We estimated the financial costs of different interventions against urogenital schistosomiasis, implemented by the Zanzibar Elimination of Schistosomiasis Transmission (ZEST) project, on Pemba and Unguja islands, Tanzania. We used available data on project activities, resources used, and costs reported in the accounting information systems of ZEST partners. The costs were estimated for all the activities related to snail control, behavior change interventions, the impact assessment surveys, and management of the whole program. Costs are presented in US$ for the full duration of the ZEST project from 2011/2012 to 2017. The total financial costs of implementing snail control activities over 5 years, excluding the costs for donated Bayluscide, were US$55,796 on Pemba and US$73,581 on Unguja, mainly driven by personnel costs. The total financial costs of implementing behavior change activities were US$109,165 on Pemba and US$155,828 on Unguja, with costs for personnel accounting for 47% on Pemba and 69% on Unguja. Costs of implementing biannual mass drug administration refer to the estimated 2.4 million treatments provided on Pemba over 4 years (2013–2016), and do not include the costs of donated praziquantel. The total cost per provided treatment was, on average, US$0.21. This study showed the value of exploiting administrative data to estimate costs of major global health interventions. It also provides an evidence base for financial costs and main cost drivers of implementing multiple combinations of intervention sets that inform decisions regarding the feasibility and affordability of implementing schistosomiasis control and elimination strategies. These conclusions are partially related to the lack of studies that embedded economic evaluations in implementation studies. Clearly, conducting these studies is time consuming, highly complex, and often requires accessing sensitive information that may highlight inefficient use, let alone misuse, of resources.8 There are, however, underused information and data available on the resources used in implementation research and in conducting disease control programs. The information is, for instance, comprised in budget data, financial reports, and activity reports by different stakeholders involved in implementing control interventions. This information can be used to conduct relatively basic but informative costing analyses that can be useful to assess the affordability of implementing such interventions.Evidence regarding the financial implications of implementing schistosomiasis control and elimination interventions is important to inform program decisions on their feasibility and affordability for scaling up of activities.Schistosoma haematobium infection prevalence and intensity in children from Zanzibar, and to compare the effect between these alternative approaches.9 At the end of the study in 2017, urogenital schistosomiasis was eliminated as public health problem from most of the 90 study sites.10 Moreover, the overall S. haematobium prevalence was significantly reduced from 6.1% to 1.7% in children aged 9–12 years and from 3.9% to 1.5% in adults aged 20–55 years from 2012 to 2017.10 However, 10 rounds of MDA with praziquantel over 5 years failed to interrupt S. haematobium transmission, even when supplemented by the implementation of additional elimination measures.10The Zanzibar Elimination of Schistosomiasis Transmission (ZEST) project, implemented on the two main islands of the Zanzibar archipelago , from November 2011 to May 2017 is an example of a multiyear effort to eliminate schistosomiasis. Embedded in the ZEST project was a large-scale study of various interventions. In brief, a cluster-randomized trial—funded by the Schistosomiasis Consortium for Operational Research and Evaluation (SCORE)—associated with the ZEST project aimed to assess biannual mass drug administration (MDA) applied alone or in combination with either snail control or behavior change interventions for the reduction of In this study, we used the programmatic and financial information made available by the ZEST project partners to conduct a cost analysis aimed at estimating the financial costs of the interventions against urogenital schistosomiasis on the two main islands of Zanzibar. Experiences and lessons learned are important for managers of schistosomiasis and other neglected tropical disease (NTD) control programs.11 The two islands are divided into 11 districts, which are subdivided into smaller administrative areas . According to the 2012 Tanzania Population and Housing Census, there were a total of 339 shehias: 212 on Unguja and 127 on Pemba. In 2018, there were 239 public primary schools and 102 private primary schools registered.11 The primary school net enrollment rate in the 2014/2015 household budget survey was estimated at 84.2%.11The two main islands of the Zanzibar archipelago, Pemba and Unguja, are located offshore the mainland of the United Republic of Tanzania in the Indian Ocean. The projected population of Zanzibar for 2019 was approximately 1.6 million inhabitants.S. haematobium, used to be highly prevalent on the Zanzibar islands in the twentieth century.16 In the 1990s and early 2000s, large-scale preventive chemotherapy campaigns, where anthelmintic treatment was provided without prior diagnosis to large parts of the population, consolidated by socioeconomic growth and improved access to clean water, reduced the prevalence and intensity of infection, and hence, morbidity due to S. haematobium in the at-risk population.19 In 2011, the ZEST alliance was formed with the goal to eliminate schistosomiasis as a public health problem on Pemba and to interrupt S. haematobium transmission on Unguja over 5 years.20 The cluster-randomized trial within the ZEST project was implemented from November 2011 to May 2017. The trial was funded by SCORE via a major grant from the Bill & Melinda Gates Foundation.21Urogenital schistosomiasis, caused by the parasitic blood fluke 20 In brief, 10 rounds of MDA with praziquantel against urogenital schistosomiasis  were implemented across the whole islands biannually from 2012 to 2016 as part of the routine activities of the NTD program of the Zanzibar Ministry of Health (ZNTD). An exception was the South district in Unguja, where, because of the absence of intermediate host snails and, hence, no S. haematobium transmission, there was no praziquantel distribution. With the exception of the sixth round on Pemba, each MDA round contained a community-wide treatment (CWT) approach.10 In CWT, trained community drug distributors (CDDs) were supposed to visit each household on the islands and, in a door-to-door approach, distribute the anthelmintic drugs to the eligible population. All individuals aged > 3 years were eligible to receive praziquantel (40 mg/kg administered by using a dose pole), with the exception of severely sick people and pregnant women. In the sixth round on Pemba, instead of the described CWT approach, health posts were installed, where people could visit and receive the drugs on request. In addition to CWT, to improve the coverage of the school-age population, school-based treatment (SBT) was implemented in five of the treatment rounds  on both islands and on Pemba also in the sixth round. In SBT, children attending public primary schools, and from 2015, also nurseries, private, and religious schools , received the anthelmintic drugs by their teachers and staff of the ZNTD by directly observed treatment. Treatment coverage data for all targeted shehias and schools were provided by the ZNTD through the registers of the CDDs and teachers, respectively.The three main ZEST interventions were MDA, snail control, and behavioral change.For the implementation of the cluster-randomized trial, on each island, 45 shehias were randomized to one of three study arms: biannual MDA alone, biannual MDA plus snail control, or biannual MDA plus behavior change.S. haematobium, which is Bulinus globosus. In case B. globosus were found, the snail control team sprayed the shoreline with a molluscicide  using a petrol sprayer or Hudson backpack sprayers to kill the snails. Snail control activities started in August 2012 and were implemented throughout the study years until February 2017, with exception of the rainy seasons. If no B. globosus were found, that area was not sprayed during that round. In Pemba, a total of 167 human–water contact sites were identified that were visited by the snail control team over the study period on a total of 556 days.22 At 71 among the 167 sites, B. globosus snails were found at least once, and 60 sites were treated with Bayluscide. On Unguja, a total of 121 human–water contact sites were identified that were visited by the snail control team over the study period on a total of 346 days.22 At 91 among the 121 sites, B. globosus snails were found at least once, and 65 sites were sprayed with Bayluscide.Snail control was implemented in 15 shehias per island, as part of the cluster-randomized trial from late 2012 until early 2017. Once the natural freshwater bodies in the study shehias were identified, they were regularly visited by trained snail control teams on the two islands. A visit included a snail survey at a specific site of the water body readily used by the local population for domestic and recreational activities . For the snail survey, snail control team members searched the shoreline of the water body for snails, including the intermediate host snail of 25 Four main behavior change activities were carried out over time from late 2012 until early 2017: 1) classroom-based interactive teaching about schistosomiasis by school teachers who had received specific training by the staff of the behavior change teams on each island and were provided with a schistosomiasis tool kit containing teaching materials such as lesson plans, blood fluke picture, flip charts tailored to the local perceptions, and snail boards26; 2) school-based interactive “Kichocho days,” where the whole school was involved in games, dramas, and songs with health education messages about the prevention, control, and treatment of schistosomiasis; 3) community-based installment of 30 urinals on Pemba and 28 urinals on Unguja; and 4) community-based installment of 21 washing platforms on Pemba and 25 washing platforms on Unguja.9In 15 randomly selected shehias per island, in addition to MDA, behavior change interventions were implemented. The interventions were developed in a participatory human-centered design approach, together with the local communities.am and 2 pm and subsequently examined in the laboratory for S. haematobium eggs by single urine filtration microscopy of 10 mL of urine per participant and for blood in urine by reagent strips  by trained laboratory technicians.27The impact of biannual MDA alone, or in combination with snail control or behavioral change interventions, was assessed in cross-sectional surveys conducted annually in 45 study schools and 45 shehias on Pemba and Unguja, respectively, as part of the cluster-randomized trial. Annually, from 2011/2012 to 2017, approximately 100 children from grades 3 and 4 (aged 9–12 years) in the primary schools and 50 adults (aged 20–55 years) living in the shehia communities of each of the 90 study sites were randomly selected and invited for participation in the study by experienced field-workers. Their urines were collected between 10 Supplemental Materials).The study presented here is