In July 2010, WHO published new recommendations on providing antiretroviral therapy to adults and adolescents, including starting ART earlier, usually at a CD4 count of 350 or lower, specific regimens for first- and second-line therapies, and other recommendations. This paper estimates the potential impact and cost of the revised guidelines by first, calculating the number of people that would be in need of antiretroviral therapy (ART) with different eligibility criteria, and second, calculating the costs associated with the potential impact. Results indicate that switching the eligibility criterion from CD4 count <200 to <350 increases the need for ART in low- and middle-income countries (country-level) by 50% (range 34% to 70%). The costs of ART programs only to increase coverage to 80% by 2015 would be 44% more (range 29% to 63%) when switching the eligibility criterion to CD4 count <350. When testing and outreach costs are included, total costs increase by 62%, from US$26.3 billion under the previous eligibility criterion of treating those with CD4 <200 to US$42.5 billion using the revised eligibility criterion of treating those with CD4 <350. In July 2010, the World Health Organization (WHO) published new recommendations on providing antiretroviral therapy (ART) to adults and adolescents in resource-limited settings that revised the guidelines previously published in 2006. The new recommendations encourage starting ART earlier, usually at a CD4 count of 350 or lower, specifies regimens for first and second line therapies, and contains other recommendations regarding laboratory monitoring and other elements . The. The2]. λ1, λ2, λ3, and λ4 represent the probability of progressing from one CD4 category to the next; the derivation of these probabilities is discussed in detail below. In each category there is some probability of death from HIV-related causes, designated as μ1, μ2, μ3, μ4, and μ5 as well as a chance of death from non-AIDS causes, μ0 (not shown in the figure). The probability of HIV-related death increases as CD4 counts decrease.We assume that all newly infected people start with CD4 counts above 500, and that their CD4 counts decline over time. The transition probabilities The number of people in the different CD4 count categories represents the HIV-infected population that is not on ART. The number of people eligible for treatment is the number in each CD4 count category that is below the recommended level for initiating ART. α1, α2, α3, α4, and α5.Depending on the eligibility criterion and the level of first-line ART coverage a percentage of those eligible for treatment will start first-line ART . Those on ART are categorized by their CD4 count at the initiation of treatment. The model does not track the temporal decline of CD4 counts of those on treatment. Those on first-line ART have a probability of failure depending on their CD4 count at initiation, The number starting ART each year is determined by the assumed coverage and the number of people eligible for treatment. We assume that those starting on ART will be distributed among the eligible CD4 categories such that an equal percentage of people in each eligible CD4 category initiate treatment.β1, β2, β3, β4, β5). Those failing on first line ART will either start on second line ART  or die from HIV-related causes. Those on second line have some probability of dying from HIV-related causes each year , Cape TData are also available from several cohort studies on the overall progression from HIV infection to HIV-related death. The Analysing Longitudinal Population-based HIV/AIDS data on Africa (ALPHA) network has conducted a pooled analysis using data from several cohorts to estimate the proportion surviving by the number of years since infection . Only thWe fit the model to both sets of data simultaneously. One version of the model was set up for Kenya and used the Spectrum estimates of the number of new infections from 1980 to 2007 and the reported number of people on ART from 2000 to 2007. We compared the data on distribution by CD4 count from the Kenya AIDS Indicator Survey (KAIS) with the model projection for 2007. Another version of the model followed a cohort of 1000 new HIV infections as they progress through the various CD4 categories and to death. The resulting proportions surviving were compared with the ALPHA network survival curve for East and Southern Africa. We searched for the single set of transition probabilities that provided the best fit in both cases. The model used a time step of one-tenth of a year in order to accommodate the short duration in the 200-250 category that could be less than one year. The fits are shown in Figures The fit of the model to Karonga  and Orange Farm (South Africa) data sets using the parameter values derived from the fit to the Kenya data and the annual number of new infections in Malawi and South Africa is shown in Figures Four categories of cost are considered: antiretroviral (ARV) drugs, laboratory costs, service delivery costs, and identification (outreach and testing). The cost of ARV drugs is determined from the number of people on first and second line, the distribution of patients by regimen and the costs of each regimen. Following previous work, we examine two sets of alternative regimens: one that contains a fast phase-out of d4T, and another that contains a slower phase-out of d4T .Drug costs may be different for patients in low and middle income countries. Current costs are based on WHO and Clinton Foundation reports . Laboratory costs are calculated separately for new and continuing patients and can vary by regimen. Currently, laboratory costs are calculated as the annual median cost for lab tests across recent literature. Recent studies in various countries  are used as the basis –20. The Service delivery costs are based on a standard number of inpatient days and outpatient visits per patient per year and country specific costs for inpatient days and outpatient visits. For this analysis we used the same studies referenced above for laboratory costs (with the exception of Cote d'Ivoire and the addition of another South Africa study ) to calcOutreach and testing costs vary primarily by the type of population reached. The model considers 10 population categories for testing:patients with symptoms of HIV,sexually-transmitted Infection (STI) patients,tuberculosis patients,pregnant women,other health system contacts,sex workers,men who have sex with men (MSM),injecting drug users (IDU),voluntary counseling and testing (VCT),general population. Due to lack of data, those coinfected with hepatitis B are not included here.The unit cost of VCT services average about $16 per client. We have used this cost also for provider-initiated testing and counseling. No additional testing and counseling costs are included for pregnant women since the costs of testing and counseling are already covered in the Prevention of Mother-To-Child Transmission (PMTCT) programs. Similarly we assume that outreach and counseling for sex workers, IDU, and MSM are already covered in prevention programs for those populations, and add only $1 for the costs of the test itself. For general population testing we have doubled the personnel costs associated with VCT to allow for additional outreach programs in addition to the testing and counseling costs. The resulting cost is $23 per person tested.The number of tests for each population group will depend on the eligibility criterion and the coverage. We assume that patients with symptoms who are found to be HIV+ will be in the lowest CD4 count category. We assume that those who are found to be HIV+ in the other population groups will be distributed by CD4 count according to the distribution of all HIV+ people excluding those <200. The model has been applied to all low- and middle-income countries (LMIC) and to seven countries individually: Burkina Faso, Mexico, Nigeria, Russia, Tanzania, Ukraine, and Vietnam. The number of new infections each year and the number of people on ART through 2008 were taken from the Spectrum projections for each country. Estimates of the population sizes are based on national estimates prepared as part of the effort to estimate global resource needs . Our estimates suggest that switching the eligibility criterion from CD4 count <200 to <350 increases the number of person-years of ART from 40.7 million to over 61 million, a 50% increase. There is a concomitant reduction in the number of AIDS deaths, with the number decreasing by 21% when the eligibility criterion switches to CD4 count <350. The number of new HIV infections is also reduced, due to the lower infectivity that occurs when people receive ART; new HIV infections are reduced by 11% when the eligibility criterion changes.The financial costs of providing ART to meet the new need from increasing the eligibility criterion also increase in a similar way to the increase displayed in the number of person-years of ART. The costs of providing ART would be 44% higher with a switch to providing ART to those with CD4 count <350. Although the overall costs are higher with the fast phase-out of d4T relative to the slower phase-out of d4T, the difference is quite small. Note that the slightly lower percentage increase in costs versus the number of person-years on ART reflects the relatively greater numbers of people on first-line therapy with the increase in eligibility criterion. When the additional testing costs incurred in order to identify the new patients are included, however, total costs increase relatively more than the number of person-years of ART. Total costs increase from US$26.3 billion (US$27.0 billion) to US$42.5 billion (US$43.5 billion) if the eligibility criterion is CD4 count <350 and there is a fast (slower) phase-out of d4T, an increase of 62% (61%). Combining the results for incremental costs and deaths averted suggests that the cost per AIDS death averted is approximately US$9,700 if the eligibility criterion switches to CD4 count <350.In order to perform a sensitivity analysis, we vary the costs of laboratory testing and service delivery costs using the interquartile distribution of laboratory testing costs from the studies cited above. Using the first quartile function result, laboratory and service delivery costs are reduced by 31%, while using the third quartile function result increases laboratory and service delivery costs by 64%. Overall, this translates to a range in total costs (not presented here) of US$42.5 billion to US$55.5 billion for the scenario with a slower phase-out of d4T, and a range in total costs of US$37.2 billion to US$56.5 billion for a fast phase-out of d4T.  In order to compare results for different epidemic types and different regions, we performed the analysis for seven countries: Burkina Faso, Mexico, Nigeria, Russia, Tanzania, Ukraine, and Vietnam. Results indicate that there is not a great deal of variation across countries see . While tIn this paper, we model both the impact and cost of the new 2010 WHO recommendations for providing antiretroviral therapy to adults and adolescents in resource-limited settings. We examine the impact of changing the eligibility criterion for antiretroviral therapy from CD4 count <200 to CD4 count <350 on the number of person-years on ART, the number of AIDS deaths averted, and the costs of the change including the costs of additional tests and recruitment costs. We also examine the financial impact of switching away from d4T towards other recommended regimens.We find that, although the total costs for providing ART increase, the percentage increase is slightly less than the increase in number of person-years on ART. The number of person-years on ART increases for LMIC by 50%, varying between 34% and 70% at the country level, while the cost of providing ART increases by 44% for LMIC, varying between 30% and 63% at the country level when the eligibility criterion changes to CD4 count <350. There is minimal impact on the incremental cost when phasing out d4T either fast or more slowly when the eligibility criterion varies. When testing and outreach costs are included, total costs increase by 62%, from US$26.3 billion under the previous eligibility criterion of treating those with CD4 <200 to US$42.5 billion using the revised eligibility criterion of treating those with CD4 <350.In addition, the number of AIDS deaths decreases at the global level by 21% when the eligibility criterion switches to CD4 count <350, with country-level results varying between decreases of 16% and 23%. Combining the data results in a cost per AIDS deaths averted varying between approximately US$7,100 and US$9,700  depending on the change in eligibility criterion.


