Loa loa microfilariae in the blood preclude onchocerciasis elimination through community-directed treatment with ivermectin (CDTI) in Central Africa. We measured the cost of a community-based pilot using a test-and-not-treat (TaNT) strategy in the Soa health district in Cameroon.Severe adverse events after treatment with ivermectin in individuals with high levels of Based on actual expenditures, we empirically estimated the economic cost of the Soa TaNT campaign, including financial costs and opportunity costs that will likely be borne by control programs and stakeholders in the future. In addition to the empirical analyses, we estimated base-case, less intensive, and more intensive resource use scenarios to explore how costs might differ if TaNT were implemented programmatically.The total costs of US$283 938 divided by total population, people tested, and people treated with 42% coverage were US$4.0, US$9.2, and US$9.5, respectively. In programmatic implementation, these costs (base-case estimates with less and more intensive scenarios) could be US$2.2 ($1.9–$3.6), US$5.2 ($4.5–$8.3), and US$5.4 ($4.6–$8.6), respectively.Loa loa. Although it is more expensive than standard CDTI, costs vary depending on the setting, the implementation choices made by the institutions involved, and the community participation rate. Research on the required duration of TaNT is needed to improve the affordability assessment, and more experience is needed to understand how to implement TaNT optimally.TaNT clearly provides a safe strategy for large-scale ivermectin treatment and overcomes a major obstacle to the elimination of onchocerciasis in areas coendemic for  Loa loa. Costs per person were US$4.0 (population), US$9.2 (tested), and US$9.5 (treated).We measured the costs of a community-based pilot using a safe test-and-not-treat strategy to treat onchocerciasis with ivermectin in areas coendemic for  Loa loa  [Onchocerca volvulus nodules exceeds 20% (hyperendemic and mesoendemic areas), albeit with enhanced adverse event (AE) surveillance. However, fear of SAEs has caused individuals in such areas to refuse treatment, leading to suboptimal drug coverage and continued O. volvulus transmission. Moreover, CDTI is not recommended in Loa-coendemic areas where O. volvulus is hypoendemic , as the risk of SAEs is thought to outweigh the benefits of CDTI. This jeopardizes onchocerciasis elimination in Africa [Community-directed treatment with ivermectin (CDTI) has been used since 1999 as the main strategy to combat onchocerciasis in Africa . While g Africa) , 4. CDTIn Africa , 6.A test-and-not-treat (TaNT) strategy using a smartphone-based videomicroscope (LoaScope) enabled the safe implementation of ivermectin treatment in a pilot study in the Okola health district (HD) in Cameroon where all individuals ≥5 years of age , 8 were Although TaNT is a promising strategy for onchocerciasis elimination in loiasis-coendemic areas, its implementation is expected to be costlier than that of CDTI. Consequently, there are affordability concerns at a wider scale. Thus, we measured the cost of community-based TaNT in the above-mentioned Soa pilot study . In addiA microcosting approach  was usedThe Soa HD in Cameroon  is an ivThe census, testing/treating, and monitoring of SAEs in the TaNT round took place from September 2017 to February 2018. Costs were collected beginning with the preparation of the round in August 2017 until the last payments in March 2018.The “supplies” category includes (but is not restricted to) fuel, car maintenance, food, transportation, office materials, field materials, and drugs to treat AEs. The “personnel” category includes per diems of community drug distributors (CDDs) and other staff, project staff salaries, and fringe costs for study personnel, when applicable. Overhead costs were not explicitly measured but were assumed to be 15% of direct costs, as reported in the project budget. Costs of LoaScopes and capillaries were provided in US dollars (US$); all other costs were reported in Central African francs (XAF) and converted to US$ using the US Treasury Reporting Rates of Exchange of 31 December 2017 (US$1 = XAF 568) .Costs were based on actual payments or booked transfers collected from all invoices and bank transactions provided by project managers. External MoH staff working in the project and CDDs were interviewed using structured questionnaires to obtain information about their time spent on the project and opportunity cost of their salaries. A summary of the design, structure, and dissemination protocol for each questionnaire and English versions of these can be found in The invoices were analyzed and compared to the data provided by monthly financial reports and bank transactions for consistency and quality checks. The value of each invoice or bank transaction was labeled according to the input category, the health area in which the cost was incurred , and the activity to which the cost could be attributed . Data from invoices and questionnaires were entered into a Microsoft Excel spreadsheet for further analysis. Overhead costs were added to the subtotal and the final result was divided by the project outputs.To assess the effect of different implementation strategies on the overall cost of TaNT, costs were calculated using 3 hypothetical alternative TaNT implementation scenarios: “base-case,” “less intensive resource use,” and “more intensive resource use.” The scenarios were designed based on the literature and our own expertise in conducting CDTI in Cameroon, using the same HD for context  , 15. TabThe population size and numbers of individuals tested, treated, or excluded, as well as the occurrence of AEs, per health area are given in The costs of the pilot, disaggregated by programmatic activity, are presented in The majority of the personnel costs were not related to a specific activity but to actions related to the implementation of the round as a whole. The same holds true for district and health area level MoH personnel. The main costs of all other categories were related to the delivery of the intervention .The total cost for the entire pilot was US$283 938 , with a The absolute costs of the Soa pilot and each of the implementation scenarios disaggregated by programmatic activity, as well as the percentages of each activity, are given in O. volvulus and L. loa. Based on actual expenditure during the pilot, this strategy cost US$4.0 per person in the population, US$9.2 for each person tested, and US$9.5 for each person treated. In the alternative implementation scenarios, these costs (shown as base-case estimates with less and more intensive scenarios) were estimated to be US$2.2 ($1.9–$3.6), US$5.2 ($4.5–$8.3), and US$5.4 ($4.6–$8.6), assuming that population participation remained unchanged.This study provides both empirical cost estimates of a pilot and projected costs based on scenario analyses using the new TaNT strategy in regions coendemic for Several limitations apply to the internal validity of our study. Despite the careful collection of available data from invoices, bank transfers, and questionnaires, we had to adapt to the system usually used by financial managers of the implementing institution, without explicit reporting of the cost of previously acquired capital items or building rental. Using a retrospective questionnaire investigating opportunity costs of MoH staff was also not ideal, as it allows recall bias. Opportunity costs of time spent by school staff when helping during the treatment phase and of vehicles and furniture acquired for other projects were not included in this study, leading to a slight underestimation of the costs of the pilot. These limitations should be addressed in future studies to increase the accuracy of cost estimates and enable policymakers to anticipate their costs more reliably. This would also allow economic costs to be presented separated from financial costs.Because we measured costs in a study setting, alongside a pilot implementation of community-based TaNT, our results may not be fully representative of a programmatic setting, limiting the external validity. Some costs may have been higher than expected under programmatic implementation . Furthermore, best implementation practice still remains to be determined, and future programs may be implemented somewhat differently. The programmatic operationalization scenarios were designed to address the external validity limitations. They still do not cover all possible variations in implementation choices. We built the base-case scenario aiming to reflect Cameroonian reality as much as possible. As shown in the scenario analyses, costs heavily depend on implementation choices, and cost-savings compared to the pilot can be achieved through reductions in supervisory personnel, health education and sensitization, surveillance and management of AEs, and purchase prices of the LoaScopes and capillaries. The last 2 categories can be further reduced by implementing NGDOs/MoH if external funding agencies opt to donate these supplies to countries choosing to implement the TaNT strategy, as already done with the donation of ivermectin.Overall study coverage was relatively low, particularly in the semiurban and urban areas. Higher coverage is essential to onchocerciasis elimination. Achieving high coverage in urban areas is a known problem, even in standard CDTI, due to lack of trust in public programs, migrant populations, and disorganized poor urban settlements , 17. YetOur estimates, derived from a community-based single study, are not directly transferable to other settings due to many factors, such as variations in local prices, organization and availability of healthcare, and geographic characteristics such as remoteness, accessibility, and level of “urbanness” , 19.L. loa microfilarial densities remain low after treatment for at least 18 months, people proven to be treated in the previous round (using reidentification methods) would not need to be retested [To eliminate onchocerciasis, treatment would have to be repeated yearly (or more frequently) for many years with sufficient treatment coverage  . The cosretested . CapillaL. loa microfilarial densities before treating for onchocerciasis to which we could compare our results. We cannot directly estimate the difference in costs between TaNT and CDTI without empirical comparisons, as it is impossible to disentangle costs of treatment from those of testing in the TaNT strategy. A unit cost benchmarking study by Fitzpatrick et al [To date, there are no studies of the costs of alternative strategies of measuring ck et al  showed tck et al  that proThrough TaNT, community treatment with ivermectin can be expanded into the many areas in central Africa that are hypoendemic for onchocerciasis and coendemic for loiasis, where standard CDTI cannot be applied for safety reasons. Implementing treatment in these regions is crucial for the continent-wide elimination of onchocerciasis, with associated health and socioeconomic benefits estimated at more than US$6 billion . The popLoa-free areas if the coverage is sufficiently high and the proportion of the population excluded from treatment (because of high-density loiasis) is as low as observed in Soa and Okola (1% on average). The effect of excluding people from treatment may be more important where the proportion of people excluded due to loiasis is higher, but this can be mitigated by offering alternative treatment for onchocerciasis, such as a course of doxycycline. Although we did not assess the costs associated with such treatment, these should be weighed against the impact on program duration and success.Costs of TaNT are higher than costs of standard CDTI. How much higher will depend on how subsequent treatment rounds will be implemented and the required program duration. The duration will not be much longer than in L. loa. Our empirical study shows that TaNT using LoaScopes remains affordable, given the enormous potential economic benefits of reaching elimination of onchocerciasis in Cameroon and in other countries where L. loa is coendemic with O. volvulus. Various implementation scenarios show that costs could be further reduced. Future studies in this field are needed to investigate the costs of a programmatic implementation.Even though TaNT is undoubtedly more expensive than MDA, onchocerciasis elimination will not be reached without a strategy that can be safely used in areas coendemic for Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciz461_suppl_Supplementary_MaterialClick here for additional data file.


