To explore geographic and host-taxonomic patterns of hantaviruses in Paraguay, we established sampling sites in the Mbaracayú Biosphere Reserve. We detected Jaborá virus and Itapúa37/Juquitiba–related virus in locations ≈20 m apart in different years, which suggested sympatry of 2 distinct hantaviruses. Calomys laucha (little laucha) caused an outbreak of hantavirus pulmonary syndrome in western Paraguay  in western Paraguay; and Ape Aime virus (AAIV) harbored by Akodon montensis (Montane akodont), Itapúa virus strain 37 (IPV37) harbored by Oligoryzomys nigripes (black-footed colilargo), and Bermejo virus strain Ñeembucu harbored by O. chacoensis (Chacoan colilargo) in eastern Paraguay  placed on the ground, and another in branches or vines 2–3 m above ground to capture arboreal species. Grids were sampled for 8 nights, with at least 2 months between sampling sessions. Rodents captured in the mark-recapture grids were individually marked with passive integrated transponder tags, and ≈100 μL of blood was collected from the retroorbital sinus (once per trapping session). Animals were identified to species, age class, reproductive condition, sex, and weight and released.Rodents were also collected in a series of traplines, each of which contained 50 traps placed ≈10 m apart. Animals collected in traplines were killed, standard collecting information was recorded, and liver, lung, heart, kidney, muscle tissues, and blood specimens were collected. All samples were snap-frozen in liquid nitrogen, transported to the Museum of Texas Tech University (TTU), and stored at –80°C. Standard voucher specimens were prepared from these animals, and have or will be deposited in the Museum of TTU or the Museo Nacional de Historia Natural del Paraguay. All field protocols followed American Society of Mammologists guidelines for the use of wild mammals in research  (A. montensis (55.7%), Necromys lasiurus (hairy-tailed akodont) (10.8%), C. callosus (big laucha) (6.5%), and O. fornesi (Fornes’ colilargo) (6.3%).A total of 1,150 small mammals from animals) . The domA. montensis, N. lasiurus, O. fornesi, O. nigripes, Oligoryzomys sp., Oryzomys megacephalus (Azara’s broad-headed Oryzomys sp.), and Oxymycterus delator (Paraguayan hocicudo). Antibodies to hantavirus antigens were 3× more abundant in blood samples from males than females  hantavirus RNA segment  were selected for additional PCR, sequencing, and phylogenetic analysis. PCR-amplified cDNAs of a 1,014-nt amino terminus region of the S segment were purified by agarose gel electrophoresis and cloned into pCRII  (http://darwin.uvigo.es/software/modeltest.html.), maximum-likelihood estimation, and Bayesian inference  from southern Brazil, and strains from RNBM in Paraguay  from Brazil and Itapúa virus strain 37, which was originally detected in O. nigripes from Itapúa Department in eastern Paraguay (Akodonmontensis clade at RNBM and more closely related to Andes (clade C2b) and Bermejo-Ñeembucú (clade C2a) viruses. This finding suggests that spillover infection of JUQV-related viruses is actively occurring among oryzomyine rodent species at RNBM, as reported for other hantaviruses in oryzomyines  in close proximity (collected ≈20 m apart on the same grid in the same sampling session) on 2 occasions, in sites separated by ≈30 km ; Table 2A. montensis) (A. montensis in Paraguay. To address host-jumping of hantaviruses among sympatric rodent species in RNBM and other regions in South America, future longitudinal studies are warranted. Such studies are critical to understanding evolutionary adaptation of hantaviruses in rodents in South America, the ability of these viruses to adapt to new rodent reservoirs, and their emergence and maintenance in the environment.Coexistence of hantaviruses in 2 rodent species at mark-recapture sites has been observed (Small mammals collected at Mbaracayú Reserve, Paraguay, 2003-2007*


Eidolon helvum in West Africa, and from two species of New World leaf-nosed fruit bats (Phyllostomidae), Artibeus jamaicensis and Artibeus lituratus in Central America. A sequence-independent virus discovery technique (VIDISCA) was used in combination with high throughput sequencing to detect two novel parvoviruses: a PARV4-like virus named Eh-BtPV-1 in Eidolon helvum from Ghana and the first member of a putative new genus in Artibeus jamaicensis from Panama (Aj-BtPV-1). Those viruses were circulating in the corresponding bat colony at rates of 7–8%. Aj-BtPV-1 was also found in Artibeus lituratus (5.5%). Both viruses were detected in the blood of infected animals at high concentrations: up to 10E8 and to 10E10 copies/ml for Aj-BtPV-1 and Eh-BtPV-1 respectively. Eh-BtPV-1 was additionally detected in all organs collected from bats  and spleen and kidneys were identified as the most likely sites where viral replication takes place. Our study shows that bat parvoviruses share common ancestors with known parvoviruses of humans and livestock. We also provide evidence that a variety of Parvovirinae are able to cause active infection in bats and that they are widely distributed in these animals with different geographic origin, ecologies and climatic ranges.Bats, a globally distributed group of mammals with high ecological importance, are increasingly recognized as natural reservoir hosts for viral agents of significance to human and animal health. In the present study, we evaluated pools of blood samples obtained from two phylogenetically distant bat families, in particular from flying foxes (Pteropodidae),  Parvoviridae family in fecal samples of different species of bats Bats  and the subfamily Densovirinae (viruses that infect arthropods), are non-enveloped viruses, containing a single stranded DNA genome of approximately 5 kb. The Parvovirinae subfamily is currently subdivided into 5 well established genera  and a new genus (PARV-4 like viruses) that contains 5 viruses that have recently been identified: Human Parvovirus 4  Parvovirinae viruses require a co-infection with a helper virus to be perpetuated (the majority of the viruses within the genus Dependovirus), but most can replicate autonomously.The Parvovirinae subfamily, in particular viruses that are closely related to the human ones in search for ancestral viruses and new potentially zoonotic viruses. So far virus discovery in bats was predominately performed in stool samples Eidolon helvum and Artibeus jamaicensis/A. lituratus, frugivorous bats from Ghana and Panama, respectively. A sequence independent virus discovery technique (VIDISCA) was used in combination with high throughput sequencing In the present study we evaluated whether bats carry viruses that belong to the Eidolon helvum  in Kumasi, Ashantia Region, Ghana, in 2009. For all capturing, sampling and exports permission was obtained from the Wildlife Division of the Ministry of Lands, Forestry, and Mines in Ghana . Ethics approval was given by the Kwame Nkrumah University of Science and Technology . Research samples were exported under a state agreement between the Republic of Ghana and the Federal Republic of Germany, represented by the City of Hamburg. Additional export permission was obtained from the Veterinary Services of the Ghana Ministry of Food and Agriculture. Whole EDTA blood samples from Panamanian Artibeus jamaicensis and A. lituratus fruit bats were collected during an ecological study on haemoparasites (November 2005) in a tropical lowland forest (Barro Colorado Nature Monument) in Panama Serum and organ samples were collected from wild Samples for virus discovery were tested in pools: 10 pools each containing 2 serum samples collected from Ghanaian bats, one pool containing 50 EDTA-plasma samples derived from Panamanian bats.VIDISCA-454  was performed with 110 µl of pooled serum/EDTA-plasma samples as previously described Primer and MID (Multiplex Identifier) sequences were trimmed from every read. Sequences were then assembled with CodonCode Aligner software version 3.5.6. Contigs and unassembled sequences were compared to known nucleotide and protein sequences contained in GenBank database using different BLAST tools  Identified viral fragments were used as a template for primer design (sequences available on request) to obtain the nearly complete genome sequences. Sequencing was performed employing Big Dye terminator chemistry .Eidolon helvum bats was isolated by the QIAamp MinElute Virus Spin Kit  according to the manufacturer's recommendations. The input volume was 200 µl, the elution volume was 120 µl. Up to 200 µl of whole EDTA blood collected from 92 Panamanian Artibeus jamaicensis bats and from 18 Panamanian Artibeus lituratus bats were mixed with 500 µl lysis buffer  and DNA was prepared by using the QIAamp Blood DNA Mini Kit  according to the manufacturer's instructions except for an initial 15 sec incubation with proteinase K Eidolon helvum serum samples and 22 pools (5 eluates each) were made with 5 µl of each DNA preparation from Panamanian Artibeus bats specimens.DNA from 100 serum samples collected from Ghanaian Eidolon helvum bats found to be parvovirus-positive. Samples collected from brain (n = 7), lungs (n = 7), liver (n = 6), kidneys (n = 7), spleen (n = 7), small intestine (n = 5), and large intestine (n = 4) were analyzed. Nucleic acid was prepared from 20 mg of tissue by using the TissueLyser method with steel beads  and proteinase K in ATL buffer followed by the QIAamp DNA Blood Mini Kit procedure . DNA was eluted in 100 µl.To test the organ distribution of the virus and to detect the potential replication site, DNA was also isolated from organ tissues of DNA preparations from single samples and from pools were tested for the presence of the newly identified viruses through homemade hemi-nested or nested PCRs . Plasmid DNA was prepared by using the QIAprep Spin Miniprep Kit . Nucleic acid concentration was determined spectrophotometrically.Absolute quantification of viral DNA  was achieved by real-time qPCR using plasmid-based standards and primers and probes listed in http://www.ncbi.nlm.nih.gov/tools/cobalt/) for nucleotide and amino acidic sequences, respectively. Only complete coding sequences were included in the analysis. Alignments were manually edited when needed with BioEdit software version 7.0.5.3 Fifty-nine reference nucleotide sequences and amino acid sequences were downloaded from the GenBank database and aligned together with the ones obtained in this study using ClustalX software version 2.0.12 http://www.ncbi.nlm.nih.gov/projects/gorf/).To examine whether the viruses were recombinant viruses, Bootscanning analysis All sequences obtained in this study have been deposited in the GenBank database under accession numbers JQ037753–JQ037754 (reference strains) and JQ037755–JQ037761 .Eidelon helvum bats from Ghana and in one pool of 50 EDTA blood samples collected from frugivorous Artibeus jamaicensis bats from Panama 136 parvoviral hits  and 168 parvoviral hits  were identified. Respectively 42.5% and 52.5% of the complete genome were obtained with VIDISCA-454.VIDISCA is a virus discovery technique that can identify RNA and DNA viruses, without prior knowledge of its genome sequence. The technique was successfully used to discover human coronavirus NL63 Parvovirinae subfamily (5065 nt for the Ghanaian virus and 4595 nt for the Panamanian virus), although sequences of the terminal hairpins are missing.The identified viral fragments were used as template for primer design and