a retrospective cost analysis of the ZEST project that used available data on the project activities, resources used, and costs reported in the accounting information systems of multiple ZEST partners. To classify the costs, we first developed a reference cost model and a cost classification tool. The reference cost model was informed by the existing literature and exchanges with experts from the wider SCORE network. The reference cost model was structured into two hierarchical levels  and used—to the extent possible—mutually exclusive and complete activity/process and associated expense/cost categories. For each category, be it at the higher or the lower hierarchical level, a short written definition and explanation was developed. Based on this reference cost model, a cost classification tool was established, which included all the details from the reference cost model and additional information considered relevant and useful for estimating the costs . All data obtained from the ZEST partners were classified according to the reference cost tool  Public Health Laboratory-Ivo de Carneri (PHL-IdC) data that included the costs and activities related to snail control, behavioral change interventions, parasitologic surveys, and the correlated management activities for the whole duration of the project on Pemba (2011–2017); 2) ZNTD data that included the costs and activities related to snail control, behavioral change interventions, parasitologic surveys, and the correlated management activities for the whole duration of the project on Unguja (2011–2017); 3) Schistosomiasis Control Initiative data that included the costs of the implementation of biannual MDA in Pemba between 2013 and 2016; and 4) Natural History Museum (NHM) data, which corresponded to procurements performed in the United Kingdom for equipment and material for the parasitologic and snail surveys on Pemba and Unguja, respectively, that were not available locally for the whole duration of the project. Whenever possible, for PHL-IdC, ZNTD, and NHM data, we assigned the costs to only one of the activities. When the costs referred to a generic management definition , we kept it as an extra single category. If the costs referred to two activities , we split the costs equally.Following the same pattern for each of the main activities, the costs were assigned to four categories: 1) personnel, 2) equipment, 3) material, and 4) travel. Personnel costs depend on the income that a staff received and included the following subcategories: top-up for project involvement and allowances. As regards equipment costs, these included capital costs that were represented by means of transport , microscopes, information technology (IT) infrastructures , and other capital equipment that varied from building material to a simple bucket. Material costs refer to consumables goods, which were represented by laboratory material  and office material . With regard to transport costs, these included gasoline and public transport . Of note, costs of gasoline in the PHL-IdC and ZNTD databases were not attributable to a specific activity. Hence, we summed up all the costs for gasoline related to any of the three activities  carried out by the PHL-IdC and ZNTD and reallocated them to each of the three activities according to the days of work per month for each of them .The costs of praziquantel tablets for MDA and of Bayluscide for snail control were not included in our analysis because these were donated by the WHO and Bayer Crop Science, respectively. The costs of external experts that supported the implementation of the study were not included, as they were essentially related to the research activities but not to the program interventions.The cost estimates of the activities related to snail control, behavior change, the parasitologic surveys, and for the management of the whole ZEST project are presented for the full duration of the project from 2011/2012 to 2017. The costs of the implementation of biannual MDA on Pemba are presented for the years 2013–2016 only. All costs are expressed in US$ and not adjusted for inflation, separately for Pemba and Unguja, and the following interventions and activities: snail control, behavior change, MDA, parasitologic surveys, and program management.The costs of snail control are presented in terms of total costs, cost per day in the field, and cost per shehia covered. Of note, the costs of Bayluscide were not included, as this molluscicide was donated. The costs of behavior change interventions are presented in terms of total cost and cost per shehia covered. The costs of the parasitologic surveys are presented in terms of total costs, cost per shehia monitored, and cost per survey conducted, including both school surveys and community surveys. The costs of biannual MDA are presented in terms of total costs and cost per person treated. The cost of the anthelmintic drug praziquantel was not included, as the drug was donated. The costs attributed to managing the whole ZEST project locally in Zanzibar were presented only in terms of total costs, as these are costs attributed to all activity categories implemented on Pemba and Unguja.The total estimated financial costs for implementing snail control activities over 5 years, excluding the costs for Bayluscide, were US$55,797 on Pemba  and US$7The total estimated financial costs for implementing behavior change activities over 5 years were US$109,166 in Pemba  and US$1Cost estimates for MDA were only available for Pemba and do not include the costs of donated praziquantel. The costs refer to the estimated 2.4 million treatments provided on Pemba over 4 years within eight rounds of MDA. The total cost of MDA amounted to US$484,887. The average cost for a single treatment was US$0.20 . More thThe total costs of six parasitologic surveys were estimated to be US$112,110 on Pemba  and US$1The estimated total costs of managing the ZEST project over the 5 years (from 2011/2012 to 2017) were US$82,831 on Pemba  and US$220 Despite the limitations of using administrative data for estimating the costs of global health interventions,28 the findings of this study are relevant not only for schistosomiasis control and elimination efforts on Zanzibar but also for other NTD control programs elsewhere in sub-Saharan Africa.We estimated the financial costs of the ZEST project, analyzing programmatic and financial data made available by the project partners. The ultimate goal of the ZEST project was to eliminate urogenital schistosomiasis as a public health problem on the two islands of Zanzibar and to interrupt transmission of the disease in the longer run.28 Our findings indicate, for example, the considerable costs related to project management activities. In fact, the estimated costs of managing the whole ZEST project were slightly higher than the estimated costs of behavioral change activities. It should be noted that managing a large-scale schistosomiasis elimination project is a complex undertaking that requires the availability of well-trained personnel. In addition, the estimated costs of the cross-sectional parasitologic surveys to assess longitudinally the impact of the interventions were relatively high. These findings point to the importance of considering the costs of monitoring activities and surveillance systems in the context30 of NTD elimination strategies. There are only a few studies that estimated the costs of surveillance systems, and most of them only provided aggregated cost estimates.34First, the financial cost estimates include all the activities implemented by the various ZEST partners and stakeholders. Specifically, costs of the different interventions , large-scale repeated cross-sectional parasitologic surveys that are necessary to assess the impact of interventions, and for managing the project were included. Such a comprehensive approach is rarely pursued in costing studies of global health interventions. Indeed, prior studies mainly estimated the costs of a single intervention or, in case of a package of intervention, did not provide disaggregated estimates by interventions, and lacked inclusion of ancillary but crucial activities such as those related to project management and monitoring.Second, this study estimated the costs of the ZEST project disaggregating them according to how the interventions were geographically distributed. Although the disaggregated cost estimates for Pemba and Unguja have limitations due to the predefined budgets both islands had for implementing the surveys and interventions, these disaggregated estimates can be useful for informing decisions on the resources required for future schistosomiasis control and elimination efforts. In the ZEST project, the costs per shehia covered for snail control and for behavior change activities were higher on Unguja than on Pemba. The differences were mainly driven by the cost of transport and personnel. In Unguja, more people and at a higher level  were involved in managing the ZEST project, which included both research and programmatic parts. Taken together, the cost of personnel was 2.6-fold higher in Unguja than on Pemba.36 around 80% of the non–medicine-related costs of MDA was accounted by personnel. Similarly, personnel emerged as a major cost driver for snail control and behavior change interventions. Our findings further revealed that for snail control and behavior change interventions, travel costs were considerable and, for the behavior intervention, budgeting for consumable goods , was important. In addition, the project management cost estimates show that besides personnel, to manage the implementation of schistosomiasis control interventions, a substantial budget to cover travel expenses is required.Third, the cost estimates disaggregated by cost category provide important information of the relative importance of the different types of costs and related activities. Consistent with the results of previous studies,8 estimated the costs of praziquantel used in schistosomiasis control programs for MDA to represent a large part of the total economic cost of the intervention.Our findings should be interpreted with caution, as the study has several limitations. First, the costs estimates are based on a retrospective analysis of administrative data made available by the various ZEST partners. Hence, the analysis required considerable efforts to identify various resources used and reclassification of budget data to be attributed to the specific interventions and activities. Although this exercise proved to be feasible, we cannot guarantee that all reclassifications were accurate. This may be particularly relevant to the estimates of the project management costs allocated to the two islands. Second, using administrative data of a project supported by external partners may lead to cost estimates that are affected by budgeting rules and practice, and budgets predefined by external funders. Third, the costs presented in this study should be considered as conservative estimates, as they do not include support from external experts who nevertheless played a crucial role in the design, implementation, and longitudinal monitoring of the project. For example, the costs of behavior change interventions presented here do not include the costs of the consultant, who was crucial to develop the behavior change interventions and to train the staff of the behavior change team for implementation of interventions, and hence need to be interpreted with care. The cost estimates presented here include only financial costs and, hence, do not consider the costs of infrastructure that was already in place. In addition, the costs of MDA did not include the cost of praziquantel and the costs of snail control did not include the cost of Bayluscide. Both products were donated, and they therefore did not generate any financial expense. Praziquantel was donated to the ZNTD by the WHO, whereas Bayluscide was donated by Bayer Crop Sciences. Including these costs was beyond the scope of this study. Moreover, we consider the estimation of the economic value of donated products prone to methodologic challenges and uncertainties. Yet, it is worth noting that a recent literature reviewDespite these limitations, our study showed the value of exploiting administrative data to estimate costs of a package of global health interventions. The granular cost estimates presented here could, for example, be used by program managers to estimate the financial costs of scaling up a control program in Zanzibar or similar settings. Our study strengthens the evidence base of the financial costs and cost drivers of implementing a comprehensive package of schistosomiasis control and elimination interventions in different socio-ecological settings that can be used to inform decisions regarding the feasibility and affordability of schistosomiasis elimination strategies.Supplemental materials