It is well known that stretching interventions are effective in improving age-related changes in range of motion (ROM) and muscle stiffness. We investigated the effects of various stretching interventions, such as static stretching and hold–relax stretching, on ROM and muscle stiffness in older adults to establish the most effective stretching technique. Our results showed that static stretching and hold–relax stretching increased ROM, which could be contributed by not change in muscle stiffness, but stretch tolerance. Conversely, medial gastrocnemius muscle stiffness decreased only after a static stretching intervention and not after hold–relax stretching. Our results indicated that static stretching intervention improved ROM and muscle stiffness in older adults.Various stretching techniques are generally recommended to counteract age-related declines in range of motion (ROM) and/or increased muscle stiffness. However, to date, an effective stretching technique has not yet been established for older adults. Consequently, we compared the acute effects of hold relax stretching (HRS) and static stretching (SS) on dorsiflexion (DF) ROM and muscle stiffness among older adults. Overall, 15 elderly men and nine elderly women  were enrolled, and both legs were randomized to either HRS or SS stretching. We measured DF ROM and muscle stiffness using a dynamometer and ultrasonography before and after 120 s of HRS or SS interventions. Our multivariate analysis indicated no significant interaction effects, but a main effect for DF ROM. Post-hoc tests revealed that DF ROM was increased after both HRS and SS interventions. Moreover, multivariate analysis showed a significant interaction effect for muscle stiffness. Post-hoc tests revealed that muscle stiffness was decreased significantly after only SS intervention. Taken together, our results indicated that both HRS and SS interventions are recommended to increase ROM, and SS is recommended to decrease muscle stiffness in older adults. Generally, range of motion (ROM) is known to decrease with age ,2, and pStatic stretching (SS) has generally been recommended to counteract age-related decline in ROM. Additionally, previous studies have shown that older adults exhibited an increase in ROM after SS intervention ,7,8. SS Recently, studies have suggested that the perception of passive tension, called stretch tolerance, could influence ROM. Hirata et al. investigated the association between dorsiflexion (DF) ROM and muscle stiffness or stretch tolerance among both young and older men . Their sA quasi-randomized experimental design was used to compare the acute effects of HRS and SS on DF ROM and MG muscle stiffness. Both legs were randomly allocated to receive either HRS or SS intervention, and the left and right sides were treated in a random order. All measurements were performed prior to (PRE) and immediately after (POST) HRS and SS. Participants were familiarized with the procedure before measurements and were asked to be relaxed throughout the measurements. In this study, all variables were measured during the passive stretching test .DF ROM and muscle stiffness in both legs were measured in 15 elderly men and nine elderly women. Baseline characteristics (mean ± standard deviation (SD)) were as follows: age, 70.2 ± 3.9 years; height, 160.8 ± 7.8 cm; and body mass, 59.6 ± 9.7 kg. Inclusion criteria were age >60 years, living in the community, and able to walk independently. Exclusion criteria were cognitive impairment, severe cardiac or musculoskeletal disorders, previous diagnosis of pulmonary disease, and hearing impairment. In this study, we used G* power 3.1 software  for calculating sample size for a split-plot analysis of variance (ANOVA) . Thus, >22 participants were required for this study. All the participants were informed of the purpose and procedure of the study and gave written informed consent prior to participation. The study was approved by the university (#18193) and performed in accordance with the Declaration of Helsinki.Participants were instructed to sit on an isokinetic dynamometer  with the hip at 50° flexion and the knee at 0° . The upper body was fixed using straps, and the ankle was fixed in a footplate at the neutral ankle position. The ankle was then passively moved to the maximum DF position, until the participant experienced discomfort or pain ,20,21, wPrior to the study, we confirmed that the test–retest reliability for DF ROM, and the passive torque at DF ROM was determined using coefficient variation (CV) and intraclass correlation coefficient (ICC) in 10 young adults . CV was 6.2 ± 5.0% and 7.1 ± 5.9%, and ICC  was 0.903 and 0.937, respectively.B-mode ultrasound apparatus  was used to determine the displacement of the muscle–tendon junction (MTJ) during passive ankle DF. The MTJ was identified and visualized as a continuous sagittal plane on ultrasound image using an 8-MHz linear-array probe. To ensure that the probe did not move during passive DF, a marker was placed on the skin ,25,26,27We confirmed the test–retest reliability for muscle stiffness in 10 young adults . CV was 2.8 ± 2.2% and ICC  was 0.973.Both HRS and SS were performed using the dynamometer in a sitting position, similar to the previous passive assessments. During HRS, the ankle underwent initially passive DF at a constant velocity of 5°/s, starting from 10° plantar flexion to DF ROM by the dynamometer, after which participants were asked to accomplish submaximal maximal voluntary isometric contraction plantar flexions for 10 s in the same position . After tDuring SS, the ankle underwent passive DF, starting from 10° plantar flexion to DF ROM, and was held at the end angle for 30 s. This 30 s SS application was then repeated three times, resulting in a total stretching time of 2 min .t-test. A two-way ANOVA with time (PRE vs. POST) and technique (HRS vs. SS) was used for multivariate comparison. Furthermore, a post-hoc paired t-test was used to determine differences between PRE and POST in each technique. Differences were considered significant at an alpha level of 0.05. Effect sizes (ES) were calculated and interpreted according to the suggestions of Cohen [SPSS Statistics for Windows version 24.0  was used for statistical analysis. Differences between HRS and SS techniques at PRE were assessed using the unpaired of Cohen . If ANOVp = 0.362, ηp2 = 0.02 and F = 0.35, p = 0.555, ηp2 = 0.01, respectively), but a significant main effect was seen for DF ROM . Post-hoc test revealed that POST DF ROM values were significantly higher than PRE values in both techniques . Moreover, split-plot ANOVA indicated a significant interaction effect for muscle stiffness . Post-hoc test revealed that POST muscle stiffness values were significantly lower than PRE values during SS , whereas no significant difference was observed during HRS .PRE and POST values during HRS and SS are summarized in s = 0.497, p = 0.016 and rs = 0.628, p < 0.01, respectively). However, no significant correlation was observed between changes in DF ROM and changes in muscle stiffness .In addition, Spearman’s rank correlation coefficients showed a significant correlation between changes in DF ROM and changes in passive torque at DF ROM after HRS and SS , whereas no significant difference had been noted during HRS . This can be underlined with the findings of further studies, who reported that muscle stiffness among older adults decreased after SS [ANOVA indicated a significant interaction effect for muscle stiffness. Moreover, the post-hoc test revealed that muscle stiffness decreased following SS (after SS . Sobolewafter SS . Moreoveafter SS , which wRegarding the increase in DF ROM, Spearman’s rank correlation coefficient showed significant associations between the changes in DF ROM and the changes in passive torque at DF ROM after both HRS and SS. Conversely, no significant associations had been observed between the changes in DF ROM and change in muscle stiffness. Our results support those of Kay et al., who investigated the effects of HRS and SS among young adults . On the As outlined, age-related ROM decline leads to a decline in locomotion and balance ,4 and anOur study had some limitations. First, we investigated only acute effects of HRS and SS on gastrocnemius muscles in older adults. Second, the effects of stretching interventions on locomotion, balance function, and risk of falls were not investigated in this study. Therefore, future studies should investigate the chronic effect of HRS and SS intervention on ROM and muscle stiffness as well as on physical functions and the risk of falls in older adults.The current study investigated the acute effects of 120-s HRS and SS interventions on DF ROM, stretch tolerance, and muscle stiffness of the MG among older adults. Accordingly, our results showed that both HRS and SS intervention were effective in improving DF ROM, with the change in DF ROM being related to the change in stretch tolerance. On the other hand, our results suggested that SS was required to decrease MG stiffness instead of HRS.