with specific PCRs the original positive samples were detected. Via genome walking it was possible to amplify and sequence the viral genome from 2 previously unknown members of the No other reads with clear identity to any known viruses were detected in any of the samples.Eidolon helvum bats is similar to the other members of the Parvovirinae, with two large ORFs. The 5′ ORF (nucleotides 174–2252) putatively encodes for the non structural protein  and the 3′ ORF (nucleotides 2279–5038) for structural viral proteins VP1 (919 aa) and VP2 (544 aa). Moreover, by using ORF finder an additional ORF  and the corresponding putative protein (77 aa) were also identified . Phylogenetically, Eh-BtPV-1 was located between the Porcine Parvovirus 2 (PPV2) and the PARV4-like groups resembling a separate branch near the root of the PARV4-like genus thus potentially sharing a common ancestor with human PARV4, the Porcine (PoHV), the Bovine (BoHV), and the Chimpanzee (ChimpPTV) PARV4-like viruses . The NS1 protein of Eh-BtPV-1 is equally distant from NS1 of all the other PARV4-like viruses, being on average 41.4% amino acid identical to primates viruses, 42.4% identical to the bovine virus and 43.8% identical to porcine virus, and is thus the most dissimilar within the genus  coded by ORF2 appears to be larger than VP1 of the primate PARV4-like viruses (914 aa) but smaller than the one of the porcine hokovirus (925 aa) and the VP1 of the bovine virus (931 aa) . Like NS2 motif, which is typically present in most of the parvoviruses but absent in other viral families. This motif  is located in the N-terminal VP1 region that does not overlap the VP2 protein (VP1 unique region). As expected, this motif was conserved among all PARV4-like viruses. The catalytic domain of PLA2 (HDXXY) had only one difference in Eh-BtPV-1 compared to all the other viruses (HDRRY vs. HDERY) whereas the sequence of the Ca++ binding loop (YXGXG) was identical in all the viruses (YVGPG).The VP1 of Eh-BtPV-1 contains the phospholipase A2 motif in the VP1 unique coding region. In all PARV4-like viruses the MARF is in frame +1 in respect to the VP1 gene  resulted positive for the presence of Eh-BtPV-1. Viral concentrations ranged from 2.84E+4 to 1.47E+10 copies/ml .Additionally, Eh-BtPV-1 was found in all types of tested organs  and, in most cases, at high titers . Based oThe sequences obtained from the Eh-BtPV-1-positive bats (both from blood and organs) were 98.7–100% identical to each other, demonstrating the circulation of a single type in the analyzed bat population (data not shown). Beyond this, all the sequences obtained from samples belonging to the same bat (but different body districts) were identical, showing no apparent variability during the infection.Artibeus jamaicensis bats, exhibits characteristics common to the other vertebrate parvoviruses.Like Eh-BtPV-1, the genomic organization of the novel parvovirus identified in Panamanian Parvovirinae subfamily. The virus was named Artibeus jamaicensis Bat Parvovirus 1, Aj-BtPV-1.Phylogenetic analysis showed no clustering in the different trees and no clearly identifiable closest relative in any of the known genera . In all Parvovirinae a Simplot analysis was performed. In the first putative ORF  it was possible to identify a more conserved region of approx. 500 nucleotides. This region (nucleotides 835–1320 of Aj-BtPV-1 NS1 ORF) was then used to perform additional alignments and create further phylogenetic trees (both at nucleotide and amino acid level). The top identity percentages of Aj-BtPV-1 were found with Dependoviruses (between 53.4 and 59.6% at the amino acid level). However, all trees were consistent with the previous ones and Aj-BtPV-1 was still forming a separate lineage (branching autonomously from the others) confirming its independence from all the other known genera.To identify conserved parts between Since this virus represents the first member of a potentially new parvoviral genus and the transcriptional profile is not yet available, two alternative start codons could be hypothesized as the transcriptional origin of the VP1 protein. The first one (at position 1965 of the sequenced genome) is only 3 nucleotides after the stop codon of the first putative ORF and it would give origin to a protein of 876 aa (stop codon starts at nucleotide 4593); the second start codon is located at position 2025 and the product of this ORF would be a protein of 856 aa.2 domain with the conserved catalytic site (HDRGY) and the Ca++ binding loop (YTGPG) was identified at amino acids 259–303 of VP1 protein.The PLAArtibeus jamaicensis  and one animal .Out of 110 whole EDTA blood samples, 8 bats (7.3%) tested positive for the presence of Aj-BtPV-1, with a viral concentration ranging from 8.3E+03 to 4.87E+08 copies/ml. Seven of the Aj-BtPV-1-positive animals belonged to the frugivorous bat species  #174 in  belongedArtibeus lituratus belonged to the type 1 cluster.Based on phylogenetic analysis of a 740 nt fragment (nucleotides 4329–4561) amplified from different bats, three Aj-BtPV-1 types were distinguishable . Types 1Globally, bats are, among mammals, one of the most diverse and species-rich groups of vertebrates and, together with humans and rodents, they are the most broadly dispersed mammalian species. They live frequently in large groups and can reach very high densities in roosts where up to millions of bats may cluster in tight spaces Among the most important molecular characteristics that are likely to contribute to the emergence of a virus in a novel host system is the ‘evolutionary potential’: a rapid mutation rate and the tendency to recombine make a virus more prone to spread in a new host species Parvovirinae subfamily, we analyzed blood samples from two phylogenetically distant bat families Parvovirinae: Eh-BtPV-1, a PARV4-like virus, identified in Ghanaian Eidelon helvum bats and Aj-BtPV-1, initially identified in Panamanian Artibeus jamaicensis bats and representing the first member of a putative new parvoviral genus.To evaluate whether bats carry viruses belonging to the Parvovirinae identified in bat blood. Eh-BtPV-1 exhibits monophyletic origin with human PARV4 and the other primate, bovine and porcine viruses more recently identified 2 motif, but in a different reading frame. MARF was subsequently detected in all PARV4-like viruses so far known suggesting that the presence of this ORF characterizes this putative genus. Besides, Eh-BtPV-1 resembles a separate branch within the genus and, amongst the PARV4-like viruses identified until now it is the virus closest to the root of the genus. These data may suggest that a possible zoonotic transmission could have occurred in the past allowing the ancestor virus to be transmitted from bats to other mammals, as it has been already proposed for SARS-CoV To our knowledge, Eh-BtPV-1 is the first potentially autonomously replicating member of the subfamily Eidolon helvum, the most widely distributed fruit bat species in Africa, is a transcontinental migrant seasonally forming huge colonies of 5 to 10 million individuals. Its primary habitat is equatorial Africa but flying-foxes can migrate more than 2500 km Eidolon helvum in Ghana Artibeus jamaicensis bats from Panama. Aj-BtPV-1 was circulating in the respective A. jamaicensis population at a rate of 7.6% and, based on phylogenetic analysis, it clearly appears that at least three different lineages are currently circulating in this bat species. Interestingly, Aj-BtPV-1 was also found in blood collected from a bat belonging to the closely related species A. lituratus. Although the number of analyzed A. lituratus bat is low (18), the prevalence of viraemia (5.5%) was close to that detected in Artibeus jamaicensis bats. Aj-BtPV-1 is the first member of a potential new genus within the Parvovirinae subfamily, although its existence needs first to be accepted by the advisory board of the ICTV. Still, its discovery could open new research paths aiming to search for similar viruses in other mammals including humans. The presence of high viral variability may also increase the chance that further mutations could favor a viral transmission to other species. Artibeus jamaicensis, a bat from the endemic family of New World leaf-nosed bats (Phyllostomidae) that only occurs in Central and South America, forages mainly on fruit and occasionally nectar, pollen and leaves The second virus identified in this study, Aj-BtPV-1, was initially found in Dependovirus that mainly includes non-autonomous viruses. Additionally, in one of these studies a virus (TM-2p) was identified which is topologically located in the phylogenetic tree between the two parvoviral subfamilies Parvovirinae and DensovirinaeBeyond these newly identified bat viruses, two recent studies reported the discovery of new parvoviruses in fecal samples from insectivore bats  The two parvoviruses reported here were identified from blood samples, indicating that these viruses cause active infections in bats. Beyond the 2 parvoviruses, no other viral sequences were identified during our study, suggesting that both viruses may replicate autonomously and may not need other viruses to reach high titers (up to 1.47E+10 copies/ml). Our data also indicate that the viruses can spread easily throughout the body and that they circulate within the bat population with frequencies up to 8%. Furthermore, the presence of bat parvovirus in the intestinal tissue suggests that those viruses might be present in feces, as already reported for adeno-associated bat parvoviruses. Taken together, the data from the present and previous studies


Objectives. To explore health care costs associated with ankylosing spondylitis (AS) in Turkey. Methods. Research-identified data from a system that processes claims for all Turkish health insurance funds were analyzed. Adult prevalent and incident AS patients with two AS visits at least 60 days apart, identified between June 1, 2010 and December 31, 2010, with at least 1 year of continuous health plan enrollment for the baseline and follow-up years were included in the study. Pharmacy, outpatient, and inpatient claims were compiled over the study period for the selected patients. Generalized linear models were used to estimate the expected annual costs, controlling for baseline demographic and clinical characteristics. Results. A total of 2.986 patients were identified, of which 603 were incident cases and 2.383 prevalent cases. The mean ages were 39 and 41 years, respectively, and 44% and 38% were women for incident and prevalent cases. Prevalent patients had higher comorbidity scores  and were more likely to be prescribed nonsteroidal anti-inflammatory drugs (NSAIDs)  or biologics  relative to incident patients. Seventy-seven percent of prevalent patients were prescribed NSAIDs, followed by biologic and disease-modifying antirheumatic drugs (DMARDs). Total annual medical costs for incident AS patients were €2.253 and €4.233 for prevalent patients. Pharmacy costs accounted for a significant portion of total costs , followed by physician office visit costs. Prior comorbidities and treatment type also significantly contributed to overall costs. Conclusion. Annual expenditures for AS patients in Turkey were comparable relative to European countries. Pharmaceutical expenditures cover a significant portion of the overall costs. Comparative effectiveness studies are necessary to further decrease health care costs of AS