To understand the emergence of vaccinia virus Cantagalo strain in the Amazon biome of Brazil, during 2008–2010 we conducted a molecular and epidemiologic survey of poxvirus outbreaks. Data indicate that animal movement was the major cause of virus dissemination within Rondônia State, leading to the establishment and spread of this pathogen. VACMato Grosso State is partially inserted into the southeastern Amazon biome  and has From June 2008 through June 2010, we investigated outbreaks of poxvirus-related disease on 56 dairy farms of Mato Grosso and Rondônia . A proba,,,,,,A total of 52 samples of scabs were collected from cattle on 23 farms. DNA was isolated as described ,,,,,, were poAn epidemiologic investigation was conducted by using data available from the 56 epidemiologic investigation forms, analyzing cattle movement forms issued during this period, and interviewing farmers and animal health agency veterinarians. In addition to the 23 farms with positive samples, 33 other farms had epidemiologic links to CTGV-positive farms. Therefore, our data suggest that CTGV infection was the cause of all outbreaks.>100 cattle (mean=253), in contrast to 90% of southeastern farms, which had <100 cattle  . These rBR-364 is a 4,141.5-km highway with a major veterinary inspection station, the Amazon Gate, at the Mato Grosso–Rondônia border. Affected farms were mostly located along this main road connecting Mato Grosso with the Amazon region , panel A,2 in contrast to 17.44 cattle/km2 in Mato Grosso. The occurrence of new CTGV outbreaks in 2010 supports a nonsporadic pattern of infection in Rondônia, highlighting the establishment of this zoonotic infection within the state.Cattle trade during the auction launched virus spread throughout the state, which was amplified by subsequent trading and human translocation . Animal –This study investigated the appearance, establishment, and spread of CTGV infection into the Amazon biome. Our findings suggest that animal movement was the main cause of virus long-distance dissemination in Rondônia, whereas the migration of dairy workers was involved in the focal spread. Animal movement plays an essential role in cross-border spread of several disorders worldwide, including bovine tuberculosis, foot-and-mouth disease, and bovine diarrhea viruses –, panel B


Yet, the continuous replication of influenza virus using a two-stage bioreactor setup is a novel tool to study aspects of viral evolution and the impact of DIPs.Influenza viruses are a major public health burden during seasonal epidemics and a continuous threat due to their potential to cause pandemics. Annual vaccination provides the best protection against the contagious respiratory illness caused by influenza viruses. However, the current production capacities for influenza vaccines are insufficient to meet the increasing demands. We explored the possibility to establish a continuous production process for influenza viruses using the duck-derived suspension cell line AGE1.CR. A two-stage bioreactor setup was designed in which cells were cultivated in a first stirred tank reactor where an almost constant cell concentration was maintained. Cells were then constantly fed to a second bioreactor where virus infection and replication took place. Using this two-stage reactor system, it was possible to continuously produce influenza viruses. Surprisingly, virus titers showed a periodic increase and decrease during the run-time of 17 days. These titer fluctuations were caused by the presence of defective interfering particles (DIPs), which we detected by PCR. Mathematical modeling confirmed this observation showing that constant virus titers can only emerge in the absence of DIPs. Even with very low amounts of DIPs in the seed virus and very low rates for  Orthomyxoviridea. They are enveloped viruses with a segmented single-stranded RNA genome of negative polarity. The genome of influenza A viruses consists of eight genome segments encoding up to 13 polypeptides Influenza viruses belong to the family of The continuous avian cell line AGE1.CR was designed for the production of vaccine viruses that replicate in avian hosts Typically, cell culture-based processes for the production of influenza vaccines rely on batch cultivations Here, we explored the possibility to establish a continuous production process for influenza viruses based on a two-stage bioreactor setup. AGE1.CR cells were cultivated in a first stirred tank bioreactor (STR) such that a stable cell concentration was maintained. From this bioreactor, the suspension cells were continuously fed into a second bioreactor where virus infection and replication took place.3 IGF, 10 ng/mL final concentration, Sigma).The duck-derived suspension cell line AGE1.CR 50)  = 6.6×107 virions/mL (used for the first continuous cultivation); TCID50 = 1.6×108 virions/mL (second run)) was stored in aliquots at −80°C.The human influenza virus A/Puerto Rico/8/34 H1N1  was adapted to the AGE1.CR cell line over several passages. The resulting AGE1.CR adapted virus seed  at a multiplicity of infection (MOI) of 0.025 based on the viable cell count and the TCID50 of the seed virus. Additionally, the virus inoculum was supplemented with 1×10−6 U/cell trypsin . One hour after infection, the two STRs were operated in continuous mode with flow rates depicted in 50 assay Two small scale stirred tank bioreactors  were used. The first lab-scale bioreactor was inoculated with AGE1.CR cells and cultivations were carried out at 37°C, pH 7.2 and a stirring speed of 120 rpm with a working volume of 1 L. Aeration was controlled to 40% DO by pulsed aeration with pure oxygen through a microsparger. When cell concentrations reached levels of more than 4×10To analyze viral genomes, bioreactor samples were centrifuged for 5 min at 1000 g and viral RNA was purified from 150 µL supernatant using the NucleoSpin RNA Virus extraction kit (Macherey-Nagel) according to the manufacturer's instructions.A reverse transcription was performed by using the Uni12-primer 2 (10 mM), 1 µL dNTPs (10 mM each), 1 µL forward primer (10 µM), 1 µL reverse primer (10 µM), 0.2 µL Phusion DNA polymerase (2 U/µL) (Thermo Scientific) and 8.8 µL nuclease-free water. Initial denaturation was performed at 98°C for 3 min followed by 25 cycles with 98°C for 25 sec, 60°C (or 55°C for segment 6) for 45 sec and 72°C for 1–2 min (depending on segment length). The final elongation was performed at 72°C for 10 min. PCR products were visualized using gel electrophoresis.For the segment-specific PCR eight primer pairs were used . 2 µL ofTo systematically study our continuous virus production system, we developed a segregated mathematical model based on an existing description of a batch process proposed by Kirkwood and Bangham The concentration of uninfected target cells  reduces to a three dimensional model of virus growth if the seed virus is free of DIPs , it has been demonstrated that AGE1.CR cells maintain permissivity and high virus yields after long-term cell cultivation with up to 318 serial passages. At this high passage level, AGE1.CR cells also preserved the expression of the introduced E1A gene 6 cells/mL was reached. Then 0.375 L were transferred to the second STR. Subsequently, fresh medium was added to adjust the first STR to 1 L again and the second STR to 0.5 L working volume. One day after the transfer of cells, the influenza virus A/Puerto Rico/8/34 was added to the second STR (virus bioreactor) at a multiplicity of infection (MOI) of 0.025. The continuous culture was started by a constant feed of 0.22 mL/min of fresh medium into the cell bioreactor where the cell concentration was maintained between 3.8–4.8×106 cells/mL. Cells were constantly fed from the cell bioreactor into the virus bioreactor . In addition, 0.11 mL/min of fresh medium was added to the virus reactor and up to 0.33 mL/min was harvested to maintain the 0.5 L working volume.A setup with two stirred tank bioreactors (STRs) was chosen  to estab6 cells/mL at day 4 post infection (p.i.). This was caused by an extensive production of infectious viruses that reached concentrations of 7.6×108 virions/mL already 16 h p.i. (50) assay takes three days. Instead, the hemagglutination (HA) titer was monitored on a daily basis, which represents the total virus particle concentration (infectious as well as non-infectious). As HA titers decreased by day 7 p.i. while cell concentrations increased, additional trypsin was injected into the virus bioreactor. At this point, it was assumed that the trypsin supplementation of 3600 units/L in the feed medium of the virus bioreactor might not be sufficient. However, HA titers kept declining showing that trypsin activity was not limiting virus propagation. Hence, additional seed virus was injected into the virus reactor at day 9 p.i. as an attempt to restore virus propagation. Yet, the subsequent analysis of virus titers by the TCID50 assay revealed that infectious titers were already at high levels before we added the new seed virus which indicates that infectious virus particles were also not a limiting factor. Also at later time points, the injection of either extra seed virus alone or in combination with additional trypsin had no impact on the decrease of HA titers.During the first continuous influenza virus production, cell concentrations in the cell bioreactor only fluctuated within the error range of the cell count instrument (maximum relative standard deviation 5%) . In cont6 h p.i. . SurprisIn order to establish more stable continuous virus propagation, the cultivation procedure was slightly modified in the second run. This time, instead of trypsin being added to the feed medium of the virus bioreactor, fresh trypsin was injected directly into the STR on a daily basis to avoid loss of trypsin activity due to self-degradation. Furthermore, another batch of seed virus with a higher infectious virus titer was used and no additional seed virus was added after the initial infection of the virus bioreactor (MOI 0.025). However, a similar periodic increase and decrease of virus titers was observed during this second continuous virus cultivation . In gene−3 to 10−4. Overall, because variations in cell concentrations were low, the dynamics of MOIs matched the course of infectious viral titers and fluctuated within six orders of magnitude.Based on the infectious virus titer and corresponding viable cell concentration, the MOI that occurred in the virus reactor was calculated for every sampling time point. Both cultivations were infected at an MOI of 0.025 and therefore low MOIs were obtained at the first sampling time point directly after infection and the start of continuous operation mode . HoweverSuccessive passaging of influenza virus at high MOI is known to support the accumulation of defective interfering particles (DIPs). This phenomenon was first described in the early 1950 s by von Magnus who reported the formation of what he called “incomplete” virus after undiluted serial passages in embryonated eggs To analyze whether DIPs were causing fluctuations