To decrease child mortality due to common but life-threatening illnesses, community health workers (CHWs) are trained to assess, classify and treat sick children. For pneumonia, CHWs are trained to count the respiratory rate of a child with cough and/or difficulty breathing, and determine whether the child has fast breathing or not based on how the child’s breath count relates to age-specific respiratory rate cut-off points. International organizations training CHWs to classify fast breathing realized that many of them faced challenges counting and determining how the respiratory rate relates to age-specific cut-off points. Counting beads were designed to overcome these challenges. This article presents findings from different studies on the utility of these beads, in conjunction with a timer, as a tool to improve classification of fast breathing. Studies conducted by the International Rescue Committee and Save the Children among illiterate CHWs assessed the effectiveness of counting beads to improve both counting and classifying respiratory rate against age-specific cut-off points. These studies found that the use of counting beads enabled and improved the assessment and classification of fast breathing. However, a Malaria Consortium study found that the use of counting beads decreased the accuracy of counting breaths among literate CHWs. Qualitative findings from these studies and two additional studies by UNICEF suggest that the design of the beads is crucial: beads should move comfortably, and a separate bead string, with colour coding, is required for the age groups with different cut-off thresholds—eliminating more complicated calculations. Further research, using standardized protocols and gold standard comparisons, is needed to understand the accuracy of beads in comparison to other tools used for classifying pneumonia, which CHWs benefit most from each different tool (i.e. disaggregating data by levels of literacy and numeracy) and what the impact is on improving appropriate treatment for pneumonia.  The use of age-specific and colour-coded beads enables community health workers (CHWs) to track rather than mentally count the child’s respiratory rate and eliminates the need to remember the age-specific fast breathing cut-offs for pneumonia classification.Well-designed age-specific and colour-coded counting beads, when used in conjunction with an accurate timing device, have the potential to improve correct classification of fast breathing by CHWs with limited numeracy and literacy in low-resource settings.Further research is required to demonstrate the relationship between well-designed age-specific and colour-coded beads and health outcomes, particularly when data are disaggregated by key characteristics of CHWs (e.g. by levels of literacy and numeracy) and severity of illness.Because of the overall complexity of diagnosis, the still staggering mortality, lack of diagnostic aids and the growing problem of antibiotic resistance for pneumonia, there is an urgent need for more robust data on tools for pneumonia diagnosis. Pneumonia is the leading cause of childhood mortality, accounting for 17% of global deaths of children under five . Timely There are two steps in detecting whether a child has fast breathing: (1) a CHW needs to visually count a child’s breath for 1 min and (2) the CHW has to determine how the child’s breath count relates to age-specific respiratory cut-off points . InternaWatches and timers have been used as timing aids to facilitate 1-min RR counting. The potential of the use of counting devices, such as beads, is unknown due to lack of information regarding their effectiveness and utility. However, several organizations  have conducted small-scale studies among CHWs with differing levels of literacy and numeracy, using counting beads, which contribute to the knowledge base.In this review, we compile the current evidence base of the effectiveness of counting beads to assess and classify breathing rates to guide pneumonia diagnosis. These findings are essential to further guide integrated Community Case Management (iCCM) programming aiming to decrease pneumonia deaths in young children.The findings presented are based on studies conducted by IRC, Save the Children, Malaria Consortium and UNICEF to improve iCCM programming in South Sudan, Uganda and Ghana. All CHWs in these studies were trained in iCCM using the World Health Organization (WHO) and UNICEF IMCI ‘Caring for Newborns and Children in the Community’ protocol. CHWs are named differently in various countries, e.g. in South Sudan they are called community-based distributors; however, in this article, we refer them as CHWs.What is known regarding primarily illiterate CHWs’ ability to assess and classify fast breathing without the use of counting beads?Does the use of beads improve the ability of CHWs, particularly those with limited or no literacy and numeracy, to correctly classify fast breathing ?Does the use of beads improve the ability of literate CHWs to correctly assess the breath count?What are CHWs’ perceptions of these tools, and does this differ by literacy level?We compiled all research findings on the use of counting beads within these programmes. Although most findings presented are part of larger research initiatives, in this review we focused on the following questions:A brief description of the studies is provided below and a summary of the key methodological elements of the different studies can be found in As part of a larger assessment evaluating the quality of care provided by CHWs, IRC compared the CHWs’ ability to assess fast breathing using the ARI timer alone vs using the timer and beads. The geographical areas (payams) were selected on the basis of their geographical accessibility to the evaluation team and recent start-up of the programme.First, CHWs were asked to describe the cut-off points for fast breathing for the two age groups (2–11 and 12–59 months) and to count the child’s RR using the ARI timer. A trained clinician who was part of the evaluation team counted simultaneously with the CHW. The RR from the clinician was written down and the CHW was asked to say his/her count. Afterwards, CHWs were given the right counting bead string for the age of the child being assessed and were shown how to move their fingers along the beads and to stop when the timer beeped. Then CHWs were asked to repeat the assessment with the same child using the timer and the beads. The clinician used the counting beads simultaneously with, but out of sight of, the CHW. The beads were counted back and recorded for both the CHW and the trained clinician. Microsoft Excel was used to analyse the data.The use of beads by CHWs with limited numeracy was part of an operational research lead by Save the Children. The research was conducted to assess the effectiveness of simulation-based training of CHWs using video technology.During the training, CHWs were shown video clips of cases with danger signs  and the interactions between CHWs and caretakers. The video also showed the assessment, classification, treatment and advice on home care. CHWs were given one set (two strings) of beads to count and classify the breath count along with the ARI timer acting as a stop watch. CHWs were taken to a hospital outpatient department for post-training skills assessment, where they assessed sick children (40 out of the 69 cases had a history of cough). Each CHW managed  two to three sick children aged 2–59 months. Each CHW also completed a knowledge questionnaire. As these particular CHWs were illiterate, one clinician trained in IMCI observed the CHWs’ management of sick children and recorded the assessment, classification and treatment findings on a study form. A senior evaluator who was also an IMCI trainer independently assessed the children at the same time and recorded his findings on a similar form. The RR was measured for all 69 children by the CHWs and the evaluator. To assess fast breathing the CHW had to choose the correct bead string for the age group. The observer marked the case as having fast breathing if the CHW reached the red beads (fast breathing for 2- to 11-month and 12- to 59-month age groups) within the minute. The evaluator used the ARI timer to count child breaths and noted the actual breath count in the form. The data were entered in CSPro and imported and analysed in Microsoft Excel.Malaria Consortium assessed whether there was a difference in accuracy in counting RR among CHWs using (1) the ARI timer alone vs (2) using the timer and beads and (3) using a mobile phone application, where the centre button on the phone was pressed for every breath observed, and which beeped after 1 min, after which the count was displayed. The sub-county was selected on the basis of its geographical location and mixture of CHWs with different age, sex and literacy levels [varying from being able to read and write well in any of the local languages (78%) to fairly well (22%)].First, each CHW received a detailed explanation of how to count the RR, while simultaneously using the ARI timer, the same timer with beads as well as the mobile phone application. The CHWs then had the opportunity to practise three different methods of counting RR on videos of children with different breath counts. After familiarizing themselves with the three methods, each CHW was observed counting using the three options, respectively, on children with different breath counts. The video case scenarios were displayed on laptop screens, enabling one CHW to assess a child at a time. The final RR was recorded for the three tools as follows: (1) when using the timer alone, the CHW was asked for the final RR count, (2) when using both the timer and beads, the beads were counted back by a research assistant who provided the final RR count and (3) for the mobile phone application, the result was read from the screen. The breathing rate of the children in the video was known and was used as the gold standard. STATA 12 was used to analyse the data. The research only assessed the impact on counting the RR and did not assess the impact of these devices on classifying the breath count against the age-specific cut-off points.Along with their quantitative research efforts, IRC and Malaria Consortium assessed CHWs’ perceptions about the beads, focusing on their opinions and proposed improvements. Data were collected through interviews. Malaria Consortium also assessed CHWs’ perceptions about how the use of counting beads helped them communicate the test results to caregivers.Other qualitative research was initiated by UNICEF, as part of a broader effort to identify CHWs’ unmet needs regarding tools to support the assessment and classification of RRs. In Uganda the focus of the research was on CHWs’ experiences assessing RR using the ARI timer and their ideas regarding tools that might be useful to help improve their assessment and the classification of RRs. The CHWs were not familiar with the use of beads. Building on these findings, a subsequent study was conducted in Northern Ghana, where the CHWs had been trained to use both the ARI timer and beads. The main objective of this study was to help UNICEF improve the design of diagnostic aids based on the challenges CHWs indicated they face while assessing, classifying and identifying treatment needs for children under 5 with rapid breathing.Non–age-specific counting beads: These counting beads are designed to help the CHW keep track of the amount of breaths taken. The CHW counts moves bead for each breath. When 1 min has passed the CHW counts back the beads to determine the RR. Because of the colour coding of the number of beads (e.g. every set of 10), the CHW can count back the beads per colour: e.g. 1 colour = 10 breaths, 2 colours = 20 breaths, etc. On the basis of the RR, the CHW compares the result against the IMCI guideline, correctly remembering the age-specific cut-off rate.Age-specific beads with colour coding: These counting beads support the CHW not only with counting but also with interpreting the RR against the IMCI guidelines. These beads remove the need to count by the CHWs to assess pneumonia  because they consist of a set of two strands or rows of beads that are colour-coded to match the thresholds for the two different age groups. Depending on the age of the child, the CHW selects the matching bead strand and moves the beads for 1 min. Once the minute has passed, the CHW can identify whether the child has pneumonia, depending on the colour of the bead she/he is holding between his/her fingers.The IRC assessed what the