treatment. Ankylosing spondylitis (AS) is a progressive and chronic inflammatory disease that affects the axial skeleton, causing characteristic back pain that can lead to structural and functional impairments and a decrease in quality of life –3.Epidemiologic studies indicate that AS is a more prevalent disease than previously thought. Overall AS prevalence in Europe is between 0.1% and 1.4% with mid-Europe being 0.3% to 0.5%, and AS incidence is calculated between 0.5 and 14 per 100,000 people per year. . There a€2.335 in The Netherlands, €2.064 in France, €1.572 in Belgium, and €1.750 in the United States [Prior to the introduction to biologics, due to the low cost of available therapy options, overall treatment costs were relatively low and estimated at d States .Recently, biological DMARDs, including tumor necrosis factor (TNF) inhibitors, have been shown to be efficacious in the treatment of AS. However, this new treatment option is expensive and emphasizes the need for information concerning the current burden of the disease. Although the economic burden of AS has been analyzed in European countries , 13, UniThis study estimated the medical costs associated with AS in Turkey for both prevalent and incident cases using nationwide real-world claims data.In 2006, Law 5502 by the Turkish Grand National Assembly aimed to unify the three existing separate social security and health insurance systems  into one, unified Social Security Institute (SSI). Enrollment in the currently existing Universal Health Insurance (UHI) Fund within the SSI will become mandatory, with contribution rates proportional to patients' ability to pay and all beneficiaries entitled to the same benefit package. MEDULA, a claims and utilization management system was established under the 2007 Health Budget Law (SUT), and all public and private facilities under contract with the SSI must submit claims through the MEDULA system. MEDULA data was used for this study. MEDULA covers 80% of the population in Turkey and is comprised of pharmacy, inpatient, outpatient, and laboratory claims from 17.800 pharmacies, 5.600 general practitioners, 4.500 medical centers, 1.200 government hospitals, and 338 private hospitals. The remaining 20% of the population not included in the data included people whose contribution rates were paid for by the government due to their income level (this data was maintained separately from the UHI Fund in the SSI until 2012). In addition, members of the Turkish Grand National Assembly and the Supreme Court, as well as foreign insurance policy holders and some military personnel, were excluded from the UHI Fund in the SSI. All patients diagnosed with AS between age 18 and 99 were included in the study for the identification period , using appropriated International Classification of Diseases Tenth Revision Clinical Modification (ICD-10-CM) diagnosis codes. In order to increase reliability of the diagnosis, patients were required to have two AS diagnoses. To ensure that individuals initially evaluated but subsequently not diagnosed with AS were correctly classified as not diagnosed with AS, a minimum of 60 days between claims was required . The datAll pharmacy, outpatient, and inpatient claims were compiled for the study period for all remaining patients.Two groups were analyzed: (1) AS incidence group and (2) AS prevalent group. For the incident group, patients were required to have no AS diagnosis during the baseline period, while patients in the prevalent group were required to have a minimum of one AS diagnosis during the baseline period.The first aim calculated annual costs of incident and prevalent AS cases in Turkey and identified how total costs were divided by inpatient, outpatient, pharmacy costs, and copayments. Inpatient and outpatient costs were categorized by service type, including surgery, consultations, office visits, laboratory tests, radiology, hospital admissions, medical devices, and other. Patient age and gender were available in the dataset. Since Turkey is divided into seven regions, flags were created according to patients' region of residence. To control for clinical characteristics, we calculated a comorbidity index score for each patient for the baseline period, using the Elixhauser method . This inGeneralized linear models (GLMs) were used to estimate risk-adjusted total annual costs for prevalent and incident cases. In particular, the expected annual cost value based on patient demographic and clinical characteristics was estimated. After applying the Park Test, Gamma distribution with log link function was used . AccordiAll statistical analyses were conducted using SAS V.9.3 and STATA V11.P < 0.001). In terms of individual comorbidities, there were no differences in diabetes or allergies, but cardiovascular  and respiratory comorbidities  were significantly higher for prevalent patients. During the baseline period, 77% (72%) of prevalent (incident) subjects received NSAIDs, 34.75% (7.46%) were prescribed biologics, and 23.29% (21.39%) were prescribed DMARDs. Note that these numbers are not mutually exclusive. A total of 2.986 patients satisfied all inclusion and exclusion criteria, of which 603 were incident cases and 2.383 prevalent cases. As presented in As €452 and €492, respectively, after excluding the aforementioned items. For both cases, a significant portion of this cost was due to physician costs . For prevalent cases, 14.03% of overall inpatient and outpatient costs were due to other costs, followed by hospital pharmacy (13.88%), laboratory tests (9.06%), surgery (9.01%), medical devices (4.90%), and hospital admissions (4.81%). For incident patients, after physician and surgery costs (17.13%), the most expensive components of inpatient and outpatient costs were other costs (15.86%), followed by laboratory tests (14.29%), inpatient pharmacy (6.11%), hospital admissions (4.02%), and surgery costs (3.00%).€3.307 for prevalent cases and €2.000 for incident cases (P < 0.001). For incidence cases, the cost of care was lower for the 18–39 age group when controlling for other factors. For prevalent cases, there were no differences in health care costs in terms of region, gender, age, comorbidities, or prior NSAID or DMARD use. Determinants of total health care costs were estimated using GLMs. After controlling for demographic and clinical factors, risk-adjusted annual total costs were calculated as nt cases . Prior b€1.678 for NSAID, €1.780 for DMARD versus None €2.852). When outpatient pharmacy costs were excluded from the overall costs, there were no differences in terms of the costs among the different types of treatments.Both models determined that the cost of biologic use was the single most important contributor to overall health care costs. Current retrospective analysis of a nationwide sample of Turkish patients not only provides solid data for economic evaluation in Turkey but also contributes to the general knowledge of how costs are distributed across health care services for AS. This study provides information regarding real-world clinical practices across patient subgroups and includes variations difficult to assess using data from trials, surveys, expert opinions, and other primary data sources.€3.566 [To our knowledge, this is the first study to investigate total medical health care costs associated with AS in Turkey using nationwide data. The only prior studies to analyze AS costs in Turkey were based on expert opinion, in which estimated direct costs were approximately €3.566 . Other s€3.566 , 24–26.€4.634 in Spain [€4.578 in Canada [Recently, health care costs of AS has been calculated at in Spain , €4.578 n Canada , and €3.n Canada . Economin Canada . TherefoIn Turkey, payment by health insurance fund is based both on a retrospective fee-for-service (FFS) and a bundled payment system depending on the disease category and services related to the particular disease. Payment procedures are outlined by health budget laws (SUT). For example, private hospitals are generally paid according to the bundled payment system. University hospitals, however, are based on the FFS system. Laboratory services can be paid separately from the bundled payment system based on certain conditions. Access to biological drugs is determined by protocols of the Ministry of Health, and payment is determined by the health budget law of the SSI . These p€3.833 in Turkey. Approximately 88% of the overall costs were associated with pharmaceutical costs. Inpatient costs were approximately 4%, and outpatient costs comprised 8% of the overall costs. The average annual copay amount was €22. These ratios are not surprising in light of the fact that health service fees such as hospital and physician costs are lower in Turkey relative to European countries. Prior medication use significantly affected the cost estimation.This study's estimates were similar to other countries' estimated AS costs. Combining the AS incident and prevalent cases, annual health care costs of AS were €4.218, whereas costs for patients with a score of lower than 2 costs were only €2.254. However, after controlling for medication use, comorbidity burden did not affect overall health care costs. A number of studies found that costs were driven by disease severity , 11, 12.€15.622 [€8.359. In the past, fewer and less expensive treatment options were available for AS patients. The recent introduction of biologic medications was a substantial development for AS treatment. However, these new efficacious treatment options are available at a high price. A British study estimated that the average annual cost for a newly approved biologic medications prescribed for AS treatment is €15.622 . The incIt is important to note that this study has limitations . First, Second, it is not uncommon in many parts of the world for mild AS patients who respond well to NSAIDs to buy these medications out-of-pocket while seeing their physicians in private practice and hospitals' settings only every few years. To the extent that these patients exist in our dataset, our cost estimates are underestimated since private practice claims would be missing. However, lower copayment amounts in Turkey mitigate this bias. Third, occurrence of a drug prescription fill does not guarantee actual consumption of the drug by the patient. Therefore, study results are biased to the extent that compliance rates are unevenly distributed among treatment options. Although a multivariate regression model was used to control for risk factors, the data did not contain any measure of disease activity, health status, or patient lifestyle. Comorbidity index scores, individual comorbidities, and baseline treatments were used to proxy for disease severity. However, they may not be a true representative of disease severity. Further studies can link the outcomes from claims data to hospital charts, where disease activity scores may be derived.Nationally representative real-world data from Turkey was used to estimate health care costs associated with AS. To ensure that economic evaluations are relevant, country-specific observational studies are necessary. These study's findings are consistent with existing research and suggest that AS health care costs in Turkey resemble those in Europe. Costs are driven by treatment choices. The current data provide a baseline to evaluate the economic effect of such treatments and assist future AS-related health care policy and analysis.


