Traffic crashes are a major cause of global morbidity and mortality disproportionately affecting low- and middle-income countries (LMICs). Motorcycle taxi (boda boda) drivers are particularly vulnerable because they are exposed to traffic risks with limited safety equipment. This study aims to characterize injury prevalence and safety habits among boda boda drivers, as well as ways to improve road traffic safety in LMICs.A cross-sectional mixed methods study was conducted with 300 boda boda drivers between 24 March and 3 April 2014 in urban Moshi, Tanzania. A convenience sample of participants was drawn from 25 of 58 registered boda boda stands and 2 of 31 unregistered stands. Data were analyzed using R, and content thematic analysis was performed and agreed upon by three investigators. Logistic regression models were used to evaluate the association between boda boda characteristics and injury risk.In total, 300 drivers participated, of whom 148 (49.3%) had experienced a crash during their lifetime, and 114 (77.0%) sustained at least one injury. Only 27 of those injured (23.4%) were hospitalized. Of all participants, 220 (73.3%) reported consistent helmet usage, despite 285 participants (95.0%) agreeing that helmet usage reduces injury severity. From the 280 helmets observed, 231 (82.5%) were either damaged or fit improperly. Having a cracked helmet was associated with higher risk of being involved in a traffic crash. Owning a helmet with a proper fit was associated with reduced risk for a traffic crash (OR = 0.06) and road traffic injuries (OR = 0.07). A thematic analysis of boda boda drivers’ suggestions to increase road safety identified four intervention areas: 1) roadway infrastructure and traffic regulation, 2) road user attitudes and safe driving behaviors, 3) education and training, and 4) law enforcement.Our study demonstrates boda boda drivers’ safety behaviors and identifies four intervention areas that can be leveraged to increase overall road traffic safety. Unfortunately, while boda boda drivers are aware of ways to improve safety, adherence to safety habits remains low. Successful multi-sectoral interventions are needed to improve road safety for boda boda drivers in Tanzania. Traffic crashes are a major cause of global morbidity and mortality, resulting in an estimated 20 million–50 million road traffic injuries (RTIs) and 1.2 million deaths per year . As LMICAmong the many types of road users, motorcycle riders are particularly vulnerable because they are exposed with limited protective equipment compared to the car and truck drivers with whom they share the road . In a stDespite the risks, many individuals in LMICs still choose motorcycles as their main mode of transportation for the benefits of convenience and low cost when compared to cost of owning an automobile. Motorcycle transportation became particularly prominent in countries affected by the Structural Adjustment Programs (SAPs) of the 1980s and 1990s. For example, structural adjustment in Nigeria increased motor vehicle prices by more than 200%, pricing car and minibus ownership out of reach for many and increasing the appeal of motorcycle transportation .Additionally, due to poor roadway infrastructure in LMICs, motorcycles are often the “only means of transport to many streets, connecting roads, or villages” . Even inThough the reported incidence of RTIs is striking, it is widely accepted that the figures are underestimated, mostly due to limitations in data collection , 24. ForThe present study aims to define overall prevalence of non-fatal RTIs among boda boda drivers in Moshi, Tanzania, and to describe their safety practices, to further understand injury epidemiology in East Africa. This increased understanding of injury epidemiology can be leveraged to identify potential areas for further research and to develop effective intervention strategies to reduce the incidence of RTIs.This was a cross-sectional mixed methods study of safety habits of boda boda drivers in Moshi, Tanzania, between 24 March and 3 April 2014. Ethical clearance was provided by the Kilimanjaro Christian Medical Center Ethics Committee as well as the Duke University Institutional Review Board. Our methods are described according to the GRAMMS guideline for mixed methods studies . We condA survey of boda boda drivers’ safety habits and traffic crash experience was conducted between 24 March and 3 April 2014 in Moshi, Tanzania. We enrolled 300 boda boda drivers, chosen at convenience from 25 of 58 registered boda boda stands and 2 of 31 unregistered stands. Despite efforts to register boda boda stands, the specific population of boda boda drivers is unregistered and the total number of drivers (targeted population) is unknown. Thus, our sample size of 300 was adopted since it would be enough to study the prevalence of non-fatal traffic crashes for an unknown population, with 95% confidence and a 5% error margin, based on an average prevalence of 30%, as suggested in previous literature –31.Stands were selected taking into account the spatial distribution that covered the Moshi Urban area, according to their locations. This approach was adopted to include stands that covered all the Moshi Urban geographical area, and also to cover areas for hotspots for motor vehicle crashes defined in a previous publication . We inclA trained native Kiswahili-speaking research assistant approached boda boda drivers at boda boda stands at a random hour of the day and offered them participation in the research study. The research assistant invited all boda boda drivers present at the stand during a one-hour period of data collection at each stand. If they were interested in the research project, participants were provided informed consent and formally enrolled in the study. If they were not interested in participating, the research assistant invited the next driver until we reached the pre-specified sample size of 300. Each questionnaire took about 20 minutes. Participants received a 5,000 TSH (~2 USD) cell phone voucher as reimbursement for their time to participate in the research. The voucher is equivalent to the average value of one boda boda ride for about the length of the Moshi Urban research area. Trained data collectors administered the survey using a computerized, Internet-based survey tool (REDCAP) and tablet computers.The questionnaire included questions about: (a) demographic information; (b) experience as a boda boda driver (e.g. number of years working as a boda boda driver); (c) injury history ; and (d) safety habits . Prior to data collection, we performed a pilot study with a sample of 5 boda boda drivers to ensure that the questions would be understood and that the survey/qualitative interviews could be done at their local stands during working hours. Some questions in the initial version of the survey were excluded after the pilot because they didn’t pertain to the Moshi setting. Additionally, piloting the survey allowed the research assistants to become familiar with the computer-based questionnaire administrate and navigate any Internet challenges. The full Swahili version of the questionnaire is included in the Appendix.Data were analyzed using R Language for Statistical Computing . DescripEach boda boda driver was also interviewed with an open-ended question about their suggestions to improve safety and decrease traffic crashes. The interview occurred after the survey was administered and was initiated with the general question. Probing questions were used when needed to clarify the respondent’s suggestion for improvement. All interviews were conducted by the same research assistant in the local language, thus ensuring a standard procedure, and digitally recorded. Record files were transcribed using naturalized standard qualitative methods .To analyze the qualitative data, we used a thematic content analysis. The qualitative interview transcripts were back translated, then independently reviewed and coded by two members (one from the United States and one from Tanzania) of the research team (TN and MP) using manual coding. Codes were grouped into categories, which were then reduced to themes through discussion and repeated review of interview scripts by the research team members  . Ambiguiall the US and Tanzanian research team members for validation purposes. This validation helped confirm our findings and minimize the influences of personal bias. We continued discussing the emerging themes with the US and Tanzanian research teams for each coded interview until we reached a saturation point where no new themes were evident. Results for each category/theme are presented, along with their frequency of appearance in the participants’ responses.For each coded interview, we shared the resultant emerging themes with Three hundred boda boda drivers operating in Moshi Urban, Tanzania, were surveyed during the study period. All drivers were male, with a mean age of 28 years. As seen in Of the 300 participants, 148 (49.3%) had been involved in a traffic crash. From the 148 traffic crashes, 118 (79.7%) occurred while the boda boda drivers were working. Crash rates increased as the day progressed, peaking in the evening hours. Of the 148 crashes, 26 (17.6%) occurred in the morning, 37 (25.0%) occurred during the day, 46 (31.1%) occurred in the evening, and 36 (24.3%) occurred at night, while 3 had missing information on the time of day. Near misses were highly prevalent, with 247 study participants (82.3%) reporting at least one near miss within the past month .The traffic crashes carried a high risk of injury, with 114 crash victims (38.0%) sustaining at least one injury and 11 crash victims sustaining multiple injuries. The most common injuries were injuries to the head and the lower and upper extremities  . DespiteOnly 220 participants (73.3%) reported consistent helmet usage despite 285 participants (95.0%) either agreeing or strongly agreeing that helmet usage is valuable in reducing injuries from a crash. Furthermore, only 176 participants (58.7%) reported consistent helmet strap usage despite 289 participants (96.3%) either agreeing or strongly agreeing that helmet strap usage helps reduce crash injuries .Of the 280 helmets observed, 60 helmets (21.4%) had cracked or dented shells, 128 helmets (45.7%) had scratches in the paint, and 44 helmets (15.7%) had broken chin straps. Only 151 helmets (53.9%) featured face shields. Scratches, paint, and graphics obscured driver vision on 53.0% (n = 80) of those face shields .In terms of boda boda conspicuity, only 109 drivers (36.3%) drove with their headlights on at all times. However, consistent headlight usage increased to 91.0% (n = 273) after dark. When asked to describe their colleagues’ driving habits, a staggering 97.3% (n = 292) believed that their colleagues were engaged in risky driving behaviors at least occasionally. Only 2.7% (n = 8) of participants believed that their colleagues never engaged in risky driving.Boda boda drivers’ responses showed that having a cracked helmet was associated with a 2 times higher risk of having been involved in a traffic crash, while drivers who owned a helmet with a proper fit were less likely to have been in a traffic crash (OR = 0.06). Regarding injury, having a helmet with a proper fit was also associated with a lower risk of RTIs (OR = 0.07). As for near misses, participants who reported using a chin strap buckle when using a helmet were less likely to report a near miss in the last month. Interestingly, participants who used headlights when driving at night showed a higher risk of near misses .When asked how road traffic safety for boda bodas could be improved, the most common suggestions were stricter enforcement of helmet usage , improved driver training for boda boda drivers , usage of reflective vests for drivers , increased awareness of boda bodas by other vehicles , and improved road conditions . Other suggestions included increased numbers of pedestrian walkways , improved traffic regulation , better road lighting , lower traffic volume , and the creation of a boda boda traffic lane . These suggested improvements can be grouped into four main themes: improved roadway infrastructure and traffic regulation, road user attitudes and safe driving behaviors, increased education and training, and stricter law enforcement .These suggestions center on the idea of creating a driving environment conducive to road safety, and point to the fact that Tanzania’s current roadway infrastructure is unsatisfactory to that end. Roads may be considered low quality due to either low design standards, poor workmanship, or both. Participants made such suggestions as increasing road lighting, which would improve boda boda visibility in low-light conditions, creating a designated boda boda traffic lane, which would reduce the likelihood of collisions with other road users, and creating more lanes to help decrease traffic volume. We define traffic volume as the count of vehicles passing by the stand during the data collection time. Responses that fell into this theme included, “Traffic lights should be installed on the roads,” “Roads should be better constructed because they have low quality,” “Roads are so narrow, so they should widen the roads,” and “Road signs should be visible.”These suggestions center on the idea of encouraging boda boda drivers to take responsibility for their own safety by adopting safer driving habits while avoiding risky driving habits. Suggestions included more consistent usage of helmets and reflective vests, careful and respectful driving, and the reduction of overall alcohol consumption in the population, all of which are factors of road safety within the driver’s control. Responses that were assigned to this category included, “Boda boda drivers should wear helmets all the time when they are on the road,” “Boda boda drivers need to put on the headlights during the day so they can be visible,” “Drivers should reduce speed and alcohol use during work hours,” and “Drivers should wear reflectors.”These suggestions center on the idea of educating road users how to drive safely and reduce their risk for injuries. Suggestions include safety seminars, improved boda boda training programs, and general education for all road users. Responses assigned to this category included, “Seminars to educate all kinds of drivers,” “Education should be provided [so road users would] drive in average speed,” “Frequent education to all drivers,” and “Education should be provided [to] all road users.”These suggestions center on the idea that stricter enforcement of existing traffic laws will increase adherence to those laws. The suggestions called for the inspection and verification of driving licenses, increased punishments and fines for violating traffic laws, and controlling police corruption. Responses that fit this theme included, “Police should fulfill their duties and stop being corrupt,” “Police should do their responsibility,” “Police should make sure that all drivers have a driving license,” and “[Boda boda] Drivers should be inspected for their driving license.”Road traffic injuries cost LMICs over $100 billion annually in both direct medical costs and lost wages . The preDue to the nature of their work, boda boda drivers are prone to high levels of fatigue and stress. A previous study found that boda boda drivers work incredibly long hours, which can result in the accumulation of physical fatigue; additionally sleepiness and inattention were named common crash causes by Tanzanian truck drivers . The stuNot only are boda boda drivers incentivized to work longer hours, but they are also incentivized to engage in riskier driving behaviors, such as speeding and weaving through traffic, which reduces gas consumption and allows boda boda drivers to serve more customers . SpeedinIn addition to the inherently risky nature of boda boda operation, the data show that the average boda boda driver has had less than three years of experience. Previous research has shown that inexperienced drivers are overconfident in their driving ability  and are Notably, most of the reported crashes occurred in the evening hours, when the glare of the setting sun may have impaired both the vision and visibility of the boda boda drivers. Previous research has shown that “periods of extreme sun glare reduce the ability to safely operate a motor vehicle,” particularly at intersections . SimilarAnother issue regarding boda boda visibility is the usage of conspicuity measures. One study found that usage of conspicuity measures such as reflective clothing, continuous headlight usage, and white helmets reduced the risk of crash by 37%, 27%, and 24%, respectively . In lighIn the event of a crash, boda boda drivers often suffer both physical and financial hardships. Our data show that the vast majority of injured crash victims do not receive the care they need; in the present study, only 23.7% of injured crash victims were hospitalized, and only 13.2% received any rehabilitation. While we were not able to compile the cost of treatment for our boda boda drivers, a tertiary hospital study in Uganda estimated the cost of medical care per injured patient to be roughly 300 USD, or the monthly salary of a registered nurse. This estimation excludes other expenses such as travel, food, medication, and loss of revenue . Crash vSurprisingly, despite the general consensus among boda boda drivers that helmets and helmet straps reduce the risk of injury in the event of a crash, only 73.3% of drivers reported wearing a helmet consistently and only 58.7% consistently wore chin straps. Regarding the quality of the helmets, many of the helmets were found to have cracks, dents, scratches, and broken chin straps. It is reasonable to assume that damaged helmets are unreliable and offer less protection in the event of a crash. Unfortunately, the data show that boda boda drivers are unlikely to replace their helmets when they are damaged. This is likely due to the cost of buying a new helmet, which is more than twice the amount boda boda drivers would ideally spend. Some helmets not only offer less protection, but may even increase the risk of a crash, as is the case with the many helmets featuring face shields that obscure driver vision with scratches, paint, or graphics.The survey data suggest four main areas of focus to increase road traffic safety, which reflect the commonly cited three E’s of road safety: engineering, education, and enforcement . The firSome limitations to this study should be acknowledged when evaluating our results. First, as this was a survey of active boda boda drivers, it is subject to a significant survivor bias. As such, we did not focus on injuries, since those with fatal injuries and severe life and job-threatening injuries would have been missed, but on safety and safety behaviors since our population was more likely to represent the best safety profile among the survivors. This survivor bias also limits our ability to make accurate disability, missed work, and lost wage estimates given more severe injuries were excluded; still, with our safer, surviving population, these estimates are conservative estimates. Secondly, our method, using self-report and personal opinion about road safety improvements, has intrinsic bias in self-reporting and understanding of road safety. Even in light of this, we found significant differences between knowledge and practice of safety behaviors highlighting that knowledge alone is insufficient for safe behavior. Similarly, many boda boda drivers cited external causes for crashes most likely due to a perspective bias, yet some participants did cite that alcohol use among boda boda drivers was a significant road safety concern. And while participants cite improvements like ‘need for road quality improvements,’ these suggestions should be taken with caution; while improved lighting has obvious safety impact, improved road quality has variable impact as it can be associated with increased speeds therefore more injuries .The present study demonstrates the high prevalence of traffic crashes and injuries that make boda boda driving an inherently risky occupation. Fatigue, stress, and inexperience can increase the risk of injury for boda boda drivers. Traffic crashes and injuries can cause both physical and financial hardship for the crash victim. Many intervention points can be leveraged to reduce the risk of injury and increase overall road traffic safety. Unfortunately, while boda boda drivers are aware of ways to improve safety, adherence to safety habits remains low. Successful interventions will bridge the gap between knowledge and practice of safety habits.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(CSV)Click here for additional data file.


Stroke remains a major global health problem. In China, stroke was the leading cause of death and imposed a large impact on the healthcare system. This study aimed to examine the hospitalization costs by five stroke types and the associated factors for inpatient costs of stroke in Guangzhou City, Southern China.This was a prevalence-based, cross-sectional study. Data were obtained from urban health insurance claims database of Guangzhou city. Samples including all the reimbursement claims submitted for inpatient care with the primary diagnosis of stroke from 2006 to 2013 were identified using the International Classification of Diseases codes. Descriptive analysis and multivariate regression analysis based on the Extended Estimating Equations model were performed.A total of 114,872 hospitalizations for five stroke types were identified. The average age was 71.7 years old, 54.2% were male and 60.1% received medical treatment in the tertiary hospitals, and 92.3% were covered by the urban employee-based medical insurance. The average length of stay was 26.7 days. Among all the hospitalizations (average cost: Chinese Yuan (CNY) 20,203.1 = $3212.1), the average costs of ischaemic stroke (IS), subarachnoid haemorrhage (SAH), intracerebral haemorrhage (ICH), transient ischaemic attack (TIA), and other strokes were CNY 17,730.5, CNY 62,494.2, CNY 38,757.6, CNY 10,365.3 and CNY 18,920.6, respectively. Medication costs accounted for 42.9, 43.0 and 40.4% of the total inpatient costs among patients with IS, ICH and TIA, respectively, whereas for patients with SAH, the biggest proportion of total inpatient costs was from non-medication treatment costs (57.6%). Factors significantly associated with costs were stroke types, insurance types, age, comorbidities, severity of disease, length of stay and hospital levels. SAH was linked with the highest inpatient costs, followed by ICH, IS, other strokes and TIA.The costs of hospitalization for stroke were high and differed substantially by types of stroke. These findings could provide economic evidence for evaluating the cost-effectiveness of interventions for the treatment of different stroke types as well as useful information for healthcare policy in China. As the second most common cause of death worldwide , stroke Stroke imposed a huge financial burden on healthcare systems in many countries, consuming about 2–4% of the total health care expenditures across the world . For exaMany developed countries have evaluated the direct medical costs associated with stroke, and they used either claims database or hospital discharge data to estimate the medical costs for different types of stroke separately , 8–12. IIn China, three studies examined the hospitalization costs of stroke –19. Wei Different from the previous literature in China - which either focused on only one or two stroke types or did not use a large-scale insurance claims data, this study aimed to examine hospitalization costs by five stroke