Analysis of uncharacterized bunyavirus isolates identified a possible reassortant virus. Orthobunyavirus species that causes febrile disease in humans reinforces the need to expand viral disease surveillance in tropical regions of South America.Our genetic analyses of uncharacterized bunyaviruses isolated in Peru identified a possible reassortant virus containing small and large gene segment sequences closely related to the Caraparu virus and a medium gene segment sequence potentially derived from an unidentified group C orthobunyavirus. Neutralization tests confirmed serologic distinction among the newly identified virus and the prototype and Caraparu strains. This virus, named Itaya, was isolated in 1999 and 2006 from febrile patients in the cities of Iquitos and Yurimaguas in Peru. The geographic distance between the 2 cases suggests that the Itaya virus could be widely distributed throughout the Amazon basin in northeastern Peru. Identification of a new  Orthobunyavirus genus, part of the group of viruses known as arboviruses, comprises several human and zoonotic pathogens known to be transmitted by mosquitoes, culicoides midges, nest bugs, and ticks and is the largest of the 5 genera within the Bunyaviridae family. Orthobunyaviruses, like other members of the Bunyaviridae family, have a trisegmented  negative-sense RNA genome. The L RNA segment encodes for the RNA-dependent RNA polymerase, the M segment encodes for the glycoproteins Gn and Gc, and the S segment encodes for the nucleocapsid protein. Many orthobunyaviruses also encode the nonstructural proteins NSm and NSs within the M and S segments, respectively; however, these proteins are not encoded in all orthobunyaviruses described  in Lima, Peru, has collaborated with the Peruvian Ministry of Health to investigate the etiology of febrile illnesses in Peru and greater Latin America . Study subjects enrolled were Aedes albopictus mosquito (C6/36) cells and maintained at 37°C and 28°C, respectively. The cell cultures were examined daily for 10 days for evidence of viral cytopathic effects (CPE); on the appearance of CPE, spot-slides were prepared and an immunofluorescence assay was done by using a polyclonal antibody against specific arboviruses that are known to circulate in Peru , filtered through a 0.45-μM pore size filter , and treated with a combination of DNases: 14 U Turbo DNase ; 20 U Benzonase (EMD Millipore); and 20 U RNase One  for 1 h at 37°C. Next, 24 mL of supernatant was loaded on top of 8 mL 30% sucrose , and centrifuged at 15,000 × g for 4 h at 4°C. Finally, the pellet was resuspended in 250 μL RNase/DNase and protease-free water (Ambion), and viral RNA was extracted using the Trizol reagent  by using the manufacturer’s protocols.For the extraction of RNA, cell culture supernatants were harvested and clarified by low-speed centrifugation  was fragmented by incubation at 94°C for 8 min in 19.5 μL of Illumina fragmentation buffer 15016648 . A sequencing library was prepared from the sample RNA by using an Illumina TruSeq RNA Sample Preparation Kit v2 using the manufacturer’s protocol (Illumina). The sample was sequenced on a HiSeq 1000 by using the 2 × 50 paired-end protocol. Reads in fastq format were quality filtered, and any adaptor sequences were removed by using Trimmomatic software (We deposited the complete genome sequences of Itaya virus and other group C viruses obtained for this study in GenBank under accession numbers KM092512-KM092514 and KM280924-KM280938. We used the neighbor-joining method available in MEGA5 (We used established methods to obtain hyperimmune ascitic fluid for classic cross-neutralization tests (Aedes, Mansonia, and Psorophora spp. in Brazil; a variety of other mosquito species in Ecuador, Brazil, Trinidad, Colombia, Argentina, and French Guiana; and from horses in Guyana and Colombia (Orthobunyavirus genus. In 1999, the virus strain IQT9646 was isolated from a 25-year-old male febrile patient who resided in Belen, Iquitos, Peru. The patient had an illness with symptoms of fever, headache, retro-orbital pain, arthralgia, chills, cough, and nasal congestion. These clinical symptoms are also characteristic of dengue, malaria, and other tropical infectious diseases common in the region (Phylogenetic trees based on the S and L gene segments placed the IQT9646 virus among isolates of Caraparu virus, a member of the group C virus serogroup , 3, a paTo determine if the IQT9646 strain is antigenically distinct from Caraparu virus, we investigated the serologic relationships of IQT9646 using cross-neutralization tests. Mouse antisera were prepared against the IQT9646 strain and the prototype Caraparu strain BeAn 3994. These antisera displayed a minimum 4-fold difference in neutralization titers between these viruses, indicating that IQT9646 is serologically distinct from the prototype Caraparu virus. Because some genetic variation exists between the prototype Caraparu and recent Caraparu isolates from Peru . In summGenomic characterization of some group C prototype viruses and other isolates found in South America were recently reported (Considering our findings with the IQT9646 strain, which, like FSL2923, possesses the S and L RNA segment sequences closely related to Caraparu virus and the M RNA segment sequence derived from an unidentified group C virus, we made genetic comparison and observed that the S, M, and L RNA segments of IQT9646 and FSL2923 shared >98% nucleotide and >99% amino acid sequence homology for all 3 viral RNA segments. These findings indicate that the FSL2923 isolate is the same virus as strain of IQT9646. The data also suggest that this reassortant virus is widely distributed throughout the Amazon basin in northeastern Peru, because it was isolated in Iquitos and Yurimaguas, in the Department of Loreto, within a 7-year time period .Arboviral diseases continue to be a frequent cause of illness and death worldwide. In recent years, a considerable number of novel arboviruses associated with outbreaks of human or livestock disease have been identified, and the expansion of known arboviruses into new geographic areas have also been reported (Surveillance studies performed by NAMRU-6 over the past 2 decades have been instrumental in characterizing the extent of many endemic tropical diseases throughout Latin America (>10 of the 13 distinct group C viruses have been associated with human disease elsewhere, including Oriboca, Itaqui, Nepuyo, Apeu, Murutucu, Restan, Ossa, Madrid, Caraparu, and Marituba viruses (Among the bunyaviruses, the average symptomatic rates in Iquitos for group C and Iquitos viruses were 14.3/100,000 and 14.2/100,000, respectively, based on participants enrolled in NAMRU-6’s febrile surveillance protocol (In this study, we aimed to expand our knowledge and understanding of bunyaviruses associated with febrile human illness in Peru, and these efforts focused on Maguari-like viruses, which were provisionally identified by using indirect immunofluorescence assays. Although indirect immunofluorescence assay is a procedure commonly used in laboratories to tentatively identify viruses, there is a degree of cross-reactivity among viruses from the same family; therefore, additional testing is needed to properly identify the viral agent. Therefore, we used a genetic and antigenic approach to characterize and identify the Maguari-like virus isolated from samples of febrile patients. These efforts yielded the identification of the group C reassortant virus that we named Itaya virus. Furthermore, we generated complete genome sequence information for prototype group C viruses, and as noted (The prototype strain of Itaya virus was isolated from a patient who resided in Iquitos. The patient visited the health post in January 1999 with a mild febrile illness characterized by fever, headache, retro-orbital pain, arthralgia, and chills, among other symptoms. During February 1999, a patient with similar signs and symptoms visited the same health post in Iquitos, and subsequent studies determined that the patient had been infected with a novel reassortant orthobunyavirus, which we subsequently named Iquitos virus (In conclusion, our report expands upon the list of known arboviruses associated with febrile illness in Peru and raises awareness about the continuous emergence of reassortant bunyaviruses with human pathogenic potential. Future studies designed to genetically characterize existing and recently isolated bunyaviruses may be able to identify the viral donor of the Itaya M segment.


