, and the presence of recorded metabolic diseases including obesity (as measured by body mass index [bmi]), diabetes mellitus, and hyperlipidemia. the presence of organ-specific disease included hypertension, coronary heart disease, congestive heart failure, chronic obstructive pulmonary disease, asthma, m a n u s c r i p t interstitial lung disease, cerebrovascular disease, chronic kidney disease, end stage renal disease, malignancy including hematologic malignancy (lymphoma, leukemia), hiv, and history of organ or bone marrow transplantation. missing data were imputed by multiple imputation using the amelia package in r. 22 the multiple-imputation models used all baseline data. bmi and smoking status for all patients were also imputed by multiple-imputation models using r, and 10 imputations were carried out in total under the assumption that data were missing at random. data for median household income and population density were obtained from census data reported by the census bureau for the state of ma, and were linked to patient by zip code of reported residential address. 23 because immunocompromised patients generally demonstrate increased susceptibility to respiratory viral infections, we analysed our study population of patients for history of solid organ transplantation (sot), lymphoma, leukemia or hiv to assess whether these factors were associated with hospitalization or icu admission. as appropriate, descriptive analyses of variables are presented as proportions or medians with interquartile range (iqr) for all endpoints (not hospitalized, hospitalized, admitted to icu). categorical data were compared using chi-squared tests, while t-test was used for continuous variables to identify univariable associations. multivariable logistic regression models were constructed to identify factors associated with hospitalization and icu admission. we tested our assumption that data were missing at random by constructing logistic regression models for missingness using all of the variables included in multiple imputations. we further examined the association between racial and ethnic background and hospital admission by comparing proportions of endpoints for each 5-year age interval from 18 to 90. we also examined the association between racial and ethnic background, pre-existing comorbidities, and hospitalization or icu admission by stratifying by number of baseline comorbidities (0 vs. ≥1) and race and ethnicity. we further performed a sensitivity analysis with adjustment for socioeconomic status as a predictor for hospitalization/icu admission for patients from massachusetts state with median household income data available. patients with median household income less than 20 th percentile ($53, 335) were classified to have low ses as a dichotomous variable. statistical significance was defined as p<0.05 for all analyses, and all statistical analysis was completed using r v 3.6.1. 24 we graphed the geographic representation of the confirmed covid patients during our study using microsoft excel version 16.36. a total of 5,190 patients were diagnosed with laboratory confirmed covid-19 in the time frame of the study and were included in our analysis (figure 1) . out of the total study population, 1,489 (28.6%) were hospitalized. overall, hospitalized patients were more likely to be male (56% vs. 42%, p<0.001) and older (median 62 vs. 47 years, p<0.001) compared to non-hospitalized patients. hospitalized patients were also more likely to be obese (34% vs. 17% bmi 30-40 kg/m 2 , p<0.001), with on average more cardiovascular and pulmonary risk factors, and more comorbid conditions compared to non-hospitalized patients ( table 1) . the most common comorbidities in the hospitalized study population were hypertension (48%), hyperlipidemia (36%), diabetes (33%), and obstructive lung disease (15%). the mortality rate was higher in hospitalized compared to nonhospitalized patients (15% vs. 0.2%, p<0.001). our test of the missing at random assumption demonstrated significant predictors of missingness for all variables for which imputation was conducted. among the total covid-19 positive patient study population, 2,404 (46%) were white, 1,309 were latinx (25%), 719 were african american (14%) and 177 were asian american (3%). ethnicity and race were significantly associated with the rate of hospitalization. latinx and african american patients were more likely to be admitted to the hospital than white patients (35.9% and 29.1%, respectively, vs. 25.8% for white patients). overall latinx, african american, and asian american hospitalized patients were younger compared to white patients (median age 52, 60 and 61, vs. 72 respectively, p<0.001, table 2 ). subgroup analyses of the ages between 18-85 at 5-year intervals showed that in each age group, latinx and african american patients were more likely to be admitted as a result of covid-19 compared to white patients (p<0.05 for each comparison, figure 2 ). for example, among those 18-40 and 40-60 years old, latinx and african american patients were admitted to the hospital at the rates of (22% and 13% vs. 6%, and 46% and 38% vs. 20%, in comparison to. white patients respectively, p<0.001 for each comparison). we observed similar, among those aged >60 (supplementary table 1) . with regard to reported comorbidities, hospitalized latinx patients were more likely to be obese in the range of 30-40 kg/m 2 (41% compared to 31% among white and 33% among african american patients, p<0.001). however, the proportions of white, latinx and african american patients who were in range of >40 kg/m 2 were similar. white patients had higher rates of reported hyperlipidemia, hypertension, obstructive lung disease, coronary artery disease, congestive heart m a n u s c r i p t failure, chronic kidney disease and malignancy compared with other groups (p<0.05, table 2, supplementary table 2) . when stratified by baseline reported comorbidities, latinx and african american patients were admitted at twice the rate of whites, regardless of whether they had reported preexisting comorbidities (p<0.001, table 3 ). compared to non-hospitalized patients, univariable logistic regression stratified by race identified pre-existing comorbidities including metabolic, cardiovascular, cerebrovascular, pulmonary and kidney disease as predictors for hospitalization in all racial and ethnic groups (p<0.05, supplementary table 4 ). most of these predictors remained significant after adjustment for socioeconomic status (supplementary table 4 ). based on patients' zip-code data, 43% of all the patients who tested positive lived in zip codes with median income between $50,000-$75,000, of which 35% were hospitalized. a higher proportion of hospitalized latinx and african american patients lived in those zip codes (65% and 50% respectively, compared to 38% of white patients, figure 3a, supplementary table 7) . interestingly, smaller proportions of patients living in zip codes with income <$50,000 were hospitalized (supplementary table 6 ). in addition, residence in a zip-code with greater density of living per household was corelated with a higher likelihood of hospitalization (figure 3b, supplementary table 8 ). in our study population, 44% of admitted patients were from areas residence with population density >10,000/square mile. a history of solid organ transplantation was associated with a significantly increased risk for hospitalization (p<0.001). of the 22 transplant patients who were covid-19 positive (12 kidney recipients, three liver recipients, four heart recipients, two lung recipients and one heart/lung recipient) seventeen (77%) were admitted. in contrast, hiv, lymphoma or a history of leukemia were not associated with increased risk for hospitalization or icu admission (supplementary table 9 ). a possible difference between sot patients and those with hiv or a history of lymphoma or leukemia, is that sot patients were universally immunosuppressed at the time of infection while the degree of immune impairment for other groups was likely more heterogeneous. m a n u s c r i p t latinx and african american patients disproportionally required admission to the icu compared to white patients. overall, 39% of hospitalized latinx patients required admission to the icu compared to 33% of african american patients, 24% of asian american patients and 30% of white patients (p<0.007, table 2 ). the presence of reported metabolic or organ-specific comorbidities was not significantly associated with need for icu admission (supplementary table 5) . in multivariable regression analysis, age greater than 60 years old (or=2.71, 95%ci: 1.44-5.09 for latinx, or=5.49, 95%ci: 1.46-20.63 for african american patients) and obesity with bmi >40kg/m 2 (or=3.43, 95%ci: 1.45-7.67 for latinx patients), and diabetes (or=2.78, 95%ci: 1.08-7.11 for african american patients) were identified as significant predictors of icu admission ( table 5 ). in addition to these predictors, low median household income was as a significant predictor for icu admission in white patients (or=2.50, 95% ci: 1.39-4.52) after adjustment for socioeconomic status (supplementary table 6 ). in spite of aggressive efforts by the medical and public health communities worldwide, understanding of the overall impact of the covid-19 pandemic remains limited. based on data from china, italy, and spain, and preliminary data from the united states, patients who are 60 years or older are more vulnerable to covid-19, with higher morbidity and mortality. [4] [5] [6] [7] [8] [9] furthermore, patients with other comorbidities, such as cardiovascular disease and hypertension, are more likely to be hospitalized and die from the infection. 2, 5, 12, 13 this evidence mostly derives from studies performed in racially and ethnically homogeneous populations. 5, 10 the impact of the disease in an ethnically and racially diverse population has not been fully explored. anecdotal and news reports and a report from the uk suggest that racial and ethnic minorities may be more likely to contract covid-19, and more likely to suffer poor outcomes as a result of infection. [15] [16] [17] [18] [19] 25 price-haywood et al. and others showed that the majority of patients hospitalized with covid-19 and of those who died in a louisiana study population were african american. 15, 26 our firsthand clinical experience with covid-19 patients indicate that in addition to african american patients, a higher percentage of covid-19 latinx patients required hospitalization and critical care admission. to examine this issue, we investigated the impact of covid-19 on the patient population covered by our group of hospitals, which serve a diverse and broad population of eastern massachusetts similar to the racial and ethnic compositions of many large metropolitan areas of the united states. 27 we confirmed that age, male gender and obesity are indeed important risk factors m a n u s c r i p t for worse outcomes after covid-19 infection, and that the presence of reported comorbid medical conditions is an important contributing factor to hospitalization among all ethnic and racial groups. three additional important findings emerged from our study. first, analysis of our large study population confirmed our firsthand clinical experience and showed indeed that latinx and african american patients are at higher risk of being hospitalized and admitted to icu level of care with covid-19, than white patients. a second important finding is that the differences observed between latinx and african american vs. white patients occur at all age groups and are not only limited to the "higher risk" older age groups identified in prior studies. a third important finding is that the observed disparities appear to be less likely related to reported pre-existing medical comorbidities, since latinx and african american patients who tested positive for covid-19 were admitted at twice the rate of whites, regardless of whether they had reported comorbidities or not. in addition, the proportion of white patients who had reported comorbidities such as hyperlipidemia, hypertension, obstructive lung disease, coronary heart disease, cerebrovascular disease was at least as great as the proportions of latinx and african american patients who have these comorbidities. the underlying etiology of such disparity in hospitalization from covid 19 between latinx and african american vs. white patients is likely multifactorial. first, patients from these historically disadvantaged racial and ethnic groups may be less likely to be insured than white patients. immigration status could also play another role in restricting access of racial and ethnic minority patients to health insurance coverage and increase their challenges in finding a source of care to get tested or accessing covid related treatments. therefore, they may have presented at a later, more severe phase of the disease and therefore required hospitalization. in support of this, when we analyzed the zip-code of residence of these patients we found a close correlation between residence in a zip code of low median income and greater density of living in the same household with higher rate of hospitalization. in general, lower income patients tend to defer seeking healthcare for fear of financial burden and/or limited health care access and quality. 28 second, it is possible that latinx and african american patients with covid 19 have a higher severity of reported comorbidities. our data do not show that such disparities in hospitalization can be explained by the presence of reported comorbidities. however, there are limitations to our interpretation of these data. because our findings are based on reported data in the medical records, they do not take into account the severity of the preexisting conditions, which is difficult to quantify in such a large study population. it is likely that the severity of certain underlying comorbidities is higher in ethnic and racial minorities than in white patients, perhaps due to previously described healthcare disparities, 29, 30 or to issues with medication use and adherence. [31] [32] [33] third, other issues of stress and allostatic load that could impact health, which were out of the scope of our observational study may also be contributing factors to the observed disparities and require further investigation. 34, 35 these include crowded housing conditions as we alluded above, or the type of employment where exposure to covid 19 could possibly be more common. our study has several possible limitations. first, this is a registry database study from a single mixed health system (with primary and tertiary institution) using structured data captured in the a c c e p t e d m a n u s c r i p t electronic medical record. this study may also underrepresent covid 19 patients who do not seek medical attention or have whose medical data are stored at other facilities. however, the strengths of this study include a large, diverse study population of covid-19 positive patients from a wide geographic region that allowed us to analyze a large number of latinx, african american, asian american in addition to white patients. we were also able to collect detailed sets of variables on each patient, including factors that predict hospitalization and icu-level care, which allows for multivariable adjustment. follow up identified a number of outcome events, including deaths and icu-level admissions. while we acknowledge the limitations of our study, reporting data based on societal understanding of race and ethnicity, using patient self-reported race and ethnicity, is an important step in highlighting existing disparities in covid-19 treatment and try to mitigate contributing factors for the future. 38, 39 our findings also could have immediate policy implications, since it would be crucial to target the most vulnerable groups when testing or vaccination strategies are devised to limit further spread of covid-19 and minimize its impact. this is especially relevant with the new surge in the numbers of covid cases in states where latinx patients constitute a significant portion of the population such as florida and texas. m a n u s c r i p t table and figure legends table 1 . characteristics of all covid-19 positive patients in the mass general brigham healthcare system. table 2 . baseline characteristics by race and ethnicity among admitted covid-19 patients. table 3 . rate of hospitalization among all covid-19 patients by baseline comorbidities and race and ethnicity. *the household income and population density reported are obtained from county-level census data. dr. ross zafonte serves on the scientific advisory board of oxeia biopharma, biodirection, elminda, and myomo. he also evaluates patients in the mgh brain and body-trust program which is funded by the nfl players association. the remaining authors declare no conflict of interests. m a n u s c r i p t iqr=interquartile range, copd=chronic obstructive pulmonary disease, chf=congestive heart failure, ckd=chronic kidney disease, esrd=end stage renal disease *missing for 38% of population **missing for 16% of population m a n u s c r i p t epidemiology of covid-19 in a long-term care facility in king county presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the an interactive web-based dashboard to track covid-19 in real time early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of 138 hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china the resilience of the spanish health system against the covid-19 pandemic coronavirus disease 2019 (covid-19) in italy facing covid-19 in italy -ethics, logistics, and therapeutics on the epidemic's front line critical care utilization for the covid-19 outbreak in lombardy, italy: early experience and forecast during an emergency response characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention severe outcomes among patients with coronavirus disease 2019 (covid-19) -united states does comorbidity increase the risk of patients with covid-19: evidence from meta-analysis epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study the changing racial and ethnic composition of the us population: emerging american identities african american covid-19 mortality: a sentinel event covid-19 and african americans kills-latinxs-and-african americans-in-new-york-city.) 20. interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease 2019 (covid-19) income statistics and demographics for states, counties, cities and zip codes risk factors for sars-cov-2 among patients in the oxford royal college of general practitioners research and surveillance centre primary care network: a cross-sectional study hospitalization and mortality among african american patients and white patients with covid-19. nejm 2020. 27. population health statistics: race and ethnicity reports socioeconomic disparities in health in the united states: what the patterns tell us mechanisms for racial and ethnic disparities in glycemic control in middle-aged and older americans in the health and retirement study contribution of major diseases to disparities in mortality racial and ethnic disparities in medication adherence among privately insured patients in the united states socioeconomic status and nonadherence to antihypertensive drugs: a systematic review and meta-analysis race/ethnicity, disability, and medication adherence among medicare beneficiaries with heart failure allostatic load burden and racial disparities in mortality protective and damaging effects of stress mediators obesity as a predictor for a poor prognosis of covid-19: a systematic review commentary: obesity: the "achilles heel" for covid-19? racial health disparities and covid-19 -caution and context covid-19 and racial/ethnic disparities a c c e p t e d m a n u s c r i p t key: cord-021655-ojfm5rt3 authors: langan, jennifer n.; jankowski, gwen title: overview of african wild dog medicine date: 2018-09-28 journal: fowler's zoo and wild animal medicine current therapy, volume 9 doi: 10.1016/b978-0-323-55228-8.00077-1 sha: doc_id: 21655 cord_uid: ojfm5rt3 nan the african wild dog (lycaon pictus)-also referred to as the african hunting dog, painted dog, and cape hunting dog-is one of africa's most endangered carnivores. 1 owing to its decreasing numbers, it holds an international union for conservation of nature (iucn) red list priority status for the conservation of canid species in africa. 2 african wild dogs were formerly distributed throughout sub-saharan africa but are now mostly confined to southern africa and the southern portion of east africa (fig. 77.1 ). 3, 4 they require large ranges and live at low population densities. the population is currently estimated at 6600 animals and continues to decline as a result of ongoing habitat fragmentation, conflict with humans, and infectious disease. 1, 5 predation by lions and competition with spotted hyenas also contribute to population suppression. 6 there are approximately 600 african wild dogs in zoos, which serve to educate the public and fulfill an important role as ambassadors aiding the recovery effort for this species. the african wild dog is a member of the family canidae in the order carnivora; it likely diverged from wolves in the pleistocene period. 1 genetic studies demonstrate that it is sufficiently different from other canid species to warrant being classified into a separate genus. adults weigh 18-35 kg, with males slightly larger than females. 2, 3, 7 the average age of survival in zoos is 10.3 years 8 ; a few animals live 12-16 years. 2, 7, 9 the african wild dog's most striking characteristic is the tricolored spotted coat, for which it received its latin name, lycaon pictus, meaning "painted wolf." it has large round ears, lacks a supracaudal (tail) gland, has four digits on each limb (lacks dew claws), and the pads of the middle digits are connected by dermal webbing. reproductive anatomy is similar to that of domestic dogs in both males and females, but females have 12-14 mammae. they have very sharp, large premolars relative to their body mass, which allow them to consume 77 overview of african wild dog medicine jennifer n. langan and gwen jankowski sizable quantities of meat and bone with impressive speed. the dental formula is i3/3, c1/1, pm3/4, m3/3 = 21, of which the last mandibular molar is vestigial and generally not visualized. 1 the typical wild pack is composed of an older dominant female paired with a young dominant male and subordinates of both sexes. dominant males may be displaced as they age or lose strength. juvenile males are most likely to stay with the pack, whereas females often emigrate. following the death of the dominant female, significant social changes occur within the group, which can result in pack dispersal in the wild. 1, 7 social management of african wild dogs under human care is challenging and can have significant health impacts. it involves working across institutions to create and maintain packs that thrive socially, support a healthy population, and sustain genetic diversity. the most stable social groups include a well-established dominant pair with male offspring of any age and young female offspring. the inability to disperse may result in conflict between female offspring above 18 months of age and the dominant female. the decreasing frequency of "hoo-calls" (long-distance communication calls) and distance between resting sites of same-sex groups suggest that unrelated individuals under human care are more likely to integrate into a pack successfully. 10 if animals need to be separated due to social incompatibility, it is recommended that individuals be split up as same-sex packs or with littermates less than 18 months of age. 7 contraception for reproductively mature individuals intended to reduce aggression has not been successful. 7 measurement of fecal corticosteroids may be a useful management tool and has demonstrated that dominant animals generally have the highest stress levels. [11] [12] [13] behavior is a key indicator of social and physical wellbeing in african wild dogs. "normal" behavior varies by an individual's status within the pack, and establishing pack hierarchy is essential for avoiding excessive aggression. have access to multiple heated areas if the temperature regularly drops below 4.4°c-7.2°c (40°f-45°f) and should have shelter from the elements. 7 additionally, a heated den should be provided if breeding is planned. facilities should have sufficient holding space to accommodate separating animals for long periods. with the exception of fish, housing african wild dogs with other species is not recommended due to their high predatory drive. african wild dogs should be moved only in sturdy metal or wood crates with good ventilation that meet us department of agriculture (usda) and international air transport association (iata) requirements for live animal transport. completing a written transport plan, health evaluation, and crate training facilitates transitions when animals are relocated. african wild dogs are generalist predators, occupying a range of habitats where they hunt medium-sized antelope. 6 in natural settings, reduced prey populations and competition from other predators inhibit population growth. 3, 17 african wild dogs in zoos are fed a nutritionally complete raw meat-based diet (1-1.36 kg/adult/day) and are supplemented with small whole prey, knuckle/rib/shank bones (one to two times week), and carcasses (pig, deer, calf, horse). lactating bitches require up to three times permanent or even brief temporary removal of an established pack member may have profound social impacts, including changes in social hierarchy with aggression so substantial that reintroduction may not be possible. 7, 15 detailed plans to reduce stress and promote normal behaviors should be implemented if a dog must be isolated, including maintaining olfactory and visual contact. if separation is required, it may be helpful to subdivide the pack and then reintroduce them all simultaneously. alternatively, introductions of subordinate dogs first, then dominant pairs, may be effective. successful implementation of enrichment has included environmental devices, sensory stimulants, and food, behavioral, and habitat variance. 16 piles of leaves, dirt, and mulch allow natural digging and rolling behaviors. rotating exhibits with other predators provides habitat diversity and promotes scent-marking behavior. offering carcass feeds hung from trees or on zip lines, feeding bones, providing several types of enrichment to the pack simultaneously, and permitting breeding when possible are recommended for this species. 15, 16 enclosures should be large and contain ample space for exercise to meet the animals' physical, social, behavioral, and psychological needs. 7 specific size and perimeter recommendations may be found in the association of zoos & aquariums (aza) large canid care manual. 7 facilities that allow the public to observe the animals should prevent close contact or inadvertent access to the enclosure. dogs should ah, inc., fort worth, texas). current estimates show 23% of females have some degree of reproductive pathology, 28 the most frequently reported of which is cystic endometrial hyperplasia (ceh) with or without pyometra and adenomyosis ( fig. 77.2 ). [27] [28] [29] adenocarcinoma, uterine rupture, and pyometra without other pathology have also been reported (kinsel, personal communication, april 10, 2017). 23, 27, 28 the species survival plan program (ssp) currently recommends that all postreproductive females (>10 years) be spayed. 8 deslorelin implants have been used for contraception and behavioral alteration in males with variable results (see table 77 .1 for a summary of reproductive information) (see also chapter 22) . 26, 30, 31 healthy adult african wild dogs are not physically restrained due to safety concerns. noninvasive procedures including visual examination, hand injections, wound treatment, venipuncture, and crate training may be accomplished with operant conditioning. restraint cages are useful and provide a safe, controlled environment to facilitate intramuscular injections of anesthetics. a quiet area away from the pack promotes quick inductions and smooth recoveries. remote injection systems are recommended for immobilizing free-ranging african wild dogs or in situations when a chute is not available. anesthetic regimens selected should take into account health status, age, and environmental conditions. in cases where cardiovascular disease has been confirmed or cardiac status is unknown, alpha-2 agonists should be avoided and alternatives such as ketamine-midazolam-butorphanol with propofol or gas anesthesia (isoflurane, sevoflurane) should be considered. chemical restraint protocols used in african wild dogs may be found in table 77 .2 and have been previously published. [32] [33] [34] drug combinations at higher dosages for free-ranging african wild dogs are available in the literature. 35, 36 reversal of alpha-2 agonists and opioids with atipamezole and naloxone decreases recovery time. to avoid dysphoria, it is advised to wait at least 60 minutes postinduction before administering reversals. recovery in a crate or nest box may reduce struggles to stand during recovery. telazol (tiletamine hcl and zolazepam hcl, zoetis, parsippany, new jersey) as a sole agent or used in combination with medetomidine is a reliable option during an emergency response but often results in a prolonged recovery time. 34 vascular access, intubation, and monitoring equipment are applied as in other canids. every wild dog anesthesia should include oxygen supplementation, electrocardiography (ecg), and monitoring of pulse oximetry, heart and respiratory rate, blood pressure, and temperature. as in other canids, the jugular, cephalic, and saphenous veins are commonly used sites for venipuncture. tables 77.3 maintenance caloric intake. specific recommendations for kilocalorie requirements may be found in the aza large canid care manual. 7 feeding african wild dogs a portion of their diet while separated from the pack aids in monitoring individual animals' food consumption. packs are generally fasted from their normal meat diet 1 day per week and may be provided with bones. african wild dogs are seasonally monoestrous obligate cooperative breeders with a brief copulatory tie. 18, 19 within a pack the alpha male and female produce the majority of surviving pups annually. 7, 20, 21 most successful reproduction occurs after 2 years of age, with senescence around 8-9 years. 8, 21 subordinate females may reproduce, but offspring typically do not survive. more often, subordinate females develop pseudopregnancy and may lactate in order to help care for the pups of the dominant pair. 1 females produce an average of six to eight pups 22,23 and up to 21 pups 8 in a den after a gestation of 69-71 days. [24] [25] [26] primiparous females have higher estrogen, which is reported to result in more male offspring. 24 hand-rearing is not recommended due to the extremely aggressive and social nature of these animals. 8 in zoos, the breeding pair is separated from the pack to prevent trauma to the pups. the group is gradually introduced when the pups begin to emerge from the nest box. a birth plan detailing responses to aggression toward the pups, large litter size, and other contingencies is recommended. newborn pups weigh about 300 g, open their eyes around 2 weeks of age, and emerge from the den to start taking solid food at 3 weeks. sex determination is similar to that for other canids. pups are weaned and start to follow the pack at 11-12 weeks of age. in free-range settings, all members of the pack raise the pups; they regurgitate food while the young are in the den and relinquish kills to the pups and yearlings. 1, 3 reproductive anatomy is similar to that of other canid species. owing to social dynamics, most reproduction has been natural; however, semen has been preserved and used for artificial inseminations. 15 captive lycaon pictus generally reproduce in the fall in the northern hemisphere. 8 estrus lasts 6-9 days and includes vulvar swelling and sanguineous discharge with interest from the male. attraction from the male may be observed for 1-2 weeks prior to tying. 14, 25 males show increased testicular development, spermatorrhea, and semen production; therefore the corresponding seasonal ability to collect sperm via electroejaculation is improved. 15 the progestin-based melengestrol acetate (mga) implant, previously used in canids, has been associated with uterine pathology. 27 the aza reproduction management center (formerly the wildlife contraception center) (www.stlzoo.org/ animals/scienceresearch/reproductivemanagementcenter) recommends gonadotropin releasing hormone (gnrh) agonists such as suprelorin (deslorelin acetate) implants or lupron depot (leuprolide acetate 4.7 mg implant, virbac extinction events. [38] [39] [40] [41] there is serologic evidence of exposure to canine parvovirus, canine distemper virus, adenovirus, rabies virus, coronavirus, rotavirus, and ehrlichia canis. 21, 42, 43 outbreaks of anthrax in kruger national park occur, but infected animals commonly survive. 43 contact with domestic dogs has been reported to increase exposure to some canid pathogens, but sylvatic viral strains also pose a significant threat. 5, 44 parasitic disease and infection has rarely been described. [44] [45] [46] toxocara canis, dipylidium caninum, spirometra sp., taeniidae, and ancylostoma spp., as well as two genera of canid protozoan gastrointestinal parasites, sarcocystis and isopora, were identified in fecal samples from free-ranging animals but were not associated with clinical disease. 44, 47 standard anthelmintics at canine dosages have been successfully used to treat internal parasites. it is recommended that african wild dogs be routinely tested and maintained on prophylactic heartworm preventative in endemic areas. over the last decade, valvular dysplasia of varying severity has been increasingly recognized as a significant concern in african wild dogs in north america. sibling groups and offspring have been affected over multiple generations, and 77.4 show normal hematologic and serum chemistry reference ranges for captive african wild dogs. 9, 37 diseases rabies and distemper have contributed to mortality in african dog populations, occasionally resulting in local neoplasia has not been extensively reported in the literature but appears to play an important role in the health of captive populations. 48 apocrine gland tumors have been documented in clinical settings, presenting as single or multiple dorsal cutaneous masses that can progress to large regionally invasive tumors (fig. 77.4) (agnew, personal communication, april 25, 2017) . cases from females are overrepresented in pathology reports submitted to the african wild dog ssp (kinsel, personal communication, april 10, 2017). surgical excision is recommended, although large tumors may require other forms of treatment. tumor growth results in ulceration and necrosis and negatively affects an animal's quality of life. other common neoplasias include hemangiosarcoma, peripheral odontogenic fibroma (fibromatous epulis), adrenocortical adenoma/carcinoma, and mammary and uterine neoplasia. other notable conditions diagnosed in african wild dogs include dental disease-particularly fractured teethpancreatitis, diabetes, spina bifida, syringomyelia, keratitis, snake bites, 49 and trauma from conspecifics (kinsel, personal communication, april 10, 2017). african wild dogs mask signs of illness and may have advanced disease by the time a change in behavior or appetite is observed. suggesting that the condition has a genetic, inheritable basis, as is well documented in domestic dogs. 8 cases range from minor to severe valvular insufficiency with congestive heart failure (fig. 77.3) . mildly affected animals exhibit no clinical signs, making the condition difficult to detect. the extent of this disease is uncertain, but it is highly probable that cardiac disease is underdiagnosed. incorporating preventative health, preshipment, and quarantine examinations should be conducted to determine an animal's health. a complete physical exam-including a dental examination, complete blood count, chemistry panel, heartworm testing, urinalysis, radiographs of the thorax and abdomen, and evaluation for endo-and ectoparasites-should be completed on a routine schedule as part of a preventive medicine plan. additionally, abdominal ultrasound examination or computed tomography in females and echocardiograms for both sexes are recommended owing to disease predilection in this species. athens, georgia) provided persistent protective titers. 57 a survey in 2006 showed that most african wild dogs maintained presumably protective titers after vaccination for canine distemper and rabies for 1 year; however, few dogs maintained titers for 2-3 years. 58 there is a paucity of scientific information regarding vaccination against canine parvovirus and leptospiral infection. protocols should be developed that take into consideration local environmental disease prevalence, animal health, and risk factors. select vaccination protocols for captive african wild dogs are listed in table 77 .5 (see also chapter 79). external and internal parasites should be treated according to domestic dog guidelines. fleas, ticks, and ear tip trauma from biting flies have responded well to products containing carbaryl or pyrethrins. good hygiene, removing standing water, fly traps, and premise sprays help control fly and mosquito populations. newly acquired animals should be quarantined away from the collection for a predetermined period based on a thorough risk assessment by the supervising veterinarian and have at least two negative fecal examinations. animals should be individually identified with microchip transponders placed subcutaneously between the shoulder blades or to the left of midline over the shoulder. currently there are no universal recommendations for vaccination protocols. the safety and efficacy of vaccines in african wild dogs have historically been unsatisfactory. vaccination strategies to conserve free-ranging populations have been reported and continue to be investigated. 50 modified live canine distemper vaccinations have failed to produce protective antibody levels in some cases 51 and have induced distemper resulting in mortality in other cases. 7, 45, 52, 53 vaccine-induced distemper can be avoided by using killed vaccines, and at least one vaccine (purevax ferret distemper, merial inc., athens, georgia) has been shown to produce measurable titers after a series of three injections at 2-to 3-week intervals. 54 subunit canine distemper vaccines (cdv-iscom, erasmus mc, rotterdam, the netherlands) stimulated appropriate titer formation, but titers did not endure relative to the purevax ferret distemper vaccine. vaccine recommendations for domestic dogs have been reduced from yearly to triennially; however, it is unknown whether nondomestic canids maintain titers in a similar manner. in one study, protective titers from the purevax vaccine persisted in 39%-85% of african wild dogs for a minimum of 1 year. 55 early studies have indicated that vaccination with killed rabies vaccines may not be sufficient for protection. 5, 50, 56 however, a recent study showed that a single intramuscular vaccination of dogs older than 14 weeks with imrab 3 (merial inc., the african wild dog: behavior, ecology and conservation canids: foxes, wolves, jackals and dogs. status survey and conservation action plan. iucn/ssc canid specialist group carnivora. in grzimek's animal life encyclopedia lycaon pictus (north africa subpopulation) birth order, estrogens and sex-ratio adaptation in african wild dogs (lycaon pictus) steroid metabolism and validation of noninvasive endocrine monitoring in the african wild dog (lycaon pictus) seasonal changes in steroid hormone profiles, body weight, semen quality and the reproductive tract in captive african wild dogs (lycaon pictus) in south africa naturally occurring and melengestrol acetate-associated reproductive tract lesions in zoo canids retrospective study of mortality of captive african wild dogs (lycaon pictus) in a french zoo cystic endometrial hyperplasia and pyometra in three captive african hunting dogs (lycaon pictus) control of reproduction and sex related behaviour in exotic wild carnivores with the gnrh analogue deslorelin: preliminary observations induction of contraception in some african wild carnivores by downregulation of lh and fsh secretion using the gnrh analogue deslorelin immobilization of wild dogs (lycaon pictus) with a tiletamine hydrochloride/ zolazepam hydrochloride combination and subsequent evaluation of selected blood chemistry parameters anesthesia of captive african wild dogs (lycaon pictus) using a medetomidine-ketamineatropine combination zoo animal and wildlife immobilization and anesthesia chemical restraint and immobilization of wild canids immobilization of freeranging african wild dogs (lycaon pictus) using a ketamine/ xylazine/atropine combination lycaon pictus, no selection by gender, all ages combined, 2013 cd.html in isis physiological reference intervals for captive wildlife: a cd-rom resource african wild dogs (lycaon pictus) endangered by a canine distemper epizootic among domestic dogs near the masai mara national reserve canine distemperrelated mortality among wild dogs (lycaon pictus) in chobe national park fatal canine distemper infection in a pack of african wild dogs in the serengeti ecosystem contact with domestic dogs increases pathogen exposure in endangered african wild dogs (lycaon pictus) diet of four sympatric carnivores in save valley conservancy, zimbabwe: implications for conservation of the african wild dog (lycaon pictus) aza canid tag: large canid (canidae) care manual population analysis and breeding and transfer plan: african painted (wild) dog (lycaon pictus) aza species survival plan yellow program general zims database reference: species360, zims.species360. org behavioural cues can be used to predict the outcome of artificial pack formation in african wild dogs (lycaon pictus) monitoring stress in captive and free-ranging african wild dogs (lycaon pictus) using faecal glucocorticoid metabolites evaluating adrenal activity in african wild dogs (lycaon pictus) by fecal corticosteroid analysis social stress and dominance reproductive hormonal patterns in pregnant, pseudopregnant and acyclic captive african wild dogs (lycaon pictus) studies of male reproduction in captive african wild dogs (lycaon pictus) enrichment options for african painted dogs (lycaon pictus) order carnivora hormonal and experiential factors in the expression of social and parenteral behavior of canids natural selection of the communal rearing of pups in african wild dogs (lycaon pictus) a molecular genetic analysis of social structure, dispersal, and interpack relations of the african wild dog (lycaon pictus) serosurvey for selected viral diseases and demography of african wild dogs in tanzania conservation role of captive african wild dogs (lycaon pictus) intrapartum uterine rupture with coincidental uterine adenomyosis in an african wild dog (lycaon pictus) vaccination strategies to conserve the endangered african wild dog (lycaon pictus) serum antibody levels before and after administration of live canine distemper vaccine to the wild dog (lycaon pictus) canine distemper in african hunting dogs (lycaon pictus) -possibly vaccine induced vaccine-associated canine distemper infection in a litter of african hunting dogs (lycaon pictus) comparison of oral and intramuscular recombinant canine distemper vaccination in african wild dogs (lycaon pictus) humoral immune response of african wild dogs (lycaon pictus) to novel canine distemper vaccines in proceedings of the attempts to reintroduce african wild dogs (lycaon pictus) into etosha national park, namibia single versus double dose rabies vaccination in captive african wild dogs (lycaon pictus) immunization and antibody persistence to canine distemper and rabies vaccination in captive african wild dogs (lycaon pictus rabies in african wild dogs (lycaon pictus) in the serengeti region serologic survey for selected microbial pathogens in african wild dogs (lycaon pictus) and sympatric domestic dogs (canis familiaris) in maasai mara an investigation into the health status and diseases of wild dogs (lycaon pictus) in kruger national park a survey of internal parasites in free-ranging african wild dogs (lycaon pictus distemperlike disease and encephalitozoonosis in wild dogs (lycaon pictus) molecular detection of babesia rossi and hepatozoon sp. in african wild dogs (lycaon pictus) in south africa african wild dog (lycaon pictus) and spotted hyaena (crocuta crocuta) in the luangwa valley, zambia multilobular tumor of bone in an african wild dog (lycaon pictus) successful snakebite treatment in three juvenile african wild dogs (lycaon pictus) with polyvalent antivenom: a namibian case report this chapter is dedicated to the researchers, veterinarians, biologists, and animal care staff that have contributed to our collective knowledge, helped conserve wild populations, and have improved the care of african wild dogs around the world. we thank drs. anneke moresco and mike kinsel for their contributions to reproduction, pathology, and disease presented in this chapter and drs. sathya chinnadurai and matthew lenyo for sharing their expertise and thoughtful review. key: cord-260305-pl2ditn7 authors: nyika, aceme title: the ethics of improving african traditional medical practice: scientific or african traditional research methods? date: 2009-11-30 journal: acta tropica doi: 10.1016/j.actatropica.2009.08.010 sha: doc_id: 260305 cord_uid: pl2ditn7 abstract the disease burden in africa, which is relatively very large compared with developed countries, has been attributed to various factors that include poverty, food shortages, inadequate access to health care and unaffordability of western medicines to the majority of african populations. although for ‘old diseases’ knowledge about the right african traditional medicines to treat or cure the diseases has been passed from generation to generation, knowledge about traditional medicines to treat newly emerging diseases has to be generated in one way or another. in addition, the existing traditional medicines have to be continuously improved, which is also the case with western scientific medicines. whereas one school of thought supports the idea of improving medicines, be they traditional or western, through scientific research, an opposing school of thought argues that subjecting african traditional medicines to scientific research would be tantamount to some form of colonization and imperialism. this paper argues that continuing to use african traditional medicines for old and new diseases without making concerted efforts to improve their efficacy and safety is unethical since the disease burden affecting africa may continue to rise in spite of the availability and accessibility of the traditional medicines. most importantly, the paper commends efforts being made in some african countries to improve african traditional medicine through a combination of different mechanisms that include the controversial approach of scientific research on traditional medicines. traditional medicine (tm) is found virtually on all continents where it has been practiced for many centuries. in most developing countries tm is easily accessible and affordable to the larger proportion of the populations relative to western medicine (wm). in africa, it is estimated that about 80% of the populations rely on tm for their primary health care needs (who, 2008) . in the developed world tm is used by a relatively smaller proportion of the populations than the proportion that relies on wm. consequently, in the developed countries tm is referred to as "complementary" or "alternative" medicine (cam) since it complements wm which is accessed by a larger proportion of the populations. according to the world health organisation (who, 2008) , "traditional medicine refers to health practices, approaches, knowledge and beliefs incorporating plant, animal and mineral based medicines, spiritual therapies, manual techniques and exercises, applied singularly or in combination to treat, diagnose and prevent illnesses or maintain well-being." * tel.: +255 22 2700018; fax: +255 22 2700380. e-mail address: anyika@amanet-trust.org. one major characteristic of african traditional medicine (atm) is that it has spiritual and non-spiritual components (jolles and jolles, 2000) . the non-spiritual component is generally referred to as herbalism. it should be made very clear that this paper is not discussing the spiritual component of atm that involves divine healing based on religious faith which is believed to involve supernatural spiritual powers. however, in most cases in practice there is lack of clarity as to whether a traditional healer is practicing as a herbalist or as a divine healer. it is such ambiguity that tends to shroud the practice of atm with so much sacredness that any attempt to question anything associated with the practice is perceived by some people not only as a taboo but also as one of the manifestations of neocolonialism. a paper that touched on some ethical and regulatory issues surrounding african tm in the context of hiv/aids (nyika, 2007) stimulated some debate in the public domain (knapp van bogaert, 2007; tangwa, 2007) . according to tangwa, ethical principles that emanated from atrocities committed by western medical practitioners should not be used to assess whether some actions of african traditional practitioners are right or wrong because they did not take part in the atrocities (tangwa, 2007) . the current paper, which focuses on atm, will address the issue of whether or not the african traditional healers conduct any research, be it scientific or 'african traditional', before they come up with new treatments. if they do, then there are ethical requirements pertaining to conduction of research in patients which should be met (cioms, 2002; world medical association (wma) declaration of helsinki, 2008) . two case studies that capture research approaches that were used during edward jenner's era in western medicine and approaches that are currently being used by some african traditional healers will be discussed. the aim is to highlight firstly the need for properly designed research to improve both traditional and western medicines and secondly, to flag some ethical issues surrounding the practice of and 'research' in atm. the case studies illustrate the fact that research methods that were considered acceptable centuries of years ago should always be reviewed and improved as part of efforts to enhance protection of the well being of patients in particular and mankind in general. 2.1. case study 1: discovery of vaccination by edward jenner when edward jenner tested the concept of using cowpox virus as a vaccine against small pox, he used a small boy, james phipps, as an experimental subject (http://en.wikipedia. org/wiki/edward jenner). there was a serious outbreak of measles, and as a medical practitioner whose duty was to save human lives, he used his observation that milk maids exposed to cowpox virus were less susceptible to infection with small pox virus as the rationale for testing cowpox virus in the small boy. edward jenner used his experience and knowledge in his capacity as a medical practitioner and demonstrated the concept of vaccination. he became very famous and globally, vaccines have contributed towards successful control of many diseases such as measles and polio. however, research methodologies for the development of vaccines have since evolved and the approach used by jenner is now considered to be poor scientific design and unethical. instead of one person conducting the research, multidisciplinary research teams conduct research so as to minimize the risks of bias and errors in the research process as well as in the analysis of collected data. in addition, a research protocol has to undergo review to ensure minimum acceptable scientific and ethical standards before the research starts. 2.2. case study 2: discovery of hiv/aids treatment and cure by a traditional healer a well-known, highly respected african traditional healer used to treat many patients suffering from a variety of illnesses. when the hiv/aids pandemic started affecting his country, he also started 'diagnosing' hiv infection in some of his patients. using his knowledge and experience as a traditional healer, he tried some of his traditional medicines on his hiv/aids patients but without informing them that the medicines were being tested. after the 'trials', the healer was convinced that the medicines were effective not only for treating but curing hiv/aids patients. since his medicines were very affordable compared with the western medicines, he soon became the first port of call for many patients seeking treatment and cure for hiv/aids. he became a very rich and famous person in his country and in neighbouring countries. however, the traditional healer was totally against the idea of subjecting his medicines for hiv/aids to any form of tests involving other stakeholders and insisted that his observations and deductions were sufficient to guarantee safety and efficacy. the traditional healer and those who shared his view argued that scientists and pharmaceutical companies wanted to hijack the traditional knowledge for their own commercial benefits. on the other hand, people calling for properly designed scientific research to be conducted on the traditional medicines argued that if the traditional medicines that were being claimed to treat or cure hiv/aids were truly effective, the ever rising disease burden and mortalities due to hiv/aids in africa would have been brought under control since the traditional medicines were widely accessible to those who needed them. most importantly, they argued that the risk of bias in the observations and deductions made by the healer who would benefit from payments by patients was unacceptably high. 3.1. western medical practice and research: from edward jenner type of research to randomised controlled trials case study 1 captures research methodology that was used ages ago, and was acceptable then but is no longer acceptable nowadays. today, a single medical doctor may not simply decide to test something in a patient or in patients without involving other people with relevant expertise that the doctor may not have. there is now a requirement that any testing of anything in human beings should be explained in a protocol in order to enable peer review (cioms, 2002 guideline 2; world medical association (wma) declaration of helsinki, 2008) . the review of the protocol is meant to ensure that firstly there are no flaws in the planned methodology that may compromise the usefulness of the findings and secondly that the safety of the humans who will take part will not be compromised. the most important requirement for research nowadays is the informed consent. many international and national ethical guidelines or legal frameworks clearly stipulate that obtaining informed consent is a prerequisite for research. for instance, the cioms guideline 4 states that "for all biomedical research involving humans the investigator must obtain the voluntary informed consent of the prospective subject or, in the case of an individual who is not capable of giving informed consent, the permission of a legally authorized representative in accordance with applicable law. waiver of informed consent is to be regarded as uncommon and exceptional, and must in all cases be approved by an ethical review committee (cioms, 2002) ." thus when a patient presents to a medical doctor seeking treatment and the doctor happens to be involved in a study that is testing an alternative treatment, the doctor must inform the patient so that s/he decides whether to take part in the study or not. the patient may decide not to take part in the study but should still be given the standard treatment. if there is no proven standard treatment for the disease, then the doctor has to explain to the patient that the medicine available is still being tested. that is the distinction between medical practice and research. indeed, both the treatment and research may be done in the same hospital settings, but the patients must be informed if they are to be used in a study. edward jenner conducted his experiment alone, which during his era was most probably acceptable to the communities, the medical fraternity and perhaps to the parents of james phipps for medical practitioners to use their discretion to conduct 'research' as they saw fit without any peer review. it was fortunate that edward jenner's observations and deductions happened to be correct and that his 'experiment' yielded positive results without serious adverse reactions. but nobody knows whether or not he had conducted other 'experiments' detrimental to the health of his research subjects. nowadays it is no longer acceptable, no matter how knowledgeable the medical doctor is, to conduct such tests using people without voluntary informed consent. we are in a new era in which people should be adequately informed to enable them to make informed decisions. precautions should always be taken to safeguard the welfare of patients or research participants, and also to be sure that the research methodology is such that the research questions can be answered objectively and accurately. it should be acknowledged that atm has evolved over centuries of years, and has involved systematic methods of assessing medicinal or detrimental properties of herbs. although one school of thought may want to refer to that systematic assessment of traditional herbs as 'african traditional research methods', it could be argued that the systematic approach was basically scientific, more or less similar to the approach that jenner used, and was devised by the african traditional healers themselves. for instance, one approach used by the traditional healers long time ago was to observe herbs that were never eaten at all by animals, and such herbs were suspected to be poisonous and would be avoided by the traditional healers. long time ago there were no means of testing toxicity; hence such an approach was logical during that era. the point is that centuries of years ago african traditional healers developed methods of assessing safety and medicinal properties of herbs that were logical and acceptable during that era, but the methods should change with time. traditional knowledge was therefore not gathered haphazardly, there were systematic ways of gathering the knowledge which was then passed from one generation to the next. was this approach of gathering knowledge not scientific research methodology that was acceptable during that era, just like jenner's approach? if it was not scientific but an african research methodology, should it remain the same as it was ages ago even when circumstances are changing and the diseases affecting africa are dynamic? just as there were limitations in the research methodologies used by jenner, the research methodologies used by traditional healers had limitations which should be gradually overcome as time goes by. in other words, the research approaches used by traditional healers should be dynamic, the aim being to continuously improve and overcome limitations. in contrast to case study 1 which depicts what used to happen ages ago in the context of wm, case study 2 shows what is still happening today in the context of atm. in the advent of the hiv/aids pandemic, the african traditional healer in case study 2 tried some of his medicines in patients and came to the conclusion that his medicines were effective. the implication is that there was some form of research that involved testing of the traditional medicines for hiv/aids, albeit without disclosure of such 'research' activities to the patients. the traditional medicines that were being given to people living with hiv/aids were not free of charge; the traditional healer benefited financially from the sale of the medicines. it therefore means that there was conflict of interest since the traditional healer testing the medicines was the one who interpreted the findings and eventually benefited financially from the sale of the medicines. although the medicines were affordable to the majority of the people needing them, the high prevalence of hiv/aids in most african countries meant that there were large numbers of people needing such medicines, which translated into huge financial gains for the healer relative to ordinary members of the community. another point to be highlighted is that when the traditional medicines are being tested, the patients are not informed about the testing aspects. it is important that any necessary procedural differences in the process of obtaining informed consent are allowed in order to respect different cultural and traditional practices. for instance, empirical studies conducted in some developing countries showed that most people prefer to discuss with their spouses or relatives before deciding whether or not to take part in health research (decosta et al., 2004; nyika et al., 2009) . thus obtaining informed consent should not be an event, but a process that allows ample time for consultations as per the wishes of the prospective participants. another issue is that patients have to buy the traditional medicines even if they are still being 'tested' and may have side effects. there is need to assess potential side effects, and such assessments may require laboratory tests in addition to clinical assessments that the traditional healers may perform. it is therefore critical that medical practice, be it traditional or western, is clearly distinguished from medical research, especially when medical practitioners play both roles as service providers and researchers. it also means that there should be well thought out plans of how the research would be done, instead of anecdotal ways of testing medicines, be they western or traditional. most importantly, the experimental people should be informed that the medicines are being tested in them. since no person is omniscient, there may be need to include people with expertise in various relevant fields in the research team. instead of having a one-man research project, like the one edward jenner or the traditional healer conducted, bringing on board other experts such as epidemiologists, microbiologists, statisticians, pharmacists, physicians and people experienced in conducting clinical trials could significantly increase the quality of research. the ultimate goal of health care systems is to reduce the burden of diseases and improve the quality of life. thus it negates the purpose of having health care if availability and affordability of the health care do not effectively contribute towards the achievement of good health of people and reduced disease burden. it would be unethical to focus only on availability and affordability of african traditional medicines to the poor african populations relying on the medicines without assessing whether or not the disease burden of the populations is being reduced. it should be acknowledged that there are other factors such as poverty, poor nutrition and environmental factors that affect disease burden. however, if some african traditional medicines and some western medicines can make positive impact even in the presence of such factors, then the aim should be to improve the medicines so that they can make as much impact as possible. the question that should be asked is whether or not the access to african traditional medicines by the estimated 80% of the african populations is effectively tackling the disease burden of the populations. in the advent of emerging and re-emerging diseases, it is imperative that atm keeps improving in order to rise up to the new challenges. organizations such as the cdc have realized the need for public health systems to be ready for such diseases and are always working on prevention strategies in light of environmental, societal and technological factors (centers for disease control and prevention (cdc), 1998). examples of emerging diseases are hiv/aids, ebola, severe acute respiratory syndrome (sars), multidrug resistant tuberculosis (mdrtb) and extreme drug resistant tuberculosis (xdr tb). examples of re-emerging diseases include plague caused by yersinia pestis, cholera caused by vibrio cholerae and dengue hemorrhagic fever caused by flaviruses. as is the case with western medicines, research is needed to better understand potentially harmful side effects that may be associated with some traditional medicines. it should be emphasized that harms are not always caused deliberately; they may be caused inadvertently. hence the need for checks and balances to minimize potential harms. in cases where crude mixtures of traditional medicines are used, the possibility of identifying and separating active components from other non-therapeutic components which may have harmful side effects should be explored. it means therefore that existing traditional medicines should be improved and new ones should be developed in order to match the dynamics in the disease burden of the global population. african traditional medicine should reach a stage where it competes with the western medicines, thus becoming a source of revenue for countries that are bestowed with such valuable natural resources. such competitiveness could go a long way in lowering the costs of drugs and making effective health care accessible to poor communities. africa should not bemoan the unavailability or unaffordability of western medicines developed by pharmaceutical companies when african traditional medicines are abundantly available and accessible to the african populations to the extent that it has been proposed that atm should be considered the 'orthodox/conventional' medicine for africa (tangwa, 2007) . for instance, hiv/aids affects africa more than other continents in terms of deaths and prevalence; this is in spite of the availability of affordable traditional medicines in africa. instead of scrutinizing the effectiveness of the abundantly accessible african traditional medicines, unavailability or unaffordability of western medicines are sometimes blamed for the high prevalence and mortality rates prevailing in africa. it follows therefore that there is need for research in order to improve the efficacy of the african traditional medicines. if there are african 'traditional' research methods that are different from scientific methods of research but can improve the efficacy of the traditional medicines, then such methods should be used. what is important is to develop safe and effective medicines, and any method that is appropriate and effective should be used, provided people who participate in the process are informed. it could be argued that scientific research methods are the most effective, if the impact arguably being made by medicines developed by pharmaceutical companies is anything to go by. the involvement of such organizations as the world health organization (who) and the african union (au) in promoting scientific research into atm has enhanced conduction of various types of scientific studies aimed at improving the safety, efficacy and quality of traditional medicines (aachrd, 2002) . consequently, several african countries are intensifying efforts to improve traditional medicines through scientific research. for instance, laboratory analyses have been done in who-supported scientific studies conducted on traditional medicines intended for treatment of hiv/aids in burkina faso and zimbabwe (aachrd, 2002) . the laboratory analyses done include cd4/cd8 counts and viral load measurements to assess efficacy as well as liver and kidney function tests to assess potential side effects of the medicines. in addition, in ghana, kenya and nigeria who has sponsored pilot clinical trials to test the efficacy of traditional medicines used to treat malaria. the control arms in the pilot trials were treated with chloroquine or fansidar for comparison (aachrd, 2002) . the clinical trials of the traditional medicines are being conducted by african scientists based at institutions in african countries. it should be pointed out that informed consent is obtained from people who participate in the scientific studies testing the traditional medicines, which should always be done when anything is being tested in humans. in an effort to regulate, promote, develop or standardize the practice of atm, some african countries are reviewing existing legal frameworks or putting in place news ones (who, 2001) . for instance, the south african parliament recently passed the traditional health practitioners act, no 35 (traditional health practitioners act, 2004) . there are also efforts in various african countries such as south africa to promote collaboration between traditional healers and biomedical researchers (south african medical association task team, 2006) , which could go a long way in enhancing ethical and scientific standards and sharing of knowledge. if a traditional healer is not sure of the efficacy of the herbs s/he wants to try/test on a patient, but believes for one reason or another that it should work, patients should be informed about the uncertainty. informing the person whose body is to be exposed to the 'experimental' traditional medicine, which in most cases is not for free but is paid for, that the medicine is being tested demonstrates respect for the person. whether we want to call the 'testing' of the traditional medicines in patients 'research', and the disclosure of such 'testing' informed consent or something else is a matter of semantics. the fact of the matter is that a patient or customer should be given such information in order to make an informed decision. when african traditional medicines are being tested in clinical trials, the participants are informed and they give informed consent; the same should be done when the traditional medicines are being tested by african traditional healers. if it is unethical for 'western' medical practitioners to conduct the edward jenner type of 'testing' (which was poorly designed research) in patients, it follows that it is also unethical for traditional medical practitioners to conduct the same edward jenner-type of 'testing' (which is similarly poorly designed research) in their patients without first obtaining their informed consent. if such an action is considered ethically and morally wrong when the moral agent is a 'western' medical practitioner and the patient is a poor, desperate african person, it should not be considered ethically and morally right when the moral agent is an african traditional medical practitioner and the patient is the same poor desperate person. if the 'western' medical practitioner was to secretly test the african traditional medicines in the poor desperate african patients without their informed consent would it be considered to be ethically and morally right? the fact that most of the ethical codes and guidelines being used nationally and internationally were a result of human abuses committed by scientists in western countries does not make them completely irrelevant to atm in africa. one argument put forward is that "african tm was not an accomplice in, let alone the one responsible for, the medical atrocities that resulted in the nuremberg trials and the nuremberg code" (tangwa, 2007) . do we need to first have an african version of the nazi atrocities so that we can then develop african versions of the nuremberg code and african versions of all the subsequent ethical codes and guidelines? is some form of disaster a prerequisite for existing mechanisms of protecting the clientele of african traditional healers to be strengthened and/or new ones to be put in place? is it ethical and logical to require that humans should be harmed by atm first, in large numbers like in the nazi atrocities, before precautions could be taken or enhanced? would that be an objective, proactive and ethical way of protecting human beings? are the ethical and moral principles on which the 'western' codes and guidelines are based different from the ethical and moral values that prevail in the african communities that rely mainly on atm? such african ethical and moral values may not be written or given the same terms as the western codes, but they are practiced in the day-to-day lives of the communities. the ethical principles of autonomy, beneficence, non-maleficence and justice have been practiced in the african communities in the absence of the ter-minology that was eventually coined by the west. what then is wrong if the same principles and guidelines are used to assess the actions of african traditional healers as they practice or test their medicines either to improve efficacy or to tackle new diseases? the paucity of documented harms caused by atm does not necessarily mean that such harms never occur. the global trade in traditional medicines is increasing in both developing and developed countries. for instance, us$17 billion was spent on cam in 2000 in the usa alone, with the global market for herbal medicines being over us$60 billion (who, 2003) . thus traditional medicines are natural resources which could be developed to a level that significantly complements exports that generate the much needed foreign currency for most developing countries. the ultimate goal should be to have the improved african traditional medicines developed and manufactured in the developing countries so as to export them as finished products. efforts to develop african traditional medicines into value-added medical products are being intensified in some african countries. for instance, a pharmaceutical company in south africa has developed a local traditional medicinal herb called sunderlandia into standardized tablets (aachrd, 2002) . in nigeria two traditional medicines, dopravil and conavil, that local traditional healers claim to be effective for the management of hiv/aids have been standardized and have reached clinical trial phase ii (aachrd, 2002) . the developing countries stand to benefit to some extent from employment created by such endeavours to develop traditional medicines. in addition, there could be significant financial savings due to reduced imports of medicines from developed countries. the challenge to protect the owners of traditional knowledge, who may not be adequately protected by the existing current intellectual property rights (ipr) frameworks, has been pointed out (timmermans, 2003; nyika, 2007) , and there is need to review or expand the current ipr policies in order to address the peculiar nature of traditional knowledge. the majority of the african populations rely on atm because it is easily accessible and is affordable. this fact makes the need to ensure that atm is made as effective and as safe as possible urgent because of the large numbers of people who make use of it in the wake of high disease burden. efforts to improve atm should not be perceived as evidence of condemnation of the practice of atm or as neocolonialism because dynamics in the disease patterns and socioeconomic status of the global populations, including african populations, dictate that atm has to respond to the changes in order to effectively serve the purpose for which it has been developed. the ultimate goal should always be to make various complementary efforts that are ethical and are aimed at reducing the disease burden in african countries. the efficacy and safety of african traditional medicines could be enhanced through well designed research that is conducted ethically in africa. any methods, be they african, western, chinese, or any other, that are capable of further improving the safety and efficacy of the african traditional medicines should be used as a matter of urgency because diseases are wrecking havoc in africa in spite of the accessibility and affordability of the african traditional medicines in their current state. if it so happens that scientific methods are the most effective in terms of improving the safety and efficacy of medicinal products, be they african or western, then let african scientists and traditional healers use the scientific methods to improve the african traditional medicines. what is the point of african govern-ments investing a lot of resources in educating africans in sciences and then consider use of scientific research to develop african traditional medicines to be unacceptable? however, whatever methods are used to develop the african traditional medicines, patients should be informed that research is going on and secondly ways of protecting the custodians of the traditional knowledge should be put in place. when the actions of scientists are subjected to scrutiny, it is not because scientists are all evil people whose aim is to deliberately harm patients or research participants. similarly, scrutiny of african traditional healers should not be viewed as condemnation of the practice of atm in its entirety. whereas it is to a reasonable extent possible to distinguish between practice and research in western medicine, it is generally not very clear in the context of african traditional medicine. if african traditional medicines are being tested in humans, the traditional healers should inform the patients that the traditional medicines are being tested for effectiveness against the diseases that the patients are suffering from. that is respect for the patients who have a right to know that they are in a study to test some traditional medicines. african advisory committee for health research and development (aachrd) preventing emerging infectious diseases: a strategy for the 21st century international ethical guidelines for biomedical research involving human subjects community based trials and informed consent in rural north india zulu ritual immunisation in perspective ethical consideration in african traditional medicine: a response to nyika ethical and regulatory issues surrounding african traditional medicine in the context of hiv the effect of relationships on decisionmaking process of women in harare bridging the gap: potential for a health care partnership between african traditional healers and biomedical personnel in south africa how not to compare western scientific medicine with african traditional medicine intellectual property rights and traditional medicines: policy dilemmas at the interface republic of south africa legal status of traditional medicine and complementary/alternative medicine: a worldwide view traditional medicine fact sheet number 134 ethical principles for medical research involving human subjects i am grateful to the bill and melinda gates foundation for grant id #37350 awarded to amanet for building institutional capacities in health research ethics in africa. amanet also receives major support from the danish development agency (danida), the european commission (dg-research and aidco), the netherlands ministry of international cooperation (dgis), the european developing countries clinical trials partnership (edctp), and the african caribbean pacific secretariat for various project activities. key: cord-031316-yvid6qps authors: bisimwa, patrick n.; ongus, juliette r.; tiambo, christian k.; machuka, eunice m.; bisimwa, espoir b.; steinaa, lucilla; pelle, roger title: first detection of african swine fever (asf) virus genotype x and serogroup 7 in symptomatic pigs in the democratic republic of congo date: 2020-09-03 journal: virol j doi: 10.1186/s12985-020-01398-8 sha: doc_id: 31316 cord_uid: yvid6qps background: african swine fever (asf) is a highly contagious and severe hemorrhagic viral disease of domestic pigs. the analysis of variable regions of african swine fever virus (asfv) genome led to more genotypic and serotypic information about circulating strains. the present study aimed at investigating the genetic diversity of asfv strains in symptomatic pigs in south kivu province of the democratic republic of congo (drc). materials and methods: blood samples collected from 391 asf symptomatic domestic pigs in 6 of 8 districts in south kivu were screened for the presence of asfv, using a vp73 gene-specific polymerase chain reaction (pcr) with the universal primer set ppa1-ppa2. to genotype the strains, we sequenced and compared the nucleotide sequences of ppa-positive samples at three loci: the c-terminus of b646l gene encoding the p72 protein, the e183l gene encoding the p54 protein, and the central hypervariable region (cvr) of the b602l gene encoding the j9l protein. in addition, to serotype and discriminate between closely related strains, the ep402l (cd2v) gene and the intergenic region between the i73r and i329l genes were analyzed. results: asfv was confirmed in 26 of 391 pigs tested. however, only 19 and 15 ppa-positive samples, respectively, were successfully sequenced and phylogenetically analyzed for p72 (b646l) and p54 (e183l). all the asfv studied were of genotype x. the cvr tetrameric repeat clustered the asfv strains in two subgroups: the uvira subgroup (10 trs repeats, aaaabnaaba) and another subgroup from all other strains (8 trs repeats, aabnaaba). the phylogenetic analysis of the ep402l gene clustered all the strains into cd2v serogroup 7. analyzing the intergenic region between i73r and i329l genes revealed that the strains were identical but contained a deletion of a 33-nucleotide internal repeat sequence compared to asfv strain kenya 1950. conclusion: asfv genotype x and serogroup 7 was identified in the asf disease outbreaks in south kivu province of drc in 2018–2019. this represents the first report of asfv genotype x in drc. cvr tetrameric repeat sequences clustered the asfv strains studied in two subgroups. our finding emphasizes the need for improved coordination of the control of asf. pigs are increasingly contributing to improved nutrition and household incomes in regions of africa where pork consumption and pig keeping are culturally acceptable [1] . despite the importance of pig farming, this sector is facing several constraints, with infectious disease burden being the major problem [2] . african swine fever virus (asfv) causes an acute, highly contagious, and fatal disease in domestic pigs, with clinical signs such as fever and haemorrhagic lesions [3] . there are currently no vaccines available to combat african swine fever (asf). the first recorded asf outbreaks were reported in pigs belonging to european settlers in kenya in 1914 [4] . the disease continues to spread throughout eastern europe since 2007 [5] and was reported in belgium and china in 2018 [6] [7] [8] . asfv is a large, enveloped, doublestranded dna arbovirus belonging to the genus asfivirus, and the only member of the family asfarviridae [2, 9, 10] . warthogs, bush pigs and the soft tick of the genus ornithodoros are the major reservoirs of asfv [3] . the contagious nature and the ability to spread rapidly in pig populations over long distances makes it the most feared disease of domestic pigs [11, 12] . the genome size varies from 170 to 193 kbp and encodes between 150 and 167 open reading frames, depending on the virus strains [13] . to date, 24 asfv genotypes have been reported worldwide based on the b646l gene, which encodes the capsid protein p72, and all of them are known to circulate in africa [14] [15] [16] . using the serotype-identification approach [17] , an additional 8 asfv serotypes have been reported based on the ep402r gene encoding the cd2v protein [17] [18] [19] . distinct antigenic types of asfv were identified based on haemadsorption inhibition (hai) serological typing, where asf protective immunity was shown to be serotype-specific, and viruses belonging to identical serogroups cross-protected against each other [20] . this has significant importance for vaccine development. the cd2v protein, encoded by the asfv ep402r gene, is a transmembrane glycoprotein located in the viroplasm (around viral factories) and in the plasma membrane of infected cells. it is among the most variable genes in the asfv genome [21, 22] . haemadsorption involves adhesion of pig erythrocytes to the surface of asfv infected cells, a key requirement is expression of cd2v in asfv-infected cells [20] . control of the disease is relying on surveillance, restriction of pigs and pork products movement, and rapid diagnosis and culling of asfv infected animals. the implementation of these measures is particularly difficult for african pig farmers of which many can be characterized as smallholders, due to limited capacity and appropriate policy. in 2011, asf outbreaks were reported in more than 25 african countries with the highest number of outbreaks (84) registered in the democratic republic of congo (drc) causing a loss of 105,614 pigs [23, 24] . previous studies have reported circulation of genotype i, ix and xiv in drc, encouraging the need for continued characterization of asfv strains responsible for outbreaks to better understand the spread of this economical important disease in drc. several variable regions of the asfv genome have been extensively used as targets for molecular epidemiology studies of asfv strains [25] [26] [27] . however, previous studies achieved high resolution for discrimination between different virus strains when combining p72, p54 and b602l (central variable region or cvr) proteins [28, 29] . moreover, the ep402r gene encoding the cd2v protein and the intergenic region between the i73r and i329l have also been used to characterize asfv from various locations and to track virus spread [25, 26] . the first report of the presence of asf in drc was in 1939 [30] . south kivu province is an area in the eastern drc where suspected cases of asf appear regular. reports from the provincial ministry of agriculture livestock and fishery (pmalf) and the local veterinary body indicated the death of 1600 pigs out of 1608 that presented clinical signs of asf in 2015 (report of the pmalf, unpublished data). more recently, we have used a combination of p72 and p54 proteins to characterize asfv genotype ix in apparently healthy pigs in south kivu province sampled in 2016 [31] . it was the first study of asfv in the south kivu province. however, despite report of frequent incidences of asf in the south kivu province by the pmalf, information about asfv strains in circulation in suspected infected animals is lacking. therefore, the present study was set up to identify and characterize asfv strains in infected domestic pigs with symptoms of asf from different smallholder farms in the south kivu province in order to increase epidemiological knowledge of asfv, and to generate information for improvement of control strategies. ethical approval for the study reported here and the permission for the collection of samples was provided by the interdisciplinary centre for ethical research (cire) established by the evangelical university in africa, bukavu, dr congo, with reference (uea/sgac/km 132/2016). a consent form which described the aim of the study was signed by farmers willing to participate in the study after translation into local languages. the study was carried out in south kivu province of the democratic republic of congo (drc), situated in the eastern part of the country. it is a large region with an area of 66,814 km 2 , located between longitudes 26°10′ 30″ and 29°58′ east, latitudes 00′ 58″ and 4°51′ 21″ south. comparatively, the south kivu province is over two times the size of burundi (27,834 km 2 ) and rwanda (26,338 km 2 ) put together. the province experiences two main seasons: a 9-month long rainy period, from september to may, and a 3-month dry period (june to august). the annual average rainfall is 1300 mm. six out of eight territories were selected for purposes of this study including; fizi, kabare, kalehe, mwenga, uvira and walungu (fig. 1) . a key factor in selecting the sample sites was the inclusion of the main pig-producing, marketing, and consuming areas, with a particular focus on locations where suspected asf outbreaks had been recently reported by the provincial ministry of agriculture livestock and fishery. a cross-sectional study was conducted where the target population was households that keep pigs, and where suspected asf cases were notified based on the reports from the local veterinary and provincial ministry of agriculture livestock and fishery during december 2018 to january 2019. in general, the number of pigs per farm varied between 1 and 5 and they shared housing. thus, farms with pigs presenting symptoms such as high fever, reddening of the skin, particularly ears and snout, coughing and difficulties in breathing, hemorrhagic diarrhea and vomiting, inability to walk, loss of appetites, general weakness, were considered for sampling and all the pigs were sampled. drc has a pig population of approximately 1 million. the recommended sample size for a population of that size, using a confidence level of 95% and a margin of error of 5% would be 384 [32] . based on this, a total of 391 blood samples from suspected asfv infected pigs were collected in edta (anticoagulant) tubes and were used for pcr analysis. all blood samples were collected from the jugular vein of pigs of over 3 months of age. after collection, all collected blood samples were transported to the université evangélique en afrique (uea) and stored at − 20°c before being shipped on ice packs to the pan african university institute of science technology and innovation (pauisti) in nairobi then to beca-ilri hub, for subsequent analysis. total dna was extracted directly from 200 μl of whole blood using the dneasy blood and tissue kit (qiagen, usa) following the manufacturer's recommendations. to detect the presence of asfv dna, polymerase chain reaction (pcr) amplification assay was carried out using the asfv diagnosis primers ppa1/ppa2 (peste porcine africaine) that target the virus vp73 (p72) coding region to generate an amplicon of 257 bp [33] . pcr products were confirmed using a 2% agarose gel electrophoresis. all ppa-positive samples were characterized in subsequent analyses. five separate pcr experiments were set up to amplify ppa-positive samples: (i) for p72 genotype classification, the c-terminal region of the p72 protein gene was amplified using the primers p72-u/d [15] ; (ii) for p54 genotyping, the gene e183l encoding the p54 protein was amplified using the primers ppa722/ppa89 [11] ; (iii) the b602l gene characterized by the central variable region (cvr) was amplified using the primer pairs cvr-fl1 and cvr-fl2 as previously reported [24] ; (iv) for determining the origin, and to distinguish between closely related asfv strains circulating in the south kivu province, a 356 bp fragment, specific for identification of tandem repeat sequences (trs), located between the i73r and i329l genes was amplified using the primer pairs eco1a and eco1b [34] ; (v) to determine the serogroups of the strains, the partial ep402r gene encoding the cd2v protein [27] was amplified and sequenced using two sets of primers to generate two overlapping fragments. the primers used for the diagnosis and genotyping are presented in the table 1. pcr amplicons were confirmed using 2% agarose gel electrophoresis in the presence of molecular weight markers. for sequencing, pcr products were purified using quick pcr purification kit (qiagen, usa) following the manufacturer's instructions and sent to macrogen europe bv (amsterdam, the netherlands) for sanger sequencing. open reading frames present within the sequences generated from the amplified cvr dna fragments were translated into amino acid sequences using emboss-transeq software [19] . both strands of purified amplicons were sequenced using the 7 primer sets for genotyping described above. to verify similarity with known sequences, the amplicon sequences obtained were submitted to blast (basic local alignment search tool) [35] against non-redundant genbank database. multiple sequence alignments of sequences were generated using clustal w [36] , whereas for each locus, the unrooted maximum likelihood method (ml) phylogenetic tree with 1000 bootstrap replications was estimated by mega 7 program and kimura 2-parameter model [37] was selected based on the best-fit substitution model (ml) with the lowest bayesian information criterion (bic) score. asfv sequence data of strains and isolates available in the genbank were included as references. sequences from this study have been submitted to genbank with accession numbers mn689307 to mn689322 (for p72) and mn704903 to mn704917 (for p54). a total of 391 blood samples collected from symptomatic pigs were screened for the presence of the asf viral dna using conventional pcr with the diagnostic primers ppa1/ppa2. a total of 26 blood samples showed clear amplicons of the expected size (257 bp) and data were distributed as shown in table 2 , with the highest number of positive samples found in the uvira territory 9/68 (13.2%), while the lowest was in mwenga 2/65 (3.07%). sequence analysis of asfv based on the b646l (p72), e183l (p54) and b602l (cvr) genes of the 26 asfv positive samples using ppa diagnostic primers, we successfully amplified and sequenced 19 (73.07%) samples for p72 and 15 (57.69%) samples for p54 ( table 2 ). the p72 amplicon sequences shared 99-100% identity due to some few synonymous mutations while the p54 sequences were 100% identical (data not shown). thus, clustal protein sequence alignment showed 100% identity between all the p72 and p54 sequences in the study samples. sequences of p72 and p54 amplicon were compared with 25 other p72 and p54 asfv sequences retrieved from the gen-bank database and the phylogenetic analysis revealed that the south kivu asf virus strains analyzed clustered with p72 genotype x including strains reported in previous studies in burundi (af449463), kenya (ay261360) and tanzania (jx403648, af301546, mf437291) ( fig. 2a and b) . this is the first report of genotype x in the drc. in addition, the predicted amino acid sequences of the cvr nucleotide sequences were generated from 15 samples (2 fizi, 2 kabare, 2 kalehe, 2 mwenga, 5 uvira, 2 walungu) and specific features based on the previously reported asfv tetrameric amino acid repeats within the cvr [38, 39] were obtained. analysis of the cvr signature of the b602l gene showed two different signatures when compared with sequences of strains of the same genotype from burundi, tanzania and uganda. all asfv strains contained a cvr with 3 tetrameric amino acids, namely cast (a), cadt (b) and nvdt (n). however, in strains from the uvira territory the cvr sequence was repeated 10 times with the profile aaaabnaaba. in contrast, in strains from the five other territories, the cvr sequences contained only 8 repeats with the signature aabnaaba (table 3 ). both cvr signatures were different from the strains tan13/moshi, bur 84/1, bur84/2 and ug95/3 [38] [39] [40] . 15 15 no. strains positive for asfv using the 3 pcr assays 13 a pcr assays were performed on the 26 samples positive for ppa pcr sequence analysis of the intergenic region between i73r and i329l genes and the ep402r gene amplification of the ep402r gene (encoding cd2v protein) was performed, and pcr amplicons of 8 strains from 4 territories were successfully sequenced. comparative analysis of the 8 sequences obtained was carried out together with 20 other asfv sequences retrieved from the genbank database and previously characterized as serogroups. in this study, the phylogenetic analysis showed that the south kivu strains belonged to serogroup 7 and were grouped with the uganda strain (km609361), the only available serogroup 7 in the gen-bank. this research suggests that the strains from this study may have a similar hemadsorption inhibition (hai) characteristics as the only known strain serogroup 7 (fig. 3) . the analysis of whole-genome sequences of asfv has facilitated identification of several regions containing tandem repeat sequences, important for discriminating between closely related asfv strains and for predicting the origin of the virus [26] . in this study, the intergenic region between the i73r and i329l genes was analyzed for 15 strains from the 6 territories studied. the south kivu asfv strains were compared to the kenyan 1950 strain (ay261360), which was identified from a domestic pig. the sequence alignment showed an indel of 33 bp (5′-cctatatacctataatcttataccctataa tct-3′) between nucleotide position 226 to 258 (fig. 4 ). african swine fever constitutes the major obstacle to the development of the pig industry in the drc, with sporadic outbreaks occurring across various areas throughout the year [24] . despite recurrent occurrence of suspected asf outbreaks in south kivu province, information on the virus characterization remains scarce. to determine the prevalence of asf and genotypes of asfv in circulation in the south kivu province, a study was carried out in the south kivu province from january to august 2016, a period with no report of asf outbreaks a b fig. 2 phylogenetic relationships of p72 and p54 genotypes. the evolutionary history was inferred by the maximum likelihood method based on the kimura 2-parameter model [33] . phylogeny was inferred following 1000 bootstrap replications, and the node values show percentage bootstrap support. scale bar indicates nucleotide substitutions per site. scale bar indicates nucleotide substitutions per site. a p72 genotypes. the analysis included 19 b646l (p72) sequences from this study (plain circle ) and sequences from the genbank database. the genbank accession numbers for the different b646l (p72) genes are indicated in parenthesis. b p54 genotypes. the analysis involved 15 e183l (p54) gene sequences of african swine fever viruses from this study (black diamond ◆) and sequences from the genbank database. the sequences for the different b646l (p72) and e183l (p54) genes are starting by genbank accession numbers or cases in the sampled area [31] . we conducted a cross-sectional study in 5 of the 8 districts of the province and 267 pig blood from 250 smallholder pig farms were screened for presence of the asfv antibody and viral genome using indirect enzyme linked immunosorbent assay (elisa) and polymerase chain reaction (pcr), respectively, on asymptomatic domestic pigs. we found that 37% of pigs contained asfv antibodies whereas virus dna was present in 22.8% of pigs. sequence analysis revealed that all the asfv detected from asymptomatic pigs belonged to the genotype ix. continuous characterization of asfv strains is key in endemic regions to better understand disease outbreak patterns and map the different strains according to their geographical regions, in which they circulate [41] . the present study was targeting domestic pigs showing asf clinical signs with the aim to characterize asfv in symptomatic animals in the south kivu province. we confirmed the presence of asfv in domestic pigs with clinical signs of asf in the six studied territories of south kivu province: kabare, kalehe, fizi, mwenga, uvira and walungu. although this study was not designed to determine the prevalence of asfv, low rate of infection was observed in mwenga and kalehe (3 and 3.1%, respectively), whereas the highest infection rates were registered in walunga and uvira (9 and 13.2%, respectively). in our previous study which included asymptomatic pigs [31] , walungu had the highest prevalence of asfv (33.7%) while the lowest asfv prevalence was found in kalehe (15.8%). the overall low infection rate may be attributed to the sensitivity of the assay used. indeed, the conventional pcr method used in this study is less sensitive than other molecular methods such as nested-pcr [42] and real-time pcr [43] and may fail to detect potentially positive samples containing very low amount of viral genetic material. in addition to the low sensitivity of the conventional pcr used, other conditions may have contributed to the observed low prevalence including: (i) most pigs sampled may not have been infected or may have low virus load not detectable by the pcr technique used; and (ii) suspected pigs may have been affected by other diseases and conditions with similar symptoms to asf such as porcine reproductive an respiratory syndrome, porcine dermatitis and nephropathy syndrome, salmonellosis. our results confirmed asf viral infections in pigs with clinical signs of asf in the south kivu province. from the 26 ppa-pcr positive samples, 19, 15 and 15 samples were successfully amplified and sequenced for b646l (p72), e183l (p54) and b602l (cvr) genes, respectively. the combination of these three viral regions is to ensure a high-level resolution for asfv discrimination. the p54 genotyping study corroborated the p72 analysis. both p72 and p54 phylogenetic analyses clustered asfv strains in circulation in symptomatic domestic pigs during the december 2018 -january 2019 outbreaks in the south kivu province with asfv genotype x, which is the major genotype associated with asf outbreaks throughout burundi, in some parts of tanzania, kenya and uganda [38, 39] . genotype x has been reported to be a sylvatic cycle-associated genotype that include asfv identified from domestic pigs, warthogs as well as ticks in these three countries [15, 27, 44] . furthermore, alignment of the 433 bp long sequence from the variable 3′-end of the b646l (p72) gene in the south kivu viruses showed 100% identity with asfv strains from burundi 1984 (data not shown). it is a possibility that viruses in this study may originate from, or could have expanded to burundi. this scenario seems plausible as the south kivu province is bordering burundi through the river rizizi and lake tanganyika, and uncontrolled cross-border movements of pigs and pork products are observed in the region and may constitute a major route of transmission of asf in this endemic area [45] . our current result contrasts with our previous finding of circulating asfv strains of genotype ix in asymptomatic domestic pigs in the studied area during a period with no asf outbreaks or cases [31] . it is unlikely that data from both studies suggest that asfv of genotype ix may not cause disease in pigs whereas [40] . although the report did not have any cases in the south kivu province, it identified asfv of genotype i in the neighboring province of maniema. nevertheless, further investigation in relation to both the host and virus genetics will be important to understand our findings. we are currently working on the lab-isolation of viruses of genotype ix and x in circulation in asymptomatic pigs and symptomatic pigs, respectively, for complete genome sequencing and comparative genomic analysis. data obtained will improve our understanding of this contrasting finding in pigs within the south kivu province. to the best of our knowledge, this is the first report of asf virus genotype x in the dr congo. as all the strains were p72/p54 genotype x, we further characterized them at a higher resolution using the intra-genotypic central variable region (cvr) of the bl602l gene. based on the tetrameric repeat sequences (trs), our analysis identified two different cvr variants, clustering the strains into two subgroups. subgroup 1 was composed only of strains from uvira characterized by 10 trs whereas all other strains formed the subgroup 2 and had only 8 trs. the profile of the subgroup 2 (aabnaaba) was almost identical to the cvr amino acid sequence of uganda 95/3 (aabnbaba), having the b code (cadt) in place of a at the 5th repeat [39] . the number of trs repeat observed is relatively small compared to reports from some studies in the same geographic region describing the trs motif repeated over 20 to 50 times [38, 46] . however, mulumba-mfumu et al. also observed this sequence repeated only 5 or 6 times in some dr congo strains [24] . the two cvr variants found in our study were different from the previously reported variants in dr congo asfv strains [40] and to any other known viruses causing outbreaks or asf cases, thus suggesting that the asfv genotype 10 in circulation in the south kivu province of dr congo identified in this study may be unique [45, 47] . within the vaccine field, it has been suggested that protective immunity is serotype-specific, as defined by asfv hemadsorption inhibition (hai) serological assay, with viruses within a serogroup cross-protecting against one another [17, 18] . the hai assay can be used to type asfv of a given genotype into distinct and individual serogroups, based on the asfv proteins cd2v (ep402r) and/or c-type lectin (ep153r). thus, hai-based serogroup classification has been suggested as a better correlate for in vivo cross-protection among strains compared to the p72 genotyping [17] . in our study, we obtained cd2v sequences of 8 strains from 4 territories and comparative sequence analysis revealed that they were all identical. moreover, phylogenetic analysis showed that the uganda strain (genbank acc. no. km609361.1), which represents the only member of the serogroup 7, was closest related to the south kivu viruses, suggesting that the asfv strains, identified in this study, may belong to serogroup 7. the high bootstrap value of 99% grouping the south kivu strains with the uganda serogroup 7 and the fact that strains from this study showed 99.2% amino acid identity with the uganda serotype 7 strain strongly support the genetic relatedness between these two groups. it is worth noting that strains of serogroups 1 and 2 have been reported in drc [18] . overall, our data showed that these south kivu asfv strains are serologically different from other strains reported so far. analysis of the intergenic region between the i73r and i329l genes has previously been used for distinguishing between closely related asfvs [26] . characterization of fig. 4 partial nucleotide sequence alignments of the intergenic region between i73r and i329l genes. sequences of african swine fever virus (asfv) strains from the south kivu province, eastern drc, showing tetrameric repeats of representative genotypes, including a reference sequence of a virus isolated in 1950 in kenya (kenya 1950; genbank accession no. ay261360.1). the indel that results from the insertion of the nucleotide sequence cctatatacctataatcttataccctataatct in the asfv from kenya is boxed this intergenic region genes did not identify any genetic diversity among the south kivu strains. however, all the 15 strains analyzed had high sequence identity with the kenyan strain 1950 (genbank acc. no. ay261360) but lacked an insertion of 33 bp. indels have also been reported in a similar analysis [26] . altogether, our study provided evidence of circulating asfv genotype x which were antigenically related to serogroup 7 in domestic pigs with clinical signs of asf in eastern drc. the genotyping approach was also supported with the hai serotyping for improved diversity analysis and finer discrimination of asfv strains. this represents the first report of asfv genotype x in drc. in this study, asfv isolated from symptomatic domestic pigs in the south kivu province of the democratic republic of congo were characterized for the genetic diversity. all the asfv strains analyzed in this study belonged to the p72 genotype x and the cd2v serotype 7. this is the first report of circulating asfv genotype x in drc. the genetic similarity of these strains suggests that they may originate from a common source. however, cvr tetrameric sequence repeat analysis clustered the strains into a subgroup with 10 tsr (uvira strains) and a subgroup with 8 trs (strains from other territories), thus underlining genetic variation among these asfv. therefore, a better understanding of asfv evolution and spread throughout the south kivu province will need further in-depth comparative sequence analyses including whole genome sequencing of asfv strains circulating in the area. domestic pigs in africa risk factors for african swine fever in smallholder pig production systems in uganda role of wild suids in the epidemiology of african swine fever history of "swine fever" in southern africa african swine fever: how can global spread be prevented? phylogeographic analysis of african swine fever virus evolution in europe of african swine fever genotype ii viruses from highly to moderately virulent molecular characterization of african swine fever virus pig empire under infectious threat: risk of african swine fever introduction into the people's republic of china empres watch: african swine fever in the caucasus epidemiology of african swine fever in poland since the detection of the first case african swine fever virus replication and genomics identification of a new genotype of african swine fever virus in domestic pigs from ethiopia genotyping field strains of african swine fever virus by partial p72 gene characterization genetic characterization of african swine fever virus isolates from soft ticks at the wildlife/domestic interface in mozambique and identification of a novel genotype comparative analysis of african swine fever virus genotypes and serogroups african swine fever virus cd2v and c-type lectin gene loci mediate serological specificity emboss: the european molecular biology open software suite antigenic diversity of african swine fever viruses processing and localization of the african swine fever virus cd2v transmembrane protein african swine fever virus african swine fever. in: pan african animal health yearbook. nairobi: interafrican bureau for animal resources, african union african swine fever: update on eastern, central and southern africa enhanced discrimination of african swine fever virus isolates through nucleotide sequencing of the p54, p72, and pb602l (cvr) genes genetic variation among african swine fever genotype ii viruses, eastern and central europe molecular epidemiology of african swine fever in east africa co-circulation of two genetically distinct viruses in an outbreak of african swine fever in mozambique: no evidence for individual co-infection african swine fever, manual of standards for diagnostic test and vaccines la peste porcine africaine. paris: institut d'élevage et de médecine vétérinaire des pays tropicaux evidence for the presence of african swine fever virus in apparently healthy pigs in south kivu province of the democratic republic of congo veterinary epidemiologic research jm: highly sensitive pcr assay for routine diagnosis of african swine fever virus in clinical samples african swine fever: a global view of the current challenge gapped blast and psi-blast: a new generation of protein database search programs clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice molecular evolutionary genetic analysis version 7.0 for bigger datasets genetic characterization of african swine fever viruses from a 2008 outbreak in tanzania. transb and emerg dis molecular epidemiology of african swine fever virus studied by analysis of four variable genome regions genetic assessment of african swine fever isolates involved in outbreaks in the democratic republic of congo between 2005 and 2012 reveals co-circulation of p72 genotypes i, ix and xiv, including 19 variants molecular characterization of african swine fever viruses from outbreaks in peri-urban kampala, uganda development of a nested pcr and its internal control for the detection of african swine fever virus (asfv) in ornithodoros erraticus molecular diagnosis of african swine fever by a new real-time pcr using universal probe library african swine fever viruses with two different genotypes, both of which occur in domestic pigs, are associated with ticks and adult warthogs, respectively, at a single geographical site transmission routes of african swine fever virus to domestic pigs: current knowledge and future research directions prevalence of african swine fever virus in apparently healthy domestic pigs in uganda molecular characterization of african swine fever virus in apparently healthy domestic pigs in uganda publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we acknowledge the biosciences eastern and central africa authors' contributions bnp collected samples for dna isolation, performed laboratory work and wrote the manuscript; jro supervised the study, conceived and designed the study, and edited the manuscript; ckt performed the laboratory work and edited the manuscript; emm performed the laboratory work and sequence analysis; ebb conceived and designed the study and edited the manuscript; ls supervised the study and edited the manuscript; rp supervised the study, performed sequence analysis, wrote and edited the manuscript. all authors read and approved the manuscript. this work was funded by the biosciences eastern and central africa -international livestock research institute (beca-ilri) hub, through africa biosciences challenge fund (abcf). the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate see material and methods section. not applicable. the authors declare that they have no competing interests.author details 1 department of molecular biology and biotechnology, pan african university, institute of basic sciences, technology and innovation, nairobi, kenya. key: cord-032552-rjuug7er authors: umviligihozo, gisele; mupfumi, lucy; sonela, nelson; naicker, delon; obuku, ekwaro a.; koofhethile, catherine; mogashoa, tuelo; kapaata, anne; ombati, geoffrey; michelo, clive m.; makobu, kimani; todowede, olamide; balinda, sheila n. title: sub-saharan africa preparedness and response to the covid-19 pandemic: a perspective of early career african scientists date: 2020-07-08 journal: wellcome open res doi: 10.12688/wellcomeopenres.16070.1 sha: doc_id: 32552 cord_uid: rjuug7er emerging highly transmissible viral infections such as sars-cov-2 pose a significant global threat to human health and the economy. since its first appearance in december 2019 in the city of wuhan, hubei province, china, sars-cov-2 infection has quickly spread across the globe, with the first case reported on the african continent, in egypt on february 14 (th), 2020. although the global number of covid-19 infections has increased exponentially since the beginning of the pandemic, the number of new infections and deaths recorded in african countries have been relatively modest, suggesting slower transmission dynamics of the virus on the continent, a lower case fatality rate, or simply a lack of testing or reliable data. notably, there is no significant increase in unexplained pneumonias or deaths on the continent which could possibly indicate the effectiveness of interventions introduced by several african governments. however, there has not yet been a comprehensive assessment of sub-saharan africa’s (ssa) preparedness and response to the covid-19 pandemic that may have contributed to prevent an uncontrolled outbreak so far. as a group of early career scientists and the next generation of african scientific leaders with experience of working in medical and diverse health research fields in both ssa and resource-rich countries, we present a unique perspective on the current public health interventions to fight covid-19 in africa. our perspective is based on extensive review of the available scientific publications, official technical reports and announcements released by governmental and non-governmental health organizations as well as from our personal experiences as workers on the covid-19 battlefield in ssa. we documented public health interventions implemented in seven ssa countries including uganda, kenya, rwanda, cameroon, zambia, south africa and botswana, the existing gaps and the important components of disease control that may strengthen ssa response to future outbreaks. transmission dynamics of the virus on the continent, a lower case fatality rate, or simply a lack of testing or reliable data. notably, there is no significant increase in unexplained pneumonias or deaths on the continent which could possibly indicate the effectiveness of interventions introduced by several african governments. however, there has not yet been a comprehensive assessment of sub-saharan africa's (ssa) preparedness and response to the covid-19 pandemic that may have contributed to prevent an uncontrolled outbreak so far. as a group of early career scientists and the next generation of african scientific leaders with experience of working in medical and diverse health research fields in both ssa and resource-rich countries, we present a unique perspective on the current public health interventions to fight covid-19 in africa. our perspective is based on extensive review of the available scientific publications, official technical reports and announcements released by governmental and non-governmental health organizations as well as from our personal experiences as workers on the covid-19 battlefield in ssa. we documented public health interventions implemented in seven ssa countries including uganda, kenya, rwanda, cameroon, zambia, south africa and botswana, the existing gaps and the important components of disease control that may strengthen ssa response to future outbreaks. 08 jul 2020 report report the views expressed in this article are those of the author(s) and do not reflect those of their employers or institutions. publication in wellcome open research does not imply endorsement by wellcome. covid-19 is caused by a novel beta-coronavirus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that was first reported in december 2019 in the city of wuhan, hubei province, china 1 . sars-cov-2 infection has quickly spread across the globe 2 , with the first case reported on the african continent, in egypt on february 14 th 2020 3 . covid-19 was declared a public health emergency of international concern by the world health organization (who) on march 11 th 2020 4 . just a few months into the pandemic, covid-19 has ravaged developed countries, with significant case fatality rates in europe and the usa 5 . considering the large number of people that live in poor and crowded informal settings in sub-saharan africa (ssa) 6, 7 , the mode of transmission of sars-cov-2, the severity of the disease as well as the existing fragile health systems [8] [9] [10] it was hypothesized that africa may markedly be affected by the covid-19 pandemic resulting in disastrous consequences with a large number of patients overwhelming hospitals and high case fatality rates 8, 11 . as of june 6 th covid-19 global infections recorded in 215 countries were 6 12 and only one of these countries is located in the ssa region. although the global number of sars-cov-2 infections increased exponentially since the beginning of the pandemic due to ongoing transmission, the low number of infections and deaths recorded in ssa countries have raised suspicions on whether they represent a slow progression of the pandemic in africa, a lower case fatality rate, or simply a lack of testing or reliable data. notably, there is no significant increase in unexplained pneumonias or deaths recorded on the continent which could possibly indicate the effectiveness of interventions introduced by several african governments. however, there has not yet been a comprehensive assessment of ssa's preparedness and response to the covid-19 pandemic that may have contributed to prevent an uncontrolled outbreak so far. here, we -a diverse group of early career researchers (graduate students and post-doctoral scientists) and the next generation of african scientific leaders with experience working in various fields of health research including medicine, immunology, molecular biology, microbiology, virology and public health in both ssa countries and in developed countries -conducted an assessment of african preparedness and response to the covid-19 during the early (first 3 months) of the pandemic on the african continent. the early career researchers involved are fellows of the african academy of science's sub-saharan african network for tb/hiv research excellence (santhe) 13 , (part of the deltas africa initiative) and some were at the frontline of the battle against covid-19 in their respective countries during the time of this assessment. as of june 6 th 2020, the seven countries represented had reported the following caseloads of covid 12 . even though the covid-19 pandemic unfolded rapidly, and the undertaken public health measures interrupted our studies, our careers and other usual activities, this pandemic has also offered a blueprint on how to deal with epidemics. this analysis presents a unique perspective on the currently developed public health interventions to fight covid-19 on the african continent. we discuss the challenges and opportunities that exist to improve african capacity to fight future epidemics from our perspective as the next generation of scientists that will oversee these responses in the future. our evaluation of sub-saharan africa preparedness and response to covid-19 reviewed country specific preventive measures and critically examined the response to the pandemic in seven african countries. we assessed the public health measures and other crucial interventions that were put in place in the control of covid-19 in uganda, kenya, rwanda, cameroon, zambia, south africa and botswana. we argue that these strategies may have helped to prevent a disastrous outcome by reducing rapid transmissions that may happen in clusters and minimizing the number of patients seeking medical assistance at the same time. we also identified and summarized in three categories of biomedical, sociocultural and economic factors; the challenges encountered at different levels of the health system. we presented the gaps existing in the public health intervention programs that may result in delays/failure to halt the spread of the disease as well as the important components of disease control that may strengthen sub-saharan africa preparedness and response to future outbreaks. the rapid rise of sars-cov-2 infections sent a clear message to the world that quick action was needed to prevent the spread of the disease. who warned on february 22 nd 2020 that all member states of the african union should develop an early strong plan of action to tackle the growing threat 14 . as emerging africa leaders in health research and fellows of the santhe consortium, we convened a virtual meeting to discuss the threat that the pandemic posed to ssa and agreed that we had a responsibility to critically analyze the response of our governments so far and to offer our own perspective on how this and other similar epidemics should be tackled on the continent. in the meeting, participants discussed the origin of the new sars-cov-2 virus, infection preventive measures, diagnostic tests, clinical management of the disease and the development of vaccines and therapeutics by critically reviewing the available scientific literature. we discussed the need for scientific and evidence-based responses that considered africa's unique healthcare, sociocultural and economic challenges. we therefore decided to review sars-cov-2 infection dynamics in africa and the responses that african governments were taking, relying primarily on published scientific articles, publicly available government technical reports, media announcements and our own personal experiences and perspectives. considering that the response to the epidemic has been very variable from country to country, we agreed to focus our analysis from reports emanating from uganda, kenya, rwanda, cameroon, zambia, south africa and botswana, countries that were represented among us. an extensive review of the steps taken in preparedness and response to covid-19 guided by a representative from each of the seven ssa countries was conducted and a conclusive report encompassing our perspectives was generated. since the beginning of the covid-19 pandemic, the world's top priority was to contain the spread of sars-cov-2, to reduce disease fatalities and to limit the patient burden on health systems. despite the uncertainty and unanswered questions around the management of the newly emergent sars-cov-2 infection, african countries joined the global effort to battle the covid-19 pandemic as we outline in detail below. it has been reported that the experiences of ssa countries in handling ongoing outbreaks and managing infectious diseases such as ebola, tuberculosis, malaria and hiv came in handy in the fight of covid-19 15-17 . pre-existing emergency plans on public health interventions, community engagement programs and the work force composed of emergency medical experts and trained health care workers were quickly re-directed to ensure a fast response to covid-19 15-19 . as early as january 2020, prior to the identification of the first case of sars-cov-2 infection on the african continent on february 14 th 2020 3 the majority of african countries lack specialized medical capacity that is critical for handling severe cases of covid-19, such as intensive care unit (icu) beds 10,56 and mechanical ventilators 57 . therefore, the main priority on the african continent was to contain the infection, initiate immediate testing for suspected cases and to start medical intervention prior to development or progression to severe clinical disease. the countries we assessed focused their efforts on prevention, early identification of new infections and mitigating mass spread of the virus by quickly tracing case contacts based on the available information. all seven countries initiated border screening at ports of entry by march 2020 and a 14-day self-quarantine was recommended for all incoming travellers 51,58-63 . although as a result of few or unavailable laboratory technology, some ssa countries could not test for covid-19 locally, the early established collaborative model among african countries, coordinated by the africa cdc, increased testing capacity across the continent 20,64,65 as an example early samples of suspected covid-19 cases from the central african republic were shipped to rwanda for testing until the local capacity became available. nonetheless, testing constraints still remain in most countries 66 , and therefore testing priority was given to most at risk persons such as returning travellers, or the people who have been in contact with confirmed cases, identified through contact tracing by health care workers. all the seven countries have initiated early testing of suspected cases and established designated facilities for testing and clinical care of covid-19 patients 28,34-39 . the early implementation of covid-19 preventive measures delayed the rapid spread of the virus within the african population, but these procedures could not completely halt the spread of the virus in all seven countries. soon after each country had identified the first case of covid-19, new infections were reported, with the majority related to returnee travellers or contacts of index covid-19 cases. to reduce the risk of imported cases, these countries, with the exception of zambia, swiftly closed borders, shut airports and reduced incoming travellers to essential workers and returning residents 25,60-63,67-70 . in order to mitigate further spread of the disease, individuals diagnosed with covid-19 were admitted in designated isolation areas for care and medical assistance while case contact tracing was immediately initiated. additionally, mass gatherings and non-essential travels were prohibited, government and private business staff were encouraged to work from home and schools were closed 63,69-75 . with the exception of zambia, the assessed ssa countries implemented a dusk to dawn curfew and nation-wide lockdown to enforce social distancing measures, limiting movements to essential service providers 69,76-81 . these measures were mainly put in place to prevent large volumes of new infections that would result in a high demand for hospital services, potentially leading to overwhelming of the fragile medical infrastructure 82,83 . contrary to what was initially expected, the spread of sars-cov-2 has been relatively slower in africa 11 , and covid-19 infections have been generally mild to moderate, leading to more recoveries and lower fatality rates in the seven ssa countries 84 compared to western countries 5 . it should also be mentioned that this pandemic started earlier on the other continents, suggesting that it may be too early for ssa to celebrate its relative success as africa may have not yet faced the highest phase of the disease. however, a comparison of the early phases of the pandemic in some african and european countries has shown a positive impact of early interventions initiated by ssa countries resulting in distinct disease trajectories, for example a comparison of the infection dynamics in the united states, united kingdom, italy and spain vs south africa and cameroon has shown a continual exponential peak in non-african countries but slow and gradual increase in both of the ssa countries 85 . our analysis suggests that early initiation of preventive measures, a faster response by timely testing of suspected cases and immediate contact tracing done by ssa countries has mitigated a faster and more extensive spread of the virus in the population. additionally, a contemporary warm climate may have impacted the dynamics of the sars-cov-2 transmission in these countries [86] [87] [88] . we posit that the predominantly young demography could be a contributing factor to a mild disease and low case fatality observed in africa 18, 89 . furthermore, there are suggestions that cross reactive-immunity resulting from previous infections that are predominant in the region or the universal bcg vaccine policy 90 , widely recommended for infants vaccination in the assessed countries, may have offered some health benefits such as enhanced lung cells immunity against infections contributing to better clinical outcome of the disease. however, these observations have not yet been confirmed by rigorous evaluations. altogether, the prevention programs that were put in place and the early response implemented by ssa countries may have mitigated the widespread dissemination of the sars-cov-2 virus and fatality due to covid-19 in ssa countries 67,91 . the interventions implemented in all seven ssa for prevention and control of the covid-19 are summarized in table 1 . the mode of transmission of sars-cov-2 has led to enforcement of social distancing measures by restrictions of mass gatherings and a national lockdown in six of the seven countries that we assessed 60-63,69,70,72-75 . this method of prevention has specifically disrupted the school programmes and created economic crises that resulted in hunger 101 and other hardships for the large ssa population who depend on casual labor and rely on daily income 6 . the fragile health systems coupled with lockdown measures have inadvertently reduced access to health care for non-emergency and other pre-existing medical conditions. for example with a re-prioritisation of human resources, it is estimated that an additional 6.5 million tb cases will occur over the next five years 102 . although the impact on other diseases such as hiv and malaria has not been assessed, it is likely that the covid-19 pandemic will set back some of the gains made in the countries' responses to these killer diseases. recommended solutions to bridge the gaps for improved outbreak preparedness and response based on our assessment of the challenges and the gaps that were found in the approach used by the seven ssa to prevent/ respond to covid-19 outbreak, we summarized the potential solutions in figure 1 . we classified these into three interconnected table 1 . summary of interventions implemented for prevention and response to covid-19. strengthening medical capacity and testing technology. in collaboration with who, africa cdc and member states the following steps were taken: • january 27 th : africa cdc activated its emergency operations center incident management system (ims) for the 2019 n-cov outbreak. • procurement of sars-cov-2 testing kits • establishment of collaborative model among african countries, setting up a specimen referral system. preparedness and preventive measures (january-march 2020) all the seven sub-saharan african countries implemented similar responses to covid-19 except zambia that didn't close borders or enforce a national lockdown. the different interventions and the date the implementation date in each country are presented below: border closure for non-citizen and non-essential workers. (except for zambia) covid-19 screening at port of entry for all seven countries 14-dayself-quarantine recommended for all incoming travellers. isolation of covid-19 patients at designated facilities and close medical monitoring immediate contact tracing and testing. prohibit mass gathering and non-essential travels inside the country recommendation to work from home for private and government institutions dusk to dawn curfew and a national lockdown (except zambia) only screening at the port of entry and self-quarantine were implemented in zambia. a curfew was first implemented in kenya in march 28 th followed by a national lockdown in april 6 th 2020 • delayed testing that may result in increased disease spread due to late detection of covid-19 cases. shortage of medical/research and clinical laboratory personnel and space • overworked medical personnel • focused medical attention to covid-19 delaying non-essential medical services during the pandemic such as the recommended regular medical check-ups and non-life-threatening interventions. • interruption or delay of non-covid-19 related medical/research activities (example: minor/elective surgeries). lack of local biotech capacity to conduct advanced biomedical research studies such as transmissibility of sars-cov-2 in the african climate conditions, antibody-based therapy, or vaccine and treatment research in the african population. • relying on responses from countries that have the capacity to create solutions. • unavailability of accurate information relevant to the local context that is important for development of adequate preventive measures. borders closure and reduced frequency of international trade • delay of transport of essential materials that are initially imported (example shortage of infant vaccines, anesthesia used for minor surgeries or dentistry). • increased cost for medical supplies and imported food items • unavailability of needed materials locally interrupted supply chain due to market scarcity/ priority given to non-african countries • incapacity to obtain suppliers for the african market even when there are available funds. • shortage of frequently used reagents that need to be imported. poor infrastructure, poverty, informal housing and high population density • increased risk to get the infection due to unavailability of essential sanitary services • nearly impossible to comply with social distancing • hardly able implement safety measures reduced job security due to lockdown measures • increased unemployment during covid-19 pandemic • loss of income for most of the families who depend on casual labor, informal market that have been severely affected by the lockdown. lockdown resulting in reduced movements between cities, unavailability of public transportation and discontinued non-essential work activities including stopping work for researchers working on non covid-19 projects • interruption of pre-existing programs (ex: hiv prevention programs such as prep, art treatment, tb programs, cancer, maternal health care or non-lifethreatening surgeries), • ironically, the emergence of a new virus has prevented virologists to go to the lab! lack of income due to discontinued earning activities, inability to buy food leading to starvation • countries unplanned mobilization of emergency fund to feed poor families. • early ease of the lockdown that may result in new infections • re-opening work activities to avoid hunger related deaths. categories of biomedical, sociocultural and economic aspects that we recommended to help improve the preparedness and response to future outbreaks. in response to the current covid-19 pandemic, africa cdc has started planning for the coordination of a centralised procurement system to reach the target of 55 million tests across the continent. tens of thousands of test kits, ppe and thermometers have already been distributed to countries through a donation from the jack ma foundation 97 . however, sustainability of a strong supply chain requires african governments to mobilize resources and to avail funding for health emergency response and research development on the continent including funded education in speciality fields. this will allow the countries to generate adequate interventions and to maintain a rapid response to outbreaks without overreliance on expertise from non-african countries and urgent importation of supplies. ssa countries should leverage novel medical/research capacity upon the existing structures that were put in place over many decades of fighting other public health threats such as ebola, hiv, tb and malaria. the establishment of a network of multidisciplinary health care workers competent in various tasks such as community education and testing, in a multi-disease focused approach would allow management of staff shortage rather than having to prioritize the new life-threatening disease over those that were already prevailing on the continent. some of the limitations encountered during the development of this assessment were mainly related to the lack of sufficient documentation to address the actual reality in ssa countries, such as the status of health systems or informal housing structure. documentation on physical capacity on the african ground is needed. some essential documents lacked the important information such as the date signed and released. improvement on good record keeping especially for health data of this kind is essential for future references. our assessment was not designed to demonstrate with certitude that the implemented interventions were directly linked to the number of infections or covid-19 deaths in the assessed ssa countries. clinical and biomedical research studies may be more appropriate to confirm these observations. further, systematic reviews of effects would be informative. nevertheless, we believe that our unique perspective on the ssa countries preparedness and response to a great health threat such as the covid-19 pandemic has provided a valuable contribution to the future interventions. we assessed the sub-saharan africa preparedness and response to covid-19. based on an extensive review of the available scientific publications, the government technical reports and the announcements released by governmental and nongovernmental health organizations as well as our personal experiences as workers on the covid-19 battlefield in ssa countries. we documented the preventive measures and the response put in place to contain the sars-cov-2 in seven ssa countries including uganda, kenya, rwanda, cameroon, zambia, south africa and botswana. we have shown the strengths of early initiated interventions that may have contributed to modest and slower dynamics of covid-19 in ssa countries. we discussed the need for scientific and evidence-based responses that considered africa's unique healthcare, sociocultural and economic challenges. while efforts to bridge some of the gaps have been initiated, we recommend that ssa countries develop continued funding streams to support these initiatives as well to increase south to south/north-south collaborations to enhance the capacity of the existing health systems. therefore, if these problems are addressed in a timely manner, there is no doubt that in the next five years ssa countries will have developed a reliable-strong health system to prevent the newly emerging viral infections to spread at a large scale. no data are associated with this article. this is an open letter by umviligihozo et al. that aims at documenting the public health interventions against covid-19 implemented in seven sub-saharan african countries. overall, the paper is well written and covers the most pertinent issues on the topic. it highlights the diverse implementation strategies employed by the seven ssa countries but notes the relative similar low mortality experienced in ssa. however, the article could do with a bit of balancing of viewpoints, especially discussing why despite the relatively different implementation strategies of pubic health measures the mortality is relatively low but similar among the ssa countries. in countries like malawi (not discussed in the article), that have struggled to implement public health interventions, the mortality has not been very different from the other ssa countries. could this observation mean that it is not necessarily the public health interventions that have driven the low mortality in ssa but potentially other factors? the authors have speculated that other factors including population demographics, crossreactive immunity, and climate could have contributed to the less severe outcomes, but this has been relegated to a short paragraph. in its current state, the article puts a lot of weight on public health interventions but could be strengthened with a bit more discussion on the other factors. the article would also benefit from a discussion on whether the planned public health interventions in the different countries were successfully implemented. experiences from other countries show that what was planned on paper is not what happened or is happening on the ground. a discussion on this within the article will aid the reader in the interpretation of the findings and recommendations. all in all, i commend the authors for a well-thought-out open letter and for their confidence as young scientists in driving opinion in africa. is the rationale for the open letter provided in sufficient detail? yes work. the following summary discussing the challenges faced in the implementation of the interventions described was added to the text. "it should be noted that implementation of the public health interventions for preparedness and response to the covid-19 pandemic in sub-saharan africa faced many challenges. overall, the assessed countries lacked the local biotechnological capacity for the production of biomedical supplies, 4 they had a limited workforce with the expertise to specifically address the pandemic using evidence, 5 there was lack of community trust 6 , poor infrastructure 7 , inability to manage remote education programs, 8 failure to maintain the measures implemented due to the population economic instability 9,10 and insufficient funds. 11 formal studies of the extent of these challenges and how to address them in future will be required." of international concern on january 30 th , 2020 and a global pandemic on march 11 th , 2020 by the world health organization (who). 1 " we agree with the reviewer's comment on the need to draw the reader's attention to the changing dynamics of the covid-19 pandemic in ssa, to address this concern we updated the introduction to include that recent reports from south africa indicate a surge in weekly deaths from natural causes that may be attributable to covid-19. 2 we also clarified in conclusion that "this assessment was conducted during the first three month of the covid-19 pandemic before the virus spread widely in ssa and our conclusions were drawn based on statistical information on the disease presented in this study" ○ the sociocultural challenges mentioned in table 2 refer to the misinformation about the consequences of the safety recommendations. as an example, misinformation has led to some interventions being termed harmful or unethical among some communities. examples include interventions such as wearing a mask which some have suggested is detrimental to health due to carbon dioxide poisoning, 3 numerous faith-related rumors, whereby some religious leaders have spread misleading information that they are able to cure covid-19 4 . table 2 was updated to clarify these points. outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle pubmed abstract | publisher full text | free full text a novel coronavirus outbreak of global health concern pubmed abstract | publisher full text | free full text 3. egypt today: egypt announces first coronavirus infection who declares covid-19 a pandemic pubmed abstract | publisher full text 5. world health organization (who): who coronavirus (covid-19) dashboard the state and the informal in sub-saharan african urban economies: revisiting debates on dualism. cris states res cent work pap the urban informal sector in sub-saharan africa: from bad to good (and back again?) preparedness and vulnerability of african countries against importations of covid-19: a modelling study pubmed abstract | publisher full text | free full text identifying key challenges facing healthcare systems in africa and potential solutions pubmed abstract | publisher full text | free full text intensive care unit capacity in lowincome countries: a systematic review pubmed abstract | publisher full text | free full text identifying future disease hot spots: infectious disease vulnerability index santhe: sub-saharan african network for tb/hiv research excellence. 2020. reference source 14. the journal.ie: who warns africa is ill-equipped to deal with coronavirus due to "weaker health systems. 2020; published february 22. reference source 15. tasamba j: rwanda uses ebola experience to combat covid-19 south africa hopes its battle with hiv and tb helped prepare it for covid-19. science (80-). 2020. reference source 17. africa renewal: who:how the lessons from ebola are helping africa's covid-19 response reference source bbc news: coronavirus: most africans "will go hungry in 14-day lockdown high sars-cov-2 seroprevalence in health care workers but relatively low numbers of 2 deaths in urban malawi. covid-19 sars-cov-2 prepr from medrxiv biorxiv why is there low morbidity and mortality of covid-19 in africa? access to lifesaving medical resources for african countries : covid-19 testing and response , ethics , and politics deadly masks" claims debunked intensive care unit capacity in low-income countries: a systematic review the state and the informal in sub-saharan african urban economies: revisiting debates on dualism. cris states res cent work pap identifying key challenges facing healthcare systems in africa and potential solutions reference source is the rationale for the open letter provided in sufficient detail? references 1. world health organization (who). world health organization: who director-general's statement on ihr deadly masks" claims debunked pastor who claimed he cured coronavirus with faith dies from illness we gratefully acknowledge the contributions of health care workers for their helpful discussions. we thank professor thumbi ndung'u for his guidance, dr victoria kasprowicz and dr denis chopera for their invaluable contribution to this work. we thank the reviewer for the constructive comments and important suggestions for improving this article. our responses are presented below:we agree with the reviewer that there are aspects of the covid-19 presentation in sub-saharan african that remain unresolved and require additional research. however, our goal here was to review that response in selected african countries and this preliminary analysis suggests a robust and effective response, notwithstanding other factors that may have influenced transmission dynamics and mortality. in response to the reviewer, we have under the conclusion the following clause that highlighted the importance of other factors driving the observations on infection dynamics of the covid-19. "it was noted that zambia, which did not fully implement the interventions described in this study as well as other countries not included in our review that have struggled to put into action public health interventions, 1,2 reported no significant increases in deaths compared to countries with more robust responses reported here. there, consistent with the recent study by m. njenga et al 3 which investigated the causes of low morbidity and low mortality of covid-19 in african countries, we also suggest that other factors such as a warm climate, young population, pre-existing cross-reactive immunity may have considerably contributed to the evolution of the covid-19 pandemic in ssa countries."we agree with the reviewer that further studies of the efficiency of implementation of public health interventions is required. this was beyond the scope of the current the authors write that this article is "based on an extensive review of the available scientific publications, the government technical reports and the announcements released by governmental and nongovernmental health organizations …". however, the authors do not provide a description of the search and selection of the literature used in this article. i suggest that they should provide this information in the article or as a supplementary document.the world health organization declared covid-19 a public health emergency of international concern on 30 january 2020 and not in march 2020. please refer to reference 1 1 .the authors state that "notably, there is no significant increase in unexplained pneumonias or deaths recorded on the continent which could possibly indicate the effectiveness of interventions introduced by several african governments". while i generally agree with this statement, especially given the time they submitted the article, i would like to alert the authors to reports by the south african medical research council of a recent upsurge in weekly deaths from natural causes in south africa. please refer to reference 2 2 .in table 2 , what does the sociocultural challenge "suspicion of harmful and unethical interventions" mean? what are the harmful and unethical interventions and why and who offered the harmful and unethical interventions to whom?what are "faith-related rumors"? are all factual statements correct, and are statements and arguments made adequately supported by citations? partly where applicable, are recommendations and next steps explained clearly for others to follow? yescompeting interests: no competing interests were disclosed.reviewer expertise: systematic reviews and meta-analysis, implementation science, evidenceinformed policymaking, vaccinology.i confirm that i have read this submission and believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however i have significant reservations, as outlined above.author response 07 sep 2020gisele umviligihozo, simon fraser university, burnaby, canadawe thank the reviewer for the positive comments and suggestions for improving this article, our responses are as follows:to elaborate on the search criteria and to describe the research methodology used in the selection of the literature for this article, the following statement under "assessment and findings" was updated to reflect these steps. "we therefore decided to review articles focusing on sars-cov-2 infection dynamics in africa and the responses that african governments were taking. specifically, we used the free search engine pubmed to identify articles that discussed the transmission dynamics of the new sars-cov-2 virus, or described the sociocultural, economic and the state of health care systems in africa and their impact on sars-cov-2 transmission. moreover, we searched for popular media reports and performed internet searches at official websites for any documents or articles highlighting interventions against covid-19 implemented by african governments, local health authorities or nongovernmental health organizations. we also relied on our own personal experiences and perspectives." ○ we agree with the reviewer's comment on this date, a correction has been made to this statement which now reads "covid-19 was declared a public health emergency ○ competing interests: no competing interests were disclosed. key: cord-313574-8t5y9gqq authors: roy, siddhartha; dickey, sabrina; wang, hsiao-lan; washington, alexandria; polo, randy; gwede, clement k.; luque, john s. title: systematic review of interventions to increase stool blood colorectal cancer screening in african americans date: 2020-06-24 journal: j community health doi: 10.1007/s10900-020-00867-z sha: doc_id: 313574 cord_uid: 8t5y9gqq african americans experience colorectal cancer (crc) related disparities compared to other racial groups in the united states. african americans are frequently diagnosed with crc at a later stage, screening is underutilized, and mortality rates are highest in this group. this systematic review focused on intervention studies using stool blood crc screening among african americans in primary care and community settings. given wide accessibility, low cost, and ease of dissemination of stool-based crc screening tests, this review aims to determine effective interventions to improve participation rates. this systematic review included intervention studies published between january 1, 2000 and march 16, 2019. after reviewing an initial search of 650 studies, 11 studies were eventually included in this review. the included studies were studies conducted in community and clinical settings, using both inreach and outreach strategies to increase crc screening. for each study, an unadjusted odds ratio (or) for the crc screening intervention compared to the control arm was calculated based on the data in each study to report effectiveness. the eleven studies together recruited a total of 3334 participants. the five studies using two-arm experimental designs ranged in effectiveness with ors ranging from 1.1 to 13.0 using interventions such as mailed reminders, patient navigation, and tailored educational materials. effective strategies to increase stool blood testing included mailed stool blood tests augmented by patient navigation, tailored educational materials, and follow-up calls or mailings to increase trust in the patient-provider relationship. more studies are needed on stool blood testing interventions to determine effectiveness in this population. although all population groups in the u.s. are affected by cancer, minority communities experience a higher cancer burden. notably, african americans exhibit higher cancer death rates and lower survival rates compared to other racial or ethnic groups [1] . minority communities are more likely to be poor, medically underserved, and have limited access to quality health care, leading to cancer health disparities. these persistent disparities may be attributed in part to lower rates of completing recommended cancer screenings and increased risk factors, which are more prevalent among low-income and uninsured individuals living in minority communities [1] . colorectal cancer (crc) is the third most commonly diagnosed cancer in men and women in the united states with an estimated 101,420 new cases [2] . crc continues to affect african americans at disproportionately higher rates than non-hispanic whites despite improved screening and treatment (jackson, oman, patel & vega, 2016). the crc incidence rate between 2011 and 2015 was 24% higher for african american men and 19% higher for african american women [1] . moreover, crc mortality rates were 47% higher in african american men and 34% higher in african american women than in non-hispanic whites [1] . the cancer health disparity in mortality is decreasing at a faster rate in african american women than in men, possibly due to differences in risk factors (e.g., physical activity and diet) and from higher crc screening rates in women which has improved early detection [1] . other factors associated with crc disparities across the cancer continuum may include genetic and microbiomic influences, differences in tumor biology, environmental causes, structural barriers such as costs, transportation and availability of healthcare facilities, and individual barriers including lack of knowledge, low self-efficacy, low health literacy, and mistrust of providers [3, 4] . patient factors, provider-related factors, health care system-related factors, and structural factors all contribute to crc disparities for african americans [4] . in the current era of covid-19, elective surgeries are being postponed and cancer screenings deferred because of fear of exposure to the virus in hospitals and clinics; therefore, mail-based screenings such as stool blood crc screening are a safer alternative. the u.s. preventive service task force recommends that screening for crc start at age 50 and continue until age 75 [5] . crc screening options generally include a high-sensitivity stool blood test [fecal immunochemical test (fit), guaiac-based fecal occult blood test (gfobt), or multitarget stool fit-dna test)] or a direct visualization test (colonoscopy, flexible sigmoidoscopy, or ct colonography). early detection with colonoscopy can detect precancerous polyps and abnormalities in the colon and rectum to reduce mortality with this test which is done every 10 years. crc screening may also involve annual stool blood tests, such as the fit or gfobt, and follow-up with diagnostic colonoscopy if needed for abnormal findings. communication of abnormal fit results are associated with improved adherence to follow-up colonoscopy [6] . the american cancer society (acs) recently updated its screening recommendations with a "qualified recommendation" for beginning crc screening for adults beginning at 45 years old and a "strong recommendation" for beginning screening at 50 years and older until the age of 75 in may 2018. a recent study reported that crc screening more than doubled among people between 45 and 49 years since the acs guideline change [7] . in 2009, the american college of gastroenterology (acg) recommended that african americans begin screening at 45 years old [8, 9] . the difference in age recommendations is related to the type and quality of evidence for individuals aged 45-49 years old. currently, the most common tests used in the u.s. to adhere to crc screening recommendations include either a colonoscopy every 10 years or an annual stool blood test (fit or gfobt). there is no consensus on which test recommendation will yield higher adherence rates to crc screening recommendations; however, studies suggest that stool blood tests may be more acceptable and beneficial for african american communities to reach recommended screening levels [10] . moreover, there are many regions of the u.s. where access to colonoscopy is suboptimal [11] . a previous systematic review focused on crc screening by colonoscopy in african americans concluded that patient, provider, and systematic barriers to screening could be modifiable to improve screening rates [12] . these barriers were classified into the following three domains: (1) patient factors-psychological (fear) and lack of knowledge; (2) provider factors-confusion about age recommendations, low knowledge of patient barriers, and lack of counseling; and (3) system factorscosts, insurance coverage, and limited primary care visits [12] . other barriers to crc screening adherence include difficulty in scheduling appointments, provider preferences for one screening modality over another which may limit access, poor healthcare infrastructure including low access to gastroenterology specialists, and available health education resources. while many crc screening interventions focus on increasing colonoscopy screening, stool blood tests can be done in the privacy of a person's home, are more cost effective, and can yield higher rates of adherence [13, 14] . while there are many different intervention possibilities to ameliorate these cancer health disparities, focusing on increasing colorectal cancer screening in minority and underserved communities is one that has been widely implemented in both health care system and communitybased contexts using both outreach and inreach strategies [15] [16] [17] [18] [19] [20] [21] . many of these programs emphasize colonoscopy screening, which is an example of an invasive screening, but several other methods of noninvasive screening tests exist, but require more frequent screening intervals. mailed invitations to complete stool blood testing is an effective outreach approach frequently used by medical providers. to critically assess and synthesize the available evidence for the effectiveness of colorectal cancer screening programs in primary and community health care, this systematic review aims to review quality studies testing different strategies for increasing stool blood colorectal cancer screening rates in african americans. there have been no systematic reviews focused specifically on clinical and behavioral interventions to increase stool blood colorectal screening. studies might include both endoscopic and stool blood methods for their outcomes, but this review sought to identify studies focused on stool blood testing, as a less expensive alternative to colonoscopy in minority communities. the objective of this systematic review is to assess the effectiveness of colorectal cancer screening using stool blood testing approaches, specifically in studies which included either exclusively african americans, or were conducted in health systems with a high proportion of african american patients. the inclusion criteria were the following items: 1. studies using a randomized controlled trial or quasiexperimental study design. control groups could include both usual care or other types of clinical or behavioral interventions to increase colorectal cancer screening such as may be used in comparative effectiveness study designs. 2. studies had to include substantial participants of african americans (> 50% of the study sample) and be conducted in the u.s. 3. studies needed to include a screening completion outcome involving stool blood testing such as high sensitivity and high specificity gfobt, fobt, or fit. 4. only peer-reviewed studies published in scientific journals were included. the exclusion criteria were the following items: 1. studies that were literature reviews, case studies, or quality improvement initiatives without an experimental research design. 2. studies that did not include a comparison group. 3. studies that duplicated the findings from another previously published study to report findings on a subset of participants from the original study. 4. studies that did not measure a colorectal cancer screening outcome, such as studies which only measured changes in knowledge or screening intentions. 5. studies that did not include greater than 50% african americans in the participant sample. 6. studies conducted outside of the u.s. a research librarian searched bibliographic databases which included scopus, web of science, and cinahl from database inception until march 16, 2019. the following syntax illustrates the search strategy used: ((colon or colorectal or sigmoid or rectum or rectal or colonic or anus) n5 (cancer or neoplasm or tumor or tumour)) and (((stool or feces or fecal or faeces or faecal) n5 ( screen* or specimen)) or immunochem* or immunoassay* or "occult blood" or fobt) and (black or blacks or "african american" or "african americans") after the search of bibliographic databases, 650 citations were identified. eight references were eliminated since there were no author names and were either published guidelines or news articles that included no screening outcome or intervention. next, 269 duplicates were identified, and the database was reduced to 381 references. two independent reviewers examined the remaining articles using the inclusion and exclusion criteria and 25 articles met criteria for full text review. articles published before 2000 were eliminated. the rationale for excluding older articles was to account for recent advances in stool blood crc testing technology, such as the introduction of stool dna (sdna) technology in 2000 and the later approval of fit by the centers for medicare and medicaid services (cms) in 2003 [13] . a third reviewer examined all decisions of the two independent reviewers to resolve disagreements on article inclusion and conducted the full text review of the 25 articles ( fig. 1) . after a thorough review of each article, 14 articles were additionally excluded, leaving a final group of 11 articles for article abstraction [14, [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . the most common reasons for excluding articles included: (1) less than 50% of the study sample were african american; (2) did not meet study design parameters; (3) no study outcomes included stool blood test outcomes; and (4) review articles or qualitative studies. to abstract information from each study, the reviewers employed a form adapted from a previous systematic review [32] . the form included questions on sample setting, sample size, study design, randomization procedures, follow-up time periods, target population, intervention description, control group description, participant demographics, type of screening tests, means of screening confirmation (chart versus self-report), and study outcomes by study arm and screening adherence. two reviewers collected information from the included articles independently on the article abstraction form and discussed any discrepancies in data abstraction to come to consensus. there was substantial heterogeneity among the studies identified based on study design, sample size, follow-up time period to measure outcomes, and study site/setting. for example, several studies used more conventional experimental designs with two study arms, other studies were efficacy studies comparing two variations of an intervention, while other studies used three or more study arms or factorial designs. for study site, some studies were clinic-based while others were community-based, so the target population for these studies were considerably different. for each study, an unadjusted odds ratio (or) for the crc screening intervention compared to the control arm was calculated based on the raw data reported in each study to determine the effectiveness. given the substantial heterogeneity between included studies, the decision was made not to conduct a meta-analysis since it was not possible to calculate an overall summary or estimate for the odds of receiving a stool blood test at time of primary study completion. moreover, for a few studies, it was not possible to disaggregate specific types of crc screening test completion in the outcome data (e.g., colonoscopy versus gfobt), which is acknowledged in the study limitations. for the 11 identified studies, eight of the studies included african american study participants of greater than 80% of the total sample ( table 1) . three of the studies included study samples of african americans ranging between 62 and 70% [22, 23, 31] . the 11 included studies had greater proportion of african american women participants compared to men (range 59-88%). most of the 11 studies included participants in the age range for recommended colorectal cancer screening (i.e., 50 to 75 years). however, of the 11 studies, one study focused on only older participants between 65-75 years [28] , and two studies enrolled older individuals up to age 80 years [26] or 94 years [30] . education level varied among participants in the included studies. four of the studies reported that > 70% of participants had completed high school [14, [22] [23] [24] , whereas four other studies reported < 45% [14, 22-24, 27-29, 31 ]. the other three included studies did not report on education level [25, 26, 30] . included studies varied in study designs ( table 2) . six of the studies were randomized controlled trials with two study arms. many of these studies were comparing an enhanced intervention in clinical settings, such as using patient navigation or telephone outreach compared to a standard intervention approach using mail-delivered stool blood tests. other two-arm studies were efficacy studies, for example comparing different combinations of educational materials for fit completion [14] or comparing variations of a church-based community intervention [27] . three of the other included studies employed three study arms or a factorial design. the interventions in these studies included community-based approaches (e.g., lay health advisors) [24] and educational approaches in clinics (e.g., pamphlets and videos) [22, 30] . two of the included studies conducted interventions in rural areas [22, 24] . the results for the two-arm randomized controlled studies ranged in effectiveness (table 3) . some studies reported modest improvements. for example, one study reported a 27% increase in crc screening from baseline using a telephone outreach strategy [23] and another study reported a 44% increase in crc screening from baseline using a video intervention [25] . however, a few studies reported more dramatic increases in crc screening participation, with one study reporting an 82% screening adherence postintervention from implementing a tailored print intervention and fit kit [14] . the five studies using two-arm experimental designs ranged in effectiveness with odds ratios (or) ranging from 1.1 to 13.0 using interventions such as mailed reminders, patient navigation, and tailored educational materials [23, 25, 26, 28, 29] . while the study by goldberg et al. [26] had a large or (13.0; ci 3.7-46.5), there was a small sample size of only 119 participants. two studies with large samples which focused on increasing stool blood testing were found to have findings that did not reach statistical significance, such as the study by friedman et al. [25] with an or of 1.4 (ci 0.7-2.7) and the study by horne et al. [28] with an or of 1.1 (ci 0.7-1.6). the myers et al. study [29] tested a tailored navigation intervention compared to standard mailed reminders and found significant results with an or of 1.5 (ci 1.0-2.2) for the fit screening outcome only. the results for the three-arm studies also ranged in effectiveness (table 4 ). similar to the two-arm studies, some studies reported modest differences between intervention and control. for example, one study reported a 17% increase in crc screening from baseline using a tailored print and video intervention [24] ; a second study reported a 30% increase in crc screening from baseline using a decision aid intervention [31] ; and a third study reported a 49% increase in crc screening from baseline using different variations of a mailed intervention [22] . however, one study reported a more dramatic increase in crc screening of 61% screening adherence post-intervention following receipt of a tailored group intervention using video education in senior centers [30] . while several of these studies used either 6-month or 12-month primary outcomes, a few studies examined repeat stool blood testing outcomes during a longer time period, such as the study by arnold et al. which examined third-year stool blood testing outcomes [22] . for nine of the 11 studies included in the review which used random assignment of participation to intervention or control arm, there was relatively low attrition of participants. for the clinical studies, outcomes could be tracked for virtually all participants through electronic patient records. a few studies reported higher research participant attrition between 33 and 42%. the most common limitation reported was the higher number of african american female participants than male in several studies. in addition, a few studies cited crc screening outcomes based on self-report, which is less reliable than patient chart verification (table 5 ). this systematic review examines summary findings from 11 experimental studies designed to test the effectiveness of interventions to increase colorectal cancer screening, specifically using stool blood crc screening approaches such as fit or gfobt in african american communities. the eleven studies together enrolled a total of 4364 participants (88% african american). based on these summary findings which tested effectiveness of clinical and behavioral interventions, a tailored navigation approach either by telephone or in person, might be a potentially effective strategy for increasing crc screening in african american communities as these interventions tested in two arm studies to control conditions were associated table 5 methodological quality of included studies allocation methods attrition other potential limitations arnold et al. [22] quasi-experimental low attrition reported (9%) method of determining fobt receipt not reported. differences between arms in sociodemographics, and sample was predominately composed of african american female participants in fqhcs basch et al. [23] random assignment low attrition (7% in intervention arm; 4% in control arm) all participants had health insurance and a physician, and stool blood test kits were not provided campbell et al. [24] random assignment medium attrition reported (42%) method of determining crc screening was based on self-report. there was a small sample size and factorial design limited comparative analysis christy et al. [14] random assignment not reported not possible to isolate providing the fit kit from print educational materials since both study arms used educational materials in the efficacy study friedman et al. [25] random assignment not reported sample was predominately composed of african american female participants goldberg et al. [26] random assignment no attrition reported (0%) study was completed at one clinical primary care setting site. study was completed only in one single year, so repeat fobt card return data were not collected holt et al. [50] random assignment low attrition reported (10%) method of determining crc screening based on self-report. participation was not limited based on not being up-to-date with crc screening horne et al. [28] random assignment low to medium attrition reported (33% in intervention arm; 23% in control arm) method of determining crc screening based on self-report. high percentage of participants were up-to-date at baseline myers et al. [29] random assignment low attrition reported (< 1%) the sample was predominately composed of african american female participants. all participants were patients at primary care practices powe et al. [30] quasi-experimental not reported the sample was predominately composed of african american female participants. there was a small sample size and 3-arm study design limited comparative analysis schroy et al. [31] random assignment no attrition reported (0%) no blinding of providers to one of two intervention arms (decision aid plus personalized risk assessment or decision aid alone) with percentage point differences ranging from 6 to 36% [23, 25, 26, 29] . the outcomes for the three-arm studies testing variations of interventions in efficacy studies or testing two different variations of an intervention compared to controls, did not report markedly different findings from the two-arm studies. the study by arnold et al. [22] was unique because it reported 3-year outcomes for stool blood testing, an outcome worth examining because of the recommendation for annual screening. except for the study by schroy et al. [31] , the studies using experimental designs other than two-arm experimental designs had relatively small sample sizes. the study by schroy et al. [31] reported that a decision aid improved overall crc screening rates, but did not report outcomes separately for stool blood testing. the study by christy et al. [14] was particularly notable for the very high fit kit return of 87% of all participants, and the finding that prior screening status did not predict uptake. in this study, african american participants either received a fit kit plus a cdc standard crc screening educational brochure or fit kit plus a tailored photonovella, as well as reminder letters for those not returning the fit kit within a month-long timeframe, and both approaches were effective. two of the included studies, both three-arm studies, were identified as taking place in rural areas [22, 24] . previous literature has found that the types of screening tests used for colorectal cancer may differ between rural and urban settings [33] . screening rates by colonoscopy or sigmoidoscopy have increased in both rural and urban populations; however, screening rates via fobt have decreased in urban populations but increased in rural populations, possibly due to greater access to colonoscopy in urban areas and lack of access to colonoscopy services in rural settings [33] . the results from the two included studies in rural areas reported increases in crc screening uptake, specifically fobt screening, which provides further support for the preference for fobt screening in rural areas. this systematic review included studies that implemented interventions in clinic or community settings. our results showed that the two-arm studies that were implemented in clinic-based settings had higher odds ratios than the two-arm studies implemented in community-based settings. based on these results, implementing crc screening interventions in clinic-based settings may lead to greater crc screening uptake among african americans than community outreach strategies outside of a clinical setting, possibly due to clinical interactions, audit and feedback systems, or other electronic means to remind patients of screening appointments. another finding in this systematic review indicated females were more likely to engage in fit and fobt, which is consistent with the results of studies examining stool based testing in articles that were eventually excluded after thorough review [34] [35] [36] [37] [38] . however, in the included studies, there were larger samples of women than men, which might partially explain these findings, as well as highlight the challenges of recruiting african american men to cancer screening studies. the findings from this group of articles also suggests the promise of culturally tailored interventions to improve crc screening, such as the use of trusted community venues (e.g., churches, barber and beauty shops) to recruit participants and the implementation of patient navigation approaches to increase trust in providers. more research is needed to test effective interventions for increasing crc screening in minority communities and to test whether combined modalities or offering stool blood testing as a frontline strategy increases overall crc screening rates. unfortunately, some african american communities have higher levels of uninsured individuals, poor access to healthcare services, lower perceived benefits of crc screening, lower perceived risk of colorectal cancer, and lower engagement in preventive health behaviors [39, 40] . the previously listed factors have also been cited as barriers for engaging minorities in crc screening [12, 41] . in light of the economic burden of crc screening, utilizing fit and gfobt can provide a low-cost first-line screening test and be the deciding factor for medically underserved communities to engage in colorectal cancer screening [42] . an examination of the studies in this review identified this frontline screening strategy with fit or gfobt can be cost effective. a previous study by hester et al. [42] indicated stool blood testing presented fewer barriers for completion such as lack of transportation to the facility, bowel preparation, and absence from work, which may impede crc screening with colonoscopy. when the barriers to crc screening are removed, there is an opportunity to increase rates of screening among minority and non-minority groups. consistent with the literature, our findings indicated the feasibility and acceptability of fit and gfobt in african american communities as a primary screening strategy (baker et al. 2014; davis et al. 2010; rawl et al. 2008) . this systematic review focused on stool blood testing as one convenient method to increase crc screening in medically underserved communities. other studies using experimental designs reported similar findings but were excluded because of smaller african american patient samples [37, 38, 43] . some identified studies employed other minority samples in their interventions. for example, the experimental study by muller et al. [44] reported the effectiveness of the low-cost strategy of text messaging for increasing crc screening among alaska native patients. even though the current systematic review only included studies with a substantial representation of african american study participants, other studies examining diverse populations also reported the need for innovative interventions to increase crc screening in different community and clinical settings. based on the healthy people 2020 objectives, the 2020 target for adults between 50 and 75 years to receive crc screening was 70.5%; however, 2018 data indicated only 65.2% of adults had completed screening [45] . this systematic review provides some evidence for future strategies to promote crc screening, especially for increasing the use of fobt among african americans. behavioral interventions may be more efficient if delivered through mail reminders or by using patient navigators. the content of such interventions should include tailored messages. if tailored materials are not available, a standard crc screening educational brochure may also be effective. for rural african americans, if primary care settings are not easily reachable, fobt may be a more accessible choice and preferable method to promote crc screening behavior. furthermore, the screening rates achieved by these interventions fall well short of the goal of 80% screening rate (by 2018 and in every community) proposed by the national colorectal cancer round table ( nccrt), which includes the american cancer society and the centers for disease control and prevention (cdc) [46] . the rigor of the current systematic review lies in the methodology for the article search protocol pertaining to inclusion criteria (e.g., type of study design, peer-reviewed studies in scientific journals, presence of comparison groups) and search strategy. however, some studies may not have been included in the initial search because of the particular search terms used or article indexing procedure that may have excluded an article from the search. the included studies varied in study quality. seven of the 11 studies included randomization of study participants, and five of the 11 studies were randomized controlled trials. because of variations in reporting study findings and not stratifying by screening modality, for a few studies it was not possible to isolate the effect of the stool blood testing method. it is recommended that screening outcomes are disaggregated in the reporting of study outcomes. a meta-analysis was not possible for this review because of the heterogeneity in study designs and reported outcomes. some studies did not report odds ratios so the decision was made to calculate odds ratios for all studies based on the data presented in each article. a systematic review of u.s.-based crc screening interventions reported that crc screening interventions which include a combination of screening methods might be most effective; however, most clinic-based interventions might need to provide the array of screening options if they are part of their menu of services [47] . some clinics, such as federally-qualified health centers (fqhc), might only provide stool blood testing and referral to colonoscopy, so these types of clinics would be more likely to be study sites for a stool blood testing approach only. future reviews might include all stool blood testing interventions regardless of population served to determine preferences in actual clinical practice settings to test whether they may be differences by geography or racial group. the option to choose stool blood testing is part of a comprehensive cancer education approach to increase crc screening. our findings align with the healthy people 2020 objective, which seeks to increase colorectal cancer screening based on current guidelines. recent guidelines from leading medical and community health organizations (i.e., united states preventative task force, the american cancer society, the american academy of family physicians, and task force on community preventive services) recommend the use of stool blood testing for colorectal cancer screening, ideally leading to early detection and a reduction in mortality rates [48, 49] . the low-cost and decreased barriers compared to colonoscopy (e.g., time off work, access to a medical facility, and transportation) might provide an opportunity for some minority populations experiencing crc disparities to increase participation in screening. however, without sufficient knowledge of these tests and the recommended screening intervals for such screening, minority communities cannot receive the full benefits of crc cancer prevention. strategies to increase stool blood testing in african americans include standard mailed blood stool tests augmented by patient navigation, tailored educational materials, and follow-up calls or mailings to increase trust in the patient-provider relationship. to increase overall crc screening in african americans also requires decreasing barriers to access for preventive screenings by increasing insurance coverage and lowering out of pocket healthcare costs. cancer statistics for african americans cancer statistics clinical and genetic factors to inform reducing colorectal cancer disparitites in african americans toward the elimination of colorectal cancer disparities among african americans screening for colorectal cancer: us preventive services task force recommendation statement standardized workflows improve colonoscopy followup after abnormal fecal immunochemical tests in a safety-net system colorectal cancer screening patterns after the american cancer society's recommendation to initiate screening at age 45 years colorectal cancer screening: recommendations for physicians and patients from the u.s. multi-society task force on colorectal cancer american college of gastroenterology guidelines for colorectal cancer screening exploring perceptions of colorectal cancer and fecal immunochemical testing among african americans in a north carolina community. preventing chronic disease challenges and possible solutions to colorectal cancer screening for the underserved explaining persistent under-use of colonoscopic cancer screening in african americans: a systematic review colorectal cancer screening: stool dna and other noninvasive modalities a community-based trial of educational interventions with fecal immunochemical tests for colorectal cancer screening uptake among blacks in community settings a program to enhance completion of screening colonoscopy among urban minorities a culturally tailored navigator program for colorectal cancer screening in a community health center: a randomized, controlled trial formative research on knowledge and preferences for stool-based tests compared to colonoscopy: what patients and providers think outreach invitations for fit and colonoscopy improve colorectal cancer screening rates: a randomized controlled trial in a safety-net health system the colorectal cancer screening process in community settings: a conceptual model for the population-based research optimizing screening through personalized regimens consortium colorectal cancer screening at community health centers: a survey of clinicians' attitudes, practices, and perceived barriers reducing colorectal cancer incidence and disparities: performance and outcomes of a screening colonoscopy program in south carolina third annual fecal occult blood testing in community health clinics telephone outreach to increase colorectal cancer screening in an urban minority population improving multiple behaviors for colorectal cancer prevention among african american church members compliance with fecal occult blood test screening among low-income medical outpatients: a randomized controlled trial using a videotaped intervention mailings timed to patients' appointments: a controlled trial of fecal occult blood test cards spiritually based intervention to increase colorectal cancer screening among african americans: screening and theory-based outcomes from a randomized trial effect of patient navigation on colorectal cancer screening in a community-based randomized controlled trial of urban african american adults. cancer causes and control increasing colon cancer screening in primary care among african americans an intervention study to increase colorectal cancer knowledge and screening among community elders aidassisted decision making and colorectal cancer screening: a randomized controlled trial systematic review of mammography screening educational interventions for hispanic women in the united states urban-rural disparities in colorectal cancer screening: cross-sectional analysis of 1998-2005 data from the centers for disease control's behavioral risk factor surveillance study fobt completion in fqhcs: impact of physician recommendation, fobt information, or receipt of the fobt kit colorectal cancer screening among low-income african americans in east harlem: a theoretical approach to understanding barriers and promoters to screening increasing colorectal cancer screening at an urban fqhc using ifobt and patient navigation a randomized controlled trial of the impact of targeted and tailored interventions on colorectal cancer screening a randomized trial of two print interventions to increase colon cancer screening among first-degree relatives. patient education and counseling the cascade of social determinants in producing chronic disease in low-income african-american men male role norms, knowledge, attitudes, and perceptions of colorectal cancer screening among young adult african american men the role of perceived benefits and barriers in colorectal cancer screening in intervention trials among african americans decisional stage distribution for colorectal cancer screening among diverse, low-income study participants implementation intentions and colorectal screening: a randomized trial in safety-net clinics text message reminders increased colorectal cancer screening in a randomized trial with alaska native and american indian people along the way to developing a theory of the program: a re-examination of the conceptual framework as an organizing strategy creating and implementing a national public health campaign: the american cancer society's and national colorectal cancer roundtable's 80% by 2018 initiative a systematic review of u.s.-based colorectal cancer screening uptake intervention systematic reviews: available evidence and lessons learned for research and practice recommendations for client-and provider-directed interventions to increase breast, cervical, and colorectal cancer screening colorectal cancer screening and prevention spiritually based intervention to increase colorectal cancer awareness among african americans: intermediate outcomes from a randomized trial publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-342588-berrojmq authors: burri, christian title: sleeping sickness at the crossroads date: 2020-04-08 journal: trop med infect dis doi: 10.3390/tropicalmed5020057 sha: doc_id: 342588 cord_uid: berrojmq human african trypanosomiasis (hat; sleeping sickness) is a disease with truly historic dimensions [...]. these activities marked the start of incredible efforts of the colonial powers to control the disease, but particularly also of the vertical approach towards disease control, with special programs run in parallel with the public health system-which is now one of the challenges in the elimination "end game" with very few patients remaining requiring an integrated public health approach. the detection of the causative agent, the mode of transmission and the first documented major epidemics coincided with the advent of modern pharmacology. it is, therefore, no surprise that there was an interest to find drugs against this disease. in 1908, the colonial powers, in a joint conference, decided to give drug development a high priority and in course, several molecules were developed. the history of the oldest drug still in use against hat, suramin, is described in the context of the history in the article of madeja et al., not only is the drug still manufactured, but also the continued support of bayer since the year 2000 is a major contributing factor allowing us to write about the elimination of hat today. the activity of inorganic arsenic-based compounds had already been recognized in the mid-19th century and the treatment of "nagana" (animal trypanosomiasis) was described by david livingstone in 1848 and david bruce in 1895. this knowledge led to the development of the first organo-arsenic compound atoxyl ® in 1905 by paul ehrlich, who used trypanosomes as a model to screen molecules, but was mainly searching for drugs against syphilis. wolferstan thomas in liverpool subsequently showed that atoxyl ® was effective against t. b. gambiense. however, atoxyl ® , meaning 'non-toxic', caused severe adverse drug reactions particularly affecting the optic nerve; it was only active against early stage hat and was followed by tryparsamide in 1919. tryparsamide was developed in the usa and was the first drug to be active against the late stage, although not very active and also prone to extensive resistance development. tryparsamide was also very toxic, with a dose dependent risk damaging the optic nerve. this is documented in a horrifying report from 1930 when a lieutenant of the french army in cameroon doubled the prescribed dose of tryparsamide to speed up the recovery of 800 patients. two days later, all these patients were blind. this event prompted the swiss chemist and physician dr. friedheim to investigate alternative drugs. the introduction of a triazine ring lead to the development of melarsen (disodium p-melaminyl-phenyl arsonate) in 1940, which proved to be a very efficient drug against t. b. gambiense, but was still very toxic. in 1944, friedheim first described the trivalent arsenoxide form of melarsan, known as melarsenoxide. he reported its use in treatment of human sleeping sickness in 1948. the main achievement was the reduction of the duration of therapy to six weeks, with two weeks of seven daily injections of 1.5 mg/kg each, spaced by an interval of one month. in a further step, capping the arsenic in melarsen oxide with british anti-lewisite, an antidote to the arsenical warfare agent lewisite, reduced the toxicity by a factor of the order of 100, but the trypanocidal activity only by a factor 2.5. the new drug was called melarsoprol (mel b; arsobal ® ) [5] . it remained the mainstay of second stage hat treatment until the early 2000s despite the long treatment duration of 35 days, the known related adverse drug reactions, particularly the encephalopathic syndrome that occurs in about 10% of the patients treated and leads to their death in about 50% of cases, and the potential for drug resistance. another line of research was the molecules with a diamidine structure discovered in the 1930s. these molecules were detected by serendipity in the search for hypoglycaemic compounds, with the idea in mind that this effect might compromise the very prominent and particular glucose metabolism of trypanosomes. several compounds, however, proved to have a direct trypanocidal effect with the three compounds stilbamidine, pentamidine, and propamidine identified to have the highest activity [6] . pentamidine does not penetrate the blood brain barrier so its use is limited to first stage hat; despite this drawback it is still is in use today, for treatment of children below six kilograms. the distinctly better safety profile of the drugs only active against first stage disease (pentamidine and suramin) versus melarsoprol was also the advent of the consistent performance of lumbar puncture to determine the disease stage and make a treatment decision. the need for a lumbar puncture was, for over 50 years, a characteristic of hat treatment, a source of patient distress, stigma and technical limitation of treatment. some 70 years after the discovery of pentamidine, the diamidines again became the focus of drug development, although not successful until today. the development of the organo-arsenicals, diamidines, and also drugs developed much later and still in use like eflornithine and nifurtimox, including their drawbacks related to the potential for drug resistance are described in detail in the contribution of de koning. the "scramble for africa" was an investment with very high political and economic stakes, and sleeping sickness was not just a disease; it had become the colonial disease. besides drug development, the responses of the anglophone and francophone colonial powers to trypanosomiasis differed significantly. francophone countries chose to concentrate directly on the medical problems presented by the disease in humans. this included the introduction of "mobile teams" actively searching and screening population for hat cases. this method of systematic case detection and treatment with the aim of elimination of the parasite reservoir was suggested by the french military surgeon eugène jamot and such activities started in 1926 in cameroon ("atoxylisation") [7] . subsequently, the prevalence of hat declined from as high as 60% in 1919 to 0.2-4.1% in 1930, leading to an expansion of the methodology to other countries. after the second world war, atoxyl ® was replaced by pentamidine and a regular application of the drug every six months to the population at risk was introduced ("pentamidinisation"). in the 1950s in the belgian congo alone, some two million people were subjected to this preventive mass drug administration [3] . due to the partial presence of t. b. rhodesiense in their territories, the anglophone countries were confronted with the more widespread problem of disease in domestic livestock, which also presented a reservoir for human disease. their approach included vector control (traps, spraying), bush clearing, and game destruction [3, 7] , and later the chemopreventive use of veterinary drugs (diminazene, isometamidium, and homidium). the control measures were overall very successful and progressively controlled the disease, reaching a very low, generalized transmission by the mid-1960s, with a minimum of 4435 cases declared in africa in 1964 [8] . however, the measures taken were very costly, but above all very unpopular in the communities. this led to concealment where possible, and made the longterm goal of elimination by chemotherapy difficult, if not impossible [3] . we could observe similar tendencies when conducting clinical trials in the 2000s; potential patients were mostly hiding away or fleeing the villages at the beginning of mobile team campaigns for reasons of fear of stigmatization, lumbar puncture, pain, and the possibility of being treated with the dangerous drug melarsoprol. the in-depth understanding of the communities' beliefs, needs, and approaches is therefore key in a successful elimination attempt; insight on these topics are presented in the papers by and falisse et al., lee et al., and palmer et al. a factor mentioned by winslow in 1951, which may still be under-researched today, is the relationship between the disease and poverty, particularly inadequate food supply, as the disease leads to unused land, which creates malnutrition [3] . such a view would require an even more integrated approach towards disease control and elimination. when the colonial powers withdrew from africa between 1960 and 1975, a new era began. the young nations created their own institutions with the goal of continuing research towards the elimination of hat (e.g., kenya trypanosomiasis research institute (ketri), nigerian institute for trypanosomiasis research (nitr), uganda trypanosomiasis research organisation (utro), and programme sur la recherche sur la trypanosomiase in côte d'ivoire (prct)). these institutions were reinforced since the 1970s by internationally funded institutes dedicated wholly or partly to trypanosomiasis research (e.g., international laboratory for research on animal diseases (ilrad) in kenya, international centre of insect physiology and ecology (icipe) in kenya, international trypanotolerance centre (itc) in the gambia). however, in the course of monetary adjustments in the 1980s, the decreasing funds available, and the emergence or increase of other health priorities, institutions devoted to a single disease were no longer sustainable and they were continuously integrated or transformed into multilateral institutions. overall, in the years after the independence process, the expenditures for hat were reduced, and awareness and surveillance of the disease decreased. the number of mobile teams was decreased, and it was attempted to transfer activities to the public health system-without having the respective tools, approaches, and knowledge [9] . this, together with social instability, conflicts, and insecurity constraining disease control interventions led to a significant resurge of gambiense hat in the 1980s and 1990s [8] , mainly affecting angola, congo, southern sudan, and the west nile district of uganda [1] . at the end of the 1990s, the situation was comparable to the one in the 1920 and 1930; the number of reported cases was almost 30,000 with 300,000 cases suspected [10] . in 2001, we published a special issue in the journal tropical medicine and international health [11] , asking ourselves whether there were new approaches to roll back hat. not only was this the time of 300,000 hat cases suspected in 19 countries of sub-saharan africa, it was also the time when the organo-arsenic drug melarsoprol was still the only treatment available for second stage hat. treatment with melarsoprol required around 35 days of hospitalization with numerous and very painful injections, very severe adverse drug reactions like an encephalopathic syndrome were common and the mortality rate under treatment was as high as 2-10%. in those days, an oncologist tried to comfort us, saying that a 95% treatment success rate for a disease with an inevitably fatal course was fantastic. we did not share this view, and rather expressed a dream: to make hat "an ordinary" disease, which follows the usual pattern "test, treat, track"-without the need of a lumbar puncture to make treatment decisions, without a high mortality rate under treatment, without the pain, without the stigma. elimination was a very far-fetched goal at this time, but there were some first positive signals that the dimensions of the problem and its impact on society and development were being recognized. the conclusions adopted by the international scientific committee of trypanosomiasis research and control (isctrc) in 1999 reflected a new awareness of the disease. the african union member states were urged to give highest priority ranking to african trypanosomiasis in their development programs, and it was recommended that urgent and particular attention should be given to surveillance and intervention in epidemic areas, to drug availability and resistance, and to the implementation of operational research to respond to the needs of control programs. at their meeting in lomé in july 2000 the oau heads of state and governments signed a declaration of intent to eradicate tsetse flies on the african continent-something that will likely not happen, but it was the turning point towards manifold activities which make us today work towards elimination of hat as a public health problem of 2020 and the interruption of transmission by 2030. at the same time, médecins sans frontières' access to medicines campaign were able to make a compelling case that society needed to rethink drug discovery paradigms for neglected diseases. aventis (now sanofi) was persuaded to repurpose and develop the failed anti-cancer drug eflornithine for use against hat and to donate it at no cost to the who for distribution in africa. millions of dollars were also provided by aventis/sanofi to the who, who could now develop new screening and intervention programmes [9] . bayer signed a similar contract with the who, a success story and joint effort which has been renewed by both companies until today, and which is one of the strong drivers in control and now elimination. in 2001, the bill and melinda gates foundation selected hat to be one of the first diseases they targeted through the consortium of parasitic drug development (cpdd) and shortly thereafter, the drugs for neglected diseases initiative (dndi) was founded. the beginning of the century was truly an exciting time for neglected diseases and for hat in particular. the changes and significant impact on funding were later summarized in the landmark publication "the new landscape of neglected disease drug development" [12] . the changed situation immediately led to the initiation of several large scale activities in drug development and the term "elimination" in the context of hat was mentioned for the first time by dr. jannin, leading the anti-hat efforts at the who in 2004 [13] . drug development had been virtually dormant for about 50 years. although the cultivation and test methods for drug screening for anti-trypanosomal drugs had been developed in the 1980s and 1990s [14] , the money for pursing lead compounds in preclinical work, translational studies and large-scale trials was too scarce in these days. during the 1990s, some initial limited drug activities were carried out on shoestring budgets: eflornithine, which had initially been developed in the 1970s as a potential anti-cancer drug, was found to be active against second stage gambiense hat. this discovery in the 1980s was a scientific breakthrough [15] and eflornithine was shown to be much safer compared to melarsoprol. the drug received orphan drug status by the us food and drug administration in 1990; however, production was stopped after a few years and only resumed after significant public and political pressure. eflornithine, however, was only introduced for treatment in a limited number of centres by msf in 2000, but not until 2006 by the national sleeping sickness control programs because of its limited availability, the initial high costs, and particularly the logistical challenge to transport the drug and its associated 56 bottles of sterile water per treatment. the turning point was when who launched a kit format and coordinated training of staff from national sleeping sickness control programs [16] . furthermore, in the 1990s nifurtimox, developed against chagas disease was used in experimental settings mainly to treat melarsoprol refractory cases [17] . in the mid-1990s, the pharmacokinetics of melarsoprol was elucidated. the assessment of a subsequently proposed abridged 10 days regimen in a large scale trial with 550 patients in angola (impamel i) allowed the replacement of the empirically derived complex schemes lasting from 25-36 days in 2003 [18] [19] [20] . whereas the new regimen had major socio-economic advantages, the disappointment was that the frequency of the worst adverse drug reaction, the encephalopathic syndrome, remained at levels of 5-10% of patients treated, still resulting in death in 10-50% of those in whom encephalopathy developed. the metabolism of melarsoprol was elucidated somewhat later [21] . the finding that the major metabolite melarsenoxide covalently bound to a midsize protein triggered another large-scale clinical trial, which led to the elucidation of the nature of the encephalopathic syndrome. the early 2000s were dominated by the development of the oral prodrug pafuramidine against first stage hat; the program failed at a late point of development, but it contributed much to the understanding of hat chemotherapy and the conduct of clinical trials against hat, which it is described in detail by the paper of dickie et al. in parallel, several trials assessing combinations of eflornithine, melarsoprol, and nifurtimox were conducted. in all trials, the efficacy was better in the combination arms compared to the monotherapies. however, combinations containing melarsoprol resulted in very high frequencies of severe adverse drug reactions and were rapidly abandoned [16] . a multiple-centre trial, conducted in the republic of congo and the democratic republic of the congo (drc) compared nifurtimox-eflornithine combination therapy (nect) with the standard eflornithine therapy. nect reduces the number of eflornithine infusions from 56 to 14, the total amount of eflornithine by half and the hospitalization time by one-third [22] . based on the favourable results of the trials conducted, nect was included for treatment of second stage gambiense hat into the who's essential medicines list in 2009 [23] , and for children in 2013 [16] . nect can be considered a milestone improvement: under optimal conditions, fatality during treatment is 0.5% compared to 5-6% under melarsoprol [24] . the complexity of its application still restricts the use to the second stage disease, meaning that the lumbar puncture for diagnostic staging is still required [24] , continuing until today. to identify better alternatives, the drugs for neglected diseases initiative initiated a major compound mining effort in 2005 to explore new and old nitroimidazoles as drug leads against human african trypanosomiasis. one of the 830 compounds screened, fexinidazole, proved to be orally active against t. b. gambiense and t. b. rhodesiense in animal studies and had an excellent safety profile. the development of this orally active compound is described in detail in the papers of neau et al., and dickie et al., fexinidazole received a positive scientific opinion from the european medicines agency for treatment of gambiense hat in late 2018, it was approved by the drug regulatory authority of the drc and added to the who list of essential medicines in 2019, and the first official application in the drc happened at the end of january 2020 on world ntd day in a public ceremony. this deliberate coincidence of the date depicts the new integrated thinking of hat control and elimination in the framework of ntds clearly. fexinidazole will be an essential component towards hat elimination. however, it has some limitations, which will hamper its widespread use in the field: its absorption is dependent on simultaneous food intake, or else only subtherapeutic drug levels are reached; based on the observation of a lowered efficacy in patients with advanced disease, a lumbar puncture for staging still is necessary in such patients; and the drug has not been tested yet for children below six years [25] . hence, the search for "the magic bullet" [26] continues-with an excellent starting position compared to 20 years ago: for the first time in history, we can speak of a modest pipeline of anti-hat drugs. one most promising candidate is in late clinical development, several compounds are well advanced in pre-clinical stages, and medicinal chemistry and lead selection work is continued as currently, the leading novel class of molecules are the boron-containing benzoxaboroles. one candidate, scyx-7158, acoziborole, entered phase ii/iii assessment in 2016 [27] . the compound is described in the publication of dickie et al. should the development program be successful, acoziborole would further revolutionize the efforts to eliminate and sustain elimination of hat. due to its long half-life of 400 h, it can be potentially used as a single-dose treatment and should it be well tolerated this would provide further options for decentralized use, and maybe even for "ring-treatment" of patient contacts following the example of ring-vaccinations used, e.g., in the control of the ebola virus. with fexinidazole, and potentially even more with acoziborole, the focus will turn away from the discovery and development of better tools, to the understanding of the implementation, optimal use, including the needs and perception of patients. the clinical research programs have contributed to the reduction of cases: new strong partnerships were formed as described by taylor et al. and the conduct of clinical trials in a number of endemic areas per se has had an impact through staff training, attention to disease, and intensified active case search and treatment of a large number of patients as described by mbo et al. besides the improvements of the renewed interest of governments and improved drug treatment, there are several other reasons for the decrease of hat prevalence: the advances in diagnostics are one of the major factors. the serological card agglutination test for trypanosomes (catt) first published in 1986 [28] had a paramount impact on how patients could be screened by mobile teams. the test was adapted and improved several times, and despite its disadvantages (insufficient specificity to confirm diagnosis, only available in larger batches, cold chain necessary), it has kept its place in hat diagnosis. the mini-anion exchange chromatography for trypanosomes (maect) which increases the sensitivity to detect the parasite in the blood significantly was already published in 1976 [29, 30] , however, only the increased funding available allowed its more consistent use and therefore detection of cases with low parasitemia. the introduction of rapid diagnostic tests is a true advancement, but also lacks the specificity needed to make a final treatment decision [31] . additional tools were recently developed but so far only introduced to a limited extent into routine use (e.g., loop-mediated isothermal amplification (lamp) [32, 33] ; immune trypanolysis test [34] ), which will both play a role in the "end-game". the question, however, is how such tests will be used in the future, and in what settings. the currently ongoing research program ditect-hat is set up do exactly that: it seeks to validate the performance of diagnostic tools and algorithms for early and rapid diagnosis of gambiense hat for passive case detection, post-elimination monitoring, and for assessing the therapeutic response [35] . in addition to the optimization of the technical aspects, however, it is of paramount interest to know about local settings, preferences, and the loss of skills in areas with decreasing patient numbers. the paper by palmer et al. reports on such investigations carried out in uganda. benhamou et al., through a case report on a repeatedly misdiagnosed patient, gives us an insight on future challenges for rapid diagnosis if knowledge and interest in the public health system is not maintained and broadened. another unresolved caveat of diagnosis is that a number of patients are determined to be seropositive, but thereafter hat cannot be confirmed. it will be one of the leading discussions when defining future strategies, and what to do in such cases. nkieri et al. investigated the extent of this phenomenon in the still affected regions of drc. on one hand, relapses have always been a major challenge in the treatment of hat and have made follow-up periods of up 24 months after treatment necessary [23] . on the other hand, until a few years ago, the dogma was "infected, but not treated inevitably leads to the death of the patient". reports on patients surviving for longer periods despite infection with trypanosomes emerged in the past few years [36] . one of the compartments where trypanosome may survive seems to be the skin [37, 38] . this might also explain how hat can re-emerge in so-called silent foci as illustrated by a nine-year-old child, who was diagnosed with gambiense-hat in ghana in 2013, 10 years after the last detected case [39] . in this light, the findings of mudji et al. also have importance: over ten years after treatment in the framework of clinical trials, a number of patients revisited presented continued signs and symptoms seen in hat (lymphadenopathy, severe headaches, sleep disturbances); since no trypanosomes could be identified by any means, the implication of these findings remain open at this point. in any case, the existence of such long-term cases has a sudden and dramatic impact on the view of hat epidemiology and hat elimination [39] . besides further epidemiological, parasitological, and molecular research, mathematical modelling may help to improve our epidemiological knowledge and inform about elimination strategies [40] and their related costs [41] . this field has significantly developed against all odds in the past years: trypanosomiasis with its extremely focal distribution and the many external factors influencing its transmission has been a true headache over two decades for all modellers and predictive mappers. studies of existing gambiense-hat models in a few foci (i.e., drc, guinea, and chad) suggest that some type of additional infection reservoir is needed to match the observed dynamics of reported hat cases [42, 43] . this could arise from another human reservoir (including undiagnosed and latent infections), an animal reservoir, and/or heterogeneities in human risk exposure and surveillance coverage [39] . the french colonial forces had completely dismissed the value of vector control due the successes of the treatment strategy proposed by laveran. however, vector control may play a larger role in gambiense hat elimination than anticipated. historical investigations, practical intervention studies, and modelling demonstrate the significant role that vector control can play in the control of gambiense hat. recent models suggest vector control will be essential if we are to reach the set target of elimination of the diseases as a public health problem by 2020 and beyond [44, 45] . the fact that neither modelling nor vector control are represented in this edition does not represent a valuation of these topics. this special issue comes in a very timely moment, because it is now important to secure what has been achieved, to understand missing pieces, and to finish the work. however, several challenges have to be overcome to not to end up, again, in disaster. in 2012, the world health organization, which has played an instrumental role in the control, set the goal for the elimination of human african trypanosomiasis (hat), caused by trypanosoma brucei gambiense (ghat), as a public health problem for 2020 and for the total interruption of transmission to humans for 2030. the efforts to maximise output and optimize innovation by the who has intensified since, and several stakeholders and expert groups have been created and convened [46] . since 2012, the spectacular decrease of the case number has continued: some 2,164 cases were reported in 2016, far fewer than the targeted 2016 milestone of 4000 cases, and 660 in 2018 [47, 48] . first of all, "donor fatigue" must be avoided. the elimination to "no transmission" in the drc where over 95% of the cases are nowadays occurring is a herculean task, which will not happen without considerable and continued funding. the conventional measurements of success (e.g., us$ spent per daly prevented) inevitably fail in an elimination scenario. naturally, the amount of money spent per patient identified and treated will soar, so the question to the health economists is, rather how much money do we lose in case efforts would not be continued, factoring in the needed future efforts to re-start and control the disease again. decisions on priorities will be necessary, too: whereas the total number of patients has massively decreased, the area in the drc they are coming from has not. therefore, a vast area still has to be kept under surveillance; this area has to be gradually reduced by safely "closing" focus by focus in order to not jeopardize the efforts. the elimination of hat, malaria, and guinea worm were all believed at a certain point to only be a matter of time-before new reservoirs became known (guinea worm), pestidicide, and drug resistance set in (malaria), and interest was decreased in a premature belief in success (malaria and hat). secondly, the human factor will start to play a key role: in theory, fexinidazole could be applied in 1,338 fixed health facilities (2017) an increase up by 52% from 2015 [47] , and it will be many more, should acoziborole make it to application in a few years. however, hat is a massively stigmatized disease, linked to many beliefs and bad spirits. traditionally, patients after treatment were excluded from working and sexual intercourse for six months [49] . therefore, the questions are: "will the disease be recognized by the younger physicians and nurses who have never seen a case of hat?" "is the medical staff willing to recognize a suspected case, given this will create a massive workload including trouble with the relatives of the patient and village, paperwork, an invasion of specialists for diagnosis and follow up activities?" "is the medical staff that was told for over 100 years that hat belongs into the specialized hands of the vertical programs willing to assume this task and challenge?" "are the patients willing to accept hat as a diagnosis anymore?" "can we overcome wrong dogma and information?" and "will patients falling into the respective category accept a lumbar puncture?" these questions can only be addressed through the thorough understanding of beliefs, perceptions, preferences, and decision-making processes. therefore, the social and anthropological science, as well as health economics, will start to play a key role in the "end game". the articles presented in this issue by falisse et al., and lee et al. are contributing to this area. thirdly, peace, stability, and a minimal standard of living for the people in the remote regions most affected are necessary to achieve disease elimination-this condition has not yet been met. there is little the scientific community can directly contribute to this-however, knowing that disease and poverty are inextricably linked [50] , our efforts have to continue. finally, rhodesiense hat (the east african form of the disease) was reduced to as little as 54 cases in 2016 [47] , goals have been reached and it may be seen as a quantité négligable. however, the disease with a zoonotic reservoir has the potential for spectacular returns, and this real danger still exists as described in the contribution of matovu et al. the surveillance and the knowledge of local medical staff has dwindled, innovation was absent for t. b. rhodesiense for decades, and serological instead of microscopic tests are used for diagnosis of other diseases making accidental diagnosis impossible. from january to october 2019, a total of 2-8 cases were reported per month from all treatment centres in malawi; in november 2019 to january 2020, this number surged to 25 and higher. the cases were reported from populations around the two geographically separate wildlife reserve areas, vwaza and nkhotakota; the reason for this increase is, so far, unknown (personal communication, world health organization, control of neglected tropical diseases, geneva). this outbreak causes major concern and should be a serious warning to everyone who is of the opinion that sleeping sickness has been conquered. similarly, other unexpected priorities, such as the current sars-cov-2 epidemic, may at all times derail a fragile health system. as soon as the mental and financial attention and priority is on another disease, signals of a hat resurgence may well be overlooked-we should now be well aware about the consequences and impact of late reactions and exponential transmission. compared to when i wrote the conclusion of the 2001 hat special issue in [11] , we are at a completely different point today. we celebrated several marvellous scientific successes in the meantime, tools were improved, patients numbers down-but to reach the set goals and to get completely rid of this horrible disease, the conclusion is the same again: "the goal must now be to maintain the momentum" and "even the biggest efforts of the research scientists, field workers, development agencies, and companies will fail if they are not paralleled by achievements in the political field bringing peace, stability, and a minimal standard of living to the people in the remote regions most affected". the author declares no conflict of interest. the history of african trypanosomiasis. parasites vectors the history of sleeping sickness african trypanosomiasis-centennial review the elusive trypanosome pharmacological aspects of the trypanocidal drug melarsoprol chemotherapy of protozoal infections: amebiasis, giardiasis, trichomoniasis, trypanosomiasis, leishmania and other protozoal infectious the development of drugs for treatment of sleeping sickness: a historical review epidemiology of human african trypanosomiasis control of human african trypanosomiasis: back to square one sleeping sickness-a growing problem? are there new approaches to roll back trypanosomiasis (editorial) the new landscape of neglected disease drug development commentary: sleeping sickness-a growing problem? new developments in human african trypanosomiasis effects of the ornithine decarboxylase inhibitors dl-α-difluoromethylornithine and α-monofluoromethyldehydroornithine methyl ester alone and in combination with suramin against trypanosoma brucei brucei central nervous system models treatment options for second-stage gambiense human african trypanosomiasis. expert rev. anti-infective ther nifurtimox in late-stage arsenical refractory gambiense sleeping sickness efficacy of new, concise schedule for melarsoprol in treatment of sleeping sickness caused by trypanosoma brucei gambiense: a randomised trial efficacy of 10-day melarsoprol schedule 2 years after treatment for late-stage gambiense sleeping sickness effectiveness of a 10-day melarsoprol schedule for the treatment of late-stage human african trypanosomiasis: confirmation from a multinational study investigations of the metabolites of the trypanocidal drug melarsoprol nifurtimox-eflornithine combination therapy for second-stage african trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase iii, non-inferiority trial control and surveillance of human african trypanosomiasis: report of a who expert committee; world health organisation update on field use of the available drugs for the chemotherapy of human african trypanosomiasis new who guidelines for treatment of gambiense human african trypanosomiasis including fexinidazole: substantial changes for clinical practice a 'magic bullet' for african sleeping sickness ) for the serological diagnosis of t. b. gambiense trypanosomiasis the separation of trypanosomes from blood by anion exchange chromatography: from sheila lanham's discovery 50 years ago to a gold standard for sleeping sickness diagnosis miniature anion exchange centrifugation for detection of low parasitemias: adaptation for field use sensitivity and specificity of hat sero-k-set, a rapid diagnostic test for serodiagnosis of sleeping sickness caused by trypanosoma brucei gambiense: a case-control study loop-mediated isothermal amplification for detection of african trypanosomes using detergent-enhanced lamp for african trypanosome detection in human cerebrospinal fluid and implications for disease staging immune trypanolysis test as a promising bioassay to monitor the elimination of gambiense human african trypanosomiasis diagnostic tools for human african trypanosomiasis elimination and clinical trials (ditect-hat). 2020. available online untreated human infections by trypanosoma brucei gambiense are not 100% fatal the skin is a significant but overlooked anatomical reservoir for vector-borne african trypanosomes the dermis as a delivery site of trypanosoma brucei for tsetse flies do cryptic reservoirs threaten gambienses-leeping sickness elimination? mathematical models of human african trypanosomiasis epidemiology seeing beyond 2020: an economic evaluation of contemporary and emerging strategies for elimination of trypanosoma brucei gambiense quantitative evaluation of the strategy to eliminate human african trypanosomiasis in the democratic republic of congo evaluating long-term effectiveness of sleeping sickness control measures in guinea the impact of vector migration on the effectiveness of strategies to control gambiense human african trypanosomiasis human african trypanosomiasis control: achievements and challenges all past events/information related to human african trypanosomiasis monitoring the elimination of human african trypanosomiasis: update to the flipside of eradicating a disease; human african trypanosomiasis in a woman in rural democratic republic of congo: a case report whose elimination? frontline workers' perspectives on the elimination of the human african trypanosomiasis and its anticipated consequences this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-001274-vz0qvp01 authors: chitray, m.; de beer, t. a. p.; vosloo, w.; maree, f. f. title: genetic heterogeneity in the leader and p1-coding regions of foot-and-mouth disease virus serotypes a and o in africa date: 2013-11-13 journal: arch virol doi: 10.1007/s00705-013-1838-9 sha: doc_id: 1274 cord_uid: vz0qvp01 genetic information regarding the leader (l) and complete capsid-coding (p1) region of fmd serotype a and o viruses prevalent on the african continent is lacking. here, we present the complete l-p1 sequences for eight serotype a and nine serotype o viruses recovered from fmdv outbreaks in east and west africa over the last 33 years. phylogenetic analysis of the p1 and capsid-coding regions revealed that the african isolates grouped according to serotype, and certain clusters were indicative of transboundary as well as intra-regional spread of the virus. however, similar analysis of the l region revealed random groupings of isolates from serotypes o and a. comparisons between the phylogenetic trees derived from the structural coding regions and the l region pointed to a possibility of genetic recombination. the intertypic nucleotide and amino acid variation of all the isolates in this study supported results from previous studies where the externally located 1d was the most variable whilst the internally located 1a was the most conserved, which likely reflects the selective pressures on these proteins. amino acids identified previously as important for fmdv structure and functioning were found to be highly conserved. the information gained from this study will contribute to the construction of structurally designed fmdv vaccines in africa. electronic supplementary material: the online version of this article (doi:10.1007/s00705-013-1838-9) contains supplementary material, which is available to authorized users. foot-and-mouth disease (fmd) is a highly contagious disease that affects domestic and wild cloven-hoofed animals [2, 77] . despite all the information accumulated over the years on many aspects of fmd basic biology, there is still a lack of information regarding fmd virus transmission, maintenance, virulence and host range. although fmd is referred to as a single disease [18] , the causative agent of the disease, fmd virus (fmdv), consists of seven immunologically distinct serotypes [23, 24] . the fmdv serotypes, i.e., a, o, c, asia 1 and the south african territories (sat) types 1, 2 and 3, have different global geographical distribution patterns [8-10, 18, 44, 73, 88] and are endemic in many countries. even on the african continent, the distribution of serotypes is variable, with the sat serotypes occurring in most regions of sub-saharan africa but a and o confined mostly to the central and northern parts of the region [88] . mortality is usually low, but morbidity can reach 100 % and therefore remains a major economic concern for livestock health in many developing countries and a continued threat to disease-free countries [44] . the eradication and control of fmdv in africa is complex and difficult due to the role of wildlife in virus spread and maintenance [82] and the presence of six of the seven serotypes, i.e., a, o, c, sat1, sat2 and sat3. serotype c has not been reported since 2004 [22] . fmdv is a non-enveloped virus containing a singlestranded rna genome of positive polarity in the genus aphthovirus of the family picornaviridae [1, 2, 27] . the large open reading frame (orf) of *6,996 nt, which differs in length between the different serotypes [20] , encodes a single polypeptide, which is co-and posttranslationally cleaved by viral proteases to give rise to the structural and non-structural proteins [3, 13, 55, 67] . ten of the 13 cleavage events are catalysed by the virally encoded 3c protease [15, 58, 67, 78] . translation takes place from a single open reading frame by a cap-independent mechanism at the internal ribosome entry site (ires) [49] , located in the 5' untranslated region (utr). there are two different sites on the rna at which the initiation of protein synthesis occurs, resulting in the generation of two forms of l proteinase (l pro ), lb and the less abundant lab, where lb is the truncated version, which arises after the initiation of translation at the second aug start codon [13] . lab and lb can cleave the l/p1 junction and ensure the proteolytic degradation of the cellular cap-binding protein complex (eif4g), which results in the shutoff of host translation [22] . the p1 region is the viral capsid precursor and consists of the proteins 1a (vp4), 1b (vp2), 1c (vp3) and 1d (vp1). the antigenicity of the viral particles is dependent on the amino acid (aa) residues that are exposed on the surface of the capsid [56, 85] . furthermore, it has been shown that the external capsid proteins play a role in binding to the fmdv cell-surface receptors, i.e., the rgddependant integrins [14, 25, 37-39, 59, 60] and heparan sulphate proteoglycans (hspgs) [4, 36, 68] . the genetic heterogeneity of the virus, which is due to the lack of a proofreading mechanism during virus replication, has resulted in the occurrence of extensive variability as well as different lineages and antigenic variants within a serotype that have established themselves in different geographical regions [reviewed in refs. 8-10, 44, 70, 71, 75, 76, 88] . this has resulted in the need for multiple vaccine strains required for each serotype to cover the antigenic diversity when using vaccination as a control option [26] . in africa and countries bordering europe, the disease is mainly controlled using vaccination and restriction of animal movement. thus, it is imperative to obtain as much information as possible regarding the fmdv prevalent on the african continent to further our knowledge on fmd epidemiology, define genetic relationships of viruses causing outbreaks [45, 47] and to enable better control strategies by successful vaccine development. genetic information regarding the leader (l) and complete capsid-coding (p1) region of serotype a and o viruses prevalent on the african continent is lacking, although the sat isolates have been broadly studied in the past [8] [9] [10] 86] . for this study, the l and p1 coding regions for eight fmdv a and nine fmdv o viruses isolated between 1975 and 2003 were successfully sequenced and analysed using phylogenetic analysis, examination of sequence variability, and identification of highly conserved genomic regions relating to previously identified fmdv functional and structural biological capabilities. non-conservative substitutions were mapped to the available o (o1bfs) [53] and a (a 10 /hol/61) [29] capsid structures, and amino acid substitutions that may be involved in antigenic divergence were identified. the sub-saharan african isolates included in this study belong to different topotypes of fmdv serotypes a and o as defined by 1d sequencing and represent a broad geographical distribution of viruses within east and west africa. the nine fmdv serotype o isolates and eight serotype a isolates were obtained from the institute for animal health, pirbright laboratory, pirbright, united kingdom (table 1) . for the purpose of analysis, a select few complete l and p1 fmdv sequences currently available in genbank were included (table 1) . the fmdv type o viruses were passaged for a previous study and were used directly in this study for processing, whereas the fmdv a isolates were first propagated on ib-rs-2 cells (instituto biologico renal suino cell line, a pig kidney cell line) to obtain a high viral titer. the ib-rs-2 cells were maintained in rpmi medium (sigma) supplemented with 10 % foetal calf serum (fcs; delta bioproducts) and 1x antibiotic-antimycotic (1009, gibcoò), invitrogen). virus was added to prepared cells containing rpmi supplemented with 1 % (v/v) fcs and 19 antibiotic-antimycotic mixture and incubated at 37°c until complete cpe was attained (after 48 h). clarified cell culture supernatant containing virus was stored at -80°c until further use. chinese hamster ovary (cho) cells strain k1 (atcc ccl-61) were maintained in ham's f-12 medium (invitrogen) supplemented with 10 % fcs. plaque assays were performed by infecting monolayer cells with the virus for 1 h, followed by the addition of a 2-ml tragacanth overlay [66] and staining with 1 % (w/v) methylene blue [54] . total viral rna was extracted using a modified guanidinium thiocyanate (guscn)-silica method [17] . the viral rna template was reverse transcribed at 42°c for 1 h using 10 u of amv reverse transcriptase (promega) and the antisense p1 primer (wda; 5'-gaagggcccaggg ttggactc-3') [12] as described previously [7] . amplification of the l-p1 region was undertaken using the antisense p1 (wda) primer and the sense ncr1 primer (5'-taccaagcgacactcgggatct-3') followed by pcr reactions using long-template taq dna polymerase (roche) and thermal cycling conditions described by van rensburg et al. [86] . pcr products of ca. 2,820 bp were excised from a 1 % agarose gel and purified using a nucleospin ò extract kit (macherey-nagel). purified pcr products were sequenced using a genome-walking approach with genome-specific oligonucleotides and an abi prism tm bigdyeò terminator cycle ready reaction kit v3.1 (applied biosystems). sequences were analysed using an abi prism 3100 genetic analyser (applied biosystems). ambiguous nucleotides (nt) of the l-p1 sequences were resolved manually and assembled into a contig using the sequencher tm 4.7 dna sequence analysis software (gene codes corporation, ann arbor, mi, usa). a consensus sequence representing the most probable nt for each position of the sequence was obtained for each isolate. consensus sequences were translated in bioedit 5.0.9 dna sequence analysis software [32] , and the complete l-p1 nt and aa sequences were aligned using clustalx 1.8.1 [83] . hypervariable regions in the complete aa alignment were defined as a linear 10-aa region containing more than 50 % variable residues. the phylogenetic analysis included the newly determined sequences as well as sequences of non-african serotype a and o isolates obtained from genbank (table 1) . maximum-likelihood analysis of the aligned sequences was carried out in paup [79] under the aikake information criterion. phylogenetic trees were constructed using the neighbour-joining (nj), minimum-evolution (me) and maximum-parsimony (mp) methods included in the mega 4.0 program [50] for the l, 1a, 1b, 1c, 1d-coding regions separately as well as the full p1-coding region. node reliability was estimated by 1000 bootstrap replications for nj, me and mp trees, whilst the nucleotide substitution model of kimura 2-parameter was employed for the nj and me trees and close-neighbour-interchange (cni) with search level 1 in effect for the mp and me trees. mega 4.0 [50] was utilised to determine the nt and aa variation. plots representing the aa variation, hydrophobicity and secondary structures for each protein were drawn using python (http://python.org) and the matplotlib package (http:// matplotlib.sourceforge.net). the number of different amino acids occurring at a specific position was used as a measure of variation, and the hydrophobicity scale of kyte and doolittle [51] was used to measure relative aa hydrophobicity. the crystallographic protomers of the capsid proteins of o 1 bfs (1fod) [53] and a 10 /hol/61 ((1zbe) [29] were visualized and the surface-exposed residues identified with pymol v1.1rc2pre (delano scientific llc). phylogenetic trees based on the p1 (fig. 1) (fig. 1) , the latter belonging to the middle east-south asia (me-sa) topotype based on 1d phylogeny [44] . the exception is o/sar/ 19/2000, which was isolated in south africa in 2000 during an outbreak caused by illegal feeding of swill to pigs [74] . (fig. 1) . furthermore, these p1 groupings were also observed when me and mp phylogenetic models were utilised (not shown). clustering similar to that of the p1 region was observed for the separate gene regions, but with low bootstrap support except for 1b ( (fig. 1) . the nt sequence differences in the p1-coding region between members of each topotype were typically more than 15 %, similar to the cutoff defined for a topotype [47] . globally, fmdv serotype a exists in three geographically distinct topotypes, asia, africa and europe-south america (euro-sa), based on the genetic relationships of 1d sequences [44] . using the sequence information of the african a isolates together with p1 sequences of serotype a viruses available in the genbank database, at least two separate clusters were observed for the type a viruses, i.e., non-african and african a isolates, supported by 100 % bootstrap values for all phylogenetic methods used for the p1 (fig. 1) , 1b, 1c and 1d gene regions (supplementary data, s1-s3). two east african isolates, a/tan/4/80 and a/som/1/78 formed a well-supported subgroup for the p1 (fig. 1 ) and 1d nj trees (supplementary data, s3). in addition, there was a consistently strong grouping for three west african isolates, a/nig/4/79, a/civ/4/95 and a/sen/1/97, in the p1 (fig. 1) , 1b, 1c and 1d nj analyses (supplementary data, s1-s3). the serotype a non-african and african viruses displayed similar genetic variability when compared to serotype o. the intratypic nt sequence variation in an alignment of the 2222-nt p1-coding region for type a was calculated to be 40.4 %, whilst the corresponding region (2202-2205 nt) of type o only revealed 38.5 % variable nucleotides. analysis of the 1a gene region resulted in phylogenetic groupings that differed from those of the p1, 1b, 1c and 1d analyses. when performing phylogenetic analysis on the combined o and a dataset, the fmdv a and o isolates did not group strictly according to serotype (supplementary data, s4). for example, three non-african fmdv a strains, isolated from brazil and venezuela (a17/agua-rulbos/iso83, a18/zulia/iso48 and a13/brazil/ iso75), grouped with o viruses from the me-sa, sea and ea topotypes, but with low bootstrap support. as expected, the region encoding 1a was the most conserved, exhibiting 37.9 % variant nucleotides and was the only capsid-coding region with the highest average %ts/tv rate of 1.0 % ( table 2 ). in contrast, 1d had the highest variability of 58.7 % and lowest average %ts/tv rate of 0.28 % ( table 2) . the phylogenetic trees based on the l pro -coding region for the combined serotype o and a dataset had similar tree topologies for the a and o isolates, independent of the phylogenetic methods employed. the nj tree of the l procoding region (fig. 2) showed that the viruses did not group strictly according to serotype, in contrast to those areas of fmdv hydrophobicity and aa variation are represented by blue and green lines, respectively. regions of variability or hypervariable sites were defined as sites on the p1 that had five or more variable aa residues within a window of 10 residues based on the structural proteins. the non-african a and o isolates that form a part of the euro-sa lineage [44] formed separate subgroupings in the l pro -coding sequence nj tree (fig. 2) . the pan-asian isolates formed a separate grouping with high bootstrap support (100 % the l pro aa sequence displayed significant variation for a functional protein: 46.6 % for the serotype a alignment and 29.6 % for the serotype o isolates (table 2) . at least 30.3 % (224 of 739 aa) of the aa residues were variable in the alignment of the structural proteins (translated from the p1 region) of the 26 serotype a isolates, whilst the (table 2) . a systematic analysis of the capsid proteins revealed the variation not to be random but focused in local regions of hypervariability. the most variable capsid region, 1d, displayed the most regions of hypervariability. figure 3a shows the hypervariable regions of type o at aa positions 34-60, 76-87, 135-147, 152-160, 196-213 . at least seven discrete hypervariable regions (21-63, 80-87, 97-104, 135-146, 150-163, 167-176, 193-207) were identified in 1d of type a (fig. 3b) . the conserved n-terminal motif of 1b, dkkteettl-ledril-ttrnghttsttqssvg, described by carrillo et al. [20] , was present in the african a and o sequences (results not shown). two hypervariable sites, residues 72-85 within the bb-bc loop and 131-141 in the be-bf loop, were mapped within 1b of type o (fig. 3c) . 1b of type a displayed the same two hypervariable regions, residues 61-92 and 129-139, and a third hypervariable region, 188-198 (bh-bi loop; fig. 3d ). most of the 1c aa substitutions for type o were concentrated in one hypervariable region, i.e. 68-80. a second region with significant variability worth mentioning was residues 175-181, where three residue positions displayed high entropy and were located within a surface-exposed loop of 1c (fig. 3e) . the latter was situated in the b-b 'knob' of 1c and included the epitope site 4 for serotype o [43] . at least three hypervariable regions were identified in the type a alignment, i.e. residues 58-72, 132-142 and 197-211 (fig. 3f) . the 1a protein of serotype o was most conserved, with only four variable residues and hypervariable regions that were not common for 1a (not shown). the amino acids that have previously been identified as critical for fmdv were compared to the complete aa sequence alignment of the african and non-african a and o isolates from this study and are summarized in supplementary data s5, showing that the aa residues important for fmdv function are conserved. [68] and fry et al. [28] confirmed the importance of the r56 residue of 1c for hs binding and cell culture adaptation. fmdv plaque assays in cho-k1 cells (table 3) confirmed that o/ken/10/95 was the only virus that was able to infect and replicate in this cell line. taking all of the serotype o capsid-sequence data together, 25 of the 27 o isolates had a his residue at position 56 of 1c, and they might therefore require integrins to replicate in cell culture. amino acid sequence variation in relation to structure vaccines based on a22/iraq/64, a/eri/98 and o1manisa are recommended for the control of fmd in africa [33] . we examined the variation within the deduced amino acid sequences of the capsid proteins of the african o and a isolates and compared the surface-exposed regions with those of the three recommended vaccine strains. regions with high aa variability in an alignment of the capsid proteins were mapped onto the x-ray crystallographic structures of type a (a 10 /hol/61; 1qqp) [29] and o (o 1 bfs; 1fod) [53] viruses. figure 4 shows that the regions of variability were mostly located on surfaceexposed regions of the virion. not all of the aa side chains within a variable region were exposed on the surface. closer inspection of each aa position within a region of hypervariability indicated that positions with high variability had side chains exposed to the microenvironment of the virion. for serotype a viruses, most of the hypervariable regions outside the 1d bg-bh loop were concentrated around the 5-fold and 3-fold axes of the virion and the c-terminus of 1d (fig. 4 ) and correlated to a large extent with residues previously found to be involved in escape from neutralization by monoclonal antibodies (table 4) . furthermore, many of the putative epitopes were probably discontinuous. for example, there was close proximity of 1b residue 2191 and 1c residues 3068-3071 and 3197-3198 around the 3-fold pore of the virion (fig. 4) . similarly the regions of variability for type o correlated strongly with epitopes previously identified with distribution around the 5-fold and 3-fold axes of the virion ( fig. 4 ; table 4 ). the data from the analysis of the complete capsid-coding region, p1, as well as the individual capsid-coding regions indicated that very similar tree topologies existed for the different genomic regions when comparing the african a and o viruses with those from other regions of the world. in general, analysis of the entire structural protein-coding region improved bootstrap values relative to 1d analysis alone. the longer the capsid-coding region included in the analysis, the more accurate the relationship conclusion. this supports the view that sequencing of the entire capsidcoding region, rather than 1d alone, is desirable in molecular evolution studies. phylogeny based on the nj trees of the p1, 1b, 1c and 1d sequences resulted in the grouping of viruses according to serotype. in addition, the a and o virus clusters could be further divided into separate groupings of the african and non-african a and o isolates, which were observed for the p1, 1b, 1c and 1d nj, me and mp trees. the separate groupings of the african and non-african a viruses support previous findings for type a viruses. these could be grouped into three major restricted genotypes, i.e., euro-south america, asia and africa, based on 1d phylogeny (this study only included fmdv a viruses from euro-south america and africa) [44, 46, 57] . similarly, based on 1d phylogeny, type o viruses were divided into three groups: those originating from asia, europe-south america and the far east [44, 69, 73, 74] . the p1 phylogeny therefore supports the three major virus groups within serotype o. the eastern and western african o viruses were grouped together with the sea and me-sa lineages, together with the pan-asia strain [44, 45, 73] , albeit as lineages restricted to geographic regions (east africa-1, 2, 3, 4 and west africa). furthermore, the phylogeny is indicative of the transboundary spread of fmdv in africa among the east african countries, uganda, kenya, somalia and tanzania, that are in close proximity to each other, which is also true for the west african countries, i.e. nigeria, ivory coast and senegal. the groupings also indicated that the east african and west african viruses fall into separate large groups. another well-supported grouping was observed for the p1, 1b and 1c trees (all methodologies) for o/uga/1/75, o/uga/6/ 76 and o/uga/17/98, with a maximum of 15.1 % nt and 6.5 % aa substitutions in any pairwise alignment. this grouping most likely signifies that the 1998 outbreak strains re-emerged from older strains that have been maintained in the endemic area since the early 1970s, i.e. from 1975 to 1998 (23 years). there was a difference in the groupings for the 1a trees when compared to the p1 and other capsid-coding gene regions where three non-african a isolates clustered with the non-african o viruses (for all phylogenetic methodologies). the phylogenetic tree representing the region encoding the l protein differed from that of the structural proteins where sub-grouping according to serotype was much less apparent, which was consistent with previous findings for this region [81, 86] . interestingly, certain a and o african viruses clustered together and also did not separate into geographical regions such as east and west africa as observed for the structural coding regions. for example, bootstrap support of 73 % for the l-region nj tree was observed for the grouping of o/uga/17/98, o/uga/1/75, o/uga/6/76 & a/eth/7/92, which was not observed with the 1a, 1b, 1c, 1d and p1 phylogenetic analysis. this suggests that the african viruses share similarities or are closely related when comparing the l sequences, irrespective of serotype. taking into account the extensive, uncontrolled movement of animals across the borders and the ease of virus spread and infection of multiple serotypes in one animal, the role of recombination events in the genetic diversification of fmdv cannot be excluded. although we did not perform a study on the occurrence of recombination, the similarities present between fmdv a and o l sequences could be due to the occurrence of intertypic recombination events [30, [40] [41] [42] 86] . due to the high mutation rates of fmdv, it is likely that even brief epidemics might result in the generation of substantial antigenic variability [35] . however, the adaptive significance of this variation remains unclear [34] . the antigenicity of fmdv is attributed to the aa residues that are exposed on the surface of the capsid [56] . an important immunogenic determinant, the 1d g-h loop [3] , exhibited a high degree of variation for the a and o isolates included in this study. consequently, aa changes in this region are most likely involved in the appearance of novel antigenic types. analyses of antigenic sites of picornaviruses have been carried out using neutralising monoclonal antibodies (mabs) to select and screen mab-resistant mutants. sequence analysis of these mutants resulted in the identification of five antigenic sites of serotype o virus, i.e., o 1 kaufbeuren [21, 44] , and six sites for the fmdv a viruses [44] . alignments of the aa sequences of the african a and o viruses indicated that the regions of variability identified corresponded to the known antigenic sites, which points to the fact that the location of antigenic sites are structurally conserved for the african a and o viruses. in addition to these sites, other regions of variability were identified for both the fmdv o and a african isolates from the aa variability plots. these regions could potentially be antigenic determinants, which may be difficult to map by the classical methodology of mab-resistant escape mutants. we have recently shown that an approach combining sequence variation with structural data and antigenic variation results in the reasonably accurate identification of novel antigenic determinants on the virion surface [65] . the aligned l pro aa sequences displayed marked variation in both the lab and lb regions (not shown); however, despite this variation, the aa residues identified as being critical for the l pro function were highly conserved, i.e., the residues c53, h153 and d168 required for l pro catalytic activity, the e81 residue required for l pro autocatalysis, and two his residues (h114 and h143) important for cleavage of the translation initiation factor, eif4g, [31, 48, 62, 63] . a comparison of the l/p1 cleavage sequence at the c-terminus of the l protein and n-terminus of the 1a protein of the fmdv non-african a types revealed a sequence of r(q/w)klk*gagq (* indicates cleaved peptide bond), whereas the african a types included in this study had the sequence k(r)r(k)lk*gagq (results not shown). both the fmdv non-african and african o types revealed a sequence of (k/r)(k/r)l(k/r)*gagq (* indicates cleaved peptide bond) (results not shown). these observations compared well with the l pro /1a junction previously described for serotypes a, o and c [76] , where the residues k(r)r(k)lk(r) at the l pro c terminus and the gagq at the 1a n terminus were observed. these results suggest that for all the a and o types included in this study, the conserved sequence xxlk(r)*gagq (where x is either k or r) is sufficient for l/p1 cleavage by l pro . the degree of hydrophobicity/hydrophilicity of the loops connecting the b chains varied between the african a and o surface proteins. hydrophilic b-b loops tend to be exposed on the protein surface, sometimes protruding from the protein core, and are candidates for antibody binding [87] . overall, the aa sequence variation observed for the fmdv a and o viruses included in this study showed that the a viruses exhibited more variation, possibly indicating that the a viruses evolved rapidly, which supports studies by bachrach [2] and brooksby [19] . additionally, tully and fares [84] showed that among all of the fmdv serotypes, serotype a is the most divergent and that adaptive evolution has occurred in the 3c protease (involved in rna replication and processing of the polyprotein) and 2b (involved in membrane rearrangements), which supports the hypothesis of selection for faster replication in serotype a. neff et al. [59] showed that a variant of the type o1 virus containing an arg at residue 56 of 1c required only hs binding to replicate in cho-k1 cells but that another variant with a his residue at this position required integrins to replicate in cell culture. interestingly, in this study, it was shown that o/ken/10/95 was the only african virus to have this arg residue at residue 56 of 1c, and it was indeed able to replicate in cho-k1 cells. however this virus has been passaged three times on ib-rs-2 cells, and it is possible that the mutation arose during cell culture passage. additionally, various aa residues that were previously identified as important for playing a role in various functions for fmdv were found to be conserved for the a and o isolates (see ''results''). it is clear from the outbreaks of fmd during the last two decades that there is a continuing threat to the livestock industry. the results presented here show distinct geographical grouping of serotype a and o viruses in africa, although common ancestry with the euro-south american-asian topotypes is clear. the natural diversification of fmdv occurs during replication in infected animals and results in the rapid generation of mutants and the ability to persist and to spread amongst livestock. thus, continuous surveillance and an active molecular epidemiology program increases our knowledge with regard to fmdv phylogenetic relationships, virus antigenicity, and the ability of existing vaccine strains to provide protection against emerging and re-emerging viruses. the pathogenesis and diagnosis of foot-and-mouth disease foot-and-mouth disease robertson bh (1982) foot-and-mouth disease virus: immunogenicity and structure of fragments derived from capsid protein vp and of virus containing cleaved vp multiple virulence determinants of foot-andmouth disease virus in cell culture neutralization epitopes of type o foot-and-mouth disease virus. i. identification and characterization of three functionally independent, conformational sites monoclonal antibodies, against o 00 serotype foot-and-mouth disease virus, from a natural bovine host, recognize similar antigenic features to those defined by the mouse detection and characterisation of foot-andmouth disease in sub-saharan africa genetic heterogeneity of sat-1 type foot-and-mouth disease viruses in southern africa the implications of virus diversity within the sat 2 serotype for control of foot-and-mouth disease in sub-saharan africa molecular epidemiology of sat3-type footand-mouth disease analysis of neutralising antigenic sites on the surface of type a12 foot-and-mouth disease virus subtyping of european foot-andmouth disease virus strains by nucleotide sequence determination distinctive features of foot-and-mouth disease virus, a member of the picornavirus family; aspects of virus protein synthesis, protein processing and structure antibodies to the vitronectin receptor (integrin avb3) inhibit binding and the infection of foot-and-mouth disease virus to cultured cells crystal structure of foot-and-mouth disease virus 3c protease: new insights into catalytic mechanism and cleavage specificity epitope mapping of foot-and-mouth disease virus with neutralizing monoclonal antibodies rapid and simple method for purification of nucleic acids comparison of two 3abc enzyme-linked immunosorbent assays for diagnosis of multipleserotype foot-and-mouth disease in a cattle population in an area of endemicity portraits of viruses: foot-and-mouth disease virus comparative genomics of foot and mouth disease virus identification of a fifth neutralisable site on type o foot-and-mouth disease virus following characterization of single and quintuple monoclonal antibody escape mutants combining livestock trade patterns with phylogenetics to help understand the spread of foot-and-mouth disease in sub-saharan africa, the middle east and southeast asia the quasispecies (extremely heterogeneous) nature of viral rna genome populations: biological relevance-a review footand-mouth disease virus foot-and-mouth disease virus receptors: comparison of bovine av integrin utilization by type a & o viruses report of the session of the research group of the standing technical committee of the european commission for the control of foot-and-mouth disease (eufmd) held at chania nucleotide sequence and genome organization of foot-and-mouth disease virus the structure and function of a foot and mouth disease virus-oligosaccharide receptor complex the structure of foot-andmouth disease virus. foot-and-mouth disease virus isolation and characterization of recombinants between attenuated and virulent aphthovirus strains putative papainrelated thiol proteases of positive strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alphaand coronaviruses bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt oie/fao fmd reference laboratory network annual report characterising sequence variation in the vp1 capsid proteins of foot-and-mouth disease virus (serotype o) with the respect to virion structure evidence for positive selection in foot-and-mouth disease virus capsid genes from field isolates efficient infection of cells in culture by type o foot-and-mouth disease virus requires binding to cell surface heparan sulfate the epithelial integrin avb6 is a receptor for foot-andmouth disease virus integrin avb1 is a receptor for foot-and-mouth disease virus integrin avb8 functions as a receptor of foot-and-mouth disease virus: role of the b-chain cytodomain in integrin-mediated infection mosaic structure of foot-andmouth disease virus genomes multiple sites of recombination within the rna genome of foot-and-mouth disease virus sequence analysis of monoclonal antibody resistant mutants of type o foot and mouth disease virus: evidence for the involvement of the three surface exposed capsid proteins in four antigenic sites molecular epidemiology of footand-mouth disease virus pandemic strain of foot-and-mouth disease virus serotype o foot-and-mouth disease virus serotype a in egypt recent spread of a new strain (a-iran-05) of foot-and-mouth disease virus type a in the middle east foot-and-mouth disease virus leader proteinase: a papain-like enzyme requiring and acidic environment in the active site functional analysis of the internal translation initiation site of foot-and-mouth disease virus mega: molecular evolutionary genetics analysis version 3.0. pennsylvania state university a simple method for displaying the hydropathic character of a protein the complete genome sequence of foot-and-mouth disease virus o/akesu/58 strain and its some molecular characteristics structure of a major immunogenic site on footand-mouth disease virus mapping of amino acid residues responsible for adhesion of cell culture-adapted foot-and-mouth disease sat type viruses comparisons of the complete genomes of asian, african and european isolates of a recent foot-and-mouth disease virus type o pandemic strain (panasia) direct evaluation of the immunodominance of a major antigenic site of foot-and-mouth disease virus in a natural host genetic and antigenic analysis of type a foot-and-mouth disease viruses isolated in india during 1987-1996 role of rna structure and rna binding activity of foot-and-mouth disease virus 3c protein in vpg uridylylation and virus replication foot-and-mouth disease virus virulent for cattle utilizes the integrin avb3 as its receptor high efficiency utilization of the bovine integrin avb3 as a receptor for foot and mouth disease virus is dependant on the bovine b3 subunit the nucleotide sequence of foot-and-mouth disease virus o/fra/1/2001 and comparison with its british parental strain o/ukg/35/2001 the foot-and-mouth disease virus leader proteinase gene is not required for viral replication identification of the active-site residues of the l-proteinase of foot-and-mouth disease virus evidence of genetic recombination in footand-mouth disease virus sequence-based prediction for vaccine strain selection and identification of antigenic variability in foot-and-mouth disease virus genetically engineered foot-and-mouth disease viruses with poly(c) tracts of two nucleotides are virulent in mice picornaviridae: the viruses and their replication tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle molecular epidemiology of serotype o foot-and-mouth disease virus isolated from cattle in ethiopia between 1979-2001 comparison of sat-1 foot-and-mouth disease virus isolates obtained from east africa between 1971 and 2000 with viruses from the rest of sub-saharan africa study of the genetic heterogeneity of sat-2 foot-and-mouth disease virus in sub-saharan africa with specific focus on east africa. onderstepoort identification of neutralising antigenic sites on vp1 and vp2 of type a5 foot-and-mouth disease virus, defined by neutralisationresistant variants foot-and-mouth disease type o viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes) molecular epidemiology of serotype o footand-mouth disease virus with emphasis on west and south africa retrospective genetic analysis of sat-1 type foot-and-mouth disease outbreaks in west africa (1975-1981) the structures of picornaviral proteinases control and eradication of foot-and-mouth disease structural and mutagenic analysis of foot-andmouth disease virus 3c protease reveals the role of the b-ribbon in proteolysis paup: phylogenetic analysis using parsimony (and other methods) antigenic sites on foot-and-mouth disease virus type a10 molecular phylogeny of leader proteinase gene of type a of foot-and-mouth disease virus from india infectious diseases of livestock with special reference to southern africa the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools shifts in the selection-drift balance drive the evolution and epidemiology of foot-and-mouth disease virus lymphocyte recognition elements on the vp1 protein of theiler's virus genetic heterogeneity in the foot-and-mouth disease virus leader and 3c proteinase a similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation review of the status and control of foot and mouth disease in sub-saharan africa sequencing and analysis for the full-length genome rna of foot-and-mouth disease virus china/99 genetic heterogeneity of fmdv african types a and o 961 acknowledgments this work was supported by the sa-uk collaboration initiative via the department of science and technology. the authors would like to express their gratitude to the personnel at the arc-ovi (tadp) for their contributions to virus isolation. we would like to thank dr. b. blignaut for assistance with plaque titrations. we also gratefully acknowledge dr. o. koekemoer, dr. m. van kleef and dr. n. singanallur for critical reading of the manuscript.open access this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. key: cord-352670-21r0cwsc authors: muyingo, rajab idd; mpoza, abdulhamid; kasadha, juma title: coronavirus in the era of digital connectivity: opportunities and challenges date: 2020-08-02 journal: j public aff doi: 10.1002/pa.2246 sha: doc_id: 352670 cord_uid: 21r0cwsc in this article, we examine the opportunities and challenges digital connectedness creates in the fight against epidemics, in particular the coronavirus (covid‐19). we deduce that digital connectedness of individuals and organizations eased sharing of information on the causes and measures aimed at controlling and avoid the rapid spread of the epidemic in developing economies of africa. we conclude that digital connectedness enabled african countries to access; share and implement globally suggested mechanisms aimed at controlling the spread of the covid‐19 epidemic. future, studies should examine how african economies use digital connectedness to increase individual and organizational participation in curbing epidemics and also boost health management. in this article, we examine the opportunities and challenges digital connectedness creates in the fight against epidemics, in particular the coronavirus . we deduce that digital connectedness of individuals and organizations eased sharing of information on the causes and measures aimed at controlling and avoid the rapid spread of the epidemic in developing economies of africa. we conclude that digital connectedness enabled african countries to access; share and implement globally suggested mechanisms aimed at controlling the spread of the covid-19 epidemic. future, studies should examine how african economies use digital connectedness to increase individual and organizational participation in curbing epidemics and also boost health management. organization officially named the coronavirus as covid-19 (aljazeera, 2020) . covid-19 is currently estimated to have registered over 50,000 cases in over 24 countries and resulted in over 1,381 deaths (ica, 2020). in developing economies (kasadha, 2018 (kasadha, , 2020 , digital connectivity has increased community mobilization in response to their which has not yet been reported in africa, the continent has often engaged its communities in the fight against other epidemics such as swine fever and ebola viruses. for instance, the "african swine fever (asf) a viral hemorrhagic disease, caused by a large double-stranded dna virus with an icosahedral symmetry, many african nations reacted to it during its infancy level. since its first description in kenya, the disease was reported in various countries around the world such as china, but remaining endemic in sardinia, east africa, and southern africa, where it represents a major threat for development of the pig industry" (kalenzi, ochwo, afayoa, norbert, kokas, arinaitwe, et al., 2013; montgomery, 1921; mwiine, nkamwesiga, ndekezi, & sylvester, 2019; rowlands, michaud, heath, hutchings, oura, vosloo, et al., 2008; sánchez-cordón, montoya, reis, & dixon, 2018) . we further, connote that digital connectivity has enabled china to meet its social responsibility (bowd, bowd, & harris, 2006) coronavirus: australian scientists first to recreate virus outside china the logic of connective action bill & melinda gates foundation commits $10 million to global response to communicating corporate social responsibility: an exploratory case study of a major uk retail centre chinese officials race to contain anger over virus. the new york times as coronavirus explodes in china, countries struggle to control its spread. the new york times ica 2020 conference and coronavirus provisions. international communications association epidemiological overview of african swine fever in uganda information technology to support digitally networked action in developing economies. a case of nyanzi's #pads4girlsug campaign. electronic journal of information systems in developing countries does social media matter in developing democracies? examining its impact on citizens political participation and expression in uganda social media taxation and its impact on africa's economic growth how ai could combat the spread of china's deadly coronavirus. the telegraph on a form of swine fever occurring in british east africa (kenya colony) molecular characterization of african swine fever viruses from outbreaks in peri-urban kampala, uganda the virus wars. the atlantic african swine fever virus isolate, georgia african swine fever: a re-emerging viral disease threatening the global pig industry infection prevention and control during health care when novel coronavirus (ncov) infection is suspected. world health organization key: cord-309327-eham6trt authors: lor, aun; thomas, james c.; barrett, drue h.; ortmann, leonard w.; herrera guibert, dionisio j. title: key ethical issues discussed at cdc-sponsored international, regional meetings to explore cultural perspectives and contexts on pandemic influenza preparedness and response date: 2016-05-17 journal: int j health policy manag doi: 10.15171/ijhpm.2016.55 sha: doc_id: 309327 cord_uid: eham6trt background: recognizing the importance of having a broad exploration of how cultural perspectives may shape thinking about ethical considerations, the centers for disease control and prevention (cdc) funded four regional meetings in africa, asia, latin america, and the eastern mediterranean to explore these perspectives relevant to pandemic influenza preparedness and response. the meetings were attended by 168 health professionals, scientists, academics, ethicists, religious leaders, and other community members representing 40 countries in these regions. methods: we reviewed the meeting reports, notes and stories and mapped outcomes to the key ethical challenges for pandemic influenza response described in the world health organization’s (who’s) guidance, ethical considerations in developing a public health response to pandemic influenza: transparency and public engagement, allocation of resources, social distancing, obligations to and of healthcare workers, and international collaboration. results: the important role of transparency and public engagement were widely accepted among participants. however, there was general agreement that no "one size fits all" approach to allocating resources can address the variety of economic, cultural and other contextual factors that must be taken into account. the importance of social distancing as a tool to limit disease transmission was also recognized, but the difficulties associated with this measure were acknowledged. there was agreement that healthcare workers often have competing obligations and that government has a responsibility to assist healthcare workers in doing their job by providing appropriate training and equipment. finally, there was agreement about the importance of international collaboration for combating global health threats. conclusion: although some cultural differences in the values that frame pandemic preparedness and response efforts were observed, participants generally agreed on the key ethical principles discussed in the who’s guidance. most significantly the input gathered from these regional meetings pointed to the important role that procedural ethics can play in bringing people and countries together to respond to the shared health threat posed by a pandemic influenza despite the existence of cultural differences. before the 2014 ebola outbreak in west africa captured the world's attention, one of the most feared yet widely anticipated events in public health was a pandemic of highly pathogenic influenza. in the 20th century, there were three notable influenza pandemics -the "spanish flu" (h1n1 virus) in 1918 and 1919, which resulted in approximately 50 million deaths worldwide 1 ; the "asian flu" (h2n2 virus) in [1957] [1958] , which resulted in 1-2 million deaths worldwide 2, 3 ; and the "hong kong flu" (h3n2 virus) in 1968, which resulted in 1 million deaths worldwide. 