types - 1) subarachnoid haemorrhage (SAH), 2) intracerebral haemorrhage (ICH), 3) ischaemic stroke (IS), 4) transient ischaemic attack (TIA) and 5) other strokes using the urban health insurance claims data from the largest city in southern China and investigate the factors that were associated with hospitalization costs.Data were obtained from the Urban Employee-based Basic Medical Insurance (UEBMI) and the Urban Resident-based Basic Medical Insurance (URBMI) claims databases of Guangzhou City for the years 2006 through 2013, which contained sociodemographic information, medical conditions and hospitalization costs based on actual payments to providers. The UEBMI (launched in 1998 covering urban employees) and URBMI (launched in 2007 covering the urban unemployed residents)  are two This was a cross-sectional study conducted in China to estimate the hospitalization costs of stroke. A retrospective, prevalence-based approach was used to determine the medical costs of inpatients with stroke. We obtained all the reimbursement claims submitted for inpatient care with the single primary diagnosis of stroke from 2006 to 2013 using the International Classification of Diseases (ICD) Codes ninth version (ICD-9 used for the years 2006 and 2007) and tenth version (ICD-10 used for the years from 2008 to 2013), which included SAH , ICH , IS , TIA  and other types of stroke  and out-of-pocket (OOP) payment from the enrolees. The total direct hospitalization costs were separated into laboratory and diagnostic costs, non-medication treatment costs, medication costs, bed fees and the costs of other services, including special caring fees and air-conditioning, based on the classification of costs used in the UEBMI and URBMI schemes. Laboratory and diagnostic costs were the costs of physical examinations and biochemical tests. Medication costs were divided into traditional Chinese medicine and Western medicine. Non-medication treatment costs referred to the costs for any other treatments except for medication, which included blood transfusions, surgery fees, anaesthesia charges, and medical consumables. All costs from 2006 to 2012 in this study were adjusted to 2013 Chinese Yuan (CNY) value using the urban resident’s consumer price index (CPI) of Guangzhou City . The excThe outcome variable in the analysis was total hospitalization expenditures per inpatient. The primary independent variable was types of stroke and measured in five categories according to the ICD codes described above: IS, SAH, ICH, TIA, and other strokes. Additional covariates included in the expenditure model were age, gender, insurance type, comorbidities, severity of disease (intensive care unit (ICU) admission, readmission in 15 days and referral from other hospitals), hospital levels , and length of stay (LOS), and years. The information on seven common comorbidities  was matched using personal identifiers with a chronic patient registry under the Outpatient Chronic Disease Program. If not matched, we assumed the patients had no comorbidities. In this study, there were 59,388 patients who had registered to this program.Those predictors of total hospitalization costs for patients with stroke were chosen in reference to the Andersen’s behavioural model  as the cGender was dichotomized as male vs female, and insurance type was dichotomized as UEBMI vs URBMI. Age was categorized into five groups: 18–44 years old, 45–64, 65–74, 75–79, 80 and older. We used three dummy variables as proxy measures for the severity of disease – whether having an intensive care unit (ICU) admission, having a readmission in 15 days, and having a referral from other hospitals. Hospital level was classified into three levels – primary, secondary and tertiary. The LOS was grouped into three categories: less than 10 days, 10–19 days, 20 days and longer. Comorbidities were measured as binary variables for the following conditions that are available in the registry – whether having a diagnosis of coronary heart disease, hypertension, diabetes, Alzheimer’s disease, Parkinson’s disease, mental disorder, chronic kidney disease. Years were measured as binary variables for controlling the impact of policy changes across the years.Descriptive statistics ) were used for demographic information and costs. Since the value of medical expenditure usually has a skewed distribution, the Kruskal-Wallis test was used to identify the differences in inpatient costs by types of stroke. To identify the predictors of total inpatient costs, the extension of generalized linear model (GLM) - the extended estimating equations (EEE) approach  was perfA total of 114,872 hospitalizations were identified , ICH (60.8%), TIA (52.0%) and others (52.5%), except for SAH among which 44.9% of the patients were male. SAH is a more severe and life-threating stroke and related with a higher percentage of ICU admission (2.2%) and referral rate (3.5%) than all other types of stroke included. Patients from the SAH subgroup were younger (60.3 ± 17.1) than those from the other four subgroups. On average, patients with ICH tended to have the longest LOS in hospitals (41.0 ± 73.2).P < 0.01). In addition, out-of-pocket (OOP) spending imposes a high burden to stroke patients. The OOP spending accounted for 24.2% of the total hospitalization costs. The OOP expenses among patients with SAH occupied the biggest proportion of total inpatient costs (31.6%) among five types of stroke.The mean total direct inpatient cost was CNY 20,203.1 (US $3212.1) for all stroke patients . When comparing cost composition across different types of stroke, medication costs accounted for the biggest proportion of hospitalization costs for patients with IS (42.9%), ICH (43.0%), TIA (40.4%). With regards to patients with SAH, the biggest proportion of total inpatient costs was non-medication treatment costs (57.6%).P < 0.001)  (Table P < 0.05). The ICU admission, hospital referral and readmission in 15 days were all significantly correlated with higher inpatient costs. Compared with patients with the URBMI scheme, the total inpatient costs of stroke were CNY 910.4 higher than patients with the UEBMI scheme (P < 0.01). Patients with a longer LOS and higher hospital levels  had significantly higher hospitalization costs (P < 0.01). We conducted the sensitivity analysis and found that the subgroup regression results on the cost differences across stroke types from the sensitivity analysis and our total sample regression results reported above were similar, which suggested that the assumption that some comorbidity information might be incomplete was not a concern since it did not significantly affect the cost estimates and did not threaten the robustness of the findings.Compared with TIA, the inpatient costs for patients with four other subtypes of stroke were significantly higher, CNY 27,497.1 (SAH), CNY 9655.8 (ICH), CNY 1959.1 (IS) and CNY 848.3 (other strokes), after controlling for other covariates , while SAH predicted the highest average inpatient costs, followed by ICH, IS, other strokes, and TIA. The types of stroke, insurance types, age, comorbidities, severity of disease, LOS and hospital levels were significantly associated with hospitalization costs of stroke. This is the first study using a large urban health insurance claims database from an entire city to estimate the costs of hospitalization for stroke and compare the inpatient costs among five different stroke subtypes in China.When comparing our results with findings in other countries, a large variation in costs are observed. The average hospitalization cost  reported in this study was much lower than that in other countries, compared to US$20,396 2008 price) in the United States , €9004 , followed by ICH CNY 38,757.6), other strokes CNY 18,920.6), IS CNY 17,730.5) and TIA CNY 10,365.3). The regression analysis in this study also suggested that compared with TIA patients, the inpatient costs among patients with SAH, ICH, IS and other strokes were significantly higher. The variation in costs of different subtypes of stroke might be attributable to disease severity and cost composition. Regarding SAH that was reported for the first time in China, the hospitalization cost was the highest, the same as previous studies reported in other countries [4.2, foll,365.3. T,757.6, o,920.6, I,730.5 anFurthermore, the type of health insurance was found to be a significant determinant for the hospitalization costs of stroke in the present study. We found that the UEBMI enrolees with stroke had higher mean costs for hospitalization than the URBMI enrolees for all stroke subtypes except for TIA, mostly because the UEBMI had a higher benefit level for its enrolees . There aRegarding age, younger patients with stroke had higher overall healthcare costs than their older counterparts. Wang et al. , togetheWith the help of three proxies  employed in this study to capture the patients’ state of stroke, the severity of stroke was positively related to the inpatient expenditures. The finding was consistent with the previous studies in China and countries outsides of Asia , 19. A hVariations in the costs of different types of stroke tend to be driven by the level of hospitals and LOS, with patients having medical treatment in tertiary hospitals and longer LOS being more likely to incur higher costs of care. Previous studies conducted in China and other countries also found that a longer LOS and hospitalization in tertiary hospitals were positive drivers of inpatient costs for stroke , 16, 17.There were some limitations in this study. First, the study analysed hospitalization costs. The costs of outpatient care, indirect costs due to loss of productivity and family members’ informal care were not examined since our dataset did not include this information. Thus, we likely underestimated the total medical expenditures of stroke in China. Second, clinical severity factors such as neurologic severity, an important predictor of costs, was omitted from the analysis because such data were not available in the claims data. However, we adopted three severity proxies so that stroke severity could be measured. Third, the study population was limited to urban enrolees under two major insurance schemes in one city of China and did not include rural enrolees and people who were not covered in the health insurance system because data were not available, from which the sample cannot represent the whole Chinese population. Fourth, the comorbidity information was linked to another chronic patient registry under the Outpatient Chronic Disease Program, and we had access to the patient registry of seven common chronic diseases . We did not have information on patients of other comorbidities and patients who had these chronic diseases but did not register to this program. Nevertheless, our sensitivity analysis suggested that doing this had no statistically significant impact on our cost estimates, and the missing information due to unmeasured comorbidities would not substantially alter the conclusion of this study. Fifth, the data did not allow us to identify the hospitalizations caused by first-ever or recurrent stroke. Finally, the dataset was a little bit old due to administrative restrictions on data availability, which may not reveal the stroke costs and differences at present. Further studies could consider using a more recent claims dataset, as well as including people covered by all types of insurance, comparing the costs of insured and uninsured patients, and examining the indirect costs for a more comprehensive evaluation of stroke costs.The costs of hospitalization for stroke were high and differed substantially by types of stroke. These findings could provide economic evidence for evaluating the cost-effectiveness of interventions for the treatment of different stroke types as well as useful information for healthcare policy in China.


