The major obstacle for long-term successful anti-HIV treatment is the emergence of drug resistant mutants. Current data of drug resistance was mainly obtained on HIV-1 subtype B but rarely on non-B virus, even more rare with newly emerged circulating recombinant forms (CRFs). The lack of such data limits the rational management of ART for the increasing number of patients infected by non-subtype B virus. In this study, a HIV-1 CRF07_BC strain CNGZD was isolated from a HIV patient and its genome was sequenced and deposited in GenBank (JQ423923). Potential drug resistant mutants of this CRF07_BC virus strain were selected in PBMCs cultures in the presence of Nevirapine (NVP), which is the most frequently used antiretroviral drug in China. Four combination profiles of mutations were identified in the NVP-selected mutants, which were initiated with A98G, V108I, Y181C and I135T/I382L and followed by more than two other mutations at the end of the selections, respectively. A total of seven previously reported mutations  and seven novel mutations  in the reverse transcriptase gene were found in these NVP-selected mutants. Phenotypic analysis in the NVP-selected mutants showed that all the mutations, except P4H, contribute to NVP resistance. Among them, V106M and Y181C reduce NVP susceptibility for more than 20-fold, while the other mutations cause less than 20 folds drug resistance. Although the information obtained in this  Circulating recombinant forms (CRFs) are the virus with recombinant genomes from different subtypes, which have been detected as epidemic strains and displayed as their identifying numbers and with letters presenting the involved subtypes Nevirapine (NVP), the first generation of nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI), is widely used with nucleoside RT inhibitor (NRTI) as the first line antiretroviral regimens, while NVP-containing highly active antiretroviral therapy (HAART) is the most popular drug combination in China in vitro and further analyzed phenotypes of these mutations by comparing levels of NVP resistance among the induced CRF07_BC mutants. The study results will provide important information of NVP resistant mutations for improving the management of NVP containing antiviral treatment for patients carrying CRF07_BC virus.In the present study, we cultured clinical isolated CRF07_BC virus with increasing concentration of NVP in PBMC to induce NVP associated mutations The virus strain of CRF07_BC was isolated from a 32-year-old female injection drug user (IDU) living in Guangzhou. Patient’s blood sample was collected by Guangzhou CDC in 2007 when the patient was asymptomatic and did not start antiviral treatment. Virus was isolated by co-culture of patient’s PBMC with healthy PBMC in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics and 1 µg/ml interleukin-2 (IL-2) and was named as CNGZD. Biological clones of CNGZD were also prepared using a limiting dilution protocol http://hivdb.stanford.edu). The viral genome sequence was annotated and deposited into GenBank (accession number: JQ423923).Viral RNA was extracted from primary virus culture supernatant by QIAamp Viral RNA Mini Kit . Virus cDNA was synthesized from virus RNA using SuperScript™ II Reverse Transcriptase (Invitrogen). Nearly 9.0 kb full length genome of CNGZD was recovered by four independent PCR reactions using 4 paired primers, in which the PCR products at size of about 2.0–2.5 kb were overlapping the whole virus genome except long terminal repeat (LTR) fragment. Each segment of viral PCR products was subcloned into pMD18-T Simple Vector (TaKaRa) using ligation kits (TaKaRa). Five plasmids for each PCR segment were prepared for cycle sequencing PCR. The sequence electrograms of each sample were aligned using Staden Package in vitro selection of virus variants resistant to NVP in three and two independent cultures, respectively, according to the methods described previously 6) were initially infected with 2×105 BFU/well of the virus in a 24-well tissue culture plate (in quintuple) and cultured in 1 ml medium containing diluted NVP. The starting and ending NVP culture concentration was 5 nM and 64 µM, respectively. One NVP untreated well was included as negative control. At day 3, 7 and 10, virus titers in culture supernatant of each well were detected by BFU assay. Once the BFU number in NVP treated cultures was more than 20% of the untreated control, the drug concentration was increased to 2-fold. While the BFU number in NVP treated cultures was less than 5% of the untreated control, restart the selection. The whole selection was carried out for 25 passages. Passages were performed with supernatant of preceding passage and conducted in the same rules mentioned above. Culture supernatants of passage 5, 10, 15, 20 and 25 were saved for genotypic and phenotypic resistance analysis.Primarily isolated virus strain and two biological clones of CNGZD were used for http://hivdb.stanford.edu) for the interpretation of genotypic resistance as described previously Viral RNA was extracted by QIAamp Viral RNA Mini kit  and viral cDNA was reverse-transcribed using SuperScript™ II Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. The entire protease gene and the first 440 codons of RT gene were amplified from cDNA by high fidelity