Hazardous alcohol use is detrimental to persons with HIV (PWH), impacting medication adherence and liver function, yet globally resources to target alcohol use behavior in this population are limited. Few studies have identified the costs of integrating alcohol reduction interventions into HIV care.To estimate the costs of implementing and delivering two evidence-based behavioral counseling interventions targeting hazardous alcohol use among persons with HIV and to estimate the costs of scale-up in ART clinics in Thai Nguyen, Vietnam.We undertook a micro-costing approach to determine the costs of delivering two adapted evidence-based interventions to reduce alcohol use: an intensive combined cognitive behavioral therapy and motivational enhancement therapy-informed intervention (CoI) and an abbreviated brief alcohol intervention (BI). A total of 294 participants with hazardous alcohol use were identified through a brief screening tool and received the CoI (n = 147) and the BI (n = 147) over 3 months. We estimated costs using time and motion studies, budget analysis, staff interviews, and participant questionnaires. Data were collected from 2016 to 2018 in VND and converted to USD.The total cost of implementation and administration of the intervention to 147 participants receiving the CoI was $13,900 ($95 per participant) and to 147 participants receiving the BI was $5700 ($39 per participant). Implementation and startup costs including training accounted for 27% of costs for the CoI and 28% for the BI. Counselor costs accounted for a large proportion of both the CoI (41%) and the BI (30%).Implementing and delivering alcohol reduction interventions to people with HIV in Vietnam with appropriate fidelity is costly. These costs may be reduced, particularly counselor labor costs, by using an evidence-based brief intervention format. Future research should explore the budgetary impact of brief and combined interventions to reduce hazardous alcohol use, particularly among vulnerable populations. Alcohol use is a major contributor to health problems globally. Excessive drinking, including hazardous drinking, has a substantial impact on morbidity and mortality in populations, resulting in downstream effects on work productivity and local economies . CountriIn Vietnam alcohol use is normative, and hazardous alcohol use is pervasive ,9. Few aHazardous alcohol use, defined as more than 14 drinks per week for men and more than 7 drinks per week for women , impactsCompeting health priorities limit the number of programs supported by government funds even when programs are shown to be effective. Without information about the cost of delivering alcohol interventions in Vietnam, policymakers are unlikely to scale up evidence-based alcohol reduction interventions, as such interventions may be perceived as diverting limited resources from existing health programs. Collecting data on the per-client costs associated with implementing and delivering an evidence-based alcohol reduction intervention among PWH, novel for this setting, would inform policy discussions of health programs and contribute to the information needed for disease control priority setting in this population .From 2016 to 2018, the Reducing Hazardous Alcohol Use among ART Clients (REDART) was implemented in northern Vietnam with the objective to improve abstinence from alcohol among persons receiving antiretroviral therapy (ART) at seven ART clinics in Thai Nguyen, Vietnam. Participants were randomized to one of the three arms: combined intervention (CoI), brief intervention (BI), and standard of care (SOC). At 12-month follow-up, the authors identified a significant (p =.001) increase in percent days abstinent in both intervention arms as compared to the SOC with an increase of 15% . When comparing the two intervention arms at 12-month follow-up the authors found no difference in change in percent days abstinent from alcohol as well as no difference in their secondary outcome of a reduction in heavy drinking days (defined as four or more drinks per day for men and three or more drinks per day for women). Furthermore, they did not identify a difference in intervention effectiveness for those persons with a history of injection drug use. The percent of persons who were virally suppressed, defined as <20 copies/mL, at 12 months was also significantly (p = 0.014) higher for those who received the BI (89%) as compared to those who received the SOC (78%) [Given this trial, our study objective was to estimate the costs associated with implementing two separate evidence-based alcohol reduction interventions among HIV-positive persons with hazardous alcohol use adapted for the Vietnamese setting and estimate the costs of possible scale-up throughout northern Vietnam. Because of both the lack of existing cost information in this setting and the need to collect cost data in as much detail as possible to inform future implementation, we undertook a micro-costing approach  in whichWe prospectively collected data on costs from 2015 to 2016 as part of a three-arm randomized control trial delivered from 2016 to 2018 in Thai Nguyen, Vietnam. Thai Nguyen, located 100 km north of the capital of Hanoi, is the ninth largest province in Vietnam. This semi-urban mountainous province has a population of 1.1 million and an economy based on the export of green tea and steel. Participants were residents of Thai Nguyen who were HIV-positive and attended one of the seven government-run antiretroviral treatment (ART) clinics in the province. Men screened eligible for the study by receiving a score of four or above and women screened eligible for the study by receiving a score of three or above on the brief Alcohol Use Disorders Inventory Test (AUDIT-C) , a brief questionnaire that has previously been validated in Vietnam [Individuals eligible for the trial were randomized to one of the three arms: combined intervention (CoI), brief intervention (BI), and standard of care (SOC). Each arm contained 147 participants. The SOC in Vietnam is a verbal referral to a substance use provider; given that this is the existing approach in the country it was not possible to identify the costs and thus is outside the scope of this analysis. The interventions are described in detail elsewhere . BrieflyWe undertook a micro-costing approach that included the healthcare provider and participant perspectives. In order to identify costs associated with the delivery of these two interventions, we defined four categories for costs: implementation, counselor costs, participant costs, and overhead. Each of these cost categories is described in more detail below. Briefly, implementation costs are costs associated with the activities to put the intervention in place. Trainings and manuals were included in these costs. We also determined the supervisor to be part of implementation costs given her role in supporting the counselors to develop and maintain skills in intervention delivery. We defined counselor costs as those associated with the maintenance of the intervention including time waiting for participants and time in counseling sessions as well as administrative costs such as scheduling counseling sessions. Participant costs were defined as those costs borne by the participant including travel time to and from the clinic, time spent in sessions, and other costs to them as a result of attending sessions such as paying for childcare and meals while away from home. Lastly, we included overhead costs  as a separate category.In order to deliver the intervention, several key steps were taken in the formative stage. In 2014, qualitative formative research was conducted to understand perceptions, local culture, and motivations for alcohol use among PWH receiving ART in Thai Nguyen . InterviThe findings from the formative research informed the adaptation of manuals from a previously implemented evidence-based US-based study . One intIn conjunction with the development and completion of the manual, from 2015 to 2016, a series of training sessions were conducted to provide skills and knowledge to counselors in both the BI and CoI. The training of counselors administering the CoI required 13 days and for those counselors administering the BI required 6 days; preliminary trainings were delivered to both sets of counselors.To implement the study, the team leased separate rooms that were adjacent to the regular clinic and furnished the room with equipment including a locked cabinet in which to keep the participant files.One program coordinator coordinated both the CoI and the BI. The program coordinator’s three-month salary is included in the cost of implementation (3 months is the estimated time to train staff and implement each six-week intervention). Her duties, including facilitating training sessions and continuous administrative check-ins with staff on counseling sessions, were equally split between the CoI and the BI reflecting the division of cost between the two interventions.Operational costs included telecommunication, photocopying, paper, water for staff, and refreshments for participants during individual as well as group counseling sessions.Time-and-motion data were collected from December 2016 to February 2017. Budgetary data were collected retrospectively. We also estimated costs of implementation, as described above. Finally, we evaluated costs from the participant perspective, including self-reported time traveling to and from the clinic separate from HIV appointments as well as lost wages due to attending clinic appointments.Human resource costs required to deliver the intervention were estimated using time and motion studies  in whichFrom March 2016 to March 2018 participant cost data were collected using an interviewer-administered questionnaire designed to estimate individual participant costs, based on the Stop TB Partnership’s ‘Tool to Estimate Patients’ Costs’ . Questioa priori sub-analysis, we also considered the cost of implementing the CoI and BI beyond Thai Nguyen. The team held three separate Skype calls to discuss costs to consider for scale-up. Local staff identified four nearby provinces in northern Vietnam  in which we had data on the number of ART clinics and the number of clients served within each clinic in those provinces. The team then consulted with Thai Nguyen study staff to estimate the personnel, administrative and other resources that would be required