key challenges were for primarily illiterate CHWs while classifying fast breathing. Findings indicated that 46% (21/46) of CHWs in Uganda were not able to apply the age-specific cut-off points and 33% (15/46) made an incorrect count using the ARI timer. In South Sudan, 59% (19/32) of CHWs were not able to apply the age-specific cut-off points and 72% (23/32) of CHWs made an incorrect count using the ARI timer.P = 0.002) when they used the beads together with the timer. Findings were similar in Uganda, where the ability to classify fast breathing increased from 37% (17/46), using only the timer, to 73% (24/33) , using both tools. Combining the data from both countries, the ability to classify fast breathing increased from 27% (21/78) to 68% (44/65) , see In South Sudan 13% (4 out of 32) of the CHWs were able to classify fast breathing using only the ARI timer, whereas this number increased to 63% (20/32) (Odds Ratio (OR) OR = 11.7, Save the Children’s research did not have a component of assessing a sick child without the use of beads, as they identified that CHWs had limited numeracy, and were not able to count beyond 10. A total of 69 sick children were assessed by the 27 CHWs. Using the ‘age-specified and colour-coded’ beads and ARI timer, the CHWs classified 25 cases as suffering from fast breathing. The senior evaluator who only used the ARI timer classified 23 as fast breathers. Of the 25 CHW-classified fast-breathing cases, 15 (60%) matched with the evaluator-classified fast-breathing cases.CHWs were also administered a knowledge questionnaire that included questions on the use of beads appropriate for age. All 27 CHWs picked the beads appropriate for the two age groups. With respect to classifying a child as having fast breathing, 26 out of 27 picked the right colour (red) for the 2- to 11-month group and 27 out of 27 for the 12- to 59-month age group.P < 0.001). There was no significant difference between the ARI timer and mobile phone application , implying that CHWs have a similar capacity to correctly count RR using either of the assessment methods (i.e. counting themselves vs pressing a button for every breath observed).In Uganda, Malaria Consortium assessed whether the use of tools, including counting beads, improved the ability of literate CHWs to count RR. CHWs were 5.6 times more likely to count (not classify) RR correctly (i.e. ±2 breaths) using the timer alone compared with when it is combined with beads , whereas for the mobile phone application and counting beads, the difference between the rates observed and the true rate was significantly different from 0 . Using the same test, the median difference observed between the ARI timer and mobile phone application was not significantly different (P = 0.179), whereas the difference was significantly different between the ARI timer and the counting beads (P < 0.001) as well as between the mobile phone timer and the counting beads and timer (P = 0.001).Overall, the median difference between the ‘true rate’ and the rate observed with the three methods was −1 (interquartile range(IQR) −5 to 2) for the UNICEF timer, −1 (IQR −7 to 2) for the mobile phone application and −5 (−12 to 2) for the ARI timer with beads. Using the sign test for non-parametric data on matched pairs, the differences in rates observed using the ARI timer compared with the true rate was not significantly different from 0 , it was demonstrated that all three methods performed much better on the slower breathing rates (i.e. 40 breaths/min) than the fast rates (i.e. 65 and 66 breaths/min). All three methods tended to overestimate the rate in the slow-breathing scenario, whereas they underestimated the rate in the fast-breathing scenarios.The colour-coded beads designed by IRC in Uganda match the colour of the locally available amoxicillin packages for specific age groups, and according to (mainly illiterate) CHWs, this helped them to identify the correct string needed for the child, eliminating the need to recall the cut-off points for the different age groups. Regarding the use of counting beads, CHWs interviewed by IRC were most likely to say that: (1) the use of beads eliminated the need to count or worry about forgetting the number or making mistakes while counting; (2) it was easy to move hands along the beads; (3) it was easy to know when to give the treatment and (4) it was easy to explain to the mother that her child did not need medicine. The need for more training and practice regarding the use of beads in combination with the ARI timer was mentioned by CHWs in South Sudan and Uganda.What I have liked about this method is that I don’t have to count the beads as I move them, counting usually comes last and this gives me more concentration on observing the child and moving the beads.The method was acknowledged as one that could give a quick picture of the diagnosis by identifying the colour of the bead where the hand has stopped after the alarm has gone off (i.e. whether white or red/green bead), the next steps could follow later:From what I have learnt today, the combination of both the timer and beads is very helpful because it helps me to immediately know whether the child has fast breathing by looking at the colour of beads where I have stopped and then, I count the beads to confirm what I have seen, after which I write in my book.The use of the beads also made it easier to communicate the results to the caretaker, as the colours visually flagged if the child had a high breath rate (indicating pneumonia) or not.In the study amongst literate CHWs by Malaria Consortium in Uganda, CHWs expressed their support for alternative methods to count the RR other than the timer which they were familiar with. Using the beads, in addition to the timer, was perceived as being advantageous because each breath was being represented by movement of a bead and then counting of the beads could be done afterwards, thus giving more accurate results.For me, I find this method very challenging because I have to observe three things at the same time: I have to look at the child, start the timer and also move the beads at the same time, which is a bit tasking. That is why you have seen that I have been forgetting to put on the timer as I move the beads.However, the beads were also perceived by some as disadvantageous because combining the timer and beads was considered to be more tasking, with a slow bead movement process and therefore reduced accuracy. During observations, it was noted that CHWs often found it difficult to start moving the beads immediately after starting the timer.Related to the design, CHWs often mentioned that the space between individual beads can affect the outcome of the count. The space between the beads should be small, as the bigger the distance was between the beads, the more difficult it was to move them. The CHWs also suggested that the beads should be light and unattractive in order not to be mistaken for accessories and have a strong string, which would not break easily.I would have preferred the beads because each time I count, I hold a bead so I will be able to know how many beads I left behind in case I forget where I was when countingGreen is OK. Red is for danger.Data from the assessment in Uganda by UNICEF, where mainly illiterate CHWs only used the ARI timer, revealed that CHWs would find it useful to have a device that supports them with the classification of fast breathing and that would help them communicate the findings to the caregivers. When the CHWs in Uganda were shown counting beads they mentioned that beads might be confused with a toy; however, it would help them with the assessment and the classification of fast breathing.The main concern among mainly illiterate CHWs regarding the use of non–age-specific beads used in the Northern region of Ghana was that these beads still required counting and remembering the cut-off rate. The beads also have ‘separator bead’, which the CHWs thought creates confusion because ‘we move the beads without looking at them’. Nevertheless, the use of prayer beads in the region is common; CHWs are used to counting with beads and are at ease with this. However, the CHWs reported that this similarity could reduce the acceptance by caretakers as the beads are perceived as a tool for prayer and not as a healthcare-related tool.This one has only two colours and the colours easily tell if the child has pneumonia or not. I think that this one will be more convenient to use than the current one we have.When shown the ‘age-specific and colour-coded’ beads used by IRC and Save the Children, the CHWs and their supervisors in Ghana showed a strong preference to this design as it eliminated the need to count and remember the cut-off rate.This review of studies on the utility of counting beads as a tool to improve classification of fast breathing in children with cough and/or difficulty breathing to guide pneumonia diagnosis shows that the introduction of ‘age-specific and colour-coded’ counting beads, in addition to an accurate timer, can help CHWs with limited numeracy and literacy to more accurately assess fast breathing. Although CHWs also expressed concerns on the task intensity of the method, in general, it was acceptable and applicable across different settings.There are several limitations associated with the studies included in this review. First, the studies used several research approaches, methods (including gold standards) and research questions. Moreover, programmatic settings as well as the levels of literacy and numeracy of the CHWs differed. Second, the case scenarios by the various organizations assessed children in different settings , which resulted in different breathings patterns, e.g. the children assessed at home were unlikely to have fast breathing, although a few of them happened to have it. This review suggests that these factors influence the utility of beads.Data from IRC, Save the Children and anecdotal evidence from PSI and IRC suggest that ‘age-specific and colour-coded’ beads enable and improve accuracy in the classification of fast breathing by CHWs with limited literacy and numeracy. However, if literacy and numeracy is not an issue, findings from Malaria Consortium show that the use of beads complicates the assessment, as it resulted in more inaccurate counts (i.e. CHWs were 5.6 times less likely to count correctly using the beads and timer). A reason for the breathing count inaccuracy by literate CHWs was that some perceived the use of beads as more task intensive. Although for CHWs with limited literacy and numeracy (e.g. those not able to count beyond 10), the use of beads enabled them to track the breathing rates as well as classifying the count against the age-specific cut-off points, which would not be possible without these beads.It was also found that the rate of breathing influences the accuracy of the RR count. The research conducted by Malaria Consortium shows that, regardless of the tool CHWs used, they tended to overestimate the rate in the slow-breathing scenario, whereas they underestimated the rate in the fast-breathing scenarios.Regarding the use of counting beads, these data show that it is premature to conclude to which degree the beads, in addition to a timer, would improve the ability of trained CHWs to correctly classify fast breathing. A more conclusive assessment is needed amongst sick children, disaggregating data by intensity of training and supervision, levels of literacy, numeracy and comparing the final breath count to a gold standard.Previously conducted studies on pneumonia diagnosis by CHWs indicate that even if CHWs are good in counting, they still often make mistakes in classifying the breath count (Concurrent to the need of more evidence regarding the utility of beads, there is a need for more research assessing the effectiveness of other devices, such as automated RR counters.Given the overall paucity of data, this review of recent studies provides insights on a range of issues to consider when implementing counting beads in iCCM programmes. This emerging evidence suggests that the introduction of well-designed ‘age-specific and colour-coded’ beads in addition to an accurate timer can help CHWs who have difficulty counting breaths and remembering age-specific cut-off rates to more accurately assess and classify fast breathing. It also has the potential to improve communication with the child’s caretakers—particularly regarding appropriate treatment options. However, more research is needed on these and other devices to decrease the inaccuracy in pneumonia diagnosis.