Zika virus is an emergent pathogen that gained significant importance during the epidemic in South and Central America as unusual and alarming complications of infection were recognized. Although initially considered a self-limited benign infection, a panoply of neurologic complications were recognized including a Guillain–Barré-like syndrome and in-utero fetal infection causing microcephaly, blindness, and other congenital neurologic complications. Numerous Zika virus vaccines were developed, with nine different vaccines representing five different platforms entered into clinical trials, one progressing to Phase II. Here we review the current landscape and challenges confronting Zika virus vaccine development. The Zika virus, discovered in Uganda in 1947 . IsolateIn this paper, we provide a brief update of current progress in Zika virus vaccine development and explore the challenges to vaccine assessment and eventual licensure.Zika virus infection presents with a symptom complex consisting of a diffuse maculo-papular rash, fever, asthenia, myalgias, arthralgias, headache, and retroorbital pain. The frequency and degree of symptomatology has, however, varied between studies. A retrospective study immediately following the outbreak on Yap Island found that only 19% of survey participants reported symptoms consistent with Zika virus infection , a figurIn adults, the most common reported complication of Zika virus infection is a Guillain–Barré-like illness that had a prevalence of 0.24 cases per 1000 cases of infection in the French Polynesian epidemic . Of inteIn contrast, infection during pregnancy has been associated with fetal microcephaly and a number of other congenital illnesses including visual deficits, hearing disorders, neural calcifications, learning disabilities, and arthrogryposis that may affect as many as 30% children born to mothers infected during pregnancy ,19,20,21Zika virus can also be transmitted through sexual contact, first reported for a researcher returning from Senegal in 2008 . NumerouIn mice, Zika virus infection causes testicular atrophy and significantly decreases spermatic function and fertility rates ,29,30. IFemales may also excrete Zika virus RNA for extended times. A prospective study of five women showed that Zika RNA was detected in vaginal fluid for as long as 6 months and a month or more in three of the five . Murine Thus, while Zika virus infection is mildly symptomatic and self-limited for the vast majority of individuals, with infrequent neurologic complications in adults, vaccination of the general population may not be warranted. However, vaccination of females at or entering reproductive age and their male partners is prudent. As discussed previously, vaccination during pregnancy is non-ideal due to the time to generate protective immunity and unknown vaccine safety . The onehttps://www.paho.org/). The almost complete disappearance of Zika has created significant barriers to ongoing vaccine studies as attack rates have fallen below those need to meet reasonable sample sizes [In late 2015 and through mid-2016, global concern for this spreading epidemic was increasing, however, the experience in the two prior epidemics was predictive of the subsequent epidemiology in the Americas. The Zika virus outbreaks in Yap Island and French Polynesia were characterized by rapid onset and rapid resolution over a span of 3–4 months . The ZikThe WHO removed the designation of Zika a virus of global concern in 2018 as case rates have fallen. In March 2018, the WHO and the National Institutes of Allergy and Infectious Diseases (NIAID) convened a meeting of academics, representatives from industry, and regulators to discuss the approach to Zika virus vaccine development in an era of waning incidence . AlthougVaccine development against the Zika virus began in earnest in late 2015 following the reports of microcephaly in fetuses and infants in Brazil. Of note, the first demonstration of immunoprotection was as part of a 1953 study to define the ultrastructural characteristics of Zika virus, that found intramuscular vaccination of mice with infectious viral filtrates protected against cerebral infection .Poland et al. provide a comprehensive listing of almost 40 Zika virus vaccines in development as of 2017 . DiamondA total of three DNA vaccines have entered into human testing ,42, inclGLS-5700, a DNA vaccine encoding for the Zika virus prME genes designed as a consensus based on available Zika virus sequences through December 2015 , was theTwo additional DNA vaccines, based on the French Polynesian H/PF/2013 strain were developed as chimeras that included the JEV prM signal sequence followed by the Zika envelope (E) gene (VRC5283) or a similar construct with the terminal 98 amino acids of E, representing the stem and transmembrane regions, exchanged for the analogous JEV sequence (VRC5288) . Both vaThree inactivated vaccines have entered into clinical studies, of which clinical data for only ZPIV vaccine has been published . ZPIV isClinical trial data for the other vaccines has not yet been reported as of the date of this monograph, however, pre-clinical data has been reported for three candidate vaccines. An mRNA vaccine that incorporates the prM-E genes of a Micronesian strain of Zika virus was created incorporating with or without four-point mutations in the fusion-loop segment of the DII region of the envelope gene that abolished binding of antibodies directed against the fusion-loop region to reduce the potential risk for antibody-dependent enhancement of infection . ImmunizA number of animal models have been assessed for vaccine development and have been reviewed elsewhere . ImmunocThe logistical difficulties in pursuing standard vaccine evaluation have created significant interest in the possibility of controlled human infection models (CHIM). The conduct and planning for human challenge trials raises unique medical and ethical considerations. For Zika virus trials, one must balance the relative benign and self-limited infection experienced by the vast majority against the risk for less benign complications and transmission risks.As reviewed above, published studies provide estimates that Zika virus infection is relatively asymptomatic and self-limited for the majority of individuals, however, methodology in these studies varies widely. In adults, two sets of complications warrant consideration for a proposed CHIM study. As reviewed above, a Guillain–Barre-like syndrome occurs in approximately 1 of 5000 Zika virus infections , whereasTo address safety concerns, there has been work to develop attenuated viral strains deleted for potential neurotropic regions  with a gIn summary, Zika vaccine development continues with multiple candidate vaccines in clinical trials. Because of the significant decline in incidence, evaluation of vaccine efficacy is increasingly difficult. There has been renewed interest in animal model and human infection models of infection.


The protection of ZPG beads with gastro-resistant capsules (Eudragit® L 100-55) resulted in stability in both SGM and SIM; they disintegrated immediately in simulated colonic medium containing pectinolytic enzymes. MSKE-loaded ZPG beads were stable at 4, 25 and 45 °C during the study period of four months. The present study revealed that ZPG beads in enteric-coated capsules might be a promising carrier for delivering MSKE to the colon.Pectinate gel beads containing Thai mango seed kernel extract  were developed and characterised for the purpose of colon-targeted delivery. The MSKE-loaded pectinate beads were prepared using ionotropic gelation with varying pectin-to-MSKE ratios, MSKE concentrations, and concentrations of two cross-linkers . The formulated beads were spherical in shape and ranged in size between 1.13 mm and 1.88 mm. Zinc-pectinate (ZPG) beads containing high amounts of MSKE showed complete entrapment efficiency (EE) of MSKE (100%), while calcium-pectinate (CPG) beads demonstrated 70% EE. The  Mangifera indica L.), which belong to the family Anacardiaceae, grow in tropical and subtropical regions, and their components are commonly used in folk medicine to produce a wide variety of remedies , amidated or not, can form gel beads in a presence of divalent cations, such as Ca2+ and Zn2+ [2+ and Zn2+) to obtain Ca- or Zn-pectinate gel beads without the use of organic solvents and harsh ingredients as in the method described by Aydin and Akbuğa [in vitro in dissolution conditions mimicking gastric to colon transit to check their efficacy to target the colon.The objectives of this study were to develop and characterise pectinate gel beads containing MSKE for the purpose of colon-targeted delivery. Colon drug delivery has the major advantage of utilising the colon for systemic therapy where the residence time is more than 24 h . Moreoveand Zn2+ . Throughd Akbuğa . The res2+ and Zn2+). Intermolecular cross-links (known as the “egg-box” conformation) were instantaneously formed between the negatively charged carboxyl groups of the LM pectin molecules and the positively charged counter-ions. Consequently, the pectin droplets formed gelled spherical beads. This process is known as the ionotropic gelation method. Using this method, spherical beads were easily prepared, without the use of sophisticated instruments. In this study, MSKE was homogeneously dispersed in an aqueous solution of low methoxylated (LM) pectin and added dropwise to counter-ion solutions  at 6% (w/v) with a cross-linking time of 20 min to form stable and complete cross-linked pectin-Ca networks, as described by Bourgeois et al. [In this study, we have prepared pectin beads using optimal concentrations of pectin and calcium chloride  of the dried beads prepared with different formulation variables are shown in For MSKE-loaded CPG beads, the mean diameter was increased from 1.13 ± 0.05 mm to 1.63 ± 0.10 mm as the pectin:MSKE ratio was increased from 1:1 to 1.75:1. An increase in pectin concentration generally leads to an increased mean bead diameter. When beads were prepared with a constant ratio of pectin:MSKE at 1:1 but with increases in both concentrations from 4:4% to 7:7% (w/w), the bead sizes (1.13 ± 0.05 mm–1.54 ± 0.10 mm) were in the same range of the beads prepared with an increasing ratio of pectin:MSKE. These results indicated that bead size was independent of MSKE concentration but dependent on pectin concentration.2 from 6% to 10% w/v did not significantly (p > 0.05) change the mean diameter of the CPG beads (1.54 ± 0.10 mm–1.70 ± 0.10 mm). This finding was because excess free Ca in the medium was eliminated from the beads.It was found that an increase in the concentration of CaCl3COO)2); their size ranged between 1.86 ± 0.02 mm and 1.88 ± 0.01 mm. In this study, we found no significant differences (p > 0.05) in size ranges between ZPG beads and CPG beads (1.54 ± 0.10 mm–1.70 ± 0.10 mm) prepared with the same concentrations of pectin and MSKE (7% w/v).ZPG beads were prepared with a constant ratio of pectin and MSKE (1:1), at 7% w/v each, but with increasing concentrations of zinc acetate (Zn(CHThe SEM photographs  indicate2 concentrations from 6% to 10% w/v did not significantly (p > 0.05) change the MSKE EE (65%–70%) of the CPG beads. Based on the above findings, the CPG beads demonstrated the greatest EE of MSKE at 70% with concentrations of pectin and MSKE both at 7% w/v. The viscosity of the pectin and MSKE mixture at this concentration did not cause the needle to clog during the preparation of pectin beads. These appropriate conditions were therefore chosen for the preparation of the MSKE-loaded ZPG beads. 