4, 5 in the late 1990s and in early 2000s, concern focused on the spread of avian influenza virus h5n1 to humans. the first cases of human infection with h5n1 were reported in 1997 in hong kong (18 cases of which 6 were fatal). [6] [7] [8] [9] fears about h5n1 were heightened in 2003 when the virus was found to be responsible for serious disease and death in humans. nearly 650 human cases of h5n1 have been reported from 15 countries since 2003 through october 2015. 4 other outbreaks led to questions about preparedness. the outbreak of severe acute respiratory syndrome (sars) in 2003 contributed to growing concern about the world's ability to prepare for and respond to a worldwide epidemic. sars, first reported in asia in february 2003, is a viral respiratory illness caused by a coronavirus. the illness spread rapidly to more than two dozen countries in north america, south america, europe, and asia before the sars global outbreak was contained in 2004. 10 the reemergence of the h1n1 virus during the 2009-2010 influenza season, 11 the emergence of the middle east respiratory syndrome coronavirus (mers-cov) in 2012, 12 and the cases of severe illness in humans from a new avian influenza a (h7n9) virus in 2013 13 heightened concerns about the need to be prepared for pandemics. an influenza pandemic results in a sudden surge of people with acute health needs, placing extra burden on health resources already overstretched in many places. the severity and suddenness of these burdens can create ethical tensions along a number of fronts. one such tension to which policymakers already have given considerable attention is the ethical allocation of scarce supplies of antivirals, vaccines, respirators, and personal protective equipment. [14] [15] [16] [17] healthcare workers will encounter challenging ethical dilemmas involving their professional duties to patients and their strong competing obligation to protect and care for themselves and their family. the employers of these workers will have obligations to minimize risks to their employees, while countries will have obligations relating to international collaboration that can compete with domestic priorities. at the request of member states, the world health organization (who) convened an international group in 2006 to identify common ethical concerns in preparing for and responding to a pandemic influenza and to provide preliminary guidance on how to address these issues. this resulted in the 2007 release of ethical considerations in developing a public health response to pandemic influenza. 18 in addition to discussing general ethical considerations (eg, balancing rights, interests and values, transparency and community engagement), the who ethics guidance discussed issues relating to priority setting and equitable access to therapeutic and prophylactic measures; use of isolation, quarantine, border control and social distancing measures; and the role and obligations of and to healthcare workers. an overriding theme for the who guidance was the need for international cooperation and the importance of taking into account the contextual and cultural considerations of particular countries or regions. the who document notes that "ethical considerations will be shaped by the local context and cultural values. " 18 cdc also developed ethical guidance relating to pandemic influenza. 19, 20 this guidance focused on ethical issues relating to allocation of scarce resources and use of public health interventions which may limit individual liberties. in addition to the who and cdc documents, there is considerable literature devoted to ethical considerations in pandemic influenza preparedness and response. [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] however, whereas most of this literature assumes a liberal democratic perspective in considering ethics and values, many countries that would partner in a global pandemic response are more hierarchically structured. in hierarchical societies, citizens tend to implicitly expect and trust decisions from their leaders. notwithstanding that liberal democratic societies emphasize individual autonomy, collective decision-making and the interrelatedness and interdependence of community members are fundamental to every human society. 32 because in hierarchic societies these factors have greater visibility, in such societies the success of public health interventions will depend on engaging recognized tribal, community or religious leaders in decision-making. recognizing the importance of having a broader exploration of how cultural perspectives may shape thinking about ethical considerations, cdc sponsored meetings in africa, asia, latin america, and the eastern mediterranean to explore various cultural perspectives relevant to pandemic influenza preparedness and response. the meeting in africa was held in collaboration with who and the african field epidemiology network (afenet) in kampala, uganda on august 11-15, 2008 ) in sharm el sheikh, egypt on december 5-6, 2011. the key objectives for all four meetings were to: (1) identify culturalspecific ethical challenges in pandemic influenza detection and control, (2) explore approaches for addressing these ethical challenges, including how to best integrate ethical considerations into country/regional pandemic influenza preparedness and response guidelines and implementation strategies, and (3) begin establishing a social network to foster continued discussion about ethical issues in the practice of public health. the meetings were attended by government health officials and policy-makers, public health practitioners, scientists from academic and research institutions, epidemiologists, philosophers, ethicists, religious leaders, and representatives of international aid and health organizations. the african meeting was attended by 71 people, including representatives from 12 african countries (nigeria, south sudan, south africa, zimbabwe, togo, mali, cameroun, burkina faso, tanzania, kenya, egypt, and uganda). the asian meeting was attended by 30 people, including representatives from 9 countries (cambodia, china, india, indonesia, laos, myanmar, philippines, thailand, and vietnam). the latin america meeting was attended by 33 people, including representatives from 11 latin american countries (argentina, brazil, columbia, costa rica, dominican republic, el salvador, guatemala, honduras, mexico, panama, and peru). the eastern mediterranean meeting was attended by 34 people, including representatives from 8 countries (egypt, afghanistan, pakistan, sudan, yemen, iraq, jordan, and morocco). for this paper, we reviewed the reports, notes, and stories resulted from the four regional meetings. we mapped outcomes from the meetings to five key ethical challenges identified in the who guidance: transparency and public engagement, allocation of resources, social distancing, obligations to and of healthcare workers, and international collaboration. we report here the objectives, processes, and ethical issues discussed at these meetings as they relate to the who key ethical challenges. in addition, each of the coauthors of this paper attended at least one of the four meetings. description of processes used in organizing the regional meetings to ensure that everyone had the basic knowledge needed to engage in the discussions, all meetings began with overviews of influenza biology, epidemiology, and history, including lessons learned from past influenza pandemics and the 2003 sars pandemic. the focus of the meetings was on planning for a highly pathogenic h5n1 influenza; however, the h1n1 influenza was running its course during the asian meeting and discussion of that epidemic entered the conversation. information was presented on the 2007 who 18 and 2007 cdc 14 ethics guidance documents as a starting point for discussing how ethical considerations may differ between countries. in addition, there were presentations on the basic principles of public health ethics and how it differs from more traditional clinical and research ethics approaches, and discussion of ethical challenges that are likely to arise in response to planning for and responding to pandemic influenza. the latin america meeting also included a session on human rights. out of respect for local partners, somewhat different processes were used in the four meetings to generate discussion of ethical issues. in the african meeting, participants met in small groups to discuss pre-developed case studies addressing the topics of non-pharmaceutical interventions, obligations of healthcare workers, and equitable access to treatment and prophylaxis. participants were asked to consider the case in light of specific challenges they may face in their countries. in the asian meeting, participants met in small groups and were asked to develop their own short narratives about the ethical issues they thought would be important in a pandemic influenza and how the values and cultural consideration in their counties would impact approaches for addressing these ethical issues. participants wrote short narratives answering the following prompt: prior to this meeting, a close friend explains that he does not understand why ethical issues are important in a pandemic response. what experience, either yours or someone else's would you share to illustrate the significance and importance of public health ethics in a pandemic response? during the latin america meeting, participants, grouped by country, were asked to identify key points for integrating ethics into emergency plans. during the eastern mediterranean meeting, participants were asked to share stories about ethical issues encountered during public health responses and to discuss how these issues were addressed in their response plans. organizers took minutes and notes of meeting proceedings and developed summary reports for all meetings. unpublished reports are available at request. for each of the organizing topics below, we first present some common themes discussed at the four meetings followed by more specific perspectives from each of the meetings in chronological order of when the meetings were held, beginning with african perspectives (august 2008), followed by asian perspectives (march 2010), latin american perspectives (july 2011), and eastern mediterranean (december 2011) perspectives. the who framework was used as a starting point for discussion at all meetings, but due to the characteristics, nature of events, and interests of local partners, discussions were not always focused on the same issues. discussions at the two latter meetings were by-andlarge affirming of the perspectives discussed at the two former meetings. based on the meeting reports, fewer details related to the ethical challenges emerged from the latin america and eastern mediterranean meetings than are for the africa and asian meetings. transparency, in which relevant information is made freely available, and public engagement were seen by participants at all meetings as factors critical to an effective response during a pandemic influenza emergency. many related issues were discussed, including low literacy level, poverty, and trust of and/or deference to health authorities. some cultural variations were expressed; for example, that certain societies more readily accept autocratic directives for disease control. participants at all meetings affirmed that their cultures do not tolerate corruption and indicated that a lack of transparency raises suspicions of corrupt dealings. government authorities and leaders are expected to be open and consult the community in making important decisions, including public health emergency decisions, affecting their people. factors that complicate mass communication that were discussed at all meetings include low levels of literacy, the inaccessibility of media such as television, newspapers, and the internet because of poverty; and the unavailability of the internet and cell phone towers in some rural areas. although the detail and depth of discussions regionally varied and some cultural variation was evident, transparency in decision-making was in general decisively affirmed. at the african meeting, in contrast to the general perception that "big men" and individuals with centralized power make all of the decisions, participants agreed that traditional cultures expect leaders to seek input from those they lead, through elder councils and similar institutions. participants noted that public health leaders include traditional healers who serve as both recipients of and conduits for information. because many africans will seek care from traditional healers during a pandemic, these health providers must also be informed of how to protect themselves from infection, and how to guard against spreading the infection. in addition, a wide variety of local and international non-governmental organizations (ngos), often funded by high-income countries, are active in resource poor countries in africa seeking to meet the populations' most basic needs. thus, it is important that both traditional healers and ngos be engaged in the decisionmaking process. the participants at the asian meeting varied widely in their views and practices relating to informing and engaging the public. for example, when sars broke out in one asian country, the government issued mandatory public health measures and expected public compliance. due to the culture of deference to authority, nearly all communities in this country instantly adopted the measures (eg, quarantine, isolation, and social distancing). however, not all participants reported such deference to authority. this was reflected in a story from another asian country about a boycott of a government polio immunization campaign by a minority community due to suspicions about the government's motives. others reported that when the central government was perceived as misgoverned or weak, responsibilities for informing the public about health threats and providing leadership during emergencies fell to local leaders. participants at the latin american meeting stressed that community participation and cooperation will be crucial during a pandemic influenza response, particularly for migrants or minorities who are already stigmatized. latin american participants also pointed to the importance of the media and health authorities in communicating health information and avoiding panic, as well as to convey factual information about availability and access to therapeutics. some participants were concerned about the wide disparity in resources within and between countries, which make transparency even more important. participants at the eastern mediterranean meeting emphasized the need for inclusion, accountability, and transparency in public health policies, but also noted the reluctance among countries to collaborate because of political differences and disparity in wealth and resources. participants discussed the need to establish a clear understanding of who will make what decisions during an emergency, how guidelines will be established, and the importance of considering multiple perspectives, including perspectives from individuals most at risk. allocation of scarce resources economic, demographic, geographical, and population vulnerability factors were common challenges identified as affecting resource allocation decisions. these challenges were shared in all regions where the meetings occurred. participants agreed that because of the cultural and regional variations, a "one size fits all" approach to any planning and response activity is unlikely to be optimal and should be questioned and challenged. however, although differences were acknowledged, there were also shared understanding and general agreement on the importance of the ethical values discussed at the meetings. participants felt that planning and response should take into account contextual variations and cultural differences. additionally, participants discussed the issue of resource allocation within the framework of transparency, especially when preferential treatment is given to the most powerful community members as opposed to the most vulnerable. the african meeting participants affirmed the importance of providing resources to the young, but noted important differences in perspectives among countries. many african societies give higher status to the elderly than to other age groups. with a life expectancy for some african countries in the 40s and even 30s, the proportion of the population composed of young children is much higher than in other countries. thus, in african countries, a preference of allocating scarce resources to children would leave little resources for other segments of the population. generally, african countries are much more rural than are countries of other continents, making access to villages difficult, whether by road, telephone, or internet. although meeting attendees did not feel that rural or urban habitation should be a criterion for allocation of resources, they anticipated that logistical challenges would make it so. these concerns are dwarfed, however, by the likelihood that resources such as antivirals and vaccine will be far scarcer in african countries than elsewhere because of the continent's dual challenge of weak economies coupled with the number of other endemic health challenges, such as malaria and hiv/ aids. with few resources at hand, the ethical imperative to respond to pandemic influenza may fall below that of addressing hyperendemic fatal diseases. each of the countries represented in the asian meeting had a pandemic flu preparedness plan that addressed allocation issues and maintained a national stockpile of antiviral drugs. in most cases, biological vulnerability determined priority; thus, the very young or very old, pregnant women, and immunecompromised individuals tended to be prioritized to receive antiviral drugs. however, as asian cultures exhibit more hierarchy than those in the west, more honor is accorded to the elderly, senior staff, royalty, and public service personnel in asian cultures. in addition, it is expected that relatives and friends of the powerful will be unofficially prioritized to receive limited resources, without that being considered unethical. indeed, in many asian contexts, such prioritization is viewed as a social obligation (eg, a health worker would consider offering antivirals from the limited supply to a senior official before offering the resources to a person in one of the official priority groups). nevertheless, participants expressed disapproval of officials who abuse their power and demand or extort limited resources for themselves. one participant described a shortage of n95 face masks (masks that can filter at least 95% of particles from the air) during the sars epidemic. although some people were willing to pay twice the regular price for a n95 mask, yielding to this demand would in effect favor protection for the rich over the poor. the participants felt the government had a duty to enforce price controls in order to ensure an adequate supply. as an example of enforcing price controls, one government instituted licensing for antiretroviral distribution to put a ceiling on the costs of the medications. some of the asian perspectives on ethical distribution differed by religion. an appeal to buddhist beliefs stated a priority for those who are most severely ill, and the young making sacrifices for their elders. a priority for women and children was expressed with reference to catholic values. when a choice must be made between a mother and her child, participants at the asian meeting felt that catholic values would typically give preference to the mother. some asian nations include island archipelagoes. it will be difficult for populations living on minor remote islands to access medical services and resources during a pandemic. the participants questioned whether their country plans address the challenges that certain geographical conditions may place on the equitable distribution of resources. many participants of the latin american meeting thought that individual rights were paramount and that during a pandemic a clear communication plan that includes community input into the process for drug allocation would help avoid panic. they emphasized the importance of including all sectors of society, including the private sectors, migrants, and minorities in public health decision-making process. issues such as discrimination and stigmatization of certain sectors of the population must be addressed before an emergency situation arises. meeting participants stressed that emergency plans should take into account the diversity of population, must be transparent, and favor equal distribution of resources. at the eastern mediterranean meeting, participants discussed the need to evaluate "what is good for you versus what is good for others. " this included discussing ethical challenges associated with distribution of scarce resources. questions that were explored included: which group of people should be vaccinated first? who will make decisions about distribution? one theme that was identified from this discussion was the importance of prioritizing healthcare workers for access to limited resources, including medical and psychological care and other social benefits, should they become sick during an emergency. the use of social distancing to limit transmission was widely accepted as an important tool in a pandemic influenza response, but participants warned of the many factors and challenges that complicate this traditional public health measure. these include socio-economic factors (eg, densely populated settings), and cultural factors (eg, family duty, funeral rituals). participants in the african meeting agreed with the social distancing principles described in the 2007 who document, 20 including making the measures voluntary to the greatest extent possible; ensuring "safe, habitable, and humane conditions of confinement including the provision of basic necessities (food, water, clothing, medical care, etc)"; and employment protection for workers who comply with social distancing measures against the wishes of their employers. participants stressed that isolation and quarantine measures will be more difficult to enforce in rural compared to urban areas in africa due to the isolated geography of some rural areas and the lack of healthcare workers and security officials. however, participants noted that these public health measures have been successfully employed in rural areas in prior epidemics in africa. densely populated urban slums were also noted to present a challenge to social distancing. in a typical slum dwelling, people occupy all available sleeping space at night in small and poorly ventilated homes. there is no separate space available for isolation or quarantine. the same applies in some refugee camps. in such densely populated settings, the lack of freedom of movement may lead to near-certainty of transmission. neighboring communities will be tempted to protect themselves by fencing off the slums or forcibly preventing the exit of slum residents. there was also concern among the participants that some african countries would rely heavily on military personnel to impose order, potentially with unnecessary force. participants in the asian meeting also noted challenges associated with the use of social distancing measures. duty to family is a major theme of confucian philosophy. in some asian countries, it is a tradition for friends and relatives to visit and even stay with a hospitalized person. in many instances, exhortations to family and friends about the serious nature of isolation are no match for the force of tradition: some find a way in and out of the isolation wards. due to the lack of resources, isolation wards do not have security guards, and nursing staff are not able to add policing to their already heavy workload. a common concern reported by participants at the asian meeting was the risk of stigmatization of patients and family members who were placed in isolation and quarantine. one of the participants reported that during the sars outbreak, an entire village was stigmatized because it was home to one of the cases. anybody known to have come from the village was avoided by others. workers from the village were not admitted to their place of employment outside the village. similarly, students were kept out of their schools. when the village was eventually quarantined, people feared delivering food and supplies. the stigma remained long after the epidemic subsided and the quarantine was lifted. high rates of poverty also pose a challenge for use of social distancing measures. it is difficult for patients to remain in isolation wards or for potentially exposed individuals to remain quarantined for long period of time unless compensation can be offered for lost wages. participants at the latin american and eastern mediterranean meetings reflected on the long history of human rights abuses in their countries. this made them more likely to view use of social distancing measures as something that should be considered with great caution. some even viewed these measures as human rights violations. obligations to and of healthcare workers healthcare workers have multiple obligations, including obligations to their patients, to their employers, to their governments, to themselves, and to their families. participants at all meetings understood that healthcare workers cannot completely sacrifice their and their family members' health and well-being as they fulfill their public health duties during an emergency response. participants at the african meeting felt that healthcare workers have the right to stop working if they feel they are not wellprotected. factors discussed included the challenges related to the displacement of health workers during post-election conflict, traditional or cultural practices that may increase the risk of disease spread (eg, hugging or handshaking), and conflicts between senior officials and frontline healthcare workers regarding access to resources. they felt that frontline health workers should have first priority. a complicating factor in many african countries is the presence of large numbers of health-care-related ngos from a variety of countries. what obligations would they have in a pandemic? if the workers or the organizations were to leave the country to attend to the needs of their home country or their families, the african country could lose a sizable proportion of its health workforce. and yet host governments have little authority to demand their assistance. socio-economic factors were predominant in stories told by participants at the asian meeting. for example, during sars outbreak, some private hospitals in one country were only admitting patients who could pay, while some suspected patients did not go to hospitals because they could not pay the inpatient care that could exceed $250 per day in a country where per capita annual income is less than $3000. some countries reported lack of personal protective equipment, such as face masks, for healthcare workers; or differences in the degree of protection offered according to position (eg, physicians offered more protection than nurses). participants reported that some healthcare workers refused to treat suspected cases, because either they did not have protective equipment and/ or because they were concerned for their own safety and the safety of their loved ones to whom they would return after work. the participants agreed that healthcare facilities and governments had an ethical obligation to adequately and equitably provide personal protective equipment to their employees. in addition, participants felt that education of the employees about transmission control and, in some instances, additional incentives such as hazard pay, can help overcome the hesitancy of healthcare workers to remain on duty during a pandemic surge in cases. participants at the latin american and eastern mediterranean meetings also discussed the roles of healthcare workers during influenza pandemic. participants at both meetings recognized the important responsibilities healthcare workers have to treat patients regardless of the risk to themselves, but also noted that governments have responsibilities to protect healthcare workers. some participants believed that healthcare workers have the right to refuse treatment to patients if the provider fears exposing their own family and that society has an obligation to compensate their families if they die while treating patients. other participants felt that doctors do not have the right to refuse treatment because of their oaths and duties as physicians. international collaboration international collaboration is complicated by many factors, including disparities in resources, political differences, ethnic tension, and distrust. participants, however, agreed that during a pandemic, collaboration is critical, because diseases respect no boundary. participants pointed out that no country, developed or undeveloped, has eliminated poverty and the underlying causes of ill-health, such as lower literacy among the poor and less knowledge about disease prevention. the prevalence of poverty affects not only individuals, but institutions and systems. because of the interdependence of nations, participants thought that it is in the best interest of resource rich countries to help build the capacity of poorer countries to conduct surveillance and disease control. participants in the african meeting stressed that the ability of a developing country to conduct thorough and accurate surveillance depends in large part on the assistance of developed countries in building and maintaining basic public health infrastructure well before a pandemic occurs. moreover, by its very nature, surveillance is an ongoing process, not one that can be initiated in the face of an emergency response and then terminated when things return to normal. in emergencies, international scientists may temporarily fill some personnel gaps. participants observed, however, that some international scientists providing technical assistance during an emergency seem more interested in research than in helping to control the disease outbreak. in some cases, they even diverted resources, such as healthcare workers, that could have been used for disease control. lack of wellequipped laboratories in many african countries has resulted in the transfer of human biological specimens to distant laboratories, sometimes delaying diagnosis and intervention. some surveillance resources are provided by donor nations for specific purposes such as measles eradication. strict accounting rules may prevent the shift of those resources to other purposes, even in the face of a major global threat. the asian meeting participants also expressed concern about specimen sharing. during outbreaks of sars and h5n1 influenza, for example, china shared its specimens with countries around the world for research and vaccine development. 33 during the outbreak of h1n1 in 2009, who noted that 150 countries shared specimens. 34 the asian participants noted that collaboration and communication about disease transmission requires a transparency that can be at odds with the cultural value of 'protecting honor' and 'avoiding being shamed' that is common in asia and elsewhere. reporting an outbreak to other countries can be perceived as admitting inadequate disease control and asking for help from another country may be viewed as a sign of weakness. this is complicated by often pre-existing disputes between neighboring countries. moreover, while helping others is also an important asian cultural value, offering help when a country has not asked for it may be regarded as meddling with the internal affairs of that country. moreover, two asian countries who conducted a joint outbreak investigation exercise observed that multilateral coordination can be time-consuming in ways that hinder a speedy and effective response. participants at the latin american meeting believed that it is important to clarify and disseminate guidelines for pandemic preparation and response, including those produced by who. dissemination of pertinent information and guidelines between countries was considered as an obligation countries have to one another. some noted that although wide disparity exists among latin american countries, there is a great deal of solidarity, which facilitates cross-border collaboration, such as seen in the collaboration between haiti and the dominican republic during the haiti earthquake and resulting cholera epidemic in 2010. participants at the eastern mediterranean meeting noted that countries are sometimes reluctant to collaborate because of the political and resource differences and other disparities between countries in the region. however, they agreed that plans for responding to an influenza pandemic should be shared among countries so that countries will be familiar with neighboring countries' plans. participants believed that country or even regional plans are too broad and more specific sub-regional plans should be developed and implemented. although the ethical concerns raised by participants from these four distinct regions (africa, asia, latin american, and the eastern mediterranean) describe important issues that can shape responses to an international pandemic, the similarities of the perspectives and the concerns were notable. participants reaffirmed the importance of the five key ethical issues framed by who (ie, transparency and public engagement, allocation of resources, social distancing, obligations to and of healthcare workers, and international collaboration). participant feedback can be summarized as followed: • the procedural values of transparency and inclusiveness are widely accepted and crucial for ethical decisionmaking. • no "one size fits all" approach to allocating resources can address the variety of economic, cultural and other contextual factors that must be taken into account, but engaging with communities can help both to discover these factors and to build support for public health recommendations. • although meeting participants acknowledged the importance of social distancing as a tool to limit disease transmission, they also recognized the difficulties associated with this measure. • healthcare workers often have competing obligations that can compromise their ability to fulfill public health duties during an emergency response. government has a responsibility to assist them in doing their job by providing appropriate training and equipment. • although international collaboration may be difficult, a focus on procedural ethics (ie, procedures that ensure transparency, consistency, inclusiveness, and a fair hearing of concerns in a deliberative format) make collaboration possible in efforts to combat global health threats. the discussions from the meetings offer perspectives on how countries can collaborate in the control of international pandemics while respecting different cultural values. although we initially were concerned that cultural differences could seriously impede international collaborations, we believed that anticipatory awareness of value differences would help prevent them from becoming potential stumbling blocks. given this outlook, the meeting organizers were poised to highlight cultural differences. indeed, the meeting exposed numerous cultural differences; eg, people in asian countries more readily defer decision-making to government officials, elders, or other authority figures. many of the differences that surfaced during the meetings reflected differences in how decisions are reached in the context of a country's political arrangements. moreover, as the discussion of resource allocation illustrated, differences in local contexts and traditions necessarily will play a role in how interventions will be implemented. nevertheless, the similarities in perspectives between countries challenged our initial expectation that cultural differences would seriously impede if not prevent collaboration. attempts to change traditional cultural practices frequently fail or result in unintended consequences. however, addressing procedural ethics according to established international norms can assist with overcoming cultural differences within the context of global disease pandemic, political organization or local context. for example, the complicated ritual washing of bodies became a contentious issue in the 2014-2015 ebola response, because of its role in facilitating the spread of ebola virus. culturally, this practice was considered an essential part of preparing the dead for the after-life. 35 public health and government workers contemplating halting or altering ritual practices require great cultural sensitivity and finesse in presenting alternatives that are perceived as fair and acceptable to a community already suffering from irreplaceable loss of their loved ones. the recent who ethics workgroup on ebola again illustrates the importance of a focus on procedural ethics. 36 the workgroup included, along with ethicists and subject matter experts on ebola, representatives from the three west african countries hit hardest by the ebola virus. in relatively short order, the workgroup came to agreement on prominent ethical issues, such as the use of promising experimental drugs against ebola virus, the need to conduct research on these drugs, and the importance of informed consent even during a public health emergency. their success suggests that, when a fair process is established that includes the voices of those affected by the outbreak, a pandemic involving a deadly disease can bring countries rapidly together around the shared value of health, rather than divide them on the basis of cultural differences. perhaps the consensus regarding the importance of combating a pandemic health threat was to be expected, given that cdc or its partners such as tephinet sponsored and coordinated all four meetings, and, more importantly, the 2007 who ethics guidance framed the discussions of ethics topics. perhaps the participants consciously or subconsciously stated what they thought the sponsors wanted to hear. in addition, as many of the participants were public health officials, they brought with them a shared commitment to addressing health concerns. it is possible that the input of these health professionals, more numerous and vocal than other participants, explains the observed continuity around health-related matters. however, these conjectures do not seem compelling. cdc's sponsorship and the framing of discussion around the who's topics did not of themselves preclude major differences from surfacing within any particular topic. it also seems highly unlikely that the majority of differences were to be found outside of those ethics topics discussed at the meeting. the same who framework that oriented the participants to these topics also oriented them to the theme of cultural differences. moreover, the exercises, discussions, and responses were open ended and varied rather than being highly directive. it also seems improbable that participants were merely telling us what we wanted to hear and held back from expressing profound differences when the purpose of the meetings was precisely to explore cultural differences. a simpler and more compelling explanation lies in a crosscultural continuity regarding the importance of combating the health threats that would result from a pandemic event. this continuity should come as no surprise. human rights advocates, for example, deem health so fundamental to human flourishing that they consider it a basic human right. [37] [38] [39] [40] [41] the "right of everyone to the enjoyment of the highest attainable standard of physical and mental health. " is encapsulated in article 12 of the international covenant on economic, social, and cultural rights (cescr), a covenant which 164 out of 197 countries have thus far ratified. 42, 43 similarly, the capability approach maintains that the freedom to achieve well-being is a primary human capability that creates the opportunity for people to realize other capabilities they value. campbell describes health as a liberation or freedom not only from pain or illness, but also as a freedom that allows a person to "create, inhabit a space, to simply live, and share the world around us. " 44 for campbell, the concept of health lies in this freedom. he further believes that the meaning of health reflects personal values and beliefs that are closely linked to the local community and socio-cultural group. health in this view and as a matter of common human understanding is seen as a gateway, if not precondition, for developing other human functions and capabilities. it is a matter, then, not only of ethical theory but also of practical human life that pandemics, which pose existential threats to health, could be expected to elicit similar responses across cultures. there are a number of limitations associated with this manuscript. the manuscript reflects what we found of most interest in the reports, notes, and stories generated from the four regional meetings. it does not provide a complete reporting of the meeting proceedings; rather, it focuses on the parts of the discussion that were related to the key ethical challenges identified in the who ethical framework document. the meetings were meant to initiate an international dialogue about how ethical considerations can be incorporated into pandemic influenza preparedness among members of the field epidemiology training programs, public health officials, policy-makers, scientists, ethicists, religious leaders, and representatives of international aid and health organizations. the meetings were not part of a research study meant to develop new or generalizable knowledge. participants were not recruited in a systematic fashion. cdc relied on local partners to identify and nominate participants to attend the regional meetings. meeting agendas, sessions, and structures were tailored to the local interests and circumstances. although participants were oriented to the who ethics framework at the beginning of each meeting, discussions were not always focused on the same issues. this may explain the lack of consistency in the amount and depth of the discussions on the key ethical challenges identified in the who document. if the analysis and explanations above are sound, then it indicates that cultural differences need not pose a serious challenge for collaboration between countries in addressing an international pandemic. likewise, substantive ethical differences need not pose a serious impediment to pandemic preparedness efforts especially if more attention is paid to procedural ethics, that is, to procedures that ensure transparency, consistency, inclusiveness, and a fair hearing of concerns in a deliberative format. 45 if any lesson is learned from past pandemics, it is that each one informs our response to the next. likewise, the ethical issues raised by past public health emergencies should serve to better prepare ourselves to effectively respond to the next emergency. 46 the same applies to the discussions generated by the regional meetings described in this document. they affirm the notion that, cultural differences notwithstanding, people and countries will come together to combat the health threat a pandemic influenza poses to all, when fair procedures are established that give those affected a seat at the table and a voice. pandemic flu history. department of health and human services (flu.gov) website h2n2. department of health and human services (flu.gov) website lessons from previous influenza pandemics and from the mexican response to the current influenza pandemic h5n1. department of health and human services (flu.gov) website influenza epidemics and pandemics avian influenza virus h5n1: a review of its history and information regarding its potential to cause the next pandemic influenza viruses and the evolution of avian influenza virus h5n1 avian influenza: the next pandemic? disease-a-month h5n1 avian influenza: timeline of major events -25 january. world health organization website severe acute respiratory syndrome h1n1 virus transmission and outbreaks update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov) -worldwide avian influenza a (h7n9) virus. centers for disease control and prevention website stockpiling antiviral drugs for pandemic influenza: the key ethical principles. prepared by the ethics subcommittee of the advisory committee to the director ethical considerations for decision-making regarding allocation of mechanical ventilators during a severe influenza pandemic or other public health emergency. prepared by the ventilator document workgroup, ethics subcommittee of the advisory committee to the director identifying prioritization criteria to supplement critical care triage protocols for the allocation of ventilators during a pandemic influenza public engagement on ethical principles in allocating scarce resources during an influenza pandemic ethical considerations in developing a public health response to pandemic influenza. world health organization website ethical guidelines in pandemic influenza ethical guidelines in pandemic influenza: recommendations of the ethics subcommittee of the advisory committee of the director ethics and pandemic preparedness: the importance of cross-jurisdictional and cross-cultural perspectives ethical reasoning in pandemic preparedness plans: southeast asia and the western pacific influenza pandemic preparedness: legal and ethical dimensions kayman h, ablorh-odjidja a. revisiting public health preparedness: incorporating social justice principles into pandemic preparedness planning for influenza joint massachusetts department of public health-harvard altered standards of care working group on pandemics and the duty to care: whose duty? who cares facing the challenges of influenza in healthcare settings: the ethical rationale for mandatory seasonal influenza vaccination and its implications for future pandemics a matrix for ethical decision-making in a pandemic. the oregon tool for emergency preparedness pandemic influenza preparedness: an ethical framework to guide decisionmaking an ethics framework for public health and avian influenza pandemic preparedness why china's sars legacy may give it an edge against ebola pandemic (h1n1) 2009 -update 77. world health organization website ethical challenges posed by the ebola virus epidemic in west africa ethical issues related to study design for trials on therapeutics for ebola virus disease. who ethics working group meeting report medicine and public health, ethics and human rights towards the development of a human rights impact assessment for the formulation and evaluation of public health policies the right to health in international human rights law what are health and human rights? what is a human-rights based approach to health and does it matter? health hum rights social and cultural rights status of ratification: international covenant on economic, social and cultural rights health as liberation: medicine, theology, and the quest for justice global health as a field of power relations: a response to recent commentaries ebola: the ethics of thinking ahead we acknowledge the invaluable contributions of the following individuals and organizations in organizing the regional meetings, developing meeting reports and summaries, from which this manuscript was based. we especially appreciate the work of mark white (cdc retired) who conceptualized, designed, collected, and analyzed data, and obtained funding and coordinated the regional meetings with external partners. in no particular order, we also would like to acknowledge the contributions of maria consorcia lim-quizon, david mukanga, fred wabwire-mangen, joseph ochieng, patrick nguku, rebecca babirye, dominic thomas, anant bhan, goldie macdonald, andreas reis (who), afenet, emphnet, safetynet, and tephinet. the findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention (cdc), the university of north carolina, chapel hill, nc, usa or tephinet at the task force for global health. all coauthors were involved in reviewing reports, stories, summaries, and notes from the meetings described in the manuscript. funding was provided by the united states agency for international development and cdc to support the four regional meetings described in the manuscript. funding was used for travel-related expenses of all participants, including the co-authors of this paper, to participate in one or more of the meetings. not applicable. this manuscript was based on analysis of reports and documentations generated from four cdc-sponsored regional meetings. the meetings were considered routine public health practice and not research per se. key: cord-326027-58whwspe authors: hernaez, bruno; escribano, jose m.; alonso, covadonga title: visualization of the african swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera date: 2006-06-20 journal: virology doi: 10.1016/j.virol.2006.01.021 sha: doc_id: 326027 cord_uid: 58whwspe many stages of african swine fever virus infection have not yet been studied in detail. to track the behavior of african swine fever virus (asfv) in the infected cells in real time, we produced an infectious recombinant asfv (b54gfp-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (egfp). the incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-egfp was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. the tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. in this work, diverse potential applications of b54gfp-2 to study different aspects of asfv infection are shown. by using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. additionally, we have been able to visualize for first time the asfv factory formation dynamics and the cytophatic effect of the virus in live infected cells. finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations. many aspects of the infection cycle of african swine fever virus (asfv) remain still poorly understood. some events of the infection such those viral proteins involved in the virus attachment, the intracellular transport to perinuclear areas of the nucleus for virus replication, as well as morphogenesis and transport events of the intracellular virus away from the factories have been biochemically defined. however, a clear understanding of these processes, how and when they occur, has been hampered by the inability to directly observe these events in infected cells. asfv assembly occurs in viral factories that contain high levels of viral structural proteins, viral dna, and amorphous membranous material used to produce viral envelopes. the 170-kb genome of asfv encodes some 150 open reading frames, and as many as 50 viral proteins are incorporated to the viral particle (esteves et al., 1986) . approximately 35% of the mass of the virion is provided by p72, the major capsid protein, while the structural proteins p150, p37, p34 and p14, all of them derived from polyprotein p220, provides another 25% of the virion mass (andres et al., 1997) . asfv particles assemble within cytoplasmic viral factories from endoplasmic reticulum-derived viral membranes (andres et al., 1997 rouiller et al., 1998) at perinuclear sites (nunes et al., 1975) that contain fully assembled virions seen as 200-nm-diameter hexagons in cross-section, and a series of one to six-sided assembly intermediates (rouiller et al., 1998) . morphological evidence indicates that viral membranes become icosahedral particles by the progressive construction of the outer capsid layer, which is composed mainly of viral protein p72 . biochemical data also suggest that protein p72 is assembled in a time-dependent fashion into large membrane-bound complexes that may correspond to capsid-like structures (cobbold and wileman, 1998) . concomitantly, the core shell is formed underneath the viral envelope, and subsequently the viral dna and nucleoproteins are packaged and condensed to form the electrondense nucleoid (andres et al., 1997 (andres et al., , 2002 brookes et al., 1998) . intracellular mature virions made at the assembly sites are infectious (andres et al., 2001) . a fraction of them, however are transported by microtubule-mediated transport (alonso et al., 2001; de matos and carvalho, 1993) to the plasma membrane, where they are released by budding (breese and pan, 1978) to give rise to the infectious extracellular enveloped virions. because of the high viscosity of the cytoplasm, movement of asfv particles by diffusion is likely to be very limited (for review see (luby-phelps, 2000) . some viruses overcome this obstacle by hijacking cytoplasmic motors to utilize the cellular cytoskeleton as a roadway. for instance, herpes simplex virus hsv-1, adenovirus, asfv and rabies virus are thought to use dynein motors to travel along microtubule network for intracellular transport (alonso et al., 2001; raux et al., 2000; sodeik et al., 1997; suomalainen et al., 1999) . asfv interacts with the dynein motor complex through the structural virus protein p54 (alonso et al., 2001) . to further characterize the intracellular trafficking of some of these viruses, several fluorescence-based methods have been developed allowing the detection and characterization of intracellular complexes of viral origin. importantly, several of these labeling methods allow the visualization of individual virions in living cells (smith and enquist, 2002) . these studies suggest that the intracellular trafficking of these viruses is a highly ordered process using cellular motor pathways. similarly, with tagged viruses it would be possible to visualize virus morphogenesis and cytopathic effects produced in the cells as infection progresses. in this report, we describe the construction of an asf recombinant virus, in which we have exchanged the single copy of the p54 open reading frame in the asfv genome, gene e183l (rodriguez et al., 1994) with a chimeric gene encoding p54-egfp. interestingly, this virus is fully viable and exhibits growth kinetics similar to those of its parental virus. moreover, p54-egfp is incorporated into the virus particle with the same efficiency as p54. the presence of p54-egfp in the virion results in fluorescent particles which are readily visualized with a fluorescence microscope, allowing visualization of p54-egfp within live cells. as a consequence of such sensitive detection of p54-egfp throughout infection, we have been able to use time lapse confocal microscopy to monitor the trafficking of p54-egfp within individual cells. we present those results as subsequent images and time lapse animations. thus, we have generated a reagent which will enable the visualization of several aspects of asfv infection in live cells, including virus entry, assembly, trafficking and apoptotic cell death. the main goal of the molecular manipulations described below was to generate a tagged asf virion that allowed us monitoring the infection in living cells. we selected the asfv envelope protein p54, encoded by the essential e183l gene (rodriguez et al., 1994) , to drive the egfp incorporation into the virus envelope. then, we replaced the protein p54 in the virus particle by p54-egfp fusion chimera in two steps. first, we introduced in the tk gene of the virus the sequence encoding the fusion protein p54-egfp under the control of p54 promoter (fig. 1a) . recombinant virus plaques exhibiting green fluorescence were picked and individual recombinant viruses were further plaque purified by limiting dilution and screened by pcr and southern blot to confirm the presence of the p54-egfp coding sequence in the tk locus, as expected (fig. 1b) . the recombinant virus obtained was designated as b54gfp-1 and used to infect cells to visualize the fluorescence properties of individual virus clones (figs. 1c and d). moreover, the use of egfp as marker gene allowed a faster detection of recombinant viral plaques when compared to wild-type virus (data not shown). egfp fusion to a specific protein often modifies its inherent properties, so we first determined whether the addition of egfp to the c-terminus of p54 could affect the usual subcellular distribution of this protein. then, vero cells were infected with b54gfp-1 or wild-type virus (ba71v) and subcellular distributions of p54-egfp and p54 were examined by conventional immunofluorescence microscopy (figs. 1e-j). no differences were observed when compared, demonstrating that typical perinuclear accumulation described for p54 in asfv infection was not altered by the gfp fusion. p54 is an essential protein and then to study p54-egfp functionality in the virus morphogenesis and to increase the number of copies of the tagged protein in the virions, the wildtype e183l gene was interrupted by insertion of the βgalactosidase coding sequence. this second step in recombinant virus generation was performed by homologous recombination with vector pδp54 which contains the β-galactosidase coding sequence under the control of p72 promoter and flanqued by 424 pb and 888 pb from p54 e183l right and left extremes, respectively. resulting recombinant virus plaques exhibiting β-gal activity were picked and analyzed by pcr as well as southern blot to confirm the interruption of gene e183l in the genome of asfv (fig. 1b) . after isolation by several rounds of plaque purification, all virus plaques exhibited both gfp and β-galactosidase activities. the double recombinant virus finally obtained was designated b54gfp-2 and when used to infect cells, no trace of the wild-type p54 protein could be detected by western blot in cell extracts obtained at different times of infection using a specific anti-p54 serum (fig. 3a) . (a) schematic representation of the ecorik and ecorie fragments in the genome of the wild type asfv (ba71v) and the recombinants viruses generated (b54gfp-1 and b54gfp-2) after insertion of chimera p54-egfp in tk locus by homologous recombination and disruption of the p54 gene by insertion of the β-gal marker gene, as described in methods. e: ecori site. (b) genomic analysis, after ecori digestion, of parental and recombinant asf viruses by southern blot using a digoxigenin-11-dutp labeled dna probe corresponding to the p54 complete coding sequence. size of the fragments probed was as expected after genome manipulations. (c and d) after insertion of p54-egfp recombinants viral plaques formed in vero cells monolayers exhibited fluorescence and allowed and easy detection after 5 dpi. (c) a single recombinant fluorescent plaque was photographed in a fluorescence microscope (original magnification ×100) and correspondent transmitted light image is shown below (d). fluorescence microscopy analysis of b54gfp-1 (e-g) or ba71v infected cells at 14 hpi (h-j). p54-egfp was performed by direct fluorescence (e), while p54 was detected with an anti-p54 serum and secondary antibody conjugated to alexa 488 (h). in both cases viral and cellular dna were counterstained with hoechst to visualize viral factories, indicated by arrows (f and i). as it can be observed, in the merged images (g and j), p54-egfp and p54 showed similar perinuclear distribution coincident with viral factories. scale bar, 18 μm. to determine that protein p54, a major component of the external envelope of asfv, fused to egfp protein remains incorporated to the viral particle, ba71v and b54gfp-2 virions were percoll purified and analyzed by sds-page and western blotting using specific antibodies (fig. 2a) . no reactivity of anti-gfp antibody with ba71v purified virus was found, while a 51-kda band corresponding to the expected size of p54-egfp protein was detected in b54gfp-2 purified virus. however, when an anti-p54 serum was probed, a 25-kda band appeared with ba71v virus and with anti-gfp serum, a 51-kda band was only reactive with the recombinant virus, demonstrating the incorporation of p54-egfp fusion protein into the virus particles. the typical doublet detected with anti-p54 serum in ba71v samples was not present in those from b54gfp-2 when detection of p54-egfp was carried out with monoclonal antibody anti-gfp. it is noteworthy that the levels of p54-egfp and p54 incorporated into their respective particles were apparently equivalent, since the amount of total virus protein loaded in sds-page was similar in both cases, as judged by coomasie blue staining. in order to further confirm this, we analyzed by western blotting the presence of major structural asfv protein p72 in these highly purified virions (fig. 2a) . the proportion of p54-egfp relative to p72 in b54gfp-2 virions was roughly the same as p54/p72 rate inba71v virions, indicating that the fusion protein was incorporated to virus particle at similar rates than p54. to ascertain the growth properties of the b54gfp-2 virus, a time course of infection was carried out for both, wild-type and recombinant viruses. monolayers of vero cells were infected with ba71v or b54gfp-2 at a multiplicity of infection (moi) of 5 pfu/cell and harvested at different postinfection times. total cell lysates were analyzed by sds-page and western blotting, and the kinetics of synthesis of virus proteins were assessed (fig. 3a ). western blotting with an anti-p54 serum allowed the identification of a 51-kda band, the expected size for p54-egfp, only in b54gfp-2 infected cell extracts from 8 to 24 hpi. at the same times postinfection, p54 was detected as a doublet in ba71v infected cell extracts while a single 51 kda band was detected with a monoclonal antibody against gfp, only in recombinant virus infected cell extracts at these time points. p54-egfp apparently remained intact throughout the course of the infection and sample preparation, since no doublet was observed in wb. as expected, fusion protein was synthesized late after infection (detectable from 8 hpi by wb), in contrast to the early protein p30 used as a control, which was detected from 4 hpi. to demonstrate that p54-egfp is synthesized during the late viral expression phase under the control of p54 promoter, we examined the synthesis of fusion protein in the presence and absence of a dna replication inhibitor, such as cytosine βarabinofuranoside (ara-c). in the presence of ara-c, we could detect neither p54-egfp nor p54 with anti-p54 serum, while early asfv protein p30 expression was confirmed by wb with a monoclonal antibody against p30 in the presence and absence of inhibitor (fig. 3b) . these results demonstrated that virus gene expression was similar in b54gfp-2 and wild-type virus infected cells, but it was conceivable, since p54-egfp is a structural component of the virus particle, that assembly and/or release from the cell of the double recombinant virus could be in some way restricted. to assess the rate of virus assembly and egress throughout several infection cell cycles, growth curves were carried out for both b54gfp-2 and the ba71v virus. vero cells were infected at a multiplicity of 0.1 pfu/cell and harvested every 10 h for 72 h, and both extracellular and intracellular virus yields were calculated for both viruses. the results showed that the growth curves for extracellular and intracellular virus were similar for wild-type and recombinant virus infections, not only in total infectious virus production after 72 hpi, but at every time point we examined (fig. 3c) . these results imply that the rates of both virus assembly and virus egress from the cell were the same for the two viruses. cell monolayers were infected with ba71v or b54gfp-2 at 5 pfu/, in presence (+) or absence (−) of inhibitor cytosine βarabinofuranoside (ara-c) to discriminate late from early viral protein synthesis. cells were harvested at 14 hpi and analyzed by western blot with a serum anti-p54 that recognized p54-egfp and p54 only in absence of ara-c. early asfv protein p30 was detected in presence and absence of inhibitor using monoclonal antibody anti-p30. (c) analysis of the replicative phenotype of b54gfp-2. cells were infected with 0.1 pfu/cell of ba71v or b54gfp. every 12 hpi (for a maximum of 72) extracellular and intracellular infectious virus progeny was estimated by plaque assays. growth curve obtained for recombinant virus resulted similar to the parental virus ba71v. the incorporation of p54-egfp into virions of the asfv b54gfp-2 makes possible that these particles can be detectable by fluorescence. to test if this was the case, virions purified by sedimentation through sucrose cushion were used to infect vero cells grown onto a coverslip at a multiplicity of 10 pfu/cell. after synchronizing the infection at 4°c for 2 h, cells were incubated for 30 min at 37°c and subsequently examined by fluorescence microscopy. fluorescent particles, visualized as point sources of gfp fluorescence, were readily detected inside cell cytoplasm (fig. 2b) . when target cells were washed of free virus and allowed to incubate further at 37°c, a significant proportion of the gfp signal accumulated in the perinuclear region, in a short time after infection 4 h (additional file 1). images were acquired at 30 min and 4 h postinfection, before late viral protein expression phase onset to avoid that observed gfp signal corresponds to newly synthesized p54-egfp. some obvious applications of a virus expressing a fluorescent structural protein would be to localize that protein within the cell and follow its trafficking during the infection cycle and ultimately, to visualize the pathway of virus assembly in live cells. as previously shown, wild-type p54 and p54-egfp distribution at late times in wild type and b54gfp-1 infection was shown in perinuclear viral factories (figs. 1e-j). we examined this distribution for b54gfp-2 infected cells at 12-16 hpi by confocal fluorescence microscopy in fixed cells . protein p54-egfp distribution coincided with assembly sites in the viral factories, which is characterized by a great accumulation of newly synthesized viral dna. to further confirm that p54-egfp distribution could be used as a novel viral factory marker in asfv infection we showed that the golgi complex in living b54gfp-2 infected cells was located surrounding, but not coincident with the viral factory where a great accumulation of p54-egfp is found (figs. 4e-g). the ultimate elegance of a gfp-incorporating virus is the potential for monitoring virus infection in individual living cells. the abundant incorporation of p54-egfp into asfv particles indicated that it should be possible to visualize the intracellular movement of virions in real time by laser-scanning confocal microscopy of live cells. approximately 50% confluent vero cells were infected with more than 10 pfu of b54gfp-2 virus per cell. virus infection was synchronized by incubation of cells at 4°c for 90 min and images were collected, after incubation at 37°c for nearly 2 h, to analyze the first steps of infection at high magnification. individual virus particles were readily observed and images were acquired with the confocal microscope setup at a rate of 1 frame/3s for a maximum of 2 min. with this short interval of time, we discerned that intracellular movement in the proximity of perinuclear areas was composed of frequent stops and starts, and often without any apparent direction. fig. 5 shows an example of this intermittent or saltatory movement of an individual virion. over the times and distances measured in following frames acquired from 10 different virions, an average speed ranged from 0.2 to 0.5 μm/s at this stage of infection was calculated. in order to test the role of microtubules at this stage of infection, disruption of microtubules with 10 μm nocodazole was achieved prior to infection with b54gfp-2. under these conditions we could not track any virion movement examined at 1 hpi or 4 hpi in live cells. interestingly, at mentioned times postinfection most gfp signal was localized far from perinuclear areas in contrast to infected cells with intact microtubules (additional file 2). to determine the stage of infection at which newly synthesized p54-egfp could initially be detected by direct fluorescence microscopy, and to visualize virus factory constitution, vero cells were infected with b54gfp-2 at a multiplicity of infection of 1 pfu/cell. after synchronization of the infection, cells were examined for egfp fluorescence every fig. 6 . protein p54-egfp was first observed in the majority of cells as early as 7-8 hpi. the protein was mainly localized as small, cytoplasmic and fluorescent aggregates around the nucleus (2 or 3 spots were usually observed within single cell). from this point, all of these perinuclear spots finally aggregated along the next 2 h to give rise to a unique brilliant spot close to the nucleus (fig. 6b ). this accumulation of p54-egfp, which was previously shown coincident to the viral factory, gradually increased in size and fluorescence intensity as infection proceeded. it should be mentioned that aggregation of initial perinuclear spots of p54-egfp could not be observed in every infected cell, but in most of them, probably due to changes in the optical section acquired during the monitoring of the infection. at late times postinfection, in most of the cells, it was usually to observe violent changes in viral factories location (fig. 6a) , however these changes seem to correspond to movements of infected cell prior to detachment from the dish, while factory location is conserved. in another set of experiments, we investigated the relationship, of mitochondria status and viral factory formation in a time course manner. at initial infection steps mitochondria were observed as an almost continuous succession of organelles extending throughout the cytoplasm. nevertheless, time lapse experiments with image acquisitions every 5 min in vero cells infected at a moi of 1 pfu/cell, showed the progressive alteration of the normal mitochondrial pattern and mitochondrial irregular clumping and aggregation around viral factory was observed (fig. 8) . this aggregation started at 8 h after infection, coincident with p54-egfp synthesis, suggesting the high energy requirements of the virus assembly process. apoptosis has been described to play an important role in asfv pathogenesis (ramiro-ibanez et al., 1996) , so we decided to analyze the very late stages of asfv infection in living cells infected with b54gfp-2. fig. 7 shows in detail the characteristic morphological changes that occur during programmed cell death of an infected cell. since 12 hpi an early cytopathic effect, consisting in cytoplasmic vacuolization, rounding and reduction in the size of the infected cell was be observed. from this time point onwards, the cell lost contacts with neighboring cells and detached from the monolayer, undergoing nuclear fragmentation and final cell death. this process included typical membrane blebbing of the infected cell that led to the formation of numerous vesicles containing large amounts of p54-egfp. vesicle formation has been considered a very characteristic feature of asfv infection in cultured cells and these are viruscontaining vesicles. completion of the process from the first morphological features of apoptosis lasted around 6 h. successful african swine fever virus life cycle is dependent on several and critical interactions with host cell which remain poorly understood. some of these events include virus internalization into cellular cytoplasm, transport to perinuclear areas where viral dna replication occurs, egress of viral progeny from assembly sites to cell periphery and virus induced cell death. recent improvements in optical imaging techniques combined with protein fusion to gfp variants, open new fig. 5 . analysis of the intracellular movement of b54gfp-2 viral particles. vero cells were b54gfp-2 infected with 20 pfu/cell and examined by confocal microscopy at 1 hpi, acquiring images every 3 s corresponding to 0.5 μm optical sections from the z axis. virus particles were detected as egfp point sources, and correspondent transmitted light image provided points of reference information. movement of 10 virions were individually analyzed. upper image shows an example of the trajectory followed by one of the virions analyzed (white discontinuous line) and selected area is enlarged below. total distance covered by this virion was 11.74 μm in 30 s, with an average speed of 0.5 μm/s. as can be observed in the figure, virion velocity was not constant, with a maximum speed from 12 to 15 s. scale bar, 8μm as indicated. possibilities for the direct analysis of these dynamic processes. this new methodology has been successfully applied to study different aspects of the infection of adenoviruses (lux et al., 2005) , herpesviruses (glotzer et al., 2001; sampaio et al., 2005) , poxviruses (geada et al., 2001; hollinshead et al., 2001; ward and moss, 2001) , rhabdoviruses (finke et al., 2004) or retroviruses (mcdonald et al., 2002; muller et al., 2004) . we reported here the generation of a fluorescently tagged and viable asfv expressing the structural protein p54 as an egfp fusion protein. expression of p54-egfp enables easy identification of infected cells by direct fluorescence analysis, which results useful when immunodetection of diverse antigens in the same sample is desirable. selection of p54 as the viral fusion protein was supported by several reasons. since p54 is located in the inner viral envelope (rodriguez et al., 1994) , fluorescent p54 would allow the detection of individual viral particles upon infection. as the carboxi-terminal tail of p54 is facing the surface of mature virions, it was expected to be a suitable target for egfp fusion. on the other hand, newly synthesized p54 accumulates within perinuclear areas where morphogenesis takes place at late times postinfection. thus, p54-egfp could become a very helpful tool to analyze viral factories assembly. the strategy to generate tagged virus b54gfp-2, consisted in two sequential steps. after initial insertion of p54-egfp coding sequence under the control of p54 promoter, it was found that fusion of egfp to the c-terminus of p54 did not affect the perinuclear subcellular distribution of the viral protein, as it was directly observed in b54gfp-1 infected cells. then, we interrupted e183l gene in b54gfp-1 genome by insertion of β-galactosidase coding sequence as second selection marker, to ensure that every p54 molecule that accommodates in virion inner envelope would be egfp tagged. previously, p54 has been shown to be essential for virus viability, playing and important role in asfv morphogenesis (rodriguez et al., 1996 (rodriguez et al., , 2004 , so the recovery of recombinant virus b54gfp-2 after wild type p54 interruption indicated that fusion did not apparently affect the normal function of p54. furthermore, growth curves obtained with recombinant virus indicated identical replication behavior and infectious efficiency when compared to the parental strain. furthermore, fusion to egfp did not alter the expression pattern determined by wb analysis for p54 in infection as p54egfp expression remained under control of p54 natural promoter in b54gfp-2. p54-egfp was synthesized and detected during late viral expression phase, from 8 hpi, as expected. this observation was confirmed by wb analysis in the presence of inhibitor ara-c, which demonstrated that there was not chimera protein expression when viral dna replication was inhibited. also, expression levels of p54-egfp in infection, determined by wb, did not apparently differ from those observed for p54. in order to track single fluorescent asfv particles, it is an essential requirement that p54-egfp is incorporated into viral particles. previously, p54 has been successfully used as a target to generate asfv chimeras incorporating short foreign viral epitopes such as the antigenic site a from foot-and-mouth disease virus vp1 protein and the da3 antigenic determinant from transmissible gastroenteritis coronavirus nucleoprotein n (brun et al., 1999) . in both cases, chimeric p54 proteins were successfully incorporated into the viral particles, supporting selection of p54 as the fusion protein. as expected, western blot with percoll purified b54gfp-2 particles demonstrated the association of p54-egfp with asfv virions. as deduced from detection of control protein p72 and p54 in percoll purified samples, inclusion of p54-egfp into virions appeared to occur with same effectiveness than p54. such tagged virus will enable live analysis of various stages in the asfv replicative cycle using confocal microscopy. one of these potential applications of b54gfp-2, shown in present work, would be the analysis of intracellular movements of fluorescent particles during the initial phase of infection immediately after internalization into the host cell. when collecting confocal microscopic images every 3 s, from 2 hpi, it was found that most of the viral particles exhibited a special kind of movement defined as saltatory, intermittent and apparently disorganized, making difficult the estimation of fig. 7 . live-cell analysis of the last stages of b54gfp-2 infection. series of egfp and transmitted light simultaneous acquisitions of the same b54gfp-2 infected vero cell by confocal microscopy from 10 hpi (when viral factory is already constituted) and every 5 min (see additional file 3 to complete animation sequence). rounding of the infected cell is followed by membrane blebbing and subsequent formation of p54-egfp (green) containing vesicles. at 18 hpi, detachment and final cell destruction is observed. scale bar, 20 μm. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) speed rates. this type of movement has been described as characteristic of microtubule associated movement. from the analysis of movement of 10 individual virions an average speed ranging from 0.2 to 0.5 μm/s was calculated with the applied resolution. these data are in agreement with those obtained for other large enveloped viruses. using fluorescence videomicroscopy, which allows a resolution of 1 frame/s, speed rates from 0.3 to 1 μm/s have been determined during the late phase of infection with labeled vaccinia virus (geada et al., 2001; ward, 2005; ward and moss, 2001) . recently, human cytomegalovirus has been shown to move with apparent velocity of 0.7 to 0.8 μm/ s (sampaio et al., 2005) at very early stages in infection. moreover, hiv virions transport towards host cell nucleus linked to microtubules occurs at 1 μm/s (mcdonald et al., 2002) . the saltatory movements of tagged asfv virions and the speed rates obtained are consistent with a microtubular based transport (king, 2000) and association of this virus with microtubules has been described (de matos and carvalho, 1993) . previously, we identified the lc8 subunit of cytoplasmic dynein, a minus end directed microtubule-associated motor protein, as an interacting protein with p54 (alonso et al., 2001) , resulting this interaction critical in the first stages of asfv infection. collectively, these data suggested a role for microtubules in the intracellular transport of asfv after internalization. this role is supported by data showing that nocodazole disruption of microtubules prior to infection results in stop of virion movement and lack of perinuclear accumulation of viral particles at 4 hpi that remained dispersed in the cytoplasm. also, there was no subsequent constitution of the characteristic virus factory and finally a reduction in asfv progeny was found (additional files 1 and 2 and alonso et al., 2001) . also, during the late phase of asfv infection, transport of newly assembled virions from viral factory to cell periphery along microtubules has been recently demonstrated (stefanovic et al., 2005) , similar to the exit transport described for vaccinia virus (carter et al., 2003) . further live studies involving labeled b54gfp-2 that are now in course will provide useful and detailed information to clarify the whole asfv penetration and egress process into the host cell. p54 has been previously described to localize in membrane structures within the virus factories and in immature asf virions, as judged by immunogold electron microscopy (rodriguez et al., 1994) . p54-egfp, just as p54, showed an intracellular distribution fully coincident with asfv factory at late postinfection times. this allowed us, to follow the viral factory formation process in live cells throughout the infection cycle for first time. animations obtained show that p54-egfp was first detected in cytoplasm as several discrete and bright spots representing initial viral assembly sites. as infection progresses these initial assembly sites migrated to a perinuclear area, fused and finally formed a single large accumulation of p54-egfp per cell. similar assembly sites were previously described as viral antigens inclusions containing viral precursors which remained tightly associated to microtubules (carvalho et al., 1988) . indeed, microtubule associated motors are required to transport these initial assembly sites towards the microtubule organization center (mtoc), where final asfv factory is constituted (alonso et al., 2001; heath et al., 2001) following viral replication and viral assembly takes place. first steps in asfv assembly consist in the modification of er membranes rouiller et al., 1998) . protein p54 has been involved in the recruitment to the viral factories and transformation of the er-derived envelope precursors that are finally acquired by virions (rodriguez et al., 2004) . this new role for p54 in morphogenesis supports the selection of p54 as viral fusion protein and suggests that studies about p54-egfp trafficking during infection in live cells would be helpful to analyze the acquisition of asfv envelopes from er during virus assembly. the formation of the viral factory often involves not only host cytoplasmic membranes and cytoskeletal components, but also cell organelles such as mitochondria. these are thought to play an important role in several large dna enveloped virus infections (murata et al., 2000; novoa et al., 2005; rojo et al., 1998) , supplying the energy required in the morphogenetic and assembly processes. our results, obtained with live asfv infected cells have confirmed these previous observations in fixed cells (hernaez et al., 2004a; rojo et al., 1998) , which showed that asfv infection induce the migration of mitochondria to the perinuclear assembly sites, producing the clustering of this actively respiring organelle around viral factory. this migration occurs since 8 hpi, before the definitive viral factory is fully constituted, suggesting that energy contribution from mitochondria is required since first stages of asfv morphogenesis. finally, we analyzed the destruction of the asfv infected cell. this process started at 12 hpi and usually lasted about 6 h, being identical to apoptotic cell death, which has been demonstrated to play a relevant role in asfv pathogenesis (hernaez et al., 2004a (hernaez et al., , 2004b ramiro-ibanez et al., 1996) . in live cells, it was possible to observe host cell membrane blebbing, resulting in the formation of p54-egfp containing vesicles. this finding raised the possibility that these vesicles could contain virus particles. previous publications reported that the abundant vesicle fraction released in last asfv infection stages contains virus (carrascosa et al., 1985) . then, it is conceivable that the fluorescence detected in the vesicles formed at the end of apoptosis (blebbing), could correspond to gfp labeled virus particles. this vesicle formation observed in live cells is rarely observed using common fixation methods, then it is not possible to observe it with the parental unlabeled virus. in summary, we have generated a fluorescently tagged asfv, which could be a powerful tool since it enables live analyses of diverse aspects of asfv live cycle, including viral particles intracellular transport, morphogenesis and apoptosis of the infected cell. we presented here, for the first time, live images of some of these stages in asfv infection and more detailed information will arise from future and novel studies of virus entry and egress. vero cells were obtained from ecacc and grown at 37°c in a 5% co 2 atmosphere in dulbecco's modified eagle's medium (dmem) supplemented with 5% foetal bovine serum (fbs). asfv isolate ba71v, adapted to grow in vero cells, was used in infection experiments carried out at 37°c and 5% co 2 . when synchronization of infection was required, this was performed for 90 min at 4°c after viral inoculum was added. in most cases, asfv stocks from culture supernatants were semipurified from vesicles by ultracentrifugation at 40.000 × g through a 20% (w/v) sucrose cushion in pbs for 1 h at 4°c. when needed, asfv particles were highly purified from the extracellular medium by percoll equilibrium centrifugation as previously described (carrascosa et al., 1985) . after centrifugation, percoll gradients were fractionated and fractions 2 to 8 from percoll gradients, containing virus particle essentially free of vesicles and contaminant membranes, were collected after sedimentation and gel filtered through sephacryl s-1000 (amersham) to remove percoll. the construction of the insertion vector pins-54egfp involved several steps. the expression plasmid pegfp-n1 (clontech) was used as template to obtain the egfp coding sequence by pcr with specific primers (5′-cgcggatccgc-agtaaaaaaatg-3′ and 5′-cgcggaattcatggtgag-caagggc-3′). a fragment of 800 pb corresponding to p54 promoter and complete p54 coding sequence was also obtained from ba71v genome by pcr with specific primers (5′-gcgggatcccgttgtctaggtaa-3′and 5′-gcggaatt-ccaaggagttttct agg-3′). in order to generate the vector pins-54gfp, previous pcr products were digested with ecori and bamhi and cloned into the 4.7 kpb fragment resulting from the digestion of plasmid pins72gal (gomez-puertas et al., 1995; rodriguez et al., 1992) with bamhi. the construction of the vector pδp54, used to perform the interruption of e183l gene, was described previously (rodriguez et al., 1996) . briefly, this vector contains β-galactosidase coding sequence under control of p72 promoter, flanked by two fragments from e183l that allow homologous recombination with this orf in ba71v genome. these flanking sequences correspond to 424 pb and 888 pb from right and left extremes from p54 gene, respectively. an african swine fever virus recombinant expressing an enhanced version of the gfp gene (egfp) fused to the cterminus of the p54 coding sequence was constructed in two steps: first, the insertion of the p54-egfp coding sequence into thymidine kinase locus (tk) in ba71v genome was achieved by homologous recombination after transfection of vero cells with pins-p54egfp in the presence of fugene (roche), according to manufacturer's instructions. eight to 16 h after transfection, cells were infected with ba71v at a moi of 0.1 pfu/ cell. when the cytophatic effect was complete (approximately 72 hpi) the cultures were harvested and sonicated. these transfection-infection mixtures were used to infected monolayers of vero cells seeded onto 100 mm dishes (nunc) at different dilutions. after 5-6 dpi, the recombinant virus generated was isolated from progeny virus by three rounds of plaque purification on vero cells, during which recombinant virus plaques were screened for egfp direct fluorescence. the resulting recombinant virus was named b54gfp-1. additional second step consisted in the inactivation of the original gene of p54 (e183l) in the b54gfp-1 genome to finally generate the recombinant virus b54gfp-2. the transfection of vero cells with pδp54 and following infection with b54gfp-1 allowed the insertion of the β-galactosidase coding sequence downstream of the strong p73 promoter within e183l gene. isolation of b54gfp-2 was performed as described above, screening recombinant virus plaques for egfp fluorescence and expression of β-galactosidase (gomez-puertas et al., 1995) . the genomic structure of these recombinant viruses was confirmed by dna hybridization analysis and could be observed in figs. 1a and b . briefly, dna from parental and recombinant viruses were obtained and purified as previously described (esposito et al., 1981) . resulting fragments, after eco ri digestion, were resolved by agarose gel electrophoresis and then blotted onto uncharged nylon membranes (amersham). membranes were probed with a digoxigenin-11-dutp labeled dna fragment corresponding to the complete p54 coding sequence. final detection of digoxigenin was performed with cspd (roche) after probing the membrane with specific monoclonal antibody anti-digoxigenin (roche). preconfluent monolayers of vero cells were infected with either recombinant b54gfp-2 or parental ba71v at 0.1 pfu/ cell. after 90 min, inoculum was removed and cells were washed with fresh dmem and overlaid with dmem supplemented with 2% fbs. infected cells with their culture supernatants were harvested at different times postinfection (0, 12, 24, 48 and 72 hpi) and centrifuged at 3000 × g for 10 min. cell pellets was resuspended in dmem. both the cellular fraction and the supernatant were sonicated and separately titrated by plaque assay to determine the intracellular and extracellular virus production respectively. plaques assays were performed as described previously (gomez-puertas et al., 1995) . when isolation of recombinant virus plaques was desired, detection of egfp fluorescence and expression of β-galactosidase were performed at 4 and 6 days postinfection respectively. vero cells preconfluent monolayers seeded on 6-well plates were infected with ba71v or b54gfp-2 at a moi of 5 pfu/cell. when inhibition of late viral protein synthesis was required, cells were incubated with cytosine β-arabinofuranoside (ara-c) 50 μg/ml at 3 hpi. in all cases, cells were washed with pbs and harvested at 0, 4, 8, 12 and 24 hpi and lysed in ripa buffer. after centrifugation at 10,000 × g for 20 min, proteins from cleared lysates were electrophoresed in 12% sds-polyacrylamide gels and transferred to nitrocelluose membranes (biorad). membranes were incubated for 2 h at room temperature in pbs containing 5% non-fat dry milk and then probed with corresponding primary antibodies: monoclonal antibody anti p30 diluted 1:500, monoclonal antibody anti-gfp diluted 1:2500 (clontech), and specific serum against p54 raised in rabbit diluted 1:1000, for 1 h at room temperature in pbs containing 0.05% tween 20 (pbs-t). after extensive washing with pbs-t, membranes were incubated with anti mouse igg antibody (diluted 1:5000) conjugated to horseradish peroxidase (amersham) or anti rabbit igg (diluted 1:4000) conjugated to horseradish peroxidase (amersham). in other experiments, proteins from asfv highly purified particles were analyzed by western blotting in the same way, but membranes were probed with monoclonal anti-gfp antibody, monoclonal anti-p72 antibody and polyclonal specific antibody anti-p54 raised in rabbit (diluted 1:1000). anti mouse igg antibody (diluted 1:5000) and anti rabbit igg antibody (diluted 1:4000) conjugated to horseradish peroxidase (amersham) were used as secondary antibodies respectively. in all cases, final detection on membranes was performed with ecl western detection reagent (amersham) and exposure to x-ray films (kodak). vero cells were grown on coverslips at 50-60% confluency and then infected with ba71v or recombinant viruses at different moi depending on the experiment. at desired time postinfection cells were rinsed twice with pbs and fixed with pbs containing 4% paraformaldehyde for 10 min at room temperature. when permeabilization was required, cells were incubated in presence of 0.1% triton x-100 for 15 min at room temperature. a monospecific antiserum against p54 was raised in rabbit and used to visualize p54 (diluted 1:300). to detect tubulin a monoclonal antibody against β-tubulin (sigma) was used (diluted 1:200). secondary antibodies used were an anti rabbit igg antibody conjugated to alexa 488 fluor (molecular probes) and anti mouse igg antibody conjugated to alexa fluor 647 (molecular probes). specificity of labeling and absence of signal crossover were determined by examination of single labeled control samples. p54-egfp was directly observed by detection of fluorescence at λ = 509 nm. nuclei as well as dna in virus factories were detected by staining with hoechst 33342. finally, cells were mounted onto slides using fluorsave reagent (calbiochem). conventional microscopy was carried out in a leica photomicroscope with a digital camera, and digitized images were obtained with qwin software (leica). confocal microscopy was carried out in a leica confocal microscope tcs sp2-aobs equipped with a 63 or 100× objectives. vero cells were plated at 60% confluence onto 35 mm dishes (willco wells) and infected with 1 or 20 pfu of b54gfp-2 per cell, depending on the experiment. for the majority of experiments, synchronization of virus infection was achieved by performing the adsorption at 4°c for 90 min and following removal of unbound virus. at indicated time points, cells were imaged by confocal microscopy carried out in a leica confocal microscope tcs sp2-aobs. in either case, cells were maintained on a heated 35 mm stage with the temperature set at 37°c and fresh dmem supplemented with 2.5% fetal bovine serum was perfused onto the dish at a rate of 0.1 ml/min throughout the experiment. the acquisition of images was every 5 min in long experiments (4 to 8 h length) and every 2-3 s in shorter experiments (from 1 to 4 hpi). nevertheless, in long experiments only indicated postinfection times images are shown in figures. simultaneous acquisition of egfp fluorescence emission and transmitted light was performed. microtubules depolymerization was achieved with 10 μm nocodazole (sigma) 2 h before the infection without removing it during the acquisition of images. detection of mitochondria in live cells was carried out with mitotracker red cmxros (molecular probes) 100 nm for 30 min after virus infection. golgi complex was detected in live cells with 5 μm bodipy-trc 5 -ceramide complexed to bsa (molecular probes) for 30 min before infection, and emission detected at λ = 617. manipulation and subsequent analysis of acquired images were carried out with leica confocal software v.2.0. african swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein assembly of african swine fever virus: role of polyprotein pp220 african swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum african swine fever virus protease, a new viral member of the sumo-1-specific protease family repression of african swine fever virus polyprotein pp220-encoding gene leads to the assembly of icosahedral core-less particles electron microscopic observation of african swine fever virus development in vero cells characterization of african swine fever virion proteins j5r and j13l: immunolocalization in virus particles and assembly sites design and construction of african swine fever virus chimeras incorporating foreign viral epitopes purification and properties of african swine fever virus vaccinia virus cores are transported on microtubules association of african swine fever virus with the cytoskeleton the major structural protein of african swine fever virus, p73, is packaged into large structures, indicative of viral capsid or matrix precursors, on the endoplasmic reticulum african swine fever virus interaction with microtubules the preparation of orthopoxvirus dna two-dimensional analysis of african swine fever virus proteins and proteins induced in infected cells tracking fluorescence-labeled rabies virus: enhanced green fluorescent protein-tagged phosphoprotein p supports virus gene expression and formation of infectious particles inducible gene expression from african swine fever virus recombinants: analysis of the major capsid protein p72 movements of vaccinia virus intracellular enveloped virions with gfp tagged to the f13l envelope protein microtubule-independent motility and nuclear targeting of adenoviruses with fluorescently labeled genomes improvement of african swine fever virus neutralization assay using recombinant viruses expressing chromogenic marker genes aggresomes resemble sites specialized for virus assembly the african swine fever virus dynein-binding protein p54 induces infected cell apoptosis switching on and off the cell death cascade: african swine fever virus apoptosis regulation vaccinia virus utilizes microtubules for movement to the cell surface the dynein microtubule motor cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking visualization of the intracellular behavior of hiv in living cells construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative mitochondrial distribution and function in herpes simplex virus-infected cells virus factories: associations of cell organelles for viral replication and morphogenesis ultrastructural study of african swine fever virus replication in cultures of swine bone marrow cells apoptosis: a mechanism of cell killing and lymphoid organ impairment during acute african swine fever virus infection interaction of the rabies virus p protein with the lc8 dynein light chain genetic manipulation of african swine fever virus: construction of recombinant viruses expressing the beta-galactosidase gene characterization and molecular basis of heterogeneity of the african swine fever virus envelope protein p54 the structural protein p54 is essential for african swine fever virus viability african swine fever virus structural protein p54 is essential for the recruitment of envelope precursors to assembly sites migration of mitochondria to viral assembly sites in african swine fever virus-infected cells african swine fever virus is wrapped by the endoplasmic reticulum human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements break ins and break outs: viral interactions with the cytoskeleton of mammalian cells microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus vimentin rearrangement during african swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase ii microtubule-dependent plus-and minus end-directed motilities are competing processes for nuclear targeting of adenovirus visualization and characterization of the intracellular movement of vaccinia virus intracellular mature virions visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-b5r membrane protein chimera this study was supported by grants from the spanish comisión interministerial de ciencia y tecnología projects agl2002-00668, agl2004-07857-c03-03, bio2004-00690 and bio2005-0651. supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2006.01.021. key: cord-002222-rgqwm3vb authors: olarte-castillo, ximena a.; hofer, heribert; goller, katja v.; martella, vito; moehlman, patricia d.; east, marion l. title: divergent sapovirus strains and infection prevalence in wild carnivores in the serengeti ecosystem: a long-term study date: 2016-09-23 journal: plos one doi: 10.1371/journal.pone.0163548 sha: doc_id: 2222 cord_uid: rgqwm3vb the genus sapovirus, in the family caliciviridae, includes enteric viruses of humans and domestic animals. information on sapovirus infection of wildlife is limited and is currently lacking for any free-ranging wildlife species in africa. by screening a large number of predominantly fecal samples (n = 631) obtained from five carnivore species in the serengeti ecosystem, east africa, sapovirus rna was detected in the spotted hyena (crocuta crocuta, family hyaenidae), african lion (panthera leo, family felidae), and bat-eared fox (otocyon megalotis, family canidae), but not in golden or silver-backed jackals (canis aureus and c. mesomelas, respectively, family canidae). a phylogenetic analysis based on partial rna-dependent rna polymerase (rdrp) gene sequences placed the sapovirus strains from african carnivores in a monophyletic group. within this monophyletic group, sapovirus strains from spotted hyenas formed one independent sub-group, and those from bat-eared fox and african lion a second sub-group. the percentage nucleotide similarity between sapoviruses from african carnivores and those from other species was low (< 70.4%). long-term monitoring of sapovirus in a population of individually known spotted hyenas from 2001 to 2012 revealed: i) a relatively high overall infection prevalence (34.8%); ii) the circulation of several genetically diverse variants; iii) large fluctuations in infection prevalence across years, indicative of outbreaks; iv) no significant difference in the likelihood of infection between animals in different age categories. the likelihood of sapovirus infection decreased with increasing hyena group size, suggesting an encounter reduction effect, but was independent of socially mediated ano-genital contact, or the extent of the area over which an individual roamed. environmental contamination might be an important route for fecal-oral transmission of sapovirus, for example when spotted hyenas sniff virus infected feces or ingest water contaminated with virus infected feces. if so, individuals with a limited range may be less likely to encounter virus infected feces than those with an extensive range. in the serengeti np, adult and subadult hyenas (i.e. those !12 months of age) not only range throughout the approximately 56 km 2 of their clan's territory, but also undertake long distance foraging trips (of approximately 140 km distance round-trip) from their clan territory [46, 47] . the ranges of cubs (<12 months of age) are by comparison extremely limited, being restricted to the communal den area within the clan territory [47] . if the extent of an animal's range determines its chance of encountering virus infected feces, then all else being equal, cubs should be less often infected with sapovirus than older animals. finally, sapovirus transmission might depend on basic population parameters such as population density, principally represented by clan (group) size. if the chance of transmission increases with animal density, then individuals living in larger clans should be more likely to be infected than those in smaller clans. however, if an encounter reduction effect operates [48, 49] , then we expect the chance of susceptible individuals encountering an infected animal to decline with clan size. in humans, sapovirus infection is currently thought to provide immunological protection, at least to antigenically homologous sapoviruses, although specific immunological responses are still unknown [16] . currently nothing is known about the immunological responses of spotted hyenas to sapovirus infection, or the length of immunological protection following sapovirus infection. even so, if sapovirus infection induces long-term immunity against reinfection regardless of strain-type, we would expect cubs (i.e. naïve animals) to be more prone to infection than adults, as is the case for coronavirus infection in this species [35] . however, if sapovirus infection provides only short-term immunity, we would expect re-infections among animals of all ages. if immune responses are strain-specific, re-infection would also be expected in animals of all ages, following the appearance of a divergent strain. this study aims to advance knowledge of sapovirus infection in wild carnivore communities in africa. we report the identification of sapoviruses in wild carnivores in africa and investigate the genetic diversity of strains infecting sympatric carnivore species in the serengeti ecosystem. we assess temporal changes in sapovirus infection in a large population of individually known spotted hyenas during a period spanning more than a decade and investigate whether sapovirus infection provides long-term immunity against future infection. furthermore, we test three mechanisms likely to affect the fecal-oral spread of sapovirus infection in spotted hyenas. this study was conducted in the serengeti np, from february 2001 to march 2012. fresh fecal samples (n = 514) were collected shortly after deposition from individually known spotted hyenas including 146 samples from adults (females n = 93, males n = 53), 41 samples from subadults (females n = 20, males n = 21) and 327 samples from cubs (females n = 152, males n = 175) from three large clans (denoted in fig 2 as i, p, and m). fecal samples were also collected from other carnivores in the serengeti np (african lion, panthera leo, n = 9; bat-eared fox, otocyon megalotis, n = 9; silver-backed jackal, canis mesomelas, n = 74 samples; golden jackal, canis aureus, n = 25). following collection, feces were thoroughly mixed and divided in aliquots. tissue samples (5 intestine, 2 liver, 9 lung, 10 lymph node, 12 spleen, 10 blood, 1 muscle, 1 saliva) were also collected opportunistically from dead spotted hyenas which were mostly killed by lions or when hit by motor vehicles [40] and hence were not necessarily members of study clans, and from two other carnivore species (bat-eared fox: 2 intestines; silver-backed jackal: 1 intestine, 2 liver, 3 lung). both fecal and tissue samples were stored and transported frozen at -80°c, or were preserved in rnalater (sigma-aldrich inc., st. louis, mo, usa), stored initially at -10°c, and finally stored at -80°c until analyses [35, 36] . currently, porcine enteric calicivirus (pec) cowden strain [50, 51] is the only known sapovirus that can be cultured. hence, viral detection and initial characterization involves mostly molecular methods based on sequence data of the well-conserved rna-dependent rna polymerase (rdrp) and the variable structural vp1 genes [16, 21] . in this study, sapovirus rna was detected by targeting the highly conserved rdrp gene. total rna was extracted from 200μl of 10% (wt/vol) fecal suspension in depc-treated water using the qiaamp minelute virus spin kit (qiagen, hilden, germany), according to manufacturer's instructions. sapovirus rna was detected with the broadly reactive primer pair p289 and p290 [52] targeting highly conserved motifs in the rdrp protein of caliciviruses. based on the sequences initially generated, nested primers were designed, cali2f (5'-cag tga cag cca cat cct tg-3') and cali2r (5'-agc act gca gca gca aag ta-3'), targeting the rdrp gene. rt-pcr was performed using superscript tm iii one-step rt-pcr system with platinum 1 taq dna polymerase (invitrogen, karlsruhe, germany) following the user manual's instructions in a total reaction volume of 25μl. amplicons of the expected size were purified using the qiagen pcr purification kit (qiagen, hilden, germany). in order to avoid rnases, all surfaces were cleansed with rnase away (molecular bioproducts, san diego, ca, usa). the purified products were sequenced bidirectional using the big dye terminator cycle sequencing kit 1.1 (applied biosystems [abi], darmstadt, germany) following the manufacturer's instructions. a 3130 genetic analyzer (abi) was used for the sequencing. subsequently, sequences were assembled in geneious v 9.0.2 (biomatters ltd, auckland, new zealand) or bioedit 7.0.9.0 [53] . samples that could not be sequenced were considered positive when bands of the expected size were present with both primer pairs. for these samples the rt-pcrs were run in duplicate to ensure that the results were reliable. to obtain a longer segment of the rdrp gene, the primer 90r (5'-rcc ctc cat ytc aaa cac ta-3') was used together with the primer calir2.. genbank accession numbers for 20 sequences identified by this study are designated kt777545-kt777564. these accession number are included in our phylogenetic tree (fig 2) , together with the host species, the year in which the variant was collected and for spotted hyenas also clan membership, denoted as i,m,p if know or z if not known. all partial rdrp genes sequences (210 nucleotides, 70 amino-acids) presented the characteristic caliciviral glpsg motif. one sample from a spotted hyena in 2011 was sequenced for a longer fragment of the rdrp gene (700 nucleotides, 233 amino-acids, accession number kt777560) which presented both the glpsg motif and the ygdd motif. sapovirus sequences obtained in this study for the partial rdrp gene together with others retrieved from genbank were aligned using the muscle algorithm [54] in geneious v 9.0.6 (biomatters ltd, auckland, new zealand). at least one reference sequence of each of the five genogroups of sapovirus (gi-gv) was included in the analysis (gi, n = 5, accession numbers ay237422, ay694184, dq366345, u95644, u73124, gii, n = 3, ay646855, ay237420, ay603425, giii, n = 3, fj715800, af182760, fj387164, giv, n = 1, dq058829, gv, n = 1, ay646856). additional sapovirus sequences from domestic dog (jn387135, jn387134), california sea lion (jn420370), mink (ay144337) and bats (jn899072, jn899074, jn899075) were included. viruses from other genera in the caliciviridae family known to infect carnivores were also included, such as feline calicivirus (af098931-32) and canine calicivirus (af053720, ab070225) from the vesivirus genus, and a norovirus reported from a captive african lion (genus norovirus, ef450827). average nucleotide and amino-acid similarities were calculated using discovery studio visualizer 4.0 (accelrys software inc, san diego, usa). phylogenetic relationships were reconstructed using maximum likelihood (ml) and bayesian markov chain monte carlo (mcmc) phylogenetic inferences. the ml analysis was performed in paup ã 4.0b10 [55] using 1,000 bootstrap replicates to estimate the statistical support of the branches. the bayesian analysis was carried out using mrbayes version 3.1 [56, 57] . the mcmc search was set to 10,000,000 iterations, with trees sampled every 1,000 th iteration. the nucleotide substitution model used in the ml analysis was obtained using modeltest 3.7 [58] and for the bayesian analysis using mrmodeltest 2.3 [59] . for both cases the akaike information criterion (aic) was used to select the best-fitting model. to determine factors influencing the likelihood of sapovirus infection and changes in long-term infection prevalence we screened feces from individually recognized spotted hyenas in three study clans (i,m,p). age was estimated when individuals were first sighted as cubs, to an accuracy of ± 7 days [60] using pelage characteristics, whether their ears were flattened or upright, and their coordination during locomotion [61, 62] . we classified animals as cubs when less than 12 months of age, as subadults when between 12 and less than 24 months of age, and as adults when ! 24 months of age [63] . sex was determined by the dimorphic glans morphology of the erect phallus [64] . total clan size comprised all adults, subadults and cubs of both sexes. access to food resources in clan territories is determined by social status: all immigrant males are socially subordinate to female clan members and their offspring at food resources in the clan territory [65] . we determined the rank of adults in separate female and breeding male linear dominance hierarchies using the outcome of submissive responses in dyadic interactions within each sex, as detailed in [43, 60, 63] . to compare individual ranks across clans of different sizes, we used standardized ranks. we calculated the standardized rank of each individual within its clan on the date it was sampled using the method described by [66] . this method assigns standardized ranks between -1 (held by the animal with the lowest rank) and +1 (held by the animal with the highest rank) [60, 63] . adult females with standardized ranks higher or equal to the median standardized rank of 0 were classified as holding high social status, those with standardized ranks below 0 as low social status [43] . cubs and subadults were assigned the social status of their mother [60] . all immigrant males held a social status below adult clan females [63] . if sapovirus infection depends on intra-specific contact rates, we would expect the dynamics of social interactions within each clan to determine exposure to pathogens. for this purpose we constructed an index of social (ano-genital during greeting ceremonies) contact rates in spotted hyenas as follows. we combined social status and sex in that high ranking females and their offspring were given a high score (for contact rate), low ranking females and their offspring were given a medium score, and immigrant and reproductively active natal males were given a low score. in order to assess whether the range of an animal, the size of the area over which an individual typically roams, determines the chance of exposure to pathogens, we classified adults and subadults of both sexes with an extensive geographical foraging range as 'roaming' , because they range both within their clan territory (~55-75 km 2 ) and undertake long distance foraging trips outside the clan territory [46, 47] . cubs were classified as 'den-bound' , i.e., with a small range restricted to the vicinity of the communal den inside the clan territory. for the purpose of considering the effect of basic population parameters such as population density on incidence of infection we used total clan size on the date each animal was sampled. to investigate whether sapovirus infection provided immunity against re-infections we genetically screened feces from 91 individually known spotted hyenas from which fecal samples were obtained on at least two different dates. of these, 76 individuals were screened on two different dates, 10 individuals on three different dates and 5 individuals on four different dates. using these screening results we calculated the average interval duration between two successive sampling dates. we used nonparametric models, including the mann-whitney u-test and the kruskal-wallis test, to compare medians [67] and the kaplan-meier survivorship and the logrank test in survival analyses to compare the survivorship curves of intervals between different combinations of incidences of infection [68] . to investigate differences in the prevalence of sapovirus in the spotted hyena population studied between 2001 and 2012 we first tested for differences in the prevalence of infection across years, using a log-likelihood ratio-test. for this test we only considered years with a sample size of at least 20 individuals, thus years 2001, 2002 and 2012 were excluded where sample sizes were 17, 11 and 5, respectively. we also checked for possible differences between age categories, using the same statistical test. these analyses were run in systat version 13 (systat software inc., richmond, va, usa). we then ran models to assess which of three possible mechanisms influenced the likelihood of sapovirus infection in our study population. for this purpose we used binary logistic regression models [69] , with predictor variables contact rate, lifetime range and clan size, and ran these as mixed models with animal identity as a random variable to account for the fact that some individuals contributed more than one tissue or fecal sample to the data set. if a genetic screening result was available for more than one organ or fecal sample for an individual on the same sampling date, only one result was included in the dataset for the prevalence models; if we obtained both a positive and negative result from an animal on the same day, the positive result was selected. this applied to 7 individuals where we had two fecal samples from the same day, and to 7 individuals from which altogether 16 tissue samples were examined. we included data from all individuals sampled during the years which could either be classified as outbreak or non-outbreak years (see results). models were run with the glmer function of package lme 4 version 1.1-8 in in r (r development core team, v. 3.1.1). we used log-likelihood ratio tests and information criteria (aic and schwartz's [bic s ] and raftery's bayesian information criterion [bic r ]) to check whether the final model was superior to an intercept-only or a reduced model. models were considered similar if differences in aic were less than 2.5 and preferable if the difference exceeded 6.0 [70] ; similar if differences in bic r were less than 2.0, a positive degree of preference if values of bic r varied between 2.01 and 6.0 and a strong degree of preference if values of bic r differed by more than 6 (a. raftery in [71] , p73). as the evaluation of our models with all information criteria produced similar conclusions, we report only aic values. the significance of each predictor variable was assessed in the following way. we calculated the marginal contribution of each parameter to the full model by subtracting from the full model the log-likelihood ratio of a second model with each variable removed and testing the difference against a chi-square distribution with the appropriate degrees of freedoms (see discussions in [69, 71] ). in order to illustrate the effect of clan size on the chance of infection, we proceeded as follows. we calculated "covariate adjusted estimates" of the logits for each record over the observed range of values by adjusting them to the median of the remaining covariates (contact rate, lifetime range) of their log-odds (logit) for being infected ( [69] , p80), and then converted the resulting estimates into probabilities using the logistic equation. this permitted us to show the effect of clan size on the likelihood of infection whilst controlling for the covariates contact rate and lifetime range at their middle values. the significance threshold for all tests was fixed at 5% and all tests were two-tailed. the data used for the statistical analyses is contained in s1 table. ethics statement the study was approved by the tanzanian commission of science and technology (cost-ech) and the tanzania wildlife research institute (tawiri). permission to work in the serengeti national park was granted by the tanzanian national parks authority (tanapa). the work was also approved by the internal ethics committee of the leibniz institute for zoo and wildlife research (izw), approval no. 2011-04-03. screening targeting the conserved rdrp gene revealed sapovirus rna in feces from spotted hyena (33.3%, 171/514 samples), african lion (33.3%, 3/9 samples) and bat-eared fox (22.2%, 2/9 samples). no sapovirus rna was found in fecal samples from golden (0/25) or silverbacked jackals (0/74). sapovirus rna was found in tissue samples collected opportunistically from dead spotted hyenas (spleen, 6/12 samples, liver 1/2 samples, lymph node 2/10 samples), but not in intestine (0/5 samples) or lung samples (0/9 samples). animals with positive spleen samples were negative for sapovirus rna in their other available tissues (2 lymph nodes; 1 liver; 1 lung). a total of 20 partial rdrp gene sequences (16 from spotted hyenas, 3 from african lions and 1 from bat-eared foxes) were obtained and used for the phylogenetic analysis, together with publically available sequence data from 25 representatives of all sapovirus genogroups, divergent unclassified sapoviruses, and other genera in the caliciviridae family, including norovirus and vesivirus. the sapovirus strains from wild carnivore species in the serengeti ecosystem were placed together in one independent monophyletic cluster (fig 2) , and separately from all recognized sapovirus genogroups (gi to gv) and other unclassified sapoviruses. nucleotide sequence comparison between strains within the african wild carnivore group and other sapoviruses revealed low nucleotide similarity, ranging from 70.4% ± 1.4 with two domestic dog strains to 56.2% ± 1.5 with sequences from genogroup gii strains. at the amino acid level the highest similarity was with strains within genogroup giv (84.9% ± 1.5) and the lowest with strains from one bat species in asia (74.8% ± 0.6). nucleotide sequence comparison with members of other genera in the calicivirus family known to infect carnivores also showed low similarity values (feline and canine calicivirus; 60.3% ± 1.7 and 59.3% ± 2.1, respectively, norovirus from a captive african lion; 56.1% ± 0.9). within the group of african wild carnivore strains, the sapovirus strains from spotted hyenas grouped together and separately from those obtained from african lions and bat-eared foxes (fig 2) . one strain from a member of the p clan (all from members of the p study clan), all the clusters contained variants obtained from members of at least two of the three study clans. notably, one variant obtained from a bat-eared fox in 2011 was placed separately from those obtained from spotted hyenas in that same year, but close to the three variants obtained in 2010 from african lions. comparison of nucleotide sequences revealed that the average similarity between all strains from spotted hyenas (96.1% ± 3.8) was lower than between strains from african lions and bateared foxes (99.1% ± 0.3). the percentage similarity from the variant in 2007 placed separately from the other spotted hyena variants was lower (87.4% ± 0.6) than that of all the other spotted hyena variants that grouped more closely (97.4% ± 1.6). however, comparison at the amino acid level revealed that all strains from spotted hyenas were identical. the average similarity of the sequences from african lions and bat-eared foxes was 99.2% ± 0.9 (with differences at two amino acid positions). the average nucleotide and amino acid similarity between the spotted hyena strains and those from african lions and bat-eared foxes was 85.1% ± 1.0 and 99.6% ± 0.7, respectively. overall, sapovirus infection prevalence (combining results from fecal and tissue samples) in spotted hyenas was 34.8% (180/517 samples, table 1 ). infection prevalence between 2001 and 2012 (fig 3) fluctuated substantially between years (log likelihood ratio = 69.157, df = 11, p < 0.00001). we considered infection prevalence in any given year equal to or above 40% as indicative of an outbreak of sapovirus infection in that year. by this definition, 2003, 2004, 2006, 2007, 2010 were considered 'outbreak years' , and 2005, 2008, 2009, 2011 were considered 'non-outbreak years' in which infection prevalence was below 40%. of the 16 partial rdrp gene sequences obtained from spotted hyenas, 11 of these were from outbreak years, three were from non-outbreak years and two were from years that could not be classified (2002). in the phylogenetic tree strains from non-outbreak years clustered with those from outbreak years (fig 2) . to determine whether the prevalence of sapovirus infection was affected by age, we screened feces from animals in different age categories (i.e., cubs, subadults and adults). we found no significant differences in infection prevalence between different age categories across all years (chi square test, likelihood ratio = 2.045, df = 2, p = 0.36), in non-outbreak years (likelihood ratio = 3.860, df = 2, p = 0.15), or outbreak years (likelihood ratio = 3.331, df = 2, p = 0.19). we screened for sapovirus rna in feces obtained from 91 individuals on two separate occasions (s2 table) . of these, 15 individuals were sampled on at least three separate occasions, and from five of these animals on a fourth occasion ( table 2) . results revealed 32 transitions of an individual from sapovirus rna negative to positive and 15 transitions from positive to negative. in many cases the infection status did not change between sampling dates, for both initially negative (negative to negative, 53 cases) and initially positive individuals (positive to positive, 11 cases). we found no cases of transitions from positive to negative to positive ( table 2) . transition intervals were similar between first and second, second and third, and third and fourth sampling date (kruskal-wallis test, h = 2.567, df = 2, p = 0.28). we therefore analyzed the relationship between the duration of time intervals between successive samples and changes in infection status without regard to the number sampling repeats per individual. the duration significantly varied between different categories of changes of infection status (survival analysis, log-rank test, log-likelihood ratio = 10.114, df = 3, p = 0.018). similar intervals were observed for changes in infection status from negative to positive (mean: 466.6 days, 95% c.i. 285.5-647.6 days, median 158 days, n = 32) and from positive to negative (mean: 602.8 days, 95% c.i. 185.8-1019.8 days, median 180 days, n = 15). the shortest and longest intervals were observed when there was no change in status: negative to negative (mean: 241.1 days, 95% c.i. 132.5-349.7 days, median 82 days, n = 53) and positive to positive (mean: 769.0 days, 95% c.i. 412.5-1125.5 days, median 715 days, n = 11), respectively. we used a mixed-effects binary logistic regression model to test factors influencing the likelihood of infection in spotted hyenas (log-likelihood ratio = 16.717, df = 4, p = 0.0022, n = 484 samples from 380 individuals with complete information). the results revealed that infection was not significantly altered by either contact rates or the extent of an individual's range ( table 3 ). the likelihood of infection significantly declined as clan size increased: with every additional individual in the clan, clan members were 1.02 times less likely to be infected with sapovirus ( table 3) . as the actual clan sizes ranged from 65 to 145 individuals, this implied a more than two-fold change in the likelihood of infection across the observed range of clan sizes (fig 4) . correspondingly, median clan sizes were significantly lower during outbreak (median = 77, mean = 83.6, with 95% c.i.: 81.4-85.9) than non-outbreak (median = 89, table 2 . rt-pcr fecal screening results for known spotted hyenas sampled at least three dates. this is the first report of sapovirus infection in wildlife species in africa. our results extend the host species range for this genus to include the spotted hyena, african lion and bat-eared fox. prior to our study, sapovirus infection in carnivores worldwide was not known from any species belonging to the felidae (including the domestic cat) [72] , or hyaenidae, but was reported only for species in the families otariidae (californian sea lion) [23] , mustelidae (mink) [25] and canidae (domestic dog) [26] . our phylogenetic analysis based on partial rdrp gene sequences revealed that sapovirus strains from wild carnivores in the serengeti ecosystem formed a monophyletic group that was distinct from other sapovirus strains worldwide, including strains from the three previously identified carnivore hosts (fig 2) . strains from spotted hyena formed a separate sub-group from those obtained from african lions and bat-eared foxes, even within the same sampling year (fig 2) , suggesting that strains circulating in the spotted hyena population are distinct from those in the african lion and bat-eared fox populations. evidence for a degree of speciesspecificity in host range is apparent in other viruses of carnivores in the serengeti ecosystem. genetically distinct alphacoronavirus variants infect spotted hyenas and sympatric silverbacked jackal during the same year [35] , genetically distinct strains of kobuvirus infect domestic dogs and wild carnivores [36] , and during the 1993/1994 canine distemper epidemic in the serengeti np, genetically distinct strains circulated in non-canids (african lion and spotted hyena) and canids (domestic dog and bat-eared fox) [73] . more extensive characterization of sapovirus strains infecting carnivore species in the serengeti ecosystem would clarify their host range and help identify which species in the large carnivore guild are infected with sapovirus. currently it is not known whether or not domestic dogs and domestic cats in africa are infected with sapovirus. our results support the conclusion of previous studies, which emphasize the importance of long-term monitoring when documenting the genetic diversity of sapovirus strains [20, 28, 31] . clearly we would have detected far less genetic diversity in our partial rdrp gene sequence data had our sampling of spotted hyenas been limited to a time frame of one or two years (fig 2) , and particularly if sampling was (by chance) only undertaken during non-outbreak years when infection prevalence was low (fig 3) . samples obtained during outbreak years revealed considerable genetic diversity; for example from 2006 to 2007 we obtained sequence data from five different variants, including the distinct 2007 variant (kt777556) which was the least similar to all others from this host species. as we were not able to sequence data from all rt-pcr positive samples, we cannot exclude the possibility that the genetic diversity among spotted hyena strains was higher than our results indicate. even so, in line with a previous study [74] , our result show that sequence data from the non-structural rdrp gene yields useful information on the genetic diversity of circulating sapovirus strains. some outbreaks of sapovirus infection in humans can be linked to the emergence of specific genotypes [18, 75, 76] , suggesting that herd immunity against prevailing genotypes may be evaded by the emergence of genetically novel strains. our long-term monitoring of sapovirus infection in spotted hyenas revealed significant changes in yearly prevalence during the study (fig 3) and the occurrence of three outbreaks of infection. the highest infection prevalence (above 72.4%) occurred during an outbreak from 2002 to 2004, whereas infection prevalence in two later outbreaks (from 2006 to 2007 and in 2010) was considerably lower. presumably the 2002/2004 sapovirus outbreak induced herd immunity to the genetic strains that circulated in spotted hyenas during this period, but our phylogenetic analysis (fig 2) did not reveal the emergence of genetically distinct strains in response to this. even so, our partial sapovirus rdrp gene sequence data are insufficient to draw strong conclusions. for this, a more extensive genetic investigation is needed, particularly using the vp1 gene used to place sapoviruses in genogroups [16, 21] , that may reflect the antigenic relationships between sapovirus [77] . overall sapovirus infection prevalence in spotted hyenas (34.8%) in the serengeti np was several magnitudes higher than the prevalence reported for the domestic dog (< 2%) [26, 72] and the bat h. pomona (1.6%) [28] . moreover, our long-term monitoring reveals that infection prevalence in spotted hyenas was typically high, being above 20% in most years (fig 3) . there has been much discussion about the effect of human age on sapovirus infection (reviewed by [16] ), mostly based on studies on individuals with gastrointestinal infections. however, there is growing evidence from research on humans with and without clinical symptoms which demonstrates sapovirus infection across a wide range of ages [76] , including elderly people [78] . our results on sapovirus infection across different age categories indicate that the likelihood of infections in spotted hyenas was not significantly influenced by age ( table 1) . the long-term perspective of our study allowed us to assess the sapovirus infection status of several individually known spotted hyenas on different sampling dates. several animals transitioned from positive to negative, and we interpret this to indicate that they successfully cleared the virus following infection. if, following an initial infection, spotted hyenas gained long-term immunity against further infection, infection prevalence should decline with age. as prevalence amongst adults reached almost 50% during outbreak years, our results do not provide strong evidence for long-term immunity in this species (table 1) . even so, we found no individual that changed from rt-pcr positive to negative to positive (table 2) which would have provided direct evidence of re-infection. during outbreaks of sapovirus in humans and pigs, cases of re-infection with sapovirus belonging to different genogroups have been reported in both species, suggesting genogroup-specific immunity for sapoviruses [74, 79, 80] . more extensive investigation of the genetic diversity across strains circulating in our spotted hyena population is needed to determine whether sapovirus strains induce (1) short-term immunity, which would permit re-infection with strains from the same sapovirus genogroup, (2) genogroup-specific immunity, in which re-infection would involve strains from different genogroups, or (3) possibly a complex interplay between the two, as hypothesized for the genetically and antigenically diverse norovirus which is closely related to the sapovirus genus [81, 82] . interestingly, infection status depended on the length of time between repeated samples. animals that were negative on two separate dates (table 2 ) had the shortest median period between sampling dates (82 days), those that changed from negative to positive (158 days) and from positive to negative (180 days) had an intermediate median number of days, whereas animals that were positive on both sampling days (715 days) had the longest median period. taken together, these results suggest that when a spotted hyena is infected, infection is cleared, and reinfection is unlikely within a period of several months, which is consistent with the idea that exposure to sapovirus does not provide long-term immunity against further infection. in humans, sapovirus shedding often subsided 14 days after the onset of illness [83] , but can persist for up to 38 days [74] . hence, we speculate that the spotted hyenas which were positive on two sampling dates several months apart were animals that were re-infected rather than individuals with persistent long-term infections. however, currently we cannot exclude the possibility that there may be spotted hyenas that shed sapoviruses for periods spanning several months. spotted hyena social and ranging behavior has been shown to structure transmission routes and the likelihood of infection by a broad range of pathogens [35, [41] [42] [43] [44] ]. yet when we tested whether these two factors influenced sapovirus infection in this species, neither the predicted effect of contact rates based on known patterns of greeting ceremonies, nor the extent of an individual's range significantly influenced the likelihood of infection in our study population (table 3) . clan size was a significant factor, but contrary to our expectation, the likelihood of infection declined with increasing clan size. this phenomenon is known in the behavioral literature as the attack-abatement effect [84] or as the encounter-reduction effect [48, 85, 86] . in multi-species host assemblages the same phenomenon in the ecological literature is termed a 'dilution' effect. at least five non-exclusive mechanisms have been identified [49] that can cause a reduction in infection incidence as the number of host species increases, but not all of them are relevant to intraspecific sapovirus infections in spotted hyenas. we have no evidence that sapovirus infection increases the death rate of infected individuals (mechanism 1), as no obvious clinical signs are associated with sapovirus infection in most spotted hyenas. although we lack a precise measure of the recovery rate (mechanism 2), the general absence of obvious clinical signs of sapovirus infection suggest that the recovery rate is already very high, so that a change in this factor is likely to be modest. a decrease in the density of susceptible individuals (mechanism 3) is unlikely unless this results from a substantial increase in the proportion of clan members immune to sapovirus infection, even if immunity is relatively transient. a substantial increase in the prevalence of immune clan members would result in far fewer sapovirus susceptible individuals. moreover, such an increase in herd immunity would probably also lead to a decrease in the prevalence of sapovirus excreting clan members, thereby reducing the probability of: (1) sapovirus transmission per encounter (mechanism 4) between clan members and (2) the encounter rate between susceptible and infectious individuals (mechanism 5) in a clan. further research and more detailed modeling of the interplay between clan size and the prevalence of clan members in different sapovirus infection states (susceptible, infected/excreting virus, and immune) is required to test this idea. in the context of our study, mechanism 5 (encounter rate between susceptible and infectious individuals) is similar to mechanism 3 (decrease in the density of susceptible individuals) because a reduced density of susceptible clan members caused by a rise in the prevalence of transient immunity would also curb the number of infected animals in a clan and may prevent their number growing in proportion to increasing clan size, and possibly holding them at or below a fixed number (threshold). an increase in the risk of internal pathogen infection with a decrease in a group size component has been reported by other studies [3, 42] . as sapoviruses cannot be cultured, with the exception of the strain pec cowden [50, 51] , knowledge of viral tropism and the receptor use for entry to host cells is limited. moreover, sapovirus typically infects intestinal tissue and to our knowledge is not known to infect other tissues, as illustrated by the absence of viral rna in any extra-intestinal tissues in gnotobiotic pigs inoculated with pec cowden [87] . however, in a few dead spotted hyenas (typically road casualties) from which we opportunistically obtained tissue samples, we detected sapovirus rna most often in the spleen and occasionally also in lymph nodes, intestines and the liver. to our knowledge this is the first report of sapovirus rna in extra-intestinal tissues following natural infections. the possibility that sapoviruses disseminate to extra-intestinal tissues may be of clinical importance [88] . notably, in asymptomatic mice shedding murine norovirus in feces, viral rna was also found in several extra-intestinal organs, including the liver, spleen and lymph nodes [89] . studies on caliciviruses infecting wild carnivores have focused on feline calicivirus (fcv, genus vesivirus, family caliciviridae). for example, serological surveys documented that african lions [90] and spotted hyenas in the serengeti ecosystem were exposed to fcv [91] with a high prevalence. our phylogenetic analysis shows that the variants we report from wild carnivores in africa are distinct from both fcv and canine calicivirus (fig 2) . a norovirus (genus norovirus, family caliciviridae), genetically related to human noroviruses was reported from a captive african lion [92] and subsequently identified in domestic dogs and domestic cats [93, 94] . combining these findings with our results suggests that the spotted hyena and african lion can be infected by viruses belonging to three different genera of the family caliciviridae. we provide the first report of sapovirus infection in wild carnivore species in africa, including the spotted hyena, african lion and bat-eared fox. long-term monitoring revealed considerable genetic diversity of variants from these species which were phylogenetically distinct from previously reported sapovirus strains from other geographical areas worldwide. sapovirus prevalence in the spotted hyenas varied between years and was not influenced by age. an individual's likelihood of infection significantly declined with increasing group size, consistent with an encounter reduction effect. our results reinforce the importance of long-term studies in viral epidemiology of wild species. supporting information s1 emerging infectious diseases of wildlife-threats to biodiversity and human health wildlife as a source of zoonotic infection social organization and movement influence the incidence of bovine tuberculosis in an undisturbed high-density badger meles meles population regulation and stability of host-parasite population interactions. i regulatory processes the role of evolution in the emergence of infectious diseases population genetics and evolutionary epidemiology of rna viruses the role of constrains and limitations in driving individual variation in immune response reproduction and nutritional stress are risk factors for hendra virus in little red flying foxes (pteropus scapulatus) parasites as predators: unifying natural enemy ecology increased mortality and aidslike immunopathology in wild chimpanzees infected with sivcpz the ecology of infectious disease: effects of host diversity and community composition on lyme disease risk conservation physiology comparative endocrinology, environment and global change stress and immunosuppression as factors in the decline and extinction of wildlife populations. concepts, evidence and challenges amphibian and reptile declines over 35 years at la selva, costa rica comprehensive review of human sapoviruses caliciviruses in man epidemiology and genotype analysis of emerging sapovirus-associated infections across europe zoonotic aspects of infections with noroviruses and sapoviruses human sapoviruses: genetic diversity, recombination, and classification human sapovirus classification based on complete capsid nucleotide sequences frequent detection of noroviruses and sapoviruses in swine and high genetic diversity of porcine sapovirus in japan during fiscal year 2008 the fecal viral flora of california sea lions prevalence of porcine noroviruses, molecular characterization of emerging porcine sapoviruses from finisher swine in the united states, and unified classification scheme for sapoviruses detection and molecular characterization of cultivable caliciviruses from clinically normal mink and enteric caliciviruses associated with diarrhea in mink viruses in diarrhoeic dogs include novel kobuviruses and sapoviruses detection of zoonotic pathogens and characterization of novel viruses carried by commensal rattus norvegicus in new york city discovery and genomic characterization of a novel bat sapovirus with unusual genomic features and phylogenetic position sapovirus prevalence in children less than five years of age hospitalised for diarrhoeal disease in south africa human caliciviruses detected in hiv-seropositive children in kenya diverse sapovirus genotypes identified in children hospitalised with gastroenteritis in selected regions of south africa human calicivirus diversity in wastewater in south africa serengeti past and present fatal canine distemper infection in a pack of african wild dogs in the seregenti ecosystem coronavirus genotype diversity and prevalence of infection in wild carnivores in the serengeti national park molecular characterization of canine kobuvirus in wild carnivores and the domestic dog in africa population dynamics, population size and the commuting system of serengeti spotted hyenas. sinclair are and arcese p, editors. serengeti. ii. dynamics, management, and conservation of an ecosystem crocuta crocuta spotted hyena canine distemper virus infection in serengeti spotted hyenas regular exposure to rabies virus and lack of symptomatic disease in serengeti spotted hyenas a hepatozoon species genetically distinct from h. canis infecting spotted heynas in the serengeti ecosystem factors influencing dypylidium sp. infection in a free-ranging social carnivore, the spotted hyaena (crocuta crocuta) does lactation lead to resource allocation trade-offs in the spotted hyaena? the impact of a pathogenic bacterium on a social carnivore population the erect 'penis' is a flag of submission in a female-dominated society: greetings in serengeti spotted hyenas the commuting system of serengeti spotted hyaenas: how a predator copes with migratory prey. ii. intrusion pressure and commuters space use the commuting system of serengeti spotted hyaenas: how a predator copes with migratory prey. iii. attendance and maternal care animal grouping for protection from parasites: selfish herd and encounter-dilution effects effects of species diversity on disease risk serial propagation of porcine enteric calicivirus-like virus in primary porcine kidney cell cultures serial propagation of porcine enteric calicivirus in a continuous cell line. effect of medium supplementation with intestinal contents or enzymes design and evaluation of a primer pair that detects both norwalk and sapporo-like caliciviruses by rt-pcr bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt muscle: a multiple sequence alignment method with reduced time and space complexity paup: phylogenetic analysis using parsimony (and other methods) mrbayes: bayesian inference of phylogeny mrbayes 3: bayesian phylogenetic inference under mixed models bioinformatics modeltest: testing the model of dna substitution mrmodeltest v2. program distributed by the author evolutionary biology centre behavioral processes and costs of co-existence in female spotted hyenas: a life history perspective observations on the birth and early development of the spotted hyaena tü rk a. functions of birth dens in spotted hyaenas (crocuta crocuta) sexual conflict in spotted hyenas: male and female mating tactics and their reproductive outcome with respect to age, social status and tenure sexual dimorphism in the spotted hyaena (crocuta crocuta) the spotted hyena. a study of predation and social behavior social, state-dependent and environmental modulation of faecal corticosteroid levels in free-ranging female spotted hyenas nonparametric statistical methods applied survival analysis: regression modeling of time to event data applied logistic regression logistic regression models negative binomial regression detection of norovirus and sapovirus from diarrheic dogs and cats in japan canine distemper virus in the serengeti ecosystem: molecular adaptation to different carnivore species surveillance of pathogens in outpatients with gastroenteritis and characterization of sapovirus strains between epidemiology and genotype analysis of sapovirus associated with gastroenteritis outbreaks in phylogenetic analysis of the nonstructural and structural protein encoding region sequences, indicating successive appearance of genomically diverse sapovirus strains from gastroenteritis patients antigenic diversity of human sapoviruses sapovirus outbreaks in long-term care facilities a confirmation of sapovirus reinfection gastroenteritis cases with different genogroups and genetic shifts in the evolving sapovirus genotypes bö ttiger b. repeated examination of natural sapovirus infection in pig litters raised under experimental conditions norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations genetic and antigenic diversity among noroviruses natural history of human calicivirus infection: a prospective cohort study attack abatement: a model for group protection by combined avoidance and dilution parasitism and group size in social animals: a meta-analysis parasites and the behavior of animals comparative pathogenesis of tissue cultureadapted and wild-type cowden porcine enteric calicivirus (pec) in gnotobiotic pigs and induction of diarrhea by intravenous inoculation of wild-type pec the mucosal lesion of the proximal small intestine in acute infectious nonbacterial gastroenteritis desk encyclopedia of human and medical virology prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in east africa antibodies to canine and feline viruses in spotted hyenas (crocuta crocuta) in the masai mara national reserve norovirus in captive lion cub (panthera leo). emerg infect dis detection and molecular characterization of a canine norovirus discovery and genomic characterization of noroviruses from a gastroenteritis outbreak in domestic cats in the us key: cord-273965-ma1rwkdq authors: omonzejele, peter f. title: preserving bodily integrity of deceased patients from the novel sars-cov-2 pandemic in west africa date: 2020-11-09 journal: j bioeth inq doi: 10.1007/s11673-020-10061-4 sha: doc_id: 273965 cord_uid: ma1rwkdq the outbreak of the novel coronavirus pandemic, otherwise known as covid-19 brought about the use of new terminologies—new lexical items such as social distancing, self-isolation, and lockdown. in developed countries, basic social amenities to support these are taken for granted; this is not the case in west african countries. instead, those suggested safeguards against contracting covid-19 have exposed the infrastructural deficit in west african countries. in addition, and more profoundly, these safeguards against the disease have distorted the traditional community-individuality balance. the enforcement of social distancing, self-isolation, and lockdown has made it impossible for west africans to drift to their ancestral homes and villages, as is usually the case in times of crisis, with attendant consequences for communal life and traditional burial rites. this could be one of the reasons why some covid-19 patients are escaping from isolation centres, since to die in such centres violates their bodily integrity at an ontological level. the sars-cov-2 (hereafter referred to as pandemic that started in china in december 2019 found its way to west africa in february 2019. this allowed most west africans to play the ostrich for about three months as they had thought the virus unlikely to survive in the warm climate of west africa. it is true, generally, that african countries have recorded fewer cases of covid-19 than countries in europe and america. what is not clear at the moment is if a warmer temperature is responsible for the low record of cases or simply a lack of kits for extensive testing. there is no scientific evidence to show that africans have special immunity against the virus, but at present, africa presents the least number of confirmed covid-19 cases compared to america, asia, and europe. according to the statistics provided by the european centre for disease prevention and control (2020) , as at june 26, 2020, africa had accumulated 347,779 confirmed covid-19 cases, whereas asia had 2,036,409, america 4,821,970, and europe 2,365,834. covid-19 deaths at the same date are africa 9,069, asia 51,471, america 238,690, and europe 189,812. perhaps due to the comparatively low death rate, some people in africa have argued that there was no such thing as covid-19. they have further opined that what the west refers to as covid-19 is just a form of acute malaria. this line of thinking might be premised on some similarities of symptoms (such as high temperature and fatigue) associated with both diseases, which in some cases have posed screening challenge (chanda-kapata et al., 2020, 151-153) . this is unlike the case of the ebola epidemic, which had a distinctive haemorrhagic fever and a high fatality rate. despite the scepticism expressed by some people in sub-saharan africa about the reality of covid-19, elementary epistemology teaches that there is a difference between belief and truth. for example, i couldfor whatever reason-choose to believe whatever catches my fancy. this could be true or false. however, truth or facts are independent of my belief and how i feel about something or about the truth of it. africans will have to face up to the fact that the world is dealing with a virus of a global proportion and so take appropriate steps to deal with it. in fact, most governments in africa are struggling to address challenges posed by the reality of the pandemic. the covid-19 pandemic has exposed the general infrastructural deficit in the region, particularly west africa, as governments in the region grapple to address the challenges it has posed. the immediate step taken by most west african leaders was to shut their air and land borders to countries with high incidences of covid-19. some people, especially those of the opposition parties, have argued that the decision was taken belatedly. they argued that given the weak health infrastructure in the region, those countries ought to have acted earlier-even before they recorded any incident of the disease, as all primary cases of covid-19 were imported into the region. despite those attacks, regional governments took steps to place bans on public gatherings for sporting events and in worship areas, the workplace, and schools. in short, countries in the west african region were shut down. those preventive steps are similar to those taken by other countries around the world. the world health organization (who) suggested several preventive ways to avoid contracting the disease, some of which are regular handwashing with running water and soap, social (physical) distancing, and the use of alcohol-based hand-sanitizers. west african leaders keyed into this and communicated it to their citizens through print and electronic media. however, the challenge is that while some of those measures may appear commonplace and ordinary in western countries, the same cannot be said for most west african countries where these safeguards may be considered exotic and extraordinary, depending on the density of poverty in that region. the west african "region includes some of the poorest countries in the world … as nine of the 25 poorest countries are in the region" (martinez-alvarez et al. 2020, e631) . this means that simple safeguards such as handwashing, hand sanitizers, and so on may prove difficult in many communities. let us examine the practicality of employing those safeguards. during the 2014 ebola epidemic in western africa, the challenges associated with regular hand washing with running water were also apparent. though the who has suggested it as one of the most effective ways to prevent contracting the covid-19 virus, the fact remains that many west african communities do not have access to running water, especially those in rural areas (omonzejele 2014, 418) . according to the world bank report of 2016 on water and sanitation, over 663 million people lack access to drinking water and about half of those people are in sub-saharan africa (world bank 2016). this means that the suggested use of handwashing against contracting covid-19 is not practicable for many citizens in the west african region, especially those in rural areas. the alternative, use of hand sanitizers, is also a challenge due to poverty. however, government agencies, corporate bodies and non-governmental organizations have assisted with the production and free distribution of hand-sanitizers and also face masks. it is, however, not clear whether, and to what extent, all rural dwellers benefited from the gesture. at the level of care for those who have contracted covid-19, everyone knew, ab initio, it was going to constitute a challenge. within the first month of managing people for the condition, there were shortages of test kits, ventilators, personal protective equipment, and so on. at some point, the old saying that necessity is the mother of invention came to bear. for instance, in senegal, with only fifty ventilators nation-wide, scientists in collaboration with mologic, a british biotech company, "developed a covid-19 test kit that cost $1 and a ventilator which cost $60. the kit can deliver results in 10 minutes and can be used at home like a pregnancy kit" (oguntola 2020) . in comparison, imported ventilators cost $16,000 each. nigerian engineers and scientists in tertiary institutions have also reportedly manufactured low-cost ventilators as well. in the meantime, the chinese government, the european union business organization, and wealthy individuals have provided assistance in the form of medical equipment, technical assistance, and much-needed financial support, among other things, to build standard isolation centres. however, something unusual has continued to happen in isolation centres in nigeria and perhaps in other west african countries. some patients in the isolation centres for covid-19 management have either escaped or attempted to escape from those centres. instances of patients escaping from isolation centres are so rampant that nigeria's leadership newspaper devoted a special column to it. according to morphy (2020) , in the nigerian state of osun, "six persons, who had tested positive for covid-19, had escaped from the isolation centre without trace." in addition, he reported that in abuja, the administrative headquarters of nigeria, "that a lady suspected to be infected with the novel coronavirus allegedly escaped from an isolation centre in abuja and was reportedly traced and arrested in akwanga, in nasarawa state." in a similar vein, "a commercial driver who tested positive to coronavirus in niger state had reportedly escaped from the isolation centre in bida." there are several instances that show that these successful and failed attempts to escape from covid-19 treatment centres are not isolated cases. escapes and attempts to escape by some patients from those centres have nothing to do with the quality of the isolation centres in nigeria-which meet who standards. instead, there appear to be deeper reasons for such seemingly "unreasonable" behaviour, as a patient's stay at an isolation centre does not attract any cost to the patient for medical care, nutrition, and general care. what then is the reason? one of the reasons is discoverable in the west african traditional principle of communityindividuality and traditional understanding of bodily integrity for a deceased member of a community. the phrase community-individuality was first used by iroegbu (1995, 345) to describe a way of life which has been in place in traditional african communities since time immemorial. he used the phrase to explain the relationship that exists between the community and the individual in the african context. speaking from a west african perspective, the human person is understood as a being in communion with others and not as an individual entity. for instance, it would be impossible to define the human person in isolation from that person's relationship with his kindred and ancestral community. a person's life is intertwined with that of his ancestral community, in life and in death, and there are ceremonies and rituals associated with them. one of the events in the life of the west african is burial rites. a burial rite is contingent upon having a corpse. corpses of deceased patients from covid-19 are not recoverable as they are interred by relevant government agencies to curb further transmission of the virus. this is a serious challenge for the west african mind. this is because for the west african, the human body is not a mere container but an entity of intrinsic worth. the intrinsic worth of a person is weaved into those values which are associated with all humans, such as the entitlement for all humans to be treated with respect. but in addition, another-second-layer of intrinsic worth of humans has to do with the specific talents and abilities associated with that individual. on the demise of a person, some of those attributes evaporate with the individual, except for posthumous appreciation of their achievements. the need to dispose of such a body in ceremonial rites is associated with this second level of intrinsic worth. this is the reason a more dignified public burial ceremony (this is different from traditional burial rites) is accorded a deceased prime minister over that of a peasant farmer. however, for the west african, there are other traditional rites associated with the appreciation of a deceased relative, as it is believed that such a deceased has migrated to another phase of life, where they serve as gobetween between the families and communities they left behind and the other world where the deceased now resides. for the west african, this is another level of intrinsic human worth that is only possible after physical death, especially where the deceased was elderly-and the elderly are usually the most susceptible to the covid-19 virus. it is not any wonder that west africans spend a lot of money as part of burial rites on a presumably "lifeless" body of a departed one. based on tangwa's (2000, 41) correct explanation that african themes and traditions are similar in their essentials, i will use the tradition of the esan people of nigeria to explain what constitutes traditional burial rites in western africa and the implication of its denial. on the death of an elderly man (it could be a woman): before the body was ready for burial, it was washed, a new pot being used for the water. if the dead man was well-to-do the egbele demanded the slaughtering of a goat called ebhe ihion in honour in honour of the sponge with which the body had been washed. the body was then wrapped in a new mat and brought to the front of the main building, for the ceremony with eman elinmin, the special foufou prepared simply with fish, or with goat flesh in the case of a wealthy man. the children assembled round the body that was lying with the head towards the house. on behalf of the first son, one of the iko egbele blessed the dead and cut some of the foufou at the feet. the children and all the descendants knelt beside the body and were given a bit of the food one by one. the body was then placed on a form made with seven fresh mid-ribs of palm branches. the whole corpse is then wrapped with a white piece of cloth provided by the heir. the body is then taken on the head by two men, with the feet in front. they then headed for the cemetery with the heir holding the pot that has been used during the washing. when the body has been interred a pot was placed on the tomb. (okogie 1994, 173-174) there are slight variations to burial rites allowing for peculiar circumstances of the dead and communities. based on some aspects of the burial rites indicated above, it is easy to understand why someone suffering from a potentially fatal disease such as covid-19 will escape from management centres, as it is common knowledge that bodies of the dead are not given to family members for burial rites. though the fatality rate is low, the fact still remains that the disease is deadly and has no cure, hence, if contracted, one could die from it, and corpses of the deceased will not be released by the government for traditional burial rites. it is for this reason some people prefer to go to their villages where they resort to the use of local herbs. for them, it is considered a win-win situation. if they are healed from the use of local herbs, then, that is splendid. in the event that they succumbed to the disease, their people despite the contagious nature of the disease must of necessity devise ways to accord them traditional burial rites which will accord such bodies moral and spiritual benefits. this again is a win, for to die without traditional burial rites is inconceivable and abominable. west africans believe that the departed is still in communion with the living. this is the third level of intrinsic worth of persons. hence, it is commonplace to talk of one's ancestors and their spiritual usefulness. but it is impossible to talk of an ancestral being that did not go through the purification of traditional burial rites which accords the deceased with bodily integrity. this means that beyond the body, mind, and soul, the human body on its earthly demise and after burial rites transcends to an ontological level that mbiti referred to as "the living dead" (mbiti 1969, 83-275) . in similar vein, gbenda (2006, 7) adds that "… the ancestors are believed to be the living-dead …they were good people in their earthly life and fulfilled societal obligations and duties, they are now spirits." but what qualifies the dead to be an ancestor, that is, "a living-dead"? oguejiofor (1996, 25) provided the response as: the individual must live a morally upright life. he must also be successful as a person, and his success is shown in his possession of wife (or wives) and children especially male children, and enough material possession. he must have died a good death … not of mysterious illness or by accident (onwu ekwensu). these conditions must be accompanied by a befitting burial [with which] the dead are received in the land of the spirit (ala mmuo) by his ancestors. this clearly shows that burial rites are central to the ontological transcendence to the level of the "livingdead." this in turn allows for ancestor veneration (nwanfor 2017, 41-42) . these rites cannot be undertaken in the absence of a corpse and so might be responsible for the escape of patients from isolation centres. within the west african context, the community is the placenta through which the individual derives his sustenance and being. but the placenta (the community) is meaningless without the individual it nourishes. in times of crisis, such as the covid-19 pandemic with no known cure, one simply went back to one's ancestral community to seek remedy or cure for one's condition. in the event the person succumbs to the illness, the person at least receives a befitting burial which accords posthumous bodily respect. this is the most probable reason patients continue to escape from covid-19 isolation centres. since this is the most probable case for escapes from isolation centres, governments in the west african region should, perhaps, engage with the who to seek technical expertise in the decontamination of deceased people from covid-19 isolation centres, and then make such corpses available to their families for traditional burial rites. funding no funding support for the research. conflict of interest no conflict of interest. covid-19 and malaria: a symptom screening challenge for malaria endemic countries african religion and christianity in a changing world. a comparative approach european centre for disease prevention and control metaphysics: the kpim of philosophy covid-19 pandemic in west africa african religions and philosophy danger! covid-19 patients are escaping from isolation centres. leadership the living-dead (ancestors) among the igbo-african people: an interpretation of catholic sainthood eschatology, immortality and igbo philosophy of life senegal scientists develop $1 covid-19 testing kit, $60 ventilator. leadership esan native laws and customs with ethnographic studies of the esan people ethical challenges posed by the ebola virus epidemic in west africa the traditional african perception of a person: some implications for bioethics understanding poverty-water publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-304909-3wmrjlhy authors: wicker, l. v.; canfield, p. j.; higgins, d. p. title: potential pathogens reported in species of the family viverridae and their implications for human and animal health date: 2016-06-30 journal: zoonoses public health doi: 10.1111/zph.12290 sha: doc_id: 304909 cord_uid: 3wmrjlhy the viverridae is a family of nocturnal carnivores including civets, genets and african linsangs. while a list of known organisms isolated from a species is an essential tool for population management, this review represents the first attempt to collate published reports of organisms isolated from viverrids. a wide range of organisms, including 11 viruses, eight bacterial species, one internal arthropod species, representatives from eight genera of protozoan, 21 genera of nematode, seven genera of cestode, eight genera of trematode and six genera of external arthropod (mites, ticks and louse), have been reported in literature spanning over a century of research. many of these are capable of infecting multiple hosts, including humans. this is of concern given the anthropogenic factors that bring humans and domestic species into close contact with viverrids, facilitating transmission and spillover of organisms between groups. these factors include trade in viverrids for human consumption, captive management in zoos, rescue centres or on commercial breeding farms, and the increasing overlap of free‐ranging viverrid distribution and human settlement. the family viverridae is a diverse group of small, nocturnal carnivores, including 34 species of civet, genet and african linsang distributed across southern europe, africa and asia (wilson and reeder, 2005) . viverrids are commonly exploited for human consumption, being farmed and hunted for their fur, meat, 'civet' scent, to produce civet coffee, or to be kept as zoo animals or pets (schreiber et al., 1989; balakrishnan and sreedevi, 2007; shepherd and shepherd, 2010; d'cruze et al., 2014) . while habitat loss has contributed to historical population declines, hunting for human consumption is considered the most significant threat to their global conservation (schreiber et al., 1989; shepherd and shepherd, 2010 ). an inventory of organisms known to be associated with a particular species is considered essential baseline information for species management (munson, 1991) . this information guides rational development of husbandry protocols for captive populations (hope and deem, 2006) , facilitates design of surveillance programmes to investigate change in species disease profiles over time (lonsdorf et al., 2006) and highlights the threatening role infectious diseases might play in endangered species management (gilbert et al., 2014) . given the potential for transmission of diseases between wildlife, humans and livestock (jones et al., 2008) , a complete list of pathogens and indigenous microflora also enables evidence-based risk assessment of human and animal contact with the species in question . no attempt has yet been made to collate the current knowledge of potential pathogens detected from species within the family viverridae (shepherd, 2008; bongiovanni et al., 2014) despite the family's long history of exploitation for human consumption (abebe, 2003; shepherd and shepherd, 2010) , the management of threatened viverrids for conservation (roberton et al., 2002) , the peri-urban habitation of many species within the family (king et al., 1993; ninomiya et al., 2003; sato et al., 2013) and their susceptibility to a number of important zoonotic pathogens including the novel coronavirus responsible for the outbreak of severe acute respiratory syndrome (sars) (guan et al., 2003; tu, 2004) , rabies virus (matsumoto et al., 2011) and highly pathogenic avian influenza h5n1 (hpai h5n1) (roberton et al., 2006) . while many organisms have been identified in a very wide range of viverrid species globally, this information is scattered throughout the scientific and grey literature. an accessible list of organisms isolated from viverrids does not currently exist. this review represents the first collation of currently recognized organisms isolated from the viverridae, derived from an extensive and intensive assessment of the literature. in doing so, it expands on our understanding of viverrid health and the key epidemiological factors of infectious disease involving free living and captive wildlife. more importantly, this review assesses the significance of isolation of organisms from viverrids in terms of the implications for public health. anthropogenic factors including wildlife trade, the keeping of viverrids in captivity and land use change all encourage increased contact between viverrids and other species, thereby facilitating the transmission and spillover of these organisms between humans, viverrids and other animals. a systematic review of the scientific literature resulted in this first collation of all organisms previously reported for species from the carnivorous family viverridae. all available fields of the databases google scholar, web of science, pubmed and scopus were searched using all possible combinations of the following key terms: 'viverrid*' or 'civet' or 'linsang' or 'binturong' or 'genetta' (the scientific name for the genus genetta was used to reduce the number of papers on 'genetics' returned by the search), and 'disease' or 'pathogen' or 'vir*' (to locate both virus and viral) or 'bacteria*' (to locate both bacteria and bacterial) or 'parasit*' (to locate parasite or parasitic) or 'fung*' (to locate both fungus and fungal). no limitations were placed on date of publication. additional papers were identified by scanning the reference lists and 'cited by' lists of selected papers. for all organisms, complete primary references describing original research were sought. while this review resulted in important baseline information, a number of limitations are acknowledged. it is undoubtedly incomplete as many more reports are likely to exist in the medical records of zoos and rescue centres that hold viverrids, unavailable to a database literature review. inaccuracies are also likely included due to the reliance on traditional approaches to identification, such as descriptions of morphology (nadler and de leon, 2011) . increased availability of molecular methods has significantly improved accuracy in the detection and identification of organisms (bhadury et al., 2008; thompson et al., 2009) , resulting in a revision of numerous taxonomic trees (brown-elliott et al., 2006; balajee et al., 2009) . future research may find some organisms listed here no longer considered valid species, and advancing molecular techniques mean that future revision of this list of recognized organisms of viverridae are inevitable. a total of 98 peer-reviewed publications reported the identification of organisms from species within the family viverridae. these included 10 viruses, eight bacterial species, a single internal arthropod species and representatives from 21 genera of nematode, seven genera of cestode, eight genera of trematode, six genera of external arthropod, and eight genera of protozoan, many of which were not identified to species level. these organisms are presented in detail in tables 1-7. of the ten viruses reported for viverridae (table 1) , three were zoonoses of considerable public health significance. these were the sars coronavirus (sars cov) (guan et al., 2003; poon et al., 2005) ; hpai h5n1 (roberton et al., 2006; wu et al., 2013 ) and a number of variants of the rabies virus (enurah et al., 1988a; wilde et al., 1991; tremlett et al., 1994; nel et al., 2005; susetya et al., 2008; pfukenyi et al., 2009; matsumoto et al., 2011) . three important viruses of domestic carnivores were also reported, including canine distemper virus (cdv) (machida et al., 1992; hur et al., 1999; chandra et al., 2000; l opez-peña et al., 2001; hirama et al., 2004; chen et al., 2007; takayama et al., 2009) , feline parvovirus (caused by the feline panleukopenia virus, fpv) (ikeda et al., 1999; demeter et al., 2009) and canine parvovirus (cp) (santos et al., 2009; xiao-ying et al., 2011; duarte et al., 2013) . in more recent years, the use of advanced molecular techniques has enabled the identification of five novel viruses of uncertain significance. these were an orthoreovirus (mammalian reovirus mpc/04) isolated (guan et al., 2003; tu, 2004; kan et al., 2005; wu et al., 2005; wang and eaton, 2007) family (enurah et al., 1988a; wilde et al., 1991; tremlett et al., 1994; nel et al., 2005; susetya et al., 2008b; pfukenyi et al., 2009; matsumoto et al., 2011) ikoma lyssavirus (machida et al., 1992; hur et al., 1999; chandra et al., 2000; l opez-peña et al., 2001; hirama et al., 2004; chen et al., 2007; takayama et al., 2009; techangamsuwan et al., 2014) family from masked palm civet (paguma larvata) in china (shao et al., 2008 (shao et al., , 2010 ; the ikoma lyssavirus, a novel divergent lyssavirus (marston et al., 2012) ; two picornaviruses (genet faecal theilovirus (gftv) and a kobuvirus genotype); and a picobirnavirus (genet faecal picobirnavirus) (bodewes et al., 2014; ) . reports of eight bacterial species, all of which are multihost organisms with zoonotic potential, were found (table 2 ). nine serovars of leptospira interrogans, including some known to cause leptospirosis in humans (mill an et al., 2009) , were cultured (smith et al., 1961; tsai et al., 1973) or identified on serology (mill an et al., 2009; moinet et al., 2010) , and the gram-negative bacteria bartonella henselae, responsible for cat scratch fever in humans, was isolated from a wild, peri-urban-dwelling masked palm civet (sato et al., 2013) . a number of reports also describe isolation of enteric bacteria from viverrids. these include salmonella enterica serovar glostrup (falade and durojaiye, 1976) ; klebsiella pneumoniae (enurah et al., 1988b) ; and plesiomonas shigelloides (bardon, 1999) , an escherichia coli with a broad antibiotic resistance panel from a captive masked palm civet (ahmed et al., 2007) , and salmonella enterica enterica (serovars enteritidis, nagoya and 4,12:i), yersinia pseudotuberculosis and campylobacter spp. from peri-urban, free-ranging masked palm civets (lee et al., 2011) . of the vectorborne protozoans recognized for viverrids (table 4) , those carried by ticks include hepatozoon canis (laird, 1959) and six babesia spp. (l eger and l eger, 1920; heisch, 1952; peenen et al., 1968; tolosa and regassa, 2007) . the trypanosomes, trypanosoma brucei and t. congolense (njiokou et al., 2006; tolosa and regassa, 2007) , and leishmania donovani, the protozoan responsible for visceral leishmaniasis in humans, were reported (hoogstraal and heyneman, 1969) . species from four genera of non-vector-borne protozoan were reported (table 4 ). these included the zoonotic intracellular protozoan toxoplasma gondii (janitschke and werner, 1972; lopes et al., 2011) ; the coccidian isospora spp. (colon and patton, 2013; su et al., 2013) ; eimeria spp. (colon and patton, 2013) ; and three species of giardia including giardia dasi (abraham, 1962) , g. hegneri (chu, 1930) and the zoonotic g. duodenalis, one of the few organisms confirmed using molecular (polymerase chain reaction (pcr) on faeces of a captive common palm civet, paradoxurus hermaphroditus) rather than morphological techniques (beck et al., 2011) . some of the earliest papers found during this review reported on nematode (roundworm) parasites. over two centuries of research has reported on nematodes isolated from a very wide range of viverrid species (table 5) . while many were not identified to species level, nematodes representing 21 genera from 16 families have been reported in 25 published papers. these included toxocara spp. (warren, 1972; maung, 1975; alvarez et al., 1990; colon and patton, 2013; su et al., 2013) ; ascaris spp. (xavier et al., 2000; ajibade et al., 2010; pradhan et al., 2011) ; the hookworms ancylostoma spp. and uncinaria longespiculum (baylis, 1933; chowdhury and schad, 1972; coumaranem and mohan, 2008; colon and patton, 2013; su et al., 2013) ; strongyloides sp. threadworms (su et al., 2013) ; crenosoma sp. lungworms and viverrostrongylus brauni (myers and kuntz, 1969; asakawa et al., 1986; colon and patton, 2013) ; mammomonogamus sp. (colon and patton, 2013) ; trichuris spp. whipworms (colon and patton, 2013; su et al., 2013) ; capillaria sp. (colon and patton, 2013; su et al., 2013) ; rictularia spp. (baylis, 1928; chen, 1937; schmidt and kuntz, 1967; kumar et al., 2005; mahali et al., 2010) , spirura sp. (casanova et al., 2000) ; spirocera sp. and cyathospirura seurati (su et al., 2013) ; physaloptera sp. (casanova et al., 2000) ; gnathostoma sp. (probably g. spinigerum) (colon and patton, 2013) ; brugia spp. and dirofilaria sp. (edeson and wilson, 1964; masbar et al., 1981) ; and trichinella spp. (wang et al., 2012) . nematodes were commonly identified by the examination of morphological features of adult worms, eggs, larvae or oocysts under light microscopy. only a single reference described the use of molecular diagnostic techniques, in this case pcr and genetic sequencing to accurately identify zoonotic trichinella spiralis in infected muscle tissue of a masked palm civet in china (wang et al., 2012) . platyhelminthes (flatworms) from two classes (cestoda and trematoda), many of which have zoonotic potential, were also reported ( table 6 ). cestode tapeworms from four families included echinococcus sp. and taenia spp. (mahannop et al., 1984; ndiaye et al., 2002; millan and casanova, 2007; lahmar et al., 2009 ); diplopylidium spp. and joyeuxiella pasqualei (casanova et al., 2000; millan and casanova, senegal genet (g. senegalensis) not discussed sudan (wild) (hoogstraal and heyneman, 1969) z, zoonotic potential acknowledged; m, protozoan affects multiple hosts other than viverridae. (chu, 1930; abraham, 1962; beck et al., 2011) z, zoonotic potential acknowledged; m, protozoan affects multiple hosts other than viverridae. (schrank et al., 1788; warren, 1972; maung, 1975; alvarez et al., 1990; casanova et al., 2000; colon and patton, 2013; su et al., 2013) family (baylis, 1928; chen, 1937; schmidt and kuntz, 1967; casanova et al., 2000; kumar et al., 2005; mahali et al., 2010) family (casanova et al., 2000) 2007); mesocestoides spp. (casanova et al., 2000; su et al., 2013) ; and spirometra erinaceieuropaei and diplobothrium sp. (uchida et al., 2000; xavier et al., 2000) . flukes from the class trematoda reported for viverrids included species from eight genera, in six families. these were as follows: echinochasmus hangzhouensis and artyfechinostomum sp. (xida, 1990; varadharajan and kandasamy, 2000) ; metagonimus yokagawai (uchida et al., 2000) ; opisthorchis viverrine and metorchis albidus (nicoll, 1927; casanova et al., 2000) ; schistosoma japonicum (carney et al., 1978; he et al., 2001) ; brachylaema spp. (baugh, 1962; casanova et al., 2000) ; and paragonimus spp. in a wide range of civet species (chen, 1959; dissanaike and paramananthan, 1961; sachs et al., 1986; gang et al., 1999; aka et al., 2009; colon and patton, 2013; su et al., 2013) . the vast majority of platyhelminthes reported for viverrids were identified by examining morphology of different stages within the parasite's life cycle. only one paper cited the use of a molecular biochemical test, an enzyme-linked immunosorbent assay (elisa), to identify echinococcus sp. antigen in common genet (genetta genetta) faecal samples from tunisia (lahmar et al., 2009) . both external and internal arthropods from seven genera, in four families -sarcoptidae (mites), ixodidae (ticks), trichodectidae (chewing lice) and porocephalidae (pentostomes)were reported for viverrids (table 7) . the sarcoptid mite, notoedres cati, a common parasite of domestic and wild felids, was reported for urban-dwelling masked palm civet in japan (ninomiya et al., 2003) . many species of ixodid tick, most of which are themselves carriers of zoonotic pathogens, were also reported, including amblyomma testudinarium (grassman et al., 2004) ; ixodes ovatus (tanskul et al., 1983; robbins et al., 1997) ; a number of haemaphysalis spp. (hoogstraal and trapido, 1966; hoogstraal, 1971; tanskul et al., 1983; tolosa and regassa, 2007; mediannikov et al., 2012) ; rhipicephalus spp. (grassman et al., 2004; tolosa and regassa, 2007) ; dermacentor tawianensis (hoogstraal et al., 1986) and d. auratus (hoogstraal and wassef, 1985) . the chewing louse, felicola bengalensis was collected from a wild civet (changbunjong et al., 2011) , and one internal arthropod parasite, the pentostome armillifer moniliformis, was reported for a range of civet species (stabler and self, 1967; krishnasamy et al., 1981) . identification of arthropods was based on morphology of varying life cycle stages of the organisms collected from viverrids. while previous authors have suggested that very little is known about the pathogens of viverrids (williams and thorne, 1996; shepherd, 2008; bongiovanni et al., 2014) , this review identified a wide range of viral, bacterial, knowledge of the significance of isolation of an organism for animal health builds on the baseline information provided by a simple inventory of organisms for the species. observation of clinical signs in infected individuals contributes to our understanding of pathogenicity. detailed descriptions of clinical signs seen in viverrids were included in a small number of reports, including an investigation into morbidity or mortality in viverridae (roberton et al., 2006) , and the documentation of experimental infection of viverrids with sars cov (wu et al., 2005) . however, most (chen, 1959; dissanaike and paramananthan, 1961; sachs et al., 1986; gang et al., 1999; aka et al., 2009; colon and patton, 2013; su et al., 2013) z, zoonotic potential acknowledged; m, platyhelminth affects multiple hosts other than viverridae. (hoogstraal and trapido, 1966; hoogstraal, 1971; tanskul et al., 1983; tolosa and regassa, 2007; mediannikov et al., 2012) rhipicephalus (hoogstraal and wassef, 1985; hoogstraal et al., 1986) family (stabler and self, 1967; krishnasamy et al., 1981) z, zoonotic potential acknowledged; m, arthropod affects multiple hosts other than viverridae. reports listed here provide no information on the presence or absence of clinical signs associated with infection, as many document researches carried out on stored biological samples (moinet et al., 2010) , on samples collected at necropsy (santos et al., 2009; duarte et al., 2013) , or on samples, such as faeces, collected opportunistically without seeing the individual animal (colon and patton, 2013; su et al., 2013) . in other reports, including all reported nematodes in viverrids (table 3) , clinical signs were simply not discussed. in these cases, it is not possible to elucidate whether isolation of these organisms from clinically unwell viverridae explains the presence of disease, or whether it is simply an incidental finding. knowledge of the role a host plays in the life cycle of an organism provides further understanding of the significance of its isolation, and assists in the prevention or management of outbreaks of disease caused by the organism. viverrids are understood to be infective hosts, capable of transmitting disease, for a small number of organisms. these include the sars cov, for which masked palm civets were important amplification and transmission hosts in the epidemic of respiratory disease caused by the organism in southern china in 2003 (guan et al., 2003) . viverrids also transmit the infective life cycle stage of a number of macroparasites including the sarcoptid mite, notoedres cati (ninomiya et al., 2003) and helminthes toxocara genettae (sanmartin et al., 1992) , ancylostoma sp. (coumaranem and mohan, 2008) , rictularia sp. (schmidt and kuntz, 1967; kumar et al., 2005) , taenia sp. (mahannop et al., 1984; millan and casanova, 2007) and diplopylidium monoophorum (millan and casanova, 2007) . however, for many more, the significance of viverrids as hosts is probably insignificant. for example, wild common genets are dead end hosts for leptospira interrogans serovars (icterohemorragiae and ballum) (mill an et al., 2009) ; and binturong (arctictus binturong), small-toothed palm civet (arctogalidia trivirgata), masked palm civet, common palm civet and otter civet (cynogale bennettii) are believed to be accidental hosts for the pentostome armillifer moniliformis (stabler and self, 1967; krishnasamy et al., 1981; ) . in many other cases, the opportunistic identification of organisms from a small sample size, a recognized impediment to disease surveillance in wild species (stitt et al., 2007) , hampers our ability to understand the role of viverridae in the organism's life cycle. the amount of useful epidemiological information obtained is particularly limited where a multihost organism, such as the ixodid tick amblyomma testudinarium, is identified from just one individual viverrid (grassman et al., 2004) . positive results obtained through opportunistic sampling of small numbers of individuals remain useful in that they provide information on the presence of potential new carriers for an organism, but further research is required to understand the role that viverrids play in the organism's life cycle, or their significance in the epidemiology of the diseases which they cause. some of the organisms listed in this review may be of significance for the conservation of threatened populations of viverrid or other wild carnivores. canine distemper virus (cdv), feline panleukopenia virus (fpv) and canine parvovirus (cp) ( table 1 ) are all highly contagious and cause significant morbidity and mortality in susceptible carnivore species. infected carnivores shed a high viral load in faeces and other bodily secretions (deem et al., 2000; steinel et al., 2001) , facilitating environmental contamination and rapid spread of disease. infection of susceptible animals does not require direct contact as transmission may be via inhalation of aerosolized respiratory secretions or ingestion of contaminated material from the environment (deem et al., 2000; steinel et al., 2001) . spillover of canine distemper virus from the domestic dog has already impacted several wild carnivore populations, including african wild dogs (lycaon pictus) (fanshawe et al., 1991) , santa catalina island foxes (urocyon littoralis catalinae) (timm et al., 2009) , black-footed ferrets (mustela nigripes) (williams et al., 1988) and lions (panthera leo) (roelke-parker et al., 1996) , and has been implicated as a significant concern for the conservation of amur tiger (panthera tigris altaica) in russia (gilbert et al., 2014) . multihost viruses such as those listed for viverridae may pose a threat to the conservation of threatened wild viverrids where their distribution overlaps areas inhabited by unvaccinated domestic carnivores, and must be considered in population management, preventative health measures or in case of disease outbreak. conversely, viverrids themselves may contribute to the maintenance and cycling of pathogens in an environment which, in turn, could threaten the conservation of other endangered species. in addition to the potential significance for viverrid and other animal health, many of the organisms listed for viverridae raise concerns for human health. wild species are a common source or carrier of zoonotic pathogens (wolfe et al., 2005; jones et al., 2008) , and there is a growing interest in understanding the host, pathogen and external (environmental and anthropogenic) factors that facilitate their transmission from wild animals to people and other animals (bengis et al., 2004; woolhouse and gowtage-sequeria, 2005; greger, 2007; chomel, 2008; rhyan and spraker, 2010) . the vast majority of organisms reported here for viverrids have the capacity to infect a wide range of species a characteristic which, while common amongst pathogens in general -91% of all domestic carnivore pathogens infect more than one host (cleaveland et al., 2001 )is of concern as it is a significant risk factor for outbreak of disease (cleaveland et al., 2001; greger, 2007) . given the prevalence of multihost organisms reported for viverrids, particularly those with zoonotic potential, an examination of the factors bringing these forest dwelling mammals into unnaturally close proximity with humans is warranted. for the viverridae, these include the trades (legal and illegal) in wildlife for human consumption, keeping of viverrids in captivity (wildlife farms, zoos or rescue centres), and human contact with free-ranging viverrids in areas where human and viverrid habitation overlap, or from human incursion into wild spaces. the global wildlife trade is widely regarded as an interface where close contact between humans and wild animals creates an ecosystem favouring disease emergence and expression (bengis et al., 2004; kuiken et al., 2005; karesh et al., 2007; pavlin et al., 2009; cutler et al., 2010; bausch and schwarz, 2014) . stressed, sick and injured animals are transported in mixed-species shipments (bell et al., 2004) to wet markets or other trade nodes where animals are kept in crowded, unhygienic conditions, exposed to both a wide range of other wild and domestic animals for sale (kan et al., 2005; woo et al., 2006) , and to potentially immunologically na€ ıve, urban-dwelling wildlife consumers (swift et al., 2007) . this combination of conditions facilitates emergence, transmission and amplification of pathogens (greger, 2007) . viverrids are frequently encountered in the wildlife trade in africa and asia (shepherd, 2008; nijman, 2009; shepherd and shepherd, 2010) where they are consumed for their meat (bell et al., 2004; yang et al., 2007; van song, 2008) or utilized in traditional medicines (kumara and singh, 2007; ashwell and walston, 2008; tsegaye et al., 2008) . while the number of organisms reported for this animal source (refer to 'country (source)' column, tables 1-7) is low compared to other animal sources, this is likely due to the paucity of research in this area rather than a reliable indication of the health of viverrids in the trade, or the potential significance of this source as a concern for public health. in fact, although few in number, all organisms reported for viverrids sourced from the wildlife tradethe sars coronavirus (table 1) (tu, 2004) , an ixodid tick (table 7) (robbins et al., 1997) , a giardia sp. protozoan (table 4) (abraham, 1962) , the vectorborne parasite responsible for african sleeping sickness trypanosoma brucei (table 3 ) (njiokou et al., 2006) and a trematode fluke of genus paragonimus (table 6) (chen, 1959) are zoonotic, multihost organisms with the potential to cause considerable socio-economic impact chan et al., 2013) . in fact, a viverrid played a central role in the pandemic of human respiratory disease caused by one of the most significant diseases to emerge from the wildlife trade system to datethe sars cov (table 1) . masked palm civets in this crowded, multispecies wet market in southern china were implicated as the main source of infection in humans via exposure to viral particles in bodily fluids aerosolized during slaughter and preparation for human consumption (guan et al., 2003; who, 2004; li, 2008; chan et al., 2013) . the socioeconomic impact of this outbreak, which resulted in over 8000 human cases and a fatality rate of 10%, was considerable (dong et al., 2007; chan et al., 2013) . this process of preparation of carcasses for human consumption presents a considerable risk for transmission of disease to and from viverrids (wang, 2005; woo et al., 2006) , a risk which is amplified by the illegal nature of much of the wildlife trade, meaning that slaughter and preparation for human consumption frequently occur outside government-regulated slaughterhouses, unbound by official hygiene standards (wcs, 2008) . any one of the zoonotic pathogens reported for viverrids in this review may be transmitted to humans during this process, but the rabies virus, another significant viral zoonosis commonly reported for viverrids, is one for which this process is a documented transmission risk (table 1 ). the broad tissue tropism of rabies (carey and mclean, 1978) facilitates aerosolization and environmental contamination during the butchering of rabid animals, resulting in confirmed cases of transmission to people following slaughter of domestic dog and cat in china and vietnam (kureishi et al., 1992; wertheim et al., 2009; nguyen et al., 2011) . given the conditions in which the wildlife trade currently operates, the considerable scale of trade in viverrids and their susceptibility to such a wide range of multihost organisms, it is likely that species from this family could again play a central role in the emergence, amplification and transmission of disease to people within this system (dong et al., 2007) . maintenance of wild species in captivity provides another opportunity for unnatural human-wildlife proximity, facilitating interspecies sharing of organisms (daszak et al., 2001) . viverrids are kept in captivity in zoos (cosson et al., 2007) , conservation breeding programmes for threatened species (roberton et al., 2002) and, in far greater numbers, on commercial wildlife farms. civets have traditionally been farmed for their fur (shi and hu, 2008) and to supply the demand for 'civet' scent (used in the production of perfume), a practice which drives farming of civets in ethiopia (tolosa and regassa, 2007) and india (balakrishnan and sreedevi, 2007) . more recently, farming of civet in asia has expanded to supply a growing demand for their meat in wildlife restaurants and markets (patou et al., 2009) . while published data on wildlife farming are scant, the number of individual civets on farms is likely to be large by 2003, 40,000 individual civets were registered on farms in china (patou et al., 2009) . another growing industry is the production of civet coffee, where coffee beans are fed to civets and subsequently harvested, undigested, from their faeces. traditionally collected from the faeces of free-ranging animals in the vicinity of coffee farms, coffee producers are increasingly turning to caged production to satisfy demand for this product, which has the dubious distinction of being the 'world's most expensive coffee' (d'cruze et al., 2014; techangamsuwan et al., 2014) . a wide variety of zoonoses have been reported for captive viverrids (refer to 'country (source)' columns, tables 1-7), including the very high profile zoonotic virus hpai h5n1 (table 1) . this was confirmed on pcr and serology from a number of owston's civets (chrotogale owstoni) which died following an outbreak of respiratory and neurological diseases in a conservation breeding programme in vietnam (roberton et al., 2006) . while no evidence of viverrid-to-human transmission of hpai h5n1 exists, isolation of pathogens with pandemic potential from any mammalian host is significant as it may provide conditions suitable for the virus to adapt to mammalian respiratory epithelial receptors, enabling efficient mammalto-human, and possibly also human-to-human, transmission, paving the way for a potentially devastating pandemic (rimmelzwaan et al., 2006; peiris et al., 2007) . isolation of this virus from captive civets is also significant in that some countries with the highest number of human cases and fatalities due to hpai h5n1, such as indonesia, china and vietnam (who, 2015) , overlap with those where civets are also farmed in the greatest numbers (patou et al., 2009; d'cruze et al., 2014) , a risk compounded by the poor standards of husbandry and lack of veterinary care reported for wildlife farms (wspa, 1998; wcs, 2008) . knowledge of susceptibility to zoonotic pathogens guides the development of appropriate quarantine and biosecurity protocols in captive institutions required to safeguard both human and animal health. understanding transmission routes of reported organisms also assists in identifying the practices associated with keeping viverrids in captivity which pose the greatest risk to public health. for many of the organisms reported here for captive civets, including the enterobacteriaceae klebsiella pneumoniae and salmonella enterica serovar glostrup in nigerian zoos (falade and durojaiye, 1976; enurah et al., 1988b) and escherichia coli in a japanese zoo (ahmed et al., 2007) , the faecal-oral transmission route is common. appropriate hygiene and husbandry standards can reduce the spread of these organisms amongst animals, and between animals and humans, in captive environments. however, for those workers tasked with the collection of undigested coffee beans from civet faeces in the production of civet coffee (d'cruze et al., 2014) , they present a significant, ongoing occupational hazard which must be addressed. captive viverrids have also been reported as hosts for a number of diseases listed by the world health organization (who) as 'neglected tropical diseases' (ntd), including rabies in an african civet which displayed neurological signs prior to death (table 1) (enurah et al., 1988a) , soiltransmitted helminthiasis (ascarid roundworms in large indian civet and masked palm civets and ancylostome hookworms in common palm civet and small indian civet, table 5 ) (baylis, 1933; ajibade et al., 2010; pradhan et al., 2011) and human african trypanosomiasis in a farmed african civet (table 3 ) (njiokou et al., 2006) . these pathogens are considered 'neglected' because they are poorly studied and commonly ignored by donors, policy makers and public health officials (maudlin et al., 2009) ; however, they continue to cause widespread morbidity and mortality in the poorer regions of the world (hotez et al., 2007; who, 2010) . identification of the pathogens responsible for ntds in captive viverrids contributes to our understanding of potential animal reservoirs for these diseases, knowledge which is vital to the development of interventions aimed at their control or elimination (zinsstag et al., 2007) . ecotourism is a further anthropogenic factor which brings people into contact with free-ranging viverrids, facilitating the cycling of organisms between these groups. as ecotourism, and hence visitation to national parks, expands globally, an increase in human-wildlife contact has led to a corresponding increase in the risk of transmission of disease (muehlenbein et al., 2008) , a risk which was illustrated in the serengeti national park in 2009 when an african civet, showing clinical signs consistent with rabies, bit a child in an unprovoked attack. a novel lyssavirus, the ikoma lyssavirus, was subsequently isolated from the civet (marston et al., 2012) . although the pathogenicity of ikoma lyssavirus to humans is unknown, and further research is required to understand its prevalence and distribution (marston et al., 2012) , this case highlighted the risk of disease emergence when immunologically na€ ıve people come into close and unnatural proximity with wild animals. as human populations expand, and wild spaces are lost, there is an increasing overlap between human habitation and the distribution of wild species, particularly for species which have adapted well to human settlement (campbell, 2009) . tolerance of human presence by some viverrids has led to high density urban and peri-urban populations, where they are frequently seen as pests, scavenging food from garbage bins, gardens and orchards, and living in the roofs and wall spaces of houses (ninomiya et al., 2003; campbell, 2009) . direct contact between viverrids and people in an urban environment leading to disease transmission was reported in japan, where cat scratch fever (caused by the gram-negative bacteria bartonella henselae) was transmitted to a man via a scratch from a free-ranging masked palm civet (table 2) (sato et al., 2013) . peri-urban wildlife may also serve as sylvatic reservoirs for zoonotic disease (weiss and mcmichael, 2004) , as reported for african civet, believed to contribute to the maintenance and cycling of the mongoose biotype of the rabies virus (sabeta et al., 2008) , and masked palm civet, considered a maintenance host of the enteric pathogens salmonella, campylobacter spp. and yersinia (table 2) in japan (lee et al., 2011) . while the list of known organisms for viverrids will grow as future work incorporates improved methodology and advanced molecular techniques, this review provides an important baseline inventory of organisms reported for species from the family viverridae. a number of these have clear and serious implications for viverrid health, and as such, this information facilitates sensible health management of both wild and captive viverrids, and provides a knowledge base from which future research, surveillance programmes and disease outbreak investigations can be developed. as the vast majority of organisms listed here are also capable of infecting a wide range of hosts, including humans, many may also pose a risk to public health. this is of concern given the anthropogenic factors, including trade of viverrids for human consumption, keeping of viverrids in captivity, and land use changes which bring people and other animals into closer contact with the viverridae than would naturally occur, facilitating transmission and spillover of organisms within and between these groups. however, gaps remain in our understanding of the significance of isolation for many of the organisms reported for viverrids. whether due to small sample numbers, or a failure to investigate factors which help to explain the epidemiology of the diseases they cause, we have very little appreciation of the importance of many of these organisms for viverrid, human and other animal health. future research should utilize larger host sample sizes in a range of environmental settings (wild and captive), investigate the role of viverrids in the life cycle of the organism in question, and consider the significance of known and novel organisms for both human and animal health, rather than simply reporting their presence. sustainable utilization of the african civet (civettictis civetta) in ethiopia on two new species of giardia parasitic in indian mammals zoo animals as reservoirs of gram-negative bacteria harboring integrons and antimicrobial resistance genes coprological survey and inventory of animals at obafemi awolowo university and university of ibadan zoological gardens human paragonimiasis in africa new findings on the helminth fauna of the common european genet (genetta genetta l.) first record of toxocara genettae warren, 1972 (ascarididae) in europe studies on the parasite fauna of malaysia i. a redescription of strongylus brauni linstow, 1897, and the establishment of a new genus an overview of the use and trade of plants and animals in traditional medicine systems in cambodia sequence-based identification of aspergillus, fusarium, and mucorales species in the clinical mycology laboratory: where are we and where should we go from here? captive breeding of the small indian civet viverricula indica ( e. geoffroy saint evaluation of the pathogenicity of strains of plesiomonas shigelloides isolated in animals contributions to our knowledge of digenetic trematodes iv outbreak of ebola virus disease in guinea: where ecology meets economy 1928: lxxii. some further parasitic worms from sarawak a new species of the nematode genus uncinaria from a sea-lion, with some observations on related species prevalence and molecular typing of giardia spp. in captive mammals at the zoo of zagreb animal origins of sars coronavirus: possible links with the international trade in small carnivores the role of wildlife in emerging and re-emerging zoonoses evaluation of combined morphological and molecular techniques for marine nematode (terschellingia spp.) identification viral metagenomic analysis of feces of wild small carnivores fatal hemorrhagic-necrotizing pancreatitis associated with pancreatic and hepatic lipidosis in an obese asian palm civet (paradoxurus hermaphroditus). asian pac clinical and laboratory features of the nocardia spp. based on current molecular taxonomy proximity in a ghanaian savanna: human reactions to the african palm civet nandinia binotata rabies antibody prevalence and virus tissue tropism in wild carnivores in virginia a mammalian reservoir of schistosoma japonicum in the napu valley faunistic and ecological trends on the helminthic community of genetta genetta linnaeus, 1758 (carnivora: viverridae) in the iberian peninsula interspecies transmission and emergence of novel viruses: lessons from bats and birds canine distemper virus infection in binturongs (arctictis binturong) a redescription of felicola (paradoxuroecus) bengalensis (werneck, 1948) (phthiraptera: trichodectidae) from a common palm civet (paradoxurus hermaphroditus) in thailand some parasitic nematodes from mammals of south china the occurrence of a new type of paragonimus and some clinical problems related to lung flukes in china (annual report 1958) the status of canine distemper virus in wild carnivore community in southern taiwan chapter 11: wildlife zoonoses -emerging and re-emerging zoonoses from wildlife reservoirs ancylostoma ceylanicum: a parasite of man in calcutta and environs giardia hegneri sp. from a philippine civet cat diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence a caprological survey genetic diversity of captive binturongs (arctictis binturong, viverridae, carnivora): implications for conservation mixed parasitic infection in a captive toddy cat public health threat of new, reemerging, and neglected zoonoses in the industrialized world anthropogenic environmental change and the emergence of infectious diseases in wildlife what is the true cost of the world's most expensive coffee? canine distemper in terrestrial carnivores: a review feline parvovirus infection in an asian palm civet (paradoxurus hermaphroditus) detection of a novel and highly divergent coronavirus from asian leopard cats and chinese ferret badgers in southern snapshot of viral infections in wild carnivores reveals ubiquity of parvovirus and susceptibility of egyptian mongoose to feline panleukopenia virus the epidemiology of filariasis due to wuchereria bancroft and brugia malayi rabies in a civet cat (civettictis civetta) in the jos zoo, nigeria klebsiella pneumoniae as a cause of pneumonia and septicemia in a civet kitten (civettictis civetta) in the jos zoo salmonellae isolated from captive animals in ibadan, western state of nigeria the wild dog-africa's vanishing carnivore survey of natural focuses of paragonimiasis of guangxi estimating the potential impact of canine distemper virus on the amur tiger population (panthera tigris altaica) in russia ticks (acari: ixodidae) parasitizing wild carnivores in phu khieo wildlife sanctuary the human/animal interface: emergence and resurgence of zoonotic infectious isolation and characterisation of viruses related to the sars coronavirus from animals in southern china host-parasite relationships of schistosoma japonicum in mammalian hosts new piroplasms of a genet cat and a groundsquirrel from kenya phylogenetic analysis of the hemagglutinin (h) gene of canine distemper viruses isolated from wild masked palm civets (paguma larvata) identity, hosts, and distribution of haemaphysalis (rhipistoma) canestrinii (supino) (resurrected), the postulated asian progenitor of the african leachi complex (ixodoidea: ixodidae) leishmaniasis in the sudan republic: 30. final epidemiologic report species described by supino in 1897 from burma, with special reference to h. (rhipistoma) asiaticus dermacentor (indocentor) auratus (acari: ixodoidea: ixodidae): hosts, distribution, and medical importance in tropical asia dermacentor (indocentor) taiwanensis (acari: ixodoidea: ixodidae): hosts and distribution in taiwan and southern japan retrospective study of morbidity and mortality of captive jaguars (panthera onca) in north america control of neglected tropical diseases canine distemper virus infection in binturongs (arctictus binturong) serosurvey for selected virus infections of wild carnivores in taiwan and vietnam untersuchungen € uber die wirtsspezifit€ at des geschlechtlichen entwicklungszyklus von toxoplasma gondii global trends in emerging infectious diseases molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms implications of wildlife trade on the movement of avian influenza and other infectious diseases canid and viverrid rabies viruses in south africa the natural infection of wild animals in west malaysia with nymphs of armillifer moniliformis (diesing 1934) sambon 1922 public health: pathogen surveillance in animals rictularia sp small carnivores of karnataka: distribution and sight records rabies in china: recommendations for control echinococcus in the wild carnivores and stray dogs of northern tunisia: the results of a pilot survey malayan protozoa 2. hepatozoon miller (sporozoa: coccidia), with an unusual host record for h.canis (james) prevalence of salmonella, yersinia and campylobacter spp. in feral raccoons (procyon lotor) and masked palm civets (paguma larvata) in japan structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections paragonimiasis: an important food-borne zoonosis in china using retrospective health data from the gombe chimpanzee study to inform future monitoring efforts high seroprevalence of antibodies to toxoplasma gondii in wild animals from portugal canine distemper in a genet (gennetta gennetta), associated with endogenous lipid pneumonia canine distemper virus infection in a masked palm civet (paguma larvata) incidence and seasonal variation of gastro-intestinal parasitic infections in captive carnivores in nandankanan zoological park survey of intestinal parasitism in the exotic animals at dusit zoo 2012: ikoma lyssavirus, highly divergent novel lyssavirus in an african civet blood parasites of wild and domestic animals from south kalimantan novel sylvatic rabies virus variant in endangered golden palm civet neglected and endemic zoonoses an ascaridoid nematode from a hippopotamus spotted fever group rickettsiae in ticks and fleas from the democratic republic of the congo helminth parasites of the endangered iberian lynx (lynx pardinus) and sympatric carnivores leptospirosis in wild and domestic carnivores in natural areas in andalusia. spain. vector borne zoonotic dis leptospirosis in free-ranging endangered european mink (mustela lutreola) and other small carnivores (mustelidae, viverridae) from southwestern france perceived vaccination status in ecotourists and risks of anthropozoonoses strategies for integrating pathology into single species conservation programs nematode parasites of mammals (dermoptera, primates, pholidota, rodentia, carnivora, and artiodactyla) from north borneo (malaysia) integrating molecular and morphological approaches for characterizing parasite cryptic species: implications for parasitology ultrastructure of spermiogenseis and spermatozoa of taenia parva baer, 1926 (cestoda, cyclophyllidea, taeniidae), a parasite of the common genet (genetta genetta) mongoose rabies in southern africa: a re-evaluation based on molecular epidemiology molecular epidemiology of rabies virus in vietnam 1927: a reference list of the trematode parasites of man and the primates an overview of international wildlife trade from southeast asia notoedric mange in free-ranging masked palm civets (paguma larvata) in japan wild fauna as a probable animal reservoir for trypanosoma brucei gambiense in cameroon low genetic diversity in the masked palm civet paguma larvata (viverridae) risk of importing zoonotic diseases through wildlife trade, united states hematozoa from mammals of south vietnam avian influenza virus (h5n1): a threat to human health babesiosis of wild carnivores and ungulates a retrospective study of wildlife rabies in zimbabwe identification of a novel coronavirus in bats preliminary investigation on the parasites of mammals at padmaja naidu himalayan zoological park. darjeeling emergence of diseases from wildlife reservoirs influenza a virus (h5n1) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts two noteworthy collections of ticks (acari: ixodida: ixodidae) from endangered carnivores in the lao people's democratic republic management guidelines for owston's palm civet, chrotogale owstoni (thomas, 1912) avian influenza h5n1 in viverrids: implications for wildlife health and conservation a canine distemper virus epidemic in serengeti lions (panthera leo) mongoose rabies and the african civet in zimbabwe prevalence of paragonimus uterobilateralis infection in children in a liberian village a scanning electron microscope study of toxocara genettae warren 1972 (ascaridae), with data on morphometric variation. folia parasitol serologic survey for canine distemper virus and canine parvovirus in free-ranging wild carnivores from portugal small indian mongooses and masked palm civets serve as new reservoirs of bartonella henselae and potential sources of infection for humans nematode parasites of oceanica. ii. redescription of rictularia whartoni tubangui, 1931, and notes on other species from palawan verzeichnis der bisher hinl€ anglich bekannten eingeweidew€ urmer, nebst einer abhandlung € uber ihre anverwandtschaft weasels, civets, mongooses, and their relatives: an action plan for the conservation of mustelids and viverrids isolation and identification of a reovirus from masked civet cats (paguma larvata) whole genome sequencing of mammalian orthoreovirus mpc/04 strain from masked civet cats ) with notes on legal protection the trade in viverridae and prionodontidae in peninsular malaysia with notes on conservation and legislation a review of studies on animal reservoirs of the sars coronavirus animal leptospirosis in malaya: 1. methods, zoogeographical background, and broad analysis of results the binturong: a new host for the nymphs of armillifer moniliformis (diesing, 1836) parvovirus infections in wild carnivores opportunities and obstacles to collecting wildlife disease data for public health purposes: results of a pilot study on vancouver island. british columbia leaf swallowing and parasitic infection of the chinese lesser civet viverricula indica in northeastern taiwan molecular epidemiology of rabies in indonesia wildlife trade and the emergence of infectious diseases pathological and phylogenetic features of prevalent canine distemper viruses in wild masked palm civets in japan a checklist of ticks of thailand (acari: metastigmata: ixodoidea) pathologic and molecular virologic characterization of a canine distemper outbreak in farmed civets parasite zoonoses and wildlife: emerging issues a suspected canine distemper epidemic as the cause of a catastrophic decline in santa catalina islan foxes (urocyon littoralis catalinae) the husbandry, welfare and health of captive african civets (vivera civetica) in western ethiopa disease risk analysis: a tool for primate conservation planning and decision making rabies virus typing-preliminary survey in botswana a team approach to a disease survey on an aboriginal island (orchid island, taiwan). ii. leptospirosis in man and animals on lan yu scent-marking by the african civet civettictis civetta in the menagesha-suba state forest antibodies to sars coronavirus in civets new host records of metagonimus yokogawai and spirometra erinaceieuropaei from the masked palm civet (paguma larvata) wildlife trading in vietnam: situation, causes, and solutions a survey of gastrointestinal parasites of wild animals in captivity in the voc park and mini zoo. coimbatore sars co-v infection in a restaurant from a palm civet bats, civets and the emergence of sars molecular identification and phylogenetic analysis of trichinella isolates from different provinces in mainland china two new species of toxocara from viverrid hosts commercial wildlife farms in vietnam: a problem or solution for conservation social and environmental risk factors in the emergence of infectious diseases 6, e1000044. who, 2004: who guidelines for the global surveillance of severe acute respiratory syndrome (sars) first who report on neglected tropical diseases: working to overcome the global impact of neglected tropical diseases cumulative number of confirmed human cases of avian influenza a/(h5n1)reported to who infectious and parasitic diseases of captive carnivores, with special emphasis on the black-footed ferret (mustela nigripes) canine distemper in black-footed ferrets (mustela nigripes) from wyoming bushmeat hunting, deforestation, and prediction of zoonotic disease infectious diseases emerging from chinese wet-markets: zoonotic origins of severe respiratory viral infections wspa report -civet farming: an ethiopian investigation. world society for the protection of animals civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates genetic analysis and pathogenicity of an h5n1 avian influenza virus isolated from palm-civet a coprological survey of parasites of seven mammal groups at silent valley national park. kerala genetic diversity of parvovirus isolates from dogs and wild animals in china a new species of echinochasmus parasitic in civet (trematoda: echinostomatidae) changes in attitudes toward wildlife and wildlife meats in hunan province, central china, before and after the severe acute respiratory syndrome outbreak human benefits of animal interventions for zoonosis control key: cord-301856-71syce4n authors: domínguez-andrés, jorge; netea, mihai g. title: impact of historic migrations and evolutionary processes on human immunity date: 2019-11-27 journal: trends immunol doi: 10.1016/j.it.2019.10.001 sha: doc_id: 301856 cord_uid: 71syce4n the evolution of mankind has constantly been influenced by the pathogens encountered. the various populations of modern humans that ventured out of africa adapted to different environments and faced a large variety of infectious agents, resulting in local adaptations of the immune system for these populations. the functional variation of immune genes as a result of evolution is relevant in the responses against infection, as well as in the emergence of autoimmune and inflammatory diseases observed in modern populations. understanding how host–pathogen interactions have influenced the human immune system from an evolutionary perspective might contribute to unveiling the causes behind different immune-mediated disorders and promote the development of new strategies to detect and control such diseases. the evolution of mankind has constantly been influenced by the pathogens encountered. the various populations of modern humans that ventured out of africa adapted to different environments and faced a large variety of infectious agents, resulting in local adaptations of the immune system for these populations. the functional variation of immune genes as a result of evolution is relevant in the responses against infection, as well as in the emergence of autoimmune and inflammatory diseases observed in modern populations. understanding how host-pathogen interactions have influenced the human immune system from an evolutionary perspective might contribute to unveiling the causes behind different immune-mediated disorders and promote the development of new strategies to detect and control such diseases. infectious diseases are arguably the main source of evolutionary pressure that humanity has ever confronted. the dispersion of different human communities around the globe has exposed each population to different infectious agents, exerting a selective pressure (see glossary) on them; thus, adaptation to the new environment has favored the selection of the most beneficial genetic variants for the host. as a result, infectious agents have caused the expansion of alleles behind the induction of either protection or tolerance to these diseases; heritable variations, that increased the survival to deadly infectious agents, may have been naturally selected before the hosts had the opportunity to reproduce [1] . natural selection driven by pathogens is probably more remarkable for those infectious agents that have been among us for a longer time, namely the causative agents of well-known diseases such as leprosy, smallpox, malaria, or tuberculosis. the genetic imprint of pathogen-driven selection depends on the length and the virulence of the infections and also the geographical distribution. the human genome presents more than 5000 genetic loci with traces of selective pressure [2] . this group includes more than 300 immune-related genes with functional variations between populations, which are probably behind the variability of responses to immune-related diseases reported nowadays [3, 4] . besides natural selection, other evolutionary mechanisms, such as genetic drift, greatly influence the frequencies of the genetic variants found within diverse populations throughout the world [5] (box 1 and figure 1 ). with the burst of next-generation sequencing and the development of cutting-edge technologies such as transcriptomics, proteomics, and systems biology, we are starting to witness the great impact of evolutionary processes on human immunity and how the interactions between microorganisms and humans that took place millennia ago might play a fundamental role not only in the response against modern pathogenic threats, but also in the emergence of autoimmune and inflammatory diseases observed in modern populations worldwide. in this review we offer a novel perspective on the role of infectious diseases as agents of natural selection and as forces behind the evolutionary pressure encountered by human ancestors and modern humans in their migrations around the globe. specific genetic variants selected throughout different periods of human history may have influenced immune responses of present-day populations against pathogenic microorganisms and may have played a role in the development of certain inflammatory and autoimmune diseases. the majority of experts agree that africa is where our species originated. genetic studies conducted in diverse contemporary populations suggest connections with ancestors that lived on the continent up to 350 000 years ago [6] . human evolution has been constantly influenced by pathogens; therefore, a great number of human genes linked to immune functions and immunity-related disorders have evolved along with humans. the heterogeneity in the immune response to infectious diseases across different populations is under genetic control and is the result of evolutionary processes. genetic variants that have been under evolutionary pressure can contribute to explaining the differences in the susceptibility to diseases observed across different populations. the ancestry of individuals from different populations across the globe greatly influences their possibility of developing certain autoimmune diseases and inflammatory disorders. the lifestyle of western societies affects the symbiotic relationships between humans, viruses, and other organisms and might contribute to the rise of certain autoimmune and inflammatory diseases. pathogens have played a central role as agents of natural selection from those very early days. among various infectious diseases, malaria has exerted the highest evolutionary pressure on the communities across the african continent ( figure 2 ) [7] . populations remaining in sub-saharan africa have been exposed to malaria for such long periods of time that their genetic structures have been shaped by the severity of malaria (plasmodium sp.) infections. in 1954, allison described that sickle cell disease distribution was confined to africa and was associated with the geographical presence of malaria [8] . this finding led to the more recent description of the existence of mutations in the hemoglobin-b (hbb) gene as a result of natural selection driven by evolutionary pressure for protection against malaria [9] (table 1) . similarly to hbb, some areas of west africa with a high incidence of malaria the gene variations that pass from one generation to the next are often transmitted as a random process known as genetic drift, while selection of advantageous variants tends to be preferentially transmitted. mutations, genetic drift, migration, and environmental selective pressure are among the fundamental processes behind the evolution of humans. the influence of these mechanisms in the diverse communities that were mobilized and then became isolated, as well as severe external factors such as epidemics, caused successive genetic bottlenecks in populations (see figure 1 in main text) [90] . human evolutionary studies are currently considered under 'modern synthesis', which merges darwin's ideas of natural selection with theoretical population genetics and mendelian principles, stating that evolution occurs via small genetic changes that are regulated by natural selection [91] . these beneficial adaptations subsequently expand within the members of a population and become evident in the ancestral specificity and the geographical distribution of the advantageous alleles in the genomes of contemporary humans. genetic bottlenecks occur when the number of individuals in a population is reduced drastically due to a catastrophic event such as an earthquake, a flood, a famine, or the outbreak of an infectious disease. these events limit the genetic variation of a population and can lead to genetic drift. as a result, a smaller population, with a correspondingly reduced genetic diversity, remains to transmit genes to future generations through sexual reproduction. even if this reduction in the genetic diversity is temporary, it can lead to long-lasting effects on the genetic variation of the offspring populations. genetic drift: changes in the allele frequencies of a population over generations due to chance. genetic locus: fixed position on a chromosome (e.g., the position of a gene or a genetic marker). histocompatibility complex: region of approximately 4000 kb, located on human chromosome 6, that contains a large number of genes whose products are expressed as proteins on immune cells. of these genes, the best known are hla genes. introgressive hybridization: incorporation of genes from one species into the genetic reserves of another by interspecific hybridization and backcrossing with the parent species. present a high frequency of hemoglobin-c (hbc) in their populations, which is associated with a 30-93% decrease in the possibility of developing the disease [10] . this is also the case for the duffy antigen receptor gene (darc) in erythrocytes and single nucleotide polymorphisms (snps) in human leukocyte antigen (hla), which have been associated with protection against plasmodium vivax malaria in certain areas of africa where this disease is endemic [11, 12] . another example of natural selection driven by evolutionary pressure for protection against malaria are thalassemia (a and b) pathologies, a group of hemoglobin disorders that presents an incidence of up to 30% among communities of west africa [13] . the human casp12 t 125 c snp, expression of which is restricted to the african subcontinent, south america, and certain areas of asia, can modulate immune and inflammatory responses to malaria by antagonizing interleukin (il)-1b and nf-kb signaling in innate immune cells; moreover, caspase 12-deficient mice (casp12 -/-) exhibit decreased interferon (ifn)-g production and clearance of the parasite, relative to wild type (wt) infected mice [14] . however, others have questioned these findings, given that caspase 12-deficient mice also lack caspase 11 expression, so the effects observed might not be specific to caspase 12 [15] . mycobacterium tuberculosis (mtb) has caused infections in our species and ancestors for at least 500 000 years [16] . this long-standing relationship between humans and mtb probably underlies the large variety of immune-related factors that modulate susceptibility to mtb infection, including vitamin d receptor (vdr), natural resistance-associated macrophage protein 1 (slc11a1), tir domain containing adaptor protein (tirap), hla, monocyte chemoattractant protein 1 (mcp-1), and cytokines such as il-12 and ifn-g [17] ( table 1) . patients with african ancestry present a higher frequency of mtb-related genetic variants than individuals from other populations, including variants in the gene encoding for toll-like receptor 6 (tlr6), mediating cellular responses to bacterial malaria is one of the greatest causes of morbidity and mortality in the history of humanity. most human populations with a long history of endemic malaria have evolved genetic adaptations to malaria parasites due to the strong selective pressure that this infection has exerted. since the parasite infects erythrocytes, the evolutionary pressure has selected genetic variants that affect red blood cells and, therefore, the survival of the parasite as well. genetic variants conferring resistance to the disease have spread through human populations over time, including several abnormal hemoglobins that protect against malaria but usually cause erythrocyte-associated diseases in the populations where these adaptations are prevalent. these factors include the t 125 c polymorphism in the caspase 12 gene (casp12); the hemoglobin b (hbb) and hemoglobin c (hbc) variants; mutations in the duffy antigen receptor gene (darc); thalassemias (a and b); sickle cell disease; and polymorphisms in the human leukocyte antigen (hla) loci. dna nucleotide. for example, an snp may replace cytosine (c) with thymine (t) in a certain segment of dna. selective pressure: phenomenon that alters the behavior and fitness of living organisms within a given environment. it is the driving force of evolution and natural selection. thalassemias (a and b): inherited hemoglobinopathies characterized by a failure in the synthesis of the globin alpha or beta chains. toll-like receptors: family of transmembrane pattern recognition receptors expressed by immune and nonimmune cells that recognize conserved pathogenassociated molecular patterns. they play a pivotal role in innate immunity. transgenerational inheritance: transmission of traits from generation to generation. trends in immunology, december 2019, vol. 40, no. 12 1107 lipoproteins [18, 19] . selective pressure has also shaped the mechanisms that modulate the expression of genes implicated in immune responses against lassa virus, such as il-21 (il21) and the glycosyltransferase-like protein large1 (large), suggesting that the natural selection exerted by the virus drove the expansion of genetic variants that enhance immunity against lassa fever [20] . these examples indicate that infectious diseases have contributed to shaping the genetic landscape of african populations and their descendants, and highlight the great impact of pathogens as an evolutionary force in humans. our homo sapiens ancestors were not the only species to venture out of africa, with other homo species performing a similar migration much earlier, such as homo ergaster, homo erectus, or homo heidelbergensis [21] . from these early migrations, local populations such as the denisovans and neanderthals evolved [22] . these lineages were not geographically isolated, but lived side by side with modern humans and interbred with them, leaving a genetic footprint in their common progeny. accordingly, 1-4% of the genome of european and asian populations is thought to derive from these now-extinct hominid lineages [23] . neanderthals spent close to 600 000 years adapting to their environment and their immune systems were shaped by the infections they faced. by interbreeding with archaic humans, modern humans incorporated these advantageous adaptations in the genome of their descendants. this was highlighted by different studies that showed that the introgression of diverse genes related to immune functions, such as the oas cluster, tlr1, or the histocompatibility complex from denisovans and neanderthals shaped the genetic landscape of present-day eurasian, but not african, communities. genomic sequences and expression data from lymphoblastoid cell lines from 421 individuals of european and african ancestry confirmed that the tlr1-tlr6-tlr10 genetic loci, presenting signs of local positive selection and repeated introgression from both neanderthal and denisovan genomes [24] [25] [26] , showed a significantly higher expression in individuals carrying archaic-like alleles than in individuals carrying the nonintrogressed modern human alleles [2, 26, 27] . the expression of these genes has shaped human immune responses against different types of pathogens. for example, the gp41 protein of the hiv-1 virus has been recently recognized as a tlr10 ligand [28] . in this regard, increased tlr10 expression has been correlated with higher il-8 production by the macrophage cell line thp-1 and higher titers of hiv-1 in the breast milk of hiv-1-infected nigerian women relative to controls [28] . tlr1 and tlr6 form dimers with tlr2, triggering immune responses against different types of bacteria, fungi, virus, and parasites [29] . variation in tlr1-tlr6-tlr10 is the major genetic determinant of human interindividual differences in tlr1/2-mediated responses, including cytokine production to a number of clinically relevant pathogens such as staphylococcus aureus and listeria monocytogenes [30] (table 1 ). this inheritance from archaic humans may have also left some human individuals more prone than others to developing asthma, hay fever, and other allergies (of 58 snps associated with susceptibility to allergic disease, 12 had a neanderthal or denisovan origin) [26] , although these associations remain to be fully demonstrated [31] . these reports demonstrate that by interbreeding with archaic humans, modern humans incorporated a group of advantageous adaptations to the genome of their descendants and contributed to shaping immune responses in modern human populations. the migration of our human ancestors out of africa implied the exposure to different types of infectious diseases (box 2 and figure 3 ). one study tested the responsiveness of human macrophages to pathogenic bacteria in vitro, finding that almost 10% of the genes present in human macrophages infected with the bacteria salmonella typhimurium or l. monocytogenes present different regulatory responses directly linked to the lineage of the donors and, also, that macrophages obtained from individuals of african origin display enhanced bactericidal activity compared with those from individuals of european lineage [32] . the trend towards lower inflammatory responses in european populations is strengthened by the fixation of a tlr1 gene variant that results in lower proinflammatory gene expression in populations with a european ancestry compared with those with an african one [33] . the largest population differences in gene expression between africans and europeans have been found in the macrophage receptor with collagenous structure (marco), a protein implicated in responses against viral infections and tlr-induced dendritic cell activation [34] , the chemokine receptor cx3cr1, which mediates effector lymphocyte functions, and also several ifn-stimulated genes [35] . west eurasian populations present a high frequency of tirap ser180leu snps [36] . tirap is an adaptor protein in tlr2 and tlr4 signaling pathways, involved in inflammatory responses and cytokine production. the heterozygous expression of the ser180leu snp is protective against invasive pneumococcal disease, bacteremia, malaria, and tuberculosis, as shown in a case-control study of 6106 individuals from the uk, vietnam, and several african countries, and it is associated with lower tlr2 signaling in humans [36] . this variant is considered to be a consequence of the natural selection that may have taken place in an early period following the migration of modern humans out of africa [37] . european populations present a selective adaptation of the ifn gene that allows a high production of ifn-g in infectious scenarios due to the positive selection of ifng variants +5173g and +874t; this suggests the existence of strong environmental pressures linked to higher ifn-g concentrations in plasma during mtb infection in european individuals relative to other populations [38, 39] . in line with this, a database meta-analysis showed that individuals expressing the +874t/a variant of ifng presented higher susceptibility to tuberculosis mtb infection than individuals without it, which might be considered a putative prognostic factor for the development of tuberculosis [40] , although this remains to be robustly demonstrated. one of the most interesting aspects of humans is their ability to adapt to almost every ecosystem of the planet. the history of mankind is also the history of millions of individuals wandering around the world, looking for a better place to live. a glimpse to the migratory legacy of humanity around the globe reveals the great impact that the massive population movements defined the world as we know it today (see figure 3 in main text). the distances ancient humans travelled are impressive, from the first hominids colonizing africa to the conquest of the americas in a time when the bering strait was not yet under water. the historical exodus of mankind started almost 2 million years ago with the migration of homo erectus from africa through eurasia. from this event on, relatively isolated human populations evolved separately on different continents, leading to the emergence of different human species, such as neanderthals in europe, the denisovans in asia, and, later, modern homo sapiens in africa [92, 93] . h. sapiens first colonized large areas of the continent around 300 000 years ago [6] , spread towards the middle east at some point between 150 000 and 80 000 years ago, and migrated through eurasia, reaching australia within 20 000 years [94] . asian human ancestors went through the frozen waters of the bering strait in two distinct waves to colonize the american continent approximately 20 000 years before the present time [95] . when humans ventured out of africa, they faced different types of pathogens than the communities that stayed in the african continent. with time, the series of events faced by diverse populations has generated differences in the immune responses to pathogens in the populations with an african or eurasian origin and which have spread throughout the world. the indigenous populations of south america are descendants of migrating populations of north-east asians that crossed the bering strait around 20 000 years ago [41] . five centuries ago, european settlers disembarked on the american continent, bringing a large collection of pathogens such as those causing measles, pneumonic plague, and influenza infections, which the indigenous populations had never faced before. these diseases rapidly spread and caused mortality rates above 90% [42] . the consequences of these pandemics are still visible in current populations; one report studied dna from the bones of 25 ancient humans from the tsimshian community, living in the british columbia region in canada until the 15th century, identifying marks of positive selection in a number of immune-related variants [43] . specifically, the hla-dqa1 variant was present in almost the totality of tsimshian individuals, but only in one third of present-day humans studied; this suggested that ancient american genomes were evolutionarily selected to respond to local diseases but not to fight against pathogens brought by the europeans [43] . another study compiled information on infectious diseases that have killed more than 10 000 individuals among 59 indigenous communities of the amazonia in the past two centuries, showing that the mortality rates and the incidence of infectious diseases rapidly decayed within the time following the first encounter with the pathogen, compatible with genetic adaptation [44] . european colonizers underwent purifying selection in situations of intense pressure. such scenarios were documented when dutch colonists migrated to surinam and encountered epidemics of yellow and typhoid fever that caused a 60% mortality rate among settlers [45] . variations in the frequencies of c3, glo, and hla-b genes among the descendants were not likely caused by genetic drift, but rather, it has been proposed that these populations were probably selected through genetic control of survival to the epidemics [45] (box 3). africans and americans with an african ancestry present a much higher number of genetic variants related to robust inflammatory reactions, increased cytokine secretion, and bactericidal activities compared with the other populations [32] , including more than 250 genes with traces of recent selection, such as il1a and il1b gene variants [46] . the degree of african ancestry, analyzed by fine-mapping analysis refined to the duffy-null allele of rs2814778, was correlated with an increased amount of the proinflammatory chemokines ccl2 and ccl11 in plasma relative to controls [12] . a study involving 12 000 african american and hispanic american women found that the higher values of c-reactive protein (crp) in blood found in these populations compared with european americans were related to a crp-associated variant of triggering receptors expressed by myeloid cells 2 (trem2) [47] . moreover, comparison of health record data from individuals with connective tissue diseases, including rheumatoid arthritis and systemic lupus erythematosus (sle), as well as atherosclerotic cardiovascular disease from almost 300 000 african american and european american adults was conducted; the study reported for the latter, a prevalence of atherosclerotic cardiovascular disease in 29.7% african americans (particularly high in young individuals), relative to 14.7% in european americans [48] . these studies highlight certain genetic links to inflammatory predisposition/manifestation. however, increased proinflammatory activity is a double-edged sword. in the absence of regular pathogen challenges that require maintained modulation of the balance between inflammation and suppression of the immune response, the organism can overreact to inflammatory stimuli and trigger exacerbated responses. for instance, descendants of african populations generally present higher susceptibility to a variety of autoimmune syndromes such as inflammation-associated carcinomas, lupus, asthma, and multiple sclerosis (ms), the overall prevalence of which is up to three times higher in individuals with african ancestry relative to individuals with european ancestry [31, 49, 50] . there are extensive differences in immune cell gene expression between americans with african and european ancestry. the increased proinflammatory responses observed in american individuals relative to other populations might be beneficial to combatting infections, but might also increase the chances of developing inflammatory and autoimmune disorders, which warrants further investigation. our gastrointestinal tract provides residence to both beneficial and potentially pathogenic microorganisms, harboring ten million different microbial genes in the human fecal microbiome [51] . the microbiome has its own evolutionary scenario across different populations with divergent lifestyles, nutrition, and exposure to environmental agents, generating extraordinary heterogeneity. the ongoing process of 'lifestyle westernization' of different societies has an important impact on the mutualistic relationships between humans and commensal organisms worldwide. african tribes are adopting western subsistence patterns, leading to remarkable changes in the composition of their microbiota [52] . the comparison of the intestinal flora of the baaka hunter-gatherers and the bantu agriculturalists (both from the central africa republic), with a group of us-born african americans showed a great example of the evolution of the human microbiome [52] . specifically, the bantu, still engaged in hunting, have a greater bacterial gut diversity than their baaka neighbors, who left the jungle for agriculture, and even more than urbanized westerners (us african-americans) [52] . this reduced microbiota diversity in western societies has been associated with a higher incidence of the so-called 'diseases of civilization' such as cardiovascular diseases, diabetes, obesity, and autoimmune disorders, which are very unusual in hunter-gatherer societies compared with communities living a western-type lifestyle [52, 53] . although viruses are mainly seen as pathogenic agents, they also play a fundamental role in the evolution and maturation of the human immune system [54, 55] . approximately 8% of the human genome is composed of endogenous retroviruses (ervs), sequences derived from past retroviral infections and permanently inserted into different regions of the human genome [56] . one study showed that ervs played a central role in the induction of ifn-dependent immune responses and that the removal of one or more of these viral dna elements in the hela human cell line severely impaired the recruitment of transcription factors necessary to trigger the expression of ifng against vaccinia virus infection relative to controls [57] . viruses can also influence the severity of infections caused by other viruses. for example, cytomegalovirus infection in hiv-1 seropositive humans can potentiate the effects of hiv-1 infection by expanding the pool of circulating regulatory t cells (immunosuppressive); these were shown to inhibit the proliferation of autologous peripheral blood mononuclear cell the origins of the hiv virus are still a matter of scientific discussion. the most accepted scenario argues that hiv originated in simians and was transmitted to humans in west africa in the 1920s, likely due to local ingestion of ape meat infected with the simian immunodeficiency virus. around 1960, the virus reached wide parts of the continent and finally spread overseas thanks to a group of haitian professors coming back from africa. in the following decades, the virus spread worldwide and generated the pandemic we now know. today, there are approximately 37 million people worldwide living with hiv-1/aids [110] . ccr5 is a receptor of chemokines that plays a fundamental role in hiv-1 pathogenesis and it is also one of the most promising targets to restrict the infection, since mutations in this receptor turn individuals resistant [96] . the ccr5-d32 mutation results in a deletion that eliminates the hiv-1 co-receptor on lymphocytes, providing robust protection against hiv-1 and, therefore, aids [97] . ccr5-d32 allele frequencies reach 14% in northern europe populations, whereas it is not present in populations with different ancestry, such as east asian, native american, or african groups [96] . this regional distribution of ccr5-d32 variants is most likely related to a naturally selective episode that struck european populations around 700 years ago and involved a strong infectious agent that also employed ccr5 [98]. a mathematical model studying the changes in the european populations in the middle ages suggested yersinia pestis (bubonic plague) as the most probable infectious agent behind the pressure that selected this particular genetic variant [99] . this is in agreement with the finding that european rroma populations, but not northwestern indian populations that inhabit the area where the rroma originally lived, present signatures of positive selection in tlr1-tlr6-tlr10, which influence cytokine responses in y. pestis infections [100] . (pbmc) in response to cytomegalovirus infection in vitro [58] . in one study, patients with chronic hepatitis c virus (hcv) infection and hepatitis a virus (hav) superinfection presented lower titers of hcv rna than patients harboring only hcv, suggesting that hcv replication might be potentially suppressed during hav infection [59] , although this will still require further investigation. the relationships between humans and pathogenic or nonpathogenic organisms are extraordinarily complex and include tripartite evolutionary interactions between humans and microbes competing with each other. this is the case of parasites that infect other parasites, such as bacteriophage viruses, that can influence the outcome of bacterial infections. for example, in a cohort of individuals with chronic wounds, a report showed that the phage pf, which coexists with pseudomonas aeruginosa in infected wounds, triggered the production of type i ifn, the inhibition of tumor necrosis factor (tnf) production, and the suppression of phagocytosis in human primary monocytes and mouse bone marrow-derived macrophages, dampening the antibacterial response and promoting the bacterial infection [60] . however, bacteriophages can also provide protection to the human host by directly attacking pathogenic bacteria and by upregulating in human pbmcs the expression of proinflammatory genes such as il1a, il1b, il6, tnfa, cxcl1, and cxcl5, as shown for several s. aureus and p. aeruginosa phages, including pnm, luz19, 14-1, and ge-vb_pae-kakheti25 [61] . cooperative relationships between organisms are evolutionary processes themselves. the way microorganisms and their hosts associate can lead to interactions of mutualism, in which the interplay may be so intimate as to provide benefit for each party and influence immune responses against different types of pathogens. a great number of humans live far away from the original settlements of their ancestors and are subject to radically different environmental conditions. between two and three million people with european genealogy suffer from autoimmune diseases, the prevalence of which is also increasing in other populations across the globe [62]. there is rising evidence that the emergence of autoimmune diseases is associated with the presence of a number of immune-related alleles that have been selected via evolutionary processes; and, furthermore, that the contrasting differences in the prevalence of autoimmune diseases between populations may be a result of different selective pressures [63] . alleles associated with inflammatory diseases that present marks of modern positive selection include the risk allele fut2 at rs601338 for crohn's disease (cd) or the risk variant sh2b3 at rs3184504 for celiac disease [64] ; such variants have been linked to the development of several human autoimmune diseases, such as type 1 diabetes, ms, and celiac disease [64, 65] . the analysis of the integrated haplotype score [66] of loci associated with sle that might provide protection against infections, such as tnip1, itgam, ptpn22, tnfsf4, uhrf1bp1, tet3-dguok, and blk, has suggested that these loci exhibit robust signs of positive selection [67] (figure 4 , key figure and table 1 ). african and asian human populations exposed to trypanosoma brucei or plasmodium sp. have presented positive selection of snps in the apol1 and fcgr2b genes; indeed, by enhancing human macrophage-mediated phagocytosis of infected erythrocytes, despite their association with an sle predisposition, these snps have been associated with protective roles against sleeping sickness and malaria, respectively [68, 69] . an analysis of human loci associated with inflammatory bowel disease (ibd) concluded that the majority of the loci associated with cd are also linked with a higher risk of developing ulcerative colitis [70] ( table 1 ). many of these loci were also associated with the development of other autoimmune diseases, namely psoriasis and ankylosing spondylitis [62] . pbmcs from individuals carrying the sh2b3 variant rs3184504*a (associated with a risk for developing cd) [71] presented higher production of il-6 and il-1b after stimulation with lipopolysaccharide or muramyl dipeptide due to enhanced activity of the nod2 inflammasome pathway relative to controls [72] ; this has suggested an immune-related role for sh2b3 in the context of bacterial infections, which might help explain the positive selection of sh2b3 approximately 1500 years ago [72] . others found an association between genetic variants in nod2, cd14, and increased susceptibility to cd [73] , in line with previous results showing that mutations in nod2 and tlr4/cd14 are related to an increased risk of developing ibd [74] . from another angle, changes in hygiene patterns seen in the past two centuries brought vast improvements in sanitation, drinking water, and garbage collection, which greatly reduced the exposure to many infectious diseases. however, these conditions may have reduced the exposure to viral and microbial agents that help the immune system to develop tolerance during childhood. the hygiene hypothesis proposes that the lack of exposure to microbial agents in the early stages of life is related to a higher risk of developing hypersensitivity reactions, based on the fact that children that are exposed to higher amounts of microbial stimuli (e.g., by growing on farms) are less prone to develop allergies and asthma [75, 76] . moreover, reduced exposure to infectious agents can have a much wider effect than initially believed. lack of exposure to microbes in childhood can cause aberrant responses to infection and potentiate the effects of etv6-runx1 mutations in the pathogenesis of acute lymphoblastic leukemia [77] . by contrast, a meta-analysis of six observational studies, including 1902 participants, showed a correlation between low helicobacter pylori infection and ms, suggesting that low h. pylori prevalence might be a putative protective factor in ms, although this remains to be experimentally validated [78] . one study also reported that antibodies against toxoplasma gondii were detected less often in patients with ms compared with healthy controls [79] . however, these findings warrant further and robust investigation. overall, it is clear that evolutionary processes can drive the fixation of genetic variations that increase (or decrease) our defense against infections upon sensing microbial ligands, but can also lead to a greater risk of developing certain autoimmune diseases in which endogenous ligands can cause tissue damage and inflammation. a growing number of reports suggest that inheritance is not always governed by classical darwinian evolutionary processes. exposure to certain environmental stimuli can cause effects in the progeny of an exposed individual, even though the stimuli are no longer present. this type of transgenerational inheritance might be explained through the effects of epigenetic processes, which are hypothesized to be transmitted through the germline and passed on to the offspring [80] . for example, the worm caenorhabditis elegans can transmit improved resistance to infections to pathogenic bacteria to their offspring through alterations of the histone landscape [81] . indian meal moths exposed to low doses of virus are subsequently less susceptible to viral challenge, a protection offered to their offspring as well [82] . transgenerational inheritance of diverse traits has also been observed in mice, in which the variation of the color of the coat is passed on the next generation [83, 84] . offspring of male rats subjected to a high-fat diet present glucose intolerance and reduced insulin secretion, linked to reduced methylation at the il13ra2 gene relative to controls [85] ; and mice fed scorpions are more resistant to a challenge with scorpion venom than mice on a normal diet [86] . since infections are one of the strongest factors impacting survival, it is conceivable that transgenerational transmission of traits in mammals, including humans, evolved to improve host defense. the number of studies of the potential role of epigenetic inheritance in shaping the human immune system is still scarce. however, different experiences undergone by certain communities indicate that these mechanisms might be important. for example, the babies of pregnant women who suffered during the early stages of pregnancy (the effects of the dutch hunger winter in 1944), 60 years later showed reduced dna methylation marks in several genes that control metabolism and cell differentiation during development, such as igf2, pim3, txnip, abcg1, pfkfb3, and mettl8, compared with their siblings [87, 88] . this was related with higher rates of obesity, heart disease, cancer, and depression in individuals whose pregnant mothers suffered the effects of the famine [87, 88] . some of these effects seemed to be present in the progeny of this group, that is, in the grandchildren of those who had passed the famine during pregnancy [89] . the rapid growth in the number of reports covering the impact of epigenetic mechanisms in different human processes warrants further and robust studies on the impact of epigenetic inheritance in shaping the evolution of the human immune system. human immune responses have been shaped by the evolutionary pressure exerted by microorganisms and viruses throughout history. generating a broad range of genetic variations and immune functions in different populations favors the adaptation to new environments and increases the chance of survival of the human species against potential pandemics. much remains to be learned in this exciting field over the coming years in order to identify the main regulatory forces and the time window necessary for the fixation of an advantageous genetic trait in a population (see outstanding questions). the combination of the selective pressure caused by infectious diseases with other evolutionarily relevant processes, such as genetic drift, migratory events, bottlenecks, and introgressive hybridization, contribute to driving the expansion, fixation, or elimination of characteristic immune response-related traits in different populations around the world. these specific genetic variants are able to boost the host response against pathogens by improving the sensing of microbial ligands but can also lead to the development of autoimmune diseases, in which the immune system responds to endogenous ligands and induces abnormal responses targeted against the host's own tissues. of note, it is very difficult to assign certain variants, a specific role in the protection or induction of autoimmunity. to assign changes in the genetic landscape of human populations to certain diseases is an extraordinary challenge. moreover, our species is in constant evolutionary interaction with various microorganisms and viruses. populations of bacteria and their viruses (phages) undergo, under natural conditions, reciprocal evolution in terms of resistance and infection; this, in turn, also affects the evolutionary traits of our immune system. thus, an extraordinarily complex scenario exists in which organisms of different phyla interact, compete, and coevolve, to ensure their own survival. as novel tools, the development and refinement of methods that study large sections of the human genome, epigenome, and microbiota, will help to obtain genome-wide data in diverse human populations, allowing us to follow the evolutionary trails left from the encounters with different organisms, further unveiling the roots of human immunity. high-throughput biotechnology and an expanding computational capacity can enable the study of global population genomics and might contribute to decoding the origin and consequences of functional changes in adaptive alleles down to the single cell level. however, these methods also have limitations associated with the difficulty in linking gene variations to clinical phenotypes and disease, the generation of false positives, or the high number of samples necessary to reach reliable conclusions. expanding the heterogeneity of populations studied for immune gene association studies relevant to disease will be key, as generally, a large focus is placed on certain european or american communities, thus generating results that are difficult to extrapolate to other populations. the knowledge of the evolutionary and genetic basis of human immune traits and their impact on diverse pathologies (e.g., autoimmunity, infections, inflammatory diseases, cancer) increasingly suggests that the genetic basis of disease may be derived from a large number of rare variants of modest effect. the mechanisms described here acquire special importance in the current scenario of world globalization, in which the migration fluxes and the admixture of different populations are reaching unprecedented levels, allowing faster expansion of advantageous alleles. however, these processes may also accelerate the spread of new epidemics, as seen in the cases of hiv infection, or more recently, sars-cov, ebola, and chikungunya viruses; as well as the emergence of multiresistant bacteria and fungi, such as methicillin-resistant s. aureus or candida auris. this is just the starting point to unveil the evolutionary history of the relationships between pathogens, the immune system, and humans. further investigation of the functional adaptations of human populations is warranted to provide a broad picture of the functional consequences of evolution in human immunity. acknowledgments m.g.n. is supported by a spinoza grant of the netherlands organization for scientific research and an erc advanced grant (#833247). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. we thank laszlo a. groh for which are the strongest evolutionary forces that drive the evolution of the human innate immune system? how long does the genome of a given population take to adapt to a new infectious threat? are the mechanisms of resistance to infection transmitted only via genetic modifications, or can epigenetic adaptations to resistance also be transmitted to the progeny, and under what circumstances? the development of single-cell sequencing technologies has opened new fields of study. how does genetic variation of the expression of a specific variant vary between different cell subsets and how does it affect the overall phenotype of an individual within a population? are there specific immune processes that are preferentially impacted by evolutionary pressures? in western societies we enjoy a life expectancy vastly superior to that of our predecessors, but at the same time, we suffer diseases that they did not suffer. can some of the reasons for these changes lie among some of those bacteria that we have somehow lost in our microbiome? identifying these bacteria and understanding their effects on the human body might be the first step to developing putative therapies based on bacterial restoration. since many of the variants causing autoimmune diseases are linked to an enhanced responsiveness to pathogens that is no longer needed in developed countries, could these genes and their related pathways be employed as targets for new putative therapeutic approaches against inflammatory/ autoimmune syndromes? are other newly described genetic regulatory pathways, such as interfering rna or long noncoding rnas, also influenced by pathogen-driven evolutionary processes in different populations globally? proofreading and correction of the manuscript. we additionally thank srinivas agra for providing the figure icon 'bacteria' as deposited on https://thenounproject.com. natural selection and infectious disease in human populations genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes genome-wide identification of regulatory sequences undergoing accelerated evolution in the human genome divergent selection of pattern recognition receptors in mammals with different ecological characteristics a population genetic interpretation of gwas findings for human quantitative traits new fossils from jebel irhoud, morocco and the pan-african origin of homo sapiens protection afforded by sicklecell trait against subtertian malareal infection extended linkage disequilibrium surrounding the hemoglobin e variant due to malarial selection haemoglobin c protects against clinical plasmodium falciparum malaria positive natural selection in the human lineage genetic ancestry and population differences in levels of inflammatory cytokines in women: role for evolutionary selection and environmental factors world distribution, population genetics, and health burden of the hemoglobinopathies. cold spring harb caspase-12 dampens the immune response to malaria independently of the inflammasome by targeting nf-b signaling does caspase-12 suppress inflammasome activation? first homo erectus from turkey and implications for migrations into temperate eurasia genetics of sub-saharan african human population: implications for hiv/aids, tuberculosis, and malaria the role of ancestry in tb susceptibility of an admixed south african population association of the tlr1 variant rs5743557 with susceptibility to tuberculosis genome-wide scans provide evidence for positive selection of genes implicated in lassa fever the evolutionary history of the human face human occupation of northern australia by 65,000 years ago the complete genome sequence of a neanderthal from the altai mountains variation and functional impact of neanderthal ancestry in western asia disentangling immediate adaptive introgression from selection on standing introgressed variation in humans introgression of neandertal-and denisovan-like haplotypes contributes to adaptive variation in human toll-like receptors the shaping of modern human immune systems by multiregional admixture with archaic humans tlr10 senses hiv-1 proteins and significantly enhances hiv-1 infection the role of tlr2 in infection and immunity variation in the tlr10/ tlr1/tlr6 locus is the major genetic determinant of interindividual difference in tlr1/2-mediated responses race is associated with differences in airway inflammation in patients with asthma genetic ancestry and natural selection drive population differences in immune responses to pathogens genetic adaptation and neandertal admixture shaped the immune system of human populations key role of the scavenger receptor marco in mediating adenovirus infection and subsequent innate responses of macrophages cx3cr1-dependent renal macrophage survival promotes candida control and host survival a mal functional variant is associated with protection against invasive pneumococcal disease, bacteremia, malaria and tuberculosis functional and genetic evidence that the mal/tirap allele variant 180l has been selected by providing protection against septic shock evolutionary genetics evidence of an essential, nonredundant role of the ifn-g pathway in protective immunity ifng +874t/a polymorphism and cytokine plasma levels are associated with susceptibility to mycobacterium tuberculosis infection and clinical manifestation of tuberculosis a single nucleotide polymorphism in the interferon-g gene (ifng +874 t/a) is associated with susceptibility to tuberculosis genetic evidence for two founding populations of the americas the significance of disease in the extinction of the new england indians a time transect of exomes from a native american population before and after european contact mortality from contactrelated epidemics among indigenous populations in greater amazonia genetic control of survival in epidemics differential distribution of allelic variants in cytokine genes among african americans and white americans genome-wide association and population genetic analysis of c-reactive protein in african american and hispanic american women the prevalence of atherosclerosis in those with inflammatory connective tissue disease by race, age and traditional risk factors group differences in proneness to inflammation the contribution of natural selection to present-day susceptibility to chronic inflammatory and autoimmune disease an integrated catalog of reference genes in the human gut microbiome gut microbiome of coexisting baaka pygmies and bantu reflects gradients of traditional subsistence patterns gut microbiota is associated with obesity and cardiometabolic disease in a population in the midst of the good viruses: viral mutualistic symbioses virolution: the most important evolutionary book since dawkins' selfish gene to erv is human: a phenotype-wide scan linking polymorphic human endogenous retrovirus-k insertions to complex phenotypes regulatory evolution of innate immunity through co-option of endogenous retroviruses cytomegalovirus infection in hiv-infected and uninfected individuals is characterized by circulating regulatory t cells of unconstrained antigenic specificity hepatitis a virus infection suppresses hepatitis c virus replication and may lead to clearance of hcv bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection pro-and antiinflammatory responses of peripheral blood mononuclear cells induced by staphylococcus aureus and pseudomonas aeruginosa phages host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease genetics of autoimmune diseases: insights from population genetics common risk alleles for inflammatory diseases are targets of recent positive selection signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution a map of recent positive selection in the human genome genes associated with sle are targets of recent positive selection end-stage renal disease in african americans with lupus nephritis is associated with apol1 systemic lupus erythematosus-associated defects in the inhibitory receptor fc riib reduce susceptibility to malaria inherited determinants of crohn's disease and ulcerative colitis phenotypes: a genetic association study newly identified genetic risk variants for celiac disease related to the immune response evolutionary and functional analysis of celiac risk loci reveals sh2b3 as a protective factor against bacterial infection card15/nod2, cd14 and toll-like 4 receptor gene polymorphisms in saudi patients with crohn's disease association between polymorphisms in the toll-like receptor 4, cd14, and card15/nod2 and inflammatory bowel disease in the greek population the effects of growing up on a farm on adult lung function and allergic phenotypes: an international population-based study innate immunity and asthma risk in amish and hutterite farm children a causal mechanism for childhood acute lymphoblastic leukaemia association between helicobacter pylori infection and multiple sclerosis: a systematic review and meta-analysis is toxoplasma gondii infection protective against multiple sclerosis risk? a critical view on transgenerational epigenetic inheritance in humans the pirna pathway responds to environmental signals to establish intergenerational adaptation to stress within and transgenerational immune priming in an insect to a dna virus epigenetic inheritance at the agouti locus in the mouse genetic and epigenetic variation among inbred mouse littermates: identification of inter-individual differentially methylated regions chronic high-fat diet in fathers programs b-cell dysfunction in female rat offspring physiological resistance of grasshopper mice (onychomys spp.) to arizona bark scorpion (centruroides exilicauda) venom persistent epigenetic differences associated with prenatal exposure to famine in humans dna methylation as a mediator of the association between prenatal adversity and risk factors for metabolic disease in adulthood transgenerational effects of prenatal exposure to the 1944-45 dutch famine analysis of bottlenecks in experimental models of infection haldane's the causes of evolution and the modern synthesis in evolutionary biology the genome of the offspring of a neanderthal mother and a denisovan father timing of archaic hominin occupation of denisova cave in southern siberia on the origin of modern humans: asian perspectives early human dispersals within the americas dating the origin of the ccr5-d32 aids-resistance allele by the coalescence of haplotypes long-term control of hiv by ccr5 delta32/delta32 stem-cell transplantation evaluating plague and smallpox as historical selective pressures for the ccr5-delta 32 hiv-resistance allele reappraisal of the historical selective pressures for the ccr5-d32 mutation convergent evolution in european and rroma populations reveals pressure exerted by plague on toll-like receptors small amounts of archaic admixture provide big insights into human history malaria in east african highlands during the past 30 years: impact of environmental changes common west african hla antigens are associated with protection from severe malaria full-exon resequencing reveals toll-like receptor variants contribute to human susceptibility to tuberculosis disease multiple common variants for celiac disease influencing immune gene expression epitope selection for hla-dq2 presentation: implications for celiac disease and viral defense card15/nod2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease association of nod2 leucine-rich repeat variants with susceptibility to crohn's disease nod1 and nod2: beyond peptidoglycan sensing global, regional and countrylevel 90-90-90 estimates for 2018: assessing progress towards the 2020 targe key: cord-323966-vj9za3cx authors: collier, karole t.; rothstein, david h. title: covid 19: surgery & the question of race date: 2020-05-20 journal: am j surg doi: 10.1016/j.amjsurg.2020.05.026 sha: doc_id: 323966 cord_uid: vj9za3cx nan population majority. 1 in cities like chicago, milwaukee and new orleans, death tolls are primarily concentrated in african americans neighborhoods. 2 it is no coincidence that in these same locations, residents have battled an entrenched history of segregation, underinvestment and underemployment. these factors work synergistically with poor social determinants of health to increase vulnerability to covid-19 and result in egregious disparities in infection rates and morbidity/mortality. 1 although the national discussion is appropriately focused on the disheartening reality of covid-19's effects on the african american community, the disparities seen within this demographic are illustrative of the damage covid-19 is having on other marginalized populations. the disease burden currently seen in the african american community foreshadows a deeper and unknown risk of covid-19-related fatality in other equally vulnerable minority groups, including undocumented immigrants, people of native americans and latin american descent, rural inhabitants, those living in poverty, the homeless and the incarcerated. in this moment, our response to african american covid disparities is critical and signifies our call to action for all vulnerable populations affected. race inequity has a history both stark and nuanced in the u.s. and has rightfully warranted caution in scientific discussion. for some, covid-19's emerging disparity sings an all too familiar tune and remains a harsh reminder about the legacy and deadliness of racism in the u.s. in the face of covid-related health disparities, we are reminded that race, poverty and novel disease are inextricably linked. looking back to the not so distant past, the aids epidemic gave us a clear framework of this relationship and the lasting consequences of novel epidemics to african american health. like covid-19, aids started as a novel disease with a unique clinical picture and devastated the nation with an inconceivable rate of morbidity and mortality. as aids spread across the u.s., it exposed structural vulnerability within african american communities and the ways in which poor pre-existing health infrastructure contributed to outcome disparities. today, aids continues to disproportionately affect african americans, who account for 42% of new aids cases while making up only 13% of the total u.s. population. 3 the aids epidemic is the only modern day health crisis we can compare to covid-19; its lessons require us to have truthful reflection and discussion regarding our progress with disparities and the associations of race with health outcomes. we cannot allow the lessons learned from aids to be wasted and for mistakes to be repeated. how, you might ask, does this affect us as surgeons? while covid-19 and aids are not strictly surgical diseases, their implications are noteworthy and may deepen mistrust and distance between minorities and healthcare providers. thus, our investment in the pre-operative health of minorities is more critical now than ever before. this requires our protection, advocacy, and purposeful availability to minority communities. in light of the covid-19 related disparities, the question becomes: is it enough to continue the work that we are doing, or might this moment of exposed inequalities reveal an opportunity to pursue a more equitable healthcare system? how can we facilitate and advocate for new healthcare system partnerships? and when the dust settles, how do we keep true to our commitment to racial equity in medicine? it is in our nature to seek out a clear, concise, and actionable solution; sadly with the current pandemic there is no quick fix. but we might do well to push the conversation about racial disparities, and look at our own profession and practices. given the gravity of covid-19 and the current uncertainty regarding elective surgical cases, 4,5 recommitment and reformation must be our resolve. access to surgery starts long before a patient walks into our office or into our emergency room. surgical outcomes have to do with far more than our hands and our teams, and follow-up does not end with the post-operative visit. a successful surgical outcome is tied inextricably to a patient's overall health and the health of the system that cares for them. race, poverty and insurance status are only a few of the "non-medical" factors that influence outcomes. shortly before our nation had its first surge of covid-19 patients, the american college of surgeons published a perspective piece with a specific call to action in addressing disparities and surgical access. 6 other authors have encouraged surgeons to seek interventions by addressing various factors that may contribute to disparities, such as socio-economic factors and imbalanced referral networks. 7 in the complex terrain of covid-19, it is imperative that we recognize the utility of that request. let us take this moment to outline some of the tangible steps in our purview. first, reflect on one's own practice, survey our respective implicit bias training, and be honest with our diversity initiatives. two, reach out to primary care physicians and community organizations that are well informed about the populations at risk, and aid in their outreach efforts. three, advocate at the hospital administrator level for reviewing data that include race and social determinants. finally, support community initiatives, which reach volunteers directly to improve access to care by novel approaches such as telemedicine. while some of us have laid the foundation for these requests, we must have dedicated action towards our distance from primary care and fortify our partnerships with community leaders. such dedicated action can better engineer fast track referral systems and aid surgical care to minority communities. if covid-19 has taught us anything, it has taught us that the problems of healthcare inequity cannot afford the status quo. we need a paradigm shift in how we care for patients, and in particular, minority patients. the complex nature of race, poverty, and social vulnerability are immutable characteristics of these patient populations 7 and whether it's now in the face of covid-19, or in the future as we rebuild, these characteristics will change the way in which these patients have surgery. though it is wise to precaution against generalized assumptions about race and its effects on african american health, it cannot be contested that regardless of socioeconomic status and prior health, african americans tend to have poorer outcomes and worse morbidity across a myriad of health measures. this is what makes covid-19 so devastating; we must acknowledge the impact of this disparity in this community and recognize that it is one that goes far beyond this moment. too often we have avoided "race informed" medicine. this is to mean utilization of what we know regarding race-based health disparities and appropriately augmenting care in pursuit of a more equitable system. sadly, we are well aware of the past, the exploitation of race, and the lasting harm it has created for minority communities. the call now is to not shy away from race, but instead to use it as a means of informing care; to right our wrongs and lift up those most crucially affected, so that we don't again find ourselves rewriting the same story. the coronavirus is infecting and killing black americans at an alarmingly high rate chicago's coronavirus disparity: black chicagoans are dying at nearly six times the rate of white residents, data show. chicagotribune the covid trolley dilemma elective surgery in the time of covid-19 perspective: identifying and addressing disparities in surgical access: a health systems call to action surgical disparities: beyond non-modifiable patient factors surgery & the question of race the authors whose names are listed immediately below certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript children's hospital conventus building 1001 main st 3rd floor buffalo this does not necessarily represent the views of the university at buffalo jacobs school of medicine & biomedical sciences or john r. oishei children's hospital. key: cord-308165-pk8d48hs authors: olu, olushayo oluseun; waya, joy luba lomole; maleghemi, sylvester; rumunu, john; ameh, david; wamala, joseph francis title: moving from rhetoric to action: how africa can use scientific evidence to halt the covid-19 pandemic date: 2020-10-28 journal: infect dis poverty doi: 10.1186/s40249-020-00740-0 sha: doc_id: 308165 cord_uid: pk8d48hs the ongoing pandemic of the coronavirus disease 2019 has spread rapidly to all countries of the world. africa is particularly predisposed to an escalation of the pandemic and its negative impact given its weak economy and health systems. in addition, inadequate access to the social determinants of health such as water and sanitation and socio-cultural attributes may constrain the implementation of critical preventive measures such as hand washing and social distancing on the continent. given these facts, the continent needs to focus on targeted and high impact prevention and control strategies and interventions which could break the chain of transmission quickly. we conclude that the available body of scientific evidence on the coronavirus disease 2019 holds the key to the development of such strategies and interventions. going forward, we recommend that the african research community should scale up research to provide scientific evidence for a better characterization of the epidemiology, transmission dynamics, prevention and control of the virus on the continent. the ongoing pandemic of the coronavirus disease 2019 (covid-19) has severely impacted global health, economy and politics in different patterns in various continents. the disease which is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was declared a public health event of international concern on 30th january 2020 [1] and a pandemic on 11th march 2020 [2] . as of 10th august 2020, 19 462 112 confirmed cases and 722 285 deaths of the disease had been reported from all countries, areas and territories of the world [3] . africa reported its first case in egypt on 14th february 2020 through an importation and by 10th august 2020, all 54 african countries had reported 1 035 932 confirmed cases and 22 920 deaths [4, 5] . local community transmission has been established in most of these african countries. while africa remains one of the least affected regions, recent developments and data from within the continent and other regions particularly europe and america show that the numbers of cases and deaths can grow exponentially and overwhelm even the best of systems in a relatively short time if effective prevention and control measures are not instituted on time [6, 7] . the fear, anxiety and panic induced by the rapid spread of the pandemic prompted several african countries to take drastic actions some of which are not necessarily based on scientific evidence. some countries closed their air, sea and land borders and imposed total national or partial sub-national lockdowns in a bid to prevent importation and minimize local transmission. given the weak health system in most african countries, mounting timely and robust responses to the covid-19 pandemic will be a big challenge hence the need to focus on targeted and high impact prevention and control interventions that could break the chain of transmission quickly. this becomes more pertinent given the african context where inadequate access to water, sanitation and the extended family system renders the implementation of critical preventive measures such as hand washing and social distancing challenging. this is further compounded by the global shortage of required human and material resources, which is more glaring in africa. due to the rapid spread and impact of the disease on human health, trade and travel, several research (mostly preliminary) have been conducted and published to characterize the virus and the dynamics of its transmission, prevention and control. while some of these publications have shown varying findings, conclusions and recommendations, many key and consistent evidences on the characteristics of the virus, its transmission, prevention and control are now emerging. this body of knowledge is critical to inform the development of timely, effective and context-specific prevention and control strategies in africa. in this article, we review the relevant scientific literatures on the covid-19 pandemic, and synthesize the relevant evidence that could potentially change the game in africa's fight against the disease; finally we propose strategic recommendations for prevention and control of covid-19 transmission in the africa continent specifically. the initial characteristics of covid-19 cases suggest that the disease is zoonotic [8] . however, recent scientific evidence demonstrates that the current transmission pattern globally is from human-to-human. the virus is similar to the severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) viruses with susceptibility and severity associated with older age group [9, 10] , male gender [11, 12] and underlying medical conditions such as poor immune functions, chronic diseases and surgery [13] . with a basic reproduction rate ranging from 2.6 to 4.71 [14, 15] and fatality rate from 2.3 to 11% [9, 16] it is more transmissible but less fatal compared to sars and mers [17, 18] . as is the case with most influenza viruses, the transmission of sars-cov-2 significantly reduces with an increase in temperature and humidity [19, 20] , however, this advantage may be offset by the high transmissibility of the virus. three main routes of transmission have been identified among humans namely ingestion or inhalation of contaminated droplets released into the air when a patient sneezes, coughs or talks, contact with surfaces which have been contaminated by infected persons and the inhalation of aerosols generated during some medical procedures [21, 22] . a few studies have also suggested faeco-oral transmission of the virus [23, 24] . a recent study concluded that the virus remains viable and infectious in aerosols for a few hours and up to a few days on surfaces, particularly on stainless steel and plastics [25] . while distinct signs and symptoms such as fever, dry cough, runny nose, difficulty in breathing, etc. have been associated with the disease, recent evidence suggests a high proportion of covid-19 cases are infectious but undocumented either because they have mild or no symptoms but yet continue to transmit the disease [26] . this could contribute to the high transmissibility and rapid geographic spread of the virus. based on the lessons learnt from responding to the outbreak in china, prevention and control strategies have been proposed and are in use at various levels. in the absence of a vaccine, infection prevention and control measures that include measures to reduce or prevent exposure to the virus such as identification of suspected cases through syndromic screening at points of entry into countries, public places, health facilities, prevention of shedding of virus into the environment through respiratory hygiene have been recommended as prevention and control strategies in the general population [21] . others include proper sanitation and waste management, social distancing to prevent contact with infected persons, avoidance of touching potentially contaminated surfaces, eyes, nose and mouth with contaminated hands and hand washing with soap and water or hand sanitizers which contain at least 60% alcohol [21] . available evidence suggests that social distancing may have a dramatic effect on the transmission of sars-cov-2 and other respiratory infections, [27] but there may be renewed virus transmission following relaxation of such measures due to the large proportion of susceptible people that would still be in the population [28] . there is a convergent view on the important role of face masks in reducing the transmission of covid-19 by protecting healthy persons who come into contact with an infected individual and by preventing infected persons from shedding the droplets into the environment [29, 30] . the use of face masks has also been associated with lower levels of anxiety and depression among the general population and health care workers which further supports its importance in prevention and control of the disease [31] [32] [33] . at the individual case level, timely diagnosis, isolation and supportive management of confirmed cases, identification and follow-up of their contacts, prevention of nosocomial transmission through strong infection prevention and control methods and use of personal protective equipment are recommended [34] . there is no known cure for the disease currently but several clinical trials involving various therapies are ongoing. who recommends that all laboratory confirmed cases should be isolated and managed in health facility settings but where this is not possible priority should be given to cases with the probability of poor outcomes such as those aged above 60 years and with underlying medical conditions which put them at higher risk [35] . recommendations for covid-19 strategy development specific for the africa continent putting the above scientific evidences on the characteristics and dynamics of covid-19 transmission, prevention and control into perspective against the backdrop of the social, cultural and economic context in africa, we deduce several lessons which could guide african countries to better prepare for and respond to the covid-19 pandemic on the continent. while the mostly hot and humid african weather and largely younger population may be deterrent factors for wide transmission of the disease, any advantage conferred by this is offset by the high transmissibility of the disease. this is more so given the high population density, larger families and large vulnerable populations such as refugees, internally displaced persons (idps), people living with the human immunodeficiency virus (hiv), tuberculosis (tb) and malnutrition thus emphasis in african countries should be on prevention of the spread of infection, especially to these vulnerable groups. in this regard, african countries should invest in identifying, developing and implementing tailor-made prevention strategies to protect at risk populations from infection [36] . syndromic surveillance, laboratory testing and contact tracing at the community level given the high proportion of undocumented and asymptomatic cases of covid-19, the use of syndromic surveillance for disease detection at points of entry may not be very effective [37] . while syndromic surveillance may offer some level of reassurance to governments and the general population, the cost in terms of the human and financial resources associated with conducting it may offset its benefits. african countries should rather invest in active search for cases and their contacts at the community and household levels particularly the asymptomatic contacts and transmitters through scaling up testing of all persons who may have been exposed to the virus but remain asymptomatic. to achieve this, covid-19 testing strategies which prioritize massive testing of various categories of persons based on the transmission scenario should be developed. additionally, efforts should be made to increase testing capacity and timeliness by decentralizing testing to the sub-national levels. the evidence that the virus survives much longer on surfaces such as stainless steel and plastic as compared to respiratory droplets have far reaching implications for prevention and control of the virus at the population level. first, risk communication messages should emphasize the high risk constituted by surfaces such as doorknobs, stainless steel handrails, disposable plastics etc. and encourage people to refrain from unnecessary touching of such surfaces. second, regular disinfection of such surfaces at the household and community level is advised and should be included in risk communication messages and during community engagement sessions [23] . third, in the light of new findings on asymptomatic transmission of covid-19 and recommendations on the usage of face masks by healthy individuals, african countries should define clear policies on the use of face masks. such policies should ensure that face masks are available to those who need them particularly the frontline healthcare workers, caregivers and vulnerable groups, address the issue of shortage of masks and other supplies which is already a challenge in many african countries and importantly provide clear guidance to the general population on the pros and cons, safe use, donning and doffing of face masks. furthermore, african scientists should pursue urgent researches into the use of locally available material for the production of face masks which are suitable to the african context [29, 30] . while available scientific evidence shows that the social distancing which is the ultimate aim of the current lockdowns and population movement restriction measures instituted by several african countries may reduce transmission of the virus in the short term, this strategy alone may not be enough to break the chain of transmission [28] . since there is already widespread community transmission in many of these countries, population confinement may result in a change in the transmission pattern from the community to the household level [38] . furthermore, african countries may not be able to sustain such lockdowns for a long time given their socioeconomic context thus they should focus on making the best use of the small window of opportunity that they offer. first, clear objectives should be set for lockdowns which should be to reduce transmission through the scale up of preparedness and response interventions and to control the outbreak in areas of transmission. these objectives should be communicated clearly to the general population to forestall community resistance to lockdowns which is being experienced in some of the african countries. second, the definition of areas where to impose confinement should be guided by the epidemiology and pattern of transmission of the disease. third, the lockdowns should be accompanied by intensive risk communication, active case search at the community and household levels, massive testing, contact tracing and isolation. fourth, adequate preparation should be made to ensure that confined populations have access to basic services such as food, water, healthcare etc. during lockdowns in order to reduce community resistance and ensure adherence. fifth, appropriate strategies to prevent a second wave of the pandemic following the lifting of the lockdown measures should be developed and implemented [39] . given the weak health systems in most of the infected african countries, institutional management of all laboratory confirmed cases may not be a feasible option. on the other hand, home management of such cases is constrained by several challenges due to the large household size, poor housing and high population density in many african countries. countries should therefore develop context-specific case management strategies which should classify cases according to their risk and health needs, identify places such as health facilities and non-health facilities such as repurposed hostels, schools, hotels or stadia where the various categories of cases will be isolated, define and identify the minimum package of resources such as health workers, medicines, medical equipment and other logistics which are needed to effectively manage the anticipated caseloads. such strategies should be based on the prevailing transmission scenario in the country. the covid-19 pandemic has overwhelmed even strong health systems in europe and america. a review and analysis of the impact of the 2014-2015 ebola outbreaks in west africa on health systems revealed that there was a significant reduction in access to routine health services and this led to substantially increased mortality from preventable diseases such as malaria, measles, hiv, aids and tb. african countries should learn from this experience and implement available guidance from who to ensure that essential health services are maintained during the covid-19 pandemic particularly during lockdowns to reduce excess mortality from other preventable diseases [40] [41] [42] . key to maintenance of essential services during the covid-19 pandemic is the protection of health care workers from acquiring covid-19 infection; this can be achieved by providing african health workers with the necessary equipment, information and training on how to protect themselves [43] . management of covid-19 outbreaks in the situation of population displacement such as refugee and idps camps and in prisons and urban slums which are common in africa is a major challenge. other high-risk situations include in large-scale industries which employ a large number of semi-skilled workers [44] . the high transmissibility of the virus, overcrowding and inadequate access to social services such as water and sanitation will rapidly facilitate transmission of the virus and constrain implementation of preventive measures such as social distancing in such situations. african countries should, therefore, invest in the development of special public health strategies for prevention and control of outbreaks in such settings [36] . establishment of covid-19 information and testing centres near such areas is recommended to improve rapid access of the high-risk populations to covid-19 prevention and control services [44] . importantly, the lessons from the rapid spread of the virus in china, italy, iran, republic of korea and america should be a wake-up call for african countries to rapidly scale up risk communication, community engagement, and participation. the scientific evidence described above and outcomes of anthropological studies on covid-19 should be used as the basis for development of evidence-based and context specific risk communication messages. such risk communication messages should be focused on achieving behavioural change and tailor-made to address the several sociocultural myths, stigma, misconceptions and rumours associated with the virus, its transmission, prevention and control. african countries need to act early and decisively to avert excess morbidity and mortality due to covid-19 and the associated impact it could have on their economy, public health and health system. this could be achieved by using the available global scientific evidence to inform the development and implementation of context-specific covid-19 prevention and control strategies. given that there is currently no known cure or vaccine for the disease, such strategies should prioritize prevention and other appropriate interventions in a balanced manner. the african research community should scale up research to provide scientific information for better characterization of the epidemiology, transmission dynamics, prevention and control of the sars-cov-2 and other viruses on the continent. furthermore, african countries should use the opportunity of the covid-19 preparedness and response to systematically strengthen their health system capacity for broader and longer-term epidemic preparedness and response by using platforms such as the national action plans for health security. finally, given the chronic outlook of this pandemic, african countries should explore opportunities to mainstream ongoing covid-19 response interventions into existing healthcare programmes to ensure cost-effectiveness and sustainability in the long-term. detail/30-01-2020-statement-on-the-secondmeeting-of-the-international-health-regulations accessed 29 world health organization. who director-general's opening remarks at the media briefing on covid world health organization. coronavirus disease (covid-19) situation dashboard world health organization regional office for africa. covid-19 in the world health organization african region world health organization regional office for eastern mediterranean world health organization. coronavirus disease 2019 (covid-19) situation report â��68 lessons from italy's response to coronavirus, havard business review chinese center for disease control and prevention. 585 environmental samples from the south china seafood market in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia the incubation period of 2019-ncov infections among travellers from wuhan. china euro surveill updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan modelling the epidemic trend of the 2019novel coronavirus outbreak in china breaking down of healthcare system: mathematical modelling for controlling the novel coronavirus (2019-ncov) outbreak in wuhan transmission dynamics of 2019 novel coronavirus (2019-ncov) a mathematical model for simulating the phase-based transmissibility of a novel coronavirus high temperature and high humidity reduce the transmission of covid-19 accessed 29 the efects of temperature and relative humidity on the viability of the sars coronavirus epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (covid-19) during the early outbreak period: a scoping review modes of transmission of virus causing covid-19: implications for ipc precaution recommendations. scientific brief air, surface environmental, and personal protective equipment contamination by severe acute respiratory ryndrome coronavirus 2 (sars-cov-2) from a symptomatic patient aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) how we know ending social distancing will lead to more deaths public health interventions and epidemic intensity during the 1918 influenza pandemic rational use of face masks in the covid-19 pandemic the-use-of-masks-in-the-community-during-home-care-andin-healthcare-settings-in-the-context-of-the-novel-coronavirus-(2019-ncov)-outbreak is returning to work during the covid-19 pandemic stressful? a study on immediate mental health status and psychoneuroimmunity prevention measures of chinese workforce immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china a longitudinal study on the mental health of general population during the covid-19 epidemic in china disinfection measures for pneumonia foci infected by novel coronavirus in 2019 home care for patients with covid-19 presenting with mild symptoms and management of their contacts: interim guidance covid-19 control in low-income settings and displaced populations: what can realistically be done? effectiveness of airport screening at detecting travellers infected with novel coronavirus (2019-ncov) the covid-19 pandemic in the us: a clinical update what's the way out? potential exit strategies from the covid-19 lockdown the health impact of the 2014-15 ebola outbreak effects of response to 2014-2015 ebola outbreak on deaths from malaria, hiv/aids, and tuberculosis covid-19: operational guidance for maintaining essential health services during an outbreak interim guidance coverage of health information by different sources in communities: implication for covid-19 epidemic response characterize health and economic vulnerabilities of workers to control the emergence of covid-19 in an industrial zone in vietnam we thank the global research community which has worked tirelessly to provide scientific evidence for better understanding and management of covid-19. we acknowledge the support provided by victoria awuor jura in the proof reading and copy-editing of the final version of the manuscript. the authors alone are responsible for the views expressed in this article, which do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. authors' contributions ooo conceived and wrote the first draft of the manuscript. all authors read and provided significant inputs into all drafts of the manuscript, agreed to be accountable for all aspects of the work and approved the final draft of the manuscript for publication. all the authors are members of the covid-19 preparedness and response team of who country office, south sudan. no funding was received for this manuscript.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. received: 13 april 2020 accepted: 12 august 2020 key: cord-355737-o0y4rn0z authors: ng, melinda; ndungo, esther; kaczmarek, maria e; herbert, andrew s; binger, tabea; kuehne, ana i; jangra, rohit k; hawkins, john a; gifford, robert j; biswas, rohan; demogines, ann; james, rebekah m; yu, meng; brummelkamp, thijn r; drosten, christian; wang, lin-fa; kuhn, jens h; müller, marcel a; dye, john m; sawyer, sara l; chandran, kartik title: filovirus receptor npc1 contributes to species-specific patterns of ebolavirus susceptibility in bats date: 2015-12-23 journal: elife doi: 10.7554/elife.11785 sha: doc_id: 355737 cord_uid: o0y4rn0z biological factors that influence the host range and spillover of ebola virus (ebov) and other filoviruses remain enigmatic. while filoviruses infect diverse mammalian cell lines, we report that cells from african straw-colored fruit bats (eidolon helvum) are refractory to ebov infection. this could be explained by a single amino acid change in the filovirus receptor, npc1, which greatly reduces the affinity of ebov-npc1 interaction. we found signatures of positive selection in bat npc1 concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls ebov infection in eidolon helvum cells. our work identifies npc1 as a genetic determinant of filovirus susceptibility in bats, and suggests that some npc1 variations reflect host adaptations to reduce filovirus replication and virulence. a single viral mutation afforded escape from receptor control, revealing a pathway for compensatory viral evolution and a potential avenue for expansion of filovirus host range in nature. doi: http://dx.doi.org/10.7554/elife.11785.001 ebola virus (ebov) and some of its relatives in the family filoviridae (filoviruses) cause sporadic outbreaks of a highly lethal disease. these outbreaks are thought to be initiated by viral spillover from an animal reservoir to a highly susceptible accidental host, such as a human or nonhuman primate (feldmann and geisbert, 2011; leroy et al., 2005; towner et al., 2009) . recent work suggests that some filoviruses infect bats in nature, and that these viruses may be distributed more widely than previously recognized. very short rna fragments corresponding to portions of ebolavirus genomes were detected in several frugivorous bats of the family pteropodidae ('old world fruit bats') in both africa and asia (leroy et al., 2005; jayme et al., 2015) , and longer filovirus rna fragments and near-complete rna genomes were isolated from insectivorous schreibers's long-fingered bats in asia and europe, respectively (negredo et al., 2011; he et al., 2015) . however, despite considerable efforts, infectious ebolaviruses have never been recovered from bats. by contrast, marburg (marv) and ravn (ravv) viruses were found to circulate in egyptian rousettes (rousettus aegyptiacus), indicating that these bats are susceptible to marv/ravv and encounter them frequently in nature. egyptian rousettes have been proposed as natural hosts for these viruses (amman et al., 2012; towner et al., 2009) . this progress notwithstanding, many key questions remain. for example, the biological factors that influence filovirus host range and interspecies transmission are still poorly understood, as are the virus-host relationships that determine which species of bats are susceptible to infection by ebov and other filoviruses. viral entry receptors are key determinants of tissue tropism and host range (radoshitzky et al., 2008; sheahan et al., 2008; hueffer et al., 2003; demogines et al., 2013) . niemann-pick c1 (npc1), a highly conserved endo/lysosomal protein involved in cellular cholesterol trafficking, was recently identified to be an essential entry receptor for all known filoviruses (cô té et al., 2011; carette et al., 2011; ng et al., 2014) . in this study, we uncover a pattern of virus and host species specificity in the filovirus susceptibility of bat cells, which can be explained by elife digest ebola virus and other filoviruses can cause devastating diseases in humans and other apes. numerous small outbreaks of ebola virus disease have occurred in africa over the past 40 years. however, in 2013-2015, the largest outbreak on record took place in three western african nations with no previous history of the disease. human outbreaks of ebola virus disease likely begin when a person encounters an infected wild animal. though it remains unclear precisely which animals harbor ebola virus between outbreaks, and how they transmit the virus to humans or other primates, recent work showed that some filoviruses do infect specific types of bats in nature. ng, ndungo, kaczmarek et al. sought to identify the genes that influence whether or not a type of bat is susceptible to infection by ebola virus and other filoviruses. several filoviruses, including ebola virus, were tested to see if they could infect cells that had been collected from four types of african fruit bats. these bats are all found in areas where outbreaks have occurred in the past. the tests revealed that a small change in the sequence of the npc1 gene in some bat cells greatly reduced their susceptibility to ebola virus. npc1 encodes a protein that mammals need in order to move cholesterol within their cells. in humans, the loss of the protein encoded by npc1 causes a rare but very severe disease called niemann-pick type c disease. this protein also turns out to be a receptor that the filoviruses must bind to before they can infect the cells. further analysis then revealed that npc1 has evolved rapidly in bats, with changes concentrated in the parts of the receptor that interact with ebola virus. ng, ndungo, kaczmarek et al. went on to discover some changes in the genome sequence of ebola virus that could compensate for the changes in the bat's npc1 gene. these findings hint at one way that a filovirus could evolve to better infect a host with receptors that were less than optimal. following on from this work, the next challenges will be to expand the investigation to include additional types of bats, other types of mammals, and other host genes that could influence filovirus infection and disease. further studies could also examine the other side of the arms race -that is, the evolution of viral genes in bats. however, such studies would be complicated by the lack of viral sequences that have been collected from bats, because to date most have been isolated from humans and other primates instead. changes in the affinity of the essential interaction between npc1 and the filovirus entry glycoprotein, gp. crucially, genetic analyses reveal that npc1 is under positive selection in bats, with a strong signature of selection at precisely the same residue that influences the filovirus-receptor interaction. our findings suggest that amino acid sequence changes in npc1 at these positively-selected sites represent host adaptations to resist filovirus infection, and reveal one pathway by which a filovirus could escape from receptor control. in sum, our results support the hypothesis that bats and filoviruses have been engaged in a long-term co-evolutionary relationship, one facet of which is a molecular arms race between the viral glycoprotein and its entry receptor, npc1. means ± sd (n = 3-4) from two biological replicates are shown. in panels c and d, the infectivity of each virus was normalized to that obtained in vero grivet monkey cells. means for infection of the different cell lines by each virus were compared by one-way anova (p-value indicated above each group of bars). tukey's post hoc test was used to compare infection means on hypsignathus monstrosus vs eidolon helvum cells (*p < 0.05; ****p < 0.0001; ns, no statistical significance). doi: 10.7554/elife.11785.003 the following figure supplements are available for figure 1 : we first explored the possibility that there exist virus-and/or bat species-dependent differences in the cellular host range of filoviruses. kidney fibroblast cell lines derived from three african pteropodids whose ranges overlap the locations of known african filovirus disease outbreaks ( figure 1a ,b) were exposed to authentic ebov and marv ( figure 1c ). we observed a large ebov infection defect in african straw-colored fruit bat (eidolon helvum) cells but not in cells from bü ttikofer's epauletted fruit bats (epomops buettikoferi) and egyptian rousettes. by contrast, cells from bats of all three species were similarly susceptible to infection by marv ( figure 1c) . thus, cells from african straw-colored fruit bats appear to be selectively refractory to ebov infection. an npc1-dependent block to cell entry accounts for the ebov infection deficit in african straw-colored fruit bat cells the viral spike glycoprotein, gp 1,2 (herein termed gp) mediates all steps of filovirus entry into the cytoplasm of host cells . vesicular stomatitis viruses bearing filovirus gp proteins (vsv pseudotypes) provide a highly validated surrogate system to recapitulate filovirus entry under biosafety level 2 containment (takada et al., 1997; jangra et al., 2015) . to assess whether the ebov infection defect in the african straw-colored fruit bat cells occurs at the viral entry step, we exposed an expanded panel of kidney fibroblast cell lines from four african pteropodids to vsv pseudotypes bearing gp spikes (vsv-gp) from seven filoviruses, including two non-african viruses, reston virus (restv) and lloviu virus (llov) ( figure 1d ). as observed with authentic ebov, vsv-ebov gp infection was substantially reduced in the african straw-colored fruit bat cells; however, this virus could efficiently infect cells derived from the other pteropodids, including those of a proposed ebov host, the hammer-headed fruit bat (hypsignathus monstrosus) (leroy et al., 2005) . strikingly, only vsvs bearing ebov gp, and to a lesser degree, those bearing bdbv and tafv gp, were deficient at infecting african straw-colored fruit bat fibroblasts. similar strong but ebov-specific reductions in infection were measured in two kidney and lung cell lines derived from additional african straw-colored fruit bats (figure 1-figure supplement 1) . therefore, reduced infection of these bat cells by ebov reflects a virus-and host species-specific restriction at the cell entry step. we surmised that the filovirus receptor, npc1, might explain the selective resistance of the african straw-colored fruit bat cells to ebov entry and infection. accordingly, we engineered these cells to stably express human npc1 (hsnpc1) (figure 2-figure supplements 1,2), and then exposed them to ebov ( figure 2a ). provision of hsnpc1 substantially enhanced authentic ebov infection in the african straw-colored fruit bat cells. by contrast, we found no evidence that either marv infection in these cells, or ebov/marv infection in permissive bü ttikofer's epauletted fruit bat cells was limited by receptor availability (figure 2a ). finally, similar results were obtained with vsvs bearing filovirus glycoproteins ( figure 2b ). taken together, these findings indicate that ebov infection is reduced in african straw-colored fruit bat cells because of a specific molecular incompatibility between the ebov glycoprotein and the filovirus entry receptor. npc1-dependent cell entry is reduced, but not completely eliminated, in african straw-colored fruit bat cells although ebov entry and infection in african straw-colored fruit bat cells was consistently reduced to 0.1-1% relative to that in cells from the other pteropodids, we noted that infection was not completely blocked. to determine if ebov could inefficiently infect these bat cells via an npc1-independent mechanism, we used crispr/cas9 genome engineering to derive an african straw-colored fruit bat cell line fully deficient in npc1. we identified a single cell clone (eidolon helvum npc1-#1 [ehnpc1-#1]) in which all npc1 alleles bore insertions or deletions (indels) at the expected site ( figure 3a ). these indels were predicted to frameshift the npc1 open reading frame at amino acid position 81 (homo sapiens hsnpc1 numbering), generating truncated polypeptides of 82, 83, and 109 residues that lacked the majority of the 1278-amino acid npc1 sequence. ehnpc1-#1 cells were deficient in clearance of lysosomal cholesterol, a well-established cellular function of npc1 (carstea et al., 1997) , but could be rescued by ectopic hsnpc1 expression, confirming that npc1 had indeed been disrupted in these cells ( figure 3b ). we next exposed wild-type (wt) and ehnpc1-#1 fibroblasts to vsvs bearing ebov or marv gp. no detectable infection was obtained with either virus in npc1-deficient cells, indicating that filovirus entry into these cells is absolutely dependent on the e. helvum npc1 ortholog ( figure 3c ). moreover, ebov gp-dependent infection in ehnpc1-#1 cells reconstituted with hsnpc1 was dramatically enhanced over that observed in wt cells, whereas marv gp-dependent infection was rescued by hsnpc1 expression to a level resembling that in wt cells ( figure 3c ). therefore, the low levels of ebov infection in african straw-colored fruit bat cells likely arise from the weak, but nonzero, activity of ehnpc1 as an ebov entry receptor. filovirus gps must directly engage the second luminal domain of npc1, domain c, during cell entry . accordingly, we postulated that the african straw-colored fruit bat npc1 ortholog is poorly recognized by ebov gp. to test that hypothesis, we generated and sequenced npc1 cdnas from all four pteropodid cell lines. alignment of their domain c amino acid sequences with that of hsnpc1 revealed a high degree of conservation (>90%), with identical arrangements of cysteine residues and similar predicted secondary structures suggestive of a similar overall fold (figure 3-figure supplement 1) . to examine gp-npc1 binding, we engineered and expressed soluble forms of the four pteropodid npc1 domain cs, as described for hsnpc1 (figure 4 -figure supplement 1) . a cleaved form of ebov gp could capture hsnpc1 domain c in an elisa, as shown previously . ebov gp bound with similar avidity to npc1 domain cs derived from egyptian rousettes (ranpc1), hammer-headed fruit bats (hmnpc1) and bü ttikofer's epauletted fruit bats (ebnpc1), but poorly or not at all to that of african straw-colored fruit bats (ehnpc1) ( figure 4a ). like the infection defect in african straw-colored fruit bat cells, this receptor binding defect was selective for ebov gp, since gps derived from marv and the european filovirus, llov (ng et al., 2014) , bound equivalently to all four pteropodid domain cs ( figure 4a ). these findings the restriction in ehnpc1-ebov gp binding can be mapped to a single amino acid change in ehnpc1 to define the molecular basis of the defect in interaction between ebov and ehnpc1, we generated a panel of npc1 domain c chimeras comprising sequences from permissive ranpc1 and nonpermissive ehnpc1, and tested them in the gp-binding elisa. a single chimera, ehnpc1 domain c containing four ehnpc1firanpc1 amino acid residue changes, regained the capacity to efficiently recognize ebov gp ( figure 4b ). further dissection revealed that only a single amino acid change, f502d, in a central region of npc1 domain c was needed to effect this complete restoration in gpfigure 3 . the incompatibility between ebov gp and eidolon helvum npc1 reduces, but does not eliminate, ebov entry into african straw-colored fruit bat cells. (a) crispr/cas9 genome engineering was used to knock out the npc1 gene in african straw-colored fruit bat kidney fibroblasts. wt npc1 gene sequence aligned with the sequences of all three alleles in the knockout (npc1-#1) cell clone. the grna target sequence is marked in red, and the protospacer adjacent motif (pam) sequence of the grna target site is underlined. (b) the capacity of wt and npc1-#1 cells, and npc1-#1 cells stably expressing hsnpc1, to clear lysosomal cholesterol was determined by staining with filipin iii complex from streptomyces filipensis, as described (carette et al., 2011) . . we conclude that a species-specific defect in virus-receptor interaction, caused by a single amino acid residue change in ehnpc1 relative to other, permissive african pteropodid npc1 orthologs, reduces ebov infection in african straw-colored fruit bat cells. moreover, because residues in the npc1-binding site are conserved among all available ebov gp sequences (supplementary file 1), this restriction is almost certain to be encountered by all known ebov variants and their isolates, including those detected in ebov disease patients during the recent epidemics in western and middle africa gire et al., 2014; tong et al., 2015; carroll et al., 2015; kugelman et al., 2015) . previous work has led to the hypothesis that bats in equatorial africa and elsewhere harbor filoviruses (reviewed in [wahl-jensen et al., 2013] ). these results, together with our findings for virusand host species-specific differences in cellular susceptibility to filovirus infection, hinted at the possibility of a deeper co-evolutionary relationship between filoviruses and bats. one hallmark of such a relationship between a virus and its host is the evolution, under selective pressure to resist infection, of host genes encoding proviral and antiviral factors. to evaluate whether the npc1 gene has evolved under positive selection in bats, we combined the npc1 sequences obtained in this study with those of bats from six other species (two non-african pteropodids, two phyllostomids, and two vespertilionids) compiled through assembly of publicly available rnaseq data ( a mutation in ehnpc1 reduces receptor binding to ebov gp and viral infection, a phenotype that could reasonably produce a selective advantage (figure 4) . other codons identified in only some of the tests for dn/ds>1, or at slightly lower significance levels, may still have functional significance. for example, additional codons were identified in two regions of domain c that may form a part of the recognition surface for ebov gp ( figure 5c ). our finding that signatures of accelerated sequence evolution localize to structural features in npc1 that are important for virus binding (domain c and position 502) leads us to postulate that mutations at these sites can protect bats from infection or severe disease caused by filoviruses and/or other intracellular microbes. a single mutation at residue 141 in ebov gp enhances viral entry by strengthening its interaction with ehnpc1 co-evolutionary arms races between hosts and pathogens are thought to be driven by cycles of genetic adaptation and counter-adaptation (meyerson and sawyer, 2011; daugherty and malik, 2012; demogines et al., 2013) . in this context, we postulated that mutation of residue 502 in ehnpc1 could be countered by viral mutation. to identify such putative compensatory viral changes, figure 6 . a sequence polymorphism in the npc1-binding site of filovirus gp influences gp-ehnpc1 binding and ehnpc1-dependent filovirus entry. (a) binding of ebov gp (wt and mutant v141a) to soluble npc1 domain c proteins derived from african pteropodids measured by an elisa. ranpc1, egyptian rousette; ebnpc1, bü ttikofer's epauletted fruit bat; hmnpc1, hammer-headed fruit bat; ehnpc1, african straw-colored fruit bat. (b) infection of african straw-colored fruit bat cells with vsv pseudotypes bearing ebov gp (wt or v141a). means ± sd (n = 3-4) from a representative experiment are shown in each panel. means for vsv-ebov gp wt vs v141a infection were compared by unpaired two-tailed student's t-test with welch's correction (**p < 0.01). (c) surface-shaded representation of a single gp1-gp2 monomer (pdb id: 3csy highlighting key residues in the npc1-binding site (yellow) and residue 141 (red). gp1, blue. gp2, grey. (d) alignments of gp1 sequences from a panel of filoviruses. v141, orange; a141, white text on blue shading; other residues divergent from consensus sequence, black text on green shading. (e) infection of african pteropodid cells with vsv pseudotypes bearing sudv gp (wt or a141v). means ± sd (n = 4) from two biological replicates are shown. means for vsv-sudv gp wt vs a141v infection on each cell line were compared by unpaired two-tailed student's t-test with welch's correction (*p < 0.05, **p < 0.01, ****p < 0.0001). doi: 10.7554/elife.11785.015 we screened a panel of point mutants in the npc1-binding site of ebov gp by elisa for enhanced binders to ehnpc1 domain c. while no single point mutant bound to ehnpc1 as well as it did to the other pteropodid npc1s or to hsnpc1, gp(v141a) partially restored ehnpc1 binding ( figure 6a ). infection by vsv particles bearing ebov gp(v141a) was substantially enhanced in african straw-colored fruit bat cells, commensurate with this mutant gp's increased binding affinity for ehnpc1 ( figure 6b) . examination of the x-ray crystal structure of ebov gp revealed that v141 is located at the edge of the putative npc1-binding site, where it forms part of a raised rim ( figure 6c ). the v141a mutation likely creates a more sterically favorable (open) npc1binding site that can overcome the structural mismatch at the gp-npc1 binding interface ( figure 6c ). naturally-occurring sequence variation at residue 141 in gp contributes to virus-and bat species-specific patterns of cellular susceptibility to filoviruses although no known ebov isolate contains the v141a mutation, we observed that llov and sudan virus (sudv) gp naturally possess a141 ( figure 6d ). because both gp proteins could mediate efficient viral entry into african straw-colored fruit bat cells ( figure 1d ) and bind to ehnpc1 ( figure 4a data not shown for sudv), we postulated that amino acid changes at position 141 in the gp receptor-binding site broadly influence the capacity of filovirus glycoproteins to utilize ehnpc1 for viral entry. accordingly, we exposed pteropodid kidney fibroblasts to vsv pseudotypes bearing sudv gp(wt) or sudv gp(a141v) ( figure 6e ). consistent with our hypothesis, the a141v mutation substantially reduced sudv gp-dependent infection in african straw-colored fruit bat cells. unexpectedly, this mutant virus also infected egyptian rousette cells significantly less well than wt, pointing to the existence of sequence context-dependent effects that selectively affect sudv gp(a141v) binding to ranpc1 ( figure 6e ). these findings provide evidence that gp residue 141 can influence cellular susceptibility to infection by modulating npc1 recognition in a manner that depends on the sequences of both proteins. we speculate that sequence variation at residue 141 and potentially other positions in the receptor-binding site of filovirus glycoproteins has been shaped by selective pressure to utilize restrictive npc1 receptors, with potential consequences for viral host range and virulence. the ongoing, unprecedented ebola virus disease epidemic in western africa highlights the urgent need to uncover the biological and ecological factors that underlie the distribution, evolution, and emergence of filoviruses. while a full answer to this question will require the integration of knowledge across multiple levels of biological organization, from genes to populations to ecosystems, previous work has shown that studies of molecular interactions between viruses and their host cells can contribute important pieces to this puzzle. the essential interactions between viruses and their entry receptors provide particularly cogent examples. a switch in receptor binding from the feline to the canine ortholog of the transferrin receptor drove the emergence of a new virus, canine parvovirus, and fueled a global disease pandemic in dogs (allison et al., 2014) . analyses of interactions of sars-like coronaviruses with their receptor ace2 have helped to trace the emergence of sars coronavirus from bats to humans, and its use of civets as intermediate amplifying hosts ge et al., 2013; ren et al., 2008) . in this study, we show that interactions between filoviruses and their entry receptor npc1 can influence the cellular susceptibility of bats to infection. this observation is especially striking in light of previous findings that filoviruses could efficiently infect a broad range of mammalian cells, including some derived from bats (kuhn, 2008; kuhl et al., 2011) . indeed, this prior work and the results of experimental infection studies in rodents and bats have led to the hypothesis that interactions between viral components and those of the host innate and adaptive immune systems constitute the primary molecular variables influencing filovirus host range in nature (ebihara et al., 2006; volchkov et al., 2000) . here, we propose that npc1 is also a genetic determinant of filovirus susceptibility in bats. the essential nature of npc1 for infection in cells derived from mammals of multiple species, including bats (figure 3) , and for infection and in vivo pathogenesis in lethal ebov infection mouse models argues against the existence of alternative filovirus entry receptors (carette et al., 2011; herbert et al., 2015) . therefore, strong reductions in the affinity of virus-npc1 recognition are predicted to reduce or eliminate infection in whole bat hosts, as observed in npc1deficient mice (carette et al., 2011; herbert et al., 2015) , barring viral mutation to enhance this affinity. it is conceivable that even modest defects or delays in viral multiplication through such a mechanism could help determine host range by accelerating viral immune clearance, as recently observed in npc1-heterozygous mice (herbert et al., 2015) , or by synergizing with other host-virus barriers. the highly virus-and host species-specific nature of the virus-receptor mismatch uncovered in this study warrants the determination of more bat npc1 sequences for inclusion in genetic analyses (see below), and a more comprehensive phenotypic examination of virus-bat pairs. such studies maydiscover additional interesting bat-filovirus dynamics, including incompatibilities between filoviruses and npc1 or other proviral/antiviral host factors. such discoveries have potential implications for our understanding of the molecular basis of filovirus infection, virulence, and host range. we found that a single amino acid change, at residue 502, in the african straw-colored fruit bat ortholog of npc1 (ehnpc1) greatly diminished the susceptibility of cells from multiple tissues and individuals to ebov. these migratory pteropodids are widely distributed across sub-saharan africa ( figure 1a) , roost in large colonies near human settlements, and host other rna viruses with zoonotic potential peel et al., 2013) . moreover, they are extensively hunted for bushmeat in western africa (kamins et al., 2011) , making them ideal candidates to transmit viruses directly to humans. unfortunately, there is little information currently available on the susceptibility of african straw-colored fruit bats to ebov or their potential role as filovirus hosts. serologic surveys have found some evidence for exposure to one or more filovirus; however, neither infectious virus nor coding-complete or full viral genomes-the gold standards-have been successfully obtained from these bats, indicating they may only have been exposed to filoviruses, rather than being productively infected (reviewed in [wahl-jensen et al., 2013; olival and hayman, 2014] ). while more extensive wildlife sampling and, if feasible, experimental infections of african straw-colored fruit bats will be required to clarify this picture, we can extrapolate to several possible scenarios. first, these bats are fully resistant to ebov, and therefore cannot be the source of this virus in the 2013-present ebov disease outbreak in western africa or the 2014 outbreak in middle africa. second, because african straw-colored fruit bat cells do remain weakly susceptible to ebov ( figure 3c ), it is conceivable that they support ebov replication at low levels. indeed, this is one hallmark of a sustaining viral reservoir. third, the filoviruses circulating in these bats, whether ebov or otherwise, bear one or more gp mutations (e.g., v141a) that circumvent the infection barrier imposed by ehnpc1. assessing this last hypothesis and understanding the nature of the selection pressures that drive gp evolution in vivo will require the isolation of ebolavirus gp sequences from bats-there are none currently available. although these results suggest that african straw-colored fruit bats are selectively refractory to ebov, our genetic findings indicate that this is not merely a special relationship between one host and one virus. rather, we used a diverse set of bat npc1 sequences, only one of which is from african straw-colored fruit bats, to show that a number of codons, including residue 502, have evolved under recurrent positive selection. this is a process in which resistant npc1 variants are 'serially replaced' in response to compensating viral mutations that restore susceptibility. we provide evidence that the filovirus gp interaction surface in the second luminal domain of npc1, domain c, is a hotspot for such positive selection ( figure 5 ). by contrast, the vast majority of codons in mammalian npc1 have evolved under purifying selection. we propose that this pattern of selection is the signature of a long-term genetic conflict between filoviruses and npc1 in bats, superimposed over the normal evolutionary signature of a housekeeping gene with a critical role in cellular cholesterol trafficking. similar signatures of recurrent positive selection have been identified in other housekeeping genes that encode viral receptors, including the transferrin receptor (kaelber, et al., 2012; demogines et al., 2013 ) (tfr; receptor for new world arenaviruses [radoshitzky et al., 2007] , the betaretrovirus murine mammary tumor virus [ross et al., 2002] , and parvoviruses [parker et al., 2001] ), bat angiotensin-converting enzyme-2 (demogines et al., 2012) (ace2; receptor for sars-like coronaviruses [li et al., 2003] ), and mammalian dipeptidyl peptidase-4 (cui et al., 2013) (dpp4; receptor for mers-like coronaviruses [raj et al., 2013] ). in these cases as well, the preponderance of positively-selected residues localize to virus-receptor interfaces. interestingly, the sequence polymorphism at npc1 residue 502 did not impair cholesterol clearance from lysosomes ( figure 3) , and none of the residues under positive selection were found to be mutated in niemann-pick type c disease patients (runz et al., 2008; vanier and millat, 2003) . thus, despite being constrained by its housekeeping function, npc1 appears to retains a sizeable sequence space accessible to adaptive mutation. it is tempting to speculate that sequence variation at residue 141 ( figure 6 ) and potentially other positions in the receptor-binding site of filovirus glycoproteins represents the other half of the genetic arms race, shaped by selective pressure to utilize restrictive npc1 receptors. although more data, especially filovirus sequences from bats, are needed, our findings raise the tantalizing possibility that filoviruses, including those yet undiscovered, are each adapted to specific bat hosts, with co-evolved virus-receptor interactions constituting one potential biological barrier to interspecies viral transmission. alternatively, it is conceivable that repeated contacts between unknown (non-bat) reservoir hosts carrying specific filoviruses, and bats of particular species, have driven positive selection in bat npc1 to limit infection (and selection of filoviruses with compensating sequence changes in gp). in this scenario, detection of anti-filovirus antibodies or filovirus genome-derived oligonucleotides may reflect a type of spillover event from the actual filovirus reservoir hosts into bats. our hypothesis that npc1 in bats has been genetically sculpted by filoviruses (and vice versa) presupposes not only a long-term coevolutionary relationship, but also one in which these viruses have imposed selective pressure on bats to limit or eliminate infection. the discovery of filovirus np-and vp35-related endogenous viral elements (eves) in bat genomes is consistent with such a long-term relationship (taylor et al., 2010; katzourakis and gifford, 2010) . to further investigate the deeper origins of filoviruses in bats, we screened all available bat genomes for filovirus-related eves. we obtained evidence for synteny between a filovirus nucleoprotein (np)-like eve in the genome of the big brown bat (eptesicus fuscus) and those previously identified in three, more distantly-related, myotis bats (figure 7 and supplementary file 5) (taylor et al., 2011) . this new discovery strongly suggests that all four eves resulted from a single insertion event prior to the divergence of the myotis and eptesicus genera, »25 million years ago (miller-butterworth et al., 2007) . therefore, bats may have been exposed to filovirus-like agents for far longer than previously recognized (»13 million years ago [taylor et al., 2011]) . available experimental exposure studies, although limited in number and scope, suggest that some filoviruses isolated from humans can replicate in bats without causing substantial host pathology (e.g., marv and ravv in egyptian rousettes jones et al., 2015; paweska et al., 2012] ). these observations therefore prompt a key question: what is the origin and nature of the selective pressure that has driven accelerated npc1 evolution in bats? our scant understanding admits a number of possibilities. first, it is conceivable that some filoviruses do indeed replicate in a manner that is deleterious to their specific bat hosts-we may simply not have identified the viruses and hosts in question. indeed, the filovirus llov, discovered in schreibers's long-fingered bat carcasses in spain and portugal, may exemplify this possibility (negredo et al., 2011) . alternatively, in some cases (e.g., ebolaviruses and egyptian rousettes), the human viral isolates used in challenge studies may differ from these bat isolates in important respects due to human adaptation (human ebov, bdbv, tafv, restv, and sudv isolates do not infect egyptian rousettes ). second, filoviruses may have been more virulent in bats in the past. thus, the positive selection signatures observed in bat npc1, which cannot be accurately dated, may represent fixed alleles that are the consequence of a selective process driven by ancient filoviruses with properties distinct from their modern counterparts. indeed, the lack of virulence observed in some bats may reflect a dé tente that was shaped by precisely these historic genetic conflicts between filoviruses and bats. third, we cannot rule out the (unlikely) possibility that the evolution of npc1 in bats was driven by an entirely different infectious agent that also utilizes (or utilized) npc1 to multiply in its hosts. regardless of the mechanisms that genetically shaped npc1, we propose that polymorphisms in this gene nevertheless impose host barriers that impede the colonization and spread of present-day filoviruses in bats in africa and elsewhere. our findings set the stage for broader explorations of species-specificity in filovirus interactions with proviral and antiviral host factors, with an eye to uncovering new molecular arms races between filoviruses and bats and new genetic determinants of filovirus host range and host switching. the following immortalized pteropodid fibroblast cell lines were used: roni/7.1 (kidney; rousettus aegyptiacus), hypni/1. (biesold et al., 2011) . the species origin of each cell line was confirmed in the publication in which it was first described (kuhl et al., 2011) . bat cell populations stably expressing human npc1 (hsnpc1) were generated as described previously (carette et al., 2011) . briefly, subconfluent monolayers of cells were transduced with a retroviral vector expressing hsnpc1 modified at the c-terminus with a triple flag epitope tag. transduced cells were selected by puromycin treatment (10 mg/ml). licenses for capturing and export of bats, as well as ethical review and clearances of animal handling procedures were obtained from the ghana forestry commission of the ministry of food and agriculture. bat organ samples were obtained as described (drexler et al., 2012) . bats were caught, anesthetised with ketamine/xylazine and exsanguinated by heart puncture. carcasses were transported on ice to a nearby laboratory facility, and organs were dissected and immediately snap-frozen for long-term storage. animals were typed morphologically and genetically as described previously (kuhl et al., 2011) . vero african grivet kidney cells and 293t human embryonic kidney fibroblast cells were obtained from atcc. cell lines were maintained in dulbecco's modified eagle medium (dmem) (life technologies, grand island, ny) and supplemented with 10% fetal bovine serum (atlanta biologicals, flowery branch, ga), and 1% penicillin-streptomycin (life technologies). all cell lines were maintained in a humidified 37˚c, 5% co 2 incubator. we knocked out the npc1 gene in the eidni/41.3 cell line by crispr-cas9-mediated genome editing as described previously (mali et al., 2013) . a crispr guide rna (grna) sequence to target 5'-gttgtgatgttcagcagcttcgg-3' in the e. helvum npc1 mrna was cloned into the grna cloning vector (addgene plasmid #41824). eidni/41.3 cells were co-transfected with plasmid encoding human codon-optimized endonuclease cas9 (hcas9, addgene plasmid #41815), grna cloning vector encoding the e. helvum npc1-specific grna, a monomeric red fluorescent protein (mrfp1) expression plasmid (to monitor transfection efficiency), and pmx-ires-blasti (confers blasticidin resistance to transfected cells) using lipofectamine 2000 (life technologies). at 24 hr post-transfection, transfected cells were selected with 50 mg/ml of blasticidin for 24 hr and then allowed to recover in the absence of the selection agent. total rna was isolated from surviving cells with the rnaeasy mini kit (qiagen, valencia, ca) as per the manufacturer's directions. the e helvum npc1 mrna sequence flanking the grna target site was amplified with the one-step rt-pcr kit (qiagen) and the following primers: forward: 5'-at-tctggactaccaaaatctttgcc-3', and reverse: 5'-acatggcatccaagcccaag-3'. thermocycling conditions used for the rt-pcr were: 50˚c for 30 min (reverse transcription), followed by 95˚c for 15 min (initial pcr activation), then 30 cycles of 94˚c for 30 sec, 60˚c for 30 sec, 72˚c for 1 min, then a final extension of 72˚c for 10 min. amplified pcr products were tested for indels at the target site with the surveyor mutation detection kit for standard gel electrophoresis (transgenomic, omaha, ne), as per the manufacturer's instructions. once indels were confirmed, amplified pcr products from single cell clones were cloned into a topo-ta vector (life technologies). multiple clones for each single cell population were sequenced to confirm disruption of npc1 alleles. recombinant vesicular stomatitis indiana viruses (vsvs) expressing egfp, and ebov, marv, or llov gp in place of vsv g have been described previously wong et al., 2010; ng et al., 2014) . vsv pseudotypes bearing glycoproteins derived from vsv, ebov, bdbv, tafv, sudv, and marv were generated essentially as described previously (takada et al., 1997) . vsv particles containing gp cl were generated by incubating rvsv-gp-ebov with thermolysin (200 mg/ml) (sigma-aldrich, st. louis, mo) for 1 hr at 37˚c. the protease was inactivated by addition of phosphoramidon (1 mm) (sigma-aldrich), and reaction mixtures were used immediately. infectivities of vsv pseudotypes were measured by manual counting of egfp-positive cells using fluorescence microscopy at 16-24 hr post-infection, as described (chandran et al., 2005) . infectivities of rvsvs were measured in a similar manner, except that nh 4 cl (20 mm) was added to infected cell cultures at 1-2 hr post-infection to block viral spread, and individual egfp-positive cells were manually counted at 12-14 hr post-infection. the wild-type filoviruses ebola virus/h.sapiens-tc/cod/1995/kikwit-9510621 (ebov/kik-9510621; "ebov-zaire 1995") and marburg virus/h.sapiens-tc/deu/1967/hesse-ci67 (marv/ci67) used in this study were described previously swenson et al., 2008) . cells were exposed to virus at an moi of 1 pfu/cell ( figure 1c ) or 3 pfu/cell ( figure 2a ) for 1 hr. viral inoculum was then removed, and fresh culture media was added. at 48 hr (figure 2a ) or 72 hr ( figure 1c ) post-infection, cells were fixed with formalin and blocked with 1% bovine serum albumin (bsa). ebov-infected cells and uninfected controls were incubated with ebov gp-specific monoclonal antibody kz52 (maruyama et al., 1999) . marv-infected cells and uninfected controls were incubated with marv gp-specific monoclonal antibody 9g4 (swenson et al., 2004) . cells were washed with pbs prior to incubation with either goat anti-mouse igg or goat anti-human igg conjugated to alexa 488. cells were counterstained with hoechst stain (invitrogen, carlsbad, ca), washed with phosphate-buffered saline (pbs), and stored at 4˚c. infected cells were quantitated by fluorescence microscopy and automated image analysis. images were acquired at 20 fields/well with a 20â objective lens on an operetta high content device (perkinelmer, waltham, ny). operetta images were analyzed with a customized scheme built from image analysis functions available in harmony software. from bats of four species (hypsignathus monstrosus, eidolon helvum, epomops buettikoferi, and, rousettus aegyptiacus), mrna was collected from cell lines (or spleen samples for additional eidolon helvum npc1 domain c sequences; figure 4 -figure supplement 2), cdna libraries were constructed, and the npc1 transcript was sequenced (see supplementary file 3 for primers). using available rnaseq read data (supplementary file 2), we assembled bat transcriptomes and identified npc1 sequences in bats of six additional species (myotis brandtii, artibeus jamaicensis, cynopterus sphinx, myotis lucifugus, pteropus alecto, and desmodus rotundus) . transcriptome data were cleaned with trimmomatic (bolger et al., 2014) and assembled using trinity (grabherr et al., 2011) and trans-abyss (robertson et al., 2010) . the 10-species npc1 alignment (supplementary file 4) was analyzed for positive selection using the m8 codon model in the codeml package in paml (yang et al., 2000) , rel, and fel (pond and frost, 2005) , and meme (murrell et al., 2012) available at http://datamonkey.org/ (delport et al., 2010) . all evolutionary analyses were done using both the npc1 gene tree and a species tree ( figure 5-figure supplement 1) . the species tree represents the accepted relationships amongst the bats analyzed (agnarsson et al., 2011; almeida et al., 2011) . to identify orthologous filovirus-related eve insertions, we screened bat genomes in silico for eves. a representative set of filovirus protein sequences was obtained from genbank, supplemented by the putative protein sequences of previously identified filovirus eves (taylor et al., 2014; taylor et al., 2011; taylor et al., 2010; katzourakis and gifford, 2010) . these sequences were used as 'probes' in tblastn screens of whole genome shotgun (wgs) sequence data derived from bats of ten species (eidolon helvum, eptesicus fuscus, myotis brandtii, myotis davidii, myotis lucifugus, pteropus alecto, pteropus vampyrus, megaderma lyra, pteronotus parnellii, and rhinolophus ferrumequinum) . statistically significant matches to filovirus probes were extracted, conceptually translated, and aligned with homologous filovirus proteins. orthologous flanking sequences were identified by blast comparison of eve-containing contigs. an alignment of the identified eves, along with the flanking information in the relevant bat genomes, is shown in supplementary file 5. a construct engineered to encode hsnpc1 domain c (residues 372-622) flanked by sequences that form a stable, antiparallel coiled coil, and fused to a preprotrypsin signal sequence with flag and hexahistidine tags at its n-terminus has been described (deffieu and pfeffer, 2011; . similar constructs bearing bat npc1 domain cs were generated by replacing the human domain c sequence with a sequence encoding domain c from each bat npc1 ortholog. soluble domain c was expressed in human 293-freestyle cells (invitrogen) and purified from supernatants by nickel affinity chromatography, as described previously . alternatively, cell supernatants containing soluble domain c were used directly for gp-npc1 binding elisas following calibration for domain c concentration (see below). npc1 domain c concentrations used in the elisas were normalized as follows. proteins were resolved by sds-page followed by immunoblotting with an anti-flag antibody followed by an antimouse alexa-680 secondary antibody (invitrogen). blots were visualized using the li-cor odyssey imager, and the domain c band was quantified using the li-cor image studio package (li-cor biosciences, lincoln, ne). thermolysin-cleaved vsv-ebov gp particles were captured onto high-binding 96-well elisa plates (corning, corning, ny) using kz52, a conformation-specific anti-ebov gp monoclonal antibody. plates were blocked with pbs containing 3% bsa, and serial dilutions of npc1 domain c protein were then added. bound domain c was detected with an anti-flag antibody conjugated to horseradish peroxidase (sigma-aldrich) and ultra-tmb substrate (thermofisher, grand island, ny). all binding steps were carried out at 37˚c for 1 hr or at 4˚c overnight. elisas with vsvs bearing llov and marv gp were performed as above, but with the following modifications. vsv-llov gp particles were cleaved by incubation with a reduced concentration of thermolysin (12.5 mg/ml, 37˚c, 1 hr) due to its enhanced protease sensitivity relative to ebolavirus gps, as described (ng et al., 2014) . the viral envelope was then labeled with biotin using a function-spacer-lipid construct (fslbiotin) (sigma-aldrich), as described previously (ng et al., 2014) . biotinylated viral particles were captured onto streptavidin-coated elisa plates (0.65 mg/ml). the remainder of the steps in the elisa were performed as described above for vsv-ebov gp. vsv-marv gp particles were cleaved by incubation with trypsin (150 mg/ml, 37˚c, 5 min; sigma-aldrich), modified as above using fslbiotin, and captured onto streptavidin-coated magnetic beads (spherotech, lake forest, il). beads were then aliquotted into a 96-well round-bottomed plate for the remaining elisa steps. pbs containing 5% nonfat dry milk was used for blocking and washing steps. binding curves were generated by nonlinear regression analysis using prism (4-parameter logistic equation; graphpad software, la jolla, ca). to detect npc1 in primate or bat kidney fibroblasts, whole cell lysates were prepared as previously described . briefly, cells were washed with pbs and lysed in nte-chaps buffer (10mm tris [ph 7.5], 140mm nacl, 1mm edta, 0.5% vol/vol 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) (sigma-aldrich) containing a protease inhibitor cocktail (roche, basel, switzerland), and placed on ice for 30 min. to assist in cell lysis, cell suspensions were vortexed every 10 min, and then placed on ice for 30 min. samples were spun at 14,000 âg for 10 min, and supernatants harvested for western blot. in some experiments, proteins were deglycosylated with protein n-glycosidase f (new england biolabs, ipswich, ma) according to the manufacturer's instructions. proteins were resolved in 8% sodium dodecyl sulfate (sds)-polyacrylamide gels and transferred to nitrocellulose membranes. endogenous npc1 was detected using an anti-niemann pick c1 polyclonal antibody (1:1,000 dilution; ab36983, abcam, cambridge, ma), followed by incubation with a donkey anti-rabbit antibody conjugated to horseradish peroxidase (1:5,000 dilution, santa cruz biotechnology, dallas, tx). endogenous cyclin-dependent kinase 4 (cdk4; loading control) was detected with a rabbit polyclonal antibody (1:1,000 dilution; sc-260, santa cruz biotechnology). ectopic expression of hsnpc1-flag was detected with an anti-flag antibody conjugated to horseradish peroxidase (sigma-aldrich). bands were visualized by incubation with an enhanced chemiluminescence reagent (thermofisher) followed by exposure to x-ray film. in figure 3 , cells were visualized using an inverted fluorescence microscope under illumination with a 63x high-numerical aperture oil objective ( figure 3b ) or a 10x air objective ( figure 3c ). images were captured with an axiocam mrm ccd camera using axiovision software (zeiss usa, thornwood, ny), and imported into photoshop (adobe systems, san jose, ca) for processing. images were cropped, inverted ( figure 3b) , and subjected to linear adjustment for overall brightness and contrast using the levels tool. developed x-ray films were digitized with a flatbed scanner and processed in photoshop as described above. statistical comparison of means among multiple independent groups was carried out by one-way analysis of variance (anova) with tukey's post hoc test for multiple comparisons. in some figures (see figure legends) , an unpaired two-tailed student's t-test with welch's correction for unequal variances (ruxton, 2006) was used for pairwise comparison of independent groups. all statistical analyses were performed in graphpad prism. the albert einstein college of medicine. opinions, conclusions, interpretations, and recommendations are those of the authors and are not necessarily endorsed by the us department of the army, the us department of defense, or the us department of health and human services. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. author contributions mn, en, mek, jmd, sls, kc, conception and design, acquisition of data, analysis and interpretation of data, drafting or revising the article; ash, rjg, acquisition of data, analysis and interpretation of data, drafting or revising the article; tb, aik, rmj, acquisition of data, analysis and interpretation of data; rkj, acquisition of data, analysis and interpretation of data, drafting or revising the article, contributed unpublished essential data or reagents; jah, my, acquisition of data, analysis and interpretation of data, contributed unpublished essential data or reagents; rb, acquisition of data, contributed unpublished essential data or reagents; ad, analysis and interpretation of data, drafting or revising the article; trb, conception and design, analysis and interpretation of data, drafting or revising the article; cd, mam, drafting or revising the article, contributed unpublished essential data or reagents; lfw, jhk, analysis and interpretation of data, drafting or revising the article, contributed unpublished essential data or reagents a time-calibrated species-level phylogeny of bats (chiroptera, mammalia) host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species evolutionary relationships of the old world fruit bats (chiroptera, pteropodidae): another star phylogeny? seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection oral shedding of marburg virus in experimentally infected egyptian fruit bats (rousettus aegyptiacus) novel, potentially zoonotic paramyxoviruses from the african straw-colored fruit bat eidolon helvum type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum trimmomatic: a flexible trimmer for illumina sequence data ebola virus entry requires the cholesterol transporter niemann-pick c1 niemann-pick c1 disease gene: homology to mediators of cholesterol homeostasis endosomal proteolysis of the ebola virus glycoprotein is necessary for infection adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of middle east respiratory syndrome coronavirus small molecule inhibitors reveal niemann-pick c1 is essential for ebola virus infection rules of engagement: molecular insights from host-virus arms races niemann-pick type c1 function requires lumenal domain residues that mediate cholesterol-dependent npc2 binding datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology dual host-virus arms races shape an essential housekeeping protein evidence for ace2-utilizing coronaviruses (covs) related to severe acute respiratory syndrome cov in bats bats host major mammalian paramyxoviruses molecular determinants of ebola virus virulence in mice ebola haemorrhagic fever isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor genomic surveillance elucidates ebola virus origin and transmission during the 2014 outbreak full-length transcriptome assembly from rna-seq data without a reference genome filovirus rna in fruit bats niemann-pick c1 is essential for ebolavirus replication and pathogenesis in vivo the natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor evaluation of immune globulin and recombinant interferon-alpha2b for treatment of experimental ebola virus infections filovirus entry into susceptible cells molecular evidence of ebola reston virus infection in philippine bats experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera evolutionary reconstructions of the transferrin receptor of caniforms supports canine parvovirus being a re-emerged and not a novel pathogen in dogs uncovering the fruit bat bushmeat commodity chain and the true extent of fruit bat hunting in ghana endogenous viral elements in animal genomes niemann-pick c1 (npc1)/npc1-like1 chimeras define sequences critical for npc1's function as a flovirus entry receptor monitoring of ebola virus makona evolution through establishment of advanced genomic capability in liberia filoviruses. a compendium of 40 years of epidemiological, clinical, and laboratory studies comparative analysis of ebola virus glycoprotein interactions with human and bat cells structure of the ebola virus glycoprotein bound to an antibody from a human survivor fruit bats as reservoirs of ebola virus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus rna-guided human genome engineering via cas9 ebola virus can be effectively neutralized by antibody produced in natural human infection two-stepping through time: mammals and viruses filovirus entry into cells -new insights ebola virus entry requires the host-programmed recognition of an intracellular receptor a family matter: conclusive resolution of the taxonomic position of the long-fingered bats detecting individual sites subject to episodic diversifying selection discovery of an ebolavirus-like filovirus in europe cell entry by a novel european filovirus requires host endosomal cysteine proteases and niemann-pick c1 filoviruses in bats: current knowledge and future directions ebola virus epidemiology, transmission, and evolution during seven months in sierra leone canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells virological and serological findings in rousettus aegyptiacus experimentally inoculated with vero cellsadapted hogan strain of marburg virus continent-wide panmixia of an african fruit bat facilitates transmission of potentially zoonotic viruses datamonkey: rapid detection of selective pressure on individual sites of codon alignments transferrin receptor 1 is a cellular receptor for new world haemorrhagic fever arenaviruses receptor determinants of zoonotic transmission of new world hemorrhagic fever arenaviruses dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin de novo assembly and analysis of rna-seq data mouse transferrin receptor 1 is the cell entry receptor for mouse mammary tumor virus npc-db, a niemann-pick type c disease gene variation database the unequal variance t-test is an underused alternative to student's t-test and the mann-whitney u test pathways of cross-species transmission of synthetically reconstructed zoonotic severe acute respiratory syndrome coronavirus vaccine to confer to nonhuman primates complete protection against multistrain ebola and marburg virus infections generation of marburg virus-like particles by co-expression of glycoprotein and matrix protein a system for functional analysis of ebola virus glycoprotein evidence that ebolaviruses and cuevaviruses have been diverging from marburgviruses since the miocene evolutionary maintenance of filovirus-like genes in bat genomes filoviruses are ancient and integrated into mammalian genomes genetic diversity and evolutionary dynamics of ebola virus in sierra leone isolation of genetically diverse marburg viruses from egyptian fruit bats niemann-pick disease type c molecular characterization of guinea pig-adapted variants of ebola virus roles of rodents and bats in human viral hemorrhagic fevers a forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry codon-substitution models for heterogeneous selection pressure at amino acid sites we thank tyler krause and cecelia harold for technical support, and gary crameri, shawn todd, and mary tachedjian for help with sourcing bat cdna for npc1 gene amplification. we also thank margaret kielian, jack lenz, max nibert, vinayaka prasad, deeann reeder, nancy simmons, and susan tsang for useful discussions. we thank laura bollinger, integrated research facility at fort detrick, for critically editing this manuscript.supported by grants from the us national institutes of health (ai101436 to kc, gm093086 to sls), the us defense threat reduction agency (hdtra1-11-c-0061 to sls, cb3948 to jmd), eu fp-7 antigone (grant 278976) and the ebokon project (to cd and mam). lfw is supported in part by an nrf-crp grant (nrf2012nrf-crp001-056) in singapore. jhk performed this work as an employee of tunnell government services, inc., a subcontractor to battelle memorial institute, under battelle's prime contract with niaid (no. hhs27220070016i). sls is a burroughs wellcome fund investigator in the pathogenesis of infectious disease. kc is additionally supported by a harold and muriel block faculty scholarship and an irma t. hirschl/monique weill-caulier research award at key: cord-102874-6z5f2gz3 authors: barreiro, luis b.; patin, etienne; neyrolles, olivier; cann, howard m.; gicquel, brigitte; quintana-murci, lluís title: the heritage of pathogen pressures and ancient demography in the human innate-immunity cd209/cd209l region date: 2005-11-30 journal: the american journal of human genetics doi: 10.1086/497613 sha: doc_id: 102874 cord_uid: 6z5f2gz3 the innate immunity system constitutes the first line of host defense against pathogens. two closely related innate immunity genes, cd209 and cd209l, are particularly interesting because they directly recognize a plethora of pathogens, including bacteria, viruses, and parasites. both genes, which result from an ancient duplication, possess a neck region, made up of seven repeats of 23 amino acids each, known to play a major role in the pathogen-binding properties of these proteins. to explore the extent to which pathogens have exerted selective pressures on these innate immunity genes, we resequenced them in a group of samples from sub-saharan africa, europe, and east asia. moreover, variation in the number of repeats of the neck region was defined in the entire human genome diversity panel for both genes. our results, which are based on diversity levels, neutrality tests, population genetic distances, and neck-region length variation, provide genetic evidence that cd209 has been under a strong selective constraint that prevents accumulation of any amino acid changes, whereas cd209l variability has most likely been shaped by the action of balancing selection in non-african populations. in addition, our data point to the neck region as the functional target of such selective pressures: cd209 presents a constant size in the neck region populationwide, whereas cd209l presents an excess of length variation, particularly in non-african populations. an additional interesting observation came from the coalescent-based cd209 gene tree, whose binary topology and time depth (∼2.8 million years ago) are compatible with an ancestral population structure in africa. altogether, our study has revealed that even a short segment of the human genome can uncover an extraordinarily complex evolutionary history, including different pathogen pressures on host genes as well as traces of admixture among archaic hominid populations. infectious diseases have been paramount among the threats to health and survival for most of human evolutionary history (haldane 1949; lederberg 1999; harpending and rogers 2000; cooke and hill 2001) . the interaction of the human host with a wide variety of pathogens has been accompanied by genetic adaptations to spatially and temporally fluctuating selective pressures imposed by the infectious agents. numerous studies have sought the genetic imprint of natural selection imposed by pathogen pressures in human genes involved in immune response or, more generally, in host-pathogen interactions (vallender and lahn 2004) . for example, natural selection has acted on such genes as mhc, b-globin, g6pd, il-2, il-4, tnfsf5, the duffy blood group genes, and ccr5 (ohta 1991; hughes et al. 1994; flint et al. 1998; hamblin and di rienzo 2000; tishkoff et al. 2001; bamshad et al. 2002; sabeti et al. 2002; verrelli et al. 2002) . however, little is known about genetic variation of genes involved in direct recognition of pathogens, or pathogens' products, and virtually no studies have investigated the extent to which pathogens have exerted selective pressures on the innate immune system. the phylogenetically ancient innate immune system governs the initial detection of pathogens and stimulates the first line of host defense (medzhitov and janeway 1998a janeway and medzhitov 2002) . recognition of pathogens is mediated by phagocytic cells through germline-encoded receptors, known as "pattern recognition receptors," which detect pathogenassociated molecular patterns that are characteristic products of microbial physiology (kimbrell and beutler 2001; janeway and medzhitov 2002) . this initial interaction is then translated into a set of endogenous signals that ultimately lead to the induction of the adaptive immune response (medzhitov and janeway 1998b) . in recent years, the c-type lectin receptors have rescaled diagram of the cd209/cd209l genomic region. sequenced regions are represented in gray. for cd209, we sequenced a total of 5,500 bp per chromosome, and, for cd209l, 5,391 bp per chromosome. the neck region corresponding to exon 4 and composed of seven coding repeats is also shown. ceived much attention in the area of innate immunology, the results of which were novel functional insights into the primary interface between host and pathogens (medzhitov 2001; cook et al. 2003; fujita et al. 2004; geijtenbeek et al. 2004; mcgreal et al. 2004) . in this context, two prototypic members of the c-type lectinreceptor family are particularly interesting, since they can act as both cell-adhesion receptors and pathogenrecognition receptors. these lectins include cd209 (dc-sign: dendritic cell-specific icam-3 grabbing nonintegrin [mim 604672]) and its close relative cd209l (l-sign: liver/lymph node-specific icam-3 grabbing nonintegrin [mim 605872]) (curtis et al. 1992; geijtenbeek et al. 2000b geijtenbeek et al. , 2004 soilleux et al. 2000; pohlmann et al. 2001 ). these lectin-coding genes are located on chromosome 19p13.2-3, within an ∼26-kb segment, and result from a duplication of an ancestral gene (bashirova et al. 2003; soilleux 2003 ). an additional characteristic of both cd209 and cd209l is the presence of a neck region, primarily made up of seven highly conserved 23-aa repeats, that separates the carbohydrate-recognition domain involved in pathogen binding from the transmembrane region. this neck region presents high nucleotide identity between repeats, both within each molecule and between cd209 and cd209l. it has been shown that this region plays a crucial role in the oligomerization and support of the carbohydraterecognition domain; therefore, it influences the pathogen-binding properties of these two receptors (soilleux et al. 2000 (soilleux et al. , 2003 feinberg et al. 2005) . in regard to expression profiles, cd209 is expressed primarily on phagocytic cells, such as dendritic cells and macrophages, whereas cd209l expression is restricted to endothelial cells in liver and lymph nodes (bashirova et al. 2001; soilleux et al. 2001 soilleux et al. , 2002 . as pathogen-recognition receptors, the two lectins have been shown to recognize a vast range of microbes, some of which are of major public health importance (geijtenbeek et al. 2004) . indeed, cd209 captures bacteria such as mycobacterium tuberculosis, helicobacter pylori, and certain klebsiela pneumonia strains; viruses such as hiv-1, ebola virus, cytomegalovirus, hepatitis c virus, dengue virus, and sars-coronavirus; and parasites like leish-mania pifanoi and schistosoma mansoni (geijtenbeek et al. 2000a alvarez et al. 2002; colmenares et al. 2002; halary et al. 2002; appelmelk et al. 2003; lozach et al. 2003; tailleux et al. 2003; tassaneetrithep et al. 2003; bergman et al. 2004; marzi et al. 2004) . with regard to cd209l, studies to date have shown an interaction with a variety of viruses, including hiv, hepatitis c, ebola, and coronavirus, as well as with the parasite schistosoma mansoni (bashirova et al. 2001; alvarez et al. 2002; gardner et al. 2003; jeffers et al. 2004; van liempt et al. 2004) . in this context, the efficiency of the two lectins in pathogen recognition and subsequent processing may have important consequences for the quality of host immune responses and consequent pathogen control and/or clearance. an important step forward in the understanding of human adaptation to pathogens and control of infectious diseases includes the description of quality and quantity of genetic variation in genes involved in host recognition of infectious agents. given the direct interaction of cd209 and cd209l with a large variety of pathogens, the cd209/cd209l genomic region provides an excellent model system to illustrate the extent to which pathogens have exerted selective pressures on host immunity genes. an additional feature that makes these genes highly interesting in evolutionary studies is that they are likely to have been influenced by similar genomic forces (recombination, mutation rates, etc.) because of their close physical proximity (∼15 kb), high nucleotide (73%) and amino acid (77%) identity, and identical exon-intron organization (soilleux 2003) 1 ). in addition, it has been proposed that gene duplication of immunity genes is a molecular strategy developed by the host to enlarge its defense potential (ohno 1970; trowsdale and parham 2004) . a number of immune-system gene families have evolved, by gene duplication followed by natural selection, to provide responses to a wider range of pathogens, with welldocumented examples in immunoglobulin and mhc genes (hughes et al. 1994; ota et al. 2000) . in this context, duplicated genes in cis, like cd209 and cd209l, may have undergone differential selective pressures to enlarge the defense role of these lectins. to address these complex issues, we performed a sequence-based survey of the entire cd209/cd209l region in a panel of individuals of different ethnic origins. here, we report evidence showing that these two closely related innate immunity genes have gone through completely different evolutionary processes that are reflected in their current patterns of diversity. in addition, our study provides novel insights into how pathogens have shaped the patterns of variability of immunity genes resulting from gene duplication. sequence variation of the cd209/cd209l region was determined in 41 sub-saharan africans, 43 europeans, and 43 east asians, in a total of 254 chromosomes from the human genome diversity panel (hgdp)-ceph panel ). more-detailed information about the composition of the three major ethnic groups can be found in table 1. the variation in the repeat number of the neck region of cd209 and cd209l was defined in the entire hgdp-ceph panel, comprising 1,064 dna samples from 52 worldwide populations. in addition, the orthologous regions for both genes were sequenced in four chimpanzees (pan troglodytes). the sequenced fragments of the cd209/cd209l genomic region are shown in figure 1. the entire cd209 region-including exons, introns, and ∼1 kb of the 5 utr corresponding to the promoter region-was sequenced, for a total of 5.5 kb per individual. for cd209l, we sequenced a total of ∼5.4 kb per individual, following the same approach used for cd209, with the exception of the neck region. that region was genotyped for its number of repeats, since it turned out to be highly polymorphic, which prevented the sequencing process. genotyping was performed by a single pcr amplification followed by migration in 2% agarose gels. human primers were used to both amplify and sequence the orthologous regions in chimpanzees. however, because of polymorphisms specific to the chimpanzee lineage, we could not obtain the entirety of the sequence. thus, 4.9 kb (90% of the total) of the chimpanzee cd209 sequence were obtained, and 5.3 kb (98% of the total) of cd209l. detailed information on primer sequences and pcr amplification conditions is available on request. all nucleotide sequences were obtained using the big dye terminator kit and the 3100 automated sequencer from applied biosystems. sequence files and chromatograms were inspected using the genalys software (takahashi et al. 2003 ; centre national de genotypage). as a measure of quality control, when new mutations were identified in primer binding regions, new primers were designed and sequence reactions were repeated, to avoid allele-specific amplification. all singletons observed in our data set were systematically reamplified and resequenced. on the basis of the levels of diversity observed in the cd209/cd209l genomic region, we calculated the average number of pairwise differences (p) and the watterson's estimator ( ) (watterson 1975 (fay and wu 2000) . p values for the different tests were estimated from coalescent simulations under an infinite-site 4 10 model, with use of a fixed number of segregating sites and the assumption of no recombination, which has been shown to be a conservative assumption (gilad et al. 2002) . in parallel, we estimated p values for all these tests, using the empirical distribution obtained from sequencing data of 132 genes in a panel of 24 african americans and 23 european americans (akey et al. 2004 ). all these analyses, together with the interspecies mcdonald-kreitman (mcdonald and kreitman 1991) and k a /k s (kimura 1968 ) tests, were performed using the dnasp package (rozas et al. 2003) . genetic distances between populations (f st ) and heterozygosity values were estimated using the arlequin package (schneider et al. 2000) . f st statistical significance was assessed using 10,000 bootstrap replications. to bear out a deficit or an excess of heterozygosity in the neck region of cd209 and cd209l, we used bottleneck (cornuet and luikart 1996) to compute for each geographic region, the distribution of the heterozygosity expected from the observed number of alleles, given the sample size (n) under the assumption of mutational-drift equilibrium. this distribution was obtained through simulation of the coalescent process of n genes under two mutational models, the infinite-site model and the stepwise mutation model. in addition, to obtain information on the fraction of genetic variance in the neck region that is due to intraand interpopulation differences, we performed an anal-ysis of molecular variance (amova), using the arlequin package (schneider et al. 2000) . the amova results were compared with those of 377 microsatellites analyzed in the same population panel . haplotype reconstruction was performed by use of the bayesian statistical method implemented in phase (v.2.1.1) (stephens and donnelly 2003) . we applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs. after haplotype reconstruction, linkage disequilibrium (ld) between pairs of snps was computed using lewontin's d index (lewontin 1964) . for this analysis, only markers presenting a minimum allele frequency (maf) of 10% were considered, since rare alleles have been shown to present a higher probability of being in significant ld than do common ones (reich et al. 2001) . the graphic display of the ld plots was constructed using gold (abecasis and cookson 2000; center for statistical genetics) . to support the existence of a recombination hotspot in the region under study, we used the hotspot-recombination model implemented in phase (v.2.1.1). under this model, we assumed that there was, at most, one hotspot of unknown position. we then estimated the background population-recombination rate (r) and the relative intensity of any recombination hotspot. to obtain better estimates, we increased 10 times the number of iterations of the final run of the algorithm. all our estimations were obtained by averaging results of five independent runs with use of different seed numbers. since the model used is bayesian, we could also estimate, for each population, the posterior probability of a hotspot of intensity 11 ( ) l 1 1 and 110 ( ). l 1 10 we obtained the gene tree and estimated the time of the most recent common ancestor (t mrca ) for cd209, using the maximum-likelihood coalescent method implemented in genetree (griffiths and tavare 1994) . the mutation rate m for each gene was estimated on the basis of the net divergence between humans and chimpanzees and under the assumption both that the species separation occurred 5 million years ago (mya) and of a generation time of 20 years. using this m and v maximum likelihood (v ml ), we estimated the effective population size parameter ( ). with the assumption of a n e generation time of 20 years and the estimated , the n e coalescence time, scaled in units, was converted into 2n e years. the coalescence process implemented in sim-coal2 (laval and excoffier 2004 ) allowed us to estimate the probability of the t mrca for cd209, through simulations, with use of both the number of 4 2 # 10 observed segregating sites and the estimated . n e we determined sequence diversity in the cd209 and cd209l genes ( fig. 1 ) as well as length variation of the neck region in 254 chromosomes originating from three major ethnic groups: sub-saharan africans, europeans, and east asians. in addition, the orthologous sequences were obtained in four chimpanzees, to infer the ancestral state at each site, to estimate the divergence between humans and chimpanzees, and to perform a number of interspecies neutrality tests. for cd209, we identified a total of 79 snps and 2 indels, including 5 nonsynonymous, 5 synonymous, and 71 noncoding variants. the five nonsynonymous snps were all located in the neck region (exon 4): snps 1839 (argrgln), 1888 (glurasp), and 1908 (argrgln) achieved a frequency of ∼15%, and snp 1970 (leur val), a frequency of 6%. these mutations were restricted to the african sample. snp 1472 (alarthr) was observed as a singleton in an east-asian individual. for cd209l, we identified 64 snps and 2 indels, including 4 nonsynonymous and 62 noncoding variants. the four nonsynonymous variants were located in different exons: snp 141 (thrrala) in exon 2, snp 3476 (aspr asn) in exon 5, snp 4268 (thrrala) in exon 6, and snp 5580 (argrgln) in exon 7. all these mutations were singletons except snp 3476, which presented high frequencies for its derived allele in all geographic regions: 97.6% in africans, 57% in europeans, and 77% in east asians. all variable sites were in hardy-weinberg equilibrium for both cd209 and cd209l, after bonferroni correction for multiple testing. the allelic composition of cd209 and cd209l haplotypes and their frequency distribution in the three major ethnic groups is illustrated in figure 2, along with the haplotype composed of the ancestral allelic state of each snp inferred from chimpanzee data. for cd209, we identified 42 different haplotypes, with an overall heterozygosity of 84% (table 2) . three major haplotypes (h2, h29, and h40) accounted for ∼50% of the african variability, whereas they were at very low frequency (h2 at ∼5%) or absent (h29 and h40) in europeans and east asians ( fig. 2a ). in turn, the two haplotypes (h1 and h3) that accounted for 58% and 83% of the european and east asian variability, respectively, were observed at very low frequency (h1 at 6%) or even absent (h3) in africa. however, h3, which had a frequency of 36% and 20% in europe and east asia, respectively, is just a one-step mutation (snp 871) from h2, the most frequent haplotype in the african sample. the most ininferred haplotypes for cd209 (a) and cd209l (b). the chimpanzee sequence was used to deduce the ancestral state at each position, except for the cd209l positions 1232, 1236, and 1240. for those polymorphisms, the ancestral state was considered to be the most frequent allele. dark boxes correspond to the derived state at each position. the numbers on the right of the figure indicate the absolute frequency of each haplotype in the different populations studied. repeat-number variation in the neck region of each gene is reported in the gray columns with the column heads "nr." indel polymorphisms are referred as to "1" for insertion and "0" for deletion. teresting observation of the cd209 haplotype variability was the presence of a highly divergent haplotype cluster. this cluster, which contains haplotypes 40-42 (referred to here as "cluster a"), differs from all other haplotypes (referred to here as "cluster b") by 35 fixed positions ( fig. 2a ). cluster a is africa specific and is present at a frequency of ∼15%, whereas cluster b is present in the remaining african and all non-african samples. it is worth noting that three (snps 1839 (snps , 1888 (snps , and 1908 of the five nonsynonymous mutations identified for this gene are unique to cluster a. in all cases, these three mutations were segregating together, with the exception of one haplotype, h41, which does not contain the snp 1839. samples from cluster a are geographically wide2b) , with an overall heterozygosity of 94% (table 2) . only one haplotype (h38) at a frequency of ∼15% was shared in the three continental regions. to assess the degree of population differentiation, if any, we computed wright's f st (wright 1931) , using haplotype frequencies. estimates were significant f st ( ) for all population comparisons, indicating p ! .0001 continental differentiation for both cd209 and cd209l. however, substantial differences were observed between the two genes: the overall f st for cd209 among africans, europeans, and east-asians was 0.15, whereas cd209l presented a threefold lower f st value of 0.05. for both genes, the larger f st values were observed between african and east asian populations, with f st values of 0.22 for cd209 and 0.07 for cd209l. the average nucleotide diversity (p) was strikingly different, both between the two genes and among populations (table 2) . globally, p values were three-to fivefold lower for cd209 ( ) than for cd209l ϫ4 3-7 # 10 (∼16 # 10 ϫ4 ), except for african populations, for whom the cd209 p value was unusually high ( ) be-ϫ4 26 # 10 cause of the presence of the highly divergent cluster a. indeed, when cluster a was excluded from the analysis, the african p value dropped to . to estimate ϫ4 8 # 10 the substitution rate of each region and evince possible mutational differences that could explain the strong contrast observed in nucleotide-diversity patterns, we determined the human-chimpanzee divergence for both genes. the average net number of differences between the two species was 77.3 substitutions (or 0.0157 substitutions per nucleotide) for cd209 and 90.6 substitutions (or 0.0171 substitutions per nucleotide) for cd209l. since the human-chimpanzee speciation occurred 5 mya, we obtained similar nucleotide-substitution rates per site per year (cd209, ; ϫ9 1.57 # 10 cd209l, ). ϫ9 1.70 # 10 to assess the patterns of ld in the cd209/cd209l region, haplotypes for the entire genomic region were reconstructed using markers with an maf of 10%. d measures among these markers were estimated for african and non-african populations independently; the graphical representation of ld levels is illustrated in figure 3. two distinct regions, which correspond to either cd209 or cd209l, showed strong ld and are separated by a boundary that corresponds to the intergenic region. for cd209, a block of intragenic ld was observed in both african and non-african populations. for the african sample, 89% of all pairwise comparisons indicated significant levels of ld, whereas, for non-africans, all d pairwise comparisons were significant. the magnitude of intragenic recombination (and/or gene conversion) of cd209l was slightly higher than for cd209. nevertheless, considerable and significant levels of ld were observed between sites: 83% of all ld pairwise comparisons were significant in the african group, and 99% were in the non-african sample. overall, cd209 exhibited a blocklike structure in both groups, whereas cd209l presented lower-although mostly pairwise d ld plots in non-african and african populations. european and east asian samples were plotted together as "non-africans" because they showed similar levels of ld (data not shown). red tags indicate the physical position of each snp across the genomic region studied. blue and green lines label the snps ( ) used for cd209 and cd209l, respectively, in the ld plot. for cd209, 47 maf 1 10% snps presented an in the african sample and 5 in the non-african, whereas, for cd209l, 18 snps showed an in maf 1 10% maf 1 10% africans and 20 in non-africans. the high prevalence of snps with for cd209 in africa is due to the presence of the highly maf 1 10% divergent cluster a, which presents 35 diagnostic variants with a frequency of 15%. significant-ld levels, in particular among the non-african sample. the strong decay in ld observed in the intergenic region ( fig. 3) , which spans only ∼14 kb, suggests the occurrence of a number of recombination events. to test the hypothesis of a possible recombination hotspot situated within this region, recombination parameters across the entire cd209/cd209l region (∼26 kb) were computed for the three populations, by use of the recombination model implemented in phase (v.2.1.1) ( fig. 4 ). this model (stephens and donnelly 2003) estimates the position and relative intensity of the hotspot (l) as compared with the background population recombination rate (r) (see the "material and methods" section). a l value of 1 corresponds to absence of recombinationrate variation, whereas l values 11 indicate the presence of a hotspot. the model detected the occurrence of a hotspot in the intergenic region, with africans presenting a l of 18, whereas europeans and east asians exhibited l values of 63 and 53, respectively ( fig. 4) . we estimated the posterior probabilities of a hotspot of any kind, , and of at least 10 times the background re-pr (l 1 1) combination rate, . was 100% for pr (l 1 10) pr (l 1 1) all population groups, and was 64% for af-pr (l 1 10) ricans, 97% for europeans, and 92% for east asians. thus, our data clearly indicate a relative increase of the recombination levels between the two genes, which suggests the occurrence of a hotspot of recombination, the magnitude of which varies among the major ethnic groups. however, our data do not include intergenic snps; therefore, the exact location and width of the recombination hotspot within the intergenic region remains unclear, since this observation would be consistent with either an intense narrow hotspot or a weaker but wider hotspot. the identification of a strong decay in ld between cd209 and cd209l facilitated the interpretation of neutrality tests, because the noise introduced by hitchhiking effects between the genes is reduced. we applied tajima's d and fay and wu's h tests to determine whether these statistics significantly deviated from expectations under neutrality, using both coalescent simulations and the empirical distribution obtained from akey et al. (2004) . globally, tajima's d test indicated different tendencies for the two genes (table 2) . cd209 always yielded negative values for tajima's d but never achieved significance to reject the hypothesis of neutrality, whereas cd209l yielded significantly positive values for non-african populations, with use of both estimates of the hotspot intensity (l) for africans, europeans, and east asians. estimates of the population recombination rate (r) for each population as well as the posterior probabilities of and are also reported in the key. l 1 1 l 1 10 (table 2) . to evaluate the selective pressures at the protein level, we performed two interspecies tests: k a /k s , which gives the ratio of nonsynonymous and synonymous changes between species, and the mcdonald-kreitman test, which tests the null hypothesis that the ratio of the number of fixed differences to polymorphisms is the same for both nonsynonymous and synonymous mutations. for the k a /k s test, cd209 and cd209l showed similar values, 0.34 and 0.37, respectively. for the mcdonald-kreitman test, the hypothesis of neutrality was rejected for only cd209, because of a clear lack of nonsynonymous polymorphic sites (table 3) . the identical genomic organization of cd209 and cd209l is extended to the neck region, which, in both genes, encodes a track of seven coding repeats of 23 aa each ( fig. 1) (soilleux et al. 2000) . a previous study has shown that the length of the neck region of cd209l varied between individuals of european descent (bashirova et al. 2001) . to investigate the degree of polymorphism of the neck region in both cd209 and cd209l, we genotyped it in the entire hgdp-ceph panel (1,064 individuals from 52 worldwide populations). striking differences were observed between the two genes (see fig. 5 and table 4 for detailed allele frequencies in each population). for cd209, virtually no variation was observed, and the 7-repeat allele accounted for 99% of the total variability. despite this limited variation, eight different alleles were observed, with an allele size range of 2-10 repeats, not including a 9-repeat allele. the geographic region that presented the highest variability was the middle east, with five of the eight different alleles observed ( fig. 5a and table 4) . for cd209l, a com-pletely different pattern emerged, with strong variation in allelic frequencies of different repeat numbers. of the seven alleles observed (from 4-10-repeat allele size classes), the three most common overall were the 7-(57.42%), the 5-(23.92%), and the 6-(11.37%) repeat alleles. european, asian, and pacific populations presented a mosaic composition of different allelic classes, whereas 7-and 6-repeat alleles accounted for most (96%) of the african diversity ( fig. 5b ). the strong difference in the neck-region lengths between the two genes was consequently visible in the heterozygosity values: cd209 exhibited an overall heterozygosity of only 2%, whereas cd209l presented a value of 54% (table 5) . our results showed that the levels of heterozygosity observed at cd209 were considerably lower than expected, regardless of the mutation model considered (i.e., infinite site or stepwise mutation models) (table 5). in strong contrast, although not statistically significant for individual populations, cd209l exhibited a pattern of an excess of heterozygosity in all populations. the table is available in its entirety in the online edition of the american journal of human genetics. the low levels of intragenic recombination observed in cd209 allowed maximum-likelihood coalescent analysis (griffiths and tavare 1994) for estimation of the time scale of the origin and evolution of this gene. since this method assumes an infinite-site model without recombination, the same analysis for cd209l was not conducted because of the substantial amount of recombinant haplotypes observed. for cd209, only 29 of the 254 chromosomes analyzed had to be excluded, as did a single segregating site (snp 939). the resulting cd209 gene tree estimate, rooted with the chimpanzee sequence (i.e., the chimpanzee sequence was used to define ancestral/derived status of human mutations), is shown in figure 6 . the tree is partitioned into two deep branches that correspond to haplotype clusters a and b. african samples were observed in both sides of the deepest node of the tree (i.e., in both clusters a and b), whereas non-african samples are restricted to one branch of the tree (i.e., cluster b). the maximum-likelihood estimate of v (v ml ) for cd209 was 8.4. on the basis of this v ml value and the estimated mutation rate ( per gene ϫ4 1.54 # 10 per generation), the effective population size ( ) was n e 13,636, a value comparable to most figures reported in the literature (for a review, see tishkoff and verrelli [2003] ). the t mrca of the cd209 tree was then estimated at mya, one of the oldest t mrca val-2.8 ‫ע‬ 0.22 ues estimated so far in the human genome (excoffier 2002 ). the cd209/cd209l region possesses a number of characteristics that make it a powerful tool for evolutionary inference. these two genes are not in ld, despite their very close physical vicinity (∼15 kb), and each of them behaves as an independent genetic entity. moreover, our results suggest that the cd209/cd209l region is a uniform landscape of genomic forces, since the two lectincoding genes present similar mutation rates, as well as high nucleotide identity and conserved exon-intron organization ( fig. 1 ). our diversity study revealed completely different patterns for the two genes. first, levels of nucleotide diversity (p) were found to be much lower for cd209 than for cd209l (table 2) . on the basis of 1.42 million snps, the international snp map working group defined as the average value of nucleotide di-ϫ4 7.5 # 10 versity for the human genome and showed that 95% of all bins presented p values varying from to ϫ4 2.0 # 10 (sachidanandam et al. 2001 ). in addition, ϫ4 15.8 # 10 an independent study analyzed nucleotide and haplotype diversity for 313 genes and defined the average p value as (stephens et al. 2001) . in this context, the ) are at least twofold higher than average ϫ4 16-18 # 10 genome estimates and fall into the upper limit of the 95% ci defined by the snp consortium (sachidanandam et al. 2001) . this contrast in nucleotide diversity between the two genes can be explained either by a disparity in local mutation rates or by actual differences in selective pressures. however, no major differences in mutation rates ( vs. ) were ob1.57 # 10 1.70 # 10 served between the two homologues, nor was there substantial variation in gc content, which has been positively correlated with mutation rates and levels of polymorphisms (sachidanandam et al. 2001; smith et al. 2002; waterston et al. 2002; hellmann et al. 2003) . indeed, the gc content for cd209 (53.7%) was slightly higher than that observed for cd209l (50.9%), which reinforces the idea that different selective pressures may indeed have been the driving force behind the distinct patterns of diversity observed. second, the patterns of repeat variation in the neck region also turned out to be strikingly different between the two genes. cd209 showed levels of heterozygosity of only 2%, whereas cd209l presented an extraordinarily high level of worldwide diversity, with an overall heterozygosity of 54% (table 5 and fig. 5 ). although the neck regions of both genes share 92% of nucleotide identity, nonuniform mutation rates could, again, explain the patterns observed. however, this does not seem to be the case, since mutation-rate variation should influence the number of alleles observed rather than their frequencies, which are subject either to genetic drift or to natural selection. indeed, we observed an even higher number of repeat alleles for cd209 (eight alleles) than for cd209l (seven alleles) (table 4 and fig. 5 ). overall, differences in genomic forces seem to be insufficient to explain the contrasting patterns observed at both the sequence and neck-region length variation levels; therefore, the action of differential selective pressures acting on these genes becomes the most plausible scenario. for cd209, not only nucleotide diversity but also f st intercontinental values (0.15) were in conformity with previous worldwide estimations (harpending and rogers 2000; akey et al. 2002; cavalli-sforza and feldman 2003) . for frequency-spectrum-based tests, only fay and wu's h test detected an excess of highly frequently derived alleles for the african and east asian samples, a picture that may be interpreted as the result of a selective sweep. however, the significantly negative value observed in africa is, again, exclusively due to the presence of cluster a, since 22 of the 35 fixed snps distinguishing it from cluster b corresponded to the derived allelic status in the latter cluster. because cluster b accounts for 85% of the african variability, a clear excess of frequently derived alleles was observed. the extent to which the presence of this cluster is due to either natural selection or population structure will be discussed in detail below. for east asia, the significance of the h test is also questionable when accounting for the confounding effects of demography. indeed, when we plotted our h value against the empirical distribution of 132 h values from non-african populations (akey et al. 2004) , the east asian p value became nonsignificant ( ). this observation reinforces the idea that the p p .36 h test is particularly sensitive to past bottlenecks and/ or population subdivision (przeworski 2002) . thus, regarding the global levels of sequence diversity, the cd209 locus seems to evolve under evolutionary neutrality. nevertheless, when we focused our analyses at the protein level, signs of natural selection were uncovered. indeed, the mcdonald-kreitman test rejected neutrality for this gene because of a clear excess of polymorphic synonymous sites (i.e., a lack of nonsynonymous variants). in addition, when the number of synonymous sites (146) versus nonsynonymous sites (499) was compared with the observed number of synonymous (5) versus nonsynonymous (0) mutations, we detected a significant lack of nonsynonymous mutations (twotailed fisher exact test, ). these obser-ϫ4 p p 6.3 # 10 vations point to a strong selective constraint acting on cd209 that prevents the accumulation of amino acid replacements over time. further support for a functional constraint in cd209 comes from the patterns of diversity observed in the neck region. in contrast to cd209l, virtually no variation was observed at cd209 ( fig. 5a) , with the 7-repeat allele accounting for 99% of the total variability. moreover, the low levels of heterozygosity observed resulted in a consistent rejection of mutation-drift equilibrium in almost all geographical regions (table 5). the probability of finding such a low heterozygosity value, given the overall number of alleles observed, was estimated to be !0.2%, independent of the mutational model considered (table 5) . thus, the fact that no alleles other than the 7repeat allele have increased in frequency, together with recent studies addressing the functional consequences of a populations are grouped as described by rosenberg et al. (2002) . b amova values are from our cd209l study; 95% cis are defined from 377 autosomal microsatellites in the same population panel repeat-number variation in this region (bernhard et al. 2004; feinberg et al. 2005) , strongly suggests a clear reduced fitness of any allele other than the 7-repeat allele. interestingly, it has been recently shown that a protein with two fewer repeats (a 5-repeat allele) results in a partial dissociation of the final tetramer, whereas a protein with !5 repeats exhibits a dramatic reduction in overall stability (feinberg et al. 2005) , with all these differences having a direct impact on the quality of ligand-binding functions (bernhard et al. 2004) . taken together, the patterns of diversity observed at cd209 clearly point to a strong functional constraint acting on this gene and further support the proposed crucial role of this lectin in pathogen recognition and in the early steps of immune response (geijtenbeek et al. 2000b (geijtenbeek et al. , 2004 . in clear contrast to its homologue, cd209l presented extremely elevated nucleotide-diversity levels. high levels of diversity can result either from a relaxation of the functional constraint, which allows the stochastic accumulation of new mutations, or from the action of balancing selection, which maintains over time two or more functionally different alleles (and all linked variation) at intermediate frequencies. several lines of evidence lend support to the selective hypothesis. first, if cd209l nucleotide diversity has been driven by the action of balancing selection, population-genetics relationships would have been accordingly altered. in this context, diversity studies in neutral, or assumedly neutral, regions of the genome-such as the y chromosome (underhill et al. 2000; hammer et al. 2001; jobling and tyler-smith 2003) , mtdna (wallace et al. 1999; ingman et al. 2000; mishmar et al. 2003) , alu insertions (watkins et al. 2001) , as well as some autosomal genes (stephens et al. 2001; akey et al. 2004 )-showed that african populations are genetically more diverse than are non-africans, an observation generally interpreted as a support of the "out of africa" model for the origin of modern humans (lewin 1987) . for cd209l, even if we observed 1.5 times more segregating sites in african than in non-african populations, as indicated by the higher value found in africa, similar values of nucleotide v w diversity were detected in the three groups, with europeans presenting even higher p values than do africans. this unusual scenario, which is at odds with neutral expectations, has already been described for other regions of the genome, such as the b-globin gene and the 5 cis-regulatory region of ccr5, for which the action of balancing selection has been convincingly proposed (harding et al. 1997; bamshad et al. 2002) . second, balancing selection tends to increase within-population diversity while decreasing f st , compared with neutrally evolving loci (cavalli-sforza 1966; harpending and rogers 2000; akey et al. 2002; bamshad and wooding 2003; cavalli-sforza and feldman 2003) . indeed, our data are compatible with these predictions, since the 5% f st value observed for cd209l is threefold lower than that estimated for cd209 (15%) and is similar to that found, for example, for the bitter-taste receptor gene (5.6%), for which there is compelling evidence of balancing-selection action (wooding et al. 2004) . third, results of our tajima's d analysis were significantly positive for european and east asian populations, because of the skew of cd209l frequency spectrum toward an excess of intermediate-frequency alleles (table 2), a pattern that further supports the action of balancing selection. however, since the null model used to assess significance makes unrealistic assumptions about past population demography (i.e., constant population sizes), the rejection of the standard neutral model cannot be interpreted as unambiguous evidence of selection. indeed, the observation that only non-african populations showed a significant departure from neutrality raises the question of whether these patterns could have resulted instead from the bottleneck that occurred during the out of africa exodus. a way to circumvent this conundrum is to analytically integrate the fact that demography affects all the genome equally, whereas selection directs its effects toward specific loci. thus, to correct for the confounding effects of demography, we plotted our results against the empirical distributions of akey et al. (2004) for tajima's d statistics. our values remained significant for cd209l, which therefore reinforces the idea that the pattern observed is unlikely to be the sole result of demography. last, if the patterns of variation in cd209l represent the molecular signature of balancing selection, at least in non-africans, then a functional target of such selective regime is needed. in this context, the neck region constitutes an excellent candidate, since it plays a major mediating role in the orientation and flexibility of the carbohydrate-recognition domain. since this domain is directly involved in pathogen recognition, neck-region length variation has important consequences for the pathogen-binding properties of these lectins (mitchell et al. 2001; bernhard et al. 2004; feinberg et al. 2005) . in perfect agreement with the results of our sequence-based data set, higher diversity in repeat variation was observed in the neck region among non-african populations (native americans excepted). out of africa, at least three alleles account for most population diversity, whereas, in africa, the 6-and 7-repeat alleles alone account for 96% of the global variability ( fig. 5b ). again, the higher diversity observed out of africa could be due to a higher level of relaxation of the functional constraint of the neck region in non-african compared with african populations, which would lead to a random accumulation of proteins with varying neck-region lengths among non-africans. conversely, these patterns could also be explained by the action of balancing selection in non-africans and could therefore point to the neck region as the functional target of such selective regime. to evaluate the plausibility of these two conflicting scenarios, we compared the variation in the cd209l neck region with that inferred from 377 neutral autosomal microsatellites typed elsewhere for the same population panel . we reasoned that if cd209l diversity has been shaped only by demography (i.e., bottleneck out of africa), the distribution of genetic variance at different hierarchical levels should be comparable to that inferred through the neutral markers. on the other hand, if selection has driven the cd209l neckregion diversity, population-genetics distances would be influenced accordingly and would therefore differ from neutral expectations. indeed, the amova values inferred for cd209l fell systematically outside the 95% ci defined for the microsatellite data set (table 6). we observed that populations within europe, asia, the middle east, and oceania exhibited lower-than-expected diversity among populations within the same region. a reduction of genetic distances between populations is expected under balancing selection; therefore, the results from the cd209l neck region favor, once again, the action of this selective regime in most non-african populations, in detriment of the neutral hypothesis. one may argue that the differences in the proportions of genetic variance between our data and those of could be due to differences in the pace of mutation between microsatellite loci and our neck repeated region that could be considered a "coding minisatellite." however, under neutrality, differences in mutation rate should have a similar and proportional effect in all population comparisons and should influence all values with a similar tendency (i.e., higher or lower values). indeed, this is not the case: populations within europe, the middle east, central/south asia, east asia, and oceania turned out to be genetically closer than expected, whereas populations within africa and the americas exhibited the opposite pattern (table 6) , which makes it highly unlikely that mutation-rate differences influenced our conclusions. taken together, the integration of the results from levels of nucleotide and amino acid diversity, neutrality tests, population-genetics distances, and neck-region length variation in cd209 and cd209l clearly points to a situation in which cd209 has been under a strong selective constraint that prevents accumulation of any of amino acid changes over time, whereas cd209l variability has most likely been driven by the action of balancing selection, at least in non-african populations. in apparent dichotomy with the strong selective constraint described for cd209, we observed an unusual excess of diversity of 35 fixed differences separating the two basal branches of the gene tree ( fig. 6 ). in addition, we estimated a t mrca of mya, a time that 2.8 ‫ע‬ 0.22 places the most recent common ancestor of cd209 back in the pliocene epoch, before the estimated time for the origins of the genus homo ∼1.9 mya (wood 1996; wood and collard 1999) . a number of studies have already reported loci that present unusually deep coalescent times (harris and hey 1999; zhao et al. 2000; webster et al. 2003; garrigan et al. 2005a garrigan et al. , 2005b , but our estimation for cd209 remains one of the deepest t mrca values yet reported (excoffier 2002) . the probability of finding such a deep coalescence time under a scenario of a random-mating population was estimated, through a coalescent process (laval and excoffier 2004) , to be very low ( ) (see fig. 7 ). in addition to the p p .018 unexpected antiquity of the cd209 locus, we observed a peculiar tree topology made of two highly divergent and frequency-unbalanced lineages, cluster a embracing only 2 internal haplotypes and cluster b comprising the remaining 23 ( fig. 6 ). different hypotheses can account for such elongated and divergent haplotype patterns. indeed, the high levels of nucleotide identity between cd209 and cd209l could have led to gene conversion between the two genes, an event that would explain the outlier position of cluster a in the context of cd209 phylogeny. we reasoned that if gene conversion has occurred, we expect that the derived alleles distinguishing clusters a and b in cd209 would correspond to the allelic state observed in their homologous positions in cd209l. of all positions, only four fit this criterion. in addition, these positions were not physically clustered, which therefore excludes a major gene-conversion event as the explanation of the divergent cd209 phylogeny. two other circumstances may be responsible for the topology and the time depth of the cd209 gene tree: long-standing balancing selection or ancient population structure, with africa, in both cases, being the arena of such events (i.e., cluster a is restricted to africa). several lines of evidence argue against the balancing-selection hypothesis. first, under this selective regime, one would expect that tajima's d test would also point in this direction by yielding significantly positive values, which is not the case (table 2) . second, such a long-standing balancing selection in africa would have entailed a number of recombinant haplotypes between clusters a and b, which, again, is not the case, as illustrated by the high ld levels at cd209 (fig. 3) . third, a claim of balancing selection at this locus must imply a functional difference between the two balanced alleles. indeed, three nonsynonymous mutations, situated in the neck region, separate cluster a and b, and they could correspond to the alleles under selection. but, if the neck region is the target of selection, it is more likely that the balanced alleles would correspond to different numbers of repeats rather than punctual nucleotide variation within each track, as observed for cd209l and suggested by functional studies (bernhard et al. 2004; feinberg et al. 2005) . since no variation in the number of repeats was detected between both clusters, we predict that there are no major functional differences between the two lineages. taken together, maintenance of ancient lineages by balancing selection does not seem to be responsible for the observed haplotype divergence. in this view, the patterns observed are best explained by an ancestral population structure on the african continent. indeed, several studies have already proposed that african populations must have been more strongly subdivided and isolated than non-african ones (harris and hey 1999; labuda et al. 2000; excoffier 2002; goldstein and chikhi 2002; harding and mcvean 2004; satta and takahata 2004; garrigan et al. 2005a) . in particular, a recent study of the xp21.1 locus presented convincing statistical evidence that supports the hypothesis that our species does not descend from a single, historically panmictic population (garrigan et al. 2005a ). the divergent haplotype pattern observed at the xp21.1 locus prompted those authors to explain their data under the isolation-and-admixture (iaa) model and/or a metapopulation model (harding and mcvean 2004; wakeley 2004) . indeed, as observed for cd209, under an iaa model, the two basal branches are expected to be longer than those under a wright-fisher model, depending on the length of time subpopulations spent in isolation. the extent to which the iaa model fits the data depends on the number of mutations, referred as to "congruent sites," occurring in the two basal branches of the genealogy. for xp21.1, 10 congruent sites over 24 polymorphisms were observed (i.e., ∼42% of the total number of sites). we applied the same approach to cd209 and obtained a very similar percentage of ∼45%, in good accordance with the iaa model. our observations, together with a number of autosomal diversity studies, show that modern human diversity appears to have kept genetic traces of admixture among archaic hominid populations. however, a number of questions remain unanswered, such as the time when these admixture events occurred (i.e., before or after the appearance of anatomically modern humans), the precise quantitative contribution of ancient genetic material to our modern gene pool, and the geographic provenance of these genetic vestiges. the need of continuous evolution for both the human host and the pathogens is predicted by the red queen hypothesis (van valen 1973; bell 1982) , in reference to the remark of the red queen to alice in through the looking glass (carroll 1872) : "now, here, you see, it takes all the running you can do, to keep in the same place." this metaphor provides a conceptual framework for understanding how interactions between the two species lead to constant natural selection for adaptation and counteradaptation. in this context, one feature exploited by the host immunity genes to increase their defense potential is gene duplication by retention, through conservation of one duplicate, of the currently useful function of the encoded protein, while its twin is liberated to mutate and possibly acquire novel functions (ohno 1970; trowsdale and parham 2004) . the lectins cd209 and cd209l represent a prototypic model of a duplicated progeny of ancestral genes that interact with a vast spectrum of pathogens. our results clearly indicate that these duplicated genes have evolved, and might still evolve, under completely different evolutionary pressures. whereas one, cd209, shows signals of strong conservation, its paralogue, cd209l, exhibits an excess of sequence diversity compatible with the action of balancing selection. in addition, the strong contrast observed in length variation of the neck region between the two genes may have important consequences in medical genetics. in this context, association studies are now needed that correlate length variation of the neck region and susceptibility to infectious diseases whose etiological agents are known to interact with one (or both) of these lectins. more generally, our study has revealed that even a short segment of the human genome can help uncover an extraordinarily complex evolutionary history, including different pathogen pressures on host immunity genes, as well as traces of ancient population structure in the african continent. the coming years will certainly bring unprecedented large data sets of sequence diversity, genomewide and populationwide, with each genomic region possibly revealing a different aspect of human history. the integration of all these apparently independent pieces of the same reality will provide us with a much broader and more realistic view of the demographic history of the human species, as well as of human adaptation to the different environmental conditions imposed not only by pathogens but also by other major factors such as climate and nutritional resources. gold-graphical overview of linkage disequilibrium population history and natural selection shape patterns of genetic variation in 132 genes interrogating a high-density snp map for signatures of natural selection c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific icam-3-grabbing nonintegrin on dendritic cells signatures of natural selection in the human genome a strong signature of balancing selection in the 5 cis-regulatory region of ccr5 a dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv-1 infection novel member of the cd209 (dc-sign) gene family in primates helicobacter pylori modulates the t helper cell 1/t helper cell 2 balance through phase-variable interaction between lipopolysaccharide and dc-sign proteomic analysis of dc-sign on dendritic cells detects tetramers required for ligand binding but no association with cd4 a human genome diversity cell line panel through the looking glass. macmillan, london cavalli-sforza ll (1966) population structure and human evolution the application of molecular genetic approaches to the study of human evolution dendritic cell (dc)-specific intercellular adhesion molecule 3 (icam-3)-grabbing nonintegrin (dc-sign, cd209), a c-type surface lectin in human dcs, is a receptor for leishmania amastigotes toll-like receptors and the genetics of innate immunity genetics of susceptibility to human infectious disease description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data sequence and expression of a membrane-associated c-type lectin that exhibits cd4-independent binding of human immunodeficiency virus envelope glycoprotein gp120 human demographic history: refining the recent african origin model hitchhiking under positive darwinian selection extended neck regions stabilize tetramers of the receptors dc-sign and dc-signr the population genetics of the haemoglobinopathies the lectin-complement pathway-its role in innate immunity and evolution l-sign (cd 209l) is a liverspecific capture receptor for hepatitis c virus deep haplotype divergence and long-range linkage disequilibrium at xp21.1 provide evidence that humans descend from a structured ancestral population evidence for archaic asian ancestry on the human x chromosome dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances trans-infection of t cells identification of dc-sign, a novel dendritic cell-specific icam-3 receptor that supports primary immune responses self-and nonself-recognition by c-type lectins on dendritic cells mycobacteria target dc-sign to suppress dendritic cell function evidence for positive selection and population structure at the human mao-a gene human migrations and population structure: what we know and why it matters sampling theory for neutral alleles in a varying environment human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection disease and evolution detection of the signature of natural selection in humans: evidence from the duffy blood group locus hierarchical patterns of global human y-chromosome diversity archaic african and asian lineages in the genetic ancestry of modern humans a structured ancestral population for the evolution of modern humans genetic perspectives on human origins and differentiation x chromosome evidence for ancient human histories a neutral explanation for the correlation of diversity with recombination rates in humans natural selection at the class ii major histocompatibility complex loci of mammals mitochondrial genome variation and the origin of modern humans innate immune recognition cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus the human y chromosome: an evolutionary marker comes of age the evolution and genetics of innate immunity evolutionary rate at the molecular level archaic lineages in the history of modern humans simcoal 2.0: a program to simulate genomic diversity over large recombining regions in a subdivided population with a complex history haldane (1949) on infectious disease and evolution africa: cradle of modern humans the interaction of selection and linkage. ii. optimum models dc-sign and l-sign are high affinity binding receptors for hepatitis c virus glycoprotein e2 dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus adaptive protein evolution at the adh locus in drosophila divergent roles for c-type lectins expressed by cells of the innate immune system toll-like receptors and innate immunity decoding the patterns of self and nonself by the innate immune system natural selection shaped regional mtdna variation in humans a novel mechanism of carbohydrate recognition by the c-type lectins dc-sign and dc-signr: subunit organization and binding to multivalent ligands evolution by gene duplication role of diversifying selection and gene conversion in evolution of major histocompatibility complex loci evolution of vertebrate immunoglobulin variable gene segments dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans the signature of positive selection at randomly chosen loci linkage disequilibrium in the human genome genetic structure of human populations dnasp, dna polymorphism analyses by the coalescent and other methods detecting recent positive selection in the human genome from haplotype structure a map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms the distribution of the ancestral haplotype in finite stepping-stone models with population expansion deterministic mutation rate variation in the human genome dc-sign (dendritic cell-specific icamgrabbing non-integrin) and dc-sign-related (dc-signr): friend or foe? dc-sign; a related gene, dc-signr; and cd23 form a cluster on 19p13 placental expression of dc-sign may mediate intrauterine vertical transmission of hiv constitutive and induced expression of dc-sign on dendritic cell and macrophage subpopulations in situ and in vitro haplotype variation and linkage disequilibrium in 313 human genes a comparison of bayesian methods for haplotype reconstruction from population genotype data dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells statistical method for testing the neutral mutation hypothesis by dna polymorphism automated identification of single nucleotide polymorphisms from sequencing data dc-sign (cd209) mediates dengue virus infection of human dendritic cells haplotype diversity and linkage disequilibrium at human g6pd: recent origin of alleles that confer malarial resistance patterns of human genetic diversity: implications for human evolutionary history and disease mini-review: defense strategies and immunity-related genes y chromosome sequence variation and the history of human populations positive selection on the human genome van die i (2004) molecular basis of the differences in binding properties of the highly related c-type lectins dc-sign and l-sign to lewis x trisaccharide and schistosoma mansoni egg antigens a new evolutionary law evidence for balancing selection from nucleotide sequence analyses of human g6pd metapopulation models for historical inference mitochondrial dna variation in human evolution and disease initial sequencing and comparative analysis of the mouse genome patterns of ancestral human diversity: an analysis of aluinsertion and restriction-site polymorphisms on the number of segregating sites in genetical models without recombination common 5 bglobin rflp haplotypes harbour a surprising level of ancestral sequence mosaicism human evolution the human genus natural selection and molecular evolution in ptc, a bitter-taste receptor gene evolution in mendelian populations worldwide dna sequence variation in a 10-kilobase noncoding region on human chromosome 22 we warmly acknowledge guillaume laval for useful suggestions on the use of simcoal software, laurent excoffier and francesca luca for stimulating discussions, and two reviewers for constructive comments on the first version of the manuscript. l.b.b. was supported by fundaçã o para a ciência e a tecnologia fellowship sfrh/bd/18580/2004. the urls for data presented herein are as follows: key: cord-018764-02l423mk authors: clark, ian a.; griffiths, michael j. title: the molecular basis of paediatric malarial disease date: 2007 journal: pediatric infectious diseases revisited doi: 10.1007/978-3-7643-8099-1_9 sha: doc_id: 18764 cord_uid: 02l423mk severe falciparum malaria is an acute systemic disease that can affect multiple organs, including those in which few parasites are found. the acute disease bears many similarities both clinically and, potentially, mechanistically, to the systemic diseases caused by bacteria, rickettsia, and viruses. traditionally the morbidity and mortality associated with severe malarial disease has been explained in terms of mechanical obstruction to vascular flow by adherence to endothelium (termed sequestration) of erythrocytes containing mature-stage parasites. however, over the past few decades an alternative ‘cytokine theory of disease’ has also evolved, where malarial pathology is explained in terms of a balance between the proand anti-inflammatory cytokines. the final common pathway for this pro-inflammatory imbalance is believed to be a limitation in the supply and mitochondrial utilisation of energy to cells. different patterns of ensuing energy depletion (both temporal and spatial) throughout the cells in the body present as different clinical syndromes. this chapter draws attention to the over-arching position that inflammatory cytokines are beginning to occupy in the pathogenesis of acute malaria and other acute infections. the influence of inflammatory cytokines on cellular function offers a molecular framework to explain the multiple clinical syndromes that are observed during acute malarial illness, and provides a fresh avenue of investigation for adjunct therapies to ameliorate the malarial disease process. although many species of malarial parasite exist, only plasmodium falciparum, vivax, ovale, and malariae are classically associated with human infection. the former two species are most frequently associated with malarial disease in humans, with severe malarial disease almost exclusively associated with p. falciparum infection. falciparum malaria is responsible for considerable morbidity (300-500 million annual clinical cases) and death across the globe, with a particular burden of mortality among children in sub-saharan africa. infection with p. vivax is rarely fatal, but is associated with considerable morbidity outside the african continent. it should also be recalled that malaria causes social and economic disruption on a uniquely large scale [1] . severe adult malaria is a clinical syndrome originally classified using 10 defining and 5 supportive (often overlapping) clinical features unified by the presence of asexual malarial parasites in the peripheral blood smear [2] . based on observations of children in coastal kenya, paediatric severe malaria has similarly been distilled into three main (again often overlapping) clinical syndromes, anaemia, respiratory distress (an indicator of an underlying metabolic acidosis) and impairment of consciousness [3] . these clinical syndromes are discussed below. in the review mentioned above [3] , the authors' judicious use of the term impaired consciousness, rather than cerebral malaria (cm), promoted the useful concept that the neurological features (and in-turn the underlying mechanisms) associated with severe malaria are not necessarily unique to malarial disease. indeed, over 60 years ago, it was noted that the clinical features of malaria can resemble those exhibited in patients with fulminant bacterial or viral infections [4] . severe malaria has been intensively studied, and there appears to be a complex interplay between host infection and disease. this is highlighted by the different clinical manifestations of severe malaria exhibited by children and adults. these differences are undoubtedly, in part, a function of patient age. however, age is just one of a series of interacting factors, e.g. geographical region, level of malaria transmission, degree of previous malaria exposure, length of illness prior to treatment and host immunity that may influence the clinical presentation of severe malaria. this variation in clinical presentation has been mirrored by a similar multitude of proposals regarding the functional mechanisms underlying pathogenesis of severe malaria. one concept of pathogenesis consistently articulated has been the 'mechanical theory'. historically, this theory was developed from two fundamental differences between p. falciparum and p. vivax infection. firstly, erythrocytes parasitised with p. vivax do not sequester. secondly, death following p. vivax infection is rare. consequently, pathogenesis is believed to be due to obstruction of micro-vascular flow by erythrocytes containing mature-stage falciparum parasites adhering to the endothelium (termed sequestration). more recently the 'cytokine theory of disease' has also gained credence. this theory can be applied to disease following both falciparum and vivax infection. the lower mortality associated with p. vivax being explained by a relatively milder degree of pro-inflammatory imbalance during the host's response to p. vivax infection. the main theme of this chapter is to examine the increased understanding of the functions of inflammatory cytokines gained over the past 15 years, and explore how these insights are changing attitudes in malarial disease research. we also discuss how two theories (mechanical and cytokine) can, as proposed first in a recognisable form at least 65 years ago [5] , be complementary. the majority of the clinical cases of malaria occur in sub-saharan africa. nevertheless, malaria also accounts for considerable morbidity and mortality in other continents particularly south east asia [6] . in malaria-endemic regions (e.g. sub-saharan africa), where the resident population have continuous exposure to malarial parasites, most of the severe cases are seen in children [7] . in hypoendemic regions (e.g. south east asia), where parasite exposure is more intermittent, cases of severe malaria are also common in adults (tab. 1). clinical features associated with malaria mortality vary between children and adults, but acidosis and coma are associated with malarial mortality in both populations [7, 8] . acute renal failure (arf) and pulmonary oedema, a marker for adult respiratory distress syndrome (ards), are almost exclusively reported among adults [9, 10] , whereas mortality associated with hypoglycaemia is frequently reported among children [11] . why malarial disease displays such age-related differences in pathophysiology is unclear. however, these differences are not exclusive to malaria. ards, which is more frequently observed as a complication of trauma in adults compared with children [12] , is believed to reflect an exaggerated pro-inflammatory response within the lung [9] . a possible lead for future studies on these age-related differences in malaria is suggested by a report of peritoneal macrophages collected from healthy adults producing much less interleukin (il)-10 (an anti-inflammatory cytokine), but the same levels of pro-inflammatory cytokine, than those from healthy children, giving adults a much higher pro-inflammatory status [13, 14] . the mechanism of malarial arf pathogenesis is postulated to be multifactorial, involving mechanical, haemodynamic, and immunological factors [15] . the observation that arf is more frequently observed as a complication of trauma in adults than children [12] suggests that age-related variations in cytokine response may again influence pathogenesis. hypoglycaemia is regarded as a more frequent complication of sepsis in paediatric populations compared with adults [16] . hypoglycaemia in children may, in part, be associated with a higher basal metabolic rate, and lower glycolytic [17] and gluconeogenic substrate reserves compared to adults [18] . however, these substrates are not always limiting during acute paediatric malaria, suggesting functional impairments of glucose metabolism may also occur [19] . such functional impairments may, in part, be influenced by increases in inflammatory cytokines as the infection progresses. once the malarial parasite was identified as the cause of disease, it quickly became apparent that illness and death were linked with parasite invasion into bloodstream and subsequent parasite growth within (and release from) the erythrocytes. by the start of the 20th century, two major theories, capillary blockage and toxicity of the parasites themselves, had been proposed to explain morbidity and mortality. thus, the study of malarial disease is not a settled story requiring regular updates, but one containing, from its beginning, an unresolved tension. vascular occlusion and malarial toxin (nowadays vascular occlusion and inflammatory cytokines) have been alternative approaches to understanding malarial disease as a whole, as well as the coma, for over a century, and the two have often been discussed side by side [5, 20, 21] . the presence of hyperlactataemia, hypoglycaemia, and metabolic acidosis, all three consistent with a patient being forced to rely on anaerobic glycolysis for energy production, have provided a consensus that hypoxia is central to disease pathogenesis in falciparum malaria. as sum-marised below, the modern literature offers two main theories for cellular hypoxia during infection; insufficient oxygen delivery to cells and impaired oxygen utilization within the cells. both mechanisms may be governed by the host inflammatory cytokine response to infection. this chapter focuses on how an increased understanding of the molecular functions of cytokines during disease demonstrates a closer alignment between the pathogenesis of falciparum infection and other systemic infectious diseases. one hundred and twenty years ago, golgi (of the golgi apparatus [22] ), noted onset of malarial fever and illness at a predictable short interval after the regular shower of new parasites were released from bursting red cells. the nature of the putative toxin so released was much discussed in the first decade of the 20th century [23] . it was assumed to be directly toxic, in the manner of tetanus toxin. the proposal that malarial products were not harmful in themselves, but only through causing the infected host to harm itself through generating toxic amounts of molecules (pro-inflammatory cytokines) that, in lower concentrations, inhibit growth of malarial parasites did not arise until 1981 [24] . indeed, acceptance of the broad applicability of this concept to infectious disease in general is now sufficient for its evolution to be a subject for research [25] . tumour necrosis factor (tnf) is regarded as a major player, malaria being the first disease in which it was proposed to cause systemic illness and pathology [24] . multiple tnf promoter polymorphisms have since been independently associated with severe malaria across several geographical populations [26] . a longitudinal study in burkino faso has also demonstrated several tnf promoter polymorphisms associated with the regulation of host-parasite density [27] . the tnf concept has since begun to dominate the sepsis literature [28] , and the virulence of different strains of influenza, a disease that is a standard clinical misdiagnosis for imported malaria, has recently been expressed in terms of their capacity to induce tnf [29] . the critical role of tnf in both malaria and influenza pathogenesis is consistent with the clinical similarities between the diseases. indeed, tnf infusions in tumour patients produce side effects mimicking both diseases [30] , as discussed below. although tnf is the prototype pro-inflammatory cytokine linked with severe malaria, other cytokines (and mediators) including interferon (ifn)[31] , its corresponding receptors ifn-receptor-1 [32] and ifn-receptor-1 [33] , il-1 [34] , il-4 [35] and il-10 [36] have all be identified through genetic association analysis to be linked with their potential regulation of malarial disease severity. all the above cytokines typically act as homeostatic agents, but can cause pathology if produced excessively. when this happens they also induce a late-onset, but long-acting cytokine termed the high mobility group box 1 (hmgb1) protein, which prolongs and amplifies inflammation [37, 38] . this molecule, normally in the cellular nucleus and previously known only for several physiological functions, now shows great promise as a therapeutic target in sepsis, in that countering it after the onset of illness protects well in experimental sepsis [39, 40] . it accumulates, in proportion to degree of illness, in serum from african children infected with falciparum malaria [41] . once neutralising anti-tnf antibodies became available for human use, they were tested for efficacy against malarial disease. unfortunately, a central tenet of the cytokine concept of infectious disease (that the proinflammatory cytokines that cause disease are the same mediators that, in lower concentrations, are responsible for the innate immunity that controls parasite growth) was not taken into consideration. tnf has been shown to inhibit a mouse malarial parasite in vivo [42] , and p. falciparum in vitro, provided white cells to generate the next down-stream mediator, possibly nitric oxide (no) [43] , were present [44] . this is consistent with findings in human subjects [45] . thus, it is not surprising that anti-tnf antibody, by removing inhibitory pressure from the pathogen, can enhance the disease in falciparum malaria [46] , as shown 5 years earlier in human sepsis [47] . cytokines as a disease mechanism extends beyond malaria as noted above, the idea that excessive production of inflammatory cytokines underlies the pathology of illness is used widely, from malaria across a range of conditions, infectious or otherwise. as reviewed recently [48] , this now includes the illnesses caused by rickettsias, protozoa other than malaria, and viruses. increased circulating levels of these cytokines have been detected in the serum very soon after onset of illness in virtually all those infectious diseases in which they have been sought. some cytokine increased, and consequences are shown in table 2 . when rtnf was under trial in volunteers as an anti-tumour agent [49, 50] nearly 20 years ago, virtually all of the symptoms and signs they share were reproduced as side effects. this includes headache, fever and rigours, nausea and vomiting, diarrhoea, anorexia, myalgia, thrombocytopaenia, immunosuppression, and central nervous system manifestations, all of which have been shown to be caused by a mechanism involving inflammatory cytokines. the rate, timing and intensity of cytokine release vary in different disease states, and provide them with somewhat individual clinical pictures, but the fundamentals remain. nevertheless, the clinical patterns generated are remarkably close, in that, at least in some populations, clinical features cannot predict a diagnosis of malaria from other causes of fever [51] . mature erythrocytic forms of p. falciparum are not seen in peripheral blood smears, and cause the erythrocytes they inhabit to adhere to the walls of venules and capillaries. from this observation arose the widely held view that much of the pathology following malarial infection is explained through parasite sequestration causing impairment of microvascular flow. sequestration certainly occurs, since the life cycle dictates this. however, whether the temporal and anatomical patterns of sequestration are the same in both individuals with fatal disease and in parasite tolerant individuals has not been ascertained. consequently, whether sequestration is the principal instigator of local pathology, or whether sequestration is an associated feature of all malarial infections with local pathology determined by other factors in the host response to the infection, e.g. a local imbalance of inflammatory mediators, has not been fully elucidated. erythrocyte cyto-adherence (irrespective of whether this adhesive process is directly or indirectly due to parasite sequestration) has repeatedly been shown to be mediated through a series of host-derived ligands. cd36 and thrombospondin were the first described endothelial receptors that bound infected red blood cells (rbcs) [52, 53] , with most studied wild parasite isolates demonstrating adhesion to cd36 [54] . more recently, it has been shown that p. falciparum also interacts with other host adhesion receptors, i.e. intercellular adhesion molecule-1 (icam-1 cd54), vascular cell adhesion molecule-1 (vcam-1 cd106) and e-selectin [55, 56] . certain adhesive phenotypes, such as rosetting (the spontaneous tethering of infected and non-infected rbcs) and clumping (tethering of infected rbcs through platelets) have been preferentially associated with severe malarial disease [57, 58] . cd36 is involved in both mechanisms of adhesion, and a non-sense mutation in the gene encoding for cd36 has also been associated with protection from severe malaria [59] . polymorphisms in the gene encoding icam-1 have also been associated with susceptibility to severe disease [27] . furthermore, icam-1, together with vcam and e-selectin, are up-regulated by tnf, with circulating levels of these ligands shown to be increased in severe malaria compared to uncomplicated infection [60] . sequestration during falciparum malaria appears to be concentrated in the brain and placenta. there is some evidence to suggest that the propensity of inflammatory cytokines to up-regulate cell adhesion molecules, secondary to local variation in the density of thrombomodulin, is potentially higher in the microvasculature of the brain and placenta compared to other tissues. as reviewed [61] , tnf and il-1 increase tissue factor expression on endothelial cells, thereby initiating pathways that generate thrombin [62] . when thrombin binds to thrombomodulin on the endothelial cell surface, protein c is activated, which in turn can lead to further downstream activation of the coagulation cascade. therefore vasculature with lowest thrombomodulin densities on the endothelial cell surface (brain least, placenta next least, and other organs more [63] ) will have more unbound thrombin available for its other functions on activated endothelium. these other functions include up-regulation of adhesion molecules such as selectins, icam-1, vcam-1 [64] and monocyte chemotactic protein-1 (mcp-1) [65] . therefore, up-regulation of adhesion molecules within the cerebral vessels may occur as a local endothelial response to systemic inflammation and may not necessarily be precipitated by parasite sequestration. anaemia is another obvious way in which too little oxygen reaches cells, and thus their mitochondria [66] . as recently reviewed [67] , critical illness associated with an inflammatory response invariably causes multifactorial anaemia. obviously a high parasite load in malaria indicates that the infected rbcs will soon burst when the next generation of erythrocytic forms escapes, but anaemia does not correlate with parasitaemia, and sometimes is extreme when very few parasites are, or have been, present. the severe anaemia in transgenic mice expressing human tnf [68] incriminates the inflammatory response itself, so anaemia and mitochondrial dysfunction (see mitochondrial dysfunction section below), both consequences of systemic inflammation, can be expected to coexist, and both contribute to total energy depletion. the lifespan of an rbc is, in part, limited by how long it can remain flexible enough to squeeze through fenestrations in specialised vessels in the red pulp of the spleen, and thus avoid phagocytosis by adjacent macrophages. normally this loss is balanced by erythropoiesis, and haematocrit remains normal. if rbcs develop a premature loss of deformability they are removed from the circulation earlier. this loss of deformability happens to both infected and non-infected red cells in malaria, whether caused by p. vivax or p. falciparum. under physiological conditions, erythrocytes (and other cells) control the passive influx of osmotic active solutes (especially na + ) via an active, energy-dependent elimination of these solutes using na + /k + -atpase. this prevents intracellular accumulation of osmotically active solutes, preventing a subsequent influx of water, cell swelling and loss of cell integrity. during human [69] and monkey [70] malaria infection, intracellular na + accumulates within erythrocytes (both parasitised and non-parasitised) implying that this na + /k + pump is impaired during the disease process. parallel changes in the ionic content of erythrocytes have been documented in a sepsis model of infection [71] . similarly, reduction in erythrocyte deformability was shown to be associated with increased no, an inhibitor of this membrane pump [72] , in another sepsis model [73] . since inhibition of the na + /k + pump in vitro correlates with both reduced red cell deformability and decreased red cell filterability [74] , any factor that inhibits the na + /k + pump could potentially worsen anaemia. identification of inducible no synthase (inos) activity, as one factor influencing red cell deformability, suggests that a pro-inflammatory milieu [75] may again govern the reduction in red cell deformability observed during malaria infection. originally observed in uraemic patients, poor red cell deformability was recognised in a small pilot study of malaria patients in 1985 [76] . it was reported soon afterwards in sepsis [77, 78] , and subsequently studied in falciparum malaria with a view to understanding both circulatory obstruction [79] and anaemia [80] . it seems clear that a short life (poor deformability), and a slow replacement rate (dyserythropoiesis, below) can combine to cause severe anaemia in various diseases, particularly in chronic infections such as malaria. when red cells have a shortened lifespan, e.g. secondary to reduced erythrocyte deformability, replacement by new recruits is vital to avoid anaemia. unfortunately, the same inflammatory cytokines that shorten lifespan also retard replacement. some years ago researchers began to stress the contribution of bone marrow dyserythropoiesis to the anaemia of falciparum malaria [81, 82] . a group in oxford [83] , seeking an explanation for this dyserythropoiesis through an electron microscopy study of bone marrow, observed sequestration of parasitised red cells and argued that this caused the bone marrow dysfunction in falciparum malaria by restricting blood flow and thus inducing hypoxic changes. this idea proved inadequate, however, when this same group subsequently reported dyserythropoiesis and erythrophagocytosis in vivax malaria, in which parasitised red cells do not sequester [84] . some time ago an undefined product in macrophage supernatants [85] , later identified as tnf [86] , was found to inhibit the growth and differentiation of erythroid progenitor cells. when rtnf became available, the dyserythropoiesis and erythrophagocytosis seen in terminal plasmodium vinckei-infected mice was reproduced by giving a single injection early in the course of the infection [87] . phagocytosis of erythroblasts in bone marrow, a phenomenon also reported by wickramasinghe et al [83, 84] in human malaria, also occurred. decreased erythropoiesis was subsequently reported in mice receiving continuous tnf infusions via implanted osmotic pumps, and mice expressing high levels of human tnf have been shown to become markedly anaemic during malaria infections [68] , even though parasite numbers, and therefore red cell loss post-schizogony, are considerably reduced. the past decade has seen an expansion of this line of enquiry into human malaria, and also the number of cytokines, both pro-inflammatory and anti-inflammatory [88, 89] in absolute amounts and ratios [90, 91] , that have been investigated in this context. investigations have been extended to include other pro-inflammatory cytokines, such as il-12 [92] and fasl [93] , and examined the role in anaemia of the persistence of cytokine production during malaria infection [94] . another inflammatory cytokine, macrophage inhibitory factor (mif) that is increased in malaria, and induced by tnf, has been shown to cause dyserythropoiesis in in vitro studies on bone marrow cells [95, 96] . thus, inflammatory cytokines generated during malaria are a major determinant of the degree to which anaemia influences the amount of oxygen that reaches tissues in malaria. mitochondria are vital to energy (atp) generation through cellular respiration. cellular respiration requires oxygen and pyruvate, as well as multiple cofactors and active transport molecules. within the matrix of the mitochondrion organelle, pyruvate is catabolised via the krebs cycle and oxidative phosphorylation (involving nadh and fadh2) to generate atp. when this series of reactions are 100% efficient (unlikely in vivo), 1 molecule of glucose generates 2 molecules of pyruvate, which are further catabolised to water and carbon dioxide with the concomitant generation of 36 molecules of atp. in comparison, during anaerobic glucose catabolism, pyruvate is converted to lactate with the concomitant generation of 2 molecules of atp, a process that also facilitates regeneration of nadh and fadh2. evidence is accumulating that inflammatory cytokines, as released in malaria, sepsis, and viral diseases, induce mitochondrial dysfunction and dysregulate cellular respiration, resulting in the incomplete catabolism of pyruvate. the process, termed 'cytopathic hypoxia' [97] , mimics cellular hypoxia, in that it results in the incomplete catabolism of pyruvate and accumulation of lactate. awareness of this mechanism began with oxygen tension being shown to be increased in septic rats [98] and patients [99] . a cytokine model of mitochondrial dysfunction has since been developed in which impairment of cellular respiration occurs following induction of sepsis (or exposure to pro-inflammatory cytokines), despite sufficient oxygen supply [97, 100, 101] . more recently, impairment of enzyme activity associated with the mitochondrial complexes has been demonstrated in muscle biopsies retrieved from rodent models of sepsis [102] and septic patients [103, 104] . the observation that the inflammatory cytokines implicated in mitochondrial shutdown are prominent in both sepsis and malaria [105, 106] supports such organelle dysfunction being equally plausible in malaria. researchers are also becoming aware that, beyond energy production, mitochondria also play a vital role in cell homeostasis through generation and detoxification of reactive oxygen species [107] . the accelerated oxidative damage that accompanies sepsis could be both a cause and a consequence of cytokine-induced mitochondrial dysfunction. interestingly, the ultrastructural damage reported to accompany mitochondrial dysfunction in sepsis [102] reflects maegraith's observations in monkey malaria [108] [109] [110] decades ago. metabolic acidosis, often associated with hyperlactataemia, has been described in african children with severe falciparum malaria [111, 112] . it is not unique to this disease, being seen in viral, rickettsial and bacterial infections [113] as well as acute gastroenteritis, where its prevalence is higher than in malaria [114] . the terms hyperlactataemia and lactic acidosis are often mistakenly used interchangeably in the malaria literature. as often reviewed in the basic literature [115] [116] [117] [118] , protons (h + , the basis of acidosis) are not formed when atp and lactate are generated during glycolysis, but on the subsequent hydrolysis of atp in tissues. every time a molecule of atp undergoes hydrolysis, a proton is released. if this occurs under aero-bic conditions, these protons are consumed within atp regeneration from adp, and ph remains normal, i.e. acidosis does not occur. in contrast, if the mitochondria are not functioning adequately, whether through insufficient oxygen supply or an inability to use it, atp regenerates under anaerobic condition, and the protons are not consumed. hence, once the buffering capacity of the body is exceeded, acidosis occurs. in short, metabolic acidosis requires the ratio of glycolytic (i.e. anaerobic) atp hydrolysis to mitochondrial (i.e. aerobic) atp hydrolysis to reach a point at which the buffering systems can no longer cope. pathological changes in the buffering system can be a major determinant of when this occurs. high lactate levels have traditionally been seen not only as a marker for poor oxygen delivery in disease states, but also a consequence of it, and the cause of the acidosis. for some time hyperlactataemia has been regarded as a functionally relevant marker for a poor prognosis in both sepsis [119] and malaria [66, 112, 120] . although the sepsis world now discusses several origins for the lactate increase, including inflammation-induced mitochondrial dysfunction [97] , in falciparum malaria it is still generally attributed to a reduced oxygen supply, mostly through microvascular occlusion by sequestered parasitised erythrocytes [121] . other mechanisms are known to contribute to acidosis in malaria, independent of lactate production, e.g. acute renal failure [8] . impaired hepatic clearance [8, 112] , production by parasites, and, in some areas, thiamine deficiency [122] are also argued to contribute to lactate accumulation independent of impaired cellular respiration. thus, as described below, although acidosis and hyperlactataemia can be associated, they are independent cellular mechanisms. lactate anion has complex roles in biology. hyperlactataemia may be associated with acidosis, a normal ph, or alkalosis [123] . a recent editorial in critical care medicine [124] has lucidly summarised the key points of the mechanism of metabolic acidosis in sepsis, a condition that shares systemic inflammation and a range of its consequences with severe malaria (tab. 2). these authors argue against lactate as the cause of the acidosis associated with hypoxia. instead, they note the evidence that during hypoxia, be it from limited oxygen supply or utilisation, the unconsumed protons that cause acidosis arise from the hydrolysis of non-mitochondrial atp. since these reactions are independent of lactate levels, it is difficult to see how therapeutically reducing levels of this anion, as has been proposed [125] , could increase survival rate in falciparum malaria any more than in sepsis [126] . indeed, in theory it could harm comatose patients, since there is evidence that lactate helps brain tissue survive hypoxic and hypoglycaemic episodes [127] [128] [129] , and the lactate shuttle is proving to be how astrocytes protect neurons from metabolic stress [130] . even when considerable lactate is generated in acute inflammatory states, other, unidentified, anions contribute much more than it does to the strong ion difference that, through influencing the body's buffering capacity, influences acidosis in sepsis [131, 132] and falciparum malaria [114, 133] . thus, lactate accumulation can only partially account for the high anion gap observed during the metabolic acidosis associated with severe malaria. in summary, lactate is an imprecise but useful marker for metabolic acidosis in malaria. in turn, acidosis is an imprecise but useful marker of impaired cellular respiration. whether impaired cellular respiration arises from (a) poor supply of oxygen to mitochondria (through vaso-occlusion, low circulating volume, anaemia or cardiac insufficiency) or impaired mitochondrial function (in response to severe systemic inflammation) the outcome is essentially the same. the resulting high anion gap metabolic acidosis is strongly predictive of death in severe malaria. greater understanding of the multiple factors influencing the metabolic acidosis could provide further insight into the underlying pathophysiological process and may provide additional therapeutic options. when glycolysis is enhanced for any period glycogen stores are soon depleted, and gluconeogenesis supervenes. however, its substrate supplies are limiting [134] , and the hypoglycaemia often reported in severe malaria [135] and sepsis [19, 136] occurs. hypoglycaemia is therefore a secondary cause of harm in these diseases, and is an inevitable consequence of exuberant, mostly anaerobic, glycolysis. cm is a clinical syndrome characterised by coma (inability to localise a painful stimulus) at least 1 h after termination of a seizure or correction of hypoglycaemia, detection of asexual forms of p. falciparum malarial parasites on peripheral blood smears, and exclusion of other causes of encephalopathy [137] . a relatively consistent feature of acute cm in children is raised intracranial pressure (icp). studies in african children have demonstrated a raised cerebrospinal fluid (csf) opening pressure during lumbar puncture in 80% of cm children [138] , raised icp during intracranial pressure monitoring (23/23 icp > 10 mmhg) [139] and papilloedema (a late sign of raised icp) in 44% of cm patients who died [140] . where computer tomography has been performed, there was evidence of diffuse brain swelling in 40% of patients [139] . the cause of the raised icp is likely to be multi-factorial and has been postulated to involve both vasogenic and cytotoxic patterns of cerebral oedema. vasogenic oedema is characterised by accumulation of interstitial fluid within the brain secondary to increased permeability of the blood-brain barrier (bbb). it has been demonstrated in bacterial cerebral infections, but evidence of significant disruption of the bbb is not conclusive in cm [141] . others have proposed that icam-1 binding by infected erythrocytes may generate a cascade of intracellular signalling events that disrupt the cytoskeletal-cell junction structure and cause focal disruption to the bbb [142] . adult post-mortem analysis has shown cerebrovascular endothelial cell activation (increased icam-1 endothelial staining, reduction in cell junction staining, and disruption of junction proteins), particularly in vessels containing infected erythrocytes [143] . however, disruption of intercellular junctions is not associated with significant leakage of plasma proteins (fibrinogen, igg, or c5b-9) into perivascular areas or csf [143] . in thai adults, transfer of radioactively labelled albumin into csf was not raised during unconsciousness compared with convalescence [144] . similarly, the albumin index (ratio of concentrations of albumin in csf to those in blood) was not altered significantly in vietnamese adults [145] or significantly different between malawian children with cm who died and those who survived [143] . cytotoxic oedema is increasingly being recognised as an important mechanism of cerebral oedema in traumatic brain injury [146] . as previously discussed, this type of cell swelling involves disturbance of the "pumpleak equilibrium" maintained, under physiological conditions via active elimination of osmotically active solutes through the energy-dependent na + /k + -atpase. thus, cytotoxic oedema can occur secondary to an imbalance in supply and demand of energy within the cells. several mechanisms, such as sustained increase in neuronal activation, impaired substrate delivery (structural and functional) and impaired mitochondrial utilisation of available substrates, including oxygen, may coexist to generate this imbalance. all these mechanisms could contribute to atp depletion and na + /k + atpase failure, leading to cytotoxic oedema in cm. cm is clearly associated with increased neuronal activity. a recent review identified that 80% of african children with cm have a history of seizures, with prolonged and recurrent seizures associated with a poor outcome [147] . impaired vascular flow during acute cm may limit substrate delivery within the brain and contribute to energy imbalance. in the past, a common premise was that parasite sequestration precipitated cerebral vaso-occlusive/ischaemic (i.e. stroke-like) events that manifested clinically as cm. however, cm demonstrates several features that are atypical for stroke. in children, focal neurological signs do not tend to accompany coma, although a sub-set of patients do exhibit hemiparesis or focal brainstem deficits during the agonal period [148] . the incidence of residual neurological deficits following recovery from coma is relatively low (11% [147] ) when compared to childhood stroke (93% had residual neurological deficit [149] ). where computer tomography has been performed in children, diffuse brain swelling was observed [150] rather than focal lesions more typical of stroke. although retinal haemorrhages have been observed in 46% of malawian children with cm (and in 63% of patients who died), these lesions were also seen in 30% of children with sma in the same study [140] . consequently, although associated with cm, retinal haemorrhages do not confirm that focal cerebral vaso-occlusive/ischaemic events underlie cm. similarly, histological examination of 32 fatal cm cases of african children at autopsy demonstrated that one third had little or no evidence of local vascular change in the brain, as indicated by sequestered parasites, monocyte clusters, micro-haemorrhages, local vascular inos [151] or haemoxygenase -1 (ho-1) [152] staining. accepting that cm may occur without ischaemia does not exclude temporary or less severe reductions in vessel flow occurring during acute cm (associated or independent of parasite sequestration) that may contribute to impaired substrate delivery and lead to energy imbalance. as previously discussed, energy imbalance may also be impaired due to the uncoupling action of inflammatory cytokines on mitochondrial atp production. in gambian and ghanaian children, concentrations of tnf and its receptor were higher in those with cm than in those with mild or uncomplicated malaria [153, 154] . polymorphisms in the tnf promoter region have also been associated with increased risk of cm and death [155] or neurological sequelae [156] . cytokines may also up-regulate inos in brain endothelial cells, increasing production of no, which could then diffuse into brain tissue and disrupt neuronal (and/or mitochondrial) function [157, 158] . in the brain, mitochondrial function may also be influenced by neuronal excitotoxins. within the simplified model of dissociated neuronal culture, mitochondria appear to play a critical role in neuronal homeostasis during excitotoxin exposure. mitochondria are not only involved with maintaining atp production but also calcium homeostasis, and generation and detoxification of reactive oxygen species [107] . excitotoxin production may also be influenced by cytokine release. tnf administration has been shown to alter brain metabolism of tryptophan to produce more kynurinine [159, 160] . thus, as part of a general inflammatory reaction, increased excitotoxin generation during acute malaria may contribute to cellular energy imbalance. elevated levels of neuronal excitotoxins (quinolinic and picolinic acid) in the csf have been associated with a fatal outcome in malawian children with cm [161] . similarly, a graded increment of quinolinic acid concentration in csf was observed across patient outcome groups of increasing severity in african children [162] . although a subset of the malawaian autopsy patients [163] demonstrated negligible histological change in their brains, they did demonstrate inflammation, as indicated by inos, mif [151] and ho-1 [152] , staining in other tissues. these systemic changes were shared with the comatose sepsis cases in the study, and therefore are consistent with the premise that coma may in part be secondary to a host inflammatory response to systemic infection. below are further examples of systemic responses to infection that present with diffuse cerebral syndromes, including coma. in the past, the term cm has been restricted to falciparum malaria, and patients with p. vivax infection exhibiting symptoms of severe malaria, including coma, have been dismissed as undiagnosed falciparum co-infections. however, the use of more sensitive diagnostic techniques makes such dismissal less tenable. two such studies report adults exhibiting severe malaria with p. vivax (but not p. falciparum) infection detectable on pcr and serological and testing [142, 143] . the patients exhibited multiple organ failure including cerebral symptoms, renal failure, circulatory collapse, severe anaemia, haemoglobinuria, abnormal bleeding, acute respiratory distress syndrome, and jaundice. vivax malaria has been associated with a strong systemic inflammatory response [164] , but this was not investigated in the above studies. sepsis-associated encephalopathy (sae) syndrome has multiple features that resemble cm. it is characterised by a diffuse disturbance of cerebral function (typically impairment of consciousness) that occurs in the context of systemic response to infection without direct neuroinvasion (i.e. meningitis, macroscopic cerebritis and brain abscesses are excluded). sae is associated with generalised slow waves on the electroencephalogram (eeg), with the depth of coma linked with mortality. mild sae cases often recover completely, while survivors of severe sae may have persistent neurological deficit [165] ). in line with adult cm, the severity of encephalopathy parallels the severity of systemic organ failure [141] . inflammatory cytokines have been demonstrated to be higher in the serum than in the csf, suggesting that sepsis encephalopathy is a consequence of the systemic inflammatory response to infection [141] . an animal model in which prior administration of a neutralising antibody to tnf prevented the sepsis encephalopathy of pancreatitis [166] is consistent with this. further postulated reversible mechanisms of pathogenesis include changes in regional cerebral blood flow, neurotransmitter imbalance, mitochondrial dysfunction, bbb impairment and oxidative stress [167] . severe influenza infection can present with encephalopathy, yet as in malaria, the pathogen is not neuroinvasive [168] . seizures and coma occur after high fever [169] , commonly accompanied by thrombocytopaenia [169] , with metabolic acidosis and hyperlactataemia in severe cases (t. ichiyama, personal communication). similar to adult malaria, neurological sequelae occur concurrently with multiple organ failure [170] . tnf, il-6, stnfri, and soluble e-selectin are increased in serum and csf [171, 172] , and serum nitrite/nitrate levels are increased [173] . detailed examination of brain has revealed apoptosis of neurons and glial cell, histological evidence of active caspase-3 and caspase-cleaved parp, cerebral oedema, and bbb impairment [174] . these parallel changes are set out in table 3 . it is clear, therefore, that the presence of sequestering parasitised red cells is not necessary to generate these changes, which are also demonstrable in the falciparum malaria encephalopathy. notably, high levels of inflammatory cytokine are present in each disease. seizures are a very common component of acute malaria illness in children. a recent review documented that 80% of african children had a history of seizures, with 60% exhibiting seizures during hospital admission [175] . the molecular basis of the seizures is unclear. multiple mechanisms have been postulated, including fever, hypoxia and/or cytokine stimulation leading to an imbalance of neurotransmitters and excitotoxins or neuronal damage [11, 148] . recently, lang and co-workers [176] demonstrated that falciparum parasitaemia is associated with the generation of specific antibodies for voltage-gated calcium channels directed against neurones. higher antibody concentrations were detectable in sera from patients exhibiting cm or malaria with seizures than uncomplicated malaria, suggesting that these antibodies may influence seizure propensity. only the erythrocytic form of malaria is associated with disease, so valuable information about which african children are likely to have more, or less, severe malaria has inevitably been obtained from examining the inborn rbc abnormalities that endemic malaria has selected across the tropics. the coinciding geographic distributions of malaria transmission and the thalassaemias prompted haldane to put forward the 'malaria hypothesis', which proposed that common erythrocyte abnormalities are selected because of the fitness advantage they confer against malaria [177] . sickle cell haemoglobin (hbs) has also been repeatedly shown to be associated with malaria resistance, with heterozygotes for the hbs trait demonstrating 10% of the population at risk for severe malaria in certain populations [178] . other haemoglobinopathies (e.g. hbc [179, 180] and hbe [181] ) and deficiencies in rbc enzymes (e.g. glucose-6-phosphate dehydrogenase deficiency [182] ) have also been linked with protection against severe malaria. the mechanisms of protection afforded by haemoglobinopathies are likely to be multi-factorial. studies have demonstrated evidence to support several independent mechanisms including: reduced parasite invasion of rbcs and diminished intraerythrocytic growth of parasites in patients with the hbs trait [183] , enhanced phagocytosis of parasite-infected erythrocytes (ies) [184] and enhanced immune responses against ies [185] . recent in vitro studies observed that hbc modifies the quantity and distribution of the variant antigen p. falciparum erythrocyte membrane protein 1(pfemp1) on the ie surface. pfemp1 has been implicated in numerous ie adhesive interactions. in the latter study the authors demonstrated that hbc reduces the level of ie adhesion to endothelial monolayers, in addition to ie rosetting (the adhesion of ies to uninfected erythrocytes) and ie agglutination by sera. these findings provide the prospect that hbc pro-tects against severe malaria by mitigating the obstruction and inflammation caused by the pfemp1-mediated adherence of ies [186] . however, sequestration is believed to enhance parasite survival by enabling ies to avoid splenic clearance, so any reduction of sequestration by hbc can be expected to limit parasite fitness. multiple epidemiology studies (e.g. [179, 187, 188] ) have failed to identify any significant impact of hbc on the frequency or density of parasitaemia in naturally exposed populations. consequently, the influence of the changes in ie surface conformation needs to be confirmed and further examined in vivo [189] . a recent study re-confirmed that african children with -thalassaemia trait are significantly less likely to be hospitalised with severe malaria, particularly with coma or severe anaemia (hb < 5 g/100 ml). it is intriguing that the -thalassaemia patients did not demonstrate a lower incidence of uncomplicated malaria nor any reduction in peripheral parasite density [190] . thalassaemia has also been associated with increased incidence of clinical vivax and falciparum malaria during early life [191] . the findings raise speculation that the trait may indirectly afford enhanced immunity through increased non-lethal exposure to malarial parasites. such a mechanism is appealing, since it would be equally plausible across a range of haemoglobinopathies, including hbc. variations in erythrocyte membrane proteins also have a profound influence on malaria susceptibility. most notably the absence of duffy antigen protein confers absolute protection to p. vivax infection. more recently, the duffy antigen has also been associated with a protection against falciparum malaria [192] . enzymes involved with iron handling may also have a critical influence on malaria morbidity. a recent study from the gambia demonstrated that children in an endemic malaria area possessing the haptoglobin 2,2, isotype had a significantly increased risk of anaemia [193] . however, a lack of parallel alterations in other haematinic indices leaves the mechanism of this process unclear. malarial protection within individuals exhibiting multiple rbc abnormalities appears even more complex. a recent study observed that the concurrent presence of sickle cell and -thalassaemia trait among african children had a negative influence on the risk of malaria infection [194] . the results warn geneticists that gene epistasis may have a profound influence on overall malarial susceptibility. in tropical countries many hospital deaths from falciparum malaria happen before anti-malarial drugs have had time to kill the parasites. two approaches could help rectify this -addressing public-health problems resulting in delayed presentation, and identifying the physiological processes and molecular pathways that lead to these early deaths, with a view to developing evidence-based adjunct therapies. therapies being explored in sepsis, and based on disease pathogenesis data common to sepsis and malaria, may prove to be transferable from either of these diseases to the other. as noted above, circulating levels of a late-appearing inflammatory cytokine, hmgb1, are increased in falciparum malaria [41] as well as in sepsis. results from animal models on the role of hmgb1, although untested in humans, have inspired enthusiasm for inhibition of this molecule as a potential intervention for human sepsis. for instance, anti-hmgb1 antibodies provided dose-dependent protection [37] and reduced mortality [195] against experimental sepsis in mice. late administration of ethyl pyruvate, which inhibits hmgb1 release from macrophages, also conferred protection against endotoxaemia in mice [196] . treatments directed towards critical downstream consequences of malaria infection and inflammation, such as those intended to limit acidosis, are also a focus of investigation. one current approach is to identify which acute malaria patients most benefit from early volume expansion [197] . controlling lactic acidosis via sodium dichloroacetate (dca), an inhibitor of pyruvate dehydrogenate kinase (maintaining pyruvate dehydrogenase in its active form), is also being examined. dca reduced lactate levels in acute malaria patients [198] , although the study was unable to determine whether treatment improved outcome. an earlier large sepsis study also demonstrated that dca reduced lactate, but again with no improvement in outcome [126] . as outlined in the section 'is hyperlactataemia a cause or marker of the acidosis of malaria?', some researchers argue, in view of the strong ion difference contributing to acidosis and the postulated mitochondrial dysfunction during acute malaria infection, that lactate reduction per se may have limited impact on prognosis. other adjunct therapies are also being examined. improving rbc deformability provides one potential therapeutic approach. in vitro studies with n-acetylcysteine (nac), reported to scavenge free radicals, showed improvement in red cell deformability through in vitro studies [199] . unfortunately, an initial in vivo trial of nac in malaria patients had no effect on mortality [200] . blocking endothelial activation is also a focus of research, with initial in vitro studies providing some encouraging results [201] . in conclusion, continuing to identify the host responses to malaria infection that lead to disease is providing insights into novel molecular mechanisms. this information is beginning to guide the design of much needed additional therapies against this disease. there is little doubt that poor oxygen supply through vascular occlusion or anaemia could contribute to the body relying on excessive glycolysis to generate energy, resulting in hyperlactataemia, hypoglycaemia, and metabolic acidosis, and altered consciousness. however, inflammatory cytokines control these changes, as well as inhibit the capacity of mitochondria to use oxygen. thus, as described throughout this review, inflammatory cytokines are likely to have various pivotal roles across the multiple pathological processes involved in malarial disease (fig. 1 ). indicators of life-threatening malaria in african children the pathophysiologic and prognostic significance of acidosis in severe adult malaria malaria and the lung indicators of mortality in african adults with malaria severe falciparum malaria in children: current understanding of pathophysiology and supportive treatment the injured child is resistant to multiple organ failure: a different inflammatory response? endotoxin induces an exaggerated interleukin-10 response in peritoneal macrophages of children compared with adults il-1 beta induces an exaggerated pro-and anti-inflammatory response in peritoneal macrophages of children compared with adults current knowledge in falciparum malaria-induced acute renal failure pediatric considerations hypoglycemia in infancy and childhood glucose production and gluconeogenesis in adults with uncomplicated falciparum malaria hypoglycaemia in african children with severe malaria severe and complicated falciparum malaria in melanesian adults in papua new guinea cerebral malaria: the sequestration hypothesis the cytokine theory of human cerebral malaria golgi c (1886) sull infezione malarica the prevention of malaria possible importance of macrophage-derived mediators in acute malaria evolutionary causes and consequences of immunopathology how malaria has affected the human genome and what human genetics can teach us about malaria a high frequency african coding polymorphism in the n-terminal domain of icam-1 predisposing to cerebral malaria in kenya induction of cachectin (tumor necrosis factor) synthesis by influenza virus: deficient production by endotoxin-resistant (c3h/hej) macrophages induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease recombinant human tumor necrosis factor administered as a 24-hour intravenous infusion. a phase 1 and pharmacologic study innate immunity to malaria ifngr1 gene promoter polymorphisms and susceptibility to cerebral malaria interferon-alpha receptor-1 (ifnar1) variants are associated with protection against cerebral malaria in the gambia interleukin-1 gene cluster polymorphisms and susceptibility to clinical malaria in a gambian case-control study antimalarial antibody levels and il-4 polymorphism in the fulani of west africa analysis of il10 haplotypic associations with severe malaria hmg-1 as a late mediator of endotoxin lethality in mice high mobility group 1 protein (hmg-1) stimulates proinflammatory cytokine synthesis in human monocytes ethyl pyruvate protects against lethal systemic inflammation by preventing hmgb1 release hmgb-1 as a therapeutic target for infectious and inflammatory disorders high mobility group box 1 (hmgb1) protein: possible amplification signal in the pathogenesis of falciparum malaria inhibition of murine malaria (plasmodium chabaudi) in vivo by recombinant interferon-gamma or tumor necrosis factor, and its enhancement by butylated hydroxyanisole killing of plasmodium falciparum in vitro by nitric oxide derivatives influence of tumor necrosis factor-alpha on the ability of monocytes and lymphocytes to destroy intraerythrocytic plasmodium falciparum in vitro immunity to malaria after administration of ultra-low doses of red cells infected with plasmodium falciparum the effect of a monoclonal antibody to tumor necrosis factor on survival from childhood cerebral malaria treatment of septic shock with the tumor necrosis factor receptor: fc fusion protein disease pathogenesis in malaria and clinically similar conditions tumour necrosis factor and related cytokines a phase 1 clinical trial of recombinant human tumor necrosis factor clinical features cannot predict a diagnosis of malaria or differentiate the infecting species in children living in an area of low transmission a human 88-kd membrane glycoprotein (cd36) functions in vitro as a receptor for a cytoadherence ligand on plasmodium falciparum-infected erythrocytes thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence receptor-specific adhesion and clinical disease in plasmodium falciparum intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for plasmodium falciparum human vascular endothelial cell adhesion receptors for plasmodium falciparum-infected erythrocytes: roles for endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1 human cerebral malaria: associated with erythrocyte rosetting and lack of anti-rosetting antibodies platelet-mediated clumping of plasmodium falciparum-infected erythrocytes is a common adhesive phonotype and is associated with severe malaria a non-sense mutation in cd36 gene is associated with protection from severe malaria increased plasma concentrations of sicam-1, svcam-1 and selam-1 in patients with plasmodium falciparum or p. vivax malaria and association with disease severity possible involvement of cytokines in diffuse intravascular coagulation and thrombosis tissue factor in infection and severe inflammation transcriptional expression of tissue factor pathway inhibitor, thrombomodulin and von willebrand factor in normal human tissues thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression of intercellular adhesion molecule-1 (icam-1 cd54) and vascular cell adhesion molecule-1 (vcam-1 cd106) thrombin stimulates proliferation of liver fat-storing cells and expression of monocyte chemotactic protein-1: potential role in liver injury lactic acidosis and oxygen debt in african children with severe anaemia red blood cell physiology in critical illness anaemia and resistance to malaria in transgenic mice expressing human tumour necrosis factor physiological studies in the human malarial host alterations in red blood cell sodium transport during malaria changes in sodium, potassium, and adenosine triphosphate contents of red blood cells in sepsis and septic shock autocrine inhibition of na + /k + -atpase by nitric oxide in mouse proximal tubule epithelial cells erythrocyte deformability is a nitric oxide-mediated factor in decreased capillary density during sepsis sodium transport and red cell deformability red blood cell physiology in critical illness diminished red blood cell deformability in uncomplicated human malaria. a preliminary report alterations of capillary flow during sepsis red blood cell deformability in human and experimental sepsis prognostic significance of reduced red blood cell deformability in severe falciparum malaria red blood cell deformability as a predictor of anemia in severe falciparum malaria bone marrow changes in human malaria ineffective erythropoiesis in acute human p. falciparum malaria the bone marrow in human cerebral malaria: parasite sequestration within sinusoids dyserythropoiesis and ineffective erythropoiesis in plasmodium vivax malaria inhibiton of the growth and differentiation of erythroid precursor cells by an endotoxin-induced mediator from peritoneal macrophages identity of tumour necrosis factor and the macrophagesecreted factor cachectin tumour necrosis factor may contribute to the anaemia of malaria by causing dyserythropoiesis and erythrophagocytosis a low interleukin-10 tumor necrosis factor-alpha ratio is associated with malaria anemia in children residing in a holoendemic malaria region in western kenya reduced interleukin-12 and transforming growth factor-beta1 in severe childhood malaria: relationship of cytokine balance with disease severity plasma interleukin-10: tumor necrosis factor (tnf)-alpha ratio is associated with tnf promoter variants and predicts malarial complications absolute levels and ratios of proinflammatory and anti-inflammatory cytokine production in vitro predict clinical immunity to plasmodium falciparum malaria low interleukin-12 activity in severe plasmodium falciparum malaria severe malarial anemia associated with increased soluble fas ligand (sfasl) concentrations in gabonese children bone marrow suppression and severe anaemia associated with persistent plasmodium falciparum infection in african children with microscopically undetectable parasitaemia macrophage migration inhibitory factor release by macrophages after ingestion of plasmodium chabaudi-infected erythrocytes: possible role in the pathogenesis of malarial anemia a critical role for the host mediator macrophage migration inhibitory factor in the pathogenesis of malarial anemia cytopathic hypoxia. mitochondrial dysfunction as mechanism contributing to organ dysfunction in sepsis evaluation of the role of cellular hypoxia in sepsis by the hypoxic marker [ 18 f]fluoromisonidazole skeletal muscle partial pressure of oxygen in patients with sepsis cytopathic hypoxia in sepsis a global approach to energy metabolism in an experimental model of sepsis mitochondrial dysfunction in a long-term rodent model of sepsis and organ failure association between mitochondrial dysfunction and severity and outcome of septic shock mitochondrial dysfunction in patients with severe sepsis: an epr interrogation of individual respiratory chain components elevated tumour necrosis factor alpha and interleukin-6 serum levels as markers for complicated plasmodium falciparum malaria tnf concentration in fatal cerebral, non-fatal cerebral, and uncomplicated plasmodium falciparum malaria mitochondrial dysfunction and glutamate excitotoxicity studied in primary neuronal cultures a factor in the serum of malaria-infected animals capable of inhibiting the in vitro oxidative metabolism of normal liver mitochondria the inhibition of mitochondrial respiration and oxidative phosphorylation by serum from malaria-infected animals. ii. the inhibitory activity of serum ultrafitrates from plasmodium knowlesi-infected monkeys the pathogenesis of mammalian malaria acid-base status in paediatric plasmodium falciparum malaria lactic acidosis and hypoglycaemia in children with severe malaria -pathophysiological and prognostic significance the significance of type b hyperlactataemia in infective encephalopathy characterisation of metabolic acidosis in kenyan children admitted to hospital for acute non-surgical conditions protons in ischemia: where do they come from; where do they go to? reevaluation of the role of cellular hypoxia and bioenergetic failure in sepsis does tissue acidosis in sepsis indicate tissue hypoperfusion? association between blood lactate and acidbase status and mortality in ventilated babies ultimate survival from septic shock severe hypoglycemia and hyperinsulinemia in falciparum malaria malaria and fluids -balancing acts thiamine deficiency and malaria in adults from southeast asia lactate homeostasis and lactic acidosis lactic acidosis in sepsis: another commentary dichloroacetate for lactic acidosis in severe malaria: a pharmacokinetic and pharmacodynamic assessment a controlled clinical trial of dichloroacetate for treatment of lactic acidosis in adults protection by lactate of cerebral function during hypoglycaemia brain lactate, not glucose, fuels the recovery of synaptic function from hypoxia upon reoxygenation: an in vitro study the effect of intravenous lactate on cerebral function during hypoglycaemia lactate as a pivotal element in neuron-glia metabolic cooperation unaccounted for anion in metabolic acidosis during severe sepsis in humans unmeasured anions identified by the fencl-stewart method predict mortality better than base excess, anion gap, and lactate in patients in the pediatric intensive care unit unidentified acids of strong prognostic significance in severe malaria glucose homeostasis in children with falciparum malaria: precursor supply limits gluconeogenesis and glucose production hypoglycemia as a manifestation of sepsis hypoglycaemia is an important complication of falciparum malaria severe falciparum malaria intracranial pressure in childhood cerebral malaria intracranial hypertension in africans with cerebral malaria prognostic significance and course of retinopathy in children with severe malaria blood-brain barrier in falciparum malaria breaking down the blood-brain barrier: signaling a path to cerebral malaria? evidence of blood-brain barrier dysfunction in human cerebral malaria function of the blood-cerebrospinal fluid barrier in human cerebral malaria: rejection of the permeability hypothesis blood-brain barrier function in cerebral malaria and cns infections in vietnam edema and brain trauma clinical manifestations of severe malaria in the highlands of southwestern uganda malaria: pathogenicity and disease outcome of stroke in saudi children brain swelling and ischaemia in kenyans with cerebral malaria tissue distribution of migration inhibitory factor and inducible nitric oxide synthase in falciparum malaria and sepsis in african children induction of ho-1 in tissue macrophages and monocytes in fatal falciparum malaria and sepsis tumor necrosis factor production in falciparum malaria and its association with schizont rupture distinct patterns of cytokine regulation in discrete clinical forms of plasmodium falciparum malaria tumor necrosis factor alpha in the pathogenesis of cerebral malaria variation in the tnf-alpha promoter region associated with susceptibility to cerebral malaria possible central role of nitric oxide in conditions clinically similar to cerebral malaria nitric oxide potently and reversibly deenergizes mitochondria at low oxygen tension tumor necrosis factor-alpha and lipopolysaccharide enhance interferoninduced antichlamydial indoleamine dioxygenase activity independently lipopolysaccharide induction of indoleamine 2,3-dioxygenase is mediated dominantly by an ifn-gamma-independent mechanism metabolites of the kynurenine pathway of tryptophan metabolism in the cerebrospinal fluid of malawian children with malaria cerebrospinal fluid studies in children with cerebral malaria: an excitotoxic mechanism? does malarial tolerance, through nitric oxide, explain the low incidence of autoimmune disease in tropical africa the role of cytokines in plasmodium vivax malaria the encephalopathy associated with septic illness tumor necrosis factor alpha antibody prevents brain damage of rats with acute necrotizing pancreatitis progress in clinical neurosciences: sepsis-associated encephalopathy: evolving concepts influenza-associated encephalopathy: no evidence for neuroinvasion by influenza virus nor for reactivation of human herpesvirus 6 or 7 encephalitis and encephalopathy associated with an influenza epidemic in japan cerebrospinal fluid and serum levels of cytokines and soluble tumor necrosis factor receptor in influenza virus-associated encephalopathy analysis of cytokine levels and nf-[kappa]b activation in peripheral blood mononuclear cells in influenza virus-associated encephalopathy cytochrome c and tumor necrosis factor-alpha values in serum and cerebrospinal fluid of patients with influenza-associated encephalopathy high concentration of serum nitrite/ nitrate obtained from patients with influenza-associated encephalopathy apoptosis and microglial activation in influenza encephalopathy pathogenesis, clinical features, and neurological outcome of cerebral malaria antibodies to voltage-gated calcium channels in children with falciparum malaria the rate of mutation of human genes common west african hla antigens are associated with protection from severe malaria hemoglobin c associated with protection from severe malaria in the dogon of mali, a west african population with a low prevalence of hemoglobin s haemoglobin c protects against clinical plasmodium falciparum malaria influence of hemoglobin e trait on the severity of falciparum malaria natural selection of hemi-and heterozygotes for g6pd deficiency in africa by resistance to severe malaria cellular mechanism for the protective effect of haemoglobin s against p. falciparum malaria early phagocytosis of glucose-6-phosphate dehydrogenase (g6pd)-deficient erythrocytes parasitized by plasmodium falciparum may explain malaria protection in g6pd deficiency lymphoproliferative responses to plasmodium falciparum antigens in children with and without the sickle cell trait abnormal display of pfemp-1 on erythrocytes carrying haemoglobin c may protect against malaria a new look at the protection of hemoglobin as and ac genotypes against plasmodium falciparum infection: a census tract approach hemoglobin c and resistance to severe malaria in ghanaian children red blood cells that do and red blood cells that don't: how to resist a persistent parasite the effect of alpha+-thalassaemia on the incidence of malaria and other diseases in children living on the coast of kenya high incidence of malaria in alpha-thalassaemic children high prevalence of human antibodies to recombinant duffy binding-like alpha domains of the plasmodium falciparum-infected erythrocyte membrane protein 1 in semi-immune adults compared to that in non-immune children seasonal childhood anaemia in west africa is associated with the haptoglobin 2-2 genotype negative epistasis between the malaria-protective effects of alpha(+)-thalassemia and the sickle cell trait reversing established sepsis with antagonists of endogenous high-mobility group box 1 ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation randomized trial of volume expansion with albumin or saline in children with severe malaria: preliminary evidence of albumin benefit pharmacokinetics and pharmacodynamics of dichloroacetate in children with lactic acidosis due to severe malaria oxidative stress and rheology in severe malaria a pilot study of n-acetylcysteine as adjunctive therapy for severe malaria inhibition of endothelial activation: a new way to treat cerebral malaria key: cord-331058-ou6vqp7n authors: chireh, batholomew; essien, samuel kwaku title: leveraging best practices: protecting sub-saharan african prison detainees amid covid-19 date: 2020-06-24 journal: pan afr med j doi: 10.11604/pamj.2020.36.121.24133 sha: doc_id: 331058 cord_uid: ou6vqp7n the risk of infection and death from covid-19 is higher among older prisoners with pre-existing health conditions especially in sub-saharan african. hawks l et al. raise four concerns that need to be considered when developing public health and clinical responses to covid-19 to protect prisoners. this paper applies these concerns to the sub-saharan african context. these focus areas include 1) challenges of social distancing; 2) higher risk of severe infection and death; 3) difficulties health care systems may face in the case of covid-19 surge; and 4) recommended solutions to prevent harm and preventing a public health catastrophe. prisoners are more vulnerable and the time to take immediate actions to minimize an imminent covid-19 outbreak and its impacts is now. most prisons in sub-saharan african countries are overcrowded with prisoners living in extremely dirty and unpleasant conditions. in 2016, an estimated 668,000 people were incarcerated and actively serving prison sentences [1] . another report estimated that 50% to 90% of detainees constitute pretrial detainees of the total prison population of most countries in the continent. which means, most prisons in africa can be decongested if there is the political will [2] . amid the coronavirus pandemic, prisoners are among the vulnerable groups who are at a higher risk of contracting the deadly disease. systemic poor prison conditions will worsen the plight of most prisoners in this covid-19 pandemic era. extreme poor healthcare systems inside prisons do not provide the necessary medications and health personnel for the treatment of prisoner´s basic health needs making them vulnerable to covid-19 risk. this risk is further exacerbated among older prisoners with underlying medical conditions such as cardiovascular disease, diabetes, chronic respiratory disease, and cancer [3] . evidence also shows that institutions such as mines and prisons in which people live in proximity can act as incubators of infection [3] . there are already hundreds of covid-19 cases in subfight. therefore, sub-saharan african countries must take note of the concerns discussed below and take precautionary measures to prevent the spread of covid-19 among this vulnerable population. hawks l et al. [4] discuss four areas of concern to consider when developing public health and clinical responses to protect prisoners from covid-19. although prison conditions in the united states may be quite different from those of sub-saharan african countries, this paper applies these focus areas to the african context and provides simple and immediate measures to proactively prevent the spread of covid-19 among prisoners in the region. the first area of concern that relates to countries in sub-saharan africa as well as other low and middle countries is the challenge of social or physical distancing. the covid-19 outbreak on the diamond princess cruise ship highlights the importance of social distancing in the fight against covid-19. for example, within 4-weeks, 700 people got infected and 12 casualties were recorded out of the total number of 3700 passengers and crew held onboard the ship. the major source of infection was through kitchen staff housed together and was responsible for feeding passengers on board [5] . similar conditions are common and conducive for covid-19 spread in most african prisons given the poor infrastructure of prisons in the region. also, the fact that prison officers, other staff, and relatives of incarcerated persons often visit prison facilities and then return, it exposes inmates to a higher risk of covid-19. social distancing may be a practical impossibility considering the crowded nature of most prisons in the region. the second area of concern is that prisoners are at high risk of severe infection and death. as stated earlier older prisoners and persons with underlying medical conditions such as cardiovascular disease, diabetes, chronic respiratory disease, and cancer are more likely to report severe infection and death in low-and middle-income countries [3] . while statistics on the average age and duration of pretrial detention in africa are difficult to obtain, evidence suggests that waits are longest in central and west african nations. for example, nearly the highest rate of pretrial prisoners in prison in the world is found in liberia (97.3 percent), the second highest in the world is mali with 88.7 percent, benin is 4 th with 79.6 percent, and niger 5 th with 76 percent [1] . this leads to overcrowding and possible disease spread in ssa prisons. health data from african prisons differed by regional blocks. a study published in the lancet found that comorbid hiv and tuberculosis prevalence among prisoners in africa ranges between 2.3% to 34.9% with a regional breakdown of 2.3% to 10.8% in west africa and 4.2% to 23% in east africa [1] . also, a recent cameroonian study reported higher levels of diabetes, hypertension, and obesity among prison inmates [6] . all of which are risk factors for an increased risk of covid-19 deaths. thirdly, the effects on health care systems in an imminent covid-19 outbreak in ssa prisons are of concern. low-and middle-income countries (lmics), with fragile healthcare systems, are already stretched by the dual burden of non-communicable diseases (ncdssuch as type 2 diabetes, cancers, arthritis, hypertension, stroke, heart diseases,) and earlier infectious diseases [1, 6] . not only are these diseases increasing, but they are increasing disproportionately among women, rural residents, prisoners, and those with low income [1, 6] . these fragile health care systems may not be able to contain a surge page number not for citation purposes 3 in covid-19 in the general population as well as the prison population. the current covid-19 pandemic if not well managed in both the general population and in prisons has the potential to cause similar havoc just like the recent ebola virus disease outbreak in some west african countries. the inability of less-resourced african prison systems to bear hospital-related costs may be a disincentive for referring inmates with pre-existing conditions to seek better health care although it is their right. the fourth area of focus is to design strategies to prevent a public health catastrophe in ssa prisons. even before the advent of covid-19, civil society and other civil rights organizations have strongly advocated for the provision of basic health amenities such as personal protective equipment, routine screening or testing, and availability of medical care for inmates [2] . in this commentary, we sought to leverage best practices from other jurisdictions that have proven to have yielded positive results in the fight against infectious diseases in prisons and apply them to the african context. we therefore recommend the following solutions. first and foremost is the decongestion of prison populations by releasing non-violent inmates 50+ years with serious health issues and reducing unnecessary pretrial detention. while the covid-19 may be an albatross around the necks of most ssa countries, it opens a window of opportunity for the respective countries to decongest their prisons and drastically reduce prison populations to avoid a looming outbreak. this has been found as the most effective way of combating covid-19 in prisons [7] . lessons can be drawn from the us and other developed countries where prison programs such as probation, conditional sentences, provincial parole, and community alternative to remand or reduction in pretrial detention have shown to have effectively decongested prisons in these countries [8] . secondly, for those remaining in the detention facilities, they should be allowed access to mental health professionals and provided with a standard of healthcare that meets each prisoner´s individual needs similar to those available in the community, and that ensures the maximum possible protection against the spread of covid-19. a recent systematic review in the us found that improving health in people in jails and prisons can also improve the health of the general population, improve the safety of communities, and decrease health care costs. these researchers believed that emergency department use will be reduced if infectious diseases can be treated to limit ongoing transmission, while crime rates can be decreased through the treatment of people with mental illness, and provision of quality and accessible primary health care [9] . thirdly, ensure that prisoners have separate cells, giving them the ability to physically distant from each other. most prisons in ssa do not have the minimum space requirements for their inmates mainly due to high rates of pretrial detentions and limited infrastructure [1] . this can only happen if prisons are decongested. although this may be practically difficult to achieve in most prisons in ssa countries at this time, a recent systematic review of studies from 16 countries found that covid-19 spread was lower with the physical distancing of 1 meter or more, compared with a distance of less than 1 meter in all populations [10] . finally, we recommend the heightening of sanitation standards for all staff, inmates, and facilities, ensuring ample sinks for hiv and tuberculosis in prisons in sub-saharan africa protect detainees in sub-saharan africa against covid-19 coronavirus disease 2019 (covid-19) situation report-51. 11 th covid-19 in prisons and jails in the united states initial investigation of transmission of covid-19 among crew members during quarantine of a cruise ship-yokohama prevalence of diabetes and associated risk factors among a group of prisoners in the yaoundé central prison flattening the curve for incarcerated populations-covid-19 in jails and prisons united states prison policy initiative. responses to the covid-19 pandemic a systematic review of randomized controlled trials of interventions to improve the health of persons during imprisonment and in the year after release physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis. the lancet both authors declare no competing interests. bc and ske gathered information and wrote the commentary. both authors read and agreed to the final manuscript. key: cord-311423-8yvu9xhw authors: betson, d. n.; maitra, d. a. title: disproportionate covid-19 related mortality amongst african americans in four southern states in the united states date: 2020-06-12 journal: nan doi: 10.1101/2020.06.08.20124297 sha: doc_id: 311423 cord_uid: 8yvu9xhw background african american have been severely affected by covid-19 noted with the rising mortality rates within the african american community. health disparities, health inequities and issues with systemic health access are some of the pre-existing issues african american were subjected to within the southern states in the united states. second, social distancing is a critical non-pharmacological intervention to reduce the spread of covid-19. however, social distancing was not practical and presented a challenge within the african american community, specifically, in the southern states. objective this article assesses the effect of covid-19 on african american in the southern states. methodology this short communication queried the publicly available department of health statistics on covid-19 related mortality and underlying health conditions in four southern states (alabama [al], georgia [ga], louisiana [la] and mississippi [ms]) with a high proportion of african american residents. second, unacast covid-19 toolkit was used to derive a social distancing (sd) grade for any given state, based on three different metrics: (i) percent change in average distance travelled (ii) percent change in non-essential visits and (iii) decrease in human encounters (compared to national baseline). results across the four states, on average, as many as 54% of covid-19 related deaths are in the african american community, although this minority group comprises only 32% of the population cumulatively. this article finds that all four southern states received a social distancing grade of f. covid-19 have demonstrated that adverse outcomes are higher in individuals with underlying health conditions such as diabetes, cardiovascular diseases, or pre-existing pulmonary compromise. conclusion recognizing that there is a great need for african american representation or diversity in the health workforce would be able to better address the health disparities. in addition, the lack of diversity in the healthcare system causes the morbidity and mortality rates to increase in the african american communities because it is not able to address its primary obligations within the african american communities in the southern states during covid-19 pandemic. these primary obligations are to restore, protect, improve health and to suppress health disparities and inequalities of covid-19 within in the african american communities. keywords: covid-19; african american; mortality background african american have been severely affected by covid-19 noted with the rising mortality rates within the african american community. health disparities, health inequities and issues with systemic health access are some of the pre-existing issues african american were subjected to within the southern states in the united states. second, social distancing is a critical non-pharmacological intervention to reduce the spread of covid-19. however, social distancing was not practical and presented a challenge within the african american community, specifically, in the southern states. ) with a high proportion of african american residents. second, unacast covid-19 toolkit was used to derive a social distancing (sd) grade for any given state, based on three different metrics: (i) percent change in average distance travelled (ii) percent change in nonessential visits and (iii) decrease in human encounters (compared to national baseline). across the four states, on average, as many as 54% of covid-19 related deaths are in the african american community, although this minority group comprises only 32% of the population cumulatively. this article finds that all four southern states received a social distancing grade of f. covid-19 have demonstrated that adverse outcomes are higher in individuals with underlying health conditions such as diabetes, cardiovascular diseases, or pre-existing pulmonary compromise. recognizing that there is a great need for african american representation or diversity in the health workforce would be able to better address the health disparities. in addition, the lack of diversity in the healthcare system causes the morbidity and mortality rates to increase in the african american communities because it is not able to address its primary obligations within the african american communities in the southern states during covid-19 pandemic. these primary obligations are to restore, protect, improve health and to suppress health disparities and inequalities of covid-19 within in the african american communities. keywords: covid-19; african american; mortality . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.08.20124297 doi: medrxiv preprint we have read with great interest the recent series of commentaries in cdc on disparities in hospitalization and death rates amongst african american populations compared to caucasians 1-3 . however, a systematic quantitative assessment of these important metrics, critical for implementing public health measures like testing, tracing and isolation in the highest risk communities, is still lacking. we queried the publicly available department of health (doh) statistics on covid-19 related mortality in four southern states (alabama [al], georgia [ga], louisiana [la] and mississippi [ms]) with a high proportion of african american residents. across the four states, on average, as many as 54% of covid-19 related deaths are in the african american community, although this minority group comprises only 32% of the population cumulatively. specifically, the proportion of african americans residing in a given southern state and the proportion of covid-19 related mortality that are comprised of african americans in that state are as follows: al -26.6% and `50%, ga -31.5% and ~50%, la -32.2% and 59%, and ms -37.7% and 57%. the number of deaths in the minority population has kept at par with caucasians in three of the four states, and exceeded the latter in the hardest hit state of la (figure 1 ). there are likely to be several reasons for the disproportionate causes of mortality amongst african americans in the four southern states we have interrogated here. first, emerging clinical series on covid-19 have demonstrated that adverse outcomes are higher in individuals with underlying health conditions such as diabetes, cardiovascular diseases, or pre-existing pulmonary compromise 4, 5 . indeed, in three of the states (al, la and ms) where such data is publicly cataloged, many of the patients dying from causes related to confirmed covid-19 had an underlying chronic health condition (table 1) . secondly, social distancing is a critical non-pharmacological intervention to reduce the spread of covid-19. the unacast covid-19 toolkit (https://www.unacast.com/covid19/social-distancing-scoreboard) can be used to derive a social distancing (sd) grade for any given state, based on three different metrics: (i) percent change in average distance traveled (ii) percent change in non-essential visits and (iii) decrease in human encounters (compared to national baseline), each of is an average to create the overall grade for that state. based on these parameters, all four states received a sd grade of f. unfortunately, the suboptimal sd measures adopted statewide is even more likely to impact individuals living in crowded urban localities, those relying on public transport, or families where one or more members have been deemed as "essential" at-work personnel. according to povertyusa (www.povertyusa.org), the poverty rates in the four states stands at 18.4% on average, which is greater than the national rate of 14.1%; in addition, of the reported overall numbers, the average poverty rate amongst the african american community (29.1%) is approximately 2.3 times higher compared to caucasians (12.6%). the median household income reported by us census bureau for al, ga, la and ms were $49,861, $58,756, $47,905, and $44,717, respectively, which is, on average, more than $11,600 less than the national median household income ($61,937). while we cannot exclude an underlying genetic basis to the disproportionate burden of mortality related to covid-19 among the african american community (for example . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.08.20124297 doi: medrxiv preprint ace2 polymorphisms), the emerging data strongly suggests that african american communities face an array of social and economic inequities that impact their baseline health status and access to healthcare, and is likely driving the course of the pandemic in this population 6 . measures at mitigating the impact of covid-19 on african africans need to address these inequities in lockstep with more conventional interventions. author statement: ethical approval was not necessary because this study did not involve human subjects and animal studies . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.08.20124297 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.08.20124297 doi: medrxiv preprint * in la, the specific numbers for comorbidities associated with covid-19 related deaths are not reported, only the percentages. ** in ga, comorbidity status is only available for confirmed covid-19 cases, and not for covid-19 related deaths al, https://www.alabamapublichealth.gov/covid19/assets/cov-al-cases-042820.pdf la, http://ldh.la.gov/coronavirus/ ga, https://dph.georgia.gov/covid-19-daily-status-report ms, https://msdh.ms.gov/msdhsite/_static/14,0,420.html data accessed on june 7 th , 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.08.20124297 doi: medrxiv preprint covid-19 and african americans pérez-stable e. covid-19 and racial/ethnic disparities covid-19 and health equity-a new kind of characteristics and clinical outcomes of adult patients hospitalized with covid-19 -georgia clinical characteristics of covid-19 in new york city the covid-19 pandemic: a call to action to identify and key: cord-343962-12t247bn authors: cori, anne; donnelly, christl a.; dorigatti, ilaria; ferguson, neil m.; fraser, christophe; garske, tini; jombart, thibaut; nedjati-gilani, gemma; nouvellet, pierre; riley, steven; van kerkhove, maria d.; mills, harriet l.; blake, isobel m. title: key data for outbreak evaluation: building on the ebola experience date: 2017-05-26 journal: philos trans r soc lond b biol sci doi: 10.1098/rstb.2016.0371 sha: doc_id: 343962 cord_uid: 12t247bn following the detection of an infectious disease outbreak, rapid epidemiological assessment is critical for guiding an effective public health response. to understand the transmission dynamics and potential impact of an outbreak, several types of data are necessary. here we build on experience gained in the west african ebola epidemic and prior emerging infectious disease outbreaks to set out a checklist of data needed to: (1) quantify severity and transmissibility; (2) characterize heterogeneities in transmission and their determinants; and (3) assess the effectiveness of different interventions. we differentiate data needs into individual-level data (e.g. a detailed list of reported cases), exposure data (e.g. identifying where/how cases may have been infected) and population-level data (e.g. size/demographics of the population(s) affected and when/where interventions were implemented). a remarkable amount of individual-level and exposure data was collected during the west african ebola epidemic, which allowed the assessment of (1) and (2). however, gaps in population-level data (particularly around which interventions were applied when and where) posed challenges to the assessment of (3). here we highlight recurrent data issues, give practical suggestions for addressing these issues and discuss priorities for improvements in data collection in future outbreaks. this article is part of the themed issue ‘the 2013–2016 west african ebola epidemic: data, decision-making and disease control’. detection of a new infectious disease outbreak requires rapid assessment of both the clinical severity and the pattern of transmission to plan appropriate response activities. following the subsequent roll-out of interventions, continued evaluation is necessary to detect reductions in transmission and assess the relative impact of different interventions. surveillance data are crucial for informing these analyses, and directly determine the extent to which they can be performed. despite the unprecedented scale of the 2013-2016 west african ebola epidemic [1, 2] , detailed data were collected during the outbreak, which proved invaluable in guiding the response [3] [4] [5] [6] [7] [8] [9] [10] . multiple studies have already considered the lessons to be learned from the ebola experience with respect to coordinating international responses to health crises, strengthening local health systems and improving clinical care and surveillance tools [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . here we discuss what can be learned to improve real-time epidemiological assessment in future outbreaks via improved data collection and analyses, building on similar contributions after other epidemics [24 -26] . we focus on efforts to reduce and interrupt transmission. first, we outline analyses that are essential to inform response activities during different stages of an epidemic. second, we detail the various types of data needed to perform these analyses, with examples from the ebola experience. third, we summarize the successes and challenges of data collection experienced during this outbreak, and the implications this had for answering key public health questions. fourth, we suggest improvements that could be implemented in future outbreaks, again drawing from the ebola experience. finally, we discuss issues related to availability of data and analyses (box 1). key public health questions for any emerging infectious disease outbreak are the following: (i) what is the likely public health impact of the outbreak? (ii) how feasible is controlling the outbreak and what interventions would be appropriate? (iii) are current interventions effective and could they be improved? here, we describe statistical and mathematical analyses that facilitate epidemic response planning, focusing on these questions (figure 1). in this section, we take a general view, as these questions are recurring during most, if not all, outbreaks. we provide examples from the west african ebola epidemic in subsequent sections. (a) what is the likely public health impact of the outbreak? a key issue in the early phase of an epidemic is to determine the potential impact of the outbreak in terms of clinical severity and the likely total number of cases over different time periods. the severity of a pathogen is often characterized by the case fatality ratio (cfr), the proportion of cases who die as a result of their infection. estimating the cfr during an outbreak can be challenging due to inconsistent case definitions, incomplete case reporting and right-censoring of data [27] [28] [29] . in particular, it is critical to know the proportion of cases for whom clinical outcome is unknown or has not been recorded, which is typically easier to assess using detailed case data rather than aggregated case counts [27] . the cfr may differ across populations (e.g. age, space, treatment); quantifying box 1. recommendations for collecting and using data for outbreak response. data need to be collected at each of the three levels: individual level: detailed information about cases exposure level: information about exposure events that may have led to transmission population level: characteristics of the population(s) in which the outbreak is spreading and the interventions carried out in the population(s) although some data will be context-specific, others, in particular at the population level, will be useful for a wide range of epidemics, and should be routinely and centrally collected in preparation for the next outbreak. 2. optimizing data quality having a general framework ready in advance of the next outbreak will facilitate: quality and timeliness of data centralization and harmonization of data at all three levels preparedness to deploy training material, personnel and logistical resources there is substantial room for improvement in the quality of data collected in an epidemic context, particularly for emerging pathogens. 3. ensuring adequate data availability data need to be shared ensuring a balance between the following considerations: ethical: protecting anonymity while ensuring data are sufficiently detailed to be useful scientific: wide data access is desirable to promote independent analyses; however, mechanisms must be in place for systematic comparison of results practical: deciding on a data format for sharing, on who will be responsible for data cleaning and on how various roles will be acknowledged discussions and decisions relating to data sharing remain ongoing and guidelines should be agreed on in advance of the next outbreak. 