Violence against women and girls (VAWG) is a global problem with profound consequences. Although there is a growing body of evidence on the effectiveness of VAWG prevention interventions, economic data are scarce. We carried out a cross-country study to examine the costs of VAWG prevention interventions in low- and middle-income countries. We collected primary cost data on six different pilot VAWG prevention interventions in six countries: Ghana, Kenya, Pakistan, Rwanda, South Africa and Zambia. The interventions varied in their delivery platforms, target populations, settings and theories of change. We adopted a micro-costing methodology. We calculated total costs and a number of unit costs common across interventions (e.g. cost per beneficiary reached). We used the pilot-level cost data to model the expected total costs and unit costs of five interventions scaled up to the national level. Total costs of the pilots varied between ∼US $208 000 in a small group intervention in South Africa to US $2 788 000 in a couples and community-based intervention in Rwanda. Staff costs were the largest cost input across all interventions; consequently, total costs were sensitive to staff time use and salaries. The cost per beneficiary reached in the pilots ranged from ∼US $4 in a community-based intervention in Ghana to US $1324 for one-to-one counselling in Zambia. When scaled up to the national level, total costs ranged from US $32 million in Ghana to US $168 million in Pakistan. Cost per beneficiary reached at scale decreased for all interventions compared to the pilots, except for school-based interventions due to differences in student density per school between the pilot and the national average. The costs of delivering VAWG prevention vary greatly due to differences in the geographical reach, number of intervention components and the complexity of adapting the intervention to the country. Cost-effectiveness analyses are necessary to determine the value for money of interventions. Key MessagesInterventions to prevent violence against women and girls (VAWG) can be delivered in a multiplicity of platforms, through different components, across various settings and with different levels of intensity. The costs involved in delivering VAWG prevention services largely reflect these intervention characteristics.In the six interventions costed, the cost per beneficiary ranged widely, from US $4 in a community-based intervention in Ghana to US $1324 for one-to-one counselling in Zambia; cost-effectiveness analyses will determine whether more resource-intensive interventions generate better value for money.VAWG prevention interventions are staff intensive. Staff-related costs make up the biggest proportion of costs across all interventions, and total costs are very sensitive to staff salaries. Training frontline staff is a large investment; ensuring retention of staff is essential in maintaining costs low in the long term.The cost per beneficiary will likely change when interventions are scaled up from pilots to the national level. Cost modelling suggests that unit costs can decrease in community-based and workshop-based interventions. Conversely, unit costs can increase in interventions with fixed platforms, such as schools, when the average ratio of students-per-school is lower at the national level than in the pilot (due to high fixed costs at the delivery site). Violence against women and girls (VAWG) is a global problem with profound consequences. Between 30% and 65% of women and adolescent girls over the age of 15 years in sub-Saharan Africa, and over 40% of those in South Asia, have experienced intimate partner violence (IPV) , and hasGovernments and international donors, such as the United Kingdom’s Department for International Development (DfID) and the World Bank, have invested in VAWG prevention pilots and programmes  in low- Estimating the costs of public health and social interventions in LMICs requires substantial primary data collection given the dearth of routine financial reporting systems. To date, two economic evaluations of VAWG prevention interventions in LMICs have been published. Evidence on costs and cost-effectiveness are available for the Intervention with Microfinance for AIDS & Gender Equity (IMAGE), a combined microfinance and gender training intervention to prevent IPV in rural South Africa (What Works to Prevent Violence against Women and Girls (What Works) is a global programme building evidence on violence prevention interventions in 13 LMICs . The proThis study has two aims: first, to present the total costs and unit costs of six VAWG prevention interventions piloted through different platforms  and evaluated using randomized controlled trials as part of the ‘What Works’ programme (i.e. costing ‘implementation in a research setting’) in six countries: Ghana, Kenya, Pakistan, Rwanda, South Africa and Zambia; and second, to use these primary data to model the costs of scaling up these interventions at a national level, presenting total costs and unit costs for five of the six countries.We collected the costs of six VAWG prevention interventions in six LMICs. These interventions were piloted and assessed using randomized controlled trials . FurtherThe interventions also varied in terms of setting , coverage , theories of change approaches  and durations of implementation (12–31 months). Full information on the interventions costed is available elsewhere  programmes designed to prevent VAWG in LMICs . Our metWe estimated costs of the start-up and implementation phases. The start-up phase was divided into three sub-phases: (1) intervention development, defined as the period when the curriculum or manual of activities was initially designed, (2) adaptation, defined as the process of making the curriculum or manual specific to the target population and (3) set-up, defined as the period of community entry and training of frontline staff. The implementation phase spanned the time between when the first and last clients received the intervention.All three sub-phases were costed for IMPower/SOS (Kenya), SSCF (South Africa) and VATU (Zambia). For RTP (Pakistan) and Indashyikirwa (Rwanda), only set-up and adaption costs were included. RTP’s development sub-phase started in 2008, and resource-use data were not available; it therefore could not be costed. Indashyikirwa is a multiple-component intervention, partially based on SASA! and Journeys of Transformation (previously developed interventions) but with some newly developed components. Given the complexity of Indashyikirwa’s start-up phase, it was difficult to disaggregate resources used strictly to develop new components vs adaptation of existing components. For analytical simplicity, we classified all costs as adaptation (vis-à-vis development) and tested this assumption in sensitivity analysis. For RRS (Ghana), only the set-up sub-phase was costed. The intervention was developed over 15 years prior to the pilot, and no financial records were available. Furthermore, RRS did not go through an adaptation phase before the implementation.A full financial and economic micro-costing was carried out for each intervention. Data on resource utilization were obtained through structured interviews, review of financial records, monitoring and evaluations data and travel logbooks. For financial costs, purchase prices were used, with replacement prices for capital goods, commonly available from project records. Costs were separated by phase and sub-phase. Most costs were calculated through a bottom-up approach (measuring quantities in a granular manner at the activity and sub-activity level). When unfeasible, a top-down approach  was used to calculate some administration and management costs.Costs were broken down by input type  and allocated to a number of activities common across interventions  and intervention-specific core activities (e.g. community sensitization or counselling). Research costs were excluded. Implementing organization monitoring and evaluation costs were included as these would be expected to be incurred in routine service delivery. Allocations between activities and sub-activities were based on staff time use and financial and programmatic records   deflator and to US Dollars using average annual exchange rates . CapitalWe calculated a total cost, as well as cost per phase, and present results by input type. We calculated three common unit costs across interventions: cost per frontline worker trained, cost per session delivered and cost per beneficiary reached. We acknowledge that the term ‘unit cost’ has various definitions depending on the purpose of the costing and the methodological approach. We defined a unit cost as a mean cost per unit of output, consistent with the Global Health Costing Consortium Reference Case and VAWG-specific guidelines , this is the number of beneficiaries enrolled at baseline, irrespective of the number of sessions completed. For community- and school-based interventions , which were based on the model of social diffusion where community members (either villagers or pupils in school) were exposed directly or indirectly, the number of beneficiaries was defined as the total number of people within the relevant target population in each cluster (i.e. village or school). Indashyikirwa (Rwanda) had an initial workshop-based component with couples that fed into the community-based approach; however, in this paper, we focus on the beneficiaries at the community level.To account for uncertainty, we carried out a number of one-way deterministic sensitivity analyses. We examined the sensitivity of costs to the following parameters: staff salaries, replacement value of volunteer labour, working days per year, prices of fuel, useful life of adaptation and set-up costs and discount rates (see There were discrepancies in perceptions of time use between those reported by staff and those reported by the research team in VATU (Zambia) over which full agreement could not be reached. Due to this extra uncertainty, we carried out an additional one-way deterministic sensitivity analysis varying the four parameters around which there was disagreement: percentage of time spent on research activities, percentage of time spent on management activities, amount of time devoted by supervisors to travel, and the inclusion of a one-day feedback session in the start-up phase see . Given tWe modelled the costs at national scale in five out of the six interventions. We excluded VATU (Zambia) given the uncertainty about ‘implementation in a research setting’ costs and non-availability of information to model scale-up assumptions.Cost data from the pilots were used to estimate costs of full-scale intervention delivery at the national level . Scale-up can be conceptualized across different dimensions. The World Health Organization suggests that scaling up can be applied to inputs, outputs, outcomes or impact . While tAn increase in inputs at scale requires increased resources , but costs are not typically a linear function of the number of beneficiaries reached at scale. As interventions are scaled up, some costs remain fixed (i.e. costs that remain constant regardless of the number of outputs) such as the costs of adapting an intervention to a new country, while others should be treated as variable (i.e. costs that vary according to the level of output). Some costs vary as a function of the number of beneficiaries reached , while others vary as a function of intermediate outputs, such the number of delivery sites (e.g. costs of training teachers on the intervention in a school).Moreover, intervention modifications may be necessary