Changuinola virus (CGLV) serotype Irituia virus (BE AN 28873) isolated from a wild rodent (Oryzomys goeldi) in the municipality of Ipixuna, State of Pará, northern Brazil. All genome segments showed similarity with those belonging to members of the genus Orbivirus, family Reoviridae.We report here the first complete genome sequence of a  Orbivirus belongs to the family Reoviridae and includes 22 virus species, as well as 10 unclassified or unassigned viruses  and African horse sickness virus (AHSV) are the most economically important members of this genus. Several other orbiviruses are potentially important too, such as Equine encephalosis virus (EEV), Epizootic hemorrhagic disease virus (EHDV), Palyam virus (PALV), and Peruvian horse sickness virus (PHSV) (Kemerovo virus (KEMV) and Tribeč virus (TRBV)  , while os (TRBV) .Irituia virus (BE AN 28873) was isolated at the Evandro Chagas Institute, Belém, Brazil, from the blood of a wild rodent species, Oryzomys goeldi, caught on 29 March 1961 in the municipality of Ipixuna, Brazil (3°S 49°W). Antigenic characterization was carried out by complement fixation and neutralization tests  (3 days postinfection) the supernatant was harvested. Viral particles were concentrated using polyethylene glycol (PEG) centrifugation as previously described  adapter was ligated to the 3′ ends of each RNA segment  to be abde novo Assembler (Newbler v. 2.6). For further analysis, the software Geneious v. 6.1.4 and a set of programs  available in the DNASTAR package (Lasergene) were used. The coverage of the assembled genome was 1,522-fold. This is the first report for the complete genome sequence of a Brazilian CGLV serotype.The assembly of the obtained sequences was done using the GS KF624614 to KF624623.The complete genome sequence has been deposited in GenBank under the accession numbers


Prevalence ranged from 7.8% to 22.1%, depending on region. In Senegal, during 2002–2007, 11 outbreaks of African swine fever (ASF) were reported to the World Organisation for Animal Health. Despite this, little was known of the epidemiology of ASF in the country. To determine the prevalence of ASF in Senegal in 2006, we tested serum specimens collected from a sample of pigs in the 3 main pig-farming regions for antibodies to ASF virus using an ELISA. Of 747 serum samples examined, 126 were positive for ASF, suggesting a prevalence of 16.9%. The estimated prevalences within each of the regions  were 13.3%, 7.8%, and 22.1%, respectively, with statistical evidence to suggest that the prevalence in Ziguinchor was higher than in Fatick or Kolda. This regional difference is considered in relation to different farming systems and illegal trade with neighboring countries where the infection is endemic. Asfarviridae. The disease is highly contagious and often lethal for pigs and is of considerable economic importance, due to the direct loss of animals as well as resulting trade restrictions. No vaccine is available against the virus. The epidemiology of ASF is complex, transmission is direct and vector-borne, and the disease has well recognized sylvatic and domestic cycles.African swine fever (ASF) is a disease caused by a DNA virus in the family ASF is currently considered enzootic in eastern and southern Africa, and the epidemiologic cycles of importance in many of the countries in these regions are well understood , and in areas where tourism has increased the demand for pork, such as Sine Saloum (the Fatick region). Although ASF has been identified as one of the 6 major diseases in need of epidemiologic surveillance in Senegal, few structured surveys have been conducted  of all farms in the country were free-range systems , with a required precision of 6% and an α-error of 5%. Villages were considered as clusters of animals, and a decision was made to sample 10 pigs per village, from as many different farms as possible to maximize the representativeness of the sample. The formulas used in determining sample size, while accounting for clustering at the village level, are shown below , which is a blocking ELISA that uses a purified protein extract from the virus (VP73) as the antigen. According to C. Gallardo  the sensitivity and specificity of this test were both in the region of 95% to 98%. The apparent prevalence estimates were therefore corrected to give the true prevalence by using the following formula (www.r-project.org). Farms were nested within villages, and both were modeled as random effects. A z test was conducted to compare these random effects, to evaluate whether evidence of clustering of seropositivity could be found within villages and farms. The prevalence estimates in different regions were then compared by using the method described by Altman and Bland . The general linear mixed model method gave seroprevalence estimates of 13.3%, 7.8%, and 22.1% for the regions of Fatick, Kolda, and Ziguinchor, respectively . The preTest sensitivity and specificity were accounted for to estimate the true seroprevalence for each region. We gave the smallest and higher value taking into account the uncertainty around the true value of sensitivity and specificity. Following this process, the seroprevalence estimates for Fatick, Kolda, and Ziguinchor were expanded to range from 8.9% to 12.1%; from 3.0% to 6.2%; and from 18.3% to 21.6%, respectively. 95% confidence intervals for these estimates are shown in The results of this study are corroborated by unpublished data from the Senegalese Institute of Agricultural Research regarding the prevalence of ASF in the Ziguinchor region . Considering that ASF viruses currently circulating in West Africa are closely related to those circulating in Europe in the second half of the last century (Ornithodoros sonrai ticks containing remnants of ASF virus DNA have been identified in the Fatick region (O. sonrai ticks (,k region , the souAlthough ASF virus can persist for long periods after infection and even recovery in pigs, seroprevalence estimates for the antibodies against the virus do not estimate the percentage of pigs with current infection, or even the percentage of carrier pigs. Rather, they indicate the percentage of pigs that have been exposed to the virus at some point in their lifetime. Bech-Nielsen et al. reported the detection of ASF virus in only 4.4% of carrier animals (,Considering that only pigs from 6 months to 2 years of age were tested, the pigs that tested positive must have become infected between 2004 and 2006. During this period, 5 outbreaks were declared in Senegal, with 646 cases and 561 deaths (,Furthermore, our results suggest that these pigs survived virus infection, which contrasts with the widespread perception that mortality rates for ASF virus infection are high, approaching 80% (,,ASF virus strains of low virulence have been identified in various countries since 1984, and despite low virulence, could still maintain a high infectivity (Antibody-mediated resistance to ASF virus can be acquired through passive transfer of maternal antibodies or by previous infection with a virus of low pathogenicity or from low doses of highly virulent viruses (Significant differences in seroprevalence were observed between the 3 regions, with a higher seroprevalence identified in Ziguinchor than in Kolda or Fatick. The Ziguinchor region lies between the Gambia and Guinea-Bissau. ASF has been enzootic within Guinea-Bissau for years, and no efforts to control the disease have been reported (A study conducted in 1987 and 1988 found no evidence of seropositive animals among the 122 samples in the Fatick region (,Although ASF has been known to be enzootic in the Ziguinchor region for >10 years (


Wolbachia, which both cause cytoplasmic incompatibility and protect their hosts against viruses. Here, we carry out the first test as to whether a male-killing strain of Wolbachia also provides a direct benefit to its host by providing antiviral protection to its host Drosophila bifasciata. We infected flies with two positive sense RNA viruses known to replicate in a range of Drosophila species (Drosophila C virus and Flock House virus) and measure the rate of death in Wolbachia positive and negative host lines with the same genetic background.Insect symbionts employ multiple strategies to enhance their spread through populations, and some play a dual role as both a mutualist and a reproductive manipulator. It has recently been found that this is the case for some strains of D. bifasciata flies, with Drosophila C virus killing 43% more flies than the uninfected controls and Flock House virus killing 78% more flies than the uninfected controls. However, viral induced mortality was unaffected by the presence of Wolbachia.Both viruses caused considerable mortality to Wolbachia strain tested for antiviral effects, we found no evidence that it conferred protection against two RNA viruses. We show that although antiviral resistance is widespread across the Wolbachia phylogeny, the trait seems to have been lost or gained along some lineages. We discuss the potential mechanisms of this, and can seemingly discount protection against these viruses as a reason why this symbiont has spread through Drosophila populations.In the first male-killing  Wolbachia alone [Maternally transmitted bacterial symbionts are extremely common in insects, with over half of all species estimated to be infected by bacteria from the genus ia alone . Becauseia alone . Some syia alone . For exaia alone -6. Otheria alone . Some muia alone , while oia alone ,9).Spiroplasma bacteria provide protection from nematodes in Drosophila neotestacea [Drosophila hydei [. Recently, Wolbachia has been shown to make species of Drosophila and mosquitoes resistant to RNA viruses [D. melanogaster more tolerant to viral infection, as the survival of flies infected with flock house virus (FHV) increased despite there being no effect on viral titres [Dicistroviridae , Nodaviridae (Flock House virus), Picorna-like viruses (Nora virus), Togaviridae (Chikungunya virus) and Flaviviridae (Dengue virus and West Nile virus) [A number of recent studies have found secondary symbionts providing the host with protection against parasites and pathogens . In aphitestacea  and parala hydei . Recentl viruses -22. It cl titres . This pre virus) ,20,22,23Rickettsia in whiteflies act both to directly increase host fitness and distort the sex ratio towards the production of female offspring [Wolbachia can both act as both a mutualist and a reproductive manipulator; in Drosophila simulans, strains of Wolbachia that induce strong cytoplasmic incompatibility also protect the host from viral infection [Wolbachia in D. melanogaster are commonly below 50% [Wolbachia infections [Symbionts can sometimes employ multiple strategies to enhance their spread through populations. ffspring . It has nfection . Such dunfection ,25,26. Telow 50% ,28. Thiselow 50% , and recfections . Similarfections . Howeverfections , so siblfections  could thD. bifasciata infected with a male-killing strain of Wolbachia have greater protection from viral pathogens. This strain of Wolbachia naturally infects 5-7% of female D. bifasciata resulting in close to 100% female broods at 18°C [In this study we have tested whether  at 18°C . At elev at 18°C . In thisDicistroviridae [D. melanogaster in the wild [Drosophila species [Flock House virus (FHV), a positive sense RNA virus in the