AccuPrime™ Taq DNA Polymerase (Invitrogen) using primers AV150 and polM4 as described previously 50) of NVP was determined according to the curve which was plotted by the percentage of reduction of luciferase activity versus serial NVP concentrations. NVP resistance of virus variant was expressed as the fold change (FC) of NVP EC50. The NVP susceptibility fold-change of virus variants was defined as the ratio between the EC50 of drug resistant variants with certain mutations and the EC50 of virus without the certain mutations.Phenotypic analysis was performed in TZM-bl cells containing luciferase reporter gene induced by HIV infection as described previously gag, pol, vif, vpr, tat, rev, vpu, env and nef coding for viral proteins p17, p24, p2, p1, p6, PR, RT, IN, Vif, Vpr, Tat, Rev, Vpu, gp120, gp41 and Nef, respectively .To analyze the phenotypes of NVP-selected CRF07_BC virus variants, the NVP susceptibility of 14 virus variants selected in the cultures . Among 4Based on the results of NVP susceptibility, NVP-selected mutations were classified to high (over 20-fold), medium (between 4 to 20-fold) and low (less than 4-fold) level resistance to NVP. As summarized in Drug resistant effects of the 5 reported mutations in CRF07_BC virus were further compared with which were reported in Stanford University HIV drug resistance database . Mutatioin vitro NVP selection study. Considering that the results obtained from this study should be used for hypothetical management of RTI treatments for HIV patients infected by this CRF of the virus, we selected a clinical isolate of CRF07_BC virus strain CNGZD, which was isolated from a HIV-1 infected patient, to represent CRF07_BC. NVP was selected for this study, because it has been included in the free ARV treatment in China and currently used in AIDS patients.Since there is no experimental and clinical data for NVP resistant mutations of HIV CRF07_BC available yet, we designed and performed an The genome of CNGZD was sequenced to understand its genetic background , and 3. in vitro selection in vitro selection study Primarily isolated virus strain and biological clones of CNGZD were cultured in PBMCs with increasing concentration of NVP. We cannot exclude the possibility of selecting pre-existing minority drug resistant variants from the quasi-species which were not revealed by the Sanger-based population sequencing. However, it has been reported that the pre-existing virus variants will emerge soon after 1 or 2 passages by NVP resistance mutations are complicate and variable. Thus far, about 32 NVP-resistance associated mutations in 17 positions, including 15 major NVP-resistant mutations at 5 positions , have been summarized in HIV drug resistance database of Stanford University The other two mutations, A98G and V108I, have been reported to be polymorphic accessory mutations in patients with subtype B or C virus, while K238N is rarely selected by NNRTI and not common to be found in NNRTI-treated patients with infection of subtype B or C virus  50), while double mutations I135T/I382L were detected between passages 10 and 15 (in vitro studies, both Y181C and V106A have been reported as initial mutations resistant to NVP in both subtype B and C viruses in vitro selection may be highly possible to occur in clinics.NVP selected variants were defined to 4 combination profiles based on the initial mutations appearing in the selection period. Initial mutations A98G, V108I and Y181C appeared during the first 10 passages in the presence of 0 and 15 . In previn vitro PBMC culture system. Our study identified 7 novel mutations and 7 reported mutations selected by NVP treatment that are associated with different levels of drug resistance to NVP. The results have also suggested that major NNRTI mutations Y181C and V106M may be found in patients harboring CRF07_BC virus with high possibility when NVP was included in ART treatment and these two mutations may cause serious resistance to not only NVP but also other NNRTI. Furthermore, novel mutations have been selected by NVP treatment in diverse and variable combination with the other major mutations in our study. This phenomenon may happen when more and more patients infected with different HIV CRFs come into therapy. Although the information obtained in this in vitro selection study will not fully cover the resistant mutations which will actually occur in patients, it has still provided useful information for rational management of ART in patients infected with HIV CRF_07BC subtype.In summary, this study has showcased the evolution of virus resistance to NVP in clinical isolated CRF_07BC virus by