Background: The current disease outbreak caused by the Ebola virus Makona variant (EBOV/Mak) has led to unprecedented morbidity and lethality given its geographic reach and sustained transmission. Sodium hypochlorite and ethanol are well-accepted decontamination agents, however little published evidence supports the selection of appropriate concentrations and contact times. The present study addresses the environmental robustness of EBOV/Mak and evaluates the effectiveness of sodium hypochlorite and ethanol as disinfectants. Methods: EBOV/Mak was suspended in a simulated organic soil load and dried onto surfaces. Viability was measured at 1 hour, 24 hours, 72 hours, and 192 hours. For the evaluation of disinfectants, EBOV/Mak in a simulated organic soil was dried onto stainless steel carriers and disinfected with 0.01% (v/v), 0.1% (v/v), 0.5% (v/v) and 1% (v/v) sodium hypochlorite solutions or 67% (v/v) ethanol at contact times of 1, 5 or 10 minutes. Results: EBOV/Mak persisted longer on steel and plastic surfaces (192 hours) than cotton (<24 hours). Dilute sodium hypochlorite (0.01% and 0.1%) showed little antiviral action, whereas 0.5% and 1% sodium hypochlorite solutions demonstrated recoverable virus at one minute but sterilized surfaces in five minutes. Disinfection with 67% ethanol did not fully clear infectious virions from 3/9 carriers at 1 minute but sterilized all carriers at 5 and 10 minutes. Conclusions: Sodium hypochlorite and ethanol effectively decontaminate EBOV/Mak suspended in a simulated organic load; however, selection of concentration and contact time proves critical. Zaire ebolavirus and family Filoviridae, has caused episodic disease outbreaks with high lethality in Africa since 1976. The current Ebola virus Makona variant (EBOV/Mak) epidemic originated from Guéckédou, Guinea in early December 2013 and extensive human-to-human transmission has caused the virus to spread across six African nations. Additionally, Ebola virus disease (EVD) patients have been medically evacuated to Europe and the United States, nosocomial transmission has occurred in both Spain and the United Sates [Ebola virus (EBOV), species The field of filovirus decontamination contains limited published literature. The role of gamma irradiation and ultraviolet exposure has been relatively well studied ,3,4. TheIt is not yet known how porous surfaces impact EBOV persistence. The extended survival of EBOV suspended in serum suggests that enveloped viruses may persist better in organic solutions with high protein concentrations than protein deficient ones ,7. PatieThe issue of EBOV disinfection also requires additional examination, given its importance to outbreak management and relationship to environmental persistence. If an organic soil load was demonstrated to favor the persistence of EBOV/Mak in the tested environments then it could also affect the virus’s susceptibility to chemical inactivation. Governing bodies recommend disinfection of EBOV-contaminated surfaces with a 0.5% sodium hypochlorite solution ; howeverOverall, our study aims to elucidate the environmental persistence of EBOV/Mak in an organic soil load on clinically relevant surfaces and outline the effectiveness of sodium hypochlorite and ethanol at inactivating EBOV.2. Virus infection medium (2% FBS and 1% antibiotics in DMEM) was then added and the flasks were incubated. Once 70%–80% CPE developed at approximately 9 days, EBOV/Mak was harvested by centrifugation at 5000 × g for 10 minutes and aliquots were stored in liquid nitrogen as a passage one (P1) stock.Serum isolated from a sixteen-year-old female patient (C05) from Guéckédou, Guinea diagnosed with EVD caused by EBOV/Mak  was blind passaged in the Containment Level 4 (CL-4) laboratory , Winnipeg, MB, Canada). Vero E6 cells provided by the Centers for Disease Control and Prevention  were maintained in Dulbecco’s Modified Essential Medium (DMEM)  supplemented with Fetal Bovine serum (FBS)  and 1% penicillin and streptomycin (Gibco). One day prior to infection, Vero E6 cells were sub-cultured to 80%–90% confluence in a T150 flask , 24 hours prior to infection. The cells were infected with serum from patient C05 and incubated for 1 hour at 37 °C/5% CO2. When 70%–80% CPE developed, the flasks were frozen overnight at −70 °C. After thawing, cellular debris was removed by centrifugation at 5000 × g for 10 minutes followed by additional centrifugation  on a 20% sucrose cushion for two hours. Concentrated virus was re-suspended in virus infection medium, stored and titrated. EBOV/Mak from two separate P2 stocks was used for these experiments (P2A and P2B), as indicated in the results section.A P1 stock was removed from liquid nitrogen and used to infect, 9-T150 flasks (MOI = 0.05) of 80%–90% confluent Vero E6 cells. After absorption for 1 hour, virus infection medium was added and the flasks were re-incubated at 37 °C/5% CO2 carriers (fabricated at CSCHAH). The carriers were sterilized by submersion in 70% ethanol and heat-treatment at 160 °C for 2 hours. Hospital-grade PPE, including: surgical mask , cotton gown  and waterproof plastic gown  were prepared as 0.5 cm2 carriers and sterilized with 1 MRAD of gamma irradiation. EBOV/Mak was inoculated into a simulated organic soil load following the ASTM International  Quantitative Carrier Testing 2 international standard [50 assay. Three technical repetitions were performed for each surface at every time point (n = 3).Environmental Surfaces: lightly-scratched, stainless steel disks were prepared as 0.5 cmstandard ,12. Specstandard . Ten mic2. Following absorption, 150 µL of viral infection medium was added to each well for a final volume of 200 µL. The plates were incubated for 14 days post-infection, scored for CPE and TCID50/mL was calculated by the Reed and Muench method [Vero E6 cells were grown overnight to 80%–90% confluence in 96-well plates. Samples were thawed and 10-fold serially diluted, each dilution was added in replicates of 5 to cell monolayers for 1 hour at 37 °C/5% COh method .50.Sodium hypochlorite: On the day of each assay, fresh bleach  containing 10.8% sodium hypochlorite and 10.3% available chlorine was diluted to final concentrations of 1%, 0.5%, 0.1%, and 0.01% v/v sodium hypochlorite in hard water . Concurrently, a 1% v/v solution of sodium thiosulfate in DMEM was made to neutralize the activity of sodium hypochlorite at an established contact time. EBOV/Mak-soil load mixtures were prepared and deposited on steel carriers, as described above. Fifty µL of each sodium hypochlorite concentration was added to the carriers and neutralized with 950 µL of 1% sodium thiosulfate at . Time point 0 served as a positive control where 1000 µL of 1% sodium thiosulfate was added to the dry carriers. Five hundred microliters of each sample was removed for titration by TCID0). This made the 10−1 dilution the limit of virus detection by TCID50 assay, as we could not determine whether the observed CPE at the 100 was due to chemical damage or viral infection. To confirm an absolute kill at the 5 and 10-minute contact times for the 0.5% and 1% sodium hypochlorite samples the remaining 500 µL of each 100 dilution was pooled in replicates of three. These three 1.5 mL solutions were used to infect T150 flasks of Vero E6 cells, as the extensive dilution overcomes chemical toxicity. These flasks were incubated for 14 days and observed for CPE. No viral infection in these flasks would confirm absolute kill of EBOV/Mak.Previous use of 1% sodium hypochlorite and 1% sodium thiosulfate demonstrated cytotoxicity to Vero E6 cells when undiluted (1050 assay possessed a full range of detection (100–108). For the tested disinfectants three biological replicates of 3 technical repetitions were performed at every time point (n = 9).Ethanol: Fresh solutions of 67% ethanol were prepared in hard water . EBOV/Mak-soil load mixtures were deposited on steel carriers and dried, as described earlier. To evaluate disinfection, 50 µL of 67% ethanol was added to the carriers and neutralized with 950 µL of virus infection medium. As a control, no ethanol was added to time point 0; instead the carriers were washed with 1000 µL of virus infection medium. After gentle pipetting, 500 µL was removed for titrations. As neutralized ethanol caused no cytotoxicity the TCID50 calculations, averaging and determination of standard deviations were performed in Microsoft Excel 2011. GraphPad (Prism 5) software was used to generate figures and calculate the standard error of the mean, which was used to make positive and negative error bars. Statistical analysis by the students T-test and analysis of variance (ANOVA) were performed using the SPSS statistical software suite .All TCID10 reduction in viral titer.Non-linear regression analysis of EBOV/Mak persistence data was performed using the one phase decay equation of GraphPad (Prism 5). The accuracy of the models’ was determined by the R2 value and replicates test. The one phase decay equation (50 units/mL (SD ± 0.34). The solid surfaces of the surgical mask, plastic gown and steel carriers allowed for prolonged viral survival. There was no significant difference (p = 0.058) in the viral titer at 192 hours between the surgical mask (2.9 log10 TCID50/mL) and plastic gown (3.6 log10 TCID50/mL). The virus persisted better on stainless steel carriers (4.0 log10 TCID50/mL) than on both plastic surfaces  over the same time period. On the rough textile surface of a cotton gown a 47% reduction in viral titer occurred after one hour of exposure, followed by complete inactivation at 24 hours. Non-permeable surfaces enabled substantially longer EBOV/Mak persistence than a permeable surface such as cotton. While there was relatively rapid inactivation of the virus on cotton, it is important to note that sufficient virus to cause infection was recoverable at the one-hour mark, the estimated infectious dose ranges between 1 and 10 virions [2 value indicating closeness of fit. To additionally, examine the accuracy of the models a replicates test was performed, all of the models passed this test with the exception of stainless steel.EBOV/Mak suspended in an organic soil load showed environmental persistence on common hospital surfaces.  virions . Since t10 (SD +/− 0.34) across experimental replicates using the P2A stock; namely the 0.01%, 0.1% and 1% experiments. The 0.5% experiment was conducted with the P2B virus stock, with a lower input load (6.6 SD +/− 0.3 TCID50 units/mL). At the lowest tested sodium hypochlorite concentration (0.01%) no significant reduction in viral titer was observed (p = 0.079). A 0.1% sodium hypochlorite solution proved to be partially effective against the virus at a 10-minute contact time but 3.7 log10 (SD +/− 2.0) viable virus particles remained. The 0.5% and 1% sodium hypochlorite solutions performed similarly. At both concentrations, viable virus was recoverable at one minute , while all stainless steel carriers were sterile following a five-minute contact time. The higher viral load observed at the 1 minute mark with the 1% concentration of sodium hypochlorite compared to the 0.5% solution is likely a reflection of lower input virus on the steel carriers used for the 0.5% experiments, as the average reduction (2.2 log10 TCID50 units/mL) was the same for both concentrations. Infection of T150 flasks of Vero E6 with pooled 100 dilutions (n = 9) of the 0.5% and 1% sodium hypochlorite samples confirmed absolute kill. The efficiency of EBOV/Mak disinfection positively correlated to the concentration of sodium hypochlorite and length of contact times. Within the test conditions, higher concentrations of sodium hypochlorite at longer contact times achieved greater disinfection.The ability of  concentrations of sodium hypochlorite to inactivate suspended EBOV/Mak on steel carriers was tested at three contact times . InitialWe tested the effectiveness of 67% ethanol, a common ingredient of gel-based hand sanitizers and a clinical disinfectant, at inactivating EBOV/Mak in a simulated organic soil load on steel carriers. The unprecedented scale of the 2014 EVD outbreak has global implications with the international movement of patients and the return of infected health care workers to their home nations from Western Africa [EBOV/Mak in an organic soil load survived for lengthy periods of time on the examined surfaces. This finding agrees with previously reported EBOV environmental persistence data ,7 suggesDuring previous EVD outbreaks, health care professionals applied sodium hypochlorite to successfully decontaminate clinical instruments, laboratory materials and personal protective equipment . The WorIn some clinical situations ethanol diluted in water or as an ingredient of hand sanitizer is preferred to sodium hypochlorite as a disinfectant, as it does not cause corrosive damage to sensitive equipment and skin . There ii.e., Micro-Chem Plus). The Makona variant shows 97% nucleotide similarity to other EBOV variants, including Kikwit and Yambuku [i.e., high viral titer and an organic soil load) are stringent but clinically appropriate, thus providing confidence in the applicability and safety of conclusions.Limitations of the present study include a fairly wide choice of contact times . We conducted the experiments under the chosen conditions in order to generate a range of data and are currently working to identify ideal contact times for disinfection of several EBOV variants with a variety of chemical disinfectants  on the surface of common healthcare equipment. This makes rigorous decontamination procedures vital to outbreak management. The use of sodium hypochlorite as a primary disinfectant appears generally effective; however, the concentration of sodium hypochlorite and contact time heavily influence antiviral action. A similar trend was observed in the challenge of EBOV/Mak with 67% ethanol as viral titer decreased over the duration of contact time. Health care workers should take precaution to not leave EBOV/Mak contaminated surfaces unmanaged and when faced with decontaminating PPE or other equipment should consider an appropriate selection of disinfectant as well as contact time. This study of EBOV/Mak suspended in an organic soil load concludes that a 0.5% or 1% solution of sodium hypochlorite and 67% ethanol completely sterilized EBOV/Mak contaminated stainless steel surfaces following five minutes of contact. Overall, these findings suggest that the environmental persistence of infectious EBOV/Mak on clinical surfaces is a substantial risk, even after several days, and that choice of disinfectant concentration and contact time are important factors in remediating contaminated areas.