Outcomes data for patients who received tedizolid for acute bacterial skin and skin-structure infections (ABSSSIs) are scant. We provide a real-world analysis of economic and clinical outcomes following tedizolid use in the outpatient setting.This retrospective study of adults with skin infections treated with tedizolid (index period: 1 July 2014–31 May 2016) used data from the Optum Research and Impact National Benchmark databases.n = 18; without complications, n = 73). Some patients had > 1 complication and infection site. Among patients with complications, pre-index complications during the [index date − 30] through [index date + 1] period included osteomyelitis (44.4%), septicemia (44.4%), and prosthetic joint/device/graft infection (16.7%). For the [index date − 7] through [index date + 1] period, the infection site included abscesses (55.6%) and chronic ulcers (38.9%). Mean (standard deviation [SD]) days supplied for the index tedizolid claim was 6.8 (2.3) days. Healthcare resource utilization (HCRU) during the 30-day post-index period included ≥ 1 ambulatory visit (100.0%), ≥ 1 emergency department (ED) visit (16.7%), and ≥ 1 hospitalization (22.2%). Median 30-day post-index all-cause costs were $11,098 . Among patients without complications, the pre-index infection site from ([index date − 7] through [index date + 1]) included abscesses (60.3%), chronic ulcers (37.0%), and cellulitis (2.7%). Mean (SD) days supplied for the index tedizolid claim was 6.6 (2.5) days. Thirty-day post-index HCRU included ≥ 1 ambulatory visit (91.8%), ≥ 1 ED visit (17.8%), and ≥ 1 hospitalization (5.5%). Median 30-day post-index all-cause costs were $3230 .Ninety-one patients received tedizolid for the treatment of skin infections  contains supplementary material, which is available to authorized users. Streptococcus pyogenes, Staphylococcus aureus [including methicillin-resistant S. aureus (MRSA)] is now the predominant cause of SSTIs; the incidence of S. aureus-related SSTI hospitalizations in the US significantly increased from 2001 to 2009   through [index date − 1]. Costs expressed in US dollars ($) were adjusted to 2016 costs using the annual medical care component of the Consumer Pricing Index (CPI) to reflect inflation between 2014 and 2016.The count of subsequent claims for tedizolid during a maximum of 91 days post-index was calculated, and total days supplied was categorized as 1–6, 7–12, and ≥ 13 days. The following burden of illness outcomes were evaluated for the period of the index date through [index date + 29]: (1) HCRU , (2) CPI-adjusted healthcare costs , and (3) antibiotic therapy related to skin infection (encompassing any agent indicated primarily against gram-positive bacteria).1) and upper quartile (Q3) values for HCRU/costs]. All-cause hospital costs, ambulatory costs, and emergency services costs (pre- and post-index) were reported for users . Missing data were reported as a category where appropriate.All study variables, including outcomes and covariates of interest, were descriptively evaluated for patients with skin infections, stratified by complication status. Numbers and percentages were reported for dichotomous and polychotomous variables. Means, standard deviations (SDs), and medians were reported for continuous variables  through [index date + 1] included osteomyelitis (44.4%), septicemia (44.4%), and prosthetic joint/device/graft infection (16.7%). The pre-index infection site closest to the index date during the period of [index date − 7] through [index date + 1] included abscesses (55.6%) and chronic ulcers (38.9%).n = 8), ≥ 1 ambulatory visit , and ≥ 1 ED visit  of patients were discharged  in the 7 days before the index date. The median all-cause total costs during the period of [index date − 30] through [index date − 1] were $14,001 . Median medical and pharmacy costs were $13,531  and $483 , respectively. Median hospital costs for users comprised $11,655 . Median ambulatory and emergency services costs for users were $3506  and $423 , respectively.Pre-index all-cause HCRU during the period of [index date − 30] through [index date − 1] included ≥ 1 hospitalization  days supplied for the index tedizolid claim was 6.8 (2.3) days. Total outpatient days supplied for combined tedizolid fills for the index date through [index date + 90] included 1–6 days (72.2%), 7–12 days (11.1%), and ≥ 13 days (16.7%). Approximately half of patients (55.6%) had evidence of treatment with an anti-gram-positive agent other than tedizolid following the index date , whereas 66.7% were treated with an anti-gram-positive agent during the period of [index date − 30] through [index date − 1].n = 4) had ≥ 1 all-cause hospitalization , 100.0% (n = 18) had ≥ 1 all-cause ambulatory visit , and 16.7% (n = 3) had ≥ 1 all-cause ED visit , $14,637 ), medical costs $7983 , and pharmacy costs $2126 . Median hospital costs for users comprised $3314 . Median ambulatory and emergency services costs for users were $2916  and $274 , respectively.During the first month following the index date among patients with skin infections with complications, 22.2%  age was 58.5 (15.7) years and 46.6% were female. Patients were predominantly from the South (63.0%), followed by the Northeast (15.1%) and Midwest (12.3%). More than half (56.2%) of patients had a pre-index Quan-Charlson comorbidity score of 0. Common pre-index CCS comorbidities were skin and subcutaneous tissue infections (75.3%), other connective tissue diseases (54.8%), hypertension (45.2%), and diseases of veins and lymphatics (45.2%). The pre-index infection site closest to the index date during the period of [index date − 7] through [index date + 1] included abscesses (60.3%), chronic ulcers (37.0%), and cellulitis (2.7%).n = 10), ≥ 1 ambulatory visit , and ≥ 1 ED visit  patients were discharged  in the 7 days before the index date. Patients with skin infections without complications had median all-cause total costs during the [index date − 30] through [index date − 1] period of $3843 . Median medical and pharmacy costs were $2967  and $170 , respectively. Median inpatient costs for users comprised $7158 . Median ambulatory and emergency services costs for users were $1066  and $437 , respectively.Pre-index all-cause HCRU during the period of [index date − 30] through [index date − 1] included ≥ 1 hospitalization  days supplied for the index tedizolid claim was 6.6 (2.5) days. Total outpatient days supplied for the index date through [index date + 90] tedizolid claims was 1–6 days (75.3%), 7–12 days (15.1%), and ≥ 13 days (9.6%). Evidence of treatment with an anti-gram-positive agent other than tedizolid following the index date  was observed for 31.5% of patients, whereas 58.9% were treated with an anti-gram-positive agent during the period of [index date − 30] through [index date − 1] .n = 4) had ≥ 1 all-cause hospitalization , 91.8% (n = 67) had ≥ 1 all-cause ambulatory visit , and 17.8% (n = 13) had ≥ 1 all-cause ED visit . Median all-cause costs included total costs $3230 , medical costs $646 , and pharmacy costs $2031 . Median hospital costs for users comprised $10,836 . Median ambulatory and emergency services costs for users were $575  and $641 , respectively.During the first month following the index date among patients with skin infections without complications, 5.5%  and uncomplicated  SSSIs, hospital admissions after tedizolid treatment in the outpatient setting were similar to or lower than nationally reported 30-day readmission rates (20%) . PatientThis study revealed that patients treated with tedizolid in the outpatient setting experienced a short duration of therapy, low hospital admissions, and modest post-index HCRU indicators. This is the first real-world analysis of the economic and clinical impact of tedizolid in the outpatient setting.When interpreting this retrospective observational study, there are several limitations. It is important to note that due to the nature of the study, causality cannot be inferred. Given the retrospective nature of this study and utilization of a claims database, not all clinical and therapeutic information, such as lesion surface area and causative microorganisms, was available, which may have impacted outcomes. Furthermore, there was no comparator group, limiting both internal and external validity. This study was also performed in the US, with the pharmacoeconomic aspects likely not applicable to other countries where costs differ. In addition, the data source used in this study was a claims database intended to facilitate payment and not research. Use of International Classification of Diseases, 9th and 10th Revision, Clinical Modification diagnosis codes for cSSSI may also be considered a limitation because the codes assigned to the patient only reflect the claims that physicians/facilities submit for reimbursement for that visit and may not align with a confirmed diagnosis, in that patients may be initially miscoded or misdiagnosed. Similarly, pharmacy claims data cannot ensure the patient used the medication as prescribed: these data only document that a claim was submitted. The value of claims data lies in the efficient and effective examination of healthcare outcomes, treatment patterns, HCRU, and costs in a “real-world” setting that includes a large number of patients with varied medical histories. Finally, this study was focused on tedizolid, and other therapeutic options (including surgery) were not part of the study design. Such details should be of interest for future analyses.Among patients with skin infections, with and without complications, treated in the outpatient setting with tedizolid, hospital admissions were low, and all-cause and skin infection-related HCRU in the 30 days after receipt of tedizolid were modest and consistent with other outpatient antibiotic therapies. While comparative studies are warranted, these findings support the use of tedizolid in an outpatient setting for treatment of ABSSSIs.Supplementary material 1 (DOCX 24 kb)Below is the link to the electronic supplementary material.


