To the Editor: In March 2009, a new strain of influenza A (H1N1) virus of swine origin emerged; the virus had crossed the species barrier to humans and acquired the capability of human-to-human transmission. Soon after, the World Health Organization raised the worldwide pandemic alert to level 6 (www.who.int/en), declaring the first influenza pandemic in the past 42 years. The virus was named influenza A pandemic (H1N1) 2009 virus. The illness caused by this virus is particularly dangerous for pregnant women and for patients with chronic diseases  2009. All samples were first tested for the H275Y mutation by using an in-house real-time reverse transcription–PCR (RT-PCR) assay developed at the Central Virology Laboratory of Chaim Sheba Medical Center; positive results were confirmed by sequencing. The histidine-to-tyrosine mutation at the 275 position of the neuraminidase protein results in reduced binding of oseltamivir.During June–August 2009, ≈80 children in an institution for disabled children were suspected of being infected with pandemic (H1N1) 2009 virus. The children had influenza-like signs and symptoms, and at that time the only influenza virus circulating in Israel was pandemic (H1N1) virus. Of these 80 patients, 10 were hospitalized because of the severity of their clinical signs or disease complications, and for 7, RNA of the pandemic (H1N1) 2009 virus was detected in throat and nasal swabs by real-time RT-PCR.Patient 1 was a 13-year-old boy with cerebral palsy and partial blindness, who was treated with oseltamivir (60 mg twice a day) for 5 days . After some improvement, his condition worsened, and he was hospitalized on August 13 for breathing difficulty and high fever. Real-time RT-PCR indicated infection with pandemic (H1N1) 2009 virus. During our survey, we found that patient 1 was infected with a virus carrying the H275Y mutation, suggesting that the mutation evolved during oseltamivir treatment.Patient 2 was a 10-year-old girl who had lived in the room next to patient 1 and who also had cerebral palsy. Her signs and symptoms started on August 13, 2009, and she was hospitalized on August 15. She was treated in the hospital for 5 consecutive days with oseltamivir, steroids, and augmentin; she was discharged on August 21. Her diagnosis was made early in the clinical course of her infection, and she was infected with pandemic (H1N1) 2009 virus carrying the H275Y mutation.In contrast, none of the other 8 children who were hospitalized for pandemic (H1N1) 2009 carried the mutation. Although we cannot rule out the possibility that the virus was transmitted by a third person, we suggest that the virus carrying the resistance mutation was probably transmitted from patient 1 to patient 2. This transmission is probable because these 2 patients lived in adjacent rooms, they were in contact with each other, clinical onset of patient 1 preceded that of patient 2 by a few days, and patient 2 had the mutation at the beginning of her disease.Fortunately, despite the conditions that favor virus spread in such institutions, this virus did not seem to spread further; the other 8 hospitalized children from this institution were infected with the wild-type virus. Nevertheless, the potential for spread of pandemic (H1N1) 2009 virus carrying the oseltamivir resistance mutation exists, thereby emphasizing the urgent need for a vaccination to prevent illness and for alternative drugs to treat infected patients.


Paramyxoviridae. Fruit-eating bats of the Pteropus genus have been suggested as their natural reservoir. Human Henipavirus infections have been reported in a region extending from Australia via Malaysia into Bangladesh, compatible with the geographic range of Pteropus. These bats do not occur in continental Africa, but a whole range of other fruit bats is encountered. One of the most abundant is Eidolon helvum, the African Straw-coloured fruit bat.Henipaviruses (Hendra and Nipah virus) are highly pathogenic members of the family E. helvum roosting in an urban setting in Kumasi/Ghana were tested for Henipavirus RNA. Sequences of three novel viruses in phylogenetic relationship to known Henipaviruses were detected. Virus RNA concentrations in feces were low.Feces from The finding of novel putative Henipaviruses outside Australia and Asia contributes a significant extension of the region of potential endemicity of one of the most pathogenic virus genera known in humans. Paramyxovirinae in the family Paramyxoviridae comprises the five genera Respiro-, Morbilli-, Rubula-, Avula-, and Henipavirus, as well as a group of yet unclassified viruses The subfamily E. helvum, a fruit-eating bat that occurs in Africa but not in Asia, implicated the presence of antigenetically related viruses E. helvum is highly abundant in Africa and colonies are believed to conduct annual transcontinental migration, following the rainfall gradient to suitable feeding grounds, with reports on individual bats travelling up to 2,000 kilometres E. helvum is a rather large fruit bat that roosts exposed and at high densities in trees during daytime. The animals are frequently hunted and consumed by humans as a supplementary source of protein Interestingly, a recent serological study on E. helvum in Ghana. Jointly with recent serological data In spite of serological evidence, it has never been confirmed so far that Henipaviruses exist in African bats. In the current study we positively identified putative Henipaviruses for the first time in For all capturing and sampling, permission was obtained from the Wildlife Division, Forestry Commission, Accra, Ghana. Samples were exported under a state contract between the Republic of Ghana and the Federal Republic of Germany, and under an additional export permission from the Veterinary Services of the Ghana Ministry of Food and Agriculture.E. helvum by trained field biologists on site. Additionally, of those samples that yielded positive RT-PCR results, mitochondrial DNA was amplified and sequenced for species confirmation as described Geographic co-ordinates of the sampling site were N06°42′02.0″ W001°37′29.9″. Bats were identified as Myotis nattereri, E. helvum and Rousettus aegyptiacus bats (own unpublished data). No cytopathic effect was observed and no virus growth was seen by RT-PCR, despite repeated trials (data not shown).Based on prior findings E. helvum fruit bats was studied in the zoological gardens of Kumasi, Ghana (2) was laid out in the late afternoon and early in the morning under several trees densely occupied by bats, in order to collect fecal samples. Due to a low frequency of droppings received on the plastic film (about one dropping per 2 minutes), and due to the distance between the individual droppings on plastic film, it was assumed that individual droppings stemmed from different individual bats.During February 2008, a large colony of i, Ghana . The colParamyxoviridae reference strains, including Hendra and Nipah viruses from GenBank. Supplemental In total, three of 215 fecal samples yielded RT-PCR products. 496 base pair fragments of all products were sequenced and aligned with homologous fragments of Paramyxoviridae taxonomy. The three viral sequences from E. helvum were in close or basal association with the currently established genus Henipavirus . Individual E. helvum. The genetic distance between these viruses and Hendra and Nipah virus was compatible with the distance observed within other established Paramyxoviridae genera, suggesting that the novel viruses might constitute members of the Henipavirus genus. The considerable sequence divergence between the three novel putative Henipaviruses encountered in one huge colony might indicate substantial genetic variability of related viruses in bats. Formal taxonomic classification according to conventions for Paramyxoviridae will require the characterization of additional genomic features, such as the phylogenies of the nucleoprotein and F genes, the lengths of intergenic regions, as well as the predicted transcription start sites of subgenomic RNAs Further to serological data provided by Hayman et al. E. helvum that occupied trees in a zoological garden in the centre of Kumasi, Ghana's second largest city with a population of 1.5 million. Large colonies of E. helvum are widely observed in urban areas of Subsaharan Africa E. helvum uses urban habitats only for roosting and rarely for feeding. Nevertheless, detailed studies on the foraging behavior of E. helvum are lacking. Another way of exposition may be via contact with bat urine or fecal material. In this study we have sampled bat feces that are abundant under trees in urban E. helvum roosting sites. Interestingly, the observed virus RNA concentrations in fecal material were rather low as compared to enteric viruses transmitted via the fecal-oral route in humans The novel viruses were detected from wild E. helvum is known to be one of the preferred bat species hunted for wild game in Africa Another way of exposure to bat viruses may be via consumption of bat meat for food or medical purposes Figure S1Nucleotide alignment of Henipavirus reference strains and novel viruses. The entire 439 base pair polymerase fragment is aligned with Hendra virus and Nipah virus reference strains. Hendra virus on top serves as the comparison sequence in the alignment. Dots represent identical bases in compared sequences; deviations are spelled out.(0.02 MB PDF)Click here for additional data file.Figure S2Amino acid alignment of Henipavirus reference strains and novel viruses. The 146 amino acids resulting from translation of a 439 base pair polymerase fragment are aligned with Hendra virus and Nipah virus reference strains. Hendra virus on top serves as the comparison sequence in the alignment. Dots represent identical amino acids in compared sequences; deviations are spelled out.(0.01 MB PDF)Click here for additional data file.