to scale the intervention to include these four provinces, in addition to Thai Nguyen, totaling five provinces. These costs included, for example, training of additional staff, obtaining administrative approvals, and delivery of the intervention. We assumed that each ART clinic in each province would require a dedicated part-time staff member (counselor or nurse), drawn from existing personnel, to administer either the CoI or the BI, and that 2 months would be required to find and train a new counselor (ongoing while working). We assumed that training a newly hired staff member would take 2 weeks  and would be directed by a full-time coordinator who would also manage logistics and perform site visits across the entire program. Additionally, we assumed that existing staff (counselors and supervisors) would need to spend 3 days per year on re-training activities to ensure high-quality performance and up-to-date skills. We then used those estimates (from the five provinces) to scale to 25 provinces reflecting approximately half the size of the northern region (58 provinces) of Vietnam.In an All cost data were collected in the local currency, Vietnamese Dong (VND), and then converted to USA Dollar (USD) based on the 2015 currency conversion of 21,921 USD to VND; 2015 was the year in which training and manual development began.Details of the full trial sample have been published elsewhere . A briefThe implementation of each intervention arm constituted a large proportion of the overall costs with implementation costs accounting for 27% ($3784) of the costs of the CoI and 28% ($1629) of the costs for the BI. Additionally, the CoI had greater training costs ($2548) than the BI ($502); this is in part due to the size of the manual required for counselors and the added hours of skill-building in counselors required to deliver the six-session individualized counseling sessions.Overhead costs accounted for 13% of costs associated with the CoI ($1848) and 32% of costs associated with the BI ($1848).The counselor labor costs for the CoI accounted for more than one-third of the overall cost (40%) while the counselor labor costs for the BI accounted for more than one-fourth (30%). Counselors for the CoI spent a median time of 52 minutes (48–60) per session in individual counseling sessions and a median time of 65 minutes per session in group sessions, whereas counselors for the BI spent a median time of 50 minutes (44–73) per individual counseling session and a median time of 8 minutes (8–12) on phone counseling sessions. Furthermore, counselors in the CoI received a monthly salary of 8,500,000 VND ($390) as compared to counselors in the BI who received a monthly salary of 7,000,000 VND ($320) reflecting the additional training they received for a more complex intervention. Counselors for the CoI spent a median 19 minutes (10–35) waiting for participants whereas counselors for the BI spent a median 29 minutes (13–48) waiting for participants.Participants self-reported a mean weekly salary of 830,000 VND or 38 USD; this figure included 19% of participants who reported being unemployed or retired. The greatest cost to participants was travel; a round trip to the clinic was estimated to take an average of 1 hour and result in lost wages of 16,400 VND ($0.75). The cost to the participant of the in-person individual sessions accounted for 5% of the total cost of the CoI and 3% of the BI. Incidentals  accounted for 2% of costs in the CoI and 1% of costs in the BI.We completed a sub-analysis from the healthcare provider perspective in order to determine how the per-person cost to deliver each intervention might be impacted by scaling up to more provinces. To scale up the CoI to five provinces in northern Vietnam we estimated a total cost of 139,198 USD . This woIn this comprehensive, ingredients-based costing of alcohol reduction interventions nested within a randomized trial in northern Vietnam, we found that the overall cost to deliver the CoI to 147 participants over a three-month period was 13,900 USD ($95 per participant), and the overall cost to deliver the BI to 147 participants over the same time period was less than half or 5,700 USD ($39 per participant) . CounselParticipants who received the CoI spent more on travel to access the intervention ($1200) compared to those receiving the BI ($260). Similarly, implementation costs and counselor costs were much higher for the CoI because the intervention is more intensive  and thus requires more time and effort in both implementation and delivery. A high proportion of the costs for both the CoI and the BI was related to implementation, which reflects the amount of time required for training and preparation of the intervention and the role of the supervisor in facilitating the intervention introduction. As a result, implementation costs accounted for more than a quarter of the costs of the CoI (27%) and more than a quarter of the costs of the BI (28%).The proportion of costs dedicated to counselors reflects the length of the intervention being delivered. The costs of brief interventions (defined as fewer than four contacts with a patient) in high-resource settings to reduce alcohol use have been frequently evaluated in the literature . Brief iFuture research should seek to understand the barriers to implementation of behavioral health interventions in HIV clinic settings. Despite a wealth of evidence from randomized control trials on the effectiveness of alcohol interventions ,29, limiThere are limitations to our analysis. First, a true societal perspective would capture additional costs, such as the cost impact on family and community members if an individual commits a crime or cannot work because of alcohol use . Such daOverall the cost of the CoI was more than twice the cost of the BI, primarily reflecting additional costs for implementation and counselor and participant time. The cost to participants was also noteworthy given the expectations to attend appointments and arrange transportation to access their care. A scale-up of these alcohol reduction interventions beyond one clinic in Thai Nguyen may be feasible and could distribute the costs of implementation more broadly, thereby modestly reducing the per-person cost of the intervention. Behavioral interventions to reduce alcohol use that are delivered in settings where participants are already receiving care for HIV are ideal for convenience but do require considerations of space, staff sharing, and other unexplored barriers.To facilitate the delivery of an alcohol reduction behavioral program in an HIV clinic setting, an understanding of implementation and maintenance costs is required. This research can help decision-makers better understand the per-patient and component costs of implementing and delivering structured alcohol reduction interventions among people living with HIV in Vietnam and other similar settings. The global impact of alcohol use will strain economies and burden resource-constrained settings with competing health priorities. How we address this burden will partly depend on how well we understand the costs associated with doing so.Click here for additional data file.Click here for additional data file.


Closteroviridae family were determined. The complete coding sequence of OLYaV was obtained by high throughput sequencing of total RNA from a 35-year-old olive tree  from Brazil, showing olive leaf yellowing disease and deformations in the wood. This represents the first report of OLYaV in this country. A genomic sequence of 16,700 nt containing 11 open reading frames (ORFs) was recovered, representing the complete virus coding capacity. The knowledge of the nucleotide sequence of the genome including the gene that codes the coat protein will facilitate the development of diagnostic tests, which are limited so far to PCR-based methods targeting the HSP70h gene. Interestingly, a thaumatin-like protein (ORF2), previously reported in other unassigned viruses in the Closteroviridae family, persimmon virus B and actidinia virus 1, was identified in the OLYaV genome. Phylogenetic analysis of shared proteins  with all members of the Closteroviridae family provides new insight into the taxonomic position of these three closteroviruses and suggests they could represent a new genus in the family.Genome organization and phylogenetic relationships of olive leaf yellowing-associated virus (OLYaV) with other members of the  Closteroviridae. Its presence was confirmed in olive and in unidentified species of pseudococcid mealybugs and the psyllid Euphyllura oliviana fed on an infected cv. Biancolilla plant [Closteroviridae because more biological and molecular data is needed for its classification and because the available partial genome data do not allow one to clearly assign it to any of the genera in the family [Closterovirus (closter in Greek for thread) genus, the first recognized species being beet yellows virus (BYV) and citrus tristeza virus (CTV). Carnation necrotic fleck virus (CNFV) was later included [Closteroviridae family. The molecular organization, evolution and taxonomy of closteroviruses have been discussed in several reviews [Closteroviridae is now divided into four genera; Closterovirus, Ampelovirus, Crinivirus and the relatively recently created genus Velarivirus, in which forty-nine species are classified. Seven other species in the family remain unassigned to a genus: Blueberry virus A (BVA), Actidinia virus 1 (AV1) and Persimmon virus B, for which complete genomes are available; Mint vein banding-associated virus (MVBaV) and OLYaV, for which only partial genomic sequences are available; and Alligatorweed stunting virus and Megakepasma mosaic virus without available nucleotide sequences [Closteroviridae family opens new possibilities in the taxonomical classification of OLYaV.Olive yellowing leaf-associated virus (OYLaV) was reported for the first time in Italy on cv. Biancolilla from Sicily, in plants showing bright chrome yellow discolorations of the leaves . Partialla plant . The parla plant , and a sla plant . The devla plant ,4 has rela plant , Italy , resulting in 9714,650 reads remaining for the subsequent analysis. De novo assembly by CLC Genomics Workbench 10.1.1.  