Canthaxanthin is a naturally occurring chemical, which is most commonly utilized as a colorant for food and dyes or a skin bronzing agent. Its most prevalent impact on human health is canthaxanthin retinopathy, which appears as birefringent, yellow to red crystals surrounding the macula. This occurs with increasing, dose-dependent exposure. Generally, patients remain asymptomatic and findings may only be evident on funduscopic examination. Cessation of canthaxanthin ingestion appears to reverse the retinopathy, but the time until crystal disappearance is variable. Despite a usually favorable outcome, long-standing visual changes may occur. We report a case of an 84-year-old woman with significant visual loss secondary to canthaxanthin retinopathy that ultimately improved upon cessation of the drug. Canthaxanthin is a naturally occurring carotenoid pigment which is synthesized by microorganisms and plants. It can be found in fruits, vegetables, and fish and primarily occurs in human tissue as a result of dietary ingestion. Like other carotenoids, it is fat-soluble and intensely colored. Canthaxanthin carries a red to orange hue and is used as an agent for coloring foods, dyes, and for skin bronzing. Experimental use has been successful for photoprotection for erythropoietic protoporphyria and cosmetic improvement in vitiligo . The broAn 84-year-old woman presented with a two-month history of worsening vision in both eyes. She had a 10-year history of canthaxanthin use for tanning, at variable dosages. Estimated total dosage is greater than 100 g. She had undergone bilateral cataract extractions in the past and had no other known ocular diseases. Visual acuity was 20/200 OD and 20/300 OS. Funduscopic examination demonstrated bilateral macular pigmentary changes and gold crystalline deposits . FluoresCanthaxanthin and other carotenoids locate in biological lipid membranes and based on their orientation and location can influence membrane properties such as permeability and fluidity. They are powerful antioxidants due to their radical scavenging and singlet oxygen-quenching properties . HoweverIncidence and prevalence are difficult to predict due to the generally asymptomatic course of canthaxanthin retinopathy. Some reports state an incidence between 12 and 14% , 9. Harnμm) crystals accumulating in the perifoveal area. Daicker et al. [Funduscopic examination generally reveals highly reflective, tiny (30 r et al.  reportedr et al. . While tr et al. , 13. Decr et al. –16. The present case, to our knowledge, represents the oldest reported patient with canthaxanthin retinopathy. Also relatively unique to this case is the severe decrease in visual acuity. Such a complication is likely related to the large total ingested dose compounded by the patient's age, similar to other drug-related retinopathies such as that associated with hydroxychloroquine . Differential diagnosis for canthaxanthin retinopathy includes other crystalline disorders such as tamoxifen, methoxyflurane, and talc retinopathy. Primary ocular disorders such as Bietti's crystalline retinopathy, calcified macular drusen, and idiopathic parafoveal telangiectasis are other possibilities. Crystals on the inner retinal surface in the setting of longstanding retinal detachment have also been reported, although the pathophysiology of this finding has not been fully elucidated . FinallyTreatment for canthaxanthin retinopathy is immediate discontinuation of the drug as soon as crystals are identified, even if the patient is asymptomatic. Prognosis is very good with complete recovery occurring in the vast majority of patients. Hueber et al.  followedIn summary, canthaxanthin retinal crystal deposition is a very common finding in patients with prolonged use of the drug. Symptomatic visual loss is less common and correlates with total dosage and possibly patient age. Even with profound visual loss, prognosis for improvement is very good with recognition and discontinuation of the drug.


Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses.African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a Potato virus X (PVX) vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct.All genes encoded by four distinct begomoviruses (Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case. Plant virus infections may result in disease symptoms that can include chlorosis and/or necrosis, leaf curling, altered plant stature and morphology, presumably caused by interference with developmental processes . In recemiRNAs are endogenous, approx. 21nt RNAs that play important regulatory roles in animals and plants by targeting mRNA for cleavage or translational repression . They arGeminiviridae are divided into four genera based on insect vectors, host range and genome organization  the analysis has shown that the other (non-suppressor) proteins can affect miRNA levels, the first time this has been shown to be the case. Nevertheless, at least four of these proteins  have nucleic acid binding activities, of which only one (Rep) is sequence-specific. At the moment, there is no evidence to suggest that they can directly bind small RNAs such as miRNAs. Since these apparently do not have a direct effect on miRNAs, by binding them so as to remove them from the system and thereby up-regulate the genes they control, the implication is that their effects on miRNA levels is indirect, by affecting host factors that in turn affect miRNA levels. Consistent with the idea it has recently been shown that Oilseed rape mosaic virus infection up-regulates transcription from the miR164a promoter  (AJ496461), ACMV-[CM:DO3:98] , CbLCuV-[US:Flo:96]  and TYLCV-Mld[ES:72:97] (AF071228) were amplified with specific primers  at 28°C until the OD600 of the culture was 1. The cells were harvested by centrifugation at 3220 ×g for 8 minutes and resuspended in LB medium containing acetosyringone . For agroinoculation to N. benthamiana, plants at the 4 to 5 leaf stage were selected and