The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design. Antibodies that potently neutralize a broad range of circulating HIV strains have been described. These antibodies target a variety of sites on the envelope protein of HIV, three copies of which associate to form a trimer that decorate the membrane surface of the virus particle. Some of these antibodies target regions of the envelope protein close to the membrane, some bind to the top of the trimer, others bind via carbohydrates which cover the envelope protein and another subset binds to the same site as the human HIV receptor CD4. Despite effectively blocking 50% of infection at low antibody concentrations, for some particular virus/antibody combinations a proportion of virus particles are resistant to antibody neutralization, even at extremely high concentrations. This phenomenon is called incomplete neutralization and also frequently results in non-sigmoidal dose-response curves when antibody concentration is plotted against the level of virus infection. Previously, antibodies that target the apex of the trimer have been associated with incomplete neutralization and non-sigmoidal curves. In this study we show that representatives from all the groups of antibodies described above result in incomplete neutralization against at least one virus but that the phenomenon is more frequent for those binding the apex and the stalk of the trimer. Resistant populations of virus were seen whether the virus was produced in the natural target of HIV infection (human CD4+ T cells) or engineered human cells more commonly used to produce virus to test antibody function. Understanding this phenomenon is important for the future use of antibodies as therapeutics and for vaccine studies as a resistant population of viruses could result in failure to control the virus infection in patients. The gp120 protein includes about 25 N-linked glycans that comprise almost 50% of its mass [The HIV-1 envelope glycoprotein (Env) spike, the sole target of neutralizing antibodies (nAbs), is a heterotrimer of composition gp120)03(gp41)3its mass . Three rits mass ,8. One rits mass –12. The We have previously shown that certain bnMAbs display non-sigmoidal neutralization curves that plateau at <100% for some isolates, ,14. In t5GlcNAc2 at N160 and high mannose-type or complex-type N-linked glycans at the secondary site (N156 or N173) [PG9 and PG16 ,23–25, Por N173) . Furtheror N173) . In addior N173) .8/9GlcNAc2 glycan at residue N332 but complex or hybrid glycans at N137, N156 and N301 are also involved [The third region of gp120 targeted by bnMAbs is a set of overlapping epitopes that all appear to involve the glycan at N332 as a significant contributor to antibody binding. Some of the most potent bnMAbs to HIV, PGT121-123, PGT125-7, PGT128, and 10–1074 are amongst those targeting this region as well as 2G12, PGT130-131 and PGT135-137. 2G12 binds the terminal mannose of glycans at N295, N332, N339, and N392. PGT121-123 bind gp120 V1, V3 and several surrounding glycans [involved . PGT128 BnMAbs 4E10, 2F5, Z13e1 and 10E8 recognize the MPER on gp41 –36. WhilSeveral bnMAbs have been particularly associated with incomplete neutralization. Notably, the PGT150 series bnMAbs do not always achieve 100% neutralization even when able to potently neutralize the virus under investigation with an IC50 value of less than 1μg/ml. Maximum neutralization ranges from 50–100% with PGT151 and PGT152 achieving less than 80% neutralization against 26% and 20% of 117 viruses tested. Similarly, PG16, and to a lesser degree PG9, have also been shown earlier to display neutralization curves, for some isolates, that plateau at <100% and are non-sigmoidal ,14. Incoin vivo.Incomplete neutralization may have serious consequences for the ability of nAbs to protect against HIV exposure. If transmitted viruses are heterogeneous with respect to neutralization sensitivity, as observed for pseudoviruses and primary viruses grown in PBMCs, then the failure of an antibody to neutralize a population of viruses completely could lead to the establishment of an infection. However, it should be noted that incomplete neutralization has not been reported for serum samples from elite neutralizer donors. In the current study, we were interested in investigating the extent of non-sigmoidal slopes that can plateau at <100% for HIV bnMAbs targeting the four best-studied regions of the Env spike involved in broad neutralization. We found all bnMAbs to be subject to inhibition curves that do not reach 100%, and most bnMAbs also had neutralization curves with non-sigmoidal slopes but bnMAbs targeting certain areas of the trimer were more prone to these effects. Primary isolate and molecular cloned viruses produced in primary cells were also incompletely neutralized, suggesting this phenomenon could be significant 50 >1μg/ml were not further considered because we could not exclude the possibility that at/above this concentration the IC50 would be too high for 100% neutralization to be reached by a typical sigmoidal curve at the highest antibody concentration measured (50 μg/ml). For each bnMAb, we determined the maximum percent neutralization (MPN), i.e. the percent at which the neutralization curve plateaus for those viruses neutralized with an IC50 <1μg/ml. For the panel of bnMAbs tested, b12, 2G12, PGT136 and PGT137 had a relatively low number of viruses neutralized with an IC50 <1μg/ml  have been characterized with a TZM-bl cell based assay using a different 117-pseudovirus panel. Therefore, we determined the MPN of bnMAbs using this system. In Together with the high-mannose patch bnAbs, the CD4bs antibodies showed a very high tendency to complete neutralization against the 278-virus panel in the U87 cell assay. In line with this, the CD4bs antibody 12A12 resulted in a MPN values of >90% of all but three viruses in the TZM-bl assay , with a The V2 apex bnMAbs were shown in the U87 cell system to have a greater propensity to result in incomplete neutralization than the CD4bs or high-mannose patch binding bnMAbs. Similarly, in the TZM-bl assay PG9 and PGT143-144 resulted in a greater spread of MPN values than PGT121, 123, 125, 127, 128 and 12A12, with many viruses showing between 90–95% neutralization and a number that were neutralized to only between 75–90% . HoweverFinally, the two related specificities PGT151 and PGT152, which target a newly described cleavage-dependent gp120/gp41 glycan epitope , showed Although the U87 and TZM-bl studies reported here use different target cells and different virus panels, the trends observed for the bnMAbs tested in both assays are similar  . In the To extend these findings, the neutralization profiles of bnMAbs were evaluated with a large panel of pseudoviruses in the TZM-bl assay . Again, 8/9GlcNAc2 glycans, which suggests that the glycan at N332 may be expressed as relatively homogenous Man8GlcNAc2 and/or Man9GlcNAc2 in pseudoviruses [8GlcNAc2. This homogeneity may be a significant contributor to the ability of bnMAbs that are dependent on the glycan on N332 to bind a high proportion of the envelopes of an isolate effectively and therefore neutralize the vast majority of virions. Furthermore, some bnMAbs recognizing this region can tolerate some heterogeneity in glycan usage [The mechanism(s) of incomplete neutralization and non-sigmoidal neutralization curves were beyond the scope of the present investigation but an earlier study on the bnMAbs PG9 and PG16 showed the importance of glycosylation heterogeneity for these features , and thioviruses . Similaran usage  and/or uan usage  that mayan usage ,21 and han usage ,48.in vivo. As a first step, we investigated the effects on primary viruses in vitro by study of the incidence of incomplete neutralization in a large panel of molecular cloned viruses generated from patient swarms from the Amsterdam Cohort Studies on HIV Infection and AIDS. These clones were propagated and assayed in PBMC cultures, which increases the level of variability inherent in the assay and decreases the confidence with which we can define complete neutralization. Furthermore, Abs were titrated from a lower concentration than in the other assay systems which means, although a 1μg/ml cut-off was used as previously described, some Abs may not have reached saturating concentrations in this assay. However, among a large primary virus panel and the five bnMAbs tested there were clear examples of both incomplete neutralization (<90%) and complete neutralization (100%)  . Thus, wn (100%)  on an inAs described, a key question for further study is how incomplete neutralization impacts the ability of bnMAbs to mediate protective effects. Protection has been clearly demonstrated in macaques via passive transfer of many bnMAbs when the challenge virus was completely neutralized –55 and aHuman blood samples from healthy donors were obtained form The Normal Blood Donor service at The Scripps Research Institute. The collection of human blood samples for isolation of PBMCs and subsequent propagation of HIV-1 was approved by the institutional Review Board at The Scripps Research Institute (protocol number HSC-06-4604), all samples were analyzed anonymously. The Amsterdam Cohort Studies on HIV infection and AIDS (ACS) are conducted in accordance with the ethical principles set out in the declaration of Helsinki, and written informed consent was obtained prior to data collection. The study was approved by the Academic Medical Center’s Institutional Medical Ethics Committee.2G12, 4E10 and 2F5  were procured by the IAVI Neutralizing Antibody Consortium. The recombinant bnMAb IgG1 b12 was expressed by the Center for Antibody Development and Production  in Chinese hamster ovary (CHO-K1) cells, purified using affinity chromatography  and the purity and integrity was checked by SDS-PAGE. PGT 121–123, PGT 125–131, PGT 135–137, PGT 141–145, PGV04, PDGM1400, PGT151-153 and PG9/16, were transiently expressed with the FreeStyle 293 Expression System (Invitrogen). Antibodies were purified using affinity chromatography  and the purity and integrity was checked by SDS–PAGE.+-depleted PBMCS [Monogram Biosciences performed neutralization assays using pseudovirus that undergoes a single round of replication as previously described . Brieflyed PBMCS . Virus ped PBMCS .Statistical analyses were done with Prism 5.0c for Mac (GraphPad).S1 TextFig J displays individual MPN values for all bnMAbs and viruses from the 278-virus panel (A) and the 117-virus panel (B) for which the IC50 value <1μg/ml arrayed according to virus strain along the X-axis. Fig M shows MPN values for 18 molecular clone viruses produced in both 293T cells and PBMCs linked by black bars, the bnMAbs tested are indicated above the graph.(XLSX)Click here for additional data file.