4. analysing data and reporting results in an appropriate manner the scientific community should agree on guidelines for epidemic modelling and analyses (such as those in place for reporting experimental studies), such as: assumptions underlying analyses should be clearly stated sensitivity to these assumptions should be tested uncertainty in results should be adequately explored and reported these are particularly relevant for reporting epidemic projections. these heterogeneities can help target resources appropriately and compare different care regimens. for less severe emerging pathogens, the case definition typically only encompasses a small fraction of all infected individuals, and hence the infection fatality rate (i.e. the proportion of infected individuals who die, rather than the proportion of cases who die-as per the case definition, which may not be equivalent to infection) may be a more useful measure of severity [30] . (ii) short-and long-term incidence projections short-term impact of an outbreak can be assessed by predicting the number of cases that will arise in the next few days or weeks. this is particularly relevant for evaluation of immediate health-care capacity needs. projections of future incidence and estimates of the doubling time (the time taken for the incidence to double) can be obtained by extrapolating the early time series of reported cases either obtained from aggregated surveillance data or calculated from individual records [31] . these projections typically rely on the assumption that incidence initially grows exponentially [31] . they are subject to uncertainty, which increases the further one looks into the future. quantifying and appropriately reporting such uncertainty and underlying assumptions are crucial [22, 25, 32, 33] . overstating uncertainty can lead to inappropriately pessimistic projections, which may in turn be detrimental to the control of the outbreak [34] . on the other hand, understating uncertainty prevents policymakers from making decisions based on the whole spectrum of possible impacts. some studies have already discussed how to find the balance between these two extremes [35 -37] . here, we propose two simple rules of thumb for projecting case numbers. first, projections should not be made for more than two or three generations of cases into the future. second, central projections should be shown together with lower and upper bounds. in the future, the modelling community should agree on guidelines for reporting epidemic projections, as are already in place for reporting experimental studies [38] . a number of factors can lead to incidence not growing exponentially. in particular, this happens once herd immunity accumulates, if population behaviour changes or as a result of the implementation of interventions. dynamic transmission models, which account for saturation effects, can be used to assess the long-term impact of the outbreak such as predicting the timing and magnitude of the epidemic peak or the attack rate (final proportion of population infected) [39, 40] . however, these models are hard to parametrize as they require information on population size and immunity, interventions (if any) and potential behavioural changes over time, all of which may be subject to uncertainty [41, 42] . we discuss these issues in more detail in §3. projecting longer term is likely to be associated with a large degree of uncertainty and these projections may be more useful for evaluating qualitative trends and evaluating intervention choices rather than predicting exact case numbers. interventions would be appropriate? interventions to reduce transmission can include community mobilization, quarantine, isolation, treatment or vaccination. the potential success of these interventions is determined by general characteristics of the disease such as overall transmissibility and how this varies across populations [43] . furthermore, certain types of interventions may be more or less appropriate depending on the natural history of the disease and the context of the epidemic. the transmissibility of a pathogen determines the intensity of interventions needed to achieve epidemic control [44] . the parameter most often used to quantify transmissibility is the reproduction number (r), the mean number of secondary cases infected by a single individual. this parameter has an intuitive interpretation: if r . 1, then the epidemic is likely to grow, whereas if r , 1, the epidemic will decline [44, 45] . the final attack rate ( proportion of the population infected) of an epidemic also depends on the value of r at the start of an epidemic (termed r 0 if the population has no immunity). r can be estimated from the incidence of reported cases, given knowledge of the serial interval distribution (i.e. distribution of time between symptom onset in a case and symptom onset in his/her infector; see §2b(iii)) [46] [47] [48] [49] . heterogeneity in the number of secondary cases generated by each infected individual affects epidemic establishment and the ease of control. greater heterogeneity reduces the chance of an outbreak emerging from a single case [50] . however, this heterogeneity can make an established outbreak hard to control using mass interventions, as a single uncontrolled case can generate a large number of secondary cases [50] . conversely, heterogeneity in transmission may provide opportunities for targeting interventions if the individuals who contribute more to transmission (because of environmental, behavioural and/or biological factors [51] [52] [53] ) share socio-demographic or geographical characteristics that can be identified [50, 54] . reconstruction of transmission trees (who infects whom) can provide an understanding of who contributes more to transmission. this can be done using detailed case investigations and/or using genetic data [55] [56] [57] [58] . environmental, behavioural and biological factors may also lead to groups of individuals being disproportionately more likely to acquire infection (e.g. children during influenza outbreaks [53, 59] or health-care workers (hcws) during ebola outbreaks [60, 61] ). to identify whether such groups exist and target them appropriately, the proportion infected in each group must be estimated. this requires population size estimates for the different groups, which may be difficult to obtain, as we highlight §3c. spatial heterogeneity in transmission is particularly interesting to assess as it can inform the targeting of surveillance and interventions to the geographical areas most at risk. phylo-geographical studies based on genetic data can improve understanding of the geographical origins of the outbreak, identify and characterize sub-outbreaks and quantify whether transmission is very local or travels large distances [22, 62, 63] . results of such analyses can be used to determine the appropriate spatial scale of control measures. spatially explicit epidemic models can also be used to quantify the risk of exportation of the infection from one place to another. this can help public health officials to tailor prevention and control resources to the level of risk likely to be experienced by a given area. such models typically require detailed data on mobility patterns and immunity levels of the populations in the areas of interest [64] [65] [66] . disease natural history fundamentally affects outbreak dynamics. the generation time distribution (i.e. distribution of time between infection of an index case and infection of its secondary cases) determines-with the reproduction number-the growth rate of an epidemic [67] . most commonly, the generation time distribution is estimated from data on the serial interval distribution of an infection-the delay between symptom onset in a case and symptom onset in his/her infector. other delays between events in the natural history of infection (e.g. exposure, onset of symptoms, hospitalization and recovery or death) also affect disease transmission or have implications for control [43, 67] . delays from symptom onset to recovery (or death) will determine the required duration of health-care and case isolation. the incubation period (the delay between infection and symptom onset of a case) and the extent to which infectiousness precedes symptom onset will determine the feasibility and effectiveness of contact tracing or prophylaxis [43] . estimating these delay distributions requires detailed data on individual cases and their exposure, e.g. through transmission pairs identified in household studies [43] . other analyses can also help refine the type of interventions that should be considered. ecological associations between transmissibility (measured by r) at a fine spatio-temporal scale and any covariate measured at the same scale, may be rstb.royalsocietypublishing.org phil. trans. r. soc. b 372: 20160371 of interest. for instance, analyses of the west african ebola epidemic showed that districts with lower reported funeral attendance and faster hospitalization experienced lower transmissibility, highlighting the effectiveness of promoting safe burials and early hospitalization [10] . however, interpreting the results of such analyses can be challenging, as they might be prone to bias and confounding. efficacy (which measures the impact of an intervention under ideal and controlled circumstances) and effectiveness (which measures the impact of an intervention under real-world conditions) of an intervention (e.g. vaccine) are best measured in a trial setting (either individual-or cluster-randomized [68 -70] ). however, performing trials to evaluate the comparative impact of different multi-intervention packages is impractical. dynamic epidemic models, where the interventions of interest can be explicitly incorporated, allow the impact of such intervention packages to be predicted [71] . however, outputs of such models are strongly determined by the underlying assumptions and parameter values. hence they require careful parametrization, supported by data such as intervention efficacy and the size, infectivity, susceptibility and mixing of different groups [72, 73] . these parameters may not be straightforward to estimate, as we discuss in §3 using examples from the west african ebola epidemic. another factor determining the appropriate choice of interventions is their cost, combinations of interventions with higher effectiveness at lower cost (i.e. higher cost-effectiveness) being preferable. economic analyses combined with mathematical models can help to evaluate the optimal resource allocation among both current available interventions and potential new interventions, accounting for development and testing costs for the latter. indirect costs, e.g. those associated with a restricted workforce following school closures [74] , or trade limitations from air-travel restrictions [75] , also need to be considered. while economic analyses have played an important role in designing optimal intervention packages for endemic diseases such as hiv and malaria [76, 77] , such analyses are more difficult to perform during an epidemic, when cost data might be unavailable and uncertain, costs may vary rapidly over time and ethical considerations suggest interventions should be implemented immediately. (c) are current interventions effective and could they be improved? tracking changes in estimates of key epidemiological parameters over the course of an outbreak enhances situational awareness. it also allows the impact of interventions to be assessed as they are implemented, although disentangling the effects of different interventions carried out simultaneously may be challenging. obtaining reliable estimates of the epidemiological parameters detailed above requires a wide range of data, such as incidence time series and detailed case information (figure 1). here, we explain how these can be obtained from various sources, with the objective to help improve data collection systems in preparation for future outbreaks. we use ebola as a specific example throughout this section, commenting on the strengths and limitations of the data collected during the west african epidemic. we distinguish data needs at the individual level, the exposure level and the population level. simple analyses can be performed solely using incidence time series, from surveillance designed to capture aggregate case counts. however, individual case reports provide much richer information, essential to estimate many of the key parameters outlined above (e.g. characterization of delay distributions). such data are typically stored in a case database or 'line-list'a table with one line containing individual data for each case. the more data recorded on each case, the more detailed the analyses can be. in the ebola epidemic, demographic characteristics, spatial location, laboratory results and clinical details such as symptoms, hospitalization status, treatment and outcome, and dates associated with these were reported for at least a subset of cases. the appropriate information to collect may vary depending on the disease: for ebola, dates of isolation and funeral were relevant. comprehensive demographic information can be used to determine risk factors for transmission or severity of infection and to project case numbers stratified by demographic characteristics. detailed information also helps to identify and merge any duplicate entries in a line-list, which may occur when the same person visits multiple health centres over the course of illness, for instance. finally, information on how each case was detected-for example, through hospitalization, or via contact tracing-can aid assessment of how representative the data are and allow subsequent adjustment for bias [28] ; however, this was not available for ebola. cases in the line-list should be classified using standardized case definitions, which is sometimes difficult for outbreaks of new pathogens, or where different case definitions are provided by different organizations (e.g. world health organization (who) and us centers for disease control and prevention (cdc)) [78] . for the ebola response, although the who released official case definitions of confirmed, probable and suspected ebola cases [79] , different countries adopted different testing strategies, thereby limiting the opportunity for inter-country comparison. for example, in guinea deceased individuals were not tested for ebola, meaning these individuals were never classified as confirmed cases, unlike in liberia and sierra leone. encouraging use of a consistent testing protocol and case definition, and ensuring transparency in what is used where and when, would improve the validity of subsequent analyses. laboratory testing of clinical specimens is key for confirming cases and test results should be linked to the line-list. understanding diagnostic test performance in field conditions is important; cross-validation of diagnostic sensitivity and specificity between laboratories is useful to assess the extent to which observed differences in case incidence may be explained by variations in laboratory conditions and practices. in addition, recording raw test results with the case classification may help evaluation of diagnostic performance. ebola cases were defined as confirmed cases once ebola virus rna was isolated from clinical specimens using reverse transcription polymerase chain reaction (rt-pcr [79] temperature and humidity to which these laboratories were subjected reduced the test performance compared with manufacturer evaluation reports [82] . during the west african ebola epidemic, the case line-list contained a large quantity of data collected from reported cases. the information allowed estimation of the cfr, the incubation period distribution (and evaluation of differences in these by age and gender) and the reproduction number [3, 4, 6] . projections of the likely scale of the outbreak were also made, either from the line-list or from aggregated case counts [3, 8, 83, 84] . there were regular data updates [5] , with a total of over 19 000 confirmed and probable cases reported, which allowed analyses to be updated as more data became available. data on exposures allow cases to be linked to their potential sources of infection, and hence provide a better understanding of transmission characteristics. the relevant modes (e.g. airborne, foodborne) and pathways (e.g. animal-human, human-human) of transmission may be identified using information on exposure reported by cases. cases can report contact both with sick individuals (their potential source of infection) and healthy individuals they have contacted since becoming ill (who may need to be traced and monitored as potential new cases). these data will be more informative if the majority of infections are symptomatic (and hence easily identifiable), if individuals are mostly infectious after the onset of symptoms [43] and if the time window over which exposures and contacts are monitored is as long as the upper bound of the incubation period distribution. if exposure information is collected with enough demographic information to allow record linkage, these backward and forward contacts can be identified in the case line-list, defining transmission pairs. depending on the mutation rate of the pathogen, genetic data can also be used to identify or confirm these epidemiological links [56, 85] . the increasing availability of full genome pathogen sequences offers exciting prospects in that respect. some genetic sequence information was available during the ebola epidemic, but most sequences could not be linked to case records, limiting the use of sequence data in this context. on the other hand, individuals who were named as potential sources of infection could often be identified in the case linelist, although this process was hindered by non-unique names and limited demographic information collected on the potential sources [10] . these exposure data were used to characterize variation in transmission over the course of infection [10] , and to estimate the serial interval and the incubation period [3] . the upper bound of the incubation period distribution was estimated to be 21 days [3] , which supported monitoring contacts for up to three weeks. studies of transmission in well-defined, small settings such as households are useful to quantify asymptomatic transmission, infectivity over time and the serial interval as they capture explicitly the number and timing of secondary cases. additionally, these studies can estimate the secondary attack rate (the proportion of contacts of a case who become infected within one incubation period), which can be used to characterize heterogeneities in transmission of different groups [59] . estimates of the secondary attack rate have been obtained for the west african ebola epidemic by reconstructing household data based on information reported by cases, in particular, as part of contact-tracing activities [86, 87] . although they might not immediately appear as useful as individual-or exposure-level data, metadata are crucial for answering many public health questions. knowing the sizes of affected populations is important for quantifying the attack rate and informing dynamic transmission models. census data are likely to be the most reliable source, but may be infrequently collected. methods based on interpreting satellite imagery [88, 89] can inform population size and structure, although demographic stratifications are not always available. for the west african ebola epidemic, the most recently available age-and genderstratified population census data were from 1996 in guinea [90] , 2004 in sierra leone [91] and 2008 in liberia [92] . a particular population of interest is hcws who, due to their contact with patients, are often at high risk of infection and may also be high-risk transmitters. large numbers of hcws were infected during the west african ebola epidemic [60, 61] . however, the proportion of hcws affected at different stages of the outbreak and the relative risk of acquisition for the hcws compared with the general population could not be estimated since the total number of hcws was not systematically reported and changed during the course of the outbreak with the scale-up of interventions. note that, depending on the transmission route, the definition of hcws may need to include anyone working in a health-care setting who could be at risk (e.g. cleaners may be exposed to bodily fluids). characterizing population movement is crucial to assessing the risk of exportation of the infection from one place to another. air-travel data are the most reliable, consistently available and commonly used data source to inform models of long-distance spread [93] . such data were widely used during the west african ebola epidemic to quantify the risk of international spread of the disease, and to assess the potential impact of airport screening and travel restrictions on the outbreak [9,94 -96] . however, air travel does not cover other population movements that may play an important role in disease spread, e.g. travel by road or on foot in guinea, liberia and sierra leone and across the porous country borders during the west african ebola epidemic [97] . usually, little data are available to directly characterize these typically smaller-scale movements. gravity models, which assume that connectivity between two places depends on their population sizes and the distance between them, can be used to quantify spatial connectivity between different regions [98] [99] [100] [101] , and have proved useful to predict local epidemic spread, e.g. for chikungunya [98] . such models require data on population sizes and geographical distances. recently, mobile phone data have been explored as an alternative source of data on mobility patterns, which could be used to predict spatial epidemic spread [102] [103] [104] . however, a number of challenges (in particular related to privacy issues) meant that such data were unavailable to rstb.royalsocietypublishing.org phil. trans. r. soc. b 372: 20160371 understand the regional and local spread of the west african ebola epidemic [105] . in addition, inter-country movement is not captured from these data. at a national level, the utility of mobile phone data may depend on whether mobile phone users are representative of the population contributing to transmission, the level of mobile phone coverage in the affected population and whether population movement is likely to remain the same during an epidemic compared to the time period of the data. assessing seroprevalence in a population affected by an outbreak can provide valuable information on the underlying scale of population exposure and insight into how interventions might be targeted. for instance, if there is pre-existing population immunity prior to an outbreak that varies with age (as was the case in the 2009 h1n1 influenza pandemic), vaccination could be targeted at those with lower pre-existing immunity. dynamic transmission models incorporating such differences in susceptibility can be used to explore different targeting strategies [106] . ideally, serological surveys would be undertaken to quantify seroprevalence [107] ; however, they are expensive and not performed on a regular basis. in the absence of such data, information on historical outbreaks and vaccine use can sometimes be used to infer seroprevalence [108] . serological studies performed before and after an epidemic can also be useful to measure the attack rate and the scale of the outbreak, and hence provide information on the level of underreporting during the outbreak [109] . it was widely assumed that the population in west africa was entirely susceptible to ebola at the start of this outbreak, with no known previous outbreaks in the area. however, some studies have suggested that there might have been low levels of prior immunity [110] . during an outbreak, multiple interventions are often implemented by different groups and organizations. evaluating the role of interventions in interrupting transmission is important for revising and improving efforts, but it is challenging without detailed quantitative information of what has been implemented where and when [111, 112] . maintaining a systematic real-time record of the different interventions at a fine spatio-temporal scale would help, e.g. the number and location of health-care facilities and their personnel, number of beds, vaccine or treatment coverage and details of local community mobilization. developing centralized platforms to routinely record such data once a large-scale outbreak is underway is probably unfeasible. however, developing such tools in advance of outbreaks (such as those developed for the global polio eradication initiative [113] and those recently developed to collect health-care facility data [114] ) should be a priority since better information to evaluate intervention policies in real time will allow for more optimal resource allocation. during the west african ebola epidemic, many data on interventions were recorded at a local level by some of the numerous partners (e.g. non-governmental organizations (ngos) and other organizations) involved in the response. for example, some data were collected on the number and capacity of hospitals over time [115] and these were used in a study modelling community transmission to assess the impact of increasing hospital bed capacity [116] . however, the decentralization of the response meant that intervention data were not systematically reported or collated and these data were not shared widely with the research community. a failure to report interventions centrally and systematically can make it difficult to disentangle a lack of intervention effect from a lack of intervention implementation. this can particularly be a problem when numerous groups coordinate their own efforts, making it impossible to draw firm conclusions about interventions. in the absence of detailed data on intervention efforts in west africa, multiple studies have assessed the combined impact of all interventions in place, by comparing transmissibility in the early phase (with no intervention) to that in the later phases [87, 117] . however, this approach provides less compelling evidence of a causal effect and does not disentangle the impact of different interventions performed at the same time, and hence is less informative for future response planning. vaccine or treatment trials together with case-control and cohort studies can be useful in assessing the impact of an intervention. for example, during the west african ebola epidemic there was an urgent need to estimate the efficacy of newly developed vaccines. trials such as the guinea ebola ça suffit vaccine trial [118] provided key data on the effectiveness of the rvsv-zebov ebola vaccine [119, 120] . these trials occurred at the tail end of the epidemic and results will be useful in future outbreaks. statistical power from trials will be maximized by implementing such studies as early as possible in future outbreaks. this will be facilitated if research on diagnostics, drugs and vaccines is promoted between, and not only during, outbreaks, e.g. through new initiatives such as the coalition for epidemic preparedness innovation [121] . all of the data sources mentioned above are inevitably imperfect; what they are trying to measure is different from what they measure in practice. quantifying the mismatch between the two is vital to appropriately account for these imperfections. for instance, case line-lists are likely to contain information on only a proportion of all infected individuals: typically those with symptoms, or those who sought care. the level of reporting may also be influenced by the capacity of the local health systems, which can vary over time and space. during the west african ebola epidemic, less than a third of cases were estimated to be reported [122] and severe cases were probably over-represented compared to mild cases. at the end of 2014, health-care capacity was exceeded in many parts of guinea, liberia and sierra leone [14] , but new health-care facilities were subsequently built; hence the line-list of cases is likely to be more complete towards the end of the outbreak. underreporting might also have been higher in this compared to previous ebola outbreaks, during which the health-care systems were less overburdened. systematic evaluation of the surveillance system [123] over different spatial units and time periods could help inform the level of underreporting. in addition, joint analysis of genetic sequence and surveillance data can provide insight into the degree of underreporting [124] . quantifying completeness of, and potential biases in the line-list is important, e.g. to adequately quantify the cfr [28] . although differences in the cfr were observed across rstb.royalsocietypublishing.org phil. trans. r. soc. b 372: 20160371 different health-care facilities in the west african ebola epidemic, it was not possible to determine whether these were due to reporting differences or underlying differences between settings [125] . similarly, exposure-level data can be incomplete, depending on the available capacity (personnel and resources) to perform contact tracing and the willingness of people to share information on their contacts. complete data can be used to assess the route of transmission-animal to human or human to human-and the number of cases imported from other locations. if data are incomplete, these estimates may be incorrect. population-level data may suffer the same issues. for instance, the recording of intervention efforts (e.g. the number of personal protection equipment (ppe) kits distributed) can differ from the reality of the intervention (e.g. the number of people who used ppe in practice). quantifying this mismatch is crucial to evaluating the impact of various interventions, and requires dedicated studies, with both qualitative and quantitative components, on the acceptability of and the adherence to given interventions. such studies have been carried out in the past, e.g. to assess the potential impact of face masks on the risk of influenza transmission in households, or of condom use on the risk of hiv transmission [126] [127] [128] . it is also important to quantify delays encountered in the reporting of cases [129] . during the west african ebola epidemic, there were delays in all databases and disparities between different data sources. in particular, comparison between the line-list of cases and aggregated daily case counts reported by the affected countries highlighted reporting delays in the line-list. as a result, at any point in the outbreak, naive time series of case counts derived from the line-list suggested that the epidemic was declining, due to right-censoring. comparison between the line-list and reported aggregated case counts (which were more up-todate) and between successive versions of the line-list allowed the reporting delays to be quantified. analyses such as the projected incidence could then be adjusted accordingly [5] . in summary, the enormous quantity of detailed data collected during the west african ebola epidemic played an invaluable role in guiding response efforts. however, several analyses could not be performed. early on, severity, transmissibility and delay distributions were quantified and short-term projections were made, based on the case line-list and/or contact tracing data [3, 5, 7] . some heterogeneity in severity and transmissibility could be identified (e.g. by age and viraemia [7, 10, 116, 125, [130] [131] [132] ), but other types of heterogeneity could not be assessed. for example, it was not possible to compare the cfr between hospitalized and non-hospitalized cases, because of biases in the way cases were recorded in the line-list [28] . long-term projections were extremely challenging due to large uncertainty in population sizes, behaviour changes and changing intervention efforts. in addition, the relative risk of ebola acquisition for hcws was difficult to estimate due to the absence of reliable spatio-temporal data on the number of hcws. finally, systematic evaluation of interventions was not feasible due to multiple control measures being carried out at the same time, with little central recording of details of each intervention. data collection during the west african ebola epidemic was possible, in part, due to a pre-existing case investigation form and data management system (epi info viral haemorrhagic fever (vhf) application [133, 134] ). for outbreaks of new pathogens, such systems are not usually in place. the list of data needs we have outlined above, based on our experience during the ebola epidemic, could serve as a basis for data collection in future outbreaks. here we outline improvements that could be made to streamline data collection, minimize delays between data collection and data dissemination, and improve data quality during future outbreaks. all of these are necessary for timely analysis to inform the response in real time. we consider this particularly for line-list and exposure data. data need to be digitized before they can be analysed; streamlining this process reduces the potential for delays and errors. using electronic data capture on tablets or phones may reduce delays and errors compared with using paper forms that then require manual digitization, with its contingent issues [135] [136] [137] [138] . electronic questionnaires may not be available at the start of the epidemic, but could be quickly adopted using available tools (e.g. epicollect [139] or epibasket [140] ). interpretation problems (e.g. abbreviations) and spelling errors could also be minimized at the data collection level by using multiple choice questions rather than free text and at the data entry level by using drop-down menus. for example, for spatial information the choices could be the standard administrative levels used in the country-such as district or county. as response efforts evolve over time, such as the building of new treatment centres, the lists would be updated as required. similarly, using pop-out calendars to select dates would limit typing errors. additionally, internal consistency checks could flag problems such as the recorded date of death being before that of symptom onset, and the system could force a manual check before the record is saved. in the west african ebola epidemic, data were collected using paper forms (electronic supplementary material, figures s1 -s3) and manually entered into an electronic system at local operation centres. data entry problems were particularly noticeable in clinical dates and free text variables, such as district locations and hospital names, and required considerable cleaning (e.g. to correct spelling errors) [3, 5, 10] . with any data system there is potential for delays in data entry and dissemination due to limitations in personnel and hardware, and logistical constraints in data delivery (e.g. reliable electricity, internet access and transport). although the latter limitations are arguably outside the scope of outbreak control, delays from data collection to dissemination could be reduced by increasing training in data entry and providing more data entry facilities where possible. delays in data entry and release during the ebola epidemic meant that real-time analyses of the line-list data could not be performed on fully up-to-date data. in the world today, an epidemic emerging anywhere is a global threat due to high population connectivity [141, 142] and, as such, a response will have to operate across multiple languages. ideally, the same data entry system would be used in every affected country to allow easy collation into a single case line-list. however, a global response requires careful translation of the questions and the system to ensure they make sense and are identical in every language. additionally, different languages, dialects or alphabets could be challenging due to differing pronunciations, accents, alternate names or spellings, and these should be acknowledged in the form design. the language barrier was not a major problem during the ebola response: guinea had a form in french, while in sierra leone and liberia the form was in english, though there were some minor differences in formulations between the two (for example the english version of the form asked 'in the past one month prior to symptom onset: did the patient have contact with (. . .) any sick person before becoming ill' (see §4 in the form in electronic supplementary material, figure s1), while the french form did not specify 'before becoming ill'). analysis of laboratory results and sequence data can be much more powerful if they can be dated and linked to the epidemiological data recorded for each case. in the early stages of the ebola response, there were reports that laboratory results could not always be linked to case records as labels were incorrectly written or damaged in transit [81] . later in the epidemic, case report forms came with pre-printed unique id barcode stickers to label all records and samples for each case (electronic supplementary material, form s2). this would be useful if implemented early in future outbreaks, particularly if laboratory tests are not performed at the point of care. rapid diagnostic tests were developed during the west african ebola epidemic but not used widely [143, 144] ; similarly, mobile sequence tests were introduced later in the epidemic [145] : both of these would reduce delays and maximize the potential to link patient data. we have described a set of data needed to perform analyses to inform the public health response during an outbreak. we have proposed strategies to ensure fast and high-quality data collection, to enable robust and timely analyses. however, such analyses can only be performed if the data are accessible to data analysts and modellers. the west african ebola epidemic has prompted an ongoing public debate about the ethical, practical and scientific implications of wide data access [14, [146] [147] [148] . ethical considerations require removal of data that might compromise anonymity, but such detailed information might be required to answer important public health questions. mechanisms also need to be found to appropriately acknowledge those who collected and digitalized the data. from a scientific perspective, having several groups analysing the data is desirable, as independent analyses leading to similar results will reinforce their utility for policymaking [3, 8] . such parallelized efforts have been formalized through consortiums of highly experienced groups, e.g. for modelling of hiv [149] , influenza [150, 151] and malaria [152] . however, if results differ, understanding what assumptions drive these differences may confuse and delay the process of decision-making. a consequence of data sharing, therefore, needs to be an increased emphasis on evidence synthesis, such as systematic reviews of the different analyses. to enable this process, groups should explicitly state all assumptions underlying their analyses and which data they are based on. like the original analyses, reviews need to be timely to be useful. these issues have partly been addressed through new data availability policies [153] . furthermore, this process would ideally be performed by a group independent of those performing the primary analyses. identifying an effective system, appropriate personnel and appropriate recognition for this important role needs to be planned in advance of future outbreaks. data from the west african ebola epidemic required significant cleaning before it could be analysed, and this was necessary for every updated dataset, i.e. every few days during the peak. were outbreak data to be shared more widely, collaborative or centralized cleaning would be optimal, to avoid repetition and ensure consistency across different groups. if a centralized effort was not possible, regular sharing of the cleaning code and cleaned datasets between groups would facilitate comparison of results. even better, code could be shared on a collaborative platform such as github (https://github.com/), leading to a common clean dataset being actively maintained by the scientific community. however, for this process to be effective, a set of best practices would need to be established in advance, such as designing a transparent workflow, establishing a fair distribution of tasks and clarifying how credit will be given for this (often lengthy) task. this is important to avoid duplication of effort and allow effective collaboration. as the process of data sharing is debated further, it is critical that the practicalities of data cleaning are discussed in parallel. based on our experience, a centralized cleaning platform would be the most effective method. finally, analyses will be most useful if they are shared widely across policymakers, local health teams and other research groups. the format for dissemination could be anything from a report to a scientific publication. reports can be made available faster and regularly updated but do not undergo the peer review process of publications. peer review can delay publication, though there are now new platforms for fast-tracking this process [154, 155] . at the time of writing, as interest in zika virus is gaining momentum, there seems to be an encouraging trend to the use of pre-print servers [156,157]. by the very nature of emerging infectious diseases, we do not know which pathogen will emerge next, when or where. there have been many suggestions about how to be rstb.royalsocietypublishing.org phil. trans. r. soc. b 372: 20160371 better prepared [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] 24, 26] ; here, we argue that preparedness should include development of a broad-use data collection system that can be easily and quickly adapted to any disease (in agreement with [11] ) as well as the regular collection of population health data in centralized systems. different infectious diseases may require different types of data [140] : a single approach is not applicable to all diseases. in particular, different interventions may need to be recorded for different diseases. data collection and management may need to evolve as the outbreak progresses and/or as more is learnt about the pathogen: the ebola data collection forms changed late in 2014 to streamline collection and entry when the response effort was almost overwhelmed with cases (electronic supplementary material, figures s1 and s2). similarly, both for ebola and the recent zika outbreak, reports of sexual transmission have led to a broadening of the contact tracing and exposure information collected. the data collected during the ebola epidemic allowed many analyses to be performed, which informed the response. however, as in many outbreak situations, it has not been possible to systematically quantify the relative contribution of different interventions (such as safe burials, hospitalization, contact tracing and community mobilization) in reducing transmission. this is because data on where and when these interventions took place were not centrally recorded and released in a timely fashion. efforts have been made to collate some information from sierra leone, liberia and guinea [115] ; however, this commenced late in the epidemic. as this effort relies on different organizations to contribute data, the submissions are in different formats and are unlikely to provide comprehensive information. in the midst of a global public health crisis resources are often deployed favouring implementation rather than documentation of interventions. however, we would argue that securing some resources to monitor interventions-especially during the early stages-is critical to optimally prioritizing future control efforts. some of the data we have suggested to be collected during outbreaks may not be obviously useful at the field or case management level, e.g. detailed demographic characteristics of cases and contacts. collecting such data costs money and time as well as trained personnel. these three 'resources' are limited and, during an epidemic, should be prioritized where their need is greatest. however, from a population perspective, collecting these data may help quantify epidemic impact, assist in the design and evaluation of interventions, and help prevent new infections. further studies might examine how to appropriately balance these two considerations. we have built on the ebola experience to draw up a list of the data needed to assist the response throughout an epidemic, which should help to collect relevant data in a standardized effort in future epidemics. to make the most of these data, epidemiologists and modellers should work now to develop tools to automatically clean, analyse and report on the data in a more timely and robust manner [158] . based on critical review of past outbreak analyses, future studies could flag common methodological mistakes and propose good practice [159] . this includes clearly stating all assumptions underlying a model or analysis and ensuring that parametrization is either directly informed by relevant data or has appropriate sensitivity analyses, with corresponding uncertainty clearly reported [32, 160] . improving our ability to respond effectively to the next outbreak will require collaboration between all parties involved in outbreak response: those in the field, epidemiologists, modellers and policymakers as well as the populations affected. here we have given the data analyst perspective on what data are required to answer important policy questions. it is equally important that other perspectives should be heard to be better prepared for and improve interactions during crises, thereby minimizing the impact of future outbreaks. a review of epidemiological parameters from ebola outbreaks to inform early public health decision-making after ebola in west africa: unpredictable risks, preventable epidemics ebola virus disease in west africa-the first 9 months of the epidemic and forward projections ebola virus disease among male and female persons in west africa west african ebola epidemic after one year-slowing but not yet under control ebola virus disease among children in west africa chains of transmission and control of ebola virus disease in conakry, guinea, in 2014: an observational study estimating the future number of cases in the ebola epidemic-liberia and sierra leone effectiveness of screening for ebola at airports exposure patterns driving ebola transmission in west africa, a retrospective observational study are we learning the lessons of the ebola outbreak? the next epidemic-lessons from ebola global health security: the wider lessons from the west african ebola virus disease epidemic will ebola change the game? ten essential reforms before the next pandemic. the report of the harvard-lshtm independent panel on the global response to ebola clinical research during the ebola virus disease outbreak in guinea: lessons learned and ways forward the emergence of ebola as a global health security threat: from 'lessons learned' to coordinated multilateral containment efforts ebola in west africa: lessons we may have learned lessons learned from hospital ebola preparation ebola in west africa: learning the lessons strengthening the detection of and early response to public health emergencies: lessons from the west african ebola epidemic ebola clinical trials: five lessons learned and a way forward lessons from ebola: improving infectious disease surveillance to inform outbreak management epidemiological data management during an outbreak of ebola virus disease: key issues and observations from improving the evidence base for decision making during a pandemic: the example of 2009 influenza a/h1n1 epidemic and intervention modelling-a scientific rationale for policy decisions? lessons from the 2009 influenza pandemic emerging infections: what have we learned from sars? assessing the severity of the novel influenza a/h1n1 pandemic potential biases in estimating absolute and relative case-fatality risks during outbreaks optimizing the precision of case fatality ratio estimates under the surveillance pyramid approach influenza-related deathsavailable methods for estimating numbers and detecting patterns for seasonal and pandemic influenza in europe an introduction to infectious disease modelling six challenges in modelling for public health policy uses and abuses of mathematics in biology national center for infectious diseases sars community outreach team decisions under uncertainty: a computational framework for quantification of policies addressing infectious disease epidemics development and consideration of global policies for managing the future risks of poliovirus outbreaks: insights and lessons learned through modeling quantifying uncertainty in epidemiological models the orion statement: guidelines for transparent reporting of outbreak reports and intervention studies of nosocomial infection generality of the final size formula for an epidemic of a newly invading infectious disease modeling to inform infectious disease control nine challenges in incorporating the dynamics of behaviour in infectious diseases models nine challenges in modelling the emergence of novel pathogens factors that make an infectious disease outbreak controllable infectious diseases of humans: dynamics and control a brief history of r 0 and a recipe for its calculation epiestim: a package to estimate time varying reproduction numbers from epidemic curves (r package a new framework and software to estimate timevarying reproduction numbers during epidemics how generation intervals shape the relationship between growth rates and reproductive numbers different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures superspreading and the effect of individual variation on disease emergence mathematical models of infectious disease transmission a bayesian mcmc approach to study transmission of influenza: application to household longitudinal data risk factors of influenza transmission in households heterogeneities in the transmission of infectious agents: implications for the design of control programs bayesian inference of infectious disease transmission from wholegenome sequence data bayesian reconstruction of disease outbreaks by combining epidemiologic and genomic data unravelling transmission trees of infectious diseases by combining genetic and epidemiological data relating phylogenetic trees to transmission trees of infectious disease outbreaks household transmission of 2009 pandemic influenza a (h1n1) virus in the united states ebola virus disease in health care workers-sierra leone ebola virus disease cases among health care workers not working in ebola treatment units-liberia temporal and spatial analysis of the 2014-2015 ebola virus outbreak in west africa evolution and spread of ebola virus in liberia modeling the spatial spread of infectious diseases: the global epidemic and mobility computational model the foot-and-mouth epidemic in great britain: pattern of spread and impact of interventions large-scale spatial-transmission models of infectious disease the interval between successive cases of an infectious disease standards of evidence: criteria for efficacy, effectiveness and dissemination criteria for distinguishing effectiveness from efficacy trials in systematic reviews agency for healthcare research and quality vaccine epidemiology: efficacy, effectiveness, and the translational research roadmap modeling infectious diseases in humans and animals social contacts and mixing patterns relevant to the spread of infectious diseases modeling infectious disease parameters based on serological and social contact data: a modern statistical perspective (statistics for biology and health closure of schools during an influenza pandemic the international response to the outbreak of sars in 2003 health benefits, costs, and cost-effectiveness of earlier eligibility for adult antiretroviral therapy and expanded treatment coverage: a combined analysis of 12 mathematical models costs and cost-effectiveness of malaria control interventions-a systematic review performance of case definitions for influenza surveillance case definition recommendations for ebola or marburg virus diseases mobile diagnostics in outbreak response, not only for ebola: a blueprint for a modular and robust field laboratory reebov antigen rapid test kit for point-of-care and laboratory-based testing for ebola virus disease: a field validation study temporal variations in the effective reproduction number of the 2014 west africa ebola outbreak inference and forecast of the current west african ebola outbreak in guinea, sierra leone and liberia epidemiological and viral genomic sequence analysis of the 2014 ebola outbreak reveals clustered transmission transmissibility and pathogenicity of ebola virus: a systematic review and meta-analysis of household secondary attack rate and asymptomatic infection transmission dynamics of ebola virus disease and intervention effectiveness in sierra leone landscan tm about worldpop population statistics of guinea population and housing census national population and housing census final results the role of the airline transportation network in the prediction and predictability of global epidemics assessment of the potential for international dissemination of ebola virus via commercial air travel during the 2014 west african outbreak assessing the international spreading risk associated with the 2014 west african ebola outbreak assessing the impact of travel restrictions on international spread of the 2014 west african ebola epidemic mali case, ebola imported from guinea local and regional rstb.royalsocietypublishing.org phil gravity models for airline passenger volume estimation five challenges for spatial epidemic models dynamic population mapping using mobile phone data mapping population and pathogen movements impact of human mobility on the emergence of dengue epidemics in pakistan commentary: containing the ebola outbreak -the potential and challenge of mobile network data assessing optimal target populations for influenza vaccination programmes: an evidence synthesis and modelling study use of serological surveys to generate key insights into the changing global landscape of infectious disease key issues in the persistence of poliomyelitis in nigeria: a casecontrol study increased transmissibility explains the third wave of infection by the 2009 h1n1 pandemic virus in england undiagnosed acute viral febrile illnesses how to make predictions about future infectious disease risks new approaches to quantifying the spread of infection polio information system about healthsites the humanitarian data exchange measuring the impact of ebola control measures in sierra leone retrospective analysis of the 2014-2015 ebola epidemic in liberia the ring vaccination trial: a novel cluster randomised controlled trial design to evaluate vaccine efficacy and effectiveness during outbreaks, with special reference to ebola efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola ça suffit!) an r&d blueprint or action to prevent epidemics plan of action use of capturerecapture to estimate underreporting of ebola virus disease updated guidelines for evaluating public health surveillance systems: recommendations from the guidelines working group inference for nonlinear epidemiological models using genealogies and time series heterogeneities in the case fatality ratio in the west african ebola outbreak 2013-2016 face mask use and control of respiratory virus transmission in households barriers to mask wearing for influenza-like illnesses among urban hispanic households efficacy of structural-level condom distribution interventions: a meta-analysis of us and international studies quantifying reporting timeliness to improve outbreak control clinical features of and risk factors for fatal ebola virus disease assessment of the severity of ebola virus disease in sierra leone use of viremia to evaluate the baseline case fatality ratio of ebola virus disease and inform treatment studies: a retrospective cohort study the epi info viral hemorrhagic fever application the epi info viral hemorrhagic fever (vhf) application: a resource for outbreak data management and contact tracing in the 2014-2016 west africa ebola epidemic e-health technologies show promise in developing countries a comparison of the completeness and timeliness of automated electronic laboratory reporting and spontaneous reporting of notifiable conditions replacing paper data collection forms with electronic data entry in the field: findings from a study of communityacquired bloodstream infections in pemba, rstb.royalsocietypublishing.org phil comparison of electronic data capture (edc) with the standard data capture method for clinical trial data epibasket: how e-commerce tools can improve epidemiological preparedness. emerg who report on global surveillance of epidemic-prone infectious diseases address to the sixty-ninth world health assembly notes from the field: baseline assessment of the use of ebola rapid diagnostic tests-forecariah, guinea essential medicines and health products: diagnostics real-time, portable genome sequencing for ebola surveillance sharing data for global infectious disease surveillance and outbreak detection data sharing: make outbreak research open access providing incentives to share data early in health emergencies: the role of journal editors mathematical modelling of the pandemic h1n1 studies needed to address public health challenges of the 2009 h1n1 influenza pandemic: insights from modeling public health impact and cost-effectiveness of the rts,s/as01 malaria vaccine: a systematic comparison of predictions from four mathematical models developing global norms for sharing data and results during public health emergencies outbreaktools: a new platform for disease outbreak analysis using the r software avoidable errors in the modelling of outbreaks of emerging pathogens, with special reference to ebola seven challenges for model-driven data collection in experimental and observational studies acknowledgements. the authors give credit to and thank the many individuals who were involved in data collection, entry and management during the west african ebola epidemic. key: cord-349786-12lc3342 authors: acquah, samuel title: implications of covid-19 pandemic on evolution of diabetes in malaria-endemic african region date: 2020-10-08 journal: j diabetes res doi: 10.1155/2020/8205261 sha: doc_id: 349786 cord_uid: 12lc3342 the coronavirus disease 2019 (covid-19) pandemic continues to cause havoc to many countries of the globe, with no end in sight, due to nonavailability of a given vaccine or treatment regimen. the pandemic has so far had a relatively limited impact on the african continent, which contributes more than 93% of global malaria burden. however, the limited burden of covid-19 pandemic on the african region could have long-term implications on the health and wellbeing of affected inhabitants due to its malaria-endemic status. malaria causes recurrent insulin resistance with episodes of infection at relatively low parasitaemia. angiotensin-converting enzyme 2 (ace2) which is widely distributed in the human body is implicated in the pathogenesis of malaria, type 2 diabetes mellitus (t2dm), and covid-19. use of ace2 by the covid-19 virus induces inflammation and oxidative stress, which can lead to insulin resistance. although covid-19 patients in malaria-endemic african region may not exhibit severe signs and symptoms of the disease, their risk of exhibiting heightened insulin resistance and possible future development of t2dm is high due to their prior exposure to malaria. african governments must double efforts at containing the continued spread of the virus without neglecting existing malarial control measures if the region is to avert the plausible long-term impact of the pandemic in terms of future development of t2dm. the novel coronavirus disease pandemic that started in wuhan, china, in december 2019, has now affected 216 countries and territories as of 7 th august, 2020. covid-19 had infected 18,902,735 people worldwide with 709,511 deaths ( figure 1 ) with no sign of a definite treatment as of 10 : 00 cest on 7 th august, 2020 [1] . the global epicenter of covid-19 appears to move from one country to the next with brazil currently holding the baton as the global epicenter. the pandemic continues to exhibit inter-and intracontinental variations per million indices in terms of number of cases, deaths, and tests with no country emerging as the leader in all the three indices. this trend clearly shows the varied resilience of country-specific response to the pandemic. in terms of absolute numbers, the who american region appears to be the most affected followed by europe with africa being the least affected region of the globe after the western pacific region. however, when the available data are presented per million population, the european region of the globe becomes the most affected. although the pandemic is still raging, the interim differences in deaths and tests among countries and continents can be ascribed to variations in population dynamics, effectiveness of health systems and policies, and economic factors. since the pandemic appears to influence every aspect of human endeavor, it is pertinent that its impact is assessed from multidimensional perspectives. the current review aims at discussing the possible impact of covid-19 pandemic on the evolution of diabetes in malaria endemic regions of the globe. whereas the covid-19 pandemic and diabetes are truly global in distribution, malaria appears to be an african problem ( table 1 ). the african continent remains the hub of global infectious diseases, as there is no historical account of any infectious agent of humanity [2] [3] [4] that fails to mention the african continent. it is, perhaps based on some of this historical premise and its unique settlement, that the covid-19 pandemic is projected by the world health organisation (who) to infect 29 million to 44 million africans with associated 83,000 to 190,000 deaths if containment measures put in place by various african governments fail [5] . additionally, it is predicted that the duration of covid-19 infection in the african region could be longer than expected if african governments do not take appropriate proactive measures [5] . indeed, the covid-19 pandemic continues to cause havoc to many countries globally with no end in sight. as such, the exact impact of covid-19 on any given country is not yet fully known. fortunately, various african governments have put in place pragmatic measures to contain the spread of the virus to avert the gloomy predictions. if the measures in place work effectively, africa will enviably remain the second least affected region of the globe in terms of covid-19 morbidity and mortality. however, the potential long-term impact of the covid-19 pandemic on the health burden of africans in general and the sub-saharan african region in particular cannot be overemphasized. this has become critical due to the generally mild nature of covid-19 cases on the african continent. with the continent already responsible for over 93% of global malaria burden and associated deaths in 2018 [6] , the presence of the covid-19 pandemic does not only increase the infectious disease burden of the african region but could serve as another risk factor to the development of type 2 diabetes mellitus (t2dm) in the region. indeed, current estimates by the international diabetes federation (idf) predict the african region to experience 173% increase in incidence of diabetes by 2045 compared with a global average of 51% [7] . also, the proportion of undiagnosed diabetes cases in the african region was 59.7%, being the highest on the globe per the 2019 idf report [7] . thus, the proportion of undiagnosed cases of diabetes will increase further in the face of the covid-19 pandemic globally and most importantly in resource-constraint african region of the globe. considering the relatively high rate of infectivity and mortal-ity of the covid-19 pandemic virus, it is only logical that the highest of priority is given in terms of resource allocation to halt the continuous spread of the virus and move towards its elimination from the global health calendar. as such, as resource-constraint african countries direct available resources to combat the pandemic, not much will be left to cater for other competing health needs such as the prevention of malaria and diabetes complications through testing for early diagnosis. above all, some of the identified pathogenic mechanisms of the virus responsible for the covid-19 pandemic appear to be similar to some known risk factors to the development of diabetes such as obesity and malaria [7] [8] [9] [10] [11] . the covid-19 outbreak, which started in wuhan city in hubei province of china, was officially made known to the who on december 31, 2019 [12, 13] . since then, the viral pathogen has spread to 216 countries and territories of the globe. just like other known coronaviruses [14, 15] , the acute respiratory syndrome coronavirus-2 (sars-cov-2), responsible for the covid-19 pandemic, requires a specific receptor, angiotensin-converting enzyme 2 (ace2), for entry into host cells [16] [17] [18] [19] . indeed, the use of ace2 as a receptor by coronaviruses has long been reported [20, 21] . ace2 is a zinc-containing monocarboxypeptidase that catalyzes the conversion of angiotensin i and angiotensin ii to angiotensin 1-9 and angiotensin 1-7, respectively [22] . through angiotensin 1-7, ace2 ameliorates lung fibrosis, vascular damage, pulmonary hypertension, and pulmonary injury [23] [24] [25] . a role for ace2 has also been recognized in the development of t2dm [26] [27] [28] [29] [30] and malaria [31] [32] [33] . angiotensin ii is a known blood pressure-inducing peptide that narrows the lumen of blood vessels through persistent contraction, renal retention of water and salt with associated increased resistance, and heightened blood pressure [34] . angiotensin ii is an octapeptide produced by the hydrolytic action of angiotensin-converting enzyme (ace) on angiotensin i, a decapeptide, which is produced from angiotensinogen by renin. the overall effect of angiotensins i and ii is to increase blood pressure. as such, reduced levels of angiotensins i and ii or inhibition of their production or activities result in restoration of normotension but may increase susceptibility to malaria [31] [32] [33] 35] . thus, the angiotensin system is critical in health and disease. the main component of the renin-angiotensin system with the capability to link covid-19, malaria, and diabetes mellitus is the ace2. the action of ace2 in reducing angiotensin ii levels does not only improve cellular haemodynamics but attenuates antimalarial properties to potentially facilitate infection and inflammation which may increase the risk for development of diabetes [9, [31] [32] [33] 35] . in a study involving 518 severe acute respiratory syndrome (sars) patients and 19 patients with non-sars pneumonia, yang et al. [36] reported that binding of sars coronavirus to its receptor, ace2, in the pancreas, caused transient type 1 diabetes mellitus through damaged pancreatic islet. this observation made in this previous sars [37] and ability to take advantage of several host proteases to facilitate infection [17] [18] [19] . ace2 has been found in various cells and tissues including endothelium, heart, intestinal epithelium, lungs, pancreas, renal tubular epithelium, upper respiratory tract, and the central nervous system [38, 39] . indeed, symptomatic patients of the covid-19 virus may present dry cough, diarrhoea, dyspnea, fatigue, fever, headache, myalgia, nasal congestion, nausea, runny nose, sore throat, tastelessness, and vomiting [40] [41] [42] , reflecting the wide distribution of ace2 in the human system. hypertension is a known risk factor for t2dm, and both diabetes and hypertension are leading underlying comorbidities for unfavorable covid-19 prognosis [40, 41] . as earlier indicated, the covid-19 virus enters cells of susceptible hosts through ace2 [16] [17] [18] [19] . binding of the virus to ace2 reduces ace2 levels and its degradative effects on angiotensin ii [43] , resulting in accumulating levels of angiotensin ii and resultant negative effects on pulmonary and vascular homeostasis [23] [24] [25] . several experimental studies in animals have shown that increased levels of angiotensin ii result in abnormal hepatic lipid and carbohydrate metabolism [44, 45] . other studies have demonstrated the critical role of the renin-angiotensin system in hepatic and adipose tissue inflammation, insulin resistance, and glucose intolerance [46, 47] . indeed, santos et al. [48] demonstrated that oral administration of angiotensin(1-7) , a degradative product of angiotensin ii, prevented high-fat diet-induced obesity, inflammation, insulin resistance, and glucose intolerance through downregulation of resistin, nuclear factor kappa b (nf-κb), toll-like receptor 4 (tl4), interleukin-6 (il-6), tumor necrosis factor-α (tnfα), and mitogen-activated protein kinase (mapk) levels. inflammation and oxidative stress interact in a synergistic manner to promote the development of various health conditions including diabetes [49] [50] [51] . to this end, the covid-19 virus, which reduces ace2 levels or activities, can potentiate infected individuals for future development of t2dm through low-grade inflammation and insulin resistance. this is very critical in view of several observations that a number of infected individuals do not exhibit any signs and symptoms [52, 53] . indeed, most of the covid-19 cases in the african region fall within the mild and asymptomatic category explaining the low hospitalization rate for positive cases in the region. the presence of asymptomatic cases suggests that the virus can induce low-grade inflammatory process which can lead to the development of insulin resistance as the body takes appropriate steps to clear the virus. it has been reported that viral particles are detectable long after recovery from the infection [54] , suggesting that the body's inflammatory process can still be engaged as a homeostatic mechanism to ensure normal cellular function. thus, in both symptomatic and asymptomatic cases, inflammation is triggered and sustained, during and after infection to reestablish homeostasis. specific inflammatory molecules such as tnfα, il-6, and other inflammatory signaling pathways identified with covid-19 have been associated with the pathogenesis of type 2 diabetes mellitus [55] and malaria [56] through insulin resistance [57] . as a result, the covid-19 virus, through its interaction with ace2 may predispose affected individuals to future development of diabetes associated with low-grade inflammation and insulin resistance. data from human [9, 58] and animal [8] studies on malaria-induced insulin resistance suggest that insulin resistance can develop within a relatively short period of infection. in addition, once established, treatment of the infection does not reduce the level of the established insulin resistance to the preinfection level. as such, upon subsequent infection even at relatively low parasitaemia, insulin resistance is reestablished, suggesting that the previous infection primed affected cells to develop insulin resistance upon least reexposure. it is therefore plausible that insulin resistance caused by the covid-19 virus will also create a kind of immunological memory on affected cells, priming them for future development of insulin resistance upon exposure to other infectious agents or other favorable factors. unlike a typical immunologic memory to a given pathogen whereby a heighted immune response requires reexposure to the specific pathogen, insulin resistance priming does not require reexposure to the same pathogen that primed the cells. as such, virus-induced insulin resistance can prime affected cells for the development of insulin resistance upon later exposure sources: covid-19 estimates were obtained from who situational report 200 based on data from national authorities as of 10 : 00 cest on 7 th august, 2020. diabetes estimates were obtained from the 9 th edition of idf diabetes atlas, 2019. malaria estimates were obtained from the world malaria report, 2019. 3 journal of diabetes research to any other pathogen. this is due to the fact that the cellular factors involved in the insulin resistance development process(es) do not differ necessarily with the kind of pathogen. for instance, il-6 and tnfα released by covid-19 are also triggered by malaria and obesity as far as inflammationinduced insulin resistance is concerned. thus, the pathogenic agent could be different but the cellular mechanisms triggered, and the specific players involved in inducing the requisite pathogenic inflammatory processes may be the same. it is based on this fundamental concept that it is speculated that covid-19-infected individuals who recover from the disease may be prone to future development of insulin resistance and possibly diabetes mellitus upon exposure to favorable factors such as obesity and malaria. this is very crucial for sub-saharan africa that is already burdened with various infectious agents and struggling to deal with its increasing trend of diabesity. considering that the region has the highest proportion of undiagnosed diabetes [7] , the relatively low burden of covid-19 infections on the african continent and the mild and asymptomatic nature of the infections could be seen as a postponed burden of t2dm. as such, the continent should rather intensify efforts at preventing further spread of the covid-19 virus in order to avert a potential catastrophe in the form of future development of t2dm (figure 2) . in a recent editorial, napoli and nioi [59] postulated that malaria could be playing a protective role in covid-19 severity on the african continent apart from her relatively young population. this hypothesis was based on the relatively limited number of cases of covid-19 on the continent and other malaria-endemic regions of the globe together with antiviral properties of some antimalarial drugs. however, the postulated immune protection provided by prior malaria infection does not protect against the negative effects of inflammation. additionally, various antimalarial drugs employ oxidative stress as a mechanism for parasite clearance [60] . more so, mild inflammation coupled with drug-induced oxidative stress can cause unintended organ damage [61] . above all, there is no evidence of active malaria being protective against covid-19. in fact, active malaria, due to compromised immunity of the affected, is rather expected to facilitate covid-19 infection. on this note, active malaria in covid-19 patients should rather be treated aggressively to avert worsened outcome of covid-19. thus, the mild cases of covid-19 provide the right environment for low-grade inflammation, coupled with oxidative stress necessary for the development of insulin resistance ( figure 2 ) and increase the risk for development of t2dm. although africa remains the second least affected covid-19 region of the globe, the pandemic still poses a serious health challenge in terms of potential future development of t2dm. this is premised upon mild inflammation associated with mild cases of covid-19 infection, which can cause insulin resistance and increase the risk for development of t2dm. african governments should continue to intensify efforts at preventing the spread of covid-19 without losing focus on the need to continue to take steps to control malaria in the region. such steps have potential to protect the region from undue future development of t2dm. no conflict of interest exists between the authors. effects of malaria coronavirus disease (covid-19) situation report -200 infectious diseases-past, present, and future a história da disseminação dos microrganismos lessons from the history of quarantine, from plague to influenza a world health organisation, world malaria report, world health organization international diabetes federation, idf diabetes atlas, international diabetes federation association of insulin resistance with parasitaemia in rat malaria evidence of insulin resistance in adult uncomplicated malaria: result of a two-year prospective study virus infections in type 1 diabetes raised plasma insulin level and homeostasis model assessment (homa) score in cerebral malaria: evidence for insulin resistance and marker of virulence coronavirus disease (covid-19) situation report -1 a pneumonia outbreak associated with a new coronavirus of probable bat origin epidemiology, genetic recombination, and pathogenesis of coronaviruses coronaviruses post-sars: update on replication and pathogenesis a new coronavirus associated with human respiratory disease in china structural basis of receptor recognition by sars-cov-2 cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis for the recognition of sars-cov-2 by full-length human ace2 coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a novel angiotensinconverting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 angiotensin converting enzyme 2 abrogates bleomycin-induced lung injury angiotensin-converting enzyme 2 protects from severe acute lung failure a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury renin-angiotensin system blockers protect pancreatic islets against diet-induced obesity and insulin resistance in mice loss of ace 2 exaggerates high-calorie diet-induced insulin resistance by reduction of glut4 in mice captopril intake decreases body weight gain via angiotensin angiotensin-(1-7) mas-receptor deficiency decreases peroxisome proliferator-activated receptor gamma expression in adipocytes mas deficiency in fvb/n mice produces marked changes in lipid and glycemic metabolism angiotensin ii moderately decreases plasmodium infection and experimental cerebral malaria in mice impairment of the plasmodium falciparum erythrocytic cycle induced by angiotensin peptides gene polymorphisms in angiotensin i converting enzyme (ace i/d) and angiotensin ii converting enzyme (ace2 c→t) protect against cerebral malaria in indian adults renin-angiotensin system: biochemistry and mechanisms of action anti-plasmodium activity of angiotensin ii and related synthetic peptides binding of sars coronavirus to its receptor damages islets and causes acute diabetes structure, function, and antigenicity of the sarscov-2 spike glycoprotein neurological manifestations of hospitalized patients with covid-19 in wuhan, china: a retrospective case series study sars-cov-2: underestimated damage to nervous system clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of 138 hospitalized patients with2019 novel coronavirus-infected pneumonia in wuhan, china coronavirus disease 2019 (covid-19): current status and future perspectives angiotensin-converting enzyme 2: sars-cov-2 receptor and regulator of the renin-angiotensin system decreased hepatic gluconeogenesis in transgenic rats with increased circulating angiotensin angiotensin ii-induced non-alcoholic fatty liver disease is mediated by oxidative stress in transgenic tg(mren2)27(ren2) rats overproduction of angiotensinogen from adipose tissue induces adipose inflammation, glucose intolerance, and insulin resistance angiotensin ii participates in hepatic inflammation and fibrosis through mcp-1 expression -7) prevented obesity and hepatic inflammation by inhibition of resistin/tlr4/mapk/nf-κb in rats fed with high-fat diet linking malaria to type 2 diabetes mellitus: a review increased oxidative stress and inflammation independent of body adiposity in diabetic and nondiabetic controls infalciparummalaria low-grade chronic inflammation in the relationship between insulin sensitivity and cardiovascular disease (risc) population: associations with insulin resistance and cardiometabolic risk profile asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship post-discharge surveillance and positive virus detection in two medical staff recovered from coronavirus disease 2019 (covid-19), china diabetes mellitus and inflammation inflammatory cytokine and humoral responses to plasmodium falciparum glycosylphosphatidylinositols correlates with malaria immunity and pathogenesis molecular and metabolic mechanisms of insulin resistance and β-cell failure in type 2 diabetes asymptomatic falciparum malaria and its effects on type 2 diabetes mellitus patients in global spread of coronavirus disease 2019 and malaria: an epidemiological paradox in the early stage of a pandemic concurrent inflammation augments antimalarial drugs-induced liver injury in rats key: cord-266027-1xrq8cg9 authors: barrington, debbie s.; james, sherman a.; williams, david r. title: socioeconomic correlates of obesity in african-american and caribbean-black men and women date: 2020-07-04 journal: j racial ethn health disparities doi: 10.1007/s40615-020-00798-4 sha: doc_id: 266027 cord_uid: 1xrq8cg9 the high prevalence of obesity among black americans warrants additional investigation into its relationship with socioeconomic position (sep), sex, and ethnicity. this cross-sectional study utilizes 2001–2003 data from the national survey of american life, a nationally representative sample of 3570 african-americans and 1621 caribbean-blacks aged 18 years and older. multivariate logistic regression models stratified by ethnicity and sex describe the independent associations between obesity and multilevel socioeconomic factors after adjustment for age, other sep measures at the individual, family and neighborhood levels, and health behaviors such as physical activity, alcohol intake, and smoking. a positive relationship was observed between obesity and family income among african-american and caribbean-black men. receipt of public assistance was a strongly associated factor for obesity in caribbean-black men and women. among african-american women, inverse relationships were observed between obesity and education, occupation, and family income; residence within a neighborhood with a supermarket also decreased their odds of obesity. residence in a neighborhood with a park decreased the odds of obesity only among african-american men, whereas residence in a neighborhood with a supermarket decreased the odds of obesity among caribbean-black men. the social patterning of obesity by individual, household, and neighborhood socioeconomic resources differs for african-american and caribbean-black men and women within these cross-sectional analyses; an appreciation of these differences may be a prerequisite for developing effective weight control interventions and policies for these two populations. the high prevalence of obesity among non-hispanic black americans (38.3%), aged 18 years and older in 2018, as compared with 31.1% for the general us population, remains an important public health problem [1] . obesity has a broad range of health consequences, and its higher prevalence among non-hispanic blacks puts them at increased risk for type 2 diabetes (blacks = 13.0%, whites = 8.0%), coronary heart disease (black women = 5.2%, white women = 3.9%), asthma (blacks = 9.2%, whites = 8.0%), and stroke (blacks = 4.0%, whites = 2.7%) [1] . the important role of socioeconomic position (sep) in the obesity epidemic has long been a focus of research [2] [3] [4] [5] . sep is defined as one's location in the societal structure that determines differential access to power, privilege, and desirable resources [6] . it is a multidimensional concept that can be measured at (1) the individual level, e.g., education, earnings, and occupation; (2) the household level, characterized by familial resources, e.g., poverty, family income, and wealth; and (3) the neighborhood level, described by aspects of living conditions not captured by individual or household level variables, e.g., community structural characteristics, neighborhood poverty, and crime [7, 8] . prior research has documented correlations between lower rates of obesity and residence in communities with beneficial structural characteristics of neighborhood sep, including the presence of neighborhood parks and supermarkets [9] [10] [11] . research findings on the relationship between obesity and individual and household measures of sep, however, have been more mixed. for example, some studies have revealed a weakening of the protective effect of increasing education on obesity over the past 30 years, a consequence of the increase in obesity among the most educated [12] . the inverse association between sep and body mass index (bmi) and obesity is stronger among women compared with men [2, [13] [14] [15] [16] [17] . some studies, in fact, reported a reversal of the expected inverse sep-obesity association among men; i.e., men of high sep were more likely to have increased bmi and be overweight or obese than men of low sep [14] [15] [16] 18] . furthermore, nativity status (foreign-born vs. us-born) differentials in the association of an individual-level measure of sep on obesity have been reported among black americans, with native-born blacks being at higher risk [19] . few studies, however, have examined sex-specific associations between obesity and multilevel measures of sep within an ethnically diverse group of black americans. hence, the present study investigates the potential differential associations between obesity and individual, household, and neighborhood-level sep indicators among black american men and women (african-americans) and blacks with caribbean ethnicity (caribbean-blacks). elucidation of the differential patterning of multidimensional measures of sep on obesity among african-american and caribbean-black men and women would be beneficial for developing effective interventions to reduce obesity and obesity-related diseases within an increasingly heterogeneous us black population. this cross-sectional study utilizes data from the national survey of american life (nsal). the nsal is part of the national institute of mental health (nimh) collaborative psychiatric epidemiology surveys (cpes) initiative that also includes the national co-morbidity survey replication (ncs-r) and the national latino and asian american study (nlaas) [20] . nsal was conducted according to the guiding principles in the declaration of helsinki. all procedures involving human subjects, including oral and written consent from all participants 18 years of age and older, were approved by the institutional review board at the university of michigan [21] . the nsal includes a sample of 891 non-hispanic whites, 3570 african-americans, persons who identified as black but did not have ancestral ties to the caribbean (including 67 foreign-born blacks), and 1621 caribbean-blacks, persons who identified as black and who either were born in caribbean area countries (73% of the caribbean-black sample), had parents or grandparents who were born in the caribbean, or had indicated that they were of caribbean ethnicity. the nsal survey data includes assessments of mental, emotional, and physical health and residential, environmental, and socioeconomic characteristics. data were collected between february 2001 and june 2003 with response rates of 70.7% for african-americans, 77.7% for caribbean-blacks, and 72.3% for whites. this paper capitalizes on the growing heterogeneity of the us black population as reflected in the nsal sample and reports findings for african-american and caribbean-black men and women, aged 18 years and older within the nsal sample. the study outcome is obesity, characterized as having a body mass index (bmi, weight (kg)/height (m) 2 ) 30 or higher [22] . bmi is calculated within the nsal based on selfreported weight and height. the independent variables include self-reported measures of adult sep at the individual, family, and neighborhood levels. sep indicators at the individual level include current education specified as < high school (hs), hs and > hs, and respondent's main occupation categorized into professional/managerial, sales/administrative, skilled blue collar, unskilled blue collar, and service. sep measures at the family level consist of (1) a measure of wealth or "house value," specified as at or below the median, above the median, or "none," (2) quartiles of family income, and (3) "public support" described as currently receiving any public assistance for the family, and dichotomized into "any" or "none." finally, sep measures assessed at the neighborhood level include self-reported measures previously found to be associated with obesity within african-americans and caribbean-blacks-having a supermarket within one's neighborhood (yes vs. no) and having a park within one's neighborhood (yes vs. no) [23] . covariates utilized for statistical adjustment due to their known associations with sep and obesity include demographic variables such as age [5] , categorized into less than or equal to 29 years, 30-44 years, 45-59 years, and 60 or more years; marital status, [24] characterized as (1) married or living with a partner, (2) separated, divorced or widowed, and (3) never married; and three measures of health behaviors, (1) physical activity [22] , a continuous measure based on how often the study participants worked in the garden or yard, engaged in active sports or exercise, and walked, (2) smoking status [25] , specified as the participant never having smoked more than 100 cigarettes in his/her lifetime or "never smoker," having smoked more than 100 cigarettes in the past or "past smoker," and "current smoker," and (3) current alcohol consumption [22] , categorized for analytical purposes into consuming no alcoholic drinks within the past year or "none," having consumed less than 12 drinks within the past year, or "infrequent drinker," and moderate-to-heavy drinkers having consumed 12 or more drinks within the past year or "regular drinker." to minimize bias due to differentially distributed missing data on measures of sep and bmi by ethnicity (african-american and caribbean-black) and sex, multiple imputation was performed prior to statistical analysis within the statistical software package iveware [26] . iveware uses a sequential regression imputation method to impute values for each individual, conditional on all other values observed for that individual, consequently producing complete datasets for unbiased analyses [27] . descriptive statistics stratified by ethnicity and by sex were then calculated. chi-square tests were performed to examine group differences in categorical variables, whereas the global f test within an unadjusted linear regression model was utilized to calculate ethnicity and sex differences in physical activity. since previous studies reveal differential sep patterning of obesity by both sex and ethnicity [4, 19] , three-way interactions between measures of sep, ethnicity, and sex were tested. statistically significant three-way interactions were found for five out of the eight sep indicators, p values ranging from 0.008 to 0.024. therefore, all nested multivariate logistic regression models assessing the strength of the independent associations between multilevel measures of sep and obesity after covariate adjustment are presented stratified by ethnicity and sex. specifically, the following five models were fitted for each sep indicator: (1) odds ratios (ors) adjusted for age; (2) ors additionally adjusted for individual-level sep, i.e., education, occupation, as well as for marital status; (3) ors additionally adjusted for family-level sep, i.e., public support, house value, and family income; (4) ors additionally adjusted for neighborhood-level sep, i.e., neighborhood supermarket and neighborhood park; and (5) ors additionally adjusted for health behaviors, i.e., physical activity, alcohol intake, and smoking. data management was conducted using sas version 9, [28] and all statistical analyses were performed using sudaan [29] to account for the multiple imputation and the complex sample design of the nsal in calculating unbiased effect estimates and standard errors. sample sizes presented within tables are un-weighted; however, all other estimates, means proportions, and standard errors, as well as ors and 95% confidence intervals (cis), are weighted. table 1 describes the general characteristics of african-american and caribbean-black men and women in the nsal sample. african-american women had the highest prevalence of obesity at 41.2% followed by caribbean-black women at 31.1% and african-american men at 29.9%. caribbean-black men had the lowest prevalence of obesity at 21.6%. caribbean-black men and women were also younger than african-american men and women with 64.9% and 66.2% of caribbean-black men and women being under the age of 45 as compared with 61.0% and 58.8% of african-american men and women, respectively. caribbean-black men and women had greater educational attainment than african-american men and women, with 49.4% of caribbean-black men and 48.8% of caribbean-black women and 37.3% of african-american men and 38.4% of african-american women having > hs education. men were more likely to be married or living with a partner than women; caribbean-black men had the highest prevalence at 59.9%, while african-american women had the lowest at 35.6%. in addition, men were more likely than women to be employed in the skilled and unskilled blue collar professions; unskilled blue collar employment was highest for african-american men at 33.2%. women were more likely than men to be employed in service professions. moreover, caribbean-black men and women, at 23.0% and 23.3%, respectively, were more likely to work in professional/ managerial positions compared with african-american men and women at 15.4% and 17.9%, respectively. african-american women were the most likely to receive public support, and accordingly, most likely to have had the lowest family income compared with all other ethnic/sex groups. caribbean-black men were least likely to receive public support and to have had the highest family income. african-american men were most likely to be homeowners relative to all other ethnic/sex groups, whereas caribbean-black women were least likely to be homeowners. caribbean-black men and women were more likely than african-american men and women to live in neighborhoods that had a supermarket or a park. caribbean-black men had the highest prevalence, in that 91% lived in a neighborhood with a supermarket and 88.1% resided in a neighborhood with a park. african-american women had the lowest prevalence with approximately 71% residing in a neighborhood that had a supermarket or park. men reported higher rates of physical activity than women, with caribbean-black men having the highest average score (2.95) and african-american women having the lowest (2.56). seventy-nine percent of caribbean-black women never smoked, whereas only 49.4% of african-american men never smoked. finally, african-american women were least likely to consume alcohol in the past year where 43.5% never drank. caribbean-black men were most likely to drink alcohol in the past year and 55.7% were regular drinkers. tables 2, 3, 4, and 5 present the adjusted odds ratios (ors) and 95% confidence intervals for the associations between obesity and individual-, family-, and neighborhood-level socioeconomic position (sep) among african-american men, caribbean-black men, african-american women, and caribbean-black women, respectively. unless otherwise stated, all ors were determined from the fully adjusted models. for african-american men, no significant associations between obesity and any individual-level measure of sep were observed ( table 2) . low sep at the family level, however, was associated with a lower odds of obesity. within all adjusted models for african-american men, family income within the lowest quartile was protective against obesity relative to the highest quartile, or = 0.50, 95% ci (0.27, 0.92). furthermore, african-american male homeowners with housing equity at or below the median had a 43% increased odds of obesity compared with their non-home-owning counterparts, or = 1.43, 95% ci (1.00, 2.06). neighborhood-level sep was also associated with obesity, with all adjusted models showing lower odds of obesity for african-american men residing in neighborhoods that contained a park compared with those who lived in neighborhoods without one (or = 0.60, 95% ci (0.38, 0.94)). as shown in table 3 , none of the individual-level measures of sep for caribbean-black men was significantly associated with obesity, paralleling the findings for african-american men. nevertheless, selected family-and neighborhood-level sep measures were associated with obesity. for example, like african-american men, low family income was protective for caribbean-black men within the age-adjusted model; however, this association became non-significant (p > 0.05) with additional covariate adjustment. furthermore, caribbean-black men who received any public assistance had 3.5 times the odds (95% ci 1. 30, 9.42) of obesity compared with their counterparts who did not. finally, caribbean-black men residing in a neighborhood containing a supermarket experienced an 86% decreased odds of obesity compared with those in neighborhoods without this amenity, or = 0.14, 95% ci (0.04, 0.52). in contrast to men, table 4 shows statistically significant associations between measures of individual-level sep and obesity for african-american women. african-american women with < hs education had 1.47 times the odds (95% ci (1.04, 2.09)) of obesity than those with > hs education. though only borderline significant in fully adjusted models, african-american women with a hs education had 1.40 times the odds (95% ci (0.96, 2.05)) of obesity compared with those with > hs education. african-american women employed in the sales/administrative occupations had 27% lower odds of obesity compared with african-american women employed in service employment, or = 0.73, 95% ci (0.57, 0.94). in contrast to african-american men, higher family sep was protective against obesity for african-american women. though of borderline significance, african-american women who received public support had a 27% greater odds of obesity compared with those not receiving such support, or = 1.27, 95% ci (0.98, 1.63). though not statistically significant, african-american women with family income in the third quartile were associated with a 35% increased odds of obesity compared with women in the highest quartile, or = 1.35, 95% ci (0.97, 1.89). african-american females with housing table 5 summarizes associations between sep and obesity among caribbean-black women. occupation is the only individual-level sep measure that was associated with obesity in this group. being employed within professional/managerial occupations was associated with a decreased odds of obesity compared with employment in service occupations, after adjusting for age, marital status, and other individual-level, family-level, and neighborhood-level sep measures, or = 0.55, 95% ci (0.30, 0.99). this association became statistically insignificant, however, after adjusting for physical activity, smoking, and alcohol consumption, or = 0.61, 95% ci (0.35, 1.07). similar to african-american women, receipt of public assistance increased the odds of obesity for caribbean-black women; however, the magnitude of the association was stronger for the latter. caribbean-black women receiving public support had 1.74 times the odds of obesity compared with caribbean-black women not on public assistance, 95% ci (1.08, 2.82). yet, unlike african-american women, in ageadjusted and individual-level sep adjusted models, caribbean-black women with family income in the third quartile had a decreased odds of obesity compared with those with family income in the fourth quartile. in fully adjusted models, this relationship became statistically insignificant, or = 0.56, 95% ci (0.28, 1.14). no association was observed between this study of the association between multiple dimensions of sep (individual, family, and neighborhood) and obesity in a sample of us blacks found that associations varied by ethnicity and by sex. among african-american men, no statistically significant associations between sep and obesity were observed at the individual level, but a positive relationship was observed for family sep; namely, african-american men in the bottom quartile of family income and who had no housing equity had the lowest odds of obesity. similar to african-american men, there was no association between adulthood sep at the individual level among caribbean-black men. for caribbean-black men, more complex sep patterns for family income were observed, wherein (1) obesity increased among those receiving public assistance and (2), similar to african-american men, a positive association was observed between family income and obesity. the association between public assistance and the increasing odds of obesity among caribbean-black men is consistent with a previous report showing that participation in public assistance programs increased the risk of adult obesity [30] . in this particular study, the association was stronger among men than women and was mediated by dietary quality as assessed by higher soda consumption. moreover, in the current study, our finding of a positive association between obesity and sep at the family level among black men is also in line with previous reports [5, 14, 31, 32] . african-american elementary school boys with low sep have been reported to engage in more vigorous physical activity than high sep boys through participation in team sports such as football and basketball [33] . this engagement in vigorous team sports, if it [34] , is a possible contributing factor in lowering the risk of obesity among low sep african-american and caribbean-black men. this is a hypothesis that should be tested in prospective research. moreover, the higher obesity rate among high sep men has been postulated to be due to the positive effect increased body weight has on higher earned income and social prestige among men [35] . future longitudinal investigations should determine whether perceived and actual positive economic consequences for increasing body size drive the positive relationship between sep and obesity among men. for african-american women, the expected inverse relationship between individual-level sep and obesity was seen for education [15] ; however, educational attainment was not associated with obesity among caribbean-black women. in line with previous research [36] , our study also reveals that higher status occupations, i.e., sales and administrative jobs among african-american women and professional and managerial jobs among caribbean-black women, are associated with lower odds for obesity than lesser status service professions. finally, our results at family-level sep show that similar to caribbean-black men, receipt of public assistance increased odds for obesity among women, with a stronger association for caribbean-black women. lastly, our results show important ethnic and sex differences in the association between community sep characteristics and obesity. the lower odds of obesity among caribbean-black male and african-american female residents in neighborhoods containing a supermarket is consistent with prior research [23, 37] . in addition, the lower odds of obesity for residence within a neighborhood containing a supermarket found among caribbean-black women suggests that this relationship may hold for them as well despite the lack of statistical significance most likely due to low statistical power. the absence of any relationship between obesity and residence within a neighborhood containing a supermarket among african-american men warrants further investigation. it is important to highlight the substantial decrease in the odds of obesity exclusively among african-american men who live in neighborhoods containing a park. research has shown that increased access to parks is associated with increased moderate-to-vigorous physical activity in boys but not among girls aged 8 to 12 years [38] . for many black men, these childhood patterns of engaging in vigorous competitive team sports (e.g., basketball) in neighborhood parks extend into early adulthood, a pattern not seen historically among black women [39, 40] . additional research is needed to determine whether elevated energy expenditure due to increased access to recreational parks across the life course explains the lower prevalence of obesity among african-american men living in neighborhoods with community parks. previous research documenting a lack of association between sep and exercise frequency in black women may explain the absence of a relationship between neighborhood parks and obesity among african-american and caribbean women within our findings [41] . further theoretical and empirical investigations are needed to uncover additional mechanisms through which measures of sep at the neighborhood level independently operate to increase obesity within the various ethnic and sex groups. to our knowledge, this is the first study to document associations between multilevel and multidimensional measures of sep and obesity in a sample of us self-identified and caribbean self-identified black americans, while using an intersectionality approach centered on both within-race ethnic and sex differentiation. methodological strengths also include the employment of multiple imputation of missing data. this study has several limitations, however. multiple imputation was not performed on measures with over 40% of missing data, such as individual-level income, and so, this variable was not included within the analysis in order to minimize bias [42, 43] . furthermore, the lower sample size of caribbean-blacks within the nsal dataset limited statistical power to examine any additional variation in the sep-obesity associations by nativity status, i.e., foreign-born vs. us-born. the public-use nsal questionnaire utilized for analysis included only self-reported measures of neighborhood physical characteristics and did not allow for geocoding addresses to include census tract measures, i.e., percentages of families below the poverty, unemployed and/or low-educated adults, and homeowners within a neighborhood, for multilevel modeling. although the inability to examine objective assessments of neighborhood sep within hierarchical models is a disadvantage, perceived measures of neighborhood resources have been found in previous studies to be equally robust correlates of obesity as observed neighborhood indicators [44] . the cross-sectional nature of the nsal study design does not establish a clear, unbiased temporal relationship between multilevel sep indicators and adult obesity; therefore, neither causality nor direction of the sep-obesity associations can be ascertained with certainty. the nsal, one of the few studies that allow for an examination of the socioeconomic correlates of cardio-metabolic health by ethnicity and sex within black americans, was conducted in 2001-2003 and hence is an established dataset. notwithstanding, the stability of sep indicators over time for black americans, including median income, which was $40,573 in 2003 and $41, 361 in 2018, supports the present-day relevancy of our findings [45] . the use of self-reported weight and height to estimate obesity prevalence is also a potential bias. studies have found that these self-reported measures are reasonably valid and reliable indicators of actual weight and height, although obese individuals tend to underestimate their weight [46] [47] [48] . a dilution rather than an exaggeration of the magnitude of our associations is expected however if a systematic underreporting of weight is similar across sep categories. statistical adjustment for additional health behaviors related to obesity such as dietary quality could not be made within the analyses since this information is not assessed in the nsal. in addition, physical activity, crudely assessed within the nsal as a continuous measure of reported leisure time activity, is a less reliable measure of activity level than other methods which directly monitor the intensity of physical activity such as the use of double isotopically labeled water (d 2 o 18 ) methodology and measurement by a pedometer or tachometer [49] . the extent to which the inclusion of dietary intake and a more reliable indicator of the intensity of physical activity would explain the associations between multilevel indicators of sep and obesity cannot be determined. our study findings highlight that adulthood sep at the individual, family, and neighborhood levels plays an important role in obesity in the us black population, and this varies by ethnicity and sex. the opposite sep-obesity relationships between black men and women suggest the need for further empirical investigation into the sex-specific mechanisms of obesity, particularly among low sep families where the disparity between young adult black women and men is the largest [50] . moreover, the lower odds of obesity associated with residence in neighborhoods with a supermarket for african-american women and caribbean-black men, and with residence in neighborhoods containing a park for african-american men, suggests that continued public policy attention to increasing access to food and recreational facilities within black american communities, as was undertaken in the national "let us move!" comprehensive initiative for preventing childhood obesity [51] , is likely to be an important endeavor. evidence from this study also suggests that the development of targeted, multilevel, cultural, and sex-specific societal interventions could be vital to not only curtailing the us obesity epidemic but to reducing racial inequities in survival from emerging infectious diseases such as covid-19, for which obesity has arisen as a strong risk and prognostic factor in its severity [52] [53] [54] . funding information this study was funded by the robert wood johnson health and society scholar's program and by the national institute on minority health and health disparities (grant number k22md006133). conflict of interest the authors declare that they have no conflicts of interest. ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. informed consent informed consent was obtained from all individual participants included in the study. tables of summary health statistics for socioeconomic status and obesity: a review of the literature socioeconomic status and obesity socioeconomic status and obesity the obesity epidemic in the united statesgender, age, socioeconomic, racial/ethnic, and geographic characteristics: a systematic review and meta-regression analysis socioeconomic position in social epidemiology measuring social class in us public health research: concepts, methodologies, and guidelines us socioeconomic and racial-differences in health -patterns and explanations built environments and obesity in disadvantaged populations associations between body mass index and park proximity, size, cleanliness, and recreational facilities improving the measurement of socioeconomic status for the national assessment of educational progress: a theoretical foundation trends in the association between obesity and socioeconomic status in secular trends in body mass index and skinfold thickness with socioeconomic factors in young adult women socioeconomic inequality of obesity in the united states: do gender, age, and ethnicity matter? obesity and socioeconomic status in adults: united states cross-sectional and longitudinal associations of bmi with socioeconomic characteristics race by gender group differences in the protective effects of socioeconomic factors against sustained health problems across five domains. j racial ethn health disparities secular trends in body mass index and skinfold thickness with socioeconomic factors in young adult men racial/ ethnic disparities in obesity among us-born and foreign-born adults by sex and education sample designs and sampling methods for the collaborative psychiatric epidemiology studies (cpes) the development and implementation of the national comorbidity survey replication, the national survey of american life, and the national latino and asian american survey clincal guidelines on the identification, evaluation, and treatment of overweight and obesity in adults neighborhood environments and obesity among afro-caribbean, african american, and non-hispanic white adults in the united states: results from the national survey of american life is race associated with weight change in us adults after adjustment for income, education, and marital factors? smoking, body weight, and their effects on smoking behavior: a comprehensive review of the literature iveware: imputation and variation estimation software a multivariate technique for multiply imputing missing values using a sequence of regression models sas institute inc. sas/stat 9.1 user's guide is participation in food and income assistance programmes associated with obesity in california adults? results from a state-wide survey lifecourse socioeconomic position and weight change among blacks: the pitt county study. obesity (silver spring) socioeconomic and behavioral correlates of body mass index in black adults: the pitt county study leisure time activities of elementary school children race, class and leisure activities of urban residents health, obesity, and earnings black-white differences in social and economic consequences of obesity neighborhood risk factors for obesity. obesity (silver spring) the neighborhood and home environments: disparate relationships with physical activity and sedentary behaviors in youth parkbased physical activity in diverse communities of two u.s. cities. an observational study contribution of public parks to physical activity educational attainment and exercise frequency in american women; blacks' diminished returns principled missing data methods for researchers when and how should multiple imputation be used for handling missing data in randomised clinical trials -a practical guide with flowcharts perceived and observed neighborhood indicators of obesity among urban adults households by total money income, race, and hispanic origin of householder the reliability and validity of self-reported weight and height self-reported weight and height ethnic variation in validity of classification of overweight and obesity using self-reported weight and height in american women and men: the third national health and nutrition examination survey physical activity and its impact on health outcomes. paper 2: prevention of unhealthy weight gain and obesity by physical activity: an analysis of the evidence the female-male disparity in obesity prevalence among black american young adults: contributions of sociodemographic characteristics of the childhood family white house task force on childhood obesity report to the president. solving the problem of childhood obesity within a generation patients in a large health care system in california covid-19 is out of proportion in african americans obesity as a predictor for a poor prognosis of covid-19: a systematic review publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-318593-ni84gzg5 authors: wolf, jayanthi; bruno, samantha; eichberg, michael; jannat, risat; rudo, sharon; vanrheenen, susan; coller, beth-ann title: applying lessons from the ebola vaccine experience for sars-cov-2 and other epidemic pathogens date: 2020-06-15 journal: npj vaccines doi: 10.1038/s41541-020-0204-7 sha: doc_id: 318593 cord_uid: ni84gzg5 the world is experiencing an unprecedented global pandemic of coronavirus disease 2019 (covid-19) caused by a novel coronavirus, severe acute respiratory syndrome-coronavirus-2 (sars-cov-2). development of new vaccines and therapeutics are important to achieve long-term prevention and control of the virus. experience gained in the development of vaccines for ebola virus disease provide important lessons in the regulatory, clinical, and manufacturing process that can be applied to sars-cov-2 and other epidemic pathogens. this report outlines the main lessons learned by merck sharp & dohme corp., a subsidiary of merck & co., inc., kenilworth, nj, usa (msd) during development of an ebola zaire vaccine (ervebo®) and looks ahead to critical lessons beyond vaccine development. it highlights focus areas for public-private partnership and regulatory harmonization that can be directly applied to current vaccine development efforts for sars-cov-2, while drawing attention to the need for parallel consideration of issues beyond development that are equally important to achieve global preparedness and response goals. first discovered in the democratic republic of the congo in 1976, the highly lethal ebola virus has infected people in a number of african countries leading to sporadic outbreaks of ebola virus disease over the past 40 years. while most of the outbreaks have been limited in geographic scope and number of cases, two of the outbreaks over the last 6 years have been large, resulting in major loss of life and socioeconomic disruption in the region. in early 2014, an outbreak of ebola virus disease caused by zaire ebolavirus started in west africa and was declared a public health emergency of international concern in august of that year. by the time the outbreak ended in 2016, more than 11,000 people had died and more than 28,000 people were infected primarily in three countries, guinea, liberia, and sierra leone 1 . in the decade prior to the west african outbreak, research efforts for biodefense purposes resulted in the identification of some promising ebola vaccine candidates that protected monkeys from a lethal challenge of wild-type ebola virus 2 . these vaccine candidates were not taken into clinical development prior to the west african outbreak for several reasons, which included the inability to demonstrate clinical efficacy in the absence of an ongoing outbreak and lack of interest by the public health and vaccine development community to invest in the lengthy and costly process of vaccine development without a clear demand for an ebola vaccine 2 . the west african outbreak changed this perspective and numerous vaccines entered clinical development during the outbreak resulting in the generation of safety and immunogenicity data for many of these novel vaccine candidates and the demonstration of efficacy for one vaccine 3 . ervebo® is a live, attenuated, recombinant vesicular stomatitis virus (rvsv)-based, chimeric-vector vaccine, where the vsv envelope g protein was deleted and replaced by inserting only the envelope glycoprotein of zaire ebolavirus. extraordinary efforts were made to advance this vaccine candidate through phase 1, 2, and 3 clinical trials and the data generated in the context of the west african ebola outbreak has supported its licensure by the us food and drug administration (fda), conditional authorization by the european medicines agency (ema) and several african countries, along with prequalification by the who. the period of 5 years from the start of phase 1 trials in oct 2014 to the approval of this vaccine in nov 2019, was much faster than the typical 10-15 year timeline for vaccine development and approval 4 . a timeline of the key activities in the development of this ebola vaccine is summarized in fig. 1 and described in detail in the following sections of this article. through this ebola vaccine development effort a number of learnings have been identified, which are highly relevant for the current vaccine development efforts in response to the covid-19 pandemic. a summary of the key lessons learned can be found in fig. 2 field response and non-governmental service organizations (such as medecins sans frontieres); global public health entities (such as the world health organization, wellcome trust, and gavi); universities (e.g., the university of geneva, dalhousie university) and private sector companies (such as newlink genetics, idt biologika, and msd). each of these entities had a specific role in the broad partnership, including conducting preclinical studies, manufacturing good manufacturing practices (gmp) trial materials, conducting clinical trials, and funding portions of the research and development. msd has a well-established history in manufacturing and clinical development of vaccines, with experience in taking novel vaccines through the regulatory process to approval and providing global access. however, msd did not have prior experience with ebola virus nor clinical development expertise in the african countries in which the ebola outbreak was taking place. therefore, msd relied on the expertise of organizations that were already involved in the public health response efforts to implement and complete nearly all the clinical trials. important lessons from this effort are that leveraging the expertise of different organizations can help to accelerate product development and that broad public-private partnerships require strong leadership and coordination to facilitate cooperation of diverse partners with different expertise and missions. furthermore, concerns over evaluation of any investigational product are present in less developed parts of the world and these concerns may be further exacerbated during an outbreak 5 . therefore, clear and transparent communication with community leaders and the community at large, ethical review committees, local ministries of health, and other government agencies is critical to ensure that the purpose of the research is well understood and aligned with accepted local and international norms. failure to do so can result in delays or roadblocks to research on potentially life-saving products. ultimately the responsibility to convert the data generated by all the collaborators into a coherent regulatory submission to support licensure of a product lies with the marketing authorization holder. recognition and proactive planning for that eventuality can help to accelerate the regulatory submission to support product approval. examples of these types of activities include the following. data integration msd's role in advancing the product to licensure included integrating all the data generated by partners to support the marketing authorization applications. nine of 13 nonclinical studies, 1 of 2 clinical assay validations, and 11 of 12 clinical trials were performed by partners. msd needed to obtain all the datasets and reports from these partners to include in the license applications. data from clinical trials that were performed by partners needed to be migrated into a central database that could be used for data integration, analysis, report-writing, and submission by msd to regulatory agencies. a lesson learned during this process is to account for the time needed to convert different formats and languages used to collect the data into standard formats required by regulatory agencies, such as use of the medical dictionary for regulatory activities (meddra) and study data tabulation model (sdtm). with the clinical trials all being designed and implemented in the context of the outbreak, it was not possible to harmonize the study designs. while this is not an issue a priori for the conduct of the trials, it introduced challenges when assembling the data to support licensure. for example, it was not possible to conduct extensive integration of the safety data for the product because the data collected and methods used were different for each trial. a key lesson learned during this process is that standardization of clinical trial protocols and data collection is preferred, and that to the extent possible late stage clinical trial designs should be defined prior to outbreaks. another challenge specifically related to conducting trials in response to emerging diseases is that there is a need to build clinical development capacities, laboratory and adverse event reporting infrastructure in outbreak-prone regions. this was a challenge taken on by individual trial sponsors in the affected countries and required significant and sustained support by the trial sponsors and their local partners. establishing mechanisms to maintain the capabilities established during outbreak response to provide experienced clinical trial sites for future research is another key lesson learned. examples of this in action include the ongoing partnership between the us and liberia and the partnership for research on ebola vaccination (prevac) consortium established in the wake of the west africa outbreak. clinical assays a key immunogenicity endpoint assay was developed and validated through a collaborative effort by private and public sector partners. the intent was to provide an assay available to all vaccine developers, which might ultimately allow comparison of different vaccine candidates. while the collaborative effort was successful, balancing the input of numerous partners took time. consequently, clinical assay validation took two years to complete, resulting in a back-log of samples from clinical trials that needed to be processed. a lesson learned is that whenever possible clinical immunogenicity sample collection should be standardized and assays should be validated prior to the start of late stage clinical trials. this requires knowledge of the target pathogen well in advance of clinical trials, which is a challenge for sars-cov-2. expanded access use of the vaccine prior to approval with the rapid collection of efficacy data in support of the vaccine, it was recognized that there was the possibility that new ebola outbreak(s) could occur ahead of the licensure and availability of licensed doses and that a mechanism was needed to support access to the investigational product. for this scenario, it was necessary to manufacture doses to support outbreak response and establish regulatory frameworks needed to support vaccine deployment (e.g., compassionate use, expanded access, or emergency use). the who, msf, and other groups took the lead in implementing expanded access protocols in african countries, working closely with national governments and ministries of health. through this effort, more than 300,000 people were vaccinated during the 2018-2020 ebola outbreak in the democratic republic of the congo 6 . a lesson learned is that the deployment of an investigational product requires careful consideration and thorough planning. the manufacturing process had to be rapidly scaled-up to handle the needs for emergency use and expanded access programs, in addition to establishing a final manufacturing facility. the approach taken by msd was to take the initial manufacturing process that was developed at a contract manufacturing organization, scale it up in a clinical manufacturing facility, and transfer it to a commercial manufacturing facility. technology transfer and the establishment of a new manufacturing facility has many steps and requires significant time to execute (3-4 years is typical for a new vaccine). there is limited understanding outside of vaccine manufacturers and regulators on the rigorous requirements leading to approval of a vaccine manufacturing facility. this broad lack of understanding can lead to misperceptions of "delays" when in fact timelines are driven by the elements required for licensure. for example, although clinical data were available in 2017-2018, the vaccine could not be approved until the commercial manufacturing site was established due to expectations from regulatory agencies that the manufacturing process needs to be validated at the final manufacturing site. a lesson learned is that education is needed on how vaccines are manufactured and the time needed to establish and gain approval for a new manufacturing facility. this is particularly relevant for the clinical • exisɵng preclinical data on the vaccine plaƞorm can accelerate the start of phase 1 trials during outbreaks. • clear communicaɵon with community leaders and government agencies is criɵcal to ensure the purpose of clinical research is well understood. • standardizaɵon of clinical trial protocols and data collecɵon is needed and late stage clinical trial designs should be defined prior to outbreak. • need to build and maintain clinical development capaciɵes, laboratory and adverse event reporɵng infrastructure in outbreak-prone regions. • clinical immunogenicity sample collecɵon should be standardized and assays validated prior to the start of late stage clinical trials. • deployment of an invesɵgaɵonal product requires careful consideraɵon and thorough planning. • it takes ɵme to convert different formats and languages used to collect the clinical data into standard formats required by regulatory agencies. • educaɵon is needed on how vaccines are manufactured and the ɵme needed to establish and gain approval for a new manufacturing facility. • aggressive ɵmelines are enɵcing, but require "right-first-ɵme" manufacturing execuɵon with low/medium probability of success for new vaccines. • global and regional harmonizaɵon is criɵcal for the efficient development and licensure of pandemic vaccines. • regulatory agency collaboraɵon is criɵcal for success. • clinical data requirements for licensure need to be clearly defined and harmonized across global regulatory agencies. • frequent discussions with regulatory agencies and expedited regulatory pathways can accelerate the program. • who's leadership is needed to facilitate innovaɵve review processes and collaboraɵve mechanisms to expedite global approvals. • addiɵonal regulaɵons for recombinant live virus vaccines can lead to delays in starɵng clinical trials, manufacturing, tesɵng and shipments. • harmonized labeling and packaging requirements would facilitate pandemic vaccines distribuɵon. • public-private partnerships help accelerate product development by leveraging experɵse from different organizaɵons. • policy, program, and system innovaɵons are needed to realize the full promise of new vaccines in advancing global public health preparedness. covid-19 outbreak where all aspects of vaccine development are being accelerated at an extraordinary pace with the goal of being able to produce unprecedented quantities of vaccine to address global needs. manufacturing site selection for vaccines is a complex decision, which is further complicated when seeking to move fast with incomplete information. the company needed to manage multiple factors, such as existing space, technical capability, infrastructure, and capacity. site selection also needed to account for the regulations in the country in which the manufacturing site is located and the permits and licenses necessary to support the efforts. the country's employment environment and labor laws can also impact the ability to hire qualified staff quickly where these factors need to be considered early. a lesson learned is that aggressive timelines are enticing for public health partners, but also require "right-first-time" execution to qualify the facility and manufacturing process, for which the probability of success for a new vaccine is likely medium to low. all parties involved must work to balance the desire to be ambitious (e.g., rapid development timelines) with execution realities and stakeholder expectations. parallel work and extensive collaboration between manufacturers will be needed in order to successfully bring a sars-cov-2 vaccine to the world. regulatory agency collaboration is critical for success from the start of the west african ebola outbreak, the us fda, ema, and health canada worked closely with each other and with the national regulatory authorities of the impacted west african countries, sharing information about candidate vaccines that were being tested and reviewing the clinical protocols, available data, and benefit-risk profiles. frequent conversations with manufacturers helped the agencies expedite the start of clinical trials simultaneously in different countries. a lesson learned is that existing preclinical data and availability of clinical supplies afforded the opportunity to start phase 1 trials rapidly during the west african ebola outbreak. clinical data requirements for licensure need to be clearly defined placebo-controlled, randomized, double-blind studies are typically used to demonstrate the efficacy and safety for new vaccines. however, in the midst of the ebola outbreak, some countries considered it unethical to administer placebo to at-risk individuals. different study designs were implemented for the phase 2/3 trials and a cluster-randomized trial conducted by the world health organization in guinea was the only trial that demonstrated efficacy 7 . efficacy data from this trial, together with safety data from 12 trials and immunogenicity data with validated assays, were accepted for registration by regulatory agencies. a lesson learned is that the regulatory pathway to licensure for pandemic vaccines need to be clearly defined. novel trial designs might need to be implemented in order to maximize the possibility of evaluating efficacy during a waning outbreak, and alternative endpoints, such as using immune responses as a surrogate for efficacy, could be used to gain accelerated approval or conditional marketing authorization. frequent regulatory agency interactions are important based primarily on interim clinical efficacy data, the manufacturer applied for and was granted access to expedited regulatory pathways, such as priority medicines (prime) status from ema and breakthrough therapy designation (btd) from fda, which helped to obtain alignment on data requirements for product approval using frequent meetings. under btd, the fda accepted rolling submissions of portions of the biologics license application (bla), which resulted in approval in december 2019, three months before fda's target approval date. the fda determined that a program specific advisory committee meeting was not needed. similar frequent discussions with ema under prime status, enabled the company to gain alignment on key aspects of the development program quickly. who leadership is needed to obtain rapid global approvals who is a recognized global public health leader with regional offices in many countries, and established connections with the african vaccines regulatory forum (avaref). in order to accelerate vaccine access to african countries, the world health organization's prequalification team (who-pqt) in collaboration with the ema and avaref developed an innovative facilitated process (roadmap) for decision making on the acceptability of the vaccine for registration 8 . this allowed the company to make simultaneous submissions to ema, who-pqt, and regulatory authorities in 14 african countries, with ema acting as the reference agency. following submission, the who-pqt and members from avaref, which represented the 14 african countries, were invited to participate in teleconferences between msd and ema and in the inspection of the manufacturing facility. after a positive opinion from ema in october 2019, the european commission granted a conditional marketing authorization on november 11th. within 36 h, the who granted prequalification. one month later, african countries started to approve ervebo®, starting with burundi and the democratic republic of the congo. a lesson learned is that strong collaboration and willingness to share information with everyone involved resulted in registration nearly-simultaneously in the countries that needed the ebola vaccine. for sars-cov-2 vaccines, regulatory agencies should prepare for the simultaneous approval of candidates in multiple countries. who's leadership will be needed to facilitate innovative review processes and collaborative mechanisms to expedite approvals. recombinant viral vaccines need to overcome additional regulatory hurdles since this ebola vaccine was a recombinant virus, some countries required a detailed environmental risk assessment prior to the start of clinical trials and as part of the marketing authorization application. in some countries, the vaccine was considered a bio-safety level 2 organism, requiring special handling and permits for the manufacturing and testing sites. these permits took a significant amount of time to obtain, which prevented rapid transfers of material. samples and technology, which even included parts of the marketing authorization application, required licenses for export because this vaccine is made from the genetic sequence of two viruses that are considered "dualuse" agents and subject to trade controls. a lesson learned is that these regulations led to delays and additional costs incurred in starting clinical trials, manufacturing, testing, and shipments. for manufacturers of sars-cov-2 vaccines, countries should consider waiving these requirements, or waiving the fees and expediting these processes. harmonized labeling and packaging requirements would facilitate pandemic vaccines distribution after approval, companies face challenges with product distribution due to the need to follow country-specific product labeling regulations covering package inserts, cartons, and artwork in addition to serialization requirements. this causes a supply chain issue for a vaccine product intended to be placed in a stockpile and diverted quickly to any country. a lesson learned is that these heterogenous requirements counter the goal of flexibility, speed, and cost-efficiency relevant to emergency preparedness, and there is an urgent need for harmonized solutions. electronic labeling and quick response (qr) codes could be a possible solution, but in most countries, legislation does not currently exist to support this option. there is an urgent need to find a solution to this problem for pandemic vaccines. in addition to considering lessons related to vaccine development for epidemic preparedness, it is equally important to consider the unique challenges and opportunities related to sustainable manufacturing, supply, access, and delivery of these vaccines at the scale and speed needed to achieve preparedness and response goals. such challenges include right-sizing manufacturing capacity and production while having to accommodate leadtimes, managing uncertain demand-and-supply dynamics, ensuring equitable allocation and access, and achieving operational and economic sustainability for all partners. producing and supplying any vaccine is inherently complex. it is exponentially more complex when there are high levels of uncertainty and unpredictability across nearly every dimension of the program: unpredictable disease, unpredictable and relatively low demand, unpredictable stakeholders and customers, unpredictable timing of need, and unpredictable geographies in which needs might arise. policy, program, and system innovation-in parallel to research and development innovation-needs to be addressed to realize the full promise of innovative, new vaccines in advancing global public health preparedness. emergency preparedness and response vaccines, such as ervebo®, are critical tools in the arsenal against pathogens that can cause pandemics. public-private partnerships are a powerful and effective approach to develop these vaccines. setting clear roles, expectations, and accountability enables each partner to bring their respective strengths to the effort. shared purpose, trust, flexibility, and cooperation are critical to ensure success. as highlighted in this report and in another recent article 9 , the variability and complexity in regulatory processes and requirements interfere with the speed, flexibility, and efficiency needed to prepare for and respond to public health emergencies. there is an urgent need for global regulatory harmonization and standardization in the approach to the development of vaccines for emergency preparedness. regulatory agencies need to work together to remove roadblocks and put solutions in place to allow the rapid global development of vaccines for sars-cov-2 and other emerging pathogens. received: 19 may 2020; accepted: 3 june 2020; ebola: lessons on vaccine development accelerating vaccine development during the 2013-2016 west african ebola virus disease outbreak international federation of pharmaceutical manufacturers & associations. the complex journey of a vaccine ethics, emergencies and ebola clinical trials: the role of governments and communities in offshored research world health organization. ebola virus disease democratic republic of congo: external situation report 94 efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola ça suffit!) world health organization. roadmap for introduction and roll out of a licensed ebola vaccine developing covid-19 vaccines at pandemic speed. n. eng we thank all team members and management for their tireless efforts and contributions to this program. for their partnership and dedication, we thank external partners, collaborators, and funding organizations in addition to the study volunteers and study investigators. the authors also thank the who, fda, ema, avaref, and regulatory agencies that participated in the collaborative review procedure. we would also like to thank karyn davis of merck sharp & dohme corp., a subsidiary of merck & co., inc., kenilworth, nj, usa for editorial assistance. j.w. wrote the first draft of the manuscript. all co-authors provided critical inputs and revised the manuscript. all authors agreed to final submission. all authors are employees of merck sharp & dohme corp., a subsidiary of merck & co., inc., kenilworth, nj, usa (msd) and may own stock or stock options in the company. correspondence and requests for materials should be addressed to j.w.reprints and permission information is available at http://www.nature.com/ reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-319706-2e9jrv0s authors: ebinger, joseph e.; achamallah, natalie; ji, hongwei; claggett, brian l.; sun, nancy; botting, patrick; nguyen, trevor-trung; luong, eric; kim, elizabeth h.; park, eunice; liu, yunxian; rosenberry, ryan; matusov, yuri; zhao, steven; pedraza, isabel; zaman, tanzira; thompson, michael; raedschelders, koen; berg, anders h.; grein, jonathan d.; noble, paul w.; chugh, sumeet s.; bairey merz, c. noel; marbán, eduardo; van eyk, jennifer e.; solomon, scott d.; albert, christine m.; chen, peter; cheng, susan title: pre-existing traits associated with covid-19 illness severity date: 2020-07-23 journal: plos one doi: 10.1371/journal.pone.0236240 sha: doc_id: 319706 cord_uid: 2e9jrv0s importance: certain individuals, when infected by sars-cov-2, tend to develop the more severe forms of covid-19 illness for reasons that remain unclear. objective: to determine the demographic and clinical characteristics associated with increased severity of covid-19 infection. design: retrospective observational study. we curated data from the electronic health record, and used multivariable logistic regression to examine the association of pre-existing traits with a covid-19 illness severity defined by level of required care: need for hospital admission, need for intensive care, and need for intubation. setting: a large, multihospital healthcare system in southern california. participants: all patients with confirmed covid-19 infection (n = 442). results: of all patients studied, 48% required hospitalization, 17% required intensive care, and 12% required intubation. in multivariable-adjusted analyses, patients requiring a higher levels of care were more likely to be older (or 1.5 per 10 years, p<0.001), male (or 2.0, p = 0.001), african american (or 2.1, p = 0.011), obese (or 2.0, p = 0.021), with diabetes mellitus (or 1.8, p = 0.037), and with a higher comorbidity index (or 1.8 per sd, p<0.001). several clinical associations were more pronounced in younger compared to older patients (p(interaction)<0.05). of all hospitalized patients, males required higher levels of care (or 2.5, p = 0.003) irrespective of age, race, or morbidity profile. conclusions and relevance: in our healthcare system, greater covid-19 illness severity is seen in patients who are older, male, african american, obese, with diabetes, and with greater overall comorbidity burden. certain comorbidities paradoxically augment risk to a greater extent in younger patients. in hospitalized patients, male sex is the main determinant of needing more intensive care. further investigation is needed to understand the mechanisms underlying these findings. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) is now well recognized as the cause of the coronavirus disease 2019 (covid-19) global pandemic [1] [2] [3] . the rate of rise in covid-19 infection and its associated outcomes in the united states is now comparable to rates observed in other severely affected countries such as china, italy, and spain [4] [5] [6] [7] [8] [9] [10] . the spread of covid-19 in the united states has been especially pronounced in the states of california, new york, michigan, louisiana, and washington [11] . consistently reported across all regions is the observation that, of all individuals who become infected with sars-cov-2, a majority tend to have mild or no symptoms; however, an important minority will develop predominantly respiratory disease that can lead to critical illness and death [12] [13] [14] [15] . multiple, reports suggest that certain demographic and clinical characteristics may predispose infected persons to more severe manifestations of covid-19, such as older age, male sex, and pre-existing hypertension, pulmonary disease, or cardiovascular disease [4, [16] [17] [18] [19] . given that these traits tend to cluster among the same persons, the relative contribution of each trait to the risk for developing more severe presentations of covid-19 illness remains unclear. we conducted a comprehensive investigation of the pre-existing demographic and clinical correlates of covid-19 illness severity observed among patients evaluated for covid-19 within our multi-site healthcare system in los angeles, california. we deliberately focused our study on pre-existing characteristics for two main reasons: first, we recognize that patients with covid-19 illness can present early or late in the disease course, causing many clinical features to vary at the time of initial clinical encounter; and, second, we anticipate that ongoing public health efforts can be informed and augmented by understanding which predisposing factors may render certain segments of the population at higher risk for the most morbid sequelae of sars-cov-2 infection. the cedars-sinai health system is located in los angeles, california with a diverse catchment area of 1.8 million individuals, 33% of whom are over the age of 45 years and 80% identify as a racial or ethnic minority. the cedars-sinai health system includes cedars-sinai medical center (csmc), marina del rey hospital (mdrh), and affiliated clinics. for the current study, we included all patients who were found to have a laboratory confirmed diagnosis of sars-cov-2 infection while being evaluated or treated for signs or symptoms concerning for covid-19 at csmc or mdrh, beginning after the first confirmed case of community transmission was reported in the u.s. on february 26, 2020. subsequently, the first laboratory confirmed covid-19 case in our health system was on march 8, 2020. all laboratory testing for sars--cov-2 has been performed using reverse transcriptase polymerase chain reaction of extracted rna from nasopharyngeal swabs. all patient testing was performed by the los angeles department of public health until march 21, 2020, at which time the csmc department of pathology and laboratory medicine began using the a � star fortitude kit 2.0 covid-19 real-time rt-pcr test (accelerate technologies pte ltd, singapore). for the minority of patients in our study who had sars-cov-2 testing performed at an outside facility (3.6%), documentation of a positive test was carefully reviewed by our medical staff and considered comparable for accuracy. for all patients considered to have covid-19, based on direct or documented laboratory test result and suggestive signs and/or symptoms, we obtained information from the electronic health record (ehr) and verified data for the following demographic and clinical characteristics: age at the time of diagnosis; sex; race; ethnicity; smoking status defined as current versus prior, never, or unknown; comorbidities, including obesity, as clinically assessed and documented by a provider with icd-10 coding; and, chronic use of angiotensin converting enzyme (ace) inhibitor or angiotensin ii receptor blocker (arb) medications. chronic use of ace or arb medications was verified by confirming presence of documented ongoing medication use in an outpatient provider's clinic note along with presence of an active outpatient prescription for the medication, both dated from prior to covid-19 testing. we conducted iterative quality control and quality assurance analyses on all information extracted from the ehr; all data variables included in the main analyses were verified for completeness and accuracy through manual chart review, to avoid variable missingness or potential impact of inappropriate outliers in statistical modeling. because presenting clinical measures such as vital signs and laboratory values can be highly variable, based on timing of the original clinical presentation, we elected to focus on pre-existing traits that may predispose to covid-19 illness severity in a manner less dependent on the timing of patients presenting to medical care. to capture variation in relative comorbid status, in a way that is not captured by distinct medical history variables alone, we calculated the elixhauser comorbidity index (eci) with van walraven weighting for all patients based on all available clinical data [20] [21] [22] [23] . the eci uses 31 categories to quantify a patient's burden of comorbid conditions and has been shown to outperform other indices in predicting adverse outcomes (s1 table) [22] [23] [24] [25] [26] [27] [28] . for patients admitted to the hospital, length of stay, admission to an intensive care unit (icu) and death were ascertained from time stamps recorded for admission, unit transfers, and discharge. interventions such as intubation and prone positioning were identified through time stamped orders in the ehr and verified by manual chart review. dates and times of onset for reported or observed relevant signs and/or symptoms were also determined via manual chart review. all care was provided at the discretion of the treating physicians. our outcomes for this study included: severe illness (defined as requiring any kind of hospital admission), critical illness (defined as the need for intensive care during hospitalization), and respiratory failure (defined as the need for intubation and mechanical ventilation). the csmc institutional review board approved all protocols for the current study and waived the requirement for informed consent. for the total sample of covid-19 patients, we used parametric tests to compare normally distributed continuous variables and non-normally distributed or categorical variables, respectively. we also used histograms to display age and sex distribution for the total cohort, the patients admitted but not requiring intensive care, and patients requiring intensive care at any time during hospitalization, and the patient requiring intubation and mechanical ventilation at any time during hospitalization. we used ordinal logistic regression to examine the associations between pre-existing characteristics (based on clinically relevant, non-missing data) and a primary outcome measure of illness severity, defined as an illness severity score. we constructed the illness severity score, with higher values assigned to needing more intensive levels of clinical care, based on the following stepwise categories: 0 = clinically deemed to not require admission; 1 = required hospital admission but never required intensive care; 2 = required intensive level care but never intubation; and, 3 = required intubation during hospitalization. we constructed age-and sex-adjusted models, from which significantly associated covariates (based on p<0.20) were selected for inclusion in the final multivariable-adjusted models, where appropriate (i.e. smaller sample sizes). race was treated as a binary covariate: african american and non-african american. this approach was selected given the recently reported concerns of excess risk for african americans [29] , along with limited understanding of whether or not comorbidities contribute to this risk, in addition to the sample size for other race groups being too small for certain comparisons. because hypertension and diabetes are not calculated as substantial contributors to the elixhauser comorbidity index, we included each of these traits as separate additional covariates in all multivariable-adjusted analyses. we calculated the variance inflation factor (vif) for each of the predictor variables to confirm absence of any substantial multicollinearity. in secondary analyses, we analyzed the associations of pre-existing patient characteristics with the distinct outcomes of needing any hospital admission (severe illness) and, in the cohort of all hospitalized patients representing an especially vulnerable population, the need for intensive care (critical illness) or intubation (respiratory failure). all analyses were performed using r, version 3.5.1 (r foundation for statistical computing) and stata, version 15 (statacorp). for all final models, p values were 2-sided and considered significant at threshold level of 0.05. regional analyses showed that patients presented to our healthcare system from across a broad geographic catchment area in los angeles county (s1 fig) . the demographic and clinical characteristics of all patients in our study sample are shown in table 1 . of all patients with pharmacologically treated hypertension, a minority were taking ace inhibitor or arb class agents and a majority were taking anti-hypertensive medications from alternate classes. overall, almost half of patients (n = 214, 48%) were clinically assessed to require hospital admission, of whom over a third (n = 77; 36%) required intensive care and almost a quarter (24.3%) required intubation. in unadjusted analyses, the patients who were more likely to require higher levels of care tended to be older, male, african american, and with known hypertension, diabetes mellitus, higher elixhauser comorbidity index, and have prior myocardial infarction or heart failure ( table 1 ). the number of men with confirmed covid-19 infection outnumbered women in nearly all age groups; this sex difference was more pronounced among patients requiring hospitalization and particularly among patients requiring intensive care or intubation (fig 1) . we also observed a consistently higher rate of greater illness severity among african americans compared to persons of other racial groups (fig 2) . for the primary outcome of illness severity, categorized by escalating levels of care (i.e., hospitalization, intensive care, intubation), the pre-existing characteristics that demonstrated statistical significance in age-and sex-adjusted models included older age, male sex, african american race, obesity, hypertension, diabetes mellitus, and the elixhauser comorbidity score ( table 2 ; fig 3) . the associations that remained significant in the fully-adjusted multivariable model included older age (odds ratio [or] 1.49 per 10 years, 95% confidence interval [ci] 1.30-1.70, p<0.001), male sex (or 2.01, 95% ci 1.34-3.04, p = 0.001), african american race (or 2.13, 95% ci 1.19-3.83, p = 0.011), obesity (or 1.95, 95% ci 1.11-3.42, p = 0.021), diabetes mellitus (or 1.77, 95% ci 1.03-3.03, p = 0.037) and the comorbidity score (or 1.77 per sd, 95% ci 1.37-2.28, p<0.001). we also observed a trend towards lower severity of illness among patients chronically treated with ace inhibitor therapy, with or 0.48 (95% ci 0.22-1.04; p = 0.06). each estimated or value represents the increment in higher (or lower) odds of a patient requiring a next higher level of care, for every unit difference in a continuous variable (e.g. per 10 years of age) or for presence versus absence of a given categorical variable (e.g. male sex). in effect, every 10 years of older age was associated with~1.5-fold higher odds of requiring a higher level of care, and being male versus female was associated with a~2-fold higher odds of requiring higher level care. we used the brant method to test the proportional odds assumption for consistency of associations across our ordinal outcome; these analyses revealed no substantial qualitative violations, but did indicate that the elixhauser score was predominantly associated with the specific outcomes of admission versus non-admission (or 4.34, p<0.001) and need for intensive care versus no intensive care need (or 1.55, p = 0.008) that with the less frequent outcome of needing intubation versus no need for intubation (or 1.24, p = 0.25). for the specific outcome of needing any hospital admission, the pre-admission characteristics that demonstrated statistical significance included older age, male sex, african american race, obesity, hypertension, diabetes mellitus, the elixhauser comorbidity index, and prior myocardial infarction or heart failure (s2 table) . in the multivariable model adjusting for all key covariates, the pre-existing traits that remained significantly associated with needing any hospital admission were older age, diabetes mellitus, and higher comorbidity index. among the patients whose illness severity required hospitalization, male sex was associated with the outcome of requiring further escalating levels of care (i.e., intensive care and intubation) ( table 3 ; fig 3) . in the multivariable model adjusting for key covariates, male sex remained the single most important risk marker of requiring higher-level care (or 2.53, 95% ci 1.36-4.70, p = 0.003). the results for male sex were similar for the individual outcomes of requiring intensive care or intubation (s3 table) . we again observed a trend towards lower need for admission to the intensive care unit among patients chronically taking an ace inhibitor (or 0.38, 95% ci 0.13-1.17, p = 0.09), and greater need for intubation among african americans patients (or 2.14, 95% ci 0.99-4.64, p = 0.053). in secondary analyses, we used multiplicative interaction terms to assess for effect modification for associations observed in the main analyses (s4 table) . while considered exploratory pre-existing traits associated with covid-19 illness severity or hypothesis generating analyses, we found several interactions of potential interest (fig 4) . in particular, the associations of hispanic ethnicity, obesity, diabetes, and elixhauser comorbidity index with the primary outcome appeared paradoxically more pronounced in younger compared to older individuals (s5 table) . by contrast, the primary outcome was more pronounced among older compared to younger african americans. also paradoxically, hypertension appeared associated with greater risk in non-obese patient and with lower risk in obese patients. we repeated all main analyses with additional adjustment for smoking status in the subset of patients with available data on smoking; in these models, all significant results remained unchanged (s6 and s7 tables). we examined the pre-existing characteristics associated with severity of covid-19 illness, as observed thus far in our healthcare system located in los angeles, california. we found that almost half of patients presenting for evaluation and then confirmed to have covid-19 were clinically assessed to require hospital admission. these higher risk individuals were more likely to be older, male, african american, obese, and have diabetes mellitus in addition to a greater overall burden of medical comorbidities. notably, chronic use of an ace inhibitor appeared related to lower illness severity, in the absence of a similar finding for arb use. among all individuals requiring inpatient care for covid-19, male patients had a greater than 2.5-fold odds of needing intensive care and a 3.0-fold odds of needing intubation. all of our findings were observed even after accounting for co-existing risk factors and chronic medical conditions. recognizing that patients with covid-19 illness can present with clinical features that vary based on timing of the index encounter, we sought to identify the pre-existing traits that render some individuals at highest risk for developing the more severe forms of covid-19 illness once contracted. in our u.s. based metropolitan community, we observed that both obesity and diabetes mellitus are predisposing factors associated with a greater odds of needing hospital admission for covid-19 but not of requiring further escalation of care; this finding is consistent with emerging reports of obesity and diabetes mellitus each being associated with a greater risk for pneumonia due to covid-19 as well as other community-acquired viral agents-particularly in areas of the world where obesity is prevalent [30] [31] [32] [33] [34] [35] . also consistent with worldwide reports, we observed that older age is a significant predisposing risk factor for greater covid-19 illness severity in multivariable-adjusted models; this finding may represent an age-related immune susceptibility that is not completely captured by even a comprehensive comorbidity measure such as the elixhauser index. notwithstanding an overall age association in the expected direction of risk, we also found a paradoxical age interaction for certain key correlates. in effect, presence of obesity, diabetes, or an elevated overall comorbidity index were each associated with greater covid-19 illness severity in younger (i.e. <52 years) compared to older age groups. while unexpected, this finding is actually consistent with the known reduction of ace2 expression with advancing age, a phenomenon that has been proposed as a major contributor to the broad susceptibility to covid-19 seen in younger to middle aged individuals across the population at large [36] . consistent with worldwide reports, we found that the association of male sex with greater odds for every metric of covid-19 illness severity was especially prominent-and this was not explained by age variation, risk factors, or comorbidities [37] . reasons for the male predominance of illness severity remain unclear. although ace2 genetic expression is on the x chromosome, evidence to date would suggest relatively comparable expression levels between sexes relative risks associated with illness severity score are shown for all associations observed in the total sample (n = 442), stratified by subgroups defined by age (younger vs. older than median age 52 years), sex, and obesity (bmi �30 kg/m 2 ). � the primary outcome of covid-19 illness severity score in the total sample was defined as an ordinal variable wherein: 0 = referent, 1 = required admission but never icu level care, 2 = required icu level care but never intubated, 3 = required intubation. �� p for interaction values were calculated from likelihood ratio test between models with and without the interaction term. for each variable in the list, age (<versus �median age of 52 years), sex, and obesity interaction terms are implemented in multivariable adjusted models, with other covariates representative of the entire cohort. https://doi.org/10.1371/journal.pone.0236240.g004 [38, 39] , albeit with some potential for variation in relation to differences in sex hormones; select animal studies have shown increased ace2 activity in the setting of ovariectomy and the opposite effect with orchietomy [40, 41] . while there remains scant data currently available to explain sex differences for covid-19, male sex bias was also observed for sars and mers [42, 43] . similar to the findings in our study, this increase risk was not attributable to a greater prevalence of smoking among men. notably, prior murine studies have also demonstrated male versus female bias in susceptibility to sars-cov infection, which may be related to the effects of sex-specific steroids and x-linked gene activity on modulation of both the innate and adaptive immune response to viral infection [44] . further research specific to the sexual dimorphism seen in sars-cov-2 susceptibility is needed. in our u.s. based metropolitan community, we also observed racial and ethnic patterns of susceptibility to greater covid-19 illness severity. specifically, we found that african americans were at greater risk for needing higher levels of care overall, and this vulnerability appeared more pronounced in older age and among men. although an overall risk association was not seen for hispanic ethnicity, there was a trend towards greater covid-19 illness severity in younger aged compared to older aged hispanic/latino persons. a recent national report from the cdc also suggests overall higher rates of covid-19 susceptibility in african americans, and our findings confirm this trend exists even after adjusting for age, risk factors, and comorbidities [45] . in addition to the effects of unmeasured socioeconiomic and healthcare access variables, racial/ethnic disparities in covid-19 illness severity may relate to yet unidentified host-viral susceptibility factors that could also be contributing to heterogeneity of community transmission seen across regions worldwide and populations at large [29] . the use of ace inhibitor or angiotensin receptor blocker (arb) medications has been a focus of attention given that these agents may upregulate expression of ace2, the viral point of entry into cells [46] and alveolar type 2 epithelial cells in particular [47] . alternatively, these agents may confer benefit, given that sars-cov-2 appears to reduce ace2 activity and lead to potentially unopposed excess renin-angiotensin-aldosterone activation [36, 46, 48] . although we observed a non-significant trend in association of chronic ace inhibitor treatment with lower covid-19 illness severity, we found evidence of neither risk nor benefit with arbs. together, our findings are supportive of current recommendations to not discontinue chronic ace inhibitor or arb therapy for patients with appropriate indications for these medications. several limitations of our study merit consideration. our cohort included all individuals who underwent laboratory testing for covid-19 and not individuals who did not undergo testing; thus, our study results are derived from individuals presenting with symptoms that were deemed severe enough to warrant testing. all data including past medical history data were collected from the ehr and, thus, subject to coding bias and variations in reporting quality. to minimize the potential effects of these limitations that are inherent to ehr data, we performed iterative quality checks on the dataset and conducted manual chart review to verify values for key variables. we recognize that the illness severity outcomes defined as clinically ascertained need for hospital admission, icu level care, and intubation, may vary from practice to practice. as in many other u.s. medical centers affected by the covid-19 pandemic, our clinical staff have been practicing under institutional guidance to conserve resources and we anticipate that the thresholds for escalating care are likely comparable; thresholds for admission, transfer to intensive care, and intubation may be different in more resource constrained environments. given the relatively small number of observed in-hospital deaths (n = 11), and thus limited statistical power to detect associations, we deferred analyses of pre-existing characteristics and mortality risk to future investigations. the modest size of this early analysis of our growing clinical cohort may have limited our ability to detect potential additional predictors of covid-19 illness severity, as well as potential interactions or effect modification relevant to the outcomes; thus, further investigations are needed in larger sized samples. finally, our results are derived from a single healthcare system, albeit multi-center and serving a large catchment of the diverse population of los angeles, california. additional studies are needed to examine the extent to which our findings are generalizable to other populations affected by covid-19. in summary, we found that among patients tested and managed for laboratory confirmed covid-19 in our healthcare system to date, approximately half require admission for inpatient hospital care. these individuals are more likely to be older, male, african american, obese, and with known diabetes mellitus as well as a greater overall burden of medical comorbidities. well over a third of hospitalized patients require intensive care, with a substantial proportion needing intubation and mechanical ventilation for respiratory failure. among hospitalized patients, the highest risk individuals were more likely to be predominantly men of any age or race-for reasons not explained by comorbidities. further investigations are needed to understand the mechanisms underlying these associations and, in turn, determine the most optimal approaches to attenuating adverse outcomes for all persons at risk. (docx) s1 fig. los angeles county regional distribution of all patients with covid-19. the patients treated in our healthcare system for covid-19 illness presented from across a diverse regional distribution of residential locations across los angeles county. the map shown was generated using arcgis software by esri. (tif) the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 coronavirus covid-19 has killed more people than sars and mers combined, despite lower case fatality rate a novel coronavirus from patients with pneumonia in china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan global epidemiology of coronavirus disease 2019 (covid-19): disease incidence, daily cumulative index, mortality, and their association with country healthcare resources and economic status first case of 2019 novel coronavirus in the united states coronavirus disease 2019 (covid-19) in italy rapidly increasing cumulative incidence of coronavirus disease (covid-19) in the european union/european economic area and the united kingdom initial public health response and interim clinical guidance for the 2019 novel coronavirus outbreak-united states case-fatality rate and characteristics of patients dying in relation to covid-19 in italy an interactive web-based dashboard to track covid-19 in real time clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of coronavirus disease 2019 in china clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province. chinese medical journal clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china. intensive care med risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention a new method of classifying prognostic comorbidity in longitudinal studies: development and validation updating and validating the charlson comorbidity index and score for risk adjustment in hospital discharge abstracts using data from 6 countries comorbidity measures for use with administrative data a modification of the elixhauser comorbidity measures into a point system for hospital death using administrative data coding algorithms for defining comorbidities in icd-9-cm and icd-10 administrative data systematic review of comorbidity indices for administrative data comparison of the elixhauser and charlson/deyo methods of comorbidity measurement in administrative data comparison of the performance of two comorbidity measures, with and without information from prior hospitalizations the accuracy of administrative data for identifying the presence and timing of admission to intensive care units in a canadian province covid-19 and african americans covid 19 and the patient with obesity-the editors speak out. obesity (silver spring) covid-19 pandemic, corona viruses, and diabetes mellitus covid-19 and diabetes mellitus: unveiling the interaction of two pandemics covid-19 and diabetes diabetes is a risk factor for the progression and prognosis of covid-19 high prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (sars-cov-2) requiring invasive mechanical ventilation the dilemma of coronavirus disease 2019, aging, and cardiovascular disease: insights from cardiovascular aging science host susceptibility to severe covid-19 and establishment of a host risk score: findings of 487 cases outside wuhan age-and gender-related difference of ace2 expression in rat lung renal ace2 expression in human kidney disease sex hormones promote opposite effects on ace and ace2 activity, hypertrophy and cardiac contractility in spontaneously hypertensive rats postnatal ontogeny of angiotensin receptors and ace2 in male and female rats the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health do men have a higher case fatality rate of severe acute respiratory syndrome than women do? sex-based differences in susceptibility to severe acute respiratory syndrome coronavirus infection hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019-covid-net, 14 states renin-angiotensin-aldosterone system inhibitors in patients with covid-19 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome we are grateful to all the front-line healthcare workers in our healthcare system who continue to be dedicated to delivering the highest quality care for all patients. key: cord-310058-tp42rgmv authors: zhu, yuan-shou; shao, ning; chen, jian-wei; qi, wen-bao; li, yang; liu, peng; chen, yan-jing; bian, su-ying; zhang, yan; tao, sheng-ce title: multiplex and visual detection of african swine fever virus (asfv) based on hive-chip and direct loop-mediated isothermal amplification date: 2020-10-08 journal: anal chim acta doi: 10.1016/j.aca.2020.10.011 sha: doc_id: 310058 cord_uid: tp42rgmv african swine fever is caused by african swine fever virus (asfv), and has a mortality rate approaching 100%. it has already caused tremendous economy lost around the world. without effective vaccine, rapid and accurate on-site detection plays an indispensable role in controlling outbreaks. herein, by combining hive-chip and direct loop-mediated isothermal amplification (lamp), we establish a multiplex and visual detection platform. lamp primers targeting five asfv genes (b646l, b962l, c717r, d1133l, and g1340l) were designed and pre-fixed in hive-chip. on-chip lamp showed the limits of detection (lod) of asfv synthetic dnas and mock samples are 30 and 50 copies per microliter, respectively, and there is no cross-reaction among the target genes. the overall performance of our platform is comparable to that of the commercial kits. from sample preparation to results readout, the entire process takes less than 70 min. multiplex detection of real samples of asfv and other swine viruses further demonstrates the high sensitivity and specificity of hive-chip. overall, our platform provides a promising option for on-site, fast and accurate detection of asfv. african swine fever (asf), caused by african swine fever virus (asfv), is a highly contagious and lethal disease which manifests the symptom of acute hemorrhagic fever in domestic pigs and wild boars [1] [2] [3] . since asf was first reported in kenya in 1921, it has rapidly spread around many countries of africa, europe, america and asia [4] [5] [6] . in august 2018, the first asf outbreak in china was identified in shenyang city, liaoning province and spread to many provinces in a fast way [7] . by 5 march 2020, 165 outbreaks were reported in 32 provinces and about 1,193,000 pigs have been culled to halt further expansion, posing a devastating impact on both local and the global swine industry [8] . asfv is an enveloped double-stranded dna virus transmitted by soft ticks. asfv has a giant genome of 170-194 kb and possesses a multilayered structure and overall icosahedral morphology [9, 10] . because of complex genetic composition and high variabilities, asfv is currently classified into 24 genotypes and more subtypes [11] . there is no effective treatment or vaccine available, thus early and accurate diagnosis of asfv is crucial for rapid control of outbreaks. more recently, by comparing the genome of asfv china/2018/anhuixcgq strain and related european genotype ii strains, bao et al. found 54-107 variation sites contributing to the alteration of amino acid residues in 10-38 genes, which put forward the high requirements for accurate and mutation-tolerant detection [12] . the current methods for asfv detection can be generally divided into two types: 1) the immunology detection methods, including haemadsorption test (had) [13] , enzyme-linked immunosorbent assay (elisa) [14] , fluorescence antibody test (fat) [15] , and lateral flow assay (lfa) [16] . although had is reliable and effective, they often take several days to get the results and need high-quality tissues for preparing cells. elisa and fat are rapid and convenient methods for precisely recognizing viral antibodies, but this serological diagnosis may not suitable for accurate early diagnosis when there is very low level of asfv antibodies before seroconversion [17] . 2) the molecular biology detection methods, including many kinds of polymerase chain reaction (pcr) [18] [19] [20] , invader assay [21] , loop-mediated isothermal amplification (lamp) [22, 23] , and recombinase polymerase amplification (rpa) [24] . as a gold standard for asfv detection in the laboratory, pcr requires thermal cycling instruments and skilled operators, which is not ideal for j o u r n a l p r e -p r o o f resource-limited situations. with no need for well-equipped laboratory and professional operations, isothermal amplification has emerged as a promising on-site detection method. recently, a variety of novel isothermal amplification assays in combination with clustered regularly interspaced short palindromic repeats (crispr) and lateral flow assay (lfa) have been developed for asfv diagnosis [25] [26] [27] [28] . these methods achieved higher sensitivity and specificity, and could be adaptable for point-of-care testing (poct). nevertheless, the separation of amplification and detection in open space makes it possible to generate false-positive results due to aerosol contaminations. without amplification, a high-throughput detection system based on crispr-cas12a is also established for asfv detection, but it is inconvenient to transfer the cas12a-cleaved product from a water bath into a disposable cartridge for detection [29] . last but not least, all methods aforementioned are usually focused on detecting only one gene target at a time, such as b646l encoding viral protein p72 (vp72) [24] [25] [26] [27] [28] [29] , 9gl encoding sulfhydryl oxidase [20] , p1192r encoding topoisomerase ii [22] , or k78r encoding dna binding protein p10 [23] , which may lead to false-negative results because of possible mutations on the complex, highly variable genomes of asfv. for example, gallardo et al. found the oie (world organization for animal health)-recommended conventional pcr showed reduced sensitivity when detecting field and experimental samples because of the nucleotide mismatches between the primers and the asfv target gene [15] . to sum up, the need for rapid, sensitive, integrated, and multiplex detection of asfv without thermal cycler has not yet been satisfied. one target gene for each pathogen, which may not suitable to accurately detect variable targets, such as the viral nucleic acid. herein, we provide a multiplex and visual detection platform based on capillary array and direct lamp for rapid and accurate detection of asfv. given the complex and variable properties of asfv genomes, we choose five conserved genes (b646l, b962l, c717r, d1133l, and g1340l) as the target for detection, avoiding the false-negative results in virtue of the mutations of a certain gene. the profile of capillary array looks like a hive, so we named it "hive-chip". different sets of lamp primers are pre-fixed in each capillary. without nucleic acid extraction, naoh-treated swine blood sample is mixed with lamp reagents directly. due to the outer surface of the hive-chip is hydrophobic and the inner surface of the capillary is hydrophilic, lamp mixtures can be simultaneously loaded and dispersed into each capillary by capillary forces. then the hive-chip is incubated at 63 for 1 h with sealed optical film and the fluorescence signals are visually detected with 365 nm uv-light. to our knowledge, there is no multiplex detection platform employing multiple targets presently available for asfv detection. hive chip-based multiplex detection platform greatly enhances detection accuracy and shows superior characteristics, such as high sensitivity, compatibility with isothermal amplification, on-site detection capability, thus possesses the potential to become a major platform for animal or zoonotic epidemics monitoring. fever regional laboratory, south china agricultural university (guangzhou, china). the genomic dna samples of other swine viruses, including dsdna virus prv (pseudorabies virus), ssdna virus pcv2 (porcine circovirus type 2) and ppv (porcine parvovirus), are obtained from huazhong agricultural university (wuhan, china). according to the multiple sequence alignment of asfv china/2018/anhuixcgq (genbank: mk128995.1) with other genotype ii asfv strains available in ncbi database, we chose five target genes (b646l, b962l, c717r, d1133l and g1340l) based on the function type, conservation, and the length of sequences (data is partially shown in fig. s1 and table s1 ). meanwhile, we aligned two pairs of oie (world organization for animal health)-recommended pcr primers (i.e., oie-f/oie-r and ppa-1/ppa-2) with the target sequence (data is shown in fig. s2 ). to screen more effective primers, we used primerexplorer v5 (http://primerexplorer.jp/e/) to design four primer sets for each target gene. all primers were synthesized by sangon biotech (shanghai, china). all gene fragments were synthesized and constructed into pmv vector by qinglan biotech (wuxi, china) . the plasmid of mutated b646l gene was constructed using the quikchange site-directed mutagenesis kit (primers are listed in table s4 ). the procedure for fabricating the hive-chip mainly consists of three parts: the cleaning of capillaries, the assembly of hive-chip, and the super-hydrophobic modification. firstly, quartz capillaries with 1.0 mm outer diameter and 0.7 mm inner diameter were cut into 4 mm sections (with a volume of ∼1.6 μl) by a diamond wire cutting machine. then the short capillaries were thoroughly cleaned with piranha solution (h 2 so 4 /h 2 o 2 =3:1, v/v) for at least 0.5 h, washed by pure water and ethanol, and dried in an oven. secondly, seven preprocessed capillaries were inserted into a pdms support to 3.5 mm deep as demonstrated in fig. 1 , which carried ten arrayed through-holes and was modified by ultra-ever dry top coat. according to a previous study [30] , the pdms support was made by a tubular mold with 10 patterned columns of 1.0 mm diameter inside and cut to 4 mm in length. thirdly, the j o u r n a l p r e -p r o o f super-hydrophobic modification was the key step for simultaneously sample loading. to be specific, 15 μl ultra-ever dry top coat was loaded carefully on the top surface of the pdms support and the outer surfaces of the exposed part of capillaries. after that, the modified hive-chip was air-dried for use. all the lamp primer sets for asfv detection are listed in table s2 were also electrophoresed on a 2 % agarose gel to generate the predicted fragments. on-chip lamp adopted the same reaction system as the protocol described above, except that bst dna polymerase was 1.5× (namely 0.48 u μl −1 ) and no primers were added, because 1.6 μl of the primer set dissolved in 0.05% chitosan was already individually pre-fixed in the corresponding capillary. one blank capillary with no primers were employed as the negative control (nc). to simultaneously introduce the lamp mixture into all seven capillaries, an adapter for sample loading was produced as our previously reported [30] (fig. 1) . the adapter had a funnel-shaped inlet connected with a shallow inverted dish, which could cover the exposed parts of all capillaries in the hive-chip. when the standard 100 μl pipet tip was inserted into the inlet, the lamp mixture was gently injected and dispersed into all capillaries by capillary forces. then the adapter was removed with the locked tip and the plate well was absolutely sealed by an optical film. finally, the hive-chip was incubated at 63 °c for less than 60 min in an incubator and visually detected by a hand-held uv-light device. j o u r n a l p r e -p r o o f swine blood samples were collected in anticoagulant (edta-coated) tubes, followed by mixing of the samples with 100 mm naoh in a ratio of 1:2, with a final volume of 30 μl, and incubation at room temperature for 3 min [33] . after that, 1 μl of the mixture was pipetted into lamp reaction mixtures of 25 μl for subsequent amplification. once the lamp reactions finished, the fluorescent images of the hive-chip were captured by a digital camera or a smartphone. for quantitative analysis, the fluorescent intensity data was extracted by genepix pro 6.1 software (molecular devices, ca, usa). signal intensity was set as mean of foreground subtracted by mean of background for each capillary. signal-to-noise ratio (snr) was defined as the ratio of signal intensity of the target to signal intensity of the negative control, and the cut-off was set as snr>1.5. triplicate tests were performed for samples to verify the reproducibility of the hive-chip. to optimize the detection procedure, a direct lamp method without nucleic acid extraction was adopted [33] . as depicted in fig. 1 , minimal operation and only 3 steps are required. in the first step, swine blood samples were collected in anticoagulant tubes, followed by mixing of the samples with 100 mm naoh in a ratio of 1:2, and incubated at room temperature. the mixtures were directly used as the template for subsequent amplification. in the second step, the lamp reaction mixtures containing the pre-processed samples were injected into an inlet of the adapter that connects to the hive-chip. the mixtures could be uniformly dispersed into all capillaries by capillary forces. the adapter was then removed with the locked pipet tip and the plate was fully sealed by an optical film, providing a closed space for amplification and avoiding the aerosol contamination. in the third step, the lamp reactions were performed at 63 °c for less than 60 min in an incubator. the fluorescence signals of the amplifications were excited by a hand-held uv-light j o u r n a l p r e -p r o o f device and imaged by a digital camera or a smartphone for analysis. importantly, the entire process from sample collection to the readout of the results takes less than 70 min. the genome comparison of a dominant strain of genotype ii asfv to design the optimal primer sets for lamp with high specificity, high sensitivity, and robust for a wide range of detections, we set several principles for target genes selection: (1) since it is known that most of the variations among asfv genomes results from gain and loss of the mgf members [35] , mgfs were excluded; (2) the target genes were selected from different functional groups as diversified as possible; (3) the length of the target genes was bigger than 500 bp, the purpose is to ensure multiple sequence alignment to obtained conserved sequences in all known strains listed in genbank database. next, we performed a multiple sequence alignment analysis of all eligible genes and eventually chose five conserved genes for lamp primers design, including b646l, b962l, c717r, d1133l and g1340l (data is partially shown in fig. s1 and table s1 ). although viral protein p72 (vp72) encoded by b646l is the most frequently used gene target for asfv detection [24] [25] [26] [27] [28] [29] , many mutations are still found in different strains. besides, we also aligned two pairs of oie (world organization for animal health)-recommended pcr primers (i.e., oie-f/oie-r and ppa-1/ppa-2) with the target sequence in b646l and found many nucleotide mismatches (fig. s2) , which was consistent with the findings from other studies [15, 36] . to avoid the possible false positive detection because of gene mutations in a single gene, it is necessary to perform multi-targets detection. to assure the success of obtaining optimal primer set, we designed four primer sets for j o u r n a l p r e -p r o o f each gene (table s2 and s3). evaluation of the primers were divided into two aspects: validity tests and specificity tests. two detection methods were used to analyze the lamp products: visual detection with fluorescent reagent and gel electrophoresis. for validity tests ( fig. 2a) , all four primer sets were effective in amplification of the target gene including b646l, b962l, d1133l and g1340l. both fluorescence detection and gel electrophoresis showed the positive reaction. but only one primer set could successfully amplify the target gene c717r, in return, this prove the necessity of designing multiple sets of primers for a given target. overall, we obtained at least one set of effective lamp primers for each of the five asfv target genes. to access the specificity, every primer set was used to amplify all the five genes ( fig. 2b and table s2 ). the results clearly showed that only when a given set of primers and the corresponding template were added, expected signals were then observed for both fluorescent visual detection and gel electrophoresis, indicating that in-tube lamp was able to accurately and specifically identify the corresponding asfv targets. to verify the specificity on chip, lamp mixtures containing one of the five synthetic dnas were evenly distributed into capillaries of the hive-chip. further, we set an unrelated lamp primer set, which is specific for sad1 gene [30] , as the positive control in each capillary array to demonstrate the validity of lamp reaction. all the results were as expected (fig. 3a) . for each synthetic dna, only the capillary containing the corresponding pre-fixed primers showed positive signals and the rest were negative. for example, for b646l gene, apart from positive control (pc), the bright green signal was obtained only in capillary "1" pre-fixed with the matched primers. similarly, the other four genes also showed specific amplification results, which were consistent with in-tube lamp reactions and demonstrated high specificity. to evaluate the sensitivity, five dna templates were mixed in one-pot and loaded at the same time, resulting in positive signals from all the capillaries with asfv primers in the hive-chip. as shown in fig. 3b , serially diluted dna mixtures including 880, 88, and 30 copies per microliter were successfully detected. and a sensitivity of lower than 15 copies per microliter was only achieved for the target j o u r n a l p r e -p r o o f genes c717r and d1133l, so the limit of detection (lod) for the current hive-chip was around 30 copies per microliter, i.e., 48 copies per reaction. according to the oie (world organization for animal health) manual of standards for diagnostic test and vaccine [37] and the validation of actual samples [36, 38] , the sensitivity of oie-recommended pcr is about 600 copies per reaction. in addition, 30 copies per microliter of dna could be easily obtained from acute infection of asfv [39] . these facts demonstrating that the sensitivity of hive-chip is suitable for asfv detection in real world. by optimizing the reaction conditions and lamp primers, we believe that higher sensitivity could be achieved for hive-chip. sample treatment is one of the key steps in nucleic acid testing (nat) systems. although nucleic acid extraction can provide high-quality and relatively pure templates for the down-stream analysis, it also complicates the procedure and enhances the risk of nucleic acid loss and cross-contamination, which is not suitable for point-of-care tests (pocts). to simplify nucleic acid extraction, liu et al. [33] developed an accurate, rapid and easy-to-use snp detection method based on lamp that do not require dna extraction from whole blood, dried blood spot, buccal swab and saliva, this method is known as direct-lamp. according to their results, naoh treatment could effectively lead to cell lysis, followed by release of dna for use in lamp amplification. meanwhile, various dna polymerase inhibitors in whole blood, such as hemoglobin, igg and proteases can be inactivated by the treatment with naoh solution. to further verify the applicability of hive-chip, the best way is to test deactivated real sample in blood collected from asfv infected pigs. however, due to safety issue and highly restricted regulation of the government, it is very difficult to collect real samples. thus, we decided to mimic the real sample. we prepared a set of mock samples by spike all the five synthetic dna templates into edta-blood collected from a healthy pig. the dna templates were set as 3000, 300, 100, 50, 30 and 0 copies per microliter. because asfv is a virus with dsdna genome, and the viral particle is very easily to be disrupted [25, 26] , we believe these mock samples are very close to the real sample. by applying the mock samples, we conducted the entire analytical process from sample treatment to fluorescent readout in the hive-chip j o u r n a l p r e -p r o o f following the protocol shown in fig. 1 . as what we expected, five genes were successfully detected from this blood sample at the low limit of detection (lod) of 50 copies per microliter (fig. 4a) , which were comparable with the results acquired from the pure plasmid samples. the quantitative analysis of the signal intensity obtained from the corresponding hive-chip (e.g., 3000 copies and 30 copies per microliter) further confirmed the reliability of results. it may due to the complex background of swine blood that a sensitivity of lower than 30 copies per microliter was only achieved for the target gene b962l, c717r and d1133l. but from the perspective of detection, as long as the sensitivity of one gene reaches the lod, asf will not be missed. furthermore, to avoid that the complex background of swine blood would generate false-positive, we set the healthy blood sample with no target genes (namely 0 copies per microliter) as a control. according to the results, no capillaries showed bright green fluorescence apart from positive control, which proved the capability of performing analysis of more complicated samples of asfv. to clearly display the linear detection ranges and detection limits of different genes, we also drew sensitivity plot for each gene (fig. 4b) . although the signal advancement of the hive-chip line charts exhibited positive correlated with the concentration of the targets, they did not show good linear relationship, this could be explained by the fact that lamp is intrinsically not a linear amplification method. the fluorescence of c717r gene at 30 copies per microliter was a little bit higher than that at 50 copies per microliter, which might result from the fluctuation of low copies templates in complex background (fig. 4b) . although the relatively conserved sequence (e.g., b646l) of asfv has been chosen for detection, we can still find that the mutable viral genomes bring many mismatches between the primers and the target sequence ( fig. s1 and s2) . to further prove that the detection performance of hive-chip was comparable with commercial kits, pcr kit and lamp kit (yoyoung biotech) approved by china animal disease control center for asfv detection were also tested using mock samples of serial dilutions. as shown in fig. s3 , the limit of detection (lod) of both pcr kit and lamp kit was about 100 copies per reaction. by contrast, the sensitivity of hive-chip was 50 copies per microliter, i.e., 80 copies per reaction, which was comparable with that of these commercial kits. in addition, the sensitivity of our platform meets the international standard, i.e., the recommended oie manual of standards for diagnostic test and vaccine, i.e., approximately 600 copies per reaction [36] [37] [38] . as previously reported, zhao et al. [39] adopted qpcr to quantify vp72 gene copies of the blood samples from three infectious pigs. after 3 days infection, the vp72 gene copies can reach more than 10 7 copies per milliliter (i.e. 10 4 copies per microliter), which is far above the limit of detection of hive-chip. this also confirmed the practicability of our platform. actually, we can also increase the input of blood in the lamp mixtures to improve the sensitivity of detection when it is necessary. moreover, both oie pcr and commercial kits (yoyoung biotech) can only detect one gene (b646l) at a time and require heavy instrumentation for fluorescence detection. while the lamp kit needs an extra incubation at 80 °c for 3 min for sample preparation to release the nucleic acids. in the contrast, our platform is capable of monitoring multiple targets in a single test. the results could be directly visualized, and no sophisticated sample preparation is needed. to further demonstrate the capability of hive-chip for asfv detection, we collected three asfv genomic dna samples in complex mixtures. as shown in fig. 5a , all of them were successfully detected by hive-chip. moreover, nine genomic dna samples of other major swine viruses, including pseudorabies virus (prv), porcine circovirus type 2 (pcv2), and porcine parvovirus (ppv), were also obtained for specificity testing. as shown in fig. 5b and s4 , the assays exhibited no cross-reaction with asfv. these results indicate the high specificity of hive-chip. to clearly demonstrate the advantages of our platform, we prepared table s5 to compare hive-chip with other four representative methods for asfv detection, as well as two commercial kits. without nucleic acid extraction and heavy instrumentation, hive-chip can simultaneously detect five genes related to asfv in a one-pot reaction, which can largely guarantee the accuracy and avoid the aerosol contamination. the total testing time is less than 70 min and the limit of detection (lod) is 80 copies per reaction. moreover, compared with our previous work (table s6) , i.e., calm platform [30] and paac system [31] , the most important feature of current study is that we target multiple genes for a single pathogen (especially variable virus). to our knowledge, this concept has not been attempted for lamp and other isothermal nat technologies. meanwhile, direct-lamp was adopted to simplify the blood sample pre-treatment and facilitate the on-site detection of asfv. overall, we provided a total solution based on hive-chip for various targets and samples detection. multiplex nucleic acid detection of variable asfv is critical for accurate and early diagnosis of asf. however, current methods only target one gene of asfv, which easily brings missed inspection because of the highly mutable genomes of this virus. herein, we established a multiplex and visual detection platform based on hive-chip and direct lamp for rapid and accurate detection of asfv. this platform assured detection accuracy through monitoring several gene fragments in a single test. there are several advantages of our platform over the existing methods. firstly, the design of multi-targets detection in a single reaction plays a vital part in accurately diagnosis of the mutable viral nucleic acid. if we consider the singleplex detection as a "pistol" of shooting a target a time, hive chip-based multiplex detection platform may similar to a "shotgun", which can shoot many "enemies" (genes of asfv) a time. in this study, apart from the b646l gene included in oie-recommended pcr, the other four conserved genes (b962l, c717r, d1133l and g1340l) are also selected as the target for the first time for multiplex detection of asfv, which largely avoid the false-negative results arising from the possible mutations of a certain gene. based on this principle, hive-chip can serve as a versatile platform for various virus detection. for example, nucleic acid testing (nat) of the recent outbreak of severe j o u r n a l p r e -p r o o f acute respiratory syndrome (sars)-cov-2 mainly covers orf1ab gene, n gene, e gene and human rnase p gene as a control, which requires multiplex detection [40] [41] [42] . in this case, hive-chip can integrate multiple genes at a time to realize simultaneous detection and subsequently save a lot of time for epidemic control. moreover, to our knowledge, it has not been reported that there is a significant difference in the expression level of these five genes in different strains of asfv. currently, asfv is usually genotyped using a combination of partial sequencing of the b646l gene and analysis of the central variable region (cvr) of the b602l gene [11] . meanwhile, other genes like p30, p54, and cp204l gene are also served as a supplementary classification standard [43] . but it is hard to use the difference or ratio of these genes to identify the genotype of samples from different regions. secondly, multiplex nats are usually disturbed by cross reaction between different primer sets. hive-chip can provide physical isolation of primers by different capillaries, and the hydrophobic modification can avoid the cross contamination of capillaries in sample loading. meanwhile, the closed space for amplification and visual detection theoretically can prevent 100% aerosol contaminations. thirdly, nucleic acid extraction is the key step in nats and it often needs complex operation. integrated with direct lamp, the entire process of hive-chip from blood sample treatment to detection can be finished within 70 min independent of nucleic acid extraction and sophisticated instruments, which is suitable for on-site detection of asfv. besides, other direct amplification methods including direct rpa [34] could also be easily combined with our platform for different types of samples. last but not least, hive-chip based platform has good flexibility and compatibility. the current version of chip has ten channels for detecting as many as eight targets other than positive and negative control. as for asfv detection, we only adopt five targets and leave three channels unused. furthermore, by increasing the size of hive-chip, we can increase the channel number and locate more capillaries in a single hive-chip unit. because the current format of hive-chip is compatible with standard 96-well plate and it can be assembled into a single well, we can easily perform high-throughput sample loading by means of the multi-channel pipette [30] . based on this, hive-chip can be integrated with the current automated liquid workstation (e.g., beckman & biomek 4000) for high-throughput sample loading. furthermore, to realize massive detection, we can also employ multi-mode plate reader system (e.g., biotek & cytation 5 cell imaging multi-mode reader) to acquire fluorescence images for subsequent signal analysis. we believe that hive-chip-based platform is suitable for high-throughput screening when combined with widely accessible instruments of 96-well plate operation. unfortunately, due to the lack of biosafety laboratory for handling the infectious real samples, we didn't have chance to test swine blood sample obtained from pigs in summary, we have developed a multiplex visual detection method based on hive-chip and direct lamp with a streamlined operation procedure, which offers a feasible strategy and key reagents for rapid and accurate on-site detection of asfv. the method that we demonstrated is of high generality, it could be easily adopted for fast development of assays/ kits for animal diseases or possible future outbreaks. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f arrival of deadly pig disease could spell disaster for china african swine fever emerging in china: distribution characteristics and high-risk areas african swine fever virus: a review on a form of swine fever occurring in british east africa epidemiology of african swine fever virus interaction of historical and modern sardinian african swine fever viruses with porcine and wild-boar monocytes and monocyte-derived macrophages african swine fever in china food and agricultural organization of the united nations (2020) ictv report consortium, ictv virus taxonomy profile: asfarviridae architecture of african swine fever virus and implications for viral assembly genetic characterization of african mozambique and identification of a novel genotype genome comparison of african swine fever virus /anhuixcgq strain and related european p72 genotype ii strains african swine fever virus encodes a cd2 homolog responsible for the adhesion of erythrocytes to infected cells african swine fever virus serodiagnosis: a general review with a focus on the analyses of african serum samples assessment of african swine fever diagnostic techniques as a response to the epidemic outbreaks in eastern european union countries: how to improve surveillance and control programs development of a novel lateral flow assay for detection of african swine fever in blood simplifying sampling for african swine fever surveillance: assessment of antibody and pathogen detection from blood swabs highly sensitive pcr assay for routine diagnosis of african swine fever virus in clinical samples development of a novel quantitative real-time pcr assay with lyophilized powder reagent to detect african swine fever virus in blood samples of domestic pigs in china sensitive detection of african swine fever virus using real-time pcr with a 5′ conjugated minor groove binder probe adaptation of an invader assay for the detection of african swine fever virus dna detection of african swine fever virus by loop-mediated isothermal amplification development of a real-time loop-mediated isothermal amplification (lamp) assay and visual lamp assay for detection of african swine fever virus (asfv) a recombinase polymerase amplification-based assay for rapid detection of african swine fever virus clustered regularly interspaced short palindromic repeats/cas9-mediated lateral flow nucleic acid assay universal and naked-eye gene detection platform based on the clustered regularly interspaced short palindromic repeats/cas12a/13a system crispr/cas12a technology combined with immunochromatographic strips for portable detection of african swine fever virus cas12a-based on-site and rapid nucleic acid detection of african swine fever high-throughput and all-solution phase african swine fever virus (asfv) detection using crispr-cas12a and fluorescence based point-of-care system visual detection of multiple genetically modified organisms in a capillary array multiplex sample-to-answer detection of bacteria using a pipette-actuated capillary array comb with integrated dna extraction, isothermal amplification, and smartphone detection direct pcr amplification of forensic touch and other challenging dna samples: a review a direct isothermal amplification system adapted for rapid snp genotyping of multifarious sample types field-deployable viral diagnostics using crispr-cas13 african swine fever virus replication and genomics development of an updated pcr assay for detection of african swine fever virus african swine fever development of a taqman pcr assay with internal amplification control for the detection of african swine fever virus replication and virulence in pigs of the first african swine fever virus isolated in china detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr crispr-cas12-based detection of sars-cov-2 highly accurate and sensitive diagnostic detection of sars-cov-2 by digital pcr genomic analysis of highly virulent georgia 2007/1 isolate of african swine fever virus this work was partially supported by science and technology commission of key: cord-002994-1zjrunzc authors: faye, martin; faye, oumar; diagne, moussa moise; fall, gamou; weidmann, manfred; sembene, mbacke; sall, amadou alpha; faye, ousmane title: full-genome characterization and genetic evolution of west african isolates of bagaza virus date: 2018-04-13 journal: viruses doi: 10.3390/v10040193 sha: doc_id: 2994 cord_uid: 1zjrunzc bagaza virus is a mosquito-borne flavivirus, first isolated in 1966 in central african republic. it has currently been identified in mosquito pools collected in the field in west and central africa. emergence in wild birds in europe and serological evidence in encephalitis patients in india raise questions on its genetic evolution and the diversity of isolates circulating in africa. to better understand genetic diversity and evolution of bagaza virus, we describe the full-genome characterization of 11 west african isolates, sampled from 1988 to 2014. parameters such as genetic distances, n-glycosylation patterns, recombination events, selective pressures, and its codon adaptation to human genes are assessed. our study is noteworthy for the observation of n-glycosylation and recombination in bagaza virus and provides insight into its indian origin from the 13th century. interestingly, evidence of bagaza virus codon adaptation to human house-keeping genes is also observed to be higher than those of other flaviviruses well known in human infections. genetic variations on genome of west african bagaza virus could play an important role in generating diversity and may promote bagaza virus adaptation to other vertebrates and become an important threat in human health. bagaza virus (bagv) belongs to the flaviridae family, flavivirus genus and ntaya serological group. bagv is a mosquito-transmitted virus, which was first isolated in the bagaza district of the central african republic (car), in 1966, from a pool of mixed-species female culex spp. mosquitoes during entomological investigations [1] . as is characteristic of flaviviruses, bagv possesses a linear single-stranded, positive-sense rna genome [2] . the bagv genome is 10,941 nucleotides in length, encoding a single polyprotein (3426 amino acids) from which 11 viral proteins are derived, and flanked by 5 and 3 untranslated regions (utrs) of 94 and 556 nt, respectively [3] . bagv has been isolated repeatedly with a high titer from different species of mosquitoes in central and west african countries [4] [5] [6] , and in india, where serological investigations suggested subclinical infections in humans [7, 8] . despite this widespread circulation of bagv, outbreaks involving humans or animals have not yet been reported from these countries. subsequently, in september 2010, bagv was associated with a high mortality among game birds (partridges and pheasants) in southern spain, the first detection of bagv in europe and the first isolation from a vertebrate host [9, 10] . however, it is not surprising that bagv infects birds since it has been shown to be synonymous with israel turkey all 11 virus strains analyzed in this study were derived from the who collaborating center (http://apps.who.int/whocc/detail.aspx?cc_ref=sen-5&cc_code=sen) for arboviruses and viral hemorrhagic viruses in senegal at institut pasteur de dakar (dakar, senegal) ( table 2 ). viral stocks were prepared by inoculating viral strains into aedes albopictus (c6/36) cell line in leibovitz 15 (l-15) growth medium viruses 2018, 10, 193 3 of 26 (gibcobrl, grand island, ny, usa) supplemented with 5% fetal bovine serum (fbs) (gibcobrl, grand island, ny, usa), 10% tryptose phosphate and antibiotics (sigma, gmbh, germany). bagv infection was confirmed after 4 days of propagation by immunofluorescence assay (ifa) using specific hyper-immune mouse ascitic fluid, as previously described [15] . cultures supernatants were collected for virus rna isolation. extraction of viral rna from supernatants was performed with the qiaamp viral rna mini kit (qiagen, heiden, germany) according to manufacturer's instructions. extracted rna was frozen at −80 • c prior to downstream applications. real-time rt-pcr (reverse transcriptase-polymerase chain reaction) was performed using the quantitect ® probe rt-pcr kit (qiagen, heiden, germany) in a final volume of 25 µl following previously established protocols and primers [16] . reverse-transcription was performed using the amv kit (promega, madison, wi, usa) following manufacturer's instructions and cdna were stored at −20 • c. the polymerase chain reaction with each primer set was carried out in a final volume of 50 µl using the gotaq ® dna polymerase kit (promega, madison, wi, usa) according to manufacturer's instructions. briefly, 5 µl (around 10 µg) of cdna was added to 45 µl of a rt-pcr mix containing 25 mm mgcl2, 10 mm of dntp, 5x reaction buffer, 5 u gotaq polymerase, 16.5 µl of nuclease-free water and 40 pmols of each primer (sense and antisense). pcr was carried using the following conditions: an initial incubation at 95 • c for 5 min, followed by 40 cycles of 95 • c for 1 min, 1 min at melting temperature of primers, and 72 • c according to the length of pcr product and 72 • c during 10 min. subsequently, 5 µl of each pcr product was analyzed by gel electrophoresis on 1% agarose gels stained with ethidium bromide to check the size of amplified fragments by comparison to a dna molecular weight marker (hyperladder™ 1 kb, bioline, taunton, ma, usa). the dna bands from the pcr amplification were purified (qiaquick gel extraction kit, qiagen, heiden, germany) and sequenced from both ends for each positive sample (beckmann coulter, high wycombe, uk). sequencing of the 5 and 3 termini of the viral genome was performed using a 5 race kit (invitrogen, carlsbad, ca, usa) and a 3 race kit (roche, basel, switzerland) following the manufacturer's protocols. additional sequences representing strains from central african republic, india, strain related to spanish wild bird's outbreak in 2010, the itv and the ntaya virus were obtained from genbank, with the following accession numbers, respectively: ay632545, eu684972, hq644143, kc734549 and nc_018705. full-length genome sequences bagv isolates were obtained by assembling overlapping nucleotide sequences using the unipro ugene software (http://ugene.net/download.html) [17] . multiple alignments of full-genome sequences were carried out by using muscle algorithm (http://www.drive5.com/muscle/) [18] within unipro ugene software. based on these alignments, we investigated the genetic properties of these different isolates circulating in west africa, such as genome length and location of main conserved amino acid motifs previously described in mosquito-borne flaviviruses (mbfvs) with sometimes mutations which include no physicochemical properties changes [3] . comparatively, conservation of these motifs was also assessed in culex flavivirus (cxfv) and aedes flavivirus (aefv) (insect-specific flaviviruses; (isfs)) and in modoc virus (modv) and rio bravo virus (rbv) (vertebrate-specific flaviviruses, also known as no known vector flaviviruses (nkvfs)). we also searched for evidence of informative amino acid sites among bagv sequences using the divein web server (https://indra.mullins.microbiol.washington.edu/divein/) [19] . the genetic divergence between previously available bagv complete genomes and new characterized sequences was also assessed at the nucleotide and protein levels. prediction of n-glycosylated sites on the genome of bagv were performed by submitting complete polyproteins on online version of netnglyc 1.0 server (http://www.cbs.dtu.dk/services/ netnglyc/). n-linked glycosylation is a post-translational event whereby carbohydrates are added to asparagines, which occur in the consensus sequence asn-xaa-ser/thr, where xaa is any amino acid except proline. "potential" scores of predicted n-glycosylated sites across the protein chain from n-to c-terminal were illustrated using the default threshold of 0.5 and the "jury agreement" indicates how many of the nine networks support the prediction [20] . the rnaz method [21] implemented in the vienna rna websuite (http://rna.tbi.univie.ac.at/) [22] was used to detect thermodynamically stable and evolutionarily conserved structural rna domains on complete non-coding regions of the 11 west african bagv isolates characterized in this study and the isolates from spain and car, because complete non-coding sequences are not currently available for the isolate from india. the rnaz method use an algorithm which testing a large set of well-known conserved structural rna domains and reports a "rna classification probability" or p-value as a measure of thermodynamic stability. structural rna domains with p > 0.5 are classified as stable [21] . furthermore, the optimal secondary structures were predicted with a minimum free energy using the rnaalifold method [23] implemented also in the vienna rna websuite that use a dynamic programming algorithm with rna parameters as previously described [24] . furthermore, previously described organization of conserved sequences (cs) [3] was analyzed on predicted secondary structures of the 3 utr, considering possible repetitions of these cs. thus, a conserved sequence was considered as imperfect when it presented three or more differences with corresponding consensus sequence previously described [3] , marked by a deletion, an insertion, or a substitution. a bayesian phylogenetic analysis for estimation of data quality and selection of the best-fit nucleotide substitution model were performed using mega 6.0 (https://www.megasoftware.net/) with a discrete gamma distribution (+g) with 5 rate categories. thus, a total of 24 different nucleotide substitution models were tested and model with the lowest bic score (bayesian information criterion) was considered to describe the best substitution pattern. further parameters as aicc value (akaike information criterion, corrected) and maximum likelihood value (lnl) are also estimated [25] . a maximum likelihood tree was then constructed with complete polyprotein sequences from insect-specific flaviviruses, no known vector flaviviruses, tick-born flaviviruses, mosquito-borne flaviviruses, the 11 orfs from new characterized west african bagv isolates and bagv sequences previously available from spain (hq644143-4, kr108244-6), india (eu684972) and car (ay632545). tree was inferred using fasttree v2.1.7 (http://www.microbesonline.org/fasttree/) [26] , where nucleotide substitution was modeled using general time-reversible with a proportion of invariant sites (gtr+i). nodes were labeled with local support values, which were computed with the shimodaira-hasegawa test (sh-like) for 5000 replications. topology was visualized by figtree v.1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). to prevent potential biases during phylogenetic inference due to recombination, all polyprotein sequences were analyzed using seven methods (rdp, geneconv, maxchi, bootscan, chimaera, siscan, and 3seq) implemented in the recombination detection program (rdp4beta 4.8) to uncover evidence for recombination events [27] . the disentangle recombination signals option was "on" and the linear sequence setting was used. the remaining settings were kept at their default values. only events with p-values < 1 × 10 −6 that were detected by four or more methods were considered to represent strong evidence for recombination using 100 permutations and the bonferroni correction [28] implemented in the rdp4 program to prevent false positive results. a chi-square test was used to determine if the sequence identity between a recombinant isolate and a given parent was significantly different both inside and outside the recombinant region. in addition, a bootscan analysis including the recombinant and the parental strains determined above was also performed to confirm these putative recombination events. the occurrence of recombination in bagv genomes was also investigated with a method called genetic algorithms for recombination detection (gard) implemented in datamonkey web server (http://datamonkey.org) [29] , that estimates breakpoints based on a genetic algorithm. the statistical significance of putative breakpoints was evaluated through kishino-hasegawa (hk) tests; breakpoints were considered significant if their p value was <0.05. separate neighbor-joining (nj) trees were constructed for identified putative recombinant region and non-recombinant alignment partitions dictated by the breakpoint locations. phylogenetic trees were inferred using the percentage of 1000 bootstrap replications under the appropriate model of nucleotide substitution. recombination can mislead inference of positive natural selection if it is not properly accounted for. if recombination was identified, these potential recombinant sequences were excluded from further analyses to avoid inferential biases [30] . the non-synonymous/synonymous rate ratio (dn/ds) is a widely used method to detect positive selection. the statistical test dn/ds permitted to distinguish diversifying or positive selection (dn/ds > 1) from negative or purifying selection (dn/ds < 1). positive selection is inferred when the rate of non-synonymous (dn) substitutions is higher than that of synonymous (ds) substitutions (dn > ds). episodes of positive selection in each gene of bagv were analyzed using methods of estimation among individual sites and internal sites on branches of the phylogenetic tree. for this, a total of 9 alignment partitions were performed corresponding to c, prm, e, ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns5 proteins. as site model, we used the single-likelihood ancestor counting (slac) that estimated the difference between non-synonymous (dn) and synonymous (ds) rates per codon site at 0.1 significance level. the fast unconstrained bayesian approximation (fubar) method which evaluated episodic positive selection at each site in the alignment at posterior probability ≥0.9 was also used [31] . the mixed effects model of evolution (meme) was also conducted at a 0.1 significance level for estimation selective pressure changes among codon sites. finally, branch-site random effects likelihood (branch-site rel) analysis was used to evaluate evidence of diversifying selection on specific branches in the phylogenetic tree at a proportion of sites, considering p-values less than 0.05 as significant. all four methods were conducted with hyphy package implemented in datamonkey web server [29] . an episode of positive diversifying selection in concern of a region was considered if it was detected by at least two different methods. the evolutionary analysis was performed using a strict clock gmrf bayesian skyride coalescent tree prior [32, 33] . the gtr substitution model was used with 4 gamma rate categories. the bayesian markov chain monte carlo (mcmc) algorithms using beast v1.8.4 (http://beast.community/) [34] were employed to estimate the rate of bagv evolution from first isolation to 2014. mcmc analyses were run for 100 million generations, sampling every 100 thousand to ensure convergence of estimates. population size (ess) above 200 was assessed using the analysis program tracer v1.6 (http://beast.bio.ed.ac.uk/tracer). the posterior distribution of trees obtained from the beast analysis was also used to obtain the bayesian maximum clade credibility (mcc) tree for these sequences generated by treeannotator v.2.3.2 (http://beast.community/treeannotator) (from 100 million) after removing 10% of the runs burn-in and visualized by figtree v.1.4.2. the codon adaptation index (cai) is a measure of the synonymous codon usage bias making comparisons of codon usage preferences in different organisms and assessing the adaptation of viral genes to their hosts [35, 36] . cai was applied in many recent studies involving humans and rna viruses [37] [38] [39] . to know if there is evidence of bagv adaptation for codon usage in humans, the cai was calculated for each isolate. to calculate normalized cai, full-length polyprotein sequences of west african bagv isolates and previously available bagv sequences from spain were compared to that of human using caical v1.4 program (http://genomes.urv.es/caical/) [40] . first, we obtained a "raw" cai (rcai) and then, the cai was normalized by the "expected neutral cai" (ecai) value based on 1000 random viral sequences using similar length, codon composition, gc-content and human amino acid usage. indeed, a table for human codon usage containing the entirety of human coding genes is publicly available [41] . based on this table, we created a new table where only the 3804 identified human housekeeping genes were considered [42] . normalized cai threshold was obtained by calculating rcai/ecai values and a value above '1' is higher than neutral and considered as evidence of codon adaptation to the reference set of codon preferences [40] . cai values obtained for bagv were then compared to those of others mbfvs well known to infect humans such as dengue virus (denv), usutu virus (usuv), wnv, zika virus (zikv) and yellow fever virus (yfv), nkv flaviviruses (modv and rbv) and isfs (cxfv and aefv), using the non-parametric wilcoxon test with r program. a p-value less than 0.05 was considered as significant. sequences of tobacco mosaic virus (tmv) were compared to human codons and used as negative control to provide an example that results for codon adaptation to human house-keeping genes are robust and not false positives or anomalies. as there are no known cases of human infection, or evidence of human adaptation for tmv, we expected all sequences to have a lower cai threshold than the calculated cai. in this study, a total of 11 full-genome sequences (10,954 bp) of west african bagv isolates were obtained by sequencing overlapping pcr amplifications covering the genome and by using race (rapid amplification of cdna ends) techniques for the terminal ends and deposited in genbank (www.ncbi.nlm.nih.gov/genbank/) (accession numbers: mf380424-34) ( table 1 ). analysis of new characterized bagv complete open reading frames (orfs) was performed at nucleotide and amino acid levels including previously available sequences from car (isolate dakarb209_car_1966, accession no. ay632545) and spain (isolate spain_h_2010, accession no. hq644143) into multiple sequence alignments. the polyprotein length of the newly sequenced west african bagv isolates was determined with respect to gene sizes (table 3) . although the 5 utr was similar in length, the 3 utr of these west african isolates was either 10 nt or 137 nt longer than those of sequences from car and spain, respectively. in the 5 utr, positions 52, 55 and 93 had nucleotide changes that were distinguishable for west african isolates. nucleotide changes a to c at position 52 and t to c at position 55 were seen in west african sequences, and a t to c change at position 93 was observed only in sequences of the isolates ard54139_dakar-bango_sen_1989 and ara23139_dezidougou_ci_1988. interestingly, the 3 utr can be divided into three sections; a proximal highly variable section constituted by the 139 first nucleotides following the stop codon, a second highly conservative section located between nucleotide positions 140 and 434 and a moderately variable distal region comprising the last 142 nucleotides. in this distal section, 3 utr sequences of west african isolates presented insertions of 74 nt and 1 nt, compared to the isolates from car and spanish (kr108244-6), respectively. pairwise genetic distances of coding sequences were evaluated at nucleotide and amino acid levels between isolates characterized in this study and in comparison with previously available bagv sequences ( figure 1 ). nucleotide sequences of bagv isolated from senegal showed a mean distance of 1.9% ± 0.8% (0.3-3.4%). this lowest genetic distance was also apparent at amino acid level with a mean distance of 1.9% ± 0.6% (0.4-3.7%). here, we described location of main conserved amino acid motifs on bagv proteins using in silico analysis of complete genome sequences of the 11 west african bagv isolates characterized in this study and sequences from india, car and spain. most of highly conserved amino acid motifs localized across e, ns1, ns3 and ns5 proteins of mvfs were identified in the bagv genomes, sometimes with presence of conservative amino acid mutations (positions highlighted in black) or non-conservative amino acid mutations (positions highlighted in red) ( table 4) in protein ns1, all analyzed motifs were conserved, but nc t713p and t713a were observed in sequences of isolates ard171102_barkedji_sen_2004 and eu684972_96363_india_1996, respectively. the bagv isolate ard54139_dakar-bango_sen_1989 also contains nc p1127t. in ns3, the conserved motif identified at positions 1722-1728 contains nc a1723p and p1724l for bagv isolates ard137998_diawara_sen_2000 and ard138018_diawara_ sen_2000, whereas nc l1722s in comparison to sequence of the isolate ara23139_dezidougou_ci_1988 from côte d'ivoire, senegalese bagv isolates showed a higher mean distance of 3.4% ± 0.5% (2.7-4.1%) at nucleotide level. however, this highest genetic distance was less apparent at amino acid level with a mean distance of 1.7% ± 0.7% (2.7-4.1%). furthermore, mean distances of 6.7% (6.2-7.3%), 1.3% (0.2-2.7%), 5.8% (5.2-6.3%) were recorded at nucleotide level between senegalese bagv isolates and the isolate from car, spain, and eu684972__96363_india_1996, respectively while respective mean distances were 1.5% (0.8-2.5%), 1.7% (1.0-2.8%), 2.7% (2.0-3.8%) at amino acid level. a differentiation coefficient value of 0.17 was also observed between these west african bagv isolates and previously available sequences. here, we described location of main conserved amino acid motifs on bagv proteins using in silico analysis of complete genome sequences of the 11 west african bagv isolates characterized in this study and sequences from india, car and spain. most of highly conserved amino acid motifs localized across e, ns1, ns3 and ns5 proteins of mvfs were identified in the bagv genomes, sometimes with presence of conservative amino acid mutations (positions highlighted in black) or non-conservative amino acid mutations (positions highlighted in red) ( table 4) in protein ns1, all analyzed motifs were conserved, but nc t713p and t713a were observed in sequences of isolates ard171102_barkedji_sen_2004 and eu684972_96363_india_1996, respectively. the bagv isolate ard54139_dakar-bango_sen_1989 also contains nc p1127t. in ns3, the conserved motif identified at positions 1722-1728 contains nc a1723p and p1724l for bagv isolates ard137998_ diawara_sen_2000 and ard138018_diawara_ sen_2000, whereas nc l1722s is present in ard138018_diawara_sen_2000, and ard171075_barkedji_sen_2004. a non-conserved motif at positions 1759-1766 contained nc f1766l in all bagv sequences analyzed and isolate ard138018_diawara_sen_2000 contained additional nc t1765p and d1785y. a non-conserved motif in ns5 at positions 2734-2741 contained nc t2738n in all bagv sequences. bagv isolate ard54139_dakar-bango_sen_1989 has two supplementary mutations s2734w and s2737p. in addition, these mbfvs amino acid motifs were also mostly conserved in bagv, nkvfs and cxfv (isfs) than in aefv (isfs). non-conservative amino acid mutations on the bagv polyprotein might be associated to phenotypic differences of bagv isolates. in addition, the presence of phylogenetically informative sites was assessed on the divein web server. the identified site lap is harbored by the conserved motif laptrvv previously identified in ns3 protein of flaviviruses [3] and presents nc mutations in the genome of three bagv isolates. in addition, phylogenetically informative sites iega and griwna identified in ns4b and ns5, showed combined variations in the genome of the car isolate (dkgq and rtdmec, respectively) and the senegalese bagv isolates ard152146_diawara_sen_2001 (rraa and griwna, respectively) and ard260266_barkedji_sen_2014 (rrss and rtdmec, respectively) ( figure 2 ). non-conservative amino acid mutations on the bagv polyprotein might be associated to phenotypic differences of bagv isolates. in addition, the presence of phylogenetically informative sites was assessed on the divein web server. the identified site lap is harbored by the conserved motif laptrvv previously identified in ns3 protein of flaviviruses [3] and presents nc mutations in the genome of three bagv isolates. in addition, phylogenetically informative sites iega and griwna identified in ns4b and ns5, showed combined variations in the genome of the car isolate (dkgq and rtdmec, respectively) and the senegalese bagv isolates ard152146_diawara_sen_2001 (rraa and griwna, respectively) and ard260266_barkedji_sen_2014 (rrss and rtdmec, respectively) ( figure 2 ). prediction of n-glycosylation sites was performed using complete genome sequences of the 11 west african bagv isolates characterized in this study and sequences from india, car and spain on the divein web server. the "potential" score represents the averaged output of nine neural networks and the "jury agreement" indicates how many of the nine networks support the prediction. in total, eight n-glycosylated motifs were identified in the bagv genome (potential > 0.5) including two highly probable sites (potential > 0.5 and jury agreement of 9/9). despite high potential (0.7452) and jury agreement (9/9), the motif (asn-x-thr) nptd identified at position 603 was not considered to be glycosylated because it contained a proline known to preclude the n-glycosylation by rendering inaccessible the asparagine in the majority of cases (figure 3 ). this motif was in the domain iii region prediction of n-glycosylation sites was performed using complete genome sequences of the 11 west african bagv isolates characterized in this study and sequences from india, car and spain on the divein web server. the "potential" score represents the averaged output of nine neural networks and the "jury agreement" indicates how many of the nine networks support the prediction. in total, eight n-glycosylated motifs were identified in the bagv genome (potential > 0.5) including two highly probable sites (potential > 0.5 and jury agreement of 9/9). despite high potential (0.7452) and jury agreement (9/9), the motif (asn-x-thr) nptd identified at position 603 was not considered to be glycosylated because it contained a proline known to preclude the n-glycosylation by rendering inaccessible the asparagine in the majority of cases (figure 3 ). this motif was in the domain iii region of the e protein of all bagv isolates. however, a second (asn-x-ser) motif nfsl was highly predicted (score 0.6223 (9/9)) and suggested an n-linked glycosylation site at the residue asn-2333 in the ns4b protein. interestingly, we also found six others probable n-glycosylation at different positions on the bagv polyprotein including one site (nysi) harboring, the nys motif at the 443th position (153th position of the e protein), previously described as a virulence factor for wnv and denv. of the e protein of all bagv isolates. however, a second (asn-x-ser) motif nfsl was highly predicted (score 0.6223 (9/9)) and suggested an n-linked glycosylation site at the residue asn-2333 in the ns4b protein. interestingly, we also found six others probable n-glycosylation at different positions on the bagv polyprotein including one site (nysi) harboring, the nys motif at the 443th position (153th position of the e protein), previously described as a virulence factor for wnv and denv. the "potential" score is the averaged output of nine neural networks and the "jury agreement" indicates how many of the nine networks support the prediction. the n-glyc result column shows one of the following outputs for predictions. nglycosylated sites highly predicted by the nine networks (potential > 0.5 and jury agreement of 9/9) are highlighted in red and the site previously reported as virulence factor on e protein of flaviviruses is colored in blue. assessment of thermodynamically stable and evolutionarily conserved structural rna domains was performed using complete non-coding sequences of the 11 west african bagv isolates characterized in this study and the isolate from spain. the rnaz method implemented in the vienna figure 3 . prediction of n-glycosylation on bagaza virus genome. predictions were performed using the netnglyc 1.0 server. a position with a potential (green vertical lines) crossing the threshold (horizontal red line at 0.5) is predicted glycosylated. the "potential" score is the averaged output of nine neural networks and the "jury agreement" indicates how many of the nine networks support the prediction. the n-glyc result column shows one of the following outputs for predictions. n-glycosylated sites highly predicted by the nine networks (potential > 0.5 and jury agreement of 9/9) are highlighted in red and the site previously reported as virulence factor on e protein of flaviviruses is colored in blue. assessment of thermodynamically stable and evolutionarily conserved structural rna domains was performed using complete non-coding sequences of the 11 west african bagv isolates characterized in this study and the isolate from spain. the rnaz method implemented in the vienna rna websuite was used to identify conserved structural rna domains in the utrs of bagv characterized by a p > 0.5. using the rnaz method, highly conserved structural rna domains was not identified in the 5 utr of bagv genome while a total of four highly conserved structural rna domains were determined in the 3 region with respective classification probabilities of 0.671490, 0.994641, 0.976295 and 0.846482 ( figure s1 ). however, the rnaalifold method implemented in the vienna rna websuite server predicted that, as in the genome of other members of the genus flavivirus, bagv has a shorter 5 utr (≈100 nt), consisting of a pair of conserved stem-loops (sl-a and sl-b) (figure 4) . sl-a serves as promoter of viral polymerase activity followed by a shorter loop which contains a cyclisation sequence upstream of the 5 aug (sl-b) . the secondary structure of bagv's 3 utr could be divided in three parts; a highly variable domain 1 following the stop codon and consisting in an au-rich stem-loop (sl-i), a second domain 2 with highly conserved sequence and two stem-loops (sl-ii and sl-iii) and dumbbell structures (db1 and db2), and the moderately conserved distal domain 3 which contains the complementary cyclisation elements. in the intermediate domain, the sl-ii presented a pseudoknot pk1 preceding a short conserved loop (rcs3). this structural motif was repeated in a stem-loop sl-iii with pk2 and cs3. these stem-loops were followed by dumbbell structures db1 and db2 that presented conserved loop rcs2 connected with a pseudoknot pk3 and its repetition cs2, respectively [43] . thus, organization of conserved sequences on consensus secondary structure of bagv's 3 utr was structured rcs3-cs3-rcs2-cs2-imcs1. indeed, cs1 was imperfect (imcs1) only on sequences of west african bagv isolates with a total of nine substitutions compared to the corresponding consensus sequence previously described [3] . viruses 2018, 10, x 12 of 26 rna websuite was used to identify conserved structural rna domains in the utrs of bagv characterized by a p > 0.5. using the rnaz method, highly conserved structural rna domains was not identified in the 5′ utr of bagv genome while a total of four highly conserved structural rna domains were determined in the 3′ region with respective classification probabilities of 0.671490, 0.994641, 0.976295 and 0.846482 ( figure s1 ). however, the rnaalifold method implemented in the vienna rna websuite server predicted that, as in the genome of other members of the genus flavivirus, bagv has a shorter 5′ utr (≈100 nt), consisting of a pair of conserved stem-loops (sl-a and sl-b) (figure 4) . sl-a serves as promoter of viral polymerase activity followed by a shorter loop which contains a cyclisation sequence upstream of the 5′ aug (sl-b) . the secondary structure of bagv's 3′ utr could be divided in three parts; a highly variable domain 1 following the stop codon and consisting in an au-rich stem-loop (sl-i), a second domain 2 with highly conserved sequence and two stem-loops (sl-ii and sl-iii) and dumbbell structures (db1 and db2), and the moderately conserved distal domain 3 which contains the complementary cyclisation elements. in the intermediate domain, the sl-ii presented a pseudoknot pk1 preceding a short conserved loop (rcs3). this structural motif was repeated in a stem-loop sl-iii with pk2 and cs3. these stem-loops were followed by dumbbell structures db1 and db2 that presented conserved loop rcs2 connected with a pseudoknot pk3 and its repetition cs2, respectively [43] . thus, organization of conserved sequences on consensus secondary structure of bagv's 3′ utr was structured rcs3-cs3-rcs2-cs2-imcs1. indeed, cs1 was imperfect (imcs1) only on sequences of west african bagv isolates with a total of nine substitutions compared to the corresponding consensus sequence previously described [3] . the bayesian phylogenetic analysis for estimation of data quality and selection of the best-fit nucleotide substitution model were performed using mega 6.0 with a discrete gamma distribution (+g) with 5 rate categories. the general time-reversible with a discrete gamma distribution and a proportion of invariant sites (gtr+i) was the best nucleotide substitution model for our sequences data presenting score values of 69 the bayesian phylogenetic analysis for estimation of data quality and selection of the best-fit nucleotide substitution model were performed using mega 6.0 with a discrete gamma distribution (+g) with 5 rate categories. the general time-reversible with a discrete gamma distribution and a proportion of invariant sites (gtr+i) was the best nucleotide substitution model for our sequences data presenting score values of 69,924.128, 69,453.449 and −34,680.714 for bic, aicc and lnl criteria, respectively. the maximum likelihood (ml) tree was inferred using fasttree v2.1.7 [26] on our total data set including the 11 complete polyprotein sequences of west african bagv isolates circulating in senegal and côte d'ivoire from 1988 to 2014, the 5 bagv sequences from spain, the bagv sequences from india and car and complete polyproteins from different flaviviruses, with 10,281 bp alignment length ( figure 5 ). a gtr+i model was used, as selected by bayesian criteria. nodes were labeled with local support values computed with 5000 bootstrap replications using the shimodaira-hasegawa (sh) test. the phylogeny of complete bagv genome sequences presented evidence of a single bagv phylogenetic group. furthermore, we observed also that israel meningo-encephalitis turkey virus (itv) was closed to bagv in genetic relatedness [11] . viruses 2018, 10, x 13 of 26 respectively. the maximum likelihood (ml) tree was inferred using fasttree v2.1.7 [26] on our total data set including the 11 complete polyprotein sequences of west african bagv isolates circulating in senegal and côte d'ivoire from 1988 to 2014, the 5 bagv sequences from spain, the bagv sequences from india and car and complete polyproteins from different flaviviruses, with 10,281 bp alignment length ( figure 5 ). a gtr+i model was used, as selected by bayesian criteria. nodes were labeled with local support values computed with 5000 bootstrap replications using the shimodaira-hasegawa (sh) test. the phylogeny of complete bagv genome sequences presented evidence of a single bagv phylogenetic group. furthermore, we observed also that israel meningoencephalitis turkey virus (itv) was closed to bagv in genetic relatedness [11] . given the major implications of recombination events for evolution, pathogenicity, or diagnosis of non-segmented positive rna viruses like flaviviruses [44] , it is clearly important to determine their occurrence in the bagv genome. the rdp4beta 4.8 program used for assessment of recombination events on complete polyprotein sequences [27] revealed evidence of only one highly credible recombination event from the e protein to ns2b, with estimated breakpoints at positions 2202 and 4908 of bagv genome. this recombination event involved the isolate ard54139_dakar-bango_sen_1989 originating from saint-louis, in the north of senegal ( figure 6 ). considering the isolates ard260266_ barkedji_sen_2014 and ard171075_barkedji_sen_2004 as respective minor and major parents of the isolate ard54139_dakar-bango_sen_1989 (similarity of 98.8% and 97%, respectively), this recombination event was found by rdp, geneconv, bootscan, maxchi, chimaera, sisscan and 3seq methods and supported by significant p-values of 3.09 × 10 -16 , 9.23 × 10 −12 , 7.36 × 10 −13 , 8.45 × 10 −7 , 1.59 × 10 −7 , 3.60 × 10 −8 and 1.17 × 10 −12 , respectively. the bootscan and gard analyzes identified one significant recombination breakpoint at nucleotide position 2201 corresponding to the e protein, supported by a lhs p-value of 0.024 and a rhs p-value of 0.001. this breakpoint divides the bagv genome into two regions: one that encodes the structural proteins and another that encodes the non-structural proteins. phylogenetic trees were constructed using 1000 bootstrap replications and midpoint rooted for clarity only (figure 7 ). this recombination event led to a mismatch between nj phylogenetic trees constructed using comparison of nucleotides sequences of recombinant (positions 2202-4908) and non-recombinant genomic regions (positions 1-2201 and 4909-10,281). the tree is midpoint-rooted, nodes are labeled with local support values computed using the shimodaira-hasegawa (sh) test for 5000 bootstrap replications, species names are color-coded as follows: new characterized bagv isolatesdark blue with dots; previous sequences of bagv-dark blue; mosquito-borne flaviviruses (mbfvs)-green; dual-host affiliated isfs (disfs)-red; no known vector (nkv) flavivirusesyellow; tick-born flaviviruses (tbfvs)-light blue; classical isfs (cisfs)-orange. given the major implications of recombination events for evolution, pathogenicity, or diagnosis of non-segmented positive rna viruses like flaviviruses [44] , it is clearly important to determine their occurrence in the bagv genome. the rdp4beta 4.8 program used for assessment of recombination events on complete polyprotein sequences [27] revealed evidence of only one highly credible recombination event from the e protein to ns2b, with estimated breakpoints at positions 2202 and 4908 of bagv genome. this recombination event involved the isolate ard54139_dakar-bango_sen_1989 originating from saint-louis, in the north of senegal (figure 6 ). considering the isolates ard260266_barkedji_sen_2014 and ard171075_barkedji_sen_2004 as respective minor and major parents of the isolate ard54139_dakar-bango_sen_1989 (similarity of 98.8% and 97%, respectively), this recombination event was found by rdp, geneconv, bootscan, maxchi, chimaera, sisscan and 3seq methods and supported by significant p-values of 3.09 × 10 -16 , 9.23 × 10 −12 , 7.36 × 10 −13 , 8.45 × 10 −7 , 1.59 × 10 −7 , 3.60 × 10 −8 and 1.17 × 10 −12 , respectively. the bootscan and gard analyzes identified one significant recombination breakpoint at nucleotide position 2201 corresponding to the e protein, supported by a lhs p-value of 0.024 and a rhs p-value of 0.001. this breakpoint divides the bagv genome into two regions: one that encodes the structural proteins and another that encodes the non-structural proteins. phylogenetic trees were constructed using 1000 bootstrap replications and midpoint rooted for clarity only (figure 7 ). this recombination event led to a mismatch between nj phylogenetic trees constructed using comparison of nucleotides sequences of recombinant (positions 2202-4908) and non-recombinant genomic regions (positions 1-2201 and 4909-10,281). the structural and non-structural coding regions were analyzed separately for estimation of sites and branches under positive diversifying selection, applying four different methods to ensure consistency of these events along of bagv sequences. using this approach, we found several sites under strong negative selection and most of them were in the e, ns3 and ns5 proteins (table 5) . however, the significant evidence (p < 0.1) of episodic positive selection was obtained for all the coding genes, except for the prm, ns2b and ns4a regions. all positively selected sites estimated by the fubar model (posterior probability ≥ 0.9) were also identified by the meme method (p < 0.1). thus, an important number of positively selected sites were detected; interestingly, the majority of such sites were in the e, ns1 and ns5 proteins. branch-site analysis showed also a total of 11 branches evaluating under positive selection (p < 0.05) and the highest proportion was in the e and ns1 proteins. the structural and non-structural coding regions were analyzed separately for estimation of sites and branches under positive diversifying selection, applying four different methods to ensure consistency of these events along of bagv sequences. using this approach, we found several sites under strong negative selection and most of them were in the e, ns3 and ns5 proteins (table 5) . however, the significant evidence (p < 0.1) of episodic positive selection was obtained for all the coding genes, except for the prm, ns2b and ns4a regions. all positively selected sites estimated by the fubar model (posterior probability ≥ 0.9) were also identified by the meme method (p < 0.1). thus, an important number of positively selected sites were detected; interestingly, the majority of such sites were in the e, ns1 and ns5 proteins. branch-site analysis showed also a total of 11 branches evaluating under positive selection (p < 0.05) and the highest proportion was in the e and ns1 proteins. pervasive diversifying selection at posterior probability ≥ 0.9 with fubar model; episodic diversifying selection at 0.1 significance level with slac and meme models; episodic diversifying selection at p-value p ≤ 0.05 with branch-sites rel model. mcmc convergence was obtained for three independent runs with 100 million generations, which were sufficient to obtain a proper sample for the posterior at mcmc stationarity assessed by effective sample sizes (ess) above 200 for each gene. furthermore, the evolutionary rates (µ) and the highest posterior densities (hpd with 95% of confidence interval) were 1.226 × 10 evidence of bagv adaptation to human house-keeping genes was analyzed by calculating cai indices using complete coding polyprotein sequences of west african bagv isolates and bagv sequences available from spain, in comparison to other mbfvs such as denv, usuv, wnv, zikv and yfv, nkv flaviviruses (modv and rbv) and isfs (cxfv and aefv). cai values > 1 were obtained for polyprotein sequences of all bagv isolates. thus, there is evidence that bagv could have adaptation to the human genes ( figure 9 ). modv(mean cai: 1.072 and median cai: 1.072), rbv (mean cai: 1.059 and median cai: 1.059) and yfv (mean cai: 1.075 and median cai: 1.072) showed the highest cai values for human housekeeping genes and were significantly different to spanish and west african bagv isolates (wilcoxon rank sum test, p-values ranging from 0.0001 to 1.028 × 10 −7 ). compared to those of spanish isolates, sequences of west african bagv isolates presented significantly higher cai values (mean cai: 1.044 and median cai: 1.044, wilcoxon rank sum test, p-value < 0.002). in addition, they were also higher than denv serotype 2 (denv-2) (wilcoxon rank sum test, p-value < 1.4 × 10 −6 ). however, cai values of west african bagv isolates were lower than those of denv-1 (wilcoxon rank sum test, w = 231, p-value = 2.869 × 10 −6 ) and comparable to cai values given by denv-3 and denv-4 (wilcoxon rank sum test, w = 3258, p-value = 0.06477 and w = 824, p-value = 0.3463, respectively). interestingly, cai values of west african isolates were also significantly higher than those obtained for other mbfvs well known to infect human such as usuv (wilcoxon rank sum test, p-value < 6.796 × 10 −9 ), wnv (wilcoxon rank sum test, p-value < 2.718 × 10 −10 ), zikv (wilcoxon rank sum test, p-value < 1.67 × 10 −8 ) and isfs (means cai: 1.0015 and 1.0006 and median cai: 1.0015 and 1.0006 for cxfv and aefv, respectively) which showed low evidence evidence of bagv adaptation to human house-keeping genes was analyzed by calculating cai indices using complete coding polyprotein sequences of west african bagv isolates and bagv sequences available from spain, in comparison to other mbfvs such as denv, usuv, wnv, zikv and yfv, nkv flaviviruses (modv and rbv) and isfs (cxfv and aefv). cai values > 1 were obtained for polyprotein sequences of all bagv isolates. thus, there is evidence that bagv could have adaptation to the human genes ( figure 9 ). modv(mean cai: 1.072 and median cai: 1.072), rbv (mean cai: 1.059 and median cai: 1.059) and yfv (mean cai: 1.075 and median cai: 1.072) showed the highest cai values for human housekeeping genes and were significantly different to spanish and west african bagv isolates (wilcoxon rank sum test, p-values ranging from 0.0001 to 1.028 × 10 −7 ). compared to those of spanish isolates, sequences of west african bagv isolates presented significantly higher cai values (mean cai: 1.044 and median cai: 1.044, wilcoxon rank sum test, p-value < 0.002). in addition, they were also higher than denv serotype 2 (denv-2) (wilcoxon rank sum test, p-value < 1.4 × 10 −6 ). however, cai values of west african bagv isolates were lower than those of denv-1 (wilcoxon rank sum test, w = 231, p-value = 2.869 × 10 −6 ) and comparable to cai values given by denv-3 and denv-4 (wilcoxon rank sum test, w = 3258, p-value = 0.06477 and w = 824, p-value = 0.3463, respectively). interestingly, cai values of west african isolates were also significantly higher than those obtained for other mbfvs well known to infect human such as usuv (wilcoxon rank sum test, p-value < 6.796 × 10 −9 ), wnv (wilcoxon rank sum test, p-value < 2.718 × 10 −10 ), zikv (wilcoxon rank sum test, p-value < 1.67 × 10 −8 ) and isfs (means cai: 1.0015 and 1.0006 and median cai: 1.0015 and 1.0006 for cxfv and aefv, respectively) which showed low evidence for codon adaptation towards human housekeeping genes (wilcoxon rank sum test, p-value < 2.328 × 10 −16 ). although cai results for isfs were significantly lower to human housekeeping genes, we did not find any significant difference between cxfv and aefv codon adaptation. compared to codon usage of human genes, sequences of tobacco mosaic virus (tmv) showed mean and median cai values of 0.9587 and 0.9592, respectively. with an increasing number of emergent and re-emergent pathogens involved in human encephalitis, it is important to try to better understand which viruses have a potential to emerge causing human infection in the future. since its first isolation, bagv was only detected in mosquito pools collected in the field during entomological investigations in west and central africa and in india [12] . however, in 2010, bagv was identified as the cause of an encephalitis outbreak in wild birds circulating in southern spain [9] . in a possible host-switching event [45] , bagv could acquire future adaptation to other vertebrates such as humans [46] . in this study, genetic properties of bagv isolates circulating in west africa, the evolutionary phylogeny of bagv and evidence of bagv adaptation to human house-keeping genes were evaluated in comparison with different flavivirus groups. genomes of 11 west african bagv strains isolated from mosquito pools collected in the field from 1988 to 2014 showed similarities in terms of gene lengths when compared with polyprotein sequences of previously available isolates from car and spain. low amino acid distances observed between west african isolates (<2%) in comparison with previously non-west african sequences (<3%) combined with the weak coefficient of differentiation (<0.2) revealed evidence of a low genetic diversity of bagv sequences analyzed in this study as previously described [9] . in addition, the west african bagv isolates were more closely related to the car isolate. genome sequences originating from bagv isolates from other geographic locations would be helpful to understand if this low diversity is secluded to west-africa. although the 5′ utr was conserved between isolates, the 3′ utr of west african isolates varied in terms of length and structure. as in other mosquito-borne flavivirus genomes, bagv genome harbored structural rna domains both in 5′ and 3′ utrs which play a major role in flaviviral replication and interactions with host proteins and regulate cellular response to infection [47, 48] . with an increasing number of emergent and re-emergent pathogens involved in human encephalitis, it is important to try to better understand which viruses have a potential to emerge causing human infection in the future. since its first isolation, bagv was only detected in mosquito pools collected in the field during entomological investigations in west and central africa and in india [12] . however, in 2010, bagv was identified as the cause of an encephalitis outbreak in wild birds circulating in southern spain [9] . in a possible host-switching event [45] , bagv could acquire future adaptation to other vertebrates such as humans [46] . in this study, genetic properties of bagv isolates circulating in west africa, the evolutionary phylogeny of bagv and evidence of bagv adaptation to human house-keeping genes were evaluated in comparison with different flavivirus groups. genomes of 11 west african bagv strains isolated from mosquito pools collected in the field from 1988 to 2014 showed similarities in terms of gene lengths when compared with polyprotein sequences of previously available isolates from car and spain. low amino acid distances observed between west african isolates (<2%) in comparison with previously non-west african sequences (<3%) combined with the weak coefficient of differentiation (<0.2) revealed evidence of a low genetic diversity of bagv sequences analyzed in this study as previously described [9] . in addition, the west african bagv isolates were more closely related to the car isolate. genome sequences originating from bagv isolates from other geographic locations would be helpful to understand if this low diversity is secluded to west-africa. although the 5 utr was conserved between isolates, the 3 utr of west african isolates varied in terms of length and structure. as in other mosquito-borne flavivirus genomes, bagv genome harbored structural rna domains both in 5 and 3 utrs which play a major role in flaviviral replication and interactions with host proteins and regulate cellular response to infection [47, 48] . however, differences in determination of structural rna domains in 5 utr between the rnaz and the rnaalifold methods used in this study could be attributed to variations in algorithm of analysis used by each method [21] [22] [23] [24] . the small subgenomic rna (sfrna) identified in the 3 utr of bagv is generated through incomplete degradation of the viral genome by cellular 5 -3 exonuclease xrn1 [49, 50] and plays an important role in viral pathogenicity [49] and modulation of host responses [51, 52] . in addition, the stable 3' terminus region of the sfrna following the dumbbell structures (db1 and db2) and complementary to the 5 terminus of the 5 utr, was shown to be necessary in genomic rna cyclisation for viral replication and translation [46] . the sfrna can be in competition with the 3 utr of genomic rna in binding to proteins of viral replication complexes (rc) [53] and/or cellular machinery [54] . thus, it slows down the replication or translation and assembly of particles [51] . the 3 utr region is important for translation and replication of the rna genome through interactions with viral and host proteins, genome stabilization, and rna packaging [55] . a better understanding of the potential impact of 3 utr variations in replication of bagv could be important in the study of mechanisms implicated in their pathogenicity [56, 57] . most motifs linked to virulence previously described in these proteins of other mbfvs were conserved among bagv isolates. however, some non-conservative mutations were identified in e, ns1, ns3 and ns5. in general, non-conservative amino acid mutations (nc) are spontaneous, rare, and hazardous, and then represent the main causes of genetic diversity. thus, non-conservative mutations observed on bagv genome could modulate viral phenotypes of particular isolates in mechanisms such as virus cell entry, replication, production of viral particles, and assembly, and cause modifications in post-translational regulation as previously demonstrated for other flaviviruses such as denv [58] [59] [60] . the e protein is involved in cell receptor recognition, attachment, cell fusion, tropism, and virulence [58] . ns1 is the most conserved non-structural protein of flaviviruses. associated with the other non-structural proteins, the ns1 protein plays an important function in viral replication and assembly and viral escape to host innate immune response [61] . the ns3 protein is the main component of the replication machinery and ensures multiple functions in viral evasion to host antiviral response and in production and assembly of infectious viral particles [62] . the ns5 protein is the largest viral protein that serves as the rna-dependent rna polymerase (rdrp) and performs multiple functions essential for viral replication, including processing the viral polyprotein, replicating the viral rna. sharing these motifs of virulence mostly with mbfvs, nkvfs and cxfv than with aefv showed that bagv could be more closely related to mbfvs transmitted by culex mosquitoes and could explain frequent bagv isolations mainly from mosquitoes of culex genus and its capacity to infect vertebrates such as wild birds [1, 4, 5, 9, 10] . in addition, the west african bagv isolates characterized in our study were mainly isolated from culex poicilipes and culex neavei mosquitoes which have been reported as potential vectors for flaviviruses such as wnv [63] . culex neavei was also found as a competent vector able to transmit flaviviruses such as usuv and wnv [64, 65] . despite no available data on culex poicilipes competency to transmit flaviviruses, these two mosquito species belonging to culex genus could play an important role in natural transmission of bagv to vertebrates such as wild birds since another member of the culex genus, culex tritaeniorhynchus, has been found competent to transmit bagv to mice [8] . the phylogenetically informative sites identified on the bagv genome located mainly in ns3, ns4b and ns5 proteins, respectively, could have a considerable impact in viral fitness on host for corresponding west african isolates. in addition, the prediction of the n-glycosylated sites at different positions on bagv genome such as asn2333 in ns4b and the nysi motif at 153th position of the e protein showed that post-translational modifications may influence acquisition or loss of capacity in mechanisms such as pathogenicity, evasion of innate immune pathways. indeed, flaviviruses ns4b plays an important role in replication of viral rna facilitating the formation of replication complexes and modulating host innate immune response such as interferons, micrornas and rna interference, formation of stress granules and the unfolded protein responses [66] [67] [68] . a previous study had shown that n-glycosylation of ns4b of denv does not affect the protein stability but causes a considerable reduction in efficiency of viral production [69] . presence of a glycosylation site and an informative site in the viral ns4b protein could influence the efficiency of viral replication and the outer shape of the virion. the presence of the n-linked glycosylation motif nys had been previously reported at 67/153th and 154th on the e protein of denv and wnv (lineage 1 strains and some neuroinvasive lineage 2 strains), respectively, involving in receptor binding, viral morphogenesis, viral infectivity, and tropism [70] [71] [72] [73] [74] . since glycosylation is a means of evasion to immune recognition within the host by masking particular antigenic sites from recognition by neutralizing antibodies, it could increase the diversity of the glycosylation on viral proteins [75, 76] . nevertheless, it could be important in future studies to determine whether the predicted glycosylation sites are really used (asparagine-linked) using specific enzymatic digestion by endo h and peptide n-glycosidase (pngase f) [77] . our data suggest the ability of bagv to develop phenotypically important variations and potentially adaptation to new vertebrate hosts such as humans. however, to understand better the impact of variation on these predicted n-glycosylation sites and the identified phylogenetically important variations would require in vitro studies with reverse genetically engineered infectious clones on mosquito or mammalian cell lines and in vivo experiments in mosquitoes or in animal models like mouse [78, 79] . however, antibodies against bagv proteins or infectious clone are currently not available for bagv. the identification of natural recombination events between virus isolates is important for our understanding of virus evolution. in our study, we identified a recombination event in the e protein bagv. recombination was documented in other members of the mosquito-borne flavivirus group [80, 81] , but had not yet been demonstrated to occur in bagv. identification of recombination breakpoints and the graphical detection of conflicting phylogenetic signals gave confirmation of this recombination event in e protein of the senegalese isolate ard54139_dakar-bango_sen_1989 as previously described for zikv [74, 82] . nevertheless, the precise molecular mechanisms of the template switches are unknown. the e protein is highly important because it encodes the most important antigen with regards to virus biology and humoral immunity. therefore, large-scale genetic changes in this region, as might be brought about by recombination, could have significant impact on virus phenotype [44] . the estimation of the selection pressures acting on each protein of bagv demonstrated episodes of strong negative selection in functionally important proteins. these results suggested frequent purging of deleterious polymorphisms in the bagv genome that could be associated with accumulation of synonymous mutations during bagv transmission [83] . however, location of more significant episodes of positive selection in the e, ns1 and ns5 proteins indicated that they could represent preferential selection targets during bagv evolution [84] . indeed, the e protein of flaviviruses plays a crucial role in early steps of host cell binding and viral entry and represents a main target for immune responses influencing antigenic response and positive selection on the e protein is a hallmark of the emergence of flaviviruses [85, 86] . positive selection episodes have been also previously reported for the denv-3 capsid, however, the impact needs to be further investigated [87] . likewise, non-structural proteins could also be targets of positive selection. the ns1 protein is essential for viral rna replication, is involved in immune system evasion, and represents the major positive selection target during speciation of arthropod-born flaviviruses such as denv and zikv [88] . ns2a and ns4b proteins have been shown to antagonize the interferon response during denv infection [89] and changes in these regions would be evolutionary advantageous selecting for bagv strains with strong innate immunity suppression mechanisms. mutations in the ns4b protein were also seen to modulate several phenotype mechanisms of flaviviruses, such as pathogenesis [90] , viral adaptation [91] , replication [68] , neurovirulence [66] and host preferences [92] . thus, presence of positively selected sites in ns4b of bagv isolates could have major impact in its natural evolution. ns3 and ns5 proteins are crucial for viral replication, since non-conservative changes in these regions could modify process of protease and atpase/helicase functions of ns3 protein [93] and rna polymerase activity of ns5 protein [94] . these several polymorphic amino acid coding sites in the bagv genome suggest that these proteins may be experiencing relatively adaptive changes in the natural evolution and they should be prioritized in future experimental studies. despite the evidence of a single phylogenetic group for bagv sequences analyzed in our study, the evolutionary rates are expected in accordance to proteins functions; the ns5 representing the polymerase and the most conserved protein [86] . the inferred bayesian mcc trees indicated a single introduction of bagv into europe and africa from india, contrary to other african flaviviruses as wnv [95] and zikv [74] , suggesting an indian origin of bagv. estimated times from the mrca suggested a distant origin of west african bagv sequences analyzed in this study from the 15th century. thus, further phylodynamic analyzes based on more complete sequences could be interesting for determining geographical pathways and potential evolution patterns in correlation with bagv spread from india to african and european continents. the codon adaptation index (cai) represents a reliable bio-informatics approach to measure the synonymous codon usage bias and to assess the adaptation of viral genes to their hosts [96] . flaviviruses can infect and replicate in hosts of different phyla. therefore, their versatility in gene expression and protein synthesis and changes in the viral rna genome could affect the fitness of the virus in a specific host relating to dinucleotide frequencies, codon preferences, and codon pair biases [97] [98] [99] . nevertheless, ecology, different virus-host relationships, biogeographical migrations of flavivirus species and genetic differences may explain observed differences in flaviviral codon usage preference to human housekeeping genes [98, 100, 101] . in particular, nkv flaviviruses were only isolated from vertebrates and are maintained in nature by horizontal transmission between vertebrate hosts [102, 103] . although isfs were thought to sustain their populations in their respective insect vectors in the absence of mammal reservoirs, so lower translational efficiency in vertebrates could be expected [97, 104] . in addition, the highest cais of yfv and denv could be related to their long histories of infection in humans [105] . indeed, yfv and denv are maintained in endemic and sylvatic cycles, which conducted to repeated epidemics for more than one hundred years. however, yfv showed generally a higher virulence in human infections, particularly when it is compared to denv infections reported in africa [106] . this could explain the higher cai values of yfv towards codon usage of the human housekeeping genes. with evidence of adaptation to human house-keeping genes, bagv could be potential cause of infection in vertebrates, such as humans. considering the highest cai values of west african bagv isolates when compared to isolates responsible of the spanish wild bird's outbreak in 2010 [9] , bagv adaptation to vertebrate species such as birds could have led to an extension of adaptation to other species as shown in a previous virus study [46] . interestingly, west african bagv isolates showed a higher evidence of codon adaptation than mbfvs well known to infect humans, such as wnv which is a major cause of human encephalitis in usa and responsible of recent outbreaks in europe [107] and zikv associated with microcephaly in fetuses and newborns during the outbreak in brazil in 2015 [91] . thus, further comparison of codon adaptation indexes of other bagv genomic regions, such as the 3 utr, among isolates that differ in biological, ecological, and genetic characteristics could help to characterize the evolutionary adaptation of bagv genomes to vertebrate hosts [46, 108] . nevertheless, to ensure the potential of bagv to be involved in human encephalitis cases, it would be important to evaluate its pathogenicity on human induced pluripotent stem cell lines (ipsc) capable of differentiating into brain microvascular endothelial cells (bmecs) and constituting a robust model of the human blood-brain barrier [109] . otherwise, the ipsc cells can also generate primitive neural stem cells (nscs), which can differentiate into neurons, astrocytes, or oligodendrocytes [110] . these bagv sequences data obtained in our study could be used not only in future viral studies, but also in development of reverse genetic reagents or reliable diagnostic tools for investigation of this virus in human populations. the following are available online at http://www.mdpi.com/1999-4915/10/4/193/s1, figure s1 : sequences of conserved structural rna domains identified on the 3 utr of bagaza genomes used in this study. bag) strain: dak ar b 209 full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses mosquito vectors of the 1998-1999 outbreak of rift valley fever and other arboviruses (bagaza, sanar, wesselsbron and west nile) in mauritania and senegal isolations of west nile and bagaza viruses from mosquitoes (diptera: culicidae) in central senegal (ferlo) arbovirus isolations from mosquitoes collected during 1988 in the senegal river basin genetic characterization of bagaza virus (bagv) isolated in india and evidence of anti-bagv antibodies in sera collected from encephalitis patients preliminary findings on bagaza virus (flavivirus: flaviviridae) growth kinetics, transmission potential transovarial transmission in three species of mosquitoes bagaza virus in partridges and pheasants monitoring of the bagaza virus epidemic in wild bird species in spain bagaza virus and israel turkey meningoencephalomyelitis virus are a single virus species bagaza virus inhibits japanese encephalitis & west nile virus replication in culex tritaeniorhynchus & cx. quinquefasciatus mosquitoes natural bagaza virus infection in game birds in southern spain mega7: molecular evolutionary genetics analysis version 7.0 continuous cell lines and immune ascitic fluid pools in arbovirus detection development of one-step quantitative reverse transcription pcr for the rapid detection of flaviviruses unipro ugene: a unified bioinformatics toolkit multiple sequence alignment with high accuracy and high throughput divein: a web server to analyze phylogenies, sequence divergence, diversity, and informative sites prediction of n-glycosylation sites in human proteins the rnaz web server: prediction of thermodynamically stable and evolutionarily conserved rna structures the vienna rna websuite improved consensus structure prediction for rna alignments incorporating chemical modification constraints into a dynamic programming algorithm for prediction of rna secondary structure does the choice of nucleotide substitution models matter topologically? bmc bioinform fasttree 2: approximately maximum-likelihood trees for large alignments rdp4: detection and analysis of recombination patterns in virus genomes multiple significance tests: the bonferroni method hypothesis testing using phylogenies the effect of recombination on the accuracy of phylogeny estimation fubar: a fast, unconstrained bayesian approximation for inferring selection improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty accurate model selection of relaxed molecular clocks in bayesian phylogenetics bayesian phylogenetics with beauti and the beast 1.7 the codon adaptation index-a measure of directional synonymous codon usage bias, and its potential applications codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes spread of the pandemic zika virus lineage is associated with ns1 codon usage adaptation in humans importance of codon usage for the temporal regulation of viral gene expression genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on marburg virus evolution a combined set of tools to assess codon usage adaptation a novel server to estimate an expected value of codon adaptation index (ecai) human housekeeping genes, revisited berzal-herranz, a. functional information stored in the conserved structural rna domains of the flavivirus genome the extent of homologous recombination in members of the genus flavivirus family level phylogenies reveal modes of macroevolution in rna viruses clinical sequencing uncovers origins and evolution of lassa virus conserved rna secondary structures in flaviviridae genomes flaviviral rnas: weapons and targets in the war between virus and host identification and characterization of small sub-genomic rnas in dengue 1-4 virus-infected cell cultures and tissues a highly structured, nuclease-resistant, noncoding rna produced by flaviviruses is required for pathogenicity noncoding subgenomic flavivirus rna: multiple functions in west nile virus pathogenesis and modulation of host responses functional non-coding rnas derived from the flavivirus 3 untranslated region japanese encephalitis virus non-coding rna inhibits activation of interferon by blocking nuclear translocation of interferon regulatory factor 3 poly (a)-binding protein binds to the non-polyadenylated 3 untranslated region of dengue virus and modulates translation efficiency rna structure duplications and flavivirus host adaptation west nile virus encodes a microrna-like small rna in the 3 untranslated region which up-regulates gata4 mrna and facilitates virus replication in mosquito cells isolation and genome characterization of a novel duck tembusu virus with a 74 nucleotide insertion in the 3 non-translated region post-translational regulation and modifications of flavivirus structural proteins structural and functional studies of the promoter element for dengue virus rna replication a trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the ns4b protein of dengue virus type 4 flavivirus ns1: a multifaceted enigmatic viral protein a proline-rich n-terminal region of the dengue virus ns3 is crucial for infectious particle production evaluation of the efficiency of bird-baited traps for sampling potential west nile fever mosquito vectors (diptera: culicidae) in senegal vector competence of culex neavei (diptera: culicidae) for usutu virus vector competence of culex neavei and culex quinquefasciatus (diptera: culicidae) from senegal for lineages 1, 2, koutango and a putative new lineage of west nile virus flaviviral ns4b, chameleon and jack-in-the-box roles in viral replication and pathogenesis, and a molecular target for antiviral intervention a combined genetic-proteomic approach identifies residues within dengue virus ns4b critical for interaction with ns3 and viral replication evidence for a genetic and physical interaction between nonstructural proteins ns1 and ns4b that modulates replication of west nile virus mutation of putative n-glycosylation sites on dengue virus ns4b decreases rna replication biological and phylogenetic characteristics of west african lineages of west nile virus n-linked glycans on dengue viruses grown in mammalian and insect cells crucial role of the n-glycans on the viral e-envelope glycoprotein in dc-sign-mediated dengue virus infection n-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity molecular evolution of zika virus during its emergence in the 20(th) century the hiv glycan shield as a target for broadly neutralizing antibodies glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy characterization of n-glycan structures on the surface of mature dengue 2 virus derived from insect cells structure-based mutational analysis of several sites in the e protein: implications for understanding the entry mechanism of japanese encephalitis virus in vitro and in vivo evaluation of mutations in the ns region of lineage 2 west nile virus associated with neuroinvasiveness in a mammalian model why do rna viruses recombine? analysis of genotype diversity and evolution of dengue virus serotype 2 using complete genomes effects of recombination on densovirus phylogeny a large variation in the rates of synonymous substitution for rna viruses and its relationship to a diversity of viral infection and transmission modes recombination and positive selection identified in complete genome sequences of japanese encephalitis virus antigenic structure of flavivirus proteins molecular evolution of dengue 2 virus in puerto rico: positive selection in the viral envelope accompanies clade reintroduction jyh-hsiung, h. comparative analysis of full genomic sequences among different genotypes of dengue virus type 3 nonstructural proteins are preferential positive selection targets in zika virus and related flaviviruses inhibition of interferon signaling by dengue virus subcellular localization and membrane topology of the dengue virus type 2 non-structural protein 4b message in a bottle: lessons learned from antagonism of sting signalling during rna virus infection comparison of genotypes i and iii in japanese encephalitis virus reveals distinct differences in their genetic and host diversity crystal structure of the ns3 protease-helicase from dengue virus structural and functional analysis of methylation and 5 -rna sequence requirements of short capped rnas by the methyltransferase domain of dengue virus ns5 phylogeny, selection pressure and evolutionary time-scale analysis the global distribution of the arbovirus vectors aedes aegypti and ae. albopictus. elife does adaptation to vertebrate codon usage relate to flavivirus emergence potential? codon usage bias: causative factors, quantification methods and genome wide patterns: with emphasis on insect genomes bicluster pattern of codon context usages between flavivirus and vector mosquito aedes aegypti: relevance to infection and transcriptional response of mosquito genes factors shaping the adaptive landscape for arborviruses: implications for the emergence of disease arbovirus evolution in vivo is constrained by host alternation pathogenesis of modoc virus (flaviviridae; flavivirus) in persistently infected hamsters latent infection of rio bravo virus in salivary glands of bats insect-specific flaviviruses: a systematic review of their discovery, host range, mode of transmission, superinfection exclusion potential and genomic organization yellow fever virus: genetic and phenotypic diversity and implications for detection, prevention and therapy identification of host genes leading to west nile virus encephalitis in mice brain using rna-seq analysis zika virus: an update on epidemiology, pathology, molecular biology, and animal model genome-wide analysis reveals class and gene specific codon usage adaptation in avian paramyxoviruses 1 an isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes and neurons derived from human induced pluripotent stem cells efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells this research received no specific grant from any funding agency in the public, commercial, key: cord-304935-8gcmqh4e authors: chiriboga, gonzalo; de la rosa, andrés; molina, camila; velarde, stefany; carvajal c, ghem title: energy return on investment (eroi) and life cycle analysis (lca) of biofuels in ecuador date: 2020-06-28 journal: heliyon doi: 10.1016/j.heliyon.2020.e04213 sha: doc_id: 304935 cord_uid: 8gcmqh4e in ecuador, the net energy contribution of biofuels is unknown or unnoticed. to address this issue, we determined the energy return on investment (eroi) for bioethanol and biodiesel. the selection of raw materials relied on their productive capacity, export and import records, and historical yields. consequently, the scope included three raw materials for ethanol (sugar cane, corn, and forest residues) and four for biodiesel (african palm, pinion, bovine fat, and swine fat). using a method based on the life cycle analysis (lca) of each biofuel, we assessed the entire production chain through statistical processing of primary and secondary information. then we calculated the calorific values in the laboratory, compared energy inputs/outputs, and finally obtained the energetic returns. erois for bioethanol were: 1.797 for sugarcane, 1.040 for corn, and 0.739 for wood. the results for biodiesel were: 3.052 for african palm, 2.743 for pinion, 2.187 for bovine fat, and 2.891 for swine fat. these values suggest feasibility only for sugarcane in the case of ethanol. in contrast, biodiesel has better prospects because all the feedstocks analyzed had erois higher than two. nevertheless, biodiesel is not available for trading in ecuador because energy policy has overlooked systems based on higher energy return. future studies should consider more comprehensive variables such as climate change, land use, and water management. globally, the use of biofuels in the transport sector increased 7% in 2018 (international energy agency, 2019). however, this growth was insufficient to achieve the international objectives set in the sustainable development scenarios for 2030 (united nations, 2015) , since this commitment would require tripling bioethanol and biodiesel content in final blends, involving around 10% of global transport (sawin et al., 2018) . several issues have slowed the incorporation of biofuels into worldwide transportation, for example, competition with the food sector for the use of feedstocks (ho et al., 2014) , the decrease in the calorific value due to the presence of oxygen (oh et al., 2018) , stillage handling and disposal (silva et al., 2011) , and the energy return on investment (eroi) (jessica g lambert, hall, balogh, gupta and arnold, 2013) . the eroi is directly or indirectly linked to the achievement of wellbeing, energy quality, and energy sovereignty (jessica g . it describes the amount of energy a source requires to deliver a unit of energy to society, and considers the energy flows involved in all stages of the production process . several methods for calculating the eroi have been published that contain points of agreement, but also methodological differences. the main disagreements relate to the way energy flows (moeller and murphy, 2019) , system boundaries, and residual energy embedded in co-products are identified and quantified (capell an-p erez, castro, javier and gonz alez, 2019) . the ecuadorean government has sought alternatives for reducing oil derivative imports to meet the local demand. these endeavors have intensified since last year, and in april 2019, the ministry of agriculture announced the agrofuels plan, which would replace 30,000 ha of rice with sugar cane to produce bioethanol (el universo, 2019) . the program would also incorporate 140 million liters of african palm oil per year to begin producing 15% v/v biodiesel (b15) (ministry of agriculture., 2019) (astm specification d6751, 2010). reducing imports would have a significant effect on ecuador's economy because, since the 70s, the country has maintained a subsidy on gasoline, diesel, and liquified petroleum gas (rivadeneira, 2019) . this public expenditure is increasingly unsustainable with demand increasing at around 2.5% annually, fuel migrating to other consumer sectors, and the volatility of international derivative prices (espinoza and guayanlema, 2017) . in this context, ecuador has partially promoted the incorporation of biofuels into the energy supply (consejo nacional de planificaci on, 2017) (ministerio de electricidad y energía renovable, 2017b) (presidencia del ecuador, 2012) . for example, since 2012, a mixture of gasoline and anhydrous ethanol (5% v/v), has been available for trading in the local market (castro, 2012) . concerning biodiesel, the blends b5, b10, b15, and b20 have been mentioned officially (agencia de regulaci on de hidrocarburos, 2013), but none have a real share in the transport sector. currently, the only energy use of vegetable oils is project "zero fossil fuels in galapagos" (programa de las naciones unidas para el desarrollo, 2016) (ministerio de electricidad y energía renovable, 2017a). this initiative generates electricity through direct combustion of oil without previous transesterification. ecuador produces 225 million boe (barrels of oil equivalent) of primary energy; it exports 65%, and about 23% is retained as feedstock for refineries (ministerio coordinador de sectores estrat egicos, 2016) . this production is insufficient to cover domestic demand. thus, in 2018, about 40% of the local consumption of diesel, gasoline and liquefied petroleum gas was imported (c amara de comercio de quito, 2018) and from january to august 2019, these imports increased 16.8% compared to the same period of the previous year (banco central del ecuador, 2018) (planificaci et al., 2019) . consequently, the government has encouraged researchers and industrialists to design alternative fuels using economically viable and environmentally friendly feedstocks, as mentioned in the agrofuels plan (banerjee et al., 2019 ) (eduardo rosero, 2011 . the share of biomass in the energy matrix is less than 3%, and it is used mainly for cooking food and generating electricity (ministerio coordinador de sectores estrat egicos, 2016) . in second place is the production of anhydrous ethanol for the "gasolina ecopaís" project (about 100 million liters) (petroecuador, 2018) . the production per inhabitant is comparable with countries of the region such as colombia (unidad de planeaci on minero energ etica, 2018) or uruguay (ministerio de industria energía y minería, 2017). however, comparison with world leaders such as brazil (energy research office, 2018) reveals a big gap of almost 12 to 1. it is essential for ecuador to evaluate the actual energy contribution of biofuels and the degree of flexibility of their net output in relation to changes in production process and technology. eroi is a proper tool to carry out this evaluation since it considers the entire production cycle from planting until it is ready for blending (murphy and hall, 2010) . first, the study developed an extensive survey of the different raw materials with energetic potential (instituto nacional de preinversi on, 2014), namely agricultural and forestry crops with higher production records and yields, such as sugar cane, corn, wood, african palm (figueroa de la vega, 2008) , and pinion (instituto interamericano de cooperaci on para la agricultura, 2016). for livestock, the analysis included resources such as cattle, pigs, goats, and sheep, and focused on their transesterifiable fat content. then we determined the energy expenditure index (murphy et al., 2011) using the life cycle analysis (lca). this method permitted us to break down existing biodiesel and bioethanol production processes (tomi and schneider, 2017) . the analysis included the collection and statistical treatment of secondary information about industrial plants in operation in ecuador and other countries. the third part consisted of determining the lower calorific value and an elemental analysis by applying standardized methods for quantifying energy availability. finally, the relationship between the amount of energy produced and consumed was estimated using the eroi concept (jessica gail . few studies encompass the energy return of biofuels from different origins at the same time. furthermore, we included agricultural products with few records in the literature, such as animal fat, pinion oil, and forest residues. the objective was to group the most compelling possibilities and present their energy advantages in terms of eroi. this information is essential for allocating resources to programs that have the best energetic benefits. the presence of economic externalities does not influence eroi; instead, it relies on the concept that humans use the best resources first (hall, 2017) . the research includes biofuels that are being or could be used in ecuador, based on the characteristics of local industry. it incorporates the data treatment of the agricultural product to be analyzed, such as cultivation, consumption, import, and export, as well as the different levels of raw material allocation for biofuel production. the results permit us to see in a glance the effectiveness of policies implemented to meet energy requirements, given that sectors with more significant demands should be addressed first with higher eroi biofuels. this method is easy to replicate and allows the particularities of each country to be considered. the successful implementation of a biofuel program depends on technology, economics, environmental sustainability, and public policy support (saravanan et al., 2018) . governments of the region regulate their specifications by enacting minimum levels of ethanol or biodiesel content in blends (v azquez et al., 2016) (see table 1 ). the prospects of biofuels depend on the behavior of international energy markets (pro ecuador, 2013) (energy information administration, 2019). for instance, the fall in the oil prices in 2020 (usd 30 per barrel) would result in a severe decrease in the demand for biofuels, and lead to the collapse of their global price in the mid-term (getachew nigatu, kim hjort, agapi somwaru, 2015) . environmental standards, energy security, rural economic activation, and climate change will drive bioethanol consumption in the u.s. and the e.u., which, together with brazil, would remain as the leading producers worldwide (getachew nigatu, kim hjort, agapi somwaru, 2015) . their production would increase from 76 billion liters in 2012 to about 100 billion liters by 2022. additionally, emerging economies like china, thailand, and india would contribute up to 10 billion liters of ethanol by 2020 (u.s. department of energy, 2019). global expansion of biodiesel is expected to increase from 15 to 30 billion liters between 2012 and 2022 (union zur f€ orderung von oel -und proteinpflanzen e.v., 2017). again, the e.u., u.s., and brazil would dominate the market with good participation by argentina in south america. this production will utilize 15% of the world's vegetable oil (pro ecuador, 2013). ecuador began producing biodiesel from african palm in 2005, and up to 2012, all production was for export. in 2013, when national production reached 140 million liters, the government enacted executive decree 1303 establishing b5 blending for biodiesel. this proportion was supposed to increase gradually to b15, which would mean achieving a production of 480 million liters of biodiesel per year. for anhydrous ethanol, production started in 2012 with ecopaís gasoline, which was to contain up to 10% anhydrous bioethanol. the national fuel trade agency (arch) states that only ecopaís gasoline (5% v/v) is available in the current market, and biodiesel will have to wait for distribution and marketing policies (hernan, 2018) . the use of vegetable oil is limited to pilot projects such as zero fossil fuels for galapagos and the juan jos e castell o zambrano foundation, in cooperation with termopichincha; they have been burning pinion oil without transesterification to generate electricity since 2016. agricultural zoning published by the ministry of agriculture (mag) identified the provinces with highest potential for the production of sugar cane (guayas 74%, loja 12%, cañar 7%, imbabura 4% los ríos 1% and carchi <1%) and african palm (esmeraldas 58%, los ríos 11%, sucumbíos 9%, pichincha 7%, santo domingo de los ts achilas 5%, orellana 4%, and guayas 2%) (instituto nacional de preinversi on, 2014). this information is essential for evaluating the energy required to transport biofuels to blending centers (enclosed in thick lines on the maps). figure 1 shows the geographical distribution of the energy potential of sugar cane on the left (15 740 tj) and african palm on the right (87 830 tj) (instituto ecuatoriano de estadística y censos, 2017). this record includes fermentation, transesterification, direct combustion, gasification, and pyrolysis. although the potentials do not include energy return, the advantage of african palm is clear in terms of available energy. the eroi calculated in this research includes the energy associated with refining and transporting fuel to the country's main point of distribution where blending would take place. the ratio of this amount of energy to the energy obtained when burning the fuel is called the energy return on investment -point of use (eroi pou ) (j. lambert, hall, balogh, poisson and gupta, 2012) . 