We investigated 68 respiratory specimens from 35 coronavirus disease patients in Hong Kong, of whom 32 had mild disease. We found that severe acute respiratory syndrome coronavirus 2 and subgenomic RNA were rarely detectable beyond 8 days after onset of illness. However, virus RNA was detectable for many weeks by reverse transcription PCR. Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is causing a global pandemic and affecting global health and the world economy. Virus RNA might be detectable by reverse transcription PCR (RT-PCR) many weeks after clinical recovery (10 RNA copies/mL) .We attempted virus isolation in 68 specimens from 35 patients in Hong Kong. Specimens were collected at different times after symptom onset to define the kinetics of virus isolation in upper respiratory specimens. Those specimens with a viral load >5 log10 N gene copies/mL.The study was approved by the Research Ethics Committee of the Kowloon West Cluster  of the Hospital Authority of Hong Kong. We provide methods for virus nucleoprotein (N) gene copy number quantification . Virus sA total of 68 specimens from 35 patients were studied ; patient10 copies/mL to undetectable (limit of detection 10 copies/mL) (10 copies/mL and in culture-negative samples was 3.8 log10 copies/mL (p = 0.00001)  . The med0.00001) .10 copies/mL, 3 of 11 specimens with viral loads 6.0–6.99 log10 copies/mL, 1 of 7 specimens with viral loads 5.0–5.99 log10 copies/mL, and 0 of 33 specimens viral loads <5 log10 copies/mL.Virus was isolated from 12 of 17 specimens with viral loads ≥7.0 log>5.0 log10 virus genome copies/mL; 2 specimens had insufficient specimen for this testing. Of 33 specimens tested for sgRNA and by virus culture, both tests showed positive results for 12 (36.4%) specimens, both tests showed negative results for 12 (36.4%), sgRNA showed positive results and culture was negative for 7 (21.2%) specimens, and culture was positive and sgRNA showed negative results for 2 (6.1%) samples  indicating a moderate agreement between virus culture and sgRNA detection. Virus sgRNA was detectable in 18 (81.8%) of 22 specimens collected <8 days after symptom onset and in 1 (9.1%) of 11 specimens collected >9 days after onset of disease (p = 0.0003 by χ2 test with Yates correction)  . We alsorection) .10 genome copies/mL and in culture-negative specimens was 4.0 log10 genome copies/mL (p<0.00001 by Mann-Whitney 2-tailed U test). Of the 16 culture positive specimens, 15 (94%) had viral RNA load >6 log10 copies/mL (p<0.01 by Fisher exact test). All of them were collected within the first 8 days of illness (p = 0.01 by Fisher exact test )  . However>6 log10 virus N gene copies/mL of clinical specimen. We did not carry out serologic testing in parallel with viral culture, but data on a larger cohort of patients in Hong Kong showed that most patients had detectable virus neutralizing antibodies after day 9 of illness , but these guidelines do not distinguish between mild and severely ill patients . The World Health Organization has recently amended its guidelines for releasing COVID-19 patients from isolation (i.e., 10 days after symptom onset and Additional information on SARS-CoV-2 virus culture and subgenomic RNA for respiratory specimens from patients with mild coronavirus disease.


Flaviviridae family. After decades of circulation in Asia, ZIKV was introduced to Brazil in 2014–2015, associated with a rise in congenital malformations. Unlike the genetically related dengue virus (DENV), ZIKV constitutes only one serotype. Although assumed that ZIKV infection may engender lifelong immunity, the long-term kinetics of ZIKV antibody responses are unclear. We assessed long-term kinetics of ZIKV NS1-IgG response in 144 individuals from 3 different subpopulations: HIV patients, tuberculosis patients and healthy individuals first tested in 2016 and retested 1.5–2 years after the 2015–2016 ZIKV epidemic in Salvador de Bahia, Brazil, using a widely distributed NS1-based commercial ELISA. The seropositivity in 2016 reached 59.0%  50.7–66.7%), and decreased to 38.6%  1.5–2 years later. In addition, the median ZIKV NS1-ELISA reactivity for individuals that remained positive in both timepoints significantly decreased from a ratio of 4.4 (95% CI 3.8–5.0) to 1.6 (95% CI 1.6–1.9) over the 2-year interval  irrespective of the subpopulation analyzed. Initial 2016 DENV antibody response was non-significant between groups, suggesting comparable DENV background. The high 20.6% seroreversion suggest that widely used serologic tests may fail to account a considerable proportion of past ZIKV infections in flavivirus endemic countries. In addition, ZIKV immunity might be shorter-lived than previously thought, which may contribute to local ZIKV resurgence once individual immune responses wane sufficiently to reduce community protective immunity in addition to birth and migration.Zika virus (ZIKV) is a positive-stranded RNA virus within the  Flavivirus genus inside the Flaviviridae family. Unlike the ubiquitous dengue virus (DENV), which occurs as four distinct serotypes globally, ZIKV represents only a single serotype to which both the African and the Asian lineages of ZIKV belong [The Zika virus (ZIKV) is an enveloped positive-stranded RNA virus belonging to the V belong , 2. The V belong . VirologV belong . ZIKV seV belong . The envV belong  and NS1 V belong . The NS1V belong .2 only. In contrast, Brazil has 210 million inhabitants spread over 8 million km2 (latest United Nations Population Division estimates). In Brazil, as other Latin American countries, cocirculation of other flaviviruses such as DENV, Yellow fever virus, Bussuquara, Cacipacoré, Ilhéus, Rocio and Saint Louis encephalitis virus might elicit unique flaviviral antibody responses that impact ZIKV-specific antibody kinetics [ZIKV was first detected in 1947 in Uganda –12. The kinetics –17. NoneA total of 144 samples were taken from individuals on 2 occasions. The samples from the first timepoint correspond to a cross-sectional study conducted at the University Hospital Professor Edgard Santos (UHPES) in Salvador de Bahia, which is one of the biggest public hospitals in the region, between February and May 2016 during the end of the ZIKV epidemic . SamplesZ − 6.1; p < 0.001) irrespective of which subpopulation was analyzed. Among the initially seronegative individuals, median ELISA reactivity was almost identical between both time points   were HIV-positive individuals  than in HIV-positive patients   for serological testing in public health laboratories . We usedThe high level of seroreversion can be explained by three scenarios. First, seroreversion in HIV-positive individuals may be associated with lower magnitudes of ZIKV-specific immune responses compared to other subpopulations, consistent with generally lower antibody responses of HIV-positive individuals, e.g., following hepatitis A virus vaccination . HoweverIrrespective of the complex underlying reasons, the observed decrease in ZIKV NS1-specific antibody levels over time was reminiscent of the complete lack of seroconversion in follow-up samples of two different cohorts. First, from RT-PCR-confirmed ZIKV infections in travelers using the same NS1-based serologic test 42 days post onset of symptoms . Second,Our serologic data from Brazil are thus consistent with data from Pacific island populations in showing loss of ZIKV-specific antibody responses over a comparably short time span irrespective of the serologic method used for testing . Since N


E. coli strains was carried out based on the fluorescent signal of the core-shell beads. This platform can be a promising alternative for multiplex nucleic acid analyses due to its simplicity and high throughput. The platform reported here also reduces the cycling time and avoids evaporation as well as contamination of the sample during the amplification process.Multiplex polymerase chain reaction (PCR) is an effective tool for simultaneous detection of target genes. Nevertheless, their use has been restricted due to the intrinsic interference between primer pairs. Performing several single PCRs in an array format instead of a multiplex PCR is a simple way to overcome this obstacle. However, there are still major technical challenges in designing a new generation of single PCR microreactors with a small sample volume, rapid thermal cycling, and no evaporation during amplification. We report a simple and robust core-shell bead array for a series of single amplifications. Four core-shell beads with a polymer coating and PCR mixture were synthesized using liquid marble formation and subsequent photo polymerization. Each bead can detect one target gene. We constructed a customised system for thermal cycling of these core-shell beads. Phylogrouping of the  Escherichia coli (E. coli) is a bacterium that is frequently found in the environment and the intestine of humans and some animals. While most strains of E. coli are not harmful, some strains are pathogenic and can cause infectious diseases such as pneumonia, urinary tract infections, diarrhea, gastrointestinal, and extra-intestinal infections [E. coli strains identified four main groups comprising A, B1, B2, and D [fections ,2. Phylo2, and D ,4. The d2, and D ,6. Howev2, and D ,8,9.E. coli phylogroups. These approaches can be divided into two main groups: (i) digestion-based methods such as restriction fragment length evaluation, pulsed-field gel electrophoresis (PFGE) [Over the past few decades, a broad range of conventional genotyping methods were employed for the characterisation of s (PFGE) ; and (iis (PFGE) ,12, rands (PFGE)  or coupls (PFGE) , ribotyps (PFGE) , and muls (PFGE) ,17. The s (PFGE) . (ERIC)-s (PFGE) ,20. RAPDs (PFGE) , even ths (PFGE) . PCR ribs (PFGE) . E. coli