Orf virus (ORFV) is the etiological agent of contagious pustular dermatitis and is the prototype of the genus Parapoxvirus (PPV). It causes a severe exanthematous dermatitis that afflicts domestic and wild small ruminants.In the present study, an outbreak of proliferative dermatitis in farmed goats. The presence of ORFV in tissue scrapings from the lips was confirmed by B2L gene polymerase chain reaction (PCR) amplification. The molecular characterization of the ORFV was performed using PCR amplification, DNA sequencing and phylogenetic analysis of the B2L gene.The results of this investigation indicated that the outbreak was caused by infection with an ORFV that was closely related genetically to Nantou (DQ934351), which was isolated from the Tai wan province of China and Hoping (EU935106), which originated from South Korea in 2008. This is the first report of the phylogenetic analysis of ORFV from goats in China. Parapoxvirus, which also includes pseudocowpox virus (PCPV) in cattle, bovine papular stomatitis virus (BPSV) in cattle, squirrel parapoxvirus (SPPV) and parapoxvirus of red deer in New Zealand (PVNZ) [The ORFV is the prototype member of the genus d (PVNZ) ,2. Contad (PVNZ)  but alsod (PVNZ) -6. Humand (PVNZ) . The disd (PVNZ) .The disease not only has an economic impact on farmers worldwide but also has a considerable negative effect on animal welfare. Infected animals are sickly, fail to thrive, and are more susceptible to adventitious bacterial infections . The typIn recent years, outbreaks of orf have occurred worldwide . AlthougThe outbreak reported in this study originated on a goat farm  in the Hubei province of the People's Republic of China. On 23 March 2009, the farmer bought 655 goats from free-ranging herds and transported them a distance of about 800 km to the farm. On the third day, two goats presented with nodular lesions on the lips, tongue and around the mouth; there were 30 goats showed tubercular lesions three days later. To 31 July 2009, the incidence was approaching 60% and the mortality rate was 24.7% (162/655), although anti-viral and antibiotic medicines were administered in the drinking water, by intramuscular injection or orally.3, were removed carefully from the nodular lesions using a sterile scalpel and stored in a sealed plastic tube with 50% glycerin Phosphate Buffered Saline(PBS) at 20°C until analysis. Primers designed for the amplification of the full length of the B2L gene [Two goats with typical clinical signs were selected and euthanized with an overdose of pentobarbital sodium. Complete post mortem examinations were performed and the macroscopic changes were recorded. Small samples of diseased tissue, about 6-8 mmB2L gene  were synComparison of the B2L sequences with those available in the GenBank database was performed using the online BLAST programs. The sequence identities of nucleotides and amino acids were analyzed by the ClustalW method . A phyloThe clinical signs seen in the goats included: multifocal to coalescing papillary, verrucose, proliferative, and ulcerated lesions in the epidermis of the muzzle and lips Fig. . No visiGU320351.The orthodox methods of diagnosis that depend on pathologic examinations and clinical signs are inaccurate, virus isolation is thought to be a gold standard but it's time-consuming. With thUsing nucleotide sequences of the complete B2L gene stored in GenBank Table , the phyOrf is endemic in China, although a vaccination program has been performed to control the disease. Between 1980s and 1990s, orf occurred in eight provinces of China including Qinghai, Gansu, Tibet, Xinjiang, Liaoning, Jiangxi, Heilongjiang and Hebei. In recent years orf happened in the following provinces Innermongolia (Jun.2005), Guangxi (Mar.2005), Shanxi (Apr.2005), Fujian (Aug.2005), Jilin (Mar.2006), Jiangsu(Nov.2006) and Beijing city (Apr.2006). More seriously, in 2005 there were seven women and four men were infected by ORFV in Fujian province. So, orf is a national zoonoses in China. In this study, we diagnosed an outbreak of orf in Chinese goats and determined its phylogenetic characteristics on the basis of the complete gene sequence of the major envelope protein (B2L). We conclude that the Chinese ORFV involved in this outbreak was closely related phylogenetically to Nantou (DQ934351) and Hoping (EU935106). This is the first report to provide phylogenetic information about an ORFV strain in China, which will be of use for prospective studies in public health.ORFV: Orf virus; PCPV: pseudocowpox virus; BPSV: bovine papular stomatitis virus; SPPV: squirrel parapoxvirus; PVNZ: parapoxvirus of red deer in New Zealand; PCR: polymerace chain reaction; PBS: phosphate buffered solution; bp: base pair; kb: kilobase.The authors declare that they have no competing interests.XL was the leader of the project. KZ carried out most of the studies and drafted the manuscript. JH and JY amplified the complete B2L gene. HZ, ZL and YS provided consultation and preparation of the final report. All authors read and approved the final manuscript.


Ebolaviruses (EBOV) (family Filoviridae) cause viral hemorrhagic fevers in humans and non-human primates when they spill over from their wildlife reservoir hosts with case fatality rates of up to 90%. Fruit bats may act as reservoirs of the Filoviridae. The migratory fruit bat, Eidolon helvum, is common across sub-Saharan Africa and lives in large colonies, often situated in cities. We screened sera from 262 E. helvum using indirect fluorescent tests for antibodies against EBOV subtype Zaire. We detected a seropositive bat from Accra, Ghana, and confirmed this using western blot analysis. The bat was also seropositive for Lagos bat virus, a Lyssavirus, by virus neutralization test. The bat was fitted with a radio transmitter and was last detected in Accra 13 months after release post-sampling, demonstrating long-term survival. Antibodies to filoviruses have not been previously demonstrated in E. helvum. Radio-telemetry data demonstrates long-term survival of an individual bat following exposure to viruses of families that can be highly pathogenic to other mammal species. Because E. helvum typically lives in large urban colonies and is a source of bushmeat in some regions, further studies should determine if this species forms a reservoir for EBOV from which spillover infections into the human population may occur. Marburgvirus (MARV) and Ebolaviruses (EBOV) (family Filoviridae) can cause viral hemorrhagic fevers in humans and non-human primates when they spill over from their wildlife reservoir hosts FiloviridaeRousettus aegyptiacus and LeRoy et al. found serological and PCR evidence of EBOV infection in three other fruit bat species in West Africa R. aegyptiacus than in other species tested R. aegyptiacus live in very large roosts, with populations recorded over 100,000, which could facilitate persistence of infection within roosts.Eidolon helvum, is widespread and common across sub-Saharan Africa. It lives in large colonies, which sometimes number several million animals, often situated in cities The tree-roosting fruit bat, Ethical approval for this project (WLE/0467) was received from the Zoological Society of London Ethics Committee.E. helvum and 3 Hypsignathus monstrosus using indirect fluorescent tests for antibodies against EBOV subtype Zaire and MARV subtype Leiden E. helvum in Ghana between January and April 2008 (n = 173) from urban colonies in Accra (n = 141) and Kumasi (n = 10) and from Tanoboase (n = 22) and from Accra (n = 89) in January and February 2009. Samples from 3 H. monstrosus were collected from Tanoboase in January 2009. The 262 E. helvum samples comprised 2 from neonate, 43 from sexually immature and 217 from mature bats, with an approxiate 2∶1 overall male bias. The 3 H. monstrosus were adult females. Bats were trapped on return to roosting sites in mist nets. Each bat in a subsample (n = 98) of the E. helvum population in Accra was fitted with a radio transmitter .We screened sera from 262 E. helvum (#49), sampled and released in Accra in January 2008, had an IgG antibody titer against EBOV of >1∶80, but was seronegative for MARV. All other samples were seronegative to both MARV and EBOV. The positive reactivity for EBOV was confirmed using western blot against a recombinant nucleocapsid protein of EBOV-Zaire, which was cloned and produced in an E. coli expression vector with His tag Eidolon helvum lyssavirus, Lagos bat virus (LBV), but no antibodies against Mokola virus using the 1968 Nigerian shrew (Crocidura sp.) isolate One pregnant adult female E. helvum, which had specific antibodies to both EBOV-Zaire and LBV, was alive over 13 months post-sampling. This bat appeared to be healthy and showed typical migratory movements for E. helvum in this region of West Africa E. helvum, including E. helvum sympatric with R. aegyptiacus seropositive to EBOV and MARV The novel finding in this study was that an individual female H. monstrosus were found to be positive against EBOV following an EBOV epidemic in humans, and where PCR-positive bats were detected in the region shortly after the outbreak, suggesting recent virus circulation E. helvum known to be uninfected, formal investigation of specificity in our study is impossible. Similarly, the sensitivities of filovirus serological assays on bat samples are unknown. Efforts to ensure that results are accurate here were based on good laboratory approach and the use of western blot analysis to confirm IFA results.Fruit bats are recognised reservoirs of lyssaviruses and henipaviruses, and can have high viral seroprevalences E. helvum in Ghana. Prevalences of other bat viruses, such as lyssaviruses and henipaviruses, for example, are very low even when seroprevalences are relatively high (e.g. around 40%) E. helvum cannot be determined by our study, the presence of detectable anti-EBOV antibody demonstrates that there has been sufficient exposure to viral antigen for the bat (#49) to develop an adaptive immune response. The most parsimonious explanation is infection followed by sufficient virus replication for the animal to mount an adaptive immune response. Insufficient data exist to determine if infection of E. helvum with EBOV can be productive, thus enabling it to serve as an effective reservoir for EBOV, or if this species develops clinical signs when infected with EBOV. Also, studies in individual bat roosts should not be extrapolated to all bats or roosts of that species, but population level infection dynamics must be considered when determining the reservoir status of a species.One in 262 sera is clearly a low seroprevalence and it is likely that virus prevalence is extremely low in E. helvum, which roosts in urban areas, must be considered a possible public health risk. Because E. helvum typically lives in large urban colonies and is a source of bushmeat in some regions Spillover of infection into a large susceptible population, such as