〖eroi〗 pou ¼ ðenergy returned to societyþ ðenergy used to get and deliver that energyþ (1) eq. (1) described by ). the eroi is a useful decision-making tool before exploiting an energy resource or when defining public policy. for example, a value of 10 or 10:1 means that society would benefit from this energy resource by 90%, and the remaining 10% is necessary to obtain that energy. this proportion varies considerably depending on the source. for example, gas and oil can have an eroi of 20:1, but in the case of tar sands, the eroi decreases to 4:1. the value for coal is usually higher and can be up to 46:1. among the most compelling renewable energies, the eroi is 84:1 for hydroelectric generation and 18:1 for wind power (j. lambert et al., 2012) . for the specific case of biofuels, the values are less encouraging; for example, ethanol from corn has erois that range from 1.2 to 1.6 in u.s. distilleries (murphy and hall, 2010) . the source is not the only factor that determines the eroi; other inputs such as technological improvements or depletion can play crucial roles. gagnon et al. addressed this topic for oil and gas production worldwide. the eroi increased from 26:1 in 1992 to about 35:1 in 1999, and then it decreased again to 18:1 in 2006 (gagnon et al., 2009 ). here it is clear that during the first period from 1992 to 1999, the energy return increased thanks to technological advances and extraction methods; however, during the second period from 1999 to 2006, depletion of the sources exceeded the benefits achieved through technological advances. charles hall (the originator of the concept of eroi) states that to keep our civilization working in the way we know it, energy systems should have erois higher than 5 ). the eroi can vary depending on the system components; for example, boundaries and outputs/inputs assumed in the energy balance, and local particularities such as production methods, types of raw materials, and energy consumed for environmental remediation (see figure 2 ). lca starts as a horizontal analysis tool and examines the inputs and outputs of every component of the process. then it carries out a vertical analysis of the activities of each stage (petraru and gavrilescu, 2010) , while seeking not to include irreversibilities that are not useful to society (arvesen and hertwich, 2015) . in general terms, this tool includes the definition of objectives, scope, inventory analysis, impact assessment, and interpretation of results. in ecuador, ethanol comes from sugar cane only; therefore, energy consumption data are from actual industrial plants. in contrast, there is no industrial production of biofuels based on corn, wood, fat, or african palm. hence, statistical tools and case studies are needed to determine the relationship between the energy consumption "dependent variable" and the raw material "independent variable." for biofuels production, this information allows the construction of four scenarios with different levels of corn, wood, fat, and african palm designated. this approach is based on the concept of economy of scale, where the unitary production cost decreases as the scale of operation increases. all investigations that seek to determine the eroi of any resource use one of the following three techniques: process analysis, economic inputoutput, or a hybrid of both (murphy et al., 2011) . looking for the implications of energy return in society (jessica gail , reviewed the eroi of different sources. the most relevant references are presented below with a description of the technique used in each case (see table 2 ). the technique employed in each case defines the methodology and the instrument used to identify and account for energy flows. it consists of two dimensions. the first describes the boundaries of the production chain from cultivation to distribution; these are the energy outputs. the second sorts by levels and indicates the type of energy and materials directly or indirectly associated with the process. in this case, we chose level 2, which includes the energy inputs under investigation and the inputs from the other energy sectors (murphy et al., 2011) . this ensemble of dimensions results in a technique based on process analysis. the lca is the instrument that identifies the processes within the boundaries defined in dimension one, as well as the energy flows of dimension two. the aim is to define a roadmap for selecting the proper energy balance and the type of data to be collected. the scenarios vary the proportions of feedstock to estimate energy consumption through linear regression models. the variance analysis of the models and the t-test for the slopes show a statistically significant relationship between the amount of raw material destined for biofuel and energy consumption, since the p-value of each test is less than the level of significance chosen (α ¼ 0.05). moreover, this relationship demonstrates a high correlation between the variables according to the values of r (see tables 3, 4; figure 3 ). the selection criteria rely on the scenario that results in less energy consumption during the feedstock's industrial stage. the following are the best scenarios for each case. corn. -s4 (50%) with energy consumption in the industrial stage equal to 13.85 mj/kg bioethanol, which is higher than industrial plants in argentina 11.91 mj/kg, u.s. 11.80 mj/ kg, and chile 12.29 mj/kg. wood. -s4 with energy consumption of 29.880 mj/kg of bioethanol, a level comparable to other industrial plants, 30.370 mj/kg in the united kingdom, 32.921 mj/kg in canada, and 46.766 mj/kg in brazil. african palm. -s4 (5.694 mj/kg biodiesel), which represents 48.59% of total energy consumption. it is within the range of preliminary studies 3.87-6.58 mj/kg biodiesel. animal fat. -s4 (9.045 mj/kg), which represents 51.54% of total energy consumption; this is slightly higher than the values presented in preliminary studies (5.548-8.165 mj/kg). pinion. -s4 (8.259 mj/kg biodiesel), which represents 64.81% of total energy consumption; this is higher than values presented in peru 6.820 mj/kg, argentina 6.842 mj/kg, and india 6.442 mj/kg. (murphy et al., 2011) and (jessica gail lambert et al., 2013) . reference technique ethanol and biodiesel production (pimentel and patzek, 2005) process analysis oil and gas discovery and production (guilford, hall, o'connor and cleveland, 2011) process analysis oil and gas production (guilford et al., 2011) hybrid oil gas and tar sand production process analysis oil and gas production (hu et al., 2013) economic input-output the production of biofuels generally involves the generation of some co-products; for example, glycerin in biodiesel, which accounts for approximately 10% of energy consumption. likewise, bioethanol from corn has multiple co-products that include oils and meal for animal feed; their exclusion or incorporation into the analysis produces essential changes in the final values of the eroi. hall et al. (2011) addressed this issue when comparing the eroi of corn alcohol, previously obtained by (kim and dale, 2005) and (pimentel and patzek, 2005) , as 1.73: 1 and 0.82: 1, respectively. approximately 50% of this difference is due to the treatment and use of the co-products generated in the industrial process. this work excluded the subsequent treatment of co-products because of uncertainty about the allocation of the weighting factors of their energy consumption and their final use . hence, the scenarios developed in this work are conservative in this sense. similarly, we excluded the energy required for environmental remediations because it could change according to the specific obligations in each zone. finally, esmeraldas refinery was used as the final destination of biofuels, since this is where the mixing of ecopaís b5 gasoline take place. considering these methodological aspects, the calculated eroi is very close to the eroi at the point of use, with less than 5% difference ) (2% in our cases). we determined the energetic availability from the net amount of heat produced by fuel combustion under controlled conditions (cengel, yunus. boles, 2014) . local producers and research centers provided the samples. they are listed below with the respective selection criteria (see table 5 ). finally, we obtained the net calorific value (ncv) from specialized tests, such as astm d240-02 and (sader and oliveira, 2002) , for gross calorific value and elemental analysis, respectively. table 4 . energy consumption involves the entire production chain, including the stages of cultivation, distribution, chemical transformation, and delivery to blending facilities. figures 4 and 5 show the extraction of biofuels from agricultural commodities and animal fat, respectively. the following is a model for calculating the eroi at the point of use for sugar cane in scenario 1. other raw materials follow the same procedure. the desegregation of formula (3) shows the decrease in eroi through the production chain. this information is also shown as a percentage, taking the cultivation stage as a reference. eroi farming ¼ 27:7 8:365 the eroi farming represents the energy return of sugar cane, considering cultivation only. after adding the other stages, the eroi becomes 2.925, 1.886, and 1.796 for transportation, transformation, and distribution (see figure 6 ). the eroi farming begins at 3.311 and decreases by 11.68%, 43.04%, and 45.77% by the end of its production chain. that is, 3.311 x (100-45.77)% ¼ 1.796. this detail allows the analysis of consumption by subprocess. for example, the eroi of forest residues decreases by 80.60% by the final stage. as a result, we obtain an eroi at the point of use that is less than one. 2 the ncv of sugar cane, african palm, fat, and pinion was obtained experimentally, and ncv of corn and wood was theoretical. the following eight figures compare the results of the energy consumed per stage and the available ncv, to observe net benefits. the results confirm that energy consumption is strongly influenced by both the raw material and the industrial stage. in general terms, biodiesel has an advantage over ethanol (see table 6 ). the calculation of the eroi has been adjusted not only for the agricultural and industrial reality of ecuador, such as ethanol from sugarcane, but also for raw materials, whose production is part of national development plans and public policies for future projects such as biodiesel. therefore, we propose substitutive goods to generate more inputs that will allow the prediction of the net energy impact of introducing biofuels from different origins under current production conditions. besides eroi, other socioeconomic considerations can arise, such as the creation of jobs and the activation of the agricultural sector, as well as reducing refined fuel imports resulting from the lack of internal production capacity. the fossil fuel subsidy policy impacts the national government in two ways. on one hand, subsidized fuel increases consumption due to its low price, and the country has to allocate around usd 4 billion per year (4% of gdp) to meet the demand. on the other hand, since it does not have refining capacity to cover the domestic market, the government has to import derivatives at fluctuating prices, placing the local dollarized economy at risk year after year. the issue gained importance in the country after october 2019, because the ecuadorean government attempted to eliminate gasoline and diesel subsidies. the measure provoked intense social protests from several sectors, such as transport, agriculture, and industry. finally, in the face of excessive pressure, it had to withdraw the decision. therefore, all options that are considered should adopt an integrated analysis that includes technical, economic, and social aspects. the eroi could provide vital information to address this issue from a more insightful standpoint. development is strongly related to the eroi on which societies base their economies. in ecuador, the production of primary energy from fossil fuels is close to 94%, and biofuels constitute less than 1%. however, increasing this share is a subject of constant debate and pressure from various sectors. this is where the concept of eroi plays an influential role, since it allows real-net contributions to be compared with the apparent benefits of green energy, driven mainly by actions to mitigate climate change. in many cases, those actions ignore the fact that fossil fuels provide energy for developing renewable technology. the eroi values for ecuador range from 0.845 to 1.796 for ethanol and 1.500 to 3.052 for biodiesel. moreover, the results show that the greatest energy demand is for cultivation (10.1-54.2%) and industrial stages (33.8-85.0%), so we compared them with regional and world values. biodiesel from african palm la fabril commercially, oils and fats are one of the most concentrated sectors in ecuador according to the center for economic and social rights. la fabril is one of eight companies that account for 83% of the national market. since 2012, it has exported oil for biodiesel using the following method, astm d 6751/b100 (blends with diesel) (lasso, 2018) . there is no industrialized refining of fat to biodiesel yet. therefore, the department of petroleum, environment, and contamination (dpec) provided a sample that complies with technical regulations in terms of calorific value. the producer is part of the dpec customer portfolio. biodiesel from pinion iner pinion oil, as an energy resource, is a pilot program of the ministry of energy and non-renewable natural resources, and iner is the implementing branch of the project (instituto interamericano de cooperaci on para la agricultura, 2016). 2 the calculation for the other raw materials is available at https://uceedumy.sharepoint.com/personal/wgchiriboga_uce_edu_ec/_layouts/15/doc.aspx? energy consumption for growing sugar cane is 8.365 mj/kg, which on the average is 43.2% more energy than brazil, but 31.9% less than the u.s. the situation is different for corn, which in ecuador requires 11.083 mj/kg, compared to 2.923; 6.863; and 9.053 mj/kg in the u.s., chile, and argentina, respectively. for african palm, energy consumption is 5.52 mj/kg, 89.7% higher than indonesia and 62.2% higher than figure 7 . from a to g, breakdown of the energy consumed to produce biofuel (left), and the total energy released by combustion (right) according to the feedstock and assumed scenarios. h is a summary of the eroipou for all the feedstocks considered and sorted by scenarios. the advantage of biodiesel is evident. colombia. for livestock, ecuador consumes about 26% more energy than mexico and brazil, and up to 47 .7% more than the u.s. energy consumption in pig raising is significantly lower due to space management and the type of feeding, so the energy demand comes from the transesterification of fat. this investigation reports the current situation of energy management for both alcohol fermentation and dehydration and the transesterification of fat. we also considered the pretreatment and refining stages of each raw material. in ecuador, the incorporation of biofuels into the energy matrix has two main objectives. on one hand, it seeks to reduce the environmental impact of greenhouse gas emissions by using biomass, whose carbon comes from photosynthesis. on the other hand, it seeks to displace imports of petroleum-derived fuels, mainly gasoline and diesel, while encouraging national agricultural production. in this context, ethanol from sugar cane has the most favorable eroi, at 1.796, which leads to the following analysis. if the aim were to meet nationwide demand with ecopaís, the energy market for anhydrous ethanol would be 1,300 kboe (8,000,000 mj). this amount of energy would require 360,000,000 l of alcohol (energy intensity ¼ 22.2 mj per liter) and 450 km 2 of arable land (instituto interamericano de cooperaci on para la agricultura., 2007). fossil fuels could provide 57% of this energy; however, there would still be a contribution by renewables of about 3,440,000 mj. for the other raw materials analyzed, there would not be a net energy contribution by ethanol. in the case of wood, there would be a negative energy balance since its eroi is between 0.687 and 0.845. for biodiesel, we chose african palm to carry out a similar analysis, and the eroi calculated was 3.052. the production of b10 blends to meet nationwide demand would represent 3,500 kboe or 21,400,000 mj, and would involve around 665,000,000 l of biodiesel and 1,210 km2 of land (energy intensity ¼ 32.2 mj per liter). pinion is worth highlighting because its eroi is 2.743, and it constitutes an excellent opportunity because it does not compete directly with the food sector. only living fences are required to produce it, so it would therefore not jeopardize land use to a significant extent. biodiesel from animal fat (cattle 2.187:1 and pigs 2.891:1) appears to be more feasible than ethanol, as shown in figure 7h . the best production scenarios for both ethanol (sugar cane 1.796) and biodiesel (african palm 3.052) suggest that expending a unit of energy to produce biodiesel would return 1.256 more energetic units than bioethanol. besides, data from the last national energy balance showed that the demand for diesel is greater than gasoline (34,500 kboe and 25,000 kboe, respectively) due to the high energy consumption characteristics of this subsector, and the ease of fuel utilization for industrial activities. finally, it is worthwhile analyzing how ecuadorians use fuels for transportation. for example, individual or light cars consume 71% of the gasoline, and in the best case would transport five users at a time. and collective or heavy-duty transport utilizes the remaining 29%. in contrast, light and individual transport consumes 20% of the diesel and collective or heavy-duty transport utilizes up to 80%. so, since the biodiesel market would mean a higher energy return, better land use, and greater coverage of benefited users, it should be analyzed why, so far, ecuadoreans still cannot trade biodiesel locally. the study presents an initial analysis of the energy return of the biofuels considered in national development plans for transport. the energy return rate of bioethanol is feasible only for sugarcane because it has an energy surplus of 1.796. for biodiesel, the values are favorable in all the selected scenarios, since its eroipou was always greater than 2. the stages of cultivation and industrial transformation are the most energy-demanding; therefore, energy efficiency policies should begin by addressing these stages, so that we can achieve world productive yields like those of brazil and the u.s. the market for bioethanol as fuel is 94 million dollars (110 million liters). petroecuador e.p. has awarded the administration of the project to three private companies and a 4% share to small farmers. this gasoline is available in 10 of the 24 provinces of the country. in 2019, it achieved first place in sales with 45% of the total market. the numbers are encouraging but the energy ministry has questioned the project since 2018, because it is cheaper to import ethanol from neighboring countries. the reasons are the lack of local production of agricultural inputs and the high cost of labor. based on this analysis, the project seems to be maintained solely by the 3,000 direct jobs it generates. however, even if the objective is to prioritize job stability, the eroi could provide public policymakers with adequate information on each stage in order to correctly direct incentive programs. the biofuels program was launched in april 2019, and included biodiesel as a priority. that year, the organic law of energy efficiency called for the creation of the necessary policies for promoting the marketing of biofuels at the national level. as of the writing of this document, there are no clear implementation programs or plans for biodiesel. in the context of energy efficiency and renewable energy, the public policy proposes the electrification of the transport matrix. by 2025, new units incorporated into public transport must only be electrically powered. this measure covers 6% of national diesel demand, but the actions to be taken for light and heavy units, which constitute 93%, are not clearly expressed. here, encouraging eroi results provide an alternative in the medium term, mainly because biodiesel blends do not incur the drawbacks of electrical units, such as battery life, limited range, dispatchability, and recharging points. the land required to meet domestic demand with ecopaís and biodiesel (b10) shows similar energy densities, since both have yields of about 17.7 mj per km 2 . that is, the same amount of land would be committed to generating a unit of energy from sugar cane or african palm. however, this issue deserves further study that considers the water required and the net greenhouse gas emissions generated. we analyzed biofuels for use in transportation engines, which implies high-quality fuels and consequently, high energy demands during refining processes. diversifying the matrix and allocating fuels for direct combustion should be considered, such as pinion, for direct use in power generation. here, the eroipou would increase from 2.743 to 8.864. the unprecedented crisis in the oil markets due to the covid-19 outbreak has caused severe impacts on oil-exporting economies, such as that of ecuador. in this regard, some analysts suggest that prices will rise considerably in the medium term, which would imply a new increase in the cost of derivatives. currently, ecuador's social environment makes it highly unlikely that subsidies will be withdrawn. this forces the ecuadorian government to generate guidelines focused on incorporating viable options for the diversification of the energy matrix to alleviate the fiscal deficit caused by the subsidy. in the current situation of low global energy demand, ecuador should use the opportunity to generate public policies aimed at defining how, when and what quantity of derivatives to acquire in the future, as well as the amount of raw materials to be dedicated to the production of biofuels. both decisions must consider, among other things, the eroi as a technical criterion to indicate which biofuel to produce to minimize the impact on other energy resources and economic sectors. norma para el manejo y control del biodiesel. quito: registro oficial del ecuador integrating agronomic factors into energy efficiency assessment of agro-bioenergy production -a case study of ethanol and biogas production from maize feedstock more caution is needed when using life cycle assessment to determine energy return on investment (eroi) standard specification for diesel fuel oil, biodiesel blend (b6 to b20) reporte del sector petrolero global scenario of biofuel production : past , present and future dynamic energy return on energy investment ( eroi ) and material requirements in scenarios of global transition to reflexiones en torno al desarrollo de los biocombustibles en ecuador. ceda, 25. centro ecuatoriano de desarrollo ambiental -temas de an alisis termodin amica plan nacional de desarrollo. plan nacional de desarrollo. quito: consejo nacional de planificaci on embodied energy and energy return on investment analyses in maize production for grain and ethanol under center pivot, subsurface drip, and surface (furrow) irrigation with disk tillage and no-till practices energías renovables observatorio de energías renovables en am erica latina y el caribe agrocombustibles dar an impulso a palma y caña energy information administration governo federal, rio de janeiro ep petroecuador firma contrato con empresas alcoholeras para la producci on de ecopaís balance y proyecciones del sistema de subsidios energ eticos en ecuador. an alisis, quito tablero comando para la promoci on de los biocombustibles en ecuador a preliminary investigation of energy return on energy investment for global oil and gas production projecting the effect of oil price regimes on biofuel markets a new long term assessment of energy return on investment (eroi) for u.s. oil and gas discovery and production will eroi be the primary determinant of our economic future? the view of the natural scientist versus the economist what is the minimum eroi that a sustainable society must have? energies seeking to understand the reasons for different energy return on investment (eroi) estimates for biofuels an alisis del uso de biocombustibles en ecuador periodo a mini review on renewable sources for biofuel energy return on investment (eroi) of china's conventional fossil fuels: historical and future trends encuesta de superficie y producci on agropecuaria continua. inec 2 atlas de la agroenergía y los biocombustibles en las am ericas atlas bioenerg etico del ecuador life cycle assessment of various cropping systems utilized for producing biofuels : bioethanol and biodiesel eroi of global energy resources. preliminary status and trends. college of environmental science and forestry energy, eroi and quality of life large quantities of high quality energy eroi of different fuels and the implications for society la palma aceitera en el ecuador: ¿un cultivo social y sustentable? línea de fuego balance energ etico nacional plan maestro de electrictidad plan nacional de eficiencia energ etica 2016-2035. plan nacional de eficiencia energ etica ecuador marca su rumbo en la industria de los agrocombustibles comments on energy return on investment ( eroi ): reconciling boundary and methodological issues year in review-eroi or energy return on (energy) invested order from chaos : a preliminary protocol for determining the recent developments and key barriers to advanced biofuels: a short review towards an improved methodology for energy return on investment (eroi) for electricity supply pollution prevention, a key to economic and environmental sustainability informe ethanol production using corn, switchgrass, and wood informe estadístico enero agosto time series eroi for canadian oil and gas dectreto ejecutivo 1303. registro oficial del ecuador 2013. viceministerio de promoci on de exportaciones e inversiones energía verde para gal apagos breve reseña hist orica de la exploraci on petrolera de la cuenca oriente quantification of total nitrogen according to the dumas method biofuel policy in india: a review of policy barriers in sustainable marketing of biofuel issues to consider , existing tools and constraints in biofuels sustainability assessments issues to consider , existing tools and constraints in biofuels sustainability assessments municipal solid waste system analysis through energy consumption and return approach alternative fuels data center balance energ etico nacional colombiano union zur f€ orderung von oel sustainable development goals documento de trabajo no. 36, gerencia de políticas y an alisis econ omico. osinergmin, lima we acknowledge the contributions of iner, dpec, and la fabril in providing biodiesel samples and soderal s.a., for permitting us to collect information on the energy consumption of its industrial processes. the authors declare no conflict of interest. no additional information is available for this paper. key: cord-312183-zkoj5d8c authors: frydman, galit h.; boyer, edward w.; nazarian, rosalynn m.; van cott, elizabeth m.; piazza, gregory title: coagulation status and venous thromboembolism risk in african americans: a potential risk factor in covid-19 date: 2020-07-24 journal: clin appl thromb hemost doi: 10.1177/1076029620943671 sha: doc_id: 312183 cord_uid: zkoj5d8c severe acute respiratory syndrome coronavirus 2 infection (covid-19) is known to induce severe inflammation and activation of the coagulation system, resulting in a prothrombotic state. although inflammatory conditions and organ-specific diseases have been shown to be strong determinants of morbidity and mortality in patients with covid-19, it is unclear whether preexisting differences in coagulation impact the severity of covid-19. african americans have higher rates of covid-19 infection and disease-related morbidity and mortality. moreover, african americans are known to be at a higher risk for thrombotic events due to both biological and socioeconomic factors. in this review, we explore whether differences in baseline coagulation status and medical management of coagulation play an important role in covid-19 disease severity and contribute to racial disparity trends within covid-19. converting enzyme 2 receptor (ace2r), where it then undergoes proteolytic cleavage into spike protein 1 and 2 (sp1/sp2), resulting in successful host cell infection. 9 angiotensinconverting enzyme 2 receptors are expressed in a variety of tissues, including the lungs, various parts of the gastrointestinal (gi) tract, the heart, and brain/central nervous system. accordingly, the primary presentations of covid-19 in symptomatic patients include respiratory, cardiac, gi, and neurologic signs. [10] [11] [12] the range of clinical findings in covid-19 can span the gamut from asymptomatic to severe illness, requiring intensive care unit admission and mechanical ventilation. additionally, the severity of disease has been linked to the presence of various comorbidities. given the organ systems for which covid-19 has tropism (via localization of the host cell receptor expression, ace2r), patients with preexisting pulmonary and cardiovascular conditions are at increased risk. the risk is further heightened by the fact that many of these conditions, including congestive heart failure and chronic obstructive pulmonary disease (copd), are associated with increased levels of ace2r expression. [13] [14] [15] [16] although robust basic and clinical studies have yet to be published, various case series reports, clinical observations, and theories have been proposed regarding the pathogenesis of covid-19-related coagulopathies. upon exposure to a viral pathogen, such as sars-cov-2, there are multiple potential mechanisms that can trigger coagulation pathways, including leukocyte activation, complement activation, systemic inflammation, and pathological changes of infected host cells, such as endotheliitis, which may result in cellular damage and apoptosis, which may then further active inflammation and coagulation. [17] [18] [19] [20] in addition to the conditions that render specific organ systems at risk, such as copd and asthma, systemic diseases such as diabetes, obesity, autoimmune disorders, and cancer predispose patients to severe and rapid changes to their inflammatory system and, in many cases, also predispose a patient to being in a prothrombotic state. [21] [22] [23] [24] [25] individuals with these preexisting conditions, along with cardiopulmonary disease, appear to be at the highest risk for development of severe covid-19 as well as higher mortality rate. [13] [14] [15] [16] additionally, there is mounting evidence that a major pathological finding in severe covid-19 is the development of macrothrombi and microthrombi. multiple recent studies have demonstrated that there is pathological blood clot formation on a systemic level, including deep vein thrombosis (dvt), pulmonary embolisms (pe), pulmonary microthrombi, stroke, and even thrombus formation in the placenta. [26] [27] [28] [29] [30] [31] [32] [33] systemic thrombosis has been noted to result in acute bowel ischemia, leading to the need for emergency surgery. 34, 35 a growing body of literature describes the disproportionate effect of covid-19-related morbidity and mortality in minority populations, particularly african americans. according to a recent report from the chicago area, greater than 50% of covid-19 cases and 70% of covid-19-related deaths have been observed in african americans, although they make up only 30% of the study population. 7 similar trends have been noted in louisiana, michigan, and new york city. [17] [18] according to a johns hopkins university and america community survey, covid-19 rates were 3 times higher, and the death rate was at least 6 times higher, in 131 predominantly african american counties compared to infection and death rates from 2879 predominantly white counties, 6 predominantly asian counties, and 124 predominantly hispanic counties. 36, 37 health care disparities and socioeconomic status plays a major role in this increase in morbidity and mortality. however, to assume that these 2 factors explain the totality of the racial disparities in outcomes of covid-19 runs the risk of missing important therapeutic avenues for reducing burden of illness in these vulnerable populations. indeed, interrogating differences in comorbid conditions and baseline abnormalities of coagulation may provide critical inroads into mitigating the higher risk of adverse outcomes in minority racial groups such as african americans. recent studies have shown severe pulmonary and cardiac pathology, associated with increased thrombosis, is prevalent in african americans with severe covid-19. 26, 38 although more research into this area is needed to draw any conclusions, researchers and clinicians are beginning to take notice of the possibility of differences in baseline coagulation status, in conjunction with other variations, as potentially playing a potential role in the severity of covid-19. 38, 39 coagulation differences related to race or ancestry variations in coagulation activity can be dichotomized into congenital and acquired. congenital differences in coagulation status are due to genetic differences between individuals. limited studies describe genetic variation in coagulopathies, although faster and cheaper sequencing tools have changed our understanding of this topic. there are many genetic causes of both excess bleeding (eg, hemophilia) and excess clotting. although african americans have been shown to have an increased risk of thrombosis as compared to caucasians, most of the inherited mechanisms of thrombosis that have been studied are present primarily in caucasian populations, such as fv leiden, resulting in resistance to protein c, and prothrombin g20210a. 8, 40, 41 in general, strong evidence supports a tendency toward a prothrombotic phenotype in african americans. although the mechanism is not yet fully understood, this evidence includes a combination of differences in measured coagulation biomarkers as well as observations in increased thrombotic events. 42 african americans exhibit a well-documented trend toward having higher baseline levels of coagulation fviii, increased levels of vwf, increased thrombin generation, and elevated baseline levels of d-dimer ( figure 1 ). [43] [44] [45] [46] [47] [48] factor viii plays a role in the intrinsic coagulation pathway and is frequently elevated in patients with severe systemic inflammation, as it is a positive acute-phase protein. an increase in fviii can create an exaggerated coagulation response upon activation of the cascade and has been suggested to be related to resistance to heparin in critically ill patients. 49, 50 an increase in fviii has been observed in patients with severe covid-19. 4 another biomarker d-dimer is released upon the breakdown of a cross-linked fibrin clot, resultant from endogenous activation of the fibrinolytic pathway. increase in d-dimer is typically used as a marker of active thrombosis and fibrinolysis, such as for the exclusion of a pe, dvt, or stroke, in conjunction with other clinical tests, such as imaging. 51 african american women, in particular, have a higher incidence of thrombosis along with a higher level of d-dimer. 46 ,52 d-dimer has also been reported to be an important biomarker of serious covid-19 and has been shown to correlate with poor prognosis. [1] [2] [3] [4] african americans have at least a 30% higher prevalence of venous thromboembolism (vte), which is suspected to be at least in part due to variations in thrombomodulin, although there is conflicting evidence on the specific mechanism. 53, 54 thrombomodulin modulates thrombin and increases the activation of protein c. limited studies have also identified decreased levels of circulating protein c and protein s, endogenous anticoagulants, in african americans. [55] [56] [57] however, some genetic studies identify potential single-nucleotide polymorphisms (snps) that may affect the level of circulating protein c in multiple groups, including increased levels of protein c in african americans. 58 while fv leiden mutation, which results in activated protein c resistance, is a well-characterized mechanism of thrombosis in caucasian populations, direct changes in protein c, protein s, and thrombomodulin have been found in some african americans (as described above). african americans also have increased levels of lipoprotein (a), which is known to play a role in the development of atherosclerosis and the predisposition to thrombosis due to its similarity to plasminogen, as well as through platelet activation. 59, 60 these findings support the need for further research in this area to fully understand the variations in baseline coagulation status, as they relate to the development of coagulopathies. another potential cause of increased thrombotic events in african american patients is sickle cell disease (scd). sickle cell disease is an inherited blood disorder, caused by a mutation in the hbb gene, that results in production of abnormal hemoglobin and can be identified by the traditional c-shaped (sickleshaped) red blood cells in place of the traditional round cell shape. 61, 62 this morphologic change results in the cells having a higher likelihood of getting lodged in the microvasculature and more prone to aggregate than regular blood cells, causing blood flow obstruction and serving as a nidus for clot formation. 63 these morphological red blood cell changes along with activation of the coagulation system may result in a vasoocclusive crisis. 64 individuals with scd are at a significantly higher risk of developing dvt and pe. 61, 62 in addition to increasing the rate of thrombus formation, the presence of the sickle shape also appears to result in a denser thrombus and the clot itself appears to be more resistant to fibrinolysis. 65 although clinical scd is only present in an estimated 100 000 patients in the united states, the sickle cell trait is present in up to 8% of the african american population, with scd flagged as an important risk factor for vte. 42 a small number of studies have explored genetic variations in the context of coagulation and the response to anticoagulation therapy. some of these studies have suggested that african americans may require higher doses of warfarin to stay within the target prothrombin time/international normalized ratio therapeutic window. 66, 67 one study examined the vkorc1 and cyp2c9 genes in the context of the african american population and the variability in patient response to warfarin treatment. 68, 69 that study identified that a snp in the cyp2c cluster on chromosome 10 appeared to be associated with a clinically relevant effect on warfarin dose. other studies examining higher rates of d-dimer elevations in african americans have identified a potential genetic variation of the f3 loci, believed to be associated with this elevation. 24 studies evaluating metabolism of direct oral anticoagulants (doacs) have also identified potential differences in sulfation of active apixaban due to sult1a1*3, an allelic variant of sulta1 (a polymorphic variant of a sulfotransferase or sult), which may be associated with a moderate potential to affect the anticoagulation effects of apixaban. 70 genome studies also describe genetic variants in vwf specific to african american women, although the resulting phenotype of these changes is not clear. 71, 72 not all genotyping studies have demonstrated an increased risk factor in african americans; for example, a mutation in fii (prothrombin), called prothrombin g20210a, is found in 1 in 250 african americans, but is more common and represents a risk factor for caucasians. 73 genetic and phenotypic variations in accessories to the coagulation pathway, such as in platelets, among racial and ethnic minority groups have also been suggested. platelets play a major role in thrombosis and are a primary target, along with the coagulation pathway, in therapeutic regimens for the treatment of heart disease and other conditions associated with pathologic thrombosis. multiple studies have shown that african american women tend to have a higher platelet count than caucasians and latinos (no difference was seen between men of all races). [74] [75] [76] novel gene mutations linked to changes in platelet counts, primarily increased platelet counts (thrombocytosis), in african americans have been observed. additionally, potential differences in platelet sensitivity to some traditional platelet inhibitor drugs have been demonstrated with varying results. in particular, there are multiple studies showing differences in the protease-activated receptor-4 pathway, with african americans being less responsive to cyclooxygenase and p2y 12 receptor dual inhibition. 76, 77 these findings suggest that african americans may not respond to platelet inhibitors similar to other races, and this may alter therapeutic efficacy in this population. furthermore, several conditions indirectly affect coagulation status. one major example is systemic lupus erythematosus (sle), an autoimmune disease that involves the body raising antibodies against receptors on cell membrane components, such as phospholipids, which increase systemic inflammation and may heighten a patient's predisposition to thrombosis. 42 while typical symptoms of sle include systemic inflammation, swelling, and damage to the joints, kidneys, heart, and lungs, blood clotting is common as well. systemic lupus erythematosus is 3 times more common in african american women than in caucasian women, and up to 1 in every 250 african american women will develop sle. 78, 79 furthermore, its symptoms tend to be more severe in african americans than in other races. 79, 80 lupus anticoagulants, anticardiolipin antibodies, and b-2-glycoprotein i antibodies are antiphospholipid antibodies that are common in patients with sle (as well as certain other patients). it is common for women, especially african american women, to develop antiphospholipid antibodies during pregnancy, a major cause of miscarriage in african american women. 80, 81 interestingly, a few papers have demonstrated that there appears to be an increased frequency of lupus autoantibodies in patients with covid-19, although the frequency of lupus anticoagulant is currently debated. 82, 83 one of the more common conditions that is a strong predisposing factor for vte is chronic kidney disease (ckd). 42, 84 the development of mild-moderate ckd is associated with a 1.3-to 2-fold increased risk for vte, while severe ckd is associated with up to a 2.3-fold increased risk compared to the general population. the predisposition to vte formation in the face of ckd is due to many additive changes in coagulation that occur during renal impairment, including (1) elevated levels of d-dimer, c-reactive protein, fibrinogen, fvii, fviii, and vwf (likely due to increased synthesis); (2) lower levels of fix, fxi, and fxii (likely due to increased urinary loss); (3) decrease in endogenous anticoagulants, including at (likely due to increased urinary loss); (4) increase in platelet activation and aggregation; and (5) decreased fibrinolytic activity. 84 african americans have an increased rate of ckd and end-stage renal disease, with an odds ratio of 3.89, compared to 1 in caucasians, 2.74 in native americans, 1.56 in asians, and 1.45 in hispanics. 85, 86 in addition to the various factors listed above that may predispose african americans to thrombosis (although this list is by no means exhaustive), prevalence of other conditions, such as an increased rate of heart disease, hypertension, and diabetes, also increases the risk of thrombosis. some african americans may be genetically predisposed to a baseline prothrombotic state; adding on other conditions, such as atherosclerotic cardiovascular disease, heart failure, hypertension, diabetes, and other comorbidities, compounds the overall risk of coagulopathies. 87, 88 among the population of african americans who are infected with covid-19, some of these patients may already reside in a prothrombotic state prior to covid-19 because of higher baseline concentrations of fviii, vwf, and d-dimer, as well as increased platelet activation. this may potentially account for some of the increase in disease severity observed in this population. in addition to the physiologic mechanisms that may predispose african americans to a prothrombotic state, other key factors, such as socioeconomic status, may adversely affect coagulation management for this prothrombotic phenotype. although some common comorbid conditions may be inherited, access to medical care, medication, healthy food, and lifestyle habits play a key role in treatment and control of the disease. [89] [90] [91] patterns of increased covid-19 prevalence, morbidity, and mortality are concentrated in areas with lower socioeconomic status, coinciding with underserved racial and ethnic communities. 36, 37 these populations may have greater numbers of residents per household, be economically unable to miss work (even if sick) and are more likely to have multigenerational family members residing in one abode; these factors often make practicing social isolation difficult. this risk is further exacerbated by difficulties with travel or the ability to afford chronic care and long-term medications, creating a perfect storm for an infection such as covid-19. 36, 37, 92 disparities extend to treatment as well. studies have shown that african americans tend not to be managed with the same treatments as those commonly prescribed to other racial and ethnic groups. for example, some studies have noted that although african americans tend to exhibit higher rates of atrial fibrillation and thrombosis, they are less likely to be prescribed anticoagulants compared to their caucasian counterparts. 93, 94 even when prescribed, african american patients tend to spend less time within the therapeutic range for warfarin treatment. 66 similarly, prescription of doacs in the african american community has been limited compared to other racial and ethnic groups, despite the significant increase in doac use in the united states (now surpassing warfarin). interestingly, some studies have shown that these differences in anticoagulation trends are present even when controlling for socioeconomic variables. 66, [94] [95] [96] additionally, despite the evidence suggesting that genetic variations may alter the response to various anticoagulants and platelet inhibitors, as discussed in this article, african americans comprise fewer than 4% of the clinical trial population for many of these anticoagulants. 97 due to the low representation of african americans in these clinical trial populations, it is possible that any natural variation in clinical response would not be observed as the trial is likely not powered for this purpose. thus, the administration of standard anticoagulation therapy, as based on data sourced from caucasian patients, may be subtherapeutic in african american patients, potentially resulting in inadequate anticoagulation. in this report, we consider the contribution of both "nature" and "nurture" to the increased rate of covid-19-related morbidity and mortality among african americans. covid-19 produces severe inflammation and a prothrombotic state that culminates in thrombotic events. african americans are more likely than other racial and ethnic groups to be in a prothrombotic state or to be primed for a prothrombotic state. coupled with the higher rate of preexisting conditions that predispose patients to higher rates of covid-19 and disease, and the lower rate of therapeutic anticoagulation even when warranted, variation in coagulation status may be one of the factors that puts african americans at higher risk. given the potential differences in response to anticoagulant therapy, it remains unclear whether standard dosing effectively achieves the appropriate level of anticoagulation among hospitalized covid-19-positive african americans. although this work focuses on trends in african americans, further examination of covid-19 trends may demonstrate the urgent need for both personalized medicine and population-based care, particularly with respect to anticoagulation management. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: dr piazza has received research grant support from ekos, a btg international group company, bayer, the bristol myers squibb/pfizer alliance, daiichi-sankyo, portola, and janssen and consulting fees from amgen, pfizer, boston scientific corporation, and thrombolex. in addition to her academic appointments, dr frydman is the chief science officer of coagulo medical technologies, inc. galit h. frydman https://orcid.org/0000-0002-8126-8580 abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia anticoagulation treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy isth interim guidance on recognition and management of coagulopathy in covid-19 hypercoagulability of covid-19 patients in intensive care unit. a report of thromboelastography findings and other parameters of hemostasis covid19: consider cytokine storm syndromes and immunosuppression cross talk pathways between coagulation and inflammation covid-19 and african americans racial differences in venous thromboembolism sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor angiotensin receptor blockers as tentative sars-cov-2 therapeutics high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa influenza and sarscoronavirus activating proteases tmprss2 and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts clinical course and risk factors for mortality of adult patients with covid-19 in wuhan, china: a retrospective cohort study covid-19 and the cardiovascular system ace-2 expression in the small airway epithelia of smokers and copd patients: implications for covid-19 the impact of copd and smoking history on the severity of covid-19: a systematic review and meta-analysis immune mechanisms of pulmonary intravascular coagulopathy in covid-19 pneumonia endothelial cell infection and endotheliitis in covid-19 complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases thrombosis and vascular inflammation in diabetes: mechanisms and potential therapeutic targets coagulation takes center stage in inflammation contact pathway of coagulation and inflammation new insights into the role of adipose tissue in thrombosis coagulation and inflammation in long-term cancer survivors: results from the adult population cancer-associated thrombosis: an overview of mechanisms, risk factors, and treatment pulmonary and cardiac pathology in african american patients with covid-19: an autopsy series from new orleans placental pathology in covid-19 positive mothers: preliminary findings placental pathology in covid-19 venous thrombosis among critically ill patients with coronavirus disease 2019 (covid-19) acute pulmonary embolism in covid-19 patients on ct angiography and relationship to d-dimer levels pulmonary embolism in covid-19 patients: awareness of an increased prevalence thrombosis risk associated with covid-19 infection. a scoping review incidence of venous thromboembolism in hospitalized patients with covid-19 small bowel ischemia and sars-cov-2 infection: an underdiagnosed distinct clinical entity gastrointestinal complications in critically ill patients with covid-19 medicine: coronavirus resource center. 2020. accessed the coronavirus is infecting and killing black americans at an alarmingly high rate increased cardiovascular mortality in african americans with covid-19 rapid response: african americans: biological factors and elevated risk for covid-19 ethnic distribution of factor v leiden in 4047 men and women venous thromboembolism in blacks. circulation population differences in von willebrand factor levels affect the diagnosis of von willebrand disease in african-american women african-caribbean ethnicity is associated with a hypercoagulable state as measured by thrombin generation analysis of racial disparity in plasma of healthy volunteers using rotational thromboelastography reveals higher prothrombotic profile in african americans d-dimer in african americans high plasma levels of factor viii and the risk of recurrent venous thromboembolism risk factors for venous thrombosis in the black population factor viii may predict catheter-related thrombosis in critically ill children: a preliminary study elevated factor viii levels and the risk of thrombosis value of d-dimer testing on the exclusion of pulmonary embolism in patients with previous venous thromboembolism african american ethnicity is associated with higher plasma levels of d-dimer in adults with hypertension novel genetic predictors of venous thromboembolism risk in african americans failure to replicate genetic variant predictors of venous thromboembolism in african americans ethnic differences in markers of thrombophilia, implications for the investigation of ischemic stroke in multiethnic populations: the south london ethnicity and stroke study risk factors for thrombosis in an african population population-specific single-nucleotide polymorphism confers increased risk of venous thromboembolism in african americans genetic markers associated with plasma protein c level in african americans: the atherosclerosis risk in communities (aric) study lipoprotein(a) and venous thromboembolism serum distribution of lipoprotein(a) in african americans and nigerians: potential evidence for a genotype-environmental effect venous thromboembolism in adults with sickle cell disease: a serious and under-recognized complication sickle cell disease, sickle trait and the risk for venous thromboembolism: a systematic review and meta-analysis red blood cells modulate structure and dynamics of venous clot formation in sickle cell disease vaso-occlusion in sickle cell disease: pathophysiology and novel target therapies thrombin activation of par-1 contributes to microvascular stasis in mouse models of sickle cell disease use of oral anticoagulants in african-american and caucasian patients with atrial fibrillation: is there treatment disparity? race influences warfarin dose changes associated with genetic factors effect of genetic variants, especially cyp2c9 and vkorc1, on the pharmacology of warfarin warfarin therapy and vkorc1 and cyp genotype pharmacogenomics of novel direct oral anticoagulants: newly identified genes and genetic variants possible race and gender divergence in association of genetic variations with plasma von willebrand factor: a study of aric and 1000 genome cohorts common vwf exon 28 polymorphisms in african americans affecting the vwf activity assay by ristocetin factor factor ii mutation) resources: a genetic clotting condition or thrombophilia platelet counts in three racial groups a meta-analysis and genome-wide association study of platelet count and mean platelet volume in african americans racial difference in human platelet par4 reactivity reflects expression of pctp and mir-376c common variants in the human platelet par4 thrombin receptor alter platelet function and differ by race the clotting defect in sle evidence of a distinct group of black african patients with systemic lupus erythrmatosus office of women's health, cdc. lupus in women review: diagnosis and management of antiphospholipid syndrome in pregnancy lupus anticoagulant is frequent in patients with covid-19 covid-19 and systemic lupus erythematosus: a case series chronic kidney disease and venous thromboembolism: epidemiology and mechanisms unraveling the racial disparities associated with kidney disease usrds annual data report: executive summary. volume 1-ckd in the united states frequency, predictors, and impact of combined antiplatelet therapy on venous thromboembolism in patients with symptomatic atherosclerosis comorbidities of copd have a major impact on clinical outcomes, particularly in african americans socioeconomic factors and concomitant diseases are related to the risk for venous thromboembolism during long time follow-up relationship between neighborhood socioeconomic status and venous thromboembolism: results from a population-based study socioeconomic and occupational risk factors for venous thromboembolism in sweden: a nationwide epidemiological study health disparities experienced by black or african americans-united states racial differences in atrial fibrillation-related cardiovascular disease and mortality: the atherosclerosis risk in communities (aric) study association of race/ ethnicity with oral anticoagulant use in patients with atrial fibrillation: findings from the outcomes registry for better informed treatment of atrial fibrillation ii racial, ethnic and socioeconomic inequities in the prescription of direct oral anticoagulants in patients with venous thromboembolism in the united states a comparison of socioeconomic factors in patients continuing warfarin versus those transitioning to direct oral anticoagulants (doacs) for venous thromboembolism disease or atrial fibrillation food and drug administration. accessdata.fda.gov. clinical trial data for key: cord-317640-61crnh6a authors: zhu, zhaozhong; xiao, chao-ting; fan, yunshi; cai, zena; lu, congyu; zhang, gaihua; jiang, taijiao; tan, yongjun; peng, yousong title: homologous recombination shapes the genetic diversity of african swine fever viruses date: 2019-08-10 journal: vet microbiol doi: 10.1016/j.vetmic.2019.08.003 sha: doc_id: 317640 cord_uid: 61crnh6a the african swine fever virus (asfv) has severely influenced the swine industry of the world. currently, there is no effective vaccine or drugs against the asfv. how to effectively control the virus is challenging. in this study, we have analyzed all the publicly available asfv genomes and demonstrated that there was a large genetic diversity of asfv genomes. interestingly, the genetic diversity was mainly caused by extensive genomic insertions and/or deletions (indels) instead of the point mutations. further analyses showed that the indels may be attributed much to the homologous recombination, as supported by significant associations between the occurrence of extensive recombination events and the indels in the asfv genomes. besides, the homologous recombination also led to changes of gene content of asfvs. finally, repeated elements of dozens of nucleotides in length were observed to widely distribute and cluster in the adjacent positions of asfv genomes, which may facilitate the occurrence of homologous recombination. this work highlighted the importance of homologous recombination in shaping the genetic diversity of the asfvs, and could help understand the evolution of the virus. african swine fever virus (asfv), the causative agent of african swine fever (asf), is a complex, large, icosahedral multi-enveloped dna virus. it is classified as the only member in the family asfarviridae (galindo and alonso, 2017; arias et al., 2018) . the genome of the virus belongs to double-stranded dna, with the size ranging from 170 kb to 190 kb (dixon et al., 2013) . asfv mainly infect suids and soft ticks. the suids include domestic pigs and wild boars, and were reported as the natural hosts of the virus (sanchez-cordon et al., 2018; costard et al., 2013) . asfv was firstly discovered in kenya in 1921 (arzt et al., 2010) . it remained restricted in africa till 1957, when it was reported in spain and portugal. up to now, the virus has caused asf outbreaks in more than fifty countries in africa, europe, asia, and south america (costard et al., 2013) . the latest reports showed that the virus has caused outbreaks in nearly all provinces of china (ge et al., 2018; world animal health information and analysis department, 2018; zhou et al., 2018; food and agriculture organization of the united nations, 2019). because of the high lethality of asfv in domestic pigs, the most commonly used strategies to control the virus were the massive culling campaigns and the restriction of pig movement (sanchez-cordon et al., 2018) . both strategies have resulted in a huge economic loss for pig industry and affected people's livelihoods. how to effectively control the virus is still a great challenge for the globe. the large genetic diversity of asfvs, which was supposed to hinder the development of effective vaccines or drugs against the virus (sanchez-cordon et al., 2018; escribano et al., 2013; arabyan et al., 2018) , has been investigated in many studies. the asfv genome encodes over 150 proteins, including viral enzymes, viral transcription and replication-related proteins, structural proteins, other proteins involved in the virus assembly, the evading of host defence systems and the modulation of host cell function, etc (dixon et al., 2013; alejo et al., 2018; kessler et al., 2018) . for example, the transcription of the virus is independent on the host rna polymerase because the virus contains relevant enzymes and factors (dixon et al., 2013) . the viral genome contains a conservative central region of about 12 kb and two variable ends, which results in the variable size of the genome (dixon et al., 2013; chapman et al., 2008; de villiers et al., 2010) . there are significant variations among the asfv genomes due to the genomic insertion or deletion, such as the deletion of the multigene family (mgf) members (dixon et al., 2013) . although much progress have been made on genetic diversity of the virus, the extent and mechanisms are still not clear. besides, most of these studies either only investigated the genetic diversity of some common genes, such as p72 and p54 (fraczyk et al., 2016; michaud et al., 2013) , or only used one to twelve isolate genomes (dixon et al., 2013; chapman et al., 2008; de villiers et al., 2010) . the number of discovered viral genomes has increased rapidly as the development of dna sequencing technology. therefore, a comprehensive study on the genetic diversity of asfvs is necessary. homologous recombination, which has been reported to occur in several groups of viruses (roossinck, 1997; nagy and bujarski, 1996; wang et al., 2015) , such as herpesvirus, retroviruses, and coronaviruses, has played an important role in viral evolution (nagy and bujarski, 1996) . a few studies on several asfv genes have suggested the occurrence of homologous recombination in the evolution of asfvs (dixon et al., 2013; fraczyk et al., 2016) . however, a comprehensive study on the homologous recombination in asfv at the genomic scale is lacking, and the role of the recombination on the genetic diversity and the evolution of the virus is still unknown. in this study, we have systematically investigated the genomic diversity and the homologous recombination of asfvs based on the analysis on all the publicly available asfv genomes. the results demonstrated that the homologous recombination contributed much to the genetic diversity of asfvs. this work would help to understand the evolution of the asfv and thus facilitate the prevention and control of the virus. all the asfv genomic sequences with over 170, 000 bp were obtained from ncbi genbank database on february 15, 2019 (agarwala et al., 2016) . after removing the genomic sequences derived from a patent, a total of 39 asfv genomes were kept in the analysis (table s1 ). the genomic sequences were aligned by mafft (version 7.127b) with the default parameters (katoh and standley, 2013) . unless otherwise specified, all the analyses in this study were conducted based on the alignment by mafft. genes encoded in asfv genomes were predicted with the help of genemarks (version 4.28) (besemer et al., 2001) with the default parameters, which is available at http://opal.biology.gatech.edu/ genemark/. the protein sequences for these genes were also provided by genemarks (table s2 ). all the inferred proteins of asfvs were grouped based on sequence homology using orthofinder (version 2.2.7) (emms and kelly, 2015) with the default parameters. manual check was conducted to ensure that each protein group contains one type of protein. a total of 156 protein groups were obtained, including 146 groups with more than 2 proteins and 10 groups with only one protein (table s3) . to name each protein group, the proteins included in the group were blast against the asfv proteins downloaded from ncbi protein database on february 20, 2019. the names of the blast best hits were used to infer the name of the protein group (table s3 ). the functions of the protein groups were adapted from dixon's (dixon et al., 2013 ) and alejo's work (alejo et al., 2018) (table s3) . to determine insertion and deletion events of genes between two asfv genomes, an asfv gene set was defined as all the genes encoded by the asfv genome. the order of the gene, no matter which strand it was encoded, was determined by the coding region of the gene in the direction of 5′ end to the 3′ end on the plus strand. each gene set was firstly sorted by the gene order. then, each gene was named as the group name of the protein which the gene encoded. finally, the global alignment of pairwise gene sets was conducted using the needleman-wunsch algorithm. to reduce the uncertainty of grouping mgf genes, the genes of the same mgf family were considered to be the same in the alignment. rdp (version 4) (martin and rybicki, 2000) was used to detect the recombination events in the aligned asfv genomes. a total of nine methods, i.e., rdp, geneconv, bootscan, maxchi, chimaera, sisscan, phylpro, lard, 3seq, were used to infer the recombination events with the default parameters. only the recombination events with significant p-values (< 0.05) were recorded for each method. for each recombination event, rdp outputted the recombination region, the recombinant virus, the potential major and minor parents, and the support by each method. for robustness, only the recombination events which were detected by at least two methods were used for further analysis (table s4 ). all retrotransposons in the databases of repbase (version 23.10) (genetic information research institute, 2018) and trep (wicker et al., 2007) were downloaded on november 11, 2018. all asfv genomes were searched against these retrotransposons using blastn (altschul et al., 1997) . no hits were obtained under the e-value cutoff of 0.001. maximum-likelihood phylogenetic trees were inferred using mega (version 5.0) (tamura et al., 2013) with the default values of parameters. bootstrap analysis was conducted with 100 replicates. the phylogenetic tree was visualized using dendroscope (version 2.4) (huson et al., 2007) . to illustrate the recombination event, several maximum-likelihood phylogenetic trees were built based on genomic sequences with and without the recombination regions. to determine the genotype of asfvs analyzed, the c-terminal sequences (478 bp) of b646 l gene of the asfvs were used to build the maximum-likelihood phylogenetic tree. the genotype of each asfv was assigned based on previous studies (quembo et al., 2018; bishop et al., 2015; boshoff et al., 2007) . all the statistical analyses were conducted in r (version 3.2.5) (r core team, 2018). the t-test was used to test whether the ratios of the gaps in the recombination regions were similar to those in other regions, and whether the number of indels in the recombination regions was similar to that in other regions. the paired t-test was used to test whether the genomic differences caused by the insertions and deletions (indels) were similar to those caused by the point mutations, and whether the number of repeated elements in the windows (1000-10,000bp in length) including recombination was similar to those without recombination. the t-test and paired t-test was conducted by the function of t.test() in r. z. zhu, et al. veterinary microbiology 236 (2019) 108380 3. results a total of 39 genome sequences of asfvs were obtained from the ncbi genbank database, which were listed in table s1 . they were mostly isolated from africa and europe during the years from 1950 to 2016. besides, two isolates from china in 2018, i.e., 2018/anhuixcgq and sy18, were also included. the size of the asfv genomes ranged from 170,101 bp to 193,886 bp, averaged at 186,588 bp. the viral isolate kenya_1950 had the largest size, while the isolate ba71 v had the smallest size. no increasing or decreasing trend in the genome size was observed from 1950 to 2018 (fig. s1 ), suggesting the dynamic changes of the viral genomes. pairwise comparisons between asfv genomes were conducted after the multiple sequence alignment of 39 genomes. the pairwise genomic differences between asfv genomes ranged from 3 to 52,251 bp ( fig. s2) , with an average of 22,646 bp, which accounts for more than 10% of the genome alignment. interestingly, the genomic differences caused by the insertions and deletions (indels) were much larger than those caused by the point mutations (p-value < 2.2e-16 in the paired t-test) (fig. s3a) . specifically, the genomic differences caused by indels ranged from 3 to 37,837bp, with an average of 14,292bp; while that caused by point mutations ranged from 0 to 17,035bp, with an average of 8,354bp. among these point mutations, a median ratio of 78.5% happened in the coding regions, and a median ratio of 43.3% belonged to non-silent substitutions (fig. s4 ). for robustness of the results, we also conducted the analysis based on the genome alignment by clustalw (larkin et al., 2007) , and found that the indels caused larger genomic differences than the point mutations did (p-value = 3.2e-9 in the paired t-test) (fig. s3b) . the size and the number of indels in asfv genomes were also analyzed. 70% of indels were no longer than 10 bp, and about 9% of indels were more than 50 bp (fig. s5 ). the number of indels in each genome ranged from 804 to 1062, with a median of 988. the occurrence of indels was much more frequent in both ends of the genome, especially in the 5′ end (indicated by the red line in fig. 1 ). the distribution of the indel size was similar along the genome, but the large indels with over 50 bp (marked by a blue line in fig. 1 ) were mostly observed in both ends. the impact of the indels on gene function for each genome was further analyzed. the number of indels which occurred in coding regions was counted for each genome. about 30% of indels were located in the coding regions (fig. s6) . among them, about 70% of indels had length of 3 or multiple of 3 (colored in blue in fig. s6 ), which were likely to cause amino acid indels; the remaining indels were likely to cause changes of reading frames (colored in red in fig. s6 ). as numerous indels have been revealed in the asfv genomes, then, we investigated the mechanism of generating indels. according to the results in previous studies, three factors may contribute to the extensive indels in asfvs: replication slippage, retrotransposition and recombination. replication slippage mainly produced duplications of short genetic sequences (viguera et al., 2001) and may cause short indels, but it is unlikely to generate large indels observed in asfvs. retrotransposition can result in duplication of large genetic sequences or genes (wicker et al., 2007) , but no retrotransposons were observed in the analyzed asfv genomes (as described in materials and methods). finally, we investigated the role of recombination in the generation of indels in the asfv genomes. the analyses on the recombination showed that there were a total of 171 unique recombination events, and each asfv genome had 4-31 recombination events ( fig. 2 & table s4 ). the virus isolate mkuzi_1979 experienced the largest number of recombination events. on average, each virus experienced a median of 14 recombination events. the sizes of recombination region ranged from 50 to 20,010 bp. the ratio of recombination region in each genome, i.e., the proportion of genomic regions involved in the recombination events, ranged from 1% to 24%. in total, the regions in the asfv genomes involved in all recombination events covered a total of 121,107 nucleotide sites, accounting for 56% of the aligned genome. most recombination events were genotype-specific. there were 7 genotypes among the asfvs analyzed ( figs. 2a and s7 ). the genotype ii constituted nearly half of asfvs, including the isolates from east europe and china in recent years. more than ten recombination events were genotype ii-specific (fig. 2b) . the genotype ix, which included six viruses from uganda and kenya, had the least recombination events. fig. 3 illustrates the recombination event in genotype i and vii (colored in red), including two viral isolates from africa (mkuzi_1979 and benin_97/1) and eight viral isolates from europe. these 10 viral isolates formed a separate lineage in the phylogenetic tree. the recombination region ranged from 139,742to 143,561 bp of the genome alignment, located in the central conservative region of the genome (shown by the black arrow in fig. 2b ). in the phylogenetic tree built with genomic sequences without the recombination regions, the recombinants are the neighbors of a clade containing viruses from eastern europe countries (fig. 3a) ; while in the tree built with genomic sequences of the recombination regions, the recombinants are the descendants of viruses from africa (fig. 3b) . most recombination events happened at both ends, especially at the 5′ end (fig. 2b) . interestingly, the recombination event in the aligned genomes was observed to be consistent with the ratio of the gap in the genome (the bottom of fig. 2) . almost all the recombination events happened in or close to the gap-rich regions where the indels were observed. the ratios of the gaps in the recombination regions were found to be significantly higher than those in other regions (pvalue < 0.001 in the t-test) (fig. s8) . further comparison of the number of indels in the recombination regions and other regions showed that for indels of varying length, such as those greater than 5, 10, or 50 bp, the number of indels in the recombination regions was much larger than those in other regions (p-values < 0.001 in the t-test) (fig. s9) . then, we investigated the functional consequences of the recombination events in terms of gene content. in 150 of 171 recombination events, there was at least one gene included in the recombination region. the genes included in the recombination region of the recombinants were compared to those of the inferred major parent virus, which was supposed to provide the larger fraction of the recombinant sequence except the recombination region. in 41 of 171 recombination events, there were at least one gene difference in the recombination region between the recombinant and the major parent virus, either by gene insertion, deletion or replacement (table 1) . for example, in the ninth recombination event, a gene l83 l was inserted in the recombination region of the recombinant virus ken05/tk1, compared to that of the major parent virus kenya_1950. in another recombination event, the major parent virus kenya_1950 encoded the genes of dp148r and dp71 l in the recombination region, the latter of which was lost in the recombinant virus pretorisuskop/96/4. interesting to note, the genes of the mgf families were involved in the gene content variation of the recombination region in 24 of these 41 recombination events. we further compared the gene content between the asfv genomes. the asfv genome encoded 128-154 genes, with an average of 145 genes (table s2 ). after pairwise alignment of the gene set encoded by genomes, the number of different genes between genomes was calculated. on average, there were 11 different genes between the gene set of genomes, which was about 7% of the gene set. the number of gene deletions and insertions between genomes was further calculated and shown in fig. 4 . the matrix referred to the number of gene deletions when comparing the gene set of asfv genomes in the row to those in the column. it showed that in most cases there were both gene deletions and insertions when comparing pairwise gene sets of asfvs. even for the virus ken05/tk1, which encoded the largest number of genes among all asfvs analyzed, there were still gene insertions when comparing to other genomes (marked by the black star). among the 34 genes which were involved in gene insertions or deletions, the member of mgf families, especially the mgf-110 and mgf-360, accounted for nearly 70% of gene insertions or deletions (fig. s10) . unfortunately, most of them had unknown functions. besides, the genes of dp71 l, which was reported to have the function of neurovirulence, and p22, which was reported to be one of the antigen protein, were also widely involved in gene insertions or deletions. repeated elements could facilitate the homologous recombination. in this study, lots of repeated elements ranging from 5 to 100 bp were z. zhu, et al. veterinary microbiology 236 (2019) 108380 identified, and then the distribution of the repeated elements in the asfv genomes was analyzed. as shown in fig. s11 , the number of repeated elements in asfv genomes decreased monotonously as the size of elements increased. then, the distances between adjacent elements for a given repeated element was investigated (fig. 5a) . as the size of the elements increased from 5 to 10, the average distance between the adjacent elements also increased because the number of repeated elements in the genome decreased and the repeated elements became more disperse in the genome. interestingly, the average distance decreased as the size of the elements increased from 11 to 33; it reached to the minimum (136 bp) when the size was 33; then the distance kept unchanged as the size increased from 33 to 49; finally, it increased as the size of repeated element increased from 50 to 100. it should be noted that the average distance was still less than 300 bp even for the repeated elements of 100 bp. these phenomena suggested that the repeated elements of 11 bp or larger tended to cluster in the genome, especially for those of 33-49 bp. for example, when the size of elements was 30 bp, each genome had a median of 427 types of elements which repeated at least two times in the genome. some elements appeared for over ten times in the genome, such as the element "aggcgttaaacattaaaattattactactg" in the viral strain ba71 v. the region covered by repeated elements accounted for 1%-3% of the genome in asfvs. the median distance between repeated elements was 170 bp, suggesting they tended to cluster in adjacent regions. fig. 5b shows the distribution of repeated elements in the aligned genome. most repeated elements were located at both finally, the contribution of repeated elements to the recombination was investigated. for elements of 10 or more nucleotides, the number of repeated elements in the windows (1000-10,000bp in length) table 1 gene content variation in the recombination region between the major parent and the recombinant virus in two recombination events. genes highlighted in black bold referred to those different between the recombinant virus and the major parent virus in the recombination region. including recombination was significantly larger than those without recombination (p-values < 0.001 in the paired t-test) (table s5) . fig. 5c showed the comparison of the number of repeated elements (30 bp in length) in the windows of 10,000 bp with and without the recombination in viral genomes. the windows including the recombination had a mean of 72 repeated elements, which was four times of that in the windows without recombination. this work systematically analyzed the genetic diversity of asfvs. large diversity was observed among the genomes and the genes of asfvs, which may lead to diverse phenotype, such as the diversity in antigen and virulence. indels were found to have a larger contribution to the genetic diversity of asfvs than the point mutations. this was similar to that observed in a previous study by lin, during which the author found that insertion/deletion of simple sequence repeat (ssr) could cause large genetic variations in phages (lin, 2016) . compared to point mutations, indels could introduce a larger variation to the genome, and cause a more severe damage to the genome structures, which may lead to the death of viruses (wang et al., 2016; singh et al., 2019; sharma et al., 2018) . therefore, only few indels were observed in viruses with small genomes, such as influenza viruses (taubenberger and kash, 2010) and hepatitis c viruses (hcv) (torres-puente et al., 2007) . however, it was more robust for the indels to occur inside the viruses with large genomes, such as asfvs (dixon et al., 2013) , poxviruses (elde et al., 2012) and phages (lin, 2016) , because the viruses with large genomes had lots of repeated elements (such as ssrs) and duplicated genes (such as mgfs). moreover, indels may provide a more efficient way of survival than the point mutations under the natural zhu, et al. veterinary microbiology 236 (2019) 108380 selection pressure (dixon et al., 2013; lin, 2016; elde et al., 2012) , since the virus with indels could rapidly change its phenotype, such as antigen, virulence, or ability of replication and transcription. for example, the deletion of some mgf genes in asfv could reduce the viral replication or virulence, which may help with the viral infection of soft ticks (dixon et al., 2013; burrage et al., 2004) . as dixon et al. pointed out, gene families are commonly involved in the evolution of dna viruses with large genomes (dixon et al., 2013) . for example, the poxviruses can rapidly acquired fitness via recurrent amplification of a key anti-host defence gene (elde et al., 2012) . the asfv genomes have five mgf families, each of which has 3-22 mgf genes (dixon et al., 2013) . previous studies have shown that most genome variation in asfvs was as the result of gain or loss of mgf genes (dixon et al., 2013) . this study found that the member of mgf families were frequently involved in recombinations. they accounted for more than 2/3 of gene insertions or deletions when comparing the proteome of asfvs. although most of them had unknown functions until now, they are supposed to play important roles in rapidly changing the phenotypes of the virus. several factors could contribute to the indels, including replication slippage, retrotransposition and recombination (zhang, 2003) . the replication slippage may introduce short indels which were widely observed in asfv genomes, but it is unlikely to cause large indels. this study suggested that the ectopic homologous recombination, during which the segments with unequal length were exchanged (freitas-junior et al., 2000) , may contribute much to the extensive indels observed in asfv genomes (fig. 6a) . as a proof, significant associations were observed between the occurrence of recombination events and the indels. the clustered repeated elements observed in asfv genomes may facilitate the homologous recombination (fig. 5) . taken together, the homologous recombination should be the effective strategy of asfvs to generate the genetic diversity, which further leads to the diverse phenotypes, including antigen, virulence, replication and transcription ability, and the "weapons" of escaping from the host immunity (fig. 6b) . the widespread distribution of repeated elements in the asfv genomes may have important implications for the viral evolution. the short repeated elements may facilitate the replication slippage, leading to short indels. for example, dixon et al. have identified short tandem repeats within the asfv genes (dixon et al., 2013) , such as e183 l and b602 l, which cause large variations of these genes. besides the short repeated elements, there are also an abundance of long repeated elements, such as those longer than 30 bp. the clustered long repeated elements can facilitate the ectopic homologous recombination, which lead to large indels including the gene insertions/deletions. this work provides some insights into the prevention and control of the asfvs. since the virus can rapidly change its phenotypes, such as the antigen, traditional methods of developing vaccines or drugs may be ineffective, as was demonstrated in previous studies (escribano et al., 2013; arabyan et al., 2018; sanchez et al., 2019) . identification of the conservative antigenic epitopes or drug targets may help for development of effective vaccines and drugs. besides, development of drugs targeting host proteins instead of viral proteins may be an alternative strategy (han et al., 2017) . moreover, since the recombination play a large role in shaping the genetic diversity of the virus, coupling the drugs which inhibit the recombination process with the traditional drugs or vaccines, may help prevention and control of the viral infection. there were some limitations to this study. firstly, the number of asfv genomes was limited, which hindered a comprehensive analysis on the evolution of asfv genomes. previous studies have identified over twenty genotypes of asfvs (boshoff et al., 2007; bastos et al., 2003) , among which only seven genotypes were included in this study. fortunately, the isolates included in this study covered a long time period from 1950 to 2018, and also covered a large area including africa, europe and china, which were the major areas of the asfv circulation. besides, the genotype ii, the most widely spread genotype in recent years (ge et al., 2018; zhou et al., 2018; quembo et al., 2018; garigliany et al., 2019) , were also included and constituted nearly half of all asfvs. thus the results based on these isolates could reflect the genetic diversity of the asfvs to a large extent. secondly, the location and size of the indels observed in asfv genomes may be affected by the sequence alignment algorithm. two common methods, i.e., mafft and clustalw, for the alignment of asfv genomes were used in this study. in both methods, the indels were observed to contribute much more to the genetic diversity than the point mutations did (fig. s3) , suggesting the robustness of the results. lastly, the extensively repeated elements in asfv genomes could facilitate the frequent occurrence of recombination events. however, some of recombination events cannot be detected by the recombination detection method because of the exchange between the genomic segments with small indels. such kinds of recombination events are difficult to detect. increasing the sensitivity of the recombination detection method can help detect them, but may also bring false positives. therefore, the sensitivity and specificity should be balanced in the recombination detection methods.overall, this work provided a systematic view of the genetic diversity of asfvs. extensive homologous recombination detected in this study may contribute much to the widespread indels observed in asfv genomes, which further lead to the large genetic diversity of asfvs. the results on the causes of the diversity of asfvs would help with the understanding of the evolution of the virus and thus facilitate the prevention and control of asfvs. the authors have declared that no competing interests exist. (2016-i2m-1-005). database resources of the national center for biotechnology information a proteomic atlas of the african swine fever virus particle gapped blast and psi-blast: a new generation of protein database search programs genistein inhibits african swine fever virus replication in vitro by disrupting viral dna synthesis gaps in african swine fever: analysis and priorities agricultural diseases on the move early in the third millennium genotyping field strains of african swine fever virus by partial p72 gene characterisation genemarks: a self-training method for prediction of gene starts in microbial genomes. implications for finding sequence motifs in regulatory regions comparative analysis of the complete genome sequences of kenyan african swine fever virus isolates within p72 genotypes ix and x genetic characterisation of african swine fever viruses from outbreaks in southern africa (1973-1999) african swine fever virus multigene family 360 genes affect virus replication and generalization of infection in ornithodoros porcinus ticks comparison of the genome sequences of non-pathogenic and pathogenic african swine fever virus isolates epidemiology of african swine fever virus phylogenomic analysis of 11 complete african swine fever virus genome sequences african swine fever virus replication and genomics poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses orthofinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy antibody-mediated neutralization of african swine fever virus: myths and facts food and agriculture organization of the united nations evolution of african swine fever virus genes related to evasion of host immune response frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of p-falciparum african swine fever virus: a review phylogeographic analysis of african swine fever virus molecular characterization of african swine fever virus, china genetic information research institute human enterovirus 71 protein interaction network prompts antiviral drug repositioning dendroscope: an interactive viewer for large phylogenetic trees mafft multiple sequence alignment software version 7: improvements in performance and usability the intracellular proteome of african swine fever virus clustal w and clustal x version 2.0 simple sequence repeat variations expedite phage divergence: mechanisms of indels and gene mutations rdp: detection of recombination amongst aligned sequences comprehensive phylogenetic reconstructions of african swine fever virus: proposal for a new classification and molecular dating of the virus homologous rna recombination in brome mosaic virus: au-rich sequences decrease the accuracy of crossovers genetic characterization of african swine fever virus isolates from soft ticks at the wildlife/domestic interface in mozambique and identification of a novel genotype r: a language and environment for statistical computing. r foundation for statistical computing mechanisms of plant virus evolution development of vaccines against african swine fever virus african swine fever: a reemerging viral disease threatening the global pig industry comparative genetic variability in hiv-1 subtype c p24 gene in early age groups of infants a proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology mega6: molecular evolutionary genetics analysis version 6.0 influenza virus evolution, host adaptation, and pandemic formation contribution of insertions and deletions to the variability of hepatitis c virus populations replication slippage involves dna polymerase pausing and dissociation origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis crispr/cas9-derived mutations both inhibit hiv-1 replication and accelerate viral escape a unified classification system for eukaryotic transposable elements world animal health information and analysis department evolution by gene duplication: an update emergence of african swine fever in china supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetmic.2019.08.003. key: cord-310315-w4rgjsbl authors: belhadi, amine; kamble, sachin s.; khan, syed abdul rehman; touriki, fatima ezahra; kumar m., dileep title: infectious waste management strategy during covid-19 pandemic in africa: an integrated decision-making framework for selecting sustainable technologies date: 2020-10-23 journal: environ manage doi: 10.1007/s00267-020-01375-5 sha: doc_id: 310315 cord_uid: w4rgjsbl the emerging and underdeveloped countries in africa face numerous difficulties managing infectious waste during the sars-cov-2 disease, known as the covid-19 pandemic. hence, the main aim of this paper is to help decision-makers in african countries to select the best available waste management strategy during the covid-19 pandemic. the present research undertakes seamless assessment and prioritization of infectious solid waste (sw) and wastewater (ww) treatment technologies based on a criteria system involving four dimensions, i.e., environment-safety, technology, economics, and sociopolitics. a combined approach that integrates the results of life-cycle assessments and life-cycle costs (lca–lcc), analytic hierarchy process (ahp), and vikor method in an interval-valued fuzzy (ivf) environment is proposed. the results reveal that combined incineration and chemical disinfection approach, and combined chlorination and ultraviolet irradiation are the most sustainable technologies for managing infectious sw and ww treatment in the present context. the proposed approach, alongside the findings of the study, constitutes a reference to devise urgent planning for contagious waste management in african countries as well as developing countries worldwide. the new sars-cov-2 disease (commonly known as has induced a historical impact in most countries. since the beginning of the pandemic, the virus has distressed almost all countries causing infection to several million and claiming thousands of lives according to who (2020a, b) . the governments worldwide have implemented several measures to mitigate the propagation of the virus, including personal protective equipment, social distancing, and lockdown (klemeš et al. 2020) . the transmission behavior of sars-cov-2 is creating significant challenges for services related to solid waste (sw) and wastewater (ww) management. aerosols, plastics, oral/fecal, and inanimate surfaces (fomites) are identified as the transmission medium for the virus (nghiem et al. 2020) . the multiple modes of transmission emphasize implementing a safety management system to interrupt the potential propagation of covid-19 through sw and ww. the international organizations such as the world bank and who (2020a, b) are extensively stressing the use of proper waste management (wm) mechanisms to prevent the transmission of covid-19. the discharge of municipal sw and ww without appropriate treatment increases the probability of public exposure to infection who (2020a, b) . in the context of the present pandemic, there is a need to take precautionary measures to avoid the risks associated with public health and the environment. therefore, proper treatment and disposal of the infectious sw and ww are of great significance. the selection of the most efficient infectious waste treatment technology is a challenging task and depends on various criteria such as environment, safety, politics, and economics. the availability of diverse wm technologies with different levels of performance on selected criteria makes the decision-making complex. furthermore, the ambiguous, incomplete, and inconsistent information to make these decisions adds uncertainty in decision-making (valente and bueno 2019; voudrias 2016) . a reliable procedure to prioritize the wm treatment technologies will be of great utility for the decision-makers . the challenges for emerging and underdeveloped nations in africa are even more significant (belhadi et al. 2020 ). the budget for wm in most african countries is less compared to the other nations, constraining the treatment facilities, and posing severe threats to public health (idowu et al. 2019) . the waste generation during the covid-19 at the sanitary and environmental levels is gaining more interest in african countries. the municipal wm assumes significant importance in most emergency response projects announced by african countries to fight the covid-19 pandemic. although the researchers have extensively studied the different alternatives and technologies to manage general sw and ww disposal (ayodele et al. 2018) , specific studies concerning the treatment and disinfection of municipal sw and ww in the african countries in the context of the covid-19 pandemic are nonexistent. the evaluation of the sw and ww treatment is a multidimensional problem that should consider factors related to economy, safety, environment, society, technology, and politics. in recent times, multicriteria decision-making (mcdm) methods have gained high popularity in the wm area as a powerful tool to resolve decision problems involving multiple conflicting criteria. approaches such as analytic hierarchy process (ahp), vlsekriterijumska optimizcija i kaompromisno resenje (vikor), technique for order of preference by similarity to ideal solution (topsis), and preference ranking organization method for enrichment of evaluations (promethee) have been used extensively in solving wm related problems. several studies have combined fuzzy and rough sets theory with the mcdm techniques to overcome the problem of vagueness and imprecision of human thoughts (wang et al. 2019; kharat et al. 2019) . the context of covid-19 is posing an enormous challenge in data collection leading to an increase of uncertainty and complexity related to wm decisions. this lack of information in defining the membership function may involve some hesitation in membership degrees of fuzzy sets. the decision-maker might not be fully confident about the preferences, resulting in some amount of uncertainty associated with their decisions. therefore, the traditional fuzzy set theory will not be useful to describe the opinions of the decision-makers . consequently, we propose to use the interval-valued fuzzy (ivf) sets to address the complexity of wm models in the context of covid-19 and overcome the issues of vagueness (lack of sharp class of boundaries in human judgment and preference) and lack of information (gupta et al. 2018 ). with the above background, the present study seeks the answers for the following research questions (rqs). rq 1: how could wm alternatives be precisely prioritized under the high vagueness generated by the context of covid-19? rq 2: what are the best alternatives for underdeveloped countries to manage infectious sw and ww in the covid-19 pandemic context? in seeking the answers to the above rqs considering the criticality of the infectious municipal wm generated during the covid-19 pandemic, this paper contributes to the development of a wm strategy during the covid-19 pandemic by proposing a combined life-cycle assessment (lca), life-cycle cost (lcc) analysis, and ahp-vikor method to select a wm treatment technology under the effect of ivf environment. the proposed mcdm approach deals with high uncertainty related to wm data and seeks to develop an integrated management program of infectious sw and ww by incorporating various factors. the study makes the following significant contributions: (1) the use of ivf set theory to cope with the ambiguity and vagueness generated by the uncertain context of covid-19 can alter the decision-making process. (2) the use of lca-lcc for evaluating quantitative criteria based on actual data collected from the african context. (3) the combination of qualitative (secondary data) and quantitative (expert interviews) data to select the best alternative for sw and ww treatment. (4) the proposition of an integrated approach for managing sw and ww during and after the covid-19 pandemic. the study was conducted based on data collected from selected companies in morocco, africa. a fuzzy interval multiattribute decision-making technique was used to address uncertainty in decision-making during the ranking of alternatives. while ahp was used to determine the critical weighting factors, vikor was employed to screen and rank choices using quantitative insights from lca and qualitative judgment of experts. the rest of this article is organized as follows. background literature section presents the background literature explaining the different challenges related to wm during the covid-19 pandemic and the most used technologies for infectious wm. materials and methods section discusses the research methodology adopted in this study. the findings of the african case study are discussed in section an empirical study on infectious wm in africa. the outcome and implications of the study are discussed in section discussion and implications. conclusion section presents the conclusions, limitations, and future scope of the study. the continuous propagation of the covid-19 pandemic entails increased impacts and challenges upon human health, economy, and global environment. in particular, the current covid-19 pandemic outbreak raises various questions on the municipal waste handling and treatment practices related to the health and safety of workers and waste treatment facilities (saadat et al. 2020) . more specifically, two significant challenges are identified. the first challenge is related to the sharp increase in sw produced by either healthcare facilities or households. for instance, in china, the generation of solid and medical waste in hubei province has increased by 370% during the pandemic, despite the notable decrease of 30% of the total volume of municipal sw, according to the state council's joint prevention and control mechanism. furthermore, wuhan has shown an unprecedented increase in plastic waste generation in households by 500% from early january to late march 2020 (tang 2020). the rationale behind this situation is that plastic-based materials present tremendous utility in dealing with the various mitigation measures implemented worldwide. for instance, scientists and customers consider single-use plastics a safe solution to manage the virus. this results in increased utilization and release of plastic products even for nonmedical purposes. klemeš et al. (2020) argued that the conventional municipal treatment systems for handling and disposal of regular waste under normal conditions are not suitable for use in abnormal situations. the likelihood of generating biomedical waste at the residential colonies and the healthcare centers is high during the covid-19 outbreak. household waste includes infected gloves, masks, and other protective equipment. the selfisolation and home quarantine of the patients due to the limited capacity of hospitals increases the possibility of infectious municipal sw generation at personal locations. to mitigate this challenge of a widespread generation of medical waste klemeš et al. (2020) suggest a structural change in the existing wm procedures that include sorting, collection, treatment, and disposal, adhering to the current safety protocols for the waste collection workers. despite the recent announcements on the safety precaution measures in handling infectious waste (who 2020a, b) , the extensive generation of plastic and mixed waste presents logistical challenges, putting the economic and environmental concerns on a low priority. the second challenge is related to the pathogenic characteristic and viral transmissibility of ww during the covid-19 pandemic. the pathogenicity of ww can be illustrated through two perspectives. first, studies have confirmed the transmission potential of sars-cov-2 through untreated municipal ww (mallapaty 2020; ahmed et al. 2020) . second, previous studies during the sars-cov-1 epidemic in 2003 had found ww aerosols as a "high potential" way of virus transmission in contaminated cities (jack 2006) . hence, ww during the covid-19 pandemic could present a critical route of transmissibility of the virus. besides, the increase in drugs and pharmaceutical product consumption during the covid-19 outbreak would magnify the environmental impact and pathogenicity of ww. according to gogoi et al. (2018) , unmetabolized drugs and their metabolites are commonly considered as problematic trace contaminants as traditional ww treatment plants are unable to kill them entirely. the number of contaminants in ww would undoubtedly augment during the pandemic, and even more, if the drugs are consumed uncaringly (yu et al. 2020; chen et al. 2018) . therefore, specific stages of the ww treatment process, particularly in the upstream collection network, should be subject to disinfection and sterilization treatment to limit the propagation of covid-19. most african countries are suffering from an increasing share of the population living in cities that face a shortage of funds, inadequate wm facilities, and poor urban planning posing severe challenges to wm strategies. a who report states that only 24% of the rural population and 44% of the urban population in africa have access to sanitation facilities. the organization for economic co-operation and development (oecd) claims that only 56% of african cities dwellers have access to piped water, down from 67% in 2003, and only 11% have a sewer connection oecd/giz (2019). moreover, people in rural areas are reported to be twice less vulnerable than people in urban areas for access to clean and safe water who/unicef (2017). public and private operators such as local nongovernmental organizations (ngos) working for improvements in the water, sanitation, and wm in africa face difficulties because of financial, institutional, and technical problems (badi et al. 2019; friedrich and trois 2016) . thus, most cities and towns across africa lack the required resources and infrastructure to address the challenges of wm. of particular concern are the issues related to the current covid-19, i.e., dealing with the increasing volumes of wastes and preventing the human-to-human transmission of the sars-cov-2 through proper hygiene, sanitation, and efficient wm, which seem to be not fully provided in africa (world bank 2020). the fast propagation of covid-19 is likely to disproportionately influence the most destitute and vulnerable regions globally (street et al. 2020) . these countries require urgent and appropriate covid-19 responses, including hygiene and wm within complex circumstances. infectious sw and ww management have the potential to be a robust public health tool in africa. however, alternatives to handling and disinfecting sw and ww need to be selected more reliably and reasonably. the solution should incorporate local constraints and sustainability objectives. the present section reviews the leading technologies of sw disinfection and treatment during the pandemic context. inc is one of the most efficient waste-to-energy conversion treatment processes for recycling the inorganic and plasticbased proportion of waste (cobo et al. 2018) . klemeš et al. (2020) argued that inc (90 min, 120°c) shows exceptional ability to treat hazardous waste. the relatively high inc (over 800°c) results in an adequate decontamination cycle by killing most microorganisms. hence, the tailings of this process can be safely manipulated following nonhazardous sw regulation (hong et al. 2018) . besides, inc participates in air pollution through the generation of toxic air, such as chlorinated dibenzodioxins and dibenzofurans (voudrias 2016) . chd is often combined with mechanical crushing treatment (hong et al. 2018) . during the disinfection process, chemical disinfectants (chlorine dioxide, sodium hypochlorite, calcium hypochlorite, etc.) are mixed with the crushed, contaminated wastes and kept for sufficient time to inactivate infectious microorganisms. chemical disinfectants are characterized by immediate effect, steady yield, and broad neutering spectrum ranging from microorganisms and viruses to bacteria spores (voudrias 2016) . sodium hypochlorite, calcium hypochlorite, and chlorine dioxide are the widely used disinfectants because of their specific features such as being noncorrosive, odorless, tasteless, colorless, high solubility in water, and nonhazardous after treatment (wang et al. 2020a, b) . however, chd can be considered only when the volume of infected wastes is small (hong et al. 2018) . mcd is based on electromagnetic waves with a wavelength between (2450 ± 50) and (915 ± 25) mhz (veronesi et al. 2005) . the main features of mcd are low energy consumption, fast action, higher heat yield, and little harmful effect on the environment with no toxic wastes or residues after disinfection (veronesi et al. 2005) . moreover, the sterilization spectrum of bacteria is relatively broad, which can inactivate a wide range of microorganisms (veronesi et al. 2005) . nonetheless, the process requires meticulous monitoring by advanced microwave instruments (stolze and kühling 2009 ). std is a high heat-based technology efficient in disinfecting contaminated waste using saturated water steam, with a temperature exceeding 100°c (veronesi et al. 2005) . sanitary landfills are used to dispose of the treated waste at a specified temperature and duration. the water steam releases latent heat, destroying microorganisms due to coagulation and protein denaturation. the disinfection of potentially infectious wastes using steam does not release toxic gases. rp is microwave-assisted pyrolysis recognized with high lead for infectious metals and plastic waste (undri et al. 2014 ). according to voudrias (2016) , rp systems could reach 80% of volume reduction with 6 log10 pathogen inactivation. moreover, sterilized residues from rp are generally stable and appropriate for sanitary landfilling after shredding (undri et al. 2014) . the main drawback of the method is the release of ww by naoh used in the scrubber to monitor gaseous emissions. the cost of these technologies is the highest among the techniques considered acting as a primary barrier for acceptance voudrias (2016). in the literature, several specific disinfection treatments have been recommended to be included in the ww management chains. chl is the most commonly used ww disinfection treatment since the late i940s (lazarova et al. 1999 ). chl has a significant role in limiting waterborne infectious diseases worldwide; thereby, it is recommended by the who (2020a, b). the generation of toxic residues, inadequate inactivation of the pores, increased costs because of dechlorination requirements, and high investments in safety equipment and scrubbing due to stringent safety regulations act as the main barriers for the use chl technique (hu et al. 2018; lazarova et al. 1999) . uvi is increasingly used as an alternative to chl and found to be a highly efficient disinfectant for microorganisms (viruses and bacteria) (lazarova et al. 1999) . currently, numerous installations around the world use open channels fitted with low-pressure mercury arc lamps to perform uvi disinfection ). the popularity of uv technology is mainly due to its low costs and the lack of toxic residues . however, disinfection with uv is, at times, insubstantial due to insufficient penetration depth and risks on occupational health (kühn et al. 2003) . ozo is based on ozone, which is a powerful oxidizing agent and useful in deactivating microorganisms (bacteria and viruses). hence, ozo is widely utilized in water provision engineering and ww treatment (wang et al. 2020a , b, lazarova et al. 1999 ). however, ozone is a cystforming protozoan parasite, resistant to most other disinfectants (lazarova et al. 1999) . although the involved process is complex, the dissolved molecular ozone is mainly useful for destroying a wide range of bacteria in drinking water (lazarova et al. 1999) . moreover, several authors such as im et al. (2018) and von gunten (2003) confirmed that ozone is highly useful to inactivate viruses. uf is based on a physical barrier concept to remove colloids and more abundant molecular weight organics. uf pilot tests performed with hydrophilic membranes (cut-off 0.01 µm) on different effluents showed complete removal of coliforms, streptococci. salmonella, clostridium enteric viruses, and bacteriophages (lazarova et al. 1999) . given that the diameter of sars-cov viruses is between 0.01 and 0.012 µm (guy et al. 2001) , uf can be an option to treat infected ww (ahmed et al. 2020 ). studies on wm are mainly using mcdm techniques (i.e., ahp, vikor, topsis, prometthee, etc.) as a solution method. (2019) used a fuzzy set theory to treat uncertainty. however, the treatment of uncertainty was found to be relatively weak as the data used was mainly qualitative. sarkkinen et al. (2019) and ren and toniolo (2020) used lca to evaluate quantitative information but did not address uncertainty caused by qualitative judgments. the existing literature on ww management using mcdm showed that liu et al. (2020) , yao et al. (2020) , and narayanamoorthy et al. (2019) handled the issue of uncertainty using triangular, hesitant, and ivf sets while evaluating alternatives for ww treatment and reuse. however, they too considered qualitative judgment without any involvement of quantitative criteria. gherghel et al. (2020) and munasinghe-arachchige et al. (2020) used some advanced mcdm techniques to rank and evaluate ww treatment plants such as prometthe and simple additive weighting-paired comparison technique (saw-pct). nevertheless, these studies were also based on human judgment without any treatment of uncertainty. the review of recent literature on wm reveals several research gaps in the current literature. the methodologies used in the literature cannot be adopted in the current covid-19 context, which is characterized by great uncertainty needing decision-making accuracy. these gaps can be summarized as follows: (1) there are no studies in the recent literature to address sw and ww management jointly using the mcdm approach. (2) the recent studies are suffering from absence or poor uncertainty treatment, as they do not deal with an ambiguous and uncertain situation such as covid-19. (3) few studies rely on quantitative approaches to assess criteria to solve the problem. to fill the gaps identified in the current literature, we propose an integrated approach combining lca and lcc with ahp and vikor in an ivf environment. the framework for the suggested methodology integrates lca-lcc and mcdm to prioritize sw and ww treatment alternatives during the covid-19 pandemic. the procedure is based on four elementary phases, as illustrated in fig. 1. (1) identification of the purpose of the study. (2) development of the study background by identifying the system boundaries and a set of implementable alternatives derived from the sw and ww treatment technologies. the evaluation criteria are determined from the literature review. finally, the problem hierarchy, including measures and alternatives, is established. (3) data collection and assessment of criteria using (qualitative) experts' judgments and (quantitative) lca-lcc approach. (4) evaluation of the sw and ww treatment alternatives through ivf-ahp-vikor. the central aim of this study is to contribute to the global fight against the covid-19 pandemic by helping decisionmakers to select the best choice among the technology alternatives of the treatment of the hazardous sw and ww generated during this pandemic period according to their safety and sustainability performances. the significant selection of the most suitable treatment technologies for the infectious sw and ww during the covid-19 pandemic is a complex mcdm problem that implies consideration of multiple numbers of alternatives and evaluation criteria (wang et al. 2020a, b; cobo et al. 2018; chen et al. 2006) . the three dimensions of sustainability, viz., economic performance, environmental issue, and social concern, represent the ground of the selection criteria of the problem under study (wang et al. 2019; ren and lützen 2015) . furthermore, the specific features of the covid-19 pandemic require incorporating safety, qlt degree by which the process could be accepted by social ecosystem technology, and political considerations as these aspects influence the three pillars of sustainability. table 2 depicts the descriptions of the subcriteria under the considered criteria. we assume that all of the evaluation criteria are independent, suggesting the absence of any interaction and interdependence among them. the functional units for comparing the disposal of sw and ww are 1 tone and 1 m 3 , respectively. the gate-to-gate approach was used to define the system boundaries that are a partial lca, considering only the disposal process of municipal waste in the whole process of wm. fig. 2 . as the waste collection and storage practices were common to each scenario, they were excluded from the study. regarding infectious sw treatment, inc is considered a singular technique in the first alternative (sw1) and rp in the second alternative (sw2). more combined approaches, including inc with chd, std, and mcd, are considered in (sw3), (sw4), and (sw5), respectively. as for infectious ww treatment, chl is integrated with uvi in the first alternative (ww1), while uvi is considered independently in (ww2). besides, a combined approach, including uf with uvi, is considered in (ww3) whereas only uf is included in (ww4). finally, the fifth alternative (ww5) consists of ozo only. the hierarchal structure for the selection problem the problem decision hierarchy is conceptualized to integrate the criteria identified earlier for the assessment of the alternatives. as shown in fig. 3 , the problem hierarchy consists of four levels. the goal of the problem is represented by the first level and involves the selection of the most appropriate treatment technology for infectious sw and ww during the covid-19 pandemic. the second level includes the specification of the predefined criteria, such as environmental-safety, economic, technical, and social-political, which are involved for further assessment. the subcriteria for each measure are included in the third level. the fourth level specifies the treatment alternatives used to accomplish the purpose, after their evaluation using the criteria and subcriteria. lca is a methodology grounded in the life-cycle thinking concept for performance assessment of a product or process throughout its life span involving manufacturing, consumption, and end of life (heidari et al. 2020; . lca is mainly applied to assess environmental and indirect social impacts. further, zanghelini et al. (2018) stated that lca is highly compatible with mcdm techniques by enabling quantitative assessment of environmental indicators rather than being limited to qualitative evaluations. following the iso 14040 table 3 the weights and ranking of evaluation criteria table 4 the lcc estimates the relevant cost throughout the life cycle of products or processes (zanghelini et al. 2018 ). according to li et al. (2019) lcc is an engineering economic analysis technique grounded on the principles of economic analysis to assess the long-term costs of different alternatives along a selected analysis period. contrary to lca, lcc does not have independent iso standards. however, authors such as li et al. (2019) mention two different ways to accomplish lcc, i.e., probabilistic and deterministic. while the deterministic way uses an accurate estimate for model input variables, the probabilistic way controls the uncertainty of variables using probability distributions. usually, lcc variables are uncertain. the probabilistic way is thus highly appreciable. ahp under ivf environment is a commonly used mcdm approach to deal with both the intuitive and rational origins of uncertainty in determining the relative weights of the problem criteria (saaty 1980 (saaty , 1977 . therefore, fuzzy ahp is utilized to identify the relative importance of the n criteria for the safety, politics, and sustainability evaluation of sw and ww treatment denoted as {α 1 , α 2 ,…, α n } and the relative suitability of the m alternative technologies regarding each criterion denoted as {β 1 , β 2 ,…, β m } using the opinions of k decision-makers δ = {δ 1 , δ 2 ,…, δ k }. then, the output obtained from the fuzzy ahp is used in vikor method to prioritize the alternatives. the ivf-ahp is conducted in three steps (belhadi et al. 2017; ren and lützen 2015) . (1) step 1: construction of the fuzzy comparison matrices (c). the decision-making panel identifies the pairwise comparison matrix based on linguistic terms illustrated in appendix i (table 5) where e c k ij ¼ c k 1ij ; c 0k 1ij ; c k 2ij ; c 0k 3ij ; c k 3ij h i is a tivfn representing the relative importance of the ith criterion compared with the jth criterion as obtained from decision-makers. the reciprocal of ivf number e c k ij is (2) step 2: examining the consistency of the fuzzy pairwise comparison e c by evaluating the consistency of the defuzzified matrix c. the defuzzified c is calculated using the center of area method, resulting in weights of each criterion. in order to determine the consistency ratio (cr) of a matrix, eq. (3) is first used to calculate the matrix consistency index ci. where λ max is the largest or principal eigenvalue of the c decision matrix of pairwise comparison as aω = λ max ω. then, the consistency ratio (cr) is calculated by using the normalized random index, which depends on n the matrix size, as if cr ≥ 0.1, then the decision-makers must be consulted to revise their evaluations. (3) step 3: determining the ivf weights of criteria of each decision-maker using the extended ivf geometric mean (eq. (2)) developed by csutora and buckley (2001) , as presented in eqs. (2) and (3) e r j ¼ e c j1 e c j2 :::: e c jn à á 1=n ; r 2j ; r 0 3j ; r 3j h i represents the geometric mean regarding the jth decision-maker and stakeholder and e w k ¼ w 1k ; w 0 1k à á ; w 2k ; w 0 3k ; w 3k à á â ã represents the relative weight of the kth decision-maker. vikor method is introduced as an mcdm approach relied on compromise solutions (opricovic and tzeng 2007; opricovic 1998) . this method aims at determining the compromise solution while prioritizing a set of alternatives in the existence of controversial criteria. the vikor method introduces the multihierarchical index, based on the particular measure of "closeness" to the "ideal" solution. in this study, the ivf-based extension of vikor is applied to carry out the selection of infectious sw and ww treatment selection during the covid-19 pandemic. during the evaluation process, we consider a set of m alternatives represented as {β 1 , β 2 ,…, β m } and a set of the n pre-established assessment criteria {α 1 , α 2 ,…, αn}. accordingly, the steps of the ivf-based vikor are presented as (1) step 1: constructing the aggregate fuzzy evaluation matrix for the ranking e f ¼ ½ e f ij mân by using the linguistic scale illustrated in appendix i (table 5b) and the weighted ivf decision matrix e v ¼ e v ij â ã nâm based on ivf weights from ivf-ahp . . . where e f ij ¼ e f 1 ij þ e f 2 ij þáááþ e f k ij k stands for the average rating of the jth alternative β j with regard to ith criterion α i for the k decision-makers fig. 3 the hierarchy of the problem (2) step 3: calculating the positive ideal solution (p f* , p v* ), and negative ideal solution (n f− ) for upper and lower reference points of the ivfns. there are two groups of criteria. the first group with the larger value, the optimal the alternatives, is called beneficial criteria, while the second is called cost criteria with the greater value, the nonoptimal the alternative ( 3) step 4: the computation of the values of the utility (s i ) and the regret (r i ) and the vikor index (q i ) based on the weights of criteria determined by ivf-ahp using eqs. (12)-(16), respectively where and factor υ is the weight of the decisionmaking strategy of "the maximum utility", which is comprised in the interval [0, 1] and is often considered as 0.5. (4) step 5: the ranking of the alternatives by sorting the values of s i , r i , and q i in ascending order. note that the higher the values of q i , s i , or r i , the less superior the corresponding alternative will be. the alternative β 1 is suggested as a compromise solution that is ranked as the optimal by the measure q (q 1 is the smallest among q i values) if the following two conditions can be fulfilled (opricovic and tzeng 2007) : cond.1: acceptable advantage: alternative β 1 should satisfy q 2 à q 1 ! 1 mà1 : cond.2: acceptable stability in decision-making: alternative β 2 should also be ranked as the optimal by s and r. if one of the conditions is not fulfilled, then a set of compromise solutions is proposed, which consists of (1) alternatives β 1 and β 2 if cond.1 is fulfilled and cond.2 is not fulfilled (thus, both scenarios β 1 and β 2 are suggested as the optimal solutions). (2) alternatives, β 2 ,…, β m if cond.1 is not fulfilled (hence, a set of solutions β 1 , β 2 ,…, β m is suggested as the optimal choices); β m is identified by the equation q m à q 1 1 mà1 for maximum m (the positions of these alternatives are "in closeness"). municipal wm is a significant issue in most developing countries, especially in africa. yet, factors such as shortage of funds and increasing share of the population living in cities pose severe challenges to municipalities to provide 80% 85% 90% 95% 100% 0.00e+00 5.00e+03 pandemic. the world bank is approving health emergency funds to assist several african countries in their covid-19 emergency response projects, including an infection control and wm plan for the proper management of contaminated sw and ww during the current covid-19 pandemic (unicef 2020). thus, selecting the appropriate treatment for infectious sw and ww would be of paramount usefulness for african countries. the evaluation of criteria and decision-making perspective for the problem under study necessitates mcdm estimation models based on qualitative value judgments alongside quantitative data. therefore, a combined lca-lcc approach was used at an environmental and techno-economic level to quantify the quantitative criteria of our model. furthermore, experts' judgment is used to evaluate qualitative criteria. primary data were collected through interviews with organizations and internal sources from institutions in morocco, such as the ministry of mines, energy, water, and the environment, the national agency of waste management, and deutsche gesellschaft für internationale zusammenarbeit (giz). direct contact was established with 27 local and international companies and organizations in morocco. moreover, secondary data were collected from the ecoinvent v3.6 database. the impact assessment was evaluated using the impact world+ due to its compatibility with all impact categories and evaluation prowess. the results of the evaluation of quantitative environment-safety-related criteria ces1, ces2, ces3, and ces5 (excluding employees risk exposure (ces4), which is qualitative) are shown in fig. 4 (details are provided in appendix ii (table 6) ). regarding the infectious sw treatment alternatives, chd + inc (sw3) has the best energy consumption benefit, followed by inc (sw1). the most "energivorous" regarding the infectious ww treatment alternatives, energy consumption is not an issue in all alternatives. uf (ww4) is the best alternative as it relies on physical filtration. however, uf (ww4) is not advantageous in terms of disinfection efficiency and aquatic ecotoxicity. other options, such as chl + uvi (ww1) or even uf + uvi (ww3) are more efficient. besides, uf (ww4) is more beneficial in terms of toxic gas release. in this study, lcc method was employed to evaluate the techno-economic criteria of different alternatives. costrelated data of the various alternatives were identified based on the data gathered from organizations, companies, and treatment facilities consulted in morocco. data have been collected for different years from different organizations having comparable production capacity. accordingly, the lcc of an infectious sw and ww treatment consists of the operation costs (i.e., initial investment, auxiliary materials, labor, maintenance) and external costs (including an external market cost for eco-remediation, atmospheric and aerosol releases, and human health protection). the financial profit was computed using total production units and the unit price of produced products (raw materials, energy, etc.). figure 5 illustrates the quantification of the economic, technological, and social criteria (cec1, cec2, cec3, and csp1), whereas appendix iii (table 7) and internal), providing maximum profit and job creation. as for infectious ww treatment alternatives, uf (ww4) is economically the best alternative, although the low job creation due to the simplicity of processes compared to the other methods. the lca-lcc inputs cannot be enough due to the scarcity of evidential data in infectious wm in africa, especially when dealing with qualitative criteria. therefore, we have used expert judgment to strengthen and complement our analysis. a total of 12 experts have been chosen from healthcare institutions, wm companies, ngos, and governmental institutions in many african countries to reflect the assessment of different sides. the profiles of the expert's panel are provided in appendix iv ( table 7) . calculation of criteria weights using ivp-ahp using a comparison survey, the experts were requested to employ the linguistic scale (appendix i (table 5a) ) to evaluate the criteria. the ivf pairwise comparison matrix between all criteria has been constructed by using eq. (1). the consistency ratio for each evaluation matrix for the criteria has been systematically computed based on eqs. (2)-(4). after obtaining a cr < 0.1 for all main criteria, the aggregated ivf evaluation matrix for the criteria weights is calculated. the ivf geometric means of the criteria have been calculated using eq. (5). hence, the ivf weights are obtained using eq. (6). moreover, the weights of 17 subcriteria are ranked based on the defuzzified global weights. the weights of the top three factors are identified according to experts' evaluation: disinfection efficiency (ces2), employees risk exposure (ces4), treatment capacity (cec1). table 3 depicts the overall findings. after calculating the global iv weights of the criteria, the evaluation of the infectious sw and ww treatment alternatives with respect to each criterion can be obtained. the experts have been consulted again to evaluate the importance of the alternatives with respect to the criteria using the linguistic scale (appendix i (table 5b) ). similarly, the ivf evaluation matrix and ivf decision matrix were determined using eqs. (7) and (8), respectively. thereafter, the positive (p f* ), negative (n f− ), and weighted (p v* ) of ivf ideal solutions for upper and lower reference points are determined by using formulations in step 3. the final rankings are based on averages, and the worst group scores are determined using eq. (16). the maximum group utility (υ) is set as 0.5. final rankings of infectious sw and ww treatment alternatives alongside related regret and average scores are illustrated in table 4 . regarding infectious sw treatment alternatives, acceptable advantage, the cond.1 in step 5 is satisfied among line sw3 (q 3 = 0.102) and sw1 (q 1 = 0.498). hence, sw3 is selected as the best appropriate alternative. the infectious sw treatments can be sorted as sw3, sw1, sw4, sw5, and sw2 from the best to the worst alternative, based on the experts' decision. sensitivity analysis was performed to analyze the impact of factors using vikor under the ivf environment on selecting sw and ww treatment alternatives during the covid-19 pandemic. the maximum group utility (υ) was used to investigate the ranking of alternatives. the maximum group utility value (υ) was set between 0.00 and 1.00 with an increment of 0.1. the results of the sensitivity analysis are presented in fig. 6 . the sensitivity analysis shows that the ranking of alternatives remains unaffected under other cases confirming that the results of the ranking order are consistent. the sensitivity analysis establishes that the proposed approach yields good results and presents suitable outcomes for decision-makers. during the covid-19 pandemic, infectious municipal sw and ww treatment is a critical issue worldwide to tackle the exponential increase of infectious waste generation during this period. this is even true for african developing and underdeveloped countries where the municipal wm is generally more economic-oriented and practiced in an unsustainable way (idowu et al. 2019; kabera et al. 2019) . recently, many government task forces have been formed in african countries to develop guidelines and operating procedures for managing infectious waste from covid-19 in risk zones. in our study, we considered morocco as a case study for developing an integrated mcdm methodology to select the most effective sw and ww treatment technology. accordingly, fig. 7 presents the integrated infectious municipal wm system suggested in this research. based on the ultimate prioritization of the infectious sw treatment alternatives, it could be noted that a combined approach, including chd and inc, is the most suitable to the african context. notably, inc of waste in most african countries is not widely considered before covid-19 due to the technical and economic constraints and short-term vision (scarlat et al. 2015) . however, coupled with chd, infectious waste inc was explored as providing the maximum tradeoff at environment-safety, economics, technology, and socialpolitics levels on the medium to long term. practitioners and policymakers adopting this alternative should start with an experienced, separated, and specialized waste collection for chd. moreover, a shredding step is highly required before chd to enhance disinfection efficacity. finally, rigorous monitoring of the collection phase is a prerequisite to limit the propagation of the sars-cov-2. many organizations such as european centre for disease prevention and control (ecdc) (2020) and who (2020a, b) recognize the necessity of specific measures regarding the collection of household waste from infected areas to enhance the efficiency of the whole municipal wm in containing the sars-cov-2 spread. furthermore, an integrated chl-uv irradiation approach was found to be the most suitable alternative for disinfecting and enhancing the reuse of ww. this finding is consistent with prior studies on sars-cov-1, which is similar to sars-cov-2. for instance, chen et al. (2006) and wang et al. (2005) reported that uv irradiation and chl were the most effective in sars-cov-1 inactivation despite the inefficiency of ozone disinfection. hence, we propose that chlorine disinfection (50 mg/l during more than 1.5 h) is adopted in this scheme (lazarova et al. 1999) . uv irradiation and heating should be incorporated due to lesser residues and perfect disinfection (wang et al. 2020a, b; lazarova et al. 1999 ). during the covid-19 pandemic, municipal wm is one of the vital utility services supporting industrial activity and fig. 7 proposed infectious municipal waste management during covid-19 pandemic society. the implementation of a suitable wm system for infectious sw and ww treatment in developing and underdeveloped countries, especially in africa, is a challenging task requiring reliable and reasonable decisionmaking. the complexity of the problem lies in its dependency on numerous critical factors such as the environmental requirements, strategies in ecological management, statutes of the country, energy policy, technological and economic capabilities and feasibility, and education of citizens (wang et al. 2020a, b; idowu et al. 2019) . to help decision-makers in the african context ascertain the current challenges of municipal wm and devise a suitable infectious waste treatment plan, a combined ahp and vikor method under the ivf environment is proposed to evaluate and prioritize the infectious sw and ww alternatives from an lcc-lca perspective. five alternatives (i.e., rp, chd, inc, std, and mcd) for infectious sw treatment were considered in this study. similarly, five alternatives (i.e., chl, uv irradiation, uf, and ozo) for infectious ww treatment were evaluated for ww disinfection technologies. an evaluation criteria system for the alternatives is constructed, including 17 criteria in four dimensions (environment-safety, economics, technology, and social politics). furthermore, data collected from professional contacts with 27 companies and institutions in morocco and the consultation of national and international databases were used to conduct an lca-lcc analysis. the results of the lca-lcc are supplemented by judgments from a panel of 12 experts that supported to prioritize the alternatives for both infectious sw and ww treatment during the covid-19 pandemic. accordingly, the proposed system for the management of contagious municipal waste in this study considers integrated strategies, including inc with chd for infectious sw treatment and chl with uv irradiation for infectious ww treatment. the findings of the current study could provide useful and valuable insights for practitioners and policymakers in drafting a municipal wm system during the specific context of the covid-19 pandemic. the suggested approach in this study can effectively integrate the environmental, safety-related, political, and social outcomes in selecting the most optimal solution for infectious wm among multiple alternatives rather than considering only techno-economic parameters. this study can be considered as a starting point for approaching a significant and weakly addressed issue in the context of covid-19. therefore, the greatest need is to conduct empirical research on wm issues of developing and underdeveloped countries. one limitation of the proposed integrated mcdm model is that it continues to depend partially on expert opinion as the weights attributed to each criterion play a vital role in the final result. to tackle this problem, future studies must use mathematical optimization procedures such as linear programming, nonlinear programming, integer programming, etc., and perform a sensitivity analysis to assess the robustness of the results. another limitation is the behavior of sars-cov-2. there is a lack of clarity on the products and processes required to manage the pandemic and the effect these products generate on coming in contact with the persons. therefore, further studies are encouraged to compare findings in different countries' contexts, including changes in government guidance, technological development, and other factors. moreover, more in-depth engineering analysis is needed to ensure that the proposed alternatives can deal with the dynamic and changing nature of the sars-cov-2. conflict of interest the authors declare that they have no conflict of interest. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. very strong importance (5,6); 7; (8, 9) extreme importance (7,8); 9; (9,9) (b) level of performance triangular ivf number very poor (0.0); 0; (1,1.5) poor (0.0.5); 1; (2.5,3.5) moderately poor (0.1.5); 3; (4.5,5.5) fair (2.5,3.5); 5; (6.5,7.5) moderately good (4.5,5.5); 7; (8,9.5) good (5.5,7.5); 9; (9.5,10) very good (8.5,9.5); 10; (10.10) appendix ii appendix iii appendix iv first confirmed detection of sars-cov-2 in untreated wastewater in australia: a proof of concept for the wastewater surveillance of covid-19 in the community application of multi-criteria-decision approach for the analysis of medical waste management systems in myanmar recyclable resources from municipal solid waste: assessment of its energy, economic and environmental benefits in nigeria. resour evaluation of solid waste treatment methods in libya by using the analytic hierarchy process evaluation of critical success factors (csfs) to lean implementation in smes using ahp: a case study environmental management lean implementation in small and medium-sized enterprises in less developed countries: some empirical evidences from north africa prioritizing the solutions of lean implementation in smes to overcome its barriers the integrated effect of big data analytics, lean six sigma and green manufacturing on the environmental performance of manufacturing companies: the case of north africa covid-19 outbreak: migration, effects on society, global environment and prevention waste water disinfection during sars epidemic for microbiological and toxicological control the consequences of spatially differentiated water pollution regulation in china municipal solid waste management via multi-criteria decision making methods: a case study in istanbul from linear to circular integrated waste management systems: a review of methodological approaches. resour fuzzy hierarchical analysis: the lambda-max method aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 infection prevention and control in the household management of people with suspected or confirmed coronavirus disease (covid-19) the housing market impacts of wastewater injection induced seismicity risk current and future greenhouse gas (ghg) emissions from the management of municipal solid waste in the ethekwini municipality-south africa sustainable design of large wastewater treatment plants considering multicriteria decision analysis and stakeholders' involvement occurrence and fate of emerging contaminants in water environment: a review multi-attribute group decision making based on extended topsis method under interval-valued intuitionistic fuzzy environment characterization of a coronavirus isolated from a diarrheic foal integrating life-cycle assessment and life-cycle cost analysis to select sustainable pavement: a probabilistic model using managerial flexibilities life-cycle environmental and economic assessment of medical waste treatment comparison of drinking water treatment processes combinations for the minimization of subsequent disinfection byproducts formation during chlorination and chloramination an analyses of the status of landfill classification systems in developing countries: sub saharan africa landfill experiences performance of combined ozonation, coagulation and ceramic membrane process for water reclamation: effects and mechanism of ozonation on virus coagulation review of life-cycle environmental consequences of waste-to-energy solutions on the municipal solid waste management system. resour drainage design: factors contributing to sars transmission joint prevention and control mechanism of the state council benchmarking performance of solid waste management and recycling systems in east africa: comparing kigali rwanda with other major cities fuzzy multi-criteria decision analysis for environmentally conscious solid waste treatment and disposal technology selection minimising the present and future plastic waste, energy and environmental footprints related to covid-19 disinfection of surfaces by photocatalytic oxidation with titanium dioxide and uva light advanced wastewater disinfection technologies: state of the art and perspectives life cycle assessment and life cycle cost analysis of recycled solid waste materials in highway pavement: a review optimal selection of sewage treatment technologies in town areas: a coupled multicriteria decision-making mode how sewage could reveal true scale of coronavirus outbreak sewage treatment process refinement and intensification using multicriteria decision making approach: a case study prognostication of energy use and environmental impacts for recycle system of municipal solid waste management modeling of energy consumption and environmental life cycle assessment for incineration and landfill systems of municipal solid waste management-a case study in tehran metropolis of iran economic instruments for solid waste management in south africa: opportunities and constraints. resour sustainable assessment for selecting the best alternative of reclaimed water use under hesitant fuzzy multi-criteria decision making environmental management the covid-19 pandemic: considerations for the waste and wastewater services sector access to water and sanitation in sub-saharan africa multicriteria optimization of civil engineering systems extended vikor method in comparison with outranking methods multi-criteria decision analysis to mitigate the impact of municipal solid waste management services during floods. resour fuzzy multi-criteria decision-making method for technology selection for emissions reduction from shipping under uncertainties life cycle sustainability prioritization of alternative technologies for food waste to energy: a multi-actor multi-criteria decision-making approach. in: waste-to-energy environmental perspective of covid-19 a scaling method for priorities in hierarchical structures the analytic hierarchy process: planning, priority setting decision support framework for solid waste management based on sustainability criteria: a case study of tailings pond cover systems evaluation of energy potential of municipal solid waste from african urban areas treatment of infectious waste: development and testing of an add-on set for used gravity displacement autoclaves covid-19 wastewater surveillance: an african perspective. sci total environ tang w (2020) the medical waste related to covid-2019 is cleaned up every day-the medical waste treatment market needs to be standardised the amount of plastic waste is surging because of the coronavirus pandemic appraisal-environmental-and-social-review-summary-esrs-lao-pdr-covid-19-response-project-p173817 reverse polymerization of waste polystyrene through microwave assisted pyrolysis 19-infection-prevention-and-control-in-health-care-facilities-2020 the effects of pricing waste generation: a synthetic control approach nonincineration microwave assisted sterilization of medical waste ozonation of drinking water: part ii. disinfection and byproduct formation in presence of bromide, iodide or chlorine technology selection for infectious medical waste treatment using the analytic hierarchy process selecting sustainable energy conversion technologies for agricultural residues: a fuzzy ahp-vikor based prioritization from life cycle perspective. resour disinfection technology of hospital wastes and wastewater: suggestions for disinfection strategy during coronavirus disease 2019 (covid-19) pandemic in china severe air pollution events not avoided by reduced anthropogenic activities during covid-19 outbreak study on the resistance of severe acute respiratorysyndrome-associated coronavirus waste-to-energy, municipal solid waste treatment, and best available technology: comprehensive evaluation by an interval-valued fuzzy multi-criteria decision making method who (2020a) coronavirus disease (covid-19) situation report-114 who (2020b) water, sanitation, hygiene, and waste management for the covid-19 virus progress on drinking water, sanitation and hygiene: 2017 update and sdg baselines incomplete interval type-2 fuzzy preference relations based on a multi-criteria group decision-making model for the evaluation of wastewater treatment technologies can global pharmaceutical supply chains scale up sustainably for the covid-19 crisis? resour how multi-criteria decision analysis (mcda) is aiding life cycle assessment (lca) in results interpretation pilot-scale evaluation of oxidant speciation, 1,4-dioxane degradation and disinfection byproduct formation during uv/ hydrogen peroxide, uv/free chlorine and uv/chloramines advanced oxidation process treatment for potable reuse key: cord-030370-89n13hml authors: brown, colin s.; garde, diana; headrick, emily; fitzgerald, felicity; hall, andy; harrison, hooi-ling; walker, naomi f. title: ebola virus disease in the obstetric population date: 2019-04-11 journal: ebola virus disease doi: 10.1007/978-3-319-94854-6_4 sha: doc_id: 30370 cord_uid: 89n13hml the clinical management of ebola created a significant challenge during the outbreak in west africa, due to the paucity of previous research conducted into the optimum treatment regimen. that left many centres, to some extent, having to ‘work out’ best practice as they went along, and attempting to conduct real time prospective research. médecins sans frontières (msf) [1] were the only organization to have provided relatively in depth practical guidance prior to the outbreak and this manual was the basis of further planning between the who, national ministry of health and sanitation in sierra leone, and other relevant stakeholders. additionally, guidance changed over the epidemic as experience grew. this chapter will describe four key areas in the management of ebola in west africa. firstly, it outlines the most recent who guidance; secondly, it looks back at how ebola was managed in differing low and high resource settings; thirdly it outlines possible and optimal options for managing complications, paying particular attention to some of the controversies faced; fourthly it describes recent and ongoing studies into potential novel therapies that may shape future practice. the clinical management of ebola created a significant challenge during the outbreak in west africa in 2014/15, due to the paucity of previous research conducted into the optimum treatment regimen. that left many centres, to some extent, having to 'work out' best practice as they went along, and attempting to conduct real time prospective research. médecins sans frontières (msf) [1] were the only organization to have provided relatively in depth practical guidance prior to the outbreak and this manual was the basis of further planning between the who, national ministry of health and sanitation in sierra leone, and other relevant stakeholders. additionally, guidance changed over the epidemic as experience grew. this chapter will describe four key areas in the management of ebola in west africa. firstly, it outlines the most recent who guidance; secondly, it looks back at how ebola was managed in differing low and high resource settings; thirdly it outlines possible and optimal options for managing complications, paying particular attention to some of the controversies faced; fourthly it describes recent and ongoing studies into potential novel therapies that may shape future practice. there is, as yet, no specific cure for evd and the mainstay of treatment is supportive care and managing complications, alongside ring-fence vaccination of close contacts. however, due to many tropical illnesses masquerading as ebola, and thus requiring isolation and evd testing, it is worth bearing these diagnoses in mind as empirical treatment for them should be considered in the undifferentiated patient (table 4 .1). this is particularly important in contexts lacking diagnostic resources, and additionally, with the current absence of a rapid diagnostic test that has been widely used or field tested. as an example, o'shea et al. [2] reviewed 51 patients who presented with suspected evd but tested negative, at the uk ministry of defense etu at kerrytown, sierra leone. eleven had malaria (25.5%), 12 shigella spp. or e. coli, 12 a respiratory infection and 11, an undifferentiated febrile illness supporting the need for empirical malaria and antibiotic treatment. the who released their most recent guidance on the management of ebola in february 2016. this is recognised as the gold standard in the low-income setting, however, it is possible that the capabilities of many centres will not meet these targets. the who classifies the patient into three categories of severity; mild, moderate and severe. these are briefly summarised here, but for specific guidance the most up to date version of the who manual should be referred to. the patient will be haemodynamically normal, able to eat and drink, have no complications nor evidence of dehydration. empirical oral broad-spectrum antibiotics, anti-malarials and oral rehydration solution are recommended. treatment for symptoms such as pain, fever and dyspepsia can be provided orally. patients often have vomiting and diarrhoea, are weak and look dehydrated with sunken eyes and a skin pinch slow to return. antibiotics and anti-malarials should ideally be given intravenously, however, if there are contraindications to intravenous line insertion, can be administered intramuscularly. fluids should be given intravenously with additional oral rehydration solution. severe this patient will be severely dehydrated and shocked from either dehydration, sepsis or bleeding, although haemorrhage in the west african outbreak was seen in as little as 5-10% of cases. empirical broad-spectrum antibiotics should be administered intravenously within the first hour of presentation, along with anti-malarials, and effective intravenous fluid resuscitation is essential. tables are provided in the who guidelines to calculate fluid requirements, however, as a rule of thumb an adult who is severely dehydrated or shocked would be treated with a bolus of 1l in the first 30 min. monitoring the patient for acute fluid overload with regular vital sign measurement and fluid balance charts is required. in ill patients, the who advocates testing for malaria and hiv, and measuring potassium, sodium, bicarbonate, blood glucose, creatinine, and lactate. for women of a childbearing age, a pregnancy test should be performed as a priority. if possible, magnesium, haemoglobin or hematocrit, platelet count, inr and aptt should also be tested. in all instances management of symptoms and signs is required-these are described in the management of complications section below. there was great variation in evd management in practice, influenced in part by resource discrepancies, differences in case load and staffing, and disagreements in best practice that developed due to the void of evidence. here we discuss some of the different evd management settings, the type of care that was provided and the impact that these differences may have had on patients' outcomes. table 4 .2 describes the variation in care across the three most affected west african countries at low and high resource centres, and those repatriated or treated within europe and the united states. there was a wide variation in reported mortality rates. however, the heterogeneity of populations studied, demographics, geography, time period (early versus late in the outbreak), survivor bias (only trans cut pacing-1, none cpr uyeki et al. [15] patients who made it as far as hospital settings were included) most likely explain most of this variation, rather than the effect of differing management strategies. therefore, drawing a meaningful conclusion from these mortality trends about the effect of treatment strategies is very difficult, though expanded access to staffing, therapeutic monitoring, blood product support, and critical care is almost certainly contributory to improved survival. there was a range of the care delivered from differing facilities across the world. as aforementioned, the most recent who manual outlines categorizing the patients into mild, moderate and severe, however, this was not available at the start of the outbreak and many centres used their clinical judgment to determine appropriate therapy. additionally, there was variation across laboratory testing, diagnostic abilities, clinical management and discharge criteria. this was due to multiple factors including the human resource and funding allocation, geography of the unit and specific patient factors (such as time of day they presented and agitation levels) which affected the safety of procedures. a key component to the mohs operational plan for country preparedness was the establishment of isolation facilities at every hospital admitting suspected, rather than just confirmed cases. ehu's such as at connaught hospital in freetown provided a point of access to basic health care where unfortunately in practice the highest levels of management generally only reached intravenous lines and iv fluid and antibiotic therapy, due to resource constraints, leaving laboratory testing and closer monitoring to the etcs. isolation in these facilities was expected to be for less than 24 h, while waiting for blood results and transfer, however, patients were often waiting much longer due to delay in diagnostics and limited bed availability and thus received basic care for this period of time. additionally, at the height of the outbreak when the number of suspected cases exceeded bed availability, there were many patients who were forced to wait outside hospitals, etcs or at home, receiving only ors, until a beds became available. it is clear now that this time window was critical for patient resuscitation and the ability to check laboratory results and correct electrolyte imbalances would have been of significant benefit. even once a patient reached an etc, the level of care differed widely. for example kenema etc, in the east of sierra leone, one of the first etcs set up by msf and a 5 h drive away from the ehus in freetown was able to perform basic laboratory tests but often was so overburdened with cases that optimal management was challenging. however, in kerrytown, sierra leone, where the british funded two etc sites, one staffed by save the children and the other through the ministry of defense, patients could receive a higher level of care from onsite laboratory services measuring and correcting electrolyte imbalances and a lower patient to staff ratio allowing more time and a safer environment for monitoring and delivering intravenous fluid resuscitation. unfortunately, epitomizing the global delay in rapid response to the outbreak, kerrytown opened in late november 2015 providing a mere two months of care during the height of the outbreak. in addition, the uk mod etc at kerrytown limited its patients through selecting only health care workers who had become infected, and with a high level of resources were able to do all supportive interventions apart from intubation and ventilation and renal replacement therapy. from the review conducted by ukeyi et al. [15] on the care of 27 patients with ebola in the us and europe, it is clear that in all centres regular monitoring and laboratory testing were available and management was tailored to results. five patients received renal replacement therapy, nine received non-invasive and invasive ventilation and transcutaneous pacing was conducted on one patient. none of the patients in any of the settings received cardiopulmonary resuscitation as it was deemed that once a patient had lost cardiac output, resuscitation would be futile. complications (table 4 .3) at the start of the outbreak there was reluctance to provide intravenous fluid therapy mainly due to the risk to health care workers and challenges surrounding observing patients for bleeding complications, and therefore oral rehydration solutions were relied upon. however, over the course of the outbreak there developed an understanding of the mechanisms of death from ebola through shock, with high lactate (values reported as ranging from 4 to 10 mm/l [20] ), hypoxia from late multi-organ failure, renal failure and electrolye imbalances, in particular hypokalaemia from diarrhoea. this led to a consensus that the basic principles of resuscitative supportive care should be applied with aggressive management of intravascular volume depletion, the correction of electrolytes and prevention of complications associated with shock (hunt et al). in one well-documented case managed in a german intensive care setting, diarrhoeal output exceeding 8 l in 24 h was reported, with intravascular collapse, and was managed aggressively with fluid repletion (7-13 l per day). intravascular leak did occur (pleural effusions, pericardial effusion, ascites) but overall, the treatment was successful [21] . certainly dallatomasinas et al. [5] noted that 95% of deaths occurred within 10 days of admission, when supportive therapy is most likely to impact survival. there still is, however, uncertainty on how much, what type and how fast, fluid should be given promoting suggestions that further research is required in these areas, to optimize simple interventions, including resuscitation with a pre-mixed ebola specific fluid [22] . there were very few complications related to fluid overload in the west african setting, and perner et al. [22] commented on minimal capillary leak and less hypoxaemia/ards than in western sepsis, suggesting that perhaps not enough fluids were given as opposed to too much. unfortunately, the challenges faced from high patient to staff ratios, limited time inside the unit and concerns over bleeding risks through iv cannulation, (although clinically significant gastrointestinal bleeding was reported in less than 5% [9] ) meant that patients were likely not receiving optimal medication. electrolyte disturbances are common in evd, although electrolyte monitoring was not widely available in west african holding units and treatment centres. case [11] . hyperkalaemia was associated with acute kidney injury and mortality. in the same series, hyponatraemia was also prevalent, in over 30% patients. [24] . the uk's ebola referral hospital, the royal free, is now prepared to perform haemodialysis and intubation. there are still no standardised guidelines for use in high income settings, however, the current recommendations are that organ support should be available but that interventions should be limited with a do not resuscitate (dnr) order place. there are no specific treatments with proven efficacy for ebola. the rapid and unpredictable onset of evd outbreaks, together with their often remote location and hazards to health workers prove a major challenge to therapeutic trial implementation. a series of eight symptomatic patients in kikwit, in 1995, given convalescent whole blood, was the largest reported attempt at a an ebola therapeutic intervention study, prior to the west african ebola outbreak [25] . however, this has been a rapidly evolving field. the scale and duration of the west african evd outbreak, involving patients in urban and high resource settings, provided the opportunity and incentive for accelerated research into therapies and clinical trials. efficacy studies to date have consisted of: 1. in vitro and animal studies 2. case reports/small case series of occasional or "compassionate" human use 3. phase ii clinical trials interventions have been either post-exposure prophylaxis (pep) in asymptomatic exposed individuals, and/or treatment in symptomatic cases. candidate drugs that have made it into human clinical use currently fall into four categories: 1. novel specific antiviral agents e.g. small interfering rnas 2. repurposed anti-infective agents, e.g. favipiravir 3. other repurposed agents e.g. amiodarone 4. specific immune therapies, e.g. convalescent plasma, convalescent whole blood, monoclonal antibodies such as zmapp in summary, an impressive effort to generate human trial data on therapeutic inventions, the results of which are summarized in table 4 .4 (taken from brown [34] ) has to date identified no clearly efficacious agent. there was a reluctance to attempt double-blind, placebo-controlled trials, for questionable ethical and logistical reasons (see lanini [35] -this includes a good review of clinical trials and a discussion of the ethical issues). an opportunity to collect data on the efficacy of supportive interventions e.g. threshold for iv fluids, loperamide for diarrhea, was largely missed due to inadequate human resources, and no systematic controlled studies of such were performed to our knowledge. as evd is most likely to affect the low income setting in future, further research into interventions, such as the safety of intravenous lines, usual care versus laboratoryguided care, effectiveness of anti-diarrhoeals, empirical antibiotics and oral potassium supplements would be beneficial. provisional plans for trials should be agreed prior to the next outbreak, to improve rapid implementation when the need arises. this is a moving field and more trial data is likely to be available in the upcoming years. we suggest consulting the following useful resources for up-to-date information: https://ebolaclinicaltrials.tghn.org http://www.ukcds.org.uk/resources/ebola-research-database http://www.who.int/medicines/ebola-treatment/emp_ebola_therapies/en/ https://clinicaltrials.gov small interfering rnas, have shown potential in treatment and prevention, in non-human primates and humans. tkm-100802 (by tekmira), was fast-tracked due to encouraging animal and phase 1 data for a clinical study in sierra leone. however, the trial was terminated early, as demonstration of efficacy was deemed unlikely, and the product discontinued by the manufacturer [29] . other sirna molecules are in development with encouraging data efficacy in non-human primates (limiting symptoms, abrogating mortality-siebola-3, see thi [36] ). brincidovir, a small molecule nucleotide analogue, orally available lipid conjugate of cidofovir (chimerix, inc.), was used occasionally in repatriated patients and in an open-label, single arm phase ii clinical trial (in liberia) which stopped early, favapiravir (toyama, japan), is a broad spectrum anti-viral, approved for treatment of influenza, in japan. a therapeutic effect was reported in ebola virus-infected mice and supported by relatively good outcomes in some cases of occasional human use in repatriated patients (pep and treatment). since then two historically-controlled, open-label, single arm phase ii clinical trials have been undertaken: a multi-centre study in guinea (the jiki trial, see sissoko [27] ) and a single-centre study in freetown, sierra leone [28] . conclusions are limited by the chosen study design and the challenges of trial implementation under the circumstances. the former, larger study did not demonstrate a treatment effect of favapiravir on mortality, whereas the latter reported survival benefit and viral load reduction in treated patients. together these results support prioritization of a randomized placebocontrolled trial of favapiravir, when the opportunity presents. there is some evidence (and a widely held belief) that an ebola-specific antibody response is protective, providing the rationale for attempted treatment and prevention with ebola virus-specific antibodies. specific monoclonal antibodies were available but in short supply and were not easily amenable to rapid scale up. one such antibody cocktail, zmapp, was initially used compassionately in repatriated patients and when increased supply became available, the only randomized control clinical trial was performed, in us, guinea, liberia and sierra leone. the results suggested that there was some evidence of treatment effect, and again this agent will certainly be a likely candidate for prioritization in any future outbreaks [30] . other monoclonal antibodies, (zmab and mil77) were also used in selected cases as pep [37] . due to encouraging results during the earlier kikwit outbreak (albeit in selected patients, see mupapa [25] ) there was considerable enthusiasm for a trial of convalescent plasma and/or whole blood from evd survivors. phase ii trials were commenced in guinea and sierra leone, but neither trial recruited enough patients, and the guinea trial did not ascertain whether any neutralizing antibody was present [31, 32] . additionally, a pilot study of interferon β-1a for evd enrolled only 9 patients in the intervention arm (reference konde et al plos one 2017 interferon β-1a for the treatment of ebola virus disease: a historically controlled, singlearm proof-of-concept trial. it remains to be seen whether there are long term side effects or other consequences of therapeutic manipulation of the natural immune response to evd. it is difficult to assess the effect of different interventions on outcomes, due to the lack of formal studies involving rigorous prospective data collection and appropriate controls. the epidemiology of the disease and the response changed significantly, and so historical comparisons are relatively unhelpful. we have concluded that, in the absence of evidence and the lack of opportunity to generate evidence, it should be the priority to optimize supportive therapy, including intensive care therapy when available. other specific interventions may be used when they are readily accessible, likely to provide benefit based on extrapolation from other diseases, and very unlikely to do harm, in pragmatic studies. other interventions, including novel drug therapies and immunotherapies should be evaluated in randomized, adequately controlled clinical trials, prior to use. diana garde and emily headrick care for the pregnant and postpartum patient in the context of an ebola virus disease (evd) epidemic presents unique and challenging clinical, ethical and logistical considerations. since the first recognized epidemic of evd in zaire in 1976, subsequent outbreaks have occurred in sub-saharan africa (ssa) [38] where access to robust maternal health care services has been limited. an evd epidemic superimposed on an already weakened health system further exacerbates extremely poor outcomes for the obstetric population. in general, 15% of all women are expected to experience obstetric complications [39] . the 2013-2015 west african evd epidemic is the largest to date, with approximately 27,500 cases in three countries-guinea, sierra leone and liberia. prior to the 2013-2015 evd epidemic, these countries demonstrated some of the worst health care indicators due to inadequate infrastructure, human resources and lack of access to basic medical resources [40] [41] [42] [43] . in sierra leone, it is estimated that prior to the evd epidemic, one in every 21 women would die in childbirth-related incidents in her lifetime, [44, p. 34] with the maternal mortality ratio at 1630 per 100,000 live births in 2010-the highest in the world [45] . in addition, all three countries fell within the lowest density of medical personnel per capita, resulting in the smallest and least-skilled healthcare workforce in the world [46] . both the number of in-hospital deliveries and cesarean sections declined as the incidence of evd increased [47] . ultimately, it was predicted at the end of the epidemic in west africa, that the greatest impact of evd would be on maternal health due to the loss of medical staff to ebola (physicians, midwives and nurses). by september 2015, the three countries had lost 513 medical personnel from their already significantly small workforce [48] . from that data, it was estimated that there would be an increase of at least 38% (guinea) and as much as 111% (liberia) in maternal mortality [49] . it has been asserted that women are at an increased risk for evd infection, both physiologically and socioculturally. traditionally, they are the caregivers for ill family and community members, and they responsible for burying the dead, and as such have significant potential for interaction and contact with the virus via body fluids [50] . if a woman is pregnant during an evd epidemic, routine health care encounters for antepartum or intrapartum care places women at high risk for exposure to infected patients also seeking care. there is a higher prevalence of female medical staff, namely nurses and midwives, both inside government facilities and in the community who would then be more likely to have (knowingly and unknowingly) direct contact with positive evd patients in an epidemic. in addition, females in ssa have historically had less authority over their reproductive health and are at higher risk of gender based violence or coercion [51] ; sexual intercourse with an infected or surviving male also places women at increased risk of infection [52] . natural immunosuppression occurring in pregnancy [53] may play a role in a higher acuity of illness observed in presenting obstetric patients with positive evd, contributing to very poor maternal outcomes. considering the expectation of an excess of 1.3 million pregnancies per year [54] across guinea, sierra leone and liberia, a significant number of women who had direct contact with the virus were either pregnant or postpartum. during the 2013-2015 west african evd epidemic, pregnant women presenting for health care services-either for routine peripartum care or in the event of obstetric complications-often met case definition for evd (see table 4 .5).where proactive clinical management would have been appropriate in a non-epidemic table 4 .5 clinical and epidemiological factors in initial evd case definition screening epidemiological factors considerations for screening for general populations early: fever, profound weakness or malaise, headache, myalgia, arthralgia, conjunctivitis, nausea or anorexia, throat pain or difficulty swallowing, abdominal or epigastric pain, diarrhea (bloody or nonbloody) exposure/contact: infected animals, bushmeat or fruit also fed on by bats, healthcare workers/ traditional healers also treating evd, items soiled or touched by positive evd patient, deceased evd bodies sexual intercourse with evd-positive male or evd survivor late: confusion and irritability, hiccups, seizures, chest pain, diarrhea (watery or bloody), vomiting (with or without blood), skin rash, internal or external bleeding, shock, respiratory distress additional considerations for screening obstetric population vaginal bleeding of unknown origin, spontaneous abortion, premature labor and/or rupture of membranes, preterm labor, antepartum and postpartum hemorrhage, intrauterine fetal demise, stillbirth, loss of consciousness exposure to products of conception or deceased fetus of evd positive patient being a pregnant woman with history of contact with confirmed evd patient, recent evd survivor with an intact pregnancy, newborn of an evd positive mother, infant breastfed by a recent evd positive mother who library cataloguing-in-publication data [55] setting, women were often left untreated or were provided minimal intervention by frightened medical staff working in an overwhelmed, under resourced health care system in crisis. even as the emergency response resources became more established, an unfortunate number of women were isolated based on presenting symptoms; the majority of these women ultimately tested negative for evd, yet they languished and died in evd isolation centers due to the limitations of obstetric interventions available in this setting. it is in this context that clinical care for obstetric patients in an evd setting be specifically considered. culture, health infrastructure, access to resources, geography and physiology are all complex forces that must be considered when managing individuals and populations alike. treatment of the pregnant or postpartum patient who meets case definition for evd should be offered without haste and with the highest level of quality interventions possible, while maintaining safety of healthcare workers and the community. this chapter aims to achieve the following objectives, informed by previous research on evd outbreaks and by field experiences from the 2013 to 2015 west african evd epidemic: -examine the unique risks and needs of the obstetric patient in the context of the an evd epidemic -review clinical management of the obstetric patient infected with evd as well as the unconfirmed or suspect obstetric patient being managed in a red zone (rz) setting -provide technical guidance for establishing a safe and effective setting for triage, screening, isolation and treatment of the pregnant patient and mother/baby dyads. one of the key challenges when evaluating a pregnant patient in the context of evd is that symptoms suggestive of evd infection (i.e. abdominal pain, vaginal bleeding) may mask a true obstetric emergency. conversely, the clinical presentation of what initially appears to be an obstetric complication often confounds the accurate identification of potential evd infection. however, evd infection can be the precursor to obstetric complications and a patient presenting for care may in fact require appropriate interventions for both. the basic evaluation of suspected evd patient must consider both clinical presentation (signs and symptoms) and an accurate history to evaluate risk of exposure. due to this unique overlap in symptom presentation for the pregnant patient, additional considerations for the obstetric population must be made in addition to the general screening (table 4 .5). it is of note that not all evd-infected patients presented with or manifested a febrile state during their illness. it is estimated that only between 66% [56] and 89% [57] of patients actually presented with fever greater than 30 c. likewise, patients did not regularly demonstrate coagulopathies or overt hemorrhagic symptoms [57] , as is frequently assumed as a common presentation for a viral hemorrhagic fever, with only one study finding these as a presenting symptom in only 35% of patients [56] . in the 2013-2015 west african evd epidemic, it was frequently observed that evdpositive pregnant patients were presenting with tachypnea and tachycardia, sometimes in the absence of any other symptoms, and could be repeatedly seen "hitting the floor," or favoring a position on the floor rather than in bed. it is postulated that this behavior may be due to agitation, or to alleviate fever by lying on a cool surface, but was routinely observed among this population [55] . although many guidelines for screening include fever and bleeding as dominant symptoms in the algorithm, suspect cases can present without either and evd illness can be missed during initial clinical evaluation. in fact, there are multiple documented cases of pregnant women with known evd exposure and/or infection who do not manifest these common symptoms as expected. in these cases, the potential for an evd positive fetus/neonate is 100%, thus increasing the risk to unsuspecting healthcare workers during routine intrapartum care [58] . the majority of obstetric complications outside of an evd epidemic involve the following: postpartum hemorrhage (pph), antepartum hemorrhage, obstructed labor, postpartum sepsis, complications of abortion, severe preeclampsia or eclampsia, ectopic pregnancy and ruptured uterus [39] . in 2010, approximately 440 women in ssa died each day as a result of obstetric complications, the majority from pph [59] . in addition, the stillbirth rate in ssa is approximately 10 times higher than in developed countries at 29/1000 [60] . as such, an evd-negative obstetric patient can easily meet current who case definition, resulting in the isolation and testing of a high proportion of women who are actually in need of basic obstetric care. given that overlap of presenting symptoms may be commonly seen in both evd and non-evd obstetric cases, astute clinical decision making is required to ensure that obstetric patients are not being overwhelmingly screened and disproportionately isolated for evd when the absolute risk in the community is low. conversely, great effort must be made to ensure that symptomatic obstetric patients are not being treated outside of isolation when the absolute risk of evd is high. in west africa, it was estimated that only 1.5% of the pregnant patients admitted to isolation were evd positive; the 98.5% who were not merely needed intervention for obstetrical complications or normal delivery [61] . epidemic: in an epidemic setting it can be argued that the likelihood of infection transmission or the public health risk from evd is greater than the risk of morbidity or mortality from obstetric complications. you must first consider general screening for the basic symptoms of evd infection, with additional symptoms to be considered for the obstetric patient (table 4 .6): any of these additional obstetrical complications, particularly in combination with symptoms included in routine evd screening, should warrant isolation and polymerase chain reaction (pcr) testing. in addition, screening methods must capture any patient who presents to a healthcare facility who has recovered from evd with an intact pregnancy and is in labor or in the immediate postpartum period. given the absolute likelihood of viremia in products of conception from a patient who recovered from evd while pregnant, delivery must take place in isolation. in a post-epidemic setting the general screening criteria changes, but the obstetric screening considerations remain largely the same. contrary to epidemic settings, the risks of poor obstetrical outcomes are overwhelmingly thought to outweigh the risk of evd infection or transmission of disease; strong clinical judgment should be employed. in the general population, initial screening uses fever as a determinate for case definition. this tool is used for any acute hemorrhagic fever (table 4 .7). the fever must have persisted despite treatment with appropriate medications for the symptoms (i.e. treatment for presumed malaria), prescribed by a qualified medical professional. the patients meeting criteria must be isolated and pcr tested. pregnant women pose a challenging dilemma during the initial months after an epidemic has been declared over. those who conceived during the epidemic could potentially carry an evd infected fetus into a post epidemic setting given the average 40 weeks of gestation. in a post-epidemic setting however, one might argue for a higher level of scrutiny and clinical judgment prior to admission. the singular obstetric complication alone as criteria for admission for evd testing cannot be justified given the numbers of patients that present for care for pph, iufd and sab under non-epidemic conditions. in either an epidemic or post-epidemic setting, obstetric patients with highly concerning presenting symptoms should be admitted for testing in isolation, regardless of whether they fit the established criteria. consultation and collaboration is recommended with other obstetric providers, surveillance officers, and evd medical professionals who should be made aware of these cases. the evaluation and planning for a patient presenting with symptoms of evd is wholly dependent on whether there is a declared epidemic present, or if there are definitive laboratory-confirmed positive cases in the population. where there is any concern for evd or other hemorrhagic fevers, triage and screening should be established at the entry point to every tier of health care facility in the affected area per guidance of government mandates or international recommendation. during a declared epidemic or in cases where there is a high suspicion of infection, an obstetric-specific screening area should be staged at all peripheral health units, community clinics, and hospitals ( i.e. anywhere obstetric patient may present). all pregnant, postpartum and lactating women should have an accurate temperature taken, and basic and obstetric-specific screening questions addressed before entry to the facility. trained maternity nurses should complete the initial screening and evaluation to allow for experienced clinical judgment in this special population. ideally, screening should be operational for as many hours in a day as the facility is active. staff should have clear protocols for documenting patient information to ensure multiple opportunities for confirmation of triage status upon entry into the health care encounter. we recommend standardized forms that can be made in duplicate or triplicate for record keeping. staff should also have ways and means of communication to other colleagues so as not to leave the triage post in the event of a positive screen, an emergency in triage, or need for consultations or technical/ operational support. this station should have access to electricity or reliable lighting, and a source of water for hand washing stations and cleaning. while the triage area should ideally remain a calm environment for efficient yet thorough screening, the possibility of emergent screening is eminent. triage staff should be prepared to prioritize emergent or critical cases while adhering to screening protocol and ultimately prioritizing safety for themselves, colleagues and patients in the immediate surrounding area. patients may present to the facility in critical condition on foot, by private car or by ambulance from another facility. if the patient is unconscious or unable to give a clear history of the present condition, immediate isolation should be the most likely triage decision. if family members or an accompanying health care worker can provide a clear history that places the patient at a lower suspicion for meeting case definition, clinical judgement can be exercised judiciously. a secondary screening area can be used when staff determines that a patient is going to isolation, but more history taking and assessment must be made. an open window with a one-meter buffer can be placed between the staff area and patient secondary screening room. a patient can be removed immediately from the primary screening area and obstetric-specific information can then be gathered from safely behind the window. if the situation warrants, nursing staff can use the space in secondary screening to allow for observation to assist in determining if the patient meets case definition. when a patient enters the secondary screening, it should be considered a red zone until suspect status is otherwise ruled out, or the patient is transferred into the appropriate isolation ward and the secondary screening cleaned. in some situations, emergent or non-emergent, a thorough screening is not feasible prior to delivery and in cases of unknown history care must be administered as if the patient meets case definition. it is beneficial to design and stock the secondary screening facility to immediately turn into a safe red zone in the case that a patient exhibits an emergent illness (i.e. actively bleeding, loss of consciousness) or in cases of imminent delivery. if the patient cannot be transferred into the secondary screening area, triage nurses must have the ability to close the primary screening zone from pedestrian traffic, offering privacy to the patient, room for nurses to work and separation from the general public until the emergency is resolved. clinical and ipc staff must have ready access to all necessary materials in the triage/secondary screening area to safely and effective care for patients requiring immediate attention. we recommend the following: • nurses must have a donning and doffing area and the means to handle blood borne waste or body fluids. • delivery kits, fluid resuscitation supplies, comprehensive ppe and medications for pph should be maintained in the nursing area and access to a hospital bed in secondary screening is optimal for patient care. • there should be the means for transferring a non-ambulatory patient to either isolation or to the hospital after the appropriate screening is completed, or postpartum. this may include a wheelchair or a stretcher. there are known cases of pregnant women presenting to a health care facility in labor who do not meet case definition at admission, but who then develop higher-acuity symptoms during/after labor and are subsequently test positive for evd [62, 63] . it is crucial that health care administrators, staff and supporting partners collaborate to establish and implement protocols to increase the chances of early identification, rapid isolation, adequate treatment and effective ipc in the health care facility. ongoing assessments must take place for the in-patient population throughout their stay in the hospital. there must be q-shift assessments completed to determine if a patient is showing signs of becoming ill or demonstrating the classical obstetric warning signs. medical personnel must also screen neonates post-delivery q-shift, during their entire stay and discharge instructions must offered to caregivers regarding warning signs after discharge. given the potential for a scenario in which a patient has subclinical asymptomatic infection, but passes evd to the fetus, clinicians must be watchful for and proactive in isolating and treating as soon as symptoms arise. during an active evd epidemic, every patient must be treated as potentially infected, no matter where they may suddenly require care. while care should never be withheld from a patient requiring attention, appropriate ipc measures must be available before care is delivered. given the rapid progression of obstetric emergencies and precipitous labors, rapid response protocols should be established. any area can be rapidly turned into an ad-hoc red zone. all first responders should be comfortable with ipc principles and with the concept of red zone/green zone, which can theoretically be established with or without physical barriers. multiple sets of complete ppe should be available at multiple locations in and around health care facilities and in ambulances. care should not be rendered without appropriate ppe and ipc materials. we recommend delivery kits also be easily accessed or assembled for imminent deliveries. support staff should be available to secure a perimeter around an ad-hoc red zone to maintain crowd control, for delivery of additional materials and medications, to establish a line of communication between ad-hoc red zones and surrounding areas, and to initiate the next steps of transfer into a facility once immediate care is rendered. hygienists/ipc support staff should don full ppe and prepare to decontaminate the ad-hoc red zone once the patient is stabilized and transferred. in the event of potential exposure or contamination to others in the community, every attempt to identify potential contacts is crucial for ongoing contact tracing and surveillance. while this can be difficult when an ad hoc red zone must be established in a large facility, in a crowd, or involving methods of transportation, it is crucial in the midst of an outbreak. the 2013-2015 west african ebola epidemic catapulted the research and development of innovative designs for the structures and materials used to combat this virulent disease. while the relative quality of infrastructure used as ebola treatment centers ranged from sticks and tarps to military grade modular isolation units to modified existing structures to multimillion dollar bsl4 biocontainment facilities at prestigious academic medical facilities in the united states, the guiding principles of infection prevention and control remain the same across sites. the guiding principles of an ebola treatment center include: -limited entry and exit points, for both patients, staff, materials and corpses. -double barriers between red zone and external environment. -flow of movement from green zone, to suspect case areas, to confirmed case areas, to morgue, thus reducing the risk of transmitting virus from confirmed evd positive patients to possibly uninfected patients. -multiple points for decontamination between patients -adequate distance/barriers between patients and between "suspect" and "confirmed" wards -clinical principles that prioritize safety of staff, minimizing risk of contact with infected fluids. -around-the-clock bedside care is very difficult to realize given staffing constraints and limits of length of time in ppe. regularly scheduled "rounds" to administer patient care is more feasible [64] . these principles remain the same for an ebola treatment center serving the obstetric population. however, in our experience, we recommend the following considerations to the design and utility when caring for pregnant or laboring women in a red zone. • etcs are generally separated into suspect vs. confirmed wards, and/or "wet" versus "dry," meaning patients with active vomiting or diarrhea should be separated from those who do not to reduce the risk of transmission of highviremia fluids. we recommend that in an obstetric red zone, every attempt should be made to arrange patient beds so that actively laboring or unstable patients occupy an additional "ward," ideally one that can be visually monitored at all times. in the ob red zone setting, intrapartum patients are considered highest-risk in terms of potential of evd transmission (in known positive or unknown pcr status), but regardless of their evd status, will require the most focused care until they are stabilized. • it is likely that staff will not be able to provide 24 hour support in the red zone, but particularly when a patient is actively laboring, every attempt should be made to schedule teams of two to rotate through "red zone rounds" to assist with labor without interruption of bedside care. deliveries can be precipitous, and forgoing constant bedside care in active labor increases the risk that simple complications (i.e. shoulder dystocia, pph) result in death. • in the event that staffing support does not allow for constant bedside care during an active labor, we highly recommend designing the ob red zone with multiple access points to visualize patients from the green zone, either through windows or utilizing camera or video recording equipment if available. being able to visually assess the status of a patient to determine the best time to don ppe for bedside care can be crucial to improving outcomes. • every red zone should be equipped with the appropriate materials to care for the obstetric patient. there are several key items that will be necessary to have in abundance: -menstrual pads and diapers (adult and infant) -infant formula and feeding cups -cotton sheets (or suitable substitute) to cover patient for warmth and privacy, also to anticipate multiple bed-linen changes, and several rags, towels, linens for cleaning large amounts of blood/amniotic fluid. -bassinette which can serve as sleep area for neonate or set up as neonatal resuscitation surface post delivery (must have cleanable surface) -suture materials, needle holder, blunt tipped scissors, speculum or retractor -manual vacuum extractor or forceps -individual blood pressure cuff and thermometer at each bedside, able to be sanitized. -sharps container at each bedside -iv poles, both and short (short for patient who must be placed on the floor for safety) -bell, intercom or other means of calling for assistance. this is particularly helpful for women in early stages of labor who may not require constant monitoring to alert staff that assistance is needed. -wall clock to monitor contraction intervals, as well as time limits for staff in the red zone. -scale for infant weights or for measuring maternal blood loss -ready-made and easily accessible kits with necessary equipment to rapidly manage normal deliveries, postpartum hemorrhage, and eclampsia/seizures the acuity and complexity of patients entering into the red zone is such that a standard medication protocol is warranted. given the lack of immediate diagnostic capacity in many settings, it is recommended that all patients receive empiric treatment of antibiotics, antiprotozoal and antimalarial medications until bacterial, amoebic or malarial infections can be definitively ruled out, or the entire course completed. even in the case of evd, one cannot rule out co-infection with malaria or other common infectious disease. as such, evd suspect and positive patients should be continued on all medications unless testing confirms absence of co-infection. clearly, it would be optimal to have an extensive medication formulary at the disposal of clinical staff in an epidemic, however the historical outbreaks occurring in low resource countries have forced makeshift pharmaceutical supply. the following are suggestions for coverage of the potential needs in isolation or treatment centers. planning should include at minimum medications from the following categories. all medications listed are from the who model list of essential medicines [65] , unless otherwise noted. clinical judgment must be made as to the relative benefits and risks involved and the acuity of the individual patient when choosing medications (table 4 .8). for purposes of simplicity and as a model for the ideal facility, the following will be addressed as if both suspect and confirmed cases are in isolated and treated within the same site. at admission, it must be determined where the patient should be placed within the unit based on their status. "wet" suspect patients, or those with active bleeding, vomiting or diarrhea should be separated from "dry" suspect patients. all suspect women awaiting test results should be physically separated from probable confirmed patients. laboring patients should be given privacy and placed inside intrapartum rooms for delivery, containment of body fluids and a higher level of care and observation. infants should be with their mothers and not in a nursery. bassinet sharing must be avoided. it is critical for every patient who enters into an isolation or treatment center, that complete demographic and symptom history information be completed at the time of admission for care planning, data collection and tracking purposes. first name, last name, and birthdate should be verified and patients given a wrist name band including those three identifiers. patient information can be entered onto a whiteboard or other central documentation record for clinical planning with the following information: bed number, name (first and last), age, birthdate, pregnancy status (pregnant and gestational age, postpartum or lactating), presenting symptoms, and date of onset of symptoms. this record can also include pregnancy outcomes that are updated in real-time (i.e. delivery date and time, gender of neonate, complications, etc.). it is advisable to have an admission book, or means of keeping patient status updated and relevant data logged. the goal is that all staff members can quickly assess and interpret the status of all patients in the etc, as status can change rapidly. another whiteboard or central documentation record can also be used to track lab tests completed (date and time), when the next confirmatory tests are due and results. in order to create a care plan for individual patients, a complete head to toe assessment must be completed to the best ability of the clinician, given time and patient load constraints. information that should be collected can be divided into two categories-objective and subjective. a clinician with experience in obstetric care should collect the objective information. the subjective information is dependent on patient consciousness, the ability of a patient to be an accurate historian or in some cases must be pieced together from family members. the clinician inside the red zone is responsible for collecting gaps in history that has not been addressed before admission (table 4 .9). all information must be documented in patient charts after each assessment in the red zone and on the central documentation record/whiteboard. patients in labor should have a partograph started if over 4-6 cm dilation. to date, pcr has been the testing method of choice for evd infections. approval for use of a rapid screening test (rst) or genexpert for evd would allow access to results that could rule out evd quickly. choice of testing method must be made according to current international standards, national regulations and pharmacy board approvals. all patients entering into the center should be pcr tested on admission. if the symptom onset is less than 72 h, then a second test is needed at or after the 72h mark. suspect and confirmed patients having either a spontaneous abortion or iufd must have products of conception swabbed and tested (fetus, placenta-fetal side, associated tissue or amniotic fluid). an neonate delivered in isolation, or admitted with a suspect patient must also be tested. a patient who is deceased prior to serum collection should have oral swabs collected prior to burial. iufd at full term can also have oral swabs collected. like any other evd suspect or confirmed patient, pregnant, postpartum or lactating women must be assessed and treated based on presenting symptoms. given the nature of fluid loss, offer appropriate replacement fluids (ors or iv/io) based on the severity of dehydration and level of consciousness. consider differential diagnoses and offer antibiotic and antimalarial therapy as discussed above. symptomatic relief must also be included in the care plan for pain, nausea, vomiting, agitation etc. obstetrical care in the context of evd has historically been limited to expected management given extremely high viral load present in amniotic fluid, blood, and placental tissue. as a result the inherent risk to health care workers it was determined to be too high for interventional care. the following were considered high risk in the past and healthcare workers were cautioned against engaging in: cesarean section, artificial rupture of membranes (arom), episiotomy or deinfibulation of scarring related to female genital mutilation, manual vacuum aspiration, manual removal of a retained placenta, suturing, vacuum extraction, and craniotomy in the case of obstructed labor. anticipated delivery in isolation should be managed with caution ensuring the utmost safety of staff while offering the highest level of care to the patient and her fetus. adequate staffing numbers and skill level and immediate availability of needed delivery supplies and medications will eliminate some of the risks associated with deliveries in a limited resource setting. in serious cases where life is threatened or suffering is unmanaged, a higher level of intervention should be considered when qualified staff are present, adequate supplies are on hand and ipc measures can be adhered to. in this way, risks can be mediated and the potential for survival increased. for management of all deliveries in the red zone, protocols must be created with respect to the local ministry of health clinical guidelines and who recommendations for ob care in limited resource settings. additionally, the following considerations must be made to care for patients with evd filovirus or other hemorrhagic fevers: • an adequate intrapartum setting must be prepared before delivery to decrease the risk to staff and patients in the red zone. safety must always be a priority and clinicians must not place themselves at risk in the event a lack of appropriate water, lighting, or ppe should occur. • red zone staff should prepare the bedside when there is an impending delivery and have iv fluids, delivery kit, resuscitation equipment and neonate blanket and bedding at the ready. • staffing should be adequate for deliveries; ideally, there should be a nurse or midwife for the delivery, a nurse for the neonate and one other clinical staff member to monitor ipc and assist where needed. presence of a hygienist is also advisable. roles should be assigned prior to entry into the red zone. • a second team in the green zone must be ready and able to relieve red zone staff when they are exiting and there must be a system of report to update status of the patient before the change of shift. if able, the exiting ipc clinician should update the incoming team before they enter. • there must always be staff in the green zone to hand in needed medications, consult and support red zone clinicians. the green zone staff must help to monitor total time in the red zone and give adequate warning when doffing is required. a "sign in" whiteboard at the entrance to the red zone area allows for accurate monitoring of time in ppe and identification, location and duties of staff in the red zone. -it is optimal for clinical staff to visualize laboring patients from the green zone if there are not enough staff for round the clock care in ppe. a communication tool is also advised so that the patient can call for help if needed, or give status reports to staff. • elbow length gynecologic gloves are preferred for deliveries. in addition to standard ppe, a heavier and thick reusable apron is recommended for the delivering clinician to protect the front of the coverall from body fluid, and to reduce the movement of a lighter, thinner apron. have all needed ppe in the red zone and readily available. -treat all body fluids as potentially infected -place iv before delivery if possible in anticipation of likely ivf resuscitation and to reduce risks to staff associated with an urgent/emergent iv placement. • fresh 0.5% chlorine must be available for immediate decontamination of soiled gloves and gown. hands must be washed or outer gloves changes between procedures. • limit the number of vaginal exams during labor to the initial assessment and intermittent progress checks q 4 h if needed, or the to fewest number possible. • a partograph and/or detailed charting and adequate care planning must be maintained so as to monitor labor progression and anticipate potential interventions for complications. • regularly monitor fetal heart rate or movement. • refrain from using fundal pressure during second stage • a clear plastic sheet should be used as a drape during delivery to separate the clinician from the neonate and placenta. the delivering clinician is avoid sitting or standing at the end of the bed or between the legs of the laboring patient to limit contact with blood or amniotic fluid exposure. • limit the number of sharps in the red zone. use blunt tipped scissors if available for cord cutting. prioritize single use instruments over multi-use. if multi-use instruments are used, a system for cleaning must be employed. a rinse in 0.5% chlorine will ensure adequate decontamination. a fresh water rinse and immediate drying will delay corrosion of metal instruments. if available, an autoclave is optimal. • suturing should be available and employed only when adequate lighting and experienced staffing are available and only in cases where the patient is cooperative (not agitated) and there is a risk of negative outcome without intervention. -blood loss and uterine tone should be monitored closely after every delivery, regardless of gestation. • po medications are preferred, but if needed progress to iv, im or io. all postpartum patients should have 10 iu of slow iv push or im oxytocin (after confirmation of single fetus) to decrease the chances of pph. • if delivery of live baby (or with retained placenta), tie off and cut the cord under plastic. in cases of fetal demise, leave the cord intact, deliver the placenta and place both placenta and fetus together into a body bag, using recommended ipc measures. • a team of postpartum clinicians should be available to monitor vitals and infant transition in the hours after delivery -referral for hiv or tb testing or pmtct services should be offered at discharge if there is a known or suspected secondary infection. given the nature of a viral hemorrhagic fever superimposed on pregnancy, the likelihood of obstetric complications and coagulopathy is very high. safety of staff members must be prioritized, however, we recommend the consideration that more interventions may be appropriately rendered to improve patient outcomes than were recommended in the 2013-2015 evd outbreak. • adequate iv fluids must be on hand for volume resuscitation in high acuity cases. • consider induction of labor in emergent cases only; otherwise, defer induction if a suspect case is highly suspicious of having evd. this is in an effort to reduce unnecessary exposure of staff to body fluids. • deinfibulation of type 3 female genital mutilation (fgm) is discouraged, but may be performed if it is considered a life saving measure. if proceeding with procedure, perform at the patient's side, under plastic sheeting and with adequate anesthesia or pain relief. • perform fundal massage (with support of lower uterine segment) and 40 iu of oxytocin in 1 l of iv fluids (60 gtt per minute) for initial pph management and be prepared to offer higher levels of intervention if bleeding persists, including: external aortic compression and uterine balloon tamponade. -in cases of antepartum or postpartum hemorrhage, consider having an established system for basic blood typing within the unit and a process in place for collection and administration of blood products. no suspect or confirmed patient blood can leave the unit unless a process is in place to transfer to a evd specific lab. staff must be trained to type patient blood and to monitor for transfusion reactions. inadequate training would disqualify a treatment center from transfusing. • patients who are suspect and acutely ill are often placed in situations that are life threatening while they wait for pcr results. consider establishing a site for isolation surgery (dependent on trained personnel, equipment and adherence to ipc measures) so that patient needs can be addressed in emergencies and the option of cesarean section be made available. • consider administration of nifedipine over terbutaline (may have more serious side effects and requires higher level of monitoring) for delay of early onset preterm labor or onset of uterine hyperstimulation. consider antepartum corticosteroid (acs) administration in cases where preterm delivery (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) weeks gestation) appears to be unavoidable. • proactively treat prom or pprom with prophylactic antibiotics. • do not delay treatment of hypertensive emergencies. offer antihypertensives (po or iv) if systolic exceeds 160 and diastolic exceeds 110 and monitor regularly. address severe preeclampsia or eclampsia immediately with im magnesium sulfate and continue until 24 h post-delivery or last seizure (whichever is later). the risk of fluid overload must be balanced with the propensity of evd patients to be fluid depleted and anuric. monitor for symptoms of pulmonary edema and defer diuretics unless severe edema [67] . rapid delivery can resolve symptoms of eclampsia and induction or augmentation should be considered. • in cases of iufd, deliver as with a live infant under plastic sheeting. in many cases, symptoms of infection are present and mimic evd, particularly if labor has been delayed or obstructed. treat with antibiotics for chorioamnionitis and monitor. induce or augment labor to expedite delivery if the risk of puerperal sepsis is high. given the extremely high risk of fetal death with positive evd mothers, pregnancies must be monitored and ultimately delivered in isolation or in an etu. even if the patient is recovered, her products of conception will have high viral load and must be treated as infectious waste. • spontaneous abortions must also be treated with suspicion and poc from suspect or positive patients treated as infectious waste. • vacuum delivery and episiotomy can be considered for obstructed labor when it is believed to be a life saving measure. ipc measures should be maintained at all times. referral for cesarean should be made when available, if failed assisted delivery. • in the case surgical intervention is needed and not available, all attempts at supportive care and potentially palliative care must be administered until lab results are returned. transfer of a non-infected patient with confirmed negative pcr to non-isolation facility can be arranged when lab results are returned. this patient who has been identified as not having filovirus must be advocated for outside of isolation or treatment units and reassurance given to medical staff that they are uninfected and safe to be operated on. • confirmed evd-positive patients with no access to surgery must stay in the treatment center until recovery and two confirmed negative results have been received. the fetus, regardless of gestation, will very likely not survive and will need to be delivered within the isolation facility. -depending on the location of the epidemic, there may be access to high intervention medical care and drug therapy for neonates born from an evd positive mother and though the fatality rate has historically been almost 100%, priority for should be given to advances in clinical care and vaccines. however, in the absence of adequate interventions, the option of therapeutic medical abortion before discharge should be offered if an evd positive patient has an intact pregnancy, the patient lives far away from a evd treatment center and there is a risk that she will not return for delivery (thereby risking further infection in the community). given that the majority of women entering into the etc will be evd negative, the majority of care should mimic the expectations for infant care in a non-epidemic setting. fetal monitoring during labor and delivery should be provided for early detection of complications and immediate care of the infant is essential to ensure optimal outcomes and reduce the risk of infant mortality. • immediately after delivery, vigorously stimulate and dry the neonate • oropharyngeal/nasopharyngeal suction only if secretions suction only if secretions are obstructing the airway; there is no need to suction a vigorous neonate, even if meconium present • perform basic assessment (airway, breathing, circulation) and offer resuscitation if needed • allow delayed cord clamping and cutting in non-emergent settings, tie and cut cord under plastic sheet to minimize contact with blood • clean and dry infant and allow skin to skin/kangaroo care and bonding if the mother is capable • assess initial apgar score and then complete routine vital signs: respiration rate, heart rate (umbilicus or brachial pulse) and temperature q 30 min â 2 after delivery and q shift (at least with every assessment of mother thereafter). complete newborn assessments as thoroughly as possible given time restraints in ppe and check for jaundice, tone, retractions and feeding. stethoscopes cannot be utilized in a red zone. • ensure that the infant bassinet is covered with mosquito netting to decrease the risk of malaria infection • offer vitamin k injection im (1 mg) at birth, ophthalmic tetracycline ointment bilaterally, and chlorhexidine cord care per who recommendations • delay washing for 24 h is possible and dress infant in weather-suitable layers (1-2 layers more than adults) to stabilize body temperature. • offer other treatments based on individual symptoms-consider im antibiotic treatment for bacteremia for 3 days if concerns regarding infection survival of neonates delivered to evd positive patients in or out of an etu is close to 100% mortality rate. at the end of the 2013-2015 west african epidemic, one documented infant of a deceased evd positive patient survived after an intensive antibody, broad spectrum antiviral and antibiotic regimen [68] . one can assume that a neonate is exposed to filovirus in utero, during delivery or during breastfeeding (though it has appeared based on documented cases of live births that neonates seroconvert shortly after delivery with unknown data around the impact of breastfeeding [69] ). recommendations then include keeping the mother and infant as a treatment dyad and allow the mother to care for the infant if able. communication with the mother about her evd status and expected outcome of the neonate should occur as early as possible, with adequate psychosocial support as needed. there are several factors that must be considered when reviewing recommendations for breastfeeding a neonate after delivery in an isolation setting and before maternal pcr results are obtained. in resource low areas, the risk of unsanitary water to make powder formula, the unavailability of ready to use infant formula (ruif), the lack of hygienic means to sterilize bottles, or the prohibitive cost to families to obtain artificial feeding or animal milk products for the neonate for up to 2 years after delivery must be weighed with the immediate risk of continued breastfeeding and exposure to evd. who recommends that in cases where the mother is symptomatic and awaiting results that her breastfeeding be suspended. in these cases a discussion with the patient about the risks, benefits and options must be had. where the mother is unable to give informed consent one way or the other, family members must be brought into the conversation and clinical decisions be made also taking into account the current prevalence of infection in the community. if the patient's result is positive, it can be assumed that the neonate will be positive. there are questions about the initiation of breastfeeding and whether the infant is already infected and would (1) benefit from any maternal antibodies, (2) will get an increased viral load through breastfeeding, or (3) be likely to die regardless of feeding. who recommends suspending breastfeeding and starting ruif (ready to use infant formula) until breast milk samples test negative â2 by pcr testing after which the mother should be encouraged to initiate or resume breastfeeding exclusively for at least 6 months. for patients who suspend breastfeeding until results are returned, interim supply can be maintained with pumping. however, all expressed milk must be treated as contaminated material and discarded per appropriate ipc protocols. if the patient expires during or after delivery but prior to maternal serum testing, oral swabs of the corpse are to collected prior to burial. collect a serum sample for pcr testing of the neonate as soon as possible , however note that the infant must be in the care of isolation facility for 21 days following delivery. a neonate can be infectious but present as being asymptomatic 3 days prior to becoming ill and even then symptoms are atypical or non-specific. accounts of live newborns delivered to positive mothers were all documented to be deceased within the 19th day of life [69] . the 21-day cut off allows for monitoring for the entire contact exposure period. if neonate is positive, treat per pediatric evd protocol or arrange transfer to an appropriate treatment facility via local government regulations. planning the psychosocial impact of the 2013-2015 west africa ebola epidemic is an immeasurable burden for thousands of people. a common sentiment often expressed both from patients admitted to etus and health care workers alike was the dehumanization and trauma of being isolated. the human element of direct patient care was covered by multiple layers of plastic, only eyes visible behind foggy masks. while novel efforts to humanize health care workers rapidly spread by word of mouth among first responders, the experience of being cared for in an isolation center is undeniably traumatic. we strongly advocate for early and robust psychosocial resources as part of a comprehensive evd response. care for the pregnant patient also requires unique psychosocial considerations. while practices may vary across cultures, childbirth is a global phenomenon laden with tradition, ritual and social norms. universally, the laboring woman is at her strongest, and yet at her most vulnerable. effort should be taken to understand and support the unique sociocultural norms surrounding pregnancy and birth in a way that maintains dignity in delivery while adhering to all safety and ipc protocol in the context of evd care. in times of crisis, it may be common for the quality of patient care to suffer as safety or allocation of resources is prioritized. we assert, however, that quality can be incorporated into emergency response service delivery in a way that does not jeopardize safety or waste valuable resources. discharge planning and patient education are components of emergency response service delivery that may be neglected in the height of an emergency, but in the context of evd, these practices can be a crucial component to breaking the chain of transmission as well as improving the overall quality of patient care beyond the etu. the obstetric population has unique needs upon discharge from an evd treatment or isolation facility that should be incorporated into the development and implementation of any program working with women of reproductive age. resources and protocols should be developed early so that staff are prepared to guide patients when they are discharged from the facility. all patients discharged from a red zone should receive clear instructions that they are, by default, considered possible contacts and should monitor their signs and symptoms for 21 days after discharge. examples of unique obstetric patient pathways are summarized in table 4 .10. the development of clinical guidelines and practical health care delivery strategies to address the unique needs of evd survivors presented grand challenges for the community serving guinea, sierra leone and liberia in the recovery phase of the 2013-2015 west african evd epidemic. even verifying the number of evd survivors in west africa is a daunting, occasionally political task, but estimates are currently that out of 27,500 documented cases of evd, there are approximately 13,000 survivors [48] . to date, there are no strong data to approximate how many of those survivors have become pregnant since recovering from evd, but it may be asserted that the pregnant evd survivor represents the nexus of vulnerability. in 2010, before the a patient should be aware that these are similar presentations and may result in repeat isolation evd epidemic, sierra leone had a baseline maternal mortality ratio of 1630 deaths out of every 100,000 deaths [45] ; that number has almost certainly risen, with some estimates by as much as 30% simply by virtue of the decimated health care system and decreased utilization of services [70] . combining these baseline maternal health indicators with the pervasive stigmatization and fear of pregnant evd survivors lends an extraordinarily high risk for neglect and mistreatment of the pregnant evd patient, despite the fact that there have been no data to show active virus in the amniotic fluid or products of conception in the subsequent pregnancies of women who survived evd. it should be noted that the risk for stigma affecting care for evd survivors is not limited to the west african nations where the outbreak occurred; a case study detailing management of a pregnant evd survivor planning to deliver in the united states reports marked discomfort and concern from hospital staff despite no evidence of risk for transmission of virus [71] . furthermore, there are limited data about subsequent pregnancy outcomes for women who became pregnant after surviving evd, with a small cohort study in liberia showing a slightly higher incidence of miscarriage or stillbirth in liberian evd survivors as compared to the overall rate in both the developed and developing world [72] . however, national data for baseline miscarriage/stillbirth rates are not available in liberia, sierra leone or guinea, making data specifically reflecting the evd survivor population murky. recommendations addressing breastfeeding for evd survivors in subsequent pregnancies were initially limited due to lack of data regarding viral persistence in breastmilk. anecdotal evidence suggests that ebola virus may persist for several months in breastmilk of survivors, but breastmilk was not routinely included in the major viral persistence studies conducted in the immediate post-epidemic period. there are no known cases of a breastfeeding infant presenting with evd contracted from a lactating mother. given the overwhelming benefit to breastfeeding, particularly in resource-poor settings, current cdc guidelines support routine breastfeeding of the neonate born to evd survivors, with case-by-case evaluation to neonates born to suspect or confirmed evd patients [73] . there are multiple circumstances complicating the approach to and delivery of quality care for the pregnant evd survivor, largely due to emerging research regarding viral persistence, clinical sequelae in evd survivors and sociologic trends of stigma and access to resources for pregnant evd survivors in their communities. with the data available, we support the following recommendations: • evd survivors presenting with subsequent pregnancy outside of a known evd epidemic should be treated as a non-infected patient. antepartum and intrapartum care should not be delivered with any more ppe than would be used for a non-survivor patient (universal precautions). • status as an evd survivor should be considered as a relevant part the patient's history to inform any abnormal clinical presentation, and treated with astute clinical judgement. other key components of a thorough history taking for a pregnant evd survivor include: survivor status of the father of the baby, any recent illness/complications, social support status. • the heightened vulnerability of a pregnant evd survivor should inform a broader and more comprehensive approach to high-quality antepartum, intrapartum and post-partum care. treatment of the pregnant or postpartum patient who meets case definition for evd is controversial and is often an ethically charged debate due to the overlap in clinical presentation of evd and obstetric complications. the 2013-2015 west africa ebola epidemic illuminated the desperate need for adequate preparedness for infectious disease outbreaks in the obstetric population, with dedicated protocols, adequate training, and unique considerations for these extremely vulnerable patients. the safety and protection of the healthcare worker must be balanced with the commitment to deliver the highest degree of quality clinical intervention possible for the pregnant patient, with the theoretical risk of transmission of evd incorporated into every aspect of clinical and care management. we recommend that the lessons learned from the 2013-2015 west african epidemic, where an unknown, yet unfathomable number of evd negative women and infants lost their lives in ebola treatment centers due to inadequate obstetric care, be considered in the development of all future emergency preparedness and response protocols. with committed partners implementing informed protocols, safe and high-quality maternal/child health can and should be prioritized in the midst of an emergency. nearly 8000 children were confirmed or suspected to be infected with evd during the west african 2013-2015 outbreak, just under 25% of the total [74] . it appears that in this as with previous outbreaks, confirmed diagnoses were fewer in children than adults [75] [76] [77] . the reasons underlying this apparent sparing of children are poorly understood. firstly, it could be that diagnoses are being missed, due to underreporting or poor diagnostic sensitivity in children [77, 78] . it is possible that against a background of high infant mortality as with most countries affected by evd, and fear of seeking health care during an outbreak parents did not bring their unwell children for testing [78] . alternatively, it may be that the diagnostic tests such as both pcr (polymerase chain reaction for evd dna or serological testing) were less sensitive for small children [75] . for example, small children, particularly infants, are often challenging to take blood samples from so it may be that smaller samples were sent, or that less sensitive mouth swabs were used for pcr as an alternative early in the outbreak [79] . however, the difference may be true biological sparing of children, where children are either less exposed or less vulnerable to exposure, or finally that children were more likely to have asymptomatic infection. from glynn et al.'s study of seroprevalence of evd immunoglobulin g (igg, evidence of previous infection) in households of evd survivors, they found no evidence of asymptomatic infections in children under 12 years of age, and a slight excess of symptomatic undiagnosed infections in younger children: i.e. it appears that younger children were slightly more likely not to be taken to hospital despite symptoms [80] . bower et al. investigated age-specific attack rates in the same cohort of evd survivor households in sierra leone, and found that after adjustment for exposure type, children and adolescents aged 5-19 years were less vulnerable to infection than either younger children or adults [81] . therefore it appears from available evidence that in the west african evd outbreak at least, the apparent sparing of children was due to a combination of younger children not being brought for medical attention and true biological sparing in older children and adolescents, the mechanisms for which remain to be explained. routes of exposure are similar to adults with the additional exposure of breast milk for infants, and vertical transmission from mother to neonate (see obstetric chapter). it appears that close contact with a sick mother/primary caregiver is a key risk factor in children over and above other household or community exposures [82] . regarding breast feeding, ebola virus has been detected in breast milk up to 9 months post-infection [83] [84] [85] . indeed, in one case, investigation into the death of a 9-month old infant from evd led to the discovery of ebola virus in the mother's breast milk although the mother had had no preceding symptoms [86] . however, bower et al.'s study of 77 mother-child pairs (children aged < 2 years) found no excess risk from breast feeding over and above contact with a sick mother [82] . contact with an evd-infected mother was by far the greatest risk for the child, risk ratio (rr) compared with infections in the same household excluding mother 7.5, 95% confidence interval (ci) 1.9-28.9, p < 0.001) [82] . interestingly, household crowding and sanitation had little impact on transmission risk, and none of the children included had contact with a dead body, indicating close proximity to the mother/primary caregiver as the most important causal factor in acquisition of evd. the authors therefore agree with current who guidelines that asymptomatic infants and children should be separated from infected mothers to limit onward transmission [82, 87] . regarding children themselves as sources of the virus, the data is conflicting. one modelling study based on data from 200 burials indicated that children might be "super-spreaders" of the virus, but this has not been substantiated by epidemiological data from liberia or sierra leone [88] . in liberia, a contact tracing study showed no difference between children and adults in terms of transmission, and a study of transmission chains indicated that children were less likely to pass on the virus [89, 90] . this was mirrored in sierra leone where children were more likely to be infected in later generations within households, rather than being the primary source within a household [91] . it seems likely therefore that children may be less, rather than more likely to transmit the infection compared to adults. in terms of mortality, infants are the most vulnerable, with case fatality rates (cfrs) varying between 70 and 90% [92] [93] [94] [95] . the prognosis improves with age, such that mid-late teenagers have amongst the lowest case fatality rates. table 4.11 shows cfrs for children in studies from both the west african and prior outbreaks by age. risk factors for mortality are discussed further below. evd is notoriously non-specific in presentation, particularly in children. indeed, even fever which was key to the who clinical case definition in the west african outbreak was absent in 20-25% of cases in three studies [94, [96] [97] [98] . in most studies from the west african and previous outbreaks, features in children have included (in order of frequency) fever (71-99%), fatigue/weakness (64-80%), appetite loss (60-79%), vomiting (28-62%) and diarrhoea (43-60%) [94] [95] [96] [97] 99] . abdominal, muscle, joint and chest pain as well as headaches have been reported in 29-70%, although in younger children pain is difficult to localise and so recording of pain from various body sites has been compounded into the symptom of generalised distress, seen in 64% of a younger cohort [94] . conjunctivitis was recorded in 13-22% and hiccoughs in 7-12% [94, 95, 99, 100] . difficulty breathing and swallowing were seen approximately 13-20% of patients [96, 97, 99] . bleeding from various body sites tended to be rarer in the west african outbreak in children than previous outbreaks (1-10% compared with 20%) [95, 97, 99, 101] , although two cohort studies recorded bleeding in 15% [94, 96] , and a large study of guinean children recorded bleeding in 24% [100] . interestingly, one younger cohort (children aged up to 5 years) recorded cough in up to 54% of children, although this was less frequently seen in other cohorts [94] . blood tests have revealed dramatic leucocytosis, deranged liver and renal function alongside raised inflammatory markers (e.g. c-reactive protein) particularly in children who died [11, 95] . hypoglycaemia, often severe, was common among both children who died (55%) and those who survived (30%) in one cohort [95] . more detailed description of electrolyte and haematological disturbances over the course of disease can be seen in fig. 4.1 [102 the mean duration of incubation of evd is shortest in younger children: estimated to be 1 week in children <1 year compared to 9.8 days in children aged 10-15 years [99] . similarly, duration from symptom onset to death is shortest in younger children-under 6 days in those <1 year, compared with nearly 9 days in those aged 10-15 years [99] . however, care must be taken with these estimates as many children were admitted unaccompanied to treatment facilities, so data regarding [102] symptom duration may be unreliable particularly in younger children. duration between symptom onset to attendance was 4 days [94] . time from presentation at a treatment facility to death can also be short (a median of 3 days in one study) so there is a small window for intervention [95] . features at admission that are consistently associated independently with mortality across several studies include younger age and a high viral load (low cycle threshold (ct) with a viral polymerase chain reaction) as with mixed age cohorts [92, 95-97, 99, 100, 103, 104] . shah et al. record a hazard ratio of 9.2 (95% confidence interval 3.8-22.5) for death with a ct value <25 at admission [94] . however, in interpreting viral ct values, discrepancies between laboratories and assays used should be borne in mind, as there is currently no universally used assay. bleeding at admission and diarrhoea have also been reported to be associated with an increased risk of death [95, 97] . finally, in mixed age cohorts, concomitant infection with malaria has also been significantly associated with mortality [104] . during admission, development of bleeding, shortness of breath and diarrhoea at any point are all independently associated with mortality, as well as tachycardia within the first week of admission [103] . dysphagia was more common those who died and in shah et al.'s younger cohort (children under 5 years), hiccoughs, confusion and bleeding were only present in children who died [94] . for those patients who recovered, there was a period of defervescence over 7-10 days [103] . no children died after day 13 of admission in one study [96] . median duration of admission those who survived varies between 2 and 3 weeks [95, 97] . palich et al. have thoroughly documented the progress of a 6 year old with severe evd managed at a treatment centre with facilities of laboratory monitoring and intravenous fluid and electrolyte replacement. late complications during admission include tonic-clonic seizures, which can be prolonged and severe [95, 102] . in at least two cases, children were left with severe disabilities after a prolonged seizure including blindness and paraplegia [95] (howlett et al. in review). in bower et al.'s study of late deaths within a cohort of 151 evd survivors, one 6 year old and one 17 year old died after discharge from an ebola treatment centre [92] . the 6 year old had symptoms consistent with tuberculosis, although also had a post-mortem mouth swab that was borderline positive for ebola virus [92] . the 17 year old died 5 weeks after discharge with weight loss, night sweats and dysphagia with a post-mortem swab negative for ebola virus [92] . it appears that true recrudescence of virus as has been seen in adult patients may be very rare although possible [18, 105] as can be seen by both the breadth of symptoms exhibited by children with evd, the variations in frequency of symptoms between cohorts even within the west african outbreak, and the non-specificity of these symptoms against a backdrop of high malaria prevalence and other childhood illnesses, the clinical case definition for evd is key. a sensitive and specific clinical case definition for evd in children would allow rapid access to appropriate treatment for children with evd, crucial when early mortality is so high, and also protect children without evd (with a different illness) from exposure to evd while awaiting laboratory test confirmation. to date there has been only one attempt at deriving a paediatric-specific case definition using multicentre data on over 1000 children from the west african outbreak, and this case definition has not yet been validated on wider datasets [106] . it also does not include malaria rapid diagnostic test results which have been shown to be an important discriminator in a mixed age study [107] . however, these limitations aside, the paediatric ebola predictive score (pep) score derived had excellent discrimination (area under receiver operating characteristics curve (auroc) ¼ 0.8). the scores for each clinical feature within the score are shown in table 4 .12, along with the coefficient from the multivariable model used to derive them and associated p values. as the score has not yet been externally validated, it is presented here for information rather than recommendation for use. it is envisaged that the pep score could be used together with evd rapid diagnostic tests, several of which were trialled during the outbreak, to expedite rapid accurate diagnosis of evd [108, 109] in the stressful environment of an evd outbreak, the needs of children can be overlooked. certain considerations should be planned for in advance to ensure initial assessment for emergency clinical signs should take place using the emergency triage and assessment treatment (etat) algorithm for children [110] . assessment for dehydration is key. weighing can be a challenge in facilities with limited resources, so we would suggest using a set of scales within a ziplock plastic bag (or similar) to promote easy cleaning, prevent contamination and prolong the lifespan of the scales when being cleaned with high concentration chlorine. signs of severe or moderate dehydration should be assessed clinically on admission and at least daily (preferably more frequently during the gastrointestinal phases of disease) during admission as per guidelines in the who pocket book of hospital care for children [110] . pain and distress is common in children with evd and should be specifically assessed for and treated both at initial admission and on reassessment. blood should be taken for evd pcr as per who recommendations for exposure prone procedures, ideally by at least two staff [9] . this is particularly important for children who need to be held still during phlebotomy. a malarial rdt should be performed if possible in high prevalence areas, as coinfection is not infrequent [107, 111] . a glucose test should also be performed as a priority if feasible. any initial emergency clinical features noted during etat assessment should be managed as per the relevant treatment algorithm. placing a laminated version of the algorithm on the wall in triage will prove useful for treating clinicians. fever should be managed with antipyretics dosed as per weight/age, and again a laminated list of age/weight appropriate commonly used medications will prove useful for clinical staff both in and outside the "red zone". adequate hydration is a mainstay of therapy for those with both mild and severe evd. all children should be offered oral rehydration solution and be supported to drink it, either by a caregiver or a dedicated staff member as they may be too weak to drink it themselves [112] . if the child is severely dehydrated or malnourished, or unable to tolerate oral fluids, intravenous fluid resuscitation and maintenance will be needed. this should be carried out according to the presence/absence of shock, severe malnutrition and severe anaemia according to the detailed protocols in the who pocket guide to clinical management of patients with viral haemorrhagic fever. these include recommendations for the volume and rate of fluid replacement and are not reproduced here in the interests of space, but we recommend that laminated versions of the fluid replacement protocols be available both in the "red zone" and outside where fluids and medications are prepared and prescribed [79] . in brief, the three signs of shock considered are: cold extremities, weak and fast pulse and a capillary refill time of over 3 s; severe anaemia is diagnosed with a haematocrit <15 or a haemoglobin less than 5 g/dl; and severe acute malnutrition is diagnosed with a muac of <115 mm [79] . although in the context of large gastrointestinal fluid losses, the benefits of intravenous fluid resuscitation is unequivocal and recommended by the who, fluid resuscitation should ideally be carried out with input/output monitoring and using a paediatric giving set [79] . aggressive fluid resuscitation in african children with signs of severe infection (excluding gastrointestinal infections) is not proven to be safe, so every effort should be made to monitor both the volume of fluid given and the clinical impact on the child [113] . ongoing assessment of hydration status is therefore key, and should be planned for in daily staffing allocations. if possible, electrolytes should also be monitored and abnormalities corrected, as large derangements in sodium, potassium and calcium have all been seen in children with evd [95, 102] . hypoglycaemia has been demonstrated to be common in children with evd and should be monitored for as a priority if feasible, but if monitoring is not feasible, glucose should be given intravenously empirically in the case of seizure, coma or lethargy [95] . five percent dextrose should be used in all maintenance fluids [79] . for persistent vomiting, ondansetron (or if unavailable, promethazine, though with care to monitor for extra pyramidal side effects) may limit symptoms and permit oral intake [79] . if a malarial rdt is not available or the result is positive, children should receive a weight-or age-appropriate dose of artesunate combination therapy (act) or intravenous/intramuscular artesunate if there are signs of severe malaria for at least hours followed by a 3 day course of oral act [79] . owing to the overlap of symptoms of evd with sepsis, it is both the who and our recommendation that all children under 5 years of age admitted with suspect evd should be treated with empirical antibiotics on admission. who guidelines are that all under 5 s should receive intravenous or intramuscular broad spectrum antibiotics (e.g. ceftriaxone), although evidence is limited as to whether in relatively well younger children parentral antimicrobials will provide a benefit over enteral [95] . for older children, the decision to start antibiotics lies with the treating clinician as evidence is lacking, but the local prevalence of other common childhood illnesses such as pneumonia and gastroenteritis should be taken into consideration. if used locally, the empirical treatment guidelines laid out in the who pocket book of hospital care for children and the integrated management of neonatal and childhood illness should be utilised [110] . all those receiving antiretroviral or antituberculous therapy should continue it, and restart as soon as possible if treatment is interrupted. pain and distress has been noted frequently in children with evd (see section on clinical features at presentation and disease progression) and should be managed expectantly with age/weight appropriate doses of simple analgesia (paracetamol), or opiates (tramadol followed by morphine if available) if there is ongoing pain or distress in younger children. dysphagia has been noted to be associated with retrosternal chest pain in adults, and should be managed in children with management of bleeding can include transfusion of packed red cells or components (e.g. fresh frozen plasma if required), and particularly for malnourished children supplementation of vitamin k (enterally or parentrally) should be considered. however evidence is limited, particularly as regards antifibrinolytics [79] . other empirical management for shortnesss of breath (oxygen via nasal cannulae if available); seizures (glucose if hypoglycaemic or monitoring unavailable and benzodiazepines); confusion (reassurance, possibly sedation if agitation severe) should be given as needed/available depending on context. in the unfamiliar and frightening environment of the treatment centre, reassurance from staff or other caregivers cannot be overemphasised as a crucial therapeutic intervention. given the gastrointestinal symptoms of evd and the background prevalence of malnutrition in the countries evd has affected, assessment for malnutrition at admission is advisable. who guidelines are that a mid-upper arm circumference (muac) should be checked and oedema assessed for at a minimum if it not feasible to assess anthropometry in more detail [87] . as weight of children is key both in assessing hydration and nutritional status, we would suggest checking weight on admission in addition to muac and presence/absence of oedema. as above, scales could be kept within a ziplock plastic bag to protect from high concentration chlorine and ease of decontamination after use. if severe acute malnutrition is present, fluid resuscitation should be given according to who guidelines using smaller volumes and resomal™ as opposed to standard oral rehydration salts [79] . for asymptomatic breastfed infants whose mother is unwell with evd, current recommendations based on available evidence are to separate the mother and infant and use replacement ready to use infant formula [82, 87] . if the infant also has signs/ symptoms of evd, or a confirmed diagnosis, the current guidelines are that the benefits of continuing breastfeeding are likely to outweigh the risks and so breastfeeding should be continued if the mother is well enough to do so [87] . the challenges of caring for unwell, frightened children within a "red zone" have been well documented during the west african outbreak [94, 96, 112, 114, 115] . this problem is amplified as many children were admitted unaccompanied by a caregiver (up to 40% in one study [95] ), either because family members had already succumbed to disease or because fear of nosocomial infection prevented either relatives from wishing to enter the "red zone" or unit policies forbade the admission of asymptomatic caregivers. apart from the risk of nosocomial infection between suspect evd patients posed by unaccompanied children (who were difficult to keep in their allocated bed space); there were also the hazards of sharps bins and high concentration chlorine within the red zone. units developed different practices to mitigate these risks. one treatment centre had a nursing round specifically dedicated to paediatric care 8 times a day, to manage intravenous infusions and ensure that unaccompanied small children were fed 8 times daily with therapeutic milk [96] . other units started to develop protocols to employ survivors (believed to be at low risk of re-infection with evd) to care for unaccompanied children, but none, to our knowledge put the protocols into practice before the end of the west african outbreak. the possibility of either dedicated paediatric clinicians or a specific paediatric area with risks e.g. sharps and chlorine buckets minimised have both been discussed, and were put into limited practice towards the end of the west african outbreak. one study collected data post-discharge from caregivers admitted to ebola holding units with children who subsequently tested negative for evd [116] . it was possible to contact approximately 25% of caregivers admitted, and of those 125 contacted, none were subsequently readmitted with evd. for the overwhelming majority of those who it was not possible to contact, this was due to a lack of contact details. this indicates that it may not be as dangerous as anticipated to admit asymptomatic caregivers with their children, although this should be done in the context of very clear instructions about hand hygiene and strict isolation between patients. in the event that admitting caregivers with children is not possible or not felt to be safe, alternative mechanisms for managing the pastoral care needs of children have included using stretches of clear plastic sheeting between "red" and "green" zones to enable frequent communication with staff/relatives outside with children inside (heldermann t., personal communication); or frequent ward rounds of dedicated clinical staff as above. post discharge, consideration should be given to the mental health and developmental impact on children who, though they may have made a full physical recovery, have endured the traumatic experience of disease, separation from family, treatment within a facility and often witnessing sickness and death of parents and other close family members [84] . finally, the long term plight of ebola orphans should be considered. the west african outbreak resulted in an estimated 9600 ebola orphans across the three countries which bore the brunt [117] . although these children comprise a small proportion of the total number of orphans across the three countries, (1.4% of 711,600), whether they themselves were infected with evd or not, they are likely to be the victims of considerable stigma and may not be welcomed into fostering families as other orphans might be [117] . filovirus haemorrhagic fever guidelines. brussels: médecins sans frontières diagnosis of febrile illnesses other than ebola virus disease at an ebola treatment unit in sierra leone clinical management of patients with viral haemhorrhagic fever. geneva: who clinical illness and outcomes in patients with ebola in sierra leone ebola outbreak in rural west africa: epidemiology, clinical features and outcomes clinical features of patients isolated for suspected ebola virus disease at connaught hospital clinical presentation of patients with ebola virus disease in conakry, guinea ebola in freetown area, sierra leone-a case study of 581 patients ebola virus disease in west africa-clinical manifestations and management clinical features of patients with ebola virus disease in sierra leone clinical presentation, biochemical, and haematological parameters and their association with outcome in patients with ebola virus disease: an observational cohort study targeted electrolyte replacement in patients with ebola virus disease a health care worker with ebola virus disease and adverse prognostic factors treated in sierra leone ebola virus disease managed with blood product replacement and point of care tests in sierra leone clinical management of ebola virus disease in the united states and europe ebola and provision of critical care loperamide therapy for voluminous diarrhea in ebola virus disease ebola virus disease complicated by late-onset encephalitis and polyarthritis severe ebola virus infection with encephalopathy: evidence for direct virus involvement caring for critically ill patients with ebola virus disease. perspectives from west africa a case of severe ebola virus infection complicated by gram-negative septicemia ebola care and research protocols ebola clinical care authors group. ebola and provision of critical care extracorporeal virus elimination for the treatment of severe ebola virus disease-first experience with lectin affinity plasmapheresis treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee experimental treatment of ebola virus disease with brincidofovir experimental treatment with favipiravir for ebola virus disease (the jiki trial): a historically controlled, single-arm proof-of-concept trial in guinea clinical and virological characteristics of ebola virus disease patients treated with favipiravir (t-705)-sierra leone experimental treatment of ebola virus disease with tkm-130803: a single-arm phase 2 clinical trial prevail ii writing group multi-national prevail ii study team. a randomized, controlled trial of zmapp for ebola virus infection evaluation of convalescent plasma for ebola virus disease in guinea evaluation of convalescent whole blood for treating ebola virus disease in freetown interferon beta-1a for the treatment of ebola virus disease: a historically controlled, single-arm proof-of-concept trial what do we know about controlling ebola virus disease outbreaks? are adaptive randomised trials or non-randomised studies the best way to address the ebola outbreak in west africa? lipid nanoparticle sirna treatment of ebola-virus-makonainfected nonhuman primates post-exposure prophylaxis against ebola virus disease with experimental antiviral agents: a case-series of health-care workers outbreaks chronology: ebola virus disease, ebola hemorrhagic fever. cdc monitoring emergency obstetric care, 164 by category, maternal mortality -data by country. global health observatory data repository: maternal mortality data by country. world health organization world health organization. who, guinea. who. world health organization world health organization. who, liberia. who. world health organization estimates by who, unicef, unfpa, world bank group and the united nations population division world health organization. density of doctors, nurses and midwives in the 49 priority countries the impact of the west africa ebola outbreak on obstetric health care in sierra leone health-care worker mortality and the legacy of the ebola epidemic the pathology of inequality: gender and ebola in west africa the african women's protocol: bringing attention to reproductive rights and the mdgs new evidence of long-lasting persistence of ebola virus genetic material in semen of survivors evolution of the immune system in humans from infancy to old age the state of the world's midwifery 2014 clinical management of patients with viral haemorrhagic fever. a pocket guide for front-line health workers clinical features of and risk factors for fatal ebola virus disease clinical illness and outcomes in patients with ebola in sierra leone delivery of an ebola virus-positive stillborn infant in a rural community health center towards 2015: post-partum haemorrhage in sub-saharan africa still on the rise world health organization. who | stillbirths ebola virus screening during pregnancy in west africa: unintended consequences shedding of ebola virus in an asymptomatic pregnant woman guidance paper ebola treatment centre (etc): pregnant & lactating women. table of content world health organization. who/oms. setting up an ebola treatment center (etc) world health organization. who model list of essential medicines 19th list who model list of essential medicines world health organization. guidelines for the treatment of malaria core topics in obstetric anaesthesia first newborn baby to receive experimental therapies survives ebola virus disease live neonates born to mothers with ebola virus disease: a review of the literature how did ebola impact maternal/child health in liberia and sierra leone? center for strategic and international studies pregnancy, labor, and delivery after ebola virus disease and implications for infection control in obstetric services, united states pregnancy outcomes in liberian women who conceived after recovery from ebola virus disease recommendations for breastfeeding/infant feeding in the context of ebola virus disease heterogeneities in the case fatality ratio in the west african ebola outbreak 2013-2016 age-specific incidence of ebola virus disease transmission of ebola hemorrhagic fever: a study of risk factors in family members ebola hemorrhagic fever: why were children spared? did ebola relatively spare children? clinical management of patients with viral haemorrhagic fever: a pocket guide for front-line health workers. geneva: world health organisation asymptomatic infection and unrecognised ebola virus disease in ebola-affected households in sierra leone: a cross-sectional study using a new non-invasive assay for antibodies to ebola virus exposure-specific and age-specific attack rates for ebola virus disease in ebola-affected households effects of mother's illness and breastfeeding on risk of ebola virus disease in a cohort of very young children ebola virus in breast milk in an ebola viruspositive mother with twin babies sequelae of ebola virus disease: the emergency within the emergency clinical care for survivors of ebola virus disease 22 ebola virus persistence in breast milk after no reported illness: a likely source of virus transmission from mother to child nutritional care of children and adults with ebola virus disease in treatment centres spatial and temporal dynamics of superspreading events in the 2014-2015 west africa ebola epidemic characterizing risk of ebola transmission based on frequency and type of case-contact exposures decreased ebola transmission after rapid response to outbreaks in remote areas variability in intrahousehold transmission of ebola virus, and estimation of the household secondary attack rate deaths, late deaths, and role of infecting dose in ebola virus disease in sierra leone: retrospective cohort study ebola virus disease among male and female persons in west africa inpatient signs and symptoms and factors associated with death in children aged 5 years and younger admitted to two ebola management centres in sierra leone, 2014: a retrospective cohort study ebola virus disease in children clinical features and outcome of ebola virus disease in pediatric patients: a retrospective case series characteristics and outcomes of pediatric patients with ebola virus disease admitted to treatment units in liberia and sierra leone: a retrospective cohort study clinical management of patients in the ebola treatment centres and other care centres in sierra leone: a pocket guide. interim emergency guidelines. sierra leone adaptation ebola virus disease among children in west africa ebola virus disease in children during the 2014-2015 epidemic in guinea: a nationwide cohort study ebola haemorrhagic fever among hospitalised children and adolescents in northern uganda: epidemiologic and clinical observations a 6-year-old child with severe ebola virus disease: laboratory-guided clinical care in an ebola treatment center in guinea the natural history of acute ebola virus disease among patients managed in five ebola treatment units in west africa: a retrospective cohort study predicting ebola severity: a clinical prioritization score for ebola virus disease late ebola virus relapse causing meningoencephalitis: a case report development of a pediatric ebola predictive score predicting ebola infection: a malaria-sensitive triage score for ebola virus disease evaluation of a point-of-care blood test for identification of ebola virus disease at ebola holding units diagnosis of ebola virus disease: past, present, and future pocket book of hospital care for children: guidelines for the management of common childhood illnesses ebola outbreak in conakry, guinea: epidemiological, clinical, and outcome features ebola response in sierra leone: the impact on children mortality after fluid bolus in african children with severe infection moving towards a more aggressive and comprehensive model of care for children with ebola being a pediatrician in an ebola epidemic risk in the "red zone": outcomes for children admitted to ebola holding units in sierra leone without ebola virus disease west african ebola crisis and orphans key: cord-029480-3md13om6 authors: meix-cereceda, pablo title: educational values in human rights treaties: un, european, and african international law date: 2020-07-21 journal: hum rights rev doi: 10.1007/s12142-020-00599-6 sha: doc_id: 29480 cord_uid: 3md13om6 while human rights treaties provide a formidable set of principles on education and values, domestic courts often tend to adjudicate claims in terms of local arguments for or against each particular educational practice. this article explores how international human rights law could inspire the interpretation of domestic law and educational practice, without neglecting specific cultural aspects. firstly, the article reviews the sociological debate on values in education and shows its importance for the legal discussion. secondly, some critical contestations of international cultural human rights are outlined, as well as certain arguments to justify the importance of this model. the study of international law follows: the un, the european court of human rights, and three relevant african charters, as well as every reference to education made by the african commission on human and peoples’ rights and by the african court is examined. lastly, a comparative section reveals a certain cultural commonality inspired by the un treaties, but also reflects some cultural and institutional differences between the european and the african regional systems. educational action in schools. botha (2019, 185) has advocated the importance of international law as a reference and inspiration for interpreting domestic law and practice on educational matters involving human rights. with regard to national legal systems, du plessis (2019, 49 and 2020) has pointed out the exemplarity of the preamble of the south african national education policy act 27 of 1996 when it proclaims that "the education system must be transformed into one which serves the needs and interests and upholds the fundamental rights of all the people of south africa." nevertheless, the concrete result in each learner's education will vary according to a complex interaction of all the aforementioned factors. using a sculptural metaphor, constitutions and international human rights treaties would provide a sediment of values to the educational clay. however, this clay will be modeled, to a greater or a lesser extent, by the hands of all the aforementioned actors. the starting point of this piece is that education always implies the transmission of certain values which, nevertheless, are sometimes in a tension with each other or even contradict each other. therefore, the absorption of values in each learner's case is not easily controlled by one single force but is the result of many influences. the child's own freedom will in all probability play a major role in how these values intertwine and weigh on their moral decisions. the notion of human rights culture has been contested as reflecting a eurocentric philosophy of the individual. indeed, as stoppioni (2020, 2) has recently reminded, "the fundamental problem of the european conception of human rights would be to focus exclusively on the individual and to forget the group". a harsher critique considers that "'human rights' are, as such, a false ideological universality, which masks and legitimizes a concrete politics of western imperialism, military interventions and neocolonialism" (žižek 2005) . these arguments criticize what bonfiglio (2018) has called "human rights colonialism," which the same author has sought to overcome by proposing an intercultural theory of human rights. from an african perspective, justice mokgoro (1998, 8) , of the constitutional court of south africa, has claimed the proximity between human rights and the traditional values of ubuntu, while acknowledging that the communal dimension of ubuntu could enrich the interpretation of western law. in this regard, she mentions in particular the following ideas: -the original conception of law perceived not as a tool for personal defense, but as an opportunity given to all to survive under the protection of the order of the communal entity -communalism which emphasizes group solidarity and interests generally, and all rules which sustain it, as opposed to individual interests, with its likely utility in building a sense of national unity among south africans -the conciliatory character of the adjudication process which aims to restore peace and harmony between members rather than the adversarial approach which emphasizes retribution and seems repressive. the lawsuit is viewed as a quarrel between community members and not as a conflict. the importance of group solidarity requires restoration of peace between them -the importance of public ritual and ceremony in the communication of information within the group -the idea that law, experienced by an individual within the group, is bound to individual duty as opposed to individual rights or entitlement. closely related is the notion of sacrifice for group interests and group solidarity so central to ubuntu(ism) -the importance of sacrifice for every advantage or benefit, which has significant implicants for reciprocity and caring within the communal entity even if they were not written apropos of education, these reflections seem especially relevant to the debate on educational values, in particular with respect to which values the school system ought to promote and the related problem of conflict and restorative justice in schools. the abovementioned critical views, however, cannot undermine the importance of international protection. as will be discussed especially in "african union: commonality and specificities", regional international bodies have both the necessary independence from states and the ability to deliver a relevant view in terms of idiosyncratic proximity. in addition, the lack of adequate solutions in certain domestic cases would prove the necessity of international protection for at least certain categories of subjects. this seems of particular relevance in the case of children, arguably the most vulnerable subjects of human rights. some examples from southern african domestic courts may be helpful to illustrate the latter claim. in some cases, national courts have selected international rulings that may be isolated in a court's body of case law or not applied as a meaningful precedent by the international court itself. for instance, in the arundel school case 2 the constitutional court of zimbabwe ruled against the application of certain pupils who had been obliged to attend school prayer by the new headmistress. the pupils' parents, who were jehova's witnesses, had expressly declared their faith upon the application for admission of their children, and the children had been attending the school before the arrival of the new headmistress. despite the fact that the application had been filed by the parents, the constitutional court expressly declared the right to expel the pupils, which the school had not yet carried out. even more surprising, however, was the fact that the constitutional court based on the european court's reasoning in the case of valsamis of 1996, which has been changed in more recent rulings on similar claims, such as the cases of folgerø (2007), zengin (2007), or yalçın (2014) (see below, "european court of human rights" for details). in other cases, national courts simply refrain from using international law as a source of inspiration, even when it could reinforce the authority of their own rulings. in this regard, the generally protective constitutional court of south africa rarely integrates arguments from international law when ruling on matters concerning children and education. in the case of the juma musjid primary school, 3 the principle of the child's best interest was only discussed in terms of national constitutional law in spite of the importance that international law has in this field (see below "full development of the human personality and best interest of the child"). in this case, an eviction order was sought by the owner of the land where a school was based. the constitutional court mentioned certain international treaties and soft law on the right to education, 4 and even a us supreme court ruling, 5 but no international decisions by african or un bodies were considered. the constitutional court held that the kwazulu-natal high court had failed to take account of the interests of the learners and, instead, had simply "privileged the right to property over the learners' right to a basic education" (para 71). nevertheless, the court considered that the children had been successfully accommodated by other schools and therefore ruled for the owners and granted the order of eviction. another important case from south africa concerned the pregnancy policies adopted by several schools. in the welkom and harmony high schools case, 6 the constitutional court declared these policies unconstitutional for being contrary to a number of rights including non-discrimination, the right to education, and the respect of the child's best interest. 