when interventions are scaled up from pilot to national levels. Consequently, we consulted with senior members of each implementation team to elicit their expert opinion on the potential scale-up. Specifically, we elicited input on: (1) potential modifications in inputs at scale , (2) potential intervention delivery modifications at scale (e.g. changes in field worker supervision) and (3) potential magnitude of scale-up according to delivery platform . A final list of included modifications, and associated costs, was consequently reviewed and approved by implementers.A large number of proposed modifications involved changing the staff used to deliver the intervention: in IMPower/SOS (Kenya) service delivery shifted from implementing organization staff to local school teachers; and in Indashyikirwa (Rwanda) training shifted from being led by the implementer organization staff to community volunteers. There was also a reduction in stipends for frontline staff workers , a decrease in supervisory activities (Ghana and South Africa), a reduction in the intensity of frontline worker training (Pakistan) and a shortening of the curriculum (Rwanda) between the pilot phase and the national scale-up. We modelled two scenarios: the first with all suggestions incorporated and a second, more cautious, scale-up scenario, where we calculated costs at national scale only accounting for potential changes in inputs. A complete list of assumptions is found in It should be noted that the impact of modelled changes in inputs or intervention delivery on the interventions’ effectiveness is uncertain; our scale-up costs should therefore be considered exploratory and be monitored carefully during implementation.We excluded intervention development costs at scale, and adaptation costs were retained but treated as a fixed cost. Set-up costs, which included costs associated with community entry, stakeholder engagement and training of local frontline staff, would need to be incurred for every new delivery site so were assumed to vary as a function of the number of delivery sites.Implementation costs were divided between those incurred in programme offices  and delivery sites. Costs of programme offices were divided into direct and indirect costs. Direct costs  were multiplied by the number of delivery sites at scale and indirect costs  were multiplied by the number of projected programme offices at scale. Average implementation costs per delivery site were multiplied by the estimated number of delivery sites at scale.To determine the total number of delivery sites at scale for most interventions, we divided the total number of beneficiaries at scale by the average number of beneficiaries per site in the pilot phase . For IMPower/SOS and RTP, we assumed that the number of sites was determined by the total number of public primary schools in Kenya and by the total number of public middle schools in Pakistan.The level of expansion of the programme offices necessary at scale was determined by treating indirect costs as step costs (i.e. fixed cost until a threshold is crossed), and we assumed that costs would remain fixed up to the point where the number of delivery sites tripled. After that point, a duplication in the resources needed for indirect activities was considered necessary. This assumption was tested in sensitivity analysis.To calculate the number of beneficiaries at scale, we first defined the criteria of inclusion in the target population for each pilot interventions (e.g. age range or employment status). We then calculated the total number of people meeting said criteria at the national level. In the cases of community-based interventions in Ghana and Rwanda, this meant all adults age 20–59 years in the country: 12 210 626 and 4 976  600 people, respectively . AlthougThe capital costs of implementation  were low across all interventions , while recurrent costs made up the majority of total costs (68–89%). Staff costs  were the largest cost driver for all interventions, ranging from nearly 30% of total costs in RRS (Ghana) to 81% in VATU (Zambia). In community-based interventions (Ghana and Rwanda), volunteer costs made up 23–27% of staff costs.The importance of other recurrent costs varied by intervention. Recurrent transport costs  made up between 3% and 9% of total costs across interventions. Per diem and allowances (for both paid staff and volunteers) made up nearly one-quarter of total costs in RRS (Ghana).There was a wide range of costs per participant reached: 2016 US $4 in RRS, a community-based intervention in Ghana, 2016 US $11 in IMPower/SOS, a school-based intervention in Kenya, 2016 US $17 in Indashyikirwa, a couples and community-based intervention in Rwanda, 2016 US $18 in RTP, a school-based intervention in Pakistan, 2016 US $307 in SSCF, a small group intervention in South Africa and 2016 US $1324 in VATU, a largely one-to-one intervention in Zambia.Deterministic sensitivity analyses are presented in Total costs of national scale-up varied widely. For the first scenario, the total costs at scale were: 2016 US $32.4 million for RRS (Ghana), 2016 US $54.9 million for Indashyikirwa (Rwanda), 2016 US $86.6 million for IMPower/SOS (Kenya), 2016 $US 104.3 million for SSCF (South Africa) and 2016 $US 168.1 million for RTP (Pakistan). For the second scenario, the total costs at scale were: 2016 US $36.2 million for RRS (Ghana), 2016 US $57.7 million for Indashyikirwa (Rwanda), 2016 $US 114 million for SSCF (South Africa) and 2016 US $168.5 million for RTP (Pakistan). Total costs were the same in both scenarios for IMPower/SOS (Kenya) as no modifications to service delivery were assumed.Total costs have been broken down to show cost changes due to suggested modifications to inputs, modifications to service delivery and delivery at scale. Suggested input changes in RTP (Pakistan), SSCF (South Africa) and Indashyikirwa (Rwanda) led to decreases in total costs  and to a minor increase in IMPower/SOS (Kenya) (<1%). No changes in inputs were suggested for the intervention in Ghana.Changes in service delivery led to further reductions in total costs in the other interventions: 8% in Ghana (reduction in supervision), 7% in South Africa (reduction in meetings with facilitators and senior facilitators), 3% in Rwanda  and <1% in Pakistan (reduction in training). No changes in delivery were suggested for the intervention in Kenya.Unit costs per beneficiary reached at national scale in the first scenario were found to be 2016 US $2.65 in RRS (Ghana), 2016 US $12.03 in Indashyikirwa (Rwanda), 2016 US $26.14 in IMPower/SOS (Kenya), 2016 US $41.44 in RTP (Pakistan) and 2016 US $212.68 in SSCF (South Africa). Unit costs in the second scenario were 2016 US $2.97 in RRS (Ghana), 2016 US $12.65 in Indashyikirwa (Rwanda), 2016 US $41.53 in RTP (Pakistan) and 2016 US $232.54 in SSCF (South Africa). Unit costs did not change between the two scenarios in IMPower/SOS (Kenya).Unit costs at scale were lower than those calculated in the pilot projects across all interventions, except for the two school-based ones. The cost per beneficiary reached decreased by 30%, 31% and 37% for RRS (Ghana), SSCF (South Africa) and Indashyikirwa (Rwanda), respectively, in the first scenario and 22%, 24% and 33%, respectively, in the second scenario. On the contrary, unit costs more than doubled for RTP (Pakistan) and IMPower/SOS (Kenya) in both scenarios.We assumed that indirect costs at the programme office-level would double for every 3-fold increase in the number of delivery sites. A proportional increase in indirect costs based on the number of sites (i.e. doubling indirect costs for every 2-fold increase in sites) increased total costs at scale by between 0.1% in IMPower/SOS (Kenya) and 0.7% in Indashykirwa (Rwanda). Conversely, a doubling of indirect costs for every 10-fold increase in number of sites decreased total costs at scale from 0.1% in RRS (Ghana) and RTP (Pakistan) to 2.1% in Indashyikirwa (Rwanda).Our study presents the total costs and unit costs of developing, adapting, setting up and implementing six different interventions to prevent VAWG and the modelled costs of scale-up of five interventions. This is the first comprehensive cost dataset for VAWG prevention, and it demonstrates the application of a method for estimating costs in line with global health costing reference cases. The interventions costed vary in terms of delivery platforms, methods and target populations. Given the dearth of evidence in this area, our work represents a sizeable and valuable contribution for decision-makers and policymakers wishing to plan and advocate the scale-up of VAWG prevention programmes globally.We divided start-up costs into three sub-phases: intervention development, adaptation and set-up, to inform other pilots and the expansion of services across countries. Development was costed in Kenya, South Africa and Zambia  and costs ranged from ∼2016 US $37 000 to 2016 US $107 000. These figures are higher than those reported for IMAGE (2004 US $13 877 adjusted to 2016 US $17 353) and lower than those reported for SASA! (2011 US $139 000 adjusted to 2016 US $149 944). There was a wider range in adaptation costs: from ∼2016 US $1800 SSCF (South Africa) to 2016 US $783 000 in Indashyikirwa (Rwanda), reflecting different intensities in the process. Whereas, in South Africa, adaptation involved 1 day of post-pilot work in making content and delivery more context specific, the process of adapting SASA! to Rwanda was complex and time-consuming  and to reflect changes in the intervention (e.g. more explicit emphasis on couples taking action together).There was also a wide range of set-up costs across the interventions, from ∼2016 US $43 000 in SSCF (South Africa) to 2016 US $1 363 000 in Indashyikirwa (Rwanda). Our set-up cost estimates are in all cases (except for SSCF in South Africa), higher than those published in the literature; total cost estimates for set-up for IMAGE were 2004 US $61 000 (adjusted to 2016 US $76 280) in the trial phase and 2004 US $58 000 (adjusted to 2016 US $72 529) in the scale-up, although these costs only include training and exclude community entry costs.Our set-up costs include training and other community entry activities, such as recruitment of frontline staff, meetings with stakeholders and demand creation, all of which have to be replicated in each geographical area where the implementation occurs. The high costs in Rwanda can be partially explained by Indashyikirwa’s wide geographical reach: unlike the other interventions, which were rolled out in one or two districts or cities, Indashyikirwa was implemented across seven districts in three regions of the country. Indashyikirwa also required the training of four distinct cadres of volunteers per district ; in other interventions, only one type of frontline staff was recruited and trained. Lastly, Indashyikirwa was set up and implemented by three non-profit organizations, each with specific overhead cost structures.We made an assumption that the useful life of the set-up phase was 5 years, based on an average political cycle. When tested in sensitivity analysis, costs were sensitive to this assumption in Ghana, Rwanda and Pakistan, where community members were trained to deliver services, a process requiring substantial investment. Avoiding attrition of volunteers may be an important factor in ensuring a longer useful life of the set-up sub-phase and, consequently, maintaining lower costs in the long term.The cost per frontline worker trained varied between 2016 US $58 in Indashyikirwa (Rwanda) and 2016 US $272 in RRS (Ghana), both interventions where volunteers were