in viral titers during continuous cultivations, we looked for defective interfering RNAs in virus samples using a PCR. Therefore, cell culture samples were centrifuged for 5 min at 1000 g and 150 µL of the supernatant was used for viral RNA purification. Subsequently equal volumes of each sample were used for the reverse transcription and for the PCR reaction. Since DI RNAs arise from internal deletions and accordingly contain both the 5′ and 3′ termini of a gene segment, primers binding to those ends are able to amplify full-length as well as DI RNAs. A segment-specific PCR revealed that the used virus stock contained all eight genome segments as expected. However, for the polymerase segments 1–3 encoding PB1, PB2 and PA additional smaller PCR products in the range between 500 to 700 base pairs (bp) could be detected as faint bands or as less distinct smear . In addiImmediately after the infection the full-length genome accumulated and represented the dominant PCR product for all segments . HoweverTo further analyze the process dynamics and to provide additional evidence that DIPs can cause the observed fluctuations in virus titers; we developed a mathematical model of our continuous virus infection system. In the past, similar models were successful in showing that cyclic variations can occur in serial passage infections due to the presence of defective interfering viruses To test this hypothesis, we first analyzed a simple model of continuous influenza A virus infection in the absence of DIPs . The modde novo generation of DIPs by cells infected with STV alone, which in addition to producing large quantities of STVs release a small amount of DIPs. Note that we do not distinguish between particles with defects in different segments but rather consider a general effect of an average DIP on virus production. Intriguingly, this simple model readily showed periodic oscillations even for D>µ and was able to reproduce the frequency observed in our experiments  minimizing the DIP concentration in the seed virus, e.g. by serial low-MOI passaging , and  using undiluted serial passaging in embryonated eggs de novo generation or replication of DI genomes A continuous production process for rubella virus was successfully established et al. suggested that different populations of co-infected cells exist, which may produce a mixture of standard and defective viruses Our simple mathematical model of DIP replication during continuous infection captured the qualitative features of the measurements surprisingly well. Nevertheless, a comprehensive understanding of influenza virus infection may require further modeling work. For instance, Thompson in vitro DIP replication. Additionally, the continuous influenza virus cultivation using a two-stage bioreactor setup can serve as a novel tool to study aspects of viral evolution.Influenza A virus can be propagated in continuous culture using the robust and fast-growing suspension cell line AGE1.CR. However, a stable long-term virus production with constant high titers is impeded by defective interfering particles. Our experimental data as well as our modeling approach demonstrate that DIPs rapidly accumulate during continuous virus propagation and, thus, represent a severe challenge for the productivity of the system. Since virus titers during continuous infection show regular oscillations, as opposed to chaotic fluctuations during serial passaging, continuous systems lend themselves well to the investigation of Table S1Parameters and non-zero initial conditions used for numerical simulations.(DOCX)Click here for additional data file.Text S1Theoretical analysis for the models (1) and (2).(DOCX)Click here for additional data file.


Prolonged passage of the KFDV strain P9605 in monkey kidney tissue culture resulted in a markedly reduced virulence of the virus for both species; peak viremia titers in monkeys decreased by 2.5 to 4.0 log LD 50 (p= 0.001), and the mortality decreased to 10% (p= 0.001). In challenge experiments, monkeys previously infected with tissue-culture-adapted KFDV, or with the related Langat virus from Malaysia, were fully protected against virulent KFDV. These studies in non-human primates lend support to the idea that a live virus vaccine from a member of the TBEV complex may be broadly protective against infections by other members of the TBEV complex.Kyasanur Forest Disease Virus (KFDV), discovered in 1957, is a member of the tick-borne encephalitis virus (TBEV) complex. Diseases caused by members of the TBEV complex occur in many parts of the world. KFDV produces a hemorrhagic fever in humans in South India and fatal illnesses in both species of monkeys in the area, the black faced langur (Presbytis entellus) and the bonnet macaque (Macaca radiata). Experimental infection of the langur and the bonnet macaque with early mouse passage KFDV strain P9605 resulted in a viremia of up to 11 days duration, peak viremia titers as high as 10 Kyasanur Forest Disease Virus (KFDV), a member of the hemorrhagic fever group, was discovered in 1957 in the Shimoga district of Karnataka State, India, where it produced a febrile illness and deaths in humans, and deaths in both species of monkeys, the black-faced langur (Presbytis entellus) and the red-faced bonnet macaque (Macaca radiata), that inhabit the endemic area2. Most of the human infections with KFDV occur in the drier months of the year, with peaks in March and April, and the number of cases is variable, but may reach over 500 in some years. Alkhurma Hemorrhagic Fever Virus (AHFV), described in 1997, a cause of severe hemorrhagic fever in Saudi Arabia and other regions of the Middle East4 and Nanjianyin Virus recovered from a febrile patient in Yunnan, China5, are closely related to KFDV. Omsk Hemorrhagic Fever Virus (OHFV), which is endemic in western Siberia, Russia, and recognized as a tick-borne flavivirus in 1947, is distantly related to KFDV1. In addition to the hemorrhagic fevers noted above, viruses of the TBEV complex are also responsible for over 10,000 cases of encephalitis, annually, in Europe and Asia, and for localized illnesses in different parts of the world1.Flaviviruses of the tick-borne encephalitis virus (TBEV) complex are widely and focally distributed in nature. New viruses of the complex and illnesses associated with them are frequently being described from different parts of the world. Human illnesses associated with infections with these viruses include mild or severe encephalitis and hemorrhagic fevers6, it was completely ineffective in preventing human KFDV infections in the endemic area7. It also failed to prevent KFDV infections in laboratory personnel9. Serologically, individuals immunized with the RSSE vaccine showed a barely detectable immune response to KFDV antigens10. In contrast to the lack of effectiveness of an inactivated RSSE vaccine, an inactivated KFDV vaccine reduced the incidence of KFDV in the affected area11. Thus, KFDV infections in humans were preventable by an inactivated homologous vaccine, but not by an inactivated heterologous vaccine.In 1958–59, a formalin-inactivated mouse-brain virus vaccine prepared with the heterologous Russian spring-summer encephalitis virus (RSSE) was administered to over 10,000 residents in the KFDV-endemic area. Although this vaccine was protective against KFDV challenge in the mouse model12. The results of these studies were described only in the annual reports of the VRC and in a doctoral thesis published in 19636, so they are not readily available. Because data obtained from experimental infection and challenge of non-human primates with viruses of the TBEV complex are valuable and may help efforts aimed at developing prophylactic vaccines, we have combined the information from the doctoral thesis and from an unpublished manuscript  for this report.In the late 1950s and 1960s, investigators from the Virus Research Centre (VRC) [now the National Institute of Virology (NIV)] in Pune, India, conducted infection and challenge experiments in non-human primates using virulent and tissue-culture-adapted (TC-adapted) strains of KFDV as well as Langat virus (LGTV), a related virus of the TBEV complex isolated from ticks in MalaysiaWe show here that 1) KFDV produces a high-titered viremia and death in both species of monkeys that inhabit the endemic area, 2) a TC-adapted KFDV has markedly reduced virulence for both monkey species, and 3) monkeys previously infected with TC-adapted KFDV or with heterologous LGTV completely resist challenge with virulent KFDV. Together, these data lend support to the idea that in contrast to the inactivated vaccines that do not cross-protect, an attenuated live virus vaccine derived from a member of the TBEV complex may be broadly protective against the different viruses of the complex.Studies described here were conducted at the VRC between 1958 and 1970.The animals were trapped by professional catchers in the forested areas of Shimoga district, Karnataka State in south India, outside the known area of KFD infection, and were transported to the laboratory in Pune by train or by truck. The animals were held for observation for several weeks prior to inoculation. At the commencement of the experiments, the weight range (and mean weight) of the animals was 2.0–7.2 kg (4.6 kg) for langurs, and 1.8–5.6 kg (3.3 kg) for bonnets. Animals were first classed by weight and then individuals in each weight class were allocated randomly to the different groups in the experiment.13. Sera that had HI antibodies to any of the viruses (less than 10% of the sera) were screened for neutralizing antibodies to KFDV10 and the rare animal that had KFDV neutralizing antibodies was not included in the experiment. The animals were inoculated with KFDV by the subcutaneous (SC) or the intravenous (IV) route and with LGTV by the IV or intracerebral (IC) route. The inoculated animals were monitored daily for viremia and general state of health. Rectal temperature was taken daily but these data were found to be uninformative and are not described in this report. Tissues of some of the animals that died during the observation period were tested for virus. Pathological studies of tissues were not routinely performed.Pre-inoculation serum specimens from the animals were screened for antibodies to KFDV, LGTV, West Nile and dengue 2 by hemagglutination inhibition (HI) testsData are reported from a total of 72 animals: 42 langurs and 30 bonnets. Three animals that did not develop either viremia or antibodies following inoculation are excluded from analyses.The virus preparations used and their designations are listed below.Virulent KFDV. Early passage KFDV is designated as virulent KFDV. Two strains of virulent KFDV were used. Most of the experiments were performed with strain P9605 derived from serum of a febrile individual in the endemic area and isolated and maintained by infant mouse brain passage in Swiss albino strain of mice. Passages 4–12 in infant mouse brain were employed with passage 9 used for a majority of the animals. These preparations are referred