The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus.Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell–deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus–neutralizing antibody reach infected cells in the cerebellum of B cell–deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus-specific antibodies.The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus–specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues. Every year over 50,000 people die from rabies worldwide, primarily due to the poor availability of rabies vaccine in developing countries. However, even when vaccines are available, human deaths from rabies occur if exposure to the causative virus is not recognized and vaccination is not sought in time. This is because rabies virus immunity induced by the natural infection or current vaccines is generally not effective at removing disease-causing rabies virus from brain tissues. Our studies provide insight into why this is the case and how vaccination can be changed so that the immune response can clear the virus from brain tissues. We show that the type of immune response induced by a live-attenuated rabies virus vaccine may be the key. In animal models, live-attenuated rabies virus vaccines are effective at delivering the immune cells capable of clearing the virus into CNS tissues and promote recovery from a rabies virus infection that has spread to the brain while conventional vaccines based on killed rabies virus do not. The production of rabies-specific antibody by B cells that invade the CNS tissues is important for complete elimination of the virus. We hypothesize that similar mechanisms may promote rabies virus clearance from individuals who are diagnosed after the virus has reached, but not extensively spread, through the CNS. Rabies viruses spread from peripheral sites of entry to the central nervous system (CNS) tissues via axonal transport thereby bypassing the specialized features of the neurovasculature known as the blood-brain barrier (BBB). Once the virus reaches CNS tissues three alternative outcomes are likely: (1) the BBB remains intact and the infection is lethal due to the absence of an antiviral CNS immune response (2) immune effectors cross the BBB and mediate a CNS antiviral immune response with extensive immunopathology that contributes to the disease, or (3) immune effectors cross the BBB and clear the virus from the CNS without significant pathological consequences. It is well known that in humans naturally infected with rabies virus the latter outcome is exceedingly rare. In addition, CNS inflammation is generally limited in individuals who succumb to rabies Unlike humans where rabies viruses may take weeks to reach the CNS from the site of exposure −/− mice on a C57BL6 background were obtained from the in-house breeding colony at Thomas Jefferson University. RAG-2−/− mice on a 129/SvEv background were obtained from Taconic . Mice were infected or immunized intranasally (i.n.) with 105 focus forming units of CVS-F3, CVS-N2c or UV-inactivated CVS-F3 in PBS as previously described −/− mice were treated intraperitoneally with 1 mg of the monoclonal, rabies virus glycoprotein-specific, virus-neutralizing antibody 1112 in 500 µl of saline or with the vehicle alone at the time points noted in the figure legends. All procedures were carried out according to the protocols approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University.Eight to 12-week old wild-type control 129/SvEv and C57BL6 mice and JHD™II fluorimeter. The amount of Na-fluorescein in the CNS tissue is normalized to its level in serum by (µg of Na-fluorescein in CNS tissue/mg of tissue)/(µg of Na-fluorescein in serum/µl of serum) and is expressed as a fold increase in fluorescence uptake by comparison with the results obtained from naïve controls.BBB integrity was assessed by quantifying the leakage of a low molecular weight fluorescent marker  from the circulation into CNS tissues as previously described For immunohistochemical analysis, brains from perfused mice were snap frozen in Tissue-Tek O.C.T. Compound , sectioned using a Thermo Shandon cryostat , and fixed in either 80% acetone or 95% ethanol. Immunoglobulin (Ig) was detected using either biotinylated monoclonal rat anti-mouse kappa light chain (1 hour at 1∶50)  followed by Alexa Fluor 568 streptavidin (1 hour at 1∶1000)  or the VECTASTAIN ABC-AP KIT with polyclonal rabbit anti-mouse biotinylated IgG (1∶200) developed using the peroxidase antiperoxidase method and 3′3-diaminobenzidine as substrate  according to the manufacturer's protocol. For the additional staining shown in one of the figure panels (#3D), a 1 hour incubation with 1 mg/ml 1112 was performed prior to detection of Ig. To assess virus infection sections were stained for 1 hour with FITC-conjugated anti-rabies virus nucleoprotein monoclonal antibody (1∶50) . Photographs were taken with a Nikon digital camera on an Olympus BX-60 microscope.Total RNA was isolated from CNS tissue samples and mRNA expression levels of rabies virus nucleoprotein, CD4, CD8, IFN-γ and L13 in CNS tissues were measured by quantitative reverse-transcriptase (RT)-PCR as previously described 2) and incubated with anti-CD16/32 (1 ug/106 cells)  antibody to prevent non-specific binding. Cells were washed in PBS and incubated with anti-mouse CD19 (1∶1000)  and MHC class-II (1∶1000)  antibodies. Phenotypic characterization of antibody-labeled cells was performed on a BD-FacsCaliber Flowcytometer. CD19-MHC class II double-positive cells were defined as B cells. Numbers of rabies virus-antigen specific antibody secreting B cells were assessed using Millipore Multiscreen HA® ELISPOT plates coated with 5 ug/mL of UV-inactivated whole rabies virus. Peripheral blood or brain derived mononuclear cells were suspended in RPMI media supplemented with 25 mM HEPES and 10% FBS and 200,000 cells were incubated in each well for 18 hours. Plates were washed and bound rabies virus-specific antibodies were detected by addition of alkaline-phosphatase conjugated anti-mouse IgG antibody (1∶500)  followed by BCIP/NBT substrate. Spots were counted using a dissecting microscope.Mononuclear cells were prepared from peripheral blood collected by retro-orbital bleeding in heparinized capillary tubes by centrifugation at 300 g for 20 minutes. The white cell layer was washed in PBS twice before analysis. Mononuclear cells were isolated from CNS tissues as described elsewhere by isolation at the interface of a 30/70 Percoll (Sigma) step gradient centrifuged at 800 g for 25 minutes Results are expressed as the mean±standard error mean (S.E.M.). Statistical significance of the differences between groups was tested using the Mann-Whitney test and the symbol * indicates a p value<0.05.Mouse models are not considered to be particularly suited to studies of post-exposure prophylaxis (PEP) with rabies due to the rapid spread of the viruses to the CNS. However, our prior studies suggest that the lethal outcome of rabies in mice is more a consequence of the inability to deliver immune effectors into CNS tissues than its spread −/− mice lack B cells but have functional T cells and, unlike RAG-2−/− mice, which lack both T and B cells, are capable of elevating fluid-phase BBB permeability in response to the infection . This raises the possibility that T cell activities may be able to partly control the virus infection independently of antibody. To gain insight into the contributing T cell subsets, we compared the levels of CD4, CD8 and IFN-γ mRNAs in CNS tissues from wild-type conventional mice 24 days after CVS-F3 infection when there is little virus replication remaining (see below) and JHD−/− mice 40 days after infection. As can be seen in −/− mice but IFN-γ mRNA levels have remained relatively high and may therefore be contributing to the control of virus replication.JHDnfection . These mogresses . Howeverght gain . At the −/− mice . These J−/− mice to clear CVS-F3 from the CNS reaffirms the importance of rabies virus-specific antibodies in this process. However, little is known with respect to how these antibodies may be delivered to infected CNS tissues. Our studies of mice clearing CVS-F3 suggest that the leakage of naturally developing antibodies from the circulation into the CNS tissues may be minimal since elevated BBB permeability occurs before serum antibody titers peak −/− mice were treated with 1 mg of the mouse IgG1 monoclonal rabies virus-neutralizing, glycoprotein-specific antibody 1112, which is highly effective in post-exposure treatment models −/− mice  and after BBB permeability has peaked y higher . This suPrompt administration of PEP is the recommended course for an individual who has come in contact with a rabid animal. Since this prevents the development of clinical rabies it is impossible to be certain how many of the tens of thousands of people who receive PEP on an annual basis have actually been infected with the virus. It is also impossible to know how far the rabies virus may have spread before being cleared by the immune effectors provided or induced by PEP and the infection. The commonly held view that pathogenic wildlife rabies virus that has spread to the CNS cannot be cleared by immune mechanisms is supported by the absence of significant immune cell infiltration into the CNS tissues of individuals who die from rabies −/− mice, which lack B cells but have functional CD4 and CD8 T cells, often survive CVS-F3 infection over extended periods despite being unable to clear virus from CNS tissues and exhibiting neurological symptoms. This leads us to conclude that elements of the T cell response, likely including IFN-γ production by CD4 and CD8 T cells, can control certain features of the infection that make significant contributions to its lethality but that antibody is required for virus clearance. To examine the contribution of circulating antibody to virus clearance from CNS tissues, we administered high levels of the rabies virus neutralizing mouse monoclonal 1112 antibody to CVS-F3-infected JHD−/− mice during the stage of infection when BBB permeability is maximal. While leakage of a 150 kDa molecular weight marker from the circulation into the CNS tissues of CVS-F3-infected mice is minimal over a 4-hour period in vivo appears to be relatively low as considerably greater amounts of the antibody can bind to the cells when applied to tissue sections in vitro. While it is possible that even low levels of virus-neutralizing antibody may impact the replication and spread of the virus while BBB permeability is enhanced, treatment of CVS-F3-infected JHD−/− mice with 1112 antibody failed to clear the virus.To gain further insight into the mechanism of rabies virus clearance from the CNS tissues, we have used gene-deleted mice to study the role(s) of different antiviral immune effectors in the CNS tissues of mice clearing the attenuated rabies virus CVS-F3. Mice lacking T and B cells cannot clear this virus and die from the infection in vitro. This leads us to conclude that the high levels of antibodies required for rabies virus clearance from the CNS tissues are produced at the site of infection rather than diffusing in from the circulation. In this case, passively administered antibody during PEP would primarily impact virus in the periphery and an active immune response leading to elevated BBB permeability and immune effector delivery to the CNS tissues would likely be required to clear virus from the CNS.It should be noted with respect to the origin of the antibodies that participate in rabies virus clearance that serum rabies virus-specific antibody titers peak some time after BBB integrity has been restored As certain of the aspects of BBB function required for immune cell infiltration are unchanged by CNS infection with pathogenic rabies viruses