Carnation mottle virus (CarMV) is a single positive-strand RNA virus belonging to the genus Carmovirus. The major natural host for CarMV is the carnation. In this study, using transcriptome data, we provide for the first time a nearly complete genome sequence of CarMV infecting hop plants.The  Carnation mottle virus (CarMV) is a member of the genus Carmovirus in the family Tombusviridae. The genome of CarMV is composed of a single positive-strand RNA virus (Dianthus caryophyllus) and pinks (D. barbatus), and infection of CarMV in a wide range of plants  has been reported (Chenopodium species (The NA virus . The majreported . CarMV i species . CarMV-iHumulus lupulus), we screened several hop transcriptomes. Of them, one recent study provided the draft genome and transcriptome for two hop cultivars and a wild hop that were sequenced by Illumina HiSeq 1500  (To obtain information on viruses infecting hop plants (Seq 1500 . The tran 2.0.2) . Interesn 2.0.2) . In summCarnation mottle virus isolate Japan has been submitted to GenBank under the accession number KR002041.The genome sequence of


There is an economic burden associated with hypertension both worldwide and in Vietnam. In Vietnam, patients with uncontrolled high blood pressure are hospitalized for further diagnosis and initiation of treatment. Because there is no evidence on costs of inpatient care for hypertensive patients available yet to inform policy makers, health insurance and hospitals, this study aims to quantify direct costs of inpatient care for these patients in Vietnam.A retrospective study was conducted in a hospital in Vietnam. Direct costs were analyzed from the health-care provider’s perspective. Hospital-based costing was performed using both bottom-up and micro-costing methods. Patients with sole essential or primary hypertension (ICD-code I10) and those comorbid with sphingolipid metabolism or other lipid storage disorders (ICD-code E75) were selected. Costs were quantified based on financial and other records of the hospital. Total cost per patient resulted from an aggregation of laboratory test costs, drug costs, inpatient-days’ costs and other remaining costs, including appropriate allocation of overheads. Both mean and medians, as well as interquartile ranges (IQRs) were calculated. In addition to a base-case analysis, specific scenarios were analyzed.230 patients were included in the study (147 cases with I10 code only and 83 cases with I10 combined with E75). Median length of hospital stay was 6 days. Median total direct costs per patient were US$65 (IQR: 37 -95). Total costs per patient were higher in the combined hypertensive and lipid population than in the sole hypertensive population at US$78 and US$53, respectively. In all scenarios, hospital inpatient days’ costs were identified as the major cost driver in the total costs.Costs of hospitalization of hypertensive patients is relatively high compared to annual medication treatment at a community health station for hypertension as well as to the total health expenditure per capita in Vietnam. Given that untreated/undetected hypertension likely leads to more expensive treatments of complications, these findings may justify investments by the Vietnamese health-care sector to control high blood pressure in order to save downstream health care budgets.The online version of this article (doi:10.1186/s12913-014-0514-4) contains supplementary material, which is available to authorized users. Hypertension represents a health and economic burden worldwide. In 2000, approximately one quarter of the adult populations had hypertension, equaling to approximately 972 million adults . FurtherAmong the group of developing countries, the prevalence of hypertension in Vietnam can be rated as intermediate with estimates ranging from 20% to 30% in adults . In receCompared to surrounding countries, hypertension may impact even higher costs to society in Vietnam because diagnosis and initiation of treatments for hypertension often take place in the hospital. This may be explained by several factors, including the absence of some specific services in primary care for all regions of the country, the perception that hospital care offers a higher quality of care and the preference for specific services to be in a hospital setting for administrative and convenience reasons. At present, the inpatient and outpatient economic burdens of hypertension in Vietnam have not been estimated precisely. In addition, studies on the topic are scarce. One study at the community-health station level on the cost of drug treatment for the whole population over a 10-years period estimated costs of 9,808 billion VND associated with grade 1 hypertension and 11,192 billion VND associated with grade 2 and 3 hypertension ,13. NotaThe health-care provider payment system in Vietnam is currently being reformed. The fee- for-service system will be replaced by case-mix payments and eventually diagnostic-related groups . Thus, tIn addition, hospitals in Vietnam are gaining greater autonomy. Having knowledge on their expenditures for inpatient care services will help these hospitals to improve their financial management as well as to adequately issue fees for both insured and non-insured patients. Currently, neither information on the costs of inpatient care for the treatment of hypertension and its consequences nor solid estimates on the comprehensive costs of hypertension management is available in Vietnam.To address these gaps of information, we conducted this study to quantify the direct costs of inpatient care for hypertensive patients. Furthermore, these results may 1) contribute to a better understanding of the economic burden of hypertension in Vietnam, 2) help with reimbursement decision making for insured patients, 3) support setting potential fees and charges to be issued for non-insured patients and 4) inform on potential impacts of preventive policies.We conducted a retrospective study in the Thai Nguyen hospital. Data was collected from the financial records during October 1st 2010 to September 30th 2011.The study was conducted at a regional hospital with 800 beds in the city of Thai Nguyen, which is located in a mountainous area that is approximately 100 km North of Hanoi. It serves patients from Thai Nguyen and neighboring provinces.All costing of resource utilization was adjusted to 2011 levels and presented in US$ using the exchange rate of US$1 to VND20.830 .th version (ICD-10), we identified and retrieved information on all patients with codes I10 alone  or comorbid patients with I10 combined with E75 (sphingolipid metabolism and other lipid storage disorders). The latter group was included because hypertension often coincides with these disorders. The combination of diagnoses might reflect a relatively large share of the overall hypertensive patient group with deviating costs. ICD-10 codes were taken from hospital databases and individual patients’ records.Using the International Classification of Diseases 10Information on age, sex and ICD code was available for all of the selected patients with ICD code I10 or I10 combined with E75.However, data might be missing as administrators may forget to enter classification data or misclassify; i.e. patients and/or patient-related data might be missing. In addition, by doing this retrospectively, we had no certainty on whether each financial record was included or not. Nurses should enter all consumed items such as drugs, tests, medical materials into the database system before she/he can get them from store or lab services. Other cost items such as patient-days and examinations were automatically recorded for every patient. This ensures that all these items were recorded and financial records are likely complete. Whenever financial records seemed grossly incomplete or absent at all, patients were excluded. This was however not the case. It was therefore plausibly assumed that missing data was limited and random. Thus, no specific bias was expected.Direct costs were analyzed from the provider perspective. Data on costs included all charges to patients for drugs, materials  and laboratory testing, all crucial elements in the financial records and reflecting adequate charging. In particular, hospital-based costing was performed using both bottom-up and micro-costing methods and aggregation was subsequently conducted for all costs related to medical services used by a group of hospitalized patients ,17. For Inpatient – days costs and other costs . Costs of inpatient care can be expressed as:The step-down allocation method- partially adjusted for interaction between overhead departments- was applied for allocating overhead costs . In the Labor costs were calculated based on the actual payment for labor by the hospital every month during a year; funding was provided by government or specific funds for services organized by the hospital. Material/infrastructure costs were calculated by items used at each department multiplied with the price market which the hospital paid based on again monthly updated records of the hospital. Capital costs were calculated based on assets’ records of the hospital, which is updated every year.Notably, non-medical costs of materials/infrastructure primarily comprise of power, telephone, uniforms, stationary and cloths. In the absence of any detailed information, an assumption was made that equal costs for an inpatient day would apply for every disease within each department.The inpatient–day costs per department resulted from the total costs divided by the total number of inpatient days for that department:The cost-to-charge ratio method was applied to calculate laboratory testing costs . While tIn this study, standard national prices for drugs and materials were not available. As Thai Nguyen hospital is a non-profit hospital, we assumed that the use of drug and material charges from this hospital would well approach actual costs. Cost of drugs and materials were calculated based on the current market prices, which the hospital paid. Therefore, we could validly impute these charges into our cost analysis for each drug and material used . Both drAdmission cost, which is required for every intake examination for admission to the hospital, was calculated similarly as was done for inpatient – day’ costs. Admission costs per patient is equal to the total costs of outpatient department divided by the total number patient visits at outpatient.Examination costs at specialist departments were calculated for patients who had specific examinations. Examinations were counted and monetized using charges as determined by the recommendations of the Ministry of Health.Total costs per patient resulted from the aggregation of inpatient-days’ costs multiplied by the length of stay, laboratory test costs, drug costs, medical material costs, admission costs, and costs of examinations at specialist departments. Example of calculating total costs for a patient is presented in the Additional file Labor costs  were quantified based on financial records of the hospital. Material (not charged to patients directly) and capital  were quantified based on the financial records, administration of materials used and capital inventories of the hospital.Age, sex, department, length of stay, total numbers of each laboratory test, numbers of each drug and medical materials charged to patients directly were retrieved from patient-based databases of the hospital. Prices for specific services such as medication, materials/disposables and laboratory tests were available from the specific databases of the hospital. Furniture and land costs were not included in this study as no information was available.The number of tests completed for each patient and its charges were obtained from the individual patient sheets. We selected all tests, including test for the diagnosis and/or treatment of hypertension and other condition. Total number of all tests in 6 months was multiplied by 2 and then divided by the total number of patients in 2011, equating 11 in the whole hospital. For the hypertensive in-patient group, the average number of tests per patient was 16.8, 16.3 and 17.1 in both groups, I10 and I10 + E75, respectively. Of course for costing, each individual test was priced separately.Means, medians and Inter Quartile Range (IQRs) of costs were measured as outcome in this study. Univariate sensitivity analyses were performed to explore the robustness of the analyses . NotablyIn this context, it is very important to rule out the costs resulting from diagnosing and treating these other diseases. Therefore, we conducted one sensitivity analysis with a scenario that excluded all costs resulting from diagnosing and treating comorbid diseases. We limited to specific costs for diagnosing and treating I10 and E75 diseases in this scenario. In addition, the discount rate for capital was varied from 1% to 5%. As data on furniture were not available, these costs were not included in the base case. Additionally, a scenario where an estimated 5% furniture cost was added to the total capital cost was explored . FinallyThis study was approved by the ethical committee of the Thai Nguyen General hospital. Databases of this study were used under the permission of the Planning department of the Thai Nguyen General hospital.The analyses were based on 230 patients who met the inclusion criteria, including 147 cases with essential (primary) hypertension (I10) and 83 cases with hypertension combined with sphingolipid metabolism and other lipid storage disorders (I10 + E75). Mean age was 64.3 (SD +/-14.7) and 53.5% was female. Characteristics of patients are indicated in Table Hypertensive patients were admitted in 3 departments of the hospital; i.e., Cardiovascular Internal Medicine, Geriatric Internal Medicine and Neurology. For these departments, base- case inpatient– day costs were US$4.99, US$5.05 and US$5.33, respectively. Notably, the most expensive per day costs were associated with patients admitted to Neurology department.Costs of treatment for these hypertensive inpatients are presented in Table Results of the sensitivity analyses, including 12 scenarios reflecting relevant alternative options, are presented in Figure Hypertension is a major and increasing public health problem in Vietnam, including 5.7 million patients unaware of their status, 2.1 million aware but untreated, 2.0 million treated but uncontrolled and 1.2 million treated and controlled . NotablyOur analysis estimates that the median total cost of inpatient treatment and care is US$65 per hypertensive patient per hospital stay. To put this in perspective, the total health expenditures in Vietnam per capita in 2010 was only US$83 . As costAs expected, the costs of the sole hypertensive patients US$53) were lower than the costs for the combined hypertensive and lipid patients (US$78). It is quite plausible that comorbid conditions can drive up costs, particularly due to increases in length of stay, compared to a single disease condition. In this study, median lengths of stay were 5 and 7 days for I10 and I10 combined with E75, respectively. These inpatients days were associated with high costs, representing 41% of the total cost. This finding differs from the study conducted in the Philippines, which reported medication costs  as the highest cost component [3 were loIn this study, the number of inpatient days for sole-hypertensive patients was 5 days. These patients may have undetected and uncontrolled hypertension and now requires hospitalization to be diagnosed for primary or secondary hypertension. In addition, there is no ambulatory blood-pressure monitoring service available in the community. Patients must be admitted to a hospital for diagnosis. As an alternative to expensive hospital care, community level health services may serve as a cheaper option, but these centers need to be strengthened to enable blood-pressure monitoring for the broader population.The present findings must be interpreted in the context of potential limitations. Firstly, the study was conducted in one hospital, which may not be representative hospitals across the country. However, it is a governmental and not-for-profit hospital that is potentially similar to other hospitals regarding governmental investments in hospital beds . Thus, iIn comparison to annual medication treatment at a community health station for hypertension and total health expenditure per capita in Vietnam, the costs of hospitalization of hypertensive patients is high. The main driver of the costs is related to inpatient days rather than to treatment, laboratory or other cost categories. Our findings have important implications for health policies. Costs of treatment for hypertension and combined disorders of sphingolipid metabolism and other lipid storage disorders in this study could become the reference case for reimbursement when health insurance companies apply reimbursement by fee for diagnostic-related groups. The findings in this study, particularly the high cost of hospitalization for untreated and uncontrolled hypertension, justify increasing current expenditures by the Vietnamese health-care sector on effective interventions to control high blood pressure, which may produce savings to the health care budget by preventing expensive complications.


Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [<virus name> (<strain>)/<isolation host-suffix>/<country of sampling>/<year of sampling>/<genetic variant designation>-<isolate designation>], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences. The National Center for Biotechnology Information (NCBI) RefSeq project was initiated to create a nonredundant and curated set of genomic, transcript, and protein sequence records . GenomicIn the case of virological RefSeq records, each viral species was initially represented by only one genome sequence record, and all other genome records for members of the same species, or for different strains, variants, and isolates of the same member of this species were linked to this record as “genome neighbors” . The ratde novo complete genome.” The RefSeq <strain> field was cleared; and the RefSeq <isolate> field was filled with “Lloviu virus M.schreibersii-wt/ESP/2003/Asturias-Bat86.” The same changes should be applied to GenBank #JF828358. .Accordingly, in RefSeq #NC_016144 the definition line “Lloviu virus, complete genome” was changed to “Ebolavirus includes five species, each of which is represented by one virus.The genus Full name: Bundibugyo virus H.sapiens-tc/UGA/2007/Butalya-811250Shortened name: BDBV/H.sap/UGA/07/But-811250Abbreviated name: BDBV/But-811250Bundibugyo virus (BDBV) is the second least characterized ebolavirus. Although at least eight isolates of this virus are available ,22, all Bundibugyo ebolavirus isolate Bundibugyo virus H.sapiens-tc/UGA/2007/Butalya-811250, complete genome.” The RefSeq <isolate> field was filled with “Bundibugyo virus H.sapiens-tc/UGA/2007/Butalya-811250.” The same changes should be applied to GenBank #FJ217161.Accordingly, in RefSeq #NC_014373, the definition line “Bundibugyo ebolavirus, complete genome” was changed to “Full name: Ebola virus H.sapiens-tc/COD/1976/Yambuku-MayingaShortened name: EBOV/H.sap/COD/76/Yam-MayAbbreviated name: EBOV/Yam-MayEbola virus (EBOV) is the most thoroughly characterized ebolavirus. Dozens of EBOV isolates are available, but the vast majority of published experiments have been performed with isolates “Mayinga” and “Kikwit” (reviewed in ). The “MZaire ebolavirus isolate Ebola virus H.sapiens-tc/COD/1976/Yambuku-Mayinga, complete genome.” The RefSeq <strain” field was cleared; the RefSeq <isolate> field was filled with “Ebola virus H.sapiens-tc/COD/1976/Yambuku-Mayinga;” and the <organism> field was corrected to “Zaire ebolavirus.” The same changes should be applied to GenBank #AF086833.Accordingly, in RefSeq #NC_002549 the definition line “Zaire ebolavirus, complete genome” was changed to “Full name: Reston virus M.fascicularis-tc/USA/1989/Philippines89-PennsylvaniaShortened name: RESTV/M.fas/USA/89/Phi89-PenAbbreviated name: RESTV/Phi89-PenReston virus (RESTV) has caused multiple epizootics among captive macaques  and domestic pigs in 2008 (reviewed in ). At leaReston ebolavirus isolate Reston virus M.fascicularis-tc/USA/1989/Philippines89-Pennsylvania, complete genome.” The RefSeq <strain> field was cleared; and the RefSeq <isolate> field was filled with “Reston virus M.fascicularis-tc/USA/1989/Philippines89-Pennsylvania”. The same changes should be applied to GenBank #AF522874.Accordingly, in RefSeq #NC_004161, the definition line “Reston ebolavirus, complete genome” was changed to “1,2 were determined for both viruses complete genome,” and the RefSeq <isolate> field was filled with “Taï Forest virus H.sapiens-tc/CIV/1994/Pauléoula-CI.” The same changes should be applied to GenBank #FJ217162.Accordingly, in RefSeq # NC_014372, the definition line “Tai Forest ebolavirus, complete genome” was changed to “Marburgvirus includes a single species, which is represented by two divergent viruses.The genus Full name: Marburg virus H.sapiens-tc/KEN/1980/Mt. Elgon-MusokeShortened name: MARV/Hsap/KEN/80/MtE-MusAbbreviated name: MARV/MtE-MusMarburg virus (MARV) is the most thoroughly characterized marburgvirus. Some 70 MARV isolates are available, but the majority of published experiments have been performed with isolate “Musoke” (reviewed in ). HoweveMarburg marburgvirus isolate Marburg virus H.sapiens-tc/KEN/1980/Mt. Elgon-Musoke, complete genome.” The RefSeq <strain” field was cleared, and the RefSeq <isolate> field was filled with “Marburg virus H.sapiens-tc/KEN/1980/Mt. Elgon-Musoke.” The same changes should be applied to GenBank #DQ217792.Accordingly, in RefSeq #NC_001608, the definition line “Marburg marburgvirus, complete genome” was changed to “Full name: Ravn virus H.sapiens-tc/KEN/1987/Kitum Cave-810040Shortened name: RAVV/H.sap/KEN/87/KiC-810040Abbreviated name: RAVV/KiC-810040Ravn virus (RAVV) is a largely uncharacterized marburgvirus that belongs to the same species as MARV. At least three human  and four Egyptian rousette isolates  have been obtained. Virtually all RAVV characterization experiments have been performed with “Ravn” = “810040,” which was obtained after at least two passages in SW-13 cells and four passages in Vero E6 cells. Since RAVV is a phylogenetically distinct marburgvirus, we created a RefSeq entry for the “Ravn” isolate, for which we propose the variant designation “Kitum Cave” (after Kenya’s Kitum Cave on Mount Elgon where RAVV first emerged) and the isolate designation “810040”:Marburg marburgvirus isolate Ravn virus H.sapiens-tc/KEN/1987/Kitum Cave-810040, [coding-]complete genome.” The RefSeq <isolate> field contains “Ravn virus H.sapiens-tc/KEN/1987/Kitum Cave-810040.” The deposited sequence (NC_024781) is identical with GenBank #DQ447649, which should be updated accordingly.Accordingly, the RefSeq entry was created with the definition line “A summary of the proposed designations and RefSeq accession numbers can be found in


The predicted residues were distributed on the surface-exposed capsid regions, VP1–VP3. The significance of residue changes at eight of the predicted epitopes was tested by site-directed mutagenesis using a cDNA clone resulting in the generation of 12 mutant viruses involving seven sites. The effect of the amino acid substitutions on the antigenic nature of the virus was assessed by virus neutralization (VN) test. Mutations at four different positions, namely VP1-43, VP1-45, VP2-191 and VP3-132, led to significant reduction in VN titre . This is the first time, to our knowledge, that the antigenic regions encompassing amino acids VP1-43 to -45 , VP2-191 and VP3-132 have been predicted as epitopes and evaluated serologically for serotype A FMDVs. This identifies novel capsid epitopes of recently circulating serotype A FMDVs in East Africa.Epitopes on the surface of the foot-and-mouth disease virus (FMDV) capsid have been identified by monoclonal antibody (mAb) escape mutant studies leading to the designation of four antigenic sites in serotype A FMDV. Previous work focused on viruses isolated mainly from Asia, Europe and Latin America. In this study we report on the prediction of epitopes in African serotype A FMDVs and testing of selected epitopes using reverse genetics. Twenty-four capsid amino acid residues were predicted to be of antigenic significance by analysing the capsid sequences ( Picornaviridae, genus Aphthovirus. The virus exists as seven distinct serotypes -1, SAT-2 and SAT-3) that differ genetically and antigenically with multiple strains in different continents. Globally, most outbreaks of FMD are caused by serotype O followed in frequency by serotype A (Foot-and-mouth disease (FMD) is a highly infectious, rapidly spreading and internationally important livestock disease. It has significant socio-economic consequences due to losses in production and constraints on export of live animals and associated products to disease-free countries. FMD is caused by FMD virus (FMDV) that belongs to the family rotype A . As an Rin vitro by epitope mapping using mAb  that may improve viral fitness. These sites are commonly investigated blast searches for closely related sequences. DiscoTope, Epitopia and SEPPA were recently applied to FMDV epitope prediction by Epitopes can also be predicted from three-dimensional (3D) structural data alone, from aligned sequence data alone, or by using both sequence data and three-dimensional structural data. More than 90 % of B-cell epitopes are conformational  studies ] and (ii) by correlating differences in neutralizing serum titres between virus pairs with capsid amino acid sequence changes.Studies on the critical amino acid residues for neutralization of serotype A FMDV have used mainly mar-mutant approaches with viruses from the Middle East (A22)  of the 24 residues  between the two prediction methods . All thein silico to be of antigenic importance but their relevance so far could not be confirmed by other methods. The amino acid at position VP2-191 is located at the threefold axis of the capsid and is among the top four amino acids predicted by both in silico methods. This residue has been recently reported to be a neutralizing epitope linked to antigenic site 2 in serotype O FMDV . A total of 12 single mutant plasmids involving seven residues were generated (The capsid-coding region of serotype A FMDV (A-EA-2007) was cloned successfully into the plasmid pT7S3-O1Kwt to generate the full-length genome plasmid pT7S3/A-EA-2007. This plasmid was used as the template to introduce further mutations in the capsid-coding region. A total of eight residues  were selected for this purpose as they were indicated to have an impact on the antigenicity of the virus by comparison of capsid sequences with enerated . The capLive infectious viruses were recovered successfully from all the cDNA clones following electroporation. FMDV-specific cytopathic effect (CPE) was observed 18–24 h post-electroporation. Extensive CPE was observed at both the first and second passages. At least two independent clones of each virus were rescued. However, only one clone in each case was used for further characterization. In order to establish that the expected viruses had been rescued, reverse