allowed the recovery of a long contig of 16,700 nucleotides covered by 17,764 reads and with an average coverage of 128x. A BLASTN analysis with an e-value of 1.05 × 10−151 was obtained with the partial sequence of HSP70h of the isolate 7Oblonga3 from USA (HQ286495). The 98 nucleotides of the 5′ untranslated region (UTR) were covered with 45 reads of an average length of 121 nt and with the 10 extreme 5′ nucleotides covered by 11 non-redundant reads. The 159 nucleotides of the 3′UTR were covered by 172 reads of an average length of 121 nt, while the 10 extreme 3′ nucleotides were covered by 25 non-redundant reads, suggesting in both cases that the genomes ends have been reached or that minimal non-coding sequences were missing.A nearly complete genomic sequence of OLYaV, isolate CS1 (deposited in GenBank under the MT809205 accession number) was recovered by HTS. A total of 51,587,867 reads were obtained after trimming of adapters and the quality control of the sequences. A host genome subtraction step was performed using the caffolds  and OleaClosteroviridae members  while others have no homologous counterpart in the GenBank database, ORF3-p7, ORF7-p17, ORF8-p10, ORF9-p7, ORF10-p23 and ORF11-p10 Computational analysis of the 5′UTR by RNAfold implemented in Geneious Prime 2020  based on Vienna package 2.4.15  reveals ORF1ab (nt 99–9792)ORF1a (nt 99–8285)Closteroviridae available in the MEROPS database (https://www.ebi.ac.uk/) was used  is located at amino acids (aa) 738–1038, while the viral helicase domain (pfam01443) is located at aa 2402–2609. In order to identify the possible cleavage site of the papain-like leader protease, a multiple alignment of the amino acid sequences of papain-like proteinase domains from various representative members of the family was used . This co2.ORF1b (8287–9793 nt)Closteroviridae [Closteroviridae members, persimmon virus B  and actinidia virus 1 .ORF1b codes for the virus RNA dependent RNA polymerase (RdRp) and, similar to other members of the family oviridae , is exprA large deletion was identified in the Brazilian isolate as compared to the partial sequence of OLYaV previously deposited in GenBank (NC_043417). Thus, when aligning OLYaV-CS1 with the reference isolate NC_043417, a deletion of 182 nt between positions 9689 and 9690 nt of the Brazilian isolate was found. The analysis by BLASTN of the 182 nt sequence present in the reference sequence NC_043417 did not identify any homologous sequence in GenBank. Sanger resequencing of a RT-PCR product covering this region in the Brazilian isolate confirmed that the nucleotide sequence recovered by HTS was correct. The indel is located in the reference sequence NC_043417 nine aa upstream of the ORF1b stop codon and extends to the initial part of the non-coding region between ORF1b and ORF2. As a consequence of this deletion, the RdRp of the two isolates have very different C-terminal ends and the protein of OLYaV-CS1 is 26 aa longer than that of the previously reported OLYaV isolate .ORF2 https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?) revealed the presence of a thaumatin-like protein domain (superfamily smart00205) at aa positions 73 to 189. BLASTP analysis gave the highest percentages of identity with the previously reported OLYaV proteins  and with proteins from AV1  and PVB . These three viruses are the only members of the Closteroviridae family to possess such a protein with a thaumatin-like domain [The analysis of the predicted 21 kDa protein (190 aa) encoded by ORF2, using the online tool for conserved protein domains search (CDD) at National Center for Biotechnology Information (NCBI) Closteroviridae members.ORF 3 codes for a 7 kDa protein of 62 aa. Analysis with InterPro Scan implemented in Geneious Prime 2020 suggest that this is a putative transmembrane protein, predicted by Phobious , with aaORF4 Closteroviridae members are AV1 (37.6% aa identity) and PVB (35.2% aa identity).ORF4 codes for a heat shock protein 70 homolog (HSP70h) of 593 aa with a predicted molecular mass of 66 kDa. BLASTP analysis showed highest identities with the partial or complete proteins from other OLYaV isolates present in GenBank (81.6% to 94.2% aa identity). The most similar isolate was the 5Mission isolate from the USA and the most divergent was the OLYaV-Gr/Ms isolate from Greece. In all cases, the amino acid divergence level was lower than 25%, remaining within the species discrimination criterion and further confirming that the Brazilian CS1 isolate is indeed an OLYaV isolate. The closest ORF5 Closteroviridae members with higher levels are again AV1 and PVB (respectively 24.5% and 24.1% aa identity).ORF5 codes for a heat shock protein 90 homologue (HSP90h) of 515 aa with a predicted molecular mass of 60 kDa. BLASTN analysis gave the highest homologies with the corresponding partial protein from OLYaV isolates, with identity values ranging from 82.6% to 79.1%; the most similar isolate being 0G9 from Italy and the most divergent one LM3, from Italy as well. In fact, only partial sequences from Italian isolates are available in GenBank. BLASTP analysis showed amino acid identity levels of 76.6–90.6% with these various OLYaV isolates. The closest ORF6 This ORF codes for a protein of 233 aa with an estimated molecular mass of 26 kDa. A conserved domain search found clear similarity to closteroviral coat proteins (superfamily pfam01785) in the region corresponding to aa 52–232. BLASTP analysis identified only similarity with the coat proteins of AV1  and PVB .ORF7 , ORF8 , ORF9 , ORF10  and ORF11 .These ORFs encode hypothetical proteins of respectively 160 aa (18 kDa), 93 aa (10 kDa), 65 aa (7 kDa), 225 aa (26 kDa) and 92 aa (10 kDa) of unknown function and without BLASTP-detectable identity with any protein in GenBank.3′UTR Computational analysis of the 3′UTR by RNAfold implemented in Geneious Prime 2020  revealedClosteroviridae species were included. The results showed that OLYaV proteins clustered systematically and with high bootstrap support with those of AV1 and PVB, the two unassigned Closteroviridae members that are similar to OLYaV and that code for a thaumatin-like protein. This cluster of three viruses was systematically removed from the four known Closteroviridae genera, Closterovirus, Ampelovirus, Velarivirus and Crinivirus. The closest affinities were nevertheless with the Closterovirus genus, but with strong bootstrap support only in the P1b (RdRP) and CP trees. The unassigned blueberry virus A tended to cluster, albeit distantly, with the Closteroviridae genus, but grouped together with OLYaV, AV1 and PVB in the HSP90h tree, with solid (81%) bootstrap support.In all cases, the phylogenetic analyses were performed using the best substitution model computed by MEGA X  with theThe acronyms used in all maximum likelihood phylogenetic trees are as follows: AiPoV1: air potato virus 1; APV1: areca palm velarivirus 1; ArV1: arracacha virus 1; AV1: actinidia virus 1; BCCV1: blackcurrant-associated closterovirus 1; BcLRaV1: blackcurrant leafroll-associated virus 1; BeYDV: bean yellow disorder virus; BPYV: beet pseudo-yellows virus; BVA: blueberry virus A; BVBaV: blackberry vein banding associated virus; BYSV: beet yellow stunt virus; BYV: beet yellows virus; BYVaV: blackberry yellow vein-associated virus; BYVaV: blackberry yellow vein-associated virus; CCYV: cucurbit chlorotic yellows virus; CNFV: carnation necrotic fleck virus; CoV1: cordyline virus 1; CoV2: cordyline virus 2; CoV3: cordyline virus 3; CoV4: cordyline virus 4; CTV: citrus tristeza virus; CYLV: carrot yellow leaf virus; CYSDV: cucurbit yellow stunting disorder virus; DVCV: diodia vein chlorosis virus; FLMaV2: fig leaf mottle-associated virus 2; FMMaV: fig mild mottle-associated virus; GLRaV1: grapevine leafroll-associated virus 1; GLRaV2: grapevine leafroll-associated virus 2; GLRaV3: grapevine leafroll-associated virus 3; GLRaV4: grapevine leafroll-associated virus 4; GLRaV7: grapevine leafroll-associated virus 7; GLRaV13: grapevine leafroll-associated virus 13; LChV1: little cherry virus 1; LChV2: little cherry virus 2; LeCV: lettuce chlorosis virus; LIYV: lettuce infectious yellows virus; MV1: mint virus 1; MVBaV: mint vein banding-associated virus; OLYaV: olive leaf yellowing-associated virus; PBNSPaV: plum bark necrosis stem pitting-associated virus; PeAV: persimmon ampelovirus; PiAVA: pistachio ampelovirus A; PMWaV1: pineapple mealybug wilt-associated virus 1; PMWaV2: pineapple mealybug wilt-associated virus 2; PMWaV3: pineapple mealybug wilt-associated virus 3; PMWaV4: pineapple mealybug wilt-associated virus 4; PVB: persimmon virus B; PYVV: potato yellow vein virus; PYVV: potato yellow vein virus; ReV1: rehmannia virus 1; RLMoV: raspberry mottle virus; RLRaV: rose leaf rosette-associated virus; SCFaV: strawberry chlorotic fleck associated virus; SCFaV: strawberry chlorotic fleck associated virus; ScMMV: sugarcane mild mosaic virus isolate; SPaV: strawberry pallidosis-associated virus; SPCSV: sweet potato chlorotic stunt virus; TICV: tomato infectious chlorosis virus; ToCV: tomato chlorosis virus; ToV1: tobacco virus 1; TwVCV: tetterwort vein chlorosis virusORF1aORF1b (RdRp)HSP70hHSP90hCPClosteroviridae members have among the largest genomes of plant viruses  composed of one, two or three positive-sense, single-stranded RNA segments [Closterovirus, Ampelovirus, Velarivirus and Crinivirus. All the members of the family share some common features in their genome organization. They all have the ORF1ab, that contains one or two papain-like leader proteases, the conserved domains for methyltransferase and helicase and an RNA-dependent RNA polymerase expressed through +1 ribosomal frameshift. In addition, another characteristic of the family is the presence of a homolog to the cellular HSP70h and, in most members, the presence of a diverged, duplicate copy of the capsid protein called minor capsid protein (CPm). The genus demarcation criteria in the family include phylogenetic relationships in the RdRp, CP, HSP70h aa sequences, the identity of vectors, and the number of genomic RNAs, number and organization of ORFs and virion length [Closteroviridae species remain unassigned to a genus, including OLYaV, for which only partial sequences were available until now.segments . Currentn length . In the Closteroviridae members, AV1 and PVB, have somewhat longer non-coding regions: respectively 305 and 184 nt (5′UTR) and 404 and 282 nt (3′UTR). This suggests that the extreme genome ends of OLYaV may not have been reached. Nevertheless, the full coding potential of the OLYaV genome has been established.The OLYaV isolate CS1 herein