7 as will be discussed below, the african commission on human and peoples' rights has often sought to convince states and schools of the need to ensure school attendance of girls in general, and very especially in the case of pregnancy. nevertheless, and despite a specific allegation by one of the parties, the constitutional court did not mention any rule of international law in its judgment. other situations and cultural practices may deeply affect the full development of children and their right to education, as demonstrated by the achpr's appeals against genital mutilation, child labor, child marriage, and other forms of denial of the right to schooling, as will be discussed in the following sections of this article. while it is difficult to conceive international remedies as a valid instance for reviewing every domestic ruling, they are certainly useful for both denouncing and inspiring educational and legal practice. state authorities-but also individualsshould take notice of the problems detected by international bodies and, with the help of available or new domestic remedies, gradually work toward the improvement of such problems in daily practice. in this regard, the article seeks to provide a comprehensive view of the educational values advanced in three international human rights jurisdictions: the un, the european court of human rights, and the african instruments. part of the elements protected by these jurisdictions is shared by the other systems, but each of them has developed certain specificities. concerning the latter, an effort has been made to examine every allusion to education made by the african court on human and peoples' rights (hereinafter african court) and the african commission on human and peoples' rights (hereinafter achpr). lastly, some comparative considerations on the three systems will be outlined. the universal declaration of human rights (hereinafter udhr) 8 includes a remarkable set of educational values. they have been further developed by some of the major human rights treaties sponsored by the un. four kinds of values seem relevant in this regard. the "full development of the human personality" principle is expressly mentioned in art 26 para 2 of the udhr and has been adopted as the main goal of education by many treaties and even constitutions. in order to grasp the meaning of this principle, it may be worth exploring some interesting variations across different international treaties and declarations. in 1959, 11 years after the udhr, the declaration of the rights of the child 9 would state that education "will promote (the child's) general culture, and enable him, on a basis of equal opportunity, to develop his abilities, his individual judgement" (principle 7). this declaration acknowledged the relationship between a person's awareness of the world that surrounds them ("general culture") and the development of their personality both in terms of their talents ("abilities") and their moral sense ("individual judgment"). thirty years later, the convention on the rights of the child (crc) would rephrase and widen these ideas stating the following: (…) the education of the child shall be directed to: (a) the development of the child's personality, talents and mental and physical abilities to their fullest potential (…). 10 the main purpose thus became the development of talents and abilities, both mental and physical, as part of the more complex concept of the "child's personality." the 1959 declaration, on the other hand, included what is nowadays considered the main goal that should guide not only education but generally any measure concerning children. indeed, after the abovementioned statement, principle 7 went on as follows: the best interests of the child shall be the guiding principle of those responsible for his education and guidance; that responsibility lies in the first place with his parents. full development of the personality and a child's best interest thus appear clearly connected if we consider the udhr and the declaration of the rights of the child jointly. this connection may be useful in order to construe the principle of best interest in some difficult cases. indeed, when the 1959 declaration referred to the best interests 8 adopted 10 december 1948 unga res 217 a(iii) (udhr). 9 declaration of the rights of the child (proclaimed by the general assembly, resolution 1386 (xiv), a/res/ 14/1386, 20 november 1959) (drc). 10 convention on the rights of the child (adopted on 20 november 1989, entered into force 2 september 1990) 1577 unts 3 (crc) art 29 para 1. of the child as "the guiding principle of those responsible for his education", it also specified that this responsibility lies "in the first place with his parents". this phrase certainly means that parents are, prima facie, considered the fittest to decide what that interest may be. nevertheless, there may be some cases where parents may not be able or willing to distinguish between their own interest or belief, on one hand, and the child's best interest on the other: child labor, 11 underage marriage, 12 female genital mutilation (girls' circumcision), 13 or recruitment into militias 14 are only some of the most dramatic examples. in these cases, it may be difficult to justify an intervention of public authorities based solely on the principle of "the child's best interest," as parents might claim that international law enables them "in the first place" to determine that interest in each case. nevertheless, a joint interpretation of this principle and the full development of the child's personality may prove useful in this regard. an individual's personality may keep developing through all stages of life, which means that full development is almost an ideal, or, from a legal perspective, a principle permanently subject to the possibility of progression. this seems even clearer in the case of a child. therefore, if a decision from the parents may pose a clear threat to the child's future development, intervention from public authorities would then be justified insofar as necessary (and not any further) to safeguard the child's future possibilities of personal development. another interesting variation can be found in the international covenant on economic social and cultural rights 15 : (…) education shall be directed to the full development of the human personality and the sense of its dignity (…) human dignity has been considered as a single human right 16 and as a theoretical foundation of the entire legal system (solozábal echavarría 1994 , 2489 and garcía guerrero 2014 and even of the state (häberle 2004, 319) . from a practical perspective, however, if a person's human rights are violated or even threatened, that person may in all probability not lead a decent life. this paragraph from the icescr hence reminds that the right to education is almost literally a "key right," which raises in children and adults awareness of their own dignity, and eventually allows them to fight for their rights by any means acceptable in their society. from an economic 11 the african commission for human and peoples' rights (achpr) sent on 23 march 2018 a letter of urgent appeal to the authorities of uganda "concerning alleged child labour in extractive industries, particularly gold mining." 12 association pour le progrès et la défense des droits des femmes maliennes (apdf) and the institute for human rights and development in africa (ihrda) v republic of mali african court of human and peoples' rights 11 may 2018, especially paras 71-78. in addition, the achpr's 40th activity report (of 2016) acknowledged that "in january 2016, child marriage was declared illegal in zimbabwe by the constitutional court" (para 27.a.ix). 13 the achpr's 34th activity report (2013) found positive developments for human rights in this regard in senegal and the achpr's 36th activity report (of 2014) in four states of the sudan. 14 the achpr's 34th activity report (2013), para 21, denounced the situation in the republic of congo. 15 perspective, education would allow individuals from poorer origins to improve their situation-which is often described with the "social lift" metaphor. 17 these ideas can be summarized in the words of the committee on economic, social and cultural rights (cescr), which conceives education as "an empowerment right". 18 one last important aspect that un-promoted treaties often link to full individual development is the "strengthening of respect for human rights and fundamental freedoms". this phrase is used to describe one of the two basic aims of education according to art 26 para 2 of the udhr (the other one being the full development of the human personality). the phrase between quotes has thus come to represent, in international legal terms, the effort of the society to create a conscience in the child. in this regard, an early but powerful wording can be found in the 1924 geneva declaration of the rights of the child: the child must be brought up in the consciousness that its talents must be devoted to the service of fellow men. it is however the convention on the rights of the child that enshrines the richest catalog of educational and developmental aims. from the perspective of this paper, there are at least two interesting references in its text. firstly, the convention's preamble reminds that the "child should be fully prepared to live an individual life in society". secondly, and elaborating on this idea, art 29 para 1 even claims that "the education of the child shall be directed to: (…) (d) the preparation of the child for responsible life in a free society (…)". two different aspects should be underlined here. on the one hand, this sentence reminds that the child is to be educated in order to become a healthy adult, which prevents exploitation and generally any abusive treatment, considering that a child is not yet an adult. but, on the other hand, the fact that the child will eventually develop into an adult also stresses the need to gradually promote a certain responsibility and maturity. therefore, education should prepare a child to live autonomously in a free society. preparing a child for freedom also means preparing it for responsibility, for making its own rules and taking its own decisions as a useful member of the society, to quote principle 7 of the 1959 un declaration of the rights of the child. this link between individual development and respect for human rights paves the way for the second of the aims mentioned in the general international law. the second aim of education would be clearly political. with different variations, most of the texts include understanding, tolerance, and peace among their political aims for education. a good example can be found in art 26 para 2 of the udhr, according to which, education "shall promote understanding, tolerance and friendship among all nations, racial or religious groups, and shall further the activities of the united nations for the maintenance of peace". the promotion and maintenance of peace has been the most important principle of international law at least since the establishment of the united nations in 1945 (white 1997, 3) . it was enshrined in the charter of the united nations 19 as the maintenance of "international peace and security". the reference to "understanding, tolerance and friendship among all nations, racial or religious groups" would enable the primary goal of maintaining peace. nevertheless, it can be seen as a more ambitious purpose, too. in this respect, it seems that the udhr expects, or even promotes, the emergence of a global society, which in turn would be expected to achieve much more than preventing its peoples from going to war against each other. over 40 years later, the convention on the rights of the child would repeat and widen the ideals of the universal declaration in this respect. the reference to nations and racial and religious groups was then completed with the more flexible "friendship among all peoples, ethnic, national and religious groups and persons of indigenous origin." moreover, the equality of sexes also joined the previous understanding, peace and tolerance. it is probably needless to remind that between the 1948 udhr and the 1989 crc, the international convention on the elimination of all forms of racial discrimination 20 and the convention on the elimination of all forms of discrimination against women 21 had been adopted, putting these problems on the international agenda. international law thus acknowledges the importance that children are made aware of the society's complexity as part of their growth and education process, and of the need to respect and equally treat every individual regardless of their social circumstances. indeed, the relationship between human rights and education can be conceived in dialectic terms. in this regard, promoting human rights in education by celebrating difference 22 could in turn make education a force for social cohesion and understanding. the school system could enhance the child's awareness of different cultural approaches to sensitive or controversial matters, bearing in mind the age and maturity of the learners. for instance, discussion could be encouraged on the role of religion in society, the importance of art, the relationship between freedom of speech and political correctness, environmental conscience, sex or drugs, to name but a few of these controversial matters. a number of authors have underlined these ideas by naming human rights education a human right itself (alfredsson 2001, 273; benedek 2007, 1) . despite the claim for universality of human rights and the importance of promoting mutual understanding, the social circumstances surrounding a child are also important for education, as the third aim from the un treaties conveys. 19 charter of the united nations (adopted 26 june 1945, entered into force 24 october 1945) (un charter) art 1 para 1. 20 international convention on the elimination of all forms of racial discrimination (adopted 7 march 1966, entered into force 4 january 1969) 660 unts 1. 21 convention on the elimination of all forms of discrimination against women (adopted 18 december 1979, entered into force 3 september 1981) 1249 unts 1. 22 mittler (2000, 10) has developed a model of inclusive education. in his words, "inclusion implies a radical reform of the school in terms of curriculum, assessment, pedagogy and grouping of pupils. it is based on a value system that welcomes and celebrates diversity arising from gender, nationality, race, language of origin, social background, level of educational achievement or disability" (emphasis added.) the third kind of values that are found in most declarations and treaties have to do with what sociologists would call the "meso" or middle level of societies. international law thus reveals an awareness of the importance that intermediate bodies (groups) have in a person's life. the family-despite its diminishing size in many societies-is considered the main channel for the flow of values from the groups that make a society to the child. language, cultural values, and moral and religious references, as well as many other traditions, are gradually absorbed by the child in an environment of such high affectivity as the family. all of these ideas could be summarized as the protection of cultural pluralism in education. indeed, even a treaty as sparing in educational matters as the international covenant on civil and political rights includes a reference to religious and moral education, if only to proclaim the right of parents to ensure that such education is delivered "in conformity with their own convictions". 23 nevertheless, it is very difficult to construe this principle as a right to ensure that any education involving moral or even religious contents can only be attempted in conformity with the convictions of families. the reasons would be twofold. on the one hand, the scope of contents potentially affected by moral or religious views is so wide that it would be practically impossible to carry out an institutionalized education. thus, equal access to formal education 24 and other principles that are part of the right to education would be made void. on the other hand, the aforementioned principles of the full development of the human personality and the best interest of the child justify that certain contents of public interest be addressed in a non-indoctrinatory manner, even against parental wishes. sex education has frequently been a controversial matter, 25 but teaching in certain languages in some parts of the world can be just as disputed. 26 however, it is the crc that best captures the essence behind the need to ensure pluralism in education. in its preamble, the states parties acknowledge "taking due account of the importance of the traditions and cultural values of each people for the protection and harmonious development of the child". also, when discussing placement of the child outside its original family, art 20 para 3 mandates that "(…) due regard shall be paid to the desirability of continuity in a child's upbringing and to the child's ethnic, religious, cultural and linguistic background." moreover, art 29 para 3.c places the following values among the aims of education: the development of respect for the child's parents, his or her own cultural identity, language and values, for the national values of the country in which the child is living, the country from which he or she may originate, and for civilizations different from his or her own (emphasis added). however, the importance of all these cultural values does not prevent art 14 para 1 from proclaiming "(…) the right of the child to freedom of thought, conscience and religion". we may inquire about the purpose of stressing cultural specificity in international human rights treaties. this does not seem an appropriate occasion for developing all the complexity of the tension between the universality of human rights and their implementation in different cultural environments. but it may be useful to point out a few considerations on the matter, in particular concerning the topic of this article. cultural traditions may have a great value in collective terms, for example as a way of preserving different valid approaches to the world and to mankind. according to the african court, culture should be construed in its widest sense encompassing the total way of life of a particular group, including the group's languages, symbols such as dressing codes and the manner the group constructs shelters; engages in certain economic activities, produces items for survival; rituals such as the group's particular way of dealing with problems and practicing spiritual ceremonies; identification and veneration of its own heroes or models and shared values of its members which reflect its distinctive character and personality. 27 however, this does not appear as the main argument to ponder here. when discussing child education and values, other considerations seem to take precedence. the rights of parents to instill certain values and, generally, the protection of specific groups and their traditions find a strong justification in the need of a certain psychological stability of the child, a feeling of safety which is crucial for a healthy development of the young who will, over time, become adults. childhood, once again, is not only protected on account of its intrinsic value-which it clearly has, as the right to the child's freedom of conscience underlines-but also because it is the seed of tomorrow's adults that will shape society. a fourth kind of values, still not very common in international law on education, refers to a growing concern in other domains of international law. art 29 para 1 of crc shows this concern in the following terms: [states parties agree that the education of the child shall be directed to (…)] e. the development of respect for the natural environment. given the overwhelming scientific analyses that place human activity at the origin of global warming, and of air, water, and land pollution among many other environmental hazards, the fact that this fundamental value is mentioned as a goal of education in only one of the major conventions on human rights can only be received with surprise. nevertheless, it is a perfectly valid obligation under international law, enshrined in a treaty that is binding for 196 states parties, three more than the member states of the united nations. the usa seems to be the only state not to have ratified or adhered to this convention (of which, however, it is a signatory). the european court of human rights (hereinafter ecthr) has produced approximately 40 judgments and decisions on educational matters. concerning values in education, the court's rulings stem from the second sentence of article 2 of the first additional protocol 28 to the convention. 29 this sentence reads as follows: in the exercise of any functions which it assumes in relation to education and to teaching, the state shall respect the right of parents to ensure such education and teaching in conformity with their own religious and philosophical convictions. in spite of this rather open wording, the ecthr has nevertheless managed to develop a rich case law. it is not possible to examine all of these rulings in detail or to discuss some of the ecthr's excesses. i will therefore limit the analysis to a broad summary of three principles before focusing on how they apply to human rights education, given its relevance for the discussion. firstly, the ecthr has displayed a rather extensive conception of the states' "margin of appreciation". this is especially true in matters of religious symbols, either personal (swiss teachers, 30 turkish students, 31 or french learners 32 ) or institutional. among the latter, the italian "crucifix case" 33 is widely known, but earlier rulings dealt with religious freedom and national celebrations in greek schools. 34 secondly, the ecthr has frequently applied the "best interest of the child" doctrine whenever this interest could be endangered by parental religious or philosophical 28 convictions: the abovementioned cases of kjeldsen 35 and jiménez alonso, 36 or the decision in the konrad v germany case, 37 are some of the most prominent examples on sex education (the first two) and compulsory schooling (the third one). the best interest of the child principle, however, has occasionally been subordinated to a state's margin of appreciation whenever there has been a conflict between the two. for instance, a conflict between the best interest of the child and the french principle of secularism, or the italian defense of the crucifix, or the greek celebration of the national day. most of the cases mentioned in the previous paragraph exemplify this statement. a third group of cases has developed from 2007. these rulings deal with indoctrination in the classroom. they address the instilment of certain religious values through specific ritual practices in compulsory classes for underage learners 38 or a disproportionate attention to certain religious doctrines and a practical neglect of others. 39 the ecthr has upheld the applicants in all such cases. human rights education (hre) was mentioned above as one of the key instruments for education to perform as an empowerment right (see above "promoting understanding, tolerance, and peace"). it can be provided as an autonomous subject or transversally through different subjects, or even as non-formal education. 40 in some cases, however, a vivid controversy has been prompted by the introduction of a specific subject in the school system. this was the case of spain after 2006, when the new subject on "education for citizenship and human rights" was denounced and legally challenged by the political opposition and the catholic church. the subject was deemed constitutional in a number of rulings by both the supreme court 41 and the constitutional court, 42 but the central claim of the opponents to the bill deserves some consideration nevertheless. according to this claim, the curriculum of the subject would have been indoctrinating because it reflected an intent of the state to instill certain moral values. in the opinion of the applicants, such an objective would be the prevalent responsibility of the families and therefore the state should refrain from interfering with it. while the highest domestic courts held that certain values could be legitimately taught by the school system, the case never reached the ecthr and hence the question may remain whether the spanish subject would have been sanctioned by the european court or, on the contrary, whether it would have been declared indoctrinating. however, the ecthr has delivered a number of rulings on the legitimacy of teaching values within the education system. in this respect, the abovementioned cases from norway and turkey are useful precedents. 43 as explained, parts of the curricula in these cases were considered indoctrinating on account of the prevalence granted to certain religious beliefs and rites. interestingly, however, the ecthr made it clear that the teaching of values in school entails a valuable opportunity for young people to appreciate and respect difference and even to experience the commonality of many moral principles that underlie various systems of beliefs. 44 concerning the spanish case, and given that no specific aspects of the curriculum had been challenged before the national courts, it seems arguable that the ecthr would have upheld the decision to introduce the education for citizenship and human rights subject. the misgivings of the applicants concerning the spanish subject stem perhaps from the understanding that state values essentially equal a particular government's values. nonetheless, this is precisely the boundary between acceptable hre and unlawful and indoctrinating ideological reeducation. openness and debate should be part of hre, and teachers should refrain from conveying their own views where the discussion goes beyond the common principles underlying society. this can certainly be a thin line and may not necessarily avoid the risk of complaints by specific parents, but a teacher or educator who adopts a meticulous and open approach would in all probability be in accordance with the view of the ecthr. human rights treaties fostered by the organization of african unity (oau) and its successor the african union (au) include certain interesting obligations from the perspective of values in education. in addition, the african commission on human and peoples' rights (hereinafter the achpr) has provided, and continues to do so, valuable resolutions and other soft law instruments as well as case-based decisions through its communications procedure. the achpr's activity reports are also an invaluable source of information to keep track of both "positive developments" and "areas of concern" for human rights in the continent. 43 see n. 38 and n. 39. 44 case of folgerø, cit. (see n. 38), para 88: "the intention was that the school should not be an arena for preaching or missionary activities but a meeting place for different religious and philosophical convictions where pupils could gain knowledge about their respective thoughts and traditions (…). in the view of the court, these intentions were clearly consonant with the principles of pluralism and objectivity embodied in article 2 of protocol no. 1". and para 89: "(…) from the drafting history it emerges that the idea was that the aim of avoiding sectarianism and fostering intercultural dialogue and understanding could be better achieved with an arrangement, such as here, bringing pupils together within the framework of one joint subject (...) moreover (…) the second sentence of article 2 of protocol no. 1 does not embody any right for parents that their child be kept ignorant about religion and philosophy in their education." a similar reasoning in paras 58 & 59 of the zengin case (see n. 39). the key document of african instruments on human rights is the african charter on human and peoples' rights (hereinafter, african charter). provisions on education, however, appear scarce in the african charter, and there is only one specific reference on art 17 para 1: "every individual shall have the right to education". apparently, and when compared to un provisions on education's goals and values, there is no reference to such values in the basic african instrument. nevertheless, it should be noted that article 17 also refers to free participation in the community's cultural life as a right of every individual (para 2) and to the duty of the state to promote and protect "morals and traditional values recognized by the community" (para 3). this systematic proximity induces the consideration that education, participation in the community's cultural life, and the protection of moral and traditional (non-colonial) values are deeply connected in the african charter's spirit. this interpretation will be further discussed in "african morals, traditional values and cultures, and other specific political principles" below. in contrast to the african charter on human and peoples' rights, the african charter on the rights and welfare of the child 45 displays a rich wording when dealing with educational values. this treaty has been so far ratified by 49 states and signed by another five. only one state (morocco) has done neither. 46 in addition, some provisions of the african charter on democracy, elections, and governance will be discussed. 47 up to the present, 34 states have become parties to it and fifteen others have become signatories. six states, however, have accomplished neither yet (botswana, egypt, eritrea, libya, morocco, and tanzania). 48 let us now focus on the values that should guide education according to these three charters. although this fundamental aim does not differ in substance from the one set in the udhr and the core international human rights instruments, african instruments have included certain elaborations that deserve specific consideration. art 11 para 2 of the african child charter includes a phrase that is almost identical to another in the convention on the rights of the child, which seems a reasonable outcome given the proximity of their respective adoptions (1989 for the crc and 1990 for the acc). the first of the goals mentioned reads as follows: the promotion and development of the child's personality, talents and mental and physical abilities to their fullest potential. 45 african charter on the rights and welfare of the child (adopted 11 july 1990, entered into force 29 november 1999) (african child charter or acc). 46 however, and beyond this initial commonality with un-fostered treaties, the african conception seems particularly concerned about the importance of education-and leisure (udombana 2006 49 )-for individual self-development in general and for promoting awareness of the own rights in particular. not by chance, in 2013, the achpr identified among its "areas of concern" the fact that "many children are not in school despite the provision of free and compulsory education in some state parties, due to socio-cultural and political considerations, among others 50 ; moreover, the achpr has included as a "positive development," among other measures taken to protect the rights of children, "the introduction of school feeding programs in south africa to encourage parents to send their children to school". 51 as well as the fact that certain states "(…) have put in place educational systems that are specifically tailored to suit the mobile lifestyles of their indigenous populations/ communities (namibia) (…)". 52 despite these achievements, certain "socio-cultural" patterns seem to hinder some specific groups, in particular women and girls. many examples can be found in the achpr's activity reports. among these, in 2012, the special rapporteur on the rights of women in africa informed the commission that "the situation of women in the rural areas remains dire, and in the area of education, the problem of girls' access to education despite the progress made by some countries" (sic). 53 more recently, the achpr intensified its appeal in order to denounce the "continuing discriminations and practices against women and girls, including the exclusion of pregnant girls from the education system and refusing them to take public examinations, which violates their right to education and serves to perpetuate other discriminations against them". 54 in this regard, the achpr has also addressed letters of urgent appeal to certain governments that seemed to embrace these convictions, as revealed by "the statement made by the tanzanian authorities on 22 june 2017 to the effect that pregnant girls and teen mothers would no longer be allowed to attend school and continue their education". 55 fully aware of these hindrances, one of the most recent legal instruments in african human rights, the african charter on democracy, elections, and governance, directed its state parties "to provide free and compulsory basic education to all, especially girls, rural inhabitants, minorities, people with disabilities and other marginalized social groups", 56 as well as to ensure "the literacy of citizens above compulsory school age, particularly women, rural inhabitants, minorities, people with disabilities, and other marginalized social groups". 57 49 art 12 of the acc refers to the right "to engage in play and recreational activities appropriate to the age of the child." see also n.j. udombana (2006, 190) . 50 achpr, 34th activity report (2013), para 21.vii. emphasis added. 51 achpr, 36th activity report (2014), p. 11, para v. emphasis added. 52 achpr, 30th activity report (2011), para 251. 53 achpr, combined 32nd and 33rd activity reports (2012), para 251. 54 achpr, 38th activity report (2015), para 41.b.vi. emphasis added. 55 achpr, 43rd activity report (2017). 56 acdeg art 43 para 1. 57 acdeg art 43 para 2. thus, an instrument apparently intended for political rights mandates the establishment of a free and compulsory education system and grants special attention to disadvantaged groups. nevertheless, it must not surprise that a charter on democracy, elections, and governance refers to education in such detail. to quote john dewey's classic book democracy and education, "the realization of a form of social life in which interests are mutually interpenetrating, and where progress, or readjustment, is an important consideration, makes a democratic community more interested than other communities have cause to be in deliberate and systematic education" (dewey 1916, 100-101). more recently, and from the perspective another regional system for the protection of human rights, the inter-american court of human rights considered education as the "epitome of indivisibility and interdependence of all human rights". 58 despite the political importance of education, however, complaints brought before the achpr through the communications procedure have often addressed insufficiency of funding or facilities, as well as a lack of dedicated teaching staff. 59 so far, it may be concluded that african instruments on human rights consider the very accessibility to school education as a key element for the first value that should guide education: the full development of the child's personality. therefore, great interest is placed in countering social and cultural convictions that limit such accessibility. despite its clear connection with the previous aim, the importance that different african instruments attach to this matter recommends devoting a specific subsection to its discussion. after the development of the child's personality, the african child charter considers that education should be directed to: fostering respect for human rights and fundamental freedoms with particular reference to those set out in the provisions of various african instruments on human and peoples' rights and international human rights declarations and conventions 60 the concept of education as an "empowerment right," already mentioned above, has been received in the african instruments on human rights. indeed, this idea was already present in the african charter despite the scarcity of its provisions on education. 61 as such empowerment right, education helps the child-and future adult-to take informed decisions (thus increasing individual freedom) and live in better conditions (improving a certain equality and fostering solidarity). one specific example of better 58 gonzales lluy and others v ecuador inter-american court of human rights series c no 298 (1 september 2015) para 234. see also hevia rivas (2008, 143 and 2010, 29) . 59 socio economic rights and accountability project v. nig., comm. 300/2005, 25th achpr aar annex (may-nov 2008) . the complaint was declared inadmissible due to lack of proof concerning the exhaustion of domestic remedies. 60 acc, art 11 para 2.b. 61 african charter, art 25: "state parties (…) shall have the duty to promote and ensure through teaching, education and publication, the respect of the rights and freedoms contained in the present charter (…)." living conditions would be "understanding of primary health care" (art 11 para 2.e), which is of great importance given the threat of epidemics and viral infections in the continent (yellow fever, malaria, ebola, hiv, or the covid-19 pandemic). the wording of the acc, however, does not aim so much at raising awareness of the own human rights as it does at fostering respect for human rights. both aims (raising awareness and fostering respect) are not completely diverse, but the second might seem more ambitious. moreover, from a political perspective, democracy is enhanced if citizens are more and better educated, and in particular with regard to human rights. education on human rights and democracy (more commonly "human rights education") thus appears as a highly valuable resource. to be sure, the achpr has long been aware of its importance, as it showed with its resolution on human and peoples' rights education 62 of 1993 and other statements. 63 the member states of the african union have, for their part, displayed a clear interest in promoting this kind of education, as the african charter on democracy, elections, and governance demonstrates. according to this instrument, state parties undertake to implement programs and carry out activities designed to promote democratic principles and practices as well as consolidate a culture of democracy and peace. to this end, state parties shall: (…) 4. integrate civic education in their educational curricula and develop appropriate programs and activities. 64 the importance of this obligation is further underlined by another provision. indeed, civic education shall be "systematic and comprehensive" and, more importantly, it shall aim "to encourage full participation of social groups with special needs in democracy and development processes". 65 these provisions of the acdeg allow for three considerations. firstly, education is considered essential for the political participation of social groups (in this case, specifically those with special needs). once again, the idea of education as empowerment, now applied to groups with special needs. thus, the educational aim of fostering respect for human rights appears linked with the value of solidarity within society. secondly, the reference to "(social) groups" reminds of the abovementioned "intermediate bodies." among these, political parties are of particular importance in a parliamentary democracy. political parties, however, would be hardly democratic if their militants were unable to grasp the significance-and debate on the substance-of complex decisions advanced by their leaders. in a presidential democracy, the political 62 resolution on human and peoples' rights education, adopted by the achpr at its 14th session, december 1993. the text is available at https://www.ohchr.org/en/issues/education/training/compilation/pages/5 resolutiononhumanrightseducation (1993) .aspx. 63 in its 35th activity report, of 2013, the achpr highlighted "lesotho's (…) new curriculum for schools which includes components of human rights issues affecting children." 64 acdeg, art 12. emphasis added. 65 acdeg, art 31 para 2. party does not necessarily enjoy the same significance, but the education of those called to vote (especially a pluralistic and non-indoctrinating human rights education) remains of paramount importance. according to experts, "political representation means choosing, electing representatives, selecting the political class" (bonfiglio 2013, 90; similarly, randall 2007, 85-86) , which confirms the importance of education as a crucial element for democracy. however, historically, in african politics, this has not excluded the possibility that the power of granting educational opportunities (e.g., through scholarships) was used, conversely, as an "enormously effective instrument of oneparty consolidation" (coleman and rosberg 1964, 666) or, more brutally, that teachers and health workers "were forced to attend political education meetings" under mugabe's rule in zimbabwe (laakso 2007, 243) . this confirms how strongly education can affect the political system. thirdly, the african conception of civic education appears in close connection with that of "development." if the notion of development may come as a novelty when discussing educational values, other principles deeply connected with it have been present in african human rights instruments since its early days. the ideas of independence, decolonization, and the highly interesting principle of african solidarity have more recently led to an emphasis on economic and social development. this, however, should not be deemed incompatible with the preservation of traditional values, territorial integrity, and african unity, as will be discussed below ("african morals, traditional values and cultures, and other specific political principles"). despite the strong connection of this principle with the full development of the personality, the authors of the african child charter have chosen to present it as a different goal. this principle is closely linked with the "universal" political aims of the core international human rights instruments as well. 66 nevertheless, the preparation for responsible life in freedom does not only apply to children, but also to adults, and, importantly, even to those incarcerated. 67 let us turn to the wording of the acc. its art 11 para 2 provides the following: the education of the child shall be directed to: (…) (d) the preparation of the child for responsible life in a free society, in the spirit of understanding, tolerance, dialogue, mutual respect and friendship among all peoples, ethnic, tribal and religious groups. while the general drafting and many of the elements appear very close, or even identical, to the relevant paragraph of the crc's, 68 some differences should be underlined. the african wording thus features some new elements but, strangely enough, others have been erased. among the latter, "in the spirit of" the crc appeared 66 see above "promoting understanding, tolerance and peace." 67 achpr, 31st activity report (of 2011), para 30. 68 crc, art 29 para 1.d. see above "full development of the human personality and best interest of the child". the references to "peace," "equality of sexes," "friendship among all (…) national groups," and "persons of indigenous origin". the removal of the reference to national groups may be explained by the introduction, instead, of two new and more elaborate paragraphs on the matter (namely, e and f). however, the reasons behind the other three suppressions appear obscure, and the result, difficult to approve. the additions, nevertheless, enrich the text of the provision with some aspects of african legal and political thinking. perhaps the most evident would be the reference to "tribal" groups as a basic organizational form of many african societies, a notion criticized for the "lack of conceptual groundwork and empirical testing" on whether and how ethnicity affects voting preferences (elischer 2013, 25) . moreover, the ideas of "dialog" and "mutual respect" are also an important part of african culture, as the traditional notion of ubuntu expresses well (venter 2004, 149 and le roux 2000, 43) . as vervliet (2009, 64) has written, "in (…) ubuntu, the human person does not stand on his own, but becomes more human in relation with other people". lastly, the reference to the "peoples" is not an innovation of the african instrument, but it certainly acquires a new significance given the importance of this notion in the continent's legal tradition of human rights. it should be noted that the african charter, the achpr, and the african court on human and peoples' rights all bear the reference to the peoples in their very names, thus showing their importance as a subject of rights. human and peoples' rights thus appear closely connected in the african conception. unfortunately, the study of collective rights would require an in-depth approach that clearly exceeds the scope of this article. the un convention on the rights of the child referred to the development of respect for the child's "own cultural identity, language and values, for the national values of the country in which the child is living, the country from which he or she may originate, and for civilizations different from his or her own". receptive to the influence of its immediate un precedent, the african child charter picked up the baton and included four different provisions that expand these ideas. the relevant parts of art 11 para 2 mandate the following: the education of the child shall be directed to: (…) (c) the preservation and strengthening of positive african morals, traditional values and cultures; (…) (e) the preservation of national independence and territorial integrity; (f) the promotion and achievement of african unity and solidarity; article 12, in turn, further elaborates on the right to culture: state parties shall respect and promote the right of the child to fully participate in cultural and artistic life and shall encourage the provision of appropriate and equal opportunities for cultural, artistic, recreational and leisure activity. firstly, from the perspective of moral education, the acc is unorthodox but honest when it accepts that not all african morals, traditional values, and cultures are necessarily "positive" and hence deserve to be promoted. among the positive aspects, the abovementioned notion of ubuntu requires a socially valuable orientation of individual rights. in their definition of the african child, nthontho and ogina (2020) highlight the collective effort behind the education of the young. they illustrate this cultural pattern with the traditional saying that "it takes a village to raise a child". this communalist approach to human rights and education is genuinely african and is not to be found-at least not with the same intensity-in other conceptions. 69 another example of traditional values inspiring human rights can be found in the south african constitutional court's ruling on the khosa case. 70 according to kamga (2018, 641) , when the constitutional court held that "everyone's right to access social security encompasses permanent residents in the country" (including children), it was inspired by the values of ubuntu. on the other hand, a more negative aspect would be the use of corporal punishment. while it was banned by the south african schools act in 1996, this ban was subsequently challenged by 196 independent christian schools. the constitutional court, however, upheld the act in a memorable ruling by justice albi sachs. 71 by limiting its advocacy to the positive inheritance, the acc separates itself from the wording of the older african charter, which did not include any differentiation but rather mandated the protection of any "morals and traditional values recognized by the community." still, despite the more advanced contents of the acc in this regard, basing on the african charter may include some advantages, too. an important one concerns the possibility of using the rather open communications procedure of article 55, thus attracting the attention of the achpr and, perhaps, driving it to use its "good offices" in search of a friendly settlement, or a recommendation, or even to submit the case to the african court. 72 in this regard, certain complaints have been addressed to the achpr despite the strict wording of its article 25 ("every individual shall have the right to education"). among these, for example, a case of 2003 concerning cameroon reflected an attempt by the complainants to demonstrate a linguistic, cultural, and educational undermining of the anglophone parts of the country by the government, thus trying to push the achpr to ponder on the argument of respect for cultural specificity and the protection of linguistic minorities. the achpr, nevertheless, rejected the complaint on article 17 due to lack of sufficient proof 73 (but accepted several others, including certain forms of discrimination such as that of the english language in business transactions 74 ). the notion of culture, in the second place, is not only destined to the stationary status of preservation. the use of the term strengthening in art 11 para 2.c and especially that of participation (linked in article 12 with artistic and with recreational and leisure activities) bring the educational treatment of culture closer to its dynamic essence. education should help the children to understand and preserve their cultural heritage but also to enjoy and develop it. the african charter confirms this interpretation when it includes the right to "freely" "take part in the cultural life of his community" next to the very recognition of the right to education. 75 thirdly, the references to "national independence," "territorial integrity," "african unity," and "african solidarity" are of a more political nature and reflect the historical circumstances to which the rise of the human rights movement in africa was bound. indeed, all of these ideas seem in line with the basic orientations of the organization of african unity since its inception. it should be interesting to remind that in 1963, the african heads of states and governments declared themselves "determined to safeguard and consolidate the hard-won independence as well as the sovereignty and territorial integrity of our states, and to fight against neo-colonialism in all its forms". 76 a purpose later reinforced in the preamble of the very african charter on human and people's rights, where the member states of the oau again showed themselves conscious of their duty to achieve the total liberation of africa, the peoples of which are still struggling for their dignity and genuine independence, and undertaking to eliminate colonialism, neo-colonialism, apartheid, zionism and to dismantle aggressive foreign military bases and all forms of discrimination, particularly those based on race, ethnic group, color, sex, language, religion or political opinions. summarizing, the protection of african cultural values in human rights' instruments reveals the struggle of the continent to become politically independent and economically developed while (dynamically) preserving the best of its traditional identity. one last aim of education according to the african child charter concerns the development of respect for the environment and natural resources. 77 once again, the acc takes up the baton of the un convention on the rights of the child, until then the only un instrument on human rights to have mentioned environmental awareness as an educational value. given the rich resources of the african continent, also from an economic perspective, the african instrument expands the scope in order to instill respect for natural resources as well. indeed, natural resources have historically been over-exploited, either by colonial powers 78 or under self-rule. the latter, nevertheless, were often responsible for some of the first conservation efforts as well (van eeden 2014, 640). 75 african charter art 17 para 2. 76 organization of african unity charter, 25 may 1963, (oau charter) preamble. 77 acc art 11 para 2.g. 78 c.w. de kiewiet (1941, 188) , in his history of south africa, wrote that "in all the great colonial regions of the world the history of the ruthless exploitation of natural resources is a full one." this provision, therefore, points to the aim of delivering the continent from economic exploitation. in doing so, it is perfectly coherent with the african charter's proud declarations that "all peoples shall freely dispose of their wealth and natural resources. this right shall be exercised in the exclusive interest of the people. in no case shall a people be deprived of it." 79 some of the most infamous cases of recent history are closely linked with this defense of the environment against large transnational companies backed by some governments. the most notorious is probably the niger delta dispute, 80 but the aforementioned achpr/kenya case concerning the rights of the ogiek community of the mau forest is a more recent example. both un and regional systems reflect certain humanistic values that, despite different wordings and circumstances, may be considered essentially common to all three conceptions. perhaps the best example is the principle of the full development of the personality (often linked with the child's best interest doctrine), but clearly not the only one. the promotion of understanding, tolerance, and of specific cultural values is also present in every human rights system. on the darker side, the risk of cultural indoctrination has made its appearance in both the european and the african contexts. despite these common aspects, regional systems also reveal specific conceptions that are due to each continent's own history and current challenges. in the african system, the different charters reflect a rather open understanding of cultural values and traditions by granting every individual the right to "freely participate" in cultural life. the historical struggle for political independence, economic development, and environmental protection has also left its mark on educational values. concerning the enforcement of the charters, however, the communications procedure before the achpr may only lead to the adoption of decisions that are technically not binding for the states parties involved. this lack of binding force when discussing individual communications may explain why certain statements throughout the achpr's periodical activity reports have adopted a strongly critical style, for example with regard to access to education for pregnant girls. a more frequent intervention of the african court might be desirable, but both the institutional framework and the system's budget entail limitations. the ecthr, on the other hand, may seem less demanding than the achpr in its requirements to states parties. examples of this are the frequent recourse to the margin of appreciation doctrine and the ecthr's acquiescence to france's broad interpretation of secularism or italy's defense of the crucifix as a symbol of universal values. however, this acquiescence in matters that are highly controversial within certain societies could paradoxically reflect a stronger institutional system and hence a smaller need to react to all challenges. this institutional strength (at least when compared to other human rights systems) arises from the solid position of the ecthr as the only international protector of the echr and from the mandatory nature of its rulings for the 79 african charter art 21 para 1. 80 soc. and econ. rights action ctr. v. nig, comm. 155/96, 15th achpr aar annex v (2000 -2001 . states parties involved. nevertheless, and most importantly, the ecthr receives and adjudicates a far larger number of cases than any other human rights system. this grants the european court the opportunity to rule on each case considering the social, political, and legal specificity of the state concerned, rather than motivated by the need to develop a notion that is valid for the whole continent. it therefore seems to have more occasions to develop a nuanced body of principles. such a system, however, requires greater funding by states parties. the importance of international human rights law: contestations and reasons" and stoppioni protocol to the african charter on human and peoples' rights on the establishment of an african court on human and peoples' rights comm. 266/2003, 26th achpr aar annex the right to human rights education human rights education intercultural constitutionalism: from human rights colonialism to a new constitutional theory of fundamental rights in search of ways of curtailing aggression and violence in schools: a human rights-based approach la reproduction. éléments pour une théorie du système d'enseignement. minuit, paris. english version (1990) as reproduction in education reprint 2015) democracy and education. forgotten books, london du plessis a (2019) school governance and management decentralisation and school autonomy in the south african education system. dissertation the emergence of decentralised-centralism in the south african education governance system political parties in africa. ethnicity and party formation tirant lo blanch, valencia glenn cl (2017) what is at stake in debates over education and religion verfassungsprinzipien als erziehungsziele die menschenwürde als grundlage der staatlichen gemeinschaft pobreza y derecho a la educación el derecho a la educación y la educación en derechos humanos en el contexto internacional education, social mobility and social reproduction in sweden: patterns and changes cultural values as a source of law: emerging trends of ubuntu jurisprudence in south africa insights into electoral violence in africa the concept of 'ubuntu': africa's most important contribution to multicultural education new york martín criado e (2010) la escuela sin funciones. bellaterra conceptualisation of an "african child" in the context of basic education in south africa political parties in africa and the representation of social groups dignidad de la persona stoppioni e (2020) some reflections on the human rights discourse in the eu-japan strategic partnership agreement. e-conference on eu-japan relations social rights are human rights: actualizing the rights to work and social security in africa history of south africa. from the distant past to the present day the notion of ubuntu and communalism in african educational discourse the human person. african ubuntu and the dialogue of civilisations keeping the peace. the united nations and the maintenance of international peace and security acknowledgments the author wishes to kindly thank prof johan beckmann, prof everard weber and dr. andré du plessis, from the university of pretoria's department of education management & policy studies. this research was made possible by the university of pretoria's visiting professors program. conflict of interest the author declares that he has no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-253466-7gpije5d authors: netherton, christopher; moffat, katy; brooks, elizabeth; wileman, thomas title: a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication date: 2007-08-31 journal: adv virus res doi: 10.1016/s0065-3527(07)70004-0 sha: doc_id: 253466 cord_uid: 7gpije5d virus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to the “cytopathic effect” that has been the hallmark of virus infection in tissue culture for many years. recent studies are beginning to redefine these signs of viral infection in terms of specific effects of viruses on cellular processes. in this chapter, these concepts have been illustrated by describing the replication sites produced by many different viruses. in many cases, the cellular rearrangements caused during virus infection lead to the construction of sophisticated platforms in the cell that concentrate replicase proteins, virus genomes, and host proteins required for replication, and thereby increase the efficiency of replication. interestingly, these same structures, called virus factories, virus inclusions, or virosomes, can recruit host components that are associated with cellular defences against infection and cell stress. it is possible that cellular defence pathways can be subverted by viruses to generate sites of replication. the recruitment of cellular membranes and cytoskeleton to generate virus replication sites can also benefit viruses in other ways. disruption of cellular membranes can, for example, slow the transport of immunomodulatory proteins to the surface of infected cells and protect against innate and acquired immune responses, and rearrangements to cytoskeleton can facilitate virus release. viruses are obligate intracellular parasites. unlike their hosts, they cannot replicate by growth or division but use their genomes to redirect host cell processes to produce all the components needed to make new viruses. virus replication and assembly are often confined within specific intracellular compartments called virus factories, viroplasm, or viral inclusions. these are thought to provide a physical platform to concentrate new genomes and proteins involved in replication and assembly, and this is likely to increase the efficiency of virus production. the formation of specialized sites of replication can involve extensive reorganization of cellular cytoskeleton and membrane compartments. this can lead to cell rounding and swelling and a ''cytopathic effect'' that has been documented for many years (reissig et al., 1956; robbins et al., 1950) . recent advances in microscopy, such as live cell imaging and tomography, combined with the power of reverse genetics, are now allowing the cytopathic effect to be redefined in terms of specific effects of viral proteins on specific cellular processes rather than an overwhelming assault on the cell in preparation for cell lysis. there is considerable interest in understanding how virus infection leads to the large changes in cellular organization required to produce complex replication sites. in the simplest model, virus replication sites would form passively through self-association of viral components and exclusion of host organelles. viruses, however, require a considerable number of host proteins to facilitate replication, and there is increasing evidence that these are specifically transported to sites of replication. host proteins may move to replication sites because they are actively recruited by binding to specific viral proteins. alternatively, viruses may transport viral and host material to replication sites by subverting host defences against infection [reviewed by kirkegaard et al. (2004) and wileman (2006) ]. the large scale changes in cellular membrane and cytoskeletal organization, which occur during the formation of replication sites, can offer further benefit to viruses. rearrangement of the cytoskeleton can, for example, facilitate virus release, and the block in the secretory pathway seen during infection with positive-stranded rna viruses can reduce release of inflammatory mediators and protect against innate and acquired immune responses. this is a broad subject of considerable interest to virologists and cell biologists, and we have benefited from excellent reviews that have been published (mackenzie, 2005; novoa et al., 2005) . in writing this chapter, we have concentrated on describing sites of virus replication in the context of the cell in which its replication takes place. we have illustrated these concepts with reference to replication sites produced by many different viruses and, where possible, described how virus replication impacts on the functioning of the host cell. virus replication sites have been studied for many years and have evolved their own terminology. early studies of poxvirus replication (dales and siminovitch, 1961; morgan et al., 1954) describe electron-dense aggregates and amorphous material induced early during infection called viroplasm. viroplasm has also been used to describe similar structures induced during infection with poliovirus (dales et al., 1965a) . viroplasm is often concentrated within perinuclear areas that exclude host organelles. viroplasm is thought to indicate sites of virus replication, and concentrations of viroplasm have been called virosomes, or virus factories, to reflect an organelle involved in virus production. virus infection also produces inclusion bodies. as a working definition, these can be considered to form later during infection. they can form virus factories once virus production has peaked, and/or at other sites in the cell they probably arise from an accumulation of viral proteins that do not become incorporated into viruses. the positive-stranded rna viruses encode nonstructural proteins (nsp) that cause proliferation and modification of membranes of the host secretory pathway. the membranes are thought to provide a physical framework or ''replication complex'' that concentrates the cellular and viral components required for virus replication (bienz et al., 1987; egger et al., 2002; froshauer et al., 1988; gazina et al., 2002; magliano et al., 1998; schlegel et al., 1996; van der meer et al., 1998) . assembly of the replicase on membranes, rather than the cytosol, may also help viruses evade host defence pathways that monitor cells for double-stranded rna (dsrna) intermediates indicative of virus replication. the replicase complexes of all the positive-stranded rna viruses contain an rna-dependent rna polymerase (rdrp), a protein with ntpase and helicase activity, and in many cases a methyl transferase to cap viral rna. these proteins are generated from the viral polyproteins by viral proteases, and are then targeted to membranes in ways that differ depending on virus family (fig. 1 ). the replicase proteins of positive-stranded rna viruses are directed to membranes by nsp with membrane-targeting information. (a) picornavirus. the replication complex contains 3d, the rdrp (red), and 2c which has ntpase and helicase motifs (purple). the 3d polymerases do not have membrane-targeting information but are synthesized as a 3abcd precursor. 3abcd is processed to 3ab by the 3c protease (red triangle) and a hydrophobic domain in 3a targets 3ab to the cytoplasmic face of er membranes. 3ab binds directly to 3d and this targets the polymerase to the replication complex. the replication complex also requires 2bc and 2c proteins that are targeted to membranes via their own hydrophobic domains (black lines). (b) flavivirues. the replication complex is encoded at the c-terminus of a polyprotein that is processed by the ns2 protease (red triangle). ns5b is the rna-dependent polymerase (red), and ns3 acts as helicase (purple). ns4b is a polytopic membrane protein inserted into the er cotranslationally. ns4a, 5a, and 5b have hydrophobic domains (gray lines) that allow posttranslational insertion into the cytoplasmic face of the er membrane. ns3 is recruited into the complex by associating with ns4a. (c) alphavirus. the nsp1234 polyprotein is processed by a protease activity in the c-terminus of p2 (red triangle). the polyprotein is anchored to the cytoplasmic face of endosome and lysosome membranes membranes that lie between the er and the golgi apparatus called the er-golgi intermediate compartment (ergic), or tubulovesicular structures, and specific fusion with ergic membranes is determined by a complex of proteins called transport protein particle 1 (trapp1). trapp1 proteins tether the vesicles on ergic and golgi membranes, allowing interactions between vesicle and target snare (soluble n-ethylmaleimidesensitive factor attachment protein receptor) proteins to facilitate membrane fusion. the snare interactions are controlled by vesicle-specific small gtpases called rab proteins (fig. 2) . further sorting events in the ergic and early golgi involve a second complex of coat proteins called copi. the copi complex contains seven proteins (a, b, b 0 , g, d, e, and z cop proteins), which generate vesicles that take proteins from the ergic and golgi apparatus back to the er through a retrieval pathway (fig. 2) . the copi proteins are recruited from the cytosol by the arf1-gtpase. activation of arf1 requires binding to gtp and is facilitated by gtp exchange protein, arf-gef. arf1-gtp inititates coat assembly while hydrolysis of gtp by arf1 leads to coat disassembly. this disassembly is stimulated by an arf1-gtp-activating protein (arf-gap) that promotes gtp hydrolysis by arf1. a possible role for arf1 in the generation of vesicles during picornavirus replication has been the focus of much work following the observation that poliovirus replication is blocked by brefeldin-a (bfa), a drug that inhibits the recruitment of arf1 onto membranes (maynell et al., 1992) . membrane vesicles are also produced in cells in response to starvation. this pathway, known as autophagy, is used as a part of a quality control system that removes long-lived proteins and damaged organelles from the cytoplasm and has been shown to provide a defence against intracellular pathogens (deretic, 2005; kirkegaard et al., 2004; levine and klionsky, 2004; shintani and klionsky, 2004) . the origins of the membranes formed during autophagy are unclear but may be derived from the er (reggiori and klionsky, 2005) . autophagy is suppressed by the target of rapamycin (tor) kinase and is activated by conditions that lead to inactivation of tor. this leads to the production of membrane crescents in the cytoplasm, called isolation membranes, which mature into doublemembraned vesicles of 500-to 1000-nm diameter called autophagosomes. this maturation engulfs small quantities of cytoplasm, and any organelles or pathogens present at sites of autophagy become trapped within autophagosomes. the autophagosomes ultimately fuse with lysosomes resulting in degradation of their content. autophagosomes are of interest because infection of cells with picornaviruses and coronaviruses (covs) can generate double-membraned vesicles that may be related to autophagosomes. in addition to supplying membrane and proteins to the secretory pathway, the er acts as a major site of lipid synthesis. as a consequence, the er contains a large quantity of membrane, and this is organized into a complex reticulum made from tubular and lamella structures (borgese et al., 2006) . the smooth er increases in response to a buildup of er membrane proteins and can be organized into lamellae or concentric whorls called organized smooth er (oser). structures similar to oser are also seen during virus replication. b. picornavirus replication induces numerous membrane vesicles picornaviruses are nonenveloped positive-stranded rna viruses. the genome encodes a large polyprotein that is processed to generate capsid proteins from the p1 region and nonstructural replicase proteins from the p2 and p3 regions. picornavirus 3d contains the rdrp, while 2c has ntpase and helicase motifs. the 3d polymerase does not have membrane-targeting information but is synthesized as a 3abcd precursor. 3abcd is processed to 3ab by the 3c protease, and a hydrophobic domain in 3a targets 3ab to the cytoplasmic face of the er. 3d binds directly to 3ab, and this targets the polymerase to the replication complex. the 3d polymerase of poliovirus is believed to self-assemble into a large ordered array on membranes, which is critical for binding rna and rna elongation (lyle et al., 2002) . the replication complex also requires 2bc and 2c proteins that are targeted to membranes via their own hydrop hobic dom ains ( fig. 1a ). the accumulation of large numbers of densely packed membrane vesicles in the cytoplasm is characteristic of a picornavirus infection (bienz et al., 1983 (bienz et al., , 1987 cho et al., 1994; dales et al., 1965a; schlegel et al., 1996; stuart and fogh, 1961; suhy et al., 2000) . studies have suggested that vesicles induced by poliovirus are derived from the er, either from copii-coated vesicles or from er-derived autophagic double-membraned vacuoles (bienz et al., 1987; jackson et al., 2005; rust et al., 2001; schlegel et al., 1996; suhy et al., 2000) . however, the detection of er, golgi, and lysosomal markers in membranes induced at later stages of infection by poliovirus suggests that more than one organelle may contribute membranes to the replication complex (schlegel et al., 1996) . in interpreting these studies, it is important to consider if the vesicles observed are involved in replication, or if they represent a bystander response to virus infection. evidence for a role of specific membranes in replication is provided by the presence of replicase proteins, or better still dsrna or negative-stranded intermediate viral rna (egger and bienz, 2005) . examination of cells infected with poliovirus for the first appearance of negative-stranded rna suggests that this initial stage of replication starts on the er. this is consistent with high-resolution immunofluorescence microscopy (rust et al., 2001) showing the poliovirus 2b protein associated with eres containing the sec13-sec31p proteins of the copii complex. these sites exclude resident er proteins, suggesting colocalization of 2b with copii-coated transport vesicles. replication complexes containing negative-stranded rna then move on microtubules to a perinuclear area to initiate synthesis of positive-stranded rna (egger and bienz, 2005) . membrane rearrangements have been studied by expressing individual, or combinations, of picornavirus proteins in cells. most of this work has involved studies of poliovirus proteins, and membrane rearrangements are reported for the 2b, 2c, 2bc, 3a, and 3ab proteins. poliovirus 2b causes fragmentation of the golgi complex (sandoval and carrasco, 1997) . the 2bc and 2c proteins lead to vesiculation and tubulation and sometimes myelin-like swirls of er-derived membranes (aldabe et al., 1996; cho et al., 1994) . similar structures are induced by 2c and 2bc of hepatitis a virus (teterina et al., 1997) . expression of the poliovirus 3a protein causes swelling of er cisternae and the disappearance of vesicles budding from the er, while the 3ab protein also induces myelin-like swirls of er (egger et al., 2000) . the membrane rearrangements induced by expression of single proteins do not, however, mirror those observed in infected cells, and since myelin-like modifications to the er are also seen following overexpression of er proteins [reviewed by borgese et al. (2006) ], their relevance to viral replication is unclear. importantly for poliovirus, it is a combination of 2bc and 3a protein expression that induces membrane structures morphologically similar to those seen in infected cells (suhy et al., 2000) . gazina et al. (2002) have studied replication complexes formed by several different picornaviruses. encephalomyocarditis virus (emcv), parechovirus 1, and echovirus 11 induce clustered vesicles containing dsrna in the perinuclear region of the cell. the precise nature of the vesicles varied with virus. parechovirus 1 produced homogeneous vesicles of 70-100 nm, while membranes produced by emcv and echovirus 11 were heterogeneous but more compact and associated with electron-dense material. differences for parechovirus 1 have also been reported by krogerus et al. (2003) who suggest that replication may occur on membranes derived from the late golgi rather than early er and ergic compartments. all three viruses, however, cause loss of ribosomes from the er and lack of visible golgi apparatus. the copi coat protein b-cop was found to colocalize with echovirus 11 replication complexes, but not with replication complexes produced by emcv, again suggesting that vesicles produced by different picornaviruses may differ. infection with foot-andmouth disease virus (fmdv) also results in loss of ribosomes from the er and an accumulation of heterogeneous vesicles to one side of the nucleus . high-pressure freezing can be used to increase the preservation of cellular ultrastructure during processing for electron microscopy. such analysis of cells infected with poliovirus shows that the vesicles have two membranes suggestive of autophagosomes ( jackson et al., 2005; suhy et al., 2000) . double-membraned structures containing electron-dense material, and possibly viruses, were also revealed by the early work on poliovirus (dales et al., 1965a) . high-pressure freezing has been used to compare fmdv and bovine enterovirus (bev). bev produced heterogeneous membrane clusters similar to the rosettes described for poliovirus (egger et al., 1996) . many of the vesicle membranes have high electron density suggestive of double membranes and lie adjacent to accumulations of virus-like particles. clusters of fmd viruses were also associated with vesicles and electron-dense material, but there were fewer doublemembraned vesicles . immunofluorescence analysis of poliovirus vesicles shows colocalization of replicase protein 3a and autophagy marker lc3, suggesting assembly of the replicase on autophagosomes. similar work suggesting the use of autophagosomes during replication of covs will be described below. for poliovirus, expression of 3a and 2bc, which produces vesicles similar to those seen in infected cells (suhy et al., 2000) , can induce autophagy (jackson et al., 2005) , and inhibition of autophagy reduces yields of extracellular virus. the results suggest that the autophagy pathway may facilitate the release of poliovirus from cells, and it will be interesting to see if this is true for other enteroviruses that are resistant to the low ph and proteases present in lysosomes and autophagosomes. evidence that different members of the picornavirus family vary in the way that they interact with host membranes is provided by studies of virus sensitivity to bfa. bfa completely inhibits poliovirus and echovirus 11 replication (cuconati et al., 1998; gazina et al., 2002; irurzun et al., 1992; maynell et al., 1992) and partially inhibits parechovirus 1 replication (gazina et al., 2002) but not other picornaviruses such as emcv (gazina et al., 2002) or fmdv o'donnell et al., 2001) . bfa prevents assembly of copi coats and this has generated considerable interest in understanding how copi and copii coats contribute to formation of the replication complex, and how bfa inhibits picornavirus replication. in cells infected with the highly bfa-sensitive virus echovirus 11, b-cop was recruited into the replication complex; in contrast, the replication complex formed by the bfa-resistant emcv did not contain b-cop. this correlation suggests that bfa-sensitive viruses may require copi coats for replication (gazina et al., 2002; mackenzie, 2005) . since copii coats are resistant to bfa (lippincottschwartz et al., 2000; orci et al., 1993; , it is suggested that copii coats may provide the membranes for replication complexes formed by bfa-insensitive viruses. the observation that poliovirus replicase 2b protein is seen in eres containing copii proteins, but poliovirus is sensitive to bfa, can be reconciled if this association of 2b with eres is considered to be an early step in generation of membrane for the replication complex that precedes recruitment of copi coat proteins. this is supported by work showing the movement of poliovirus replication complexes containing negative-stranded rna from the er to perinuclear sites (egger and bienz, 2005) . direct evidence that copi coat proteins are required for picornavirus replication comes from studies of drosophila c virus (dcv). dcv is a positive-stranded rna dicistronic virus that is similar to poliovirus and replicates in a cytoplasmic compartment containing virus-induced membrane vesicles. a genome-wide rna silencing screen identified six (a, b, b 0 , g, d, and z) of the seven copi coat proteins as essential for virus replication. furthermore, the formation of virus-induced vesicles required b-cop, but not copii protein, sec23p. notably, small interfering rnas against a-cop, but not sec23p, also slowed poliovirus replication (cherry et al., 2006) . the formation of copi-coated vesicles is regulated by the arf1-gtpase. the observation that bfa inhibits the replication of enteroviruses such as poliovirus, and also inhibits the function of the arf1-gtpase, provides a second link between virus replication and copi coats. arf proteins are regulated by arf-gefs that facilitate binding of gtp by removing gdp, and by arf-gaps that increase hydrolysis of gtp by arfs. arf1-gefs are inhibited by bfa, and bfa therefore reduces levels of arf1-gtp in cells. the gefs affected by picornavirus infection are golgi-associated bfaresistant protein (gbf1) and bfa-inhibited protein (big1/2). work by belov et al. (2005 belov et al. ( , 2007 indicates that infection of cells with poliovirus increases intracellular arf-gtp levels fourfold, suggesting increased activity of arf1-gefs or inhibition of arf1-gap proteins. in the absence of virus, arf1 is concentrated in the golgi apparatus, but during infection with poliovirus arf1 staining fragments and colocalizes with replicase protein 2c. this suggests that infection leads to a redistribution of arf proteins from the golgi apparatus to the replication complex. the binding of arf proteins to membranes is dynamic, with arf-gdp being released from membranes following hydrolysis of gtp. cytosolic arf1-gdp would redistribute naturally to membranes enriched for the arf1-gefs that facilitate loading of new gtp. significantly, poliovirus infection causes enrichment of gefs in membranes containing replicase proteins, and this would provide a mechanism for increasing levels of arf1-gtp at sites of virus replication. translation of poliovirus rna on membranes in vitro provides an alternative means of studying the role of arf proteins in virus replication. replication is inhibited by bfa and peptides that function as competitive inhibitors of arf (cuconati et al., 1998) , and for the most part, the assay mimics what is observed in infected cells. translation in vitro leads to recruitment of arf3 and arf5 but not arf6 (belov et al., 2007) onto membranes. suitable antibodies recognizing the er-associated arf1 were not available for these experiments, so it is not known if arf1 is also recruited to membranes during translation. membrane recruitment of arf proteins can be reconstituted by translation and expression of poliovirus 3a or 3cd. poliovirus proteins do not show intrinsic gef activity, but 3a and 3cd will induce association of gbf1 and big1/2, respectively, with membranes in vitro. this raises the possibility that recruitment of 3a and 3cd to the replication complex during infection targets arf-gef to virus-induced membranes, which in turn increases local levels of arf1-gtp. this is thought to be necessary for replication because inhibition of arf1-gef by bfa blocks replication, and replication can be rescued by overexpression of gbf1 (belov et al., 2007) . high levels of arf1-gtp would also increase recruitment of copi proteins and be consistent with the work on dcv showing that copi proteins are required for replication and vesicle production (cherry et al., 2006) . a poliovirus 3a mutant with a serine insertion at position 16 is unable to cause translocation of arf to membranes (belov et al., 2005) . poliovirus carrying the 3a mutation does not, however, show defects in replication, suggesting that arf1-gef recruitment to membranes by 3a is not essential for replication. it is possible that during infection the defect in 3a is compensated for by 3cd. interestingly, a bfa-insensitive poliovirus with mutations in the 2c and 3a proteins (crotty et al., 2004) induces vesicles and dispersal of the golgi apparatus, which begs the questions, does this mutant use a different process for forming the replication complex, or do the mutations in 3a allow the proteins to compete with bfa for gbf1 recruitment? the role of arf proteins during coxsackievirus infection has also been studied. in common with poliovirus, coxsackieviruses are enteroviruses and their replication is inhibited by bfa. expression of coxsackievirus 3a causes loss of copii coats from eres, and an accumulation of 3a, copii and a model secreted protein in both the er, and tubular-vesicular post-er structures containing ergic marker proteins. these effects closely resemble the effects of adding bfa to cells, suggesting coxsackievirus 3a may affect the function of arf proteins. coxsackievirus 3a affects the regulation of arf proteins (wessels et al., 2006b) . interestingly, the process differs to that described by belov et al. (2005 belov et al. ( , 2007 for poliovirus 3a translated in vitro. expression of coxsackievirus 3a in cells caused loss of copi and arf1 from membranes, and there was redistribution of big1/2 and gbf1 from the golgi apparatus into the cytoplasm. this suggests that coxsackievirus 3a reduces, rather than enhances, levels of arf1-gtp. coxsackievirus 3a also caused redistribution of arf1-gap to punctate structures suggestive of the ergic. a block in arf1-gef activity, combined with recruitment of arf1-gap, would reduce the levels of arf-gtp and inhibit membrane recruitment of copi. wessels et al. (2006a) examined the effects of the 3a proteins of other picornaviruses and found that only the 3a proteins of enteroviruses bound gefs. intriguingly, wessels' work contrasts with belov in that they found the interaction of 3a with gefs lead to a loss of arf proteins from membranes. why these differences are seen is, as yet, unknown but may be due to differences in cell type/methods used or differences in levels of 3a protein expression. poliovirus and coxsackievirus slow protein movement through the secretory pathway (doedens and kirkegaard, 1995; wessels et al., 2005) . expression of 2b, 2bc, and 3a individually were all able to slow secretion (cornell et al., 2006; doedens and kirkegaard, 1995; doedens et al., 1997; van kuppeveld et al., 1997; wessels et al., 2005 wessels et al., , 2006a , but for both viruses the 3a protein was found to have the greatest impact on er-to-golgi transport. poliovirus infection, and the 3a protein expressed alone in cells, reduces surface expression of mhc class i, the tnf receptor, and secretion of b-ifn, il-6, and il-8 (choe et al., 2005; deitz et al., 2000; dodd et al., 2001; neznanov et al., 2001) , and this may offer an immune evasion strategy to the picornaviruses. this is consistent with the observation that the ability of the coxsackievirus 3a protein to slow secretion may be important for virulence (wessels et al., 2006b) and has led to studies of the mechanism of action of 3a in blocking er-to-golgi transport. deletion analysis has identified residues in the unstructured n-terminal region of poliovirus and coxsackievirus 3a as important for the block in host protein secretion (choe et al., 2005) . an n-terminal proline-rich region (particularly pro19) is important for coxsackievirus block in trafficking (wessels et al., 2005) . in poliovirus, lys9 appears important, and in the triple-proline motif (positions 16-18), only the pro18 is indispensable for inhibition of protein secretion (choe et al., 2005) . a serine insertion in 3a protein between thr14 and ser15, creating the 3a-2 mutant virus (berstein and baltimore, 1988) , was found to abolish the er-to-golgi inhibition of protein trafficking but has little effect on virus replication or membrane rearrangements (dodd et al., 2001; doedens et al., 1997) . this important observation shows that the ability of 3a to inhibit protein secretion is separate from its role in membrane rearrangements and viral replication. there is continuing interest in understanding how picornavirus proteins block secretion. poliovirus 3a and 3cd, and coxsackievirus 3a, can interact with arf-gef, but the downstream events are unclear. the recruitment of arf-gef by poliovirus 3a and 3cd would increase recruitment of arf-gtp to membranes of the replication complex. this would increase recruitment of copi coat proteins into sites of virus replication and reduce the pool of copi proteins available to the ergic and golgi apparatus. alternatively, inhibition of arf-gef and recruitment of arf-gap onto ergic membranes by enterovirus 3a would decrease membrane association of arf-gtp and again reduce recruitment of copi onto ergic and golgi membranes. both mechanisms would reduce the formation of copi vesicles, and as seen for bfa, block secretion. poliovirus 3a also binds and inactivates l1s1, a component of the dynein-dynactin motor complex (kondratova et al., 2005) , which is required to move copiiderived vesicles from eres to the ergic. as seen for expression of 3a, mutant l1s1 leads to disruption of the er-to-golgi traffic and reduction in plasma membrane receptors such as tnf receptor. it is possible that 3a may also slow er-to-golgi transport by binding l1s1. a. picornaviruses differ in the use of nonstructural proteins to block secretion the ability of 3a to inhibit er-to-golgi trafficking has not been conserved in all picornaviruses (choe et al., 2005; cornell et al., 2006; deitz et al., 2000; moffat et al., 2005) . for example fmdv infection leads to reduced surface expression of mhc class i (sanz-parra et al., 1998) , but the fmdv 3a protein does not inhibit er-to-golgi transport . a lack of inhibition of secretion has also been reported for 3a proteins of human rhinovirus, hepatitis a, theiler's virus, human enterovirus, and emcv (choe et al., 2005; wessels et al., 2006a) . the 3a protein of human rhinovirus is unable to bind gbf1, or inhibit copi recruitment to membranes, and this may explain its inability to slow secretion. importantly, studies on fmdv have shown that the 2bc protein, or a combination of the processed products, 2b and 2c, inhibits protein movement from the er to the golgi apparatus (moffat et al., , 2007 , and this may be similar for other picornaviruses with 3a proteins that do not block er-to-golgi transport. a lack of effect of fmdv 3a on secretion does not result from an inability to bind membranes. fmdv 3a is recovered from postnuclear membrane fractions, and when expressed alone in cells it colocalizes with resident er proteins. in common with 3a, picornavirus 2b, 2c, and 2bc proteins also contain membrane-binding sequences. sequence alignment of the 2b, 2c (2bc), and 3a proteins of different picornaviruses showed a high level of conservation between the 2c proteins, which contain an ntp-binding site and predicted helicase motifs (gorbalenya et al., 1990) but large variations in the sequences of the 2b and 3a proteins (choe et al., 2005; moffat et al., 2005) , and these may explain their different abilities to block secretion. the fmdv 3a protein is, for example, much longer than 3a of enteroviruses, such as poliovirus, and it does not contain the n-terminal sequences thought important for poliovirus 3a to block the secretory pathway. the 2b protein of fmdv also locates to er membranes but shows a more reticular pattern than the fmdv 3a protein and can be seen in punctate structures aligned along the er suggestive of eres (fig. 3) . this is similar to the 2b of poliovirus that colocates with both figure 3 subcellular location of foot-and-mouth disease nsp encoded in the p2 region of the fmdv genome. vero cells expressing fmdv 2b (top), 2bc (middle), or 2c (bottom) were fixed and permeabilized and processed for immunofluorescence. 2c and 2bc were located using antibodies specific for 2c (3f7) and 2b was located using an antibody raised against an epitope tag in 2b. cells were counterstained using antibodies against er luminal protein erp57 (top and middle panels), or copi protein b-cop (bottom). merged images are shown at higher magnification on the far left. see moffat et al. (2005) for more details. reprinted from moffat et al. (2005) with permission from american society for microbiology. sec13p and sec31p of the copii coat. as expected, fmdv 2c is also membrane associated. when expressed in cells, 2c produces faint er staining, but mainly locates to bright punctate structures in a perinuclear region close to b-cop, reminiscent of golgi staining. the b-cop staining is, however, fragmented suggesting dispersal of the golgi apparatus, and there is not complete colocalization since 2c structures negative for b-cop protein can also be seen (moffat et al., 2007) . a similar location of fmdv nsp within the area of the cell occupied by the golgi apparatus is seen in cells infected with fmdv, and again they do not colocalize with golgi markers . the 2bc protein of fmdv is also recovered in postnuclear membrane fractions, but when expressed in cells, 2bc staining differs from that seen for the processed products, 2b and 2c (fig. 3) . fmdv 2bc locates to punctate cytoplasmic structures and larger structures surrounding the nucleus that contain er markers suggesting swelling of the er. 2bc shows partial overlap with luminal er markers but, unlike poliovirus 2bc, does not colocate with the copii marker sec13p. the er markers also appeared punctate in cells expressing 2bc, suggesting disruption of the er . interestingly, coexpression of 2b and 2c blocks secretion within post-er compartments, similar to those containing 2c. the site of block therefore seems to be determined by the subcellular location of 2c (moffat et al., 2007) and is consistent with the observation that the block in the presence of 2b can be redirected to the er, if 2c is tethered to the er by an er retention sequence. sindbis virus (sbv) and semliki forest virus (sfv) are the best studied examples of alphavirus replication in mammalian cells [reviewed by salonen et al. (2005) ]. early electron microscopy studies showed that vesicular structures called cytopathic vacuoles between 600-and 2000-nm diameter, accumulated in infected cells. the vacuoles contained 50-nm-diameter vesicles called spherules, many of which were aligned along the inside face of the vacuole and attached by a neck to the limiting membrane. the neck was often seen connected to an electron-dense matrix extending into the cytoplasm. the observation that the cytopathic vacuoles contained nsps required for rna replication, cofractionated with lysosomal enzymes, and could be labeled with endocytic markers (froshauer et al., 1988) , led to the conclusion that they are sites of viral replication derived from endosomes and lysosomes. in many cases, the vacuoles were also connected to the rough er by filaments and granular material containing the rna polymerase. alp havirus ns ps are syn thesized in the cytop lasm and bind to endosom es and lysos omes to generate a replication compl ex. the rep licase pro teins are syn thesized as a polyprote in (p1234) . the p4 domai n is the rdrp wh ile p2 has ntpase and helicase activi ties, and p1 is the methytran sferase require d to cap rna ( fig. 1c) . the p12 34 poly protein locate s to endosome or lysosome membranes via an amphipathic peptide sequence in p1 (salonen et al., 2003) . at this stage the p4 polymerase is cleaved from the polyprotein and functions with the remaining p123 protein to generate negative-stranded rna. interestingly, once the p123 is processed to individual nsps, the polymerase preferentially produces positive-stranded rna. expression of individual nsps does not lead to the formation of a cytopathic vacuoles or spherules. formation of spherules requires interactions between nsp p1, p3, and p4 and the p123 polyprotein precursor complex (salonen et al., 2003) . rubella virus is a member of the togaviridae family within the alphavirus genus. cells infected with rubella virus also contain vacuoles containing spherules and these colocalize with lysosomal markers, suggesting use of lysosomes for replication. a fibrous material connects the vacuoles to the er (lee et al., 1994; magliano et al., 1998) , again suggesting strong similarities with sfv and sbv. members of the alphavirus superfamily share homologies between proteins required for rna replication, and this extends to plant viruses. alfalfa mosaic virus replicase proteins colocalize with the plant vacuole (van der heijden et al., 2001) , and turnip yellow mosaic virus uses the chloroplast outer envelope as a site for replication. replication of tobacco mosaic virus, a tobamovirus, is dependent on arabdopsis proteins tom1 and tom2a that are integral membrane proteins of the tonoplast (hagiwara et al., 2003) . the tonoplast is a membrane compartment within plants that surrounds the vacuole/lysosome, suggesting plant alphaviruses also use the endosome/lysosome system as a site of replication. infection of plants with alphavirus-like superfamily viruses can also induce the formation of spherules (prod'homme et al., 2001) . there is evidence that tobacco mosaic virus also uses the er as a site of replication because the replicase enzyme and viral rna are located on the er of infected cells, and infection causes major changes in er morphology (reichel and beachy, 1998) , including er aggregation and formation of lamella structures. flock house virus replicates in spherules in the outer membrane of mitochondria. the rna polymerase (protein a) of flock house virus is the only protein required for rna replication and is targeted directly to the mitochondrial outer membrane by hydrophobic amino acids at the n-terminus. this sequence contains a mitochondrial localization signal and transmembrane domain that leaves the bulk of the protein exposed to the cytoplasm (miller and ahlquist, 2002) . brome mosaic virus replicates in yeast and has been studied extensively. the 1a and 2a replicase proteins are produced from separate viral rnas. the 1a protein contains a c-terminal helicase domain and an n-terminus required for rna capping. 1a is targeted to the cytoplasmic face of er membranes and recruits the 2a polymerase to the replication complex (schwartz et al., 2002) . importantly, replication of brome mosaic virus on the cytoplasmic face of the er in yeast induces membrane invaginations of 50 nm that are very similar to the spherules produced in endosomes and lysosomes during alphavirus infection of mammalian cells. it has been suggested that the active formation of spherules to separate viral rna from host responses is analogous to the coordinated assembly of viral proteins, which leads to capsid assembly, genome packaging, and budding (ahlquist, 2006; schwartz et al., 2002) . the brome mosaic virus replication complex contains viral 1a and 2a pol proteins within spherules. expression of 1a alone produces a shell containing hundreds of copies of 1a on the inside of 50-nm spherules. in a capsid assembly model (schwartz et al., 2002) , vesicles of uniform size would arise if the 1a protein first made a planar lattice with hexameric symmetry on membranes and achieved curvature by localized rearrangement of 1a into pentamers. interestingly, the formation of spherules is dependent on the relative levels of 1a and 2a pol . when levels of 2a pol are high, the spherules are lost, and 1a and 2a pol assemble into flat lamella structures associated with the er (schwartz et al., 2004) . one explanation for a failure to achieve curvature is that high levels of 2a pol may interfere with this hexamer to pentamer transition. this is supported by the observation that when domains that allow association of 1a and 2a pol are deleted, the 2a pol is unable to alter the structure of spherules formed by 1a. the correct ratio of 1a and 2a pol is clearly important for replication complex assembly and may be maintained during infection through inhibition of translation initiation of the 2a rna. d. the flaviviridae replicate in vesicular packets and membraneous webs in the flaviviridae family, which includes the flavivirus, pestivirus, and hepacivirus genera, the rna genome encodes a polyprotein precursor that is cleaved by viral proteases to produce structural proteins from the n-terminal region. the replicase of the flaviviridae is made from nsps, ns5a, ns5b, ns4b, and ns3-4a, found at the c-terminus. wi th the exce ption of the polytop ic ns4b membr ane prote in, whic h is ins erted co transl ationall y into the er, the membr ane-an chored co mponent s of the compl ex are inserted into the cytopla smic face of the er after tran slation ( fig. 1b) . the ns 5b is the rdrp, and a c-termi nal stretch of 21 hydrophobic amino acids directs ns5b to the cytoplasmic face of the er (dubuisson et al., 2002; moradpour et al., 2004) . the ns3 protein has ntpase/helicase activity. ns3 is not a membrane protein but is recruited to the complex through association with membrane-anchored ns4a. ns5a is also membrane associated, and association is mediated via 31 amino acids at the n-terminus that form an amphipathic a-helix (brass et al., 2002; elazar et al., 2003) . replication of flaviviruses (e.g., dengue, west nile, and yellow fever viruses) takes place in membrane invaginations. for historical reasons, these are called vesicular packets [reviewed in mackenzie (2005) ]. they are larger (80-to 100-nm diameter) than the 50-nm alphavirus spherules, and form from the limiting membrane of the trans-golgi network (tgn) (uchil and satchidanandam, 2003; westaway et al., 1997b) . infection by kunjin virus leads to unique membrane structures thought to be derived from both the early and late secretory pathways. these include convoluted membranes and paracrystalline arrays derived from the rough er and ergic, and vesicle packets derived from the tgn (mackenzie et al., 1999; ng, 1987; roosendaal et al., 2006; westaway et al., 1997b) . the detection of dsrna and viral nsps (ns1, ns2a, ns3, and ns4a) within the vesicle packets points strongly to this being the site of rna replication (mackenzie et al., 1998; westaway et al., 1997b) . the vesicle packets associate closely with the convoluted membranes and paracrystalline arrays, which are thought to be the sites of proteolytic processing of ns3 and ns2b (westaway et al., 1997b) . these modified membranes are linked with the er, and ultrastructural studies have shown virions present in the er, cytoplasmic vesicles, golgi cisternae, and vacuoles. the results suggest that membranes containing the spherules responsible for replication may become associated with the er to facilitate delivery of genomes to viruses, budding into early compartments of the secretory pathway (mackenzie and westaway, 2001) . hepatitis c virus (hcv) is closely related to the flaviviruses, and its importance as a human pathogen has generated great interest in its mechanism of replication. until, recently infection models have not been available to study the replication complex of hcv, and the studies discussed here have focussed on the expression of the entire polyprotein from replicons gosert et al., 2003) . however, the recent production of a hcv that rep licates ef ficiently both in vivo and in cell culture (li ndenbac h et al. , 2006; wakita et al., 2005; zhong et al., 2005) will exp and the possi bilities for studying and understanding the viral replication cycle. hcv replication is thought to occur on membranes derived from the er as all studies of nsps have found them localized to this organelle (dubuisson et al., 2002; hugle et al., 2001; kim et al., 1999; wolk et al., 2000) . studies have also identified a ''membraneous web'' of membrane vesicles of $85-nm diameter associated with the er and a population of irregular doublemembraned vesicles. the web resembled the ''sponge-like inclusions'' seen in the liver of chimpanzees infected with hcv, suggesting it is physiologically relevant. interestingly, the great majority of the nsp synthesized by full-length genomes or subgenomic replicons may not be involved in rna replication (quinkert et al., 2005) . the bulk of the nsps associated with membranes isolated from cells expressing replicons is sensitive to protease, while in vitro replicase activity is resistant to protease and nuclease activity (el-hage and luo, 2003; quinkert et al., 2005) . the results suggest that replication of hcv takes place within membrane vesicles, rather than on the surface of the membraneous web. these vesicles may be associated with the membraneous web, but the similarity between hcv and the flaviviruses leaves open the possibility that the membrane invaginations responsible for replication may also form in the tgn but be closely associated with the er. studies have investigated which viral proteins are responsible for membrane rearrangements seen in cells infected with flaviviruses. the ns4a of kunjin virus induces the characteristic convoluted membranes and paracrystalline arrays seen in flavivirus infections. the ns4a-b protein also causes membrane rearrangement, but the highly condensed structures seen in infected cells are not produced until the ns2b-3 protease cleaves ns4a free from ns4b (roosendaal et al., 2006) . the ns4b then translocates to the nucleus (westaway et al., 1997a) . interestingly, this contrasts with hcv where ns4b (and ns4a-b) konan et al., 2003) rather than ns4a is able to induce the membranous structures. flaviviruses have been found to upregulate cell surface expression of mhc class i and ii in response to interferon (king and kesson, 1988; liu et al., 1989; lobigs et al., 2004) . this is not caused by effects of the ns4a or ns4b proteins on membrane traffic; instead flavivirus infection increases expression of the er peptide transporter, tap1. this increases the supply of peptides that are necessary for the folding and export of newly synth esized mhc proteins from the er. inc reased tap expression is media ted by increas ed transcrip tional activ ity of p53 and can be induc ed in liver hepg2 cells by express ion of the hcv core/cap sid pro tein alon e ( herzer et al., 20 03; mombu rg et al ., 2001 ) . whil e the cap sid/cor e prote in is able to in crease cell surface expression of mhc clas s i through increase expres sion of tap1, exp ression of the hc v polyp rotein has been sho wn to slow the moveme nt of prote ins thr ough the secretory pathway of hos t cells ( konan et al ., 2003 ) . the rate of delive ry of mhc cl ass i to the plasm a me mbrane in cells infected with hc v was reduced three-to five fold relative to cu red contr ol cells. exp ression of the pre cursor ns4a-b was fou nd to red uce er-to-g olgi traf fic two-to threefo ld (kon an et al., 2003 ) , while the ot her ns proteins of hc v inclu ding ns4a and ns4b, indiv idually or comb ined, were unabl e to interf ere with the traf ficking pathway . ns 4b a lone indu ces a memb raneo us web in ce lls , and both ns4a-b and ns4b indu ce, and locat e to, clust ered and aggregate d membr anes looking v ery similar to the me mbraneo us web seen in cells expre ssing rep licons. in addition to agg regated me mbrane s, ns4a/b also ind uces, but does not coloca lize with , swol len vesicul ar structure s. thes es swo llen vesicl es have a similar morphology to the vesicles induced by the 3a protein of p o li ov ir us , wh ich swe lls er memb ranes and blocks sec retio n betwee n the er and the golgi appar atus (doe dens et al., 1 997 ). konan et al . (2003) hypo thesize that the ns 4a/b could be func tioning in a sim ilar man ner to poliovi rus 3a. e. the nidovira les replicate in association with doub le-membraned vesicles 1. the nidovirus replicase is generated from two polyproteins the nidovi rales order comp rises the arterivi ridae, coron aviridae , and ron iviridae famil ies. the rep licase gene is co mposed of two ope n read ing fram es termed orf1a and orf1b. orf1b is gen erated from a fram eshift in 1a, and both reading frame s encode co mplex poly proteins pro cessed by viral prote ases (go rbalenya et al., 2006 ; ziebu hr, 2006 ) . the arter ivirus orf1b encode s nsps 9-12, incl uding the rdrp (nsp 9) and helicase (nsp 10). the orf1b, however , lacks hydroph obic dom ains able to target the rep licase to membr anes. intere stingl y, the hydroph obic domains necessar y for membr ane targetin g are enco ded by orf1a in nsp2, 3, and 5, sugge sting that orf1a pr oteins produc e a scaffold to locate the viral rep lication -transcr iption compl ex to membr anes ( fig. 1d ) ( pedersen et al., 1999; van der meer et al., 1998) . a similar strategy is used by cov, for example mouse hepatitis virus (mhv) and severe acute respiratory syndrome-cov (sars-cov) (prentice et al., 2004a,b) , where transmembrane domains are located in nsp3, 4, and 6, and helicase and polymerase proteins are nsp12 and 13, respectively, and nsp16 encodes the methytransferase. the nidovirales have the largest coding capacity of the single-stranded rna viruses, and not all the 16 nsps have been studied in detail. it is possible that other proteins encoded by orfs1a and 1b, such as rna processing enzymes, are incorporated into the replication complex. several studies have investigated the intracellular sites of replication of equine arterivirus (eav), mhv, and sars-cov. such studies are difficult because during nidovirus infection, the processes of replication and envelopment occur on different membranes, and these may merge during encapsidation. furthermore, late during infection cells infected with mhv can form syncitia. newly synthesized mhv viral rna has been found in perinuclear sites colocalized with the rdrp (shi et al., 1999) , and depending on whether human or murine cells were infected, these sites colocalized with golgi or er membranes, respectively. similar studies in mouse l cells report that the polymerase and newly synthesized rna locate to late endosomes and endocytic carrier vesicles . this discrepancy is in part reconciled by later work showing that the subcellular distribution of the replicase proteins can change during the course of infection, since replicase proteins move to sites of envelopment in the ergic (bost et al., 2001) . this is supported by the finding that individual replicase proteins distribute differently following cell membrane fractionation (sims et al., 2000) . membrane fractionation has also been carried out by gosert et al. (2002) , who showed that several proteins encoded by orf1a and b were associated with membranes, and when observed by immunogold electron microscopy, these were associated with rosettes of double-membraned vesicles 200-350 nm in diameter. the role of these vesicles in viral rna replication was confirmed by in situ hybridization of labeled riboprobes. double-membraned vesicles are also seen in cells infected with eav (pedersen et al., 1999) . eav replicase proteins accumulate in perinuclear regions containing ergic and er markers and colocalize with newly synthesized viral rna, again suggesting sites of genome replication. notably, similar structures can be produced by expression of arterivirus orf1a-encoded proteins nsp2-7, which contain the membrane proteins thought to tether the replicase to membranes. double-membraned vesicles are usually rare in cells but are induced during autophagy. a role for autophagy during mhv infection is suggested because autophagy is induced in cells infected with mhv. furthermore, in cells lacking atg5, a protein required for the formation of autophagosomes, there is a 99% reduction in virus yield and mhv fails to induce double-membraned vesicles (prentice et al., 2004a) . electron micrographs show that the double-membraned vesicles induced by sars-cov extend from the er and can be labeled with antibodies specific for replicase proteins. this suggests that, in common with mhv, the vesicles are a site of replication . even though all sars-cov replicase proteins tested colocalize to punctate structures that accumulate near the nucleus, there are conflicting reports about their relationship with autophagosomes. in monkey vero cells, the replicase proteins colocalize with autophagosomes identified using antibodies against lc3 (prentice et al., 2004a) . however, when autophagosomes are identified by expression of gfp-lc3, the replicase proteins do not colocalize with the gfp signal . the vesicles induced by sars-cov are smaller at 100-to 300-nm diameter than autophagosomes (500-1000 nm) and are labeled with er markers. this has lead snijder and colleagues to suggest that they are virus-induced extensions to the er, rather than bona fide autophagosomes (pedersen et al., 1999; snijder et al., 2006) . the precise origins of the membrane crescents that form at the start of autophagy are unclear, and a number of studies have suggested they may form from the er. this makes it possible that the double-membraned structures may be autophagosomes that have been modified by an accumulation of viral protein. determining if autophagy is beneficial to sars-cov replication will have to await studies in cells where key proteins in the autophagy pathway have been removed or suppressed by gene silencing. the asfiviruses, poxviruses, iridoviruses, and the phycodnaviruses are large dna viruses encoding hundreds of proteins from genomes ranging between 150 and 350 kbp. a comparison of protein sequences encoded by these viruses has suggested that they should be grouped together in a family of viruses called the nucleocytoplasmic large dna viruses (ncldv) (iyer et al., 2001) . sequence similarities are seen in the major capsid proteins, redox enzymes that maintain disulphide bonds in the cytosol, and proteins that regulate apoptosis; and the family has been extended to include the giant mimivirus isolated from the ameba acanthamoeba polyphaga (la scola et al., 2003) . even though these viruses infect a diverse range of hosts from different phyla, including vertebrates [poxviruses, african swine fever virus (asfv)], arthropods (entomopox, asfv, chloriridoviruses), amphibians and fish (ranavirus, megalocytivirus, and lymphocystivirus genera of the iridoviridae family), marine algae (phycodnaviruses), and protozoa (mimivirus), they all generate cytoplasmic factories as major sites of virus assembly and replication (illustrated in fig. 4 ). the factories share many similarities with one another, again suggesting that this diverse group of viruses may be related and that the need to produce a virus factory in the cytoplasm was generated early in virus evolution. 1. asfv factories form next to the microtubule organizing center asfv is the sole member of the asfivirus genus, family asfarviridae but shares striking icosahedral similarity with the iridoviruses, phycodnaviruses, and mimivirus. asfv is a large double-stranded dna (dsdna) virus with a genome size ranging from 170 to 190 kbp. gene expression is a regulated cascade and immediate early, early, early/late, intermediate, and true late gene types have been characterized to date. the virion has multiple concentric layers with an electron-dense core at the center that contains the viral genome. a protein matrix surrounds the core, which in turn is enclosed by a lipid bilayer. finally, the bilayer is surrounded by a protein capsid layer. asfv can gain a third envelope when it buds from the plasma membrane at the tip of actin-rich projections that resemble filopodia (jouvenet et al., 2006) . asfv probably enters cells by receptor-mediated endocytosis, but the steps following entry are poorly understood. it is possible that a viral core is delivered into the cytoplasm intact; alternatively, cores may dissociate in endosomes requiring some mechanism of genome delivery across the endosome membrane. genome replication occurs both in the nucleus and cytoplasmic factories. transfer to the nucleus may involve microtubule transport since late gene expression is inhibited by agents that depolymerize microtubules and the dominant-negative dynein motor protein p50-dynamitin (alonso et al., 2001; heath et al., 2001) . asfv does not produce nuclear inclusions analogous to those seen in herpesvirus and adenovirus infection, but there is evidence that small fragments of viral dna are synthesized in the nucleus. the major site of asfv dna replication is, however, the virus factory (rojo et al., 1999) . a. cytoplasmic factories formed during asfv infection are assembled at the microtubule organizing center asfv induces one principal factory in the cytoplasm during infection. electron microscopy shows that the virus factory excludes obvious cellular organelles and contains mostly viral dna, viral proteins, virus-induced membranes, and partially and fully assembled virions (table i; fig. 5a ; brookes et al., 1996; moura nunes et al., 1975; rouiller et al., 1998) . the mechanisms that target viral proteins, virus-induced membranes, and viral dna to the asfv factories are poorly understood. immunofluorescence staining for viral structural proteins generally reveals a strong signal at the factory and a weaker signal in the cytoplasm. the b602lp protein (cap80), which is a viral chaperone involved in folding and membrane recruitment of the major capsid protein, p73, is, for example, absent from the virus factories (cobbold et al., 2001; epifano et al., 2006) . this suggests that p73 is synthesized and folded in the cytoplasm and then recruited to factories. similarly, the viral dutpase, which is necessary for efficient replication, is excluded from the viral factory (oliveros et al., 1999) . since the bulk of viral dna synthesis occurs in the factory (garcía-beato et al., 1992) , it is not easy to explain how the viral dutpase edits uracil from progeny viral genomes, without being present at the site of viral dna synthesis and encapsidation. asfv factories disperse when cells are incubated with drugs that depolymerize microtubules (heath et al., 2001) suggesting their formation involves microtubule motors. this may involve dynein motor proteins since p50-dynamitin, a dominant-negative version of the dynein motor, prevents both late asfv gene expression (heath et al., 2001) and vimentin recruitment to factories (see below and stefanovic et al., 2005) . yeast-twohybrid screens and in vitro pull-down experiments show that one asfv structural protein, p54/j13lp, interacts with dynein (alonso et al., 2001) . while direct binding of p54/j13lp to the motor protein has not been observed in infected cells, it is possible that the protein is involved in transporting some viral proteins into factories. the protein locates to virus factories and deletion of the e183l gene encoding p54/j13lp generates factories that lack viral membranes, the major capsid protein p73, and the polyprotein precursors (pp220, and pp62) of the viral matrix (epifano et al., 2006; rodríguez et al., 2004) . p54/j13lp is a membrane protein with the bulk of the protein, including the dynein-binding motif, exposed to the cytosol. the p73 capsid protein and pp220 polyprotein associate with membranes before assembly into viruses (cobbold and wileman, 1998; cobbold et al., 1996; heath et al., 2003) . if these membranes contain p54/j13lp, it would provide a means of allowing recruitment to factories by retrograde transport along microtubules. the formation and morphology of asfv factories closely resemble the formation of aggresomes (heath et al., 2001) , a cellular response to accumulation of misfolded protein aggregates (johnston et al., 1998) . aggresomes are microtubule-dependent inclusions containing protein aggregates that human herpesvirus 6 induces nuclear tegusomes (t). herpesviruses induce cytoplasmic assembly sites where envelopment and some tegument are acquired (env) in human herpesvirus 5, these sites include electron-dense bodies (db). iridoviruses induce multiple cytoplasmic virus factories (vf) and crystalline arrays (ca), both of which associate with mitochondria. reoviruses also induce multiple cytoplasmic virus factories (vf) and crystalline arrays (ca) that are enclosed within lysosomal membranes. alcamí et al., 1993; alonso et al., 2001; andrés et al., 1997 andrés et al., , 2001 borca et al., 1996; brookes et al., 1998a,b; carrascosa et al., 1986; chacó n et al., 1995; cobbold et al., 1996; galindo et al., 2000; garcía-beato et al., 1992; heath et al., 2001; hingamp et al., 1992; jouvenet and wileman, 2005; jouvenet et al., 2004; martinez-pomares et al., 1997; moura nunes et al., 1975; rodríguez et al., 2006; rouiller et al., 1998; sanz et al., 1985; simón-mateo et al., 1997; sun et al., 1996; vigário et al., 1967 contents of cellular origin ubiquitin, hsp70 chaperone, g-tubulin, pericentrin, p21, mdm1 surrounded by: er membranes, vimentin, p230 golgin, mitochondria, and tubulin. granja et al., 2004; heath et al., 2001; hingamp et al., 1992; jouvenet and wileman, 2005; netherton et al., 2004 netherton et al., , 2006 rojo et al., 1998; rouiller et al., 1998; stefanovic et al., 2005 poxviridae almazán et al., 2001; beaud and beaud, 1997; betakova et al., 2000; chiu et al., 2005; cudmore et al., 1996; da fonseca et al., 2000; davis and mathews, 1993; de silva and moss, 2005; domi and beaud, 2000; krijnse-locker et al., 1996; murcia-nicolas et al., 1999; nerenberg et al., 2005; ojeda et al., 2006; palacios et al., 2005; pedersen et al., 2000; reckmann et al., 1997; resch et al., 2005; risco et al., 1999; roper, 2006; salmons et al., 1997; senkevich et al., 2002; sodeik et al., 1995; szajner et al., 2004a,b,c; tolonen et al., 2001; vanslyke and hruby, 1994; welsch et al., 2003; wolffe et al., 1995; yeh et al., 2000; yuwen et al., 1993 (continued) contents of cellular origin hmg20a viral genome binding protein, hsp90; transient association, ubiquitin, ying-yang 1 transcription factor, tbp transcription factor, sp1 transcription factor, rna polymerase ii, sumo-1, ergic-53 c surrounded by: vimentin and mitochondria. broyles et al., 1999; dales and siminovitch, 1961; hsiao et al., 2006; hung et al., 2002; husain and moss, 2003; nerenberg et al., 2005; oh and broyles, 2005; palacios et al., 2005; risco et al., 2002; wilton and dales, 1989 iridoviridae, ranavirus appearance and contents f electron lucent, virus, viral dna, 108k early protein, 57k, 55k major capsid protein (orf 90r in fv3), 38k, 17k, 16k rana grylio virus dutpase (orf 63r in fv3). surrounded by vimentin, rough er, mitochondria and polysomes. chinchar et al., 1984; darlington et al., 1966; huang et al., 2006; goorha, 1983, 1989; zhao et al., 2007 herpesviridae barnard et al ., 1997; de bruyn kops et al ., 1998; everett and maul, 1994; goodrich et al ., 1990; jahedi et al ., 1999; knipe et al., 1987; lamberti and weller, 1998; leopardi et al ., 1997; liptak et al., 1996; markovitz and roizman, 2000; olivo et al ., 1989; randall and dinwoodie, 1986; reynolds et al ., 2000; taus et al ., 1998; ward et al ., 1996 contents of cellular origin rna polymerase ii, eap ribosome component, proliferating cell antigen, retinoblastoma protein, p53, dna ligase 1, dna polymerase a, promyelocytic leukemia (pml), dna-pkcs, ku86 nonhomologous end joining, bloom syndrome gene product, breast cancer-associated gene 1 protein, msh2, rad50, wrn recq helicase family member, brg1 or brm-associated factor 155, brahma-related gene-1 protein, brahma protein, histone deacetylase 2, hsnf2h, msin3a, tata binding protein (tbp), tbp-associated factors. leopardi et al., 1997; quadt et al., 2006; taylor and knipe, 2004; wilcock and lane, 1991 nuclear sites of capsid assembly or assemblons contents of viral origin d ul7 (hhv-2), ul14 (hhv-2) tegument, ul16 capsid, ul19 icp5 major capsid protein, ul26.5 icp35 dna packaging, ul27 dna packaging, ul35 vp26 p12 capsid, ul38 vp19c capsid assembly, ul43.5, ul55. de bruyn kops et al., 1998; goshima et al., 1998; nalwanga et al., 1996; nozawa et al., 2002; wada et al., 1999; ward et al., 1996a,b; yamada et al., 1998 contents of cellular origin actin, myosin 5a actin motor feierbach et al., 2006 cytoplasmic assembly and envelopment site contents of viral origin d membranes, vacuoles, capsids and enveloped virus ul19 (hhv-2) vp5 major capsid protein ul27 (hhv-2) gb vp7 ul36 (hhv-2) icp1-2, tegument ul46 (hhv-2) tegument, ul48 (hhv-2) tegument kato et al., 2000; murata et al., 2000; nozawa et al., 2004; watanabe et al., 2000 contents of cellular origin mitochondria, g-tubulin, hsp40 chaperone, hsp70 chaperone, gm130 golgi marker murata et al., 2000; nozawa et al., 2004 (continued) becker et al., 2001 becker et al., , 2003 broering et al., 2004; cashdollar, 1994; dales et al., 1965b; miller et al., 2004; sharpe et al., 1982; silverstein and schur, 1970 reoviridae, rotavirus appearance and contents electron-dense viroplasm, assembling and complete double-shelled particles vp2, vp6, vp9, nsp2, nsp5, nsp6 altenburg et al., 1980; gonzález et al., 2000; petrie et al., 1982 petrie et al., , 1984 silvestri et al., 2004 silvestri et al., , 2005 a african swine fever virus gene nomenclature is based on that for the badajoz 1971 vero adapted strain with that of the malawi lil 20/1 strain in parentheses. b vaccinia virus gene nomenclature is based on that for the copenhagen strain with that of the western reserve strain in parentheses. c one report places in ergic-53 within the virosome (risco et al., 2002) , one report places it outside (husain and moss, 2003) . d open reading frames from human herpesvirus 1 (herpes simplex virus 1) unless specified otherwise. e open reading frames from human herpesvirus 5 (human cytomegalovirus) unless specified. f proteins specified by frog virus 3 unless indicated otherwise. form next to the microtubule-organizing center (mtoc). aggresomes recruit cellular components needed to deal with the problems associated with a buildup of aggregated misfolded protein. these include cellular chaperones and proteasomes to facilitate protein folding and/or degradation and mitochondria that may provide the atp required for folding and proteolysis. the most striking structural changes seen during aggresome formation are the collapse of the intermediate filament protein, vimentin, into a cage surrounding the protein aggregates and the gross fragmentation of the golgi apparatus. asfv factory formation shows many similarities with this response to protein aggregation. factory formation is preceded by clearance of cytoplasmic proteins from perinuclear areas around the mtoc. vimentin then concentrates at the mtoc where it forms an aster aligned along microtubules (stefanovic et al., 2005) . following the onset of virus dna replication and synthesis of late structural proteins, the vimentin aster is rearranged into a cage around the factory ( fig. 5b ; heath et al., 2001; monaghan et al., 2003; stefanovic et al., 2005) . during this period, mitochondria and cellular chaperones are recruited to the factory (heath et al., 2001; rojo et al., 1998) . formation of vimentin cages in asfv-infected cells is linked to phosphorylation of vimentin at serine 82 by calcium calmodulindependent protein kinase ii (camkinase ii) (stefanovic et al., 2005) , and drugs that inhibit camkinase ii activity block late gene expression and vimentin rearrangement. as will be discussed for poxviruses and iridoviruses, the vimentin cage may form a physical scaffold within the factory, or act as a cage to prevent movement of viral components into the cytoplasm. chaperones recruited to the factory may facilitate folding of viral structural proteins during assembly, as has been shown for other viruses. the proximity of mitochondria to viral factories may provide the atp that is required for asfv assembly (cobbold et al., 2000) or be indicative of an antiviral response as mitochondria are effectors of apoptosis. taken together these results suggest that a cellular response originally designed to deal with the buildup of protein aggregates in cells is used by asfv to generate a site specialized for virus assembly. as will be described later, similarities between aggresomes and virus assembly sites are also seen for the iridoviruses and poxviruses. following the onset of asfv dna replication, the microtubule network becomes disorganized. microtubules are partially excluded from virus factories and form bundles and concentric rings in the cytoplasm (jouvenet and wileman, 2005) . asfv infection leads to disassembly of g-tubulin and pericentrin from the centrosome, and the centrosome becomes less able to nucleate microtubules. at the same time microtubules are stabilized by acetylation (jouvenet et al., 2004) . since pericentrin and g-tubulin play key roles in microtubule organization and nucleation at the mtoc, their loss from the centrosome, coupled with acetlylation of tubulin, may explain the rearrangement of microtubules induced by asfv. the reasons for these profound effects on microtubules are not known but they may facilitate disruption of the virus factory allowing release of assembled viruses into the cytoplasm. c. membrane rearrangements caused by asfv infection perturb the secretory pathway current models for asfv envelopment in virus factories predict that viral membranes are obtained from the er. the major structural proteins are recruited from the cytoplasm onto the cytoplasmic face of the er, and after which protein-protein interactions between these, and possibly viral proteins targeted to the er lumen, lead to constriction of er cisternae and clearance of host proteins from the er lumen prior to envelopment (andrés et al., 1998; netherton et al., 2004 netherton et al., , 2006 rouiller et al., 1998) . this is consistent with low levels of er proteins observed at asfv assembly sites by immunoelectronmicroscopy (rouiller et al., 1998) and standard fluorescence microscopy where er proteins appear to be actively excluded from areas of viral replication (andrés et al., 1998; netherton et al., 2004) . in addition to effects on the er, asfv also affects the structure and function of later golgi compartments of the secretory pathway (mccrossan et al., 2001; netherton et al., 2006) . golgi structure is linked to microtubule organization and the changes seen during infection may in part be related to effects of asfv infection on centrosome and microtubule function listed above. asfv infection causes dispersal of ergic marker protein ergic-53, the peripheral golgi protein gm130, and late golgi protein galnac-t2 transferase, suggesting disruption of ergic and golgi membrane compartments. most striking is the complete loss of the tgn. tgn loss is dependent on microtubules and involves dispersal of the tgn into separate vesicle populations containing either peripheral golgi proteins or the integral membrane protein, tgn46. not surprisingly, this dispersal slows the transport of proteins through the secretory pathway. asfv slows the delivery of newly synthesized lysosomal enzymes to lysosomes (mccrossan et al., 2001) , and in macrophages reduces transport of newly synthesized mhc class i to the plasma membrane (netherton et al., 2006) . thus, in common with picornaviruses, disruption of the secretory pathway by asfv has the potential to slow the transport of important immunomodulatory proteins to the surface of infected cells and may mask them from immune surveillance. poxviruses are large dsdna viruses with genomes ranging from 130 to 375 kbp. poxvirus gene expression follows the regulated cascade of other large dsdna viruses with early, intermediate, and late transcripts described. poxvirus progeny genomes are replicated exclusively in the cytoplasm in virus factories. the virus encodes all the enzymes necessary for transcription and replication of its genome. genetic analysis has identified a minimum of five viral genes necessary for genome replication, these are a20r, b1r, d4r, d5r, and e9l encoding the dna polymerase processivity factor, serine/threonine protein kinase, uracil dna glycosylase, dna-independent nucleoside triphosphatase, and the dna polymerase, respectively (de silva and moss, 2005; evans et al., 1995; millns et al., 1994; punjabi et al., 2001; rempel et al., 1990; sridhar and condit, 1983) . only the product of the d4r gene, encoding the viral dna glycosylase, has been confirmed to localize to the site of genome synthesis (de silva and moss, 2005) , and it would be interesting to discover the subcellular location of the other members of the minimum replicase. when viewed by electron microscopy, infectious virions have a striking brick-shaped morphology, and different forms of virus are documented which vary in degree of complexity [for review, see condit et al. (2006) ]. the interior of all poxvirus particles contains the virus core which houses the viral genome. cores are enveloped in virus factories to produce the intracellular mature virus (imv), which is fully infectious. additional envelope layers gained at the tgn give rise to intracellular enveloped viruses (iev), which after budding through the plasma membrane form cell-associated and extracellular enveloped viruses (cev and eev). poxviruses induce two principal inclusions during infection, the a-type inclusion that is nonreplicative and the b-type inclusion where virus replication and assembly occur in the virus factory ( fig. 4 ; kato et al., 1959). a. poxvirus a-type inclusions contain the mature intracellular virus but not enveloped viruses a-type inclusions are cytoplasmic bodies of dense homogeneous matter that contain mature virus particles and are studded with polyribosomes (fig. 6a) (ichihashi et al., 1971) . a-type inclusions are extremely rare in vaccinia, variola, and rabbit pox infections but are prominent in cowpox, ectromelia, fowlpox, and canarypox infections where they are also referred to as downie, marchal, bollinger, and burnet bodies, respectively (kato et al., 1959) . the major component of a-type inclusions is the product of the a26l gene or its equivalents. in vaccinia, a26 is truncated and produces a protein of 92-94 kda whereas the fulllength gene in cowpox encodes a protein of 160 kda (patel et al., 1986) , both versions are myristylated (martin et al., 1999) . immunfluorescence analysis of cells infected with vaccinia virus with antibodies raised against a26 does reveal multiple a-type inclusions in the cytoplasm, but they are much smaller than those seen in cells infected with cowpox, and do not contain virus particles (patel et al., 1986) . in cells infected with wild-type cowpox, only imv particles were observed within a-type inclusions, but treatment with rifampicin, a drug that blocks poxvirus maturation at an early stage in morphogenesis, caused aberrant immature virus particles to integrate into the inclusions (ichihashi et al., 1971) . the factor necessary for occlusion of viral particles in a-type inclusions has been identified as the 4c core protein (mckelvey et al., 2002; shida et al., 1977; ulaeto et al., 1996) . it has been hypothesized that 4c retains vaccinia virions within the cell as imvs in a-type inclusions preventing their transport to the tgn for envelopment and maturation to the iev types of virion (mckelvey et al., 2002) . a-type inclusions are predicted to protect imvs during transport between hosts akin to that of the polyhedra that occlude entomopox and baculoviruses (rohrmann, 1986) . therefore, eevs may be important for cell-to-cell spread, while imvs (whether occluded or not) may be more important for host-to-host spread (mckelvey et al., 2002) . b. poxvirus b-type inclusions are factories and are the main sites of replication and assembly b-type inclusions originally called guarnieri bodies (guarnieri, 1893) are the primary replication centers of the poxviruses, now generally referred to as virosomes or virus factories (fig. 6c) . electron microscopic analysis of b-type inclusions revealed a granular matrix that was denser than the surrounding cellular material and in a defined area of the cytoplasm called viroplasm (dales and siminovitch, 1961; higashi, 1973) . the factories also contain viral crescents consisting of membrane and viral proteins associated with viroplasm, spherical immature virus, and imvs (dales and siminovitch, 1961) . factories are surrounded by mitochondria, increase in number and size during the replication cycle and can occupy the majority of the cytoplasm at late times of infection (dales and siminovitch, 1961) . the assembly and envelopment of vaccinia virus within virus factories has been the subject of many studies and is discussed in papers and reviews (griffiths et al., 2001; heuser, 2005; hollinshead et al., 1999; sodeik and krijnse-locker, 2002) . here, we will review some of the early steps that lead up to the start of genome replication and factory production. these have also been described in a review (schramm and krijnse-locker, 2005) . it is generally believed that infection results in the delivery of viral cores into the cytoplasm. cores are seen associated with microtubules (carter et al., 2003; mallardo et al., 2001; ploubidou et al., 2000) and may use microtubules to reach perinuclear sites that will eventually house the virus factories. viral cores can transcribe as many as 100 early mrnas before the onset of dna replication, and these early mrnas appear in discrete foci that associate with microtubules, contain polyribosomes and other translational machinery. it is unlikely that foci involved in transcribing early rnas mature into viral replication sites because they do not initiate dna synthesis (mallardo et al., 2002) . it is likely that each infecting virus can induce its own replication center (cairns, 1960) , but it is not clear where in the cell the cores initiate dna synthesis. it has been suggested that the onset of dna synthesis may occur at peripheral sites and therefore precedes delivery to the perinuclear region of the cell. when cells are incubated with hydroxyurea to prevent the onset of viral dna replication, it is possible to localize viral dna released into the cytoplasm. under these conditions, viral genomes are seen at several discrete sites that contain b1 protein kinase, e8 membrane protein, i3 ssdnabinding protein, and h5 late transcription factor (domi and beaud, 2000; welsch et al., 2003) . after removal of hydroxyurea, these foci begin to make new viral dna, showing that they are sites of dna replication. live cell imaging studies have shown that these initial sites of dna replication form in the cell periphery and then move toward the nucleus where they coalesce into large structures (schramm and krijnse-locker, 2005) . electron micrographs suggest that sites of dna release from cores are intimately associated with er membranes and become completely enclosed by them during the initial stages of dna replication (mallardo et al., 2002) . this process is likely facilitated by the e8r gene product which is a membrane protein localized to the er and early golgi membranes, has dna-binding activity, and is able to capture viral genomes tolonen et al., 2001) . these er-enclosed genomes are short-lived structures because they are not seen once viral crescents, iv and imvs, appear in factories (tolonen et al., 2001) . the sites of dna replication are also separate from the foci involved in transcribing early rnas, and it is interesting to consider how the cores are separated from newly transcribed rna. viral cores and sites of rna transcription both align on microtubules and partially colocalize with the l4 core dna-binding protein (mallardo et al., 2001) . the l4 protein is able to bind microtubules (ploubidou et al., 2000) and may be involved in separating rna from cores along microtubule tracks (mallardo et al., 2001) . inducible recombinants or temperature-sensitive mutants grown under nonpermissive conditions can give further insight into the early stages of inclusion formation. electron micrographic analysis of the factories formed under these conditions yield striking images of distinct inclusions of homogeneous electron-dense viroplasm next to empty spherical immature virions ( fig. 6b and c) (szajner et al., 2001 (szajner et al., , 2003 (szajner et al., , 2004a . a seven-protein complex comprising the gene products of the a15l, a30l, d2l, d3l, f10l g7l, and j1r open reading frames has been identified as being necessary for association of viral membranes with the viroplasm (szajner et al., 2004a) . consistent with this role, all of these proteins are known to localize to the virus factory except d2 and d3 (table i) ; however, these have been identified as core proteins (dyster and niles, 1991) so are likely to reside at viral assembly sites. localization of d13l to the virus factory is sensitive to the antibiotic rifampicin (miner and hruby, 1989) , and treatment with this drug induces irregular shaped viral membranes instead of the well-defined hemispherical viral crescents seen in natural infection (moss et al., 1969; pennington et al., 1970) . therefore, it was suggested that d13l may act as a scaffold on which the viral membrane is shaped, allowing correct association with the viroplasm (mohandas and dales, 1995) . deep etch electron microscopy has confirmed this role for d13l, as it forms the honeycomb lattice identified as the outer coat of the viral membrane of immature virions (heuser, 2005; szajner et al., 2005) . interestingly, d13l shares a structural similarity with structural proteins from many other virus families, including those of the other large dsdna viruses (benson et al., 2004) . it will be interesting to see if the structural similarities to d13l translate to functional similarities in the assembly strategies of other viruses. to the viral factory. ying-yang 1 (yy1), tbp, sp1 transcription factors, and rna polymerase ii are recruited from the nucleus to the factory (broyles et al., 1999; oh and broyles, 2005; wilton and dales, 1989) . yy1 is a nuclear transcription factor that can activate late viral promoters and although poxviruses encode most of the genes necessary for transcription, there is evidence that cellular factors may be required for intermediate and late gene expression (lackner and condit, 2000; rosales et al., 1994; wright et al., 2001) . the function of the other transcription factors in viral replication is unknown. they may be necessary for viral transcription like yy1, or perhaps they are sequestered into the factory to divert them from their normal roles in the nucleus, or their presence may represent an antiviral response by the cell. the presence of rna polymerase ii in the viral factory is a surprise because the virus encodes its own rna polymerase activity which accounts for at least 9 orfs and $7% of the genome capacity [western reserve (wr) strain]. another cellular protein recruited from the nucleus to the cytoplasm is the hmg20a protein. this protein can bind the viral genome and has been implicated in host range restriction of vaccinia virus in chinese hamster ovary cells (hsiao et al., 2006) . during unproductive infection by vaccinia virus, hmg20a is recruited from the nucleus to the factory where it binds viral dna. if the cowpox host range gene cp77 is artificially introduced into vaccinia virus then cp77 also enters the virus factory and binds to hmg20a; the cellular protein then dissociates from the viral genome and replication proceeds (hsiao et al., 2006) . as seen for iridovirus and asfv replication sites, vaccinia factories are surrounded by a vimentin cage (risco et al., 2002; schepis et al., 2006) and recruit molecular chaperones (hung et al., 2002) , suggesting similarity with aggresomes. many proteins targeted to aggresomes are ubiqutinated, and most poxviruses encode a ring protein that is both a functional ubiquitin ligase and a virulence factor (nerenberg et al., 2005) . exceptions to this are the two most common laboratory strains of vaccinia, copenhagen and wr. the ring protein from the ihd-w strain of vaccinia is capable of directing transfected tagged ubiquitin to wr virus factories (nerenberg et al., 2005) ; however, it is unknown if native ubiquitin is localized to wr factories. the product of the a40r gene of vaccinia is tagged with the ubiquitin-like protein sumo-1, and this modification is necessary for a40 targeting to viral factories, where it associates with er membranes and may play a role in the formation of i3 sites (palacios et al., 2005) . it is not known if movement of sumolyated a40 and ubiquitinated protein is directed along microtubules in a manner analogous to hdac6mediated targeting of misfolded proteins to aggresomes (kawaguchi et al., 2003) . as reported for asfv (see above) and cells infected with herpes simplex virus (avitabile et al., 1995) , infection of cells with vaccinia virus also leads to disruption of microtubule organization and centrosome function and dispersal of the golgi apparatus (ploubidou et al., 2000) . whether these are bystander effects of the production of virus factories close to the centrosome or induced deliberately to facilitate virus egress is not known. imv exit from the factory and transport to envelopment sites at the tgn is nonetheless dependent on microtubules (sanderson et al., 2000) and has been reported to be dependent on the a4l and a27l gene products (sanderson et al., 2000; ward, 2005) . following envelopment, the a35l and f12l gene products then regulate microtubule-dependent movement of intracellular enveloped viruses from the tgn to the plasma membrane (herrero-martínez et al., 2005; ward and moss, 2001 ). crystalline arrays a. iridoviruses iridoviruses are large dsdna viruses with genomes ranging from 100 to 210 kbp in length encoding between 100 and 230 proteins (williams et al., 2005) . much of the work on iridovirus replication has been carried out on the ranavirus frog virus 3 (fv3). fv3 genome synthesis occurs in the nucleus and cytoplasm. no nuclear inclusions have been reported during fv3 infection, and as such it is unclear how the nuclear replication stage is mediated. however, viral dna is initially synthesized as units that are 1-2 genomes in length and then transported to the cytoplasm where multiple length concatemers are produced (goorha, 1982) . b. cytoplasmic factories formed during iridovirus infection resemble aggresomes infection induces two cytoplasmic inclusions. viral factories form in the cytoplasm and become the major site of viral dna replication. fv3 also induces large crystalline arrays of viral particles which give rise to the iridescent coloring of purified virus, and hosts, that are characteristic of iridovirus infections. virus factories are electron lucent relative to the cytoplasm and contain viral membranes, partially assembled viruses, and are surrounded by rough er membranes and polysomes. fv3 factories also resemble aggresomes since they recruit intermediate filaments (fig. 7a) and mitochondria, some of which show darlington et al. (1966) with permission from elsevier. signs of damage (darlington et al., 1966; granoff et al., 1966; huang et al., 2006; tripier et al., 1977) . crystalline arrays of virus are associated with virus factories and can induce nuclear deformations that lead to kidneyshaped nuclei similar to those seen in asfv infection (fig. 7b ) and after aggresome formation (darlington et al., 1966; heath et al., 2001; johnston et al., 1998) . as seen for asfv and poxviruses, the intermediate filament vimentin plays an important role in replication (murti and goorha, 1983) . vimentin is phosphorylated during fv3 infection, prior to factory formation (chen et al., 1986; willis et al., 1979) , and temperature-sensitive mutants that are unable to phosphorylate vimentin do not form vimentin cages and are unable to proceed to late gene expression. drug treatment with taxol or colchicine (murti et al., 1988) showed that recruitment of vimentin to assembly sites requires dynamic, but not polymerizing microtubules, and microinjection of anti-vimentin antibody prevented recruitment of vimentin to factories. this allowed intrusion of cell components into assembly sites and reduced virus growth by 70-80% (murti et al., 1988) . vimentin may therefore provide a scaffold for iridovirus replication, maintaining a barrier between the cytoplasm and the contents of the virus factory. consistent with this hypothesis is the observation that during infection, polyribosomes and most newly synthesized viral proteins associate with intermediate filaments (murti and goorha, 1989) . fv3 factory formation may also be dependent on the early 108k protein, as it is recruited to factories in the absence of late protein synthesis (chinchar et al., 1984) . phycodnaviruses and the recently described giant virus mimivirus (la scola et al., 2003) induce replication complexes in the cytoplasm of infected ameba (meints et al., 1986; raoult et al., 2007) . the factories of the phycodnavirus paramecium bursaria chlorella virus 1 (pbcv-1) are electron translucent areas of the cytoplasm and contain viral membranes, electron-dense viroplasm, and assembling viruses. unlike many viral factories, a distinct order appears to be present in pbcv-1 virosomes. the assembling viruses are arranged at the periphery of the virosome/ factory, giving the appearance of a rosette (meints et al., 1986) . phycodnavirus replication and factory formation are not affected by a wide range of pharmacological disruptors of the cytoskeleton, including microtubule depolymerization by nocodazole and taxol, and depolymerization of actin by cytochalasin d (nietfeldt et al., 1992) . in this way, they differ from factories formed by large dna viruses such as asfv, vaccinia, and fv3. the successful cultivation of algae in the laboratory has allowed studies of the intracellular sites of replication of large icosahedral mclav-1 and hincv-1 viruses (wolf et al., 1998 (wolf et al., , 2000 . these viruses produce a latent infection that becomes apparent once the algae produce reproductive organs that become host to millions of virus particles. replication of these viruses begins in the nucleus, but the first evidence for virus assembly is provided by the appearance of electron-dense bodies next to the nucleus at sites of breakdown in the nuclear envelope. infection leads to stacking of er cisternae that may provide membranes for virus envelopment. the dense bodies remain next to the nucleus in large inclusions, and take on the angular shape characteristic of capsid assembly seen for iridoviruses and asfv. the nucleus eventually disintegrates, and the virus factory occupies most of the cytoplasm. herpesviruses are large dsdna viruses with genomes ranging in size from 120 to 250 kbp. herpesvirus genes are expressed in a regulated cascade starting with the immediate early a genes, then early b genes, and finally two subsets of late g genes, g 1 and g 2 . complete herpesvirus particles have four main layers, the core containing dna, an icosahedral capsid, a poorly defined layer of protein called tegument, and finally the viral envelope containing several glycoproteins. genome synthesis and packaging and capsid assembly occur in inclusions in the nucleus. nucleocapsids then obtain tegument in either the nucleus or the cytoplasm, or both, and the viral envelope is acquired exclusively in the cytoplasm [see mettenleiter (2002) and for more thorough analysis]. the transfer of virus from the nucleus to the cytoplasm and acquisition of tegument appears well defined for human herpesvirus 6 (hhv-6) (roffman et al., 1990) but is controversial for the alphaherpesviruses (campadelli-fiume and roizman, 2006; mettenleiter and minson, 2006) . the subcellular organization of herpesvirus replication complexes formed in the nucleus during the early stages of productive infection has been described in considerable detail. the inclusions function as sites of virus replication and contain the virally encoded proteins and host proteins needed for virus replication. interestingly, nuclear inclusions formed during herpes virus replication also contain cellular proteins involved in the control of dna damage and repair. these may be recruited into inclusions in response to virus genome replication, and whether they are beneficial or detrimental to virus replication is a subject of considerable interest [reviewed by everett (2006) ]. herpesviruses enter the cell by fusing their envelopes with the plasma membrane, whereon the naked nucleocapsids migrate to nuclear pores, possibly along microtubules (granzow et al., 1997; sodeik et al., 1997) [reviewed by smith and enquist (2002) ]. nuclear inclusions housing herpesvirus dna replication are globular and can occupy the majority of the nucleus (de bruyn kops and knipe, 1988; randall and dinwoodie, 1986; taylor et al., 2003) . they are identified through the presence of the viral dna-binding protein encoded by the ul29 gene, which is also known as infected cell protein 8 (icp8). a minimum set of seven genes, ul5, ul8, ul9, ul29, ul30, ul42, and ul52, has been identified as necessary for viral dna replication (challberg, 1991) . a plasmid transfection system has shown in vitro these can form globular nuclear compartments that are sites of 5-bromo-2 0 -deoxyuridine (brdu) incorporation and visually are similar to those formed during infection zhong and hayward, 1997) . nuclear inclusions organizing viral dna replication have been followed in real time by a recombinant virus expressing a gfp-icp8 fusion protein. small inclusions merge with adjacent replication complexes and increase in size to form globular replication complexes, which eventually fill most of the nucleus (randall and dinwoodie, 1986; taylor et al., 2003) . replication compartments are formed from a number of different discrete foci that are induced early in infection and whose interrelatedness is not fully understood. the initial stages of productive herpesvirus infection are, however, intimately linked with nuclear structures called nd10 bodies (illustrated in fig. 8 ) , maul et al. (1996) , review by borden (2002) ]. live cell studies have shown that the immediate early regulatory protein icp4, which binds viral dna, forms discrete foci as early as 30-min postinfection (fig. 8a) . these initially appear close to the nuclear envelope, possibly at sites where the genome first enters the nucleus following capsid disassembly at nuclear pores (everett and murray, 2005) , and are then seen throughout the nucleus (everett et al., 2004) . icp4 foci are seen juxtaposed to the nd10 marker promyelocytic leukemia protein (pml) some 60-min later. the early and late regulatory protein icp27 is recruited to icp4 foci 2-h postinfection and facilitates efficient early gene expression (everett et al., 2004) . during the same period, the immediate early regulatory protein, icp0, colocalizes with nd10 bodies, some of which are likely juxtaposed to icp4 bodies (everett et al., 2003) . icp0 mediates the ubiquitin and/or sumo-1-targeted proteasomal degradation of nd10 components (chelbi-alix and de everett, 2000; everett and maul, 1994; everett et al., 2004) . finally, parental genomes localize to icp4 foci (everett and murray, 2005) , and the icp4 foci enlarge into structures that resemble early icp8 replication compartments (everett and murray, 2005; everett et al., 2003) . formation of icp8 replication compartments (taylor and knipe, 2004) is also known to involve redistribution of nd10 bodies (burkham et al., 1998) . the relationship between the early icp4 structures associated with parental genome and the later icp8 compartments associated with replication and production of progeny genome is not clear; however, icp4 and icp8 both localize to late replication compartments (knipe et al., 1987) . a description of the relative and temporal distribution of the two proteins at early times awaits live cell studies following both proteins simultaneously. a second prominent nuclear inclusion induced by herpesvirus infection is the assemblon (ward et al., 1996b) . this is the site where capsid proteins accumulate and assemble into nucleocapsids (fig. 8b) . the assembly of herpesvirus nucleocapsids has been researched in great detail at the ultrastructural level facilitated by a cell-free system for reconstituting the particles (heymann et al., 2003; newcomb et al., 1994 newcomb et al., , 1996 . the mature herpesvirus capsid is icosahedral with a t ¼ 16 symmetry and is composed of 150 hexons and 11 pentons of the major capsid protein ul19. the place of the remaining penton is taken by a 12-mer of the portal protein ul6, which by analogy with bacteriophage may be the site of genome entry. nucleocapsids mature from fragile procapsids, through b capsids that lack dna and contain the internal scaffold protein ul26.5, to c capsids that contain the viral genome. the relationship between assemblons and sites of viral dna replication has been a topic of some controversy as some reports show direct colocalization (taus et al., 1998) , whereas others have shown a proximity (nalwanga et al., 1996; ward et al., 1996b) , similar to that seen between nd10 bodies and icp4 foci during the initial stages of infection. clearly, the dna has to reach the capsid in order to complete assembly, and it is likely that the different results are indicative of the dynamic interactions between different herpesvirus nuclear inclusions. the dna cleavage and packaging proteins encoded by the ul17 and ul32 genes are required for colocalization of viral dna and capsids (lamberti and weller, 1998; taus et al., 1998) . cells infected with a virus encoding a faulty ul32 gene exhibit nuclear localization of the capsid protein vp5 that is separate from replication sites (lamberti and weller, 1998) . similarly, in cells infected with mutants that lack functional ul17, the icp8 protein fails to colocalize with icp5 and icp35 (taus et al., 1998) . actin also plays an important role in the correct nuclear subcompartmentalization of viral proteins. infection with hhv-1 1 or suid herpesvirus-1 2 causes actin filaments to assemble in the nucleus, prior to the accumulation of capsid proteins (feierbach et al., 2006) . depolymerization of actin with latrunculin a inhibited correct nuclear compartmentalization of a representative capsid protein (vp26). vp26 colocalizes with the actin motor myosin va (feierbach et al., 2006) , and capsid movement within the nucleus is inhibited by the myosin motor inhibitor 2,3-butanedione monoxime (forest et al., 2005) . this suggests that the organization of nuclear inclusions involved in herpesvirus assembly is dependent on cellular actin filaments, and it will be interesting to see if the organization of inclusions housing viral dna replication sites is similarly dependent. other inclusion bodies have been reported in the nucleus of cells infected with herpesvirus. the tegument proteins vp22 and vp13/14 localize to inclusion bodies that align closely but do not overlap icp0/nd10/icp8 pre-replication complexes or assemblon inclusions (hutchinson et al., 2002) . ul55 also localizes to structures that overlap but are distinct from assemblons and dna replication complexes (fig. 8b ) . ul11 localizes to type iv and type v intranuclear dense bodies as well as virions and cytoplasmic ribbon structures (baines et al., 1995) . the alkaline dnase encoded by the ul12 gene localizes to discrete electron-dense bodies within the nucleus that also contain b-36 nucleolar protein (lopez-iglesias et al., 1988; puvion-dutilleul and pichard, 1986) . it is unknown whether these different structures are related to each other, whether they are homogenous accumulations of the individual herpesvirus protein(s), or if they are simply dead-end accumulations of protein. a large number and variety of cellular proteins accumulate at nuclear sites of herpesvirus replication and assembly. a comprehensive proteomic analysis of icp8 interacting proteins revealed more than 50 viral and cellular proteins that maybe recruited to dna replication sites (taylor and knipe, 2004) . a number of these interacting proteins were confirmed to localize to replication sites by microscopy experiments (taylor and knipe, 2004) , and these as well as proteins identified in other studies (leopardi et al., 1997; quadt et al., 2006; wilcock and lane, 1991) reveal that at least 23 cellular proteins are known to localize to nuclear inclusions involved in dna replication during herpesvirus infection (table i) . the functions of these proteins span the expected functions of nuclear genes, including dna replication, transcription, chromatin remodeling, dna repair, recombination, and nonhomologous end joining. of particular importance is the recruitment of rna polymerase ii, which is required to transcribe the viral genome. rna polymerase ii is phosphorylated during viral infection by icp22 and icp27, and the latter modification is required for targeting to replication complexes (dai-ju et al., 2006) . the role of all of these cellular genes in the viral replication cycle is poorly understood; however, cells deficient in wrn, a recq helicase family member, produced reduced virus yields while cells lacking ku86, part of a nonhomologous end-joining protein complex, produced increased yields of virus (taylor and knipe, 2004) . the implication therefore is that some cellular proteins may be actively recruited to replication complexes to aid viral replication, and some may be recruited by the cell as part of an antiviral response or sequestered by the virus in inclusions to subvert their antiviral nature. pml is induced by interferon, suggesting an antiviral role. many of the genes shown to be required for recruitment of pml to viral pre-replication sites are part of the minimal set of genes required to synthesize viral dna. recruitment of pml to viral replication sites is, for example, dependent on the viral dna polymerase (ul30), the origin binding protein (ul9 gene) and the helicase-primase complex (ul5, ul8, and ul52) (burkham et al., 2001) . recent evidence has suggested that this may be the reason why icp0 causes dispersal of pml early in infection. pml knockdown by short interfering rnas (sirna) facilitates productive replication of icp0 null mutants of herpesvirus (everett et al., 2004 ; moreover, icp0 null mutants are hypersensitive to interferon in a manner dependent on pml (chee et al., 2003) . this is of particular importance because icp0 plays a role in determining whether herpesvirus induces a quiescent or a productive, lytic infection (mossman and smiley, 2002) . the tegument layer of alphaherpesviruses is composed of at least 15 different proteins (mettenleiter, 2002) . us11, ul17, ul47, ul48, and ul49 are components of the tegument, and all are localized to the nucleus (if not exclusively) during the productive life cycle of the virus hutchinson et al., 2002; kopp et al., 2002; roller and roizman, 1992; taus et al., 1998) . ul48 may play a role in egress from the nucleus, though this has not been unequivocally established (mossman et al., 2000) . therefore, it is likely that some tegument proteins are acquired in or during viral egress from the nuclear inclusions. recently, cytoplasmic aggresome-like struct ure s have been desc ribed in ce lls infected with hhv-2. 1 these contain the major capsid protein, tegument proteins, envelope glycoproteins, and markers for the golgi complex (nozawa et al., 2004) . the latter finding is particularly interesting because herpesvirus envelopment involves membranes from the tgn turcotte et al., 2005) . hhv-5 3 is a betaherpesvirus and late during infection produces a juxtanuclear ''assembly compartment'' that again contains tegument proteins (pp150, pp28, and pp68), the major capsid protein, and viral envelope proteins (gb, gh, and gp65), suggesting a cytoplasmic site specialized for tegumentation and envelopment (fig. 8c) ; (adair et al., 2002; sanchez et al., 2000) . the precise role of the cytoplasmic assembly compartment is unclear. on the one hand, the concentration of glycoproteins and tegument proteins in one site may facilitate final stages of assembly prior to release from the cell. interestingly, in common with aggresomes induced by asfv and misfolded proteins, the cytoplasmic assembly compartment recruits chaperones and mitochondria and is dependent on microtubules and localizes to the microtubule organizing center. at present, the assembly compartments are not considered to be bona fide aggresomes because they are not surrounded by a collapsed cage of intermediate filaments (nozawa et al., 2004; sanchez et al., 2000) . it is nevertheless possible that these structures are related to aggresomes and are produced in response to a buildup of products resulting from nonproductive assembly pathways that occur late during infection. they may also contribute to the cytopathic effect seen in cells infected with hhv-5. hhv-5 infection results in cytomegaly characterized by increased cell size and intracellular water content. cytomegaly and virus replication are both dependent on the presence of extracellular na þ , and infection results in sequestration of the plasma membrane na-k-cl-cotransporter protein in large perinuclear structures that resemble the assembly compartment/ viral aggresome (maglova et al., 2004) . electron-dense bodies can be seen by electron microscopy within the cytoplasmic assembly compartments induced during hhv-5 infection (craighead et al., 1972) . dense bodies are enveloped and obtain viral glycoproteins but do not contain dna and are noninfectious. as can be seen in fig. 8c , dense bodies bud into membranes and appear as oversized enveloped viral particles without a dna containing core. dense bodies exit the cell to become extracellular dense bodies (craighead et al., 1972) . interestingly, hhv-5 immediate early ie1 proteins also become associated with extracellular dense bodies despite no reported localization to their intracellular relations (tsutsui and yamazaki, 1991) . purified extracellular dense bodies are mostly composed of ul83 but have a full complement of viral glycoproteins (irmiere and gibson, 1983) . the function of dense bodies remain unclear, and they may represent the end point of a nonproductive assembly pathway resulting from attempts to envelope capsids lacking genomes or may be used to deliver viral components to neighboring cells. interestingly, for human herpesvirus 6 (hhv-6), the tegument layer appears to be acquired within a dedicated structure that has been dubbed the tegusome (roffman et al., 1990) . this work is based on electron microscopy of cells infected with hhv-6 and shows tegusomes as intranuclear membrane compartments that abut the nuclear envelope (fig. 8d ). tegusomes may be cytoplasmic invaginations of the nuclear envelope into the nucleus because they appear to contain ribosomes and are sometimes in continuity with the cytoplasm. nucleocapsids appear to bud into the tegusome, capsids obtain a tegument layer, and then bud into cytoplasmic vacuoles where they acquire envelopes and exit the cell. adenovirus are medium-sized, nonenveloped dsdna viruses with genomes ranging from 26 to 45 kbp in length and virions of the order of 70-100 nm in diameter. like other dna viruses, they have an ordered cascade of transcripts, early, delayed early, and late types having been described. adenovirus transcripts are spliced to generate multiple transcripts from a given transcriptional unit. viral replication occurs in the nucleus, and adenovirus infection was utilized extensively as a model system for exploring different nuclear subcompartments. a productive infection of lytic adenovirus induces profound rearrangement of existing subcompartments and the induction of several new ones within the host nucleus. a study on the localization of the human adenovirus5 iva2 protein described 10 distinct nuclear and nucleolar subcompartments induced or associated with virus replication (lutz et al., 1996) , and these are listed in table i . earlier studies carried out before markers for specific nuclear subcompartments were available have described the structures in terms of shape and location (see table i ). during the initial stage of infection, viral rna , single-stranded dna (ssdna), and dsdna (puvion-dutilleul and pichard, 1992) are all synthesized in small fibrillar regions termed early replication sites. by the intermediate stage of replication, the ssdna is deposited in the center of these structures, while transcription and dsdna synthesis occur on the outside and begin to form an inclusion. the inclusion has a characteristic doughnut shape, and has been called the fibrogranular network. at late stages of infection, dsdna, viruses, and trace amounts of ssdna appear in large viral inclusions (besse and puvion-dutilleul, 1994; puvion-dutilleul and pichard, 1992) . targeting of the initial replicon is dependent on a dcmp modification of the preterminal protein (ptp), which enables ptp to form a complex with the dna polymerase and the genome (temperley and hay, 1992) . ptp mediates targeting of the heterotrimeric complex to the nuclear matrix (fredman and engler, 1993) , possibly through an interaction with cad (carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase) (angeletti and engler, 1998) . transcription and splicing are mediated by host proteins and viral rna, and non-snp rna splicing factor, hnrnp c proteins, and rna polymerase ii all colocalize with viral rna in nuclear inclusions. splicing small nuclear ribonucleoproteins (snrnps) colocalize with viral rna but not replication foci (pombo et al., 1994) , and snrnps then move to interchromatin granules late in infection, which is blocked by mutations in e4 (bridge et al., 2003) . a. rearrangement of host nuclear compartments during adenovirus replication like herpesvirus described above, adenovirus infection redistributes the components of nd10 bodies. prior to infection, pml is associated with interchromatin granules but is redistributed to the fibrillogranular matrix within the nucleus along with sp100, another nd10 component (carvalho et al., 1995) . later in infection, pml is redistributed once again from the fibrillogranular matrix to clear amorphous inclusions and protein crystals (puvion-dutilleul et al., 1995) . another study reported that sp100 and ndp55, but not pml, were relocated from nd10 bodies to viral inclusions (doucas et al., 1996) . while this is confusing, it is clear that adenovirus employs multiple mechanisms to reorganize pml. the initial movement of pml, sp100, and ndp55 to the fibrillogranular matrix occurs prior to viral dna synthesis and is dependent on the e4-orf3 11-kda protein (carvalho et al., 1995; doucas et al., 1996) . it may also be mediated by e1a proteins that colocalize with pml (carvalho et al., 1995) . e1b-55-kda protein also colocalizes with pml early on in infection, then associates with the periphery of replication centers; these interactions are mediated by the orf6 protein of the e4 transcriptional unit (lethbridge et al., 2003) . interestingly, e1b-55k and e4-orf3 target the mre11-rad50-nbs1 (mrn) complex to aggresomes for degradation (araujo et al., 2005; liu et al., 2005) . polyoma-and papillomaviruses are small double-stranded tumorigenic dna viruses with genomes of 5 and 8 kbp, respectively. replication and assembly of these two viruses follow similar strategies, and both involve nd10 bodies. the vp1 capsid protein of human polyomavirus jc is targeted to nd10 domains by vp2, vp3, and agnoprotein where they are assembled into virions (shishido-hara et al., 2004) . a similar process occurs during papillomavirus infection where the minor capsid protein, l2, is responsible for targeting capsomeres of the major capsid protein, l1, to nd10 domains (florin et al., 2002a) . this process involves l2-induced redistribution of nd10 bodies by targeting sp100 for proteasomal degradation. at this point the cellular daxx protein is recruited (florin et al., 2002b) . daax has multiple functions in the nucleus including transcriptional activation and modulating fas-mediated apoptosis [reviewed by salomoni and khelifi (2006) ]. its role in virus replication is at present unclear. one characteristic of papillomavirus infections is the appearance of nuclear and cytoplasmic inclusions in cells contained within warts. the size and number of inclusions is dependent on the type of papillomavirus and the site of infection. human papillomavirus 1 (hpv-1), for example, induces many small inclusions while hpv-4 induces one single inclusion that takes over most of the cytoplasm (croissant et al., 1985) . in vivo these structures label strongly with antiserum raised against e4 gene products which are the 17-kda e1 ∧ e4 and 16-kda e4 proteins (doorbar et al., 1986; rogel-gaillard et al., 1993) . inclusions can be induced in certain cell types in vitro by expressing e4 gene products. hpv-1 e4 staining reveals an initial association with the intermediate filament keratin and subsequent formation of inclusion bodies in the cytoplasm and nucleus (roberts et al., 2003; rogel-gaillard et al., 1993) . the hpv-1 cytoplasmic inclusions retain their association with keratin and appear to induce small cages surrounding e4 protein that are interconnected by keratin filaments (roberts et al., 2003) . the e4 gene gives rise to two proteins, the 17-kda e1 ∧ e4 which can induce cytoplasmic and nuclear inclusions whereas the 16-kda e4 can induce inclusions solely in the cytoplasm (rogel-gaillard et al., 1993) . interestingly, expression of e1 ∧ e4 gene product from hpv-16 induces the complete collapse of the keratin network, but not that of the microtubule or actin networks (doorbar et al., 1991) . it is unclear what the role of the inclusions is in viral replication or the pathology of infection. however, hpv-1 e4 expression induces the redistribution of nd10 to the periphery of nuclear inclusions in cells in culture, and similar signals are seen in vivo (roberts et al., 2003) . the temporal and functional connection between e4 and l1 redistribution of pml is unknown. members of the reoviridae family are dsrna viruses with segmented genomes and include the clinically important rotavirus and orbiviruses that cause diseases in human and animals. reoviruses are nonenveloped viruses with genome segments contained inside a virion $80 nm in diameter. the genome is encapsidated by two protein shells, an outer capsid and an inner core shell. the core contains the rdrp, capping enzymes, and the dsrna genome segments [reviewed in yue and shatkin (1998) and furuichi and shatkin (2000) ]. viruses are taken up by receptor-mediated endocytosis, the outer capsid is lost and the core is delivered into the cytoplasm. the core does not disassemble on entering cells and imports ribonucleoside triphosphates and s-adenosyl-l-methionine from the cytosol to synthesize and then export viral mrnas. in this way the core particle functions as a self-contained transcriptional unit and as such represents the replication complex. viral mrna transcribed in the cytoplasm make viral proteins that eventually form large perinuclear inclusions, called virus factories that function as sites of further virus replication and assembly. the reoviridae family contains 13 genera, and this chapter will concentrate on the two best characterized of these, the orthoreoviruses and rotaviruses. 1. formation of factories during orthoreoviruses replication and assembly a. the shape of orthoreovirus factories is determined by association with the cytoskeleton orthoreoviruses contain 10 genome segments which are classed by size and then numbered, that is l1 is large segment 1. large segments encode l genes, medium (m) segments encode m genes, and small (s) segments encode s genes. virus replication occurs in the cytoplasm in virus factories, and the majority of the virus-encoded proteins have been shown to localize completely or partially with factories (table i ). early observations revealed that different strains of orthoreoviruses induced factories with different appearances; orthoreovirus type 1 lang factories were filamentous while the factories of the dearing isolate of orthoreovirus type 3 were globular ( fig. 9a and b) . this difference maps precisely to a serine-proline switch at residue 208 of the m2 core protein . control of the localization of orthoreovirus factories reflects the degree of association m2 has with the microtubule network. filamentous virus m2 stabilizes microtubules to a greater relative degree than globular virus m2, and depolymerizing microtubules with nocodazole convert filamentous factories to globular ones. many of the events of orthoreovirus factory formation have been successfully reconstituted in vitro. a screen of orthoreovirus proteins revealed that mns, sns, and s3 were the first viral proteins to localize with viral mrna prior to the synthesis of progeny dsrna (antczak and joklik, 1992) . subsequently, it was discovered that expression of the mns protein of isolate dearing in the absence of other viral proteins induced a phasedense structure that was indistinguishable in appearance from that observed during wild-type infection . the shape of the artificial mns inclusion could be altered from globular to filamentous by coexpressing a m2 protein from a filamentous virus . similar experiments showed that coexpression of l1, l2, and s2 core surface proteins with mns caused them to localize to the mns inclusion (broering et al., 2004) . furthermore, the shape of the mns structure that the core proteins colocalized to could be altered to filamentous by coexpressing m2 from a filamentous virus (broering et al., 2004) . mns can also recruit sns, but not s3, to artificial inclusions (becker et al., 2003) , so other factors or conditions are necessary for complete assembly of an orthoreovirus factory. the precise domains involved in initiating factory formation are beginning to be elucidated. the minimal region of mns necessary for inclusion like body formation in vitro is the region composed of 250 c-terminal amino acids of the 721 residue proteins (broering et al., 2005) . residues 1-11 of sns are important for the interaction between sns and rna (gillian and nibert, 1998) , and treatment with rnase dissociates a proportion of mns from sns in coimmunoprecipiation experiments (miller et al., 2003) . interaction between mns and m2 is dependent on residues 1-40 or 41 of mns and residues 1-13 are necessary for interaction between mns and sns (miller et al., 2003) . it is likely that factory formation occurs through an interaction between mns and a sns-rna complex; this can then recruit m2 that will determine the globular or filamentous localization of the factory and hence the localization of the other viral proteins. orthoreovirus factories are clearly intimately associated with the microtubule network (fig. 9c) and have also been suggested to interact with intermediate filaments. orthoreovirus type 3 infection induces a redistribution of vimentin and viral inclusions reported to contain filamentous structures (sharpe et al., 1982) . it will be interesting to see if the in vitro factories induced by mns can also alter the distribution of the intermediate filament network. orthoreovirus factories are also ubiquitinated, and interestingly the nature of the factory determined the degree of ubiquitination; globular factories are prone to contain more ubiquitinated protein than filamentous ones . ubiquitination of orthoreovirus factories has been mapped to the m2 protein but is independent of the filamentous/globular factory determinate of m2, that is converting a filamentous factory to a globular factory does not lead to an increase in ubiquitinated m2. 2. formation of factories during rotavirus replication and assembly a. virus nonstructural proteins organize factory formation and virus assembly rotaviruses contain 11 genome segments of dsrna and like the orthoreoviruses replicate in cytoplasmic factories. rotavirus virions are composed of three protein layers. these are the core which contains the genome and polymerase, an inner capsid layer, and an outer capsid layer. the core and inner capsid layer comprise the double-layered particle (dlp), while the addition of the third capsid layer forms the mature triple-layered particle (tlp). the acquisition of the third capsid layer occurs after the virus buds into the er, and in doing so obtains a transient envelope. rotavirus factories are composed of electron-dense viroplasm often in proximity to membranes derived from the er (fig. 9d) (altenburg et al., 1980) . viroplasm contains high levels of nsp2 (fig. 9e ) and nsp5 which are thought to coordinate assembly of the factory and recruitment of structural proteins such as the inner core protein vp2 and viral polymerase vp1. the factory also contains double-layered rotaviruses, whereas the er membranes associated with the factory contain enveloped intermediates and tlp (arrowed in fig. 9d ). virus factories grow in size and decrease in number during the course of infection as neighboring factories merge (eichwald et al., 2004) . rotavirus factories appear to have an internal structure, as their centers occasionally appear more electron lucent than the periphery, giving a doughnut-like appearance (fig. 9e ). electron microscopy shows dlp at the periphery of the factory and this is (altenburg et al., 1980) consistent with fluorescent microscopy showing that the nonstructural protein nsp2 localizes to the center of the virus factory, whereas nsp5 and inner capsid protein vp6 localize to the periphery (eichwald et al., 2004; gonzález et al., 2000) . these different localizations could have functional relevance because vp6 binds the er-targeted nsp4 membrane protein and is implicated in the budding of dlps into er membranes associated with factories (silvestri et al., 2005) . therefore, a localization to the exterior of the factory may represent an organized progression of virus maturation from the interior of the viroplasm to the exterior. however, things are probably not that straightforward because vp6 is also part of the viral rna complex along with nsp2 (aponte et al., 1996) which, as noted above, is localized to the center of the viroplasm. virus factory-like structures can be introduced in vitro by coexpressing nsp2 and nsp5 (fabbretti et al., 1999) , and this is regulated by domains in the n-and c-termini (fabbretti et al., 1999) as well as the central portion of nsp5 (eichwald et al., 2002) . the process is also dependent on phosphorylation of nsp5, possibly by cellular casein kinase ii (eichwald et al., 2002) . structures similar to factories can also be induced by expressing the inner capsid protein vp6 in vitro (nilsson et al., 1998) . these structures look similar to factories in the electron microscope but lack electron-lucent areas and dlps. interestingly, expression of vp6 of group a simian rotavirus sa-11 induced globular structures, whereas expression of vp6 from group c porcine rotavirus cowden/amc-1 induced filamentous structures (nilsson et al., 1998) analogous to the difference between type 1 and type 3 orthoretroviruses. it is not clear if the difference in factory shape is solely determined by vp6 and if this involves differences in association of the factory components with microtubules. b. virus factories organize viral rna replication and translation the factory does provide the virus with a mechanism to organize viral rnas. positive-stranded viral rna is utilized as the template for synthesizing progeny dsrna genomes and as mrna for translating viral proteins. interestingly, sirna-targeted degradation of nsp1 rna blocks translation of the protein but does not block genome synthesis (silvestri et al., 2004) . furthermore, rna synthesis occurs in factories, but viral rna transcribed in vitro and introduced to infected cells after infection does not localize to factories. the implication of these experiments are that the factory enables rotavirus to sort viral rna into separate pools, one within the factory to act as a template for the rna polymerase and genome replication, and the other outside the factory where it translated on ribosomes to make viral proteins. it likely that this organization allows the virus factory to protect dsrna genomes from antiviral responses. arenaviruses are negative-stranded rna viruses that have two singlestranded genome segments which are packaged into 60-to 200-nmdiameter enveloped virions. lassa, junín, and manchupo viruses are responsible for emerging hemorraghic fevers in humans. arenaviruses induce moderately electron-dense inclusions in the cytoplasm that are composed of 20-to 25-nm-diameter granules identical to those seen within virus particles in the electron microscope (murphy et al., 1970) . the granules represent host ribosomes and between 2 and 10 are packaged into virions (pedersen, 1979) . the inclusions increase in size and density during infection until cytopathic effects are observed in cells (buckley, 1965; buckley and casals, 1970) and stain positive for viral antigens (young et al., 1987) ; however, it is unclear if they represent true virus factories. arenavirus replication is believed to occur in the cytoplasm but also requires a nuclear step as limited growth is observed in enucleated cells (banerjee et al., 1975) . the viral z protein may play a role in this as it is sufficient in vitro to shuttle pml from the nucleus to cytoplasmic inclusion bodies as occurs in vivo (borden et al., 1998) . n protein also localizes to discrete nuclear foci, as well as in the cytoplasm (young et al., 1987) , but the relationship to nd10 bodies and z protein is unknown. rabies virus is a neurotropic lyssavirus of the rhabdovirus family. rhabdovirus virions are bullet-shaped 180 â 75 nm 2 particles containing a single negative strand of rna. rabies induces two types of inclusion body in vitro, neither of which have been proven as replication sites. negri bodies are induced by street rabies viruses in infected neurons of the brain (negri, 1903) and are a good indicator for the presence of an infection site in tissue ( jackson et al., 2001) . different neuronal cell types appear to be more prone to negri bodies ( jackson et al., 2001) . negri bodies contain innerbodies (negri, 1909) and electron microscopic studies suggest the subcompartments may be cytoplasmic material engulfed by the coalescence of several smaller negri bodies (matsumoto, 1970) . the role of negri bodies in infection is poorly understood. initial em observations showed virions localized to some bodies in some cells (matsumoto et al., 1974) , and cytological staining show they contain genetic material, indicating they may be replication complexes. however, 3 h-thymidine or 3 h-uridine fail to label the structures, arguing against this conclusion (matsumoto, 1970) . fixed (brain-adapted laboratory strains) rabies can infect nonneuronal cell lines and in these cell types induce fuchsin-stained cytoplasmic structures (fcps) as well as negri-like bodies (ni et al., 1996) . fcps increase in size during infection that correlates with cytopathic effects and are composed of rabies glycoprotein and matrix protein, whereas negri bodies contain nucleocapsid (ni et al., 1996) . this chapter has described the changes to cell architecture that are induced during virus replication. we have focused on viruses that induce new cellular structures, such as inclusion bodies, virus factories, or replication complexes, to concentrate virus and host factors necessary for replication and assembly. much progress has been made in identifying which cellular components are used to generate these structures, and in some cases specific virus proteins have been identified that are able to induce them. virus inclusions often result in rearrangement of cellular membrane compartments and/or cytoskeleton. the functions of these organelles are carefully regulated in cells, and it is a challenge for the future to determine how viruses disrupt them for use as sites of replication and assembly. changes in cellular architecture may represent bystander responses to the stress associated with virus infection, and some viruses may replicate perfectly well without them. alternatively, viruses may have evolved to target key stages in the regulatory pathways that control organelle structure and function to generate sites that are essential for replication and assembly. given the coevolution of viruses with the cells that carry them, changes in cell structure induced during infection are likely to involve a combination of the two. it is also important to appreciate that many of the structures that have been studied to date have been generated by infecting tissue culture cells with attenuated viruses, often with disregard to the host range and tropism. it is possible that in the natural setting, changes in cell structure induced by viruses will be more subtle, particulary during persistent infections that occur without inflammation or cell lysis. the products of human cytomegalovirus genes ul23, ul24, ul43 and us22 are tegument components parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses mapping and sequence of the gene encoding the african swine fever virion protein of m r 11500 membrane permeabilization by poliovirus proteins 2b and 2bc the vaccinia virus superoxide dismutase-like protein (a45r) is a virion component that is nonessential for virus replication african swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein ultrastructural study of rotavirus replication in cultured cells assembly of african swine fever virus: role of polyprotein pp220 african swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum african swine fever virus protease, a new viral member of the sumo-1-specific protease family adenovirus preterminal protein binds to the cad enzyme at active sites of viral dna replication on the nuclear matrix reovirus genome segment assortment into progeny genomes studied by the use of monoclonal antibodies directed against reovirus proteins recovery and characterization of a replicase complex in rotavirus-infected cells by using a monoclonal antibody against nsp2 adenovirus type 5 e4orf3 protein targets the mre11 complex to cytoplasmic aggresomes redistribution of microtubules and golgi-apparatus in herpes-simplex virusinfected cells and their role in viral exocytosis the herpes simplex virus 1 ul11 proteins are associated with cytoplasmic and nuclear membranes and with nuclear bodies of infected cells requirement of cell nucleus for the replication of an arenavirus expression and characterization of a novel structural protein of human cytomegalovirus, pul25 preferential virosomal location of underphosphorylated h5r protein synthesized in vaccinia virus-infected cells reovirus sns protein is required for nucleation of viral assembly complexes and formation of viral inclusions reovirus sns and mns proteins form cytoplasmic inclusion structures in the absence of viral infection poliovirus proteins induce membrane association of gtpase adp-ribosylation factor hijacking components of the secretory pathway for replication of poliovirus rna does common architecture reveal a viral lineage spanning all three domains of life? deletion mutants of the herpes simplex virus type 1 ul8 protein: effect on dna synthesis and ability to interact with and influence the intracellular localization of the ul5 and ul52 proteins poliovirus mutant that contains a cold-sensitive defect in viral rna synthesis high resolution localization of replicating viral genome in adenovirus-infected hela cells the vaccinia virus a14.5l gene encodes a hydrophobic 53-amino-acid virion membrane protein that enhances virulence in mice and is conserved among vertebrate poxviruses intracellular distribution of poliovirus proteins and the induction of virus-specific cytoplasmic structures association of polioviral proteins of the p2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography the mechanisms of vesicle budding and fusion a structural dna binding protein of african swine fever virus with similarity to bacterial histone-like proteins pondering the promyelocytic leukemia protein (pml) puzzle: possible functions for pml nuclear bodies an arenavirus ring (zincbinding) protein binds the oncoprotein promyelocyte leukemia protein (pml) and relocates pml nuclear bodies to the cytoplasm endoplasmic reticulum architecture: structures in flux nuclear factor i is specifically targeted to discrete subnuclear sites in adenovirus type 2-infected cells mouse hepatitis virus replicase protein complexes are translocated to sites of m protein accumulation in the ergic at late times of infection an amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis c virus nonstructural protein 5a adenovirus early region 4 promotes the localization of splicing factors and viral rna in late-phase interchromatin granule clusters mammalian reovirus nonstructural protein mns forms large inclusions and colocalizes with reovirus microtubule-associated protein m2 in transfected cells reovirus nonstructural protein mns recruits viral core surface proteins and entering core particles to factory-like inclusions carboxyl-proximal regions of reovirus nonstructural protein mns necessary and sufficient for forming factory-like inclusions assembly of african swine fever virus: quantitative ultrastructural analysis in vitro and in vivo intracellular virus dna distribution and the acquisition of the nucleoprotein core during african swine fever virus particle assembly: ultrastructural in situ hybridisation and dnase-gold labelling characterization of african swine fever virion proteins j5r and j13l: immuno-localization in virus particles and assembly transcription factor yy1 is a vaccinia virus late promoter activator junín and tacaribe work in hela cells lassa fever, a new virus disease of man from west africa. 3. isolation and characterization of the virus nd10 protein pml is recruited to herpes simplex virus type 1 prereplicative sites and replication compartments in the presence of viral dna polymerase interactions of herpes simplex virus type 1 with nd10 and recruitment of pml to replication compartments the initiation of vaccinia infection the egress of herpesviruses from cells: the unanswered questions localization of structural proteins in african swine fever virus particles by immunoelectron microscopy vaccinia virus cores are transported on microtubules targeting of adenovirus e1a and e4-orf3 proteins to nuclear matrix-associated pml bodies characterization and structural localization of the reovirus l3 protein the african swine fever virus iap homolg is a late structural polypeptide herpes simplex virus dna replication promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated pml and sp100 proteins interaction of frog virus 3 with the cytomatrix. iv. phosphorylation of vimentin precedes the reorganization of intermediate filaments around the virus assembly sites copi activity coupled with fatty acid biosynthesis is required for viral replication early proteins are required for the formation of frog virus 3 assembly sites effects of a temperature sensitivity mutation in the j1r protein component of a complex required for vaccinia virus assembly membrane rearrangement and vesicle induction by recombinant poliovirus 2c and 2bc in human cells inhibition of cellular protein secretion by picornaviral 3a proteins the major structural protein of african swine fever virus, p73, is packaged into large structures, indicative of viral capsid or matrix precursors, on the endoplasmic reticulum involvement of the endoplasmic reticulum in the assembly and envelopment of african swine fever virus biochemical requirements of virus wrapping by the endoplasmic reticulum calcium store during envelopment of african swine fever virus a virally encoded chaperone specialized for folding of the major capsid protein of african swine fever virus in a nutshell: structure and assembly of the vaccinia virion inhibition of protein trafficking by coxsackievirus b3: multiple viral proteins target a single organelle nonviral microbodies with viral antigenicity produced in cytomegalovirus-infected cells specificity of cytopathic effect of cutaneous human papillomaviruses the poliovirus replication machinery can escape inhibition by an antiviral drug that targets a host cell protein brefeldin a inhibits cell-free, de novo synthesis of poliovirus a vaccinia virus core protein, p39, is membrane associated characterization of the vaccinia virus h3l envelope protein: topology and posttranslational membrane insertion via the c-terminal hydrophobic tail icp27 interacts with the c-terminal domain of rna polymerase ii and facilitates its recruitment to herpes simplex virus 1 transcription sites, where it undergoes proteasomal degradation during infection analysis of intracellular and intraviral localization of the human cytomegalovirus ul53 protein the development of vaccinia virus in earle's l strain cells as examined by electron microscopy electron microscopic study of the formation of poliovirus the uptake and development of reovirus in strain l cells followed with labeled viral ribonucleic acid and ferritin-antibody conjugates viruses and renal carcinoma of rana pipiens: ii. ultrastructural studies and sequential development of virus isolated from normal and tumor tissue acidic c terminus of vaccinia virus dnabinding protein interacts with ribonucleotide reductase formation of dna replication structures in herpes virus-infected cells requires a viral dna binding protein comparison of the intranuclear distributions of herpes simplex virus proteins involved in various viral functions origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors mhc i-dependent antigen presentation is inhibited by poliovirus protein 3a autophagy in innate and adaptive immunity poliovirus 3a protein limits interleukin-6 (il-6), il-8, and beta interferon secretion during viral infection inhibition of cellular protein secretion by poliovirus proteins 2b and 3a inhibition of endoplasmic reticulum-to-golgi traffic by poliovirus protein 3a: genetic and ultrastructural analysis the vaccinia virus e8r gene product: a viral membrane protein that is made early in infection and packaged into the virions' core the punctate sites of accumulation of vaccinia virus early proteins are precursors of sites of viral dna synthesis identification of the human papilloma virus-1a e4 gene products specific interaction between hpv-16 e1-e4 and cytokeratins results in collapse of the epithelial cell intermediate filament network adenovirus replication is coupled with the dynamic properties of the pml nuclear structure interaction of hepatitis c virus proteins with host cell membranes and lipids genetic and biochemical characterization of vaccinia virus genes d2l and d3r which encode virion structural proteins intracellular location and translocation of silent and active poliovirus replication complexes reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral rna synthesis formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral rna synthesis expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex rotavirus nsp5: mapping phosphorylation sites and kinase activation and viroplasm localization domains characterization of rotavirus nsp2/ nsp5 interactions and the dynamics of viroplasm formation replication of hepatitis c virus rna occurs in a membranebound replication complex containing nonstructural viral proteins and rna amphipathic helix-dependent localization of ns5a mediates hepatitis c virus rna replication the african swine fever virus non-structural protein pb602l is required for the formation of the icosahedral capsid of the virus particle the vaccinia virus d5 protein, which is required for dna replication, is a nucleic acid-independent nucleoside triphosphatase icp0 induces the accumulation of colocalizing conjugated ubiquitin interactions between dna viruses, nd10 and the dna damage response hsv-1 ie protein vmw110 causes redistribution of pml nd10 components relocate to sites associated with herpes simplex virus type 1 nucleoprotein complexes during virus infection recruitment of herpes simplex virus type 1 transcriptional regulatory protein icp4 into foci juxtaposed to nd10 in live, infected cells formation of nuclear foci of the herpes simplex virus type 1 regulatory protein icp4 at early times of infection: localization, dynamics, recruitment of icp27, and evidence for the de novo induction of nd10-like complexes pml contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by icp0 two non-structural rotavirus proteins, nsp2 and nsp5, form viroplasm-like structures in vivo alpha-herpesvirus infection induces the formation of nuclear actin filaments assembly and translocation of papillomavirus capsid proteins reorganization of nuclear domain 10 induced by papillomavirus capsid protein l2 active intranuclear movement of herpesvirus capsids adenovirus precursor to terminal protein interacts with the nuclear matrix in vivo and in vitro alphavirus rna replicase is located on the cytoplasmic surface of endosomes and lysosomes the ul48 tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions viral and cellular mrna capping: past and prospects characterization of the african swine fever virus protein p49: a new late structural polypeptide role of the host cell nucleus in the replication of african swine fever virus dna differential requirements for copi coats in formation of replication complexes among three genera of picornaviridae amino terminus of reovirus nonstructural protein sns is important for ssrna binding and nucleoprotein complex formation relative localization of viroplasmic and endoplasmic reticulum-resident rotavirus proteins in infected cells localization of the herpes simplex virus type 1 65-kilodalton dna-binding protein and dna polymerase in the presence and absence of viral dna synthesis frog virus 3 dna replication occurs in two stages a new superfamily of putative ntpbinding domains encoded by genomes of small dna and rna viruses nidovirales: evolving the largest rna virus genome rna replication of mouse hepatitis virus takes place at double-membrane vesicles identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons subcellular localization of the us3 protein kinase of herpes simplex virus type 2 modulation of p53 cellular function and cell death by african swine fever virus viruses and renal carcinoma of rana pipiens. i. the isolation and properties of virus from normal and tumor tissue ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment structure and assembly of intracellular mature vaccinia virus: thin-section analyses recherches sur la pathologie et étiologie de l'infection vaccinique et varioleuse subcellular localization of host and viral proteins associated with tobamovirus rna replication aggresomes resemble sites specialized for virus assembly membrane association facilitates the correct processing of pp220 during production of the major matrix proteins of african swine fever virus vaccinia virus intracellular enveloped virions move to the cell periphery on microtubules in the absence of the a36r protein upregulation of major histocompatibility complex class i on liver cells by hepatitis c virus core protein via p53 and tap1 impairs natural killer cell cytotoxicity deep-etch em reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic ''honeycomb'' surface coat dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy electron microscopy of viruses in thin sections of cells grown in culture a ubiquitin conjugating enzyme encoded by african swine fever virus vaccinia virus intracellular mature virions contain only one lipid membrane a poxvirus host range protein, cp77, binds to a cellular protein, hmg20a, and regulates its dissociation from the vaccinia virus genome in cho-k1 cells electron microscopic examination of the viromatrix of rana grylio virus in a fish cell line the hepatitis c virus nonstructural protein 4b is an integral endoplasmic reticulum membrane protein molecular chaperone hsp90 is important for vaccinia virus growth in cells evidence against an essential role of copii-mediated cargo transport to the endoplasmic reticulum-golgi intermediate compartment in the formation of the primary membrane of vaccinia virus sequential localization of two herpes simplex virus tegument proteins to punctate nuclear dots adjacent to icp0 domains biogenesis of poxviruses: role of a-type inclusions and host cell membranes in virus dissemination isolation and characterization of a noninfectious virionlike particle released from cells infected with human strains of cytomegalovirus involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin a the periphery of nuclear domain 10 (nd10) as site of dna virus deposition common origin of four diverse families of large eukaryotic dna viruses quantitative study of the infection in brain neurons in human rabies subversion of cellular autophagosomal machinery by rna viruses colocalization of the herpes simplex virus 1 ul4 protein with infected cell protein 22 in small, dense nuclear structures formed prior to onset of dna synthesis aggresomes: a cellular response to misfolded proteins african swine fever virus infection disrupts centrosome assembly and function transport of african swine fever virus from assembly sites to the plasma membrane is dependent on microtubules and conventional kinesin african swine fever virus induces filopodia-like projections at the plasma membrane a study on the morphological and cyto-immunological relationship between the inclusions of variola, cowpox, rabbitpox, vaccinia (variola origin) and vaccinia ihd and a consideration of the term ''guarnieri body synthesis, subcellular localization and vp16 interaction of the herpes simplex virus type 2 ul46 gene product the deacetylase hdac6 regulates aggresome formation and cell viability in response to misfolded protein stress subcellular localization of hepatitis c viral proteins in mammalian cells interferon-independent increases in class i major histocompatibility complex antigen expression follow flavivirus infection cellular autophagy: surrender, avoidance and subversion by microorganisms stages in the nuclear association of the herpes simplex virus transcriptional activator protein icp4 foot-and-mouth disease virus replication sites form next to the nucleus and close to the golgi apparatus, but exclude marker proteins associated with host membrane compartments nonstructural protein precursor ns4a/b from hepatitis c virus alters function and ultrastructure of host secretory apparatus poliovirus protein 3a binds and inactivates lis1, causing block of membrane protein trafficking and deregulation of cell division identification and characterization of the pseudorabies virus tegument proteins ul46 and ul47: role for ul47 in virion morphogenesis in the cytoplasm the role of a 21-kda viral membrane protein in the assembly of vaccinia virus from the intermediate compartment replication complex of human parechovirus 1 a giant virus in amoebae vaccinia virus gene a18r dna helicase is a transcript release factor the herpes simplex virus type 1 cleavage/packaging protein, ul32, is involved in efficient localization of capsids to replication compartments human cytomegalovirus structural components: intracellular and intraviral localization of p38 characterization of rubella virus replication complexes using antibodies to double-stranded rna association of herpes simplex virus regulatory protein icp22 with transcriptional complexes containing eap, icp4, rna polymerase ii, and viral dna requires posttranslational modification by the u(l)13 proteinkinase nuclear matrix localization and sumo-1 modification of adenovirus type 5 e1b 55k protein are controlled by e4 orf6 protein development by self-digestion: molecular mechanisms and biological functions of autophagy cell culture-grown hepatitis c virus is infectious in vivo and can be recultured in vitro secretory protein trafficking and organelle dynamics in living cells functional order of assembly of herpes simplex virus dna replication proteins into prereplicative site structures flavivirus infection up-regulates the expression of class i and class ii major histocompatibility antigens on and enhances t cell recognition of astrocytes in vitro adenovirus exploits the cellular aggresome response to accelerate inactivation of the mrn complex evidence that a mechanism for efficient flavivirus budding upregulates mhc class i herpes simplex virus type 1-induced modifications in the distribution of nucleolar b-36 protein formation of herpes simplex virus type 1 replication compartments by transfection: requirements and localization to nuclear domain 10 herpes simplex virus type 1 prereplicative sites are a heterogeneous population: only a subset are likely to be precursors to replication compartments nucleoplasmic and nucleolar distribution of the adenovirus iva2 gene product visualization and functional analysis of rna-dependent rna polymerase lattices wrapping things up about virus rna replication assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns2a and ns4a markers for trans-golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells rubella virus replication complexes are virus-modified lysosomes perinuclear localization of na-k-clcotransporter protein after human cytomegalovirus infection microtubule-dependent organization of vaccinia virus core-derived early mrnas into distinct cytoplasmic structures relationship between vaccinia virus intracellular cores, early mrnas, and dna replication sites small dense nuclear bodies are the site of localization of herpes simplex virus 1 u(l)3 and u(l)4 proteins and of icp22 only when the latter protein is present novel acylation of poxvirus a-type inclusion proteins characterization of the african swine fever virus structural protein p14.5 a dna binding protein rabies virus further studies on the replication of rabies and rabies-like viruses in organized cultures of mammalian neural tissues nuclear domain 10 as preexisting potential replication start sites of herpes simplex virus type-1 inhibition of poliovirus rna synthesis by brefeldin a the trans golgi network is lost from cells infected with african swine fever virus identification of the orthopoxvirus p4c gene, which encodes a structural protein that directs intracellular mature virus particles into a-type inclusions assembly site of the virus pbcv-1 in a chlorella-like green alga: ultrastructural studies herpesvirus assembly and egress egress of alphaherpes viruses herpesvirus assembly: a tale of two membranes flock house virus rna polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion reovirus sns protein localizes to inclusions through an association requiring the mns amino terminus increased ubiquitination and other covariant phenotypes attributed to a strain-and temperature-dependent defect of reovirus core protein m2 the vaccinia virus-encoded uracil dna glycosylase has an essential role in viral dna replication rifampicin prevents virosome localization of l65, an essential vaccinia virus polypeptide effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: 2bc but not 3a blocks endoplasmic reticulum-to-golgi transport inhibition of the secretory pathway by the foot-and-mouth disease virus 2bc protein is reproduced by co-expression of 2b with 2c and the site of inhibition is determined by the subcellular location of 2c involvement of spicules in the formation of vaccinia virus envelopes elucidated by a conditional lethal mutant modulation of transporter associated with antigen processing (tap)-mediated peptide import into the endoplasmic reticulum by flavivirus infection high-pressure freezing in the study of animal pathogens the ultrastructure of the developing replication site in foot-and-mouth disease virus-infected bhk-38 cells membrane association of the rna-dependent rna polymerase is essential for hepatitis c virus rna replication structure and development of viruses observed in the electron microscope. ii. vaccinia and fowl pox viruses rifampicin: a specific inhibitor of vaccinia virus assembly herpes simplex virus icp0 and icp34.5 counteract distinct interferon-induced barriers to virus replication evidence that herpes simplex virus vp16 is required for viral egress downstream of the initial envelopment event ultrastructural study of african swine fever virus replication in cultures of swine bone marrow cells mitochondrial distribution and function in herpes simplex virusinfected cells identification by mass spectroscopy of three major early proteins associated with virosomes in vaccinia virusinfected cells arenoviruses in vero cells: ultrastructural studies interaction of frog virus-3 with the cytoskeleton. i. altered organization of microtubules, intermediate filaments, and microfilaments synthesis of frog virus 3 proteins occurs on intermediate filament-bound polyribosomes a functional role for intermediate filaments in the formation of frog virus 3 assembly sites localization of adenovirus-encoded dna replication proteins in the nucleus by immunogold electron microscopy the ul 16 gene product of herpes simplex virus 1 is a virion protein that colocalizes with intranuclear capsid proteins beitrag zum studium de aetiologie der tollwuth ü ber die morphologie und den entwicklungszyklus des parasiten der tollwut the poxviral ring protein p28 is a ubiquitin ligase that targets ubiquitin to viral replication factories the subcellular distribution of multigene family 110 proteins of african swine fever virus is determined by differences in c-terminal kdel endoplasmic reticulum retention motifs african swine fever virus causes microtubule dependent dispersal of the trans-golgi network and slows delivery of membrane protein to the plasma membrane cell-free assembly of the herpes simplex virus capsid assembly of the herpes simplex virus capsid: characterization of intermediates observed during cell-free capsid formation poliovirus protein 3a inhibits tumor necrosis factor (tnf)-induced apoptosis by eliminating the tnf receptor from the cell surface ultrastructural studies of kunjin virus-infected aedes albopictus cells studies on unusual cytoplasmic structures which contain rabies virus envelope proteins chlorella virus pbcv-1 replication is not affected by cytoskeletal disruptors assembly of viroplasm and virus-like particles of rotavirus by a semliki forest virus replicon virus factories: associations of cell organelles for viral replication and morphogenesis identification and characterization of the ul7 gene product of herpes simplex virus type 2 formation of aggresome-like structures in herpes simplex virus type 2-infected cells and a potential role in virus assembly subcellular distribution of the foot-and-mouth disease virus 3a protein in cells infected with viruses encoding wild-type and bovine-attenuated forms of 3a host cell nuclear proteins are recruited to cytoplasmic vaccinia virus replication complexes entry of vaccinia virus and cell-cell fusion require a highly conserved cysteine-rich membrane protein encoded by the a16l gene african swine fever virus dutpase is a highly specific enzyme required for efficient replication in swine macrophages herpes simplex virus 1 gene products required for dna replication: identification and overexpression bfa bodies'': a subcompartment of the endoplasmic reticulum quantitative sumo-1 modification of a vaccinia virus protein is required for its specific localization and prevents its self-association reovirus core protein m2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules isolation of cowpox virus a-type inclusions and characterization of their major protein component structural components and replication of arenaviruses open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex characterization of vaccinia virus intracellular cores: implications for viral uncoating and core structure events in vaccinia virus-infected cells following the reversal of the antiviral action of rifampicin localization of rotavirus antigens in infected cells by ultrastructural immunocytochemistry ultrastructural localization of rotavirus antigens using colloidal gold immunoelectron microscopy analysis of hcmv gpul73 (gn) localization vaccinia virus infection disrupts microtubule organization and centrosome function adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells coronavirus replication complex formation utilizes components of cellular autophagy identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins detection and subcellular localization of the turnip yellow mosaic virus 66k replication protein in infected cells clustered charge-to-alanine mutagenesis of the vaccinia virus a20 gene: temperaturesensitive mutants have a dna-minus phenotype and are defective in the production of processive dna polymerase activity simultaneous detection of highly phosphorylated proteins and viral major dna binding protein distribution in nuclei of adenovirus type 5-infected hela cells viral alkaline nuclease in intranuclear dense bodies induced by herpes simplex infection segregation of viral double-stranded and singlestranded dna molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization replicating single-stranded adenovirus type 5 dna molecules accumulate within well-delimited intranuclear areas of lytically infected hela cells distribution of viral rna molecules during the adenovirus type 5 infectious cycle in hela cells rearrangements of intranuclear structures involved in rna processing in response to adenovirus infection adenovirus infection induces rearrangements in the intranuclear distribution of the nuclear body-associated pml protein deletion of the fiber gene induces the storage of hexon and penton base proteins in pml/sp100-containing inclusions during adenovirus infection tatabinding protein and tbp-associated factors during herpes simplex virus type 1 infection: localization at viral dna replication sites quantitative analysis of the hepatitis c virus replication complex intranuclear localization of herpes simplex virus immediate-early and delayed-early proteins: evidence that icp 4 is associated with progeny virus dna the discovery and characterization of mimivirus, the largest known virus and putative pneumonia agent the vaccinia virus f17r protein interacts with actin autophagosomes: biogenesis from scratch? tobacco mosaic virus infection induces severe morphological changes of the endoplasmic reticulum sequence of morphological changes in epithelial cell cultures infected with poliovirus temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication vaccinia virus nonstructural protein encoded by the a11r gene is required for formation of the virion membrane characterization of the ul33 gene product of herpes simplex virus 1 the vaccinia virus 39-kda protein forms a stable complex with the p4a/4a major core protein early in morphogenesis endoplasmic reticulum-golgi intermediate compartment membranes and vimentin filaments participate in vaccinia virus assembly cytopathogenic effect of poliomyelitis viruses 'in vitro' on human embryonic tissues the nd10 component promyelocytic leukemia protein relocates to human papillomavirus type 1 e4 intranuclear inclusion bodies in cultured keratinocytes and in warts african swine fever virus structural protein p54 is essential for the recruitment of envelope precursors to assembly sites african swine fever virus pb119l protein is a flavin adenine dinucelotide-linked sulfhydryl oxidase putative site for the acquisition of human herpesvirus 6 virion tegument cytopathic effect in human papillomavirus type 1-induced inclusion warts: in vitro analysis of the contribution of two forms of the viral e4 protein polyhedrin structure migration of mitochondria to viral assembly sites in african swine fever virus-infected cells replication of african swine fever virus dna in infected cells the herpes simplex virus 1 rna binding protein us11 is a virion component and associates with ribosomal 60s subunits regulated cleavages at the west nile virus ns4a-2k-ns4b junctions play a major role in rearranging cytoplasmic membranes and golgi trafficking of the ns4a protein characterization of the vaccinia virus a35r protein and its role in virulence functional analysis of adenovirus protein ix identifies domains involved in capsid stability, transcriptional activity, and nuclear reorganization adenovirus protein ix sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation a cellular factor is required for transcription of vaccinia viral intermediate-stage genes african swine fever virus is wrapped by the endoplasmic reticulum cellular copii proteins are involved in production of the vesicles that form the poliovirus replication complex vaccinia virus membrane proteins p8 and p16 are cotranslationally inserted into the rough endoplasmic reticulum and retained in the intermediate compartment daxx: death or survival protein? properly folded nonstructural polyprotein directs the semliki forest virus replication complex to the endosomal compartment viral rna replication in association with cellular membranes accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly the vaccinia virus a27l protein is needed for the microtubule-dependent transport of intracellular mature virus particles poliovirus infection and expression of the poliovirus protein 2b provoke the disassembly of the golgi complex, the organelle target for the antipoliovirus drug ro-090179 infection with foot-and-mouth disease virus results in a rapid reduction of mhc class i surface expression monoclonal antibodies specific for african swine fever virus proteins vaccinia virusinduced microtubule-dependent cellular rearrangements cellular origin and ultrastructure of membranes induced during poliovirus infection cytoplasmic organization of poxvirus dna replication a positive-strand rna virus replication complex parallels form and function of retrovirus capsids alternate, virusinduced membrane rearrangements support positive-strand rna virus genome replication expression of the vaccinia virus a2.5l redox protein is required for virion morphogenesis the interaction of mammalian reoviruses with the cytoskeleton of monkey kidney cv-1 cells colocalization and membrane association of murine hepatitis virus gene 1 products and de novo-synthesized viral rna in infected cells mechanism of virus occlusion into a-type inclusion during poxvirus infection autophagy in health and disease: a double-edged sword major and minor capsid proteins of human polyomavirus jc cooperatively accumulate to nuclear domain 10 for assembly into virions immunofluorescent localization of double-stranded rna in reovirus-infected cells rotavirus replication: plus-sense templates for double-stranded rna synthesis are made in viroplasms rotavirus glycoprotein nsp4 is a modulator of viral transcription in the infected cell proteolytic processing in african swine fever virus: evidence for a new structural polyprotein pp62 mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes break ins and break outs: viral interactions with the cytoskeleton of mammalian cells ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex assembly of vaccinia virus revisited: de novo membrane synthesis or acquisition from the host? assembly of vaccinia virus: incorporation of p14 and p32 into the membrane of the intracellular mature virus microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus adenovirus infection targets the cellular protein kinase ck2 and rna-activated protein kinase (pkr) into viral inclusions of the cell nucleus selection for temperature-sensitive mutations in specific vaccinia virus genes: isolation and characterization of a virus mutant which encodes a phosphonoacetic acid-resistant, temperature-sensitive dna polymerase vimentin rearrangement during african swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase ii micromorphology of fl cells infected with polio and coxsackie viruses remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles characterization of the african swine fever virion protein j18l vaccinia virus a30l protein is required for association of viral membranes with dense viroplasm to form immature virions vaccinia virus g7l protein interacts with the a30l protein and is required for association of viral membranes with dense viroplasm to form immature virions a complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions evidence for an essential catalytic role of the f10 protein kinase in vaccinia virus morphogenesis physical and functional interactions between vaccinia virus f10 protein kinase and virion assembly proteins a30 and g7 external scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice the herpes simplex virus 1 ul 17 gene is required for localization of capsids and major and minor capsid proteins to intranuclear sites where viral dna is cleaved and packaged proteomics of herpes simplex virus replication compartments: association of cellular dna replication, repair, recombination, and chromatin remodeling proteins with icp8 herpes simplex virus replication compartments can form by coalescence of smaller compartments recognition of the adenovirus type 2 origin of dna replication by the virally encoded dna polymerase and preterminal proteins induction of intracellular membrane rearrangements by hav proteins 2c and 2bc vaccinia virus dna replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei frog virus 3 morphogenesis: effect of temperature and metabolic inhibitors subcellular distribution of the major immediate early proteins of human cytomegalovirus changes during infection herpes simplex virus type 1 capsids transit by the trans-golgi network, where viral glycoproteins accumulate independently of capsid egress architecture of the flaviviral replication complex. protease, nuclease, and detergents reveal encasement within double-layered membrane compartments the vaccinia virus 4c and a-type inclusion proteins are specific markers for the intracellular mature virus particle alfalfa mosaic virus replicase proteins p1 and p2 interact and colocalize at the vacuolar membrane orf1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication immunolocalization of vaccinia virus structural proteins during virion formation identification and localization of genetic material of african swine fever virus by autoradiography identification and characterization of the ul14 gene product of herpes simplex virus type 2 production of infectious hepatitis c virus in tissue culture from a cloned viral genome visualization and characterization of the intracellular movement of vaccinia virus intracellular mature virions vaccinia virus intracellular movement is associated with microtubules and independent of actin tails a novel herpes simplex virus 1 gene, ul43.5, maps antisense to the ul43 gene and encodes a protein which colocalizes in nuclear structures with capsid proteins assemblons: nuclear structures defined by aggregation of immature capsids and some tegument proteins of herpes simplex virus 1 maintenance of golgi structure and function depends on the integrity of er export identification of nuclear export signal in ul37 protein of herpes simplex virus type 2 the vaccinia virus i3l gene product is localized to a complex endoplasmic reticulum-associated structure that contains the viral parental dna a proline-rich region in the coxsackievirus 3a protein is required for the protein to inhibit endoplasmic reticulum-to-golgi transport effects of picornavirus 3a proteins on protein transport and gbf1-dependent cop-i recruitment a viral protein that blocks arf1-mediated cop-i assembly by inhibiting the guanine nucleotide exchange factor gbf1 proteins c and ns4b of the flavivirus kunjin translocate independently into the nucleus ultrastructure of kunjin virus-infected cells: colocalization of ns1 and ns3 with doublestranded rna, and of ns2b with ns3, in virus-induced membrane structures localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells aggresomes and autophagy generate sites of virus replication a decade of advances in iridovirus research macromolecular synthesis in cells infected by frog virus 3. xi. a ts mutant of frog virus 3 that is defective in late transcription relationship between rna polymerase ii and efficiency of vaccinia virus replication virus assembly in hincksia hincksiae (ectocarpales, phaeophyceae) an electron and fluorescence microscopic study assembly of a large icosahedral dna virus, mclav-1, in the marine alga myriotrichia clavaeformis (dictyosiphonales, phaeophyceae) a myristylated membrane protein encoded by the vaccinia virus l1r open reading frame is the target of potent neutralizing monoclonal antibodies subcellular localization, stability, and trans-cleavage competence of the hepatitis c virus ns3-ns4a complex expressed in tetracycline-regulated cell lines vaccinia virus late transcription is activated in vitro by cellular heterogeneous nuclear ribonucleoproteins characterization of the ul55 gene product of herpes simplex virus type 2 the vaccinia virus a9l gene encodes a membrane protein required for an early step in virion morphogenesis localization of an arenavirus protein in the nuclei of infected cells enzymatic and control functions of reovirus structural proteins nuclear localization of a double-stranded rna-binding protein encoded by the vaccinia virus e3l gene characterization of an early gene encoding for dutpase in rana grylio virus assembly of complete, functionally active herpes simplex virus dna replication compartments and recruitment of associated viral and cellular proteins in transient cotransfection assays robust hepatitis c virus infection in vitro the coronavirus replicase: insights into a sophisticated enzyme machinery key: cord-002774-tpqsjjet authors: nan title: section ii: poster sessions date: 2017-12-01 journal: j urban health doi: 10.1093/jurban/jti137 sha: doc_id: 2774 cord_uid: tpqsjjet nan food and nutrition programs in large urban areas have not traditionally followed a systems approach towards mitigating food related health issues, and instead have relied upon specific issue interventions char deal with downstream indicators of illness and disease. in june of 2004, the san francisco food alliance, a group of city agencies, community based organizations and residents, initiated a collahorarive indicator project called rhe san francisco food and agriculture assessment. in order to attend to root causes of food related illnesses and diseases, the purpose of the assessment is to provide a holistic, systemic view of san francisco\'s food system with a focus on three main areas that have a profound affect on urban public health: food assistance, urban agriculture, and food retailing. using participatory, consensus methods, the san francisco food alliance jointly developed a sec of indicators to assess the state of the local food system and co set benchmarks for future analysis. members collected data from various city and stare departments as well as community based organizations. through the use of geographic information systems software, a series of maps were created to illustrate the assets and limitations within the food system in different neighborhoods and throughout the city as a whole. this participatory assessment process illustrates how to more effectively attend to structural food systems issues in large urban areas by ( t) focusing on prevention rather than crisis management, (2) emphasizing collaboration to ensure institutional and structural changes, and (3) aptly translating data into meaningful community driven prevention activities. to ~xplore the strategies to overcome barriers to population sample, we examined the data from three rapid surveys conducted at los angeles county (lac). the surveys were community-based partic· 1patory surveys utilizing a modified two-stage cluster survey method. the field modifications of the method resulted in better design effect than conventional cluster sample survey (design effect dose to that if the survey was done as simple random sample survey of the same size). the surveys were con· ducte~ among parents of hispanic and african american children in lac. geographic area was selected and d1.v1ded int.o small c~usters. in the first stage, 30 clusters were selected with probability proportionate to estimated size of children from the census data. these clusters were enumerated to identify and develop a list of households with eligible children from where a random sample was withdrawn. data collectmn for consented respondents involved 10-15 minutes in-home interview and abstraction of infor· ma~ion from vaccine record card. the survey staff had implemented community outreach activities designed to fost~r an~ maintain community trust and cooperation. the successful strategies included: developing re.lat1on .w.1th local community organizations; recruitment of community personnel and pro· vide them with training to conduct the enumeration and interview; teaming the trained community introduction: though much research has been done on the health and social benefits of pet ownership for many groups, there have been no explorations of what pet ownership can mean to adults who are marginalized, living on fixed incomes or on the street in canada. we are a community group of researchers from downtown toronto. made up of front line staff and community members, we believe that community research is important so that our concerns, visions, views and values are presented by us. we also believe that research can and should lead to social change. method: using qualitative and exploratory methods, we have investigated how pet ownership enriched and challenged the lives of homeless and transitionally housed people. our research team photographed and conducted one-on-one interviews with 11 pet owners who have experienced home· lessncss and live on fixed incomes. we had community participation in the research through a partnership with the fred vicror centre camera club. many of the fred victor centre camera club members have experienced homelessness and being marginalized because of poverty. the members of the dub took the photos and assisted in developing the photos. they also participated in the presenta· tion of our project. results: we found that pet ownership brings important health and social benefits to our partici· pants. in one of the most poignant statements, one participant said that pet ownership " ... stops you from being invisible." another commented that "well, he taught me to slow down, cut down the heavy drugs .. " we also found that pet ownership brings challenges that can at times be difficult when one is liv· ing on a fixed income. we found that the most difficult thing for most of the pet owners was finding affordable vet care for their animals. conclusion: as a group, we decided that research should only be done if we try to make some cha.nges about what we have learned. we continue the project through exploring means of affecting social change--for example, ~eti.tions and informing others about the result of our project. we would like to present our ~mdmgs and experience with community-based participatory action resea.rch m an oral. presentarton at yo~r conference in october. our presentation will include com· mumty representation ~f. both front-hne staff and people with lived experience of marginalization and homdessness. if this is not accepted as an oral presentation, we are willing to present the project m poster format. introduction the concept of a healthy city was adopted by the world health organization some time ago and it includes strong support for local involvement in problem solving and implementation of solutions. while aimed at improving social, economic or environmental conditions in a given community, more significantly the process is considered to be a building block for poliq reform and larger scale 'hange, i.e. "acting locally while thinking globally." neighbourhood planning can he the entry point for citizens to hegin engaging with neighbours on issues of the greater common good. methods: this presentation will outline how two community driven projects have unfolded to address air pollution. the first was an uphill push to create bike lanes where car lanes previously existed and the second is an ongoing, multi-sectoral round table focused on pollution and planning. both dt•monstrate the importance of having support with the process and a health focus. borrowing from traditions of "technical aid"• and community development the health promoter /planner has incorporated a range of "determinants of health" into neighbourhood planning discussions. as in most urban conditions the physical environment is linked to a range of health stressors such as social isolation, crowding, noise, lack of open space /recreation, mobility and safety. however typical planning processes do not hring in a health perspective. health as a focus for neighbourhood planning is a powerful starti_ng point when discussing transportation planning or changing land-uses. by raising awareness on determmants of health, citizens can begin to better understand how to engage in a process and affect change. often local level politics are involved and citizens witness policy change in action. the environmental liaison committee and the dundas east hike lanes project resulted from local level initiatives aimed at finding solutions to air pollution -a priority identified hy the community. srchc supported the process with facilitation and technical aid. _the processs had tangible results that ultimately improve living conditions and health. •tn the united kmgdom plannm in the 60's established "technical aid" offices much like our present day legal aid system to provide professional support and advocacy for communities undergoing change. p2-15 (c) integrating community based research: the experience of street health, a community service agency i.aura cowan and jacqueline wood street health began offering services to homeless men and women in east downtown ~oronto in 1986. nursing stations at drop-in centres and shelters were fo~lowed by hiv/aids prevent10~, harm reduction and mental health outreach, hepatitis c support, sleeping bag exchange, and personal tdennfication replacement and storage programs. as street health's progi;ams expanded, so to~ did the agency:s recognition that more nee~ed t~ be done to. address the underl~ing causes of, th~ soct~l and economic exclusion experienced by its clients. knowing t.h~t. a~voca~y ts. helped by . evtd~nce , street he.alt~ embarked on a community-based research (cbr) initiative to 1dent1fy commumty-dnven research priorities within the homeless and underhoused population. methods: five focus groups were conducted with 46 homeless people, asking participants to identify positive and negative forces in their lives, and which topics were important to take action on and learn more about. findings were validated through a validation meeting with participants. results: participants identified several important positive and negative forces in their lives. key positive forces included caring and respectful service delivery, hopefulness and peer networks. key negative forces included lack of access to adequate housing and income security, poor service delivery and negative perceptions of homeless people. five topics for future research emerged from the process, focusing on funding to address homelessness and housing; use of community services for homeless people; the daily survival needs of homeless people and barriers to transitioning out of homelessness; new approaches to service delivery that foster empowerment; and policy makers' understanding of poverty and homelessness. conclusions: although participants expressed numerous issues and provided much valuable insight, definitive research ideas and action areas were not clearly identified by participants. however, engagement in a cbr process led to some important lessons and benefits for street health. we learned that the community involvement of homeless people and front-line staff is critical to ensuring relevance and validity for a research project; that existing strong relationships with community parmers are essential to the successful implementation of a project involving marginalized groups; and that an action approach focusing on positive change can make research relevant to directly affected people and community agency staff. street health benefited from using a cbr approach, as the research process facilitated capacity building among staff and within the organization as a whole. p2-16 (c) a collaborative process to achieve access to primary health care for black women and women of colour: a model of community based participartory research notisha massaquoi, charmaine williams, amoaba gooden, and tulika agerwal in the current healthcare environment, a significant number of black women and women of color face barriers to accessing effective, high quality services. research has identified several issues that contribute to decreased access to primary health care for this population however racism has emerged as an overarching determinant of health and healthcare access. this is further amplified by simultaneous membership in multiple groups that experience discrimination and barriers to healthcare for example those affected by sexism, homelessness, poverty, homophobia and heterosexism, disability and hiv infection. the collaborative process to achieve access to primary health care for black women and women of colour project was developed with the university of toronto faculty of social work and five community partners using a collaborative methodology to address a pressing need within the community ro increase access to primary health care for black women and women of colour. women's health in women's hands community health centre, sistering, parkdale community health centre, rexdale community health centre and planed parenthood of toronto developed this community-based participatory-action research project to collaboratively barriers affecting these women, and to develop a model of care that will increase their access to health services. this framework was developed using a process which ensured that community members from the target population and service providers working in multiculrural clinical settings, were a part of the research process. they were given the opportunity to shape the course of action, from the design of the project to the evaluation and dissemination phase. empowerment is a goal of the participatory action process, therefore, the research process has deliberately prioritized _ro enabling women to increase control over their health and well-being. in this session, the presenters will explore community-based participatory research and how such a model can be useful for understanding and contextualizing the experiences of black women and women of colour. they will address. the development and use of community parmerships, design and implementation of the research prorect, challenges encountered, lessons learnt and action outcomes. they will examine how the results from a collaborative community-based research project can be used as an action strategy to poster sessions v61 address che social determinants of women health. finally the session will provide tools for service providers and researchers to explore ways to increase partnerships and to integrate strategies to meet the needs of che target population who face multiple barriers to accessing services. lynn scruby and rachel rapaport beck the purpose of this project was ro bring traditionally disenfranchised winnipeg and surrounding area women into decision-making roles. the researchers have built upon the relationships and information gachered from a pilot project and enhanced the role of input from participants on their policy prioriries. the project is guided by an advisory committee consisting of program providers and community representatives, as well as the researchers. participants included program users at four family resource cencres, two in winnipeg and two located rurally, where they participated in focus groups. the participants answered a series of questions relating to their contact with government services and then provided inpuc as to their perceptions for needed changes within government policy. following data analysis, the researchers will return to the four centres to share the information and continue che discussion on methods for advocating for change. recommendations for program planning and policy development and implementation will be discussed and have relevance to all participants in the research program. women's health vera lefranc, louise hara, denise darrell, sonya boyce, and colleen reid women's experiences with paid and unpaid work, and with the formal and informal economies, have shifted over the last 20 years. in british columbia, women's employability is affected by government legislation, federal and provincial policy changes, and local practices. two years ago we formed the coalition ior women's economic advancement to explore ways of dealing with women's worsening economic situations. since the formation of the coalition we have discussed the need for research into women's employabilicy and how women were coping and surviving. we also identified how the need to document the nature of women's employability and reliance on the informal economy bore significanc mechodological and ethical challenges. inherent in our approach is a social model of women's health that recognizes health as containing social, economic, and environmental determinants. we aim to examine the social contexc of women's healch by exploring and legitimizing women's own experiences, challenging medical dominance in understandings of health, and explaining women's health in terms of their subordination and marginalizacion. through using a feminist action research (far) methodology we will explore the relationship between women's employability and health in 4 communities that represent bricish columbia's social, economic, cultural/ethnic, and geographic diversities: skidegate, fort st . .john, lumhy, and surrey. over the course of our 2 year project, in each community we will establish and work with advisory committees, hire and train local researchers, conduct far (including a range of qualitative methods), and support action and advocacy. since the selected communities are diverse, the ways that the research unfolds will 1·ary between communities. expected outcomes, such as the provision of a written report and resources, the establishment of a website for networking among the communities, and a video do.:umentary, are aimed at supporting the research participants, coalition members, and advisory conuniuces in their action efforts. p2t 9 (c) health & housing: assessing the impact of transitional housing for people living with hiv i aids currently, there is a dearth of available literature which examines supporrive housing for phas in the canadian context. using qualitative, one-on-one interviews we investigace the impact of transitional housing for phaswho have lived in the up to nine month long hastings program. our post<'r pr<·senta-t1on will highlight research findings, as well as an examination of transitional housing and th<· imp;kt it has on the everyday lives of phas in canada. this research is one of two ground breaking undertakings within the province of ontario in which fife house is involved. p2-20 (c) eating our way to justice: widening grassroots approaches to food security, the stop community food centre as a working model charles l.evkoe food hanks in north america have come co play a central role as the widespread response to growing rates of hunger. originally thought to be a short term-solution, over the last 25 years, they have v62 poster sessions be · · · 1· d wi'thi'n society by filling the gaps in the social safety net while relieving govemcome mst1tut1ona 1ze . . . t f the ir responsibilities. dependent on corporate donations and sngmauzmg to users, food banks men so th' . ·11 i i . are incapable of addressing the structural cause~ of ~u~ger. 1s pres~ntation w1 e~~ ore a ternanve approaches to addressing urban food security while bmldmg more sustamabl.e c~mmumt1es. i:nrough the f t h st p community food centre, a toronto-based grassroots orgamzanon, a model is presented case 0 e 0 h'l k' b 'id · b that both responds to the emergency food needs of communities w 1 e wor mg to. u1 ~ sustama le and just food system. termed, the community food centre model. (cfc), ~he s~op is worki?g to widen its approach to issues of food insecurity by combining respectful ~1rect service wit~ com~~mty ~evelop ment, social justice and environmental sustainability. through this approach, various critical discourses around hunger converge with different strategic and varied implications for a~ion. as a plac~-based organization, the stop is rooted within a geographical space and connected directly to a neighbourhood. through working to increase access to healthy food, it is active in maintaining people's dignity, building a strong and democratic community and educating for social change. connected to coalitions and alliances, the stop is also active in organizing across scales in connection with the global food justice movement. inner city shelter vicky stergiopoulos, carolyn dewa, katherine rouleau, shawn yoder, and lorne tugg introduction: in the city of toronto there are more than 32,000 hostel users each year, many with mental health and addiction issues. although shelters have responded in various ways to the health needs of their clients, evidence on the effectiveness of programs delivering mental health services to the home· less in canada has been scant. the objective of this community based research was to provide a forma· tive evaluation of a multi-agency collaborative care team providing comprehensive care for high needs clients at toronto's largest shelter for homeless men. methods: a logic model provided the framework for analysis. a chart review of 56 clients referred over a nine month period was completed. demographic data were collected, and process and outcome indicators were identified for which data was obtained and analyzed. the two main outcome measures were mental status and housing status 6 months after referral to the program. improvement or lack of improvement in mental status was established by chart review and team consensus. housing outcomes were determined by chart review and the hostel databases. results: of the clients referred 75% were single and 98% were unemployed. forty four percent had a psychiatric hospitalization within the previous two years. the prevalence of severe and persistent men· tal illness, alcohol and substance use disorders were 60%, 26% and 37% respectively. six months after referral to the program 37% of clients had improved mental status and 41 % were housed. logistic regression controlling for the number of general practitioner and psychiatrist visits, presence of person· ality or substance use disorder and treatment non adherence identified two variables significantly associ· ated mental status improvement: the number of psychiatric visits (or, 1.92; 95% ci, 1.29-2.84) and treatment non adherence (or, 0.086; 95% ci, 0.01-0.78). the same two variables were associated with housing outcomes. history of forensic involvement, the presence of a personality or substance use disorder and the number of visits with a family physician were not significantly associated with either outcome. conclusions: despite the limitations in sample sire and study design, this study can yield useful informa· tion to program planners. our results suggest that strategies to improve treatment adherence and access to mental health specialists can improve outcomes for this population. although within primary care teams the appropriate collaborative care model for this population remains to be established, access to psychiatric follow up, in addition to psychiatric assessment services, may be an important component of a successful program. mount sinai hospital (msh) has become one of the pre-eminent hospitals in the world by contributing to the development of innovative approaches to effective health care and disease prevention. recently, the hospital has dedicated resources towards the development of a strategy aimed at enhancing the hospital's integration with its community partners. this approach will better serve the hospital in the current health care environment where local health integration networks have been struck to enhance and support local capacity to plan, coordinate and integrate service delivery. msh has had early success with developing partnerships. these alliances have been linked to programs serving key target populations with _estabhshe~. points of access to msh. recognizing the need to build upon these achievements to remain compe~mve, the hospital has developed a community integration strategy. at the forefront of this strategy is c.a.r.e (community advisory reference engine): the hospital's compendium of poster sessions v63 community partners. as a single point of access to community partner information, c.a.r.e. is more than a database. c.a.r.e. serves as the foundation for community-focused forecasting and a vehicle for inter and intra-organizational knowledge transfer. information gleaned from the catalog of community parmers can be used to prepare strategic, long-term partnership plans aimed at ensuring that a comprehensive array of services can be provided to the hospital community. c.a.r.e. also houses a permanent record of the hospital's alliances. this prevents administrative duplication and facilitates the formation of new alliances that best serve both the patient and the hospital. c.a.r.e. is not a stand-alone tool and is most powerful when combined with other aspects of the hospital's community integration strategy. it iscxpected that data from the hospital's community advisory committees and performance measurement department will also be stored alongside stakeholder details. this information can then be used to drive discussions at senior management and the board, ensuring congruence between stakeholder, patient and hospital objectives. the patient stands to benefit from this strategy. the unique, distinct point of reference to a wide array of community services provides case managers and discharge planners with the information they need to connect patients with appropriate community services. creating these linkages enhances the patient's capacity to convalesce in their homes or places of residence and fosters long-term connections to neighborhood supports. these connections can be used to assist with identifying patients' ongoing health care needs and potentially prevent readmission to hospital. introduction: recruiting high-risk drug users and sex workers for hiv-prevention research has often been hampered by limited access to hard to reach, socially stigmatized individuals. our recruitment effom have deployed ethnographic methodology to identify and target risk pockets. in particular, ethnographers have modeled their research on a street-outreach model, walking around with hiv-prevention materials and engaging in informal and structured conversations with local residents, and service providers, as well as self-identified drug users and sex workers. while such a methodology identifies people who feel comfortable engaging with outreach workers, it risks missing key connections with those who occupy the margins of even this marginal culture. methods: ethnographers formed a women's laundry group at a laundromat that had a central role as community switchboard and had previously functioned as a party location for the target population. the new manager helped the ethnographers invite women at high risk for hiv back into the space, this time as customers. during weekly laundry sessions, women initiated discussions about hiv-prevention, sexual health, and eventually, the vaccine research for which the center would be recruiting women. ra.its: the benefits of the group included reintroducing women to a familiar locale, this time as customers rather than unwelcome intruders; creating a span of time (wash and dry) to discuss issues important to me women and to gather data for future recruitment efforts; creating a location to meet women encountered during more traditional outreach research; establishing the site as a place for potential retention efforts; and supporting a local business. women who participated in the group completed a necessary household task while learning information that they could then bring back to the community, empowering them to be experts on hiv-prevention and vaccine research. some of these women now assist recruitment efforts. the challenges included keeping the group women-only, especially after lunch was provided, keeping the membership of the group focused on women at risk for hiv, and keeping the women in the group while they did their laundry. conclusion: public health educators and researchers can benefit from identifying alternate congregation sites within risk pockets to provide a comfortable space to discuss hiv prevention issues with high-risk community members. in our presentation, we will describe the context necessary for similar research, document the method's pitfalls and successes, and argue that the laundry group constitutes an ethical, respectful, community-based method for recruitment in an hiv-prevention vaccine trial. p3-0t (c) upgrading inner city infrastructure and services for improved environmental hygiene and health: a case of mirzapur in u.p. india madhusree mazumdar in urgency for agricultural and industrial progress to promote economic d.evelopment follo_wing independence, the government of india had neglected health promotion and given less emphasis on infrastructure to promote public health for enhanced human pro uct1v_ity. ong wit r~p1 m astrucrure development, which has become essential if citie~ are to. act ~s harbmger.s of econ~nuc ~owth, especially after the adoption of the economic liberalization policy, importance _is a_lso ~emg g1ve.n to foster environmental hygiene for preventive healthcare. the world health orga~1sat10~ is also trj:'1!1g to help the government to build a lobby at the local level for the purpose by off~rmg to mrroduce_ its heal.thy city concept to improve public health conditions, so as to reduce th_e disease burden. this pape~ 1s a report of the efforts being made towards such a goal: the paper descr~bes ~ c~se study ?f ~ small city of india called mirzapur, located on the banks of the nver ganga, a ma1or lifeline of india, m the eastern part of the state of uttar pradesh, where action for improvement began by building better sanitation and environmental infrastructure as per the ganga action plan, but continued with an effort to promote pre· ventive healthcare for overall social development through community participation in and around the city. asthma physician visits in toronto, canada tara burra, rahim moineddin, mohammad agha, and richard glazier introduction: air pollution and socio-economic status are both known to be associated with asthma in concentrated urban settings but little is known about the relationship between these factors. this study investigates socio-economic variation in ambulatory physician consultations for asthma and assesses possible effect modification of socio-economic status on the association between physician visits and ambient air pollution levels for children aged 1 to 17 and adults aged 18 to 64 in toronto, canada between 1992 and 2001. methods: generalized additive models were used to estimate the adjusted relative risk of asthma physician visits associated with an interquartile range increase in sulphur dioxide, nitrogen dioxide, pm2.5, and ozone, respectively. results: a consistent socio-economic gradient in the number of physician visits was observed among children and adults and both sexes. positive associations between ambient concentrations of sul· phur dioxide, nitrogen dioxide and pm2.5 and physician visits were observed across age and sex strata, whereas the associations with ozone were negative. the relative risk estimates for the low socio-«onomic group were not significantly greater than those for the high socio-economic group. conclusions: these findings suggest that increased ambulatory physician visits represent another component of the public health impact of exposure to urban air pollution. further, these results did not identify an age, sex, or socio-economic subgroup in which the association between physician visits and air pollution was significantly stronger than in any other population subgroup. eco-life-center (ela) in albania supports a holistic approach to justice, recognizing the environ· mental justice, social justice and economic justice depend upon and support each other. low income cit· izens and minorities suffer disproportionately from environmental hazards in the workplace, at home, and in their communities. inadequate laws, lax enforcement of existing environmental regulations, and ~ea.k penalties for infractions undermine environmental protection. in the last decade, the environmental 1ust1ce m~ve~ent in tirana metropolis has provided a framework for identifying and exposing the links ~tween irrational development practices, disproportionate siting of toxic facilities, economic depres· s1on, and a diminished quality of life in low-income communities and communities of color. the envi· ~onmental justice agenda has always been rooted in economic, racial, and social justice. tirana and the issues su.rroun~ing brow~fields redevelopment are crucial points of advocacy and activism for creating ~ubstantia~ social chan~~ m low-income communities and communities of color. we engaging intensively m prevcnnng co'.'1mumnes, especially low income or minority communities, from being coerced by gov· ernmenta~ age_nc1es or companies into siting hazardous materials, or accepting environmentally hazard· ous_ practices m order to create jobs. although environmental regulations do now exist to address the environmental, health, and social impacts of undesirable land uses, these regulations are difficult to poster sessions v65 enforce because many of these sites have been toxic-ridden for many years and investigation and cleanup of these sites can be expensive. removing health risks must be the main priority of all brown fields action plans. environmental health hazards are disproportionately concentrated in low-income communities of color. policy requirements and enforcement mechanisms to safeguard environmental health should be strengthened for all brownfields projects located in these communities. if sites are potentially endangering the health of the community, all efforts should be made for site remediation to be carried out to the highest cleanup standards possible towards the removal of this risk. the assurance of the health of the community should take precedence over any other benefits, economic or otherwise, expected to result from brownfields redevelopment. it's important to require from companies to observe a "good neighbor" policy that includes on-site visitations by a community watchdog committee, and the appointment of a neighborhood environmentalist to their board of directors in accordance with the environmental principles. vancouver 1976-2001 michael buzzelli, jason su, and nhu le this is the second paper of research programme concerned with the geographical patterning of environmental and population health at the urban neighbourhood scale. based on the vancouver metropolitan region, the aim is to better understand the role of neighbourhoods as epidemiological spaces where environmental and social characteristics combine as health processes and outcomes at the community and individual levels. this paper builds a cohort of commensurate neighbourhoods across all six censuses periods from 1976 to 2001, assembles neighbourhood air pollution data (several criterion/health effects pollutants), and providing an analysis to demonstrate how air pollution systematically and consistently maps onto neighbourhood socioeconomic markers, in this case low education and lone-parent families. we conclude with a discussion of how the neighbourhood cohort can be further developed to address emergent priorities in the population and environmental health literatures, namely the need for temporally matched data, a lifecourse approach, and analyses that control for spatial scale effects. solid waste management and environment in mumbai (india) by uttam jakoji sonkamble and bairam paswan abstract: mumbi is the individual financial capital of india. the population of greater mumbai is 3,326,837 and 437 sq. km. area. the density of population 21, 190 per sq. km. the dayto-day administration and rendering of public services within gr. mumbai is provided by the brihan mumbai mahanagar palika (mumbai corporation of gr. mumbai) that is a body of 221 elected councilors on a 5-year team. mumcial corporation provides varies conservancy services such as street sweeping, collection of solid waste, removal and transportation, disposal of solid waste, disposal of dead bodies of animals, construction, maintance and cleaning of urinals and public sanitary conveniences. the solid waste becoming complicated due to increase in unplanned urbanization and industrialization, the environment has deteriorated significantly due to inter, intra and international migration stream to mumbai. the volume of inter state migration to mumbai is considerably high i.e. 20.89 lakh and international migrant 0.77 lakh have migrated to mumbai. present paper gives the view on solid waste management and its implications to environment and health. pollution from a wide varity of emission, such as from automobiles and industrial activities, has reached critical level in mumbai, causing respiratory, ocular, water born diseases and other health problems. sources of generation of waste are -household waste, commercial waste, institutional waste, street sweeping, silt removed from drain/nallah/cleanings. disposal of solid waste in gr. mumbai done under 1 incineration 2. processing to produce organic manure. 