trained to carry out community outreach activities. However, the unit cost for Indashyikirwa is potentially underestimated; this figure only includes the costs of community activism training and excludes costs of a prior workshop-based training with couples, from which the community activists were subsequently selected . Should funders consider implementing only a sub-selection of components, further work could explore costs disaggregated by activity in multi-component interventions.The cost per beneficiary-facing session delivered also varied between 2016 US $5 per school session in RTP (Pakistan) and 2016 US $93 in SSCF (South Africa). Differences in cost can be explained by the fact that sessions in Pakistan were delivered by volunteers with relatively low stipends and were short in duration, whereas in SSCF the target audience was young adults, requiring more intensive support in livelihoods development.The cost per beneficiary reached varied greatly: between ∼2016 US $4 in RRS (Ghana) and 2016 US $1324 in VATU (Zambia). Cost per beneficiary in Indashyikirwa were within the same range as in SASA! (2011 US $15–23 adjusted to 2016 US $16–25 in the latter). The wide range in unit costs are primarily explained by the intensity of interaction and staff type required to deliver each intervention and, in particular, by the frontline staff-to-beneficiary ratio. More resources are needed to teach a small number of people (or one person) vs holding public, open-air activities for, at times, hundreds of people at once.We have presented the modelled total and unit costs of the interventions’ national scale-up. Cost per beneficiary reached ranged from 2016 US $3 in RRS (Ghana) to 2016 US $233 in SSCF (South Africa). The costs per beneficiary reached at scale were lower than those in pilot programmes in three interventions: RRS (Ghana), Indashyikirwa (Rwanda) and SSCF (South Africa). The only current ex-post evidence of scale-up costs comes from IMAGE, which was expanded from the trial setting covering 855 women (in four study villages) to 3453 within the local area; the cost per participant reached fell by ∼70% . Comparatively more modest decreases (22–37%) were estimated in RRS, Indashyikirwa and SSCF. However, in IMAGE, expansion occurred within the same geographical area as the pilot and so additional costs required were proportionally smaller. In our model, expansion occurs nationwide.The reduction in unit costs in the scale-up scenario in three of our interventions is in line with potential economies of scale: unit costs decrease as unit of outputs increase due to the smoothing of fixed costs across a greater number of units . HoweverSome have speculated that VAWG prevention interventions could be delivered at scale in an efficient manner given large fixed costs (e.g. developing training curricula)  could help IMPower/SOS and RTP lower unit costs and achieve economies of scope (i.e. reductions in unit costs as the types of services increase). In addition, in schools with fewer students, facilitators could deliver each session to more than one class at a time, thus reducing the cost per student reached.Alternatively, different models of delivery could shift activities from volunteers to school teachers, as has been done in other settings by the implementers of RTP in Pakistan. However, this model has not been evaluated in a trial setting and, therefore, there is uncertainty around its effectiveness. A shift from head coaches to teachers could reduce financial costs for the implementing organization; whether this reduces or increases economic costs depends on the degree to which teachers have idle time or whether VAWG prevention activities displace other teaching duties creating a need for further staffing.Our study suffers from several limitations. Development costs were not captured in three of the interventions costed as records were unavailable. While we report total development costs where available, we exclude annuitized development costs from implementation calculations to ensure comparability. It was difficult to differentiate between development and adaptation costs in Indashyikirwa. However, sensitivity analysis showed that assumptions on the allocation of these costs had little effect on total costs.We collected costs at different time points, and some data were captured retrospectively. While not problematic when reviewing financial records, capturing time use data post-facto through interviews can be subject to recall bias.In our scale-up analysis, we assume that costs of adaptation will not be replicated when the intervention is scaled up within the same country. However, this presupposes a high degree of in-country homogeneity, which varies by setting.Furthermore, we model the costs of expanding each intervention in the country in which it was trialled. However, scale-up may prove more complex, involving a combination of different interventions targeting different types of beneficiaries through different platforms depending on need .Our data could be used to plan scaling up programmes in the countries where they were piloted. However, while they could also be used to estimate costs in other countries, it is important to reflect on issues of transferability. We have already mentioned the high adaptation costs involved in Indashyikirwa; whether further adaptations in other countries would be as costly is uncertain. Staff costs made up the majority of total costs in our study and, thus, understanding how salaries vary between countries is important. Furthermore, these interventions require different types of skilled and semi-skilled workers who may not be readily available in some countries, potentially increasing training costs. Changes to the intervention’s length and intensity of treatment, which would have cost implications, could also potentially be necessary depending on the receptiveness of the target population. Costs at scale may also vary in other countries depending on the availability of similar delivery platforms.Our work provides a substantive contribution to the economic literature on VAWG prevention but further work is needed. While unit costs are relevant for purposes of budgeting and ensuring financial sustainability, they do not explain whether the intervention has ensured good value for money. More resource-intensive interventions  may be more effective; and so cost-effectiveness analyses are required to shed light on whether the additional costs represent good value for money.As part of the process of calculating scale-up costs, we engaged implementers to propose service adaptations potentially implementable at scale. However, whether changes to programme inputs and delivery could lead to changes in effectiveness is unknown. Further empirical work is needed to see if our estimated costs are achieved at scale and understand the trade-off between reduced costs in programme delivery and sustained effectiveness in VAWG prevention interventions. However, if resources required to carry out empirical work, particularly trials, are not available, other techniques such as decision analytic modelling could be considered.While our analysis gives an indication of changes in unit costs at scale, additional costs needed to deliver services to the hard to reach are not explicitly modelled. Further work is needed to understand the optimal scale to which service delivery should be extended. Moreover, although increasing financial, human and capital resources is a necessary part of scaling up interventions, these increases on their own may not guarantee a successful scale-up . CreatioVAWG presents a substantial human rights and societal challenge globally. Interventions that prevent VAWG demonstrate a wide range of costs, varying according to the geographical reach, number of intervention components, platforms and the complexity of adapting the intervention to the country. ‘What Works’ has proved a unique opportunity to estimate and compare these costs using standardized methods across different intervention models, providing a substantial source of costs to assist those planning the scale-up of VAWG interventions going forward.Health Policy and Planning online.czaa024_supplementary_dataClick here for additional data file.


Each subject performed five ROM tests in a randomized order—measured either via a tape measure or a digital inclinometer: Tape measure was used to evaluate the Fingertip-to-Floor test (FtF) and the Lateral Inclination test (LI). Retroflexion of the trunk modified after Janda (RF), Thomas test (TT) and a Shoulder test modified after Janda (ST) were evaluated with a digital inclinometer. In order to show general acute effects within 20 repetitions we performed ANOVA/Friedman-test with multiple comparisons. A non-linear regression was then performed to identify a plateau formation. Significance level was set at 5%. In seven out of eight ROM tests  significant flexibility gains were observed . A non-linear regression with random effects was successfully applied on FtF, RF, LI-left/right, ST-left and TT-left and thus, indicate a gradual decline in the amount of gained ROM. An acute effect was observed in most ROM tests, which is characterized by a gradual decline of ROM gain. For those tests, we can state that the acute effect described in the stretching literature also applies to the performance of typical ROM tests. Since a non-linear behavior was shown, it is the decision of the practitioner to weigh up between measurement accuracy and expenditure. Researchers and practitioners should consider this when applying ROM assessments to healthy young adults.In the application of range of motion (ROM) tests there is little agreement on the number of repetitions to be measured and the number of preceding warm-up protocols. In stretch training a plateau in ROM gains can be seen after four to five repetitions. With increasing number of repetitions, the gain in ROM is reduced. This study examines the question of whether such an effect occurs in common ROM tests. Twenty-two healthy sport students (10 m/12 f.) with an average age of 25.3 ± 1.94 years (average height 174.1 ± 9.8 cm; weight 66.6 ± 11.3 kg and BMI 21.9 ± 2.0 kg/cm The aim of any ROM test is to determine the maximum joint mobility. Hence, in every execution of the test, a stretching stimulus is applied on the connected muscle–tendon units. It has been shown, that in stretch training, such an acute effect occurs after only a few repetitions and manifests itself among other things in increased mobility, stretch tolerance and reduced passive torque9. It can be expected that this acute effect also occurs during the ROM test. Therefore, differences in the measurement protocol with regard to the measured repetitions and previous warm-up exercises, could lead to different results. However, the current evidence does not allow a determination of precise measurement protocols.In the application of range of motion (ROM) tests there is little agreement on the number of repetitions to be measured and the number of preceding warm-up protocols. In general, ROM tests are tools to measure joint mobility on the basis of routine (scientific) procedures12. In general, ROM assessments depict either the active ROM or the passive ROM. To perform active ROM, the person to be tested moves the assessed joint without assistance using the agonistic musculature. In passive ROM, the therapist, the investigator, or another external force,moves the body part of the person to be tested through the ROM1. However, changes in passive torque, stretch tolerance and isometric muscle force influence the ROM value.With a reproducible test procedure, the