reported was detected from a 35-year-old olive tree with typical leaf yellowing disease symptoms, as well as unusual woody cylinder deformations. Neither de novo assembling nor the mapping against other known olive viruses  were able to detect any of them. Thus, OLYaV is a candidate for the unusual woody cylinder symptoms as well as for the more typical leaf yellowing symptoms observed, although further studies are required to fully clarify this issue. The recovered sequence of OLYaV-CS1 included non-coding regions of respectively 98 nt (5′UTR) and 159 nt (3′UTR) that could not be further extended because no overlapping reads were found. Other closteroviruses such as BCCV1 (NC_040834) or BcLRaV1 (MH460557) have similar 5′UTR (97 nt) sizes and others such as BVA (NC_018519) or BYV (AF AF056575) present similar in 3′UTR (176 and 166 respectively) sizes. However, the genomes of the closest Closteroviridae family. It contains 11 ORFs, among them the complex ORF1ab encoding for the three typical conserved domains, papain-like leader protease, methyltransferase and helicase, and the RdRp. ORF1a codes for only one papain-like leader protease, as other members of the family do, such as BYV, MV1, LChV1 or LIYV [The complete coding sequence of the genome herein reported for OLYaV-CS1, shows a genome structure typical of the  or LIYV ,27. Ther or LIYV , has bee or LIYV ,12, whicClosteroviridae genera, which had contributed to its unassigned species status, OLYaV exhibits two original genomic features. First, ORF2 encodes for a thaumatin-like protein and second it does not code for a CPm protein. Interestingly, these two genomic features are shared by two other unassigned Closteroviridae species, AV1 and PVB [Closteroviridae members and encapsidates the 600–700 5′-terminal nucleotides, and are believed to contribute to the initiation of encapsidation. There are however some exceptions, as the ampeloviruses with the smallest genomes, such as GLRaV-4 or PMWaV-1, do not appear to possess a CPm [Closteroviridae evolved from a common ancestor and diverged with the adaptation to specific vectors; aphids in the case of closteroviruses, mealybugs in the case of ampeloviruses, whiteflies in the case of criniviruses [Besides its significant phylogenetic remoteness from members of the four  and PVB ,23. It i and PVB , are inv and PVB , and pro and PVB . The lacss a CPm . How theiviruses .Closteroviridae conserved proteins as these three viral species systematically cluster together with high bootstrap support. Taken together, the remoteness of these three viruses from extant Closteroviridae genera, their shared original genomic features and their phylogenetic relatedness provide clear markers and strong support for the creation of a novel genus within the family to gather these three species.The genome properties shared between PVB and AV1, the presence of a thaumatin-like protein, and the lack of a CPm, as well as the close phylogenetic relationship between them in the HSP70 genomic region, have been previously reported ,23. HoweStems of a 35-year-old olive tree (CS1) cv. Zarzaleña, showing typical leaf yellowing disease symptoms and also unusual woody cylinder deformations like stem pitting and woody gall symptoms  were colTotal RNA was purified from phloem scrappings using the Plant/Fungi total RNA purification kit  according to the manufacturer’s protocol. Complementary DNA was synthesized and the sequencing library prepared using the TruSeq Stranded Total RNA LT Sample Prep Kit (Plant) and the library protocol TruSeq Stranded Total RNA Sample Prep Guide, Part #15031048 Rev. E. RNA quality control, library construction and sequencing in a NextSeq 500 platform (paired 2 × 150 nt) were performed at Macrogen Inc. .Olea europaea genome Oe6 scaffolds [Olea europaea subsp. europaea plastid complete genome (NC_015401) were used for genome subtraction. The parameters used included match score 1, mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.7 and similarity 0.8. De novo assembly was performed using CLC Genomics Workbench v.10.1.1 with automatic word size, automatic bubble size, minimum contig length of 200 nt, map reads back to contigs, mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.7, similarity fraction 0.8 and updating contigs. De novo contigs were annotated by BLAST analysis (BLASTN/X) against local and online virus, viroids and nt/nr databases.Reads were subjected to trimming of adapters and quality control using CLC Genomics Workbench v.10.1.1 . caffolds  and Oleahttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?) were used to search predicted proteins for conserved domains. Nucleotide and amino acid percentages of identity were obtained using the online BLASTN and BLASTP tools from the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi).Geneious Prime 2020 was used to analyze the complete genome of the CS1 isolate. ORFs were determined with ORF finder included as a plugin in the software. The RNAfold plugin was used to predict the secondary structure of the 5′UTR and 3′UTR. Computational analysis with InterPro Scan implemented in the Geneious Prime 2020 and the online tool from the National Center for Biotechnology Information (NCBI) (Closteroviridae available from the EMBL-EBI and GenBank databases was included. Alignment of conserved proteins, ORF1a, ORF1b, HSP70, HSP90 and CP were performed using the ClustaW implemented in MEGA X [For the phylogenetic analysis of the sequence determined in this study, a representative isolate of each species belonging to the family n MEGA X  using thn MEGA X  aa substClosteroviridae, revealed that it is closest to AV1 and PVB. These three viruses have so far remained unassigned in the family and share several common genomic features. They uniquely code for a thaumatin-like protein and do not encode a CPm duplicate. Phylogenetic analyses of the five broadly conserved closteroviral proteins showed that OLYaV, AV1 and PVB systematically cluster together, supporting their close phylogenetic relationship, suggesting that these unassigned species could be grouped together in a new genus inside the Closteroviridae family.The genomic characterization of OLYaV, an unassigned member of the family


Rhabdoviridae), Equid herpesviruses (Herpesviridae), Borna disease virus (Bornaviridae) and West Nile virus (Flaviviridae), as well as exotic viruses, will be presented.Neurological disorders represent an important sanitary and economic threat for the equine industry worldwide. Among nervous diseases, viral encephalitis is of growing concern, due to the emergence of arboviruses and to the high contagiosity of herpesvirus-infected horses. The nature, severity and duration of the clinical signs could be different depending on the etiological agent and its virulence. However, definite diagnosis generally requires the implementation of combinations of direct and/or indirect screening assays in specialized laboratories. The equine practitioner, involved in a mission of prevention and surveillance, plays an important role in the clinical diagnosis of viral encephalitis. The general management of the horse is essentially supportive, focused on controlling pain and inflammation within the central nervous system, preventing injuries and providing supportive care. Despite its high medical relevance and economic impact in the equine industry, vaccines are not always available and there is no specific antiviral therapy. In this review, the major virological, clinical and epidemiological features of the main neuropathogenic viruses inducing encephalitis in equids in Europe, including rabies virus ( Neurological disorders represent an important sanitary and economic threat to the equine industry worldwide. Even mild nervous deficits can result in poor performances and long recovery of athletic horses, while severe clinical signs can induce life-threatening injuries in infected horses and may expose owners, veterinarians and care providers to significant risks . Few surRabies virus, Equid alphaherpesvirus 1 (EHV-1), West Nile virus and related flaviviruses , Mammalian 1 orthobornavirus and neurotropic alphaviruses  are the most largely described neuropathogenic viruses . Bacterial meningitis are common neurological infections in foals, while neuroborreliosis, listeriosis and Equine Protozoal Myeloencephalitis are rare and difficult to diagnose in equines . As far  viruses .Equine neuropathogenic viruses generally induce encephalitis or myeloencephalitis, which is an inflammation of the central nervous system  and/or of the spinal cord characterized by large or multifocal infiltrations of mononuclear cells d. InfectEpidemiological parameters including knowledge on the holding conditions, horse condition and nutritional needs, prevalent pathogens in a specific region  will helRhabdoviridae), equid herpesviruses (Herpesviridae), Borna disease virus (Bornaviridae) and West Nile virus (Flaviviridae), as well as exotic viruses. The most relevant information on the diagnosis and prevention from equine encephalitis viruses enzootic in Europe will also be presented in this review.Most equine neuropathogenic viruses will induce similar clinical presentations and laboratory testing, either by indirect  or direct assays  must be provided in order to confirm the etiology of the disease . For theEquine viruses causing encephalitis can be classified into two groups: viruses transmitted indirectly through the bites of an infected arthropod  or by direct transmission . Direct Rhabdoviridae), Equid herpesviruses (Herpesviridae), Borna disease virus (Bornaviridae) and neurotropic flaviviruses transmitted by mosquitoes and ticks, e.g., West Nile, tick-borne encephalitis and Louping ill viruses (Flaviviridae).In this section, we will address several viral equine encephalitis inducing the most important clinical and economic consequences in Europe: rabies virus , and neurological diseases . Nine herpesviruses have been described in the family Equidae, which includes horses, ponies, donkeys and zebras and two natural hosts have been identified key for EHV epidemiology: horses for equid herpesviruses 1 (EHV-1), EHV-2, EHV-3, EHV-4, and EHV-5 and donkeys for EHV-6, EHV-7, and EHV-8 . ConcernEquid alphaherpesvirus 1 (EHV-1) was recognized as a neuropathogenic virus in horses in 1966 . Within Varicellovirus genus, in the Alphaherpesvirinae sub-family in the family Herpesviridae  was often associated