3COO)2 was used to prepare MSKE-loaded pectinate beads  and simulated intestinal medium (SIM) were carried out to determine the feasibility of different beads (CPG or ZPG) to protect against the release of MSKE in gastric and intestinal media from the upper part of the gastrointestinal tract and allow release specifically in the colon target. The p > 0.05) at 2 h of incubation. The majority of the entrapped MSKE (88%–95%) was already released from the CPG and ZPG beads in SGM after 2 h of incubation. Moreover, it was observed that the release of MSKE from these beads into SGM was not affected by the concentrations of the cross-linking agents (CaCl2 and Zn(CH3COO)2). These results may be attributed to a drastic and sudden swelling of the dried pectinate beads, which occurred after they were exposed to SGM. However, the beads apparently kept their structure intact due to the decreased solubility of pectinic acid, a product from the ion exchange in the SGM. p < 0.05), as shown in et al. [et al. [2+ and Na+ or K+ ions could occur, which might result in a swelled bead structure [2+ ions by phosphate ions present in the SIM changed the equilibrium towards this medium which further accelerated the disintegration of the pectinate bead structure [When the pectinate beads were incubated in SIM for 8 h, the percentage release of MSKE from the CPG beads was significantly different than from the ZPG beads 2 while only 3%–4% of MSKE was released from the ZPG beads prepared with 2, 4, and 6% Zn(CH3COO)2. This result implies that the ZPG beads prepared with 1% Zn(CH3COO)2 were inappropriate for colon-specific delivery of MSKE. In the presence of pectinolytic enzymes, the percentage release of MSKE occurred to a greater extent (25%) from the ZPG beads prepared with 2% Zn(CH3COO)2 compared to the 3% release of MSKE from the ZPG beads prepared with 4 and 6% Zn(CH3COO)2. These results indicated that the release of MSKE from the ZPG beads into SCM was dependent on the concentrations of pectinolytic enzymes in the medium and of Zn(CH3COO)2 used to prepare the beads. 3COO)2 after 5 h of incubation in SIM. Nevertheless, the pre-incubation of these beads in SIM contributed to the loosening of the gel-network structure in SCM where 57%–80% of the entrapped MSKE was released after 6 h of incubation. Because beads were disintegrated in SCM by the activity of pectinolytic enzymes, the pre-incubation of the beads in SIM would lead to an ion exchange between Zn2+ and Na+ or K+ from SIM. As a result, the egg-box structure of the beads would loosen and form soluble pectin regions, which are more permeable to pectinolytic enzyme access, resulting in the degradation of the pectinate network.It is well known that the oral dosage formulations must pass through the small intestine before reaching the colon. To mimic the GI tract conditions, the release of MSKE from MSKE-loaded ZPG beads was investigated by pre-incubation in SIM for 5 h before transfer to SCM containing pectinolytic enzymes (650 U/mL) for the next 6 h of incubation at 37 °C. ® L 100-55 and containing the ZPG beads prepared with 2% Zn(CH3COO)2 were resistant to SGM without disintegration of the beads or the release of MSKE after 2 h of incubation  after storage for 4 months at the three different temperatures studied . This fiTM OF400, DE from 27% to 32%) was obtained from Cargill . Calcium chloride dihydrate and ethylenediaminetetraacetic acid (EDTA) were obtained from Acros Organics . Zinc acetate dihydrate, pepsin, pancreatin from porcine pancreas, pectinolytic enzymes , which is a mixture of pectinases from Aspergillus aculeatus, and other simulated fluids constituents  were purchased from Sigma-Aldrich . Eudragit® L 100-55 (methacrylic acid-ethyl acrylate copolymer) was obtained from Evonik . Pentagalloylglucopyranose  was obtained from Endotherm GmbH . Purified water (18.2 M Ω cm at 25 °C) was obtained from a Millipore Synergy® ultrapure water system . High-performance liquid chromatography (HPLC) grade solvents were obtained from Fisher Scientific . Simulated digestive media were prepared according to the USP28-NF23 (2005) as follows: Low methoxylated (LM) non-amidated pectin (UnipectineSimulated gastric medium (SGM) was prepared by dissolving 2 g of NaCl and 3.2 g of purified pepsin in 7 mL of HCl and sufficient water to make 1,000 mL. The final pH was 1.2.Simulated intestinal medium (SIM) was prepared by dissolving 6.8 g of KH2PO4, 0.4% (w/v) Tween® 80, 77 mL of 0.2 N NaOH and 10 g of pancreatin in sufficient water to make 1,000 mL. The pH was adjusted to pH 6.8 with 0.2 N NaOH.Simulated colonic medium  was prepared by dissolving 6.8 g of KH2PO4, 0.4% (w/v) Tween® 80, 77 mL of 0.2 N NaOH and 62.18 mL of pectinolytic enzymes (10454 U/mL) in sufficient water to make 1,000 mL. The pH was adjusted to 6 with concentrated HCl solution. The SCM without pectinolytic enzymes was prepared in the same manner.Mangifera indica L. cultivar ‘Fahlun’, Anacardiaceae) were purchased from a local market. The seed kernels were removed and extracted following the method described by Nithitanakool et al. [Fully grown, unripened Thai mango fruits  with the Spectra System SN4000 unit. The Gemini-NX column  with a guard column was used as a stationary phase. The elution gradient for the HPLC analysis consisted of two solvent compositions: 0.48% phosphoric acid in a mixture of 80/20 (v/v) water/acetonitrile (solvent A) and 0.48% phosphoric acid in a mixture of 70/30 (v/v) water/acetonitrile (solvent B). Total running time was 42 min, and the gradient program was set as follows: 100% A (5 min), 100% A to 38.4% A (22 min), 38.4% A to 100% A (15 min). The flow rate was 1 mL/min at room temperature (25 °C). The samples  were dissolved in dimethylsulfoxide (DMSO), and the sample injection volume was 20 µL. UV detection was carried out by monitoring the absorbance signal at 280 nm. Identification of PGG in MSKE was based on the comparison of the retention time and UV spectrum of the unknown peaks with the reference standard, PGG  equipped with a P1000XR pump, a SCM1000 vacuum degasser, an AS3000 autosampler and a UV6000 LP detector. The data were recorded and analysed with Chromquestard, PGG . The amo2 dihydrate and Zn(CH3COO)2 dihydrate as cross-linking agents [2+ and Zn2+ retained in the beads. Then, these beads were dried at 37 °C for 5 h in an oven. The beads were prepared by differing the formulation variables 2 concentration) as follows:The MSKE-loaded pectinate beads were prepared by ionotropic gelation using CaClg agents ,17,18. B2 constant at 4 and 6% w/v, respectively.(i) Varying the pectin-to-MSKE ratios , while keeping the concentrations of MSKE and CaCl2 concentration (6% w/v) constant.(ii) Varying the MSKE concentration , while keeping the pectin-to- MSKE ratio (1:1 w/w) and CaCl2 concentration , while keeping the pectin and MSKE concentrations constant at 7% w/v(iii) Varying the CaCl3COO)2 concentration , while keeping the pectin and MSKE concentrations constant at 7% w/v.(iv) Varying the Zn. In each batch, thirty beads were randomly selected for the study. After images of the beads were captured through a digital camera (Moticam 2300) connected with a microscope, the length and breadth of each bead were measured using the pre-calibrated image analysis program . The size of each bead was calculated from the average of these two dimensions [The size and shape of the dry beads were measured using an optical microscope , and images were recorded at magnifications of 54× and 130×.ent) was calculated from the content analysis of PGG in MSKE (Bcont):rel = amount of PGG released from the beads into the supernatant, and Bcont = amount of PGG content in MSKE (100 mg of PGG per g of MSKE).Ten beads (Ca- or Zn-pectinate network) were dissolved in 10 mL of 1% w/v EDTA aqueous solution with continuous stirring by a magnetic stirrer. DMSO (3 mL) was added to the system to ensure solubilisation of MSKE and the mixture was mixed well using a magnetic stirrer. The mixture was centrifuged at 10,000 rpm to remove pectin. An aliquot (1 mL) of the supernatant was diluted with DMSO (5 mL). PGG content in the supernatant was determined by HPLC. The actual amount of PGG present in the beads was calculated from the calibration curve constructed by plotting various concentrations of the reference standard PGG in DMSO (0.5–32 µg/mL) versus the peak areas. The linear correlation coefficient was 0.9999 for the line. The actual amount of MSKE entrapped in the beads  containing 25 mL of releasing medium (preheated at 37 °C). For each releasing medium, separate bottles were employed for withdrawing samples at each specified time interval during the study periods.The 3COO)2 . The coating of the capsules was performed by dipping and drying capsules 5 times in an acetone solution containing 10% (w/w) Eudragit® L 100-55. The coated capsules containing pectinate beads were first incubated for 2 h in SGM bottles before being transferred into SIM bottles and were then incubated until the disintegration of coated capsules was observed. The beads were further incubated in SIM for 5 h before being transferred into SCM containing pectinolytic enzymes for the next 6 h of incubation. At each incubation period, samples were taken at specified time intervals for PGG content analysis.In the first set of experiments, beads were placed in SGM bottles for 2 h in a water bath with continuous tangential stirring using a magnetic stirrer. Samples (2 mL) were withdrawn at each specified time interval  and centrifuged at 10,000 rpm for 5 min before analysing the PGG content by HPLC. Similarly, for the second and the third sets of experiments, the PGG-release tests of beads in SIM and SCM bottles were carried out during incubation periods of 8 h and 13 h, respectively. For the fourth set of experiments, beads were first incubated for 5 h in SIM bottles before being transferred into SCM bottles containing pectinolytic enzymes (650 U/mL) for the next 6 h of incubation. The last set of experiments was carried out with gastro-resistant coated capsules (size 0) containing MSKE-loaded ZPG beads prepared with 2% Zn. Ai was the initial amount of MSKE entrapped in the beads (~8 mg). All of the above experiments were performed in triplicate.The PGG content in each releasing medium was calculated from the calibration curves constructed by plotting various concentrations of the reference standard PGG in SGM (4–64 µg/mL), SIM (4–96 µg/mL) and SCM containing pectinolytic enzymes (8–96 µg/mL) versus the peak areas. The linear correlation coefficients were 0.9996, 0.9996 and 0.9999 for the lines in SGM, SIM and SCM, respectively. The percentage release of MSKE was calculated by the following equation:3COO)2 were transferred into 5 mL glass vials with rubber stoppers  and were stored at three different controlled temperatures: cold (4 °C), room temperature (25 °C), and accelerated temperature (45 °C) for 4 months (120 days). On day 121, ten beads from the vials at the different temperatures were used to measure MSKE EE by using the same procedure described in the EE study. Stability was assessed by comparing the initial EE with that obtained after storage for 4 months at each temperature. All samples were performed in triplicate. The MSKE-loaded ZPG beads prepared with 2%, 4% and 6% of Zn(CHp < 0.05) among the corresponding mean values were determined by using one-way analysis of variance (ANOVA) followed by Tukey’s pairwise comparison test. SPSS 14.0 for Windows was used for statistical analysis.All data were expressed as the mean value ± SD. Significant differences  resulted in stability against both SGM and SIM but disintegrated immediately in SCM containing pectinolytic enzymes. MSKE-loaded ZPG beads were stable at all temperatures  during the study period. The results obtained implied that ZPG beads may be a promising carrier for colon-targeted delivery of MSKE.For formulations containing high amounts of MSKE at pectin:MSKE = 1:1, ZPG beads showed higher entrapment efficiency of MSKE than CPG beads. The