were not watered for 24 hours before inoculation. Young leaves were infiltrated by gently appressing a 5 ml syringe to the abaxial surface and depressing the plunger until a water-soaked appearance was achieved.pGR107 and constructs were electroporated into N. benthamiana were grown at 28°C with 16 hours dark period/8 hours light period and 65% humidity in small 5 inch diameter plastic pots containing clay, silt, sand and compost in equal proportions. All plants were watered on daily basis and with Hoagland solution 2.4H2O, 0.5 mM KH2PO4, 1.25 mM KNO3, micronutrients  and Fe-EDTA  once a week if required. Samples were collected 12 days post inoculation (dpi) and photographed with a high resolution camera. Systemic young leaves of inoculated plants were collected, kept in plastic bags labelled with permanent marker, transported on ice and stored at -80°C until utilized.Arabidopsis miRNAs were end-labelled, as described earlier , the coat protein (CP), the (A)V2 protein [(A)V2], the nuclear shuttle protein (NSP) and the movement protein (MP) Additionally the gene encoding the hypothetical protein C5 (C5*) of Cotton leaf curl Kokhran virus and the green fluorescence protein (GFP) were expressed from the PVX. Shown below the northern blots in each case are the rRNA bands of the ethidium bromide-stained agarose gels that were used to normalize the data for loading.click here for filegfpfor cloning in the PVX vectorTable S1: Primers used to PCR amplify begomovirus genes and .click here for file


Aix sponsa) was evaluated. Pre-exposure of wood ducks to different LPAI viruses did not prevent infection with H5N1 HPAI virus, but did increase survival associated with H5N1 HPAI virus infection. The magnitude of this effect on the outcome of the H5N1 HPAI virus infection varied between different LPAI viruses, and was associated both with efficiency of LPAI viral replication in wood ducks and the development of a detectable humoral immune response. These observations suggest that in naturally occurring outbreaks of H5N1 HPAI, birds with pre-existing immunity to homologous hemagglutinin or neuraminidase subtypes of AI virus may either survive H5N1 HPAI virus infection or live longer than naïve birds and, consequently, could pose a greater risk for contributing to viral transmission and dissemination. The mechanisms responsible for this protection and/or the duration of this immunity remain unknown. The results of this study are important for surveillance efforts and help clarify epidemiological data from outbreaks of H5N1 HPAI virus in wild bird populations.Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI) virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. In this study, the effect of pre-exposure to homosubtypic (homologous hemagglutinin) and heterosubtypic (heterologous hemagglutinin) low pathogenic avian influenza (LPAI) viruses on the outcome of a H5N1 HPAI virus infection in wood ducks ( Wild birds, especially those in the Orders Anseriformes and Charadriiformes, are the natural reservoirs for all known hemagglutinin (HA) (H1–H16) and neuraminidase (NA) (N1–N9) subtypes of avian influenza (AI) viruses Anas platyrhynchos domesticus) primary inoculation with a LPAI virus induced partial protection against repeated inoculation with the same virus Branta canadensis) Cygnus olor) Anas platyrhynchos) Concurrent or successive infections with different low pathogenic avian influenza (LPAI) virus subtypes have been reported in both wild bird populations and poultry Aix sponsa).To better understand the epidemiology of H5N1 HPAI virus infections in wild bird populations, we experimentally investigated the effects that pre-exposure with homosubtypic (homologous HA) (H5N1 or H5N2) or heterosubtypic (heterologous HA) (H1N1) LPAI viruses had on the outcome of a Eurasian H5N1 HPAI virus challenge in wood ducks  approved by the Institutional Animal Care and Use Committee at The University of Georgia and the Southeast Poultry Research Laboratory (SEPRL), United States Department of Agriculture, Agricultural Research Service .ad libitum. The wood duck was selected as the animal model due to the availability of data on the susceptibility, viral shedding patterns, and pathobiology associated with the H5N1 HPAI virus used in this study Twenty-five 4-month-old wood ducks were purchased from a waterfowl breeder. Both males and females were included in approximately equal numbers. Food and water were provided Three North American and one Eurasian wild bird-origin LPAI viruses were selected for the pre-exposure portion of this study. The LPAI virus strains included: A/mallard/MN/355779/00 (H5N2) and A/blue-winged teal/LA/B228/86 (H1N1) , A/mute swan/MI/451072-2/06 (H5N1) , and A/mallard/Netherlands/2/05 (H5N2) . The HPAI isolate A/whooper swan/Mongolia/244/05 (H5N1) (Mongolia/05)  was used as challenge virus. The Mongolia/05 virus is a clade 2.2 virus and is genetically representative of H5N1 HPAI viruses that have been reported in wild birds in Asia, Europe, and Africa 50) titers were calculated using previously described methods 6 EID50 per 0.1 mL for LPAI viruses and 104 per 0.1 mL for the H5N1 HPAI virus (single bird inoculum).Individual stocks of the LPAI and the HPAI viruses used in this study were produced by second passage in 9-day-old specific