family Nodaviridae [Drosophila , a positive sense RNA virus in the family oviridae  that natthe wild ,36. DCV  species , and can species . Secondlaviridae . It is neopteran ), but wieopteran -44. Wolb viruses ,18.Wolbachia-infected line of Drosophila bifasciata was collected in Japan in 1998 [Wolbachia infection rate was 100% . The flies were reared on agar-malt medium at ~18°C.The  in 1998 . Since tWe used reverse transcription (rt) PCR to check that the fly stocks we were using were not infected with DCV or FHV before the experiment. Total RNA was extracted from 40 individuals per line using Trizol reagent  as described previously . RNA was50 of 1.35 x 106 TCID50 in 69nl for FHV and 1000 TCID50 in 69nl for DCV.We then tested the effect of fly Wolbachia infection status on viral pathogenicity. The viral isolates have been described previously ,46  was calculated. Starting from the 10-4 dilution, serial dilutions to 10-10 were made in Schneider’s medium, and each dilution was added to 8 wells of a plate. After 7 days the wells were examined and classed as “infected” when cell death and cytopathic effects were clearly visible. The TCID50 was calculated by the Reed-Muench end-point method [50 were consistent.To produce the virus, Schneider Drosophila line 2 (DL2) cells were cultured at 26.5°C in Schneider’s Drosophila Medium (Invitrogen) supplemented with 10% Fetal Bovine Serum, 2mM L-Glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin . The cells were infected with DCV, and after they showed cytopathic effect they were filtered through a 0.45 μm filter and centrifuged at 13.500 rpm for 10 minutes to remove any bacteria or cellular components. Aliquots of a 10t method . The Poit method . The expAs a negative control we also injected flies with Drosophila Ringer’s solution  for the Wolbachia on survival rates was analysed using a Cox’s proportional hazards mixed effect model, which accounted for between vial variation in survival rates. The hazard for the ith individual from vial j at time t was modelled as:The flies were examined each day and the number of dead individuals in each vial was recorded. The effect of H0(t) is the baseline hazard at time t, iX is a vector of the fixed effects, β is the corresponding vector of coefficients, and jb is a random effect of vial j. The fixed effects consisted of treatment (virus or negative), Wolbachia (infected or uninfected) and their interaction. Flies alive at the end of the experiment were censored. The model was fitted by maximum likelihood using the coxme package in R .Where D. bifasciata lines tested positive for DCV-like viruses by rtPCR, 454 flies were injected with DCV , with the death rate of infected flies accelerating after ten days, such that 59% of the DCV injected flies had died by day 16 in comparison to 16% in the uninfected controls. However, the presence of Wolbachia did not affect the rate at which DCV kills flies , nor was there an overall effect of Wolbachia on survival .DCV: Having established that neither of the z=-8.72, P<0.001). Again the presence of Wolbachia had no affect on the rate at which FHV killed flies , nor was there any main effect of Wolbachia . Neither of the fly lines tested positive for FHV-like viruses by rtPCR.FHV: The results from the FHV experiment were similar. In this experiment 539 flies were injected  [Wolbachia strains that do not protect their hosts against viruses may be common, and that each strain will require independent evaluation.It has recently become clear that secondary symbionts have often evolved multiple strategies to spread through host populations, and tests on a small number of parasite . For theimulans) -19. Our Wolbachia strains provide antiviral protection, whereas the D. bifasciata male killer strain does not. The difference could be caused by genes in the host, but results from other species suggest that this may not be the most likely explanation, as wMel retained its antiviral effect even when it was transferred between different dipteran families [Wolbachia strains suggest that protection is effective against a diverse range of RNA viruses with positive sense genomes [D. bifasciata male killer may lack the antiviral effect seen in other strains is due to genetic factors in the bacteria. Phylogenies of Wolbachia place the D. bifasciata male killer within the A clade, along with the other Wolbachia strains in Drosophila that offer protection against viruses [Wolbachia strains from mosquitoes with antiviral effects belong to the B clade [Wolbachia genome is known to recombine [Hamiltonella defensa, the only case where the genetic basis of symbiont-mediated protection is known, a protection of aphids from parasitoid wasps is encoded on genes carried by a phage [There are a number of possible explanations as to why many families . We may  genomes ,18,20,23 viruses ,50,51. I B clade ,23. The ecombine ,53 and cecombine . In Hami a phage .D. simulans [Aedes albopictus [Wolbachia strains offering the greatest protection to viruses have significantly greater densities of Wolbachia than those that did not.Regardless of whether host or bacterial genes determine whether different strains have antiviral effects, it is possible that these genes may not encode the antiviral factors themselves, but may simply control bacterial density. In both simulans  and Aedebopictus  the WolbDrosophila and some butterflies [D. bifasciata it seems the spread of the male-killer has not been aided by any antiviral effect against the two viruses examined here.In many cases the spread of male-killing bacteria through host populations is surprising. Male-killing bacteria are only expected to invade insect populations when the death of males benefits the surviving females who will transmit the infection to their offspring . For exaterflies , the benBL, GDDH and FMJ designed the study. BL and DKF carried out the experimental work. BL and FMJ analysed the data and drafted the manuscript with comments from GDDH and DKF. All authors read and approved the final manuscript.The authors declare they have no competing interests.Number of flies injected per treatment, figure in brackets is number of vials per treatment. There was a mean of 19 flies per vial.Click here for file


Dianna Edgil and colleagues evaluate the supply chain and waste management costs needed to deliver mobile medical male circumcision services to 152,000 men in Swaziland, finding that per-procedure costs almost double when these factors are taken into account. The global HIV prevention community is implementing voluntary medical male circumcision (VMMC) programs across eastern and southern Africa, with a goal of reaching 80% coverage in adult males by 2015. Successful implementation will depend on the accessibility of commodities essential for VMMC programming and the appropriate allocation of resources to support the VMMC supply chain. For this, the United States President’s Emergency Plan for AIDS Relief, in collaboration with the World Health Organization and the Joint United Nations Programme on HIV/AIDS, has developed a standard list of commodities for VMMC programs.This list of commodities was used to inform program planning for a 1-y program to circumcise 152,000 adult men in Swaziland. During this process, additional key commodities were identified, expanding the standard list to include commodities for waste management, HIV counseling and testing, and the treatment of sexually transmitted infections.The approximate costs for the procurement of commodities, management of a supply chain, and waste disposal, were determined for the VMMC program in Swaziland using current market prices of goods and services.Previous costing studies of VMMC programs did not capture supply chain costs, nor the full range of commodities needed for VMMC program implementation or waste management. Our calculations indicate that depending upon the volume of services provided, supply chain and waste management, including commodities and associated labor, contribute between US$58.92 and US$73.57 to the cost of performing one adult male circumcision in Swaziland.Experience with the VMMC program in Swaziland indicates that supply chain and waste management add approximately US$60 per circumcision, nearly doubling the total per procedure cost estimated previously; these additional costs are used to inform the estimate of per procedure costs modeled by Njeuhmeli et al. in “Voluntary Medical Male Circumcision: Modeling the Impact and Cost of Expanding Male Circumcision for HIV Prevention in Eastern and Southern Africa.” Program planners and policy makers should consider the significant contribution of supply chain and waste management to VMMC program costs as they determine future resource needs for VMMC programs.Please see later in the article for the Editors' Summary About 33 million people (including 22.5 million in sub-Saharan Africa) are currently infected with HIV, the virus that causes AIDS. Although antiretroviral drugs keep HIV in check, there is no cure for HIV/AIDS. Consequently, prevention of HIV transmission is extremely important. Because HIV is usually spread through unprotected sex with an infected partner, individuals can reduce their risk of becoming infected with HIV by abstaining from sex, by having only one or a few partners, and by always using male or female condoms. In addition, trials in sub-Saharan Africa have shown that male circumcision—the removal of the foreskin, the loose fold of skin that covers the head of the penis—reduces the risk of HIV infection in men by 60%. In 2007, the World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS) recommended that voluntary medical male circumcision (VMMC) should be part of HIV prevention programs in regions with a generalized HIV epidemic and a low level of male circumcision. Together with the United States President’s Emergency Plan for AIDS Relief (PEPFAR), WHO, and UNAIDS also prioritized 14 countries in eastern and southern Africa for VMMC program scale-up. Mathematical models suggest that, if 80% VMMC coverage is reached by 2015 (which will entail performing 20.33 million circumcisions between 2011 and 2015) and sustained thereafter, VMMC programs in these priority countries will avert more than 4 million HIV infections among adults between 2009 and 2025.Soka Uncobe [Circumcise and Conquer] in SiSwati), a one-year program to circumcise 152,000 men in Swaziland.Successful VMMC scale-up will depend on the commodities that are essential for VMMC services being accessible and on the appropriate allocation of resources to support VMMC programs . To help program planners and policy makers, costing studies have been undertaken in several African countries. These studies considered the costs of a standard list of commodities prepared by PEPFAR, WHO, and UNAIDS and estimated that, on average, one male circumcision costs about US$53. However, these studies did not include the costs of the supply chain, waste management, HIV counseling and testing, treatment of sexually transmitted infections, or the temporary infrastructure needed to deliver mobile VMMC services. Here, the researchers estimate these hitherto ignored costs for the Accelerated Saturation Initiative  and is further discussed in a PLoS Medicine Review Article by Hankins et al. .This study is part of a PLoS collection of articles on VMMC WHO, UNAIDS, and PEPFAR on all aspects