Late-life depression is a leading cause of disability in older adults and is associated with significant economic burden. This article draws from the existing literature and publicly available databases to describe the relative importance of the indirect costs associated with late-life depression. The authors found that unpaid caregiver costs represent the largest component of the indirect costs of late-life depression, with the highest level of economic burden attributed to the majority of care recipients who have fewer depressive symptoms. Other indirect costs, such as productivity losses related to early retirement, reduced ability to fulfill work and family functions and diminished financial success were mostly under-appreciated in the literature. Also, mortality cost estimates provided little clarity, employing variable methodologies and revealing mixed results. With respect to late-life suicide studies, studies approximated both economic costs and savings. More rigorous efforts to evaluate the indirect costs of late-life depression would afford a better understanding of the social and economic toll of this disorder and could influence the allocation of resources for research and treatment. Late-life depressive disorders are prevalent and associated with substantial economic burden. Approximately 10%–15% of older persons in primary care settings suffer from a depressive illness , a condiAs the population of older adults in the United States expands, it is possible, if not likely, that the total costs associated with late-life depression will increase as well. Estimates of the economic burden of a disease to society as a whole have relied on cost-of-illness studies that employ a wide range of methodologies. The principal aim of these cost-of-illness studies is to inform resource allocation by policymakers. Numerous reports have demonstrated high direct costs associated with late-life depression, arising mainly from the increased utilization of medical resources across healthcare settings ; other direct costs include expenses for pharmaceuticals and related but non-medical resources  ,16,17. HIndirect costs of late-life depression are defined as productivity losses due to illness-associated morbidity and mortality that are endured by the individual, family, employer, and society . IndirecThe authors conducted a comprehensive literature review of peer-reviewed journal articles published in English using the databases PubMed, Web of Science, Google Scholar, and CDC.gov. The search was conducted without time limit, and the date last searched was 9 July 2016. Bibliographies of the articles obtained in the initial search were examined to identify additional relevant studies. Combined searches using the following terms were performed: “cost” or “economic burden” or “cost-of-illness” or “indirect cost” or “caregiver burden” or “mortality costs” or “productivity loss” and “late-life” or “geriatric” or “elderly” or “older adult” and “depression” or “depressive disorder”. To be included in the review, the selected studies were required to present a methodology section sufficient for evaluation and to include a monetary estimate of an indirect cost of late-life depression. Publications were excluded if they did not address late-life depression, if they assessed the burden of late-life depression in non-monetary terms, if they focused only on direct costs, or if they were derived from a non-United States population. Costs in all studies were reported in US dollars and then inflated to the year 2016.The combined search generated 1480 non-duplicate journal articles. Of these, 1436 records were excluded after initial screen of abstracts and 44 full texts were assessed for eligibility. Thirty-eight articles were excluded because they did not provide a monetary estimate of the indirect cost of late-life depression, and two articles were excluded because they were derived from a non-United States population. Only four articles provided a monetary estimate of an indirect cost associated with late-life depression in the United States see , with thFew articles specifically examine productivity losses associated with late-life depression such as those attributable to early retirement , reducedProductivity losses among older adults may be under-estimated in part due to methodological barriers. Cost-of-illness studies most often employ the human capital method as a proxy for estimating a healthy individual’s future contributions to a society. To the extent that these future contributions are sacrificed by illness or disability, the human capital method offers an indirect measure of productivity losses . This apThe cost burden of depressed individuals’ family members is significant. Depressive symptoms adversely impact older adults’ independent functioning, and are consequently associated with increased levels of unpaid caregiving from family and friends . In factHowever, the cost of $12.7 billion year-2016 dollars estimated by Langa et al. , were noThere are several studies that estimate the cost of suicide, using the general approach of combining the direct costs of medical expenditures with the indirect costs of the net present value of productivity losses ,22,23 s. The CenThese calculations should be interpreted with caution because of their several limitations. Most notably, the cost estimates are based on an assumption of average productivity across different demographics, and do not subtract for the diminished productivity of depressed individuals. In fact, suicide as an outcome of late-life depression may result in economic savings for a society via the avoidance of future illness-related direct and indirect costs . This puThis article delineates the indirect costs associated with late-life depression as described in the medical literature. Unpaid caregiver costs represent the largest component of the indirect costs, with the highest level of economic burden attributed to the majority of care recipients who have fewer or diagnostically subthreshold depressive symptoms. Work loss productivity costs in depressed older adults are under-appreciated, particularly in the “young old” population (aged 65–74) in which many individuals remain engaged in the workforce. Finally, mortality cost estimations reveal mixed results, with studies approximating both economic costs and savings associated with late-life suicide ,22,23,35There are several limitations to this analysis. First, studies that include estimations of indirect costs associated with late-life depression are scarce, and those available use varying methodologies, thereby confounding the tasks of comparison and interpretation. Furthermore, of the studies that do include indirect cost estimations, the calculations mostly neglect the value of leisure. A methodological approach that incorporates the intrinsic value of life, such as a willingness-to-pay approach, may provide a better approximation of the morbidity costs of late-life depression .The analysis in this article suggests that caregiver costs represent the largest component of the indirect costs associated with depression in older adults, in contrast to findings in the general population in which workplace costs represent the largest component of the indirect costs of depression . Further


We isolated Getah virus from infected foxes in Shandong Province, eastern China. We sequenced the complete Getah virus genome, and phylogenetic analysis revealed a close relationship with a highly pathogenic swine epidemic strain in China. Epidemiologic investigation showed that pigs might play a pivotal role in disease transmission to foxes. Alphavirus, family Togaviridae) is a mosquitoborne RNA virus that causes death in young piglets, miscarriage in pregnant sows, and mild illness in horses . No We used supernatants of homogenized brain and lung tissues from each dead fox to inoculate Vero cells, as described previously (https://blast.ncbi.nlm.nih.gov/Blast.cgi) identified the sequence as that of GETV. During the investigation, farmers reported that the foxes had been fed on organs from symptomatic pigs. We therefore tested for the presence of African swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, Japanese encephalitis virus, porcine circovirus type 2, porcine circovirus type 3, porcine cytomegalovirus, and alphavirus by using the primers for those viruses  Table 2.,2 = 0.952; p<0.01).To further investigate the epidemic GETV infection, we performed quantitative RT-PCR by using RNA from all fox samples, as described elsewhere , Table 3.We obtained the complete genome of the novel GETV SD1709 strain (GenBank accession no. MH106780) by using a conventional RT-PCR method  Table 4.Culex tritaeniorhynchus, Anopheles sinensis, and Armigeres subalbatus) collected in the same region. The infection rate in pigs detected by quantitative RT-PCR was 20.0% (4/20) and by serum neutralization was 75.0% (15/20). The minimum infection rate in mosquitoes was ≈1.09%; C. tritaeniorhynchus mosquitoes had a higher minimum infection rate (2.31%) compared with other mosquito species (0–0.80%). These results suggest that pigs and C. tritaeniorhynchus mosquitoes might play a role in transmitting highly pathogenic GETV to captive foxes in this region (We also detected GETV infection in pig serum samples and in mosquitoes (,In China, the disease caused by GETV has only been reported in pigs in Hunan Province, although the virus has been detected in mosquitoes in >10 provinces (Additional information regarding highly pathogenic swine Getah virus in blue foxes, eastern China, 2017.