pathogen free (SPF) embryonating chicken eggs (ECE). Viral stocks were titrated in SPF ECE and median embryo infectious dose  group; A/blue-winged teal/LA/B228/86 (H1N1) group; A/mute swan/MI/451072-2/06 (H5N1) group; and A/mallard/MN/355779/00 (H5N2) group. One group was not experimentally exposed to a LPAI virus and served as a negative control (naïve group). Blood was collected from all birds prior to LPAI virus inoculation and at 21 days post-LPAI virus exposure (LPAIV-dpe) to test for the presence of antibodies to AI virus. In addition, cloacal and oropharyngeal (OP) swabs were collected from each bird for virus isolation prior to the study and at 2, 4, 7, and 21 LPAIV-dpe. After LPAI virus inoculation, birds were observed daily for behavior changes and/or the development of any clinical signs. At 21 LPAIV-dpe, the four LPAI virus exposed groups and the naïve group were inoculated via choanal cleft with 0.1 mL of the Mongolia/05 virus. Cloacal and OP swabs were collected from all the birds at 0, 1, 2, 4, 8, and 10 days post-HPAI virus challenge (HPAIV-dpc), and from any birds that were found dead or were euthanized due the severity of their clinical condition. Blood samples were collected at 10 HPAIV-dpc to test for the presence of antibodies to AI virus. Birds were evaluated daily as described above. Wood ducks exhibiting severe clinical signs  were euthanized with intravenous administration of sodium pentobarbital (100 mg/kg), and a full necropsy was performed. The experiment was terminated at 10 HPAIV-dpc at which time all the remaining birds were euthanized, as described above, and full necropsies were performed. All experimental infections were performed in a Biosafety Level-3-Enhanced facility at the SEPRL.All blood samples were collected from the right jugular vein. Serum samples were stored at −20°C until tested for the presence of influenza A antibodies using two serologic assays. Samples were tested for antibodies directed against the conserved internal nucleoprotein of influenza A viruses using a blocking enzyme-linked immunosorbent assay (bELISA)  following the manufacturer's instructions. Additionally, all samples were further analyzed for presence of antibodies directed against specific HA subtypes using the hemagglutination inhibition (HI) test following procedures previously described Cloacal and OP swabs were collected in sterile BHI medium with antimicrobial drugs . Samples were stored at −70°C until testing was performed. Isolation of virus was performed by inoculating the allantoic sac of four 9-day-old SPF ECE with the clarified BHI media containing the swab (0.25 mL per egg). Allantoic fluid was harvested from all inoculated SPF ECE after 4 days of incubation at 37°C and tested for the presence of hemagglutinating activity Samples of cerebrum, cerebellum, heart, lung, trachea, liver, spleen, esophagus, proventriculus, ventriculus, small intestine, large intestine, pancreas, adrenal, ovaries/testis, kidney, cloacal bursa, pectoral muscle, femur, nasal turbinates and sinus were collected from all the animals and fixed in 10% buffered formalin for histopathology and immunohistochemistry (IHC). After fixation, the tissues were processed and embedded in paraffin, and 5µm sections were stained with hematoxylin and eosin using standard histopathology protocols. Femur and nasal turbinates were decalcified with Kristensen's decalcifying solution before being processed. Duplicate sections of all tissues were immunohistochemically stained by using a mouse-derived monoclonal antibody (P13C11) specific for type A influenza virus nucleoprotein antigen as the primary antibody . Procedures used to perform the immunohistochemical testing have been previously described 2) distribution with α of 0.05 was employed to analyze the relation between survival following Mongolia/05 virus challenge and the serological status upon arrival.Fisher Exact Test with a level of significance (α) of 0.05 was used to evaluate the association between pre-exposure to each of the LPAI viruses and the survival following Mongolia/05 virus challenge. Chi-square (χPrior to the start of the study (0 LPAIV-dpe), 12 of the 25 wood ducks tested positive for influenza A virus nucleoprotein antibodies with the bELISA . These 1All wood ducks in each of the five groups remained clinically and behaviorally normal, and had an average weight gain of 8.9% (2.0–15.3%) during the 21 days following the LPAI virus pre-exposure (data not shown).Detection of viral shedding  and seroViral shedding was detected in four of five wood ducks inoculated with the A/blue-winged teal/LA/B228/86 (H1N1) virus. Virus was reisolated from wood ducks in this group until 4 to 7 LPAIV-dpe (mean 4.75 days). Three out of the four ducks that excreted the A/blue-winged teal/LA/B228/86 (H1N1) virus seroconverted, as detected by H1 specific HI test; two of these three wood ducks also had detectable antibodies on bELISA at 21 LPAIV-dpe. The A/mallard/MN/355779/00 (H5N2) virus replicated efficiently in all the five inoculated wood ducks independently of the serological status at 0 LPAIV-dpe. Viral shedding in this group was detected until 2 to 4 LPAIV-dpe (mean 2.4 days). Seroconversion was detected in four of five wood ducks in this group with both the bELISA and H5 HI test.Serological status