The development of antimalarial drug resistance has led to increasing calls for the introduction of artemisinin-based combination therapy (ACT). However, little evidence is available on the full costs associated with changing national malaria treatment policy. This paper presents findings on the actual drug and non-drug costs associated with deploying ACT in one district in Tanzania, and uses these data to estimate the nationwide costs of implementation in a setting where identification of malaria cases is primarily dependant on clinical diagnosis.Detailed data were collected over a three year period on the financial costs of providing ACT in Rufiji District as part of a large scale effectiveness evaluation, including costs of drugs, distribution, training, treatment guidelines and other information, education and communication (IEC) materials and publicity. The district-level costs were scaled up to estimate the costs of nationwide implementation, using four scenarios to extrapolate variable costs.The total district costs of implementing ACT over the three year period were slightly over one million USD, with drug purchases accounting for 72.8% of this total. The composite (best) estimate of nationwide costs for the first three years of ACT implementation was 48.3 million USD (1.29 USD per capita), which varied between 21 and 67.1 million USD in the sensitivity analysis (2003 USD). In all estimates drug costs constituted the majority of total costs. However, non-drug costs such as IEC materials, drug distribution, communication, and health worker training were also substantial, accounting for 31.4% of overall ACT implementation costs in the best estimate scenario. Annual implementation costs are equivalent to 9.5% of Tanzania's recurrent health sector budget, and 28.7% of annual expenditure on medical supplies, implying a 6-fold increase in the national budget for malaria treatment.The costs of implementing ACT are substantial. Although drug purchases constituted a majority of total costs, non-drug costs were also considerable. It is clear that substantial external resources will be required to facilitate and sustain effective ACT delivery across Tanzania and other malaria-endemic countries. For over a decade, malaria treatment in sub-Saharan Africa and other endemic regions has been in crisis. Conventional antimalarial drugs such as chloroquine and sulphadoxine/pyrimethamine (SP) have become increasingly obsolete in the face of growing drug resistance. Current debates favour using artemisinin-based combination therapy (ACT) regimens ,2. ACTs By early 2006, 34 countries in sub-Saharan Africa had adopted ACTs as first-line treatment for malaria ,6. The rThere is relatively little evidence on the actual costs associated with changing national malaria treatment policies to accommodate ACT. Three studies have provided estimates of ACT procurement costs for Tanzania or Africa more generally, but none have included other activities such as training, developing and printing clinical guidelines, behaviour change communication (BCC) and publicity materials, or other supplies -11. MoreTanzania is facing the challenge of increasing antimalarial drug resistance. By the late 1990's chloroquine resistance had reached unacceptable levels, failing to provide an adequate clinical and parasitological cure in more than half of the children studied ,16. In 2IMPACT-Tz was a large-scale study intended to evaluate the implementation of ACT in a typical rural district with intense malaria transmission. It involved a comparison of ACT implementation in Rufiji District (using SP plus artesunate), with continued use of the national first-line drug, SP, in two adjacent districts. IMPACT-Tz was one of the first large scale ACT evaluations in sub-Saharan-Africa under 'real life' conditions in which ACT was delivered through existing public health infrastructures with minimal alterations. It aimed to produce robust results, applicable in similar health resource constrained settings in rural sub-Saharan Africa. The study was explicitly designed to answer key operational research questions including those related to: antimalarial drug resistance, malaria transmission, safety, efficacy, costs and affordability, health seeking behaviour, perceptions and community attitudes towards ACT use.In 2004, following initial results from the IMPACT-Tz study and other sites participating in the East African Network for Monitoring Antimalarial Treatment , the TanThis report presents findings from the IMPACT-Tz evaluation on the district-wide costs for three years of activities deploying ACT in Rufiji District. In addition, the projected costs of various ACT policy implementation options at the national level are estimated.2, with an estimated population of 212,144 [Rufiji District is located in Coast Region 178 km south of Tanzania's commercial capital, Dar es Salaam. The District covers an area of 13,339 km 212,144 . See FigAnnual rainfall ranges between 800–1,000 mm in the Rufiji River Basin and malaria transmission occurs all year round, with an entomological inoculation rate of 79–1209 infectious bites per person per year . Malariacascades' with between four to nine health facilities per cascade. Non-governmental health care providers are urged to operate within the Ministry of Health and Social Welfare's standard operating procedures, while receiving supervision from the CHMT.The district has a total of 59 health facilities of which 46 are public, 10 mission-run and only three private for profit. Public health facilities are managed by the Council Health Management Team (CHMT) which is based in the district administrative town of Utete. The health facilities are managed through eight administrative blocks called 'Artesunate (AS) plus SP was introduced for first-line treatment of uncomplicated malaria at all health facilities in Rufiji District from February 2003. This particular combination was introduced not because it was expected to be a future option for first-line treatment, but because it was immediately available, provided an ideal opportunity to gain experience with the ACT approach and could be evaluated against SP monotherapy, the recommended first-line treatment. The design and implementation of the intervention involved a broad spectrum of activities encompassing consultation, building consensus, policy formulation, developing treatment guidelines and other communication materials, training, procurement, repackaging, drug supplies management, and sensitization campaigns. The chronology of these activities is summarized in subsequent Table A stakeholders' meeting was held in Dar es Salaam in October 2002, and key decisions regarding the implementation process were discussed and agreed . The meeIn late 2002, an artist worked with the implementation team among communities in Rufiji District to develop communications and dosing instruction illustrations to aid care-seekers and health workers during drug prescription. Five community meetings of between 10 to 30 people were held across the district during the design of these information, education and communication (IEC) materials. Dosing envelopes showing prescription instructions were developed for four age groups: children less than one year, children under-five, school aged children and adults . These wTraining of Rufiji District health personnel began in January 2003. The first training involved at least two staff (prescribers and drug dispensers) from all 56 public and mission health facilities. The training was carried out in a centrally located primary school over two days. Training focused on indications and exclusion criteria for the use of ACT, age specific dosing instructions, and the importance of emphasising adherence to dosage regimens. Clinicians were trained to identify severe or cerebral malaria cases and to abide by national treatment guidelines for such cases. The training also included a module on identifying clinical signs of adverse drug reactions resulting from use of prescribed antimalarial drugs. All training activities were conducted by the CHMT with assistance from the IMPACT-Tz project including the principal investigators, some of whom had experience with national level training during the 2001 change of national malaria treatment policy. Following this initial workshop, participants were charged with conducting similar training sessions with health facility staff under their supervision upon returning to their posts. A follow-up training (refresher course) was conducted 18 months after initial ACT introduction, and later that year a separate training course was organized on adverse drug reactions for supervising health workers at all facilities.Training for community leaders and primary school teachers was conducted two years after ACT introduction to increase awareness in the community and sensitize communities to the health and socio-economic consequences of malaria. Project staff trained supervising clinicians at each of the eight administrative blocks (cascades) who then went on to train 104 primary school teachers and 16 ward community leaders as well as volunteer village health workers.