Indonesia has one of the fastest growing HIV epidemics in Asia, which mainly concentrates within risk groups. Several strategies are available to combat this epidemic, like outreach to Men who have Sex with Men (MSM) and transgender, Harm Reduction Community Meetings (HRCMs) for Injecting Drug Users (IDUs), and Information, Education and Communication (IEC) programs at Maternal & Child Health Posts (MCHPs). Reliable cost data are currently not present, hampering HIV/AIDS priority setting. The aim of this study thus is to assess the societal costs of outreach programs to MSM and transgender, HRCMs for IDUs and IEC at MCHPs in Bandung, Indonesia in 2016.The societal costs were collected in Bandung from April until May 2017. Health care costs were collected by interviewing stakeholders, using a micro-costing approach. Non-health care costs were determined by conducting surveys within the target groups of the interventions.The societal costs of the outreach program were US$ 347,199.03 in 2016 and US$ 73.72 per reached individual. Moreover, the cost of HRCM for IDUs were US$ 48,618.31 in 2016 and US$ 365.55 per community meeting. For the IEC program at MCHPs, US$ 337.13 was paid in 2016 and the cost per visitor were US$ 0.51.This study provides valuable insights in the costs of outreach to MSM and transgender, HRCMs for IDUs and IEC at MCHPs. Policy makers can use these results in setting priorities within Indonesia. Data on effectiveness of interventions is necessary to make conclusive statements regarding cost-effectiveness and priority of interventions. With one of the fastest growing incidence rates in Asia and a total of 690,000 people living with HIV, HIV is a big problem in Indonesia –3. DespiSeveral strategies are available to bring a halt to the HIV epidemic in Indonesia, including outreach, harm-reduction community meetings (HRCMs) and Information, Education and Communication (IEC) programs. Outreach focuses on reaching hidden populations of HIV risk groups to engage them in the process of reducing HIV risk behaviors . HRCMs aAlthough many HIV/AIDS interventions are available, not all of them can be implemented. In fact, when implementing all HIV interventions described in the strategy of Indonesia in 2014, an amount of US$ 208 million would have been required, whereas only US$ 97 million was available .One important criterion considered in an MCDA, is cost-effectiveness. Determining this criterion requires comprehensive and reliable data on the costs of implementing HIV/AIDS interventions. This type of data is however scarce and highly dependent on the specific type and setting of a program. Thus, a reliable cost analysis of all available HIV/AIDS interventions in Indonesia is needed. This study therefore aims to assess the societal costs of outreach programs to MSM and transgenders, harm-reduction community meetings for IDUs, and information, education and communication programs at maternal and child health posts in Bandung, Indonesia in 2016.This study was conducted in Bandung, the capital of West Java province. The HIV/AIDS epidemic that Bandung is facing is a concentrated epidemic comparable to the national estimate .The outreach to both MSM and TG living in Bandung is conducted by a non-governmental organization (NGO) in Bandung, called Srikandi Pasundan (SP). The outreach program of SP is focussing on informing these key populations about HIV/AIDS and referring them to HIV testing facilities. The program is performed 26 times a month by both paid workers and volunteers, who visit HIV hotspots like malls, parks, and gyms and have a chat, hand out information packages, or refer individuals at risk to HIV testing facilities.The HRCMs in Bandung are organized by a NGO called Grapiks for IDUs from around Bandung with the main goal of increasing IDU’s knowledge about the effects of drug use. Several topics are discussed, including HIV/AIDS, tuberculosis, hepatitis, legal issues of drug use, and sterilization of needles. The meetings are provided by staff members of Grapiks and community health centres  at either community-health centres (11 times a month) or HIV/AIDS clinics (once a month).Maternal & child health posts (MCHPs)  are monthly-organized health facilities in Indonesia, where pregnant women or mothers with their children can come for a health check. Besides health measurement, the MHCP also offers IEC about a wide range of health topics, among which HIV/AIDS. The HIV/AIDS IEC program consists of a monthly IEC stand and an IEC presentation, which is organized three to four times a year. The program is coordinated by Warga Peduli AIDS (WPA), a civil society organization concerned with HIV/AIDS, with each district having one or more WPA volunteers who provide the program.Data collection took place in April and May 2017 and focused on the costs made in 2016. Costs were estimated from a societal perspective according the World Health Organization’s (WHO) guidelines for cost analysis in primary health care . We madeData on health care costs were collected by interviewing stakeholders involved in the program, being staff from the coordinating organization  or staff from the Bandung Aids Commission (KPA Bandung). Written informed-consent was obtained prior to all interviews with these stakeholders and participants received a reimbursement of IDR 100.000 for their participation afterwards. Health-care costs were further divided into capital costs and recurrent costs. Capital costs were defined as costs incurred for resources that last longer than one year. Recurrent costs were defined as costs incurred for resources that are purchased regularly. All inputs related to the programs were identified, classified, and quantified using a micro-costing approach .Capital costs were estimated annually with a discount rate of 3% . Annual Recurrent costs were calculated by multiplying the costs of a resource unit by the yearly quantity of usage of the resource unit. Data on quantity of usage were based on documented data or expert opinion alternatively. Costs of a resource unit were based on documented data, salary registers, market prices or expert opinion.Household costs were the only included non-health care costs and were further divided into productivity loss costs and travel costs. Productivity loss costs were defined as the income that the visitors miss because of spending their time at the program and were based on the value of their leisure time. Data on these subjects were obtained with a survey conducted among 16 MSM and 13 TG of the outreach program, 23 IDUs visiting the HRCMs and 35 visitors of the MCHPs  with a plausible 30% uncertainty range being applied. Assumptions included for outreach were duration of meetings, number of referrals and distance travelled, whereas expert opinion and duration of meetings were included for HRCMs. For the IEC program, assumptions on the value of leisure time of WPA volunteers and unemployed visitors were analysed.In 2016, the SP outreach program reached 4500 MSM and 210 TG, resulting in a total number of 4710 people being reached. When looking at HRCMs, a total number of 133 meetings were conducted in 11 community-health centres and one HIV/AIDS clinic. The HIV/AIDS IEC stand was visited by 8 people a month and each presentation was visited by 22 people on average. The average yearly number of visitors per MCHP of both the stand and the presentation was 641.The societal costs paid in 2016 for all programs and the share of total costs for all categories are shown in The costs for reaching out to one individual every week during the entire year of 2016, were US$ 73.72. HRCMs costed US$ 365.55 per meeting and the societal costs for providing IEC to one visitor were US$ 0.51. Non-health care costs accounted for the biggest share in total costs in all three programs, followed by personnel costs for outreach and IEC, and by transport cost for the HRCMs.The additional upscale scenario of the IEC program at MCHPs showed that when intensifying the frequency of the presentation from three to four times a year to 12 times a year, the annual societal costs of the program will become US$ 788.76, which is US$ 451.63 more than in 2016. Moreover, the societal costs paid for providing the IEC program to one visitor, will become US$ 0.47, compared to US$ 0.51 in 2016.A 15% over- or underestimation in duration of outreach resulted in a deviation of US$ 5.18 (7.0%) in reaching out to one individual and US$ 24,396 in societal costs. Both number of referrals and distance travelled seemed to have no significant effects on costs. For HRCMs, a deviation of 15% in values estimated by experts resulted in an in- or decrease of societal costs with US$ 4,275.00 (8.8%). A 15% and 30% decrease of the duration of HRCMs resulted in a decrease of the societal cost of respectively US$ 3,589 (7.4%) and US$ 7,179 (14.8%). In the societal costs of IEC, a 15% over- and underestimation of the value of leisure time of unemployed volunteers and visitors resulted in a US$ 18.88 and US$ 0.03 deviation of total costs and social costs per visitor respectively.This study examined the societal costs of outreach to MSM and TG, harm reduction community meetings for IDUs and information, education and communication programs at maternal and child health posts in 2016. The societal costs for the interventions in 2016 were US$ 347,199, US$ 48,618 and US$ 337.13 respectively. Besides, this study discovered that the costs for reaching out to one MSM or TG for one year were US$ 73.72. For organizing one harm reduction community meeting, US$ 365.55 had to be paid. Besides, the costs for providing IEC to one visitor of the MCHP were US$ 0.51.For both the outreach- and IEC program, the second largest cost category was personnel. Despite being a big cost item of the SP outreach program, saving on personnel costs should be done with caution as it might reduce the effectiveness of the intervention. Cutting on personnel would likely result in outreach workers being less able to really take time for their clients and by means of that affect the special bond between workers and clients. Saving costs of personnel, or in fact any cost category of IEC, does not seem very preferable as costs are already low and savings could hamper effectiveness.Transport costs could be a more suitable category for cutting costs in both outreach and HRCMs. These costs are mostly determined by transportation fees given to either outreach workers or participants of HRCMs. These fees are often higher than the transport costs that are actually made. Adjusting this system of transportation fees in a way that fees approximate the actual money spent, seems therefore a reasonable way of cutting costs. For the HRCMs however, reducing this transportation fee might make the HRCMs less attractive for the IDUs to visit and might therefore reduce the effectiveness of the program. Savings should thus be done with caution.Productivity loss costs formed a large component of the overall societal costs in all three interventions, eliciting an ongoing economic debate on the value of leisure time. In order to gain a comprehensive societal cost overview, we chose to base visitors’ leisure time values on market wage rates. Although this decision largely determined the productivity loss costs, the financial burden on visitors should not be neglected when adopting a societal perspective.The low annual frequency combined with the expressed desire by IEC visitors to increase the intensity of the IEC program, raised questions about the costs of intensifying the program. Upscaling IEC resulted in an extra amount of US$ 451.63 to be paid annually. However, when considering the costs per visitor, it was shown that the upscale scenario would be as efficient as current practice, paying US$ 0.47 per visitor compared to US$ 0.51 in current practice. Nevertheless, the question remains whether the increase in annual societal costs is affordable. Upscaling of outreach and HRCMs was deemed irrelevant, since these interventions were already implemented on a frequent basis and large scale.Although the methods of our study were largely based on the guidelines as described in the WHO manual for cost analysis in primary health care , this stFirst, some cost components were solely based on expert opinion as this was often the only way of calculating costs in absence of documented data. Prices that were the result from expert opinion were however checked with local market prices or follow-up interviews if possible and, for HRCMs, expert opinion was included in the sensitivity analyses to show influences on outcomes.Secondly, the cost analysis of outreach and HRCMs were both conducted for one single organisation in Bandung, meaning conclusions on costs can only be drawn for this single organisation. It is however expected that the way outreach and HRCM activities are undertaken in Bandung is comparable to other regions. The results of this study might therefore be considered a rough cost estimate of other comparable programs in Indonesia. This does not apply to the IEC program as this was done for an organization which implements the program throughout the whole country. The small sample size of three MCHPs in this study however means extrapolation should be done with caution as well.Thirdly, the non-health care costs calculations were based on questionnaires filled in by 16 MSM and 13 TG, 23 IDUs, and 35 women visiting MCHPs meaning non-health care costs found in this study might not represent the health care costs of the entire population.Lastly, assumptions needed to be made to complete the cost analysis. This might cause a deviation from the true costs if assumptions were not in line with reality. However, sensitivity analyses showed the influence of over- or underestimations of the biggest assumptions being made is low (<10%).No other cost analyses of these specific HIV/AIDS interventions in Indonesia have been done so far, hampering any comparison to other results in similar settings. A comparable micro-costing study in the African republic of Benin however reported the costs of outreach through peer educators to be 38,95 US$ per person reached . These cMore importantly however, only three out of the many available HIV/AIDS interventions in Bandung were included in this study . SeveralMCDA however often considers cost-effectiveness instead of costs, meaning data on effectiveness of all interventions should be available as well. In contrast to cost data, there is hardly any data on effectiveness of interventions meaning studies determining the effectiveness of all interventions should be conducted as well as soon as possible.Additionally, Siregar et al. showed that support of peers/family is important in avoiding loss-to-follow-up of HIV/AIDS patients . EncouraLastly, other studies support our finding that scaling up of a HIV/AIDS intervention within Bandung could prove to be a valuable step with low additional costs , 26.The total societal costs of outreach to MSM and Transgender, HRCMs to IDUs, and IEC at MCHPs are US$ 347,199, US$ 48,618, and US$337.13 respectively. It can be concluded that the IEC program is a low-cost program and that costs of the outreach program are relatively high with the HRCMs being not the most expensive nor the cheapest program.As this study was performed to inform the currently ongoing MCDA process in Bandung, our first recommendation is to combine our cost data with results on the effectiveness of the interventions and use them for this process. Similarly, cost and effectiveness data of other HIV/AIDS interventions in Bandung should be included in the MCDA as well.Considering costs and the contribution of each cost category to the societal costs, a few recommendations can be made as well. First, it is recommended to critically examine and possibly revise the system of transportation fees for both outreach workers and participants of HRCMs. The effectiveness of both programs should however always be in scope to e.g. prevent the scenario in which less clients will come to HRCMs. As the costs of the IEC program are already relatively low, it is not recommended to reduce any cost category. In fact, we recommend to implement the upscale scenario as you reach more visitors and by means of that possibly increase the effectiveness of the program. Before doing so, it should however be investigated if any organization involved in the program is willing and/or able to fund the upscaling of IEC presentation.S1 Appendix(DOCX)Click here for additional data file.