ROM value is a parameter to show changes in flexibility in an intervention. Depending on the joints to be measured, measuring tapes, goniometers or inclinometers are usually applied9. The acute effect of stretching on ROM shows the greatest improvement in the first repetitions, whereas the ROM gain is reduced with increasing repetitions7. Consequently, four to five repetitions are recommended for the practical implementation within a stretch training session since subsequent gains are only minimal7. Accordingly, a logarithmic behaviour can be attributed to the acute effect of stretching on ROM. This is valid for stretching training, but how does it behave when the ROM is to be determined in an assessment setting?In static and dynamic stretching, an acute effect occurs within the first five stretches for ROM, stretch tolerance, passive torque and energy, which then return to baseline after 1 h17 or with no information about the number of repetitions19. In most reliability studies, either one repetition was primarily carried out21 or a warm-up on a bicycle ergometer employed, followed by one to two trials of warm-up prior to the actual measurement being conducted23. In a reliability study investigating trunk mobility, each subject was instructed to perform five repetitions of each tested motion in advance of the measurement being taken24.The current literature provides no answers on whether such an effect occurs in the performance of common ROM tests. Scientific practice uses mixed approaches for the problem of acute effects. For example, in interventions and normative data surveys, one to three repetitions were carried out with no to two trials' warm-ups, mostly without any information on rest times7. Due to the inconsistent implementation, in both the practical application and in reliability studies, the presence of an acute effect harms the comparability of ROM test results. Also, if it can be assured, that subjects are in a warmed up state, possible stiffening factors (e.g. sports prior to measurement) can be controlled for.In conclusion, there is no homogeneity regarding warm-up, the number of repetitions and averaging in the context of interventions, normative data surveys and reliability studies. Particularly, in light of a possible acute effect of stretching on ROM, a standardized procedure is necessary, since in common stretching the effect is very large, especially in the first five repetitions25. Five frequently used and evaluated multi-joint ROM tests were selected, which mainly evaluate the mobility of the trunk: Fingertip-to-Floor test26, Lateral Inclination test (left and right side)27, Retroflexion of the trunk after Janda1, Shoulder test after Janda (left and right side)28 and the modified Thomas test (left and right side)29. On this basis, recommendations can be derived for the practical application of quality criteria.Thus, the aim of this study was to investigate the repetition-dependent acute effect of stretching on the ROM in the application of ROM tests and whether an equal behaviour can be derived. One further goal was to test whether there is a plateau formation after several repetitions. Therefore, multi-joint movements were chosen because they are restricted by the muscle–tendon unit. Single-joint movement are on the other hand limited by bones (e. g. elbow extension), mass (e. g. knee flexion) or ligaments (e. g. knee extension)2) volunteered in this prospective study. Exclusion criteria were relevant operations or surgical stiffening of the musculoskeletal system, a relevant artificial joint replacement, severe diseases such as ankylosing spondylitis, chronic destructive joint diseases, multiple sclerosis, myodystrophic or neurodegenerative diseases, congenital malpositions of the musculoskeletal system or an acute herniated disc. In addition, the intake of muscle relaxants or other drugs that influence the elasticity of the musculature and pregnancy were considered as contra indicators. Two sports students with experience in exercise physiology carried out the measurements; both raters were instructed on the methods and practised until the execution was satisfactory.Twenty-two healthy sports students  in Frankfurt am Main, Germany and was conducted in accordance with the 1964 Helsinki Declaration and its later amendments.33 or a digital inclinometer 41 1  was performed28 Fig. c. The pa28 10. The intrarater parallel-forms reliability is also very high with correlations of r = 0.91–0.93; ICC = 0.89–0.9210.The pelvic inclination must be controlled to obtain valid results29 Fig. e. In ordThere was no standardized warm-up, because every repetition represents a specific warm-up. All subjects performed all testing in one day. There was no familiarization prior to the testing. Each subject performed 20 repetitions in each ROM test listed above. The test order was randomized. Each test repetition had to be held on the active or passive maximum ROM (according to the test) for about three seconds. The investigator counted down from three, when the maximum ROM position was reached. At zero, the examiner recorded the ROM according to each test protocol. There was a break of 3 s between each repetition in each testing session. All measurements were carried out by two raters.43 and figures were produced using the package ggplot244. Since no such study is known to date from which expected results can be derived and, in particular, no information on the dispersion of the data is available, no formal power analysis is carried out. The study described here is therefore to be regarded as a pilot study. To determine if an acute effect occurs at the individual level, a regression was performed on every individual’s performance. Subsequently, the number of subjects with a slope sign corresponding to an ROM gain were counted for each ROM test. According to normal distribution of the data, either univariate ANOVA with repeated measurements45 or the Friedman-test46 with multiple comparisons were conducted. These tests were performed in order to identify an overall effect and changes within 20 repetitions. Bonferroni correction was used to counteract the problem of multiple comparisons. The mean of the standard error of measurement of each repetition served as a parameter for the measurement accuracy. If the sign test was positive, non-linear regression was applied. Random effects took repeated measurements into account, thus describing the behavior of a potential acute effect of ROM: a*exp(-b*x) + c.Statistical analysis was performed using BiAS version 11.08  and R (R Core Team 2019)With "a" being the amplitude at the very beginning, "b" the coefficient describing the change from trial to trial and "c" the asymptotic measure. In case, the model was not adequate (i.e. parameter estimations were non-significant), linear regression with random effects was performed to quantify the trend: a·x + b.Significance was set at 5%.All participants provided written informed consent to take part in the study in advance. This study was approved by the ethics research committee of the Goethe-University (2018–46) in Frankfurt am Main, Germany.p = 0.93), all ROM tests revealed significant flexibility gains within 20 repetitions, indicating changes of ROM due to repetitive stretching   and TT (1.185/1.564), whereas LI shows almost identical mean values of the standard error of measurement (0.664/0.665) . Having successfully applied a non-linear regression on six out of the eight ROM tests, it is possible to postulate predictions on the number of repetitions needed to achieve a certain amount of maximal ROM gain  showed a gradual decline in the amount of ROM gained, indicating a plateau formation Fig. . The resFor the practical application of these six ROM tests, the regression provided information on the number of repetitions above which the test offers reliable values for the evaluated ROM Table . This isWith respect to the practical application, we recommend to assess in the area of the plateau of each test. Yet a concrete naming of a plateau should be avoided since, even in higher stretch repetitions, small gains were recognizable. However, increasing the ROM from 50% of maximal to 60% of maximal required only one or two repetitions , which were mostly determined in the first trial over two days21. Due to mostly good reliabilities, it can be assumed that the warm-up state of the subjects was standardized. Nevertheless, it is unclear as to how sensitive the measurement of the first repetition is for changes, e.g. in the context of an intervention. Does the first repetition represent the actual mobility? In addition, the question arises as to what extent sample measurements, different warm-up states, special warm-ups or faulty measurements with subsequent second repetitions falsify the results. Since the influence of additional repetitions on the ROM in the context of ROM tests has scarcely been investigated so far, reference must be made at this point to further, future investigations. Angle values obtained from protocols that differ in the application of the number of repetitions, preliminary warm-up routines or averaging must not be compared. It should be noted that the functions (Fig. Tests such as FtF, TT and LI displayed high intrarater reliabilities (FtF: r = 0.76–0.9957. Furthermore, discrepancies in the test protocol existed in TT and ST compared to FtF, LI and RF. In TT there was no offset visible between the measurements during the test execution. The subjects remained in a permanent strain with sub-maximal intensity. The duration of stimulus exposure was, therefore, significantly longer than in the active ROM tests, in which the stimulus was maintained for about 3 s. In addition, in ST the arm to be measured was moved through and repositioned after each measurement repetition. However, these discrepancies do not explain different contralateral results, but may lead to different intertest outcomes There are further differences between the ROM tests in the different strain methods implicitly used in the test execution that may lead to different outcomes. If the ROM tests are categorized according to the properties and effects of stretching, then differences between the tests become apparent (Table No exponential regression could be performed in ST-right and TT-right, whereas it could be applied for the contralateral side. Considering the mean values of the standard error of measurement for each ROM test, Table 1. While FtF, LI and RF tested at maximum torque, ST and TT tested at sub-maximal torque. The source of torque in ST and TT derived solely from gravity and depended on the weight, length and weight distribution of the lever-arm. As the effects between maximal and sub-maximal stretching on the ROM differed47, this, may explain the discrepancies.Differences in either active or passive stretching are particularly remarkableAs our subjects were young healthy adults, further studies are needed to exploit whether this effect does also occur in impaired populations or in the elderly.An acute effect was observed in most ROM tests, which is characterized by a gradual decline in the amount of ROM gain. Thus, the same number of repetitions is required for the increase in ROM of 0–50% as for the increase of 50–75% of the total ROM increase. The behaviour of this acute effect could be determined using a non-linear regression for most of the ROM tests. For these tests, we can state that the acute effect described in the stretching literature of ROM also applies to the performance of typical ROM tests. Researchers and practitioners should consider this when applying ROM assessments to young healthy adults.