transcription (RT)-PCR was carried out on the RNA extracted from infected BHK-21 cells using primer pair L460F and EUR2B52R/NK72 that produced a 2500 bp fragment  of expected size (data not shown). No PCR products were generated in parallel reactions in which the enzyme reverse transcriptase was omitted, indicating that the products amplified were not generated from the transfected plasmid DNA. The PCR products were sequenced on both the strands and no additional nucleotide substitutions were observed in any of the mutants generated in this study.The parent virus A-EA-2007 represents genotype VII of serotype A FMDV circulating in East Africa. Previously, chimeric viruses containing capsid-coding regions derived from serotype A/Turkey 2/2006 or O/UKG/34/2001 FMDV with the backbone of serotype O1K cDNA clone (pT7S3-O1K) have been generated successfully . The ability of FMDVs to tolerate changes at these positions is consistent with the observation of high amino acid variability at these residue positions in the 115 field viruses analysed [56 sequences reported before those of The mutants were stable genetically at least up to third passage as confirmed by full capsid sequencing. Standard multi-step growth curves were carried out to compare the growth of the recombinant viruses with that of the parent virus. All the viruses grew at a similar rate and to a similar titre, indicating the mutations in the antigenic sites had no adverse effects on the replication efficiency of these viruses 50). Therefore, a two-dimensional micro-neutralization test (2D-VNT) was carried out using five different doses of the virus encompassing 100 TCID50 for this purpose. The resultant VN titres at each virus dose were used to calculate the serum titre at 100 TCID50 by regression analysis. Because getting consistent results was very important for the evaluation of the mutant viruses, each test was conducted in duplicate and repeated at least eight times. Test results showing evidence of a reduction in serum titre after mutagenesis were repeated eight more times for further confirmation. Among the 12 mutants generated in this study, only five mutants, i.e. rO1K/A-EA-2007M1 (VP1-L43A), rO1K/A-EA-2007M4 (VP1-L45P), rO1K/A-EA-2007M5 (VP2-T191A), rO1K/A-EA-2007M6 (VP2-T191D) and rO1K/A-EA-2007M10 (VP3-T132S), exhibited significant reductions in serum titre (The impact of the amino acid substitutions on sero-reactivity was assessed by VN test using the pooled post-vaccination serum (bovine) raised against rO1K/A-EA-2007 antigen. The main goal was to quantify the reduction in neutralization following mutations in the capsid of FMDV. Therefore, it was crucial to determine the VN titre of the sera against all the mutant viruses at a fixed virus dose  for serotype A FMDVs that is equivalent to antigenic site 3 in serotype O. Substitution of threonine (amino acid found in genotype IV viruses) at position VP2-191 with either alanine or aspartic acid confirmed the antigenic significance of this residue as well as mutations at VP1-43, VP1-45 and VP3-132. These residues are novel epitopes that have not been reported previously for serotype A FMDVs.The 56 East African type A viruses and their corresponding capsid sequences and serology data against seven vaccine strains described previously  according to the manufacturer’s recommendation. Reverse transcription (RT)-PCR to amplify the capsid-coding region, nucleotide sequencing and sequence analysis were carried out as described by AflII F/A-EA-2007SpeI R containing AflII and SpeI restriction sites, respectively (Table S1). The amplified product was cloned in to the intermediate vector pT7Blue (Promega) resulting in generation of pT7Blue-A-EA-2007. The capsid was excised from the intermediate vector using AflII and SpeI restriction enzymes. The excised capsid-encoding region was used to replace the capsid-encoding region of pT7S3-O1Kwt to generate pT7S3-O1K/A-EA-2007. The capsid-encoding region of the full-length genome plasmid was sequenced on both the strands to ensure they were from the desired virus. pT7S3-O1K/A-EA-2007 was used in all subsequent experiments.Standard molecular biological techniques were used for the cloning of the serotype A capsid into vectors. The A-EA-2007 FMDV capsid-encoding region was amplified from the total RNA isolated from the plaque-purified virus using specific primer set A-EA-2007 http://consurf.tau.il), the prediction of epitopes was performed by calculating the evolutionary conservation rate (inverse of evolutionary substitution rate) using a Bayesian method  Shannon entropy  and (b) The locations of residues involved in antibody binding sites (epitopes) were inferred by correlating the antibody cross-reactivity of viruses to their capsid sequence similarities as described previously (Standard site-directed mutagenesis (SDM) technique was used to introduce mutations at specific positions in the capsid of pT7S3-O1K/A-EA-2007. A total of eight sites , all in surface-exposed capsid regions and newly predicted (not previously reported), were selected for further investigation. These residues were substituted either to alanine or with a specific amino acid whose presence in other isolates had been correlated with higher or lower antibody reactivity. Specific overlapping primers were designed to introduce mutations either individually or in combinations to make recombinant viruses containing either single or double substitutions in the capsid (Table S1). The cDNA backbone containing the serotype A capsid was manipulated according to the method described by HpaI enzyme and full-length RNA transcripts were synthesized as described previously (The parent plasmid (pT7S3-O1K/A-EA-2007) or its derivatives were linearized by digestion with In order to characterize the recombinant viruses, RT-PCR was carried out on the total RNA isolated from virus-infected BHK-21 cells. The whole capsid was amplified and then sequenced on both strands to ensure all the mutations were present in the respective viruses. Virus titres for each virus were determined on IB-RS2 cells as described by For use in serological assays antisera were prepared in cattle against the recombinant parent virus recovered from the cDNA clone (rO1K/A-EA-2007), which is the parent to all other mutant viruses generated in this study. Briefly, the rO1K/A-EA-2007 virus was grown in BHK-21 cells. When the CPE was complete, the culture supernatant was harvested, clarified by centrifugation, and inactivated with 5 mM binary ethylenimine (BEI) at 25 °C for 24 h , 1990. T10 reciprocal antibody dilution required for 50 % neutralization of 100 tissue culture infective units of virus (log10SN50/100 TCID50). The antigenic relationship of a mutant virus to its parent is given by the ratio: ‘r1’ = neutralizing antibody titre against the mutant virus/neutralizing antibody titre against the recombinant parental virus. The significance of differences between ‘r1-values’ obtained by the polyclonal serum was evaluated according to the method of 1-value ≥0.3 as representing an expectation of adequate cross protection. Each test was conducted in duplicate and repeated at least eight times. Test results showing evidence of a reduction in serum titre after mutagenesis were repeated eight more times for further confirmation.The 2D-VNT was carried out using the pooled 28th day post-vaccination bovine sera from five animals to determine the sero-reactivity of the mutant viruses according to minitab (version 16) software. A paired t-test was used to compare the differences in sero-reactivity between the homologous and the mutant viruses.The data were analysed using


Detection of influenza virus in oropharyngeal swabs collected during wild bird surveillance is assumed to represent respiratory infection, although intestine is the main site of infection. We tested this assumption by histological examination of the respiratory tract of wild Mallards with virus-positive oropharyngeal swabs. Thirty-two of 125 Mallards tested had viral-RNA positive oropharyngeal swabs. The respiratory tracts of four Mallards with the most virus were examined in detail by immunohistochemistry. None had detectable virus antigen in the respiratory tract, suggesting it was not infected. An alternative explanation is that the oropharynx was contaminated with virus through feeding in surface water or through preening. Anas platyrhynchos in both North America and Europe [Influenza A viruses (IAV) have long been known to circulate in wild birds ,2. The md Europe -5. Thesed Europe  and wated Europe ,6. An ald Europe ,8, whichd Europe ,10. Bothd Europe -14. Furtd Europe -15. Suppd Europe ,17. Alsod Europe ,19. Howed Europe . The goaWild, free-living Mallards were routinely sampled as part of a long-term surveillance program at Ottenby Bird Observatory, located in the southern Baltic Sea (56°13´ N 16°27´ E). We selected Mallard as it is an important maintenance host of LPAI virus, and as it is the most common reservoir host at the study site. The study was carried out in November (2012), one of the peak months of Mallard migration and influenza prevalence at the study site . Across n = 2) and H5N3 (n = 1). In one individual, the same virus subtype, H5N3, was isolated both from the oropharyngeal swab and the cloacal swab . All tissues were fixed in 10% neutral-buffered formalin (Sigma) for 30 days prior to further processing.After fixation, we mounted multiple sections from the length of each tissue to increase the chance of detecting infection, even if localized, as follows: four cross-sections of the nasal mucosa, 10 of the trachea, three of each bronchus, eight of each lung, seven of the esophagus, three of the proventriculus, one of the ventriculus, seven of the duodenum, seven of each jejunal segment , three of the colon, three of the cloacal bursa, and one of the cloaca. Following embedding in paraffin, 3-μm-thick sections were made and stained for the detection of IAV antigen as previously described ,18. CellDespite testing multiple tissue