3. vermi-composting 4. landfill the study shows that the quantity of waste disposal of through processing and conversion to organic ~anure is about 200-240 m.t. per day. the processing is done by a private agency m/s excel industries ltd. who had set up a plant at the chincholi dumping ground in western mumbai for this purpose. the corporation is also disposal a plant of its waste mainly market waste through the environment friendly, natural pro-ces~ known as vermi-composing about 100 m.t. of market waste is disposed of in this manner at the various sites. there are four land fill sites are available and 95 percent of the waste matter generated m mumbai is disposed of through landfill. continuous flow of migrant and increa~e in slum population is a complex barrier in the solid waste management whenever community pamc1pat1on work strongly than only we can achieved eco-friendly environment in mumbai. persons exposed to residential craffic have elevated races of respiratory morbidity an~ ~ortality. since poverty is an important determinant of ill-health, some h~ve argued that t~es~ assoc1at1ons may relate to che lower socioeconomic status of those living along ma1or roads. our ob1ect1ve was to evaluate the association between traffic intensity at home and hospital admissions for respiratory diagnoses among montreal residents older than 60 years. morning peak traffic estimates from the emmej2 montreal traffic model (motrem98) were used as an indicator of exposure to road traffic outside the homes of those hospitalised. the influence of socioeconomic status on the relationship between traffic intensity and hospital admissions for respiratory diagnoses was explored through assessment of confounding by lodging value, expressed as the dollar average over road segments. this indicator of socioeconomic status, as calculated from the montreal property assessment database, is available at a finer geographic scale than socioeconomic information accessible from the canadian census. there was an inverse relationship between traffic intensity estimates and lodging values for those hospitalised (rho -0.23, p 3160 vehicles during che 3 hour morning peak), even after adjustment for lodging value (crude or 1.35, cl95% 1.22-1.49; adjusted or 1.13, cl95% 1.02-1.25). in montreal, elderly persons living along major roads are at higher risk of being hospitalised for respiratory illnesses, which appears not simply to reflect the fact that those living along major roads are at relative economic disadvantage. the paper argues that human beings ought to be at the centre of the concern for sustainable development. while acknowledging the importance of protecting natural resources and the ecosystem in order to secure long term global sustainability, the paper maintain that the proper starting point in the quest for urban sustainability in africa is the 'brown agenda' to improve che living and working environment of che people, especially che urban poor who face a more immediate environmental threat to their health and well-being. as the un-habitat has rightly observed, it is absolutely essential "to ensure that all people have a sufficient stake in the present to motivate them to take part in the struggle to secure the future for humanity.~ the human development approach calls for rethinking and broadening the narrow technical focus of conventional town planning and urban management in order to incorporate the emerging new ideas and principles of urban health and sustainability. i will examine how cities in sub-saharan africa have developed over the last fifty years; the extent to which government policies and programmes have facilitated or constrained urban growth, and the strategies needed to achieve better functioning, safer and more inclusive cities. in this regard i will explore insights from the united nations conferences of the 1990s, especially local agenda 21 of the rio summit, and the istanbul declaration/habitat agenda, paying particular attention to the principles of enablement, decentralization and partnership canvassed by these movements. also, i will consider the contributions of the various global initiatives especially the cities alliance for cities without slums sponsored by the world bank and other partners; che sustainable cities programme, the global campaigns for good governance and for secure tenure canvassed by unhabit at, the healthy cities programme promoted by who, and so on. the concluding section will reflect on the future of the african city; what form it will take, and how to bring about the changes needed to make the cities healthier, more productive and equitable, and better able to meet people's needs. heather jones-otazo, john clarke, donald cole, and miriam diamond urban areas, as centers of population and resource consumption, have elevated emissions and concentrations of a wide range of chemical contaminants. we have developed a modeling framework in which we first ~stimate the emissions and transport of contaminants in a city and second, use these estimates along with measured contaminant concentrations in food, to estimate the potential health risk posed by these che.micals. the latter is accomplished using risk assessment. we applied our modeling framework to consider two groups of chemical contaminants, polycyclic aromatic hydrocarbons (pah) a.nd the flame re~ardants polybrominated diphenyl ethers (pbde). pah originate from vehicles and stationary combustion sources. ~veral pah are potent carcinogens and some compounds also cause noncancer effects. pbdes are additive flame retardants used in polyurethane foams (e.g., car seats, furniture) 105fer sessions v67 and cl~ equipm~nt (e.g., compute~~· televisio~s). two out of three pbdes formulations are being voluntarily phased by mdustry due to rmng levels m human tissues and their world-wide distribution. pbdes have been .related to adv.erse neurological, developmental and reproductive effects in laboratory ijlimals. we apphed our modelmg framework to the city of toronto where we considered the southcattral area of 21 by 21 km that has a population of 1.3 million. for pah, local vehicle traffic and area sources contribute at least half of total pah in toronto. local contributions to pbdes range from 57-85%, depending on the assumptions made. air concentrations of both compounds are about 10 times higher downtown than 80 km north of toronto. although measured pah concentrations in food date to the 1980s, we estimate that the greatest exposure and contribution to lifetime cancer risk comes from ingestion of infant formula, which is consistent with toxicological evidence. the next greatest exposure and cancer risk are attributable to eating animal products (e.g. milk, eggs, fish). breathing downtown air contributes an additional 10 percent to one's lifetime cancer risk. eating vegetables from a home garden localed downtown contributes negligibly to exposure and risk. for pbdes, the greatest lifetime exposure comes through breast milk (we did not have data for infant formula), followed by ingestion of dust by the toddler and infant. these results suggest strategies to mitigate exposure and health risk. p4-01 (a) immigration and socioeconomic inequalities in cervical cancer screening in toronto, canada aisha lofters, rahim moineddin, maria creatore, mohammad agha, and richard glazier llltroduction: pap smears are recommended for cervical cancer screening from the onset of sexual activity to age 69. socioeconomic and ethnoracial gradients in self-reported cervical cancer screening have been documented in north america but there have been few direct measures of pap smear use among immigrants or other socially disadvantaged groups. our purpose was to investigate whether immigration and socioeconomic factors are related to cervical cancer screening in toronto, canada. methods: pap smears were identified using fee codes and laboratory codes in ontario physician service claims (ohip) for three years starting in 1999 for women age 18-41 and 42-66. all women with any health system contact during the three years were used as the denominator. social and economic factors were derived from the 2001 canadian census for census tracts and divided into quintiles of roughly equal population. recent registrants, over 80% of whom are expected to be recent immigrants to canada, were identified as women who first registered for health coverage in ontario after january 1, 1993. results: among 397,967 women age 18-41 and 328,885 women age 42-66, 55.3% and 55.5%, rtspcctively, had pap smears within three years. low income, low education, recent immigration, visible minority and non-english language were all associated with lower rates (least advantaged quintile:most advantaged quintile rate ratios were 0.84, 0.90, 0.81, 0.85, 0.83, respectively, p < 0.05 for all). similar gradients were found in both age groups. recent registrants comprised 22.5% of women and had mm;h lower pap smear rates than non-recent registrants (37.2 % versus 63. 7% for women age 18-41 and 35.9% versus 58.2% for women age 42-66). conclnsions: pap smear rates in toronto fall well below those dictated by evidence-based practice. at the area level, immigration, visible minority, language and socioeconomic characteristics are associated with pap smear rates. recent registrants, representing a largely immigrant group, have particularly low rates. efforts to improve coverage of cervical cancer screening need to be directed to all ~omen, their providers and the health system but with special emphasis on women who recently arrived m ontario and those with social and economic disadvantage. challeges faced: a) most of the resources are now being ~pent in ~reventing the sprea.d of hiv/ aids and maintaining the lives of those already affected. b) skilled medical ~rs~nal are dymg under· mining the capacity to provide the required health care services. ~) th.e comphcat1o~s of hiv/aids has complicated the treatment of other diseases e.g. tbs d) the ep1dem1c has led. to mcrease number of h n requiring care and support. this has further stretched the resources available for health care. orp a s d db . . i methods used on our research: 1. a simple community survey con ucte y our orgamzat1on vo · unteers in three urban centres members of the community, workers and health care prov~ders were interviewed ... 2. meeting/discussions were organized in hospitals, commun.ity centre a~d with government officials ... 3. written questionnaires to health workers, doctors and pohcy makers m th.e health sectors. lessors learning: • the biggest-health bigger-go towards hiv/aids prevention • aids are spreading faster in those families which are poor and without education. •women are the most affected. •all health facilities are usually overcrowded with hiv/aids patients. actions needed:• community education oh how to prevent the spread of hiv/aids • hiv/aids testing need to be encouraged to detect early infections for proper medical cover. • people to eat healthy • people should avoid drugs. implications of our research: community members and civic society-introduction of home based care programs to take care of the sick who cannot get a space in the overcrowded public hospitals. prl-v a te sector private sector has established programs to support and care for the staff already affected. government provision of support to care-givers, in terms of resources and finances. training more health workers. introduction: australian prisons contain in excess of 23,000 prisoners. as in most other western countries, reliance on 'deprivation of liberty' is increasing. prisoner numbers are increasing at 7% per annum; incarceration of women has doubled in the last ten years. the impacts on the community are great -4% of children have a parent in custody before their 16th birthday. for aboriginal communities, the harm is greater -aboriginal and/or torres strait islanders are incarcerated at a rate ten times higher than other australians. 25% of their children have a parent in custody before their 16th birthday. australian prisons operate under state and territory jurisdictions, there being no federal prison system. eight independent health systems, supporting the eight custodial systems, have evolved. this variability provides an unique opportunity to assess the capacity of these health providers in addressing the very high service needs of prisoners. results: five models of health service provision are identified -four of which operate in one form or another in australia: • provided by the custodial authority (queensland and western australia)• pro· vided by the health ministry through a secondary agent (south australia, the australian capital territory and tasmania) • provided through tendered contract by a private organization (victoria and northern territory) • provided by an independent health authority (new south wales) • (provided by medics as an integral component of the custodial enterprise) since 1991 the model of the independent health authority has developed in new south wales. the health needs of the prisoner population have been quantified, and attempts are being made to quantify specific health risks /benefits of incarceration. specific enquiry has been conducted into prisoner attitudes to their health care, including issues such as client information confidentiality and access to health services. specific reference will be made to: • two inmate health surveys • two inmate access surveys, and • two service demand studies. conclusions: the model of care provision, with legislative, ethical, funding and operational independence would seem provide the best opportunity to define and then respond to the health needs of prisoners. this model is being adopted in the united kingdom. better health outcomes in this high-risk group, could translate into healthier families and their communities. p4-04 (a) lnregrated ethnic-specific health care systems: their development and role in increasing access to and quality of care for marginalized ethnic minorities joshua yang introduction: changing demographics in urban areas globally have resulted in urban health systems that are racially and ethnically homogenous relative to the patient populations they aim to serve. the resultant disparities in access to and quality of health care experienced by ethnic minority groups have been addressed by short-term, instirutional level strategies. noticeably absent, however, have been structural approaches to reducing culturally-rooted disparities in health care. the development of ethnic-specific h~alth car~ systems i~ a structural, long-term approach to reducing barriers to quality health care for eth· me mmonty populations. methods: this work is based on a qualitative study on the health care experiences of san francisco chinatown in the united states, an ethnic community with a model ethnic-specific health care infrastrucrure. using snowball sampling, interviews were conducted with key stakeholders and archival research was conducted to trace and model the developmental process that led to the current ethnic-specific health care system available to the chinese in san francisco. grounded theory was the methodology ijltd to analysis of qualitative data. the result of the study is four-stage developmental model of ethnic-specific health infrastrueture development that emerged from the data. the first stage of development is the creation of the human capital resources needed for an ethnic-specific health infrastructure, with emphasis on a bilingual and bicultural health care workforce. the second stage is the effective organization of health care resources for maximal access by constituents. the third is the strengthening and stability of those institutional forms through increased organizational capacity. integration of the ethnic-specific health care system into the mainstream health care infrastructure is the final stage of development for an ethnic-specific infrastructure. conclusion: integrated ethnic-specific health care systems are an effective, long-term strategy to address the linguistic and cultural barriers that are being faced by the spectrum of ethnic populations in urban areas, acting as culturally appropriate points of access to the mainstream health care system. the model presented is a roadmap to empower ethnic communities to act on the constraints of their health and political environments to improve their health care experiences. at a policy level, ethnic-specific health care organizations are an effective long-term strategy to increase access to care and improve qualiiy of care for marginalized ethnic groups. each stage of the model serves as a target area for policy interventions to address the access and care issues faced by culturally and linguistically diverse populations. users in baltimore md: 1989-2004 noya galai, gregory lucas, peter o'driscoll, david celentano, david vlahov, gregory kirk, and shruti mehta introduction: frequent use of emergency rooms (er) and hospitalizations among injection drug users (idus) has been reported and has often been attributed to lack of access to primary health care. however, there is little longitudinal data which examine health care utilization over individual drug use careers. we examined factors associated with hospitalizations, er and outpatient (op) visits among idus over 14 years of follow-up. methods: idus were recruited through community outreach into the aids link to lntravenous experience (alive) study and followed semi-annually. 2,551 who had at least 2 follow-up visits were included in this analysis. outcomes were self-reported episodes of hospitalizations and er/op visits in the prior six months. poisson regression was used accounting for intra-person correlation with generalized estimation equations. hits: at enrollment, 73% were male, 95% were african-american, 33% were hiv positive, median age was 35 years, and median duration of drug use was 15 years. over a total of 37,512 visits, mean individual rates of utilization were 11 per 100 person years (py) for hospitalizations and 123 per 100 py for er/op visits. adjusting for age and duration of drug use, factors significantly associated with higher rates of hospitalization included hiv infection (relative incidence [ri(, 1.4), female gender (ri, 1.2), homelessness (ri, 1.6), as well as not being employed, injecting at least daily, snorting heroin, havmg a regular source of health care, having health insurance and being in methadone mainte.nance treatment (mmt). similar associations were observed for er/op visits except for mmt which was not associated with er/op visits. additional factors associated with lower er/op visits were use of alcohol, crack, injecting at least daily and trading sex for drugs. 10% of the cohort accounted for 45% of total er/op visits, while 11 % of the cohort never reported an op visit during follow-up. . . . lgbt) populations. we hypothesized that prov1dmg .appomtments .for p~t1~nts w1thm 24 hours would ensure timely care, increase patient satisfaction, and improve practice eff1c1ency. further, we anticipated that the greatest change would occur amongst our homeless patients.. . methods: we tested an experimental introduction of advanced access scheduling (usmg a 24 hour rule) in the primary care medical clinic. we tracked variables inclu~ing waiting ti~e fo~ next available appointment; number of patients seen; and no-show rates, for an eight week penod pnor to and post introduction of the new scheduling system. both patient and provider satisfaction were assessed using a brief survey (2 questions rated on a 5-pt scale). results and conclusion: preliminary analyses demonstrated shorter waiting times for appointments across the clinic, decreased no-show rates, and increased clinic capacity. introduction of the advanced access scheduling also increased both patient and provider satisfaction. the new scheduling was initiated in july 2005. quantitative analyses to measure initial and sustained changes, and to look at differential responses across populations within our clinic, are currently underway. introduction: there are three recognized approaches to linking socio-economic factors and health: use of census data, gis-based measures of accessibility/availability, and resident self-reported opinion on neighborhood conditions. this research project is primarily concerned with residents' views about their neighborhoods, identifying problems, and proposing policy changes to address them. the other two techniques will be used in future research to build a more comprehensive image of neighborhood depri· vation and health. methods: a telephone survey of 658 london, ontario residents is currently being conducted to assess: a) community resource availability, quality, access and use, b) participation in neighborhood activities, c) perceived quality of neighborhood, d) neighborhood problems, and e) neighborhood cohesion. the survey instrument is composed of indices and scales previously validated and adapted to reflect london specifically. thirty city planning districts are used to define neighborhoods. the sample size for each neighborhood reflects the size of the planning district. responses will be compared within and across neighborhoods. data will be linked with census information to study variation across socio-eco· nomic and demographic groups. linear and gis-based methods will be used for analysis. preliminary results: the survey follows a qualitative study providing a first look at how experts involved in community resource planning and administration and city residents perceive the availability, accessibility, and quality of community resources linked to neighborhood health and wellbeing, and what are the most immediate needs that should be addressed. key-informant interviews and focus groups were used. the survey was pre-tested to ensure that the language and content reflects real experiences of city residents. the qualitative research confirmed our hypothesis that planning districts are an acceptable surrogate for neighborhood, and that the language and content of the survey is appropriate for imple· mentation in london. scales and indices showed good to excellent reliability and validity during the pre· test (cronbach's alpha from 0.57-0.96). preliminary results of the survey will be detailed at the conference. conclusions: this study will help assess where community resources are lacking or need improve· ment, thus contributing to a more effective allocation of public funds. it is also hypothesized that engaged neighborhoods with a well-developed sense of community are more likely to respond to health programs and interventions. it is hoped that this study will allow london residents to better understand the needs and problems of their neighborhoods and provide a research foundation to support local understandmg of community improvement with the goal of promoting healthy neighborhoods. p4-08 (a) hiv positive in new york city and no outpatient care: who and why? hannah wolfe and victoria sharp introduction: there are approximately 1 million hiv positive individuals living in the united sta!es. about. 50% of these know their hiy status and are enrolled in outpatient care. of the remaining 50 yo, approx~mately half do not know their status; the other group frequently know their status but are not enrolled m any .sys~em of outpatient care. this group primarily accesses care through emergency departments. when md1cated, they are admitted to hospitals, receive acute care services and then, upon poster sessions v71 di5'harge, disappear from the health care system until a new crisis occurs, when they return to the emergency department. as a large urban hiv center, caring for over 3000 individuals with hiv we have an active inpatient service ".'ith appr~xi~.ately 1800 discharges annually. we decided to survey our inpatients to better charactenze those md1v1duals who were not enrolled in any system of outpatient care. results: 18% of inpatients were not enrolled in regular outpatient care: 2% at roosevelt hospital and 35% at st.luke\'s hospital. substance abuse and homelessness were highly prevalent in the cohort of patients not enrolled in regular outpatient care. 84% of patients not in care (vs. 33% of those in care) were deemed in need of substance use treatment by the inpatient social worker. 74% of those not in care were homeless (vs. 15% of those in care.) patients not in care did not differ significantly from those in me in terms of age, race, or gender. patients not in care were asked "why not:" the two most frequent responses were: "i haven't really been sick before" and "i'd rather not think about my health. conclusions: this study suggests that there is an opportunity to engage these patients during their stay on the inpatient units and attempt to enroll them in outpatient care. simple referral to an hiv clinic is insufficient, particularly given the burden of homelessness and substance use in this population. efforts are currently underway to design an intervention to focus efforts on this group of patients. p4.q9 (a) healthcare availability and accessibility in an urban area: the case of ibadan city, nigeria in oder to cater for the healthcare need of the populace, for many years after nigeria's politicl independence, empphasis was laid on the construction of teaching, general, and specialist hospital all of which were located in the urban centres. the realisation of the inadequacies of this approach in adequately meeting the healthcare needs of the people made the country to change and adopt the primary health care (phc) system in 1986. the primary health care system which is in line with the alma ata declaration of of 1978, wsa aimed at making health care available to as many people as possible on the basis of of equity and social justice. thus, close to two decades, nigeria has operated primary health care system as a strategy for providing health care for rural and urban dwellers. this study focusing on urban area, examimes the availabilty and accessibility of health care in one of nigeria's urban centre, ibadan city to be specific. this is done within the contest of the country's national heath policy of which pimary health care is the main thrust. the study also offers necessary suggestion for policy consideration. in spite of the accessibility to services provided by educated and trained midwifes in many parts of fars province (iran) there are still some deliveries conducted by untrained traditional birth attendants in rural parts of the province. as a result, a considerable proportion of deliveries are conducted under a higher risk due to unauthorised and uneducated attendants. this study has conducted to reveal the pro· portion of deliveries with un-authorized attendants and some spatial and social factors affecting the selection of delivery attendants. method: this study using a case control design compared some potentially effective parameters indud· ing: spatial, social and educational factors of mothers with deliveries attended by traditional midwifes (n=244) with those assisted by educated and trained midwifes (n=258). the mothers interviewed in our study were selected from rural areas using a cluster sampling method considering each village as a cluster. results: more than 11 % of deliveries in the rural area were assisted by traditional midwifes. there are significant direct relationship between asking a traditional birth attendant for delivery and mother age, the number of previous deliveries and distance to a health facility provided for delivery. significant inverse relationships were found between mother's education and ability to use a vehicle to get to the facilities. conclusion: despite the accessibility of mothers to educated birth attendants and health facilities (according to the government health standards), some mothers still tend to ask traditional birth attendants for help. this is partly because of unrealistic definition of accessibility. the other considerable point is the preference of the traditional attendants for older and less educated mothers showing the necessity of changing theirs knowledge and attitude to understand the risks of deliveries attended by traditional and un-educated midwifes. p4-12 (a) identification and optimization of service patterns provided by assertive community treatment teams in a major urban setting: preliminary findings &om toronto, canada jonathan weyman, peter gozdyra, margaret gehrs, daniela sota, and richard glazier objective: assertive community treatment (act) teams are financed by the ontario ministry of health and long-term care (mohltc) and are mandated to provide treatment, rehabilitation and support services in the community to people with severe and persistent mental illness. there are 13 such teams located in various regions across the city of toronto conducting home visits 1-5 times per week to each of their approximately 80 respective clients. each team consists of multidisciplinary health professionals who assist clients to identify their needs, establish goals and work toward them. due to complex referral patterns, the need for service continuity and the locations of supportive housing, clients of any one team are often found scattered across the city which increases home visit travel times and decreases efficiency of service provision. this project examines the locations of clients in relation to the home bases of all 13 act teams and identifies options for overcoming the geographical challenges which arise in a large urban setting. methods: using geographic information systems (gis) we geocoded all client and act agency addresses and depicted them on location maps. at a later stage using spatial methods of network analysis we plan to calculate average travel rimes for each act team, propose optimization of catchment areas and assess potential travel time savings. resnlts: initial results show a substantial scattering of clients from several act teams and substan· rial overlap of visit travel routes for most teams. conclusions: reallocation of catchment areas and optimization of act teams' travel patterns should lead to substantial savings in travel times, increased service efficiency and better utilization of resourc_~· ~e l'<!tenri~i modifications to catchment areas must be conducted with appropriate attention to specific clients servtce needs, service continuity and applicable regulations by the mohl tc. venice lido is a popular island within and outside italy because it hosts the international cinema festival yearly. only few people remember the island for its hospital, which, in the fifties, was one of the most important hospitals in italy and in europe, being a modern center for wind-, sun-and thermal-therapy. since the sixties, its fame became to drop away, since its structure was no longer adequate for the evolving medicine science. at the end of the seventies all the wards were moved into a new big building in the same area. at present the hospital is made up by about forty 'pavilions' (mostly partially used or empty) and a big building, disseminated in a 10 hectares area, on the sea front, on the 500 m long beach. the aim of my research is to suggest a realistic solution, sustainable on the functional use of the area according to basic local needs and economically realizable. i used a philological and multi-disciplinary method, more in detail: -from historical documents, i found what was the very first call of the property and how it adapted to the needs of the demographic development and the sanitary supply over time; -by contacting some operators from different fields, i realized which functions were suitable to the buildings in existence, to the population's needs and to the present sanitary supply. moreover the planning idea considers: -the debate going on between local people who strongly want to preserve the property, and die ones who want to renew it by different new opportunities; and -the regional sanitary politics, aiming at rationalizing the regional hospitals dislocation, and the budget needs. this plan analyzes and proposes some new alternative solutions to satisfy different needs. the different activities will be dealt out and the environmental fall will be limited as much as possible. after having analyzed its basic call, considered its environmental peculiarities and its position, verified the emerging needs of the local population, the plan proposes 5 different activities: the hospital; the social-rehabilitation center; the elderly house; the thermal center; the residential center; and other activities. such a plan wants to share in the solution of a basic problem by giving architectonic and functional dignity to an historical completely degraded area and by starting again a social-economical broken off process, by bringing in venice lido some fresh necessary elements for its so wished new throw. p4-14 (c) dilemma of free health care in spokane, wa david bunting introduction: the paper reports on the activities of project access spokane [pas], a physician sponsored initiative to provide uncompensated health care to low income, uninsured residents of spokane county wa. program providers included all county hospitals, over 600 physicians, other medical professionals, and specialized clinics. each prescription requires only a $4 co-payment. sponsored and administered by the county medical society, pas is funded by local and national foundations, private corporations, municipalities, civic organizations and individuals. method: the paper is based on a first year assessment report funded by pas, using hcfa [health care financing administration] insurance claim data documenting utilization rates, disease categories and donated charge information and patient cares [centralized applications, referrals and enrollment status) data. results: while essentially free for enrollees, the program involved real costs. an administrative organization to refer over 700 patients to any of over 800 providers had to be developed and staffed, using both paid and unpaid personnel. methods to identify potential patients and veri.fy eligibility. had to ~devised. patient appointments and transportation had to be scheduled and monitored. pu~hc relanons, provider solicitations and fundraising activities were continuous. information requirements regarding program operation were also significant. data reporting the volume. ~nd value of dona~ed medical services were necessary to demonstrate accomplishments and attr~ct addmon~l support. providers required assurances their contributions were both significant and eqmtable. funding sources sought evidence that their support had important consequences. however, generating this ~~ta proved difficult. many providers failed to submit appropriate insurance claims forms because .of a~dmonal donated effort and administrative cost, thereby not only reducing their own apparent conmbut1ons but also the overall significance of the program. conclusions: during its initial year, the estimated cost to deliver free health care was ~bout 5500 per enrollee. the lesson is that without an elaborate administrative structure and record keeping s.yst~~· efforts to provide free health care probably will fail. eligible enrollees c~n not be ~parated from in~hgi ble ones, care will not be accessed or delivered in an efficient manner, neither fundmg nor ~upport "". 1 11 be offered without evidence of tangible benefits, and providers will withdraw if they perceive mequ1table tteannent. v74 p4-1s (c) mobility in prostitution and the impact of health therese van der helm and henk sulman poster sessions introduction: many of the estimated 8.000 commer~ial ~ex wor~ers (c~w)in a'.'1~terdam ~~me from developing countries and eastern europe. to gain ins~ght mto their workmg and_ hvmg condinons the intermediary project (ip) at the municipal health serv1~e _contacts these women via ?utreach work on regular basis. since the new brothel law in october 2000 1s 1~troduc~d, ~sw ~rom outside the eu and who have no dutch residence permit are not allowed to work m prost1tut10n. smee then, many of these csw therefore have gone "underground." the consequences of the new law in the netherlands will be discussed with regard to the accessibility of csw and their risk for stl/hiv acquisition and unplanned pregnancies. methods: topics during outreach work are the interventions to increase the knowledge of safe sex and birth control for csw. written information is handed out in relevant languages and with addresses of health and social services. in conversations with the women, it appeared that many are not aware of these services. to provide easier access to health care for women, staff of the ip carries out free sti control csw working places, in windows brothels and sex houses. also, women are offered vaccination against hepatitis b (hbv). results: from january 2004 till july 2005 we approached 900 csw for first contact. one third was dutch; others were from developing countries, other eu countries and eastern europe. 350 sti consul· rations were done among non-iv drug using sex workers, of whom 50 % was dutch. of these, 2 % was diagnosed with syphilis, 1 % with gonorrhea, and 9% with chlamydia. of 234 women tested for hiv, none were infected. due to the high turnover of csw in .brothels, most wnmen were tested only once. among 845 csw tested for hbv -of whom one third is dutch -22 % had antibodies for hbv, 12 were carrier of hbv, most of whom came from hbv endemic areas. conclusions: sti control and hbv vaccination in brothels is an important support in health care. our findings suggest that csw do not play an important role in the transmission of sti. many csw are highly mobile and often not aware of the existing health and social services. continuous outreach is important to remain in contact with this mobile population. regulations in the new brothel law should not hamper contacts with (illegal) csw. heart disease (cho) is projected to become the leading cause of death. studies conducted in europe and the united states have proven a higher rate of cho in south asians who have migrated from south asia, most notably india, pakistan and bangladesh. approximately 50% of all heart attacks among south asian men occur under the age of 55; 25% of them occur under the age of 40-unheard of in any other population. associated risk factors such as diabetes, high blood pressure and cholesterol are also com· mon in this group. the population of south asians residing in nyc has more than doubled over the past decade to over 300,000, the majority being young, working-class and with limited health care access. many south asian men are employed as taxi drivers (4 out of 10 nyc taxi drivers are from south asial, working daily12 hour plus shifts. methods: acknowledging that this vulnerable, at-risk group was unavailable for outreach through conventional venues, three of the hospitals of the new york city health and hospital corporation, (elmhurst, queens and bellevue) conducted a one day outreach effort at nyc's jfk international air· ~rt's taxi holdin~ ar~a, ~here over one thousand taxis regularly assemble. bringing an outreach event directly to the taxi dnvers workplace, a tent was set up where cardiovascular-related health screenings, in~luding bl~ pressure, sugar, cholesterol, body mass index (bmi), were provided. results were shared with and explamed to the drivers and each participant received a south asian health education brochure. a total of 84 taxi drivers who identified as south asian were involved. rau/ts: participants were all male, ranging from 20 to 61 years; mean age, 41.5. screenings revealed 57% hypertensive; 46% had cholesterols over 200; 26% blood glucose over 160; 91 % had a bmi greater than 25. conc~on: a unique outreach activity, adapting to logistical, occupational limitations, is pre· se~ted. on-site f~back and explanation of results, reiterating and reinforcing the concern that south asia~. men are at mcreased danger for early coronary heart disease, and the dissemination of a culturally sensmve and r~levant brochure, may heighten awareness of prevailing cardiac risk factors in this immi· grant community. p4-17 (c) the community-hospital integration program framework: community-hospital panoenhips to improve the population's health john stevenson, richard blickstead, ann-marie marcolin, and sandi kendal v75 introduction: st. josephs health centre is a catholic community teaching hospital located in the heart of southwest toronto. st. joseph\'s was founded from a community-expressed need for hospital services, and since then has stayed committed to fostering a healthy community through collaboration and parmership. st. joseph's has developed an innovative model, the community-hospital integration program (chip), to strengthen st. joseph's partnerships with community stakeholders, and facilitates the building of links between community needs, service provision, and public policy outcomes to improve the health and wellness of the diverse neighbourhoods they together serve. methods: development of the chip framework st. joseph's health centre developed the chip framework through a multidisciplinary approach that included extensive international literature reviews, consultations, and key informant interviews with community service agencies and academics. to ensure active community voice, st. joseph's has hosted a number of community consultations including a community outreach forum attended by over ninety representatives from 68 community partners. results: the chip framework the chip framework aims to improve the health and wellness of the urban communities served by st. josephs health centre through four intersecting pillars: • raising community voices provides an infrastructure and process that supports community stakeholder input into health care service planning, decision-making, and delivery by the hospital and across the continuum of care; • sharing reciprocal capacity promotes healthy communities through the sharing of our intellectual and physical capacity with our community partners; • cultivating integration initiatives facilitates vertical, horizontal, and intersectoral integration initiatives in support of community-identified needs and gaps; and • facilitating healthy exchange develops best practices in community integration through community-based research, and facilitates community voice in informing public policy. the chip framework drives the complex inter-relationships between community-hospital engagement, reciprocal capacity-building, integration initiatives, and community-based research and evaluation, to create an interconnected network of health care services. the fluidity and simplicity of this framework, and its dedication to balancing community needs and hospital mandate, posits the st. josephs health centre's chip model as an integral component in building healthy and thriving communities. p4-18 (c) home based care promotion: improving acess to quality services and livelihood in the face of aids home based care is a service provided to persons affected/living with hiv/aids, a menu of care/support services at home/community. it is a prominent alternative approach. in uganda, hospital bed ocupacny is high, patient health care worker ratio deteriorating. empowering communities offers solutions to the problems; adressing cost, effectiveness of care. hiv/aids demands changes in attitudes, behaviour, approaches which is enhanced in home/community settings. for example art requires high levels of adherecnce, which medical workers have vety little opportunity to enforce. it also demands other support mechanisims in terms of nutrition, personal displine, etc which work best in stigma free environments. hence the relevance of home based care. this paper highlights the gaps that call for increased action in terms of home based care, examples of whre it has worked key challenges and recommendations for the future. p4-19 (c) health care for one ... health care for alli katharina kovacs burns introduction: at the surface, it appears that every person in canada _has. ~ccess to a publicly funded health care system. those living in urban centers should have the best ava1l~b1hty, chmce, and access to a variety of health care services because of the distribution of health care services, fac1lmes, and health professionals in concentrated in urban centers. this is not true for everyone -people with low income or who are homeless experience the challenges of social exclusion and being marginalized: there are many factors at play making it difficult for the latter group of people to access health care. service they need,.when they need them. health care is not as accessible or inclusive as intended. the quesuon to be explore.cl 1~: if health care is available and accessible to one person, what makes it not available to all people? the ~1gmfic~nce of having marginalized people accessing health care and other services includes e~hanced quality of hfe and decreased risks associated with being homeless, and cost savings in ion~ ter~ w1rh acute health care. methodology: community-based participatory research is applied m a case study on health care access and challenges experienced by low income and homeless people in one urban centre, edmonton, edmonton, and their challenges. the data is being gathered and ana_lyzed usmg basic m1x~d methods. results: the results will focus on how services can be more mtegrated and accessible to all people with low income and who are homeless. there will also be discussion about specific perceptions of not only the service providers but also of people with low income an~ who are homeless~ and vario~s decision makers. the results will be compared to the literature regardmg health care services access mother urban centers in canada and elsewhere. conclusions: recommendations will need to address social exclusion and other factors which can make health care and other services more available to all people in the community. the implications for health care will also be discussed. additional information will be gathered in the next phases of the study to develop a community services access model. p4-20 (c) availability and access exemplified: a case study beth hayhoe and ruth ewert introduction: access to health care in canada requires either the correct documentation or money. street youth have neither. in addition, health services in canada are designed by adults for adults. youth in general are often wary of having trust in adults with respect to their health concerns and frequently feel misunderstood. street youth are even more mistrustful of adults and public organizations, making their contact with health care professionals minimal. this combined with their risky behaviours and dangerous environment creates a population at risk for numerous health issues but with no place to have them investi· gated. at our non-government, not for profit health service designed specifically to provide a broad range of health care to street youth, professional services are provided almost entirely by volunteers. methods: using a retrospective analysis of the 11 years of data gathered from this organization and reviewing the initial reasons gathered from youth about their needs, the success of the health centre was examined. results: all the things youth had listed as being important in a health centre have been implemented, and even more things have been added to improve the breadth and quality of services offered. the use of the health centre has increased by more than six times since its opening in 1994. in addition, tens of thousands of visits have been paid to the health centre, costing the government and taxpayers nothing. as far as we know there is nothing else in canada that offers so many diverse and innovative services to youth in need. conclusion: it is clear from this case, that health care targeted at the needs of specific disadvan· taged populations can successfully provide them with appropriate health care. a model of accessible health care for marginalized populations can easily be developed for high impact and low cost from this successful case. toronto is one of the most ethno-racially diverse cities in the world of the estimated 2,529,280 toronto residents, 24% (597,218) live in scarborough population growth in scarborough is faster than toronto. scarborough's population increased by 6% from 1996 to 2001 while toronto increased only by 4%. toronto's population is projected to increase by 10% (252 000) in 2011 while scarbor· ough's will increase by 12%. (census canada 2001) low income, pove;ty, seniors, hidden homeless, new~omers to canada, the uninsured, are just a few of the issues concerning health care in our community. health care needs in scarborough are greatly impacted by diversity of ethnic and racial groups, poverty and settlement issues. this paper will share how the scarborough hospital (tsh) an urb~n communir_y hospital cares f~~ it~ community. tsh recognizes: •the changing community• b_arriers t~ accessing care • lnequalmes m health care the hospital in caring for its community pro· v1~es services that '!'e~e cu~turally, racially, and linguistically sensitive to our community. the paper will share the hospitals unique program on access and equity. the paper will share the needs in scar· borough and tsh's response to these needs: working in partnership with the local health committee, i:ne scarborough homeless co.mminee, the scarborough network of immigrant services organiza· nons a~d others. 1:'1e paper will also share the many initiatives this urban community hospital has taken viz. community and home programs in service areas such as mental health, haemodialysis day care, t~~ home oxyg~n program, the palliative at-home program, and above all support for the volunteer clinic for the uninsured. j"'"1tllu:lion: in canada's universal health care system it is generally assumed that everyone has equal access to health care. however, some immigrant groups in canada have historically been uninswed. these groups include failed refugee claimants, those overstaying their visas, and new immigrants in transition who are waiting to become eligible for health insurance. some estimates have suggested that at the present time there may be as many as one hundred thousand undocumented and thus uninsured immigrants in toronto alone. understandably, information on the characteristics of this marginalized population is very limited, since undocumented immigrants tend to have little contact with formal institutions. knowledge of the demographic characteristics of this population may aid in the development of health and social programs to better identify and meet the needs of undocumented immigrants in toronto. we conducted a review of all undocumented, uninsured patients, 15 years of age or older at access alliance multicultural community health centre (aamchc) between 1994 and 2004. demographic information such as age, gender, preferred language, length of time living in canada, selfreported education level and household income were recorded. rmdts: 72% of all adult patients at access alliance were undocumented and uninsured. 80% were under40years of age (with a median age of 30 years), 72% were women and only 19% spoke english. this group lived in canada for an average period of 2.2 years before they were first seen at aamchc. 74% of undocumented clients reported having completed at least a secondary or post-secondary education. 81% had self-reported household incomes of less than 20,000$/year, while 97% of households reported earning less than $30,000/year. the mean income per person in an average household was 425$/month. conchuions: uninsured individuals at aamchc were predominantly young females with limited knowledge of the english language. despite having attained a high level of education most were living well below the poverty line. recognition of these characteristics may assist in the development of health and social programs that are better adapted to meet the needs of undocumented immigrants. p4-23 (c) sharing expertise: a role for the hospital lactation consultant in the community. badrgrmuul: st. michael's hospital is a tertiary care hospital that serves a large inner city population in downtown toronto. in order to provide care to this population, the hospital has developed a number of unique outreach roles. one such role was developed to work with pregnant and breastfeeding women living in regent park, a social housing complex located near the hospital. the population of regent park includes new immigrants, refugees and the socially disadvantaged. approach: the outreach lactation consultant provides care, as part of the canada prenatal nutrition program, in the community prenatally, in the hospital during their inpatient stay and postnatally when they return to the community. the continuity of care enhances the probability of long term successful breastfeeding. aside from the health benefits of breastmilk, breastfeeding is especially important for women who are financially needy and must depend on food banks for formula. . . lason learned: aher two years of partnership, the relationship between the an-hospital ~stpar tum experience for breastfeeding mothers and the community postpartum support, the breastfeeding rate ~thin this community is high. the professional support and visibility of the 1:8ctation consultant, both m the hospital and in the community, contributes to the support of breastfee~m~.. . in light of ongoing funding constraints, this ha1son between hospital and community could be at risk and discontinued. in light of the benefits to the mother/baby dyad, the lactation consultant as a community partner in the canada prenatal nutrition program should be safeguarded. . lldpodiu:tion: although the importance of adequate health care in pregna~cy is well recognised, ethnic disparities in utilization of prenatal care still exist in many western coun~~es. a fun~amental factor in these disparities may be language proficiency, as it influences women's ab1hty. to ob~m and understand health information, to find the way in the health care system and to communicate with health care v78 poster sessions providers. we investigated the role of language proficiency in use and knowledge of folic acid as aspect of prenatal care in an urban multi-ethnic pregnancy c?hort. . . design: prospective cohort study. setting and parnc1pants: amsterdam ~regnant women attending obstetric care providers for their first antenatal visit (n= 8050). country of birth defined ethnicity: the netherlands, surinam, antilles, turkey, morocco, ghana, other non-~est~m (nw) and other western (w) country. main outcome measures: knowledge about and use of.fohc a~d (fa) supplements in pregnancy, and determinants of these in diffe~~t ethnic ~~ups. deterrrunants mcl~ded age, ed~ tion, parity, pregnancy intention and dutch prof1c1ency. stansncs: ~2 to co~p~re e~c groups, stranfied logistic regression (forward stepwise method) to explore determmants "'.'thm ethmc groups .. use of fa supplements was significantly lower among ghanaian, moroccan, turkish and other nw women (21%to41 %) than among dutch (86%) or other w women (78%). use amongsurinames and antillean women was intermediate (51%and60%). ethnic differences in fa knowledge were similar. knowledge was the strongest determinant of use in all ethnic groups, with odds ratios (ors) ranging from 10.9 to 42.0. language proficiency was the strongest determi~a~t of kno~ledge in ethnic groups with a mother tongue different from dutch (ors good vs. low proficiency ranging from 3.2 ro 16.0). educational level had a modifying role, as shown by an interaction effect between education and language proficiency in the nw group. here, the odds of having fa knowledge given a high education and good dutch proficiency was 20 times the odds given a good proficiency but low education. conclusions: appropriate periconceptional use of folic acid supplements in non-western ethnic groups is low, reflecting an absence of knowledge that is largely determined by the inability to speak and understand the language of the habitual country. tailored interventions using communication channels most likely to address (pregnant) women from ethnic minority groups are necessary. apan from specific interventions, our results are in support of strong incentives on language education despite current political debate. introduction: recent research suggests living in an economically disadvantaged neighborhood is asso· ciated with decreased likelihood of undergoing mammography and increased risk of late-stage breast can· cer diagnosis. long distances and travel times to facilities offering low-or no-cost mammography may be imponant barriers to adherence to mammography screening recommendations for residents of economically disadvantaged neighborhoods. the purpose of this study was to examine whether facilities providing low-and no-cost screening mammography were less spatially accessible in low-income neighborhoods in chicago, and the extent to which the relationship between neighborhood income and the spatial accessibility of facilities varied by the proponion of african-american residents in the neighborhood. methods: the sample consisted of 343 chicago neighborhoods. for each neighborhood, we con· structed three measures of the spatial accessibility of facilities: street network distance to the nearest facility, public transportation travel time to the nearest facility, and shortest automobile travel time to a facility. using 2000 decennial census data, we characterized the neighborhoods according to proportion of residents with incomes below the poverty line, proportion of residents in each of four raciavethnic groups (african-american, latino, white, and other), and population density. we used ordinary least squares (ols) and spatial exogenous lag regression to examine relationships. model 1 estimated the relationship between neighborhood poverty and the spatial accessibility of facilities, adjusting for raciaveth· nic composition and population density. model 2 added a multiplicative interaction term between neighborhood poverty and african-american. restllts: we identified deven facilities that provided low-or no-cost screening mammography to chicago residents in 2004. we found that the distance and travel times via automobile and public trans· portation to facilities generally decreased as neighborhood poveny increased. however, we also found that the ~egative associations between neighborhood poverty and two of the spatial accessibility mea· sures -distance and public transportation travel time -were less strong in neighborhoods with the highest proponions of african-american residents. among neighborhoods in the highest tertile for poveny, th~ mean distance and mean public transportation travel time to facilities were over twice as long in neighborhoods in the highest tertile than in those in the lowest tenile of african-american residents. . ~ persistent socioeconomic and racial/ethnic disparities in breast cancer stage at diagno-s11 ~nd su~1val suggest that ensuring an equitable distribution of affordable mammography is a worth-w~e policy .goal. the ~~dy sugges~ that ~ture investigations should consider both neighborhood soc1oecono1d1c charactensbes and rac1avethmc composition when examining the spatial distribution of health resources. , 2004) . epidemiological data suggest tha~ hiv prevalence among msm is over 1 % in some metropolitan cities, such .as beijing (ibid.). due to widespread homophobia in chinese society, however, this population remains invisible within current health/social services. lack of research on sexuality, specifically homosexuality, has impaired our understanding of health and health practices of chinese msm. focusing on the illness experiences of chinese msm with hiv/aids, this paper explores the socio-cultural impacts of hiv/ aids on this group. the data used for this paper were collected through semi-constructed in-depth face-toiace interviews with 21 adult plwhas (including 11 msm) in beijing, china. with the permission of the participants, the interviews were audio-taped or recorded in notes. the transcribed interviews and interview notes were analyzed by using n-vivo, a software program for qualitative data analysis. this phenomenological study aimed to understand the experiences chinese plwhas (including msm) from their own perspectives. results: it is found that the illness experiences of chinese msm with hiv/aids are profoundly shaped by the socio-cultural meanings of homosexuality, which are further complicated by the dominant discourses on hiv/aids in china. at a macro level, homophobia in the larger society has decreased the capability of this group to respond to this epidemic in a more efficient way. at a meso level, aids-phobia within the chinese msm circuits has prevented this group from openly confronting this disease and offering support to those who are already affected by it. at a micro level, however, these hiv-infected/ affected msm have tried their best to locate spaces to live and to construct hope for their future lives despite various barriers within family, community, and the larger society. this study suggests that facilitating chinese msm's response to the aids crisis urgently requires bridging awareness, commitment, knowledge and resources both within and without this group. it also illustrates the importance of understanding homosexuality in the local context of hiv i aids, which will be helpful for developing culturally sensitive hiv/aids programs for this population on the community, national and international levels. introduction: having a regular family doctor (rfd) is known to be positively correlated with a good medical follow up, including access to preventive and/or chronic care. inversely, a lack of primary care may lead to high rates of avoidable hospitalizations, especially among poor people and/or in underserved neighbourhoods. in such a matter, a recent comparative study showed that paris was better ranged than other cities, such as nyc, tokyo or london. nevertheless, despite a quasi universal health insurance and a high density of general practitioners, a noticeable fraction of the paris population does not have any regular physician and we aimed to understand who they are and for what reasons rhey don't have any rfd. mdbods: cross sectional population survey among a random sample of households in 2 ~nderpriv ileged neighbourhoods in paris inner city, performed in 2003, using a face-to-face quest10nna1re collecr-1ng more than 400 social and health characteristics. ruults: a quarter (26.3 %) of rhe study popularion did not have any rfd. this proporrion was iignificantly higher among male (or= 2.00, 95%ci = [ 1.41-2.781), younger (e.g. or 18 -29/> .s l _= 4._oo, 95"1.ci = (2.13-7.69)), and/or unemployed (or =2.01, 95%ci = ij .21-3.3411_ people. in multtvanate analysis, after a full adjustment on gender, age, health status, health insura~ce, income, occupat10n and tducation level, we observed significant associations between having no rfd and: ~arrtal and_ pare~t hood status (e.g. or single no kids/in couple+kids = 2.12, 95%ci = ( 1.26-3.59()~ quality of relattonsh1ps with neighbours (or bad/good= 3 .82, 95%ci = [ 1.84-7.94)), and length of residence m the neighbourhood (with a dose/effect statistical relationship). . co11clusion: gender, age, employment status, mariral and parenthood stat~s as well as ~e1gh bourhood anchorage seem to be major predictors of having a rfd, even when um.versa! health i~sur ance has reduced most of financial barriers. in urban contexts, where residential migrattons and single lift (or family ruptures) are frequent, specific information may be conducted to encourage people to ket rfd. :tu~y tries to assess the health effects and costs and also analyse the availability and accessibility to health care for poor. . methods: data for this study was collected by a survey on 300 households of the local community living near the factories and 100 households where radiation hazard w~s n?~ present. ~~art from mor· bidity status and health expenditure, data was collected ~n access, a~ail~b1.hty and eff1c1ency of healrh care. a discriminant analysis was done to identify the vanables that d1scnmmate between the study and control group households in terms of health care pattern. a contingent valuation survey was also undertaken among the study group to find out the factors affecting their willingness to pay for health insurance and was analysed using logit model. results: the health costs and indebtedness in families of the study group was high as compared to control group households and this was mainly due to high health expenditure. the discriminant analysis showed that expenditure incurred by private hospital inpatient and outpatient expenditure were significant variables, which discriminated between the two types of households. the logit analysis showed !hat variables like indebtedness of households, better health care and presence of radiation induced illnesses were significant factors influencing willingness to pay for health insurance. the study showed that study group households were dependent on private sector to get better health care and there were problems with access and availability at the public sector. conclusion: the study found out that the quality of life of the local community is poor due to health effects of radiation and the burden of radiation induced illnesses are so high for them. there is an urgent need for government intervention in this matter. there is also a need for the public sector to be efficient to cater to the needs of the poor. a health insurance or other forms of support to these households will improve the quality for health care services, better and fast access to health care facilities and reduces the financial burden of the local fishing community. the prevalence of substance abuse is an increasing problem among low-income urban women in puerto rico. latina access to treatment may play an important role in remission from substance abuse. little is known, however, about latinas' access to drug treatment. further, the role of social capital in substance abuse treatment utilization is unknown. this study examines the relative roles of social capital and other factors in obtaining substance abuse treatment, in a three-wave longitudinal study of women ages 18-35 living in high-risk urban areas of puerto rico, the inner city latina drug using study (icldus). social capital is measured at the individual level and includes variables from social support and networks, familism, physical environment, and religion instruments of the icdus. the study also elucidates the role of treatment received during the study in bringing about changes in social capital. the theoretical framework used in exploring the utilization of substance abuse treatment is the social support approach to social capital. the research addresses three main questions: ( t) does social capital predict parti~ipating in treatment programs? (2) does participation in drug treatment programs increase social capital?, and (3) is there a significant difference among treatment modalities in affecting change in ~ial capital? the findings revealed no significant association between levels of social capital and gettmg treatment. also, women who received drug treatment did not increase their levels of social capital. the findings, however, revealed a number of significant predictors of social capital and receiving drug ~buse treatment. predictors of social capital at wave iii include employment status, total monthly mcoi:rie, and baseline social capital. predictors of receiving drug abuse treatment include perception of physical health and total amount of money spent on drugs. other different variables were associated to treatment receipt prior to the icldus study. no significant difference in changes of social capital was found among users of different treatment modalities. this research represents an initial attempt to elucidate the two-way relationship between social capital and substance abuse treatment. more work is necessary to unden~nd. ~e role of political forces that promote social inequalities in creating drug abuse problems and ava1lab1hty of treatment; the relationship between the benefits provided by current treatment poster sessions v81 sctrings and treatment-seeking behaviors; the paths of recovery; and the efficacy and effectiveness of the trtaanent. and alejandro jadad health professionals in urban centres must meet the challenge of providing equitable care to a population with diverse needs and abilities to access and use available services. within the canadian health care system, providers are time-pressured and ill-equipped to deal with patients who face barriers of poverty, literacy, language, culture and social isolation. directing patients to needed supportive care services is even more difficult than providing them with appropriate technical care. a large proportion of the population do not have equitable access to services and face major problems navigating complex systems. new approaches are needed to bridge across diverse populations and reach out to underserved patients most in need. the objective of this project was to develop an innovative program to help underserved cancer patients access, understand and use needed health and social services. it implemented and evaluated, a pilot intervention employing trained 'personal health coaches' to assist underserved patients from a variety of ethno-linguistic, socio economic and educational backgrounds to meet their supportive cancer care needs. the intervention was tested with a group of 46 underserved cancer patients at the princess margaret hospital, toronto. personal coaches helped patients identify needs, access information, and use supportive care services. triangulation was used to compare and contrast multiple sources of quantitative and qualitative evaluation data provided by patients, personal health coaches, and health care providers to assess needs, barriers and the effectiveness of the coach program. many patients faced multiple barriers and had complex unmet needs. barriers of poverty and language were the easiest to detect. a formal, systematic method to identify and meet supportive care needs was not in place at the hospital. however, when patients were referred to the program, an overwhelming majority of participants were highly satisfied with the intervention. the service also appeared to have important implications for improved technical health care by ensuring attendance at appointments, arranging transportation and translation services, encouraging adherence to therapy and mitigating financial hardship -using existing community services. this intervention identified a new approach that was effective in helping very needy patients navigate health and social services systems. such programs hold potential to improve both emotional and physical health out· comes. since assistance from a coach at the right time can prevent crises, it can create efficiencies in the health system. the successful use of individuals who were not licensed health professionals for this purpose has implications for health manpower planning. needle exchange programs (neps) have been distributing harm reduction materials in toronto since 1990. counterfit harm reduction program is a small project operated out of a community health centre in south-east toronto. the project is operated by a single full-time coordinator, one pan-rime mobile outreach worker and two peers who work a few hours each week. all of counterfit's staff, peers, and volunteers identify themselves as active illicit drug users. yet the program dis~rib utes more needles and safer crack using kits and serves more illicit drug u~rs t~an the comb1~e~ number of all neps in toronto. this presentation will discuss the reasons behind this success, .s~1f1cally the extended hours of operation, delivery models, and the inclusion of an. extremely marg1~ahzed community in all aspects of program design, implementation and eva.luat1?n. ~ounterfit was recently evaluated by drs. peggy milson and carol strike, two leading ep1dem1olog1st and researchers in the hiv and nep fields in toronto and below are some of their findings: "the program has experienced considerable success in delivering a high quality, accessible and well-used program .... the pro· gram has allowed (service users) to become active participants in providing. services to others and has resulted in true community development in the best sense. " ... counterf1t has ~~n verr succe~sful attracting and retaining clients, developing an effective peer-based model an.d assisting chen~s ~1th a vast range of issues .... the program has become a model for harm red~ctmn progr~ms withm the province of ontario and beyond." in june 2004, the association of ?ntano co~mumty heal~~ <:en· ires recognized counterfit's acheivements with the excellence m community health initiatives award. in kenya, health outcomes and the performance of government health service~ have det~riorated since the late 1980s, trends which coincide with a period of severe resource constramts necessitated by macro-economic stabilization measures after the extreme neo-liberalism of the 1980s. when the govern· ment withdrew from direct service provision as reform trends and donor advocacy suggested, how does it perform its new indirect role of managing relations with new direct health services providers in terms of regulating, enabling, and managing relations with these health services providers? in this paper therefore, we seek to investigate how healthcare access and availability in the slums of nairobi has been impacted upon by the government's withdrawal from direct health care provision. the methodology involved col· leering primary data by conducting field visits to 8 health institutions located in the slum areas of kibera and korogocho in nairobi. purposive random sampling was utilized in this study because this sampling technique allowed the researcher(s) to select those health care seekers and providers who had the required information with respect to the objectives of the study. in-depth interviews using a semi-structured ques· tionnaire were administered ro key informants in health care institutions. this sought to explore ways in which the government and the private sector had responded and addressed in practical terms to new demands of health care provision following the structural adjustment programmes of the 1990s. this was complemented by secondary literature review of publications and records of key governmental, bilateral and multilateral development partners in nairobi. the study notes a number of weaknesses especially of kenya's ministry of health to perform its expected roles such as managing user fee revenue and financial sustainability of health insurance systems. this changing face of health services provision in kenya there· fore creates a complex situation, which demands greater understanding of the roles of competition and choice, regulatory structures and models of financing in shaving the evolution of health services. we rec· ommend that the introduction of user fees, decentralization of service provision and contracting-out of non-clinical to private and voluntary agencies require a new management culture, and new and clear insri· tutional relationships. experience with private sector involvement in health projects underlines the need not only for innovative financial structures to deal with a multitude of contractual, political, market and risks, but also building credible structures to ensure that health services projects are environmentally responsive, socially sensitive, economically viable, and politically feasible. purpose: the purpose of this study is to examine the status of mammography screening utilization and its predictors among muslim women living in southern california. methods: we conducted a cross-sectional study that included 202 women aged ::!: 40 years. we col· leered data using a questionnaire in the primary language of the subjects. the questionnaire included questions on demography; practices of breast self-examination (bse) and clinical breast examination (cbe); utilization of mammography; and family history of breast cancer. bivariate and multiple logistic regression analyses were performed to estimate the odds ratios of mammography use as a function of demographic and other predictor variables. . results: among the 202 women, 78% were married, 68% were 40-50 years old, and 20% had family h1story of breast cancer. thirty-two percent of the participating women never practiced bse and 32% had not undergone cbe during the past two years. the data indicated that 46% of the women did not have mammography in the last two years. logistic regression analysis showed that age (0r=5.1, 95% confi· dcnc~ interval (cl)=l.8-14.2), having clinical breast examination (0r=24.9, 95% cl=8.4-73.7), and practtce of self-breast examination (0r=2.6, 95% cl= 1.1-6.2), were strong predictors of mammography use . . conclusions: the data point to the need for intervention targeting muslim women to inform and motivate th.cm a~ut practices for early detection of breast cancer and screening. further studies are needed to investigate the factors associated with low utilization of mammography among muslim women population in california. we conducted a review of the scientific literature and° government documents to describe ditnational health care program "barrio adentro" (inside the neighborhood). we also conducted qualiurivt interviews with members of the local health committees in urban settings to descrihe the comm unity participation component of the program. rtsmlts: until recently, the venezuelan public health system was characterized by a lack or limited access w health care (70% of the population) and long waiting lists that amounted to denial of service. moit than half of the mds worked in the five wealthiest metropolitan areas of the country. jn the spring oi2003, a pilot program hired 50 cuban mds to live in the slums of caracas to provide health care to piople who had previously been marginalized from social programs. the program underwent a massive expansion and in only two years 20,000 cuban and 6,500 venezuelan health care providers were working acmss the country. they provide a daily average of 20-40 medical consultations and home visits, c1lly out neighborhood rounds, and deliver health prevention initiatives, including immunization programs. they also provide generic medicines at no cost to patients, which treat 80% of presenting ill-ij!m, barrio adentro aims to build 8,000 clinics (primary care), 1,200 diagnostic and rehabilitation ctnrres (secondary care), and upgrade the current hospital infrastructure (tertiary care). local health committees survey the community to identify needs and organize a variety of lobby groups to improve dit material conditions of the community. last year, barrio adentro conducted 3.5 times the medical visits conducted by the ministry of health. the philosophy of care follows an integrated approach where btalrh is related to housing, education, employment, sports, environment, and food security. conclusions: barrio adentro is a unique collaboration between low-middle income countries to provide health care to people who have been traditionally excluded from social programs. this program shows that it is possible to develop an effective international collaboration based on participatory democracy. low-income americans are at the greatest risk of being uninsured and often face multiple health concerns. this evaluation of the neighborhood health initiative (nh!), an organization which uses multiple programmatic approaches to meet the multiple health needs of clients, reflected the program's many activities and the clients' many service needs. nh! serves low-income, underserved, and hard-to-reach residents in the des moines enterprise community. multiple approaches (fourth-generation evaluation, grounded theory, strengths-and needs-based) and methods (staff and client interviews, concept mapping, observations, qualitative and quantitative analysis) were used to achieve that reflection. results indicate good targeting of residents in the 50314 zip code and positive findings in the way of health insurance coverage and reported unmet health needs of clients. program activities were found to match client nttds, validating the organization\'s assessment of clients. important components of nhi were the staff composition and that the organization had become part of both the formal and informal networks. nhi 1 1 positioned as a link between the target population and local health and social sc:rvice agencies, working to connect residents with services and information as well as aid local agencies in reaching this underserved population. p4-36 (c) welfare: definition by new york city maribeth gregory for an individual who resides in new york city, to obtain health insurance under the medicaid policy one must fall under certain criteria .. (new york city's welfare programs 2003) if the individual _is on ssi or earns equal to or less than $934 per month, he is entitled to receive no more than $5,600 m resources. a family the size of two would need to earn less than $942 per month to qualify for no greater than ss,650 worth of medicaid benefits. a family of three would qualify for $5,650 is they earned less than $942 per month and so on. introduction: the vancouver gay communiry has a significant number of asian descendan!l. because of their double minority status of being gay and asian, many asian men who have sex with men (msm) are struggling with unique issues. dealing with racism in both mainstream society and the gay communiry, cultural differences, traditional family relations, and language challenges can be some of their everyday srruggles. however, culturally, sexually, and linguistically specific services for asian msm are very limited. a lack of availability and accessibiliry of culturally appropriate sexual health services isolates asian msm from mainstream society, the gay community, and their own cultural communities, deprives them of self-esteem, and endangers their sexual well-being. this research focuses on the qualita· tive narrative voices of asian msm who express their issues related to their sexualiry and the challenges of asking for help. by listening to their voices, practitioners can get ideas of what we are missing and how we need to intervene in order to reach asian msm and ensure their sexual health. methods: since many asian msm are very discreet, it is crucial to build up trust relationships between the researcher and asian msm in order to collect qualitative data. for this reason, a community based participatory research model was adopted by forming a six week discussion group for asian msm. in each group session, the researcher tape recorded the discussion, observed interactions among the participants, and analyzed the data by focusing on participants' personal thoughts, experiences, and emotions for given discussion topics. ra11lts: many asian msm share challenges such as coping with a language barrier, cultural differ· ences for interpreting issues and problems, and westerncentrism when they approach existing sexual health services. moreover, because of their fear of being disclosed in their small ethnic communities, a lot of asian msm feel insecure about seeking sexual health services when their issues are related to their sexual orientation. conclflsion: sexual health services should contain multilingual and multicultural capacities to meet minority clients' needs. for asian msm, outreach may be a more effective way to provide them with accessible sexual health services since many asian msm are closeted and are therefore reluctant to approach the services. building a communiry for asian msm is also a significant step toward including them in healthcare services. a communiry-based panicipatory approach can help to build a community for asian msm since it creates a rrust relationship between a worker and clients. p4-38 (c) identifying key techniques to sustain interpretation services for assisting newcomers isolated by linguistic and cultural barriers from accessing health services s. gopi krishna lntrodaetion: the greater toronto area (gta) is home to many newcomer immigrants and other vulnerable groups who can't access health resources due to linguistic, cultural and systemic barriers. linguistic and cultural issues are of special concern to suburbs like scarborough, which is home to thousands of newcomer immigrants and refugees lacking fluency in english. multilingual community ~nterpreter. service~ (mcis) is a non-profit social service organization mandated to provide high quality mterpretanon services. to help newcomers access health services, mcis partnered with the scarborough network of immigrant serving organizations (sniso) to recruit and train volunteer interpreters to accompany clienrs lacking fluency in english and interpret for them to access health services at various locati?ns, incl~~ing communiry ~c:-lth centres/social service agencies and hospitals. the model envisioned agencies recruin~ and mcis ~.mm.g and creating an online database of pooled interpreter resources. this da.tabase, acces&1bl~ to all pama~~g ?rganization is to be maintained through administrative/member · ship fees to. be ~1d by each parnapanng organization. this paper analyzes the results of the project, defines and identifies suc:cases before providing a detailed analysis for the reasons for the success . . methods:. this ~per~ q~ntitative (i.e. client numben) and qualitative analysis (i.e. results of key •~ormant m~rv1ews with semce ~sers and interpreters) to analyze the project development, training and 1mplementanon phases of the project. it then identifies the successes and failures through the afore· mentioned analysis. poster sessions vss resljts: the results of the analysis can be summarized as: • the program saw modest success both ia l?lllls of numbers of clients served as well as sustainability at various locations, except in the hospital iririog. o the success of the program rests strongly on the commitment of not just the volunteer interprmr, but on service users acknowledgments through providing transponation allowance, small honororia, letter of reference etc. • the hospital sustained the program better at the hospital due to the iolume and nature of the need, as well as innate capacity for managing and acknowledging volunceers. collc/llsion: it is possible to facilitate and sustain vulnerable newcomer immigrants access to health !ul'ices through the training and commitment of an interpreter volunteer core. acknowledging volunteer commitment is key to the sustenance of the project. this finding is important to immigration and health policy given the significant numbers of newcomer immigrants arriving in canada's urban communities. nity program was established in 1993 to provide support to people dying at home, especially those who were waiting for admission to the resi<lential hospice. with the advent of haart and corresponding challenges for people living with hiv/aids in the community, the community program has developed a unique case management approach. this model features an interdisciplinary team providing a clientdrivcn service. the case manager provides continuity of care to clients in a variety of settings both within and outside of the health care system. a case study is used to demonstrate how formal and informal networks of support facilitate efficient and appropriate resource utilization to promote client health. this case study demonstrates the value of consistent coordination with a client who has complex physical, spiritual, substance use and mental health needs. the authors will show how they liaised with the housing authority, police, social services as well as the family physician, clinic staff and a hospital emergtncy room to ensure this client did not fall between the cracks. tarek hussain recognition of the importance of community involvement and sectoral cooperation in health and !ocial services, formalized in alma-ata declaration in 1978, was reinforced at riga meeting held at the mid·point between alma-ata and the year 2000. then at the 20th anniversary of alma-ata declaration, ir was declared that 'primary health care is everybody's business'. health does not exist in isolation. health cannot be defined only as an outcome of 'medical care' but also as one of 'social action'; the responsibility of disease prevention and health promotion rests not only on governments, but also with don·govemmentorgan izations, the community, and individuals. the major accomplishment to date may be that rhere is growing realization in the health sector that community involvement is more than a political right-it is absolute necessary. lessons have learned during the implementation of disease-specific l'cltical programmes, such as cdd, ari, and epi; first, integrated and holistic approaches to childcare art needed, and second, the need for community involvement has become evident. imc! is a broad inte-gra1ed strategy with an overall objective contributing to reducing child morbidity and mortality in developing countries. and one of the imci components, 'community !mci', which is now broadening ~s an entry point for child-focused community development initiatives. community jmcl/c~1ld health is ~n mtcgrated approach to the promotion of key family and community practices that have impact on: child growth and development, disease prevention, home care for sick, malnourished child~en, a.nd care-seekmg behavior and compliance with advice and treatment. the presentation addres~es, m brief, the ~evel opinent of community !mci, operational aspect of community/im cl, key strategies and tools ava1la~le for implementation of c/imci. the paper draws some successful programme examples on working logether for imc! with the community in various countries which had substantial benefns on programme outcomes. p4-4 t (c) healthy child screening: an innovative service initiative ann-marie marcolin and joyce allen . introduction: with mounting attention for creative and integrated models of c~re .that are populallon and community focused, the healthy child screening initiative achiev.es t~ese pnncipl~s and more! . • parkdale high park rotary (sponsorship) the heal~h ~h1ld scree?1?g is a ~opl~-centred model of care. agency and professional sector-specific silos are ehmmated, ~rov1dmg f~~1hes wit~ one-sto~ access to primary prevention services in a local school gym! children at nsk are rece1vmg early mtervent10n and appropriate referrals to the right care provider, at the right time a~d in t~e right place. further, with a complimentary focus on prevention, the program serves to keep at nsk children healthy. healthy child screening results: the service model is developed around four distinct phases: 1) planned outreach; 2) coordinated intake and registration; 3) interdisciplinary screening; and 4) targeted referral. since the fall 2003 and through six collaborative healthy child screenings 158 children ranging in age from 2 to 6 have benefited from this unique service model of care. conclusion: the presentation will provide practical information on the development and implementation of the model. there will also be a focus on lessons learned in building community service provider-hospital relationships. according to statistics canada (2001) 49% of the toronto population was born outside of canada and 21 % were new immigrants. immigrants often arrive in canada in superior health compared to the canadian born population, but they lose this health advantage over time. changes in immigrant health status over time may be attributed to a variety of factors including barriers to accessing and receiving care as well as limitations in the mainstream health service organizations and their approaches to health care delivery (hyman, 2001 ) . for example, immigrant women are less likely to receive pap tests, which places them at a greater risk for developing cervical cancer. similarly, immigrant women receive less mammography screening than their canadian counterparts. the immigrant women's health centre mobile health unit (mhu) was created as an alternative care delivery model to address the need for increased accessibility as well as culturally and linguistically appropriate reproductive health care. toronto western hospital and the immigrant women's health centre have formed a collaborative partnership which incorporates a primary care nurse practitioner and lay ethnocultural counselors providing care on the mhu. currently, a qualitative ethnonursing study is underway to understand the unique experiences of women using the mhu for their reproductive health care as well as the perspectives of the mhu health care providers. based on a literature review and preliminary findings from provider and participant interviews, the proposed presentation will address the themes of health status for immigrant and refugee women, accessibility issues as well strategies to improve their reproductive health care. we will discuss benefits as well as limitations to health care provision using this alternative service delivery model. michael carden, brian edlin, andrew talal, elizabeth getter, and marla shu lntrod.ction: to date most active drug users have not had access to treatment for hepatitis c virus (hcv) infection and substantial barriers continue to exist that interfere with treatment availability for this population. methods: active illicit drug users interested in pursuing hcv care and treatment were recruited in partnership w~th co~munity-based organizations (cbo's) in new york city. baseline data were collected on quahty of hfe, substance use patterns, depression, health care utilization, hcv health beliefs and other relev.ant variables. medical evaluations were conducted, including liver biopsy when indicated, an~ treatment ts ~ffered when no contraindications are present. participants also receive psychiatric evaluahons to determme the appropriateness of interferon treatment. renlts: fifteen individuals have been recruited to date and received an initial medical evaluation. the mean age was 40 years and _the average age of initial heroin or cocaine use was 16.8. eight (53%) ha~ been homeless at least once m the last 6 months and s (33%) were homeless for most or all of this penod. fourteen (93%) had a history of injection drug use with the mean age at first injection being 21 111s1frsewons v87 ,an. elrven persc.!ns (79%) reported inje~~ng .heroin or cocaine within the last 30 days while the .wing four (21 l'o) reported regular non-m1ect1on use of cocaine and street-obtained benzodiazepines. sijiy seven percent of the sample (n= 10) reported ever being referred to hcv treatment while 2 ( t 3 % ) ftponeddiattreann~nt had been offered by~ ~e.dical provider. none had ever received a liver biopsy or araanent. most believed that hcv was a s1gmf1cant threat to their health (80%) and that there was a pm)dchance they would eventually die if their hepatitis c was not treated (67%). though 7 individuals 147%1 believed that there was no cure for hcv, 13 (87%) reported that they would initiate treatment if i was recommended by their doctor. thirteen (87%) had a current psychiatric diagnosis including «pmsion, anxiety disorder, schizophrenia, schizoaffective disorder, and borderline and antisocial persooaliry disorders. among eight individuals who had the beck depression inventory administered, the mean score was 29.75. attendance rates were 83% (83/100) at scheduled medical and psychiatric 1ppointments and 50% (2/4) at liver biopsy. <andtuions: active drug users, despite experiencing multiple psycho-social problems, can be mgaged in hcv care using a multidisciplinary approach, but require intensive follow-up support. hcv aunnent of active drug users should not be dismissed. n-44 (c) antiretroviral therapy in mv infected infants: when to initiate therapy an african experience kingsley okonkwo, ademola adeyemo, israel sokeye, and nwaife umeike /""°""ction: as technology for early diagnosis of human immunodeficiency virus [hiv] improves, m to commence highly active antiretroviral therapy [haarn is yet to be fully evaluated. this prospective study describes our initial experience with early haart in hiv infected nigerian infants and rqions the outcome. we also analyzed the socioeconomic implications of early haart therapy in infants in the african setting. method: hiv infected infants were started on haart before 6 months and followed up at 4 mkly intervals.we monitored baseline and 3 monthly cd4 . conclusion: initial results suggest early haart before 6 months resulted. in improv~d out~ome in african children. treatment appears to be as effective as in developed counmes. in. a~nca as. m most developing countries with limited resources it is possible and effective to use haart m 1~~ants if p~oper llloniroring facilities are available. however the occurrence of long-term drug related tox1c1ty and mk of emergence of resistant viral strains create need for further evaluation of haart in infants. background: most risk factors for cardiovascular disease (cvd) can be mitigated through ~th clinical interventions and lifestyle change. we used data from a telephone survey of new york city (nyc) adults to characterize health care access and healthy behavior among those with three or more cvd risk factors. methods: the study population included respondents to the nyc community health survey 2002 (n= 9,674) self-reporting~ 3 of the following: smoking, diabetes, high cholesterol level, high blood pres· sure, body mass index >25, and age >45 (males) or >55 (females) (n=2,439). results: based on self-report, an estimated 1.447,000 (24%) of nyc adults have~ 3 or more cvd risk factors. this population is 51 % male, 47% white, 25% black, and 53% with s 12 years of education. most report good access to health care, indicated by having health insurance (95%), regular doctor (89%), their blood pressure checked within last 6 months (91 %), and their choles· terol checked within the past year (90% ). only 29% reported getting at least 20 minutes of exercise ~ 4 times per week and only 9% eating ~ 5 servings of fruits and vegetables the previous day. among current smokers, 59% attempted to quit in past 12 months, but only 32% used medication or counseling. implications: these data suggest that most nyc adults known to be at high risk for cvd have access to regular health care, but most do not engage in healthy lifestyle or, if they smoke, attempt effective quit strategies. more clinic-based and population-level interventions are needed to support lifestyle change among those at high risk of cvd. introduction: recently, much interest has been directed at "obesogenic" (obesity-promoting) (swinburn, egger & raza, 1998) built environments, and at geographic information systems (gis) as a tool for their exploration. a major geographical concept is accessibility, or the ease of moving from an origin to a destination point, which has been recently explored in several health promotion-related stud· ies. there are several methods of calculating accessibility to an urban feature, each with its own strengths, drawbacks and level of precision that can be applied to various health promotion research issues. the purpose of this paper is to describe, compare and contrast four common methods of calculating accessibility to urban amenities in terms of their utility to obesity-related health promotion research. practical and conceptual issues surrounding these methods are introduced and discussed with the intent of providing health promotion researchers with information useful for selecting the most appropria1e accessibility method for their research goal~ ~ethod: this paper describes methodological insights from two studies, both of which assessed the neighbourhood-level accessibility of fast-food establishments in edmonton, canada -one which used a relatively simple coverage method and one which used a more complex minimum cos1 method. res.its: both methods of calculating accessibility revealed similar patterns of high and low access to fast-food outlets. however, a major drawback of both methods is that they assume the characteristics of the a~e~ities and of the populations using them are all the same, and are static. the gravity potential method is introduced as an alternative, since it is ·capable of factoring in measures of quality and choice. a n~mber of conceptual and pr~ctical iss~es, illustrated by the example of situational influences on food choice, make the use of the gravity potential model unwieldy for health promotion research into sociallydetermined conditions such as obesity. co.nclusions: i~ ~ommended that geographical approaches be used in partnership with, or as a foun~ation for, ~admonal exploratory methodologies such as group interviews or other forms of commumty consultation that are more inclusive and representative of the populations of interest. qilhl in los angeles county ,,..ia shaheen, richard casey, fernando cardenas, holman arthurs, and richard baker ~the retinomax autorefractor has been used for vision screening of preschool age childien. ir bas been suggested to be used and test school age children but not been validated in this age poup. ob;taiw: to compare the results of retinomax autorefractor with findings from a comprehensive i!' examination using wet retinoscopy for refractive error. mllhods: children 5-12 years old recruited from elementary schools at los angeles county were iaml with snellen's chart and the retinomax autorefractor and bad comprehensive eye examination with dilation. the proportion of children with abnormal eye examination as well as diesensitiviry and specificity of the screening tools using retinomax autorefractor alone and in combinalion wirh snellen's chart. results of the 258 children enrolled in the study (average age= 8.5± 2.1 years; age range, 5-12 years), 6?% had abnormal eye examination using retinoscopy with dilation. for the lerinomax, the sensitivity was 85% (95% confidence interval [ci] 78%-90%), and the specificity was 31% (95% ci, 22%-41 o/o). simultaneous testing using snellen's chart and retinomax resulted in gain in 111sitiviry (94%, 95% cl= 89, 97), and loss in specificity (28%, 95% cl= 19%-38%). the study showed that screening school age children with retinomax autorefractor could identify most cases with abnormal vision but would be associated with many false-positive results. simuhaneous resting using snellen's chart and retinomax maximize the case finding but with very low specificiry. mdhotjs: a language-stratified, random sample of 2366 members of the college of family physicians of canada received a confidential survey. the questionnaire collected data on socio-demographic characteristics, medical training, practice type, setting and hcv-related care practices. the self-adminisratd questionnaire was also made available to participants for completion on the internet. batdti: response proportion was 33%. median age was 41 years (47% female) and the proporlionoffrench questionnaires was 26%. approximately 88% had completed family medicine residency lllining in canada; median year of training completion was 1995. sixty-seven percent, 38% and 29% work in private offices/clinics, community hospitals and emergency departments, respectively. regarding ~practices, 94% had ever requested a hcv test and 87% of physicians had screened for hcv iafrction in rhe past 12 months· median number of tests was 10. while 17% reported having no hcv-uaed patients in their practic~, 44% had 1-5 hcv-infected patients. regarding the level of hcv care provided, 4.3% provide ongoing advanced hcv care including treatment and dose monitoring for ctmduions: in this sample of canadian family physicians, most had pro~ided hcv screening. to •least one patient in the past year. less than half had 1-5 hcv-infected patients and 41 % provide ~:relared care the role of socio-demographic factors, medical training as wel_i as hcv ca~e percep-lldas 10 rhe provision of appropriate hcv screening will be examined and described at the time of the canference. '4-50 (c) healthcare services: the context of nepal meen poudyal chhetri """1tl.ction healthcare service is related with the human rights and fundamental righ~ of the ci~ ciaaiuntry. however, the growing demand foi health care services, quality heal~care service, accessib1b~ id die mass population and paucity of funds are the different but interrelated issues to .be ~ddressed. m nepat.1n view of this context, public health sector in nepal is among other sectors, which is struggling -.i for scarce resources. . . . nepal, the problems in the field of healthcare servic~s do not bnut ~o the. paucity of faads and resources only, but there are other problems like: rural -urban imbalance, regional unbalance, poster sessio~ f the ll ·m1 ·ted resources poor healthcare services, inequity and inaccessibility of the poor management o , . poor people of the rural, remote and hilly areas for the healthcare services and so on.. . . . · . i f ct the best resource allocation is the one that max1m1zes t e sum o m ivi ua s u11 · ea t services. n a , · h d' ·b · · · h . ·t effi.ciency and efficient management are correlated. it might be t e re istn utmn of mes. ence, equi y, . . . . 1 . income or redistribution of services. moreover, maximizanon of available resources, qua tty healthcare services and efficient management of them are the very important and necessary tools and techmques to meet the growing demand and quality healthcare services in nepal. p4-51 (a) an jn-depth analysis of medical detox clients to assist in evidence based decision making xin li, huiying sun, ajay puri, david marsh, and aslam anis introduction: problematic substance use represents an ever-increasing public health challenge. in the vancouver coastal health (vch) region, there are more than 100,000 individuals having some probability of drug or alcohol dependence. to accommodate this potential demand for addiction related services, vch provides various services and treatment, including four levels of withdrawal management services (wms). clients seeking wms are screened and referred to appropriate services through a central telephone intake service (access i). the present study seeks to rigorously evaluate one of the services, vancouver detox, a medically monitored 24-bed residential detox facility, and its clients. doing so will allow decision makers to utilize evidence based decision-making in order to improve the accessibility and efficiency of wms, and therefore, the health of these clients. methods: we extract one-year data (october 1, 2003 -september 30, 2004 from an efficient and comprehensive database. the occupancy rate of the detox centre along with the clients' wait time for service and length of stay (los) are calculated. in addition, the effect of seasonality on these variables and the impact of the once per month welfare check issuance on the occupancy rate are also evaluated. results: among the 2411 clients (median age 40, 65% male) who were referred by access! to vancouver detox over the one-year period, 1448 were admitted. the majority (81 %) of those who are not admitted are either lost to follow up (i.e., clients not having a fixed address or telephone) or declined service at time of callback. the median wait time was 1 day [q3-ql: 3-1], the median los was 5 days iq3-qt: 6-3], and the average bed occupancy rate was 83%. however, during the threeday welfare check issue period the occupancy rate was lower compared to the other days of the year 175% vs. 84%, p conclusion: our analysis indicates that there was a relatively short wait time at vancouver detox, however 40% of the potential clients were not served. in addition, the occupancy rate declined during the welfare check issuance period and during the summer. this suggests that accessibility and efficiency at vancouver detox could be improved by specifically addressing these factors. background: intimate partner violence (ipv) is associated with acute and chronic physical and men· tal health outcomes for women resulting in greater use of health services. yet, a vast literature attests to cultural variations in perceptions of health and help-seeking behaviour. fewer studies have examined differences in perceptions of ipv among women from ethnocultural communities. the recognition, definition, and understanding of ipv, as well as the language used to describe these experiences, may be different in these communities. as such, a woman's response, including whether or not to disclose or seek help, may vary according to her understanding of the problem. methods: this pilot study explores the influence of cultural factors on perceptions of and responses to ipv among canadian born and immigrant young women. in-depth focus group interviews were con· ducted with women, aged 18 to 24 years, living in toronto. open-ended and semi-structured interview questions were designed to elicit information regarding how young women socially construct jpv and where they would go to receive help. interviews were transcribed, then read and independently coded by the research team. codes were compared and disagreements resolved. qualitative software qsr n6 was used to assist with data management. . ruu~ts_: res~nses_abo~t what constitutes ipv were similar across the study groups. when considering specific ab.us1ve ~1tuanons and types of relationships, participants held fairly relativistic views about ipv, especially with regard to help-seeking behaviour. cultural differences in beliefs about normaive m;ile/femal~ relations. familial.roles, and customs governing acceptable behaviours influenced partictpants perceptions about what 1n1ght be helpful to abused women. interview data highlight the social l05ter srnfons v91 11111 suucrural _impact these factors ha:e on you?g women and provide details regarding the dynamics of cibnocultur~ m~uences on help-~eekmg behav10ur: t~e ro~e of such factors such as gender inequality within rtlaoo?sh1ps and t_he ~erce1ved degree of ~oc1al 1solat1on and support nerworks are highlighted. collc~ the~ findmgs unde~score the _1mporta_nc_e of understanding cultural variations in percrprions of ipv ~ relanon to ~elp-seekmg beha~1':'ur. th1s_mformation is critical for health professionals iodiey may connnue developmg culturally sensmve practices, including screening guidelines and protorol s. ln addition, _this study demonstr~tes that focus group interviews are valuable for engaging young romen in discussions about ipv, helpmg them to 'name' their experiences, and consider sources of help when warranted. p4-s3 (a) health problems and health care use of young drug users in amsterdam .wieke krol, evelien van geffen, angela buchholz, esther welp, erik van ameijden, and maria prins /11trod11ction: recent advances in health care and drug treatment have improved the health of populations with special social and health care needs, such as drug users. however, still a substantial number dots not have access to the type of services required to improve their health status. in the netherlands, tspccially young adult drug users (yad) whose primary drug is cocaine might have limited access to drugrreatment services. in this study we examined the history and current use of (drug associated) treatmmt services, the determinants for loss of contact, and the current health care needs in the young drug mm amsterdam study (yodam). methods: yodam started in 2000 and is embedded in the amsterdam cohort study among drug mm. data were derived from y ad aged < 30 years who had used cocaine, heroin, ampheramines and i or methadone at least 3 days a week during the 2 months prior to enrolment. res11lts:of 195 yao, median age was 27 years (range: 18-30 years), 72% was male and 83% had 1dutch nationality at enrolment. nearly all participants (97%) reported a history of contact with drug llt.lnnent services (methadone maintenance, rehabilitation clinics and judicial treatment), mental health car? (ambulant mental care and psychiatric hospital) or general treatment services (day-care, night-care, hdp for living arrangements, work and finance). however, only 61 % reported contact in the past six l!xlllths. this figure was similar in the first and second follow-up visit. among y ad who reported no current contact with the health care system, 87% would like to have contact with general treatment serl' icts. among participants who have never had contact with drug treatment services, 67% used primarily cocaine compared with 22% and 8% among those who reported past or current contact, respectively. saied on the addiction severity index, 70% reported at least one mental health problem in the past 30 days, but only 11 % had current contact with mental health services. concl11sion: results from this study among young adult drug users show that despite a high contact rm with health care providers, the health care system seems to lose contact with yao. since 87% indicatt the need of general treatment services, especially for arranging house and living conditions, health m services that effectively integrate general health care with drug treatment services and mental health care might be more successful to keep contact with young cocaine users. mtthods: respondents included adults aged 18 and over who met dsm-iv diagn?snc criteria for an anxiety or depressive disorder in the past 12 months. we performed two sets of logisnc regressmns. thtdichotomous dependent variables for each of the regressions indicated whether rhe respondenr_vis-ud a psychiatrist, psychologist, family physician or social worker in the _past_ 12 months. no relationship for income. there was no significant interaction between educatmn an mco~:· r: ::or respondents with at least a high school education to seek help ~rom any of the four servic p were almost twice that for respondents who had not completed high school. th . d ec of analyses found che associacion becween educacion and use of md-provided care e secon s · · be d · · ·f· ly 1 ·n che low income group for non-md care, the assoc1anon cween e ucatlon and was s1gm icant on -· . . . . use of social workers was significant in both income groups, but significant only for use of psychologists in che high-income group. . . . conclusion: we found differences in healch service use by education level. ind1v1duals who have nor compleced high school appeared co use less mental he~lt~ servi~es provided ~y psyc~iatrists, psycholo· gists, family physicians and social workers. we found limited e.v1dence _suggesting the influence of educa· tion on service use varies according to income and type of service provider. results suggesc there may be a need to develop and evaluate progr~ms.designe~ to deliver targeted services to consumers who have noc completed high school. further quahtanve studies about the expen· ence of individuals with low education are needed to clarify whether education's relationship with ser· vice use is provider or consumer driven, and to disentangle the interrelated influences of income and education. system for homeless, hiv-infected patients in nyc? nancy sahler, chinazo cunningham, and kathryn anastos introduction: racial/ethnic disparities in access to health care have been consistently documented. one potential reason for disparities is that the cultural distance between minority patients and their providers discourage chese patients from seeking and continuing care. many institucions have incorporated cultural compecency craining and culturally sensicive models of health care delivery, hoping co encourage better relacionships becween patients and providers, more posicive views about the health care system, and, ulcimacely, improved health outcomes for minority patients. the current scudy tests whether cultural distance between physicians and patients, measured by racial discordance, predicts poorer patient attitudes about their providers and the health care system in a severely disadvantaged hiv-infected population in new york city that typically reports inconsistent patterns of health care. methods: we collected data from 396 unscably housed black and latino/a people with hiv who reported having a regular health care provider. we asked them to report on their attitudes about their provider and the health care system using validated instruments. subjects were categorized as being racially "concordant" or "discordant" with their providers, and attitudes of these two groups were compared. results: the sample consisted of 256 (65%) black and 140 (35%) latino/a people, who reported having 80 (20%) black physicians, 49 (12%) latino/a physicians, 167 (42%) white physicians, and 100 (25%) physicians of another/unknown race/ethnicity. overall, 260 (75%) subjects had physicians of a different race/ethnicity than their own. racial discordance did not predict negative attitudes about rela· tionship with providers: the mean rating of a i-item trust in provider scale (lo=high and o=low) was 8.0 for both concordant and discordant groups, and the mean score in 13-icem relationship with provider scale (4=high and !=low) was 3.5 for both groups. however discordance was significantly associated with distrust in che health care syscem: che mean score on a 7-icem scale (5=high discrust and l=low distrust) was 3.4 for discordant group and 3.0 for che concordant group (t= 2.66, p= 0.008). we further explored these patterns separacely in black and lacino/a subgroups, and using different strategies ro conceptualize racial/ethic discordance. conclusions: in this sample of unscably housed black and latino/a people who receive hiv care in new york city, having a physician from the same racial/ethnic background may be less important for developing a positive doctor-patient relationship than for helping the patients to dispel fear and distrust about the health care system as a whole. we discuss the policy implications of these findings. ilene hyman and samuel noh . .abstract objectiw: this study examines patterns of mental healthcare utilization among ethiopian 1mm1grants living in toronto. methods: a probability sample of 342 ethiopian adults ( 18 years and older) completed structured face-to-face interviews. variables ... define, especially who are non-health care providers. plan of analysis. results: approximately 5% of respondents received memal health services from mainstream healthcare providers and 8% consulted non-healthcare professionals. of those who sought mental health services from mainstream healthcare providers, 3.1 % saw family physicians, 2.1 % visited a psychiatrist. and 0.6% consulted other healthcare providers. compared with males, a significantly higher proportion 1gsfer sessions v93 ri ftlnales consulted non-healthcare_ professionals for emotional or mental health problems (p< 0.01 ). tlbile ethiopian's overall use of mamstream healthcare services for emotional problems (5%) did not prlydiffer from the rate (6%) of the general population of ontario, only a small proportion ( 12.5%) rjerhiopians with mental health needs used services from mainstream healthcare providers. of these, !oj% received family physicians' services, 4.3 % visited a psychiatrist, and 2.2% consulted other healthll/c providers. our data also suggested that ethiopian immigrants were more likely to consult tradioooal healers than health professionals for emotional or mental health problems ( 18.8% vs. 12.5% ). our bivariate analyses found the number of somatic symptoms and stressful life events to be associated with an increased use of medical services and the presence of a mental disorder to be associated with a dfcreased use of medical services for emotional problems. however, using multivariate methods, only die number of somatic symptoms remained significantly associated with use of medical services for emooonal problems. diu#ssion: study findings suggest that there is a need for ethnic-specific and culturally-appropriate mrcrvention programs to help ethiopian immigrants and refugees with mental health needs. since there ~a strong association between somatic symptoms and the use family physicians' services, there appears robe a critical role for community-based family physicians to detect potential mental health problems among their ethiopian patients, and to provide appropriate treatment and/or referral. the authors acknowledge the centre of excellence for research in immigration and settlement (ceris) in toronto and canadian heritage who provided funding for the study. we also acknowledge linn clark whose editorial work has improved significantly the quality of this manuscript. we want to thank all the participants of the study, and the ethiopian community leaders without whose honest contributions the present study would have not been possible. this paper addresses the impact of the rationalization of health-care services on the clinical decision-making of emergency physicians in two urban hospital emergency departments in atlantic canada. using the combined strategies of observational analysis and in-depth interviewing, this study provides a qualitative understanding of how physicians and, by extension, patients are impacred by the increasing ancmpts to make health-care both more efficient and cost-effective. such attempts have resulted in significantly compromised access to primary care within the community. as a consequence, patients are, out of necessity, inappropriately relying upon emergency departments for primary care services as well as access to specialty services. within the hospital, rationalization has resulted in bed closures and severely rmricted access to in-patient services. emergency physicians and their patients are in a tenuous position having many needs but few resources. furthermore, in response to demands for greater accountability, physicians have also adopted rationality in the form of evidence-based medicine. ultimately, ho~ever, rationality whether imposed upon, or adopted by, the profession significantly undermines physu.:1ans' ability to make decisions in the best interests of their patients. johnjasek, gretchen van wye, and bonnie kerker introduction: hispanics comprise an increasing proportion of th.e new york city (nyc) populanon !currently about 25%). like males in the general population, h1spamc males (hm) have a lower prrval,nce of healthcare utilization than females. however, they face additional access barriers such as bnguage differences and high rates of uninsurance. they also bear a heavy burden of health problems lllehasobesity and hiv/aids. this paper examines patterns of healthcare access and ut1hzat1on by hm compared to other nyc adults and identifies key areas for intervention. . . . 148 9 01 5 8 9 01 and older are significantly lower than the nhm popu anon . 10 v. . 10, p<.05), though hi\' screening and immunizations are comparable between the two groups. conclusion: findings suggest that hm have less access t? healthcare than hf or nhm. hown1r, hm ble to obtain certain discrete medical services as easily as other groups, perhapsdueto!rtor are a hm. i i . subsidized programs. for other services, utilization among 1s ower. mprovmg acc~tocareinthis group will help ensure routine, quality care, which can lead to a greater use of prevennve services iii! thus bener health outcomes. introduction: cancer registry is considered as one of the most important issues in cancer epidemiology and prevention. bias or under-reporting of cancer cases can affect the accuracy of the results of epidemiological studies and control programs. the aim of this study was to assess the reliability of the regional cancer report in a relatively small province (yasuj) with almost all facilities needed for c3llcll diagnosis and treatment. methods: finding the total number of cancer cases we reviewed records of all patients diagnoicd with cancer (icd 140-239) and registered in any hospital or pathology centre from1999 until 2001 i n yasuj and all (5) surrounding provinces. results: of 504 patients who were originally residents of yasui province, 43.7% wereaccoulll!d for yasuj province. the proportion varies according to the type of cancer, for exarnplecancetsofdiglstive system, skin and breast were more frequently reported by yasuj's health facilities whereas cancmoi blood, brain and bone were mostly reported by neighbouring provinces. the remaining cases (56.3%1 were diagnosed, treated and recorded by neighbouring provinces as their incident cases. this is partly because of the fact that patients seek medical services from other provinces as they believed that the facil. ities are offered by more experienced and higher quality stuffs and their relative's or temporary acooiii' modation addresses were reported as their place of residence. conclusion: measuring the spatial incidence of cancer according to the location of report ortht current address affected the spatial statistics of cancer. to correct this problem recording the permanm! address of diagnosed cases is important. p4-60 (c). providing primary healthcare to a disadvantaged population at a university-run commumty healthcare facility tracey rickards the. c:ommuni~y .h~alth ~linic (chc) is a university sponsored nurse-managed primary bealthwt (p~c:l clime. the clm1c is an innovative model of healthcare delivery in canada that has integrated tht principles of phc ser · · h' . vices wit ma community development framework. it serves to provide access to phc services for members of th · · illi · dru is ii be . . e community, particularly the poor and those who use or gs, we mg a service-learning facil'ty f d · · · · · · d rionll h . . .,m.:. · t · . meet c ient nee s. chmc nursing and social work staff and srudents r·--· ipa em various phc activities and h .l.hont" less i . f . outreac services in the local shelters and on the streels to'"" popu auon o fredericton as well th chc · model iii fosterin an on oi : . • e promotes and supports a harm reduction . · local d!or an~ h ng ~art:ersh1p with aids new brunswick and their needle exchange program, w1tha ing condoms and :xu:t h:~~~e e~aint~nance therapy clients, and with the clie~ts themselves ~_r; benefits of receiving health f ucation, a place to shower, and a small clothing and food oai~· care rom a nurse p · · d d · --""~'i"· are evidenced in th r research that involved needsaans mvo ves clients, staff, and students. to date the chc has unacn-1 · sessment/enviro i . d ; •• '""""1ll eva uanon. the clinic has also e . d nmenta scan, cost-benefit analysis, an on-go...,,1"".'i'~ facility and compassionate lea x~mme the model of care delivery' focusing on nursing roles wi~ cj rmng among students. finally, the clinic strives to share the resu•p v95 . -arch with the community in which it provides service by distributing a bi-monthly newsletter, and plllicipating in in-services and educational sessions in a variety of situations. the plan for the future is coolinued research and the use of evidence-based practice in order to guide the staff in choosing how much n~ primary healthcare services to marginalized populations will be provided. n-61 (c) tuming up the volume: marginalized women's health concerns tckla hendrickson and betty jane richmond bdrotbu:tion: the marginalization of urban women due to socio-economic status and other determinants negatively affects their health and that of their families. this undermines the overall vitaliry of urban communities. for example, regarding access to primary health care, women of lower economic surus and education levels are less likely to be screened for breast and cervical cancer. what is not as widely reported is how marginalized urban women in ontario understand and articulate their lack of access to health care, how they attribute this, and the solutions that they offer. this paper reports on the rnults of the ontario women's health network (owhn) focus group project highlighting urban women's concerns and suggestions regarding access to health care. it also raises larger issues about urban health, dual-purpose focus group design, community-based research and health planning processes. mdhods: focus group methodology was used to facilitate a total of 30 discussions with 55 urban and 54 rural women across ontario from 2003 to 2005. the women were invited to participate by local women's and health agencies and represented a range of ages, incomes, and access issues. discussions focussed on women's current health concerns, access to health care, and information needs. results were analyzed using grounded theory. the focus groups departed from traditional focus group research goals and had two purposes: 1) data collection and dissemination (representation of women's voices), and 2) fostering closer social ties between women, local agencies, and owhn. the paper provides a discussion and rationale for a dual approach. rax/ts: the results confirm current research on women's health access in women's own voices: urban women report difficulty finding responsive doctors, accessing helpful information such as visual aids in doctors' offices, and prohibitive prescription costs, in contrast with rural women's key concern of finding a family doctor. the research suggests that women's health focus groups can address access issues by helping women to network and initiate collective solutions. the study shows that marginalized urban women are articulate about their health conctrns and those of their families, often understanding them in larger socio-economic frameworks; howtver, women need greater access to primary care and women-friendly information in more languages and in places that they go for other purposes. it is crucial that urban health planning processes consult directly with women as key health care managers, and turn up the volume on marginalized women's voices. women: an evaluation of awareness, attitudes and beliefs introduction: nigeria has one of the highest rates of human immunodeficiency virus ihivi seroprrvalence in the world. as in most developing countries vertical transmission from mother to child account for most hiv infection in nigerian children. the purpose of this study was ro. determine the awareness, attitudes and beliefs of pregnant nigerian women towards voluntary counseling and testing ivct! for hiv. mnbod: a pre-tested questionnaire was used to survey a cross section '.>f.240 pregnant women ~t 2 (lrlleral antenatal clinics in awka, nigeria. data was reviewed based on willingness to ~c~ept or re1ect vct and the reasons for disapproval. knowledge of hiv infection, routes of hiv transm1ssmn and ant1rnroviral therapy iart) was evaluated. hsults: 72% of the women had good knowledge of hiv, i 5% had fair knowledge while 1.1% had poor knowledge of hiv infection.48% of the women were not aware of the association of hreast milk feeding and transmission of hiv to their babies. majority of the women 87% approved v~t while 13% disapproved vct, 93% of those who approved said it was because vct could ~educe risk of rransmission of hiv to their babies. all respondents, 100% who accepted vc.i ~ere willing to be tnted if results are kept confidential only 23% accepted to be tested if vc.t results w.111 be s~ared w1.th pinner and relatives 31 % attributed their refusal to the effect it may have on their marriage whale 69 '-gave the social 'and cultural stigmatization associated with hiv infection for their r~fusal.s 9 % wall accept vct if they will be tested at the same time with their partners.81 ~0 of ~omen wall pref~r to breast feed even if they tested positive to hiv. women with a higher education diploma were 3 times v96 more likely to accept vct. knowledge of art for hiv infected pregnant women as a means of pre. vention of maternal to child transmission [pmtct) was generally poor, 37% of respondents wm aware of art in pregnancy. conclusion: the acceptance of vct by pregnant women seems to depend on their understanding that vct has proven benefits for their unborn child. socio-cultur al factors such as stigmatizationof hiv positive individuals appears to be the maj_or impedi~ent towards widespread acceptanee of ycr in nigeria. involvemen t of male partners may 1mpro~e attitudes t~wa~ds vct:the developmentofm novative health education strategies is essential to provide women with mformanon as regards the benefits of vct and other means of pmtct. p4-63 (c) ethnic health care advisors in information centers on health care and welfare in four districts of amsterdam arlette hesselink, karien stronks, and arnoud verhoeff introduction : in amsterdam, migrants report a "worse actual health and a lower use of health care services than the native dutch population. this difference might be partly caused by problems migrants have with the dutch language and health care and welfare system. to support migrants finding their way through this system, in four districts in amsterdam information centers on health care and welfare were developed in which ethnic health care advisors were employed. their main task is to provide infor· mation to individuals or groups in order to bridge the gap between migrants and health care providers. methods: the implementat ion of the centers is evaluated using a process evaluation in order to give inside in the factors hampering and promoting the implementat ion. information is gathered using reports, attending meetings of local steering groups, and by semi-structu red interviews with persons (in)directly involved in the implementat ion of the centers. in addition, all individual and groupcontaets of the health care advisors are registered extensively. results: since 2003 four information centers, employing 12 ethnic health care advisors, are implemented. the ethnicity of the health care advisors corresponds to the main migrant groups in the different districts (e.g. moroccan, turkeys, surinamese and african). depending on the local steering groups, the focus of the activities of the health care advisors in the centers varies. in total, around 2000 individual and 225 group educational sessions have been registered since the start. most participants were positive about the individual and group sessions. the number of clients and type of questions asked depend highly on the location of the centers (e.g. as part of a welfare centre or as part of a housing corporation). in all districts implementa tion was hampered by lack of ongoing commitment of parties involved (e.g. health care providers, migrant organization s) and lack of integration with existing health care and welfare facilities. discussion: the migrant health advisors seem to have an important role in providing information on health and welfare to migrant clients, and therefore contribute in bridging the gap between migrants and professionals in health care and welfare. however, the lack of integration of the centers with the existing health care and welfare facilities in the different districts hampers further implementation . therefore, in most districts the information centres will be closed down as independent facilicities in the near future, and efforts are made to better connect the position of migrant health advisor in existing facilities. the 2005 who report ranks the philippines as ninth among 22 countries with a high tb prevalence. about a fourth of the country's population is infected, with majority of cases coming from the lower socioeconomic segments of the community. metro manila is not only the economic and political capital of the philippines but also the site of major universities and educational institutions. initial interviews with the school's clinicians have established the need to come up with treatment guidelines and protocols for students and personnel when tb is diagnosed. these cases are often identified during annual physical examinations as part of the school's requirements. in many instances, students and personnel diagnosed with tb are referred to private physicians where they are often lost to follow-up and may have failure of treatment due to un monitored self-administered therapy. this practice ignores the school clinic's great potential as a tb treatment partner. through its single practice network (spn) initiative, the philippine tuberculosis initiatives for the private sector (philippine tips), has established a model wherein school clinics serve as satellite treatment partners of larger clinics in the delivery of the directly observed treatment, short course (dots) protocol. this "treatment at the source" allows school-based patients to get their free government-suppl ied tb medicines from the clinic each day. it also cancels out the difficulty in accessing medicines through the old model where the patient has to go to the larger clinic outside his/her school to get treatment. the model also enables the clinic to monitor the treatment progress of the student and assumes more responsibility over their health. this experience illustrates how social justice in health could be achieved from means other than fund generation. the harnessing of existing health service providers in urban communities through standardized models of treatment delivery increases the probability of treatment success, not only for tb but for other conditions as well. p4-66 (c) voices for vulnerable populations: communalities across cbpr using qualitative methods martha ann carey, aja lesh, jo-ellen asbury, and mickey smith introduction: providing an opportunity to include, in all stages of health studies, the perspectives and experiences of vulnerable and marginalized populations is increasingly being recognized as a necessary component in uncovering new solutions to issues in health care. qualitative methods, especially focus groups, have been used to understand the perspectives and needs of community members and clinical staff in the development of program theory, process evaluation and refinement of interventions, and for understanding and interpreting results. however, little guidance is available for the optimal use of such information. methods: this presentation will draw on diverse experiences with children and their families in an asthma program in california, a preschool latino population in southern california, a small city afterschool prevention program for children in ohio, hiv/aids military personnel across all branches of the service in the united states, and methadone clinic clients in the south bronx in new york city. focus groups were used to elicit information from community members who would not usually have input into problem definitions and solutions. using a fairly common approach, thematic analysis as adapted from grounded theory, was used to identify concerns in each study. next we looked across these studies, in a meta-synthesis approach, to examine communalities in what was learned and in how information was used in program development and refinement. results: while the purposes and populations were diverse, and the type of concerns and the reporting of results varied, the conceptual framework that guided the planning and implementation of each study was similar, which led to a similar data analysis approach. we will briefly present the results of each study, and in more depth we will describe the communalities and how they were generated. conclusions: while some useful guidance for planning future studies of community based research was gained by looking across these diverse studies, it would be useful to pursue a broader examination of the range of populations and purposes to more fully develop guidance. background: the majority of studies examining the relationship between residential environments and cardiovascular disease have used census derived measures of neighborhood ses. there is a need to identify specific features of neighborhoods relevant to cardiovascular disease risk. we aim to 1) develop methods· data on neighborhood conditions were collected from a telephone survey of s,988 fesi· dents in balth:.ore, md; forsyth county, nc; and new york, ny. a sample of 120 of the i.ni~~l l'elpondents was re-interviewed 2-3 weeks after the initial interview t~ measure the tes~-~etest rebab1~1ty of ~e neighborhood scales. information was collected across seven ~e1ghborho~ cond1~ons (aesth~~ ~uah~, walking environment, availability of healthy foods, safety, violence, social cohesion, and acnvmes with neighbors). neighborhoods were defined as census tracts or homogen~us census tra~ clusters. ~sycho metric properties.of the neighborhood scales were accessed by ca~cu~~.ng chronba~h s alpha~ (mtemal consistency) and intraclass correlation coefficients (test-r~test reliabilmes) .. pear~n s .corre~anons were calculated to test for associations between indicators of neighborhood ses (tncludmg d1mens1ons of race/ ethnic composition, family structure, housing, area crowding, residential stability, education, employment, occupation, and income/wealth) and our seven neighborhood scales. . chronbach's alphas ranged from .73 (walking environment) to .83 (violence). intraclass correlations ranged from .60 (waling environment) to .88 (safety) and wer~ high~~~ .7~ for ~urout of the seven neighborhood dimensions. our neighborhood scales (excluding achv1hes with neighbors) were consistently correlated with commonly used census derived indicators of neighborhood ses. the results suggest that neighborhood attributes can be reliably measured. further development of such scales will improve our understanding of neighborhood conditions and their importance to health. childhood to young adulthood in a national u.s. sample jen jen chang lntrodfldion: prior studies indicate higher risk of substance use in children of depressed mothers, but no prior studies have followed up the offspring from childhood into adulthood to obtain more precise estimates of risk. this study aimed to examine the association between early exposure to maternal depl'elsive symptoms (mds) and offspring substance use across time in childhood, adolescence, and young adulthood. methods: data were obtained from the national longitudinal survey of youth. the study sample includes 4,898 mother-child/young adult dyads interviewed biennially between 1992 and 2002 with children aged 4 to 16 years old at baseline. data were gathered using a computer-assisted personal interview method. mds were measured in 1992 using the center for epidemiologic studies depression scale. offspring substance use was assessed biennially between 1994 and 2002. logistic and passion regression models with generalized estimation equation approach was used for parameter estimates to account for possible correlations among repeated measures in a longitudinal study. rnlllta: most mothers in the study sample were whites (42%), urban residents (79%), had a mean age of 31 years with at least a high school degree (82%). the mean child age at baseline was 9 years old. offspring cigarette and alcohol use increased monotonically across childhood, adolescence, and young adulthood. differential risk of substance use by gender was observed. early exposure to mds was associated with increased risk of cigarette (adjusted odds ratio (aor) = 1.52, 95% confidence interval (0): 1.12, 2.08) and marijuana use (aor = 1.46, 95% ci: 1.02, 2.08), but not with alcohol use across childhood, adolescence, and young adulthood, controlling for a child's characteristics, socioeconomic status, ~ligiosity, maternal drug use, and father's involvement. among the covariates, higher levels of father's mvolvement condluion: results from this study confirm previous suggestions that maternal depressive symptoms are associated with adverse child development. findings from the present study on early life experi-e~ce have the potential to inform valuable prevention programs for problem substance use before disturbances become severe and therefore, typically, much more difficult to ameliorate effectively. the ~act (~r-city men~ health study predicting filv/aids, club and other drug transi-b~) study 15 a multi-level study aimed at determining the association between features of the urban enyjronment mental health, drug use, and risky sexual behaviors. the study is randomly sampling foster sessions v99 neighborhood residents and assessing the relations between characteristics of 36 ethnographically defined urban neighborhoods and the health outcomes of interest. a limitation of existing systematic methods for evaluating the physical and social environments of urban neighborhoods is that they are expensive and time-consuming, therefore limiting the number of times such assessments can be conducted. this is particularly problematic for multi-year studies, where neighborhoods may change as a result of seasonality, gentrification, municipal projects, immigration and the like. therefore, we developed a simpler neighborhood assessment scale that systematically assessed the physical and social environment of urban neighborhoods. the impact neighborhood evaluation scale was developed based on existing and validated instruments, including the new york city housing and vacancy survey which is performed by the u.s. census bureau, and the nyc mayor's office of operations scorecard cleanliness program, and modified through pilot testing and cognitive testing with neighborhood residents. aspects of the physical environment assessed in the scale included physical decay, vacancy and construction, municipal investment and green space. aspects of the social environment measured include social disorder, social trust, affluence and formal and informal street economy. the scale assesses features of the neighborhood environment that are determined by personal (e.g., presence of dog feces), community (e.g., presence of a community garden), and municipal (e.g., street cleanliness) factors. the scale is administered systematically block-by-block in a neighborhood. trained research staff start at the northeast corner of an intersection and walk around the blocks in a clockwise direction. staff complete the scale for each street of the block, only evaluating the right side of the street. thus for each block, three or more assessments are completed. we are in the process of assessing psychometric properties of the instrument, including inter-rater reliability and internal consistency, and determining the minimum number of blocks or street segments that need to be assessed in order to provide an accurate estimate of the neighborhood environment. these data will be presented at the conference. obj«tive: to describe and analyze the perceptions of longterm injection drug users (idus) about their initiation into injecting. toronto. purposive sampling was used to seek out an ethnoculturally diverse sample of idus of both genders and from all areas of the city, through recruitment from harm reduction services and from referral by other study participants. interviews asked about drug use history including first use and first injecting, as well as questions about health issues, service utilization and needs. thematic analysis was used to examine initiation of drug use and of injection. results: two conditions appeared necessary for initiation of injection. one was a developed conception of drugs and their (desirable) effects, as suggested by the work of becker for marijuana. thus virtually all panicipants had used drugs by other routes prior to injecting, and had developed expectations about effects they considered pleasureable or beneficial. the second condition was a group and social context in which such use arose. no participants perceived their initiation to injecting as involving peer pressure. rather they suggested that they sought out peers with a similar social situation and interest in using drugs. observing injection by others often served as a means to initiate injection. injection served symbolic purposes for some participants, enhancing their status in their group and marking a transition to a different social world. concl111ion: better understanding of social and contextual factors motivating drug users who initiate injection can assist in prevention efforts. 10 ma!onty of them had higher educational level (57%-highschool or higher).about 20.2 yo adffiltted to have history of alcohol & another 12.4% had history of smoking. only 3.2% people were on hrt & 3.1 % were receiving steroid. majority of them (81.2) did not have history of osteoporosis. 13.6% have difficulty in ambulating. only 8.8% had family history of osteoporosis. bmd measurements as me~sured by dual xray absorptiometry (dexa) were used for the analysis. bmd results were compare~ w1~ rbc folate & serum vitamin b12 levels. no statistical significance found between bmd & serum v1taffiln b12 level but high levels of folate level is associated with normal bmd in bivariate and multivariate analysis. conclusion: in the studied elderly population, there was no relationship between bmd and vitamin b12; but there was a significant association between folate levels & bmd. introduction: adolescence is a critical period for identity formation. western studies have investigated the relationship of identity to adolescent well-being. special emphasis has been placed on the influence of ethnic identity on health, especially among forced migrants in different foreign countries. methodology: this study asses by the means of an open ended question identity categorization among youth in three economically disadvantaged urban communities in beirut, the capital of lebanon. these three communities have different histories of displacement and different socio-demographic makeup. however, they share a history of displacement due to war. results and conclusion: the results indicated that nationality was the major category of identification in all three communities followed by origin and religion. however, the percentages that self-identify by particular identity categories were significantly different among youth in the three communities, perhaps reflecting different context in which they have grown up. mechanical heart valve replacement amanda hu, chi-ming chow, diem dao, lee errett, and mary keith introduction: patients with mechanical heart valves must follow lifelong warfarin therapy. war· farin, however, is a difficult drug to take because it has a narrow therapeutic window with potential seri· ous side effects. successful anticoagulation therapy is dependent upon the patient's knowledge of this drug; however, little is known regarding the determinants of such knowledge. the purpose of this study was to determine the influence of socioeconomic status on patients' knowledge of warfarin therapy. methods: a telephone survey was conducted among 100 patients 3 to 6 months following mechan· ical heart valve replacement. a previously validated 20-item questionnaire was used to measure the patient's knowledge of warfarin, its side effects, and vitamin k food sources. demographic information, socioeconomic status data, and medical education information were also collected. results: sixty-one percent of participants had scores indicative of insufficient knowledge of warfarin therapy (score :s; 80%). age was negatively related to warfarin knowledge scores (r= 0.27, p = 0.007). in univariate analysis, patients with family incomes greater than $25,000, who had greater. than a grade 8 education and who were employed or self employed had significantly higher warfarm knowledge scores (p= 0.007, p= 0.002 and p= 0.001 respectively). gender, ethnicity, and warfar~n therapy prior to surgery were not related to warfarin knowledge scores. furthermore, none of t~e. m-hospital tea~hing practices significantly influenced warfarin knowledge scores. however, panic1~ants who _rece1v~d post discharge co~unity counseling had significantly higher knowledge scores tn comp~r1son with those who did not (p= 0.001 ). multivariate regression analysis revealed that und~r~tandmg the ~oncept of 1?ternational normalized ratio (inr), knowing the acronym, age and receiving ~ommum1!' counseling after discharge were the strongest predictors of warfarin kn~wledge. s~1oeconom1c status was not an important predictor of knowledge scores on the multivanate analysis. poster sessions v101 ~the majority of patients at our institution have insufficient knowledge of warfarin therapy.post-discharge counseling, not socioeconomic status, was found to be an important predictor of warfarin knowledge. since improved knowledge has been associated with improved compliance and control, our findings support the need to develop a comprehensive post-discharge education program or, at least, to ensure that patients have access to a community counselor to compliment the in-hospital educatiop program. brenda stade, tony barozzino, lorna bartholomew, and michael sgro lnttotl#ction: due to the paucity of prospective studies conducted and the inconsistency of results, the effects of prenatal cocaine exposure on functional abilities during childhood remain unclear. unlike the diagnosis of fetal alcohol spectrum disorder, a presentation of prenatal cocaine exposure and developmental and cognitive disabilities does not meet the criteria for specialized services. implications for public policy and services are substantial. objective: to describe the characteristics of children exposed to cocaine during gestation who present to an inner city specialty clinic. mnbods: prospective cohort research design. sample and setting: children ages 5 to 15 years old, referred to an inner city prenatal substance exposure clinic since november, 2003. data collection: data on consecutive children seen in the clinic were collected over an 18 month period. instrument: a thirteen (13) page intake and diagnostic form, and a detailed physical examination were used to collect data on prenatal substance history, school history, behavioral problems, neuro-psychological profile, growth and physical health of each of the participants. data analysis: content analysis of the data obtained was conducted. results: twenty children aged 6 to 14 years (mean= 9.8 years) participated in the study. all participants had a significant history of cocaine exposure and none had maternal history or laboratory (urine, meconium or hair) exposure to alcohol or other substances. none met the criteria of fetal alcohol spectrum disorder. all were greater than the tenth percentile on height, weight, and head circumference, and were physically healthy. twelve of the children had iqs at the 19th percentile or less. for all of the children, keeping up with age appropriate peers was an ongoing challenge because of problems in attention, motivation, motor control, sensory integration and expressive language. seventy-four percent of participants had significant behavioral and/or psychological problems including aggressiveness, hyperactivity, lying, poor peer relationships, extreme anxiety, phobias, and poor self-esteem. conclusion: pilot study results demonstrated that children prenatally exposed to cocaine have significant learning, behavioural, and social problems. further research focusing on the characteristics of children prenatally exposed to cocaine has the potential for changing policy and improving services for this population. methods: trained interviewers conducted anonymous quantitative surveys with a random sample (n= 148) of female detainees upon providing informed consent. the survey focused on: sociodemographic background; health status; housing and neighborhood stability and social resource availability upon release. results: participants were 70% african-american, 16% white, 9% mixed race and 5% native american. participants' median age was 37, the reported median income was <s2,000 year, and 53% reported receiving a high school diploma or equivalent. women reported a number of health conditions rypical of underserved populations, including asthma (42%), sexually transmitted infections (39%), high blood pressure (19%), hcv (14%), diabetes (5%) and hiv (4%). nearly half (46%) did not anticipate having a place to stay for at least 30 days upon release. factors significantly associated housing stability upon release were: high family support score (adjusted odds ratio [aor) 6.15; 95% confidence interval (95% cl) 1.76, 21.61), high neighborhood stability score (aor 4.41; 95% cl 1.25, 15.52), wanting a commercial sex worker (csw) support group (aor 0.25; 95% cl 0.10, 0.62); and identifying as bisexual (aor 0.24; 95% cl 0.07, 0.75). women desired employment (95%), housing (84%), education (77%), drug treattnent (74%), help with custody of children (31 %), childcare (28%), and domestic violence suppon (20%) services. conclusions: female detainees represent one of urban centers' most marginalized pcjpwatioos. short periods of detention represent a unique opportunity for _interven?~ns bri~ co~s and public health institutions. service providers should collaborate ~1th local ~ails to .p~de a con1111uwnof care for female detainees upon release that includes transportation , housing, residential drug treallllcnt, employment, education, family reunification, childcare and domestic viol~nce su~rt. ~pecial attenlion is warranted to lesbian and bisexual women and csws, who may be especially margmalized &om family and service-based support networks. introduction: "health care and ethnic minority immigrants" examines how health care policy dil· ferences between canada and the united states impact the health-related hardships, access and use of preventative care, and health outcomes of urban ethnic minority immigrants in both countries. methods: the findings of this paper build on the results of my qualitative comparative study of hotel workers in vancouver, bc and seattle, wa that revealed greater health related hardships for similarly matched employees in seattle compared to vancouver. in this paper, i examine the generalizability of these findings at the cross-national level for similar ethnic minority immigrant groups. i compare the impact of health care policy differences on specific groups that have emigrated to cities in both canada and the united states. these include immigrants from china, viemam, philippines, west indies, africa, sri lanka, pakistan, and latin america. comparative statistical analysis -utilizing rel· evant data from statistics canada and the u.s. census -reveal the extent to which health policy differ· ences help explain how current health policy in the united states disadvantages urban ethnic minority immigrants. results: many ethnic minority immigrants are a part of the working poor, a group which disproportionately lacks health insurance coverage in the united states. their position in the labour market makes them most vulnerable to exclusion from health care services. the data reveals how current u.s. health policy creates barriers for recently arrived ethnic minority immigrants to access health insurance and care. it remains difficult to ascribe health outcome differences directly to policy differences because of the challenge in disentangling the complex interacting interventing variables, including income inequality, that determine health outcomes. yer suggestive evidence suggests that health policy in the united states is harming the health of urban ethnic minority immigrants. the current health policy regime in the united states harms the health access, care, and outcomes of urban ethnic minority immigrants. comparing the fortunes of similar immigrants in cana· dian and u.s. cities reveals these patterns of disadvantage and some of their consequences. these barriers are an understudied factor in the sociological literature on "segmented assimilation" and social exclu· sion. the paper ends with policy recommendatio ns to improve access to health care among ethnic minor· ity immigrants in both countries, with the goal of reducing health related hardships, and improving health outcomes. mass index deyu pan, richard baker, and keith norris badtgrou~ over the past 3 decades, there has been dramatic increase in prevalence of obesity in the us population. however, the relationship between obesity and the prevalence of cardiovascular dis· ease (cv~) risk factors in different race/ethnic groups over time is not well known. . ik~rgn: _we use 2 cross-sectional , nationally representative surveys: national health and nutri· t10nal examination survey (nhanes iii 1988 -1994 (n= 14 029) and nhanes 1999 nhanes -2002 , including white, black, and hispa~ic races who were no~preg~ant, aged 20 to 74 years. res11lt1: the preval~nce of high cholesterol level (~ 240 mg/dl), untreated high blood pressure (2: l40/90 mm hg) an~ diabetes were calculated according to bmi group (lean, <25; overweight, 25-29; and obese, 2: 30) for different race/ethnicity groups. over the approximately 10 years period (from 1988l ~94 ~o [1999] [2000] [2001] [2002] , reducrion in 3 cvd risk factors in non-hispanic white has been observed. for mmonty race/~hnicity groups, black and hispanic, there had been increases in prevalence in high blood fa pressure and diabetes. the lean group experienced larger increase in prevalence of those cvd risk ctors. . fo~~ the prevalence in cvd risk factors over time had reduced in most of the bmi ca1egoes r t e w ~~e population. however, for black and hispanic, prevalence of 2 of 3 cvd risk factors ave generauy uncreased, especially in overweight and obese groups. methods: strategies identified to achieve these goals include: developing meaningful neighbourhood and district boundaries for comparing health information, providing free access to neighbourhood health profiles (including relevant data at the smallest level for which valid rates could be calculated); mapping relationships of inequality at a variety of nested levels; providing a range of formats (maps, tables) to meet the needs of users; providing technical support; seeking user input to advance and improve the site; and conducting workshops to foster access and use of data for decision-making , advocacy and collaboration. an evaluation strategy is being developed to assess the efforts and impact of these strategies. a preliminary assessment of the results of the first six months of activity will be completed in october 2005. results: significant differences in health are shown at all the geographic levels indicating success in developing the boundaries. results tabulated for the first six months use of the site include: site visits; media use; reference/acknow ledgement of the site in other documents; number of contacts through presentations and workshops; feedback from users identifying strengths and limitations; and, response from health decision makers. members of the partnership are identifying additional tools and services to further support actions that reduce health inequalities. the assessment will be used to develop more specific goals, targets and monitoring mechanisms. conclusions: our experience with paper-based health profiles indicates that they have been used extensively for program planning and advocacy. the greater availability of information in a publiclyavailable web-based format is expected to translate into even greater usefulness and wider application. the web site has had considerable usage to date, extensive media coverage. site users and workshop participants have provided positive feedback and suggestions for next steps. the six month review will be available in october 2005. in india, more than 4 and a half people are hiv-positive and half a million have aids. india currently has the largest number of positive people in the world, oumumbering south africa and accounting for nearly one tenth of the global hiv/aids prevalence. the world bank predicts that by 2015, there will be 35 million hiv/aids cases in india. the central challenge facing hiv prevention efforts in south asia today is learning how to respond to the societal determinants of vulnerability to hiv. hiv/aids is an issue largely engulfed by social stigma. stigma and discrimination are often considered the foremost barriers to effective poster sess10ns v104 prevention and care initiatives. stigma can make a person afraid of disclosing their status~ ~yone el se, and may make them feel depressed or alone. stigma can .also le~d to poor adherence to medicanon, ~ likelihood to access health and social services, and less desire to hve. hiv+ men and women may beafraidtotd! their co-workers that they have hiv for fear of their reactions or for fear of losing their jobs. in a study 1 conducted of people living with hiv/aids in delhi, india ov~r. 2003-~, many respo~ts shared their experiences of discrimination in their families, in their communmes, an~ m health ~e settmgs. these findings will be shared in the workshop, and will be compa~ed to m~ ex~nences workmg as an ~ co~l~ at the center for aids prevention studies in san francisco, cahforma and congreso de lannos unidos m philadelphia, pennsylvania. the workshop will include an overview of hiv/aids in urban contexts in tht united states (los angeles, san francisco, new york, philadelphia) and south asia (delhi, hyderabad, chennai). differences in methods for outreach, prevention and treatment efforts between devdoped world and developing world contexts will be discussed. a large portion of the workshop will be centered on discussion. participants will engage in an exercise to categorize their ideas of stigma. the workshop will also include a powerpoint presentation on the quantitative data gathered from the survey. the number of homeless and runaway youth is increasing in toronto, which is currently home to 3 out of 4 youth in the gt a who live alone, and it is the preferred locale for the street-involved and homeless youth. the city's youth population is expected to grow by nearly 20% by 2011, the school dropout rate is rising, and there are is a paucity of empirical data on what works with these at-risk youth. over a two-year period, (2002) (2003) (2004) , youthlink-inner city, conducted an evaluation of the impact of a harm reduction program aimed at reducing street youth's risk of contracting/infecting others with the hep c virus, which has both short-term and long-term deleterious effects. demographic data from 102 street-youth, along with resources/services used, employment methods, type and frequency of drug use, health status, police contacts, and their views of program impact were analyzed using a standardized sur· vey. additionally, focus groups with youth, agency staff and community key informants were conducted, as well as in-depth interviews with three youth, over a sixth-month period. this paper details study results, which both inform both practice and future study about what works, why it works, and how best to work with these vulnerable youth. sho~ed .that the wasteland in this area was contaminated by as cd zn pb cu simultaneously. the con-ta~matton of as ~d was quit significant. the contamination problems and environmental issues in this region are very. serious. the awareness on environmental and health issues was investigated by spot survey and analysis. overall awareness among the residents in the region is very poor. the measures must be taken to assu~e t~e .sustainable economy development by local and central gorvernments. keywords nandan guangx1; mmmg area; environmental awareness. concern like, epileptic fits, vision problems, earache, cough of more than 3 weeks and urinary problems. ninety-five subjects did not take bath regularly and 42 were exposed to sex. the health problems identified are only of a particular point of time and it may be difficult to interpret the depth of "iceberg" of the street children's world also draw the health-illness continuum. periodic interaction and follow-up is very difficult as they generally disappear from the research setting due to their frequent mobility for survival measures. as per the prediction of the iceberg theory, only some problems may have been identified, larger problems in respect to their health may not have been identified. in spite of various limitations, this attempt had proven that, it is possible to enter the street children world, explore the iceberg of illnesses, and establish data of health-illness continuum through an effective nursing agency. it is recommended to deworm these children, provide nutritional education, establish "condom corners" and "street children help line" in the city and urban slum areas and undertake action research on their health. malnutrition is a serious problem in developing countries like bangladesh. malnutrition causes a great deal of child suffering. malnutrition is one of the major causes of morbidity and mortality among the child. the aim of present study was to investigate the nutritional status and its relation to socioeconomic conditions of urban disadvantaged child of under five years age. nutritional status was determined by anthropoemetric measurement(heig ht, weight, mid-arm circumference etc.). determination of total protein and serum albumin were done for biochemical examination. haematological examination was done by blood haemoglobin profile estimation by cynmethhaemoglob in method and determination of haematocrit value or packed cell volume (pcv). stool examination for ova of hook worm, round worm, trichuris species and other species were also done. relevant information on socioeconomic characteristics was recorded by interviewing the house hold head using pretested questionnaire. our study demonstrated that nutritional status of the children are positively correlated with family income, parents level of formal education and negatively related to the family size. the effect of family income on the nutritional status, haematological and biochemical indices of urban disadvantaged child under five will be discussed in detail which will help us to determine proper way of utilization of health care facilities exists in developing countries. introduction: with the increase in morbidity and reduction in mortality and with the introduction of highly active antiretroviral therapy (haart), there is increasing focus on the determinants of healthrelated quality of life (hrqol) in hiv-infection. the focus on the present study is to examine the relationship between social support, depression, and hiv disease severity, and the extent to which these variables impact on hrqol in adult men with hiv-infection. methods: as part of a larger ongoing natural history study focusing on the neurobehavioural complications of hiv and aids, we administered questionnaires to assess depression (beck depression inventory), social support, and hrqol (mos-hiv) to 366 adult men with hiv-infection. a structured interview was used to collect health information and data on hiv disease severity. physical and mental health summary scores (phs and mhs) were derived through principal components analysis. participants were grouped according to level of social support: "well supported" (n= 180), "moderately supported" (n=90), "little or ineffective support" (n=96). analysis of variance was conducted to assess the effects of three levels of perceived social support, depression status (present vs absent), and hiv disease severity (aids vs non-aids) on mental and physical health dimensions of hrqol. potential interaction effects were also evaluated. results: social support was found to have a significant effect on both mhs and phs. presence of depression was found to have a significant effect on mhs but not phs. hiv disease severity (presence of aids diagnosis) was found to have a significant effect on phs but not mhs. manov a analyses revealed no significant interactions effects. conclusions and implications: level of social support, presence of depression and hiv disease severity were shown to have independent effects on hrqol. feeling "well supported" appears to have significant benefits for mental and physical health. biological indicators of hiv disease appear to have a selective impact on physical health while presence of depression is related to significant reductions in mental health. the development and evaluation of behavioural interventions to improve perceived social support network and depression will likely result in significant improvements in health outcomes of adults with hiv and aids. introduction: ongoing medical advances have greatly enhanced the longevity of penons living with aids (plwa). however, for certain subgroups living with hiv/aids, including, members of minority groups, women and the economically underprivilege d, aids outcomes have not changed so favorably. one obvious factor to consider when explaining disparities in aids outcomes is the environ· ment within which an individual resides. few studies of aids outcomes have focused on how commu· nity setting influences the effectiveness of care delivery. the principle research questions were: 1) what is the relationship between the locations of areas with concentrations of pl wa and the distribution of both primary and ancillary service providers? 2) how has the widespread availability of haart (after 1996) impacted aids outcomes at the community level? 3) how do aids mortality and fatality rates vary when related community characteristics, such as household income, ethnic mix, and geographic access to primary and ancillary hiv/aids services are considered? we examine this issue across residential communities in the los angeles area. methods: los angeles county's comprehensive aids service providers database (compiled and maintained by aids project los angeles) was geo-referenced and distances between weighted mean population centers and the nearest hiv/aids service providers were calculated to provide a measure of access which was then merged with aids diagnosis and mortality data along with relevant socioeconomic and population indicators and for analysis using bivariate and multivariate statistics at the zip code level. results: there is a growing disparity in hiv/aids outcomes between low-income minority areas and affluent non-minority areas in the post-haart era (p = 0.002). ancillary aids services such as case management and counseling and testing are located near the places where low income hiv/aids service consumers are likely to live (p =.000). in spite of having more access to ancillary services low income areas continue to be associated with smaller decreases post-haart aids mortality rates (p=0.301). conclusions: given the increasing disparities in aids outcomes between lower and upper income communities in the post -haart era, more specific research into the capacities of aids services providers and the efficiency of their interventions is required. although it is clear that ancillary services are operating in the areas of greatest need, their effectiveness remains limited. understanding the reasons for t~is, be they lack of adequate resources for inner city service providers, failure to reach target popula· tmns or other causes, requires additional research. in 20?3, y.out~ who were able to speak either french or english and had been absent from their parent's/ c~regivers ~es.1dence for at least three consecutive nights took part in the survey which consisted of inter· viewer-adm1ms tered question~aires. p.arti~i~an~s were recruited from drop in centres in 7 cities across <?-nada.y~uth self-report as either using in1ect1on or non-injection drugs. statistical analyses were car· r1ed out using sas version 8. the 'home' is a contested site for many women living in urban areas. women living in poverty and using illicit drugs are largely excluded from participation in dominant 'home-base' gender roles of wife and mother because of economic disadvantage. they thus 'make home' in ways that are specific to their everyday experiences. while research has established links between housing, health and drug use, this literature does not tend to consider the meanings that home may have within the lives of women who lack access to adequate tenured living spaces. this presentation reports the findings of my master's thesis work. using grounded theory methodology within a poststructuralist feminist conceptual framework, i conducted in-depth, open-ended interviews with 11 women living in the toronto area. the women were recruited from within the community through posters at different social service agencies (e.g., drop-in centres, community health centres). they were asked to describe their housing experiences and what home meant to them over the course of their lives. through the data analysis process, a substantive theory of 'making home' for women living in poverty and using illicit drugs emerged. the central concept of making home is a continual process of learning about and interacting with the environment. the process of making home entails different subprocesses: finding home (knowing what is available and accessible, and accessing home), adaptation, and leaving home. the different meanings of home that women developed from their experiences and knowledge emerged as critical components for the process of making home. the women i interviewed were very mobile and experienced constant change with regards to home. home was not considered only in relation to physical living space. home could be made within a relationship or with regards to a possession. while dominant meanings of home relation to tenured living spaces are generally positive, my research indicated that home could also be a negative context. the presentation will describe my research process and findings as well as the theoretical and policy implications of my results. in the western democracies, social citizenship bundles the right to social provision (defined as a share of the social good, not necessarily proportionate to the market value of a citizen's contribution) with participation in the paid labour force. for this reason, social citizenship rights, which usually involve some kind of redistributive measures, are a key mechanism by which states ensure economic equality, or at least, economic equality of opportunity, but are also a mechanism of social exclusion. social rights are inscribed around a single kind of relationship, that which exists between a male family breadwinner and the paid labor market; those who make economic contributions that fall outside this relationship -such as unpaid care-work, or, work that occurs in shadow or underground economiesare to a greater or lesser degree barred from accessing citizen-based social provisions. furthermore, social provision that occurs outside of the breadwinner/market-eco nomy dyad is very often meanstested and is accompanied by regulatory state apparatuses (this includes a range of social services, from welfare payments, to child-care subsidies, to disability pensions). social citizenship is thus stratified; the legal relationship that is at the heart of social citizenship acts as a form of social closure against claims for a share of the social good made by those who are 'lesser' citizens than others. of particular interest in this paper is how citizenship inequalities translate into inequalities in the social provisions that emerge from economic participation: the right to safety on the job, protection from employer harassment, protection from the vagaries of the market as well the right to adequate, comprehensive, and appropriate health services. drawing on mixed methods, longitudinal data from adult sex workers (n= 170) located in two research sites with different social welfare regimes -one in canada and one in the u.s. -we examine the consequences of working in a "shadow" or "underground" economy on various facets of health and access to health services, taking into consideration the mediating role that citizenship constructs and social welfare regimes play in accessing other key social and economic determinants of health. methods: the study population included methadone patients (re)admitted at the mhs in the period from 1/1/1996 to 31/1/2002, born in the netherlands, morocco, surinam, dutch antilles or turkye, and officially registered in the population register of amsterdam. the population register was used to ascer· rain the vital status. causes of death were provided by the central bureau of statistics. mortality was categorised as hiv related, directly drug related (overdose) and other.mortality. results: the methadone patients had the following characteristics: 38 years of age at entry; 81% male; 37% ethnic minority; 37% ever injected. in total, 9558 personyear (py) of observation time and 173 deaths (18% hiv related, 14% overdose; 57% other) were observed. hence, the crude mortality rate was 18/1000 py (95% ci 16-21/1000 py). the percentage of (ever) injectors was higher among the native dutch than among the ethnic minorities, 51 % and 14 % respectively. higher rates were observed among native dutch drug users compared to those belonging to an ethnic minority (age adjusted hazard ratio (hr) 1.9 (95% ci 1.4-2.7). the age adjusted hr of (ever) injecting drug users versus non injecting users was 3.0 (95% cl 2.2-4.1 ). after additional adjustment for route of administration a non significant difference between the dutch and ethnic minorities remained (hr 1.3, 95% cl 0.9-1.9, p-value 0.151. conclusion: the mortality rate among the heroin users in amsterdam is strongly affected by the high prevalence of non-injecting drug use. moreover, this study confirms that differences in mortality between the native dutch and the ethnic minorities is related to differences in route of administration. the ongoing reduction of injecting drug use (due tot selctieve mortality, and switching route ofadministration and popularity of crack cocaine) may lead to a further reduction of mortality rates among heroin users in amsterdam. interventions preventing the initiation of injecting should be encouraged. . introduction: food insecurity is an inequality that is central in the lives of many low-income cana· d1ans. people lack food security when regular access to nutritious food is limited or variable due to high prices'. low income, lack of transportation or inadequate food distribution, or they become disadvan· taged m other w~ys through the acquisition of food. a group comprising academics, health profession· als, and commumty workers who have experience in food insecurity, developed a pilot study in ottawa that sought to und~rstand the lived experience of food insecurity. the study also used the united states department of agriculture (usda) community food security module. methods: a series of group meetings determined the best research approaches; questionnaires and consent forms were developed by a working group. twenty-three self-identified food insecure respon· dents were interviewed. the data were analysed using frequency distributions· the food security module was coded according to the usda coding guide, and open-ended responses w~re coded using a grounded approach. renlts: preliminary results from our pilot study have given the research team cause for considerable concern. out of 14 households without children, 11 experienced food insecurity with hunger; 5 ~ere at the severe l~vel. out of 9 households with children, 7 experienced food insecurity with hunger. ~•rst person reflecnons commonly featured feelings of depression low self-esteem and despair. participants' account tha d . an 1 cu ty gettmg to the store, severely limited putting knowledge and skills mto practice. l'oster sessions v109 conclusion: based on these early findings, our research team is looking to challenge what appear to be assumptions about poverty and food insecurity and for best ways to use this information. some policy approaches and interventions that encourage people to eat better and exercise more, may be missing the mark for low-income individuals, and may serve to maintain food insecurity. future research will build on this pilot. with methods augmentation, and a comprehensive knowledge dissemination plan, we hope co contribute to research and policy frameworks at all levels. ps-25 (a) mental health and the corrections system: population-based analyses in urban, semi-urban, and rural settings julian somers, robert watts, and michelle patterson introduction: according to recent epidemiological research, rates of mental disorders vary considerably across countries and across regions within countries. nevertheless, rates of mental disorders (including substance use disorders) are consistently higher in populations involved in the corrections sys-1em. courts have long recognized the heavy burden of mental illness within their purview, and have developed innovative programs to better manage the needs of such individuals. the majority of these innovations (e.g., specialized courts) exist in urban settings. however, few studies have examined the degree of variability in rates of different mental disorders between courts in urban and other settings (e.g., semi-urban, rural). variability between courts in different settings, if present, holds implications for needs-based resource planning. the present study was conducted as part of a long-term inter-ministerial collaboration in british columbia (bc). analyses were conducted on linked administrative data regarding population health and correctional services. a database was constructed including all individuals sentenced in bc in a single year (n=49,142). corrections records were matched to records of health services utilization (hospital and outpatient services) for mental disorders and substance use disorders in the years preceding sentencing. services for mental health and substance-related problems were aggregated into three groups: severe mental illness (smi); less-severe mental illness (lsmi); alcohol/other drug (ad). courts were grouped, based on their annual volumes, inro three categories: "urban"; "semi-urban" and "rural." rates of service use for mental disorders were compared between groups for 1-year and 5-years preceding sentencing. results: there were significant differences in the rates of mental disorders in courts of different size (urban, semi-urban, rural) . however, differences between the rates of disorders within each of these groups exceeded the discrepancies between groups. comparatively high and low rates of disorders were observed in courts of each size. moreover, the courts with the highest rates of certain disorders (e.g., ad) did not have the highest rates of other types of mental disorders (e.g., smi). conclusions: rates of mental disorders differed significantly among the annual populations sentenced across courts in bc. urban courts (which process comparatively large numbers of people) have implemented services to address the needs of the mentally ill. the present analyses strongly suggest that program development should be closely linked to the demonstration of need. the results caution that a uniform approach to court programs for the mentally ill is likely to be inefficient, over-supplying services in some settings and underestimating need in others. results: public settings used for injection are characterized by highly unhygienic conditions, the presence of unsafely disposed syringes, limited availability of sterile syringes and a lack of sterile water for injecting. a number of unsafe injection practices observed were common among public injectors. the presence of police officers nearby and the potential of assault by street predators fostered rushed injecting among those consuming drugs in public spaces. the ecological features of these environments encourage unhygienic and unsafe injecting practices, heighten risk for overdose and the transmission of blood borne viruses. introduction: approximately 13% of women experience depression in the first weeks or months after giving birth. although it has been argued that postpartum depression (ppd) is a culture-bound p~ nomenon, experienced only in western, industrialized countries, recent international research studies have confirmed that the prevalence of ppd is similar around the world. however, little is known about variables that may influence the experience of ppd in women from diverse cultures and immigrant women. research is needed to identify the role culture may play in the presentation of ppd (i.e., types of symptoms most commonly reported), how women from various racial, ethnic and cultural backgrounds perceive and attribute their symptoms of ppd, and finally, how culture can be appropriately addressed in ppd treatment programs. method: to examine the role of culture in ppd, semi-structured interviews and self-report question· naires were administered to clients of the women's health centre of st. joseph's health centre, tor· onto. participants were identified as either first generation canadians (relative newcomen) (n=8) or second generation canadian (parents were immigrants to canada) (n=6). data were collected qd these two groups of women based on: severity of depression, symptom presentation, perceived role of social supports and culturally-specific traditions, and barriers and responses to treatment. the interview data were analyzed using qualitative methods (thematic content analysis), and the following themes were identified: 1) stress about passing on their culture to their children, 2) lack of social support and feelings of isolation, 3) perceived importance or lack of importance of cultural based tra· ditions, 4) conflicts with family members. about cultural traditions and beliefs, 5) mental health stigma within ethnic communities and beyond, 6) race-and sex-based discrimination, and 7) language barriers. conclrllion: identifying the role culture plays in the presentation of ppd, and how women from diverse backgrounds perceive and attribute their symptoms of ppd, is critical in order to establishcultur· ally appropriate guidelines for treatment options. furthermore, information gathered from this study can be used to develop policies and resources for delivering culturally competent care for newcomer wo~ who are dealing with ppd as well as the issues around acculturation (i.e., language barriers, racism). llus is of particular importance for urban health centers which provide postpartum care to diverse groups of women, and particularly recent immigrants or newcomers. ps-~8 (al. contaminated 'therapeutic landscape': perceptions of the aamjiwnaang fint nation keyln smith and isaac luginaah . in~: this. paper pres;ents the findings of an ongoing study among the aamjiwnaang f!rst nanon. aam11wnaang is located m the heart of samia's 'chemical valley', surrounded by cherrucal ~(ants such as esso, imperial oil, shell, suncor energy, and canada's largest hazardous waste disposal sate. safety kleen. this fint nation community is located within the st. clair river •area of concem'. as destgna~ by health canada for the population of samia and the surrounding region. although this comm_umty, by way of. its proximity to the numerous chemical plants is already exposed to high levels pollu~on, recent che?ucal dumping in ~mjiwnaang, as well as a failed proposal for another ethanol plant m the community, only helped to mtensify community concerns about potential health impacts of exposure. research on the cultural beliefs and traditions of first nation communities has established that th~ ~isting rel~tionship between first nations people and 'mother earth' is not only physical, but also_ spmtual. in this study we explore how the complex relationship between the aamjiwnaang first naaon and 'mother earth' be ha · · · i .. may c ngmg 111 a contammated 'therapeutic' landscape. the study a so ~lores h?w aam11wnaang residents are coping with these changes and with the probable conramina· boa of their community. ra!jts: aamjiwnaang residents acknowledge that they are living in a contaminated environment and are being impacted by emissions from the surrounding 'chemical valley' especially for future generations, and have expressed concerns that members of the community and mother earth are 'sick' as a result of this exposure. while moving from that 'place' has been discussed, residents have strong personal and generational connections to the land and insist however, that aamjiwnaang is their 'home' and will remain that way despite growing community concerns about the impact from industry. the contribution of this study lies in the direct involvement of community leadership in designing and conducting this research and distributing the results to further local policy development regarding community concerns on the reserve. background: truck driver are at very high risk for drug abuse because the factor contributing to drug abuse among driver are multiple. it is important to gain an understanding of the key factors that contributing to drug abuse among truck drivers. methods: seventy-truck driver aged 25-35 years were conveniently selected from the population. all the participants were male. an interview schedule was developed in the light of literature data and data were collected by personal interview with informed consent in selected urban area of eastern part of nepal. results: a considerable percentage of truck driver have less knowledge about drug abuse, its effect on body and their complication. majority of the participants (90%) explained that lack of education, financial crisis were leading factors of drug abuse. the average numbers of respondents were believed that lack of meaning in life, marital disharmony. friendship with drug abuser, stress, tiredness, modernization of life pattern and freedom from the home pressure were the influencing risk factors to drug abuse. conchuion: this study reveals those truck drivers are at risk in urban area for developing drugs abuse, mostly influenced by social and environmental factors. hence, these groups should undergo certain educational programme to prevent not only drug abuse, but also prevent transmission of infectious disease among these groups. introduction: food insecurity is an inequality that is central in the lives of many low-income canadians. our group of university researchers, health professionals, and community developers conducted a study in ottawa with community workers who work with people identified as food insecure. the purpose of the study was to understand the nature of their work, and their perspectives, perceptions and experiences of the causes and consequences of food insecurity. (a parallel study was conducted with food insecure participants). methods: sampling was through local knowledge of organizations involved in the provision of food to community members. written permission for the researchers to contact workers in confidence was obtained from each organization. thirteen in-depth interviews were conducted. the interviews consisted of a number of open-ended questions. the data were collated, and analysed using a grounded approach. results: workers interviewed represented organizations that offered diverse programming and methods of food distribution intended to address the needs of the community. the provision of food was seen as a necessary response to address hunger that arose for the majority from poverty caused by low income, or levels of government income support and safety nets that were insufficient to support food requirements. workers reported that food requirements varied according to different. ethnic backgrounds, ages of recipients and family configurations, and with food preferences, and d'.etary (health) requirements. workers' observations of the effects of insufficient food on adults and children such as depression, low energy and non-participation or social disengagement matched those of food insecure respondents. food insecurity was cited as "another srressor among the mountain of problems." overall, workers presented a picture of a silent, stigmatized, poor population including children, and a lack of advocacy to address the issues. be tha th · ·ty the results suggest that workers and organizations, like the commuruty mem rs t ey msecun • · · · · f ilab'l' f food · t d ·n thei·r ab1'l1'ty to address food insecurity. l1m1tanons m terms o ava 11ty o , serve, are restnc e 1 . . funding, and donations appeared to set the para?1eters of pr~g:am r.espon~ to commuruty food msecurity. workers were concerned with program des1g": and a~m1mstranon that m general addr~d hunger on an emergency short-term basis, although food msecunty was long-ter_m for ~any. sustainable solutions that include knowledge translation strategies, and health and social pohcy responses were suggested and will be explored in future research that hopes to build on this pilot. background: socioeconomic deprivation as well as low levels of soc!al s.uppoi:r have .been associated with poor outcomes following cardiac surgery. pre-operative depression m ~anents with coron~ry artery disease awaiting cabg ranges from 27-47% and is an independent predictor of post-operanve mortality. since st. michael's hospital has a large inner city population, this study was designed to determine the both the prevalence of depressive symptomatology (ds) in patients awaiting cabg as well as its relationship with socioeconomic status (ses) and social support. methods: consecutive patients referred for isolated cabg were invited to complete the centre for epidemiological studies depression scale (ces-d) as well as a social support scale and a life stressors inventory. a ces-d score of 2: 16 represents mild ds and a score of 2:27 represents moderate to severe ds. participants also recorded information on functional status, perceived progression of heart failure, demographic and ses variables. all participants with ces-d scores 2: 16 were considered to have ds. results: of the 75 patients (75%) who returned the survey, 22 (29.3%) of participants had a ces-d score 2: 16 with 8 (10.7%) of those having scores suggestive of severe depression. females and those with ongoing medical concerns tended to have more ds (p= 0.06 and p= 0.07 respectively). age was not related to ds. perceived worsening of symptoms and increased ccs angina class were significantly related to the presence of ds (p= 0.03 and p= 0.03 respectively) but not the number of diseased heart vessels. satisfaction with personal relationships including having someone to confide in (p=0.01), feeling lonely (p= 0.04) and not having a partner (p= 0.007) were significantly related to ds. being very upset by a recent breakup in the immediate family was also significantly related to ds (p= 0.02). having greater than a grade 9 education was the only ses variable related to ds (p=0.06). multivariate logistic regression suggested that not being partnered and having low education were the most significant predictors of ds. conclnsion: pre-operative depression is present in at least one quarter of patients referred for cabg. since ds is related to poor outcomes following cabg, single patients with few social supports and low education should be considered at high risk for ds. these findings support the need for the development of supportive interventions in patients at risk in order to decrease post-operative morbidity. this study examines the relationship between childhood sexual abuse, sexual assault during adolescence, and hiv-risk-related behaviours in a sample of 919 homeless youths. over the past decade, there has been increasing research devoted to the impact of childhood sexual abuse. some studies have suggested that the experience of childhood sexual abuse is associated with substance use and abuse, at· risk sexual behaviour and subsequent higher rates of hiv infection (ompad et al., 2005; cinq-mars et al., 2003; brown et al., 1997) . other studies have found that childhood abuse, especially sexual abuse, is c_o~n among stree~ youth (noell et al. 1999 ). this study compares sexually abused males and females livtng on the street ~th non-sexually abused street youth, with regards to unsafe sexual behaviours and e~ures to potmnal t:dv sources (e.g., tattooing, body piercing and injection drug use). the hypothe-sis oft~ ~nt study is that the prevalence of hn-risk-related behaviours will be higher among street youth vtctuns of sexual abuse than those who have not been sexually abused. the sample includes 919 youths aged 13 to 25 who met specific criteria's for itinerancy (649 male and 270 female, with a mean ~of 19.4 y· and a standard deviation of 2.9). they were recruited through five organisations working ~th~ you~ and were~ of an ongoing cohort study. all youth completed a 45 to 60 minute ques· ~onnaue on their sexual behaviours, use of drugs and alcohol and other hiv risk behaviours. oral spec· unens for hiv testing were. also collected. a total of 357 (38.9%) youth reported at least one incident of ~i •~use/assault (1~1 girls (67.0% of all the girls) and 176 boys (27.2% of all the boys]). preliminary descnpnve analyses pomt to sexually abused/assaulted youth showing the highest prevalence of sexual poster sessions v113 at-risk behaviours. for example, 43.3% of sexual abuse/assault victims have engaged in prostitution in their lives compared with 13.4% in the non-sexually abused/assaulted group. in addition, 42.6% of sexual abuse/assault victims reported having injected drugs, compared with 32.0% in the non-sexually abused/assaulted group. more statistical analyses will bring other significant factors to light, especially when we analyze separately those victim of childhood sexual abuse as compare to those victim of sexual assault after childhood. elucidating the risk factors for getting involved in hiv-risk-related behaviours is important for the development of comprehensive and appropriate prevention and treatment intervention strategies. introduction: new immigrants and refugees to canada frequently emigrate from regions of the world where intestinal parasitic infections with worms are endemic. of note, some of these parasites area capable of surviving for years to decades within a given host and can have life threatening consequences many years after initial infection. thus, early diagnosis and treatment of intestinal parasitic worms is considered important in high risk immigrant populations, however there remains a lack of consensus regarding the best method of accomplishing this. diagnosing worm infections through stool examinations is costly and has limited sensitivity, while presumptive treatment of all immigrants, although advocated by some, is expensive and results in healthy individuals being unnecessarily treated. herein, we examine the utility of a hematologic marker (i.e. peripheral blood eosinophilia) as a screening instrument to identify parasitic worm infections in new immigrants to toronto. we identified 192 adults, 15 years of age or older, who were screened for intestinal parasitic infections by stool examination as part of a recently developed screening protocol at a downtown toronto community health centre. we examined the prevalence of infections with worms and subsequently evaluated the impact of several demographic factors on the presence of worm infections. we also determined the sensitivity and specificity of peripheral blood eosinophilia (defined as greater than 500 eosinophils per ml) as a marker for intestinal parasitic worm infections. results: overall, 21 % of the 192 immigrants tested had evidence of at least one worm infection on a single stool examination, while 4% of such individuals bad infections with multiple worms concurrently. age did not appear to be associated with the presence of worms, however 29% of males had evidence of worm infections compared with 16% of females (p= 0.03). immigrants from asia, sub-saharan africa, and central america had the highest burden of overall infection. the sensitivity and specificity of peripheral blood eosinophilia for worm infections was 24% and 92% respectively. conclusions: one in five recent immigrants presenting to a community health centre in downtown toronto had evidence of at least one intestinal parasitic worm infection. the highest burden of illness was in men and in those emigrating from less developed regions of the world. peripheral blood eosinophilia is a poor screening test for worm infections however its presence is highly suggestive of the existence of worms in new immigrants. more than a decade ago, the journal of american college health recommended that a national study be conducted to measure the health status of african american college students, 'both health disparities and [their] positive healthful behaviors' (fennell, 1994) . it was later decided that when exploring the level of health risk behaviors for african american students, gender is an important variable and should be included in the research (fennell, 1997) . at historically black college and universities (hbcus) college health researchers must tackle gender disparities through highly-effective health promotion and disease prevention programs. the purpose of this review is to examine the literature that addresses the overarching health behaviors (i.e. risky sexual behavior, poor diet, smoking, physical inactivity, etc.) of african american men who attend hbcus. despite the various social, cultural, and academic benefits of african american students who attend hbcus, the number of research studies that adequately address their health and health behaviors is limited. studies indicate that african american men who attend hbcus are actively engaged in poor health behaviors. compared to females at hbcus, african american men are more likely to behave in ways that could result in injuries and the use tobacco, alcohol, and ~ther dru~. the city of toronto has recently proposed to conduct a street count of the homeless. like proposals that have come before, this most recent proposal is controversial among many homeless advocates and service providers because it is perceived as intrusive, likely to undercount the target population, and unnecessary for addressing the problem. advocates of the count view it as necessary to gauge the scope of the problem and advise city leaders about how to most effectively direct resources ro.the various homeless services. this paper first reviews the history of efforts to count homeless populat10ns m toronto, describing the motivations and arguments of people on both sides of the issue. second, the paper reviews the methodological complexities of counting homeless populations, describing approaches that use shelter-counts, administrative data and street observations. those interested in counts for toronto and elsewhere can benefit from this review of approaches that have been proposed and carried out in other municipalities. third, the paper proposes a new community-base d strategy that joins the skills of methodologists, local experts, and service providers to ensure a fair and accurate count. the method of systematic social observation avoids some of the problems of early counts by utilizing a repeated identification approach to minimize undercount bias. the method also has the advantage of involving and compensating homeless individuals for assisting with the street count estimates. the estimation approach can be implemented on a rolling basis throughout the year. drug transitions) is a multi-level study aimed at examining the associations between features of the urban environment and mental health, drug use, and risky sexual behaviors. research participants are being recruited in 36 new york city (nyc) neighborhoods. an essential first step in the implementation of this study was to delineate boundaries for each target neighborhood that could then be used to establish sampling frames and as key units of analytic interest. although many neighborhood studies rely on pre-established definitions (e.g. those used for the census or administrative purposes), such definitions do not always reflect contemporary settlement patterns. we felt that street level observations were a neces· sary component of the definition process. methods: the procedures used to delineate neighborhoods were: (1) development of census block maps of targeted areas; (2) review of land use maps and census tract data to ensure, prior to going into the field, tha.t particular blocks are residential and likely to have sufficient population for recruitment purposes; and (3) field vislts and observation in each of the targeted areas. field observations focused on: housing characteristics, including housing type and condition; characteristics of commercial areas, such as likely customer base (e.g. local or no~, socio-economic status and ethnicity of customers); pedestrian volume (including con· gregauons of people m parks and outside stores or residences); obstructions to pedestrian traffic (e.g. ma1or thoroughfares, multi-block industry or institutions); and maintenance and use of open spaces. results: in making the final decisions regarding neighborhood boundaries and the inclusion or exclusion of particular. blocks, we considered a broad range of factors including evidence of homogeneity an~ heterogeneity (socmeconomic, ethnic, housing type, environmental) across blocks within and blocks ad1acent to a nei.ghborhood, and across the 36 sample neighborhoods; the potential for efficient recruit· ment of appropriate particip.ants (i.e. sufficient local pedestrian traffic); and boundaries used in reporting census data (s? that population data can be used in our analysis). . altho~gh utilization of field observations for neighborhood definitions is relatively time consuming (ap~~ox1mately 2 hours for a 12 block area, with more diverse neighborhoods taking even longer), we annc1p~t~ that it will substantially improve the quality and consistency of our data gath· ~rmg ~nd analyse~. spec1f1c e?'amples from neighborhoods defined through this process will be given dur· m~ this presentatmn. we will also discuss the merits and limitations of characterizing neighborhoods usmg street level observations. has been no consideration for how social networks are associated with sse. the objective of this study was to identify personal and social network characteristics associated with being a recipient of sse. in this cross-sectional study, idus who injected in the past 6 months were recruited from syringe exchange programs in montreal, canada, between april 2004 and january 2005. information on each participant and on the persons with whom contact had occurred in the past month were elicited using a structured, interviewer-administe red questionnaire. participants provided detailed demographic and drug use information on up to 5 idu members with whom drugs or injecting materials were shared. using logistic regression, personal and network characteristics were examined in relation to receipt of sterile syringes. res.its: of 277 idus, 39% reported receiving sterile syringes in the past month from another idu. the sample mean age was 33 years (range 18-55), 73% male, 91 % caucasian, and 85% cocaine injectors. in multivariate analyses adjusted for age and gender, recipients of sterile syringes were more likely than non-recipients to share drugs after preparation (0r=2.39(1.06-5.42 )), to require help or to have helped inject another idu (or= 3.48(1.60-7.60)), to have asked someone to get sterile syringes from a syringe exchange program (or=2.54[1.20-5.40 )), to lend syringes (or=2.44[1.16-5.16) ) and to use drug preparation water that was previously used by another iou (0r=2.46(1.42-4.28) ) during the past 6 months. recipients also had a higher rate of change (p=0.01) of idus in their drug injecting network during the past month. with respect to social networks, 45% ( 171/348) of idu network members were providers of sterile syringes and were more likely than non-providers to inject everyday (or= 2.50( 1.51-4.15)). when stratified by their role as a sexual partner or as someone who provides support (e.g. friend, family member), further risk behaviours were observed in relation to sharing injecting materials with the participant. conclusion: recipients of sterile syringes and their idu network members had several high-risk behaviours for infection with bloodborne diseases. sse may afford an opportunity for the dissemination of other salutary behaviours such as information sharing on safe injecting practices between recipients and distributors. furthermore, drug injecting networks may be an avenue to reach idus who may not otherwise be exposed to preventive measures. there are numerous sources of stigma and labelling of mental and physical illness. first, the sociocultural dimension to stigma and labelling clearly influences patients, families, health care professionals and society's perception and treatment of illness. this creates multiple challenges related to illness identification and recovery, particularly in culturally diverse societies. second, the media is a major source of stigma and stereotyping with regard to a variety of mental and physical illnesses. despite increased public knowledge of many illnesses, studies have shown that stigma has intensified over the years in certain cases. a third element of stigma and labels appears with the use of diagnosis as a solution to human problems with the result that difficult to treat patients are repudiated and social issues are unaddressed. this presentation will serve to outline the development of stigma, various expressions of stigma, and consequences of stigma. it will provide some practical recommendations for the reduction of stigma related to mental and physical illness. purpose: to assess the knowledge, attitude, and practice about immunization for parent of 2-3 years old hispanic and african american children in los angeles county (lac). methods: we conducted two cluster surveys in lac. the sample was selected with probability proportionate to estimated size at the first stage and simple random sample of children at the second stage. data were collected through interview. we absuacted vaccine coverage data from immunization record card (irc) at home or from clinic records. the participation was 81 % in hispanics and 83% in african americans populations. irc was not available at home for 26% of african american and 16% of hispanic children. results: all parents perceived their children got all necessary vaccine~. fa~orable attitudes to.w~rd immunization were reported by 82 % of african american and 90% of h1s?amc parents. '.he m~1ority of the parents (90% african american and 95% hispanic) agreed that vaccines prevent senous d1se~ses among children. regarding vaccine safety, significantly more hispanic parents (91 %) than african american parents (75%) mentioned that vaccines are safe. introduction: cervical cancer has been shown to be preventable by papanicolau tests in heterosexual women; however, the utility of papanicolau test in lesbians is controversial. cervical cancer is caused by the human papillomavirus (hpv) which is transmitted by sexual _intercourse; howev_er, its' transmission by homosexual intercourse is also controversial. the goal of this study was to review the evidence for papanicolau tests in lesbians. methods: a pub med, ovid ( 1996 ovid ( to 2005 , and cochrane library search was performed using the mesh headings "homosexuality , female," "vaginal smears," and "papillomavirus, human." in pub med, the clinical queries feature was used with "diagnosis" and "broad, sensitive" filters. a google advanced search of the internet was conducted with the terms "lesbian," "cervical cancer," and "pap rest." the url was limited to ".ca," ".edu," or ".gov." results: the search yielded 8 original articles and 4 reviews, but no cochrane reviews. none of the publications were canadian. 111 website hits were found and selected websites were reviewed. studies showed that lesbians have fewer papanicolau tests than heterosexual women; however, many reported risk factors for cervical cancer, including multiple past or current sexual partners (male and female), early age of first coitus, history of sexually transmitted diseases, and cigarette smoking. hpv has been detected in 13-30% of lesbians and 6-19% of lesbians who have never had sex with men. case studies of abnormal papanicolau tests in lesbians who have never had sex with men have also been reponed. sexual transmission of hpv among lesbians can be explained by several factors: 1) 53-99% of lesbians have had sex with men and 21-30% of lesbians continue to have sex with men. 2) hpv may be transmitted by sexual behaviours among women, like digital-vaginal sex, digital-anal sex, oral sex, and the use of insertive sex toys. 3) hpv transmission requires only skin-to-skin contact. genital hpv has been identified on human fingers. some professional organizations, like the society of obstetricians and gynecologists of canada and the american cancer society, recognize the need for papanicolau tests in lesbians. other organizations, like the canadian cancer society, do not have official policy statements. the current ontario cervical screening program does not specifically address lesbians. conclusion: although the data was limited, most studies recommended that lesbians should receive papanicolau tests. the frequency of these tests was controversial. policy makers and professional organizations should address the needs of this special population and make specific recommendations . monique chaaya, ahia sibai, hiam chemaitelly, and zana el roueiheb . literature concerning the mental and physical effect of work at an old age is contradictory. in addinon, urban environments are physically and socially harsh with reduced potential for income generation and employment and increased potential for depression. the present paper is an attempt to study how do work, or lack of work, link to the experience of depression among older adults in underprivileged lebanese urban communities. the data comes from a cross-sectional survey, where 740 elderly residing in 3 underprivileged communities in beirut, including a palestinian refugee camp, were successfully inter-v1ewed through face-to-face interviews. logistic regression was performed to assess the effect of work on depression ad_justing for many other covariates. data showed that 17.5% of people aged 60 years and more were still workmg. there was a highly protective effect of work on depression in elderly males (or= 0.342, 95% cl= 0.128-0.909). the current study is the first of its kind in the arab region and more work shou_ld be _don_e in this area. it is important for the lebanese government to consider elderly rights for social mclus10n m terms of employment opportunities. amam nuru-jeter, nancy adler, and burton singer . l~u~on:_ the socioeconomic gradient is perhaps the most persistent and significant finding in social epidem1ological research. the insistent nature of the ses effect is evidenced by its significance after acc?untmg for ~umerous confounds. the significance and magnitude of the effect, however, varies by ::•al group. evidence_ o_f the relative effect of race and ses is inconclusive. further, less attention has f ~ ptid to the specific interactions of race and ses than to examinations of which is a better predictor 0 d ~ th. ~sues are further complicated by the explanatory complexity of the various measures of ses anl . owht ey relate to the health of various social groups. understanding the specific nature of these re a11ons ips 1s necessary for guiding he ith d · i 1· · h a an soc1a po mes and programs aimed at improving t e poster sessions v117 health of our most disadvantaged groups, and therefore population health, overall. this study examines the synergistic effects of ses with both race and gender in predicting group differences in mortality. methods: analyses use data from the national longitudinal mortality survey (n= 559, 715) for the years 1979, 1980, and 1981-85; and were linked to the national death index for deaths occurring through 1985. results were confirmed using data from the national mortality followback survey for people who died in 1986 (n= 13,491) and the 1986 national health interview survey (denominator). we used correlation analysis and the standardized mortality ratio to examine ses (education and income), race, and gender as predictors of group differences in changes in mortality. results: for the total population, analyses support the income gradient showing significant declines in mortality ratios from low to high income along the income strata. education shows a significant drop in mortality with completion of high school and completion of college, exhibiting a threshold rather than a gradient effect. compared to whites, blacks receive diminishing health returns for each subsequent level of income and education; with whites closely mirroring the total population in ses thresholds. similarly, compared to men, women do not experience the same health gains at comparable ses levels. for education, black men and women only show significant mortality declines upon completion of high school. the ses gradient operates differently among race and gender subgroups. threshold effects suggest resource gains in life opportunities at particular milestones along the ses gradient. further, ses matters less for blacks and women compared to whites and men suggesting differing implications for health and social policy aimed at reducing disparities. objective: we assessed the impact of diabetes in a large puerto rican community of chicago by measuring the prevalence of diagnosed diabetes and calculating the diabetes mortality rate. methods: we analyzed data from a comprehensive health survey conducted in randomly selected households in community areas. questions on diagnosed diabetes and selected risk factors were asked. in addition, mortality data were analyzed in order to calculate the age-adjusted diabetes mortality rate. when possible, rates were compared to those found in other studies. results: the diabetes prevalence located in this community (20.8%: 95% cl= 10.1 %-38.0%) is one of the highest ever reported for puerto ricans. for instance, it is more than three times higher than the prevalence for the us (6.1 %) and twice the prevalence for puerto ricans in new york ( 11.3%) and puerto rico (9.3%-9.6%). diagnosed diabetes was found to be significantly associated with obesity (p=0.023). the prevalence was particularly high among older people, females, those horn in the us, and those with a family history. the diabetes mortality rate (67.6 per 100,000 population) was more than twice the rate for all of chicago (31.2) and the us (25.4). conclusions: understanding why the diabetes and mortality rates for puerto ricans in this commu· nity are so much higher than those of other communities is imperative. collaboration between researchers, service providers and community members can help address the issues of diabetes education, early screening and diagnosis and effective treatment needed in this community. introduction: sars is a respiratory illness that is spread from person to person through dose contact. this infectious disease outbreak was unusual due to the high rate of infection among health care workers. the aim of the study is to explore the demographic and attitudinal determinants of psychological distress due to sars among health care workers of a toronto hospital. methods: a total of 997 adult participants completed questionnaires co assess psychological distress (impact of event scale -jes) and attitudes to sars. of these, 845 participants completed the questionnaire during the sars i outbreak, and 152 participants completed the questionnaire during the sars ii outbreak. participants also provided information on demographic variables, including occupation and exposure to sars. principal components analysis was used to derive eight attitudinal scales: fear: system, protection, prevention, job stress, stigma, instrumental coping, and psychological copm~. l111ear regression analyses were applied to evaluate the associations of the demographic and amtudmal variables, as well as time, with psychological distress. . . regression analyses showed that time and higher scores on fear, 1ob stress, stigma'. and instrumental coping were associated with a higher total ies score: (r2 =.30). contrary to expectations, none of the demographic variables was associated with total ies score. conclusion: the presence of psychological distress in health care workers is indicative of intrusive or avoidant responses to thoughts, feelings, and memories associated with the sars outbreak. this study shows that ( 1) fear for one's health and the he~lth of.others, (2) ex~erien~in~ job stress, (3)_tbe per· ception of stigma, (4) usage of instrumental copmg skills, ~n~ (5) t1m_e s1grufi~an~ly c~nmb~te to increased psychological distress in health care workers. potential mterventmns during mfecttous diseaie outbreaks in health care settings should be targeted to minimize the impact of these factors. purpose: the purpose of this study was to expand knowledge regarding feeding practices, know!· edge and nutritional beliefs of mothers currently residing in the dominican republic (dr). the rising incidence of obesity in children is a major national health concern and obesity in first and second-generation immigrant children also continues to rise. we know that parents (particularly mothers) shape children's early diet patterns and that immigrant mothers experience additional challenges related to acculturation and assimilation into a new culture, however there is a paucity of literature concerning the effect of immigrant adaptation to american culture on the nutrition of children. in addition, new immigrant knowledge about the causes of obesity and the resulting health implications for childhood obesity has not been assessed and reported. this qualitative study used focus group methodology to discuss views and practices related to the introduction of food for infants and feeding practices for young children, along with a discussion on knowledge and beliefs related to the causes and health implications of child· hood obesity. ten dominican mothers' of young children (birth to 6 years old) who reside in a small town in the western frontier area of the dr were participants in the focus group. results from this srudy will be compared to results from an identical study that will be conducted in fall 2005 with new immi· grant mothers from the dr to the boston area. we will compare feeding practices, beliefs and knowledge about nutrition for young children between these two groups. methods: using participatory principles, a focus group design was used to interview a group of mothers in the dr. the investigator along with a bilingual translator conducted the focus group. the session was tape recorded and is currently in the process of being transcribed and translated. transcribed data will be systematically analyzed themes will be identified. a qualitative data analysis software will be used to organize and code the data according to the specific aims of the study. supporting quotations will be selected to substrate each theme. lmpli~tions for urban health practice: an ultimate goal of this study is to lay a foundation for understanding the process of acculturation on feeding practices of mothers when they immigrate to the us. understanding beliefs, knowledge and feeding practices that mothers use with young children will help m ~he ~evelopment of culturally and linguistically appropriate educational strategies and materials for use m primary prevention of pediatric obesity. in canada more than seventy-five percent of immigrants choose to stay in three major urban centers of toronto, montreal and vancouver. approximately fifty percent of the immigrants eventually set· tie m ~~e greater tor<_>nto area. there is mounting evidence that the increasing immigrant population has a_ sigmfic~nt health disadvantage over canadian-born residents. this health disadvantage manifests particularly m the ma1"ority of 1"mm1"gr t h h d be · · h . . . . an s w o a en m canada for longer than ten years. this group as ~n associ~te~ with higher risk of chronic disease such as cardiovascular diseases. this disparity twccb n ma1onty of the immigrant population and the canadian-born population is of great importance to ur an health providers d" · i i · b as isproporttonate y arge immigrant population has settled in the ma1or ur an centers. generally the health stat f · · · · · · h h been . us 0 most 1mm1grants 1s dynamic. recent 1mm1grants w o av_e ant •;ffca~ada _for less ~han ~en years are known to have a health advantage known as 'healthy imm1• ~ants r::r · ~:s eff~ 1~ defined by the observed superior health of both male and female recent immiimmigrant participation in canadian society particularly the labour market. a new explanation of the loss of 'healthy immigrant effect' is given with the help of additional factors. lt appears that the effects of social exclusion from the labour market leading to social inequalities first experienced by recent immigrant has been responsible for the loss of healthy immigrant effect. this loss results in the subsequent health disadvantage observed in the older immigrant population. a study on patients perspectives regarding tuberculosis treatment by s.j.chander, community health cell, bangalore, india. introduction: the national tuberculosis control programme was in place over three decades; still tuberculosis control remains a challenge unmet. every day about 1440 people die of tuberculosis in india. tuberculosis affects the poor more and the poor seek help from more than one place due to various reasons. this adversely affects the treatment outcome and the patient's pocket. many tuberculosis patients become non-adherence to treatment due to many reasons. the goal of the study was to understand the patient's perspective regarding tuberculois treatment provided by the bangalore city corporation. (bmc) under the rntcp (revised national tuberculosis control programme) using dots (directly observed treatment, short course) approach. bmc were identified. the information was collected using an in-depth interview technique. they were both male and female aged between 4-70 years suffering from pulmonary and extra pulmonary tuberculosis. all patients were from the poor socio economic background. results: most patients who first sought help from private practitioners were not diagnosed and treated correctly. they sought help form them as they were easily accessible and available but they. most patients sought help later than four weeks as they lacked awareness. a few of patients sought help from traditional healers and magicians, as it did not help they turned to allopathic practitioners. the patients interviewed were inadequately informed about various aspect of the disease due to fear of stigma. the patient's family members were generally supportive during the treatment period there was no report of negative attitude of neighbours who were aware of tuberculosis patients instead sympathetic attitude was reported. there exists many myth and misconception associated with marriage and sexual relationship while one suffers from tuberculosis. patients who visited referral hospitals reported that money was demanded for providing services. most patients had to borrow money for treatment. patients want health centres to be clean and be opened on time. they don't like the staff shouting at them to cover their mouth while coughing. conclusion: community education would lead to seek help early and to take preventive measures. adequate patient education would remove all myth and conception and help the patients adhere to treatment. since tb thrives among the poor, poverty eradiation measures need to be given more emphasis. mere treatment approach would not help control tuberculosis. lntrod#ction: the main causative factor in cervical cancer is the presence of oncogenic human papillomavitus (hpv). several factors have been identified in the acquisition of hpv infection and cervical cancer and include early coitarche, large number of lifetime sexual partners, tobacco smoking, poor diet, and concomitant sexually transmitted diseases. it is known that street youth are at much higher risk for these factors and are therefore at higher risk of acquiring hpv infection and cervical cancer. thus, we endeavoured to determine the prevalence of oncogenic hpv infection, and pap test abnormalities, in street youth. ~tbods: this quantitative study uses data collected from a non governmental, not for profit dropin centre for street youth in canada. over one hundred females between the ages of sixteen and twentyfour were enrolled in the study. of these females, all underwent pap testing about those with a previous history of an abnormal pap test, or an abnormal-appearing cervix on clinical examination, underwent hpv-deoxyribonucleic (dna) testing with the digene hybrid capture ii. results: data analysis is underway. the following results will be presented: 1) number of positive hpv-dna results, 2) pap test results in this group, 3) recommended follow-up. . the results of this study will provide information about the prevalence of oncogemc hpv-dna infection and pap test abnormalities in a population of street youth. the practice implic~ tions related to our research include the potential for improved gynecologic care of street youth. in addition, our recommendations on the usefulness of hpv testing in this population will be addressed. methods: a health promotion and disease prevention tool was developed over a period of several years to meet the health needs of recent immigrants and refugees seen at access alliance multicultural community health centre (aamchc), an inner city community health centre in downtown toronto. this instrument was derived from the anecdotal experience of health care providers, a review of medical literature, and con· sultations with experts in migration health. herein we present the individual components of this instrument, aimed at promoting health and preventing disease in new immigrants and refugees to toronto. results: the health promotion and disease prevention tool for immigrants focuses on three primary health related areas: 1) globally important infectious diseases including tuberculosis (tb), hiv/aids, syphilis, viral hepatitis, intestinal parasites, and vaccine preventable diseases (vpd), 2) cancers caused by infectious diseases or those endemic to developing regions of the world, and 3) mental illnesses includiog those developing among survivors of torture. the health needs of new immigrants and refugees are complex, heterogeneous, and ohen reflect conditions found in the immigrant's country of origin. ideally, the management of all new immigrants should be adapted to their experiences prior to migration, however the scale and complexity of this strategy prohibits its general use by healthcare providers in industrialized countries. an immigrant specific disease prevention instrument could help quickly identify and potentially prevent the spread of dangerous infectious diseases, detect cancers at earlier stages of development, and inform health care providers and decision makers about the most effective and efficient strategies to prevent serious illness in new immigrants and refugees. lntrodmction: as poverty continues to grip pakistan, the number of urban street children grows and has now reached alarming proportions, demanding far greater action than presently offered. urbanization, natural catastrophe, drought, disease, war and internal conflict, economic breakdown causing unemployment, and homelessness have forced families and children in search of a "better life," often putting children at risk of abuse and exploitation. objectives: to reduce drug use on the streets in particular injectable drug use and to prevent the transmission of stds/hiv/aids among vulnerable youth. methodology: baseline study and situation assessment of health problems particularly hiv and stds among street children of quetta, pakistan. the program launched a peer education program, including: awareness o_f self and body protection focusing on child sexual abuse, stds/hiv/aids , life skills, gender and sexual rights awareness, preventive health measures, and care at work. it also opened care and counseling center for these working and street children ar.d handed these centers over to local communities. relationships among aids-related knowledge and bt:liefs and sexual behavior of young adults were determined. rea.sons for unsafe sex included: misconception about disease etiology, conflicting cultural values, risk demal, partner pressur~, trust and partner significance, accusation of promiscuity, lack of community endorsement of protecnve measures, and barriers to condom access. in addition socio-economic pressure, physiological issues, poor community participation and anitudes and low ~ducation level limited the effectiveness of existing aids prevention education. according to 'the baseline study the male children are ex~ to ~owledge of safe sex through peers, hakims, and blue films. working children found sexual mfor~anon through older children and their teachers (ustad). recommendation s: it was found that working children are highly vulnerable to stds/hiv/aids, as they lack protective meas":res in sexual abuse and are unaware of safe sexual practices. conclusion: non-fatal overdose was a common occurrence for idu in vancouver, and was associated with several factors considered including crystal methamphetamine use. these findings indicate a need for structural interventions that seek to modify the social and contextual risks for overdose, increased access to treatment programs, and trials of novel interventions such as take-home naloxone programs. background: injection drug users (idus) are at elevated risk for involvement in the criminal justice system due to possession of illicit drugs and participation in drug sales or markets. the criminalization of drug use may produce significant social, economic and health consequences for urban poor drug users. injection-related risks have also been associated with criminal justice involvement or risk of such involvement. previous research has identified racial differences in drug-related arrests and incarceration in the general population. we assess whether criminal justice system involvement differs by race/ethnicity among a community sample of idus. we analyzed data collected from idus (n = 1,084) who were recruited in san francisco, and interviewed and tested for hiv. criminal justice system involvement was measured by arrest, incarceration, drug felony, and loss/denial of social services associated with the possession of a drug felony. multivariate analyses compared measures of criminal justice involvement and race/ethnicity after adjusting for socio-demographic and drug-use behaviors including drug preference, years of injection drug use, injection frequency, age, housing status, and gender. the six-month prevalence of arrest was highest for whites (32%), compared to african americans (25%) and latinos (27% ), in addition to the mean number of weeks spent in jail in the past 6 months (7.0 vs. 5.8 and 4.2 weeks). these differences did not remain statistically significant in multivariate analyses. latinos reported the highest prevalence of a lifetime drug felony conviction (48%) and mean years of lifetime incarceration in prison (13.3 years), compared to african americans (48%, 10.7 years) and whites (34%, 6.9 years). being african american was independently associated with having a felony conviction and years of incarceration in prison as compared to whites. the history of involvement in the criminal justice system is widespread in this sample. when looking at racial/ethnic differences over a lifetime including total years of incarceration and drug felony conviction, the involvement of african americans in the criminal justice system is higher as compared to whites. more rigorous examination of these data and others on how criminal justice involvement varies by race, as well as the implications for the health and well-being of idus, is warranted. homelessness is a major social concern that has great im~act on th~se living.in urban commu?ities. metro manila, the capital of the philippines is a highly urbanized ar~ w.1t~ the h1gh~st concentration of urban poor population-an estimated 752,229 families or 3,005,857 md1v1duals. this exploratory study v122 is the first definitive study done in manila that explores the needs and concerns of street dwdlent\omc. less. it aims to establish the demographic profile, lifestyle patterns and needs of the streetdwdlersindit six districts city of manila to establish a database for planning health and other related interventions. based on protocol-guid ed field interviews of 462 street dwellers, the data is useful as a template for ref!!. ence in analyzing urban homelessness in asian developing country contexts. results of the study show that generally, the state of homelessness reflects a feeling of discontent, disenfranchisem ent and pow!!