The COVID-19 pandemic developing rapidly in 2020 is triggered by the emergence of a new human virus—SARS-CoV-2. The emergence of a new virus is not an unexpected phenomenon and has been predicted for many years. Since the virus has spread all over the world, it will be very difficult or even impossible to eradicate it. A necessary condition for complete or partial elimination of the virus is to have an effective vaccine. It is possible that SARS-CoV-2 will become milder in the next few years and COVID-19 will then only threaten individuals from risk groups. The appearance of a new human virus is an interesting but not surprising phenomenon. Many predictions and scenarios of such an event have been published in recent years. They mainly referred to the expected influenza pandemic, but coronaviruses were also taken into consideration. It was also predicted that the next pandemic might be caused by a pathogen of unknown origin and taxonomic affiliation. The emergence of such a virus in the near future would result in the Disease X pandemic  and its viruses. Mutual adaptation leading to the establishment of viruses with low pathological potential is most likely. As a consequence of this balance between host and virus, mostly asymptomatic cases of infection can be observed.Human viruses could have arisen in two ways. First, there could be a co-evolution of the host  virus is believed to be the ancestor of the human virus. Divergence of the two viruses has been estimated between the eleventh to the twelfth century AD  are considered the ancestors of HIV-1. HIV-2, in turn, are probably derived from SIVsmm (SIV of sooty mangabey)  are believed to be the result of the introductions of simian viruses in the first half of the twentieth century. Chimpanzee and/or gorilla simian immunodeficiency viruses   is potentially dangerous.In the case of SARS-CoV-2, it is assumed that the bat virus has directly or indirectly become the human virus, maybe after recombination with a coronavirus of another mammalian species  and human papillomavirus type 16 (HPV-16) are currently under consideration. In all of these cases, we have an effective vaccine and there are no animal reservoirs of the viruses. However, chronic infections occur with HBV and HPV-16. Vertical transmission for HBV also makes the eradication plan difficult to follow. Of course, high financial costs are also involved.Without an effective vaccine, it is impossible to imagine eradicating any virus. In the current COVID-19 pandemic, only when we have hundreds of millions of doses of the vaccine we will be able to consider the feasibility of SARS-CoV-2 eradication.This was the case with SARS-CoV-1. The nature of the infection made it possible to isolate all infected persons, and the lack of chronic infections allowed us to eliminate the virus from the human population. However, it should be noted that this was an exceptional situation, which is unlikely to be repeated.Nowadays, the course of SARS-CoV-2 infection  made such a scenario impossible to implement.One may wonder if a drastic lockdown had been introduced in Wuhan at the beginning of December 2019, the virus could have been stopped. However, it does not seem likely. Looking at how other countries dealt with the pandemic, one can see that there is a predominantly defensive strategy. Only those countries that have adopted an offensive strategy based on mass testing have been partially successful. However, this strategy could not be used from the very beginning, when we had not yet recognized the virus, so there was no way to test.It is not excluded that viruses can stop infecting humans without conscious human action. However, one should not count on it too much, although many historical sources say that “the plague came and went”. Unfortunately, usually, or perhaps always, the plague would go away only for a while.Assuming actual virus elimination, mechanisms on the host side and on the virus side may be considered.Such a virus elimination mechanism could be genetic selection leading to the population becoming insensitive to infection. This may have happened in the past, at times for which we do not have data, but is unlikely today. The reason is the lack of selection mechanisms leading to an increase in the frequency of the resistance genes in the population.CCR5-Δ32 homozygous individuals are practically resistant to HIV-1 infection. People with such a genotype are most common in Northern Europe , less often in Central Europe (about 1% in Poland), and rare or absent in other regions of the world  in a homozygous state. The In the case of SARS-CoV-2, it is not excluded that part of the population is insensitive and more likely some individuals are less susceptible to infection due to genetic reasons. However, the spread of the virus shows that these factors seem to be of little importance considering the development of the pandemic. Genetic factors suspected of affecting the severity of COVID-19 identified so far do not appear to have a strong effect, which can indicate a group of people resistant to the infection. However, it should be noted that the search for genetic factors is still poorly documented and is focused mainly on factors favoring the severity of the infection  do not constitute more than 50% of the population, which could slow down the spread of the virus.In the event of influenza virus outbreaks, antigenically distinct virus strains cause larger outbreaks every few years when previous strains can no longer find susceptible hosts. However, coronaviruses, unlike the flu virus, do not appear to induce durable immunity. Thus, there may not be the selection pressure described above on the virus, and therefore the virus may not change significantly and in its current or similar form will become the fifth human commonly occurring coronavirus forever.The initial development of the pandemic, related to the spread of an airborne- or droplet-transmitted virus in the world, broadly corresponds to the course of the influenza pandemic in the twentieth and twenty-first centuries  and different predicted pandemic models. However, the further development of the current pandemic may differ from an influenza pandemic due to differences in genetic and antigenic properties between orthomyxoviruses and coronaviruses. The most important factors are the lower genetic variability of coronaviruses and the likely short-term duration of immunity following SARS-CoV-2 infection.Projected models for the future course of the pandemic were presented by Kissler et al. . The autHowever, the course of the pandemic could be quite different if the following current assumptions prove to be incorrect: (1) SARS-CoV-2 is a new human virus that has permanently entered the list of human viruses. (2) Post-infection immunity is relatively short-lived. By analogy with known human common cold coronaviruses, it may not exceed 2–3 years. (3) The virus will not be under much pressure and there will be no significant mutational changes in the short term. This will distinguish it from influenza viruses, and it will resemble the situation with other coronaviruses and e.g. paramyxoviruses or picornaviruses.Analyzing the pandemic's course to date and our knowledge about the virus, it can be expected that in the future, after the situation stabilizes, we will pass primary infection at a young age as a usually mild childhood disease. Later in life, cases of primary infection will pose a similar problem, as is the case of SARS-CoV-2 infection today. One analogy arises with primary adult infections with varicella, measles or mumps viruses. With the extinction or weakening of specific immunity after primary infection, multiple reinfections may occur. However, in such a case some partially preserved immunity may prevent severe complications. As a result, in a dozen or so years, SARS-CoV-2 infection will not pose such a threat as at present. Perhaps the natural history of the known common cold coronaviruses was similar, having an initial period of significant mortality. It was proposed that HCoV-OC43 entered humans around 1890 as the zoonotic transmission of bovine coronavirus resulting in pandemic (Vijgen et al. Only vaccine development and mass prophylactic vaccinations can change created by different authors, but quite compatible scenarios.It is important to realize that the current pandemic does not mean, of course, that there will not be another pandemic for another new pathogen in the future. And there is no reason why it might not happen soon.


We tested the activity of aged-GTE to inhibit human norovirus in a human intestinal enteroids (HIEs) model and Tulane virus in LLC-monkey kidney (LLC-MK2) cell culture. HIE monolayers pretreated with aged-GTE at different temperatures showed complete inhibition of human norovirus GII.4 replication at concentrations as low as 1.0 mg/ml for 37°C, 1.75 mg/ml for 21°C, and 2.5 mg/ml for 7°C. In contrast, a moderate decrease in Tulane virus infectivity of 0.85, 0.75, and 0.65 log TCID50/ml was observed for 2.5 mg/ml aged-GTE at 37, 21, and 7°C, respectively. Our findings demonstrate that GTE could be an effective natural compound against human norovirus GII.4, while only minimally effective against Tulane virus.Human noroviruses are the leading cause of epidemic and sporadic acute gastroenteritis worldwide and the most common cause of foodborne illness in the United States. Several natural compounds, such as aged-green tea extract (aged-GTE), have been suggested as ingestible antiviral agents against human norovirus based on data using murine norovirus and feline calicivirus as surrogates. However,  Human noroviruses are the leading cause of epidemic and sporadic acute gastroenteritis worldwide and the most common cause of foodborne illness in the United States and Europe, with a societal cost of $60 billion dollars . The gloNorovirus infections are difficult to prevent due to their low infectious dose, high shedding titer, and environmental stability. The virus can spread through multiple transmission routes, even though the most common are the person-to-person and the foodborne transmissions. Most norovirus outbreaks are reported in long-term care facilities, schools, hospitals, restaurants, and cruise ships.Currently there are no licensed norovirus vaccines available  and contSeveral antiviral molecules have shown to inhibit norovirus replication by preventing either the viral binding to cell receptor , the maturation of viral proteins , or the replication of virus genome , is rich in polyphenols and has demonstrated antimicrobial activity against a wide range of pathogens such as Zika virus, dengue virus, and foodborne pathogens , a derivative of the cultivated evergreen tea plant  binding patterns similar to human noroviruses . In thisGTE  was dissolved in PBS (pH 7.2) to obtain a concentration of 10.00 mg/ml. The GTE solution was aged for 24 h at room temperature as reported previously . Fecal f2, differentiation of the HIE was initiated by replacing IntestiCult™ Organoid Growth Medium  with differentiation medium.Three-dimensional HIEs were derived from human jejunal biopsies (J3 cell line) and maintained as described previously . BrieflyTo assess potential cytotoxicity of aged-GTE on HIE and LLC-MK2 cells, cells were incubated with different concentrations of aged-GTE (up to 2.5 mg/ml) stained with trypan blue and visually inspected for cytotoxicity by using a Zeiss Axiovert 200 microscope. The proportion of live cells was determined using a Countess™ II cell counter (Thermo-Fisher). Moreover, the viral replication capability of HIEs was assessed by infecting monolayers pretreated with aged-GTE with human norovirus and measuring virus replication by RT-qPCR.10 gc/100 μl and GII.4 Sydney[P4 New Orleans]: 4.16 ± 0.0 log10 gc/100 μl) were mixed 1:1 v/v with aged-GTE, incubated 1 h at 37, 21, and 7°C and neutralized by mixing with an equal volume of CMGF−. CMGF− medium alone, virus and CMGF, and virus without aged-GTE were included as negative controls. To determine viral infectivity, two sets of 96-well plates with 100% confluent 4–6 day-old-differentiated HIE monolayers were inoculated in triplicate with 100 μl of aged-GTE-treated human norovirus samples and incubated at 37°C for 1 h. After the inoculum was removed, monolayers were washed twice with CMGF− medium and 100 μl of differentiation medium containing 500 μM GCDCA and 50 μmol/l ceramide was added to each well. For each set of infections, one 96-well plate was immediately frozen at −70°C (1 hpi) and the second plate was incubated at 37°C and 5% CO2 for 24 h and then frozen (24 hpi).Aged-GTE was diluted in CMGF− medium to 0.10, 0.40, 0.80, 1.00, 1.75, 2.50, and 5.00 mg/ml. Clarified human norovirus-positive stool suspensions  according to the manufacturer’s instructions. Norovirus RNA was detected by GI/GII TaqMan RT-qPCR . A 10-fo10 TCID50/ml or 4.76 ± 0.02 genomic copies/ml) was exposed to aged-GTE at final concentrations ranging from 0.05 to 2.5 mg/ml for 1 h at 37, 21, and 7°C and neutralized by mixing with an equal volume of Opti-MEM. An untreated sample (media only) was included as negative control. Then, 10-fold serial dilutions of Tulane virus were prepared in Opti-MEM and inoculated on eight wells with 100% confluent LLC-MK2 monolayers in 96 well plates and 20 μl of aged-GTE-treated Tulane virus per well. After incubation for 1 h at 37°C, 180 μl Opti-MEM plus 2% FBS was added. After 3–5 days, cells showing cytopathic effect (CPE) were enumerated and the Tulane virus titer was calculated as log10 (Nx/N0), where N0 is the infectious virus titer for untreated samples and Nx is the infectious virus titer for aged-GTE-treated samples. To determine effect of aged-GTE on the viral genome, Tulane virus RNA was extracted as described above and detected by RT-qPCR (Tulane virus (4.02 ± 0.2 log RT-qPCR . Genome p was <0.05. The nonlinear regression (curve fit) function from GraphPad Prism version 7  was used to obtain EC50 values.All data were compiled from three independent experiments with three technical replicates for each variable. Data are presented as mean ± SD. Significant differences in mean infectivity were determined by using one-way ANOVA followed by Dunnett’s multiple comparisons test. Differences in means were considered significant when the 2 showed no cytotoxic effect. Similarly, no differences on cell viability were detected for samples treated with aged-GTE at concentrations up to 1 mg/ml (mean cell viability 94%), while slight decreases were observed at 1.75 and 2.5 mg/ml .The human norovirus initial concentrations and the control titers  at 24 hpi were consistent across all experiments, allowing the comparison among GTE treatments and exposure temperatures.50/ml reduction when exposed to GTE at 2.5 mg/ml at 37, 21, and 7°C, respectively (50) resulted as 0.16 and 0.31 mg/ml aged-GTE tested at 37°C, 0.26 and 0.16 mg/ml at 21°C, and 0.12 and 0.21 at 7°C for human norovirus GII.4 Sydney[P16] and GII.4 Sydney[P4 New Orleans], respectively  are used for the HIE system, which is significantly higher than the amount needed to cause human infection and the amount of virus that is typically detected in environmental samples. Therefore, increasing the sensitivity of the HIE system should be explored. In addition, strain-specific replication requirements such as bile acids have been identified for several non-GII.4 genotypes (Our study has several limitations. Unlike surrogate viruses which can be grown to high viral titers, the maximum increase in replication of human norovirus is between 2 and 3 logathogens . Moreoveenotypes . This isenotypes . Finallyenotypes .These limitations hampered further evaluation of presumed active ingredients and their potential synergistic effects since more than nine catechins and derivatives have been reported as responsible of the antiviral activity .in situ capture RT-qPCR (In future studies, the HIE model could be used to validate the performance of alternative and more affordable methods such as  RT-qPCR  and viab RT-qPCR  to assesIn conclusion, we confirmed the antiviral activity of GTE at different temperatures and expanded this observation by showing not only reduction on viral genome copies, but also demonstrate that aged-GTE completely inactivates human norovirus GII.4 replication in a laboratory setting. In addition, we have showed that Tulane virus could likely be used as a conservative surrogate for measuring inactivation of norovirus with natural compounds.All datasets presented in this study are included in the article/supplementary material.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.EM was employed by Cherokee Nation Assurance.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Myotis brandtii) tested positive for rabies and based on partial sequence analysis, the novel Kotalahti bat lyssavirus (KBLV) was identified. Because the bat was in an autolyzed state, isolation of KBLV was neither successful after three consecutive cell passages on cells nor in mice. Next generation sequencing (NGS) was applied using Ion Torrent ™ S5 technology coupled with target enrichment via hybridization-based capture (myBaits®) was used to sequence 99% of the genome, comprising of 11,878 nucleotides (nt). KBLV is most closely related to EBLV-2 (78.7% identity), followed by KHUV (79.0%) and BBLV (77.6%), supporting the assignment as phylogroup I lyssavirus. Interestingly, all of these lyssaviruses were also isolated from bat species of the genus Myotis, thus supporting that M. brandtii is likely the reservoir host. All information on antigenic and genetic divergence fulfil the species demarcation criteria by ICTV, so that we recommend KBLV as a novel species within the Lyssavirus genus. Next to sequence analyses, assignment to phylogroup I was functionally corroborated by cross-neutralization of G-deleted RABV, pseudotyped with KBLV-G by sera from RABV vaccinated humans. This suggests that conventional RABV vaccines also confer protection against the novel KBLV.There is a growing diversity of bat-associated lyssaviruses in the Old World. In August 2017, a dead Brandt’s bat ( Chiroptera) with the unique trait of true flight are a fascinating order of mammals which encompasses more than 1200 species [Bats  and Pond bats (M. dasycneme) [Miniopterus schreibersii) in 2003 [M. nattereri), first in Germany [Initially, rabies in bats in Europe was detected in 1954. Subsequent molecular characterization demonstrated that viruses isolated from rabid European bats and associated human rabies cases were distinct from classical rabies virus (RABV) and belong to two genetically separate lyssaviruses, European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2) . Since 1to Spain . Bat rabsycneme) . In rece in 2003 . Eight y in 2003 ,26. LLEB in 2003 . Since 2 Germany , but lat Germany ,30,31.M. brandtii) was found in Eastern Finland in the village of Kotalahti . The bat tested positive for rabies in the fluorescent antibody test (FAT) and while virus isolation was unsuccessful, RT-PCR and subsequent phylogenetic analysis revealed that the virus differed from other known lyssaviruses and was designated as Kotalahti bat lyssavirus (KBLV) [In August 2017, a dead Brandt’s bat (s (KBLV) . Here, wg for 3 min). Virus isolation attempts followed previously published protocols [2 for 72 h, cells of control dishes were fixed and stained with a rabies anti-nucleocapsid FITC-conjugated antibody . Attempts to isolate virus were stopped after three consecutive passages with negative results. Prior to virus isolation attempts at the Friedrich-Loeffler-Institute, scarce remaining brain material and material from the spinal cord of the above-mentioned bat was homogenized in MEM using the TissueLyzer  and then clarified by centrifugation  and six suckling mice  were inoculated with homogenized, clarified sample (5–10 µL) intra-cerebrally. Mice had access to food and water ad libitum and were monitored daily for 21 days using an established lyssavirus clinical scoring system to evaluate any behavioural changes consistent with lyssavirus infection. Brains of euthanized mice were tested by direct fluorescent antibody test (FAT) . Because of limited original brain material, the precipitate from the inoculum for isolation was further used for molecular detection. The precipitate was disintegrated using the cryoPREP CP02  and the pulverized material was subsequently lysed in 1 mL pre-heated (56 °C) AL Buffer . For RNA extraction, a threefold volume of Trizol LS  was added and the aqueous phase of the resulting mixture further processed utilizing the RNeasy Mini Kit  including an on-column DNase I  digestion step. In preparation for the metagenomic analysis, the sample was further treated as previously described . BrieflyTarget enrichment was performed via hybridization-based capture using RNA baits  according to the myBaits manual v.4.01 (April 2018). Briefly, a pre-designed diagnostic bait set of 80mer biotinylated oligonucleotides targeting sequences of epizootic and zoonotic viral pathogens containing about 11,500 baits specific for RABV  was adde® and the associated NCBI nucleotide database (blastn) [In order to acquire the KBLV full genome sequence, an initial de-novo assembly of raw sequencing data was conducted  and the generated contigs were taxonomically assigned using BLAST(blastn) . The so In order to phylogenetically assign KBLV within the genus lyssavirus, a multiple sequence alignment encompassing reference sequences (where available) of all 17 classified lyssavirus species  togetherThe expression plasmid pCAGGS L16G comprised the sequence of the RABV vaccine strain SAD B19 . pCAGGS-in trans complementation of G gene deleted SAD ∆G GFP, in which the G ORF was replaced by the green fluorescence protein (GFP) reporter ORF [6 BSR T7/5 cells in a T25 cell culture flask were infected at multiplicity of infection (MOI) of 1. After 2 h of incubation at 37 °C, cells were trypsinized and the cell suspension was transferred to 6-well plates. Following over-night incubation, the infection was controlled by GFP autofluorescence and expression plasmids for G proteins of SAD B19 and KBLV . Briefly, 2.5 to 5.0 × 105 cells were seeded in 3.5 cm dishes. After overnight incubation, the cell culture medium was replaced by 1 mL serum free DMEM medium and 1 h later a mixture of 6µg plasmid and 9µg PEI in 800 mL DMEM was added. For preparation of the transfection solution, plasmid DNA and PEI were separately diluted in 400 µL DMEM and then mixed after 5 min incubation at room temperature (20–22 °C). After further 20 min incubation, the mix was added to the cell culture. After further 3.5 h of incubation at 37 °C, medium and transfection mix was removed and replaced by 2 mL fresh culture medium.Antibodies, sera and immunofluorescence microscopy. Mouse monoclonal antibody E559 against RABV G protein have been described previously [E. coli expressed and purified partial G protein purified MBP-EBLV-2G protein ). For indirect immunofluorescence detection, on glass covers slips growing cells were fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). After 30 min incubation at room temperature, the PFA was removed and the specimens were blocked by incubation with 0.025% milk powder in PBS for 20 min at RT. Immunostainings were performed by two hours incubation with primary antibody, two wash steps with PBS and subsequent 45 min incubation with Alexa-fluorophore conjugated secondary antibodies. Images were acquired with a Leica SP5 confocal laser scan microscope  with sequential acquisition of the fluorophores in double fluorescent specimens. Images were processed with the ImageJ software version 1.48b [eviously . The polon 1.48b  N = 27) from humans vaccinated against RABV were used. The virus neutralizing titre was initially established using the Rapid Fluorescent Foci Inhibition Test (RFFIT) with CVS-11 as test virus [For cross-neutralization, serum samples  and the PCR positive bat was used for virus isolation attempts. Tissue cultures after three consecutive passages were negative in the FAT. In addition, all mice survived until the termination of the observation period at day 21 post infection and brain impression smears tested negative in FAT.Viral RNA could be isolated from the remaining material, albeit the quality of RNA was low as shown by a high level of fragmentation see . NonetheMyotis brandtii.Phylogenetically, KBLV is closest related to EBLV-2 and clusters together with KHUV and BBLV . The genTo assess expression, intracellular distribution, and antigenicity of the predicted KBLV glycoprotein, expression plasmid vectors were constructed by insertion of the respective ORF as a synthetic cDNA. At one day after plasmid transfection, the KBLV and RABV G proteins were comparably detected at the cell surface by the monoclonal antibody E559 and intracellularly by a serum raised against EBLV-2 G . In order to test whether sera from human RABV vaccinees would cross-neutralize KBLV, G-deleted RABV pseudotyped with KBLV-G were used. All