® 50 mg, Sanofi-Aventis, Gentilly, France) were purchased from a single European supplier at an internationally negotiated discount price. The first consignment of artemisinin drugs was delivered to Kibiti Health Centre in January 2003. Kibiti was chosen as a delivery point because all MSD drugs were delivered to this centre, from where they were collected by the cascades and taken to their respective health facilities. For this initial consignment, the district health management team was responsible for ensuring drug distribution to each health facility.Between January 2003 and September 2005 over 450,000 adult doses of ACT were deployed to public and mission health facilities in Rufiji District. MSD continued to supply SP in the district, while the project procured artesunate. Because no co-formulated or co-packaged product was available, the drugs were co-administered at the health facility level. Artesunate tablets  health facilities in Rufiji District.In April 2003, following reports that artesunate tablets purchased for the project had been found in shops in Dar es Salaam, an inventory management consultant was hired to work with the project manager. After careful evaluation of the whole drug delivery process, the consultant recommended measures to reduce the risk of future misdirection of medicines. These included the introduction of drug stock audit books for all health facilities receiving project drugs, routine and random audits of drug stock inventories at all levels of the system, and the delivery of artesunate drugs directly to the eight cascades by project vehicles from Dar es Salaam rather than the central point in Kibiti.Another measure to curb drug leakage involved hiring part time workers in Dar es Salaam to pack the artesunate into large boxes marked with health facility names and the total number of doses the facility was to receive. A 'mystery shopper' also visited shops and private health facilities in Rufiji and Dar es Salaam to identify any vendors illegally stocking project drugs.In August 2003, a major public launch of ACT was held in Ikwiriri, the largest market center in Rufiji District. This public event was attended by diplomats, politicians and public health officials, journalists and media representatives. The event included community sensitization activities, involving live music, theatre, performing artists and road show vehicles that visited communities across the district. The public launch was followed by a series of community sensitization meetings carried out by the project team in collaboration with the CHMT and community leaders. The last sensitization meeting in August/September 2005 was judged to mark the end of ACT implementation activities in Rufiji District and the start of the transition to ARLU.The main objectives of the costing study were to a) document the financial costs of introducing an ACT in a typical malaria endemic district of rural Tanzania, and b) estimate the costs of national implementation. The costing approach was incremental in the sense that it excluded the costs of health facility infrastructure, supervision and staff, which were assumed not to change as a result of the introduction of a new drug. All costs are presented in 2003 prices in Tanzanian shillings and US dollars, based on an exchange rate of 1 USD: TZS 1065 . The 200Costs were calculated from the provider perspective, including only those costs incurred by the public health sector or the project. No Ministry of Health and Social Welfare costs above the district level were included, reflecting the emphasis on decentralization of management functions to districts. Costs to households were excluded, but are not expected to have been significantly different from those incurred under monotherapy.The timeframe for the costing exercise was defined as the three year period from the initial stakeholders' meeting in October 2002 to the last sensitization meeting held in August/September 2005. This period was selected as it covered all the set up activities needed to implement ACT, and avoided overlap with preparations for nationwide introduction of artemether-lumefantrine, for which initial preparations began in late 2005. Including drug costs for the three year period allowed for year on year variation in malaria prevalence, health facility utilization and drug use to be included, an important element seldom considered when estimating national ACT needs from models alone.This study presents both ACT programme implementation set-up costs and recurrent costs. Set-up costs were defined to include all activities required to introduce the new policy, which were not repeated regularly. They included costs related to the project implementation team , purchase of office equipment , development of IEC materials , and training of health personnel . In addition, set-up costs incorporated communication and publicity activities mainly directed to the general public which included road shows, community sensitization meetings, the programme launch and sensitization materials such as stickers, road signs, posters and t-shirts. Most set-up costs were incurred in the first year, with the exception of some additional training courses and community sensitization meetings. Recurrent costs encompassed drug purchase and distribution and other supplies including stationery and office supplies, drug repackaging materials including prescription envelopes, water treatment solution, buckets, scissors, and drug audit books.The ACT used in the study was not co-formulated. As described above, artesunate was bought separately by the project and co-administered together with SP drugs which were delivered through normal drug delivery channels within the district. SP quantities were collected from drug supply log books across Rufiji District and cross-checked against numbers obtained at the MSD headquarters. The SP drugs were valued at prices provided by MSD, plus 14% transport and distribution charges (standard MSD charging practice for mainland Tanzania).Cost data were drawn from both expenditure and budget records, routinely documented and maintained by the project accountant who was informed from the start that comprehensive records would be required for the costing exercise. Additional information was obtained through interviewing the IMPACT-Tz project implementer, the accountant, the principal investigators and key district health personnel. Care was taken to exclude research costs, but to include all activities necessary for implementation, including the time of project staff spent on implementation and monitoring activities.Annualized costs for goods with a lifespan of more than a year were calculated using a 5% discount rate. Vehicles were estimated to have a lifespan of 10 years, motorbikes and the mobile radio base station five years, and computers and printers three years.The district costs were then extrapolated to estimate the costs of national level implementation for the Tanzanian mainland. It was assumed that all mainland Tanzania would need ACT because the entire country is at risk of malaria . The isl® Pharma, Basel, Switzerland), ranging from 0.45 USD for children under five to 1.8 USD per adult dose [Since the Ministry of Health and Social Welfare has adopted ARLU as its nationwide treatment recommendation, all national cost estimates were based on ARLU procurement costs recently announced by the manufacturer . Fixed costs were those that were not expected to vary by scale of implementation, and included the project implementation team, office equipment and the development of IEC materials. Costs for IEC materials and office equipment were based directly on those incurred in Rufiji District. For the costs related to the project team, the approach used in the 2001 study on antimalarial drug policy change from chloroquine to SP was adopted , assuminNationwide variable costs related to drug purchase and distribution, other supplies, training, communication and publicity were estimated by scaling up variable costs incurred in Rufiji District according to four different scenarios, as described below. The one exception was drug distribution costs. As distribution on a nationwide scale would necessarily be undertaken through MSD, distribution costs were based on the MSD standard rate of 14% of drug purchase costs.Rufiji District variable costs were scaled up by the number of districts in mainland Tanzania (119). This approach assumes that districts are uniform, ignoring variations in population, outpatient malaria diagnoses, number of health facilities etc.Rufiji District variable costs per capita were scaled up by the total estimated population of mainland Tanzania: 37,491,094 in 2007  . The majThe three scenarios above use a common base to scale up all costs. This has the advantages of simplicity and clarity. However, in reality different bases will be appropriate for different cost components, depending on their key cost drivers. For example, drug costs will be mainly dependent on number of patients, but training costs will be driven more by staff numbers. A composite scenario was therefore calculated as follows, using the most appropriate base for each cost category:• Project team costs based on 2001 policy change from chloroquine to SP (adjusted for inflation) • Office equipment costs based on Rufiji District costs• Development of IEC materials based on actual costs incurred in Rufiji District• Training of health personnel costs scaled up by multiplying Rufiji District costs per health facility by the total number of registered public and mission health facilities in mainland Tanzania• Drug purchase and other supplies costs scaled up by multiplying Rufiji District costs per reported malaria outpatient diagnosis by the total national outpatient malaria diagnoses per year reported by the Health Management Information System for all registered public and mission health facilities in mainland Tanzania• Communication and publicity costs scaled up by multiplying Rufiji District per capita costs by the total population in mainland Tanzania.From a theoretical perspective we would consider this scenario to be the best estimate of nationwide costs.In addition, one-way sensitivity analyses were carried out with respect to three key areas: implementation team personnel, increase in health facility utilization, and the use of an alternative less costly ACT. All parameters in the scenarios and sensitivity analyses are presented in Table This study received ethical approval from the institutional review boards of the Ifakara Health Research and Development Centre, the Tanzanian Medical Research Coordinating Committee, the London School of Hygiene and Tropical Medicine, and the US Centers for Disease Control and Prevention.The costs of deploying ACT in Rufiji District are presented in Table Newly announced prices for ARLU are lower than what the project was able to negotiate for the purchase of artesunate plus SP. As ARLU has been chosen to be the national first-line antimalarial drug, the results in Rufiji were re-estimated by replacing artesunate plus SP costs with those of ARLU. This led to a reduction of drug purchase costs by 32%, from 755,659 to 529,982 USD. Substituting present-day prices of ARLU for the actual costs incurred purchasing artesunate and SP reduced overall costs of ACT implementation in Rufiji by 22% from 1,037.787 to 807,820 USD over the three year period.Estimated national level costs based on the four scenarios described above are presented in Table In all four scenarios the single greatest cost was for drug purchase, accounting for between 68.6% and 77.0% of total costs. In scenarios one, two and three, drug purchase costs were followed by drug distribution (around 10.8%). In Composite Scenario four communication and publicity was the second largest cost category (15.5%), followed by drug distribution (9.6%). All