for each of the exposure groups, as determined by the HI test at 21 LPAIV-dpe (against the homosubtypic LPAI viruses used for pre-exposure) and at 10 HPAIV-dpc (against Mongolia/05 virus), is summarized in Following the Mongolia/05 virus challenge (at 10 HPAIV-dpc), all of the surviving wood ducks in the A/mute swan/MI/451072-2/06 (H5N1) and A/mallard/MN/355779/00 (H5N2) groups had an increase in the HI titer against the LPAI virus used for pre-exposure. Three wood ducks that succumbed to Mongolia/05 virus infection  group and one bird in the A/mute swan/MI/451072-2/06 (H5N1) group) had negative HI titers (<8) against the LPAI virus used for pre-exposure at 21 LPAIV-dpe .Wood ducks that succumbed to the Mongolia/05 virus challenge (n = 13) progressively lost weight, while the ducks that survived (n = 12) experienced an initial weight loss during the first days after Mongolia/05 virus challenge, followed by a progressive weight gain throughout the remainder of the trial (data not shown). All wood ducks that succumbed to infection either exhibited premonitory neurologic signs or were found dead without overt clinical signs. The onset of morbidity or death ranged from 4 to 8 HPAIV-dpc. Clinical signs varied from mild to severe and included lethargy, incoordination, paresis, walking in circles, and head circling and tremors. One wood duck in the naïve group showed a unilateral cloudy blue eye one day before it was found dead at 5 HPAIV-dpc.P = 0.0079) and A/mute swan/MI/451072-2/06 (H5N1) (P = 0.0476) groups. The groups pre-exposed to the other two LPAI viruses  did not have a statistically significant level of protection against the Mongolia/05 virus (P>0.05).Mortality varied greatly among groups, ranging from 0 to 100% . All fiv2 = 0.987, P>0.05).The serological status of the wood ducks upon arrival had minimal if any effect on the outcome of Mongolia/05 virus infection, as demonstrated by similar mortality rates for birds within each group . StatistAfter challenge, the Mongolia/05 virus was isolated from every wood duck in each of the five groups. In general, viral shedding via oropharynx was greater than via cloaca, as demonstrated by a higher number of isolations from OP than cloacal swabs . Viral sGross lesions were not detected in any of the wood ducks that survived the Mongolia/05 virus challenge and were necropsied at 10 HPAIV-dpc. All the wood ducks that succumbed to the Mongolia/05 virus infection had one or more of the following gross lesions: multifocal to coalescing areas (1- to 7-mm-diameter) of red mottling in the pancreas (10/13), congestion of meningeal vessels of the cerebrum and cerebellum (7/13), splenomegaly (5/13), multiple red foci in the adrenal glands (4/13), and a small tan spleen (2/13).The histopathologic lesions observed in wood ducks that succumbed to Mongolia/05 virus infection and those that survived are described in Positive IHC staining in association with histological lesions was consistently observed in nervous, digestive, and endocrine systems  of the wAmong the 12 wood ducks that survived Mongolia/05 virus challenge and were necropsied at 10 HPAIV-dpc, the microscopic lesions included periportal/centrilobular lymphoplasmacytic hepatitis (10/12), splenic white pulp hyperplasia (4/12), severe nonsuppurative encephalitis (associated or not with meningitis) and marked perivascular cuffing (3/12) , neuritiWood ducks used in this study were acquired from a duck farm where several species of Anseriform and Galliform birds were raised outdoors and had direct contact with wild birds. Outdoor rearing of poultry is a major risk factor for exposure to AI virus The influence of pre-exposure to a LPAI virus on the outcome of a HPAI challenge varied from total protection  to no protection  . All theAn adequate humoral immune response to HA seems to be fundamental for the protection against challenge with H5N1 HPAI virus in multiple avian species The Mongolia/05 virus replicated systemically in wood ducks and had a strong tropism for the CNS, pancreas, and adrenal gland. Failure and/or dysfunction of one or multiple of these organs were likely the cause of death. The cloudy eye observed in one wood duck in the naïve group has been frequently reported as a clinical sign of H5N1 HPAI virus infection in birds Consistent with other experimental studies, oropharyngeal shedding in all five wood duck groups was more pronounced than cloacal shedding  [17], [2In summary, this study demonstrates that previous exposure to homosubtypic LPAI viruses in wood ducks may: prevent disease but not infection or viral shedding of H5N1 HPAI virus; decrease mortality associated with H5N1 HPAI virus infection; and increase MDT, thus prolonging the duration of H5N1 HPAI viral shedding. Furthermore, the degree of protection against the H5N1 HPAI virus varies in response to the LPAI virus subtype that the bird was previously infected with. The mechanisms responsible for this protection, as well as the extent (between different subtypes) and duration of this protective immunity still need to be elucidated in order to understand the epidemiology of AI viruses in waterfowl reservoirs and to better define the potential risks of unique AI viruses, such as H5N1 HPAI virus, to infect, move with, or become established in wild bird populations.