· lessness that contribute to their difficulty in getting out of the streets. the perceived problems andlar dangers in living on the streets are generally associated with their exposure to extreme weather condirioll! and their status of being vagrants making them prone to harassment by the police. the health needs of the street dweller respondents established in this study indicate that the existing health related servias for the homeless poor is ineffective. the street dweller respondents have little or no access to social and health services, if any. some respondents claimed that although they were able to get service from heallh centers or government hospitals, the medicines required for treatment are not usually free and are beyond their means. this group of homeless people needs well-planned interventions to hdp them improve their current situations and support their daily living. the expressed social needs of the sucet dweller respondents were significantly concentrated on the economic aspect, which is, having a perma· nent source of income to afford food, shelter, clothing and education. these reflect the street dweller' s need for personal upliftment and safety. in short, most of their expressed need is a combination of socioeconomic resources that would provide long-term options that are better than the choice of living on the streets. the suggested interventions based on the findings will be discussed. . methods: idu~ aged i 8 and older who injected drugs within the prior month were recruited in 2005 usmg rds which relies on referral networks to generate unbiased prevalence estimates. a diverse and mon· vated g~o~p of idu "seeds." were given three uniquely coded coupons and encouraged to refer up to three other ehgibl~ idu~, for which they received $5 usd per recruit. all subjects provided informed consent, an anonymous 1~t erv1ew and a venous blood sample for serologic testing of hiv, hcv and syphilis anti~!· results. a total of 213 idus were recruited in tijuana and 206 in juarez, of whom the maion!)' were .male < 9 .l.4% and 92.2%) and median age was 34. melhotls: using the data from a multi-site survey on health and well being of a random sample of older chinese in seven canadian cities, this paper examined the effects of size of the chinese community and the health status of the aging chinese. the sample (n=2,272) consisted of aging chinese aged 55 years and older. physical and mental status of the participants was measured by a chinese version medical outcome study short form sf-36. one-way analysis of variance and post-hoc scheffe test were used to test the differences in health status between the participants residing in cities representing three different sizes of the chinese community. regression analysis was also used to examine the contribution of size of the chinese community to physical and mental health status. rmdts: in general, aging chinese who resided in cities with a smaller chinese population were healthier than those who resided in cities with a larger chinese population. the size of the chinese community was significant in predicting both physical and mental health status of the participants. the findings also indicated the potential underlying effects of the variations in country of origin, access barriers, and socio-economic status of the aging chinese in communities with different chinese population size. the study concluded that size of an ethnic community affected the health status of the aging population from the same ethnic community. the intra-group diversity within the aging chinese identified in this study helped to demonstrate the different socio-cultural and structural challenges facing the aging population in different urban settings. urban health and demographic surveillance system, which is implemented by the african population & health research center (aphrc) in two slum settlements of nairobi city. this study focuses on common child illnesses including diarrhea, fever, cough, common cold and malaria, as well as on curative health care service utilization. measures of ses were created using information collected at the household level. other variables of interest included are maternal demographic and cultural factors, and child characteristics. statistical methods appropriate for clustered data were used to identify correlates of child morbidity. preliminary ratdts: morbidity was reported for 1,087 (16.1 %) out of 6,756 children accounting for a total of 2,691 illness episodes. cough, diarrhoea, runny nose/common cold, abdominal pains, malaria and fever made up the top six forms of morbidity. the only factors that had a significant associ· ation with morbidity were the child's age, ethnicity and type of toilet facility. however, all measures of socioeconomic status (mother's education, socioeconomic status, and mother's work status) had a significant effect on seeking outside care. age of child, severity of illness, type of illness and survival of father and mother were also significantly associated with seeking health care outside home. the results of this study have highlighted the need to address environmental conditions, basic amenities, and livelihood circumstances to improve child health in poor communities. the fact that socioeconomic indicators did not have a significant effect on prevalence of morbidity but were significant for health seeking behavior, indicate that while economic resources may have limited effect in preventing child illnesses when children are living in poor environmental conditions, being enlightened and having greater economic resources would mitigate the impact of the poor environmental conditions and reduce child mortality through better treatment of sick children. inequality in human life chances is about the most visible character of the third world urban space. f.conomic variability and social efficiency have often been fingered to justify such inequalities. within this separation households exist that share similar characteristics and are found to inhabit a given spatial unit of the 'city. the residential geography of cities in the third world is thus characterized by native areas whose core is made up of deteriorated slum property, poor living conditions and a decayed environment; features which personify deprivation in its unimaginable ma~t~de. there are .eviden~es that these conditions are manifested through disturbingly high levels of morbidity and mortality. ban · h h d-and a host of other factors (corrupt10n, msens1t1ve leaders 1p, poor ur 1ty on t e one an , . · 1 f · 1 · · th t ) that suggest cracks in the levels and adherence to the prmc1p es o socta 1usnce. ese governance, e c . . . . . ps £factors combine to reinforce the impacts of depnvat10n and perpetuate these unpacts. by 1den· grou o . ·1 "id . . bothh tifying health problems that are caused or driven by either matena _or soc1a e~nvanon or , t e paper concludes that deprivation need not be accepted as a way. of hfe a~d a deliberate effon must be made to stem the tide of the on going levels of abject poverty m the third world. to the extent that income related poverty is about the most important of all ramifications of po~erty, efforts n_iu_st include fiscal empowerment of the poor in deprived areas like the inner c~ty. this will ~p~ove ~he willingness of such people to use facilities of care because they are able to effectively demand 1t, smce m real sense there is no such thing as free medical services. ). there were 322 men with hiv-infection included in the present study (mean age and education of 41.8 (sd=8.4) and 13.9 (sd=2.7), respectively). a series of multiple regressions were used to examine the unique contributions of symptom burden (depression, cognitive, pain, fatigue), neuropsychologic al impairment (psychomotor efficiency), demographics (age and education) and hiv disease (cdc-93 staging) on iirs total score and jirs subscores: ( 1) activities of daily living (work, recreation, diet, health, finances); (2) psychosocial functioning (e.g., self-expression, community involvement); and (3) intimacy (sex life and relationship with partner). resnlts: total iirs score (r 2 "0.43) was associated with aids diagnosis (ii= 0.11, p <0.01) and symptoms of pain (ii= -0.14, p < 0.01 ), fatigue (ji = -0.34, p < 0.001) and cognitive difficulties (p =0.30, p < 0.001 ). for the three dimensions of the iirs, multiple regression results revealed: ( 1) activities of daily living (r2=0.42) were associated with aids diagnosis (ii =0.17, p < 0.01) and symptoms of pain <p =-0j6, p < 0.01 ), fatigue (ji = -0.31, p < 0.001) and cognitive difficulties (ji =0.32, p < 0.001 ); (2) psychosocial functioning (r2=0.31) was associated with was associated with symptoms of fatigue (ii= -0.25, p <0.001) and cognitive difficulties <ii =0.19, p <0.001); and (3) intimacy (r2=0.17) was related to was associated with symptoms of fatigue (ij = -0.17, p < 0.01) and cognitive difficulties (ii= 0.19, p < 0.001 ). methods: the sif evaluation methodology involves a comprehensive database located at the sif, a randomly selected p~o.spective coh~rt of sif users, and two pre-existing external control cohorts. . . ~ts: in addmon to reporting process data and descriptions of the sif users, we report on data indicating that the establishment of the sif has been independently associated with reductions in public ~':°g ~ (p <?.001), and syringe sharing (aor =0.3[95%ci: 0.1 _ 0.7); p =0.013) and other unsafe m1ecnon pract1_ces_ (p ~ 0.010), and increased uptake of detox services (log-rank p =0.017). as well, we report on da~ md1cat1ng that the sif has not prompted adverse changes in community drug use patterns. the vancouver sif has been well accepted by the target population, and while adve~ events s~c~ as overdoses have occurred, these events have been successfully managed. externally compiled data indicate that the sif has been associated with substantial declines in public disorder !'oster sessions v125 associated with injection drug use, syringe sharing, and increased uptake of detox services. as well, the establishment of the sif has not exacerbated community drug use patterns. ongoing evaluation activities wiu involve assessing the impact of the sif on a variety of outcomes, including infectious disease transmission, fatal overdose, and utilization of health and social services. city, brazil, 1996 -2002 maria cristina almeida, waleska caiaffa, renato assum;iio, and fernando proietti dengue cases were described according to time, space, intensity, age, gender, onset of symptoms, residence address and census tract. spatial distribution of two groups (children and women over 64 years old and men aged 20-59) were compared, under assumption that they have distinct behaviors regarding their displacement around the city. analysis included local moran index, ripley\\'s k function and recurrence of census areas over time. about 99,559 cases were identified in seven epidemic waves. concentration of cases in small areas, followed by a spread either in space or time suggested endemic patterns. regarding age-gender groups, distinct patterns suggested that residence might not be a good proxy in determining the local of infection for men aged 20-59. dengue is shifting to endemic pattern in this city and transmission may not be sole related to home environment, suggesting that additional spatial information must be couected aiming efficient control measures. murukan kandamuthan and subodh kandamuthan lnl7uduction: illness or disablement of a child may affect the economic functioning of a family as it alters the employment pattern and earnings of parents this paper tries to assess the health status and the quality of life of disabled children, and how generally, severe disablement in a child affected their fami-lies\' finances, and to quantify any such effects the problems faced by children in their home and to provide information relevant to the formation of criteria for new or increased cash support. methods: a case-control study was done in the thiruvananthapuram district, capital city of kerala by selecting a random sample of 300 families with severely disabled children below 15 years and a random sample of 300 normal children matched for age and occupation of the parents. comparative and subjective data were collected. money spent on children\'s medical care, loss of earnings of the parents, and other economic burden to the family due to the disability of the child in the family. a discriminant analysis along with univariate analysis was done to assess the factors that mostly differentiate the disabled and normal children. the mean expenditure of the families with a disabled child was rs. 852/-per month, which is significantly higher than the corresponding expenditure of rs. 389/-per month of families with normal child, (t= 16.86, p <.00001). of the disabled children, 81 % were not getting any social security payments and 90% had no special concessions for medical and other educational purposes. the analysis of income and expenditure pattern indicated that financial impact of disablement in a child on the family is significant. the discriminant model results revealed that medical expenditure was a significant variable that differentiated the disabled and normal child. the study found that the health status and quality of life of the disabled children were far from satisfactory. this in fact affected the economic status of the disabled children. in addition to reduced earnings, there are extra costs for disabled children for travel, domestic help, medical care, and health care expenditures (hospital care, physician services, dentistry, drugs and others) for disabled individuals. a strong case can be made for their long-term care by improving the financial support to such families possibly through the introduction of an allowance specially to compensate for the earnings lost by parents due to the disability of their children. widows in india are considered disadvantage group of population. because they are characterized by pangs of separation from spouse, and consequently they go through mental agony, social ~s?lation, and economic dislocation. in addition to these, poverty, landlessness, homelessness, malnutr1non etc. endure serious deterioration of their health. according to census 2001, there are 44 million women are widowed and one fourth live in urban areas. there is a noticeable increase of urban widows from the previous census (almost half). the paper examines the type of disability and chronic ailment borne among elderly widows (60 years and above). fifty second round of national sample survey aske~ the individuals three sets of questions regarding their health: their status of health, whether they are physically poster sessions v126 immobile, and whether or not they have had any specific chronic illnesses. although health infrastruc-1 1 ·1 ble 1 ·n urban setting in india as compared to rural counterpart, prevalence of tures are arge y ava1 a . . . . . h · ·11 · h h"gher 1 "n urban areas analysis shows 1omt problems m old age qmte common c romc 1 ness is muc 1 • . . and nearly 44 percent elderly widows are suffering from this ail'.11ent in urban areas. one-fifth of widows are suffering from high/low bp. heart disease, diabetes and urinary problems are much ~ore pre~alent in urban areas though disabilities is comparatively lower in urban areas. however, the d~fference is not statistically significant. disability with respect to vision is more pronounced and one third_ of the total respondents have problems regarding eyesight. one fifths ofdderly widows are _ha.rd of hearing. though disability increases with age, similar trend is not observed with respect to chr~mc illness. the percepnon of self-health among elderly becomes poorer as their age increases. the perceived health status need not be always corrected to objective indicators of health. it is noted that around 27 per~en~ of those perceived their health as "excellenr" or "very good" are found to be suffering from chrome disease of1omts and 17 percent have visual disability. to conclude an elderly widow living in urban areas have similar health problems as she jives in rural areas. this can be easily explained, as they were not made aware to seek treatment from health facility. in addition, the study shows that the economic conditions are deterrent factors for their illness. it is essential that intervention should be taken up improving their economic conditions so that wellbeing of this most vulnerable group of the society can be taken care of. heart failure is a disease that affects nearly 15 million people world wide. the united states alone accounts for one third of this population. blacks are stricken with heart failure twice as often as whites. men are more likely to develop chf than women, however since the majority of the chf population are seniors, there are actually more women than men. congestive heart failure (chf) has become a pandemic. as the baby boomer generation ages, the number of chf cases will rise dramatically. (young, j. & mills, r.,2004). after the age of 45, each proceeding decade doubles the incidence level of chf. the average person has a 1 in 5 chance of developing heart failure within their lifespan according to the framingham study (nih.gov). individuals aging 65 or older compose over half of the entire documented heart failure population. in this age group, chf is the leading cause of hospitalization. many patients are continuously readmitted. it is estimated that the chf population will increase by one million within the next 25 years. mortality was at 50% for a two year period and 70% for five years (young, j. & mills, r.,2004 ). data from: aha as technology evolves, the understanding of the nature of heart failure has become clearer. in the beginning of the 20th century heart failure was diagnosed as a condition that presented with edema (swelling) in the extremities caused by water retention. treatment in the beginning of the past cenrury was limited to abdominal drainage and diuretics. the discovery of the heart's insufficient pumping ability in heart failure patients lead to new surgeries and devices such as heart transplants and ventricular assist devices (bridge until transplant is available). ventricular hypertrophy often occurs before the development of heart failure. hypertrophy is the term given for cardiac remodeling. cardiac remodeling also includes chamber dilation (young, j. & mills, r.,2004) . chf financial burden the us healthcare financing administration has ranked chf as the highest cost illness in the diagnose-related area. direct costs related to chf in the us for 2004 were nearly $26 billion. these costs included hospital admissions, physician fees, home health aids and medications. indirect costs were over $2 billion in 2004. loss of jobs or death due to chf was the criteria for this category (young, j. & mills, r., 2004). . introduction: the injection drug user quality of life scale (iduqol) is a relatively new scale ~es1gned to capture the unique and individual circumstances that determine quality of life among injection drug users (idus). the 20 life areas comprising the instrument were based on the research literature an~ ~ocus group discussions with idus. the purpose of the present study was to evaluate the content validity of t.h~ iduqol using judgmental methods based on subject matter experts' (smes) ratings. content vah~1ty refers to the de~ee to which elements of an assessment tool are representative of the construct of interest and appropnate for a given population. methods: data were obtained from six smes, from the field of idu research and practice, who ~ere. asked t.o evaluate various aspects of the iduqol, including the title of the instrument, administraon instruchons, clarity of the names and descriptions of each life area, response format, scoring instruc-no~s and examples, record form and whether each life area should be included in the instrument. sme ratings were made using either 3 or 4 point scales. sm es were also given the opportunity to comment on poster sessions v127 each section and to suggest whether important life areas had been overlooked, were redundant, not relevant, or in need of revision. (cvi) and the ~v.erage ~eviation index (ad) were used to evaluate smes' agreement on ratings of the content vahd1ty vanables. both measures provided support for the content validity of various aspects of the iduqol, although results from the stricter ad index noted some areas of disagreement, including the description of particular life areas (e.g., being useful, drugs, sex), the inclusion of some life areas (e.g., drug treatment, education, independence and free choice), and the ease of the scoring instructions. the findings from this study provide (a) content validity evidence to support the use of the iduqol as well as (b) recommendations to suengthen both the instrument (e.g., improved descriptions for some items) and the accompanying administration and scoring manual (e.g., an easier scoring template). changes made to the instrument and its manual make the iduqol even easier for researchers, practitioners, and program evaluators to use as a way of assessing, and tracking changes over time or as a result of interventions in, quality of life in injection drug users. introduction: strict adherence to prescribed regimens is required to derive maximal benefit from highly active antireuoviral therapy (haart) in persons living with hiv/aids (phas). adherence is a key determinant of the degree and durability of viral suppression. adherence is also essential in reducing the spread of hiv and ensuring that today's treatments remain effective for as long as possible. the objective of this study is to conduct a systematic review of the research literature on the effectiveness of education and patient support strategies for improving adherence to haart. methods: a systematic search of the core databases was performed from january 1996 until may 2005. randomized controlled trials examining the effectiveness of education and patient support interventions to improve the adherence to haart were considered for inclusion. only those studies that measured adherence at a minimum of six weeks were included. study selection, quality assessments, and data abstraction were performed independently by two reviewers. results: study heterogeneity with respect to differing populations, interventions, outcomes, and length of follow-up did not allow for meta-analysis. we included 19 studies involving 2, 159 ph as. sample sizes ranged from 22 to 367. study duration ranged from a single session to a variable number of sessions delivered over one year. many of the interventions involved the provision of an educational component to improve adherence and often addressed strategies to overcome barriers to adherence and the development of problem-solving skills. they ranged from simple interventions (e.g., the provision of reminder devices) to complex interventions (e.g., cognitive-behavioural therapy delivered by licensed psychologists). eleven of the 19 studies demonstrated a statistically significant advantage associated with the described adherence intervention. the advantage associated with adherence outcomes does not seem to translate into the more clinically relevant outcome of viral suppression. only 4 out of 12 studies that reported virological or immunological results found a significant effect associated with the intervention. the studies had several methodological shortcomings. conclusions: there is a need for standardization and methodological rigour in the conduct of adherence trials. some interventions may have a significant impact on improving adherence. further research is required before any specific adherence improving strategy can confidently be incorporated into standard clinical practice. focus groups were used to collect data from a purposive sample of treatmentexperienced participants. focus group data were analyzed using a grou~ded t~eory appr~ach. i:ne themes identified from the interviews were used to identify the effects of ant1rerrov1rals on quality of hfe. the content of the mos-hiv was appraised against the themes identified from our analysis. participants also completed the mos-hiv survey and were asked whether the survey covered all important aspects of quality of life on antiretroviral medications. results: five key informant interviews and five focus groups with 38 participants were used to identify effects on quality of life that are not directly ~aptu~ed by_ the mos-hiv health survey. our~~ showed that our participants described quality of hfe as mvol~mg_ a set of ~rsonal trad~ffs ~stay alive. in most cases, worsened quality of life was traded-off for gams 1~ longevlty from ~~canon use. these eff~'ts or tradeoffs included: ( 1) downstream consequences of side effects and toxlanes; (2) loss of lrufe. pende~t decision-making privileges, (3) having to choose drugs over ~areer; (~)burden of medication-taking responsibilities; and (5) living life under a pretense and havmg to hide-the net effect of the tradeoffs, or "prices" to pay led to what was described as "longevity with hn without hope and future~. conclusion: our study highlights five major themes summarizing the impact of haart on quality on life, and suggests that currently used scales for measuring quality of life do not a~atd~ cap~e these findings and the associated consequences. the conceptual framework for measuring quality of bfe in hiv should be reconsidered in order to better capture the important effects of the medications on quality of life. this may involve widening the boundaries of the definition of health-related quality oflife to include aspects such as finances, employment, and housing. we should further consider the development of a haart adverse effect specific instrument, using a broad definition of adverse effect in order to capture all types of adverse impacts of hiv treatments on quality of life. introduction: measles (rubeola) is the most contagious vaccine preventable disease of humans. while cases of measles are uncommon in canada today, the disease continues to be a leading cause of morbidity and death in the developing world. as a result of human migration, measles cases still occur in canada, primari~ among immigrants, returning travelers, and other susceptible groups. canada's national advisory council on immunizations (naci) recommends that immigrants without records of measles immunization be considered for vac:cination since these individuals may not have been appropriately vaccinated in their native country. on rhe other hand, natural immunity to measles in immigrants is likely to be high given the high incidence of disease in developing parts of rhe world. thus it remains unclear if the most efficient strategy to achieve high levels of measles immunity in immigrants should entail initial serologic screening followed by vaccination of susceptible individuals or preemptive vaccination of all persons lacking immunization records. understanding the epidemiology of measles immunity in immigrant populations could help guide such decisions. . methods: we identified 527 adults, 15 years of age or older, who were screened for serologtc immunity to measles as part of a recently developed screening protocol at a downtown toronto community health centre. we subsequently determined if these individuals had any immunization records from their native country or from within canada. we then examined the relationship between several demographic factors and immunity to measles. results: 93% of immigrants had no prior immunization records. overall, 93% of the 527 immigrants rested for immunity to measles were found to be immune; 88% of those under the age of 20, 93% of those between 20 and 39 years of age, 99% of those between 40 and 59 years of age, and 100% of those 60 years of age or older. gender, education status, household income, and immigration status were nor ~ssoc1ated wirh immunity to measles. immunity to measles was lowest among immigrants from eastern europe (8~%), whil~ immigrants from other regions of the globe had greater than 90% immunity. c~lusrons: immigrants and refugees appear to have reasonably high levels of immunity to mea· sics, possibly related to natural infection wirh the measles virus. immigrants from eastern europe appear to have slightly. lower l~vel~ of immunity to the measles virus. universal screening for immunity to meales or preemptive vac:cmauon may both be inefficient public health strategies, however further research is needed to corroborate these findings. nancy r~ss, stephane tremblay, saeeda khan, daniel crouse, mark tremblay, and jean-marie berrhelor o~ve: the speed of the rise in obesity in places like canada suggests that rather than a shift in the gcncnc_ com~sirion of the population, the root of the obesity pandemic is an environment that suprrs ~besity. this paper examines the influence of neighbourhood and metropolitan characteristics. bour~lts:. while accounting. for individual socio-demographics and behaviours, residents of neighs with a large proportmn of poorly educated individuals had higher bmis than those living in poster sessions v129 neighbourhoods with more highly educated individuals (p <.01 ). residing in a neighbourhood with a high proportion of recent immigrants was associated with lower bmi for men (p<.01), but not women. neighbourhood dwelling density, a proxy for walkability, was not associated with bm! for either sex. metropolitan sprawl was associated with higher male bmi (p=.02) but the effect was negligible for women (p=.09). bmi was significantly lower for women (p<.01) living in a city in the province of quebec, even after accounting for the influence of individual and neighbourhood covariates. conclusions: bm! was strongly patterned by individual-level social position in urban canada. the incremental influence on bmi of neighbourhood related to the neighbourhood's social conditions and not to their physical form. metropolitan sprawl was associated with higher bm! for men, a group with longer car-dependent commutes than women. the findings suggest that both individuals and their environments (both social and physical) influence the distribution of bmi in urban populations. introduction: urban environments have been linked to a range of human health issues. in singapore, prevalence of end stage renal disease (esrd) is predicted to rise (2633 in 1999 to 6000 in 2010 , kidney international, vol.67, 2005 . the clinical and economic burden of esrd has promoted development of strategies aimed at preventing the development and progression of chronic kidney disease. these include population based screening programs such as the national kidney foundation, sin-gapore\'s (nkfs) "partnership for prevention."the program, aims to reduce incidence of esrd through comprehensive intervention and screening for early detection of urinary abnormalities, blood pressure (bp), and random blood glucose (rbg). objective of this study is to examine gender, race and age wise prevalence of elevated bp, rbg and proteinuria. methodology: this current analysis includes 575,438 subjects, age 18 to 86 years, who underwent screening during september 1999 and december 2004. participants were asked to give demographic information and the history of diseases. tests were given to get clinical information such as proteinuria, blood glucose, sbp, and dbp. blood pressure abnormalities were defined according to ]nc vi criteria. proteinuria was defined as the presence of 1 plus or greater protein (equivalent to> 30 mg/di) on dipstick analysis. results: there were 296, 116 (51.5%) males. racial distribution was chinese (78.8% ), malay (8.8% ), indians (8. 7%) and others (3. 7% ).among participants, who were apparently "healthy" (asymptomatic and without history of dm, ht, or kd), gender and race wise % prevalence of elevated (bp> 140/90), rbg (> 140 mg/di) and positive urine dipstick for protein was as follows male: (20.5;6.9; 3.5) female:(13.6;5.0;3. 2) chinese:( 17.1;6.0;3) malay: (19.4;7.3;5.6) indian:( 15.9;7.5;3.0) others: (15.4;4.5;2.9) total:(l 7.1, 6.1,3.2). percentage of participants with more than one abnormality were as follows. those with bp> 140/90mmhg, 14% also had rbg> 140mg/dl and 6.4% had proteinuria> i. those with rbg> 140mgldl, 11 % also had proteinuria> 1 and 35% had bp> 140/90mmhg. those with proteinuria> 1, 18% also had rbg> 140mg/dl, and 38% had bp> 140/90mmhg. conclusion: we conclude that sub clinical abnormalities in urinalysis, bp and rbg readings are prevalent across all genders and racial groups in the adult population. the overlap of abnormalities, point towards the high risk for esrd as well as cardiovascular disease. this indicates the urgent need for population based programs aimed at creating awareness, and initiatives to control and retard progression of disease. introduction: various theories have been proposed that link differential psychological vulnerability to health outcomes, including developmental theories about attachment, separation, and the formation of psychopathology. research in the area of psychosomatic medicine suggests an association between attachment style and physical illness, with stress as a mediator. there are two main hypotheses explored in the present study: ( t) that individuals living with hiv who were upsychologically vulne~able" at study entry would be more likely to experience symptoms of depression, anxiety and phys1ca! illness over. the course of the 9-month study period; and (2) life stressors and social support would mediate the relat10nship between psychological vulnerability and the psychological ~nd physical outcomes. . (rsles), state-trait anxiety inventory (stai), beck depr~ssi~n lnvento~ (bdi), and~ _21-item pbys~i symptoms inventory. we characterized participants as havmg psychological vulnerability and low resilience" as scoring above 35 on the raas (insecure attachment) or above 120 on the das (negative expectations about oneself). . . . . . " . . ,, . results: at baseline, 55% of parnc1pants were classified as havmg low resilience. focusmg on anxiety, the average cumulative stai score of the low-resilience group was significandy hi~e~ than that of the high-resilience group ( 18.45 sd= 10.6 versus 9.57 sd= 8.6; f(l,80)= 16.74, p <.001). similar results were obtained for bdi and physical symptoms (f( 1,80)= 14.65, p<.001 and f( 1,80)= 5.50, p<.05, respec· tively). after controlling for resilience, the effects of variance in life stres".°rs averaged over time wa~ a_sig· nificant predictor of depressive and physical symptoms, but not of anxiety. ho~e_ver, these assooan~s became non-significant when four participants with high values were removed. s1id1larly, after controlling for resilience, the effects of variance in social support averaged over time became insignificant. conclusion: not only did "low resilience" predict poor psychological and physical outcomes, it was also predictive of life events and social support; that is, individuals who were low in resilience were more likely to experience more life events and poorer social support than individuals who were resilient. for individuals with vulnerability to physical, psychological, and social outcomes, there is need to develop and test interventions to improve health outcomes in this group. rajat kapoor, ruby gupta, and jugal kishore introduction: young people in india represent almost one-fourth of the total population. they face significant risks related to sexual and reproductive health. many lack the information and skills neces· sary to make informed sexual and reproductive health choices. objective: to study the level of awareness about contraceptives among youth residing in urban and rural areas of delhi. method: a sample of 211 youths was selected from barwala (rural; n= 112) and balmiki basti (urban slums; n= 99) the field practice areas of the department of community medicine, maulana azad medical college, in delhi. a pre-tested questionnaire was used to collect the information. when/(calen· dar time), by 2, fisher exact and t were appliedxwhom (authors?). statistical tests such as as appropriate. result: nearly 9 out of 10 (89.1 %) youth had heard of at least one type of contraceptive and majority (81.5%) had heard about condoms. however, awareness regarding usage of contraceptives was as low as 9.4% for terminal methods to 39.3% for condom. condom was the best technique before and after marriage and also after childbirth. the difference in rural and urban groups was statistically signif· icant (p=.0001, give confidence interval too, if you provide the exact p value). youth knew that contra· ceptives were easily available (81 %), mainly at dispensary (68.7%) and chemist shops (65.4%). only 6.6% knew about emergency contraception. only advantage of contraceptives cited was population con· trol (42.6%); however, 3.8% believed that they could also control hiv transmission. awareness of side effects was poor among both the groups but the differences were statistically significant for pills (p=0.003). media was the main source of information (65%). majority of youth was willing to discuss a~ut contraceptive with their spouse (83.4%), but not with others. 51.2% youth believed that people in their age group use contraceptives. 35% of youth accepted that they had used contraceptives at least once. 81 % felt 2 children in family is appropriate, but only 59.7% believed in 3 year spacing. . conclusion: awareness about contraceptives is vital for youth to protect their sexual and reproduc· tive health .. knowledge about terminal methods, emergency contraception, and side effects of various contraceptives need to be strengthened in mass media and contraceptive awareness campaigns. mdbot:ls: 740 elderly aged 60+ were interviewed in 3 poor communities in beirut the capital of f:ebanon, ~e of which is a palestinia~. refugee camp. depression was assessed using the i 5-item geriat· nc depressi~n score (~l?s-15). specific q~estions relating to the 3 aspects of religiosity were asked as well as questions perta1rung to demographic, psychosocial and health-related variables. results: depression was prevalent in 24% of the interviewed elderly with the highest proportion being in the palestinian refugee camp (31 %). mosque attendance significantly reduced the odds of being depressed only for the palestinian respondents. depression was further associated, in particular communities, with low satisfaction with income, functional disability, and illness during last year. condiuion: religious practice, which was only related to depression among the refugee population, is discussed as more of an indicator of social cohesion, solidarity than an aspect of religiosity. furthermore, it has been suggested that minority groups rely on religious stratagems to cope with their pain more than other groups. implications of findings are discussed with particular relevance to the populations studied. nearly thirty percent of india's population lives in urban areas. the outcome of urbanization has resulted in rapid growth of urban slums. in a mega-city chennai, the slum populations (25.6 percent) face greater health hazards due to overcrowding, poor sanitation, lack of access to safe drinking water and environmental pollution. amongst the slum population the health of women and children are most neglected, resulting in burden of both communicable and non-communicable diseases. the focus of the paper is to present the epidemiology profile of children (below 14 years) in slums of chennai, their health status, hygiene and nutritional factors, the social response to health, the trends in child health and urbanization over a decade, the health accessibility factors, the role of gender in health care and assessment impact of health education to children. the available data prove that child health in slums is worse than rural areas. though the slum population is decreasing there is a need to explore the program intervention and carry out surveys for collecting data on some specific health implications of the slum children. objective: during the summer of 2003 there was a heat wave in central europe, producing an excess number of deaths in many countries including spain. the city of barcelona was one of the places in spain where temperatures often surpassed the excess heat threshold related with an increase in mortality. the objective of the study was to determine whether the excess of mortality which occurred in barcelona was dependent on age, gender or educational level, important but often neglected dimensions of heat wave-related studies. methods: barcelona, the second largest city in spain (1,582,738 inhabitants in 2003) , is located on the north eastern coast. we included all deaths of residents of barcelona older than 20 years that occurred in the city during the months of june, july and august of 2003 and also during the same months during the 5 preceding years. all the analyses were performed for each sex separately. the daily number of deaths in the year 2003 was compared with the mean daily number of deaths for the period 1998-2002 for each educational level. poisson regression models were fitted to obtain the rr of death in 2003 with respect to the period 1998-2002 for each educational level and age group. results: the excess of mortality during that summer was more important for women than for men and among older ages. although the increase was observed in all educational groups, in some age-groups the increase was larger for people with less than primary education. for example, for women in the group aged 65-74, the rr of dying for 2003 compared to 1998-2002 for women with no education was 1.30 (95%ci: 1.04-1-63) and for women with primary education or higher was 1.19 (95%ci: 0.90-1.56). when we consider the number of excess deaths, for total mortality (>=20 years) the excess numbers were higher for those with no education ( 17 5. 7 for women and 46. 7 for men) and those with less than primary education (112.5 for women and 11-2 for men) than those with more than primary edm:ation (75.0 for women and -10.3 for men). conclusion: age, gender and educational level were important in the 2003 barcelona heat wave. it is necessary to implement response plans to reduce heat morbidity and mortality. policies should he addressed to all population but also focusing particularly to the oldest population of low educational level. introduction: recently there has been much public discourse on homelessness and its imp~ct on health. measures have intensified to get people off the street into permanent housing. for maximum v132 poster sessions success it is important to first determine the needs of those to be housed. their views on housing and support requirements have to be considered, as th~y ar~ the ones affected. as few res.earch studies mclude the perspectives of homeless people themselves, httle is known on ho~ they e~penence the 1mpacrs on their health and what kinds of supports they believe they need to obtain housing and stay housed. the purpose of this study was to add the perspectives of homeless people to the discourse, based in the assumption that they are the experts on their own situations and needs. housing is seen as a major deter· minant of health. the research questions were: what are the effects of homelessness on health? what kind of supports are needed for homeless people to get off the street? both questions sought the views of homeless individuals on these issues. methods: this study is qualitative, descriptive, exploratory. semi-structured interviews were conducted with homeless persons on street corners, in parks and drop-ins. subsequently a thematic analysis was carried out on the data. results: the findings show that individuals' experiences of homelessness deeply affect their health. apart from physical impacts all talked about how their emotional health and self-esteem are affected. the system itself, rather than being useful, was often perceived as disabling and dehumanizing, resulting in hopelessness and resignation to life on the street. neither welfare nor minimum wage jobs are sufficient to live and pay rent. educational upgrading and job training, rather than enforced idleness, are desired by most initially. in general, the longer persons were homeless, the more they fell into patterned cycles of shelter /street life, temporary employment /unemployment, sometimes addictions and often unsuccessful housing episodes. conclusions: participants believe that resources should be put into training and education for acquisition of job skills and confidence to avoid homelessness or minimize its duration. to afford housing low-income people and welfare recipients need subsidies. early interventions, 'housing first', more humane and efficient processes for negotiating the welfare system, respectful treatment by service providers and some extra financial support in crisis initially, were suggested as helpful for avoiding homelessness altogether or helping most homeless people to leave the street. this study is a national homelessness initiative funded analysis examining the experiences and perceptions of street youth vis-a-vis their health/wellness status. through in-depth interviews with 140 street youth in halifax, montreal, toronto, calgary, ottawa and vancouver, this paper explores healthy and not-so healthy practices of young people living on the street. qualitative interviews with 45 health/ social service providers complement the analysis. more specifically, the investigation uncovers how street youth understand health and wellness; how they define good and bad health; and their experiences in accessing diverse health services. findings suggest that living on the street impacts physical, emotional and spiritual well·being, leading to cycles of despair, anger and helplessness. the majority of street youth services act as "brokers" for young people who desire health care services yet refuse to approach formal heal~h care organizational structures. as such, this study also provides case examples of promising youth services across canada who are emerging as critical spaces for street youth to heal from the ravages of ~treet cultur~. as young people increasingly make up a substantial proportion of the homeless population in canada, it becomes urgent to explore the multiple ways in which we can support them to regain a sense of wellbeing and "citizenship." p5-77 (c) health and livelihood implications of marginalization of slum dwellers in provision of water and sanitation services in nairobi city elizabeth kimani, eliya zulu, and chi-chi undie . ~ntrodfldion: un-habitat estimates that 70% of urban residents in kenya live in slums; yet due to their illegal status, they are not provided with basic services such as water sanitation and health care. ~nseque~tly, the services are provided by vendors who typically provide' poor services at exorbitant prices .. this paper investigates how the inequality in provision of basic services affects health and livelihood circumstances of the poor residents of nairobi slums . . methods: this study uses qualitative and quantitative data collected through the ongoing longitudmal .health and demographic study conducted by the african population and health research center m slum communities in n ·rob" w d · · · · ai 1. e use escnpnve analytical and qualitative techmques to assess h~w concerns relating to water supply and environmental sanitation services rank among the c~mmumty's general and health needs/concerns, and how this context affect their health and livelihood circumstances. results: water (32%) and sanitation (20%) were the most commonly reported health needs and also key among general needs (after unemployment) among slum dwellers. water and sanitation services are mainly provided by exploitative vendors who operate without any regulatory mechanism and charge exorbitantly for their poor services. for instance slum residents pay about 8 times more for water than non-slum households. water supply is irregular and residents often go for a week without water; prices are hiked and hygiene is compromised during such shortages. most houses do not have toilets and residents have to use commercial toilets or adopt unorthodox means such as disposing of their excreta in the nearby bushes or plastic bags that they throw in the open. as a direct result of the poor environmental conditions and inaccessible health services, slum residents are not only sicker, they are also less likely to utilise health services and consequently, more likely to die than non-slum residents. for instance, the prevalence of diarrhoea among children in the slums was 31 % compared to 13 % in nairobi as a whole and 17% in rural areas, while under-five mortality rates were 151/1000, 62/1000 and 113/1000 respectively. the results demonstrate the need for change in governments' policies that deprive the rapidly expanding urban poor population of basic services and regulatory mechanisms that would protect them from exploitation. the poor environmental sanitation and lack of basic services compound slum residents' poverty since they pay much more for the relatively poor services than their non-slum counterparts, and also increase their vulnerability to infectious diseases and mortality. since 1991 iepas've been working in harm reduction becoming the pioneer in latin america that brought this methodology for brazil. nowadays the main goal is to expand this strategy in the region and strive to change the drug policy in brazil. in this way harm reduction: health and citizenship program work in two areas to promote the citizenship of !du and for people living with hiv/aids offering law assistance for this population and outreach work for needle exchange to reduce damages and dissemination of hiv/aids/hepatit is. the methodology used in outreach work is peer education, needle exchange, condoms and folders distribution to reduce damages and the dissemination of diseases like hiv/aids/hepatitis besides counseling to search for basic health and rights are activities in this program. law attendance for the target population at iepas headquarters every week in order to provide law assistance that includes only supply people with correct law information or file a lawsuit. presentations in harm reduction and drug policy to expand these subjects for police chiefs and governmental in the last year attended 150 !du and 403 nidu reached and 26.364 needles and syringes exchanged. in law assistance 740 (420 people living with aids, 247 drug users, 43 inject drug users, 30 were not in profile) people attended. 492 lawsuits filed 218 lawsuits in current activity. broadcasting of the harm reduction strategies by the press helps to move the public opinion, gather supporters and diminish controversies regarding such actions. a majority number of police officer doesn't know the existence of this policy. it's still polemic discuss this subject in this part of population. women remain one of the most under seviced segments of the nigerian populationand a focus on their health and other needs is of special importance.the singular focus of the nigerian family welfare program is mostly on demographic targets by seeking to increase contraceptive prevalence.this has meant the neglect of many areas of of women's reproductive health. reproductive health is affected by a variety of socio-cultural and biological factors on on e hand and the quality of the service delivery system and its responsiveness on the other.a woman's based approach is one which responds to the needs of the adult woman and adolescent girls in a culturally sensitive manner.women's unequal access to resources including health care is well known in nigeria in which stark gender disparities are a reality .maternal health activities are unbalanced,focusi ng on immunisation and provision of iron and folic acid,rather than on sustained care of women or on the detection and referral of high risk cases. a cross-sectional study of a municipal government -owned hospitalfrom each of the 6 geo-political regions in igeria was carried out (atotal of 6 ce~ters) .. as _part ~f t~e re.search, the h~spital records were uesd as a background in addition to a 3-week mtens1ve mvesuganon m the obstemc and gynecology departments. . . . : little is known for example of the extent of gynecological morbtdtty among women; the little known suggest that teh majority suffer from one or more reproductive tr~ct infect~ons. although abortion is widespread, it continues to be performed under ilegal and unsafe condmons. with the growing v134 poster sessions hiv pandemic, while high riskgroups such ascomn;iercial sex workers and their clients have been studied, little has been accomplished in the large populat10ns, and particularly among women, regardmgstd an hiv education. . . conclusions: programs of various governmentalor non-governmental agen,c1es to mvolve strategies to broaden the narrow focus of services, and more importan~, to put wo~en s reproducnve health services and information needs in the forefront are urgently required. there is a n~d to reonent commuication and education activities to incorprate a wider interpretation of reproducnve health, to focus on the varying information needs of women, men, and youth and to the media most suitable to convey information to these diverse groups on reproductive health. introduction: it is estimated that there are 250-300 youths living on the streets, on their own with the assistance of social services or in poverty with a parent in ottawa. this population is under-serviced in many areas including health care. many of these adolescents are uncomfortable or unable to access the health care system through conventional methods and have been treated in walk-in clinics and emergency rooms without ongoing follow up. in march 2004, the ontario government provided the ct lamont institute with a grant to open an interdisciplinary and teaching medical/dental clinic for street youth in a drop-in center in downtown ottawa. bringing 5 community organizations together to provide primary medical care and dental hygiene to the streetyouths of ottawa ages 12-20, it is staffed by a family physician, family medicine residents, a nurse practitioner, 2 public health nurses, a dental hygienist, dental hygiene students and a chiropodist who link to social services already provided at the centre including housing, life skills programs and counselling. project objectives: 1. to improve the health of high risk youth by providing accessible, coordinated, comprehensive health and dental care to vulnerable adolescents. 2. to model and teach interdisciplinary adolescent care to undergraduate medical students, family medicine residents and dental hygiene students. methods: non-randomized, mixed method design involving a process and impact evaluation. data collection-qualitative:a) semi-structured interviews b) focus groups with youth quantitative:a) electronic medical records for 12 months b) records (budget, photos, project information). results: in progress-results from first 12 months available in august 2005. early results suggest that locating the clinic in a safe and familiar environment is a key factor in attracting the over 130 youths the clinic has seen to date. other findings include the prevalence of preventative interventions including vaccinations, std testing and prenatal care. the poster presentation will present these and other impacts that the clinic has had on the health of the youth in the first year of the study. conclusions: 1) the clinic has improved the health of ottawa streetyouth and will continue beyond the initial pilot project phase. 2) this project demonstrates that with strong community partnerships, it is possible meet make healthcare more accessible for urban youth. right to health care campaign by s.j.chander, community health cell, bangalore, india. introduction: the people's health movement in india launched a campaign known as 'right to health care' during the silver jubilee year of the alma ata declaration of 'health for all' by 2000 ad in collahoration with the national human rights commission (nhrc). the aim of the campaign was to establish the 'right to health care' as a basic human right and to address structural deficiencies in the pubic health care system and unregulated private sector . . methods: as part of the campaign a public hearing was organized in a slum in bangalore. former chairman of the nhrc chaired the hearing panel, consisting of a senior health official and other eminent people in the city. detailed documentation of individual case studies on 'denial of access to health care' in different parts of the city was carried out using a specific format. the focus was on cases where denial of health services has led to loss of life, physical damage or severe financial losses to the patient. results: _fourte_en people, except one who had accessed a private clinic, presented their testimonies of their experiences m accessing the public health care services in government health centres. all the people, e_xcept_ one person who spontaneously shared her testimony, were identified by the organizations worki_ng with the slum dwellers. corruption and ill treatment were the main issues of concern to the people. five of the fourteen testimonies presented resulted in death due to negligence. the public health cen· n:s not only demand money for the supposedly free services but also ill-treats them with verbal abuse. five of these fourteen case studies were presented before the national human right commission. the poster sessions v135 nhrc has asked the government health officials to look into the cases that were presented and to rectify the anomalies in the system. as a result of the public hearing held in the slum, the nhrc identified urban health as one of key areas for focus during the national public hearing. cond#sion: a campaign is necessary to check the corrupted public health care system and a covetous private health care system. it helps people to understand the structure and functioning of public health care system and to assert their right to assess heath care. the public hearings or people's tribunals held during the campaign are an instrument in making the public health system accountable. ps-82 (a) violence among women who inject drugs nadia fairbairn, jo-anne stoltz, evan wood, kathy li, julio montaner, and thomas kerr background/object ives: violence is a major cause of morbidity and mortality among women living in urban settings. though it is widely recognized that violence is endemic to inner-city illicit drug markets, little is known about violence experienced by women injection drug users (!du). therefore, the present analyses were conducted to evaluate the prevalence of, and characteristics associated with, experiencing violence among a cohort of female idu in vancouver. methods: we evaluated factors associated with violence among female participants enrolled in the vancouver injection drug user study (vidus) using univariate analyses. we also examined self-reported relationships with the perpetrator of the attack and the nature of the violent attack. results: of the 346 active iou followed between december 1, 2003 and may 6, 2005, 73 (21.1 %) had experienced violence during the last six months. variables positively associated with experiencing violence included: homelessness (or= 3.46, 95% ci: 1.66-7.21, p < 0.01), public injecting (or= 3.45, 95% ci: 1.43 -8.35, p < 0.01 ), frequent crack use (or= 2.99, 95% ci: 1. 72 -5.17, p < 0.01 ), recent incarceration (or =2.81, 95% cl: 1.38 -5.72, p < 0.01), receiving help injecting (or =2.77, 95% cl: 1.54-5.00, p < 0.01 ), shooting gallery attendance (or =2.46, 95% ci: 1.22 -4.93, p < 0.01 ), sex trade work (or =2.30, 95% cl: 1.35 -3.93, p < 0.01 ), frequent heroin injection (or= 1.96, 95% cl: 1.13 -3.40, p < 0.02), and residence in the downtown eastside (odds ratio [or] = 1.85, 95% ci: 1.09 -3.13, p < 0.02). variables negatively associated with experiencing violence included: being married or common-law (or =0.47. 95% ci: 0.25 -0.87, p < 0.02) and being in methadone treatment (or =0.53, 95% ci: 0.31 -0.91, p < 0.02). the most common perpetrators of the attack were acquaintances (48.0%), strangers (27.4%), police (9.6%), or dealers (8.2%). attacks were most frequently in the form of beatings (65.8%), robberies (21.9%), and assault with a weapon (13.7%). conclusion: violence was a common experience among women !du in this cohort. being the victim of violence was associated with various factors, including homelessness and public injecting. these findings indicate the need for targeted prevention and support services, such as supportive housing programs and safer injection facilities, for women iou. introduction: although research on determinants of tobacco use among arab youth has been carried out at several ecologic levels, such research has included conceptual models and has compared the two different types of tobacco that are most commonly used among the lebanese youth, namely cigarette and argileh. this study uses the ecological model to investigate differences between the genders as related to the determinants of both cigarette and argileh use among youth. methodology: quantitative data was collected from youth in economically disadvantaged urban communities in beirut, the capital of lebanon. results: the results indicated that there are differences by gender at a variety of ecological levels of influence on smoking behavior. for cigarettes, gender differences were found in knowledge, peer, family, and community influences. for argileh, gender differences were found at the peer, family, and community l.evels. the differential prevalence of cigarette and argileh smoking between boys and girls 1s therefore understandable and partially explained by the variation in the interpersonal and community envi.ronment which surrounds them. interventions therefore need to be tailored to the specific needs of boys and girls. introduction: the objective of this study was to assess the relationship between parents' employment status and children' health among professional immigrant families in vancouver. our target communmes v136 poster sessions included immigrants from five ethnicity groups: south korean, indian, chine~e, ~ussian, and irani~ with professional degrees (i.e., mds, lawyers, engineers, ma?~ger~, and uru~ers1ty professors) w11h no relevant job to their professions and those who had been hvmg m the studied area at least for 36 months. methodology: the participants were recruited by collaboration from three local community agencies and were interviewed individually during the fall of 2004. ra#lts: totally, 109 complete interviews were analyzed: 33 from south-east asia, 59 from south asia, 17 from russia and other eastern europe. overall, 14.5% were employed, 38.5% were underemployed, 46% indicated they were unemployed. overall, 58.5% were not satisfied with their current job. russians and other eastern europeans were most likely satisfied with their current job, while south-east asians were most satisfied from their life in canada. about 53% indicated that their spouses were not satisfied with their life in canada, while 55% believed that their children are very satisfied from their life in canada. in addition, around 30% said they were not satisfied from their family relationship in canada. while most of the responders ranked their own and their spouses' health status as either poor or very poor, jut 3% indicated that their first child's health was very poor. in most cases they ranked their children's health as excellent or very good. the results of this pilot study show that there is a need to create culturally specific child health and behavioral scales when conducting research in immigrant communities. for instance, in many asian cultures, it is customary for a parent either to praise their children profusely, or to condemn them. this cultural practice, called "saving face," can affect research results, as it might have affected the present study. necessary steps, therefore, are needed to revise the current standard health and behavioral scales for further studies by developing a new scale that is more relevant and culturally sensitive to the targeted immigrant families. metboda: database: 2003 national health survey (ministry of health www.msc.es). two thousand interviews were performed among madrid population (0.04% of the whole); 593 corresponded to older adults (0.04% of the 1. 7 million aged 50 years and over). study sample constitutes 95.3% (565 out of 593) of those older adults, who live in urban areas. demographic structure (by age and gender) of this population in relation to health services use (medical consultations, dentist visits, emergence services, hospitalisation) was studied using general linear model univariate procedure. a p0.005), while age was associated with emergence services use (26% of the population: 21 %, 28% and 45% of each age group) and hos~italisation (17% .oft~~ population: 13%, 20% and 31%, of each age ~oup) (p0.005) was fou~d with respect to dennst v1s1ts (18% vs 20%), medical consultations (29% vs 36%), and emergence services use (26% vs 26%), while an association (p= 0.005) was found according to hospitalisation (20% vs 16%). age. an~ g~der interaction effect on health services use was not found (p> 0.005), but a trend towards bosp1tal1sanon (p=0.04) could be considered. concl.uions: demographic structure of urban older adults is associated with two of the four health se~ices use studi~. a relation.ship ber_ween age. and hospital services use (emergence units and hospitalisanon), but not with ~ut-hosp1tal sei:vices (medical and dentist consultations), was found. in addition ro age, gender also contnbutes to explam hospitalisation. . sexua experiences. we exammed the prevalence expenences 10 relation to ethnic origin and other sociodemographic variables as wc1i as y1j7 die relation between unwanted sexual experiences, depression and agreuion. we did so for boys and prts separately. mdhods: data on unwanted sexual expcric:nces, depressive symptoms (ce.s-d), aggrc:uion (bohi-di and sociodemographic facron were collected by self-report quescionnairc:s administettd to 35 31 students in the: 2nd grade (aged 12-16) of secondary schools in amsterdam, the netherlands. data on the nature ol unwanted sexual experiences were collected during penonal interviews by trained schoolnursn. ltaijtj: overall prevalences of unwanted sexual experiences for boys and girls were 6.5% and 5.7% respectively. unwanted sexual experiences were more often ttported by turkish ( 17.1 %), moroc· an (10.4%) and surinamese/anrillian boys (7.4%) than by dutch boys (2.2%). moroccan and turkish girls, however, reported fewer unwanted sexual experiences (respectively 2.3 and 2.7%) than durch girls did (6.9%). depressive symptoms(or=4.6, cl=3.1-7.0) covert agression (0r•4.9, cl•3.2-7.7) and cmrt aggression (or= 2.6, cl• 1.6-4.4) were more common in girls with an unwanted sexual experi· met. boys with an unwanted sexual experience reported more depressive symptoms (or= 2.2; cl• i . .l· 3.9) and oven agression (or= 1.5, cl= 1.0-2.4) . of the reported unwanted sexual experiences rnpec· timy 17.5% and 73.5% were confirmed by male and female adolescents during a personal interview. cond11sion: we ..:an conclude that the prevalence of unwanted sexual experiences among turkish and moroccan boys is disturbing. it is possible that unwanted sexual experiences are more reported hy boys who belong to a religion or culture where the virginity of girls is a maner of family honour and talking about sexuality is taboo. more boys than girls did not confirm their initial disdosurc of an lllwalltc:d sexual experience. the low rates of disclosure among boys suggcsu a necd to educ.:atc hcahh care providen and others who work with migrant boys in the recognition and repomng of 1exu.il ... iction. viramin a aupplc:tmntation i1 at .h'yo, 1till far from tafl'eted 100%. feedinit pracn~:n panku· lerty for new born earn demand lot of educatton ernpha111 a• cxdu11ve hrealt fecdtnit for dnared rcnoj of 6 months was observtd in only 6.s% of childrrn thoulh colckturm w.11 givm 1n rn% of mwly horn ct.ildrm. the proportion of children hclow-2 waz (malnounshrdl .con" a• h!jh •• 42.6% anj "rt'i· acimy tc.. 11 compared to 1998 data. mother's ~alth: from all is 10 womm in ttprod~uvr •ill' poup, 83% were married and among marned w~ .\9% only w\"rt' u1mic wmr cnntr.-:cruve mt1h· odl 44% were married bdorc thc •ar of 18 yean and 27% had thnr ftnc prcicnancy hcftitt dlt' •icr nf 21 yean. the lt'f'vicn are not uutfactory or they arc adequate but nae unh1ed opumally. of thote' l'h mothen who had deliverrd in last one year, 80% had nailed 11ntmaral eum1nat1on 11 ira" oncc, .~o-... bad matt rhan four ttmn and ma1ortty had 1heir tetanus toxotd tnin,"t1or"'" nlht "'"'"· ljn1r11ned rn· win ronductrd 12.4% dchvcnn and 26% had home deh\'t'oc'i. ~md~: the tervtcn unbud or u111led are !tu than dnarame. the wr· l'kft provided are inadequate and on dechm reprcwnttng a looun1t ~p of h11hnto good coytti\#' ol wr· ncn. l!.ckground chanpng pnoriry cannoc be ruled out u °"" of thc coatnbutory bc10f. ps-ii ia) dcpn:wioa aad anuccy ia mip'mu ia awccr._ many de wn, witco tui~bmjer. jack dekker, aart·jan lttkman, wim gonmc:n. and amoud verhoeff ~ a dutch commumry-bucd icudy thawed 12-moarh•·prc:yalm«i al 17 .44'1. kw anx1· ay daorden and 13. 7% foi' dqrasion m anmttdam. nm .. 11p1tficantly hlllhn than dwwhrft .. dw ~thew diffamca m pttyalcnca att probably rdarcd to tlk' largr populanoa of napaan 111 ..\mturdam. <turkish 5%, moroccan 9%, and surinam 10%1. lndttd. ic'wt'll ltudla hatt ~ ~ high prevalena rata among migrant poupi in rbe ncdwrlands. howc'ytt au ttlluchn iuffenod from wry low raporne rara, or med screnung talel thac lack a ~y yabdarcd ~ in order to study the prevalence of depression and anxiety, and the (barriers to) use of mental health care among the different ethnic groups the following study was recently conducted: . the study consisted of a two-step approach. the first step was mcl~ded m the ~ h ith survey of 2004 (ahs), and consisted of three screening scales for depression and aruaety (klo, g::q-12 and mhl-5). in this survey all respondents were asked_ permission for~ second app~ 1:' 18t consisted of a structured interview containing the cidi for aruaety and depression, and questtollllalres on health care seeking, amongst others. all respondents who gave permission for a second approachwere invited by jetter for a suuctured interview with multilingual interviewers at home at a preset date and time in the following week. . in total, 439 dutch, 317 turkish, 322 moroccan and 124 surinam respondents took part in the ahs and gave permission for a second approach. in the second step, fo~ 21~ tur~h, 184m~roc can 87 surinamese and 320 dutch respondents, information from the extensive interview was available (res~nse rates between 60 and 70% ). since the data collection was completed only recently (june 2005), prevalence estimates can be presented at the conference for the first time. we have shown that with this approach it is feasible to achieve an acceptable response rate in a study on mental health among migrants. therefore we expect that this study will provide reli· able estimates of depression and anxiety in the turkish, moroccan and surinam migrants in amstw!3~· for the first time. in addition, insight into mechanisms underlying the differences between and within ethnic groups and into barriers to mental health care will provide pretexts for the improvement of pre· vention and mental health care for migrant groups. this study aimed to determine spatial patterns of mortality and morbidity of five major health problems in an urban environment: homicides, pregnancy among adolescents ((<20 years old), asthma hospitalization among young children(< 5 years old) and two mosquito-borne diseasesdengue and visceral leishmaniasis, during 1999-2003. all events were obtained through the city health database and, subsequently, geoproccsscd using the address of residence and the geographical and administrative division of the municipality, composed by 80 unit of planning (up), which in tum were formed, each one, by census tract units. two research questions were investigated: are there spatial patterns to the events dis· tribution, and moreover, are these patterns overlapping across space? we use thematic maps, index of comparative mortality/morbidity by up and the overlapped rank of the 20th worse up rates for each event. a spatial pattern of high rates of homicides, proportion of young mothers and hospitalization of asthma were overlapping in areas social and economically disadvantaged. for mosquito-borne diseases, high rates with great dispersion were found in unprivileged areas in contrast with very low rares among privileged ones. these results pointed toward a coexistence of heavier burden of diseases for those living in areas of the city where misery, poverty, lack of political public health may be modulating social health problems. a possible environmental intervention in one mosquito•bome disease might be playing a role in the occurrence of other. although with limitations, this study may provide useful information for a joint urban planning under the public perspective, articulated for use in health impact assessment. jalil safaei bdrod#aion: the association of socioeconomic status (ses), usually measured by income, and health status is an established result in bcalth studies. the poor and low income individuals have lower health sta· tus then_ those with higher incomes. in general. such finding has been usually obtained from sample surveys on speafic populations. a problem with many sample survey$ is that their results are not comparable aaoss ~ndaries as they may use different sampling methods, ask different questions, categorize the answers differently, or define groups differently. moreover, the sample data need to be standardized using the demographic: katurea of a ~ population. this study, however, uses the national population health survey (nphs) cycle 3 public use mictodata files which is based on a common survey template ·~~three~ area in canada, namely montreal, toronto, and vancouver. it also utili7.es the built-m stanclatdizaaon of the nphs data which accounts for its complex survey design. . ~:the stu~ usc:s two well-known measures of health inequalitythe relative index of meqaality and die concentranon indexto capture the extent of income related health inequality among poster sessions v139 urban female and male populations in each of the three metropolitan areas. personal income is classified into ten groups by the nphs with "no income" as the first group and "more than $60,000" as the last. health status is measured by three variables -having a chronic condition (chc), self assessed health (sah), and health utility index (hui) -using appropriate indices. alternative formulations are used to calculate the standard deviations of the estimated or calculated measures. the findings of the study suggest that the measured health inequality depends on the health measure used. for chc and hui the measured inequality indices do indicate poorer health for lower income individulas, however, they are not statistically insignificant. health inequalities are more pronounced when health is measured by sah. the results do not support any systematic ranking of the three metropolitan areas in terms of health inequalities. they do not reveal any systematic pattern of inequality between men and women, either. conclusions: despite the use of highly aggregated nphs data, income related health disparities are observed in the three metropolitan areas in canada. this is more the case with self perceived (sah) health. such disparities are preventable and call for broader health policies that address poverty and low income among other social determinants of health. introduction: the analysis of health indicators under the spatial perspective is configured as an important instrument in the detection of intra-urban differentials. this study aimed to examine the spatial distribution of the births occurred in belo horizonte, in 2001, analyzing the presence of spatial clusters of health indicators for newborns (rn) and their mothers, using data from the information system on live birth database (sinasc). method: for each covered area of the basic units of health (ubs), we calculated the proportion of: adolescent mothers, mothers with less than 8 years of schooling, first pregnancy, mothers with four or more pregnancies, dead babies born on previous pregnancies, stillbirths, cesarean section, less than four prenatal care attendance, moderate and severe hypoxemia in the 1st and 5th minutes of life, low or very low birth weight. we used empiric bayesian methods for smoothing the estimates. for spatial analysis, the indicators obtained from the global moran (i) index and local indicators of spatial association (lisa) were used. maps were built to allow the visualization of the spatial clusters. in all analysis, significance statistics was considered p~ 0.05. results: a total of 36, 12 7 births of residents in belo horizonte were registered in 200 i. the estimated bayesian proportions for the entire municipality was close to the one observed for all variables. analysis using lisa showed the presence of relevant spatial clusters for adolescent and low educated mothers, dead babies form previous pregnancies, cesarean section, low attendance to the prenatal care especially in area with low socio-demographic characteristics. three areas presented consistent clusters, with important spatial auto-correlation for almost all studied indicators. conclusion: the used methodology was configured as a great instrument of detection risk areas where clustering occurs. it can easily be incorporated in any surveillance system as a mechanism for controlling events related to births in the municipality. moreover, it can be applied to discriminate target areas for prompt public health interventions, such as improving health services access and the consolidation of better obstetric practices. introduction: gentrification, the displacement of low income and non-majority people out of longtime neighborhoods and residences is a global phenomenon. it has been identified as occurring in both developed and less developed countries and as inequality persists or increases, many communities are vulnerable to disruption and loss. while there may be some benefits to gentrification, these benefits are less likely to impact those who have been forced to move out of a gentrifying community or those who remain but economically stressed. . . . . this paper lays out a theoretical framework for 1dent1fymg and ~ddressm~. the_ health consequences of gentrification on residents living in a community prior .to o.r durmg gent~1~1cat.10n. by drawing on the literature of housing, sociology, health and urban plannmg, 1t places genmf1catt~n and neighborhood change into the context of other forces affecting th~ .hea.lth of vulnerable populations. it then proposes solutions to prevent or mitigate the impacts of genmf1cat1on. results: four main categories of consequences potentiallr re~ult from ~entr~fica~~n: !'°°r housing· related issues, spatial effects, mental health impacts and contr1bunons to racial ~spanttes m heal~. the housing issues include increased susceptibility to asthma, lead exposure, allergiesl~topy an~. acetdents/ injuries. spatial effects include reduced access to health ~are, reduce~ access to 1obslttadinonal food sources, decreased physical activity, and increased obesity. the __ pnmary mental health effects_ :ire increased stress increased risk of depression and disruption of trad1t1onal support networks. in addinon to the above he~lth issues, gentrification has the potential to increase racial disparities in health by reduc· ing access to long term and multi-culturally experienced health care provide~s. conclusions: public health practice provides a framework for addressmg the health consequences of gentrification. in this context primary prevention would involve preventing gentrification in the first place along with a renewed commitment to helping distressed communities a~~ im_proving the quality ~f life for long term residents. the next layer of meeting the challenges of gentrification would be to assjst long rime residents to stay in a community and thus potentially allowing them to participate in the bene~ts of gentrification (such as better public services). only if all these efforts fail -and these other prevennon strategies must be a priority, public policy should then work to help people and instirutions move to other communities through grants and the provision of alternative safe affordable housing and neighborhoods. sarah romans, marsha cohen, and tonia forte lnh'odlletion: there has been sustained interest in urban rural (ur) differences in mental health over the last 50 years of epidemiological research, with most studies reporting higher urban rates of mor· bidity, particularly when psychotic disorders have been studied. most recently, in britain, urban rates of non-psychotic disorder were greater than rural and semi-rural rates, both before and after the more adverse circumstances of urban dwellers were considered (paykel et al 2003) . surprisingly, there were no ur differences in help seeking. in canada, wang (2004) found greater urban rates for major depression only after he controlled for the confounding effects of race, immigration, employment and marital status, a result reminiscent of work from the us (blazer et 1985) . rural participants were less likely to have sought help for their mental health problems. in this study, we sought to examine urban/rural differences in rates of depression and help seeking in canada. method; data came from the 2000 canadian community health survey 1.2, a community survey conducted by statistics canada of people aged 15 and older, with the total sample size of 36,984 respon· dents, 77% response. analyses used weighted data; differences were assessed by chi-squared. ra.its: bivariate cross-tabulations showed a modest increase in 12 month depression rates for urban (5.4%) over rural (4.3%, p= 0.03) dwellers; there were no ur differences when the sample was examined separately by gender and age group ( 15-29, 30-44, 45-69) . in general, more urban (10.6%) than rural people (8. 7%, p= 0.002) had sought mental health treatment in the past year. there was no ur difference in helping seeking amongst those with depression (u 58.9%, r 50.1 % ns). amongst the whole population, helping seeking was gendered with more urban men accessing help (7.4%) than rural men (5.4%, p=0.004); this did not apply for women (13.7% vs 12.1% p=0.1). ~ons: the urban-rural demographic continues to generate intriguing findings, even recently when internal migration is common and people can seek out the environment most conducive to their health, ~th physical and mental. people with depression are a disadvantaged, frequently stigmatized group, with ~r quality of life a_nd often impaired function. unlike many other factors associated wi~h urban or rural hfe per se, depression can be treated. the low help seeking rates is a cause for concern, m both the ur~n and_ rural populations. it behooves researchers, policy makers and service planners to ensutt effective services are available for both humane and economic reasons. methods: using united states 2000 census income data, we assigned the 12 manhattan community districts to two major socioeconomic groups, manhattan i and ii. manhattan i is composed of community districts with average household incomes above the median for new york city; manhattan ii is those below. with public new york city department of health 1999 death records and 2000 us census data, we calculated a "standard" new york city population and estimated mortality rate distributions for manhattan i and ii. we then compared these age-adjusted actual mortality distributions with a standard t-test. results: we explored premature deaths using several cut-off points (before the ages of 65, 70, and 75), and with subcomparisons for males and females. every comparison showed the wealthier area, manhattan i, to have significantly lower premature mortality rates than the lower income manhattan ii. further, we compared age-adjusted mortality rates among the city's five boroughs, and found no significant differences based on local geography alone. conclusions: these findings suggest that in manhattan, characterized by its extremes of wealth and poverty, health status, as measured by premature mortality, does vary significantly with the local income level. in manhattan, the relative average income by community district varies as much as 4: 1. this ratio is greater than that found in the other cities we have studied: for example, the range among tokyo's kus is half that in manhattan, or about 2: 1. paris has shown the longest life expectancy and changing premature mortality rates. from our preliminary work, we expect to find less variation in premature mortality rates in comparable cities. this study will continue to explore the relationship of premature mortality rates and life expectancy to income levels and different health systems among wcp cities with a view to measuring health policy options. introduction: according to the world health organization (who), smoking-related illness is currently the world's second major cause of death, currently responsible for one out of ten adult deaths worldwide. the who's framework convention on tobacco control (fctc) aims to reduce tobaccorelated disease and deaths by changing national public policies. mexico, which, according to the who, had smoking prevalences of 32% among adults in 1998 and 23% among health care providers (hcps) in 1997, ratified the fctc in 2004. objective: to examine knowledge of and attitudes towards cigarette smoking and smoking cessation among hcps, and clinical practices regarding smoking cessation. methods: in june 2005, a convenience sample of hcps from one public clinic in urban oaxaca, mexico, part of mexico's national network of public health care clinics, were interviewed in spanish using a verbally administered questionnaire that was standard among all participants. during primary care outpatient visits, hcps were observed treating patients to assess the relative importance of smoking cessation as a health care priority. results: of the 18 hcps interviewed, including ten physicians, three nurses, and five medical students, 67% reported smoking regularly. all hcps reported assessing patients' history of smoking, knowledge of the health risks associated with smoking, and feeling qualified to discuss these risks with patients. all hcps reported counseling patients who currently smoked to quit. all hcps reported recommending nicotine replacement therapy (eg., patch and/or gum) and/or behavioral therapy (eg., psychologist, support groups) to patients who smoked. all hcps reported that there was no government funding for smoking cessation, and that all costs associated with smoking cessation were patients' out-of-pocket expenses. observation of hcp interaction with patients revealed that hcps always inquired about smoking history with new patients and rarely inquired about smoking history with returning patients. hcps were not observed counseling patients who reported current smoking on smoking cessation methods and its benefits. observation of the clinical environment suggested a focus on preventative child health (eg., immunization, water-borne illnesses), family planning, and chronic diseases (eg., diabetes, hypertension). conclusions: while the mexican government has made smoking cessation a public health priority and hcps report addressing smoking cessation, observation of hcps suggests: 1) smoking status is assessed only in new patients; and 2) smoking cessation methods are not addresse~. observ_atio~ of hcps and the clinical environment in a public clinic in oaxaca suggests that smoking cessation 1s of lower priority than other heath care issues. p6-04 (a) urban agriculture and food and nutrition security in kampala, uganda fiona yeudall, renee sebastian, abdelrahman lubowa, selahadin ibrahim, and donald cole introduction: urban agriculture is an important livelihood strategy contributing to household food security by increasing access and availability to food in urban settings (koc et al., 1999) . the purpose of poster sessions v142 rhis study was to examine relationships between child nutritional securiry outcomes, household food security, and urban agriculture activiries in kampala, uga~da. . . questionnaires assessing socio-demographic, farmmg and household food secunry (hfsi characreristics were administered to 270 households. food diversiry was ~lculate~ from ~e number of foods consumed over 24 hours for one child (2-5 years) per household. heigh~ weight,. nud-upper·ann· circumference (muac) and tricep skinfolds (tsf) were measured for the mdex child. z-sc.ores for height-for·age (haz), weighr-for-age (waz) and body mass index (zbmi) were ~~ulated usmg cen· rres for disease control and prevenrion reference data. z-scores for body composition measures were calculated using nhanes i and ii data for african americans. the lms method was used to c~.t for skewed z·score indices. all dara was checked for normaliry and univariable and backward mulnvanablc linear regression analysis conducted. ruwlts: household food securiry was significantly associated with wealth (b= 0. 71, p< 'a acre were more dependenr on assets for hfs. although sex of head of household was ~ot related to j-:ifs, it modi· fied relationships in rhar female headed households had greater food securtry when fai:mmg less than 1,4 acre compared ro male headed households, and vice versa. hfs was significantly associated with food diversiry (s=0.15, p). urbanization, especially in developing countries, has substantially increased the vulnerability of rhe mass of low-income urban dwellers. the livelihoods and quality of life for many of the poor espe· cially in larin america, asia, and africa, have deteriorated significantly over the years. urban areas are increasingly unable to provide for their populations, resulting in poverry, massive unemploymenr, job cuts, poor housing, lack of or poor public services, and a compromised health status. botswana, a country rhat lies in the sourhern parr of africa, has had its share of urban problems such as rhe mush· rooming of squarters and low-income urban sertlements. despite efforts to address the substandard living conditions in low-income urban areas, these problems have continued to grow. these living con· diuons are porenrially stressful to rhe residenrs and likely affects their health. the primary aim of the 11udy wa1 to examine the complex relationship between communiry-level stressors and interveners and health-related quality of life among residents of low-income neighborhoods in francistown, botswana. u1i"" a croa1-icctional quantitative design (both descriptive and explanatory) and using primarily cloae-mded interview• with a random sample of 388 residents, this study examined the role of chrome life 1trnson and environmental quality on overall health status qualiry of life) and the physical, psy· chological and level of independence domains of health. the major hypothesis of the study was that community-levrl stre1sor1 would influence health-related quality of life and that social capital would moderate these relationships. findings indicate that neighborhood quality is a powerful predictor of healrh 1tatu1 rhan socioeconomic status and individual life stressors. social capital was also found to he a 11111ificant positive predictor of healrh and also moderator of structural factors. social capital moderated the effects of low environmental quality on level of indrpendence and on physical health outcomn, but nor on psychological and global health outcomes. these findings suggest that as the environment get1 betttr. stresson are reduced, hence promoting berter health outcomes. the study ends with implication• for social justice, public health and social work practice, and research, focusing mostly on the ~ole of social capital and the environmental quality in predicting health outcomes. spt· cafically • anenhon should be focused on political and civic society's commitment for social justice and poveny alleviation, ~uction of the threat of insecuriry and violence; cultivating social capital and good governance; and improving the health and social environments, especially housing and environ· mental 1aiutanon to name a few. urban and other uprisings by non-elites that lead to transitions, can disrupt sexual and injection "risk" networks that transmit infectious diseases by changing mixing patterns. they can also weaken urban social networks, their associated protective norms and their informal social control mechanisms which can lead to increased sexual and drug risk behaviors and violence (fullilove 2004; wallace & wallace1998, aral 2002 , friedman & reid 2000 hankins et al 2002) . for example, the 1979 revolution and transition to theocratic rule, and subsequent urban and national conflicts, have been followed by many urban youth in iran engaging in clandestine high-risk sexual and drug behaviors. in palestine, conflicts and restrictions on movement have led to increased fatalities from chronic diseases due to limited access to hospitals and modern medical facilities (union of palestinian medical relief committees); and heroin use and violence-related blood exposure have also increased. social movements "from below" that are rooted both in urban social network dynamics and in underlying patterns of injustice can have both protective and risky effects. for example, the social movements that led to the collapse of the ussr and its dependent governments in other countries-with subsequent increases in sex trade, alcoholism, drug use, tuberculosis, hiv, stis, and mortality in many localities-were based originally on such city-based movements in eastern europe. their impacts on health were mediated by urban social dynamics and structures. some urban social movements have improved health by prevent· ing the spread of hiv, hepatitis and other diseases. examples include movements of (a) gays and lesbians and (b) drug users. these have been rooted in social networks that overlap with risk networks. theories of urban health should include social conflict as a core concept. mechanisms that generate conflicts and the pathways by which conflicts affect health should be a major part of urban health research agendas. p6-07 (c) demographic characteristics of people seen with tuberculosis in lagos state university teaching hospital (lasuth) chest clinic john bako, adewale akeredolu, and wale alabi tuberculosis has become a resurgent public health problem in recent times in the world. because resources are limited, control programmes frequently must target population at greatest risk. the purpose of this study was to study the demographic characteristics of people seen with tuberculosis in lagos state university teaching hospital. a total of 76 patients who have been both radiologically and bacteriologically confirmed tuberculosis patients were used for this study between january and march. 57 (75%) were males, while 19 (25%) were females. notable risk factors among patients with tuberculosis were overcrowding (46.1 % ), homelessness ( 11.8%) cigarette smoking (22.4% ), alcoholism (30.3%), non vaccination (25%), secondary contact (36.8%) and poor knowledge (92. l 'x,). man· agement history patterns among the patients were herbs ( 13.2 'yo), orthodox medicine ( .h . .l'x.). intervention targeting early-identified groups may be an effective way to reduce the incidence of tuber· culosis. such intervention should focus on health education, modification of life style and improvement of standard of living. p6-08 (c) profiles in urban health in 9 cities of the americas in the countries of the americas, there is an increasing migration of national and international populations to urban centers. this presentation will focus on some of the issues created by this influx of populations, the ways that 9 cities in 8 countries are dealing with them, and the efforts to promote local participation and solutions in management and decision-making. the methodology used to collect this information has been to draw upon and analyze information produced by the mayor's and ministry of health and development offices in each of the cities under consideration. an analysis of the impact of urbanization on health and health determinants will be presented. the information covers issues such as health status by geographic areas within the cities, highlighting issues such as marginality, barriers and physical, economic and cultural constructs and inequities in the delivery of and access to essential services (such as health, education, water, and basic sanitation). issues of governance and citizen participation will be highlighted to provide insight in~o the balance of power and mechanisms for decision-making at the local level. part of the analysis will show the relationship between democratic processes and social participation. public ~olicies will be reviewed to indicate those that are most beneficial in promoting health and overcoming barriers to it, as well as ways of capitalizing on local assets and resources. final~y, evidence of effectiveness of various strategies will be indicated. the presentation will conclude wuh suggesuons for future strategic directions to improve the quality of life and the promotion of health in large urban centers of the americas. introduction: much of the illicit drug in mexico is concentrated in northern border areas. the 2000 mile border between the u.s. and mexico is also characterized by migration; the border crossing between tijuana, baja california, mexico and san diego, california, u.s.a. is rep~rt~dl~ the busiest land border crossing in the world. we attempt to describe the migration scene among m1ect1on drug users (idus) m tijuana and investigate service needs. methods: migration trends were investigated in a cross-sectional study conducted from february to june 2005 among idus in tijuana. enrollment criteria included informed consent, being 18 years or older, and having injected drugs within the prior month. subjects were recruited by respondent-dnven sampling and were administered a quantitative survey and serology for hiv, hcv, and syphilis. logistic regression was used to compare idus who had migrated to the u.s. versus those who had not. results: of 222 idus enrolled, 91 % were male and median age and age at first injection were 35 and 20, respectively. drug combinations injected most frequently in the past 6 months were heroin (35%1 and crystal methamphetamine mixed with heroin (53%). of those enrolled, 212 responded to quesnons on migration and drug treatment and were included in this analysis. although 76% had resided in tijuana for at least 5 years, 70% of users were born outside of baja california. working outside of mexico was common, with 38% working abroad in the last 10 years (94% in u.s.), and 17% in the past year. in the last 6 months, 10% of idus had crossed the border to the u.s., with 57% crossing at least once per month. those working outside of mexico in the past year were less likely to have ever received substance abuse treatment, [29% vs. 53%, or 0.38, ] and were marginally less likely to have received drug treatment in the past 6 months [6% vs. 18 %, or 0.28, 95% ci (0.06-1.2)]. drug use patterns, age and gender did not differ between migrants and non-migrants, although migrants were more likely to report being in need of substance abuse treatment (68% vs. 57%, respectively). conclusions: migration is common among idus in tijuana. maintaining drug treatment regimens and determining how to best target education and services to a highly mobile population pose challenges to officials on both sides of the border. these data underscore the need to develop coordinated binational prevention and treatment efforts. introduction: despite the expanding literature on the important role public policy plays in influencing the broader determinants of the public\'s health, profound differences exist among jurisdictions in rhe attention placed upon such activities. we take an international perspective on health by examining rhe d?minant public _health paradigms of canada, usa, uk, and sweden and exploring how these paradigms shape public health practice. _method: we carefully analyze governmental and public health agencies documents to discern ~he dommant para~igms driving public health approaches and practices. we also consider the unique paht1· cal and economic contexts within each nation and consider how these contexts drive public health pahcy and approaches. . . ~u~: the canadian and usa public health communities -with some exceptions --focus upon m~ividuahzed approaches to risk management. in contrast, the uk and swedish public health scenes are oriented toward broader approaches to health determinants. we find that the extent to which govern· ments, public health agencies and public health workers concern themselves with public policy approaches £<_> ~ddress ~ro.ad~r .determinants of health depends upon the particular health parad'.~ adhered. ~o withm each 1urisd1ctton. and whether a paradigm is adopted depends upon the ideologi~a and pol~ncal context of each nation. nations such as sweden that have a long tradition of public policies promonng social jus~ce an~ equity are naturally receptive to evolving population health concepts. '[he usa represen~ a ~bey en~ro~~t where such is~ues are clear!~ subordinate. ., our findings mdicate that there 1s a strong political component that influences pubh ~ealth a~proaches and practi~ within the jurisdictions examined. the implications are that those seek· m~ to raise the broader detennmants of the public's health should work in coalition to raise these issues with non-health organizations and age · ca d d th · badrgrollnd: in developed countries, social inequalities in health have endured or even worsened comparatively throughout different social groups since the 1990s. in france, a country where access to medical and surgical care is theoretically affordable for everyone, health inequalities are among the high· est in western europe. in developing countries, health and access to care have remained critical issues. in madagascar, poverty has even increased in recent years, since the country wenr through political crisis and structural adjustment policies. objectives. we aimed to estimate and compare the impact of socio· economic status but also psychosocial characteristics (social integration, health beliefs, expectations and representation, and psychological characteristics) on the risk of having forgone healthcare in these 2 dif· fercnt contexts. methods: population surveys conducted among random samples of households in some under· served paris neighbourhoods (n= 889) and in the whole antananarivo city (n= 2807) in 2003, using a common individual questionnaire in french and malagasy. reslllts: as expected, the impact of socioeconomic status is stronger in antananarivo than in paris. but, after making adjustments for numerous individual socio-economic and health characteristics, we observed in both cities a higher (and statically significant) occurrence of reponed forgone healthcare among people who have experienced childhood and/or adulthood difficulties (with relative risks up to 2 and 3.s respectively in paris and antananarivo) and who complained about unhealthy living conditions. in paris, it is also correlated with a lack of trust in health services. coneluions: aside from purely financial hurdles, other individual factors play a role in the non-use of healthcare services. health insurance or free healthcare seems to be necessary hut not sufficienr to achieve an equitable access to care. therefore, health policies must not only focus on the reduction of the financial barriers to healthcare, but also must be supplemented by programmes (e.g. outreach care ser· vices, health education, health promotion programmes) and discretionary local policies tailored to the needs of those with poor health concern .. acknowledgments. this project was supported by the mal>io project and the national institute of statistics (instat) in madagascar, and hy the development research institute (ird) and the avenir programme of the national institute of health and medical research (inserm) in france. for the cities of developing countries, poverty is often described in terms of the living standard~ of slum populations, and there is good reason to believe that the health risks facing these populations are even greater, in some instances, than those facing rural villagers. yet much remains to be learned ahour the connections between urban poverty and health. it is not known what percentage of all urban poor live in slums, that is, in communities of concentrated poverty; neither is it known what proportion of slum residents are, in fact, poor. funhermore, no quantitative accounting is yet available that would sep· arare the health risks of slum life into those due to a househoid•s own poverty and those stemminic from poveny in the surrounding neighborhood. if urban health interventions are to be effectively targeted in developing countries, substantial progress must be made in addressing these cenrral issues. this paper examines poverty and children's health and survival using two large surveys, one a demographic and health survey fielded in urban egypt (with an oversampling of slums) and the other a survey of the slums of allahabad, india. using multivariate statistical methods. we find, in both settings: ( 11 substan· rial evidence of living standards heterogeneity within the slums; (21 strong evidence indicating that household-level poverty is an imponant influence on health; and (3) staristically significant (though less strong) evidence that with household living standards held constant, neighborhood levels of poverty adversely affect health. the paper doses with a discussion of the implications of these findings for the targeting of health and poverty program interventions. p6-13 (a) urban environment and the changing epidemiological surfacr. the cardiovascular ~ &om dorin, nigeria the emergence of cardiovascular diseases had been explained through the concomitants o_f the demographic transition wherein the prevalent causes of morbidity and monality ~hangr pr~mmant infectious diseases to diseases of lifestyle or chronic disease (see deck, 1979) . a ma1or frustration m the v146 poster sessions case of cvd is its multifactural nature. it is acknowledged that the environment, however defined is the d · f · t' b tween agents and hosts such that chronic disease pathogenesis also reqmre a me 1an o mterac ion e . spatio-temporal coincidence of these two parties. what is not clear is which among ~ever~( potennal fac· · h b pace exacerbate cvd risk more· and to what extent does the ep1dem1olog1cal trans1· tors m t e ur an s ' . . . . tion h othesis relevant in the explanation of urban disease outlook even the developmg cities like nigeri~: thesis paper explorer these within a traditional city in nigeria. . . . the data for the study were obtained from two tertiary level hospitals m the metropolis for 10 years (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) . the data contain reported cases of cvd in the two facilities for the period. adopting a series of parametric and non-parametric statistics, we draw inferences between the observed cases of cvds and various demographic and locational variables of the patients. findings: about 28% of rhe cases occurred in 3 years (1997) (1998) (1999) coinciding with the last year of military rule with great instability. 55.3% occurred among male. 78.8% also occurred among people aged 31-70 years. these are groups who are also likely to engage in most stressful life patterns. ~e study also shows that 63% of all cases occurred in the frontier wards with minor city areas also havmg their •fair' share. our result conformed with many empirical observation on the elusive nature of causation of cvd. this multifactoral nature had precluded the production of a map of hypertension that would be consistent with ideas of spatial prediction. cvd -cardiovascular diseases. mumbai is the commercial capital of india. as the hub of a rapidly transiting economy, mumbai provides an interesting case study into the health of urban populations in a developing country. with high-rise multimillion-dollar construction projects and crowded slums next to each other, mumbai presents a con· trast in development. there are a host of hi-tech hospitals which provide high quality care to the many who can afford it (including many westerners eager to jump the queue in their healthcare systems-'medical tour· ism'), at the same time there is a overcrowded and strained public healthcare system for those who cannot afford to pay. voluntary organizations are engaged in service provision as well as advocacy. the paper will outline role of the voluntary sector in the context of the development of the healthcare system in mumbai. mumbai has distinct upper, middle and lower economic classes, and the health needs and problems of all three have similarities and differences. these will be showcased, and the response of the healthcare system to these will be documented. a rising hiv prevalence rate, among the highest in india, is a challenge to the mumbai public healthcare system. the role of the voluntary sector in service provision, advocacy, and empowerment of local populations with regards to urban health has been paramount. the emergence of the voluntary sector as a major player in the puzzle of urban mumbai health, and it being visualized as voices of civil society or communiry representatives has advantages as well as pitfalls. this paper will be a unique attempt at examining urban health in india as a complex web of players. the influence of everyday socio·polirical-cultural and economic reality of the urban mumbai population will be a cross cutting theme in the analysis. the paper will thus help in filling a critical void in this context. the paper will thus map out issues of social justice, gender, equiry, effect of environment, through the lens of the role of the voluntary sector to construct a quilt of the realiry of healthcare in mumbai. the successes and failures of a long tradi· tion of the active advocacy and participation of the voluntary sector in trying to achieve social justice in the urban mumbai community will be analyzed. this will help in a better understanding of global urban health, and m how the voluntary sector/ngos fir into the larger picture. ba~und: o~er. half _of n~irobi's 2.5 million inhabitants live in illegal informal settlements that compose 5 yo of the city s res1dent1al land area. the majority of slum residents lack access to proper san· iranon and a clean and adequate water supply. this research was designed to gain a clearer understand· mg of what kappr · · · h f . . opnate samtanon means or the urban poor, to determine the linkages between gender, hvehhoods, and access to water and sanitation, and to assess the ability of community sanitation blocks to meet water and sanitation needs in urban areas. m~tbojs_: _a household survey, gender specific focus groups and key informant interviews were conducted m maih saba, a peri-urban informal settlement. qualitative and quantitative research tools were u~ to asses~ the impact and effectiveness of community sanitation blocks in two informal settlements. results ropna e samtarmn me u es not only safe and clean latrines, but also provision ° adequate drainage and access to water supply for cleaning of clothes and homes. safety and cleanliness poster sessions v147 were priorities for women in latrines. levels of poverty within the informal settlements were identified and access to water and sanitation services improved with increased income. environmental health problems related to inadequate water and sanitation remain a problem for all residents. community sanitation blocks have improved the overall local environment and usage is far greater than envisioned in the design phase. women and children use the blocks less than men. this is a result of financial, social, and safety constraints. the results highlight the importance a need to expand participatory approaches for the design of water and sanitation interventions for the urban poor. plans need to recognize "appropriate sanitation" goes beyond provision of latrines and gender and socioeconomic differences must be taken into account. lessons and resources from pilot projects must be learned from, shared and leveraged so that solutions can be scaled up. underlying all the challenges facing improving water and sanitation for the urban poor are issues of land tenure. p6-16 (c) integrating tqm (total quality management), good governance and social mobilization principles in health promotion leadership training programmes for new urban settings in 12 countries/ areas: the prolead experience susan mercado, faren abdelaziz, and dorjursen bayarsaikhan introduction: globalization and urbanization have resulted in "new urban settings" characterized by a radical process of change with positive and negative effects, increased inequities, greater environmental impacts, expanding metropolitan areas and fast-growing slums and vulnerable populations. the key role of municipal health governance in mitigating and modulating these processes cannot be overemphasized. new and more effective ways of working with a wide variety of stakeholders is an underpinning theme for good governance in new urban settings. in relation to this, organizing and sustaining infrastructure and financing to promote health in cities through better governance is of paramount importance. there is a wealth of information on how health promotion can be enhanced in cities. despite this, appropriate capacity building programmes to enable municipal players to effectively respond to the challenges and impacts on health of globalization, urbanization and increasing inequity in new urban settings are deficient. the who kobe centre, (funded by the kobe group( and in collaboration with 3 regional offices (emro, searo, wpro) with initial support from the japan voluntary contribution, developed a health promotion leadership training programme called "prolead" that focuses on new and autonomous structures and sustainable financing for health promotion in the context of new urban settings. methodology: country and/or city-level teams from 12 areas, (china, fiji, india, japan, lebanon, malaysia, mongolia, oman, philippines, republic of korea, tonga and viet nam) worked on projects to advance health promotion infrastructure and financing in their areas over a 9 month period. tools were provided to integrate principles of total quality management, good governance and social mobili1.ation. results: six countries/areas have commenced projects on earmarking of tobacco and alcohol taxes for health, moblization of sports and arts organizations, integration of health promotion and social health insurance, organizational reforms, training in advocacy and lobbying, private sector and corporate mobilization and community mobilization. results from the other six areas will be reported in 01..;obcr. conclusions: total quality management, good governance and social mobilization principles and skills are useful and relevant for helping municipal teams focus on strategic interventions to address complex and overwhelming determinants of health at the municipal level. the prolead training programmes hopes to inform other processes for building health promotion leadership capacity for new urban settings. the impact of city living and urbanization on the health of citizens in developing countries has received increasing attention in recent years. urban areas contribute largely to national economies. however, rapid and unplanned urban growth is often associated with poverty, environmental degradation and population demands that outstrip service capacity which conditions place human health at risk. local and national governments as well as multi national organizations are all grappling with the challenges of urbanization. with limited data and information available, urban health characteristics, including the types, quantities, locations and sources in kampala, are largely unknown. moreover, there is n? basis for assessing the impact of the resultant initiatives to improve health ~onditions amo~g ~o":1":1um ties settled in unplanned areas. since urban areas are more than the aggregation ?f ~?pie w~th md_1v1dual risk factors and health care needs, this paper argues that factors beyond the md1v1dual, mcludmg the poster sessions v148 · i d h · i · ment and systems of health and social services are determinants of the health soc1a an p ys1ca environ . of urban populations. however, as part of an ongoing study? ~s pape~ .addresses the basic concerns of urban health in kampala city. while applying the "urban hvmg conditions and the urban heal~ pen· alty" frameworks, this paper use aggregated urban health d~ta t~ explore the role of place an~ 111st1tu· tions in shaping health and well-being of the population m kampala by understanding how characteristics of the urban environment and specific features of the city are causally related to health of invisible and forgotten urban poor population: results i~dica~e that a .range o~ urb~n he~l~h hazards m the city of kampala include substandard housing, crowdmg, mdoor air poll.ut1on, msuff1c1ent a~d con· taminated water, inadequate sanitation and solid waste management services, vector borne .diseases, industrial waste increased motor vehicle traffic among others. the impact of these on the envtronment and community.health are mutually reinforcing. arising out of the withdra"'.al of city pl~nning systems and service delivery systems or just planning failure, thousands of people part1cularl~ low-mc~me groups have been pushed to the most undesirable sections of the city where they are faced with ~ va_r1ety ~f envj· ronmental insults. the number of initiatives to improve urban health is, however, growing mvolvjng the interaction of many sectors (health, environment, housing, energy, transportation and urban planning) and stakeholders (local government, non governmental organizations, aid donors and local community groups). key words: urban health governance, health risks, kampala. introduction: the viability of urban communities is dependent upon reliable and affordable mass transit. in particular, subway systems play an especially important role in the mass transit network, since they provide service to vast numbers of ridersseven of the 95 subway systems worldwide report over one billion passenger rides each year. surprisingly, given the large number of people potentially affected, very little is known about the health and safety hazards that could affect both passengers and transit workers; these include physical (e.g., noise, vibration, accidents, electrified sources, temperature extremes), biological (e.g., transmission of infectious diseases, either through person-to-person spread or vector-borne, for example, through rodents), chemical (e.g., exposure to toxic and irritant chemicals and metals, gas emissions, fumes), electro-magnetic radiation, and psychosocial (e.g., violence, workstress). more recently, we need to consider the threat of terrorism, which could take the form of a mass casualty event (e.g., resulting from conventional incendiary devices), radiological attack (e.g., "dirty bomb"), chemical terrorist attack (e.g., sarin gas), or bioterrorist attack (e.g., weapons grade anthrax). given the large number of riders and workers potentially at risk, the public health implications are considerable. methods: to assess the hazards associated with subways, a structured review of the (english) litera· ture was conducted. ruults: based on our review, non-violent crime, followed by accidents, and violent crimes are most prevalent. compared to all other forms of mass transit, subways present greater health and safety risks. however, the rate of subway associated fatalities is much lower than the fatality rate associated with automobile travel (0.15 vs. 0.87 per 100 million passenger miles), and cities with high subway ridership rates have a 36% lower per capita rate of transportation related fatalities than low ridership cities (7.5 versus 11.7 annual deaths per 100,000 residents). available data also suggest that subway noise levels and levels of air pollutants may exceed recommended levels. . ~: there is a paucity of published research examining the health and safety hazards associated with subways. most of the available data came from government agencies, who rely on passively reported data. research is warranted on this topic for a number of reasons, not only to address important knowled~ gaps, but also because the population at potential risk is large. importantly, from an urban perspecnve, the benefits of mass transit are optimized by high ridership ratesand these could be adversely affu:ted by unsafe conditions and health and safety concerns. veena joshi, jeremy lim. and benjamin chua ~ ~rban health issues have moved beyond infectious diseases and now centre largely on chrome diseases. diabetes is one of the most prevalent non-communicable diseases globally. 9 % of adult ¥151 benefit in providing splash pads in more parks. given the high temperature and humidity of london summers, this is an important aspect and asset of parks. interviewed parents claimed to visit city parks anywhere between 1 to 6 days per week. corrduion: given that the vast majority of canadian children are insufficiently active to gain health benefits, identifying effective qualities of local parks, that may support and foster physical activity is essential. strategies to promote activity within children's environments are an important health initiative. the results from this study have implications for city planners and policy makers; parents' opinions of, and use of city parks provides feedback as to the state current local parks, and modifications that should be made for new ones being developed. this study may also provide important feedback for health promoters trying to advocate for physical activity among children. introdt1clion: a rapidly increasing proportion of urban dwellers in africa live below the poverty line in overcrowded slums characterized by uncollected garbage, unsafe water, and deficient sanitation and overflowing sewers. this growth of urban poverty challenges the commonly held assumption that urban populations enjoy better health than their rural counterparts. the objectives of this study are (i) to compare the vaccination status, and morbidity and mortality outcomes among children in the slums of nairobi with rural kenya, and (ii) to examine the factors associated with poor child health in the slums. we use data from demographic and health survey representative of all slum settlements in nairobi city carried out in 2000 by the african population & health research center. a total of 3,256 women aged 15-49 from 4,564 households were interviewed. our sample consists of 1,210 children aged 0-35 months. the comparison data are from the 1998 kenya demographic and health survey. the outcomes of interest include child vaccination status, morbidity (diarrhea, fever and cough) and mortality, all dichotomized. socioeconomic, environmental, demographic, and behavioral factors, as well as child and mother characteristics, are included in the multivariate analyses. multilevel logistic regression models are used. l'nlimin11ry rest1lts: about 32 % of children in the slums had diarrhea in the two weeks prior to the survey, compared to 16% of rural children. these disparities between the urban poor anj the rural residents are also observed for fever (64% against 42%), cough (46% versus 20%), infant mortality (91/ 1000 against 76/1000), and complete vaccination (48% against 64%). preliminary multivariate results indicate that health service utilization and maternal education have the strongest predictive power on child morbidity and mortality in the slums, and that household wealth has only minor, statistically insignificant effects. conclruion: the superiority of health of urban children, compared with their rural counterparts, masks significant disparities within urban areas. compared to rural residents, children of slum dwellers in nairobi are sicker, are less likely to utilize health services when sick, and stand greater risk to die. our results suggest policies and programs contributing to the attainment of the millennium development goal on child health should pay particular attention to the urban poor. the insignificance of socioeconomic status suggests that poor health outcomes in these communities are compounded by poor environmental sanitation and behavioral factors that could partly be improved through female education and behavior change communication. introduction: historic trade city surat with its industrial and political peace has remained a center of attraction for people from all the comers of india resulting in to pop.ulatio~ explosio~ a~d stressed social and service infrastructure. the topography,dimate and demographic profile of the city 1s threat to the healthy environment. aim of this analysis is to review the impact of managemt'nt reform on health indicators. method: this paper is an analysis of the changing profile of population, sanitary infr~s~rucrure, local self government management and public health service reform, secondary health stat1st1cs data, health indicator and process monitoring of 25 years. . . health of entire city and challenge to the management system. plague outbr~ak (1994) was the turning point in the history of civic service management including p~blic ~e~lth service management. ~ocal self government management system was revitalized by reg~lar_ field v1s1ts o~ al~ cadre~, _decentraltzanon of power and responsibility, equity, regular vigilant momtormg, commumcanon facility, ream_approach and people participation. reform in public health service management was throu_gh stan~~rd1zed intervention protocol, innovative intervention, public private partnership, community part1c1panon, academic and service institute collaboration and research. sanitation service coverage have reached nearer to universal. area covered by safe water supply reached to 98%(2004) from 40% (1991) and underground drainage to 97% (2004) from 17% ( 1991) the overhauling of the system have reflected on health indicators of vector and water born disease. malaria spr declined to 1.23 (2004) from 23.06'yo(!991) and diarrhea case report declined to 1963(2004) from 3431 (2004). except dengue fever in 2002 no major disease outbreaks are reported after 1991. city is recipient of international/national awards/ranking for these achievements. the health department have developed an evidence and experience based intervention and monitoring system and protocol for routine as well as disaster situation. the health service and management structure of surat city have emerged as an urban health model for the country. introduction: the center for healthy communities (chc) in the department of family and com· munity medicine at the medical college of wisconsin developed a pilot project to: 1) assess the know· ledge, attitudes, and behaviors of female milwaukee public housing residents related to breast cancer; 21 develop culturally and literacy appropriate education and screening modules; 3) implement the developed modules; 4) evaluate the modules; and 5) provide follow-up services. using a community-based participatory research model the chc worked collaboratively with on-site nurse case management to meet these objectives. methods: a "breast health kick off event" was held at four separate milwaukee public housing sites for elderly and disabled adults. female residents were invited to complete a 21-item breast health survey, designed to accommodate various literacy levels. responses were anonymous and voluntary. the survey asked women about their previous physical exams for breast health, and then presented a series of state· ments about breast cancer to determine any existing myths. the final part gathered information about personal risk for breast cancer, the highest level of education completed, and whether the respondents h;td ever used hormone replacement therapy and/or consumed alcohol. responses were collected for descriptive analysis. results: a total of 45 surveys (representing 18% of the total female population in the four sites) were completed and analyzed. 89% reported that they had a physical exam in the previous rwo years. 96% of respondents indicated they never had been diagnosed with breast cancer. 85% reported having had a mammogram and 87% having had a clinical breast exam. those that never had a mammogram reported a fear of what the provider would discover or there were not any current breast problems ro warrant an exam. 80% agreed that finding breast cancer early could lower the chance of dying of cancer. over 92% reported that mammograms were helpful in finding cancer. however, 27% believed that hav· ing a mammogram actually prevents breast cancer. 14% indicated that mammograms actually cause cancer and 16% reported that a woman should get a mammogram only if there is breast cancer in her family. conclusion: this survey indicates that current information about the importance of mammograms and clinical breast exams is reaching traditionally underserved women. yet there are still critical oppor· tunities to provide valuable education on breast health. this pilot study can serve as a tool for shaping future studies of health education messages for underserved populations. located in a yourh serv· ~ng agency m downtow~ ottawa, the clinic brings together community partners to provide primary medical care. and dent~i hygiene t? the street youths of ottawa aged 12-20. the primary goal of the project is to provide accessible, coordinated, comprehensive health and dental care to vulnerable adolescents. these efforts respond to the pre-existing body of evidence suggesting that the principle barrier in accessing such care for these youths are feelings of intimidation and vulnerability in the face of a complex healthcare system. the bruyere fhn satellite clinic is located in the basement of a downtown drop-in and brings together a family medicine physician and her residents, a dental hygienist and her 2nd year students, a nurse practitioner, a chiropodist and 2 public health nurses to provide primary care. the clinic has been extremely busy and well received by the youth. this workshop will demonstrate how five community organizations have come together to meet the needs of high risk youths in ottawa. this presentation will showcase the development of the clinic from its inception through its first year including reaction of the youths, partnerships and lessons learned. it will also focus on its sustainability without continued funding. we hope to have developed a model of service delivery that could be reproduced and sustained in other large cities with faculties of medicine. methods: non-randomized, mixed method design involving a process and impact evaluation. data collection-qualitative-a) semi structured interviews with providers & partners b)focus groups with youth quantitative a)electronic medical records for 12 months records (budget, photos, project information). results: 1) successfully built and opened a medicaudental clinic which will celebrate its 1 year anniversary in august. 2) over 140 youths have been seen, and we have had over 300 visits. conclusion: 1) the clinic will continue to operate beyond the 18 month project funding. 2) the health of high risk youth in ottawa will continue to improve due to increased access to medical services. p7-11 (a) health services -for the citizens of bangalore -past, present and future savita sathyagala, girish rao, thandavamurthy shetty, and subhash chandra bangalore city, the capital of karnataka with 6.5 million is the 6th most populous city in india; supporting 30% of the urban population of karnataka, it is considered as one of the fastest growing cities in india. known as the 'silicon valley of india', bangalore is nearly 500 years old. bangalore city corporation (bmp), is a local self government and has the statutory commitment to provide to the citizens of bangalore: good roads, sanitation, street lighting, safe drinking water apart from other social obligations, cultural development and poverty alleviation activities. providing preventive and promotive heahh services is also a specific component. the objective of this study was to review the planning process with respect to health care services in the period since india independence; the specific research questions being what has been the strategies adopted by the city planners to address to the growing needs of the population amidst the background of the different strategies adopted by the country as a whole. three broad rime ranges have been considered for analysis: the 1950s, 1970s and the 1990s. the salient results are: major area of focus has been on the maternal and child care with activities ranging from day-care to in-patient-care; though the number of institutions have grown from 5 to the current day 79, their distribution has been far from satisfactory; obtaining support from the india population projects 3 and 8 major upgradarions have been undertaken in terms of infrastructure; over the years, in addition to the dispensaries of modern system of medicine, local traditional systems have also been initiated; the city has partnered with the healthy cities campaign with mixed success; disease surveillance, addressing the problems related to the emerging non-communicable diseases including mental health and road traffic injuries are still in its infancy. isolated attempts have been made to address the risks groups of elderly care and adolescent care. what stands out remarkably amongst the cities achievements is its ability to elicit participation from ngos, cbos and neighbourhood groups. however, the harnessing of this ability into the health sector cannot be said totally successful. the moot question in all the above observed development are: has the city rationally addressed it planning needs? the progress made so far can be considered as stuttered. the analysis and its presentation would identify the key posirive elements in the growth of banglore city and spell a framework for the new public health. introduction: anaemia associated with pregnancy is a major public health problem all over the world. different studies in different parts of india shown prevalence of anaemia between 60-90%. anaemia remains a serious health problem in pregnancy despite of strong action taken by the government of india through national programmes. in the present study we identified th~ social beha~iors, responsible for low compliance of if a tablets consumption in pregnancy at community level and intervention was given with new modified behaviors on trial bases. . in vadodara urban. 60 anganwadies out of 289 were selected from the list by random sampling for tips (trials of improved practices) study. . . participants: 266 pregnant women (132, intervention group+ 134, control. group) registered m the above 60 anganwadies. study was conducted in to three phases: phase: 1. formative research and baseline survey (frbs). data was collected from all 266 pregnant women to identify behaviors that are responsible for low compliance of ifa tablets. both qualitative and quantitative data were collected. haemoglobin was estimated of all pregnant women by haemo-cue. phase: 2. phase of tips. behaviors were identified both social & clinical for low compliance of ifa tablets consumption in pregnancy from frbs and against those, modified behaviors were proposed to pregnant women in the intervention group on trial bases by health education. trial period of 6 weeks was given for trial of new behaviors to pregnant women in the interven· tion group. phase: 3. in this phase, feedbacks on behaviors tried or not tried were taken from pregnant women in intervention group. haemoglobin estimation was carried out again in all 266 pregnant women. at the end of the study, messages were formulated on the bases of feedbacks from the pregnant women. results: all pregnant women in the intervention group had given positive feedback on new modified behaviors after intervention. mean haemoglobin concentration was higher in intervention group (10.04±0.11 gm%) than control group (9.60±0.14 gm%). ifa tablets compliance was improved in intervention group (95.6%) than control group (78.6%). conclusion: all pregnant women got benefits after trial of new modified behaviors in the intervention group. messages were formulated from the new modified behaviors, which can be used for longterm strategies for anaemia control in the community. introduction: in order to develop a comprehensive mch handbook for pregnant women and to assess its effect among them, a pilot study was carried out at the maternal and child health training institute (mchti), in dhaka, bangladesh. methods: from mchti a sample of 600 pregnant women was selected and all subjects were women who were attending the first visit of their current pregnancy by using a random sampling method. of the 600 subjects, 240 women were given the mch handbook as case and 360 women were not given the handbook as control. data on pre and post intervention of the handbook from the 240 cases and 360 controls were taken from data recording forms between the 1st of november 2002 and 31st of october, 2003 and data was analysed by using a multilevel analysis approach. this was a hospital-based action (case-control) research, and was applied in order to measure the outcome of pre and post intervention following the introduction of the handbook. data was used to assess the effects of utilisation of the handbook on women's knowledge, practice and utilisation of mch services. results: this study showed that the change of knowledge about antenatal care visits was 77.1% among case mothers. knowledge of danger signs improved 49.2 %, breast feeding results 31.5%, vaccination 32.0% and family planning results improved 60.3% among case. results showed some positive changes in women's attitudes among case mothers and study showed the change of practice in antenatal care visits was .u.5% in the case. other notable changes were: change of practice in case mother's tetanus toxoid (ti), 55.2%; and family planning 41.2%. in addition, handbook assessment study indicated that most women brought the handbook on subsequent visits (83.3%), the handbook was highly utilised (i.e. it was read by 84.2%, filled-in by 76.1 %, and was used as a health education tool by 80.4%). most women kept the handbook (99.5%) and found it highly useful (78.0%) with a high client satisfaction rate of 88.0%. conclusion: pregnant women in the case group had higher knowledge, better practices, and higher utilisation of mch services than mothers in the control groups who used alternative health cards. if the handbook is developed with a focus on utilising a problem-oriented approach and involving the recomendations .of end~users, it is anticipated that the mch handbook will contribute significantly to ensuring the quahry of hfe of women and their children in bangladesh. after several meetmgs to identify the needs of the community, a faso clinic was opened at ncfs. health care professionals from smh joined with developmental and social service workers from ncfs to implement the faso diagnostic process and to provide culturally appropriate after-care. the clinic is unique in that its focus is the high risk urban aboriginal population of toronto. it accepts referrals of not only children and youth, but also of adults. lessons learned: response to the faso clinic at native child and family services has been overwhelming. aboriginal children with f asd are receiving timely diagnosis and interventions. aboriginal youth and adults who have been struggling with poveny, substance abuse, and homelessness are more willing to enter the ncfs centre for diagnosis and treatment. aboriginal infants prenatally exposed to alcohol born at st. michael's hospital or referred by other centres have access to the developmental programs located in both of the partnering agencies. the presentation will describe the clinic's development, and will detail the outcomes described, including interventions unique to the aboriginal culture. p7-15 (c) seeds, soil, and stories: an exploration of community gardening in southeast toronto carolin taran, sarah wakefield, jennifer reynolds, and fiona yeudall introduction: community gardens are increasingly seen as a mechanism for improving nutrition and increasing food security in urban neighbourhoods, but the evidence available to support these claims is limited. in order to begin to address this gap in a way that is respectful of community knowledge and needs, the urban gardening research opportunities workgroup (ugrow) project explored the benefits and potential risks of community gardening in southeast toronto. the project used a community-based research (cbr) model to assess community gardens as a means of improving local health. the research process included interviews, focus groups, and participant observation (documented in field notes). we also directly engaged the community in the research process, through co-learning activities and community events which allowed participants to express their views and comment on emerging results. most of the research was conducted by a community-based research associate, herself a community gardener. key results were derived from these various sources through line-by-line coding of interview transcripts and field note review, an interactive and iterative process which involved both academic and community partners. results: these various data sources all suggest that enhanced health and access to fresh produce are important components of the gardening experience. they also highlight the central importance of empowering and community-building aspects of gardening to gardeners. community gardens were thought to play a role in developing friendships and social support, sharing food and other resources, appreciating cultural diversity, learning together, enhancing local place attachment and stewardship, and mobilizing to solve local problems (both inside and outside the garden). potential challenges to community gardens as a mechanism for communiry development include bureaucratic resistance to gardens, insecure land tenure and access, concerns about soil contamination, and a lack of awareness and under· standing by community members and decision-makers of all kinds. conclusion: the results highlight many health and broader social benefits experienced by commu· nity gardeners. they also point to the need for greater support for community gardening programs, par· ticularly ongoing the ongoing provision of resources and education programs to support gardens in their many roles. this research project is supported by the wellesley central health corporation and the centre for urban health initiatives, a cihr funded centre for research development hased at the univer· sity of toronto. p7-16 (c) developing resiliency in children living in disadvantaged neighbourhoods sarah farrell, lorna weigand, and wayne hammond the traditional idea of targeting risk reduction by focusing on the development of eff~ctive coping strategies and educational programs has merit in light of the research reportmg_ that_ ~10lupl.e forms of problem behaviour consistently appear to be predicted by increasing exposure to 1den_uf1able risk factors. as a result, many of the disadvantaged child and youth studies have focused on trymg to better _unde.r· stand the multiple risk factors that increase the likelihood of the development of at nsk behaviour m ch1ldren/youth and the potential implications for prevention. this in turn has led t_o. the conclus1on that community and health programs need to focus on risk reduction by helpm~ md1v1duals develop more effective coping strategies and a better understanding of the limitations of cenam pathologies, problematic v156 poster sessions coping behaviours and risk factors potentially inheren~ in high needs co~unities. ~owever, another ai:ea of research has proposed that preventative interventions should cons1de~ .~rotecnve fa~ors alo~~ with reducing risk factors. as opposed to just emphasizing problems, vulnerab1ht1es, and deficits, a res1liencybased perspective holds the belief that children, youth and their families. have strengths, reso~ce.s and the ability to cope with significant adversity in ways that are not only effective, but tend to result m mcreased ability to constructively respond to future adversity. with this in mind, a participatory research project sponsored by the united way of greater toronto was initiated to evaluate and determine the resiliency profiles of children 8 -12 years (n = 500) of recent immigrant families living in significantly disadvantaged communities in the toronto area. the presentation will provide an overview of the identified protective factors (both intrinsic and extrinsic) and resiliency profiles in an aggregated format as well as a summary of how the children and their parents interpreted and explained these strength-based results. as part of the focus groups, current community programs and services were examined by the participants as to what might be best practices for supporting the development and maintaining of resiliency in children, families and communities. it was proposed that the community model of assessing resiliency and protective factors as well as proposed best strength-based practice could serve as a guide for all in the community sector who provide services and programs to those in disadvantaged neighbourhoods. p7-17 (c) naloxone by prescription in san francisco, ca and new york, ny emalie huriaux the harm reduction coalition's overdose project works to reduce the number of fatal overdoses to zero. located in new york, ny and san francisco, ca, the overdose project provides overdose education for social service providers, single-room occupancy hotel (sro) residents, and syringe exchange participants. the project also conducts an innovative naloxone prescription program, providing naloxone, an opiate antagonist traditionally administered by paramedics to temporarily reverse the effects of opiate overdose, to injection drug users (idus). we will describe how naloxone distribution became a reality in new york and san francisco, how the project works, and our results. the naloxone prescription program utilizes multiple models to reach idus, including sro-and street-based trainings, and office-based trainings at syringe exchange sites. trainings include information on overdose prevention, recognition, and response. a clinician conducts a medical intake with participants and provides them with pre-filled units of naloxone. in new york, funding was initially provided by tides foundation. new york city council provides current funding. new york department of mental health and hygiene provides program oversight. while the new york project was initiated in june 2004, over half the trainings have been since march 2005. in san francisco, california endowment, tides foundation, and san francisco department of public health (sfdph) provide funding. in addition, sfdph purchases naloxone and provides clinicians who conduct medical intakes with participants. trainings have been conducted since november 2003. to date, nearly 1000 individuals have been trained and provided with naloxone. approximately 130 of them have returned for refills and reported that they used naloxone to reverse an opiate-related overdose. limited episodes of adverse effects have been reported, including vomiting, seizure, and "loss of friendship." in new york, 400 individuals have been trained and provided with naloxone. over 30 overdose reversals have been reported. over half of the participants in new york have been trained in the south bronx, the area of new york with the highest rate of overdose fatalities. in san francisco, 570 individuals have been trained and provided with naloxone. over 96 overdose reversals have been reported. the majority of the participants in san francisco have been trained in the tenderloin, 6th street corridor, and mission, areas with the highest rates of overdose fatalities. the experience of the overdose project in both cities indicates that providing idus low-threshold access to naloxone and overdose information is a cost-effective, efficient, and safe intervention to prevent accidental death in this population. p7-18 (c) successful strategies to regulate nuisance liquor stores using community mobilization, law enforcement, city council, merchants and researchers tahra goraya presenta~ion _will discuss ~uccessful environmental and public policy strategies employed in one southen:1 cahf?rmna commumty to remedy problems associated with nuisance liquor stores. participants ~111 be given tools to understand the importance of utilizing various substance abuse prevention str~tegi~ to change local policies and the importance of involving various sectors in the community to a~_1st with and advocate for community-wide policy changes. recent policy successes from the commultles of pa~ad~na and altad~na will highlight the collaborative process by which the community mobilized resulnng m several ordmances, how local law enforcement was given more authority to monitor poster sessions v157 nonconforming liquor stores, how collaborative efforts with liquor store owners helped to remove high alcohol content alcohol products from their establishments and how a community-based organiz,uion worked with local legislators to introduce statewide legislation regarding the regulation of nuisance liquor outlets. p7-19 (c) "dialogue on sex and life": a reliable health promotion tool among street-involved youth beth hayhoe and tracey methven introduction: street involved youth are a marginalized population that participate in extremely risky behaviours and have multiple health issues. unfortunately, because of previous abuses and negative experiences, they also have an extreme distrust of the adults who could help them. in 1999, toronto public health granted funding to a non governmental, nor for profit drop-in centre for street youth aged 16-24, to educate them about how to decrease rhe risk of acquiring hiv. since then the funding has been renewed yearly and the program has evolved as needed in order to target the maximum number of youth and provide them with vital information in a candid and enjoyable atmosphere. methods: using a retrospective analysis of the six years of data gathered from the "dialogue on sex and life" program, the researchers examined the number of youth involved, the kinds of things discussed, and the number of youth trained as peer leaders. also reviewed, was written feedback from the weekly logs, and anecdotal outcomes noted by the facilitators and other staff in the organization. results: over the five year period of this program, many of youth have participated in one hour sessions of candid discussion regarding a wide range of topics including sexual health, drug use, harm reduction, relationship issues, parenting, street culture, safety and life skills. many were new youth who had not participated in the program before and were often new to the street. some of the youth were given specific training regarding facilitation skills, sexual anatomy and physiology, birth control, sexually transmitted infections, hiv, substance use/abuse, harm reduction, relationships and discussion of their next steps/future plans following completion of the training. feedback has been overwhelmingly positive and stories of life changing decisions have been reported. conclusion: clearly, this program is a successful tool to reach street involved youth who may otherwise be wary of adults and their beliefs. based on data from the evaluation, recommendations have been made to public health to expand the funding and the training for peer leaders in order ro target between 100-200 new youth per year, increase the total numbers of youth reached and to increase the level of knowledge among the peer leaders. p7-20 (c) access to identification and services jane kali replacing identification has become increasingly more complex as rhe government identification issuing offices introduce new requirements rhar create significant barriers for homeless people to replace their id. new forms of identification have also been introduced that art' not accessible to homekss peoplt-(e.g. the permanent resident card). ar rhe same time, many service providers continue to require identifi· cation ro access supports such as income, housing, food, health care, employment and employmt·nt training programs. street health, as well as a number of other agencies and community health centres, h,1, been assisting with identification replacement for homeless peoplt· for a number of years. the rnrrt·nr challenges inherent within new replacement requirements, as well as the introduction of new forn1' of identification, have resulted in further barriers homeless people encounter when rrring to access t:ssential services. street health has been highlighting these issues to government identification issuing offices, as well as policy makers, in an effort to ensure rhar people who are homeless and marginalized have ac'ess to needed essential services. bandar is a somali word for •·a safe place." the bandar research project is the product of the regent park community health centre. the research looks ar the increasing number of somali and afri· can men in the homeless and precariously house population in the inner city core of down~own toronto. in the first phase of the pilot project, a needs assessment was conducted to 1dennfy barners and issues faced by rhe somali and other african men who are homeless and have add1cr10ns issues. th_e second phase of rhe research project was to identify long rerm resources and service delivery mechamsms that v158 poster sessions would enhance the abiity of this population to better access detox, treatment, and post treatment ser· vices. the final phase of the project was to facilitate the development of a conceptual model of seamless continual services and supports from the streets to detox to treatment to long term rehabilitation to housing. "between the pestle and mortar" -safe place. p7-22 (c) successful methods for studying transient populations while improving public health beth hayhoe, ruth ewert, eileen mcmahon, and dan jang introduction: street youth are a group that do not regularly access healthcare because of their mis· trust of adults. when they do access health care, it is usually for issues severe enough for hospitalization or for episodic care in community clinics. health promotion and illness prevention is rarely a part of their thinking. thus, standard public health measures implemented in a more stable population do not work in this group. for example, pap tests, which have dearly been shown to decrease prevalence of cer· vical cancer, are rarely done and when they are, rarely followed up. methods to meet the health care needs and increase the health of this population are frequently being sought. methods: a drop-in centre for street youth in canada has participated in several studies investigating sexual health in both men and women. we required the sponsoring agencies to pay the youth for their rime, even though the testing they were undergoing was necessary according to public health stan· dards. we surmised that this would increase both initial participation and return. results: many results requiring intervention have been detected. given the transient nature of this population, return rates have been encouraging so far. conclusion: it seems evident that even a small incentive for this population increases participation in needed health examinations and studies. it is possible that matching the initial and follow-up incentives would increase the return rate even further. the fact that the youth were recruited on site, and not from any external advertising, indicates that studies done where youth trust the staff, are more likely to be successful. the presentation will share the results of the "empowering stroke prevention project" which incor· porated self-help mutual aids strategies as a health promotion methodology. the presentation will include project's theoretical basis, methodology, outcomes and evaluation results. self-help methodology has proven successful in consumer involvement and behaviour modification in "at risk," "marginalized" settings. self-help is a process of learning with and from each other which provides participants oppor· tunities for support in dealing with a problem, issue, condition or need. self-help groups are mechanisms for the participants to investigate existing solutions and discover alternatives, empowering themselves in this process. learning dynamic in self-help groups is similar to that of cooperative learning and peertraining, has proven successful, effective and efficient (haller et al, 2000) . the mutual support provided by participation in these groups is documented as contributory factor in the improved health of those involved. cognizant of the above theoretical basis, in 2004 the self-help resource centre initiated the "empowering stroke prevention project." the project was implemented after the input from 32 health organizations, a scan of more than 300 resources and an in-depth analysis of 52 risk-factor-specific stroke prevention materials indicated the need for such a program. the project objectives were:• to develop a holistic and empowering health promotion model for stroke prevention that incorporates selfhelp and peer support strategies. • to develop educational materials that place modifiable risk factors and lifestyle information in a relevant context that validates project participants' life experiences and perspectives.• to educate members of at-risk communities about the modifiable risk factors associated with stroke, and promote healthy living. to achieve the above, a diverse group of community members were engaged as "co-editors" in the development of stroke prevention education materials which reflected and validated their life experiences. these community members received training to become lay health promoters (trained volunteer peer facilitators). in collaboration with local health organizations, these trained lay health promoters were then supported in organizing their own community-based stroke prevention activities. in addition, an educational booklet written in plain language, entitled healthy ways to prevent stroke: a guide for you, and a companion guide called healthy ways to pre· vent stroke: a facilitator's guide were produced. the presentation will include the results of a tw<>tiered evaluation of the program methodology, educational materials and the use of the materials beyond the life of the project. this poster presentation will focus on the development and structure of an innovative street outreach service that assists individuals who struggle mental illness/addictions and are experiencing homelessness. the mental health/outreach team at public health and community services (phcs) of hamilton, ontario assists individuals in reconnecting with health and social services. each worker brings to the ream his or her own skills-set, rendering it extremely effective at addressing the multidimensional and complex needs of clients. using a capacity building framework, each ream member is employed under a service contract between public health and community services and a local grassroots agency. there are public health nurses (phn), two of whom run a street health centre and one of canada's oldest and most successful needle exchange programs, mental health workers, housing specialists, a harm reduction worker, youth workers, and a united church minister, to name a few. a community advisory board, composed of consumers and professionals, advises the program quarterly. the program is featured on raising the roors 'shared learnings on homelessness' website at www.sharedlearnings.ca. through our poster presentation participants will learn how to create effective partnerships between government and grassroots agencies using a capacity building model that builds on existing programs. this study aims to assess the effects of broadcasting a series of documentary and drama videos, intended to provide information about the bc healthguide program in farsi, on the awareness about and the patterns of the service usage among farsi-speaking communities in the greater vancouver area. the major goals of the present study were twofold; ( 1) to compare two methods of communications (direct vs. indirect messages) on the attitudes and perceptions of the viewers regarding the credibility of messengers and the relevance of the information provided in the videos, and (2) to compare and contrast the impact of providing health information (i.e., the produced videos) via local tvs with the same materials when presented in group sessions (using vcr) on participants' attitudes and perceptions cowards the bc healrhguide services. results: through a telephone survey, 545 farsi-speaking adults were interviewed in november and december 2004. the preliminary findings show that 53% of the participants had seen the aired videos, from which, 51 % watched at least one of the 'drama' clips, 8% watched only 'documentary' clip, and 41% watched both types of video. in addition, 27% of the respondents claimed that they were aware about the program before watching the aired videos, while 73% said they leaned about the services only after watching the videos. from this group, 14% said they called the bchg for their own or their "hildren's health problems in the past month. 86% also indicated that they would use the services in the future whenever it would be needed. 48% considered the videos as "very good" and thought they rnuld deliver relevant messages and 21 % expressed their wish to increase the variety of subjects (produ\:e more videos) and increase the frequency of video dips. conclusion: the results of this study will assist public health specialists in bc who want to choose the best medium for disseminating information and apply communication interventions in multi\:ultural communities. introduction: many theorists and practitioners in community-based research (cbr) and knowledge transfer (kt) strongly advocate for involvement of potential users of research in the development of research projects, yet few examples of such involvement exist for urban workplace health interventions. we describe the process of developing a collaborative research program. methods: four different sets of stakeholders were identified as potential contributors to and users of the research: workplace health policy makers, employers, trade unions, and health and safety associations. representatives of these stakeholders formed an advisory committee which met quarterly. over the 13 month research development period, an additional 21 meetings were held between resc:ar~h~rs and stakeholders. in keeping with participant observation approaches, field notes of group and md1v1~ ual meetings were kept by the two co-authors. emails and telephone calls were also documented. qu~h tative approaches to textual analysis were used, with particular attention paid to collaborattve v160 poster sessions relationships established (as per cbr), indicators of stakeholders' knowledge utilization (as per kt), and transformations of the proposed research (as per cbr). results: despite initial strong differences of opinion both among stakeho~ders .an~ between stakeholders and researchers, goodwill was noted among all involved. acts of rec~proc1ty included mu.rual sharing of assessment tools, guidance on data utilization to stakeho~der orga~1zat10ns, and suggestions on workplace recruitment to researchers. stakeholders demonstrated mcreases m concep~ual. un~erstand ing of workplace health e.g. they more commonly discussed more complex,. psychosocial md1cators of organizational health. stakeholders made instrumental use of shared materials based on research e.g. adapting their consulting model to more sophisticated dat~ analysis. sta~ehol?~rs recogni_zed the strategic use of their alliance with researchers e.g., transformational leadership trainmg as a~ inducement to improve health and safety among small service franchises. stakeholders helped re-define the research questions, dramatically changed the method of recruitment from researcher cold call to stakeholderbased recruitment, and strongly influenced pilot research designs. owing a great deal to the elaborate joint development process, the four collaboratively developed pilot project submissions which were all successfully funded. conclusion: the intensive process of collaborative development of a research program among stakeholders and researchers was not a smooth process and was time consuming. nevertheless, the result of the collaborative process was a set of projects that were more responsive to stakeholder needs, more feasible for implementation, and more broadly applicable to relevant workplace health problems. introduction: environmental groups, municipal public health authorities and, increasingly, the general public are advocating for reductions in pesticide use in urban areas, primarily because of concern around potential adverse health impacts in vulnerable populations. however, limited evidence of the relative merits of different intervention strategies in different contexts exists. in a pilot research project, we sought to explore the options for evaluating pesticide reduction interventions across ontario municipalities. methods: the project team and a multi-stakeholder project advisory committee (pac), generated a list of potential key informants (kl) and an open ended interview guide. thirteen ki from municipal government, industry, health care, and environmental organizations completed face to face or telephone interviews lasting 30-40 minutes. in a parallel process, a workshop involving similar representatives and health researchers was held to discuss the role of pesticide exposure monitoring. minutes from pac meetings, field notes taken during ki interviews, and workshop proceedings were synthesized to generate potential evaluation methods and indicators. results: current evaluation activities were limited but all kls supported greater evaluation effons beginning with fuller indicator monitoring. indicators of education and outreach services were imponant for industry representatives changing applicator practices as well as most public health units and environmental organizations. lndictors based on bylaw enforcement were only applicable in the two cities with bylaws, though changing attitudes toward legal approaches were being assessed in many communities. the public health rapid risk factor surveillance system could use historical baseline data to assess changes in community behaviour through reported pesticide uses and practices, though it had limited penetration in immigrant communities not comfortable in english. pesticide sales (economic) data were only available in regional aggregates not useful for city specific change documentation. testing for watercourse or environmental contamination might be helpful, but it is sporadic and expensive. human exposure monitoring was fraught with ethical issues, floor effects from low levels of exposure, and prohibitive costs. clinical episodes of pesticide exposure reported to the regional poison centre (all ages) or the mother risk program (pregnant or breastfeeding women) are likely substantial underestimates that would be need to be supplemented with sentinel practice surveillance. focus on special clinical populations e.g., multiple chemical sensitivity would require additional data collection efforts . . conc~ons: broad support for evaluation and multiple indicators were proposed, though con-s~raints associate~ with access, coverage, sensitivity and feasibility were all raised, demonstrating the difficulty of evaluating such urban primary prevention initiatives. interventionists. an important aim of the youth monitor is to learn more about the health development of children and adolescents and the factors that can influence this development. special attention is paid to emo· tional and behavioural problems. the youth monitor identifies high-risk groups and factors that are associated with health problems. at various stages, the youth monitor chancrs the course of life of a child. the sources of informa· tion and methods of research are different for each age group. the results arc used to generate various kinds of repons: for children and young persons, parents, schools, neighbourhoods, boroughs and the municipality of rotterdam and its environs. any problems can be spotted early, at borough and neigh· bourhood level, based on the type of school or among the young persons and children themselves. together with schools, parents, youngsters and various organisations in the area, the municipal health service aims to really address these problems. on request, an overview is offered of potentially suitable interventions. the authors will present the philosophy, working method, preliminary effects and future developments of this instrument, which serves as the backbone for the rotterdam local youth policy. social workers to be leaders in response to aging urban populations: the practicum partnership program sarah sisco, alissa yarkony, and patricia volland 1"'"1tliu:tion: across the us, 77.5% of those over 65 live in urban areas. these aging urban popu· lations, including the baby boomers, have already begun encounter a range of heahh and mental hcahh conditions. to compound these effects, health and social service delivery fluciuates in cities, whit:h arc increasingly diverse both in their recipients and their systems. common to other disciplines (medicine, nursing, psychology, etc.) the social work profession faces a shortage of workers who are well-equipped to navigate the many systems, services, and requisite care that this vast population requires. in the next two decades, it is projected that nearly 70,000 social workers will be required to provide suppon to our older urban populations. social workers must be prepared to be aging-savvy leaders in their field, whether they specialize in gerontology or work across the life span. mllhotu: in 2000, a study conducted at the new york academy of medicine d<>1:umcntcd the need for improved synchroniciry in two aspects of social work education, classroom instruction and the field experience. with suppon from the john a. hanford foundation, our team created a pilot proj~"t entitled the practicum pannership program (ppp) in 11 master's level schools of social work, to improvt" aginr exposure in field and classroom content through use of the following: i) community-university partnrr· ships, 2) increased, diverse student field rotations, ll infusion of competcn1."}'·drivm coursework, 41 enhancement of field instructors' roles, and 5) concentrated student recruitment. we conductt"d a prr· and post-test survey into students' knowledge, skills. and satisfaction. icarlja: surveys of over 400 graduates and field inltnk."tors rcflected increased numlk-n of .1rrm:y· univmity panncrships, as well as in students placed in aging agencin for field placements. there wa1 11 marked increase in student commitments to an aging specialization. onr year por.t·gradu:nion rcvealrd that 93% of those surveyed were gainfully employed, with 80% employed in the field of aginic. by com· bining curricular enhancement with real-world experiences the ppp instilled a broad exposurr for llu· dents who worked with aging populations in multiple urban settings. coltdtuion: increased exposure to a range of levels of practicr, including clinical, policy/ajvocaq, and community-based can potentially improve service delivery for older adulh who live in elfin, and potentially improve national policy. the hanford foundation has now elected to 1uppon cxpantion of the ppp to 60 schools nationwide (urban and rural) to complement other domntic initiatives to cnhalk"c" holistic services for older adults across the aging spectrum. bodrgnn.ntl: we arc a team of rcscarcbcn and community panncn working tcj8c(her to develop an in"itepth understanding of the mental health needs of homeless youth ~ages 16 to 24) (using qualiutivc and quantitative methods 8' panicipatory rncarch methods). it is readily apparmt that '-neless youth cxpcricnce a range of mental health problems. for youth living on the street, menul illnew may be either a major risk factor for homelessnal or may frequently emcsge in response to coping with rhe multitudinous stressors associated with homclcslllcsi including exposure to violence, prasutt to pamaplte in v162 poster sessions survival sex and/or drug use. the most frequent psychiatric diagnoses amongst the homeless gencrally include: depression, anxiety and psychosis. . . . the ultimate ob1ective of the pr~am of rei:e~ is to ~evelop a plan for intervention to meet the mental health needs of street youth. prior t_o pl~nnmg mtervenbons, .it is necessary to undertake a comprehensive assessment ~f mental health needs m this ~lnerable populanon. thus, the immediate objective of this research study is to undertake a comprehensive assessment of men· tal health needs. . . melbotlology: a mixed methodology triangulating qualitative, participatory acnon and quantitative methods will capture the data related to mental health needs of homeless youth. a purposive sample of approximately 60-80 subjecrs. ages 16 to 24, is currently being ~ted ~participate from the commu.nity agencies covenant house, evergreen centre fo~ srrc;et youth, turning p?1?t and street ~ serv~. youth living on the street or in short -term residennal programs for a mmimum of 1 month pnor to their participation; ages 16 to 24 and able to give infonned consent will be invited to participate in the study. o..tcomes: the expected outcome of this initial survey will be an increased understanding of mental health needs of street youth that will be used to develop effective interventions. it is anticipated that results from this study will contribute to the development of mental health policy, as well as future programs that are relevant to the mental health needs of street youth. note: it is anticipated that preliminary quantitative data (25 subjects) and qualitative data will be available for the conference. the authors intend to present the identification of the research focus, the formation of our community-based team, relevance for policy, as well as preliminary results. p7-31 (a) the need for developing a firm health policy for urban informal worken: the case of despite their critical role in producing food for urban in kenya, urban farmers have largely been ignored by government planners and policymakers. their activity is at best dismissed as peripheral eveo, inappropriate retention of peasant culture in cities and at worst illegal and often some-times criminal· ized. urban agriculture is also condemned for its presumed negative health impact. a myth that contin· ues despite proof to the contrary is that malarial mosquitoes breed in maize grown in east african towns. however, potential health risks are insignificant compared with the benefits of urban food production. recent studies too rightly do point to the commercial value of food produced in the urban area while underscoring the importance of urban farming as a survival strategy among the urban poor, especially women-headed households. since the millennium declaration, health has emerged as one of the most serious casualties consequent on the poverty, social exclusion, marginalisation and lack of sustain· able development in africa. hiv/aids epidemic poses an unprecedented challenge, while malaria, tuber· culosis, communicable diseases of childhood all add to the untenable burden. malnutrition underpins much ill-health and is linked to more than 50 per cent of all childhood deaths. kenya's urban poor people ~ace ~ h~ge burde~ of preventable and treatable health problems, measured by any social and bi~ medical md1cator, which not only cause unnecessary death and suffering, but also undermine econonuc development and damage the country's social fabric. the burden is in spite of the availability of suitable tools and re:c=hnology for prevention and treatment and is largely rooted in poverty and in weak healah •rstems. this pa~ therefore challenges development planners who perceive a dichotomy instead of con· tmuum between informal and formal urban wage earners in so far as access to health services is con· cemed. it i~ this gap that calls for a need to developing and building sustainable health systems among the urban mformal ~wellers. we recommend a focus on an urban health policy that can build and strengthen the capacity of urban dwellers to access health services that is cost-effective and sustainable. such ~ health poli<=>: must strive for equity for the urban poor, displaced or marginalized; mobilise and effect1~ely use sufficient sustainable resources in order to build secure health systems and services. special anenti_on. should ~ afforded hiv/aids in view of the unprecedented challenge that this epidemic poses to africa s economic and social development and to health services on the continent. methods: a review of the literature led us to construct three simple models and a composite model of exposure to traffic. the data were collected with the help of a daily diary of travel activities using a sample of cyclists who went to or come back from work or study. to calculate the distance, the length of journey, and the number of intersections crossed by a cyclist different geographic information systems (gis) were operated. statistical analysis was used to determine the significance between a measure of exposure on the one hand, and the sociodemographic characteristics of the panicipants or their geographic location on the other hand. restlltj: our results indicate that cyclists were significantly exposed to road accidents, no matter of where they live or what are their sociodemographic characteristics. we also stress the point that the fact of having been involved in a road accident was significantly related to the helmet use, but did not reduce the propensity of the cyclists to expose themselves to the road hazards. condlllion: the efforts of the various authorities as regards road safety should not be directed towards the reduction of the exposure of the vulnerable users, but rather towards the reduction of the dangers to which they could face. keywords: cyclist, daily diary of activities, measures of exposure to traffic, island of montreal. p7-33 (a) intra urban disparities and environmental health: some salient features of nigerian residential neighbourhoods olumuyiwa akinbamijo intra urban disparities and environmental health: some salient features of nigerian residential neighbourhoods abstract urbanization panicularly in nigerian cities, ponends unprecedented crises of grave dimensions. from physical and demographic viewpoints, city growth rates are staggering coupled with gross inabilities to cope with the consequences. environmental and social ills associated with unguarded rapid urbanization characterize nigerian cities and threaten urban existence. this paper repons the findings of a recent study of the relationship between environmental health across inrraurban residential communities of akure, south west nigeria. it discuses the typical urbanization process of nigerian cities and its dynamic spatial-temporal characteristics. physical and socio-demographic attributes as well as the levels and effectiveness of urban infrastructural services are examined across the core residential districts and the elite residential layouts in the town. the incidence rate of cenain environmentally induced tropical diseases across residential neighborhoods and communes is examined. salient environmental variables that are germane to health procurement in the residential districts, incidence of diseases and diseases parasitology, diseases prevention and control were studied. field data were subjected to analysis ranging from the univariate and bivariate analysis. inferential statistics using the chi-square test were done to establish the truthfulness of the guiding hypothesis. given the above, the study affirms that there is strong independence in the studied communities, between the environment and incidence of diseases hence health of residents of the town. this assertion, tested statistically at the district levels revealed that residents of the core districts have very strong independence between the environment and incidences of diseases. the strength of this relationship however thins out towards the city peripheral districts. the study therefore concludes that since most of the city dwellers live in urban deprivation, urban health sensitive policies must be evolved. this is to cater for the urban dwellers who occupy fringe peripheral sites where the extension of facilities often times are illegally done. urban infrastructural facilities and services need be provided as a matter of public good for which there is no exclusive consumption or access even for the poorest of the urban poor. many suffer from low-self esteem, shame and guilt about their drug use. in addition, they often lack suppon or encounter opposition from their panners, family and friends in seeking treatment. these personal barriers are compounded by fragmented addiction, prenatal and social care services, inflexible intake systems and poor communication among sectors. the experience of accessing adequate care between services can be overwhelming and too demanding. the toronto centre for substance use in pregnancy (t-cup) is a unique program developed to minimize barriers by providing kone-stop" comprehensive healthcare. t-cup is a primary care based program located in the department of family medicine at st. joseph\'s health centre, a community teaching hospital in toronto. the interdisciplinary staff provides prenatal and addiction services, case management, as well as care of newborns affected by substance use. regular care plan meetings are held between t-cup, labour and delivery nurses and social workers in the y164 poster sessions maternity and child care program. t-cup also connects "'.omen with. inpatient treatment programs and community agencies such as breaking the cycle, an on-site counselmg group for pregnant substance users. · f · d d h ith method: retrospective chart review, qualitative patient ~ans action stu ~· an ea care provider surveys are used to determine outcomes. primary outcomes mclude changes m maternal su~tance use, psychosocial status and obstetrical complications (e.g. pre-rupture of membrane, pre-eclampsia, placen· ral abruption and hemorrhage). neonatal measures ~~nsisted of .bir~h pa_rame~ers, length of h~spital st.ay and complications (e.g. feral distress, meconium stammg, resuscitation, 1aund1ce, hypoglycemia, seventy of withdrawal and treatment length). chart review consisted of all t-cup patients who met clinical cri· reria for alcohol or drug dependence and received prenatal and intra-partum care at st. joseph's from october 2003 to june 2005. participants in the qualitative study included former and current t-cup patients. provider surveys were distributed on-site and to a local community hospital. raulb: preliminary evaluation has demonstrated positive results. treatment retention and satisfaction rates were high, maternal substance use was markedly reduced and neonatal outcomes have shown to be above those reported in literature. conclusion: this comprehensive, primary care model has shown to be optimal in the management of substance use in pregnancy and for improving neonatal outcomes. future research will focus on how this inexpensive program can be replicated in other health care settings. t-cup may prove to be the optimal model for providing care to pregnant substance users in canada. lntrod11ction: cigarette smoking is one of the most serious health problems in taiwan. the prevalence of smoking in 2002 is 48.1 % in males 5.9% in females aged 18 years and older. although the government of taiwan passed a tobacco hazards control act in 1997, it has not been strongly enforced in many places. therefore, community residents have often reported exposure of second hand smoke. the purpose of the study was to establish a device to build up more smoke-free environments in the city of tainan. methods: unique from traditional intervention studies, the study used a healthy city approach to help build up smoke-free environments. the major concept of the approach is to build up a healthy city platform, including organizing a steering committee, setting up policies and indicators, creating intersectoral collaboration, and increasing community participation. first, more than 80 enthusiastic researchers, experts, governmental officers, city counselors and community leaders in tainan were invited in the healthy city committee. second, smoke-free policies, indicators for smoke-free environments, and mechanisms for inter-departmen· tal inspections were set up. third, community volunteers were recruited and trained for persuading related stakeholders. lastly, both penalties and rewards were used for help build up the environments. raults: aher two-year (2003 aher two-year ( -2005 execution of the project, the results qualitatively showed that smoke-free environments in tainan were widely accepted and established, including smoke-free schools, smoke-free workpla~es, smoke-free households, smoke-free internet shops, and smoke-free restaurants. smoke~s were. effectively educated not to smoke in public places. community residents including adults and children m the smoke-free communities clearly understand the adverse effects of environmental tobacco smoke and actively participated anti-smoking activities. conclruions: healthy city platform is effective to conquer the barrier of limited anti-smoking rc:sources. nor. only can it enlar:ge community actions for anti-smoking campaigns, but also it can provide par_merships for collaboratjon. by establishing related policies and indicators the effects of smoke· free environments can be susta1·ned a d th · · · ' · n e progression can be monitored m a commuruty. these issues are used ~· oi::c it~ goals, weuha identifies issues that put people's health at risk. presently, team com~u:c: ran ee~tion !earns. (iats) that design integrative solutions ~tesj'°~ g om six to fifteen members. methods in order to establish wo-poster sessions v165 projects for weuha, the following approach was undertaken: i. a project-polling template was created and sent to all members of the alliance for their input. each member was asked to identify thdr top two population groups, and to suggest a project on which to focus over a 12-18 month period for each identified population. 2. there was a 47% response to the poll and the top three population groups were identified. data from the toronto community health profile database were utilized to contextualize the information supplied for these populations. a presentation was made to the steering committee and three population-based projects were selected, leaders identified and iats formed. three population-based projects: the population-based projects and health care issues identified are: newcomer prenatal uninsured women; this project will address the challenges faced by providers to a growing number of non-insured prenatal women seeking care. a service model where the barrier of "catchments" is removed to allow enhanced access and improved and co-ordinated service delivery will be pilot-tested. children/obesity/diab etes: using a health promotion model this team will focus on screening, intervention, and promoting healthy lifestyles (physical activity and nutrition) for families as well as for overweight and obese children. seniors health promotion and circle of discharge: this team will develop an early intervention model to assist seniors/family unit/caregivers in accessing information and receiving treatment/care in the community. the circle of discharge initiative will address ways of utilizing community supports to keep seniors in the community and minimize readmissions to acute care facilities. results/expected outcomes: coordinated and enhanced service delivery to identified populations, leading to improved access, improved quality of life, and health care for these targeted populations. introduction: basic human rights are often denied to high-risk populations and people living with hiv/aids. their rights to work and social security, health, privacy, non discrimination, liberty and freedom of movement, marriage and having a family have been compromised due to their sero-positive status and risk of being positive. the spread of hiv/aids has been accelerating due to the lack of general human rights among vulnerable groups. to formulate and implement effective responses needs dialogue and to prevent the epidemic to go underground barriers like stigma need to be overcome. objective: how to reduce the situation of stigma, discrimination and human rights violations experienced by people living with hiv/aids and those who are vulnerable to hiv/aids. methodology and findings: consultation meetings were strm.-rured around presentations, field visits, community meetings and group work to formulate recommendations on how govt and ngos/cbos should move forward based on objective. pakistan being a low prevalence country, the whole sense of compl;u:enc.:y that individuals are not subject to situations of vulnerable to hiv is the major threat to an explosion in th•· epidemic, therefore urgent measures are needed to integrate human rights issues from the very start of the response. the protection and promotion of human rights in an integral component of ;tll responses to the hiv/aids epidemic. it has been recognized that the response to hiv/aios must he multi sectoral and multi faceted, with each group contributing its particular expertise. for this to occur along with other knowlcdg<" more information is required in human rights abuses related to hiv/ aids in a particular scenario. the ~·on sultarion meetings on hiv/aids and human rights were an exemplary effort to achieve the same ohj<..:tivc. recommendations: the need for a comprehensive, integrated and a multi-sectoral appro;u.:h in addressing the issue of hiv/aids was highlighted. the need social, cultural and religious asp•·ct' to he: prominently addressed were identified. it was thought imperative measures even in low prevalence countries. education has a key role to play, there is a need for a code of ethics for media people and h<"alth care providers and violations should be closely monitored and follow up action taken. p7-38 (c) how can community-based funding programs contribute to building community capacity and how can we measure this elusive goal? mary frances maclellan-wright, brenda cantin, mary jane buchanan, and tammy simpson community capacity building is recognized by the public health agency of canada (phac) as an important strategy for improving the overall health of communities by enabling communities to addre~s priority issues such as social and economic determinants of health. in 2004/2005 phac.:, alberta/nwf region's population health fund (phf) supported 12 community-based projects to build community capacity on or across the determinants of health. specifically, this included creating accessible and sup· portive social and physical environments as well as creating tools and processes necessary for healthy policy development and implementation. the objective of this presentation is to highlight how the community capacity building tool, developed by phac ab/nwf region, can demonstrate gains in v166 poster sessions · · the course of a pror· ect and be used as a reflective tool for project planning and community capacity over . . . . i · a art of their reporting requirements, 12 pro1ect sites completed the community caparny eva uanon. s p . . th t i ii i'd d . building tool at the beginning and end of their ~ne-year prorect. e oo ~o ects va 1 an reliable data in the context of community-based health prorects. developed through a vigorous ~nd collabora11ve research process, the tool uses plain languag~ to expl~re nine key f~atures o~ commuruty cap~city with 35 't ch with a section for contextual information, 26 of which also mdude a four-pomt raong 1 ems, ea f fu d · scale. results show an increase in community capacity over the course o the nde prorects. pre and post aggregate data from the one-year projects measure~ statistic.ally si~n~ficant changes for 17 of the 26 scaled items. projects identified key areas of commumty capacity bmldmg that needed strengthemng, such as increasing participation, particularly among people with low incomes; engaging community members in identifying root causes; and linking with community groups. in completing the tool, projects examined root causes of the social and economic determinants of health, thereby exploring social justice issues related to the health of their community. results of the tool also served as a reflec· cion on the process of community capacity building; that is, how the project outcomes were achieved. projects also reported that the tool helped identify gaps and future directions, and was useful as a project planning, needs assessment and evaluation tool. community capacity building is a strategy that can be measured. the community capacity building tool provides a practical means to demonstrate gains in community capacity building. strengthening the elements of community capacity building through community-based funding can serve as building blocks for addressing other community issues. needs of marginalized crack users lorraine barnaby, victoria okazawa, barb panter, alan simpson, and bo yee thom background: the safer crack use coalition of toronto (scuc) was formed in 2000 in response to the growing concern for the health and well-being of marginalized crack users. a central concern was the alarm· ing hepatitis c rate ( 40%) amongst crack smokers and the lack of connection to prevention and health ser· vices. scuc is an innovative grassroots coalition comprised of front-line workers, crack users, researcher! and advocates. despite opposition and without funding, scuc has grown into the largest crack specific harm reduction coalition in canada and developed a nationally recognized sarer crack kit distribution program (involving 16 community-based agencies that provide outreach to users). the success of our coalition derives from our dedication to the issue and from the involvement of those directly affected by crack use. setting: scuc's primary service region is greater toronto, a diverse, large urban centre. much ofour work is done in areas where homeless people, sex trade workers and drug users tend to congregate. recently, scuc has reached out to regional and national stakeholders to provide leadership and education. mandate: our mandate is to advocate for marginalized crack users and support the devdopmentof a com.p.rehensive harm reduction model that addresses the health and social needs facing crack users; and to fac1htare the exchange of information between crack users, service providers, researchers, and policy developers across canada. owrview: the proposed workshop will provide participants with an overview of the devdopment of scuc, our current projects (including research, education, direct intervention and consultation), our challenge~ and s~ccesses and the role of community development and advocacy within the coalition. pre-senter~ will consist of community members who have personal crack use experience and front-line work· ers-, sc.uc conducted a community-based research project (toronto crack users perspectives, 2005) , in w~ich 1 s focus groups with marginalized crack users across toronto were conducted. participants iden· t1f1ed health and social issues affecti h b · · · d " red . . ng t em, arrsers to needed services, personal strategies, an oue recommendations for improved services. presenters will share the methodology, results and recommen· datmns resulting from the research project. conc/usio": research, field observations and consultations with stakeholders have shown that cradck shmoke~s are at an. increased risk for sexually transmitted infections hiv/aids hepatitis c, tb an ot er serious health issues health · ff, · ' ' · · . · issues a ectmg crack users are due to high risk behavmurs, socio· economic factors, such as homeless d. · · · · d · 1 . 1 . ness, 1scrsmmat1on, unemployment, violence incarceraoons, an soc1a 1so at1on, and a lack of comprehe · h i h · ' ns1ve ea t and social services targeting crack users. · · sinct · s, owever arge remains a gross underesurnaoon. poster sessions v167 these are hospital-based reports and many known cases go unreported. however teh case, young age at first intercourse, inconsistent condom use and multiple partnersplace adolescents at high risks for a diverse array of stls, including hiv. about 19% of female nigerian secondary school students report initiating sexual intercourse before age 13 years. 39% of nigerian female secondary school students report not using a condom the last time they had sexual intercourse. more than 60% of urban nigerian teens report inconsistent condom use. methods: 371 adolescents were studied, ages 12 to 19, from benin city in edo state. the models used were mother-daughter(119), mother -son(99), father -son (87), and father-daughter(66). the effect of parent-child sexual communicationat baseline on child\'s report of sexual behavior, 6 to 12 months later were studied. greater amounts of sexual risk communication were asociated with markedly fewer episodes of unprotected sexual intercourse, reduced number of sexual partners and fewer episodes of unprotected sexual intercourse. results: this study proved that parents can exert more influence on the sexual knowledge attitudes and practise of their adolescent children through desired practises or rolemodeling, reiterating their values and appropriate monitoring of the adolescents\' behavior. they also stand to provide information about sexuality and various sexual topics. parental-child sexual communication has been found to be particularly influential and has been associated with later onset of sexual initiation among adolescents, less sexual activity, more responsible sexual attitudes including greater condom use, self efficacy and lower self -reported incidence of stis. conclusions: parents need to be trained to relate more effectively with their children/wards about issues related to sex and sexuality. family -based programs to reduce sexual risk-taking need to be developed. there is also the need to carry out cross-ethnicaland cross-cultural studies to identify how parent-child influences on adolescent sexual risk behavior may vary in different regions or countries, especially inthis era of the hiv pandemic. introduction: public health interventions to identify and eliminate health disparities require evidence-based policy and adequate model specification, which includes individuals within a socioecological context, and requires the integration of biosociomedical information. multiple public and private data sources need to be linked to apportion variation in health disparities ro individual risk factors, the health delivery system, and the geosocial environment. multilevel mapping of health disparities furthers the development of evidence-based interventions through the growth of the public health information network (phin-cdc) by linking clinical and population health data. clinical encounter data, administrative hospital data, population socioenvironmental data, and local health policy were examined in a three-level geocoded multilevel model to establish a tracking system for health disparities. nj has a long established political tradition of "home rule" based in 566 elected municipal governments, which are responsible for the well-being of their populations. municipalities are contained within counties as defined by the us census, and health data are linked mostly at the municipality level. marika schwandt community organizers from the ontario coaliti~n again~t pove~, .along ":ith ~edical practitioners who have endorsed the campaign and have been mvolved m prescnbmg special diet needs for ow and odsp recipients, will discuss the raise the rates campaign. the organizati~n has used a special diet needs supplement as a political tool, meeting the urgent needs o.f .poor ~ople m toront~ while raising the issues of poverty as a primary determinant of health and nutrtnous diet as a preventative health mea· sure. health professionals carry the responsibility to ensure that they use all means available to them to improve the health of the individuals that they serve, and to prevent future disease and health conditions. most health practitioners know that those on social assistance are not able to afford nutritious foods or even sufficient amounts of food, but many are not aware of the extra dietary funds that are available aher consideration by a health practitioner. responsible nurse practitioners and physicians cannot, in good conscience, ignore the special needs diet supplement that is available to all recipients of welfare and disabiliry (ow and odsp). a number of toronto physicians have taken the position that all clients can justifiably benefit from vitamins, organic foods and high fiber diets as a preventative health measure. we know that income is one of the greatest predictors of poor health. the special needs diet is a health promotion intervention which will prevent numerous future health conditions, including chronic conditions such as cardiovascular disease, cancer, diabetes and osteoporosis. many communiry health centres and other providers have chosen to hold clinics to allow many patients to get signed up for the supplement at one time. initiated by the ontario coalition against poverty, these clinics have brought together commu· niry organizers, community health centers, health practitioners, and individuals, who believe that poverty is the primary determinant of poor health. we believe that rates must be increased to address the health problems of all people on social assistance, kids, elders, people with hiv/aids -everyone. even in the context of understaffing, it could be considered a priority activity that has potentially important health promotion benefits. many clients can be processed in a two hour clinic. most providers find it a very interesting, rewarding undertaking. in 2004 the ontario coalition for social justice found that a toronto family with two adults and two kids receives $14,316. this is $21,115 below the poverty line. p7-43 (c) the health of street youth compared to similar aged youth beth hayhoe and ruth ewert . lntrod~on: street youth are at an age normally associated with good health, but due to their risky ~hav1ours and th~ conditions in which they live, they experience health conditions unlike their peer~ an more stable env1r~nments. in addition, the majority of street youth have experienced significant physical, sexual ~nd em.ot1onal abuse as younger children, directly impacting many of the choices they make around their physical and emotional health. we examined how different their health really is. . , methodl: using a retrospective analysis of the 11 years of data gathered from yonge street mis· 510~ 5 • evergreen health centre, the top 10 conditions of youth were examined and compared with national tren~s for similar aged youth. based on knowledge of the risk factors present in the group, rea· sons for the difference were examined. d' ~its: street youth experience more illness than other youth their age and their illnesses can bt . irect t ·~kc~ to the. conditions in which they live. long-term impacts of abus~ contribute to such signif· ~~nt t e t 0 d~slpl air that youth may voluntarily engage in behaviours or lack of self care in the hope at t cir 1ve~ w1 perhaps come to a quicker end. concl11non: although it has ion b k h th' dy clearly shows 3 d'fi . h g ee~ no~n t at poverty negatively affects health, ~siu be used to make ; erence m t .e health of this particular marginalized population. the infonnanon can relates to th . ecommendatio.ns around public policy that affects children and youth, especially as it e1r access to appropriate health care and follow up. p7-44 (cl why do urban children · b gt . tarek hussain 10 an adesh die: how to save our children? the traditional belief that urban child alid. a recent study (dhs d fr 17 r~n are better off than rural children might be no longer v urban migrants are highata th om h c~untn~s i demonstrates that the child survival prospects of rural· er an t ose m their r j · · ·grants. in bangladesh, currently 30 million 0 ~r~ 0~1gm and lower than those of urban non-idi million. health of the urban 1 ~ p~e are hvmg m urban area and by the year 2025, it would be so the popu at1on 1s a key a eals that urban poor have the worse h 1 h . concern. recent study on the urban poor rev ea t situation than the nation as a whole. this study shows that infant poster sessions v169 mortality among the urban poor as 120 per thousand, which are above the rural and national level estimates. the mortality levels of the dhaka poor are well above those of the rest of the city's population but much of the difference in death rates is explained by the experience of children, especially infants. analyzing demographic surveillance data from a large zone of the city containing all sectors of the population, research showed that the one-fifth of the households with the least possessions exhibited u5 child mortality almost three times as high as that recorded by the rest of the population. why children die in bangladesh? because their parents are too poor to provide them with enough food, clean water and other basic needs to help them avoid infection and recover from illness. researchers believed that girls are more at risk than boys, as mothers regularly feed boys first. this reflects the different value placed on girls and boys, as well as resources which may not stretch far enough to provide for everyone. many studies show that housing conditions such as household construction materials and access to safe drinking water and hygienic toilet facilities are the most critical determinants of child survival in urban areas of developing countries. the present situation stressed on the need for renewed emphasis on maternal and child healthcare and child nutrition programs. mapping path for progress to save our children would need be done strategically. we have the policies on hand, we have the means, to change the world so that every child will survive and has the opportunity to develop himself fully as a healthy human being. we need the political will--courage and determination to make that a reality. p7-45 (c) sherbourne health centre: innovation in healthcare for the transgendered community james read introduction: sherbourne health centre (shc), a primary health care centre located in downtown toronto, was established to address health service gaps in the local community. its mission is to reduce barriers to health by working with the people of its diverse urban communities to promote wellness and provide innovative primary health services. in addition to the local communities there are three populations of focus: the lesbian, gay, bisexual, transgendered and transexual communities (lgbtt); people who are homeless or underhoused; and newcomers to canada. shc is dedicated to providing health services in an interdisciplinary manner and its health providers include nurses, a nurse practitioner, mental health counsellors, health promoters, client-resource workers, and physicians. in january 2003 shc began offering medical care. among the challenges faced was how to provide responsive, respectful services to the trans community. providers had considerable expertise in the area of counselling and community work, but little in the area of hormone therapy -a key health service for those who want to transition from one gender to another. method: in preparing to offer community-based health care to the trans community it was clear that shc was being welcomed but also being watched with a critical eye. trans people have traditionally experienced significant barriers in accessing medical care. to respond to this challenge a working group of members of the trans community and health providers was created to develop an overall approach to care and specific protocols for hormone therapy. the group met over a one year period and their work culminated in the development of medical protocols for the provision of hormone therapy to trans individuals. results: shc is currently providing health care to 281 registered clients who identify as trans individuals (march 31 2005) through primary care and mental health programs. in an audit of shc medical charts (january 2003 to september 2004) 55 female-to-male (ftm) and 82 male-to-female (mtf) clients were identified. less than half of the ftm group and just over two-thirds of the mtf group presented specifically for the provision of hormones. based on this chart audit and ongoing experience shc continues to update and refine these protocols to ensure delivery of quality care. conclusion: this program is an example of innovative community-based health delivery to a population who have traditionally faced barriers. shc services also include counselling, health promotion, outreach and education. p7-46 (c) healthy cities for canadian women: a national consultation sandra kerr, kimberly walker, and gail lush on march 4 2005, the national network on environment and women's health held a pan-canadian consultation to identify opportunities for health research, policy change, and action. this consultation also worked to facilitate information sharing and networking between canadian women working as urban planners, policy makers, researchers, and service workers on issues pertaining to the health of women living in canadian cities. methods: for this research project, participants included front-line service workers, policy workers, researchers, and advocates from coast to coast, including francophone women, women with disabilities, racialized women, and other marginalized groups. the following key areas were selected as topics for du.bnes i1 alto kading .:auk of end·sugr ieaal clileue ia singapore, accounting for more than so% of new can singapore (nkfs) to embark on a prevention program (pp) 10 empo~r d1ahc 1j1u1f dieir condition bttter, emphasizing education and disease sdf·managemen1 lkilla a. essennal camponenn of good glycaemic control. we sought 10 explore the effects of a 1pecialijed edu.:a11on pro· pun od glycacmic conuol, as indicated by, serum hba ic values budine serum hba ic values were determined before un<k-rgmng the education progr ;am, which couisted of comprehensive dietary advice, ideal weight goals, and improving lipid profiln. serum hbaic values were obtained ar 3,6,9 months later to determine impact of the program. analy11• wu done uling paired !·tests significant reduction in hba le levels from 0 ro 9 month• was observed in females, chinese race, older age group(> so yean). ohew-ibmi ~ 27.nwm2, wai11 hip ratio> l),up to primary and above secondary level education and those having om urine iclt showed that increasing hbalc levels (9) had increasing urmary protein (38.± 117; .18 ±i ih so± 136) and crearinine (s2.s ± 64 7s ± 71; ioi± 7s) levels fbg rnults showed that the management nf d1abetn m the nkfs preven· tion programme is effec;rive. results also indicated 1har hba le leve11 have a linnr trend wnh unnary protein and creatinine which are imponant determinants of renal diseate tal family-focused cinical palbway1 promoce politivc outcollln for ua inner city canu allicy ipmai jerrnjm1 care llctivirits in preparation for an infanr'' dilchargr honlr, and art m1endnl lo improve effi.:k'fl.:tn of c.are. 11lere i11 paucity of tttran:h, and inconsi1trncy of rnulta on 1ht-•m!*-1 of f1m1ly·fc"-'uw d1nm 1a: to determinr whrthrr implrmentation of family.focuted c:pt 1n 1 ntnn.tt.tl unit w"n mg an inner city 1;ommunity drcl't'aki leftarh of lf•y (i.osi and rromclll'i family uo•fkllon and rt.1j1 nest for dikhargr. md6odt: family-focuk"d cpi 1041 data wm coll«ted for all infant• horn btrwttn 29 and 36 wft"k• 1t"lal111mi atr who wrtt .1dm111ed to the ntonatal unit lmgdl of -.y 111. 9 n. 14.8 day'o p c o.osi ind pma .11 d•mr., ho.nr 137.3 t 1.3 n. 36.4 ± i. i wb, p < o.os) wett n«01fiamly f.lfrt 1n the pre.(]' poup. ~11.fxtmon icofn for famihn wrre high. and families noctd thc:y wnr mott prepued to ah thrar t..lby "'-· thett was .a cosi uving of s 1,814 (cdn) per patient d1teharpd home 1n the pmi-cp poap c.-pated 10 the p"''lfoup· cortclaion· lmplrmrnr.rion of family·foanrd c:p. in a nrona1.1i umt tc"fyidi an 1nnn an com· muniry decre.ned length of'"'" mft with a high dcgrft of family uujamon, and wrre coll~nt at least 35% percent of the kathmandu population lives in slum like conditions with poor access to basic health services. in these disadvantaged areas, a large proportion of children do not receive treatment due to inaccessibility to medical services. in these areas, diarrhea, pneumonia, and measles, are the key determinants of infant mortality. protein energy malnutrition and vitamin a deficiency persists and communicable diseases are compounded by the emergence of diseases like hiv/aids. while the health challenges for disadvantaged populations in kathmandu are substantial, the city has also experienced various forms of innovative and effective community development health programs. for example, there are community primary health centers established by the kathmandu municipality to deliver essential health services to targeted communities. these centers not only provide equal access to health services to the people through an effective management system but also educate them hy organizing health related awareness programs. this program is considered one of the most effective urban health programs. the paper/presentation this paper will review large, innovative, and effective urhan health programs that are operating in kathmandu. most of these programs are currently run by international and national ngos a) early detection of emerging diseases in urban settings through syndromic surveillance: 911 data pilot study kate bassil of community resources, and without adequate follow-up. in november 2003 shelter pr.oviders ~et with hospital social workers and ccac to strike a working group to address some of th~ issues by mcre.asing knowledge among hospital staff of issues surrounding homelessness, and to build a stro?g workmg relationship between both systems in hamilton. to date the hswg has conducted four w~lkmg to~ of downtown shelters for hospital staff and local politicians. recently the hswg launched its ·~ool.k1t for staff working with patients who are homeless', which contains community resources and gu1dehnes to help with effective discharge plans. a scpi proposal has been submitted to incre~se the capacity of the hswg to address education gaps and opportunities with both shelters and hospitals around homelessness and healthcare. the purpose of this poster presentation is to share hamilton's experience and learnings with communities who are experiencing similar issues. it will provide for intera~tion around shared experiences and a chance to network with practitioners across canada re: best practices. introduction and objectives: canadians view health as the biggest priority for the federal government, where health policies are often based on models that rely on abstract definitions of health that provide little assistance in the policy and analytical arena. the main objectives of this paper are to provide a functional definition of health, to create a didactic model for devising policies and determining forms of intervention, to aid health professionals and analysts to strategize and prioritize policy objectives via cost benefit analysis, and to prompt readers to view health in terms of capacity measures as opposed to status measures. this paper provides a different perspective on health, which can be applied to various applications of health such as strategies of aid and poverty reduction, and measuring the health of an individual/ community/country. this paper aims to discuss theoretical, conceptual, methodological, and applied implications associated with different health policies and strategies, which can be extended to urban communities. essentially, our paper touches on the following two main themes of this conference: •health status of disadvantaged populations; and •interventions to improve the health of urban communities.methodology: we initially surveyed other models on this topic, and extrapolated key aspects into our conceptual framework. we then devised a theoretical framework that parallels simple theories of physkal energy, where health is viewed in terms of personal/societal health capacities and effort components.after establishing a theoretical model, we constructed a graphical representation of our model using selfrated health status and life expectancy measures. ultimately, we formulated a new definition of health, and a rudimentary method of conducting cost benefit analysis on policy initiatives. we end the paper with an application example discussing the issues surrounding the introduction of a seniors program.results: this paper provides both a conceptual and theoretical model that outlines how one can go about conducting a cost-benefit analysis when implementing a program. it also devises a new definition and model for health barred on our concept of individual and societal capacities. by devising a definition for health that links with a conceptual and theoretical framework, strategies can be more logically constructed where the repercussions on the general population are minimized. equally important, our model also sets itself up nicely for future microsimulation modeling and analysis.implications: this research enhances one's ability to conduct community-based cost-benefit analysis, and acts as a pedagogical tool when identifying which strategies provide the best outcome. p7-06 (a) good playgrounds are hard to find: parents' perceptions of neighbourhood parks patricia tucker, martin holmes, jennifer irwin, and jason gilliland introduction: neighbourhood opportunities, including public parks and physical activity or sports fields hav~ been. iden.tified as correlates to physical activity among youth. increasingly, physical activity among children 1s bemg acknowledged as a vital component of children's lives as it is a modifiable determinant of childh~d obesity. children's use of parks is mainly under the influence of parents; therefore, the purpose of this study was to assess parents' perspectives of city parks, using london ontario as a case study.m~~: this qualitative study targeted a heterogeneous sample of parents of children using local parks w1thm london. parents with children using the parks were asked for 5 minutes of their time and if willing, a s.hort interview was conducted. the interview guide asked parents for their opinion 'of city parks, particularly the one they were currently using. a sample size of 50 parents is expected by the end of the summer.results: preliminary findings are identifying parents concern with the current jack of shade in local parks. most parents have identified this as a limitation of existing parks, and when asked what would make the parks better, parents agree that shade is vital. additionally, some parents are recognizing the v170 poster sessions focused discussions during the consultation: 1. women in _poverty 2. women with disability 3. immi· grant and racialized women 4. the built and _physica_l environment. . . . . r its· participants voiced the need for integration of the following issues withm the research and policy :::na; t) the intersectional nature of urban women's health i~sues wh~ch reflects the reality of women's complex lives 2) the multisectional aspect of urban wo_m~n s health, 1ss~es, which reflects the diversity within women's lives 3) the interse~roral _dynamics within _womens hves and urban health issues. these concepts span multiple sectors -mdudmg health, educat10n, and economics -when leveraging community, research, and policy support, and engaging all levels of government.policy jmplicatiom: jn order to work towards health equity for women, plans for gender equity must be incorporated nationally and internationally within urban development initiatives: • reintroduce "women" and "gender" as distinct sectors for research, analysis, advocacy, and action. •integrate the multisectional, intersectional, and intersecroral aspects of women's lives within the framework of research and policy development, as well as in the development of action strategies. • develop a strategic framework to house the consultation priorities for future health research and policy development (for example, advocacy, relationship building, evidence-based policy-relevant research, priority initiatives}.note: research conducted by nnewh has been made possible through a financial contribution from health canada. the views expressed herein do not necessarily represent the views of health canada.p7-47 (c) drugs, culture and disadvantaged populations leticia folgar and cecilia rado lntroducci6n: a partir de un proyecto de reducci6n de daiios en una comunidad urbana en situ· aci6n de extrema vulnerahilidad surge la reflexion sobre el lugar prioritario de los elementos sociocuhurales en el acceso a los servicios de salud de diferentes colectivos urbanos. las "formas de hacer, pensar y sentir" orientan las acciones y delimitan las posibilidades que tienen los individuos de definir que algo es o no problema, asf como tambien los mecanismos de pedido de ayuda. el analisis permanenre del campo de "las culturas cotidianas" de los llamados "usuarios de drogas" aporta a la comprension de la complejidad del tema en sus escenarios reales, y colabora en los diseiios contextualizados de politicas y propuestas socio-sanitarias de intervenci6n, tornandolas mas efectivas.mitodos: esta experiencia de investigaci6n-acci6n que utiliza el merodo emografico identifica elementos socio estructurales, patrones de consumo y profundiza en los elementos socio-simb61icos que estructuran los discursos de los usuarios, caracterizandolos y diferenciandolos en tanto constitutivos de identidades socia les que condicionan la implementaci6n del programas de reduccion de daiios.resultados: los resultados que presentaremos dan cuenta de las caracteristicas diferenciadas v relaciones particulares ~ntre los consumidores de drogas en este contexto espedfico. a partir de este e~tudio de caso se mtentara co1?1enzar a responder preguntas que entendemos significativas a la hora de pensar intcrvcnciones a la med1da de poblaciones que comparten ciertas caracteristicas socio-culturales. (cuales serian las .motivaciones para el cambio en estas comunidades?, cque elementos comunitarios nos ayudan a i:nnstnur dema~~a? • cque tenemos para aprender de las "soluciones" que ellos mismos encuenrran a los usos problemat1cos? methods: our study was conducted by a team of two researchers at three different sites. the mapping consisted of filling in a chart of observable neighbourhood features such as graffiti, litter, and boarded housing, and the presence or absence of each feature was noted for each city block. qualitative observations were also recorded throughout the process. researchers analyzed the compiled quantitative and qualitative neighbourhood data and then analyzed the process of data collection itself.results: this study reveals the need for further research into the effects of physical environments on individual health and sense of well-being, and perception of investment in neighbourhoods. the process reveals that perceptions of health and safety are not easily quantified. we make specific recommendations about the mapping methodology including the importance of considering how factors such as researcher social location may impact the experience of neighbourhoods and how similar neighbourhood characteristics are experienced differently in various spaces. further, we discuss some of the practical considerations around the mapping exercise such as recording of findings, time of day, temperature, and researcher safety.conclusion: this study revealed the importance of exploring conceptions of health and well-being beyond basic physical wellness. it suggests the importance of considering one's environment and one's own perception of health, safety, and well-being in determining health. this conclusion suggests that attention needs to be paid to the connection between the workplace and the external environment it is situated in. the individual's workday experience does not start and stop at the front door of their workplace, but rather extends into the neighbourhood and environment around them. our procedural observations and recommendations will allow other researchers interested in the effect of urban environments on health to consider using this innovative methodology. introduction: responding ro protests against poor medical attention for sexually assaulted women and deplorable conviction rates for sex offenders, in the late 1970s, the ontario government established what would become over 30 hospital-based sexual assault care and treatment centres (sactcs) across the province. these centres, staffed around the clock with specially trained heath care providers, have become the centralized locations for the simultaneous health care treatment of and forensic evidence collection from sexually assaulted women for the purpose of facilitating positive social and legal outcomes. since the introduction of these centres, very little evaluative research has been conducted to determine the impact of this intervention. the purpose of our study was to investigate it from the perspectives of sexually assaulted women who have undergone forensic medical examinations at these centres.method: women were referred to our study by sactc coordinators across ontario. we developed an interview schedule composed of both closed and open-ended questions. twenty-two women were interviewed, face-to-face. these interviews were approximately one-to-two hours in length, and were transcribed verbatim. to date, 19 have been analyzed for key themes.results: preliminary findings indicate that most women interviewed were canadian born (79'yo), and ranged in age from 17 to 46 years. a substantial proportion self-identified as a visible minority ( 37'x.). approximately half were single or never married (47%) and living with a spouse or family of origin (53%). most were either students or not employed (68%). two-thirds (68%) had completed high school and onethird (37%) was from a lower socio-economic stratum. almost two-fifths (37%) of women perceived the medical forensic examination as revictimizing citing, for example, the internal examination and having blood drawn. the other two-thirds (63%) indicated that it was an empowering experience, as it gave them a sense of control at a time when they described feeling otherwise powerless. most (68%) women stated that they had presented to a centre due to health care concerns and were very satisfied ( 84 % ) with their experiences and interactions with staff. almost all (89%) women felt supported and understood.conclusions: this research has important implications for clinical practice and for appropriately addressing the needs of sexually assaulted women. what is apparent is that continued high-quality medical attention administered in the milieu of specialized hospital-based services is essential. at the same time, we would suggest that some forensic evidence collection procedures warrant reevaluation. the study will take an experiential, approach by chroruclmg the impa~ of the transition f m the streets to stabilization in a managed alcohol program through the techruque of narrative i~:uiry. in keeping with the shift in thinking in the mental health fie!~ ~his stu~y is based on a paradigm of recovery rather than one of pathology. the "inner views of part1c1pants hves as they portray their worlds, experiences and observations" will be presented (charm~z, 1991, ~· 38~)-"i?e p~ of the study is to: identify barriers to recovery. it will explore the exj?cnence of ~n~t1zanon pnor to entry into the program; and following entry will: explore the meanmg ~nd defirutto~s of r~overy ~~d the impact of the new environment and highlight what supports were instrumental m movmg pan1apants along the recovery paradigm.p7-st (a) treating the "untreatable": the politics of public health in vancouver's inner city introdudion: this paper explores the everyday practices of therapeutic programs in the treadnent of hiv in vancouver's inner city. as anthropologists have shown elsewhere, therapeutic programs do not siinply treat physical ailments but they shape, regulate and manage social lives. in vancouver's inner city, there are few therapeutic options available for the treatment of 1-ilv. public health initiatives in the inner city have instead largely focused on prevention and harm reduction strategies such as needle exchange programs, safe injection sites, and safer-sex education. epidemiological reports suggest that less than a quarter of those living with hiv in the downtown eastside (dtes) are taking antiretroviral therapies raising critical questions regarding the therapeutic economy of antiretrovirals and rights to health care for the urban poor.methods: this paper is drawn from ethnographic fieldwork in vancouver's otes neighborhood focusing on therapeutic programs for hiv treatment among "hard-to-reach" populations. the research includes participant-observation at inner city health clinics specializing in the treatment of hiv; semi· structured interviews with hiv positive participants, health care professionals providing hiv treatment, and administraton working in the field of inner city public health; and, lastly, observation at public meetings and conferences surrounding hiv treatment.r.awlts: hiv prevention and treatment is a central concern in the lives of many residents living in the inner city -although it is just one of many health priorities afflicting the community. concerns about drug resistance, cost of antiretrovirals, and illicit drug use means that hiv therapy for most is characterized by the daily observation of their medicine ingestion at health clinics or pharmacies. daily observed treatment (dot) is increasingly being adopted as a strategy in the therapeutic management of "untreatable" populations. dot programs demand a particular type of subject -one who is "compliant" to the rules and regimes of public health. over emphasis on "risky practices," "chaotic lives," and "~dh~rence" preve~ts the public health system from meaningful engagement with the health of the marginalized who continue to suffer from multiple and serious health conditions and who continue to experience considerable disparities in health.~ the ~ffec~s of hiv in the inner city are compounded by poverty, laclc of safe and affordable houamg, vanous 1llegal underground economies increased rates of violence and outbreaks of ~~~·~ly tr~nsmitted infections, hepatitis, and tuberculosi: but this research suggests 'that public health uunauves aimed at reducing health disparities may be failing the most vulnerable and marginal of citiztl1s. margaret malone 1~ vi~lence that occurs in families and in intimate relationships is a significant urban, ~unity, and pu~hc health problem. it has major consequences and far-reaching effects for women, ~~--renho, you~ sen1on, and families. violence also has significant effects for those who provide and ukllc w receive health care violence · · i · · . all lasses, · is a soc1a act mvolvmg a senous abuse of power. it crosses : ' : ' ~ 11 s;nden, ag~ ~ti~, cultures, sexualities, abilities, and religions. societal responseshali ra y oc:used on identificatton, crisis intervention and services for families and individuajs.promoten are only "-"--:-g to add h · ' · i in intimate relationshi with"-~"'.". ress t e issues of violence against women and vjoence lenga to consider i~ m families. in thi_s p~per, i analyze issues, propose strategies, and note c~· cannot be full -...l'-~ whork towards erad1canng violence, while arguing that social justice and equity y -.1ucvcu w en thett are people wh mnhod: critical social theory, an analysis that addresses culturally and ethnically diverse communities, together with a population health promotion perspective frame this analysis. social determinants of health are used to highlight the extent of the problem of violence and the social and health care costs.the ottawa charter is integrated to focus on strategies for developing personal skills, strengthening community action, creating supportive environments, devdoping healthy public policies, and re-orientating health and social services. attention is directed to approaches for working with individuals, families, groups, communities, populations, and society.ratdts: this analysis demonstrates that a comprehensive interdisciplinary, multisectoral, and multifaceted approach within an overall health promoting perspective helps to focus on the relevant issues, aitical analysis, and strategies required for action. it also illuminates a number of interacting, intersecting, and interconnecting factors related to violence. attention, which is often focused on individuals who are blamed for the problem of violence, is redirected to the expertise of non-health professionals and to community-based solutions. the challenge for health promoters working in the area of violence in families and in intimate relationships is to work to empower ourselves and the communities with whom we work to create health-promoting urban environments. social justice, equiry, and emancipatory possibilities are positioned in relation to recommendations for future community-based participatory research, pedagogical practices for health care practitioners, and policy development in relation to violence and urban health. the mid-main community health center, located in vancouver british columbia (bc), has a diverse patient base reflecting various cultures, languages, abilities, and socio-economic statuses. due to these differences, some mid-main patients experience greater digital divide barriers in accessing computers and reputable, government produced consumer health information (chi) websites, such as the bc healthguide and canadian health network. inequitable access is problematic because patient empowerment is the basis of many government produced chi websites. an internet terminal was introduced at mid-main in the summer of 2005, as part of an action research project to attempt to bridge the digital divide and make government produced chi resources useful to a broad array of patients. multi-level interventions in co-operation with patients, with the clinic and eventually government ministries were envisioned to meet this goal. the idea of implementing multi-level interventions was adopted to counter the tendency in interactive design to implement a universal solution for the 'ideal' end-user [ 1 ), which discounts diversity. to design and execute the interventions, various action-oriented and ethnographic methods were employed before and during the implementation of the internet terminal. upon the introduction of the internet terminal, participant observation and interviews were conducted using a motion capture software program to record a digital video and audio track of patients' internet sessions. this research provided insight into the spectrum of patients' capacities to use technology to fulfil their health information needs and become empowered. at the mid-main clinic it is noteworthy that the most significant intervention to enhance the usefulness of chi websites for patients appeared to be a human rather than a technological presence. as demonstrated in other ethnographic research of community internet access, technical support and capacity building is a significant component of empowerment (2). the mid-main wired waiting room project indicates that medical practitioners, medical administrators, and human intermediaries remain integral to making chi websites useful to patients and their potential empowerment. (1) over the past 5 years the environmental yo~th alliance has been of~ering a.youth as~t. mappin~ program which trains young people in community research and evaluation. wh1~st the positive expenenc~ and relationships that have developed over this time attest to the success of this program, no evaluations has yet been undertaken to find out what works for t.he youth, what ~ould be changed, and what long term outcomes this approach offers for the youth, their local community, and urban governance. these topics will be shared and discussed to help other community disorganizing and uncials governments build better, youth-driven structures in the places they live.p7-55 (a) the world trade center health registry: a unique resource for urban health researchers deborah walker, lorna thorpe, mark farfel, erin gregg, and robert brackbill introduction: the world trade center health registry (wfchr) was developed as a public health response to document and evaluate the long-term physical and mental health effects of the 9/11 disaster on a large, diverse population. over 71,000 people completed a wfchr enrollment baseline survey, creating the largest u.s. health registry. while studies have begun to characterize 9/11 bealth impacts, questions on long-term impacts remain that require additional studies involving carefully selected populations, long-term follow-up and appropriate physical exams and laboratory tests. wtchr provides an exposed population directory valuable for such studies with features that make ita unique resource: (a) a large diverse population of residents, school children/staff, people in lower man· hattan on 9/11 including occupants of damaged/destroyed buildings, and rescue/recovery/cleanup work· ers; (b) consent by 91 % of enrollees to receive information about 9/11-related health studies; (c) represenration of many groups not well-studied by other researchers; (c) email addresses of 62% of enrollees; (d) 30% of enrollees recruited from lists with denominator estimates; and (e) available com· parison data for nyc residents. wfchr strives to maintain up-to-date contact information for all enrollees, an interested pool of potential study participants. follow-up surveys are planned.methods: to promote the wtchr as a public health resource, guidelines for external researcher.; were developed and posted on (www.wtcregistry.org) which include a short application form, a twopage proposal and documentation of irb approval. proposals are limited to medical, public health, or other scientific research. researchers can request de-identified baseline data or have dohmh send information about their studies to selected wfchr enrollees via mail or email. applications are scored by the wtchr review committee, comprised of representatives from dohmh, the agency for toxic subst~nces and disease registry, and wtchr's scientific, community and labor advisory committees. a data file users manual will be available in early fall 2005.~suits: three external applications have been approved in 2005, including one &om a non-u.s. ~esearcher, all requesting information to be sent to selected wtchr enrollees. the one completed mail· mg~~ wtchr enrollee~ (o 3,700 wfc tower evacuees) generated a positive survey response rate. three additional researchers mtend to submit applications in 2005. wfchr encourages collaborations between researchers and labor and community leaders.conclusion: studies involving wtchr enrollees will provide vital information about the long· term health consequences of 9/11. wtchr-related research can inform communities, researcher.;, policy makers, health care providers and public health officials examining and reacting to 9111 and other disasters. t .,. dp'"f'osed: thi is presentation will discuss the findings of attitudes toward the repeat male client iden· 1 ie as su1e1 a and substance us'n p · · · · i · 'd . . -1 g. articipants will learn about some identified effective strategies or service prov1 ers to assist this group of i · f men are oft · d bl men. n emergency care settings, studies show that this group 0 en viewe as pro emaric patient d i r for mental health p bl h h 5 an are more ikely to be discharged without an assessmen 200!) ea 1 1 rofr ems t. an or er, more cooperative patients (forster and wu 2002· hickey er al., · r y resu ts om this study suggest th · · ' ' l · d tel' mining how best to h 1 . d 1 at negative amtudes towards patients, difficu nes e · as well pathways l_e_ p patientsblan ~ck of conrinuity of care influence pathways to mental health care. • uc\:ome pro emat1c when p ti k · che system. m a ems present repeatedly and become "get stuc id methods: semi-structured intervie d . · (n=5), ed nurses (n=5) other ed ;s were con ucted with male ed patients (n=25), ed phys1oans ' sta (n= 7) and family physicians (n= 7). patients also completed a poster sessions v175 diagnostic interview. interviews were tape-recorded, transcribed verbatim and managed using n6. transcripts were coded using an iterative process and memos prepared capture emergent themes. ethics approval was obtained and all participants signed a detailed informed consent form.introduction: urban settings are particularly susceptible to the emergence and rapid spread of nt•w or rare diseases. the emergence of infectious diseases such as sars and increasing concerns over the next influenza pandemic has heightened interest in developing and using a surveillance systt·m which detects emerging public health problems early. syndromic surveillance systems, which use data b,1scd on symptoms rather than disease, offer substantial potential for this by providing near-real-rime data which are linked to an automated warning system. in toronto, we are piloting syndromic data from the 911 · emergency medical services (ems) database to examine how this information can be used on an ongoing basis for the early detection of syndromes including heat-related illness (hri), and influenza-like-illness (ill). this presentation will provide an outline of the planned desi_gn of this system and proposed evaluation. for one year, 911 call codes which reflect heat-related illness or influenza-like-illness will be selected and searched for daily using software with a multifactorial algorithm. calls will he stratified by call code, extracted from the 911-ems database and transferred electronically to toronto public health. the data will be analyzed for clusters and aberrations from the expected with the realtime outbreak and disease surveillance (rods) system, a computer-based public health tool for the early detection of disease outbreaks. this 911-ems surveillance system will be assessed in terms of its specificity and sensitivity through comparisons with the well-established tracking systems already in place for hr! and ill. others sources of data including paramedic ambulance call reports of signs and . this study will introduce complementary data sources t~ the ed ch1e~ complamt an~ o~~rthe-counter pharmacy sales syndromic surveillance data currently bemg evaluated m ~ther ontar~o cltles. . syndromic surveillance is a unique approach to proactive(~ dete~tmg early c.hangesm the health status of urban communities. the proposed study aims to provide evidence of differential effectiveness through investigating the use of 911-ems call data as a source of syndromic surveillance information for hr! and ili in toronto. introduction: there is strong evidence that primary care interventions, including screening, brief advi<:e, treatment referral and pharmacotherapy are effective in reducing morbidity and mortality caused by substance abuse. yet physicians are poor at intervening with substance users, in part because of lack of time, training and support. this study examines the hypothesis that shared care in addictions will result in decreased substance use and improved health status of patients, as well as increased use of primary care interventions by primary care practitioners (pcps). methods: the addiction medicine service (ams) at st. joseph's health centre's family medicine department is in the process of being transformed from its current structure as a traditional consult service into a shared care model called addiction shared care (asc). the program will have three components: education, office systems and clinical shared care. as opposed to a traditional consult service, the patient will be booked with both a primary care liaison worker (pcl) and addictions physician. patients referred from community physicians, the emergency department and inpatient medical and psychiatric wards will be recruited for the study as well as pcps from the surrounding community. the target sample size is 100-150 physicians and a similar number of patients. after initial consult, patient will be recruited into the study with their consent. the shared-case model underlines the interaction and collaboration with the patient's main pcp. asc will provide them with telephone consults, advice, support and re-assessment for their patients, as well as educational sessions and materials such as newsletters and informational kits.results: the impact of this transition on our patient care and on pcp's satisfaction with the asc model is currently being evaluated through a grant provided by the ministry of health & long term care. a retrospective chart review will be conducted using information on the patient's substance use, er/clinic visits, and their health/mood status. pcp satisfaction with the program will be measured through surveys and focus groups. our cost-effectiveness analysis will calculate the overall cost of the program per patient..conclusion: this low-cost service holds promise to serve as an optimal model and strategy to improve outcomes and reduce health care utilization in addict patients. the inner city public health project introduction the inner city public health project (icphp) was desi.gned to explore new an~ innovative ways to reach marginalized inner city populations that par-t1c1pate m high health-nsk beha~1ors. much of this population struggles with poverty, addictions, mental illness and homelessness, creatmg barriers to accessing health services and receiving follow-up. this pro1ect was de~igned to evaluat~ .~e success of offering clinics in the community for testing and followup of communicable diseases uuhzmg an aboriginal outreach worker to build relationships with individuals and agencies. v1n(demographics~ history ~f testi~g ~nd immunization and participation in various health-risk behaviors), records of tesnng and 1mmumzat1ons, and mterviews with partner agency and project staff after one year.. results: t~e chr ~as i~strumental in building relationships with individuals and partner agencies ' .° the c~mmun_1~ re_sultmg m req~ests for on-site outreach clinics from many of the agencies. the increase m parnc1pat10n, the chr mvolvement in the community, and the positive feedback from the agen? staff de~onstrated that.the project was successfully creating partnerships and becoming increasingly integrated m the community. data collected from clients at the initial visit indicated that the project was reaching its target populations and highlighted the unique health needs of clients, the large unmet need for health services and the barriers that exist to accessing those services. ~usion: the outreach clinics were successful at providing services to target populations of high health-nsk groups and had great support from the community agencies. the role of the chr was critical to the success of the project and proved the value of this category of health care worker in an urban aboriginal population. the unmet health needs of this disadvantaged population support the need for more dedicated resources with an emphasis on reducing access barriers. building a caring community old strathcona's whyte avenue, a district in edmonton, brought concern about increases in the population of panhandlers, street people and homeless persons to the attention of all levels of government. the issue was not only the problems of homelessness and related issues, but feelings of insecurity and disempowerment by the neighbourhood residents and businesses. their concerns were acknowledged, and civic support was offered, but it was up to the community itself to solve the problem. within a year of those meetings, an adult outreach worker program was created. the outreach worker, meets people in their own environments, including river valley camps. she provides wrap-around services rooted in harm reduction and health promotion principles. her relationship-based practice establishes the trust for helping clients with appropriate housing, physical and/or mental health issues, who have little or no income and family support to transition from homelessness. the program is an excellent example of collaboration that has been established with the businesses, community residents, community associations, churches, municiple services, and inner-city agencies such as boyle street community services. statistics are tracked using the canadian outcomes research institute homes database, and feedback from participants, including people who are street involved. this includes an annual general meeting for community and people who are homeless. the program's holistic approach to serving the homeless population has been integral, both in creating community awareness and equipping residents and businesses to effectively interact with people who are homeless. through this community development work, the outreach worker engages old strathcona in meeting the financial and material needs of the marginalized community. the success of this program has been surprising -the fact is that homeless people's lives are being changed; one person at a time and the community has been changed in how they view and treat those without homes. over two years, the program has successfully connected with approximately seventy-five individuals who call old strathcona home, but are homeless. thirty-six individuals are now in homes, while numerous others have been assisted in obtaining a healthier and safer lifestyles by becoming connected with other social/health agencies. the program highlights the roots of homelessness, barriers to change and requirements for success. it has been a thriving program and a model that works by showing how a caring community has rallied together to achieve prosperous outcomes. the spn has created models of tb service delivery to be used m part~ers~1p with phannaceunca compa-. · · -. t' ns cooperatives and health maintenance orgamzanons (hmos). for example, the mes, c1v1c orgamza 10 , . · b tb d' · spn has established a system with pharmaceutical companies that help patients to uy me 1cmes at a special discounted rate. this scheme also allows patients to get a free one-_month's worth of~ dru_g supply if they purchase the first 5 months of their regimen. the sy_s~e~s were ~es1gned to be cm~pattble with existing policies for recording and documentation of the ph1hppme national tuberculosis program (ntp). aside from that, stakeholders were also encouraged to be dots-enabled through the use of m~nual~ and on-line training courses. the spn initiative offers an alternative in easing the burden of tb sc:rv1ce delivery from rhe public sector through the harnessing of existing private-sector (dsos). the learnmgs from the spn experience would benefit groups from other locales that _work no~ only on ~ but other health concerns as well. the spn experience showcases how well-coordinated private sector involvements help promote social justice in health delivery in urban communities.p7-63 (c) young people in control; doing it safe. the safe sex comedy juan walter and pepijn v. empelen introduction: high prevalence of chlamydia and gonorrhoea have been reported among migrants youth in amsterdam, originating from the dutch antilles, suriname and sub-sahara africa. in addition, these groups also have high rates of teenage-pregnancy (stuart, 2002) and abortions (rademakers 1995), indicating unsafe sexual behaviour of these young people. young people (aged 12 -30) from the so· called urban scene (young trendsetters in r&b/hip hop music and lifestyle) in amsterdam have been approached by the municipal health service (mhs) to collaborate on a safe sex project. their input was to use comedy as vehicle to get the message a cross. for the mhs this collaboration was a valuable opportunity to reach a hard-to-reach group.mdhods: first we conducted a need assessment by means of a online survey to assess basic knowledge and to similtaneously examine issues of interest concerning sex, sexuality, safer sex and the opposite sex. second, a small literature study was conducted about elements and essential conditions for succesful entertainment & education (e&e) (bouman 1999), with as most important condition to ensure that the message is realistic (buckingham & bragg, 2003) . third a program plan was developed aiming at enhancing the stl/hiv and sexuality knowledge of the young people and addressing communication and educational skills, by means of drama. subsequently a safe sex comedy show was developed, with as main topics: being in love, sexuality, empowerment, stigma, sti, hivand safer sex. the messages where carried by a mix of video presentation, stand up comedy, spoken word, rap and dance.results: there have been two safe sex comedy shows. the attendance was good; the group was divers' with an age range between 14 and 50 year, with the majority being younger than 25 year. more women than men attended the show. the story lines were considered realistic and most of the audients recognised the situations displayed. eighty percent of the audients found the show entertaining and 60% found it edm:arional. from this 60%, one third considers the information as new. almost all respondents pointed our that they would promote this show to their friends.con.clusion: the s.h<_>w reached the hard-to-reach group of young people out of the urban scene and was cons1d_ered entert~mmg, educational and realistic. in addition, the program was able in addressing important issues, and impacted on the percieved personal risk of acquiring an sti when not using condoms, as well as on basic knowledge about stl's. introduction: modernity has contributed mightily to the marginality of adults who live with mental illnesses and the subsequent denial of opportunities to them. limited access to social, vocational, educational, and residential opportunities exacerbates their disenfranchisement, strengthening the stigma that has been associated with mental illness in western society, and resulting in the denial of their basic human rights and their exclusion from active participation in civil society. the clubhouse approach tn recovery has led to the reduction of both marginality and stigma in every locale in which it has been implemented judiciously. its elucidation via the prism of social justice principles will lead to a deeper appreciation of its efficacy and relevance to an array of settings. methods: a review of the literature on social justice and mental health was conducted to determine core principles and relationships between the concepts. in particular, fondacaro and weinberg's (2002) conceptualization of social justice in community psychology suggests the desirability of the clubhouse approach in community mental health practice. a review of clubhouse philosophy and practice has led to the inescapable conclusion that there is a strong connection between clubhouse philosophy-which represents a unique approach co recovery--and social justice principles. placing this highly effective model of community mental health practice within the context of these principles is long overdue. via textual analysis, we will glean the principles of social justice inherent in the rich trove of clubhouse literature, particularly the international standards of clubhouse development.results: fondacarao and weinberg highlight three primary social justice themes within their community psychology framework: prevention and health promotion; empowerment, and a critical pnsp<"<·tive. utilizing the prescriptive principles that inform every detail of clubhouse development and th<" movement toward recovery for individuals at a fully-realized clubhouse, this presentation asserts that both clubhouse philosophy and practice embody these social justice themes, promote human rights, and empower clubhouse members, individuals who live with mental illnesses, to achieve a level of wdl-heing and productivity previously unimagined.conclusion: a social justice framework is critical to and enhances an understanding of the clubhouse model. this model creates inclusive communities that lead to opportunities for full partic1pil!ion 111 civil society of a previously marginalized group. the implication is that clubhouses that an· based on the international standards for clubhouse programs offer an effective intervention strategy to guarantee the human rights of a sizable, worthy, and earnest group of citizens. to a drastic increase in school enrollment from 5.9 million in 2002 to 7.5 million in 200.s. however, while gross enrollment rates increased to 104°/., in the whole country after the introduction of fpe, it remained conspicuously low at 62% in the capital city, nairobi. nairobi city's enrollment rate is lower .than thatof all regions in the country except the nomadic north-eastern province. !h.e.d1sadvantage of children bas_ed in the capital city was also noted in uganda after the introduction of fpe m the late 1990s_-many_ education experts in kenya attribute the city's poor performance to the high propornon of children hvmg m slums, which are grossly underserved as far as social services are concerned. this paper ~xammes the impact of fpe and explores reasons for poor enrollment in informal settlements m na1rob1 city. methods: the study utilizes quantitative and qualitative schooling data from the longitudinal health and demographic study being implemented by the african population and health research center in two informal settlements in nairobi. descriptive statistics are used to depict trends in enrollment rates for children aged 5-19 years in slum settlements for the period 2000-2005. results: the results show that school enrollment has surprisingly steadily declined for children aged 15-19 while it increased marginally for those aged 6-14. the number of new enrollments (among those aged 5 years) did not change much between 2001 and 2004 while it declined consistently among those aged 6-9 since 2002. these results show that the underlying reasons for poor school attendance in poverty-stricken populations go far beyond the lack of school fees. indeed, the results show that lack of finances (for uniform, transportation, and scholastic materials) has continued to be a key barrier to schooling for many children in slums. furthermore, slum children have not benefited from fpe because they mostly attend informal schools since they do not have access to government schools where the policy is being implemented.conclusion: the results show the need for equity considerations in the design and implementation of the fpe program in kenya. without paying particular attention to the schooling needs of the urban poor children, the millennium development goal aimed at achieving universal primary education will remain but a pipe dream for the rapidly increasing number of children living in poor urban neighborhoods.ps-04 (c) programing for hiv/aids in the urban workplace: issues and insights joseph kamoga hiv/aids has had a major effect on the workforce. according to !lo 35million persons who are engaged in some form of production are affectefd by hiv/aids. the working class mises out on programs that take place in communities, yet in a number of jobs, there are high risks to hiv infection. working persons sopend much of their active life time in workplaces and that is where they start getting involved in risky behaviour putting entire families at risk. and when they are infected with hiv, working people face high levels of seclusion, stigmatisation and some miss out on benefits especially in countries where there are no strong workplace programs. adressing hiv/aids in the workplace is key for sucessfull responses. this paper presents a case for workplace programing; the needs, issues and recommendations especially for urban places in developing countries where the private sector workers face major challenges.