Model dynamics of important aspects in disease development within TSWV-F. occidentalis-host plant interactions were developed, focusing on F. occidentalis’ life processes as influenced by TSWV. The results show that the influence of TSWV on F. occidentalis preferential behaviour leads to an estimated increase in relative acquisition rate of the virus, and up to 33% increase in transmission rate to healthy plants. Also, increased life expectancy; which relates to improved fitness, is dependent on the virus induced preferential behaviour, consequently promoting multiplication and spread of the virus in a crop stand. The development of vector–based models could further help in elucidating the role of tri-trophic interactions in agricultural disease systems. Use of the model to examine the components of the disease process could also boost our understanding on how specific epidemiological characteristics interact to cause diseases in crops. With this level of understanding we can efficiently develop more precise control strategies for the virus and the vector.Several models have been studied on predictive epidemics of arthropod vectored plant viruses in an attempt to bring understanding to the complex but specific relationship between the three cornered pathosystem , as well as their interactions with the environment. A large body of studies mainly focuses on weather based models as management tool for monitoring pests and diseases, with very few incorporating the contribution of vector’s life processes in the disease dynamics, which is an essential aspect when mitigating virus incidences in a crop stand. In this study, we hypothesized that the multiplication and spread of  Tomato spotted wilt virus (TSWV) belongs to the genus Tospovirus, the only plant infecting genus in the family Bunyaviridae [Frankliniella occidentalis (Western flower thrips) being the most efficient vector due to its equally large host range [Tospoviruses and their complex virus–vector interaction were ranked second in a recent survey by plant virologists based on the perceived economic and scientific importance [aviridae 2]. TSWV. TSWVTomst range 3]4]. T[4]. TTom. T[4]. Tportance .F. occidentalis occurs in a propagative and persistent manner [Transmission of TSWV by t manner 6]. Only. OnlyF. t manner 8]9][10[9][10F. [10[9]. An . An F. ot manner 14][3][1[3][1F. ot manner . TSWV int manner [14]15][[F. occidt manner 17]18].[18].F. o.[18].F. t manner 20]21].[21].F. o.[21].F. Tospovirus-vector-host plant interaction. Mathematical models can therefore help to quantify the virus propagation dynamics in a crop stand as influenced by virus induced vector life’s processes.Virus replication in the tissues of its thrips host suggests the likelihood for pathological effects that could affect the fitness and performance of the vector. However, consistent demonstration of such effects has proven difficult due to the complex virus-vector-host plant interaction . MoreoveF. occidentalis being great threats to the global economy, very little effort has been made to come up with a wholesome model specifically forecasting TSWV epidemiology considering the complex and mutualistic interactions in the triangle virus-vector-host plant. According to a review paper by Jones et al . The. TheF. oes et al 34], wit, witF. oes et al 36], vec, vecF. oes et al , vector es et al  and vectes et al . Initiales et al . Most rees et al  publisheF. occidentalis-TSWV) incorporating virus induced vector life processes influencing the disease epidemic development, would provide better understanding into the often aggressive spread of TSWV in crop stands, and also allow more precise forecasting for efficient management strategies for both F. occidentalis and TSWV.We therefore presume that the application of a more specific vector–centred model  the relevant pathogens have been reported 43]44][[44][43][[[44][43]ng virus 42]. The. TheF. ong virus 24]20].[20].F. o.[20].F. ng virus 51]. How. HowF. ong virus [3]11]. . F. occing virus 52]9][2[9][2F. o[2[9][2F.ng virus 53]54].[54].F. o.[54].F. Tospovirus-thrips interaction, focusing on the influences of the TSWV on F. occidentalis biology in terms of life expectancy  and preferential behaviour.This paper aims to provide a specific analysis of TSWV disease dynamics in a model framework consisting of specific transmission characteristics in the F. occidentalis developmental as well as behavioural aspects as influenced by TSWV 41][[41][40][[[41][40] by TSWV 42]..F. occidF. occidentalis’ preferential behaviour patterns, development time, survival, as well as the key aspects of the vector lifecycle such as developmental stages, forms the basis of the hereby reported model. We consider two scenarios for the host plant; TSWV presence  or absence . The basis of the assumptions is that TSWV presence influences the transmission intensity of F. occidentalis by its influence on adults’ preferential behaviour and survival, which further affects the disease incidence in a crop stand.The influence of TSWV on F. occidentalis adults status can be described as Healthy (AN); if it is not exposed to TSWV infected plant by feeding, Infected (AI); if the vector fed on infected host plant but at a less crucial stage for acquisition in view of transmission, that is the L2 (L2N) and/or the adult stage (AN), or Transmitter (AT); if the vector fed on infected host plants at the Larvae 1 (L1) stage which is essential for later transmission by adults. AT is the only group of adults that are able to transmit the virus.F. occidentalis classes and the interactions between them, we chose to use a deterministic model consisting of differential equation systems.Due to the high numbers of The model describing the disease and the vector dynamics was developed based on the following assumptions;Virus transmission to host plants is dependent on the presence of transmitter vectors and healthy host plant.Acquisition of the virus by the vector is by feeding on an infected host plant.The feeding stages of the vector are L1, L2 and adult; pre-pupa and pupa stages do not feed.Only L1 that emerge on infected host plants and acquire the virus can become transmitters as adults.L2 and adults are able to acquire the virus and become infected but not transmitters.Larval stages (L1 and L2) are mobile but restricted to the plant where they hatched from eggs due to lack of wings.Pre-pupae and pupae stages are immobile, do not feed and spend their entire time in the growth media i.e. not on the plant.The virus replicates inside the vector and is transmitted transstadially.Adults are the only stage that can move from plant to plant, therefore, essential for the virus transmission and thus spread.Transovarial transmission of the virus is not possible.Exposure of the vector to the virus leads to improved fitness, only when feeding on healthy host plant after the exposure.Preferential behaviour of the adults to the host plant is influenced by their status i.e. viruliferous adults (infected or transmitters) prefer healthy host plants, while the virus-free  prefer infected host plants.F. occidentalis . Dev. DevF. os adults . TherefoF. occidentalis is given below  and on healthy (-TSWV) host plants, in terms of TSWV influences on their life expectancy as well as preferential behaviour. There are three groups of adults as a result of the exposure status; Healthy, Transmitters and Infected adults, already described above.A flowchart illustration of selected influences of TSWV on en below , indicatF. occidentalis and TSWV . The. The24].The adults’ population dynamics is given as follows:F. occidentalis adults is modelled as;pNH) is the preference towards infected plants, and ßA is the contact rate between the insect and infected plant.Virus acquisition by healthy Ventana Systems, Inc., Harvard, USA) with a Runge-Kutta 4 scheme. Different scenarios were compared to evaluate the impact of TSWV influence on preferential behaviour and life expectancy of F. occidentalis, on the propagation and spread dynamics of the virus in a plant population. The maximum evaluation time was set at 100 days (after TSWV introduction in a crop stand) and the step sizes were fixed to minimum (0.0078125 days) to avoid any stiffness of the scheme.Numerical simulations of the model were performed using the modeling software Vensim PLE  prefer TSWV infected host plants; while the TSWV exposed ones significantly prefer healthy host plants.The considered dynamics for the interaction were based on reports that exposure of F. occidentalis relative acquisition rate and virus transmission rate to healthy host plants were modelled in the presence and absence of virus induced preferential behaviours of the vector. The results of the relative acquisition rate show that the preferential behaviour effect was beneficial above the threshold, and only up to the first 30 days after TSWV introduction into a crop stand. Thereafter, the influence of preference on acquisition is limited  to healthy plants may increase the transmission rate by up to 33.3%. The consequence can lead to up to 30% decrease in the fraction of healthy plants . On the f spread .F. occidentalis’ life cycle considered; in terms of vector abundance, for the analysis of the influence of TSWV induced preferential behaviour in the first 100 days of the virus introduction in a crop population. This was based on our listed assumptions. In the absence of preference, the population of the transmitter adults shows exponential rise compared to the healthy and the infected adults  of different vector cohorts in the first 100 days after TSWV introduction in a crop population shows a positive virus effect on the dynamics of transmitter adults’ population with a steady rise over time observed above the threshold. For the infected adults’ population, there is also a positive effect of the virus, however the rise is minimal, only slightly above the threshold, and the effects are limited to 60 days after virus introduction . For theF. occidentalis 57],[57],F. o,[57],F. ectively 58]59][[59][F. o[[59][F. ectively 61]62],[62],F. o,[62],F.  (TYLCV) . Several (TYLCV) 23]24].[24].F. o.[24].F.  (TYLCV) .F. occidentalis in response to TSWV also showed some influences on the disease spread dynamics. In the relative acquisition and the transmission rates of the vector, the virus effects on life expectancy are obviously positive primarily in the early stages of the disease development, but thereafter tending towards negative. This relates back to the TSWV induced preferential behaviour [F. occidentalis exposed to TSWV has been associated with triggered immunity [The observed increase in life expectancy of ehaviour 42], and, andF. oehaviour [42]. Forimmunity , as the immunity . This eximmunity .Tospovirus-vector-hostplant interaction like; vector gender, individual vector genetics, among others.A phenomenon termed “Vector Manipulation Hypothesis” by Ingwell et al. , was theF. occidentalis-host plant interaction, with focus on virus induced and host plant-mediated effects on the vector’s life processes, is a new contribution to the currency of the predictive models. A thorough understanding of the vector’s life processes as influenced by the virus, and their contribution to the virus-vector-hostplant interaction, is critical for a more precise prediction of TSWV epidemics.In conclusion, the dynamics of the complex and specific TSWV-