phylogrouping. In this method, the specific primers are simultaneously used for the amplification of 4 genes whose presence/absence produces a genotype, which identifies an isolate as a member of one of 8 different phylogroups [E. coli typing. However, there are still some obstacles that limit their application. For example, agarose gel-based DNA separation technique is a necessary step for both assays. Conventional agarose gel-based technique has limitations such as inter-laboratory difference in explaining banding patterns, time-consuming and complex processes, and the need for skilled personnel as well as expensive equipment [Multiplex (tri/tetra plex) is another common PCR-based molecular technique for logroups . Among tquipment ,26. Althquipment .Microfluidics was successfully utilised for nucleic acid amplification . Lab-on-Liquid marble, a liquid droplet encapsulated by hydrophobic powder, can be an alternative platform to address the above issues of conventional methods. The powder coating prevents the direct contact between the core liquid and the surrounding, thus reducing contamination . Liquid E. coli strains. The PCR mixture was inserted into a photopolymer droplet, forming a spherical bead. The resulting liquid droplet was first embedded in a hydrophobic/oleophobic powder. The powder and the shell liquid not only provide the sterile condition for the PCR mixture, but also remarkably reduce evaporation. Moreover, solidification of the polymer transforms the liquid marble into a core-shell bead, enabling easy handling with no evaporation lost.The present paper reports an amplification-based detection assay using four core shell beads for phylogrouping of The synthesis and characterization of a core-shell bead was reported previously . The synA custom-built thermal cycler was developed to provide the required conditions for PCR with the core shell beads . A 2 cm E. coli strains were isolated on nutrient agar plates from faeces samples in a clinical pathology lab. Single colonies were picked and grown overnight at 37 °C in nutrient broth. Isolates were stored at −80 °C until use in nutrient broth with 30% glycerol. The isolates were then used for phylogrouping experiment using single PCR and quadruplex PCR assays according to the method reported by Clermont et al. [E. coli strains were isolated by a clinical pathology lab in Turkey as part of the routine analyses of clinical samples. No clinical samples were directly collected from any patients, nor were the isolated strains part of any projects; therefore, there is no need for ethics approval for further testing them.t et al. . All samFor real-time analysis of the amplification, we developed a fluorescent detection platform as depicted in E. coli strains. For the phylogrouping experiment, four core-shell beads were fabricated. Each bead contained 2 µL PCR solution with a particular set of primer to detect one marker gene. The resulting core-shell beads were subsequently located on a customised PCR device for thermal cycling. A separator made of polydimethylsiloxane (PDMS) was used to prevent the beads from sticking to the heater during the thermal cycling process. The experiment was performed in a dark laboratory environment. The blue LED light source was employed to match the excitation wavelength. The light was manually turned on at five-cycle intervals. Experiments were repeated four times, one for positive control and three for unknown samples. Compared to the traditional phylogrouping method (PCR and subsequent gel electrophoresis), which take almost 3 h, our assay was carried out in around 1.5 h. One experiment with an unknown sample was repeated three times to test the reproducibility of the assay. Satisfactory results were achieved as shown in Using a core-shell bead as a microreactor , we demoE. coli isolates could not be exactly assigned to one of the known phylo-groups and reported as unknown strains. The multi-locus sequence type (MLST) technique [We used core-shell beads of A, B, C, and D for the analysis of chuA, arpA, TspE4C2, and yjaA genes, respectively, to demonstrate the capability of our method for analysing clinical (control/unknown) samples. The numerical values of the fluorescent intensities of the amplicons inside the core shell beads were evaluated using ImageJ. The values were normalised asechnique  is requiE. coli that possess chuA, yjaA, and TspE4C2 genes. E.clade I/II strain just shows amplification of yjaA gene (lane 5). The remaining genotypes (lane 4 and 6) cannot be classified into any distinct phylo-group. These results validate the accuracy of the amplification using core-shell beads.We used quadruplex PCR and gel electrophoresis to verify the phylogrouping results of the samples in a commercial platform. E. coli strains. This method potentially resolves the issues of conventional multiplex PCR in a single tube such as the intrinsic interference and the competition among primers, the requirement for optimized multiplex PCR protocols, and the difficulty in simultaneously detecting a large number of targets. Our assay demonstrated the following advantages: (i) simple fabrication and operation; (ii) fully sealed PCR microreactor that eliminates volume loss due to evaporation and minimises the risk of contamination; and (iii) reducing the PCR mixture volume to 2 µL, which decreases the cycle time. In summary, the core-shell bead platform reported here is capable of full functionality of a PCR assay due to its simplicity and potentially high throughput. This PCR microreactor can be employed in nucleic acid studies for clinical or environmental diagnostics. We believe that our method has a huge potential for commercialisation due to the reduction of expensive lab-based equipment and labour costs. Further development will focus on the automation of the platform, making it more user-friendly. Current manual fabrication of beads for a large number of samples is labour intensive. Thus, automation of bead preparation, partitioning of the PCR solution, and delivery into the fabricated beads can increase the speed and efficiency of the assay. Moreover, this platform has the potential to be fully integrated with other steps such as sample preparation, DNA extraction, amplification, and optical detection for the identification of bacteria as well as other basic research and translational applications.Four core-shell polymer beads containing the PCR mixture were prepared by forming a liquid marble and subsequent photo polymerisation. A customised thermal cycler was developed to provide temperature control of the core-shell beads. Thermal cycling of the core-shell beads as PCR microreactor was performed for phylogrouping of the


Viral diseases have affected humans since the dawn of humanity. Smallpox-now eradicated by vaccinations-serves as a particularly poignant example. More recently, the Spanish fluoutbreak claimed a heavy toll in the early 20th century. The ongoing coronavirus pandemic appears no less threatening. The possible reason of highly variable course of disease is discussed. Vaccinations presented a breakthrough in the treatment of viral diseases. Other promising drugs include inhibitors of viral enzymes. Unlike bacteria, viruses are not, strictlyspeaking, alive. They are packets of information capable of invading and exiting cells. The host cell is burdened with synthesizing and replicating the virus, whose capability forpenetrating cells determines its potency. From the epidemiological perspective, an important factor affecting the spread of the virus is the proximity between infection targets.Viruses capable of droplet transmission are particularly dangerous. Bacteria are evolutionarily ancient entities that have long been targeted by certain classes of viruses (knownas phages). As a result, some bacteria have evolved methods to combat hostile carriers of information. The bacterial genome includes a repository of viral genetic sequences-effectivelya "catalogue" of known viruses. When a virus invades, the bacterium responds by synthesizing (based on its stored DNA) a complementary RNA matrix attached to a nuclease-a DNA-degradingenzyme. This complex is capable of recognizing the viral genome and cleaving it, disrupting the invasion ,2.EukarThe coronavirus responsible for the ongoing pandemic is spreading very rapidly. Its effects appear to depend on the carrier's age: many people, particularly young ones, remainasymptomatic, while older individuals frequently develop serious-even life-threatening-symptoms. The disease also appears to affect men more frequently than women, while itsprevalence varies by region. Of course, social measures may either promote or hamper the spread of the virus; however, this factor alone does not fully explain the observed dynamicsof the disease. A convincing argument can be made for the existence of other factors, such as varying susceptibility to the virus. Variable susceptibility to infection may be aconsequence of the general state of health, which, of course, depends on the individual's age and co-morbidities-particularly metabolic ones (e.g. diabetes). Nevertheless, theobserved profile of the disease suggests that other factors are at play and may determine whether a given organism can successfully defend itself against the virus. Since the viruspreferentially attacks the lungs, we can point to tobacco smoking as a factor, which promotes infection by damaging lung tissue. However, there are other factors as well, one of whichis alcohol consumption -5. AlcohOne mechanism which exhibits clear susceptibility to external disruption  is protein folding. Folding is usually understood as the processby which a protein attains its tertiary structure. This structure is strongly dependent on environmental