Correction to: Health Economics Review (2018) 8:28https://doi.org/10.1186/s13561-018-0212-8Following publication of the original article , the autIn Tables Subsequently, the following sentences needed to be corrected. Corrected content is shown in bold:$4.8 billion. Hospital episodes accounted for 80% of the healthcare expenditure, followed by PBS (13%) and MBS (5%).The total initial cost associated with newly diagnosed cancer for the healthcare system is The words “for hospital episodes (22%)” have been removed from the sentence].Indigenous Australians had significantly higher costs for ED presentations (23% higher), but significantly lower costs for MBS rebates (8% lower), PBS rebates (18% lower), and patient co-payments (61% lower) compared to non-Indigenous Australians. .The costs for hospital episodes were significantly higher in the first 12-months post-diagnosis with increasing remoteness, quintiles 4–5 had decreasing costs associated with hospital episodes [quintiles 3–4 were previously reported].Compared to those living in the most disadvantaged areas (IRSD Q1), those in


Eonycteris spelaea bats showed similar patterns of cross-reactivity as human samples, suggesting them as the species responsible for the spillover. In contrast, sera from Rousettus leschenaultii bats reacted to two different virus glycoproteins. Our results indicate circulation of several filoviruses in bats and the possibility for filovirus transmission from bats to humans.Bats are reservoirs for several zoonotic pathogens, including filoviruses. Recent work highlights the diversity of bat borne filoviruses in Asia. High risk activities at the bat-human interface pose the threat of zoonotic virus transmission. We present evidence for prior exposure of bat harvesters and two resident fruit bat species to filovirus surface glycoproteins by screening sera in a multiplexed serological assay. Antibodies reactive to two antigenically distinct filoviruses were detected in human sera and to three individual filoviruses in bats in remote Northeast India. Sera obtained from  Focused virus surveillance at human-wildlife interfaces enables proactive detection of potentially epidemic pathogens. Filoviruses, including ebolaviruses and marburgviruses, are pathogens with epidemic potential. They were previously detected in bats and have caused disease outbreaks in humans with a high case fatality rate. Here, we tested sera obtained from bats and humans at a high-risk interface for the presence of filovirus reactive antibodies. Human participants were engaged in annual bat hunts, possibly exposing them to bat-borne viruses. We report the exposure of humans to filoviruses that were likely derived from the two sampled bat species. The bats contain antibodies raised to presumably three distinct filoviruses. Our findings suggest bats in South Asia act as a reservoir host of a diverse range of filoviruses and filovirus spillover occurs through human exposure to these bats. Ebolavirus: Ebola virus, Bundibugyo virus, Taï forest virus, Sudan virus, Reston virus and Bombali virus; Marburgvirus: Marburg virus and Ravn virus; Cuevavirus: Lloviu virus; and Dianlovirus: Měnglà virus [Filoviruses are causative agents of viral haemorrhagic disease in humans and non-human primates although virus spillover is rare . There alà virus –4.Bats are the proposed natural reservoir of filoviruses, involved in enzootic virus maintenance and zoonotic virus transmission to susceptible hosts . The majRousettus leschenaultii and Eonycteris spelaea. We conducted a serological survey of both hunted bat species and human hunters to study if humans have been exposed to filoviruses potentially originating from bats.In the Northeast Indian state of Nagaland, local ethnic groups have conducted bat harvests for at least seven generations as a source of food and traditional medicine. These bat hunters are exposed to saliva, blood, and excreta from the bat species All study participants provided written informed consent by signing a form in their native language Nagamese. All human samples and surveys were collected under National Centre of Biological Sciences (NCBS) IEC permit 7/001 and National University of Singapore (NUS) IRB permit N-17-034E. Negative control sera were collected under the NUS IRB permit number H-18-029. All bats were sampled under the NCBS, Tata Institute of Fundamental Research IACUC permit #UR-6/2014, which adheres to provisions of the Prevention of Cruelty to Animals Act (1960) and the Breeding of and Experiments on Animals Rules (1998) and the NUS IACUC permit B16-0159 under the National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines in Singapore.E. spelaea (n = 16) and R. leschenaultii (n = 30) was collected by cardiac puncture after being sacrificed by the harvesters.In 2017, 85 individuals participating in an annual bat harvest in Mimi village  were proE. spelaea (n = 34) and R. leschenaultii (n = 69) were collected and stored individually in RNALater (Sigma-Aldrich). Personnel handling potentially infectious material in the field wore N95 particulate respirators, surgical gowns, face-shields and were double-gloved. Surfaces were disinfected with 3% Virkon solution. Needles and scalpel blades were single use and disposed in sharps containers. Sealed containers were autoclaved at the Healthcare and Research Center of the Naga Hospital Authority Kohima, Nagaland, India. Small amounts of tissue  was excised, combined by individual bat and homogenized in AVL Buffer (Qiagen) at NCBS. An aliquot of homogenate was used for downstream PCR analysis, while another aliquot of homogenate was used for NGS analysis.Blood samples were centrifuged at 1,500 x g for 10 min and sera were stored at 4°C until transport to NCBS, where they were held at -80°C. All sera were gamma-irradiated at 20KGy with the Blood Irradiator 2000  and heat-inactivated at 56°C for 60 min prior to screening. Pooled kidney, lung and spleen samples obtained from E. spelaea n = 34 and R. leschenaultii n = 34). NGS libraries were made (RNA was extracted with PureLink RNA Mini Kit (Invitrogen), and cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen). Samples were tested with a nested pan-filovirus PCR assay targeting the L gene as reported previously . The posere made  and valiHuman and bat sera samples were screened in a filovirus multiplex microsphere immunoassay as previously described . Recombith percentile of the log-normal distribution , we detected filovirus-reactive sera in 5.9% (5/85) of human samples, 6.2% (1/16) of E. spelaea samples, and 13.3% (4/30) of R. leschenaultii samples. The highest MFI values, corresponding to levels of filovirus-specific serum IgG, were detected for EBOV-GPe in human and E. spelaea sera, and for MLAV- and RAVV-GPe in R. leschenaultii sera (E. spelaea serum sample (E34) reacted to the EBOV-, SUDV- and TAFV-GPe, displaying a similar cross-reactivity pattern as seen for the first group of human sera and was previously reported for filovirus-positive E. spelaea samples from Singapore [The majority of bat hunters were between 18 and 50 years of age, male, and participated at least eleven times in the harvest . All battii sera . Our resDespite the growing evidence that filoviruses are present in South and Southeast Asia, there has been a historical absence of outbreaks of filovirus haemorrhagic fever in this region. Reasons why clusters of human filovirus infections have not yet been detected in Asia, include (a) ecological barriers prevent zoonotic transmission, (b) viruses are unable to sustain transmission between humans or (c) an uncharacterized diversity of non-pathogenic, antigenically-related filoviruses exist and cause asymptomatic infection in humans. Human populations with wildlife contact and no history of Ebola virus disease in Uganda  and the Cross-reactivity between EBOV, BDBV and SUDV of the tested samples is in agreement with a previous report . The mamR. leschenaultii) mentioned in the initial report [R. leschenaultii sera suggest circulation of two distinct filoviruses within the same species, which is serologically distinct from reactivity with E. spelaea and human samples. These results are concordant with previous findings of exposure to filoviruses antigenically closely related to EBOV, BDBV, and SUDV in E. spelaea in Singapore [Furthermore, this study describes serum reactivity to MLAV, incidentally in the same bat species Click here for additional data file.S1 FigEonycteris spelaea (B), and Rousettus leschenaultii (C). Lognormal distribution representing the best fit of all samples are indicated by solid black lines. A solid black vertical lines indicates 3-fold over mean and a dotted black line denotes cutoff established by lognormal curve fitting.Histograms for each GPe (Ebola virus (EBOV), Bundibugyo virus (BDBV), Taï Forest virus (TAFV), Sudan virus (SUDV), Reston virus (RESTV), Lloviu virus (LLOV), Marburg virus (MARV), and Ravn virus (RAVV), Měnglà virus (MLAV) and mock antigen (MOCK)) and sera from humans (A), (TIF)Click here for additional data file.S2 FigEonycteris spelaea (B), Rousettus leschenaultii (C) screened in 2018 on a Bio-Plex machine. Antibodies reactive to filovirus GPe from Ebola virus (EBOV), Bundibugyo virus (BDBV), Taï Forest virus (TAFV), Sudan ebolavirus (SUDV), Reston virus (RESTV), Lloviu virus (LLOV), Marburg virus (MARV), and Ravn virus (RAVV) are quantified in a bead-based fluorescence assay. Grey dots represent individual samples. A boxplot is overlaid to indicate median, quartiles and extremes of the sample distribution. A black dashed line indicates the cutoff determined from a single lognormal curve-fit and a black solid black line the three-fold increase over the mean.MFI values for sera obtained from humans (A), (TIF)Click here for additional data file.S3 FigEonycteris spelaea (B) and Rousettus leschenaultii (C).For the 2018 Bio-Plex dataset, mean values  and spread of the two individual measurements are shown  for sera from human (A), (TIF)Click here for additional data file.S4 FigEonycteris spelaea (A) and Rousettus leschenaultii (B).For the 2019 MAGPIX dataset, mean values  and spread of the two individual measurements are shown  for sera from (TIF)Click here for additional data file.S5 FigSamples were tested in our assay with the indicated antigens in eight technical replicates.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.