to as virulent KFDV (mouse brain) in this report. A second strain, KFDV 603260–2, was isolated and maintained in the bonnet macaque after inoculation from a mixture of two KFDV-positive tick pools. Monkey serum from the second passage of this strain was used as inoculum for one bonnet and four langurs. This strain is referred to as virulent KFDV (monkey serum).a)Tissue-culture-adapted KFDV. The virulent KFDV P9605 was passaged in primary rhesus or bonnet monkey kidney tissue culture (MKTC) prepared from healthy wild-caught animals which were non-immune to KFD, and was shown to be attenuated for mice as judged by the decrease in mortality in these animals following intraperitoneal inoculation14. The four preparations used in the primate studies were all derived from P9605, which had been adapted to MKTC following one infant mouse brain and two chick embryo tissue culture passages. The 137th and 138th monkey kidney tissue culture passage virus was used in the studies described in the doctoral thesis6. This preparation is designated ‘TC-adapted KFDV MK 137–138’. Preparations employed in the experiments in the unpublished observations of Bhatt and Dandawate (1971) were i) 177th monkey kidney tissue culture passage (designated TC-adapted KFDV MK 177); ii) virus plaque purified by three passages in monkey kidney cell monolayers after 144 passages in monkey kidney tissue culture and then passaged twice again in monkey kidney tissue culture ; and iii) the plaque-purified virus passaged 41 times in chick embryo tissue culture .b)Langat virus LGTV. The TP 21 isolate of LGTV derived from a tick pool was used in its 8th and 9th infant mouse brain passage , or as the 14th chick embryo tissue culture passage following nine infant mouse brain passages .c)0.0. If it killed less than one half of the mice the titer was reported as “trace virus (T)”.Heparinized blood or serum specimens were collected daily beginning with day 1 or day 2 post-inoculation (pi) until day 10 or 11 pi. They were titrated on the day of the bleeding and, if end points were not reached, an aliquot stored at -70°C was re-titrated at a later date. Titrations for KFDV were performed in adult mice intracerebrally, except in one experiment where they were determined by inoculation of chick embryo tissue culture tubes (which have a sensitivity similar to that of adult mice). Titrations for Langat virus were performed in infant mice intracerebrally. Viremia values are reported as the positive logarithms of the LD50 titer per 0.03 ml (when titrated in adult mice or tissue culture) or 0.02 ml (when titrated in infant mice for Langat virus) of the inoculum. If the undiluted serum killed three of six mice, the estimate of the LD50 titer was 10A lowess smoother was used to generate the estimated regression lines inVirulent KFDV in the black-faced langur. Thirteen langurs were inoculated with virulent KFDV by the SC or the IV route  of 106.6.V route . The amo1.9–106.5.All 13 animals died between day 5 and day 9 pi, with an average survival time of 6.5 days. With one exception, animals died during the viremic phase and the majority died at or near the peak of viremia. The exception was animal #84 that died on day 9, but for whom viremia was not estimated on the day of or day before death. Tissues of two animals collected post mortem (#84 and #85) were titrated for KFDV; all tested organs  were positive with titers ranging from 10TC-adapted KFDV in the black-faced langur. Twenty langurs were inoculated with TC-adapted KFDV by the IV or SC route. The amount of virus in the inoculum ranged from 101.9 to 107.9 ranged from 100.6 to 105.7 with a median value of 104.5. 107.9. There w14.There were two deaths among the 20 animals, one (#225) on day 6 at the peak of viremia and the other on day 10 (#85) in a monkey which was viremic from days 1–10. In tests of brain, lung, spleen, liver, kidney and heart tissues of animal #85, KFDV was isolated only from the lung tissue. The lung isolate was identified as the TC-adapted strain on the basis of its low pathogenicity for subadult miceVirulent KFDV in the bonnet macaque. Eleven bonnet macaques were inoculated with virulent KFDV by the IV or SC route. The amount of virus in the inoculum ranged from 104.0 to 108.1 LD50 , or at the height of viremia (one death on day 11 with the monkey serum virus), or in the post-viremic phase . Tissues of five of these animals were tested for KFDV. Three of these animals  died early in the illness during the viremic phase; virus was recovered from all of their tissues  that were examined. The amount of virus was highest in spleen or blood and lowest in the brain. In the two animals that died in the post-viremic phase at the end of the third week (#281 and #302), virus was present almost exclusively in the brain with titers of 10TC-adapted KFDV in the bonnet macaque. Ten animals were inoculated either by the IV or the SC route with 105.3–106.3 LD50 of the virus , peak titers were observed on days 3 and 4 pi and they ranged from 101.6–102.In the bonnet macaque. All nine animals inoculated either IV  or IC  with Langat TP21 M8-M9 were viremic on the first two days; peak titers ranging from 101.6 to >104.0 (mean of 102.6) were observed. The proportion of monkeys circulating the virus as well as the level of viremia decreased after the second day and viremia was not demonstrable in any monkey after day 6. The average duration of viremia was 3.6 days.Groups of animals, previously infected with Langat virus or with TC-adapted KFDV, were challenged with virulent KFDV. Data are combined inEight bonnets previously infected with Langat virus, and 4 bonnets previously infected with TC-adapted KFDV were challenged IV with 7.6–8.1 log LD50 of virulent KFDV. The interval between the immunizing infection and challenge was 31–38 days. In daily tests for viremia for 10 days, no viremia was detected in the four animals previously infected with TC-adapted KFDV, but ‘trace’ viremia was detected in 3 of the 8 bonnets previously infected with Langat virus, on day 1 or 2 pi. All but one animal survived challenge. One bonnet previously infected with Langat virus died on day 10 after challenge; blood, liver, lung, kidney, spleen and brain of this animal did not yield virus. This death was considered unrelated to the challenge.In a second series of challenge experiments in the unpublished observations of Bhatt and Dandawate (1971), 9 langurs and 2 bonnets previously infected with TC-adapted KFDV were challenged with 4.2–5.0 log LD50 of virulent KFDV. The interval between the immunizing and challenge infections was 4–30 months. In daily tests for viremia for 10 days pi, none were shown to be viremic. All of the animals survived the challenge.et al.15 have stated that “one of the most serious constraints for the study of TBE pathogenesis and for vaccine development has been the absence of realistic animal models that mimic human disease”. Our results of infection of two species of monkeys with virulent and TC-adapted KFDV and with Langat virus, and challenge of survivors with virulent KFDV, address this question.Kenyon17. A large majority of the animals found dead in the forest are langurs. The results of our experimental infections showed that the langur was highly susceptible to KFDV, and that the disease was acute, of short duration and always fatal. Viremia titers were high and deaths occurred between days 5–10 pi at or near the peak of viremia. Disease in the bonnet macaque seemed to resemble more closely the biphasic human disease. In some infected humans, the acute viremic phase is followed by a second non-viremic phase in which the patient has neurological symptoms and abnormal cerebrospinal fluid, but the symptoms are not localizing and there are no neurologic sequelae19. In the bonnet macaque, some deaths occurred during the viremic phase, but others occurred in the third week, at which time virus was recovered from the brain but not from other organs. In previous investigations, Webb and Burston20 have described neurological changes in the post-viremic period in experimentally infected bonnet macaques. Kenyonet al.15 have reported that the bonnet macaques experimentally infected with KFDV have a 100% mortality and that they show significant hematological and neurological abnormalities similar to those in KFDV-infected humans as well as significant histopathological changes in the small and large intestine, spleen and lymph nodes.Both the black-faced langur and the red-faced bonnet appear to be good models to study KFDV infection. Both species are naturally infected by KFDV in the endemic forested area in south India and many succumb to the disease21.In contrast to its high pathogenicity in the bonnet macaque, KFDV does not produce illness or death in the rhesus macaque, although the pattern of viremia is similar in the two speciesReduced virulence of TC-adapted KFDV: Bhatt and Anderson14 have reported that the passage of the prototype P9605 strain of KFDV in monkey kidney tissue culture (MKTC) resulted in a decrease of the virulence of the virus for adult mice as judged by a decrease in LD50 titer of the virus following intraperitoneal (IP) inoculation. In the current investigation, the lower virulence of the virus was also clearly evident in both species of monkeys. In the langurs, the viremia levels seemed to be lower after infection with MKTC 138 passage virus as compared to titers after inoculation of the other three preparations of TC-adapted KFDV. Nevertheless, combined for all passage levels and for both species, the mortality decreased from 82–100% with the virulent virus to 10% with the TC-adapted virus. The reduced virulence was also reflected in a decrease in peak titers of viremia of 2.5–4.5 log LD50 in the two species of monkeys. Identification of changes in the KFDV genome associated with this decrease in virulence may help us to understand the genetic determinants of KFDV virulence.22. Monkeys that had survived infection with the TC-adapted KFDV or with Langat virus completely resisted challenge with virulent KFDV. There was not a single virus-related death in these animals. In addition, there was no viremia at all in the animals previously infected with attenuated KFDV and only traces of viremia in some of the monkeys previously infected with Langat virus. A similar protection against homologous as well as heterologous challenge has been demonstrated in a report by Rumyantsevet al.23 in which chimeric live virus vaccines which contained sequences of TBEV and dengue 4 (TBEV/den4) or Langat virus and dengue 4 (LGT/den 4) were tested in rhesus monkeys. Animals immunized with either of the chimeric vaccine did not develop viremia when challenged with Langat virus.Viruses of the TBE complex are distributed focally in many parts of the world and they produce diseases of varying severity and in varying numbers. So, a single vaccine which can provide protection against all members of the complex would be beneficial. Among viruses of the TBE complex, Langat virus from Malaysia is the least virulent for mice, and mice