conditions, and also on changes in the protein’s environment -10. CelAcetaldehyde readily reacts with amines, including with guanine residues present in nucleic acids, which-when placed in close proximity-may bind to each other. The resultingcomplexes are usually unstable and fall apart quickly. Even guanine pairs (relatively stable in comparison) are generally repaired in liver tissue by the appropriate enzymes. However,if a mutation knocks out reparatory mechanisms-such as in the case of Fanconi anemia-the damage incurred by DNA may become severe and lead, among others, to carcinogenesis. Notably,ε-amine groups in lysine residues are damaged by the aldehyde, and this type of damage is believed to be long lasting. Another example of aldehydes acting upon proteins involvesdamage to microtubules, which play an important role in ensuring structural stability of the cell. Genetic polymorphism undoubtedly results in individual variability, including well-knowndifferences in alcohol tolerance. Societies used to consuming large quantities of alcohol-usually in small doses-are characterized by specific public health traits, such as thoseattributable to resveratrol. Alcohol is also increasingly implicated in a variety of pathological conditions, including cancer. Regardless of specifics, its effect upon the organism-including lung tissue-is undoubtedly significant. Identifying links between alcohol use and the kinetics of viral infection remains a speculative endeavor. Further information maybe provided by statistical studies involving a large quantity of individual habits and cases.Professional literature also suggests a possible way by which alcohol may affect the reactivity of the organism and its interaction with the environment. This mechanism refers tothe so-called bacteriome, i.e. our hybrid bacterial ecosystem -19. Bact


Tecoma are found in tropical and subtropical regions around the world. Some of them are grown as ornamental plants and others can be used as medicinal plants. In the present study, ethanolic extracts from trunks and leaves of Tecoma species were tested in vitro using assays against the Zika virus.Plant species from the genus Tecoma species. The Tecoma castaneifolia, T. garrocha, T. stans var. angustata and T. stans var. stans were prepared by percolation with ethanol. The antiviral activity was assayed in vitro against the Zika virus by the MTT colorimetric method (n = 3). The UPLC-DAD-MS analysis of ethanolic extracts was performed from all the studied species. The biofractionation of T. stans var. stans trunk extract using different separation techniques led to the isolation of crenatoside compound.There was a total of 8 extracts obtained from different anatomical parts of three Tecoma species leaves were more active against the Zika virus (EC50 149.90 to 61.25 μg/mL) when compared to the trunk extracts tested (EC50 131.0 to 66.79 μg/mL and two were not active). The ethyl acetate and aqueous fractions obtained from T. stans var. stans trunk were active against the Zika virus with EC50 values of 149.90 and 78.98 μg/mL, respectively. Crenatoside is a phenylethanoid glycoside isolated from the ethyl acetate of T. stans var. stans trunk extract. This compound was tested and exhibited EC50 34.78 μM (21.64 μg/mL), thus demonstrating a better result than the original ethanolic extracts as well as others extracts of Tecoma species, and it was more active than the positive control, ribavirin (386.84 μM). Furthermore, its selectivity index was at least 2.5 times higher than the tested ethanolic extracts and 11.1 times more potent than ribavirin.Ethanolic extract from Tecoma species demonstrated interesting in vitro activity against the Zika virus. The crenatoside, phenylethanoid glycoside that was for the first time isolated from Tecoma stans var. stans, exhibited a potent and relevant anti-Zika virus activity, being more active than ribavirin (positive control). The data show that crenatoside, was a promising compound with in vitro antiviral activity against the Zika virus.The  Zika virus (ZIKV) is an infectious mosquito-borne flavivirus. It was first reported in 1947, when monkeys from the Zika Forest in Uganda were infected. Later, the first human casualties were described in Nigeria in 1954. It could be transmitted by different species of Aedes mosquitoes in tropical and subtropical areas. Several cases were described in the Americans, Asia and Africa from 1960s to 1980s. At a later date, there were outbreaks reported in different countries in the pacific. In 2015, Brazil reported the first cases of the Zika virus in Rio Grande do Norte and Bahia  were collected in Minas Gerais, Brazil, and a taxonomic determination was made by the botanist Dr. J. R. Stehman, from the Botanical Department at the Institute of Biological Sciences, UFMG, in Belo Horizonte city, Brazil. A voucher of each species was deposited at the BHCB/UFMG herbarium.The following plant species .T. garrocha and T. stans var. angustata. The protective effect during plant tissue injury is widespread by antioxidant activity of these compounds , Distictella elongate [Xylofragma myrianthum [Markhamia lutea extract presented in vitro activity against Respiratory syncytial virus [Other Bignoniaceae family species with antivirus activity have been previously reported including in vitro anti-elongate , Xylofrarianthum . Markhamal virus .Zika virus activity, that is, it presents selective and relevant activity when compared to standard criteria [50) is slightly less than 100 μg/mL in Vero cell line, the crenatoside was not cytotoxic in MRC-5 cells at the highest concentration tested, hence according to the recommendations described by Cos et al. [According to this study, the phenylethanoid crenatoside antiviral activity shows that it is a promising substance with in vitro anti-criteria , 19. Alts et al. . Thus, cTecoma species, it was at least 2.5 times more active than the crude extracts, in relation to the origin ethyl acetate fraction, and crenatoside was 4.3 times more active. Furthermore, when compared with the positive control, ribavirin, it was 4.4 times more active against ZIKV. Until today, there is no specific treatment for the Zika fever, therefore, ribavirin was chosen as a positive control because it is an antiviral approved drug used to treat hepatitis C. Like the Zika virus, the Hepatitis C virus is a member of the Flaviviridae family, and in vitro and in vivo studies have shown the ribavirin potential to inhibit the Zika virus.Comparing the crenatoside antiviral activity with the ethanolic extracts of Respiratory syncytial inhibition; antihypertensive; analgesic [Influenza virus type A [There were some crenatoside biological activities previously reported such as: antioxidant activity, platelet aggregation, nalgesic , inhibitnalgesic  among ots type A , which pZika virus than the Influenza virus type A, although the Zika virus and the Influenza virus are viruses enveloped with RNA genetic material [Influenza virus presents a single-stranded negative-sense and belongs to the Ortomyxoviridae family [The crenatoside is four times more active against the material , 45. Mormaterial , while tZika virus allows viral multiplication inside the cells leading to the cell monolayer destruction that was observed in the images  and no morphocellular deformities are observed. Therefore, they indicate that the treatment performed after infection is effective in inhibiting the virus multiplication cycle, confirming the antiviral activity observed in the MTT colorimetric assay.Zika virus activity. Moreover, the complex structure of the crenatoside allows for many chemical modifications. Thus, future studies of this molecule might provide relevant data to produce antivirus molecules.This shows crenatoside is an interesting promisor drug with selective and relevant in vitro anti-Tecoma species showed in vitro antiviral activity against the Zika virus and crenatoside. The first time it was isolated from the Tecoma stans var. stans, it exhibited a potent and relevant anti-Zika virus, being more active than ribavirin (positive control). This data suggests that crenatoside is a promisor compound against the Zika virus and future studies of this molecule might provide relevant data to produce antivirus molecules. Furthermore, the bioactivity of crenatoside should be investigated against other viruses.Our results reveal that Tecoma species allowed the detection of phenylethanoids glycosides such as verbascoside, isoverbascoside and crenatoside in the trunk and leaves, furthermore, some lignans and flavonoids were detected. The biomonitored studies with Tecoma species are being carried out by our research group to isolate other bioactive compounds with promising antiviral activity, such as crenatoside.The phytochemical investigation of Additional file 1: Fig.1S. Mass spectrum in negative mode of Tecoma castaneifolia trunk. Figure 2S. Mass spectrum in negative mode of Tecoma castaneifolia leaves. Figure 3S. Mass spectrum in negative mode of Tecoma garrocha trunk. Figure 4S. Mass spectrum in negative mode of Tecoma garrocha leaves. Figure 5S. Mass spectrum in negative mode of Tecoma stans var. angustata trunk. Figure 6S. Mass spectrum in negative mode of Tecoma stans var. angustata leaves. Figure 7S. Mass spectrum in negative mode of Tecoma stans var. stans trunk. Figure 8S. Mass spectrum in negative mode of Tecoma stans var. stans leaves. Figure 9S. 1H-NMR spectrum of crenatoside . Figure 11S. 1H-NMR spectrum expansion 5.2 to 4.5 ppm of crenatoside . Figure 12S. 1H-NMR spectrum expansion 4.2 to 3.2 ppm of crenatoside . Figure 13S. 1H-NMR spectrum expansion 1.3 to 0 ppm of crenatoside . Figure 14S. 13C-NMR spectrum of crenatoside . Figure 15S. 13C-NMR spectrum of crenatoside .