Camels carry Middle East respiratory syndrome coronavirus, but little is known about infection age or prevalence. We studied >800 dromedaries of all ages and 15 mother–calf pairs. This syndrome constitutes an acute, epidemic, and time-limited infection in camels <4 years of age, particularly calves. Delayed social separation of calves might reduce human infection risk. Camelus dromedarius) across the Arabian Peninsula and parts of eastern and northern Africa have MERS-CoV antibodies (Middle East respiratory syndrome coronavirus (MERS-CoV) causes outbreaks and isolated cases of severe respiratory disease in humans. The virus is transmissible from human to human, but the focus of infection has remained in countries on the Arabian Peninsula. Recent reports have shown that dromedaries . The higher rate of virus isolation among calves suggests increased infectivity of calves.We investigated dairy, racing, and breeding dromedaries from 3 flocks on farms 20–40 km apart. When possible, blood and nasal swab specimens were obtained from all camels in the flocks during March–June 2014. Samples were grouped according to the camels’ ages rather than sampling site because livestock ages differed between sites. Serologic testing by ELISA yielded evidence of MERS-CoV antibodies in >96% of all dromedaries >2 years of age . SeropreTo understand MERS-CoV infection in dromedary calves, we investigated 24 mother–calf pairs from the breeding flock. The investigations were all conducted in May 2014. At the time of sampling, mother camels were >12–15 years of age, and calves were 4–6 months of age. As shown in We sequenced genomes of 9 virus isolates, representing 3 different phylogenetic lineages, from dromedaries on the 3 farms. Phylogeny of full genomes showed that all viruses clustered according to their place of origin. The phylogenetic position of 1 of these clades suggested recent separation from viruses circulating in the eastern part of Saudi Arabia; some of the animals in the breeding flock from which the viruses were isolated had been moved temporarily to Saudi Arabia for grazing. The other clade separated from these viruses somewhat earlier, but it shared recent common ancestors with other viruses from the eastern part of the Arabian Peninsula. Samples collected in June from animals on the dairy farm yielded viruses from the same clade as that for viruses derived from different animals sampled on the same farm in March (dairy farm samples I and II) .,Our findings provide evidence of infection of camel flocks in Dubai with MERS-CoV of contiguous virus clade. Similar to findings from earlier studies, we found evidence of new introductions of virus in flocks, such as the flock that temporally grazed in Saudi Arabia and was infected with a virus strain typical for Saudi Arabia  but no RNA in samples tested 8 days later. An infection experiment in adult dromedaries showed shedding occurred for The restricted and highly compartmentalized social structure of livestock camels would provide population niches in which viruses can differentiate in isolation after bottleneck-type transmission events. This situation holds promise for control of the spread of MERS-CoV through flock management practices, and it also suggests a rather simple way of avoiding camel-to-human transmission by avoiding camels <2 years of age. Camel calves are not easily accessible by humans and instinctually avoid humans. They are generally separated from their mothers after 12 months of age . Humans normally come into contact with calves only after the animals have been separated from their mothers. A change in this practice  might reduce the risk for camel-to-human MERS-CoV transmission.


Rymovirus. The major natural hosts for RgMV are members of the Gramineae species, including ryegrass. Here, we report the nearly complete genome sequence of RgMV by de novo assembly using ryegrass transcriptomes.The ryegrass mosaic virus (RgMV) is a single positive-strand RNA virus belonging to the genus  Rymovirus in the family Potyviridae, is a filamentous virus replicating in the host cytoplasm  in the family Poaceae is cultivated worldwide and is an important naturalized plant utilized as pasture and forage crops in agriculture  (Ryegrass mosaic virus (RgMV), a member of the genus ytoplasm , 2. Its lomerata , 4. Of ticulture , 6. Infep yields , 8. RgMVhystrix) . RgMV haLolium species have been screened. Of these species, a recent study provided a reference transcriptome for the perennial ryegrass, which was derived from six different tissues, including the stem, root, meristem, mature leaf, inflorescence, and leaf sheath (de novo transcriptome assembly was performed for each library using the Trinity program (v.2.0.2)  . A BLASTKR061300).The genome sequence of ryegrass mosaic virus isolate Denmark has been submitted to GenBank (accession no.


Aedes aegypti mosquitoes are responsible for transmitting many medically important viruses such as those that cause Zika and dengue. The inoculation of viruses into mosquito bite sites is an important and common stage of all mosquito-borne virus infections. We show, using Semliki Forest virus and Bunyamwera virus, that these viruses use this inflammatory niche to aid their replication and dissemination in vivo. Mosquito bites were characterized by an edema that retained virus at the inoculation site and an inflammatory influx of neutrophils that coordinated a localized innate immune program that inadvertently facilitated virus infection by encouraging the entry and infection of virus-permissive myeloid cells. Neutrophil depletion and therapeutic blockade of inflammasome activity suppressed inflammation and abrogated the ability of the bite to promote infection. This study identifies facets of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of virus infection.  •Mosquito bites enhance virus replication and dissemination and increase host mortality•Neutrophil-driven inflammation retains virus in skin to drive macrophage recruitment•Recruited and resident myeloid cells become infected and replicate virus•Blocking leukocyte recruitment to bite site inhibits viral infection The inoculation of viruses into mosquito bite sites is an important and common stage of arbovirus infections. McKimmie and colleagues show that inflammation at bite sites aids viral replication and dissemination in vivo, resulting in more severe infection. These findings define additional targets for post-exposure prophylactic intervention. Aedes mosquitoes, and in particular A. aegypti, are the primary vectors. Arboviruses are an exceptionally large and diverse group of viruses  infect hundreds of millions of people each year . This in viruses . This heAll mosquito-borne viruses share a common attribute: their site of inoculation at mosquito bite sites. This aspect of their life cycle might provide a novel target for preventing diseases spread by this vector. In susceptible vertebrates, arbovirus replication in tissues results in a transient but very high level of infectious virus in the blood that is sufficient for a feeding arthropod to become infected. The high-level viraemia often induces a debilitating febrile illness and can result in the spread of virus to other tissues such as the brain, joints, and muscle. The early events of arbovirus infection are important for survival of the host, with a close relationship between early peripheral virus burden and mortality . HoweverDue to the importance of type I interferons (IFNs) and T cell responses in anti-viral immunity, their functional perturbation by mosquito saliva during infection has often been hypothesized . HoweverAedes mosquito cell-produced virus for inoculations to ensure that the first round of infection resembled the mosquito-transmitted virus as closely as possible. Unlike many human pathogens such as dengue and chikungunya viruses, SFV replicates and disseminates efficiently within both immune-competent mice and aedine mosquitoes (We developed an in vivo model system that makes use Semliki Forest virus (SFV), a mosquito-borne alphavirus that is a close relative of the chikungunya virus . We usedsquitoes . SFV4 issquitoes . After ssquitoes A–S1D. A squitoes A–S1C. Tisquitoes B–S1D. ThA. aegypti mosquitoes and then immediately infected with a defined dose of SFV4 at the bite site in a 1 μL volume  and the cytokines IL-1β and IL-6, which were not significantly induced by virus infection alone . Genes ahiCD11bhiCD45+ neutrophils , alum, and the TLR2 ligand Pam3CSK4, all of which induced cutaneous CXCL2 and IL-1β expression in our mice A and S4Bch other A. Furthech other B and 5C ch other . Pam3CSKch other D. TogethWe next determined whether it was possible to alter the outcome of infection by modulating host immune responses to bites. Because neutrophil depletion was so effective at suppressing bite enhancement of infection , we firsIl1r1−/− mice demonstrated both a deficiency in neutrophil recruitment to infected bite sites and were not susceptible to bite-mediated enhancement of virus infection  combined with post-exposure prophylaxis at bite sites as an effective strategy for limiting mosquito-borne virus infection, especially for infections transmitted by  outcome . Our fin outcome , dengue  outcome , and bun outcome , struggl outcome , we suggIn conclusion, we highlight an important aspect of host innate immunity at mosquito bite sites. These findings not only define the mosquito bite site as a putative target for post-exposure prophylactic intervention, but also pave the way for the development of in vivo models that better recapitulate an important aspect of mosquito-borne virus infection.Detailed methodology is described in the Aedes mosquito cells and BHK cells were cultured using established protocols . This approach enabled the effect of bites on concurrent virus infection to be quantifiably compared to virus infection alone in the absence of a bite.Anesthetized mice were positioned to expose a defined area of the left foot to a cage of RNA was extracted using PureLink Plus columns and converted to cDNA using the High Capacity RNA-to-cDNA kit (Life Technologies). qPCR analysis was undertaken using SYBR-green PerfeCTa (Quanta) and TLDAs were used as per manufacturer’s instructions on a 7900HT Real time machine (Applied Biosystems). ELISAs were undertaken using Duoset kits (R&D Systems). For flow cytometry, skin was enzymatically digested to release cells and stained using a subset of antibodies. Cells were stained with Fixable Viability Dye eFluor780 (eBioscience), fixed in 4% methanol-free paraformaldehyde (Thermo Scientific) or Cytofix/Cytoperm (BD), and analyzed on a MACSQuant Analyzer 10 (Miltenyi). For cell sorting, cells were labeled with CD11b beads and sorted on magnetized columns (Miltenyi). For immunohistochemistry, skin was fixed before freezing in embedding medium and sectioned. Sections were blocked in Tris-Saline-Tween (TBS)/5% fish gelatin (Sigma-Aldrich) incubated with a primary antibody against Lyve-1 and the secondary chicken anti-goat IgG Alexa Fluor 647-conjugated antibody (Life Technologies).∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns = not significant. Wherever possible, preliminary experiments were performed to determine requirements for sample size, taking into account the available resources and ethical use of animals. Animals (gender and age matched) were assigned randomly to experimental groups. For plaque assays, samples were coded and analyzed blind by a separate investigator. All results shown are representative of either two or three experiments and where possible incorporate a variety of techniques and approaches. Importantly, biological replicates were excluded from analysis if s.c. or i.d. injection of virus inadvertently punctured a blood vessel.Data were analyzed using the non-parametric-based tests Mann-Whitney or Kruskal-Wallis test with Dunn’s multiple comparison. All column plots show the median value ± interquartile range with Conceptualization, C.S.M., J.K.F., and G.J.G.; Methodology, C.S.M., E.S., A.K., J.K.F., and G.J.G.; Investigation, M.P., S.R.B., E.P., E.S., and C.S.M.; Resources, E.S., A.K., E.P., A.M., and G.J.G.; Original draft, M.P. and C.S.M.; Review & Editing, all authors; Funding acquisition, C.S.M.