infected intraperitoneally with Langat virus survive and they resist IP challenge by all of the viruses of the TBE complex that were tested24. Immunization of over 600,000 individuals with live Langat virus was reported to have reduced markedly the incidence of encephalitis in the endemic region in Russia, but the vaccine was associated with a serious adverse effect and was responsible for the occurrence of 35 cases of meningoencephalitis with permanent sequelae1. Attempts have been made to construct Langat virus which retains its immunogenicity but is less neuroinvasive25.Live Langat virus has been used in experimental treatment of human malignant disease in the United Kingdom and also as a live virus vaccine against TBE in Russia. In both cases, the neurotropism of Langat virus for humans became apparent. In the United Kingdom, two of 23 cancer patients infected with Langat virus developed encephalitis 8–11 days after the end of viremia26 in the search for a safe and broadly effective vaccine strategy against viruses of the TBEV complex.The TC-adapted strain of KFDV has a markedly decreased virulence for non-human primates and it merits consideration for further development as a live virus vaccine. It may be anticipated that an attenuated KFDV vaccine will provide significantly greater protection against KFD than inactivated KFDV vaccine, and it may also be effective against other viruses of the TBE complex. An attenuated KFDV vaccine may be less likely to produce paralytic sequelae than the Langat live virus vaccine because human KFDV infections in south India, while they cause transient neurological illness in some infected individuals, are not associated with paralytic disease. Components of the attenuated KFDV strain could also be employed to construct a genetically engineered non-infectious recombinant TBEV vaccine I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


CCR5Δ32 variant, a deletion variant which leads to a dysfunctional receptor, has been reported in several genetic association studies in Juvenile Idiopathic Arthritis (JIA), with conflicting results. CCL14 is one of the ligands of CCR5 and polymorphisms in the CCL14 gene have been reported to be associated with Systemic Lupus Erythematosus.CCR5 is expressed on Th1 cells and may play a role in Rheumatoid Arthritis by recruiting these cells to the synovium, where they drive an inflammatory process. The CCR5 and CCL14 polymorphisms are associated with susceptibility to JIA.We performed a case-control genetic association study to investigate whether CCR5Δ32 and CCL14 rs16971802 were genotyped in 667 JIA cases and 1320 healthy controls, both of North-West-European white origin. Patients with oligoarticular (persistent and extended), polyarticular (rheumatoid factor negative and positive) and systemic JIA have been included. A meta-analysis combined with three published studies on CCR5Δ32 in JIA, with comparable allele frequencies in controls, was performed.CCR5Δ32 and CCL14 rs16971802 were not significantly associated with JIA in this study, with p-values of 0.12 and 0.72 respectively. Nevertheless, meta-analysis demonstrated association of CCR5Δ32 with protection to JIA .CCR5 and CCL14 polymorphisms with JIA, but the association of CCR5Δ32 with protection from developing JIA is strengthened in a meta-analysis. It is hypothesized that function of CCR5 could influence synovial inflammation also in JIA.This study has not demonstrated significant associations of


Cervical cancer is the leading cause of female cancer-related deaths in Tanzania. Vaccination against human papillomavirus (HPV) offers a new opportunity to control this disease. This study aimed to estimate the costs of a school-based HPV vaccination project in three districts in Mwanza Region (NCT ID: NCT01173900), Tanzania and to model incremental scaled-up costs of a regional vaccination program.We first conducted a top-down cost analysis of the vaccination project, comparing observed costs of age-based (girls born in 1998) and class-based (class 6) vaccine delivery in a total of 134 primary schools. Based on the observed project costs, we then modeled incremental costs of a scaled-up vaccination program for Mwanza Region from the perspective of the Tanzanian government, assuming that HPV vaccines would be delivered through the Expanded Programme on Immunization (EPI).Total economic project costs for delivering 3 doses of HPV vaccine to 4,211 girls were estimated at about US$349,400 (including a vaccine price of US$5 per dose). Costs per fully-immunized girl were lower for class-based delivery than for age-based delivery. Incremental economic scaled-up costs for class-based vaccination of 50,290 girls in Mwanza Region were estimated at US$1.3 million. Economic scaled-up costs per fully-immunized girl were US$26.41, including HPV vaccine at US$5 per dose. Excluding vaccine costs, vaccine could be delivered at an incremental economic cost of US$3.09 per dose and US$9.76 per fully-immunized girl. Financial scaled-up costs, excluding costs of the vaccine and salaries of existing staff were estimated at US$1.73 per dose.Project costs of class-based vaccination were found to be below those of age-based vaccination because of more eligible girls being identified and higher vaccine uptake. We estimate that vaccine can be delivered at costs that would make HPV vaccination a very cost-effective intervention. Potentially, integrating HPV vaccine delivery with cost-effective school-based health interventions and a reduction of vaccine price below US$5 per dose would further reduce the costs per fully HPV-immunized girl. Cervical cancer is the second most frequent cancer in women worldwide . More thInternational studies have estimated that HPV vaccination of young girls would be cost effective in the 72 GAVI-eligible countries , and in This costing study was conducted as part of a project in Mwanza Region, Tanzania, which measured the feasibility, uptake, and acceptability of two alternative school-based HPV vaccine delivery strategies using a cluster randomized trial design (NCT ID: NCT01173900) . It aimeFirst, a top-down cost analysis of the school-based vaccination project was conducted from the project's perspective, using resource use and cost data collected alongside the cluster randomized trial. Second, based on the observed project costs, incremental costs of a scaled-up vaccination program for Mwanza Region were modeled from the perspective of the Tanzanian government, under the assumption that HPV vaccines would be delivered through the Expanded Programme on Immunization (EPI).The HPV vaccination project compared vaccine coverage in 134 primary schools randomly allocated to age-based (girls born in 1998) and class-based (class 6 of primary school in 2010) vaccine delivery in the two districts of Mwanza City and part of neighboring Misungwi district in northwest Tanzania. School-based vaccination was selected for the study because it has the best chance of achieving high coverage in Tanzania , where tet al. [et al. [The project was carried out by the Mwanza Intervention Trials Unit (MITU) of the National Institute for Medical Research (NIMR), in collaboration with the London School of Hygiene and Tropical Medicine (LSHTM).  were randomly selected for vaccination, and then randomly allocated to either age-based or class-based vaccine delivery. Information about HPV vaccination was provided through school, parent and community meetings, leaflet and poster distribution, radio messages and community drama troupes. Two 2-day training sessions were held to train 84 health workers from 42 health facilities in cervical cancer, HPV vaccination, cold-chain logistics and in management and reporting of adverse events.® Access Programme). The project covered costs of clearance and shipment of vaccines from arrival in Dar es Salaam to Mwanza. Vaccines were stored at MITU in three refrigerators under the supervision of a pharmacist. Between August 2010 and July 2011, eligible girls were offered three doses of vaccine during four vaccination rounds. On vaccination days, project vehicles took MITU supervisory nurses, EPI-trained nurses from health facilities that covered the selected schools, vaccines, and necessary supplies to the schools according to a prearranged timetable. After vaccination days, girls who missed vaccination at schools were given the opportunity to be vaccinated at health facilities during a window period of 2 to 4 weeks. At the end of the window, remaining vaccines were returned to MITU because of cold storage capacity constraints at the health facilities.The quadrivalent  vaccine (Merck & Co.) was donated by Axios Healthcare Development, Cleveland, Ohio, USA  by a researcher (WQ) during two visits to Mwanza in November 2010 and April 2011. Interviews were conducted with all relevant project staff members, as well as with regional and district EPI staff. Logbooks of project cars were evaluated to determine distances travelled. Vaccination of girls was observed by WQ during the second round of vaccination at four schools to record the time that nurses spent on vaccination activities at different types of school. During the third round of vaccination, MITU nurses recorded the time spent at schools and travel times between MITU, health facilities and schools for 40 schools. These data were complemented by time data recorded in logbooks of project cars to estimate the proportion of total time that MITU nurses spent for procurement and vaccination for each type of school . In addition, vehicle logbooks were used to determine the proportion of total km travelled for delivering vaccines to rural and urban schools .All cost data were distributed into five broad categories: (I) personnel, (II) other recurrent , (III) allowances, (IV) transport, and (V) other capital costs for equipment. Annual capital costs were estimated based on replacement costs  and assuming a life expectancy of 10 years for vehicles and refrigerators, and 5 years for other electronic equipment. Annual financial costs were calculated through straight-line depreciation, while annual economic costs were calculated using a 3% discount rate . An estiCosts of personnel were assigned to project activities based on staff estimates. Time inputs from government employees contributing to the project  were included under economic costs. Other recurrent costs  could be directly attributed to the relevant activities based on information provided in the project management accounts. As vaccines were donated, financial costs exclude costs of the vaccine. Economic costs assume a price per dose of HPV vaccine of US$5 . In someCosts of training and social mobilization/information, education, communication (IEC) were treated as start-up costs and annuitized over 5 years. All cost data were adjusted to the year 2011 using country-specific GDP deflators .Average financial and economic costs per fully-immunized girl, that is, per girl who received three doses of vaccine, were calculated by delivery strategy (age-based or class-based) and school