with EHV-1 neuropathogenicity (in 83% to 86% of cases), while absent in the majority of EHV-1 abortion outbreaks ,49. The Diagnosis: Over the past 15 years, diagnosis tools have been improved . PCR thaPrevention and control: Practitioners need to identify quickly EHV-1 infections and to apply strict sanitary measures to stop virus spreading. Isolation and quarantine measures have to be applied according to high-risk groups. There is an urgent need to screen and separate potential virus shedders (either confirmed to be infected with EHV-1 or exposed) from non-exposed and healthy animals. Specific staff caregivers would be affected to each group and would have specific supplies . A three weeks’ quarantine starts as soon as the last reported case is declared. At the end of the infected period, an eight days’ crawlspace, after carrying out cleaning and disinfection of boxes, is needed.In vitro studies are performed around the world to identify new molecules with a strong antiviral potential  in 2013) [Transmission and epidemiology: Rabies cases have been reported across the globe in more than 150 countries . The majin 2013)   . In Austin 2013) . In Euroin 2013) .Pathogenesis and clinical disease: Rabies virus is primarily transmitted to equids through the saliva of an infected animal. Contamination occurs mainly through bites or contact of a cutaneous or mucous  lesion with infected saliva. Rabies pathogenesis is characterized by three distinct phases. Phase 1 corresponds to the ascending or centripetal period during which the virus is transported toward the CNS. Phase 1 occurs after the bite of a rabid animal, and after a short-lasting replication in local muscle cells, the virus enters motor and sensory neurons. Paresthesia at the biting site may develop, which results in rubbing or automutilation through biting. Lyssavirus mainly shows axon-neuronal transport by binding with acetylcholine-receptors at motor end plate and multiply at the ventral horn of the spinal cord before CNS spreading . The virClinical signs of rabies are highly variable in horses and three forms are classically described according to the injured area: silent, paralytic or furious forms. The furious form is uncommon (10 to 17% of cases), while silent and paralytic forms are the most common. After a long incubation time (over 6 months), the disease progresses rapidly (within 3 to 6 days), including a sudden change in behaviour (depression to manic), itching at the biting site, loss of appetite, high fever, gait disorder, paralysis at the inoculation point, aggressivity, and hyperesthesia .Diagnosis: Confirmatory diagnosis is preferably undertaken through virus identification by direct fluorescent antibody (DFA) test, direct rapid immunohistochemistry test (dRIT), or pan-lyssavirus RT-PCR assays . DFA tesPrevention and control: Rabies control has been mainly afforded through the vaccination of wild and domestic susceptible animal species . VaccinaMammalian 1 orthobornavirus according to ICTV nomenclature [Bornaviridae family, within the order Mononegavirales [Mammalian 2 orthobornavirus, was associated with fatal encephalitis in humans [Bornaviridae family and the association of the new viruses with animal and human diseases revived the interest for this family and called for new classification [The Borna disease virus , nclature , is the avirales . For yeaavirales ). Amongsavirales , and a mn humans . This exfication .Virus: Bornaviruses are enveloped, non-segmented, single-stranded, negative-sense RNA viruses. Their 8.9 kb genome encodes six viral proteins, five structural  and one non-structural (X) . They haTransmission and epidemiology: BoDV is the causative agent of the Borna disease, a rare but severe, often lethal, encephalitis, which was first described in horses during a devastating outbreak in the little city of Borna in Saxony/Germany around 1894 . Later, Crocidura leucodon, is recognized as the natural reservoir host of BoDV [The bicolored white-toothed shrew,  of BoDV ,94,95. I of BoDV . Horses  of BoDV . This is of BoDV ). In hor of BoDV , indicat of BoDV  and was  of BoDV .The question of whether BoDV is a human pathogen has been debated for years. Several studies showed it may be a cause for some psychiatric disorders ,102, andPathogenesis and clinical disease: In horses, the typical course of the disease is characterized by an acute encephalitis that develops following an incubation period which lasts up to 3 months . During post-mortem analyses performed in brain, the tissue with the highest viral load, will confirm a definitive diagnostic  does not allow a reliable diagnostic from serum or cerebrospinal fluids, even when the most sensitive serological or molecular tests are used. Standardized tests, validated by inter-laboratory assays, does not exist. Although intra-vitam studies give useful indicators, only agnostic .Prevention and control: A few therapeutics (amantadine sulfate) and vaccines (attenuated and inactivated candidates) have been developed against equine BoDV infection thus far, but none are available in veterinary medicine since none proved effective in controlling or preventing the disease .Three neurotropic flaviviruses documented in horses suffering from meningoencephalitis are enzootic in Europe, West Nile virus (WNV), tick-borne encephalitis virus (TBEV) and Louping ill virus (LIV). Flaviviruses can be divided into three distinct groups according to their vectors: tick-borne viruses, mosquito-borne viruses and viruses with unknown vectors .Flaviviridae family, are enveloped, non-segmented, single-stranded and positive-sense RNA viruses. Their genome of approximately 11 kb encodes three structural proteins  and seven non-structural proteins  involved in virus replication and counteractive of immune responses  through the bite of infected mosquitoes. Horses and humans are highly susceptible to WNV infection but are considered as dead-end hosts owing to limited and short viremia that does not sustain transmission to naïve mosquitoes. In both species, asymptomatic infections are the most common, but in rare cases (approximately 1 out of 140 infections in humans and up to 10% infected horses), neuroinvasive forms with meningitis, encephalitis or myelitis may occur [n = 2083) higher than the cumulative number from the previous seven years (n = 1832) [n = 285) in comparison with earlier WNV transmission seasons  [WNV is maintained and amplified in an enzootic cycle involving birds and mosquitoes from the ay occur ,114. WNVay occur . After aay occur ,117,118.ay occur . Interesay occur . This WNay occur . Unprece period) . In Aust period) .WNV neurovirulence and neuroinvasion are typically associated with sequence variations in the flavivirus E protein ; strikinIxodes ricinus. Contrary to mosquitoes, which become infected only if there is sufficient viremia in the vertebrate host, ticks can become infected during a shared meal or “co-feeding”, not requiring a systemic infection of the host [Myodes and Apodemus although other small rodents and shrews can contribute to the natural transmission cycle. Larger animals such as sheep, goats and more rarely cattle can be additional competent hosts. Goats, sheep and cows excrete the virus in the milk. Humans, horses and game  are epidemiological dead-end hosts. Human infection can occur through a bite from a TBEV- infected tick, more rarely through the ingestion of unpasteurized milk or milk products from goats and less often from infected cows or sheep [Ixodes ricinus) also transmit LIV. The vertebrate reservoir hosts are the wood mouse (Apodemus sylvaticus), the common shrew (Sorex araneus), the red grouse (Lagopus lagopus scoticus) and the sheep (Ovis aries). A co-feeding mechanism has also been reported for LIV transmission in I. ricinus ticks feeding on mountain hares (Lepus timidus) [TBEV is the most important human tick-borne pathogen in Europe and Asia. The estimated annual incidence rate is 10,000 human infections with a case-fatality rate ranging from 1% to 20%. For TBEV, the arthropod vectors are primarily hard ticks and in Europe, the most important tick vector is the host . In factthe host . The maior sheep . Hard titimidus) . Sheep dtimidus) . Finallytimidus) ,134,135 timidus)  while lotimidus) .TBEV is reported in the northern hemisphere of Europe and Asia. There are three main subtypes of TBEV: European (TBEV-Eu), Siberian (TBEV-Si) and Far Eastern (TBEV-FE) circulating in Europe with TBEV-Si and TBEV-FE recently detected in the Baltic countries and in Eastern Finland ,139. BasPathogenesis and clinical disease: TBEV, LIV and WNV induce severe neurological syndromes, through pathogenic mechanisms that are still largely unknown. The majority of WNV and TBEV studies were done in vitro on transformed cells lines and in vivo on mouse experimental models . The exaAn equine infection by WNV can be suspected when the following symptoms are noticed: hyperthermia, ataxia, hind legs paresis, muscle tremors, teeth grinding, cranial nerve deficits, dysphagia or face paralysis . During Few publications describe TBEV infections in animals. Clinical signs of encephalitis induced by TBEV are rare in horses and include anxiety, decreased appetite, nervousness, and emaciation ,135. GenDiagnosis: The neurological symptoms and lesions are not specific to flavivirus infection, making laboratory tests compulsory to confirm or exclude viral etiology. The diagnosis of flavivirus infection is delicate and relies on the detection of viral RNA in blood and CSF, viral isolation in cell culture and/or the detection of IgM and IgG in serum and CSF. Flavivirus infection  diagnosis is primarily based on indirect methods. Indeed, viremia of an infected horse is quite low and fleeting and already vanished by the time horses develop neurological signs. It is therefore quite difficult to identify the pathogen by direct diagnostic assays, even with highly sensitive real-time RT-PCR assays. Serology tests such as ELISA for WNV and TBEV detection and hemagglutination inhibition assay for LIV are fast and allow for the identification of anti-flavivirus antibodies. These tests suffer from poor specificity because of antibody cross-reactions between flaviviruses . SeveralPrevention and control: There is no specific treatment for flaviviruses and disease control primarily relies on integrated virus surveillance and on vector control, with strategies and methodologies