of HIV/AIDSInformation is available from NAM/aidsmap provides basic information about HIV/AIDS, summaries of recent research findings on HIV care and treatment, and information on male circumcision for the prevention of HIV transmissionAvert, an international AIDS charity on many aspects of HIV/AIDS, including information on all aspects of HIV prevention, and on HIV/AIDS in Africa and in Swaziland (in English and Spanish)Information is available from Clearinghouse on Male Circumcision, a resource provided by WHO, UNAIDS, and other international bodies, provides information and tools for VMMC policy development and program implementationThe Avert, through NAM/aidsmap, and through the charity website HealthtalkonlinePersonal stories about living with HIV/AIDS are available through  Based on evidence from randomized controlled clinical trials conducted in Africa indicating that voluntary medical male circumcision (VMMC) significantly reduces male participants’ risk of acquiring HIV infection, the global HIV prevention community has supported the scale-up of VMMC programs in 14 countries Supply chain considerations and commodity costs carry significant financial implications for all public health programs, but these costs are particularly relevant for highly technical programs planned on the scale anticipated for VMMC services. The minimum package of services that should compose all VMMC for HIV prevention programs—as delineated by the World Health Organization (WHO)—encompasses an array of activities beyond circumcision surgery, including (1) HIV testing and counseling, (2) sexually transmitted infection (STI) screening and treatment, (3) condom provision and promotion, and (4) counseling on risk reduction and safer sex As a result, costing studies have been undertaken in Namibia To date, these studies have estimated the cost of a single male circumcision to be, on average, approximately US$53, ranging from US$19 in Uganda The Partnership for Supply Chain Management (PFSCM), supported by the United States President’s Emergency Plan for AIDS Relief (PEPFAR), began assisting countries that were scaling up VMMC programs to develop, strengthen, and manage reliable, cost-effective, and sustainable supply chains as early as 2009. Further, in an attempt to harness economies of scale and streamline logistical issues, PEPFAR hosted an expert consultation in 2009 on VMMC supply chain issues As a result of that meeting, a standard list of VMMC commodities was put forward by international stakeholders. The bundling of commodities into surgical instrument kits was recommended , as well as standardized equipment  Given the limitations of previous costing exercises for VMMC programs, the purpose of this paper is to present, to our knowledge for the first time, the full supply chain costs associated with the scale-up of a VMMC program in one southern African country, Swaziland. This information will also improve the standard list of commodities outlined by PEPFAR and WHO by adding commodities for waste management, counseling and testing, and temporary infrastructure for VMMC programming.To inform this analysis, we collected costing information for supply chain and waste management activities and commodities associated with the planned implementation of Swaziland’s 1-y VMMC scale-up campaign—known as the ASI The Swazi campaign goal is to increase VMMC coverage for HIV prevention purposes from 8% to 80% among 15- to 49-y-old males by the end of 2011 to avert nearly 100,000 HIV infections; reaching this target equates to circumcising approximately 152,000 men Consistent with MOVE recommendations and to meet the target of 152,000 VMMCs, it was determined that services would be delivered by a varying number of teams per month , with up to 35 teams operating simultaneously at 27 sites during peak months in 2011 At the midpoint of the VMMC campaign in Swaziland, the rate of scale-up is less than would be expected to allow the program to reach its target of 152,000 VMMCs. To reflect a lower rate of service delivery, with a target of reaching 75,000 men over 1 y, the number of operational sites was reduced to 20, with a single team operating at each site.The PFSCM provides technical and programmatic support on procurement, logistics, and waste management to the Government of the Kingdom of Swaziland in its effort to implement the ASI program. In support of the Swaziland campaign, and with input from other stakeholders, PFSCM developed a full list of commodities for the ASI campaign. This included the four modules already developed by PEPFAR and WHO, as well as commodities modules for (1) HIV counseling and testing and STI treatment and (2) temporary infrastructure necessary for the delivery of mobile VMMC services  to ensure proper waste storage, handling, and disposal; and developing training curricula for facility staff responsible for waste management In addition to the programmatic costs associated with the operation of the supply chain, we assessed the fixed and variable cost of all quantified commodities, organized into six modules: (1) VMMC kit and consumables, (2) infection prevention and waste management, (3) equipment for male circumcision sites, (4) emergency medical supplies, (5) HIV counseling and testing and other STI management, and (6) temporary infrastructure.  or the revised target  for a single year of the VMMC scale-up program.In addition to the costs identified above, we added 2% of the total direct operating costs to cover overhead costs that were neither facility- nor labor-based. For example, the 2% overhead would cover communication costs and other program-management-related expenses.Waste management costs are both fixed and variable. PFSCM performed an assessment of the national waste management system prior to developing the waste management plan for the Swazi VMMC program. Based on the volume and weight of the commodities needed for each VMMC using fully disposable kits, PFSCM assessed the needs of the waste disposal system in Swaziland, using the combined weight of the consumables required for an individual surgery to produce an estimate of approximately 0.5 kg of biologically contaminated waste to be generated per procedure. This level was more than the national waste disposal system could process.In this analysis, staffing for waste management included the provision of technical assistance by two PFSCM headquarters staff and the training and support of two local staff (a driver and a waste handler) for transportation of medical waste. As well, cost sharing of incinerator operation and staff time at regional hospitals was included in the final cost.Using the expanded list of WHO-recommended VMMC commodities, we estimated the commodity procurement, supply chain, and waste management costs associated with providing VMMC services to 152,000 adult men in 1 y in Swaziland to be US$8,956,213.46. This is the equivalent of US$58.92 per VMMC. For a reduced target of 75,000 VMMCs in 1 y, the total cost is calculated as US$5,518,393.50, the equivalent of US$73.57 per VMMC.Module 1 contains the ethylene-oxide-sterilized, fully disposable kit for performing the forceps-guided method of VMMC and supplementary consumable medical supplies. These commodities are required for the performance of each surgery, representing a variable cost of US$25.17 per circumcision. In the Swaziland context with a target of 152,000 VMMCs, Module 1 costs contribute a total of US$3,839,144.08 to the commodity and supply chain costs of the program. For a target of 75,000 VMMCs, Module 1 contributes US$1,902,572.04 to the total cost of the Swazi VMMC program, representing a per VMMC cost of US$25.18. Aligned with prior PEPFAR and WHO guidance, the VMMC kit comprises a consumables pack and an instrument set Items within the kit are packed in the order in which they will be required for the surgical procedure. Before being placed in the kit, consumable items and any necessary instruments are removed from the manufacturer’s external packaging. This reduces the total VMMC procedure time by removing the need to unwrap various commodities. Removal from the original packaging does destroy the items’ sterility, necessitating that the entire kit be sterilized using ethylene oxide gas before use. This service is provided by the kit packager and included in the total cost of the kit. The market price for an ethylene-oxide-sterilized VMMC kit with a standard consumables pack and disposable forceps-guided instrument set is currently US$17. Module 1 also includes bulk consumables that supplement those found in the VMMC kit. These include pharmaceuticals necessary for the procedure  but that were removed from the kit itself due to expiration concerns and importation restrictions. Additional sutures, gloves, and gauze also are included, in case those in the kit are not adequate. The cost of these consumables is estimated to be US$8.26 per VMMC.The second module contains standard infection prevention items as well as those required for waste management. Previously, based on WHO recommendations, this module contained only infection prevention commodities. However, given the importance of effective waste management and its potential impact on the broader health system, this estimate expands the module to include waste management commodities.Most infection prevention commodities are consumable and represent variable costs, depending on the number of circumcisions performed. Waste management commodities represent largely fixed costs that require one-time procurement at the start of program operations. In Swaziland, for a target of 152,000 VMMCs, these commodities total US$1,427,853.90 and contribute US$9.39 to the cost of each VMMC. To reach a target of 75,000 VMMCs, the total cost of commodities for Module 2 is US$819,862.91, contributing US$10.93 to the cost of each VMMC. Of these costs, US$211,872 is fixed.Infection prevention commodities, while critical to program implementation, are largely low cost and include items such as surgical masks and caps, protective eyewear, surgical scrub solution and alcohol hand wash, buckets for instrument cleaning, and bleach.In contrast, waste management commodity costs can be significant. Items such as sharps bins and biohazard bags are used to collect three types of medical waste associated with a VMMC program—sharps waste , human tissue (foreskin), and general medical waste . This waste is transported to an incinerator, where it is turned into an ash by-product. For Swaziland, the ASI program will require the provision of upgrades to the current waste disposal facilities and the procurement of two additional incinerators, as well as the generators and fuel necessary to operate these incinerators. This increases the cost of Module 2 by US$105,470 (US$0.69 per VMMC).Metal waste, generated by the use of either disposable or reusable instruments, cannot be incinerated and must be buried or recycled. These disposal methods do not include additional commodity costs, but do require that the instruments be disinfected prior to disposal. In the case of the ASI program, disinfected metal waste will be donated to a metal foundry in Swaziland. Transportation costs to the foundry are included in the cost of metal waste disposal.The third module includes equipment necessary for the set-up of one operating theater with four beds and one recovery area. This