Clinical ketosis (CK) and subclinical ketosis (SCK) are associated with lower milk production, lower reproductive performance, an increased culling of cows and an increased probability of other disorders. Quantifying the costs related to ketosis will enable veterinarians and farmers to make more informed decisions regarding the prevention and treatment of the disease. The overall aim of this study was to estimate the combined costs of CK and SCK using assumptions and input variables from a typical Dutch context. A herd level dynamic stochastic simulation model was developed, simulating 385 herds with 130 cows each. In the default scenario there was a CK probability of almost 1% and a SCK probability of 11%. The herds under the no risk scenario had no CK and SCK, while the herds under the high-risk scenario had a doubled probability of CK and SCK compared to the default scenario. The results from the simulation model were used to estimate the annual cash flows of the herds, including the costs related to milk production losses, treatment, displaced abomasum, mastitis, calf management, culling and feed, as well as the returns from sales of milk and calves. The difference between the annual net cash flows of farms in the no risk scenario and the default scenario provides the estimate of the herd level costs of ketosis. Average herd level costs of ketosis (CK and SCK combined) were €3,613 per year for a default farm and €7,371 per year for a high-risk farm. The costs for a single CK case were on average €709 , while the costs for a single SCK case were on average €150  for the default farms. The differences in costs between cases occurred due to differences between cases . High producing dairy cows experience a negative energy balance following parturition. This negative energy balance results from a fast increase in energy requirements for milk production while feed intake capacity in early lactation is limited. Subclinical ketosis (SCK), generally defined as a blood betahydroxybutyrate (BHBA) concentration of 1,4 μmol/L or higher during early lactation without clinical signs such as a disappointing milk production with an elevated fat and a lowered protein content . In someElevated concentrations of ketone bodies in blood are associated with a lower milk production and reproduction performance, and an increase in the culling of cows . While Quantifying the losses due to ketosis will enable veterinarians and farmers to make more informed decisions regarding the prevention and treatment of the disease. Some studies compared the economic consequences of various treatment strategies –9, whilsThe overall aim of this study was to estimate the combined costs of CK and SCK, using assumptions and input variables from a typical Dutch context. A further aim was to evaluate the economic consequences of five different treatment strategies for CK and SCK. For these purposes, a dynamic stochastic simulation model at herd level was developed.The biological cow simulation model was developed using Microsoft Excel with @Risk add-in software . The model used in this study is an adaptation and extension of the cow simulation model described by Inchaisri et al.  and RuttThe cow simulation model simulates a single cow place. In this study, each iteration of a cow place started with a cow at the point of calving with a randomly assigned parity  and a rAt the start of each lactation, the potential 305-day milk production for each cow was determined based on the average herd milk production of a Dutch dairy farm , a relaBased on the simulated lactation curve, feed requirements as energy requirements, expressed in feed units for lactation  as defined by Van Es  were calThe model simulated three scenarios. In the default (base risk) scenario there was a CK a probability of 0.007 and an SCK probability of 0.11 per cow per lactation. The herds under the no risk scenario had no CK and SCK, while the herds under the high-risk scenario had a doubled probability of developing CK and SCK compared to the default scenario. For the default and high-risk scenarios it was assumed that SCK and CK occurred not more than once per lactation. The probability of developing SCK and CK was adjusted according to the relative milk production factor and lactation stage in such a way that cows with a relative high milk yield were more likely to develop SCK or CK. The increased probability of SCK cows becoming CK cows  and is pruned to 0 when milk production losses in the previous week drop below 1.5%.Simulation of SCK and CK treatments resulted in a 50% recovery of the milk production losses due to SCK or CK. A cow with SCK had a 0.1 probability of being treated, with treatment always taking place in the same week that SCK occurred. A cow with CK was always treated immediately. As some cases may need veterinary intervention, the simulation included a veterinary visit and consecutive treatment for SCK and CK with a probability of 0.5 and 1.0 respectively.In each week, the occurrence of clinical mastitis depended on the ketosis status of the cow and was adjusted for the cow’s week in milk in such a way that 70% of the mastitis cases occurred in the first four weeks of lactation and 30% in a later stage. A relative risk of 1.3 was included in the model for the effect of SCK on the occurrence of mastitis. The probability of DA occurrence depended on the ketosis status of the cow in that week and was adjusted for the cow’s week in milk in such a way that 75% of DA cases occurred in the first four weeks of lactation and 25% in a later stage. The probability of DA occurrence depended on a relative risk of 5.0 of cows having SCK.Modelling of the reproductive cycle is described by Inchaisri et al. , and for1File).Cows were culled either as a result of poor fertility, CK or for other reasons. Modelling of culling did not differ between the scenario’s . If a cow was not pregnant at either 35 weeks after parturition or after six inseminations, she was culled for fertility reasons. Cows were also culled if their daily milk production was below 15 kg milk/d. On average, 20% of all culled cows were culled for fertility reasons at a replacement rate of 30%. Cows that developed CK had a probability of 0.31 of being culled in the following four weeks. The probability of culling due to SCK was modeled indirectly as cows with SCK had a reduced conception rate and consequently a higher probability of being culled due to poor fertility. The probability of culling due to other reasons was determined in such a way that the final culling rate was 30%. To make sure that the final culling rate was 30% an adjustment factor was used  and an additional 50 iterations were added to have a uniform number of cows in each of the 385 herds. The output values were always stable before the 5The model outputs for each iteration (cow place) included annual milk production, the annual number of CK and SCK cases, annual milk production losses due to CK and SCK, the annual number of calves born, the annual number of inseminations, the annual number of DA cases, the annual number of clinical mastitis cases, the annual number of culled cows and the annual feed intake measured in VEM/year .In order to investigate the costs of ketosis, the annual model outputs were used to calculate cash flows for milk produced, calves sold, treatment costs, DA costs, clinical mastitis costs, calf management costs, culling costs and feed intake costs for all three scenarios . Input values were based on the 2018 Dutch market context .The costs of milk production losses due to SCK and CK were calculated based on the level of milk production losses, the milk price and the reduced feed costs associated with reduced milk production as a result of CK and SCK, respectively.Treatment costs of ketosis included the costs for diagnostics and drugs, veterinary visits and the farmer’s labor costs. It was assumed that all cases of CK needed a veterinary visit, a diagnostic work-up, the application of drugs by a veterinarian and labor input from the farmer . Total tThe costs associated with impaired fertility as a result of ketosis included the costs of insemination, calf management and calf sales. Insemination costs were calculated as the annual number of inseminations in the herd multiplied by the cost per insemination. The price of insemination included the costs of semen, the insemination service and the farmer’s labor . The cosDA and clinical mastitis were associated with costs for milk production losses, increased culling probability and treatment costs. Costs for milk production losses and culling were already calculated for the costs of SCK and CK. To avoid double counting, only the treatment costs were calculated for DA and clinical mastitis.The net culling costs included the slaughter value of the culled cow (revenue) and the opportunity costs of the replacement heifer (cost). These values were calculated using the same approach as Rutten et al. . SlaughtFor all three scenarios  the annual net cash flow on a farm was calculated as the sum of cash flows from milk production and calves sold, and costs for treatment, DA, clinical mastitis, calf management, culling and feed. The annual net cash flows for all 385 farms in the simulation were averaged. The difference between the average annual net cash flows for the no risk scenario and the default scenario was calculated to represent the overall costs of ketosis (CK and SCK) for a 130-cow dairy farm in the default scenario. In addition, the difference between the average annual net cash flows for the no risk scenario and the high-risk scenario was calculated to represent the overall costs of ketosis (CK and SCK) for a 130-cow dairy farm in the high-risk scenario.The costs for a single CK and SCK case were calculated in two steps. First, the costs of milk production losses and treatment for CK and SCK were divided by the number of cases of CK and SCK, respectively. Subsequently, all other costs were split equally over all cases . Both values were added up for CK and SCK to calculate the costs for a single case of CK and SCK, respectively. The costs in the default scenario were used for this calculation.The economic consequences