The number of septic total hip arthroplasty (THA) revisions is increasing continuously, placing a growing financial burden on hospitals. Orthopedic departments performing septic THA revisions have no basis for decision making regarding resource allocation as the costs of this procedure for the departments are unknown. It is widely assumed that septic THA procedures can only be performed at a loss for the department. Therefore, the purpose of this study was to investigate whether this assumption is true by performing a detailed analysis of the costs and revenues for two-stage septic THA revision.Patients who underwent revision THA for septic loosening in two sessions from January 2009 through March 2012 were included in this retrospective, consecutive cost study from the orthopedic department’s point of view. We analyzed variable and case-fixed costs for septic revision THA with special regard to implantation and explantation stay. By using marginal costing approach we neglected hospital-fixed costs. Outcome measures include reimbursement and daily contribution margins.p < .001 and p = 0.019) for ex- and implantation (€4147 vs. €6680 and €429 vs. €306) while length of stay and reimbursement were comparable.The average direct costs (reimbursement) incurred for septic two-stage revision THA was €10,828 . The difference in cost and contribution margins per day was significant (This is the first detailed analysis of the hospital department’s cost for septic revision THA performed in two sessions. Disregarding hospital-fixed costs the included variable and case fixed-costs were covered by revenues. This study provides cost data, which will be guidance for health care decision makers. Currently, one million primary total hip arthroplasties (THA) are performed worldwide each year . It has This study was performed in a hospital setting being reimbursed by diagnosis related groups (DRG), which follows the fee-per-case approach. In most health care systems, hospitals are reimbursed on the basis of DRGs rather than on daily rates. In the past it got more and more challenging to ensure that at least their costs are recovered when fulfilling their obligation to treat patients. Hospitals making a deficit may not survive. Thus, hospitals and individual departments must analyze their costs to have a sound basis for decision making. For an orthopedic department, this means that the relevant costs of septic THA need to be calculated for proper resource allocation and decisions about the service portfolio.With the exception of a few highly specialized centers, most hospitals perform a wide spectrum of different procedures. Ideally, the total revenues accruing to a hospital from this mix of procedures should be high enough to cover all liabilities. For a hospital to operate economically, the reimbursement for complex cases that require high levels of resources should at least cover the variable costs per case, while the revenues from other, more common procedures should pay the hospital’s total fixed costs , 9.Most international studies published in the literature that analyze the costs of septic revision THA use a total cost approach, not separating between fixed and variable costs , 10–13. Contribution margin I refers to the difference between net revenues and variable costs. Subtraction of variable and fixed case costs from overall revenues yields contribution margin II. If the result is negative, the orthopedic department incurs a deficit from the treatment of patients with this diagnosis, which must be compensated for by the revenues from other procedures. If the result is positive, no conclusions can be drawn as to whether outcome is also positive for the hospital because hospital-fixed costs  still have to be considered. The contribution margin approach provides data on the financial outcome for a given hospital department, in our case the orthopedic department. From its internal perspective, the department can make economic, future-oriented decisions without taking into account the hospital’s overall fixed costs.We analyzed cost structures and contribution margins for septic revision THA procedures performed in two sessions. With this approach, the actual cost associated with a procedure can be calculated, and the orthopedic department performing the procedure can estimate whether the reimbursement for this treatment covers its cost. In addition, we present a detailed analysis of all steps involved in septic revision THA and the corresponding cost separately for the explantation and the implantation procedure. We consider this separate calculation important since, to the best of our knowledge, this has not been done by investigators before. In agreement with most of the published literature, we hypothesize that the relevant variable and fixed case costs associated with septic revision THA are not covered by current reimbursement, resulting in a loss to the hospital , 13, 18.The analysis was performed using data from a university-affiliated hospital in central Europe providing maximum care. In 2012, 36,345 patients were treated in the entire hospital, which provides 926 beds in 21 departments. In 2012, the Clinic and Outpatient Clinic for Orthopedics and Orthopedic Surgery provided inpatient health care for more than 2000 cases .Retrospectively, all consecutive patients undergoing revision THA in two sessions for septic loosening from January 2009 to March 2012 were included. The analysis was based on medical and financial data provided by the Department of Medical Controlling. We first identified all inpatients with a supplementary diagnosis of “Septic - infection and inflammatory reaction due to internal joint prosthesis” (ICD-10 Code T84.5) and a positive microbiological test result (positive for bacteria). In a second step, patients not treated according to OPS codes 5–821  and 5–820  were eliminated from this initially identified set. The remaining cases were assessed by two experienced hip surgeons who reviewed the surgeons’ reports using a list of predefined eligibility criteria: (i) typical pain in the leg; (ii) imaging evidence of loosening; and (iii) characteristic laboratory and pathophysiologic parameters in blood and biopsy specimens from the joint. This data was obtained from the patient record form including clinical and operation notes as well as lab tests.The reimbursement of healthcare providers on the basis of diagnosis-related groups (DRGs) all over the world means that hospitals receive a fixed payment for a procedure. The amount is calculated on the basis of the average costs of a set of similar procedures . In our We performed a cost calculation using contribution margins as described previously by Kasch et al. .Costs were analyzed using the parameters listed in Table Variable costs included cost for implant, drugs, blood transfusions, laboratory, radiology, expenditure material while case-fixed costs remained to staff, physiotherapy and ICU. Since hospitals typically negotiate prices for materials with suppliers, we used the hospital’s actual purchase prices for the analysis rather than official list prices.For the orthopedic department, the relevant parameter of economic provision of services is the daily contribution margin, which is calculated by dividing average marginal contribution II by average length of hospital stay. The smaller the daily marginal contribution, the more difficult it becomes for the entire hospital to recover its fixed costs. It is essential to know the contribution margin per day of individual DRGs for comparison with other procedures in order to identifying those with the biggest budgetary burden. Table In patients undergoing revision THA in two sessions, each of the two hospital stays  is reimbursed separately and hence we also regard them as two separate stays in our analysis. This is the only possible approach that allows identification of the factors that provide a basis for well-founded economic decisions. The two stays per patient can also be aggregated to calculate total cost for comparison with other procedures or published data.U-test for skewed data. Normally distributed data were assessed using the Chi2-test and t-test, assuming statistical significance at p < .05. All statistical tests were performed using SPSS, version 20. The local ethics committee of Ernst-Moritz-Arndt University Greifswald approved the study (BB 010/13) and waived written informed consent.All data are given as means with ranges. Most cost data are not normally distributed. We tested for normal distribution (Kolmogorow-Smirnow test), and used the nonparametric Mann–Whitney Our search retrieved a total of 357 patients who met the inclusion criteria of an ICD-10 T84.5 supplementary diagnosis. All of them were tested for infection via joint aspiration. A positive microbiological culture was the indication for the periprosthetic infection, knowing that there are also septic joints which are culture negative.n = 321) or they did not meet eligibility criteria in the surgical reports (n = 6). Thirty patients were finally included in our analysis. All of them received a complete THA revision. Further characteristics are summarized in Table Three-hundred-and-twenty-one cases were eliminated, either because they did not meet the specific eligibility criteria for the medical OPS procedures 5–821 and 5–820 n = 21 or thep = 0.048). Most patients (63.2 %) had infection with Staphylococcus aureus . The total duration of both hospital stays per patient taken together ranged from 25 to 61 days with a mean of 40.2 days. The mean duration of the first hospital stay (explantation) was 20.5 days (13–36), while that of the second stay (implantation) was 19.8 days (10–33) (p = 0.982). In the patient records, a mean of 9.3 additional ICD-10 codes were documented; there were no significant differences between men and women (p = 0.819).The 30 patients included in our analysis had a mean age of 66.1 years with women having a significantly higher mean age than men . Cost items with significant differences between explantation and implantation were costs for the implant (p < .001), sterilization (p < .001), normal ward nursing services (p < .001), and medication (p < .001).The total average costs incurred by the hospital department for each procedure were €4147.4 for explantation and €6680.7 for implantation , the variable case costs were significantly different between explantation and implantation (p < .001).Figure n =14, 46.7 %, I03B: n = 15, 50.0 %). Thus, the data set is quite homogenous. Compared with the mean hospital stay in Germany for I03A (26.7 days), the patients of our study had shorter hospital stays . However, this does not hold for the subset of less severe I03B cases  [p = .641).Total reimbursement was €24,201.4 per patient. Of the 30 explantations performed, 25 (83.3 %) were assigned to DRG I03 (revision or replacement of the hip joint with complicated diagnosis or arthrodesis or age < 16 with (I03A) or without (I03B) extremely severe complications and/or comorbidities), while 29 implantations were assigned to this DRG (96.7 %) (I03A: Table p = 0.001) and II (p = 0.004), differ significantly between explantation and implantation. In each single case, both variable and fixed case costs were covered by revenues. Our analysis yielded a marginal contribution II per day of €429.3 for explantations and of €305.5 for implantations; the difference is significant (p = 0.019).Both contribution margins, I  contributes towards the hospital’s fixed costs, which are not included in our analysis. Published data suggest that the average fixed costs of a hospital account for 65–75 % of its total cost , 17. WhiNever the less in a hospital all clinical departments are co-responsible of the economic viability. So it is also required to have a global perspective to assure the economic sustainability of the whole hospital. Managers and clinical leaders must cooperate this in line. Positive contribution margins I and II are important but not enough, as also contribution margin III and IV needs to be positive to achieve a positive over all result.p < 0.001). It should be pointed out here seriously that, while cutting cost may be essential for the economic survival of many hospitals patient safety and good outcome must always come first. Economic and medical management must work to together to find payable but medically acceptable solutions.While DRG-based reimbursement  and hospitalization duration (20.5 days for explantation vs. 19.8 days for implantation) are nearly identical for the two procedures, the department’s costs related to implantation are much higher than for explantation (difference of €2533.3). This difference is mainly attributable to implant prices, which account for a large proportion of the total cost. Hence, we may conclude that cost savings can be realized by negotiating better purchase prices with suppliers. As patients in need of septic revision THA have an above-average morbidity and need more nursing and physician care at the time of explantation surgery, staff costs on the normal ward also differ significantly between explantation and implantation despite similar hospitalization durations . Although patients are very sick at the time of explantation, the higher contribution margin might be attributable to the fact that no implant costs are involved during the first stage of septic THA revision. With regard to previously published data septic implantations seem to be comparable to aseptic THA revisions (€298.2) in terms of contribution margins . This isOur study has some limitations, which must be taken into account when interpreting our results. The retrospective nature has the usual advantages and disadvantages of this design in terms of data quality, number of patients included, and representativeness. The total number of cases analyzed is not large, but this ensures that consistent data were available for all patients included. Due to technical aspects it was not possible to calculate costs for staff from other medical disciplines, e.g., during councils, thus the real costs for the hospital stay are slightly underestimated. As we present data from Germany, the results has to be interpreted and compared with regard to the diverging international health care systems and economic circumstances. Nevertheless our approach to calculate contribution margins is transferable. Compared with published data, the operation time and length of hospital stays appear rather short in our patients, which might suggest that the patients of our study population were in less serious conditions and thus could be treated at lower costs. Comparison with future studies will show whether our population is representative.Our analysis for the first time provides orthopedic surgeons with data on those parts of hospital costs they can influence. It might also assist hospitals in scenario planning with regard to the overall economic context. In summary, our cost analysis of septic revision THA procedures in two sessions indicates that more than half of the per-case reimbursement (55.3 %) is available for covering the hospital’s fixed costs. Hence, our hypothesis that hospital departments incur an unreimbursed loss when performing septic revision THA has been refuted. None of the 30 cases included in our analysis produced a loss for the orthopedic department. Our detailed analysis of explantation and implantation procedures for the first time shows that variable costs account for a large proportion of the costs associated with implantation procedures and might be a promising target for cost-saving measures. Purchase prices for hip implants and staff cost have been revealed as major cost factors that the hospital might influence.


The cure effect of a virus model with both cell-to-cell transmission and cell-to-virus transmission is studied. By the method of next generation matrix, the basic reproduction number is obtained. The locally asymptotic stability of the virus-free equilibrium and the endemic equilibrium is considered by investigating the characteristic equation of the model. The globally asymptotic stability of the virus-free equilibrium is proved by constructing suitable Lyapunov function, and the sufficient condition for the globally asymptotic stability of the endemic equilibrium is obtained by constructing suitable Lyapunov function and using LaSalle invariance principal. Since tobacco mosaic virus, the first virus of the world discovered by Beijerinck in 1898 . HoweverGenerally, the basic process of viral infection and virus replication occurs in six main steps: attachment, penetration, uncoating, replication, assembly, and release . After tx, y, and v represent the concentrations of uninfected target cells, infected cells, and virus, respectively. For explanations of other parameters we refer to literature [αvx to represent the interaction between uninfected target x and virus v, which obey the principle of mass action. Based on model (βxv/(x + y) in [βxv/(1 + bv) in [βxv/(1 + ax + bv) in [βxv/(1 + ax + bv + abxv) in [fv and introduced cure rate (denoted by ρ) into the following model:f satisfies the following hypotheses:f = 0, for all y ≥ 0.f/∂x > 0, for all x > 0, y ≥ 0, and v ≥ 0.∂f/∂y ≤ 0 and ∂f/∂v ≥ 0, for all x ≥ 0, y ≥ 0, and v ≥ 0.∂Since mathematical models and method of mathematical analysis were used to study the dynamics of the virus, lots of models have been established to explain the evolution of the uninfected target cells, infected cells, and the free virus. In these models, early works belonged to Nowak et al. , Nowak aterature . This modxdt=Λ−dx + y) in , βxv/(1 + bv) in , 15, βxv+ bv) in , and βxvabxv) in , and forabxv) in –20. Notiabxv) in –25 and Habxv) in –32; receabxv) in  adopted f and investigated the following model:f satisfies the following hypotheses:f = 0, for all y ≥ 0 and v ≥ 0, and f = 0, for all x ≥ 0 and y ≥ 0.f/∂x > 0, for all x ≥ 0, y ≥ 0, and v > 0.∂f/∂y ≤ 0, for all x ≥ 0, y ≥ 0, and v ≥ 0.∂f/∂v ≥ 0 and v/∂v) − f ≤ 0, for all x ≥ 0, y ≥ 0, and v ≥ 0.∂Recently, Tian and Liu  improvedng model:dxdt=Λ−dxHowever, many researches show that direct cell-to-cell spread can happen in some enveloped viruses  –37, Humax, y, and v denote the number of host cells, infected cells, and free virus, respectively. And d, a, and u are the death rates of them, respectively. Free virus is produced by infected cells at a rate ky. Λ represents the regeneration rate of host cells. ρ is the cure rate. fx = (βy + αv)x represents the total infection rate of host cells, which is divided into two parts βyx and αvx. The former represents the part where infected cells infect host cells by direct contact, and the latter means that host cells are infected by the free virus. For more detail, please see [fx = βxy≢0, for all x ≥ 0 and y ≥ 0, and ∂fx/∂y = βx ≥ 0 for all x ≥ 0, y ≥ 0, and v ≥ 0, which do not satisfy conditions (H3′) in model (H1) and (H3) in model  ∈ R+3∣x + y ≤ Λ/d, v ≥ 0}.Motivated by the works –20, 48, itions H′ in modeThe main goal of the present paper is to investigate the globally asymptotic stability of the equilibria of . This woℛ, we have The basic reproduction number , 50 of mE0 = , where x0 = Λ/d. If ℛ > 1, model , where Model  always h1, model  has a unE0 and E1.In this section, we shall show the local stability of equilibria (i)E0 is locally stable if ℛ < 1 and unstable if ℛ > 1.(ii)E1 is locally stable if ℛ > 1.For model , we haveE0 is given by J(E0) has an eigenvalue λ = −μ < 0, and the other two eigenvalues λ2 and λ3 satisfy ℛ < 1, λ2 + λ3 < 0, and λ2λ3 > 0, all the eigenvalues of J(E0) have negative real parts and E0 is locally asymptotically stable. And when ℛ > 1 and λ2λ3 < 0, J(E0) has a positive eigenvalue and E0 is unstable.We firstly prove (i). Notice the Jacobian of model  evaluateE1 is A, B > 0. And noticing that x∗(uβ + αk) = u(a + ρ), we have ℛ > 1 and y∗ = Λ/a(1 − 1/ℛ) < Λ/a are used. Then, by the Routh-Hurwitz Criterion [E1 is locally asymptotically stable for ℛ > 1.Next, we prove (ii). The Jacobian of model  evaluateriterion , we know equationAλ3+Bλ2+CE0.In this section, we study the global behaviors of model  by constℛ < 1, the virus-free equilibrium E0 is globally asymptotically stable.If L1(t) on A as follows:p > 0 is a constant to be determined. It follows from x0 < u(a + ρ); then, we can choose p > 0 such that βx0/u < p < (a + ρ − αx0)/k. Hence, we have that dL1(t)/dt < 0. Then, E0 is globally asymptotically stable.Define a Lyapunov function ows from  and 13)L1(t) onE1.Next, we study the global stability of the endemic equilibrium ℛ ≤ 1 + δ, the epidemic equilibrium E1 is globally asymptotically stable, where If 1 < ℛ > 1, we define a Lyapunov function L2(t) as follows:If ows from  and 15)ℛ > 1, wex = x∗, y = y∗, and v = v∗. Clearly, if ℛ > 1 and dx∗ − βx∗y∗ − ρy∗ > 0, then dL2(t)/dt ≤ 0. Note that dx∗ − βx∗y∗ − ρy∗ ≥ 0 can be formulated as dL2(t)/dt = 0 if and only if x = x∗, y = y∗, and v = v∗, by LaSalle invariance principle [E1 is globally asymptotically stable.The above equality holds only for rinciple , the equℛ is obtained. Firstly the locally asymptotic stability of the virus-free equilibrium and the endemic equilibrium is considered. Then, the globally asymptotic stability of the virus-free equilibrium is proved by constructing suitable Lyapunov function, and the sufficient condition for the globally asymptotic stability of the endemic equilibrium is obtained by constructing suitable Lyapunov function and using LaSalle invariance principal. By analyzing the condition for the globally asymptotic stability of the endemic equilibrium, we have that if ρ = 0, from ℛ > 1 can ensure the global stability of the equilibrium E1, While if ρ > 0, by the numerical simulations (see Figures ℛ ≤ 1 + δ in In this paper, we considered the cure effect of a virus model with both cell-to-cell transmission and cell-to-virus transmission. By the method of next generation matrix, the basic reproduction number