sections along the length of the respiratory tract, none contained cells expressing IAV antigen Table A-B. ThreWe were unable to demonstrate LPAI virus infection in the respiratory tract of any of the four Mallards in this study, despite choosing conditions and methods that maximized the chance of virus detection; the study was executed at a site with high IAV prevalence and during a period of peak prevalence; we chose individuals with the highest virus concentrations by RRT-PCR in the oropharyngeal swabs; and we performed detailed immunohistochemical analysis at all levels of the respiratory tract. These results indicate that these Mallards did not have LPAI virus infection in the respiratory tract despite testing positive in oropharyngeal swabs. Rather, we hypothesize that LPAI virus detection and isolation success in the oropharynx in these birds may be due to contamination of infectious particles from dabbling in virus-contaminated water or preening ,24. The Immunohistochemistry is a sensitive method, and has been used to detect influenza A virus antigens in natural  and expeLow quantities of viral RNA may be detected by RRT-PCR in oropharyngeal/cloacal swabs for several days after abrogation of actual virus infection, based on virus isolation ,27 and iOne reason for the re-emergence of respiratory tract infection attribution, without evidence, in the literature is that these infections are present in experimental LPAIV infection  and in nIn conclusion, we have demonstrated that IAV-positive oropharyngeal swabs obtained from wild migratory Mallards do not necessarily correspond to respiratory tract infection. The implication from this study is that Mallards that test positive for IAV only in the oropharyngeal swab should not automatically be categorized as being infected, especially if the Ct-values are high. Given that this study was performed on a limited number of Mallards infected with H4N6 or H5N3, further studies in both Mallards and other host species, naturally infected with different IAV subtypes, are needed to determine how to interpret IAV-positive oropharyngeal swabs from Mallards and other wild waterbird species sampled for influenza surveillance.The authors declare that they have no competing interests.Conceived and designed the experiments: MW JW TK. Performed the experiments: MW, PvR. Analyzed the data: MW. Contributed reagents/materials/analysis tools: TK JW. Wrote the paper: MW JW TK. All authors read and approved the final manuscript.Ct values for all individuals that were positive. Influenza A virus RNA was detected by RRT-PCR in oropharyngeal swabs, cloacal swabs, or both.Click here for file


To date, the pathomechanism of soft tissue sarcomas such as synovial sarcoma remains unclear whereas even a viral etiology was suspected. Aim of this study was to analyze whether EBV, HHV-8 or HPV play a role in the development of synovial sarcomas.In total 41 synovial sarcomas were included in this retrospective study. For detection of EBV 1/2 and HHV-8, resection specimens were analyzed with regard to virus-specific sequences using a SingleStep PCR. HPV analysis was carried out by an HPV-specific multiplex-PCR and subsequent array-hybridization for HPV-typing. No virus-specific DNA of EBV, HHV-8 or HPV was detected.An involvement of these viruses in the etiology of synovial sarcoma was not detected but further studies are needed with different virus types and sarcoma entities. Synovial sarcoma (SS) is a malignant soft tissue tumor, which is mostly seen during the 3rd decade of life, but approximately 30% of cases occur in patients younger than 20 years ,2. The eIn total 41 synovial sarcomas were included in this retrospective study .As in most soft tissue sarcomas, the etiology of SS has not been completely uncovered. Although the involvement of a fusion gene resulting from a specific chromosomal translocation tX;18)  was disclosed, which is detected in more than 95%, SS remains an entity of uncertain origin ,7. There (p11;q11It is estimated, that up to 18% of cancer diseases are associated with viral infections . The humWe therefore investigated in this study, whether virus-specific DNA of EBV, HHV-8 or HPV is detectable in tumor resection specimens of patients with synovial sarcoma. We conclude that, although these viruses contribute to the oncogenesis of a considerable number of malignant tumors, an involvement in the pathogenesis of synovial sarcoma was not detected. Further investigations with different sarcoma entities and virus types are to be conducted.


Intracameral cefuroxime is recommended as prophylaxis against postoperative endophthalmitis (POE) following cataract surgery. Aprokam is the only licensed product for prophylaxis of POE, although unlicensed intracameral cefuroxime may be administered using pre-filled syringes (PFS), either prepared in hospital by reconstituting cefuroxime via serial dilution (prepared PFS), or commercially purchased (purchased PFS). This study aimed to estimate the potential budget impact of using Aprokam over unlicensed cefuroxime for intracameral administration.A budget impact model (BIM) was developed from UK NHS hospital perspective to estimate the economic impact of adopting Aprokam compared with purchased PFS or prepared PFS for the prophylaxis of POE following cataract surgery over a 5-year time horizon. The BIM incorporated direct costs only, associated with the acquisition, delivery, storage, preparation, and administration of cefuroxime. Resource utilisation costs were also incorporated; resource utilisation was sourced from a panel survey of hospital pharmacists, surgeons, and theatre nurses who are involved in the delivery, storage, preparation, quality assurance, or administration of cefuroxime formulations. Unit costs were sourced from NHS sources; drug acquisition costs were sourced from BNF. The model base case used a hypothetical cohort comprising of 1000 surgeries in the first year and followed a 5.2 % annual increase each year.The model predicts Aprokam is cost saving compared with purchased PFS, with a modest increase compared prepared PFS over 5 years. There are total savings of £3490 with Aprokam compared with purchased PFS, driven by savings in staff costs that offset greater drug acquisition costs. Compared with prepared PFS, there are greater drug acquisition costs which drive an increased total cost over 5 years of £13,177 with Aprokam, although there are substantial savings in staff costs as well as consumables and equipment costs.The lower direct costs of using Aprokam compared with purchased PFS presents a strong argument for the adoption of Aprokam where purchased PFS is administered. The additional benefits of Aprokam include increased liability coverage and possible reduction in dilution errors and contaminations; as such, in hospitals where unlicensed prepared PFS is used, modest additional resources should be allocated to adoption of Aprokam. Postoperative endophthalmitis (POE) is defined as an inflammation of the eye arising from a bacterial, fungal, or parasitic infection during the postoperative period of surgery . This inIn 2013, the European Society of Cataract and Refractive Surgery (ESCRS) updated guidelines that recommended use of intracameral cefuroxime as prophylaxis against POE following cataract surgery . This reDespite the ECSRS recommendations, use of intracameral cefuroxime has not been fully adopted. An ESCRS survey of member ophthalmic surgeons conducted in 2012 reported that the most common reasons for not using intracameral cefuroxime during cataract surgery were: the unavailability of the country/clinic specific protocol, unavailability of an approved preparation, and concern over the risk of dilution errors . These mPrior to the availability of Aprokam, unlicensed formulations of intracameral cefuroxime have been administered using pre-filled syringes of cefuroxime (PFS) from a variety of sources. Firstly, PFS is prepared in theatre by serial dilution of 250 mg (or larger) vials of cefuroxime licensed for injection or infusion. Secondly, some hospital pharmacy departments prepare 10 mg/ml PFS (1 mg/0.1 ml) , and lastly, PFS are commercially available from a variety of pharmaceutical sources.Although the availability of a licensed alternative should discourage use of these unlicensed formulations, the use of PFS is still practiced. The protocol for the preparation of PFS at the hospital is complex and varies among hospitals , 11. PreFurthermore, serious macular detachment, macular infarction, macular oedema, corneal oedema, and haemorrhagic retinal Infarction are among the potential outcomes arising from the administration of intracameral cefuroxime at doses that are higher than recommended , 15, 17.The preparation of intracameral cefuroxime of any type (Aprokam or PFS) incurs costs associated with staff, consumables, equipment, and quality assurance. Whilst the clinical arguments (lower risk of dilution errors) supporting Aprokam over PFS are unequivocal, there remains some reluctance among budget-holders to adopt the licensed product available after consideration of the relatively higher acquisition costs associated with Aprokam. However, there may be considerable cost savings associated with staff time, consumables, and/or equipment with Aprokam. There may also be additional costs associated with the rare, but serious adverse clinical events that might result from unlicensed cefuroxime use, as well as reduced liability coverage for the hospital, both of which are difficult to evaluate.In addition to the clinical arguments, economic analyses have demonstrated the cost-effectiveness of cefuroxime compared with other antibiotics in preventing POE following cataract surgery, further validating the use of cefuroxime , 20. HowThe objective of this study was to develop a budget impact model (BIM) to estimate the potential budget impact of a UK National Health Service (NHS) hospital adopting Aprokam in place of unlicensed cefuroxime formulations (prepared PFS or purchased