differing between European countries ,155. HowEquine encephalitis viruses enzootic in Europe are not the only threat to European horses. Horses could contract the disease abroad during equine competitions or international events. Moreover, because of enhanced risks of emergence of exotic viruses and increasing animal movements owing to globalization, exotic equine encephalitis viruses could be reported in the future in new naïve territories. Expert opinion and risk assessment through modelling is strongly required to identify the viruses that are prone to emergence and the most at-risk areas in Europe and at the international level and to adapt surveillance plans ,161.Flaviviridae family , Saint-Louis encephalitis virus (SLEV) and Murray Valley encephalitis virus (MVEV)), alphaviruses in the Togaviridae family  and orbiviruses in the Reoviridae family  [The main exotic equine encephalitis viruses are vector-borne, either transmitted by mosquitoes or flying midges b. They eus, EEV) . Most ofus, EEV) ,21.Culex tritaeniorhynchus or other mosquito species mainly from the Culex genus as vectors iewed in ). Finalliewed in ,169. Froiewed in .Sigmodon, Zygodontomys, Heteromys and Proechimys genus and in Culex melanoconion mosquitoes, while epizootic variants are transmitted by more diverse mosquito species  [Culiseta melanura or morsitans mosquitoes maintain this virus in nature, while Aedes bridge vectors are involved in EEEV transmission to humans and horses [EEEV, WEEV, and VEEV are alphaviruses regularly identified in equine encephalitis in America. These enveloped, non-segmented, single-stranded and positive-sense RNA viruses encode two polyprotein gene clusters, driving the translation of four non-structural proteins (NSP1-4) and virus structural proteins  at the 5′ and 3′ ends, respectively . Epidemia genus) . Regardia genus) ,177. Altd horses . EEEV cld horses ). EEEV ed horses .Equine encephalosis virus (EEV) is an orbivirus close to African horse sickness and bluetongue viruses, two arboviruses associated with unexpected emergence in Europe in 1989 and 2006 respectively ,179. It Many neurological diseases represent an important sanitary and economic threat to the horse population worldwide. Non-vector-borne equine encephalitis viruses, such as EHV-1, rabies and BoDV, are variably reported in Europe, whereas arthropod-borne infections are usually exotic diseases. Viral encephalitis therapeutic strategies are comparable, whatever the etiologic virus. Horses with neurological conditions must be isolated in a quiet box with limited stimuli  and an appropriate bedding providing warmth, comfort, and security. Slings can be used to support paretic horses and avoid long and poor prognosis recumbency . SupportClinical signs of viral equine encephalitis are not specific and overlapping geographical areas can make virus identification very challenging. One recent and striking example of delayed identification of emerging arboviruses due to similarities in clinical presentation and cross-reactive diagnostic tools was given during WNV introduction in the United States, when WNV was initially misdiagnosed with the closely related SLEV . BearingAedes, Culex and Culiseta genera), and highly invasive Aedes albopictus and Ae. japonicus have rapidly established in several European countries over the last decade [Arboviruses are the most important cause of encephalitis in horses and many of these viruses are also significant human pathogens. Some of these arboviruses have recently emerged or resurged, such as WNV, JEV, SLEV, EEEV or EEV and an increased rate of emergence of vector-borne diseases can be inferred from recent studies . A high t decade ,190. Vect decade ,192,193.t decade . On-timet decade .


The impact of mechanical ventilation on the daily costs of intensive care unit (ICU) care is largely unknown. We thus conducted a systematic search for studies measuring the daily costs of ICU stays for general populations of adults (age ≥18 years) and the added costs of mechanical ventilation. The relative increase in the daily costs was estimated using random effects meta regression. The results of the analyses were applied to a recent study calculating the excess length-of-stay associated with ICU-acquired (ventilator-associated) pneumonia, a major complication of mechanical ventilation. The search identified five eligible studies including a total of 54 766 patients and ~238 037 patient days in the ICU. Overall, mechanical ventilation was associated with a 25.8% (95% CI 4.7%–51.2%) increase in the daily costs of ICU care. A combination of these estimates with standardised unit costs results in approximate daily costs of a single ventilated ICU day of €1654 and €1580 in France and Germany, respectively. Mechanical ventilation is a major driver of ICU costs and should be taken into account when measuring the financial burden of adverse events in ICU settings. Intensive care consumes a large proportion of healthcare resources. Days in intensive care are substantially more costly than general ward days in hospitals due to increased resource utilization and labour intensity. One major driver of these costs is mechanical ventilation.Adverse events occurring during hospitalization add a substantial further burden to the healthcare system. The most prevalent hospital-onset conditions are hospital-acquired infections, which absorb substantial resources in hospitals and often require costly treatment . For patThe major aim of this review is to determine the relative impact of mechanical ventilation on the daily costs of intensive care unit (ICU) care. We believe that this information is crucial in a number of contexts but has been neglected given the unavailability of reliable estimates.We therefore conducted a systematic review regarding the costs of an ICU day and the proportion of these costs attributable to mechanical ventilation and quantified the relative increase in the daily costs using meta regression. In order to illustrate the practical relevance of these findings, we then determine the economic burden of VAP using recent cost information from France and Germany.On 24 March 2017 we searched MEDLINE via Wolters Kluwer's search interface Ovid (indexed and non-indexed databases), Web of Science via Thomson Reuters, , CINAHL via EBSCOhost and the NHS Economic Evaluation Database (NHS EED) via Centre for Reviews and Dissemination, University of York. We performed the searches restricted to the English language and publication year 2000 to the update status at date of search (2017).The search strategies varied by the database used. For details of the search strategies in all databases, see Supplementary material (Appendix). The searches returned 2839 studies of potential interest. After elimination of duplicates, 2072 studies remained.Studies were eligible if they gave data for the daily costs of ICU stays for general populations of adults (age ⩾ 18 years) and the added costs of mechanical ventilation. Screening of titles and abstracts found 257 potentially relevant papers. We were able to access full-texts of all articles of interest. Of these, 14 articles provided data on the daily costs of ICU stays, but only five articles  fulfilleTwo authors (KK and PH) checked the titles and abstracts of all potentially relevant English articles, and the full text of these was obtained. The same authors independently checked all the full-text articles for eligibility, and author KK extracted clinical data from the full texts. Details on the number of patients, ICU days, daily ICU costs and corresponding measures of uncertainty  were derived from the studies for ICU patients currently under mechanical ventilation or not. If necessary, patient days were derived from the proportion of patients ventilated and mean/median length of ICU stay (LoS). In the case of no reported variances, a standard deviation of 20% was imputed. In two cases  it was at-tests based on summary data (command ttesti in Stata) were applied on log transformed daily costs. As the latter costs were used in all analyses, resulting coefficients were exponentiated in order to show the relative effect of ventilation on the daily ICU costs. Please note that the relative increase is always taking place within a country and year, therefore making adjustment for inflation and/or currency exchange rates unnecessary.Daily ICU costs were log transformed along with their standard errors and analysed using random effects meta regression  was used.The selection of studies is summarised in et al. [Bluhmki et al.  reportedet al. [The study of Lefrant et al.  providedet al.   for patients hospitalised in German ICUs [Bock man ICUs . Assuminman ICUs  (3.52 daIntensive care absorbs a large proportion of health care spending in industrialised countries . ReliablThe results of the systematic review and meta regression analysis showed that mechanical ventilation is associated with a substantial increase in the daily costs of ICU care. Moreover, we observed a strong variability in the relative effect of mechanical ventilation across studies. However, the number of appropriate studies was rather limited and we were unable to find relevant more recent studies. As technology, procedures and indications for ventilation have changed in the meantime, our findings may be limited in scope and further studies on this topic are needed.et al. were aware of this fact and correctly estimated the excess LoS associated with VAP using multistate models accounting for the time-dynamics of ventilation status and VAP. At the same time, Bluhmki et al. [In the estimation of the additional LoS of adverse events during hospitalization, it is crucial to treat the exposure as time-varying to avoid a time-dependent bias 12. Thisi et al.  consideri et al. –22.Our cost calculations were based on the simplifying assumption that the entire excess LoS can be attributed to ventilation; thus, we may have slightly overestimated the costs. However, we refer to the result that this excess seems to be mainly triggered by ventilation . While LOur study contributes to a more detailed understanding and measuring of the costs of intensive care and mechanical ventilation by providing first estimates and discussing methodological particularities. Being an area with relatively little empirical evidence to date, more studies on the daily costs of mechanical ventilation and intensive care are duly needed.