location  using a two-step approach. First, total project costs for preparations, social mobilization/IEC, training, administration/supervision, and for most cost elements of procurement and vaccination, were assigned to schools by delivery strategy and school location. Cold storage, recurrent costs of procurement and vaccination, and waste management were allocated to schools on the basis of the average number of doses delivered in each type of school. Second, costs per school were divided by the average number of fully-immunized girls per school. Table A model was constructed in order to estimate the incremental financial and economic costs of a scaled-up vaccination program in Mwanza Region using an ingredients approach. Reflecting the plans of the Tanzanian government , the modThe model calculated total costs for Mwanza Region and per fully-immunized girl by multiplying the assumed resources per vaccinated school and girl by the estimated unit costs. First, costs were estimated for a 'base case scenario' using a number of 'base case' assumptions, which are shown in Table Base case assumptions usually reflect findings from the project cost analysis, interviews conducted with regional and district EPI staff, a standard discount rate , numbersUnivariate and multivariate sensitivity analyses (SA) were carried out using the 'high cost' and 'low cost' assumptions. For the univariate sensitivity analysis, one model parameter was varied at a time with all other parameters kept constant. For the multivariate SA, all variables were simultaneously set to the 'low cost' assumptions or 'high cost' assumptions, respectively, in order to generate the largest possible range of costs in an analysis of extremes .Total economic project costs for delivering 3 doses of HPV vaccine to 4,211 girls in Mwanza City and Misungwi district (excluding research related activities) were estimated to be US$349,000  showed that excluding costs of HPV vaccines would reduce the costs per fully-immunized girl to US$9.76 and a cost per dose given of US$3.09. At 55% coverage for the third dose of HPV vaccine, costs per fully-immunized girl would increase to US$31.99  prevention of cervical cancer is a public health priority, (2) the introduction is programmatically feasible, (3) sustained financing can be secured, and (4) the cost effectiveness is considered . HoweverThis is one of the first studies to report detailed resource-use-based cost estimates for delivering HPV vaccines to schoolgirls in sub-Saharan Africa. Using data collected alongside a cluster randomized trial, our project cost analysis demonstrated large differences in the costs of delivering HPV vaccines to girls between age-based and class-based delivery. In addition, modeled costs of a scaled-up vaccination program for Mwanza Region in Tanzania showed that three doses of HPV vaccine, when excluding costs of the vaccine itself, could be delivered to girls at an incremental economic cost of US$3.09 per dose and about US$10 per fully-immunized girl. These estimates are similar to the US$3.15 per dose that have been reported by PATH and the national EPI program for a school-based HPV vaccination program in Uganda , but sigOur study has a number of limitations. First, despite our high reliance on observed data, the analysis of project costs was based on estimates and extrapolations for certain inputs. Personnel time was allocated to different activities of the intervention on the basis of staff interviews, which are a known source of uncertainty , althougSecond, our scaled-up cost estimates, which aimed to overcome the problems of the project cost analysis by adjusting the costs for administration/supervision, transport and cold chain, have other limitations related to the adopted ingredients approach for modeling of costs. National level costs (such as administrative and supervision costs) were excluded, although these cost categories can be important; and the ingredients approach may underestimate inefficiencies. For example, for cold storage costs, the incremental costs of HPV vaccines were calculated as the costs of the additional volume required for storing the vaccines. However, if the introduction of HPV vaccines required an increase in the number of refrigerators or cold rooms, which would not be used during non-HPV-vaccination months, the incremental costs would be much higher. For cold storage, such a scenario was tested in the sensitivity analysis, which revealed that increasing the number of refrigerators in Mwanza Region to what district cold chain coordinators deemed necessary for adding HPV vaccination would increase the costs per fully-immunized girl by about 6%, that is, from US$26.41 to US$27.90 Figure .Third, our model calculated scaled-up costs based on experiences gathered over the course of the research project and adjusted observed costs based on assumptions derived from interviews with EPI staff in Mwanza City and Misungwi District. The model assumed that similarly high coverage rates would be achieved in a regional program as in the research project; that distances travelled from district stores to health facilities would be similar for all rural districts as those in Misungwi District; and that wastage rates of vaccines and materials would be similar to those in existing EPI programs . Howeveret al. [Despite these caveats, our research has major implications for policymakers and researchers. Firstly, in our project cost analysis, we found that costs per fully-immunized schoolgirl of delivering three doses of HPV vaccines using a class-based delivery strategy were about one-third below those of using age-based delivery, although the costs per school reached were not higher for age-based delivery . In a set al.  and are et al. ,34 than et al.  , a tradSecondly, we estimated scaled-up costs of US$26 per girl fully immunized against HPV. Even when excluding vaccine costs  or per  [et al. ) would b [et al.  and the  [et al.  present The introduction of HPV vaccination in sub-Saharan Africa offers a new opportunity for cervical cancer control. Our cost estimates suggest that the vaccine can be delivered at costs that would make HPV vaccination a very cost-effective intervention . PotentiDW-J has received grant support through LSHTM from GlaxoSmithKline Biologicals. WJE's partner works for GlaxoSmithKline. There are no other conflicts of interest.DW-J conceptualized the study, which was then designed in close collaboration by all authors. WQ, JC, SS, SK, and DW-J assured data collection in Tanzania. WQ, FT-P, WJE, RHu, DAR, RHa, and DW-J analyzed and interpreted the data. WQ drafted the manuscript. All authors critically revised and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/10/137/prepubAdditional tables. Table S1: activities performed during the Mwanza human papillomavirus (HPV) vaccination project. Table S2: program elements and included inputs for modeling of scaled-up regional vaccination program. Table S3a: project costs by activity: total financial and economic costs (year 2011 US$) for vaccination of 4,211 girls. Table S3b: project costs by cost category: total financial and economic costs (year 2011 US$) for vaccination of 4,211 girls. Table S4: data for Figure Click here for fileFigure S1. Economic costs (year 2011 US$) per fully-immunized girl in a scaled-up regional vaccination program and in the Mwanza Vaccine Project (class-based delivery strategy) by school location.Click here for fileFigure S2. Economic costs (year 2011 US$) per vaccinated school in the Mwanza Vaccine Project by school location and vaccination strategy.Click here for file


The purpose, methods, data sources and assumptions behind the World Health Organization (WHO) Cervical Cancer Prevention and Control Costing (C4P) tool that was developed to assist low- and middle-income countries (LMICs) with planning and costing their nationwide human papillomavirus (HPV) vaccination program are presented. Tanzania is presented as a case study where the WHO C4P tool was used to cost and plan the roll-out of HPV vaccines nationwide as part of the national comprehensive cervical cancer prevention and control strategy.The WHO C4P tool focuses on estimating the incremental costs to the health system of vaccinating adolescent girls through school-, health facility- and/or outreach-based strategies. No costs to the user  are included. Both financial  and economic costs are estimated. The cost components for service delivery include training, vaccination , social mobilization/IEC , supervision, and monitoring and evaluation (M&E). The costs of all the resources used for HPV vaccination are totaled and shown with and without the estimated cost of the vaccine. The total cost is also divided by the number of doses administered and number of fully immunized girls (FIGs) to estimate the cost per dose and cost per FIG.Over five years (2011 to 2015), the cost of establishing an HPV vaccine program that delivers three doses of vaccine to girls at schools via phased national introduction  in Tanzania is estimated to be US$9.2 million (excluding vaccine costs) and US$31.5 million (with vaccine) assuming a vaccine price of US$5 . This is equivalent to a financial cost of US$5.77 per FIG, excluding the vaccine cost. The most important costs of service delivery are social mobilization/IEC and service delivery operational costs.When countries expand their immunization schedules with new vaccines such as the HPV vaccine, they face initial costs to fund critical pre-introduction activities, as well as incremental system costs to deliver the vaccines on an ongoing basis. In anticipation, governments need to plan ahead for non-vaccine costs so they will be financed adequately. Existing human resources need to be re-allocated or new staff need to be recruited for the program to be implemented successfully in a sustainable and long-term manner.Reaching a target group not routinely served by national immunization programs previously with three doses of vaccine requires new delivery strategies, more transport of vaccines and health workers and more intensive IEC activities leading to new delivery costs for the immunization program that are greater than the costs incurred when a new infant vaccine is added to the existing infant immunization schedule. The WHO C4P tool is intended to help LMICs to plan ahead and estimate the programmatic and operational costs of HPV vaccination. Cervical cancer caused by infection with carcinogenic types of human papillomavirus (HPV) is the second most common cancer in women worldwide according to age-standardized incidence rates (ASR). In 2008, there were more than a half million new cases and 274,000 deaths due to cervical cancer . More thThe World Health Organization (WHO) recommends routine vaccination of 9- to 13-year-old girls to protect against HPV infections with types 16 and 18, which contribute to the development of approximately 70% of cervical cancers . While tAs part of developing Tanzania's national comprehensive strategy for cervical cancer prevention and control, the Ministry