PFS) for the prophylaxis of POE following cataract surgery. The model estimated costs over a 5-year time horizon. Clinical outcomes were not considered in this study and all cefuroxime formulations were assumed to have the same efficacy and safety. The results of this study are of use for NHS managers, payers, and clinicians, who can consider the results of this analysis in the wider context of cefuroxime use, the license status and risk of dilution errors of each cefuroxime formulation, when making decisions upon the introduction of Aprokam in a clinical setting.An economic model was developed in Microsoft Excel to assess the potential budget impact of using Aprokam compared with purchased PFS or prepared PFS for the prophylaxis of POE following cataract surgery. Budget impact was estimated over a 5-year time horizon (base year 2013), from the perspective of a UK NHS hospital setting. Since all three formulations are based on the same active compound (cefuroxime), their efficacy and safety profiles were assumed to be equal.The BIM incorporated direct costs only, associated with the acquisition, delivery, storage, preparation, and administration of cefuroxime. The model took into account the annual number of cataract surgeries to estimate the total costs related to the prophylaxis of post-cataract surgery POE. The analysis was carried out for a hypothetical cohort comprising of 1000 surgeries in the starting year and followed by a 5.2 % annual increase. The annual increase in the number of cataract surgeries was based upon NHS data between 1998 and 2009 .The model accounted for resources required to perform quality assurance during the preparation of PFS at the hospital pharmacy  as per the quality assurance protocols for compounding sterile preparations published by the Royal Pharmaceutical Society in the UK , the WorCost components were estimated based on resource utilisation and resource unit costs (in 2013 GB £). Resource utilisation inputs were sourced from a panel survey submitted to hospital pharmacists, surgeons, and theatre nurses who are involved in the delivery, storage, preparation, quality assurance, or administration of cefuroxime formulations used as a prophylaxis treatment against POE. Unit costs were sourced from targeted literature searches using publicly available sources.Annual acquisition costs were estimated for the number of Aprokam vials, 750 mg vials of Zinacef® (powdered cefuroxime), or ready-to-use PFS purchased each year by the hospital, and their unit costs. The annual number of Aprokam vials and ready-to-use PFS of cefuroxime purchased by the hospital were calculated from the number of cataract operations performed. The number of vials of powdered cefuroxime purchased was based on the number of cataract operations, but also the number of individual doses prepared with each vial, and the number of individual preparations used for quality assurance purposes.Cost of members of staff involved was based on their hourly wages and their working time related to the ordering, delivery, storage, preparation, quality assurance, or administration of cefuroxime. Their hourly wages were sourced from the annual salaries published by the NHS, to which the methods for hourly wage calculations published by the Personal Social Services Research Unit were applied.These costs were related to the provision of consumables required for the preparation or the administration of intracameral cefuroxime, for example syringes, syringe caps, needles, filters, connectors, sterile bags, 0.9 % NaCl solution, heat-seal sterile pouches, and sample tubes. Their unit costs were sourced from various UK suppliers.These costs were related to storage equipment (e.g. pharmacy fridge), preparation equipment (e.g. beta-lactam laminar hood), and quality assurance equipment . The model only accounted for the proportion of the equipment value specifically dedicated to the delivery, storage, preparation, or quality assurance of cefuroxime, as informed by the panel survey. The corresponding value of equipment was then extrapolated over a 5-year depreciation time period, as per the guidelines of the finance manual issued by the UK Department of Health . AdditioBase case values for the model settings are shown in Table Drug-acquisition costs were based on the unit costs of Aprokam, PFS syringes (for purchased PFS), and Zinacef (for prepared PFS), as published in the British National Formulary Table .Table 2DCost inputs, presented in Table Based on the panel survey, the model assumed weekly deliveries of cefuroxime products and the preparation of PFS by pharmacy technicians in weekly batches using 750 mg vials. Quality assurance protocols for PFS prepared at the hospital pharmacy are necessary to prevent dilution errors and contamination and every hospital is responsible for ensuring these protocols are in place. The model therefore considered a default quality assurance protocol based on the guidelines for compounding sterile preparations published by the Royal Pharmaceutical Society in the UK , the WorSince all three products contain the same active molecule, the model assumed the same efficacy between Aprokam and the comparators. Other outcomes may differ between the three products, such as the risks of complications related to preparation errors, needle stick injuries and wastage. However, due to lack of data, the model assumed these risks to be negligible for all three products.The base case analysis showed that when Aprokam replaced purchased PFS, the overall total cost over 5 years was reduced by 4.8 %    costs, there are costs associated with resource utilisation for transfers between storage areas and surgery. Furthermore, there are also costs associated with quality assurance steps that should be undertaken with purchased PFS during delivery and storage, unlike with Aprokam.While there were extra costs related to the reconstitution of Aprokam as opposed to ready-to-use purchased PFS which do not require any preparation (e.g. extra staff time such as theatre nurse) and consumables , the overall staff costs remained substantially reduced.In contrast, compared with prepared PFS, an incremental budget impact of £13,177 over 5 years was estimated with Aprokam. The key cost driver responsible for the higher total cost was greater costs incurred with the acquisition of Aprokam. These acquisition costs were estimated to be over 65 times higher with Aprokam compared with prepared PFS. Preparation of PFS in the hospital pharmacy is a complex procedure and requires numerous steps as well as high amounts of consumables, and specialised equipment (e.g. the use of a laminar hood in order to ensure a controlled atmosphere for the preparation of PFS cefuroxime).Additionally, the risk of dilution errors associated with prepared PFS  necessitThe economic impact of adopting Aprokam is just one of a number of factors that should be used to guide decision-making at the hospital. ESCRS guidelines recommend intracameral cefuroxime for the prophylaxis of POE for patients undergoing cataract surgery . FurtherThere are also patient safety considerations to take into account when selecting cefuroxime formulations. Unlicensed formulations of intracameral cefuroxime appear to be associated with a high risk of dilution error – both over- and under-dosing . DilutioWhen considering whether to adopt Aprokam, decision makers such as clinicians, NHS managers, and local payers should consider the liability status of unlicensed cefuroxime formulations and the higher probability of dilution errors with prepared PFS as well as the economic considerations. Unlicensed cefuroxime places the patient at risk in terms of increased likelihood of receiving high doses that can result in complications such as macular oedema. Furthermore, there is risk to the hospital with the use of unlicensed treatments in terms of reduced liability coverage. The lower estimated total costs with Aprokam compared with PFS purchased as ready-to-use formulation presents a strong case for the introduction of Aprokam in hospitals where purchased PFS is still used.There are a number of limitations related to the study methodology that may influence the results of this budget impact analysis, and thus should be taken into consideration when interpreting the cost estimates. Firstly, the key inputs of the analysis are resource use data provided by pharmacists, surgeons, and theatre nurses who responded to the survey. Therefore, these inputs may not be applicable to all hospitals and may vary should more robust sources be identified. Changes in these inputs are likely to have an impact on the results and their interpretation.Due to lack of data, it was not possible to consider complications due to dilution errors and contamination, in terms of management costs  and their long-term consequences. For example, dilution errors during the preparation of PFS have been linked to higher risks of POE, IGS and TASS , 17. FurFinally, the analysis was conducted on a hypothetical cohort of 1000 cataract surgeries in the starting year, followed by a 5.2 % annual increase in the number of surgeries. This setting is a key driver of the total costs reported in this analysis over the considered time horizon, although it is unlikely to impact the cost per surgery.The lower overall total direct costs with Aprokam compared with purchased PFS presents a strong argument for the adoption of Aprokam in clinical settings where unlicensed purchased PFS is still administered. Aprokam presents with cost-savings, and increased liability coverage compared with purchased PFS. Further, there is also the assumption that the Aprokam is less likely to be associated with dilution errors and contaminations, although these claims have not been empirically demonstrated. In NHS hospitals where patients are still receiving unlicensed prepared PFS, hospital managers and local payers should strongly consider allocating additional financial resources to achieve increased liability coverage with Aprokam.


Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses. Zika virus is a mosquitoborne flavivirus that, in 2015, spread throughout the tropical and subtropical regions of the Western Hemisphere. In January 2016, the first autochthonous cases of Zika fever were confirmed in Nicaragua  have been reported and were characterized by using human specimens  Zika virus genome sequences available in GenBank (n = 21) accessed March 28, 2014). Target sequences were subsequently confirmed to match strains from the Americas. All rRT-PCR reactions were performed on an ABI 7500 instrument  by using 25-μL reactions of the SuperScript III Platinum One-Step qRT-PCR kit  and 5 μL of RNA template. Cycling conditions for the ZCD assay were as follows: 52°C for 15 min; 94°C for 2 min; 45 cycles at 94°C for 15 sec, 55°C for 20 sec (acquisition), and 68°C for 20 sec. Each run included a no-template control and positive controls for Zika virus, CHIKV, and DENV.The Zika virus primers and probe Table 1 w8 to 10 copies/μL for Zika virus and DENV-3 and from 108 to 100 copies/μL for DENV-1, −2, −4, and CHIKV. The 95% LLOD for each target, in copies/μL of eluate (5 μL added to each ZCD reaction), was as follows: Zika virus, 7.8; CHIKV, 13.2; DENV-1, 11.7; DENV-2, 13.5; DENV-3, 4.1; DENV-4, 10.5.Linear range and lower limit of 95% detection (95% LLOD) for each target were determined as recommended. We determined linear range and 95% LLOD for each target as recommended . The linAssay exclusivity was established by testing genomic RNA from the following viruses: West Nile, Japanese encephalitis, tickborne encephalitis, yellow fever, Saint Louis encephalitis, o’nyong-nyong, Semliki Forest, Mayaro, Ross River, Getah, Barmah Fores, and Unas (t) values of 41.34 and 42.25. The 2 assays demonstrated good agreement for CHIKV detection (κ = 0.662). Ct for the 35 CHIKV discrepant samples were reached significantly later  than the 113 concordant samples .De-identified serum samples, collected from Nicaraguan patients with suspected Zika virus, CHIKV, and/or DENV infections, were tested . We testt<38.5 defining a positive result), these assays demonstrated only moderate agreement (κ = 0.47), and Zika virus was detected in significantly more samples by using the ZCD assay (p<0.001). Of the 31 samples positive only for Zika virus in the ZCD assay, 22 (71%) produced a Ct (>38.5) that was reached later in the comparator Zika virus rRT-PCR. These 22 samples had mean Ct of 32.06 (SD ±2.45) in the ZCD assay and 42.09 (SD ±1.41) in the comparator Zika virus rRT-PCR. If all samples in the comparator rRT-PCR with Ct >38.5 were considered positive, the assays demonstrated very good agreement , indicating that these most likely are true, late-positive results. Improved sensitivity for Zika virus is needed given the low viremia detected in clinical samples and the current lack of accurate alternative diagnostics, such as serology  for Zika, chikungunya, and dengue viruses; assay development and analytical evaluation; statistical analysis; clinical evaluation; and comparison of Zika virus detection in the ZCD assay and comparator Zika virus rRT-PCR.


To the Editor: Peste des petits ruminants virus (PPRV) is reported globally with increasing frequency. Recently, PPRV has been detected in areas where it is considered endemic and in neighboring areas where it previously has not been reported. The reporting of “first cases” in regions where PPRV has been considered endemic is of little surprise and perhaps represents increased interest both in agricultural practices and diagnostic capacity . HDuring 2014, PPRV caused extensive agricultural losses across China. Although regions within China had previously reported relatively small outbreaks, during December 2013–June 2014, the virus appears to have greatly extended its distribution. In 2007, PPRV was detected for the first time in the Ngari region of southwestern Tibet  of 17% (2%), 58% (11%), and 79% (19%), respectively. Measures to contain these outbreaks were implemented as in 2007; however, during April and May 2014, the number of PPRV outbreaks increased sharply across much of China, including in Anhui, Guizhou, Guangxi, Hubei, Hunan, Shanxi, Xinjiang, Yunnan, and Zhejiang Provinces (Three years passed without reports of PPRV infections in Tibet or elsewhere in China before the virus was detected in Xinjiang, China’s largest administrative division, in December 2013. Xinjiang, an area of 1.6 million kmrovinces . The oriOnce the current situation has been resolved, full genetic analysis of the viruses causing the outbreaks should be conducted because it might indicate the direction of spread. A further area of interest is the application and choice of control measures. Although predicting the spread of a viral pathogen is impossible, especially across the vast distances involved in the current reports, the experiences in China might influence future responses to incursions of PPRV into areas where PPRV previously has not been documented. The current lack of disease in areas where vaccination was reported in 2010 could explain the continued absence of disease from such areas while other regions are significantly affected (Maps showing the historic and current detection of peste des petits ruminants virus lineage IV across Africa.


Ethanol production from sugarcane bagasse requires a pretreatment step to disrupt the cellulose-hemicellulose-lignin complex and to increase biomass digestibility, thus allowing the obtaining of high yields of fermentable sugars for the subsequent fermentation. Hydrothermal and lime pretreatments have emerged as effective methods in preparing the lignocellulosic biomass for bioconversion. These pretreatments are advantageous because they can be performed under mild temperature and pressure conditions, resulting in less sugar degradation compared with other pretreatments, and also are cost-effective and environmentally sustainable. In this study, we evaluated the effect of these pretreatments on the efficiency of enzymatic hydrolysis of raw sugarcane bagasse obtained directly from mill without prior screening. In addition, we evaluated the structure and composition modifications of this bagasse after lime and hydrothermal pretreatments.2/g raw] for 60 min at 120 °C compared with hydrothermally pretreated bagasse (21 % digestion) under the same time and temperature conditions. Chemical composition analyses showed that the lime pretreatment of bagasse promoted high solubilization of lignin (30 %) and hemicellulose (5 %) accompanied by a cellulose accumulation (11 %). Analysis of pretreated bagasse structure revealed that lime pretreatment caused considerable damage to the bagasse fibers, including rupture of the cell wall, exposing the cellulose-rich areas to enzymatic action.The highest cellulose hydrolysis rate (70 % digestion) was obtained for raw sugarcane bagasse pretreated with lime [0.1 g Ca(OH)We showed that lime pretreatment is effective in improving enzymatic digestibility of raw sugarcane bagasse, even at low lime loading and over a short pretreatment period. It was also demonstrated that this pretreatment caused alterations in the structure and composition of raw bagasse, which had a pronounced effect on the enzymes accessibility to the substrate, resulting in an increase of cellulose hydrolysis rate. These results indicate that the use of raw sugarcane bagasse (without prior screening) pretreated with lime  may represent a cost reduction in the cellulosic ethanol production. Following the world trend for more research on alternative fuels, Brazilian sugar, and ethanol industry has shown interest in sustainable technologies that can be aggregated to its productive chain. Efforts are currently being directed toward the inclusion of sugarcane bagasse, straw, and tops in the production cycle of second-generation ethanol –6. Due tConsidering that pretreatment represents the second most expensive step in the conversion of biomass into ethanol, the great challenge of this technology is to find an appropriate strategy to disrupt the lignocellulosic complex, allowing enzymatic hydrolysis with low loads of enzymes and low conversion times, in a cost-effective manner and environmentally sustainable , 14, 17.Hydrothermal and alkaline pretreatments have emerged as effective methods in preparing the lignocellulosic biomass for enzymatic hydrolysis because they operate under mild temperature and pressure conditions, have less sugar degradation compared with acid pretreatment and also cause delignification and deacetylation depending on the pretreatment severity, greatly enhancing carbohydrate digestibility , 17. HydThe effectiveness of pretreatment to increase the digestibility of lignocellulosic biomass is dependent on substrate structure and composition well as on pretreatments conditions. In this sense, the aim of this study was to evaluate the effect of the hydrothermal and lime pretreatments on structure, composition, and susceptibility to enzymatic hydrolysis on raw sugarcane bagasse coming from mill without prior screening. Although hydrothermal and lime pretreatments have been studied on different types of lignocellulosic biomass, only one study was performed using sugarcane bagasse as it comes from mill . The useTo evaluate the efficiency of the hydrothermal and lime pretreatments to enhance the digestibility of raw sugarcane bagasse, the rates of conversion of cellulose into glucose during enzymatic hydrolysis of pretreated bagasse were measured.As shown in Fig. p < 0.05) on cellulose digestion. Time is very important parameter for an economic analysis of the process because it allows evaluating if the increase in glucose released during saccharification compensates the energy cost when using longer pretreatment periods [2/g dry raw during 8 days at 20 °C. Fuentes et al. [2/g dry biomass for 90 h at 90 °C. Chang, Holtzapple, and Nagwany [2/g dry raw, obtained yield of 300 mg/g dry bagasse after 1 h of pretreatment at 120 °C. In the present study, higher glucose yield (320 mg/g dry raw) was obtained when bagasse was treated with the same amount of lime (0.1 g Ca(OH)2/g dry raw) but within a shorter time (30 min).Our results also showed evidence that cellulose digestion depends on the pretreatment time. Sugarcane bagasse submitted to lime pretreatment exhibited an increase of 208, 223, and 280 % in glucose release compared to hydrothermal pretreatment after 7, 30, and 60 min of pretreatment, respectively. The statistical analysis of data confirmed that the variables studied (time and pretreatment) and interaction between them have a significant effect , while cellulose content increased from 44.49 to 49.56 % (Table 2/g dry bagasse for 90 h at 90 °C. We obtained lower yield (51.1 %) with low lime loading 0.1 g Ca(OH)2/g dry bagasse and also shorter pretreatment time (60 min).These alterations resulted in greater mass reduction in bagasse pretreated with lime compared to hydrothermally pretreated bagasse. The pretreatment yield, expressed as percentage of initial material, ranged from 51 to 75 % for lime pretreatment and from 70 to 89 % for hydrothermal pretreatment, being proportional to the increase in pretreatment time , and the effect of treatment, followed by the treatment/time interaction caused greater change in this fraction. For hemicellulose, only time was significant (p < 0.05) and the treatment/time interaction did not significantly