other costs were well below 10% of total costs in all scenarios.Based on the best estimate (Composite Scenario four), we undertook a number of one-way sensitivity analyses, covering strengthening the NMCP implementation team, increased facility utilization, and alternative use of a more affordable ACT. Results are summarized in Table During the implementation of the 2001 policy change from chloroquine to SP, concerns were raised about the capacity of the NMCP team to implement their programme effectively . The impHousehold surveys in Rufiji District have shown that after ACT implementation, overall health facility utilization increased from 29% of all care seeking visits for febrile illnesses in 2002, to 43% in 2004 , which is likely to have reflected awareness that a highly effective antimalarial was available at health facilities. Significant increases were especially noticed among patients aged five years and above. It is, therefore, likely that nationwide provision of a similarly effective antimalarial drug would also increase utilization of public and mission health facilities, assuming that subsidized ACT is not available in the private-for-profit sector. In the sensitivity analysis it was assumed that there would be a 50% increase in national drug purchase costs, which in turn would increase the costs of drug distribution and other supplies. This led to an increase of 39% in overall ACT implementation costs, suggesting that changes in treatment seeking could have a major impact on total programme costs.Finally, the use of a less expensive ACT such as artesunate plus amodiaquine was considered. This combination was chosen because it is currently being used in neighbouring Zanzibar as first line drug for treating uncomplicated malaria, with an average cost of 0.85 USD per dose. At this relatively cheaper purchase price, there would be proportionate reduction of drug distribution costs but all other costs would remain constant. This led to a 56% reduction in the overall costs estimated for implementing ACT nationwide.Given the rise in antimalarial drug resistance and subsequent introduction of new malaria treatment recommendations, it is important to understand the costs of changing antimalarial drug policy in order to facilitate planning by national governments and their donor partners, and as an input into evaluations of the cost-effectiveness and affordability of new drug policies. Studies to date on the national costs of ACT implementation have focused on drug costs -11,31 inThe total cost of three years of ACT implementation in Rufiji District was 1,037,787 USD or USD 4.89 per capita. Depending on the approach used to scale up costs, the nationwide costs of the first three years of ACT implementation were estimated to be between 43.1 and 121.7 million USD, with the best estimate at 48.3 million USD (1.29 USD per capita). This varied between 21.1 and 67.1 million USD in the sensitivity analyses. In all estimates drug costs constituted the majority of total costs. However, non-drug costs such as office equipment, IEC materials, drug distribution, communication, and health worker training were also substantial, accounting for 31.4% of overall ACT implementation costs in the best estimate scenario. Of these non-drug costs, the most expensive were communication and publicity and drug distribution.In terms of affordability, the implications of ACT implementation for the health budgets of countries like Tanzania are considerable. Tanzania's total health budget (recurrent and development) in 2005 amounted to 254.6 million USD of which 169.3 million USD was allocated for recurrent expenditures, with only 56 million USD remaining for medical supplies . Over 90These results can be compared with two previous studies in Tanzania. One calculated the non-drug costs of policy change from chloroquine to SP monotherapy; while the other estimated the drug costs of policy change from SP monotherapy to combination therapy . The first study by Mulligan and colleagues was a post-hoc evaluation of the 2001 policy change from chloroquine to SP, which estimated the nationwide non-drug costs of implementation at 734,441 USD , compareSecondly, a USAID commissioned study by Wolf and Derriennic estimated the projected ACT financing gap for mainland Tanzania in 2007 assuming that ACT was provided through all public and non government facilities, and that 70% of all fever cases were treated in such facilities . The stuThere are a number of reasons why nationwide cost estimates based on the experience implementing ACT in Rufiji District could be expected to overestimate costs for the country as a whole. Firstly, though care was taken to maintain 'real life' implementation conditions, the experience in Rufiji District could be considered to represent best practice as it was relatively well-funded, implemented by a highly motivated team and closely monitored. This is likely to have led to a more comprehensive implementation programme beyond what might be expected during routine implementation on a larger scale. Secondly, the ACT used in Rufiji was not co-formulated or co-packaged in one blister pack. This may have necessitated greater resources for additional packaging, IEC and communication than would be required with a co-formulated product such as ARLU. Thirdly, a significant proportion of the Rufiji District population lives in the relatively inaccessible delta area, where the costs of accessing some health facilities and communities are particularly high. Finally, potential savings arising from the use of a more effective drug were not included, such as a decline in severe cases and thus reduced costs of inpatient admissions.On the other hand, using the actual costs incurred in Rufiji District could equally have led to underestimation of nationwide costs. Firstly, it is likely that the potential for leakage of ACT supplies to private for-profit outlets would be much greater with national level implementation, thus leading to substantial increases in drug and drug distribution costs. Secondly, with national implementation there is the potential to use the mass media for publicity campaigns, which is not feasible on a district level, which in turn could increase implementation costs. Thirdly, Rufiji District is relatively close to Dar es Salaam, facilitating the management of drug distribution and other implementation activities. Moreover, Rufiji District has benefited from the presence of the Tanzania Essential Health Intervention Project (TEHIP) which has helped strengthen the district health system – greater resources might be required to ensure effective management in other settings. Finally, the costs were estimated on an incremental basis; in some settings more fundamental health system strengthening may be required to ensure efficient roll out, such as the recruitment of additional health workers, or improvement of storage facilities to accommodate the shorter life span and bulkier packaging of ACT drugs.A final limitation is the reliance on malaria outpatient diagnoses data as a base for scaling up all costs in Scenario three and some costs in Scenario four. As in many developing countries, these routine health information system reports are often inaccurate and incomplete. This has a potentially important impact on the cost estimates as the number of malaria diagnoses is the major determinant of Composite Scenario four estimate of national drug requirements, which in turn are the major driver for total ACT deployment costs.This study's ACT cost estimates were based on the current mode of ACT implementation in Tanzania which involves ACT provision through public and private not-for-profit health facilities only, in most cases on the basis of clinical diagnosis alone. Clinical diagnosis is known to lead to substantial over-treatment with antimalarial drugs ,36, and This study has improved the evidence base for governments on the incremental financial costs of ACT implementation. The estimates were based on a detailed assessment of costs incurred during district-wide implementation in a typical rural district, and the extrapolation of these data to estimate nationwide costs. They included both drug and non-drug costs, such as IEC materials, training of health personnel, communication, drug distribution, other supplies and the project implementation team.The costs are substantial, with a best estimate of 48.2 million USD (1.29 USD per capita) over the first three years. The total overall costs varied between 21 and 67.1 million USD in the sensitivity analysis. The study highlights three key messages. Firstly, drug costs constituted the majority of total costs, but non-drug costs were also considerable, accounting for 31.4% of overall costs in Composite Scenario IV estimate. Secondly, rolling out ACTs would lead to a more than 6-fold increase in the national budget for antimalarial drugs. Thirdly, average annual implementation costs over the first three years would be equivalent to 9.5% of the total health sector recurrent budget. It is thus clear that substantial external resources are required to facilitate implementation of ACT, particularly in the early years of implementation, but also to ensure the sustainability of effective provision of malaria medicines.Njau & Goodman collected, analyzed the data and drafted the paper. Njau, Goodman, Mulligan, Kachur, and Abdulla, designed the study while Munkondya and Mchomvu assisted with the data collection process. Mills and Bloland provided overall supervision. All authors contributed to the final manuscript.The Interdisciplinary Monitoring Project for Antimalarial Combination Therapy in Tanzania (IMPACT-Tz) is a multiyear implementation research evaluation project that rests on a collaborative platform comprising the US Centers for Disease Control and Prevention (CDC), Ifakara Health Research and Development Centre, the National Institute for Medical Research, Muhimbili University College of Health Sciences, the London School of Hygiene and Tropical Medicine (UK) and the Tanzanian MoH, including its National Malaria Control Programme, the Tanzania Essential Health Interventions Project, and the Council Health Management Teams of Rufiji, Morogoro, Mvomeru, Kilombero and Ulanga Districts. IMPACT-Tz is primarily supported by funding from CDC, the United States Agency for International Development and Wellcome Trust.The findings and conclusions presented are those of the authors and do not necessarily represent those of the United States Public Health Services (USPHS) nor the Centers for Disease control and Prevention (CDC). Trade names are used for identification purposes only and do not imply endorsement by USPHS nor CDC.All authors of this article declare to have no financial or other interest with any organization or entity that may influence or be considered as constituting a real, potential or apparent conflict of interest to bias whatever findings presented in this study.