of different treatment strategies were determined by comparing five different treatment strategies. The default strategy (i) includes the situation described in the default scenario model where all CK cases and 10% of the SCK cases are treated. The four alternative treatment strategies are: (ii) a strategy in which all cases of ketosis (CK and SCK) are treated, (iii) a strategy in which all CK cases and half of the SCK cases are treated, (iv) a strategy in which all CK cases are treated and none of the SCK cases are treated and (v) a strategy in which no case of ketosis (CK or SCK) is treated. These different scenarios are selected to study in particular the impact of treatments strategies, while they may also reflect the approach of farmers that are either reluctant or focused to diagnose and treat each diseased cow.Because data were not available for external validation of the model, an internal validation was performed. A large number of inputs were compared with the output to check the consistency and the credibility of the model output.A sensitivity analysis was conducted to assess the level of impact of some input parameters on the overall costs of ketosis in the default scenario. Values for input variables in the sensitivity analysis were based on information in the literature and assumptions made by the authors. Sensitivity analyses were performed for the annual probability of CK and SCK, percentage milk production losses for CK and SCK and average herd milk production. In The average simulation results of the 385 herds of 130 cows for the no risk, default and high-risk scenarios are presented in The annual economic consequences of the simulation results are presented in The overall costs of ketosis when treating all SCK and CK cases were €4,348 per year. Treating all CK cases and half of the SCK cases resulted in overall ketosis costs of €1,051 per year, while treating all CK cases and no SCK cases resulted in overall ketosis costs of €1,471 per year. Treating no ketosis cases resulted in overall ketosis costs of €2,098 per year.The sensitivity analysis shows that the overall total costs of ketosis are most sensitive to the probability of getting CK and SCK. Varying the milk production loss had only a small effect on the overall costs .The results of the current study differ from results obtained from other studies. For instance, Geishauser et al.  estimateThe current study developed a stochastic simulation model for the estimation of the costs of ketosis, thus making the variation in results available as well Tables  and 4. TThe estimated milk production losses in the current study may have been under- or overestimated because it was difficult to understand the pattern of milk losses due to ketosis e.g., –2, 19) a, 19 a2, In the no risk scenario, annual milk production  was highIn the current model it was decided that the overall replacement rate was kept at 30%, independent of the ketosis situation. As a consequence, cows with ketosis were sometimes culled whereas cows without ketosis (which otherwise would have been culled) were kept longer. The costs of culling therefore represent the costs for a less optimal culling policy. Although this is not optimal from an economic point of view, it largely represents the approach many farmers use in practice and was also used by Rutten et al. . This waA few other studies investigated the economic consequences of different treatment strategies. They compared treatment strategies in combination with the testing of cows suspected of having SCK –9. The cAverage herd level ketosis costs (CK and SCK combined) were €3,613 per year for a default farm and €7,371 per year for a farm in the high-risk scenario. The costs for a single CK case were on average €709 , while the costs for a single SCK case were on average €150  for the default farm. Results of the sensitivity analysis show that the probability of CK and SCK have the largest effect on the herd level ketosis costs and that using other percentages for production losses for CK and SCK had a relatively small effect on the herd level ketosis costs. Because of the high impact of the probability of ketosis at the herd level, from an economic point of view the prevention of ketosis on high-risk farms is an attractive option.S1 File(DOCX)Click here for additional data file.


HIV infected individuals are treated with a panoply of over 20 antiretroviral medications. Used in combination, the drugs suppress viremia in the large majority of patients but do not eradicate the virus and as a result, patients need to remain on lifelong therapy to keep virus loads in check. In the absence of a means to entirely clear the infection, development of an immunotherapy that fortifies the immune response to the virus might allow for a “functional cure” in which patients could suppress the virus load without therapy. That such an approach could work is supported by the existence of rare elite controllers who, by virtue of a strong antiviral T cell response, hold virus loads in check over several years without therapy. A hurdle in the development of a T cell immunotherapy for HIV is that the immune response to the virus tends to focus on immunodominant viral epitopes that are highly variable and from which the virus easily escapes by mutating in response to selective pressure from antigen-specific CTLs. Further complicating the matter, T cell recognition of viral epitopes is MHC restricted such that individuals respond to different viral epitopes depending on their MHC haplotype, making it difficult to devise a universal vaccine antigen.In this issue, Garcia-Bates et al. tackle the question of an effective HIV immunotherapy with a dendritic cell (DC) vaccine strategy that elicits CTLs that kill infected cells by targeting ultra-conserved, topologically constrained epitopes from which the virus cannot easily escape A caveat of the study is the use of donor cells from patients treated in acute infection. In practice, the immunotherapy will need to be used on patients who were not fortunate enough to start treatment in the acute phase. It will be important to show that the approach works using cells from donors treated later in the disease course when it may be more difficult to enhance the T cell response. It will be interesting to see how well the approach works in a clinical trial. It remains unclear as to how controllers manage to keep virus loads in-check. It will be interesting to see whether DCs can be used to enhance T cell responses to the virus in chronic infection where CTLs display tend to be dysfunctional and display signs of exhaustion Pr Landau has no conflict of interest to disclose.


The sudden collapse of honey bee colonies in California in 2005 and alarming reports about significant colony losses in the U.S. and Europe have attracted the renewed attention of researchers and the general public to the role played by honey bee viruses in this process. Thus, research focused on the elucidation of the genomic sequences of previously identified and new honey bee-associated viruses, their pathogenicity at the individual and colony levels, their structure, their interactions with other pathogens, and their relationship with the host, many times emerging from covert-asymptomatic to overt-symptomatic infections following stress challenges, has recently been conducted. These efforts have resulted in new and substantial knowledge.Viruses is to highlight recent significant advances in this area of virology to provide an updated integrated picture of honey bee-associated viruses in order to improve, facilitate, and encourage further innovative research to broaden our understanding in this fascinating field. The aim of this Special Issue of Varroa destructor mites [The first part of this issue is dedicated to three studies reporting the development of honey bee virus clones and vectors that enable follow-up and reverse genetic-based studies. Ryabow et al.  and Gusaor mites . Jin et or mites  took a sA. mellifera cell lines, as well as other cell lines that might be helpful, including a description of ways to develop new cell lines. Moreover, Tal and Chejanovsky [A. mellifera pupae.In this respect, it is worth noticing that cell culture systems capable of sustaining virus replication proved to be powerful tools in animal and insect virology to study virus replication, and its molecular components and their role in infection and spreading it. However, cell lines capable of sustained replication of honey bee viruses are very scarce. Guo et al.  provide janovsky  show thaA. mellifera by studying the response of heat-shocked honey bees towards a model virus, Sindbis-GFP. They show that six heat-shock protein-encoding genes and three immune genes were differentially expressed in heat-shocked and/ or virus-infected bees [The interaction between the invading virus and the host's defense mechanism determines the outcome of the infection. McMenamin et al.  provide ted bees .A. mellifera is reviewed by Harwood and Dolezal [The effect of pesticides as stressors affecting virus infections in  Dolezal . They exA. mellifera is addressed in a study by Al Naggar and Paxton [Transmission of viruses in the colony can highly influence its health. The impact of horizontal transmission of the Black queen virus (BQCV) to queens and adults in d Paxton , and an d Paxton . A. mellifera colonies that results from the complex interaction between honey bees, Varroa destructor mites, and viruses. What could we expect to be the outcome of the above interaction in the absence of DWV? Roberts et al. [Many studies were published on the extensive damage to s et al.  provide Vespula pensylvanica and the invasive species Vespula vellutina) close this issue, broadening our knowledge in this aspect [Finally, transmission of honey bee viruses to other insects, and vice versa, can have important ecological effects. Two papers that deal with viruses from Vespidae (s aspect ,12.I do hope that this Special Issue of Advances in Honey Bee Virus Research provides substantial new information and encourages novel research to understand honey bee viruses’ infections at the molecular level in the individual and in the colony.