influence the solubilization of this fraction (p > 0.05). Thus, this analysis confirmed that the lime pretreatment affected the lignin and cellulose fractions, inducing high lignin solubilization and cellulose accumulation proportional to the pretreatment time.In order to assess the significance of the effects of pretreatment time and type on the solubilization of lignin, hemicellulose, and cellulose fractions in pretreated bagasse, analysis of variance (ANOVA) was performed using 95 % confidence level. The analysis of data showed that the variables studied (time and pretreatment) as well as interaction between them exerted significant influence on the delignification of pretreated bagasse, with Several studies have shown that variations in the composition of biomass submitted to different pretreatments can be related to pH variations and holding time conditions, which affect the pretreatment severity and consequently have a great effect on enzymatic hydrolysis , 45, 46.Figure As can be seen in Fig. Our results confirmed that lime pretreatment had a more pronounced effect on the efficiency of enzymatic hydrolysis of raw bagasse compared to hydrothermal pretreatment Fig. . The higAccording to literature, the presence of lignin in biomass restricts enzymatic hydrolysis because it acts as a physical barrier preventing the accessibility of cellulase to substrate and also as a competitive adsorbent for cellulases, reducing the activity of adsorbed enzymes , 43. ChaSeveral studies have shown that lime pretreatments had a remarkable effect on lignocellulosic biomass structure , 23, 50.From the analysis of Fig. Rezende et al. , using aThermogravimetric analysis (TGA) was performed to better understand the effects of lime pretreatment on the structure of raw sugarcane bagasse. This analysis provides a useful tool to characterize bagasse fibers after pretreatment because the thermal behavior of lignocellulosic biomass is closely related to the chemical composition of fibers and physical characteristics of lignin, hemicellulose, and cellulose during thermal decomposition of pretreated bagasse .The Figs. As shown by the thermogravimetric analysis (TG/DTA), the distance between peaks related to the second and the third stage is smaller for pretreated samples than for untreated bagasse. These results suggest that lime pretreatment might have caused the decomposition of some components  of pretreated bagasse. This is in agreement with data obtained from compositional analysis of pretreated bagasse Fig. , which sIn this study, X-ray diffraction analyses were performed to evaluate the impact of cellulose crystallinity on the digestibility of lime-pretreated bagasse. Cellulose crystallinity has been considered a biomass recalcitrance feature that, along with specific surface area, polymerization degree, cellulose sheathing by hemicelluloses, lignin, and acetyl contents, affect enzymatic hydrolysis performance in pretreated bagasse . CrystalIc) of pretreated bagasse. Similar results were obtained by Chundawat et al. [Figure t et al. , who comt et al. . and Shet et al. , the inct et al. , 48, 59.Taking into account all results obtained in this present study, it could be inferred that they are consistent with the model proposed by Chang and Holtzapple . Accordi2/g dry bagasse) and shorter pretreatment time (60 min) at 120 °C. These results have a substantial importance for the production of cellulosic ethanol because the use of raw sugarcane bagasse (without prior screening) pretreated with lime  may represent a cost reduction in the bioconversion process.From the results obtained in this study, it could be conclude that lime pretreatment was more efficient to promote greater digestibility rates of raw sugarcane bagasse (70 % cellulose digestion) compared with hydrothermal pretreatment (21 % cellulose digestion). This increase in the cellulose hydrolysis rate was mainly due to lignin and hemicellulose removal  and the increased cellulose content (11 % enrichment) of bagasse pretreated with lime. Analysis of pretreated bagasse structure revealed that lime pretreatment caused considerable damage in bagasse fibers, including rupture of the cell wall, exposing cellulose-rich areas to enzymatic action and consequently contributing to the high conversion rate. Comparing with literature, our results showed that it is possible to obtain high yield of fermentable sugars (384 mg glucose/g dry bagasse) using raw sugarcane bagasse pretreated with low lime loading (0.1 g Ca(OH)Fresh sugarcane bagasse was kindly provided by São Martinho sugar/ethanol plant (Pradópolis/SP-Brazil). It was dried at 60 °C to constant weight and kept into plastic bags in freezer. This biomass denominated raw bagasse was used as it comes from mill with different particle sizes (not passing through any step of screening), as shown in Fig. Lime pretreatment was carried out as described by Chang, Nagwani, and Holtzapple . In 500 The chemical composition of untreated and pretreated raw sugarcane bagasse was determined according to analytical procedures established by NREL . Raw bagThe concentration of polymeric sugars (cellulose and hemicellulose) was determined from the concentration of the corresponding monomeric sugars, using an anhydro correction of 0.88 for C-5 sugars (xylose and arabinose) and a correction of 0.90 for C-6 sugars .The soluble lignin content present on liquid-phase samples was determined by measuring the absorbance at 240 nm on a UV–Visible spectrophotometer. For determination of insoluble lignin, the solid fraction was rinsed with water up to reaching neutral pH to remove acid residues and dried in oven at 105 °C until constant weight. Ash content was obtained by burning in muffle at 600 °C for 24 h. Total lignin was calculated as the sum of soluble and insoluble lignin fractions.®) kindly provided by Genencor International . The enzymatic blend consisted of cellulase (15 FPU/g substrate) and β-glucosidase (75 U/g substrate) and the activities of these enzymes were determined according to methods described by Ghose [Enzymatic hydrolysis of pretreated raw sugarcane bagasse (insoluble fiber) was performed according to standard analytical procedures (LAP) described by NREL , using cby Ghose .Pretreated raw sugarcane bagasse samples were hydrolyzed in 50 mmol/L citrate buffer (pH 4.8) at a solid:liquid ratio of 1:100 (w/v) supplemented with enzymes and sodium azide (40 mg/L) to inhibit microbial contamination. This mixture was incubated at 50 °C for 72 h on a rotary shaker (150 rpm). All assays were performed in duplicate for each indicated time . Hydrolysate samples were collected, boiled to deactivate enzymes and analyzed for glucose and total reducing sugars.The percentage of cellulose digestion was calculated by the ratio between the amount of cellulose digested and the amount of cellulose added, as shown in Eq. Glucose and total reducing sugars were colorimetrically determined at 540 nm using GOD-PAP method  and DNS R0) was used to unify data obtained at different combinations of reaction time and pH of hydrothermal and lime pretreatments with respect to lignin solubilization. The factor R0, incorporating an integration of the time period used in the pretreatment done at a certain temperature, was calculated by Eq. t is the holding time of treatment in minutes, T(t) is the treatment temperature (in the time t), which 100 °C is the reference temperature [The severity factor log and submitted to metal plating with 10 nm of gold in a sputter. Photomicrographs were obtained on Equipment TOP WITH SM 300 marks a power of electron beam 20 kV. Various images were obtained on different areas of samples in order to assure reliable results.−1, temperature range 10–900 °C, oxidizing atmosphere with flow gas of 40 mL min−1. The derivative of TG curves (DTA) was obtained using TA analysis software.The thermogravimetric curves (TG) were obtained on a Netzsch termobalance, using alumina crucible, under the following conditions: heating rate of 10 °C minθ (Bragg angle) at a 0.05° per second of scanning rate. Powder sample data were recorded at room temperature.Crystallinity of sugarcane bagasse before and after pretreatment was analyzed by X-ray diffraction in a Siemens D5000 Diffractometer employing Co-Kα radiation. Scans were obtained from 5° to 20° 2c), which was calculated by Eq. I002 is the intensity of the 002 peak (2θ = 22º) and Iam is the intensity of the peak in the amorphous phase (2θ = 16º).The percentage of crystalline material in the biomass was expressed as the crystallinity index  and type  of pretreatments on the raw sugarcane bagasse composition and saccharification performance.The statistical significance of data was evaluated by analysis of variance (ANOVA) with confidence level of 95 %. The Minitab software version 15.0  was used for the experimental design and for statistical analyses.


In other disease areas, generic drugs are normally used after patent expiry. Patents on zidovudine, lamivudine, nevirapine and efavirenz have already expired. Patents will expire for abacavir in late 2014, lopinavir/r in 2016, and tenofovir, darunavir and atazanavir in 2017. However, patents on single-tablet regimens do not expire until after 2026.The number of people taking each antiretroviral in the UK was estimated from 23,655 individuals in the UK CHIC cohort (2012 database). Costs of patented drugs were taken from the British National Formulary database, assuming a 30% discount. Costs of generic antiretrovirals were estimated using an 80% discount from patented prices, or actual costs where available. Two options were analysed: 1 – all patients use single-tablet regimens and patented versions of drugs; prices remain stable over time; 2 – all people switch from patented to generic drugs when available, after patent expiry (dates shown above).There were an estimated 67,000 people taking antiretrovirals in the UK in 2014, estimated to rise by 8% per year until 2018 (in line with previous rises). The most widely used antiretrovirals in the CHIC cohort were tenofovir (TDF) (75%), emtricitabine (FTC) (69%), efavirenz (EFV) (39%), lamivudine (3TC) (23%), abacavir (ABC) (18%), darunavir (DRV) (21%) and atazanavir (ATV) (16%). The predicted annual UK cost of generic ABC/3TC/EFV (three generic tablets once daily) was £1018 per person-year. Costs of patented single-tablet regimens ranged from £5000 to £7500 per person-year. Assuming continued use of patented antiretrovirals in the UK, the predicted total national costs of antiretroviral treatment were predicted to rise from £425 million in 2014 to £459 m in 2015, £495 m in 2016, £536 m in 2017 and £578 m in 2018. With a 100% switch to generics, total predicted costs were £337 m in 2014, £364 m in 2015, £382 m in 2016, £144 m in 2017 and £169 m in 2018. The total predicted saving over five years from a switch to generics was £1.1 billion.Systematic switching from patented to generic antiretrovirals could potentially save approximately £1.1 billion in the UK over the next five years, compared with continued use of patented versions: this money could be spent on urgently needed HIV prevention programmes. Similar savings are feasible for other European countries, given parallel patent expiry dates. More detailed economic evaluation is required to show when patented single-tablet regimens provide value for money, compared to bioequivalent generic versions of 3–4 pills once daily.