includes such items as an operating table, lamp, instrument tray, intravenous stand, diathermy machine, and recovery chair. Small equipment items, such as a stethoscope and glucometer, are also included. These items represent fixed costs, requiring procurement only once during program implementation. Glucometer strips may be considered an exception, as they will require replacement over the lifetime of a program.For a program reaching a target of 152,000 VMMCs, with service provision at 27 sites and 35 teams, the total cost of this module is US$469,202.33, or US$3.08 per VMMC, US$2,100 of which is variable costs. For a reduced target of 75,000 VMMCs achieved through service provision at 20 sites with 20 teams, this module contributes US$267,953.96  to the total cost of the Swazi VMMC program, or US$3.57 per VMMC.The final WHO-recommended module, emergency medical supplies, includes items necessary in the case of a medical emergency. These include jump bags with necessary medical equipment , emergency pharmaceuticals , and an emergency trolley to hold these items. With the exception of the pharmaceuticals, these costs are fixed. Items in the jump bag may require occasional replacement, but this cost will be minimal and dependent on the number of adverse events in a program. These replacement costs are not included in this calculation.In Swaziland, for a program targeting 152,000 VMMCs through service provision at 35 sites, the total cost of this module is US$50,022.92, or US$0.33 per VMMC. Of these costs, US$49,630 is fixed. For a VMMC program reaching 75,000 men through service provision at 20 sites, the total cost of this module is US$28,556.46, of which US$28,360 is fixed. This module contributes US$0.38 to the cost of each VMMC.This module includes HIV test kits, lancets, and pipettes, as well as pharmaceuticals for the treatment of STIs. These costs are variable, and the quantity required will be determined by the rate of consumption. For example, HIV tests should be recommended to all VMMC clients, making this cost dependent on expected demand. Confirmatory test kits should be ordered based on the HIV prevalence in the target population. Similarly, pharmaceutical costs for STI management will depend on the prevalence of various STIs in the population.In Swaziland, the total cost of this module is US$486,833.70, or US$3.20 per VMMC to reach a target of 152,000 VMMCs. To reach a target of 75,000 VMMCs, this module costs US$243,416.85, or US$3.25 per VMMC.Finally, two key commodities are included as a separate module and represent costs associated with the mobile delivery of VMMC services in Swaziland. The first comprises mobile medical (marquees) and industrial tents where VMMC mobile services will operate. The second is generators, which will be used to power diathermy machines and other electrical equipment at mobile sites. Also included are costs associated with fuel to operate the generators.We believe that these are important costs to assess, given that many VMMC programs are considering mobile delivery of services. While the delivery method remains nearly identical in these mobile sites, there are key commodity considerations. In Swaziland, where tent structures will be used for 23 out of 27 sites (the remaining sites will use existing structures), these commodities contribute US$921,380 to the total cost of VMMC service delivery. This is the equivalent of US$6.06 per circumcision. Reducing the number of sites providing VMMC services to 20 results in an overall reduction in the total cost of this module to US$835,130, with a per VMMC cost of US$11.14. Despite the reduction of the VMMC target by half, the program still requires the use of 20 sites to facilitate access to services over the entire country. This results in a near doubling of the per VMMC costs in this module despite a reduction in volume.The procurement, freight, and distribution costs associated with these commodities are significant. After estimating costs associated with each step of the supply chain, the total cost of these services amounts to US$1,759,676.52. Costs included in the supply chain calculation are found in Logistics costs include both international freight and in-country distribution to 27 service delivery sites. We also include commodity insurance and clearance costs based on current market rates, costs associated with warehousing commodities in Swaziland, and the costs of moving commodities from a central warehouse to individual surgical sites. Finally, we include the cost of transporting waste to incineration sites and performing maintenance on incinerators.Last, we assess additional costs associated with in-country operation costs, particularly personnel. We include a waste management supervisor, as well as resources for monitoring and evaluation and technical assistance, given the embryonic state of waste management services in Swaziland. We include five additional staffing positions for other supply chain and waste management services, as well as expenses associated with an office for these staff. Staffing and other operation costs amount to US$283,820.Based on the above modules, we estimate the total cost of commodities, supply chain, and waste management for the Swaziland VMMC program reaching a target of 152,000 men to be US$8,956,213.46. Approximately US$2,609,202.66 of these costs is fixed, while the remaining US$6,334,010.80 is variable, based on the number of VMMCs performed, the number of medical staff, and the number of surgical sites. Fixed and variable costs are outlined by module in The total cost of commodities, supply chain, and waste management is US$58.92 per male circumcision.For a reduced target of 75,000 VMMCs, the total cost is calculated as US$5,518,393.50, which equals a per VMMC cost of US$73.57. Fixed and variable costs for this reduced target are also included in This report represents the first attempt, to our knowledge, to quantify fully the supply chain and waste management costs associated with VMMC programming. Previous VMMC program costing studies generally have been performed at the point of service provision and thus have failed to capture supply chain and waste management costs. However, given the significant contribution to total program costs made by commodities, it is critical that these costs be considered during implementation planning to provide an accurate indication of the resources required.For example, based on previous costing studies, which indicated an average cost of US$50 per VMMC, the initial resource needs assessment for the ASI program underestimated the actual costs by approximately US$8 million. For program planners, an underestimate of this magnitude would gravely impact successful program implementation. Moreover, given Joint United Nations Programme on HIV/AIDS (UNAIDS) estimates of a US$10,000,000,000 funding gap for HIV programs globally This paper provides a quantification that represents the best international guidance on commodity procurement. For this reason, the analysis takes into account commodities not previously considered, including HIV counseling and testing, STI treatment, temporary infrastructure, and waste management; the last is particularly important, given the historical lack of attention to this area and its potential impact on strengthening the overall health system The data presented here represent rapid scale-up of VMMC in a low-resource setting with minimal supply chain systems. These assumptions impact the calculations provided and limit their generalization to other country settings. Various factors need to be considered before this calculation is used to inform costing estimations in other settings.First, better-resourced countries may incur lower program costs because of existing infrastructure and supply chain functions . For example, according to the estimates reported here, commodities make up a significant proportion of the total unit cost (US$47.34–US$49.50). Consumables contribute more than two-thirds of the supply chain costs for VMMC programs. These costs may be reduced below the estimates presented here by using existing infrastructure  and strategically placing pooled orders, which take advantage of economies of scale and allow for the negotiation of better pricing on bulk commodities. Similar use of existing infrastructure could reduce costs associated with the procurement of temporary infrastructure, which adds at least US$6.06, and as much as US$11.14, to the cost of each surgery . Additionally, the use of in-country as opposed to expatriate staff can result in considerable savings. In the case of Swaziland, staffing costs can be reduced by at least 50% through hiring skilled local staff.The implementation timeline may also alter costs. The costs associated with the ASI program in Swaziland assume the generation of demand for 152,000 or 75,000 VMMCs in 12 mo; a lengthier timeline needed to meet these targets would increase costs for rentals, warehousing and distribution contracting, staffing, and some formerly bulk-procured commodities. Countries planning to perform a larger number of VMMCs over a longer period of time will see some cost savings as certain flat-rate costs are distributed across more surgeries .Program planners will also need to consider the fact that the number of VMMC sites will impact fixed costs and change resource needs. These costs will increase with the number of sites selected by program planners. The selection of the appropriate number of sites for a particular campaign would be determined by such factors as volume over a given time frame, geography, size, difficulty of transport, and population density. However, given that many VMMC programs are expected to move quickly to full scale and reach target saturation within 2–3 y of initiation, many of the costs of increased targets simply would be multiplied by the number of teams and/or sites at a commensurate increased cost. Alternatively, the volume through an individual site (assuming a maximum capacity of 80 VMMCs per team per day based on the MOVE model) is associated with variable costs, which would increase as volume increases, while the fixed costs associated with each site would decrease as volume increases.The largest single cost in consumables is that of the disposable kit, at US$1,275,000–US$2,584,000. Prices for forceps-guided disposable kits were negotiated by PFSCM for the aggregate group of instruments (rather than by line item), including packaging and sterilization. We did this to take advantage of economies of scale, not to be directive regarding VMMC method. The disposable kit seems relatively expensive but does help to improve efficiency and avoids the need for sterilization equipment and staff training. In fact, through PFSCM, the pooled procurement of VMMC kits already has resulted in a reduction in market price from US$23 to US$17 per kit. Further reductions in prices are expected as additional VMMC kit vendors enter the market. As of March 2011, 6 mo after the procurement of VMMC kits for Swaziland, that cost had already dropped to US$15 per kit.While the Swazi ASI chose to use the forceps-guided VMMC technique, some programs have chosen to use other methods of VMMC . These techniques require different and/or additional instruments, require more time per procedure, and are less commonly used in VMMC programs. Program planners choosing