of Health and Social Welfare (MOHSW) requested technical assistance from WHO to estimate the service delivery costs of introducing HPV vaccine into the country. This request was also in response to a MOHSW agreement to accept a three-year donation of HPV vaccine from the manufacturer. To address this request, WHO and their consultants developed the Cervical Cancer Prevention and Control Costing (C4P) tool in order to facilitate the decision-making process of program managers and policy makers by generating information on the projected costs of introducing cervical cancer interventions. The WHO C4P tool consists of a HPV Vaccine Module and a Cervical Cancer Screening and Treatment Module. As the latter component is still under development, this paper will focus only on the HPV vaccination component of the tool.This paper aims to present the purpose, definitions, methods, data sources and assumptions behind the generic WHO C4P tool to assist LMICs with planning and costing their nationwide HPV vaccination program. Furthermore, Tanzania's experience and cost results will be presented in piloting the WHO C4P tool to scale up prevention interventions as part of their national comprehensive cervical cancer prevention and control plan.The generic costing tool is a country-specific costing and planning tool that facilitates data collection and enables the user to estimate and project the value of incremental  resources required to add the country-wide delivery of HPV vaccine to an existing immunization program over a five-year period. In other words, it only estimates the value of new resources needed and does not include the cost of other goods and services  already being used for other vaccines (shared costs). For example, it does not estimate the cost of transporting HPV vaccine if this is part of the same transport used to deliver other vaccines such as rotavirus or traditional Expanded Program on Immunization (EPI) vaccines, such as diphtheria, tetanus and pertussis (DTP) from the central warehouse to the periphery in the country. Since the WHO C4P tool focuses on estimating the incremental costs of vaccinating adolescent girls from a public health care provider perspective, no costs to the user  are included.Experience from the above-mentioned small-scale demonstration projects showed ,6 that tThe HPV Vaccine Module of the WHO C4P tool enables the user to estimate the additional resource requirements based on identifying different vaccine delivery scenarios that could potentially be considered for the country. The tool allows the user to define different vaccine delivery strategies, for example, through schools, health facilities or campaigns, such as national immunization days. The tool provides estimates of several cost measures: (1) total costs of introducing the HPV vaccine in specific regions/provinces or at the national level; (2) delivery cost per dose; and (3) delivery cost per fully immunized girl (FIG) defined as the cost per dose multiplied by the total number of doses delivered over three vaccination rounds divided by the total number of girls who received all three doses as a function of coverage and dropout rates over all three vaccination rounds.The generic version of the WHO C4P tool is built on the experience of Tanzania. It was further developed and critically assessed by costing experts representing five WHO regions during a WHO workshop in December 2011 in Geneva. A beta version of the MS Excel tool  and capital costs as well as financial and economic costs. It also presents expenditures required for investments necessary during the first years of HPV vaccine introduction.Recurrent costs are defined as the value of resources that last less than one year. These include program costs such as the value of personnel time, transport, maintenance, monitoring and evaluation, and supervision as well as costs of short-term training activities that last less than a year .Capital costs are the value of resources that last longer than one year, such as cold chain equipment and vehicles. The capital goods and services used in HPV vaccination include initial investments such as introduction costs  as well as additional cold chain equipment, vehicle requirements and incinerators.Both financial and economic costs are calculated in the WHO C4P tool. The user can choose which one is most appropriate depending on the objective of the analysis. If the user wants to know the additional costs incurred or actual expenditures by the Ministry of Health, for example, they should focus on the financial cost calculation. Financial costs are the value of resources to the payer and include the value of actual resources purchased for the HPV vaccine introduction such as injection supplies, outreach allowances and per diem, resources used in training, and developing new communication materials useful for budgeting purposes.Economic costs comprise the value of all outlays for vaccine introduction as well as those already paid for or owned by the Ministry of Health and other sources of financing, for example, the salaries of health personnel, vaccines paid for by partners and time of volunteers. This analysis is useful if the user is interested in evaluating the share of different sources of finance for the vaccine introduction. For example, they may want to know the share of total costs financed by the Ministry of Health, external partners, clients and the community. This analysis gives a more complete picture of resources that are tied up in the provision of the new vaccine and their opportunity costs and should be used if a cost-effectiveness or cost-benefit analysis is to be conducted.Capital costs are calculated differently depending on whether financial or economic costs are being estimated. When calculating financial costs, straight-line depreciation is used in the calculation of capital costs. In other words, the cost of the item is annualized through dividing it by the useful life years of the good. For example, cold chain equipment could be expected to last for ten years and the total cost would be divided by ten. Straight-line depreciation assumes that capital goods are used up equally over the useful time period of the item. For economic costs, capital goods are discounted (at a 3% default discount rate) as well as annualized. This type of depreciation assumes that people have time preference and prefer to use goods and services now rather than in the future.The main differences between financial and economic costing are: (1) the time spent by health personnel, school teachers and volunteers is valued in economic costing since there is an opportunity cost to this time - for example, the workers are unable to spend time on other activities when they are occupied with HPV vaccination - but are not included in financial costs since these are already paid for with government salaries; (2) the value of donated goods and services is included in economic costs but not in financial costs since there is an opportunity cost to their use; and (3) capital costs are calculated differently for financial and economist costs.For planning purposes the user is able to identify and separate introduction costs  from recurrent costs. Introduction costs are defined as resources required during the initial years of vaccine introduction (some may occur during the second or third year if the country has phased in vaccine introduction) while the recurrent costs are the running costs of the program such as transport of vaccines and health workers, allowances and per diems and monitoring and supervision.During October 2010 to May 2011, data were collected from the MOHSW/EPI on the costs of training, social mobilization/IEC, vaccination, supervision, and monitoring and evaluation. The study team collected primary and secondary data on target populations, health facilities, schools and costs of resources used to provide HPV vaccination from the MOHSW website, and Ministry of Education website  workshops, district supervisors and vaccinators. Social mobilization and IEC activities included the following: (1) a sensitization, mobilization and communication guidelines and tools preparation workshop; (2) a district campaign preparation workshop; (3) materials development and production; (4) materials printing and airing; (5) sensitization meetings with the community; and (6) a media seminar for journalists. Radio spots are projected to air all three years, but national television advertisements will only take place in the third year when the vaccination is introduced in all of the regions, including Zanzibar.The financial costs of service delivery are comprised of per diems and transport allowances for the health worker and per diems for the school teacher which have to be paid on top of their regular salaries. Procurement costs are incurred for receiving and storage of the vaccines at the port and transport of the vaccines to the zonal level, but do not include vaccine costs since these are donated commodities. The monitoring and evaluation costs are comprised of production costs for tally sheets and vaccination cards. The costs of supervision are for transport (fuel and maintenance of vehicles) and per diems incurred during the quarterly visits from the national level to the regions and districts. The waste management costs are for construction of incinerators, fuel for incinerators and/or transport of waste.Economic costs of service delivery include the value of resources for financial costs as well as the value of the time of personnel time and donated vaccines and capital items.In the scenario examined, 1.6 million girls are fully vaccinated  over five years out of a total target population of 2.4 million girls  Table The five-year cost of introducing a national phased-in HPV vaccine program during 2011 to 2015 in Tanzania is estimated to be US$9.2 million (excluding vaccine costs) and US$31.5 million (with vaccine) assuming a vaccine price of US$5 (GAVI 2011). This is equivalent to a financial cost of US$5.77 per fully immunized girl, excluding the vaccine cost.Table Figure Table Table When countries expand their immunization schedule with a new vaccine such as HPV, they face initial costs to fund critical pre-introduction activities, as well as incremental system costs to deliver the vaccines on an ongoing basis. By using the WHO C4P, the Tanzania experience of introducing HPV vaccine through a phased school-based delivery strategy in all the 26 regions shows that the five-year recurrent total costs of introducing HPV vaccine (excluding vaccine cost) are around US$9.2 million, which is equivalent to US$5.77 per FIG to the government. These recurrent costs correspond to 0.7% of the government real expenditures on health, indicating that substantial ministry of health government resources, such as health personnel, are required to deliver the vaccine effectively. Actual resources required are dramatically higher when one considers the vaccine costs and shared costs included as part of the economic costs analysis, that is, approximately US$59 million