It is under debate whether healthcare costs related to death and in life years gained (LysG) due to life saving interventions should be included in economic evaluations. We estimated the impact of including these costs on cost-effectiveness of cancer screening. We obtained health insurance, home care, nursing homes, and mortality data for 2.1 million inhabitants in the Netherlands in 1998–1999. Costs related to death were approximated by the healthcare costs in the last year of life (LastYL), by cause and age of death. Costs in LYsG were estimated by calculating the healthcare costs in any life year. We calculated the change in cost-effectiveness ratios (CERs) if unrelated healthcare costs in the LastYL or in LYsG would be included. Costs in the LastYL were on average 33% higher for persons dying from cancer than from any cause. Including costs in LysG increased the CER by €4040 in women, and by €4100 in men. Of these, €660 in women, and €890 in men, were costs in the LastYL. Including unrelated healthcare costs in the LastYL or in LYsG will change the comparative cost-effectiveness of healthcare programmes. The CERs of cancer screening programmes will clearly increase, with approximately €4000. However, because of the favourable CER's, including unrelated healthcare costs will in general have limited policy implications. Cancer screening induces both costs and savings  including the healthcare costs related to postponed death, regardless of its cause and (2) more in general of including the unrelated healthcare costs in LysG. As a proxy of costs related to death, we studied the healthcare costs in the last year of life (LastYL), discerning between deaths caused by a specific cancer (focusing on cancers for which screening is recommended or at least under discussion) from deaths due to any cause. We also investigated the healthcare costs in the LysG and how they depend on age. Finally, to understand the potential impact, we calculated the increase these costs would make on the estimated CER.vs 36.5% insured by social and private health insurance, respectively)  . Both inThe data on nursing homes and home care were linked with health insurance data at individual level, using birth date, sex, and zip code. The registration of home care was complete. The nursing homes registry covered 65% of the users of nursing home care. As the coverage appeared to be non-selective, we adjusted the average nursing home costs in our study population, using a correction factor depending on coverage per geographical area (on average: 100/65).n=2 093 748) and decedents (n=14 839), the latter stratified by cause of death. All individuals who entered or dropped out the study population in 1998 or 1999, for example due to a change in their insurance scheme, or individuals who died in 1998, were excluded from the study. Also, individuals who died in 2000 were excluded, because part of their health expenditures in 1999 could include costs related to death.Subsequently, to obtain date and cause of death, the data of the health insurance companies was linked with mortality data from Statistics Netherlands, using birth date, sex, and zip code. In the final analysis, we distinguished between costs of survivors  is comparable for both groups of insurance.We calculated the 95% confidence intervals for healthcare costs in the LastYL on the basis of a lognormal distribution. This was not possible for the costs of nursing homes, because for this cost category, only average figures on group level were available . The costs before dying were stratified by age, gender, and cause of death. We stratified the causes of death into five types of cancer that are potentially preventable due to screening  all cancer and causes . On accoWe calculated the average healthcare costs in the LysG, by gender and age category at which death is prevented. To do so, we first calculated the average annual healthcare costs by age and gender, using costs in ‘survivors’ . In thisWe calculated the increase in costs per LyG when taking into account both the unrelated healthcare costs of dying  and the As an example, we calculated the impact of the above estimated extra costs per LyG on the CER of breast cancer screening, assuming that the mean age at breast cancer death .The overall impact depends on the CER before the adjustment. For breast cancer screening in the Netherlands, for example, this ratio was estimated at approximately €2700 per LyG  screen policy may change. On the other hand, if these costs are included, for example, for the current breast cancer screening programme in the Netherlands, the costs per LyG still are acceptable according the Dutch threshold value of about €20 000 per quality-adjusted LyG. The CER (without including costs in the LastYL or in LysG) of other cancer screening programmes in the Netherlands, such as cervical cancer screening (€9500 per LyG  and 2.By including unrelated healthcare costs in the LastYL as well as in LysG, the CER of prevention of lethal illnesses can become less favourable compared with the prevention of non-lethal illnesses. Vaccination during childhood against, for example, mumps and rubella, prevents a lot of severe disabilities, but in developed countries, individuals rarely die of those diseases in the absence of vaccination. As a consequence, there are no healthcare costs related to postponed death or in LysG in this situation. Therefore, the CER of prevention of these non-lethal illnesses will not change.In the meantime, when cost-effectiveness analyses are used for policy decisions, it is important that any type of costs are either consistently excluded or consistently included (The strength of our study is that we linked healthcare costs to the official mortality register on the individual level, and were, therefore, able to analyse cost differences per cause of death. Also, we included all medical care costs, whereas most other studies focus on hospital care only. Thereby, due to the fact that the government heavily regulates the Dutch healthcare system, the claims on the health insurance are very comparable to the actual healthcare costs that makes the estimation of the medical costs reliable.A potential weakness is that we used average annual healthcare expenditures as an estimate of the yearly healthcare costs of individuals whose cancer death is prevented by cancer screening, whereas individuals whose cancer death is prevented may tend to have a more active healthcare-seeking pattern. Also, there is some evidence that individuals diagnosed with cancer have an increased risk of other diseases due to the fact that the risk factor that played a role in the development of cancer can cause other diseases as well . The symFinally, as we mentioned before, it is argued that for an evaluation from a societal perspective non-medical costs and benefits in LysG, that is, productivity gains, paid pensions, and non-health-related quality of life need to be included in economic evaluations (In conclusion, taking the unrelated healthcare costs in the postponed LastYL or during all LysG into account in cost-effectiveness analyses will change the relative cost-effectiveness of healthcare programmes and may influence priority settings. The CERs of cancer screening programmes will clearly increase, with approximately €4000. However, because of the favourable CERs, including unrelated healthcare costs in LysG will, in general, have limited policy implications.


Our study suggests that viruses closely related to Nipah or Hendra viruses are more widespread in Southeast Asia than previously documented.Serum specimens from fruit bats were obtained at restaurants in Cambodia. We detected antibodies cross-reactive to Nipah virus by enzyme immunoassay in 11 (11.5%) of 96 Lyle’s flying foxes  Japanese encephalitis virus (JEV), a mosquito-borne pathogen endemic to the region, was suspected as the causative agent. However, a new paramyxovirus, Nipah virus, which is closely related to Hendra virus (HeV), was later implicated as the cause. Unlike JEV, Nipah virus predominated in adults rather than children. Nipah virus cases clustered in members of the same household, suggesting a high attack rate; in contrast, JEV causes symptomatic encephalitis in approximately 1/300 infected persons. A high proportion of Nipah virus patients had direct contact with pigs, unlike others in the same neighborhood who did not have the virus (providing evidence against a mosquito-borne disease); in addition, many of the pigs belonging to affected farmers had an associated history of illness  had neutralizing antibodies to Nipah virus (Pt. hypomelanus) Pteropus spp. that occur in Australia, with a moderate (20%–25%) prevalence of HeV-neutralizing antibody Pt. poliocephalus) and black flying fox (Pt. lecto) Pteropus spp. . Comprehensive studies of domestic animals and wildlife showed that a substantial proportion of Malaysian fruit bats . To further investigate the distribution of this new group of viruses, we investigated the prevalence of virus antibodies in other members of the genus Pt. lylei), 11 (11.5%) were positive (>1/10) for Nipah virus antibodies by EIA. All 11 were confirmed by serum neutralization test. Nine additional sera were found positive (low titers) only by neutralization assay. We also screened sera (when sufficient quantities were available) by neutralization test against HeV. In general, results were equivalent between the two tests. No sera were found positive for HeV and negative for Nipah virus. Our results suggest that the virus circulating in Cambodia is neither Nipah nor HeV, but another closely related virus. Of 96 serum specimens from the fruit bat (Pteropus show serologic evidence of Nipah or HeV infection. Attempts by several groups to recover virus from tissues of serologically positive bats have been unsuccessful, as have immunohistochemical tests to detect the infection in tissues Pteropus bats with a related paramyxovirus resulted in findings that the virus replicates, causes microscopic lesions, and is shed; the virus appears to clear as the antibody response appears ( Several species of the genus  appears ,14. We dPt. lylei is limited to sites where they are protected from hunting, including urban areas and temples, where the human-bat interaction may be increased. The fact that these large bats are caught and used for food further increases the risk for exposure and infection in humans. We observed no evidence that HeV  Future studies should include an evaluation of the risk of Nipah virus infection among populations intensely exposed to bats, such as those who capture, transport, slaughter, and butcher bats, as well as bat rehabilitators, animal caretakers, and wildlife conservationists. We suggest that future studies also include a cross-sectional survey of swine in Cambodia. Unlike workers on the large, commercial swine production farms of Malaysia, typical swine farmers in Cambodia raise several swine for their own use and for the local market. The potential for amplification of the virus, unlike that observed in the large concentrated pig population in Malaysia, remains very limited. Finally, a systematic study of encephalitis causes may show whether Nipah virus causes disease in humans in Cambodia and elsewhere in the region.