sera from human vaccinees cross-neutralized the supernatant of RABV pseudotyped with KBLV-G. The neutralizing activity of sera was lower against KBLV than against the standard RABV test virus (Challenge Virus Standard, CVS, M. brandtii bats had been part of surveillance activities across Europe [M. brandtii is likely a reservoir host and not infected as a result from a spill-over infection by another bat species, although this latter possibility cannot be excluded given the available data. The Myotis genus, which belongs to the family Vespertilionidae and comprises more than 100 species [M. brandtii as a western Palaearctic bat species is actually part of the Myotis New World clade, suggesting that lyssavirus-bat host co-evolution occurred after the split between New and Old World within the Myotis radiation that occurred about 19 million years ago [After the detection of rabies in a bat from Finland, sequence analyses of the partial viral genome suggested that a novel lyssavirus was discovered . For anys Europe . Previou species . Interesears ago .In the absence of viable virus, we used the viral genetic information combined with molecular methods to further characterize the virus. Initially, it was planned to clone and rescue a full length KBLV, but the different genes could not be cloned successfully into one plasmid. Also, in other attempts including the swapping of UTR and LTR with KHUV, the insertion of KBLV-G into recombinant BBLV and RABV, viable virus could not be rescued.However, expression of the predicted KBLV-G from transfected plasmids  and succM. brandtii as the likely host are initiated to detect eventually another KBLV infected bat, thus increasing the chance of virus isolation. While our attempt to isolate replication competent KBLV or recombinant viruses did not yield viable virus, in the future, other avenues to rescue viruses may be more successful. Functional assays based on molecular biology and sequence information were suitable to further characterize the novel lyssavirus KBLV in the absence of virus isolation. Further intensified surveillance targeting All information on antigenic and genetic divergence fulfill the species demarcation criteria by ICTV , so that


Accurately determining the normal range of early pregnancy markers can help to predict adverse pregnancy outcomes. The variance in ovulation days leads to uncertain accuracy of reference intervals for natural pregnancies. While the gestational age (GA) is accurate estimation during in vitro fertilization-embryo transfer (IVF-ET). Thus, the objective of this research is to construct reference intervals for gestational sac diameter (GSD), yolk sac diameter (YSD), embryonic length  and embryonic heart rate (HR) at 6–10 gestational weeks (GW) after IVF-ET.From January 2010 to December 2016, 30,416 eligible singleton pregnancies were retrospectively recruited. All included participants had full records of early ultrasound measurements and phenotypically normal live neonates after 37 GW, with birth weights > the 5th percentile for gestational age. The curve-fitting method was used to screen the optimal models to predict GSD, CRL, YSD and HR based on gestational days (GD) and GW. Additionally, the percentile method was used to calculate the 5th, 50th, and 95th percentiles.2] = 0.796), CRL = − 11.960 - 0.147 GD + 0.011 GD2 (R2 = 0.976), YSD = − 2.304 + 0.184 GD - 0.011 GD2 (R2 = 0.500), HR = − 350.410 + 15.398 GD - 0.112 GD2 (R2 = 0.911); and GSD = − 29.180 + 7.492 GW (R2 = 0.796), CRL = − 11.960 - 1.028 GW + 0.535 GW2 (R2 = 0.976), YSD = − 2.304 + 1.288 GW - 0.054 GW2 (R2 = 0.500), HR = − 350.410 + 107.788 GW - 5.488 GW2 (R2 = 0.911), (p < 0.001).There were significant associations among GSD, CRL, YSD, HR and GD and GW, the models were GSD = − 29.180 + 1.070 GD (coefficient of determination [RReference intervals for GSD, YSD, HR and CRL at 6–10 gestational weeks after IVF-ET were established. Accurately determining the normal range of early pregnancy markers can help to predict adverse pregnancy outcomes, such as miscarriage. It is also useful to determine the number of foetuses and their viability, type of twinning, and presence of gross fetal abnormalities, placental problems, and uterine or adnexal problems. Some studies have constructed reference intervals that mostly depend on natural conceptions of women with regular menstrual cycles and known dates of their last menstrual periods (LMPs) –3. HowevDuring in vitro fertilization-embryo transfer (IVF-ET), the day of oocyte retrieval and ET are known; thus, the GA estimation is accurate. We speculate that reference intervals derived from IVF-ET data are more accurate than those derived from natural conception and be more suitable for IVF populations. With the rapid development of artificial reproductive technology, especially after the implementation of the two-child policy in mainland China, more infertile couples conceive with this treatment , 11. HowThis study analysed data from a large cohort of 30,416 singleton pregnancies with normal outcomes from a Chinese population, aiming to construct reference intervals for gestational sac diameter (GSD), yolk sac diameter (YSD), heart rate (HR) and CRL at 6–10 gestational weeks (GW) following IVF-ET. The optimal models for predicting GSD, CRL, YSD and HR based on GA were also analysed.The institutional review board approved this study before data collection LL-SC-2019-015). The study was conducted using anonimized dataset of patients for research purposes and that it was conducted in agreement with Helsinki declaration for research ethics. STROBE Guidelines were followed for reporting this observational study . This re9-015. ThPregnancy and perinatal outcomes were tracked by a specified team via telephone call or fax at our centre. The inclusion criteria were as follows: (1) live singleton pregnancy following IVF-ET; (2) embryonic GSD, YSD, HR were fully measured and recorded at 6–10 GW; and (3) live birth after 37 GW of a phenotypically normal neonate with a birth weight > 5th percentile for GA . All enrWe collected the first ultrasound examination results from each patient during 6–10 GW. The ultrasound scans were performed by 4 experienced sonographers with a GE VOLUSON E8/730  equipped with a 5–9 MHz transvaginal probe. The measurements referred to the ISUOG practice guidelines  and confIntra- and interobserver reliability of measurements was tested on a random selection of 30 pregnancies at day 28 after ET. Each observer performed two measurements of GSD, YSD, CRL and HR on separate occasions and was unaware of others’ results. Written informed consent was obtained from all test patients before ultrasound scanning. The reference intervals were analysed according to the gestational days (GD) and GW. The GD can be deduced by adding 17 to the day of ET for cleavage stage embryos or adding 19 for blastocysts (Day 5 or Day 6), and the corresponding GW was obtained by dividing the GD by 7 . The cal2). The model with the largest R2 was ultimately selected as the best model. Additionally, the percentile method was used to calculate the 5th, 50th, and 95th percentiles for each time point. Scatter plots of GSD, CRL, YSD and HR compared with GD and GW were obtained. Correlation coefficients were calculated to analyse the intra- and interobserver reliability. A p value < 0.05 was considered significant.All statistical analyses were performed using SPSS software version 17.0 . Measurements are presented as the mean ± standard deviation (SD), and the enumerated data are expressed as numbers (percentages). The curve-fitting method was used to screen the optimal models for predicting GSD, CRL, YSD and HR based on GD and GW. We determined the optimal model based on the size of coefficient of determination (Rp < 0.001).From January 2010 to December 2016, 100,718 infertile patients obtained clinical pregnancies via IVF-ET in our hospital. After data exclusion, a total of 30,416 singleton pregnancies with normal outcomes were included in this study. The clinical characteristics of the study population were shown as Table P < 0.001) and GSD = -29.180 + 7.492 GW . Figure There was a significant linear association between GSD and GA. The best fit models were as follows: GSD = -29.180 + 1.070 GD  and CRL = − 11.960 - 1.028 GW + 0.535 GW2 . Figure There was a significant quadratic association between CRL and GA. The most appropriate fit models were as follows: CRL = − 11.960 - 0.147 GD + 0.011 GD2 , and YSD = − 2.304 + 1.288 GW - 0.054 GW2 . Scatter plots with the 5th, 50th, 95th percentiles of YSD against GD are presented in Fig. A significant association between YSD and GA was found. The following quadratic models showed the most appropriate fit: YSD = − 2.304 + 0.184 GD - 0.011 GD2  and HR = − 350.410 + 107.788 GW - 5.488 GW2 . Scatter plots with the 5th, 50th, 95th percentiles of HR against GD are presented in Fig. A significant association between HR and GA was found. The following quadratic models showed the best fit: HR = − 350.410 + 15.398 GD - 0.112 GDAdditionally, the details of the reference intervals for GSD, CRL, YSD and HR based on GD and GW are shown in Tables In this study, we constructed reference intervals for GSD, YSD, CRL, and HR at 6–10 GW for an IVF population with a large sample of Chinese women. The optimal models for predicting GSD, CRL, YSD and HR based on GA were also presented.In this study, a high proportion of CS is noted in Table 2 (0.500 for both GD and GW) than the other models, suggesting that the prediction models can only explain 50% of the changes in YSD; thus, in addition to GA, there are other factors to be explored.Optimal models for predicting GSD, CRL, YSD and HR based on GA were established and showed that GSD linearly increases with GA. CRL, YSD, and HR had significant quadratic associations with GA. These models can be conveniently used in clinical practice to calculate the corresponding values of GSD, CRL, YSD and HR according to GA. However, the YSD models showed relatively lower RThe reference intervals for GSD, YSD, HR and CRL at 6–10 GW were constructed from a large sample in this study. This data can provide clinicians a reliable reference to analyse the development of early embryos after IVF-ET and facilitate monitoring of pregnancy outcomes at an early stage. GSD, YSD and CRL were found to gradually increase from 6 to 10 GW. However, HR increased from 6 GW, reaching a peak at 9 GW (176.0 bpm) and decreasing from there. This trend in HR was consistent with the results of previous studies , 19 and For comparison with previous studies, we performed a literature search of PubMed, and representative literature is listed in Table The most popular formula for pregnancy dating originated from the study by Robinson and Fleming , and sevn = 12,687); however, much fewer patients underwent ultrasound on other days, particularly on later days. However, it is impractical to perform ultrasound for each patient every day to evenly distribute the sample. Therefore, future studies are needed to verify our reference intervals. Thirdly, to compare normal data with abnormal outcomes and try to understand whether the measurements may be somehow function as prognostic factors for abnormalities would be an interesting future work. Fourthly, since we collected the data retrospectively from the hospital database, some baseline data such as pharmacological treatments uses, parity, significant maternal diseases and smoking status were missing.Our study has several strengths. The large sample size allowed us to establish special reference intervals and construct optimal models for GSD, CRL, YSD and HR for IVF populations, which may be helpful for accurately analysing and monitoring the development of early pregnancy following IVF-ET. However, one potential weakness was that all the data were confined to one reproductive centre; although it is the largest centre in China, territorial limitations exist. Future studies with multi-centre samples are necessary to establish nationwide or worldwide references. Secondly, although the total sample was quite large, the patients were unevenly distributed. Most patients underwent their first ultrasound on day 28 after ET   decreaseIn conclusion, this study involving a large number of normal pregnancies presented the reference intervals for GSD, CRL YSD and HR at 6–10 GW. These data can be used as reliable references for analysing the development of early embryos after IVF-ET and for monitoring pregnancy outcomes at early stages.