over a five-year period.This study shows that, other than procurement, the most important costs of service delivery are social mobilization/IEC and service delivery operational costs. Social mobilization and IEC activities are particularly important to ensure that broad coverage is achieved by informing the population about the benefits of HPV vaccination and supporting the service delivery strategy used. Governments need to plan ahead for these non-vaccine costs so that they will be financed adequately and human resources need to be re-allocated appropriately for the program to be successfully implemented in a sustainable and long-term manner.a. These studies demonstrate that introduction costs for HPV vaccines are higher than those for existing vaccines such as meningitis A campaigns and combination DTP-HepB-Hib vaccine via routine infant EPI vaccination programs [To date, data availability on national HPV vaccination in LMICs is limited, which makes it difficult to validate the tool. However, the findings from the WHO C4P tool for Tanzania are consistent with existing information on recurrent costs from various small-scale demonstration projects from the Program for Appropriate Technology in Health (PATH) in India, Peru, Uganda and Vietnam  and the programs ,9 due toFrom the nationwide-modeled experiences based on PATH demonstration projects, the average introduction costs per eligible girl are US$2.99 (range US$2.82 to US$3.07) and the recurrent costs to deliver three doses per eligible girl are US$4.17 (range US$3.51 to US$4.78) [The resource requirements of IEC activities are a large component of total costs since these are considered to be important aspects for a successful introduction of HPV vaccination, a new vaccine that targets a non-traditional population of adolescent girls. The population will need to be assured of its safety and benefits and be provided with an explanation of why this vaccination is given only to girls and not boys. The costs of IEC activities are calculated for the following activities: (1) sensitization meetings with community leaders to inform them of the benefits of the intended vaccination activities; (2) production of leaflets and posters on the benefits of HPV vaccinations to be placed by service providers in clinics, schools and public locations in their catchment areas; (3) design and production of radio and/or television announcements on the HPV vaccine for the population; and (4) briefings with writers, journalists, editors, publishers and other media personnel to inform them about the benefits of the vaccine. As HPV vaccination is scaled up in these countries, more IEC activities will be required, given that airing of radio and TV announcements will be more effective once the vaccination is scaled up nationally.et al. in preparation). Secondly, monitoring and evaluation costs are restricted to production of tally sheets and vaccination cards. In reality, additional quality control or evaluative measures, such as cost of administrative personnel to evaluate coverage levels might be required.The WHO C4P tool in its current version has several limitations. First, depending on the countries' characteristics, additional sizable costs might be expected from a societal perspective that are not included in the WHO C4P tool, such as private costs to schoolgirls, parents and caregivers and additional costs for the cold chain. Variation in the incremental cost to the health system of vaccinating adolescent girls by different countries is expected and can potentially be explained by country characteristics, such as size of the country, population density and proximity of health facilities to schools, current infrastructure of schools and health facilities and national income level as well as the intensity of the HPV vaccine introduction effort , while 17.4 thousand women out of 60 thousand VIA-positive women receive treatment. This estimate is based on screening at the health dispensary level and above and treatment at the health center level and above . CompletFinally, HPV vaccines are not the only new vaccines being considered for introduction in LMICs. There are also pneumococcal and rotavirus vaccines as well as several older vaccines that have not yet been broadly adopted in developing countries. Although intended for national HPV vaccination costing and planning purposes, the economic cost results of the WHO C4P tool can be used as an input for cost-effectiveness analysis and/or budget impact analysis in order to assist countries in setting their priorities between competing vaccines or other cervical cancer control options. In-country cost estimates of the programmatic costs of delivering an adolescent vaccine and scaling up of cervical cancer screening and treatment interventions as an input for cost-effectiveness analysis are rare. For instance, a health and economic impact study of HPV vaccination and cervical cancer screening in five Eastern African countries would have benefited from a country-specific data collection and projection costing tool such as WHO C4P to estimate these programmatic costs  among otThe financial delivery costs of nationwide HPV vaccination are higher than those of infant vaccines and can be substantial in resource-poor settings since it requires building up new delivery channels. As a consequence, governments need to plan ahead for these non-vaccine costs so that they will have adequate finances in place for vaccine introduction.As GAVI's vaccine portfolio is expanding and countries are expected to introduce new vaccines at an increasing rate, it is recommended to include  funding for operational health system costs in order assist GAVI eligible countries in introducing HPV vaccines. Together with other decision-making tools, the WHO C4P tool could facilitate both low- and middle-income countries in demonstrating their ability to deliver HPV vaccines nationwide to the target population in an effective, sustainable and affordable manner.ASR: age-standardized incidence rates; C4P: cervical cancer prevention and control costing; CEA: cost-effectiveness analysis; cMYP: comprehensive multi-year plan; EPI: Expanded Program on Immunization; FIG: fully immunized girl; GAVI Alliance: formerly the Global Alliance for Vaccines and Immunisation; HPV: human papillomavirus; IEC: information: education and communication; LMICs: low- and middle-income countries; LSHTM: London School of Hygiene and Tropical Medicine; M&E: monitoring and evaluation; MOHSW: Ministry of Health and Social Welfare; NIP: national immunization program; NUVI: new and under-utilized vaccines implementation; PATH: Program for Appropriate Technology in Health; UNDP: United Nations Development Program; VIA: visual inspection with acetic acid; WHO: World Health Organization.The authors declare that they have no competing interests.RH, AL, SW and NB contributed to the conception and design of the study and the C4P tool. AL and WM developed the C4P tool under supervision and directions from RH, SW and NB. RH, SW, AL, MA and TJ were responsible for the acquisition of data in Tanzania. RH, SW, AL, WM and MA analyzed and interpreted the data; all co-authors have been involved in drafting the manuscript or revising it critically for important intellectual content. All authors have read and approved the final manuscript.aPersonal communication with Mr. Tandin Dorji, Chief Program Officer, Department of Public Health, Ministry of Health, Bhutan; and Mr. Maurice Gatera, Head of Vaccine Preventable Disease Division, Institute of HIV/AIDS Disease Prevention and Control, Rwanda Biomedical Center, Ministry of Health, Rwanda. Permission was obtained to quote their names.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/10/136/prepub


To the Editor: In February 2008, a 2-year-old female gray brocket deer (Mazama gouazoubira) at Pomerode Zoo in Santa Catarina, Brazil, exhibited sublingual swelling, drooling, lethargy, prostration, glossitis, slight cyanosis, and blood on the perineum. The animal showed progressive anorexia, ataxia, dyspnea, marked cyanosis, hypothermia (34.5°C), and severe anemia (hematocrit 4.6%). The deer died 5 days after the first clinical signs. Necropsy indicated that the gray brocket deer had pulmonary congestion and edema, mild renal congestion, and renal focal necrosis. Some intestinal vessels along the serosa presented with hyalinization of the walls with mild leukocytic infiltrate of neutrophils featuring vasculitis.Mazama nana) from the same zoo suddenly died. Findings on necropsy were cyanosis and petechiae in the oral mucosa, tongue, and gastrointestinal mucosa. There was bloody intestinal content, petechiae in the mucosa of the urinary bladder, and also petechiae and ecchymoses in the pericardium and epicardium. The spleen was contracted; lymph nodes and kidney medullae were hemorrhagic. The lungs showed congestion and petechiae, and the airways had a frothy fluid content. Histopathologic lesions included mild diffuse congestion in the pygmy brocket deer’s kidneys and extensive subendocardial hemorrhage. Seventeen days after this first case, a 1-year-old male pygmy brocket deer (Bluetongue virus or Epizootic hemorrhagic disease virus), we performed virus isolation and reverse transcription PCR on tissues  from the pygmy brocket deer. (Virus isolation was not carried out on specimens from the gray brocket deer because brain tissue samples were inadequate and results of PCR were negative for epizootic hemorrhagic disease virus [EHDV] or bluetongue virus [BTV].) Infection with EHDV and BTV was first diagnosed by virus isolation. Pooled specimens from spleen-liver and heart tissue were inoculated in embryonated chicken eggs and thereafter in BHK-21 cells  are involved in outbreaks of hemorrhagic disease, which occur mainly in late summer and early autumn in various parts of the world, probably because of the increase in the Culicoides population, the biologic vectors of these viruses. Clinical signs of EHDV and BTV infections are indistinguishable in deer and are characterized by severe depression, respiratory distress, anorexia, and blood-tinged oral and nasal discharge. Gross lesions are mainly edema and hemorrhage caused by vascular injury (EHDV and BTV (Blastocerus dichotomus) populations in São Paulo and Mato Grosso do Sul revealed that 74% of animals were seropositive for EHDV, 88% were seropositive for BTV, and 60% were seropositive for both viruses (Culicoides biting midges. This low serologic prevalence probably produces an area of enzootic instability in which epidemic cases occur that are associated with the presence of infected vectors and susceptible animals (Clinical reports of EHDV infection have occurred in Australia, Asia, Africa, and North America (M. nana deer are the most threatened species in Brazil (In summary, we describe the isolation of EHDV that is involved in hemorrhagic disease affecting pygmy brocket deer in Brazil. The hazard that this virus infection could pose to the local and national Cervidae populations remains unknown, but it is noteworthy that