to use one of these methods can expect an increase in commodity costs for the prepackaged kit, which is currently priced at US$19 per kit. As well, increasing the time required per surgery results in a decrease in the volume of clients serviced per team at the site level, resulting in increased fixed costs.As discussed, some programs may opt for reusable rather than disposable instruments. However, reusable instrument “lifetimes” vary significantly because of variations in instrument quality and differences in cleaning and maintenance practices. In addition, there are costs for reusable instrument use , so one cannot assume that reusable instruments are more cost-effective in every context. Programs with pre-existing autoclave facilities and staff that can be expanded  are the most likely to derive cost savings from using reusable instruments. Some programs might choose a combination of reusable instruments at sites with autoclaves and disposable kits at less well-equipped mobile and outreach sites In addition, this report assumes the use of a diathermy machine for cauterization, which changes costs associated with instruments by decreasing the number of sutures required. Programs considering the use of diathermy machines should consider costs associated with additional staff training required to operate these machines safely, as such costs are not reflected in this paper. Only programs with a reliable electricity supply, stable voltage, and demand for high-volume VMMC services are likely to derive significant benefit from the investment in diathermy machines.Finally, in Swaziland, the costs of waste disposal were based on the use of incineration for biological waste disposal. For other countries, costs will be impacted for programs choosing alternative disposal methods. As mentioned previously, countries can adapt cost estimates to reflect the reality of their infrastructure (which might vary from site to site). As in the case of Swaziland, improvements made to the health care waste management system for the disposal of VMMC programmatic waste can greatly improve waste management abilities throughout the health care system.VMMC programs implemented at the scale and pace required to stem HIV transmission and save lives as quickly as possible are somewhat at odds with the global health community’s focus on strengthening health systems. The WHO MOVE recommendations attempt to mitigate demands on the health care system by task shifting, creating efficient patient flows, and simplifying the complex list of commodities needed for a successful program. Still, much like vaccination campaigns, the implementation of VMMC programs often involves the creation of a parallel supply chain for VMMC commodities outside of the national essential medicines supply chain; this parallel supply chain is generally managed by the implementing partner providing VMMC services. These commodities are varied , demand large well-ordered storage facilities, and must be distributed to a range of dedicated and mobile clinics with seasonal operating schedules and locations.In the case of Swaziland, and given the lack of precedent in the literature, supply chain and logistics needs to support the ambitious program implementation plan were vastly underestimated and under-budgeted. Commodities included in the initial action plan were limited to VMMC kits and HIV rapid test kits; the plan for the management of the supply chain in Swaziland was limited to procurement, delivery to a small warehouse for temporary cross docking, and near immediate distribution to VMMC service provision sites. The staffing plan included only a logistics advisor and an administrative assistant.The exercise documented here helped program planners address this challenge, vastly improving estimation and budgeting for the Swaziland program. Several key lessons learned from this process will be useful to other VMMC programs in Africa to ensure efficient resource allocations.First, proper assessment of programmatic needs can prevent programs from being under-resourced. For example, because over two-thirds of the total supply chain budget is devoted to the procurement of nearly 100 different commodities, the act of quantifying program needs allowed for a more realistic budget to be created (an increase from US$2.5 million to US$8.9 million). Without this, the VMMC program in Swaziland would have been under-resourced, stalling before a single procedure was performed.Second, assessment of needs and cost estimation also ensure that existing resources are properly allocated. In Swaziland, the waste management system assessment conducted prior to launch of the program was critical to ensuring appropriate resource allocation. The assessment was conducted out of a recognition that because most African waste management programs are unable to meet current waste disposal demands Finally, despite extensive needs assessment and cost estimation, program implementation can still result in unexpected supply chain costs associated with the implementation of a VMMC program. In the Swaziland context, funding delays, the reality of lead times associated with any procurement process, and slow community engagement led to a late start to program activities (start delayed from January to April). This meant that at the midpoint of the campaign, communication efforts had generated less demand for service than anticipated. This has significantly impacted supply chain and logistics needs on the ground.Commodities were initially ordered to support a program meeting a target of 152,000 VMMCs over a 1-y period. With a lower than expected demand for services, the supply chain for these commodities has required a great deal of change. From a cross-docking operation that quickly moved commodities to the field for immediate consumption, at the country level, the Swazi VMMC supply chain has evolved into a complicated operation involving warehouse management, distribution, oversight of commodities consumption and waste disposal practices at the service delivery site, forecasting of needs, and reverse logistics of program-generated waste. Ultimately, from an initial plan of hiring two staff, five were hired to manage the supply chain, in addition to the expansion of the scopes of contracts for warehousing and distribution of supplies.In addition, for VMMC kits (US$17 per kit) procured in January 2011 for a 1-y program, a 2-y shelf life was rapidly being spent. At the current rate of service provision, the program was projected to reach 75,000 VMMCs at the end of the 1-y period. Given this forecast, it was decided that of 100,000 kits currently in country, 40,000 should be sold internationally. This required packaging, transportation, and customs brokerage, but allowed a savings of nearly US$1 million in programmatic costs that might have been wasted had the commodities expired in the warehouse.This implementation experience speaks to an important lesson learned regarding the necessity of staging the importation of commodities into a country based on real consumption data. A balance exists between staging orders and a desire to save money by pooling small orders into a much larger one; for untested VMMC programs, this desire should be tempered by the uncertainty of demand in country.While we include line item costs in this paper for one VMMC scale-up program, commodity and service costs can vary significantly, depending on location, order volume, and other market forces. The widely variable setting in which VMMC scale-up is being undertaken has such a significant impact on line item values as to make them almost impossible to estimate for broad applicability. However, this paper provides a framework for supply chain and waste management cost considerations. In addition, varying quality of such items can impact cost (and usable lifetime) significantly, and it is difficult to differentiate specific commodities in terms of quality .In addition, although certain commodities are listed as fixed costs , there may be additional costs not included here, such as the cost of maintenance and repair. Such costs are difficult to estimate and will depend on such factors as the volume of clients, climate, quality of routine maintenance, and total time used. Due to the significant variability of such costs from country to country, programs will need to make procurement decisions based on their particular settings and take into account the additional costs associated with such repairs in their specific context. Additionally, costs for resupply of emergency drugs in case of adverse events are not included.Another limitation to this paper is that the data for commodities are derived from current market prices for quantities calculated at an estimated rate of consumption, rather than expenditure reporting. It is quite likely that prices of certain commodities will be impacted by supply/demand issues both locally and internationally. Moreover, if there is a wide variation in the specific commodities ordered by program planners, the potential economies of scale can be lost or diluted.This variation in commodity costs is a particular limitation for the cost estimates provided for the performance of 75,000 VMMCs. Because PFSCM negotiated prices based on an initial volume of 152,000 VMMCs, the line item costs used for this estimate reflect this higher bulk pricing. It is possible that if 75,000 VMMCs had been the program target at the outset, rather than a function of decreased demand, commodity costs would have been higher because of lower volumes during pricing negotiations. This may have resulted in a further increase in the per VMMC price.Also, this paper does not take into account the impact of scale-up strategies (such as numbers of sites and VMMC volumes per site over time) or the need for and cost of different commodities over time. However, where possible, we have outlined fixed and variable costs associated with VMMC scale-up. These calculations should be assessed carefully based on local context to avoid over- or underestimation of costs. For example, if a site is not operating at full capacity, high fixed costs will increase the per procedure cost at that facility.The implementation time period  is not included as a variable in the calculations in this paper. The time over which services are delivered can be leveraged to maximize cost efficiencies. For example, as VMMC coverage goals are met, the demand drops and low-volume sites can be closed. The remaining usable commodities at these sites  can be used to replace at active sites those commodities that are worn out or in need of repair, thus reducing repair/replacement costs.Finally, this report estimates costs associated with VMMCs performed according to current WHO guidelines As VMMC programs mature, further efforts regarding costing should be undertaken to improve on the current estimates of the cost of VMMC. Experience from implementing the ASI VMMC program in Swaziland indicates that supply chain and waste management add an additional approximately US$60 per VMMC, which adds nearly 100% in additional costs per procedure to program costs. This analysis provides a framework to inform program planning with a more accurate representation of the critical supply chain and waste management costs of VMMC services.Table S1Commodities and price list.(DOCX)Click here for additional data file.