Flavivirus genus pose public health problems around the world. Increasing cases of Dengue and Japanese encephalitis virus in Asia, frequent outbreaks of Yellow fever virus in Africa and South America, and the ongoing spread of West Nile virus throughout the Americas, show the geographical burden of flavivirus diseases. Flavivirus infections are often indistinct from and confused with other febrile illnesses. Here we review the specificity of published primers, and describe a new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups.Species within the Flavivirus genomes revealed conserved regions not previously targeted by primers. Two degenerate primers, Flav100F and Flav200R were designed from these regions and used to generate an 800 base pair cDNA product. The region amplified encoded part of the methyltransferase and most of the RNA-dependent-RNA-polymerase (NS5) coding sequence. One-step RT-PCR testing was successful using standard conditions with RNA from over 60 different flavivirus strains representing about 50 species. The cDNA from each virus isolate was sequenced then used in phylogenetic analyses and database searches to confirm the identity of the template RNA.Bioinformatic analysis of 257 published full-length Comprehensive testing has revealed the broad specificity of these primers. We briefly discuss the advantages and uses of these universal primers. Most current molecular assays for flaviviruses use highly specific primers, which may only amplify from one species, or a range of closely related species -4. In a Flavivirus genus, this need is becoming more pressing [Many attempts to develop a systematic means for identifying flaviviruses have been made, including serology and non-serology based tests -8. Due tpressing .The first report of a reverse transcriptase-PCR (RT-PCR) for the detection of multiple species was published in 1990, with the use of species-specific probes targeting the nucleocapsid and envelope coding regions from four different Dengue virus genomes  publisheThe idea of designing primer sets relevant for diseases found in specific geographic regions has also been investigated by several groups. Meiyu  developeFlavivirus genus [Flavivirus detection and taxonomy has recently become more difficult with the determination of the nucleotide sequence of Tamana bat virus (TABV), and Cell fusing agent virus (CFAV) -14, and us genus , even thus genus . They pous genus  addresseIn the present study, we identified conserved sites and developed a universal, non-nested primer pair that amplifies cDNA from each of the major subgroups of flaviviruses, and also TABV, under standard reaction conditions. The region of the NS5 gene amplified contained sufficient variability to allow differentiation of individual viruses. We discuss the advantages of this approach, over the known detection regimes for flaviviruses.No potentially useful conserved sites were identified in the first complete alignment, utilising all available sequences. However, the sequences of TABV, CFAV and KRV were identified as a divergent cluster, and once removed several conserved sites were found. The Flav100F and Flav200R primers were designed to complement sites in the NS5 gene that begin at residues 8276 and 9062 relative to the YFV genome (NC_002031). The conserved sites encoded amino acid sequences starting at residues 2720 and 2982 in the YFV polyprotein (NP_041726), which do not correspond to any known conserved sites in flavivirus genomes. The primers have relatively low levels of degeneracy, with 8 and 12 different permutations respectively, discounting inosine positions, or with 512 and 48 permutations when inosines are counted as equivalent to four base degeneracy. To compensate for the primer multiplicity, a slightly higher primer concentration (50 pmole per 50 uL reaction) was used in the PCR.A cDNA product approximately 800 base pairs long was amplified from the RNA of each of the 65 viruses tested Figure . As expeAll amplified products were sequenced and, on average, sequences from three reactions were used to traverse each cDNA in both directions. Full length sequence was obtained for 55 viruses, and truncated sequence was obtained for DENV2 (771 bp), UGSV (742 bp), BSQV (700 bp), MVEV (684 bp), USUV (675 bp), TYUV (620 bp), TABV (500 bp), YOKV (380 bp). cDNA products of the expected size were obtained from AROAV, BAGV, BOUV and LGTV although reliable sequence data was unavailable; thus these viruses have been excluded from this phylogenetic analysis.Each product yielded sequence from a flavivirus NS5 gene as shown by BLASTN searches. Flavivirus NS5 sequences occupied the top places in every BLASTN output. The majority of the sequences from the cDNAs differed by 5 to 50 single nucleotide polymorphisms from the closest sequence with the same name in GenBank. Some viruses amplified had no relevant sequence data available on GenBank, the identities of these viruses were further tested by phylogenetic analysis.The primers were tested on, and amplified cDNA from, 24 of the 27 virus species listed in the mosquito-borne group, 10 of the 12 virus species in the tick-borne group and 13 of 14 viruses in the no known vector group . In totaDespite this amplification involving mismatching with Tamana bat virus RNA, no cDNA was amplified from the alphaviruses Barmah Forest virus, Ross River virus or the nine respiratory viruses tested: Influenza A virus, Human coronavirus NL, Human coronavirus OC43, Human adenovirus, Human bocavirus, Human rhinovirus 1, 2 or 3 (data not shown).Phylogenetic trees found using the sequences largely agreed with previously published trees ,17,18 inFlavivirus. Since the amplified products represent 8% of the genome, this is sufficient sequence to determine the species of the virus and thus potentially to identify unrecognised flaviviruses. One major problem with degenerate primers is that the concentration of some permutations in the mixture is so small, due to their great multiplicity, that amplification is effectively inhibited. For any given viral RNA target only a proportion of the primer may participate in the initiation of high efficiency extension in the early rounds of PCR. We believe that the redundancy of the Flav100F and Flav200R was insufficient to cause this problem [We have described a novel primer set capable of amplifying 800 bp from the NS5 genes from almost every recognised member of the genus  problem .Traditional serological methods based on neutralisation and fixed cell ELISA have proven effective for identifying flaviviruses and indeed classifying them . HoweverGaunt and Gould, developed primers targeting the E gene . These pPrimers targeting the NS3 gene have been developed and tested on a number of viruses including KUNV, JEV and YFV . BioinfoThe FU1 and cFD3 primers were tested on a large number of viruses; although six, covering the mosquito-borne KOKV and SOKV, tick borne (KSIV) and no known vector viruses RBV and SVV, were unable to be reproducibly amplified using these primers. These viruses are highly divergent within the three major subgroups currently recognised in this genus ,15. The Flavivirus genus may demonstrate their capacity to define novel species. The protocol is robust and tolerates a range of template concentrations (greater than five orders of magnitude), primer concentrations, and PCR-cycle conditions (data not shown). The capacity of this reaction to amplify all flaviviruses tested provides a potential tool capable of rapidly identifying endemic and exotic viruses, in a timely, cost effective manner, thus facilitating an appropriate response to epidemic outbreak, or surveys that may result in the discovery of new or novel flaviviruses. These primers also provide researchers with a tool to re-analyse archived samples that may no longer be infectious.The primers designed in the present work have been widely tested, but there are six recognised viruses not included in the analysis; the BSL4 viruses, Kyasanur Forest disease virus and Omsk hemorrhagic fever virus, the BSL3 viruses Kedougou virus, San Perlita virus and Yaounde virus and the tentative members of the genus, CFAV and KRV. The primers amplified products from all tested flaviviruses. The ability of these primers to amplify previously 'unidentified' members of the In recent years viruses have been isolated from regions outside their known geographic distribution. JEV was isolated in Australia for the first time in 1995. Until this time the closest location to report human JEV cases was Bali. The 1999 outbreak of WNV in New York reinforces the importance of accurate and rapid diagnosis of exotic viral agents, as the virus was originally mis-diagnosed in serological tests.Flaviviruses are emerging in new geographic regions as potential epidemic pathogens. Thus, the importance of an accurate, rapid and reliable method for virus identification is becoming increasingly important. A major expansion of arbovirus surveillance and reporting systems has been implemented inNorth America following the appearance of WNV. For example, ArboNet reports surveillance data from humans, mosquitoes, birds, mammals and sentinel chicken flocks and the dataare integrated into a single reporting system . Broad sThe changing global epidemiological environment is characterized by incursions of human populations into new environments, increasing overlap of the range of disease vectors with human habitation and concomitant exposure to a wider range of infectious agents . Not onlThe Flav100F and Flav200R primers have the potential to detect emerging, related flaviviruses without prior serological evidence or additional primer design. Our approach should help reduce the confirmation time for viral infections. Rapid detection at the genus level would enable informed policy measures to be implemented and this, in turn, may help disease management.et al [Primers were designed using a strategy similar to that used by Vercruysse et al . All avaet al ,31 and aet al . Severalet al , and theet al unpublishet al . StandarVirus stocks produced in the molecular virology laboratory at the University of Queensland, Australia were prepared from the supernatant medium of infected PS-EK cell cultures Table . PS-EK cOne-step RT-PCR was performed using Superscript III in a 50 μL volume  with touch-down cycling conditions . The finThe specificity of the primers was investigated by attempting amplification from cultures infected with viruses that are not flaviviruses, including Barmah Forest virus, Ross River virus, Influenza A virus, Human coronavirus NL, Human coronavirus OC43, Human adenovirus, Human bocavirus, Human rhinovirus 1, 2 or 3 and RNA from virus free cell cultures.-1 ampicillin. The plasmid was purified using a spin column kit  according to the manufacturer's protocol. Colony PCRs were performed using a step down protocol as described above although the extension temperature was 72°C . RT-PCR and PCR products were analysed on a 1% agarose gel containing ethidium bromide, and visualised using a UV transilluminator.RT-PCR products were cloned into the pGEM-T easy vector  according to the manufacturer's protocol. Colonies were PCR screened for the presence of an insert. Positive colonies were grown overnight in LB with 1 μg mLPurified plasmid was sequenced using ABI BigDye Terminator Version 3.1 chemistry, on the AB3730xl sequencing platform. SP6 and T7 promoter primers were used for sequencing. Each virus clone was sequenced twice or more in the forward and reverse directions.Sequence data were assembled using Contig Express . Sequences were then compared to the GenBank non-redundant nucleotide database using BLASTN ; the proSequences of known species and strains, identified by the ICTV using their Genbank accession codes, were compiled with the sequences from the amplified products; sequences were then aligned using the default single step progressive method of the program MAFFT version 6.0 ,39. Maxi3'UTR: 3' Untranslated region; BSQV: Bussuqara virus; cDNA: complementary Deoxyribonucleic acid; CFAV: Cell fusing agent virus; DENV: Dengue virus; DJA: primer; DJS: primer ; E gene: Envelope protein encoding gene; ELISA: Enzyme Linked Immuno-sorbent assay; EMF1: primer; ICTV: International Committee on Taxonomy of Viruses; JEV: Japanese encephalitis virus; KOKV: Kokobera virus; KRV: Kamiti River virus; KSIV: Karshi virus; KUN: Kunjin virus; L: Ladder; NS1: Non-Structural 1; NTC: No template control; NS5: Non-Structural 5; NTE: a type of buffer; PAUP: a Phylogenetic programme; PBS: Phosphate buffer Saline; PCR: Polymerase Chain Reaction; PEG: Poly-ehtylene glycol; PhyML: Phylogenetic programme; PSEK: Porcine stable Equine kidney cells; RBV: Rio Bravo virus; RNA: Ribonucleic Acid; RT-PCR: Reverse-Transcription Polymerase Chain Reaction; SOKV: Sokoluk virus; SVV: Sal Vieja virus; TABV: Tamana Bat virus; TBEV: Tick-borne encephalitis virus; UV: Ultra Violet; VD8: primer; YF1: Primer; YF3: Primer ; YFV: Yellow fever virus;RTB is a Director of Biochip Innovations Pty Ltd.SLM planned and performed the experiments and drafted the manuscript. MJG designed the primers with assistance from PJW and SLM. MJG, SLM, RH and RB planned the project and edited the manuscript along with NLF and EAG. All authors read and approved the final manuscript.