Hip fracture is the most serious complication of osteoporosis and imposes a significant financial burden on countries' economy. This study aimed to assess the hospitalization costs and length of stay associated with osteoporosis hip fractures and identify the major cost components in a referral hospital in Kermanshah city, Iran.In a prospective study, from May 21 2007 to May 21 2008, all patients with osteoporotic hip fracture admitted to a referral hospital for operation were recruited as the study sample. For each patient, information such as age, gender, length of stay (LOS) in hospital and intensive care unit (ICU), medical and diagnostic procedures and cost of surgery and implant were collected both through interview with the patient or a family member and the patients’ hospital records.A total of 103 patients (56 men and 47 women) were studied. The average hospital length of stay (LOS) for the patients was 9.7 days, ranging from 5 to 38 days. The average total hospitalization costs was 7,208,588 IRR (US$774). The main components of the costs were ward stay (16.3%), operative (54.6%), implant (26%) and medical and diagnostic procedures (3.1%). The results of this study demonstrate that the hospital resource burden associated with osteoporotic hip fractures in Iran is substantial and expected to rise with the projected increase of life expectancy and the number of elderly in Iran. Estimating the economic burden of osteoporotic hip fractures provide information that can be of importance in the planning and design of preventive strategies. It is associated with deterioration in skeletal micro architecture, which in turn increases the risk of bone fracture.15 there is little data reported on costs and resource utilization associated with osteoporotic hip fractures. Very few studies have previously tried to assess the economic burden of hip fractures in Iran.17 The current study aimed to assess the hospitalization costs and length of stay associated with osteoporotic hip fractures and identify the major cost components in a referral hospital in Kermanshah city.In Iran, where epidemiological information on osteoporotic hip fractures has been well documented in previous studies,Kermanshah, the capital city of Kermanshah province, is located in the west of Iran. In Kermanshah city, there are six hospitals with orthopedic facilities .The main referral hospital in the city is Taleghani hospital. In a prospective study, from May 21 2007 to May 21 2008, all patients with osteoporotic hip fracture admitted in Taleghani hospital for operation recruited as sample. Hip fracture is defined as clinical and radiological evidence of fracture of the proximal femur (ICD-10: S72.0). For each patient, information such as age, gender, length of stay (LOS) in hospital and intensive care unit (ICU), medical and diagnostic procedures and costs of surgery and implant were collected both through interview with the patient or a family member and the patients’ hospital records. According to the patients’ medical history, data of discharged sheets and X-ray results, those patients who had car accidents or pathologic fractures were excluded from the study sample. During the two-year study period, 116 patients with osteoporotic hip fracture were admitted to the hospital. Among these patients, non-surgical cases were also excluded from the study and the remaining cases (i.e.103 patients) were considered as the final sample. The costs of medical treatment were divided into four major components: ward stay costs, operative  costs, implant costs and cost of medical and diagnostic procedures performed. The ward stay costs were included the costs of stay in Emergency Department (ED), orthopedic ward and Intensive Care Unit (ICU). The operative costs were included operating room costs, surgeons’ and anesthetists' fees. The therapeutical, surgical and all diagnostic procedures  performed for each patient were carefully investigated and verified using the patients’ hospital records. After identifying and measuring resource used by the patients, the mean total hospitalization costs for patients was calculated. This study was approved by the ethical committees of Kermanshah University of Medical Science.A total of 103 new cases (56 men and 47 women) of osteoporotic hip fracture met the inclusion criteria. The average age of the patients was 72 years-old (ranging from 50 to 91 years-old). In general, 87% of the patients were transported to the hospital from their homes either by ambulance or commercial vehicles and 12.6% referred from other governmental healthcare facilities.The mortality rate during hospitalization was 10.7% . Surgical operations were included 64 dynamic hip screws and 39 hemiarthroplasties. Twelve patients (11.6%) were admitted to ICU during hospitalization.The average hospitalization costs for the patients was 7, 208,588 Iranian Rials (IRR) (US$774). The average LOS for the patients was 9.7 days, ranging from 5 to 38.There is little data reported on costs and resource utilization associated with osteoporotic hip fractures in Iran. The current study assessed the total hospitalization costs and LOS associated with osteoporotic hip fractures and identified the main cost components in a referral hospital in Kermanshah city. The average hospitalization costs and the average LOS for the patients in this study were US$774 and 9.7 days, respectively. Most studies measuring the costs of hip fractures have focused on the costs of the acute care. The costs of initial hospitalization have been reported vary significantly across different countries 17 However, the average costs and LOS reported in the current study and the previous Iranian studies are significantly lower compared with the average costs and LOS reported in the studies from other settingsThe estimated hospitalization costs and LOS in the current study are similar to the previous studies conducted in Iran. 22The results of studies from various countries suggest that the osteoporotic hip fractures impose a high financial burden on the societies, which is mostly associated with the duration of hospital stay.4 Furthermore, Lee et al in their study found that the patient's LOS had a significant positive correlation with the hospitalization costs.18 As it has been shown in Table II, the average LOS for the patients in the current study and the previous studies in Iran were lower compared to the studies from other countries, which might explain to some extent the lower costs for osteoporotic hip fractures in Iran.In a study by Lawrence et al, ward stay costs contributed to 84% of the total osteoporotic hip fracture costs. They reported that 40% of patients experienced a delay before undergoing surgery and the majority of hospitalization days were spent for recovery after surgery.A further explanation for the lower costs of osteoporotic hip fractures in Iran compared with other countries, can be the subsidized health care system and also the general low service costs in Iran. The operation costs in our study, for example, were lower than the costs in most countries. For better understanding, we have compared the major components of hospitalization costs including the surgical costs, implant and hospital or ward stay costs in the studies from the selected countries, shown in 15 For instance, Ahmadi-Abhari et al.15 in their study of measuring the burden of hip fracture in Iran have concluded that the osteoporotic hip fracture does not contribute significantly to the disease burden in the country, considering its low incidence. However, as the life expectancy for the Iranian population is increasing,24 the rise of financial burden of hip fracture will be anticipated. Previous studies in Iran have shown that the incidence of osteoporotic hip fracture in Iran was lower than the developed countries.We acknowledge a few limitations with the current study. This study only included hospitalization (inpatient) costs associated to osteoporotic hip fractures, and other direct medical costs such as rehabilitation and outpatient visit costs were not measured. Moreover, the hospitalization costs in this study are charged billed by the hospital and may not reflect actual payments nor true hospital costs due to factors such as government subsidies to hospital services and medicines, discounts and exemptions given to the patients and also not including out-of-pocket payments by the patients. Furthermore, the non-medical direct costs such as travel costs and the indirect costs related to informal care, as well as the costs associated with the pain and suffering by the patients and their families are not included in this study. Measuring these costs was beyond the scope of this study. The results of this study demonstrate that the hospital resource burden associated with osteoporotic hip fractures in Iran is high. It is expected that the costs of osteoporotic hip fractures to rise with the projected increase of life expectancy and the number of elderly in Iran. This study only assessed the hospitalization costs of osteoporotic hip fractures which are only a small part of the costs. Further studies are required to investigate the full economic burden of osteoporotic hip fractures in Iran. Estimating the economic burden of osteoporotic hip fractures provide information that can be of importance in the planning and design of preventive strategies.