When facing an emerging virus outbreak such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a quick reaction time is key to control the spread. It takes time to develop antivirals and vaccines, and implement vaccination campaigns. Therefore, preventive measures such as rapid isolation of cases and identification and early quarantine of cases’ close contacts—as well as masks, physical distancing, hand hygiene, surface disinfection and air control—are crucial to reduce the risk of transmission. In this context, disinfectants and antiseptics with proven efficacy against the outbreak virus should be used. However, biocidal formulations are quite complex and may include auxiliary substances such as surfactants or emollients in addition to active substances. In order to evaluate disinfectants’ efficacy objectively, meaningful efficacy data are needed. Therefore, the European Committee for Standardisation technical committee 216 ‘Chemical disinfectants and antiseptics’ Working Group 1  has developed standards for efficacy testing. The European tiered approach grades the virucidal efficacy in three levels, with corresponding marker test viruses. In the case of SARS-CoV-2, disinfectants with proven activity against vaccinia virus, the marker virus for the European claim ‘active against enveloped viruses’, should be used to ensure effective hygiene procedures to control the pandemic. When facing an emerging infectious disease outbreak, a quick reaction time is crucial to control the spread. However, it takes time to develop and produce therapeutics and/or vaccines . The chaMeasures are needed to control COVID-19, whether these are non-pharmaceutical, a pharmaceutical prophylactic or a pharmaceutical treatment ,9. ThougThe use of antiseptics and disinfectants has long been a widely accepted part of infection prevention and control to support healthcare professionals, patients and people at higher risk of serious illness with rapid and efficacious measures. In general, biocidal disinfectant or antiseptic formulations are complex and may include auxiliary substances such as surfactants or emollients in addition to active substances, which can influence the effect of the disinfectant. Therefore, recommending to use a product that contains a certain biocidal active substance is not adequate to identify suitable products.In general, it is important to have a standardised test protocol for comparing the efficacy of disinfectant and antiseptic products . In ordeEuropean standards for testing antiseptics and disinfectants are based on a tiered approach, using relevant surrogate test organisms. This helps to facilitate efficacy tests and to develop adequate practical recommendations regarding the disinfectants that can be used immediately in an ongoing outbreak, which will add confidence to the bundle of measures necessary to prevent spread of infectious diseases.Information on the efficacy of disinfectants and antiseptics against emerging enveloped viruses such as Ebola virus (EBOV) or SARS-CoV-2 is limited because of the high biosafety level (BSL) of these viruses or because they are not available in laboratories, particularly at the beginning of an outbreak. To avoid this problem, vaccinia virus (modified vaccinia virus Ankara (MVA) or vaccinia virus strain Elstree, respectively) was introduced as a surrogate virus for the European claim ‘active against enveloped viruses’ in 2015, after in vitro studies comparing MVA with EBOV, SARS-CoV-1 and MERS-coronavirus (CoV) using povidone-iodine (PVP-I) showed that vaccinia virus was the most resilient virus ,14. ThisThe European approach with surrogate organisms has been effective for more than 60 years against bacteria and for about 40 years against viruses, e.g. in German national standards. The choice of surrogate viruses is of great importance when establishing a standardised test. The requirements for standardised surrogate viruses are: high resistance to disinfectants and drying, combined with simple virus propagation in cell culture, availability in virus collections and a low BSL (BSL 1 and 2). For example, 30 years ago, European virucidal activity tests focused only on the most resistant virus: hepatitis A, a picornavirus that it is difficult to handle in a routine testing laboratory. The poliovirus 1 Sabin vaccine strain Lsc-2ab, another picornavirus, can easily be handled in cell culture for virus propagation and therefore became the first surrogate virus for the claim ‘virucidal activity’.Presently, the tiered approach on the virucidal standard EN 14476  consistsThe three European virucidal activity levels take into account that enveloped viruses contain lipids and have lipophilic properties (Box). These viruses are sensitive to disinfectants that contain active substances that disrupt their lipid bilayer envelope. In contrast, non-enveloped viruses are usually hydrophilic and less susceptible to disinfectants. Thus, viruses can be grouped according to these properties .BoxVirucidal activity against enveloped viruses: Activity against enveloped viruses (e.g. influenza (including avian influenza A (H7N9)), herpes viruses, HIV, HBV, HCV, Zika virus, Ebola virus, coronaviruses (including SARS-CoV-2 and MERS-CoV) and flaviviruses). This claim is for hand disinfection, surface disinfection and pre-cleaning products for instruments, which are combined cleaner/disinfectants.• Limited spectrum virucidal activity: Activity against non-enveloped noroviruses, rotaviruses, adenoviruses and enveloped viruses. This claim is for hand disinfection and surface disinfection.• Virucidal activity: Activity against viruses, including non-enveloped  and enveloped viruses. This claim is for hand disinfection, surface disinfection, instrument disinfection and textile disinfection.• HBV: hepatitis B virus; HCV: hepatitis C virus; MERS-CoV: Middle East respiratory syndrome coronavirus; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.The CEN/TC 216 requires a stepwise procedure for efficacy testing of disinfectants, i.e. first a test in suspension , followed by a test simulating practical conditions in the laboratory . This tiered approach, including organic soiling, is described in the framework standard EN 14885 . The quaViruses may persist on surfaces for several days or even months  and can Escherichia coli K12. Since most emerging viruses are enveloped viruses, a relevant surrogate for products with activity against enveloped viruses should be sought. More than 30 years ago, tests with vaccinia virus were performed on the fingertips of volunteers [In order to further improve our knowledge of the time/concentration relationships of handrubs, taking into consideration practical conditions for use in clinical surroundings, more practical testing  are necessary, such as the EN 1500 for evaluating the bactericidal efficacies of hygienic handrubs . In suchlunteers ; howeverlunteers  that emplunteers .Disinfectants and antiseptics are part of the bundle of measures necessary to achieve infection prevention and control in healthcare settings and the community. The European tiered approach of grading the virucidal efficacy as ‘virucidal activity against enveloped viruses’, ‘limited spectrum of virucidal activity’ and ‘virucidal activity’ helps to select the most appropriate claim for emerging or re-emerging virus outbreaks, with a proven concentration and contact time. This leads to the correct choice of test method to determine the appropriate use, concentration and contact time of antiseptics and disinfectants.The standards outlined in


To the Editor: Alera et al. described a 2012 case of Zika virus infection in the Philippines (50) . The sample tested negative for IgM against dengue and Japanese encephalitis viruses but positive for IgG against Zika virus nonstructural protein 1. These results suggest local Zika virus infection in the Philippines since at least 2010, 2 years earlier than the previously reported infection (We detected Zika virus infection in 1 (0.75%) of 134 febrile, non–dengue infected patients at St. Luke’s Medical Center  during 2010–2015 by subjecting patient serum samples to serological and molecular tests. Ethics clearance (reference no. 19042) for this study was given by St. Luke’s Medical Center Institutional Ethics Review Committee. The only patient who tested positive for Zika virus was a 31-year-old woman diagnosed with an upper respiratory tract infection in 2010. Because of her work, she might not have traveled internationally. We obtained her serum sample on day 3 of fever. She did not have a rash or arthralgia. Although we did not isolate Zika virus according to guidelines (