In 2012, a female wildlife biologist experienced fever, malaise, headache, generalized myalgia and arthralgia, neck stiffness, and a sore throat shortly after returning to the United States from a 6-week field expedition to South Sudan and Uganda. She was hospitalized, after which a maculopapular rash developed and became confluent. When the patient was discharged from the hospital on day 14, arthralgia and myalgia had improved, oropharynx ulcerations had healed, the rash had resolved without desquamation, and blood counts and hepatic enzyme levels were returning to reference levels. After several known suspect pathogens were ruled out as the cause of her illness, deep sequencing and metagenomics analysis revealed a novel paramyxovirus related to rubula-like viruses isolated from fruit bats. Paramyxoviruses comprise a large family of viruses, including pathogens that cause severe disease in humans  revealed no abnormalities. No rash or synovitis was noted. Treatment with intravenous ceftriaxone was begun for possible typhoid fever, and artemether/lumefantrine was continued for presumptive malaria.On hospital day 2, a maculopapular rash erupted over the patient’s trunk , panel AThe possibility of hemophagocytosis was considered, and a bone marrow biopsy sample was obtained on day 4. The sample showed a mild increase in macrocytic hemophagocytosis and pancytopenia with a hypocellular marrow with myeloid hyperplasia and erythroid hypoplasia , panel BThe fever slowly but progressively decreased, and the patient improved over the next few days; the last recorded fever was on hospital day 9. Abdominal pain and diarrhea resolved. Ceftriaxone was discontinued after 8 days. When the patient was discharged on hospital day 14, arthralgia and myalgia had improved, oropharynx ulcerations had healed, the rash had resolved without desquamation, and blood counts and hepatic enzyme levels were returning to reference limits. Considerable sequelae  persisted for several months.The initial suspected diagnosis was hematophagocytic syndrome (hemophagocytic lymphohistiocytosis). This clinical syndrome has been associated with a variety of viral, bacterial, fungal, and parasitic infections, as well as collagen–vascular diseases and malignancies. Initial diagnostic testing for various infectious diseases included blood screening for respiratory viruses, HIV, cytomegalovirus, and malaria parasites; all results were negative.On hospital day 2, a diagnosis of a viral hemorrhagic fever was considered, and blood specimens were sent to the Centers for Disease Control and Prevention (CDC) for testing. Molecular testing results were negative for rickettsiae, filoviruses (Marburgviruses and Ebolaviruses), selected bunyaviruses , arenaviruses , and several arboviruses .www.ncbi.nlm.nih.gov/blast/Blast.cgi). The sequence reads linked to the BLASTx results were distributed into taxa by using MEGAN (Paramyxoviridae) recently isolated from Rousettus leschenaultii fruit bats in southern China , V protein (V), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin–neuraminidase (HN), and polymerase (L). The sequence of the RNA editing site in the V/P gene is identical to that of Tuhoko virus 3 . The complete genome of Sosuga virus was 15,480 nt long and conformed to the paramyxovirus rule of 6   , panel DHN and L genes. Consistent with virus replication and observed neurologic signs, viral RNA was found in the brain but not in liver or spleen samples  with patient blood and serum collected 3 days after symptom onset, but no virus isolate was obtained. As an alternative, 10 μL of the blood sample was also inoculated intracranially and intraperitoneally into 2-day-old suckling mice, which were then observed for 28 days for signs of illness. Neurologic signs developed 9–10 days after inoculation in 2 of the 20 mice; these 2 mice were euthanized 2 days later. To confirm the presence of the virus, we extracted total RNA from liver, spleen, and brains of the euthanized animals and used it as input in a specific RT-PCR designed to amplify a 2,188-bp fragment partially spanning the virus  samples , panel ABrain homogenates from the euthanized mice were inoculated into fresh monolayers of Vero-E6 cells and H292 cells; 12 days after infection, a cytopathic effect, with cell rounding but no syncytia formation, became evident. Virus antigen was detected by immunofluorescence in both cell lines by using patient’s convalescent-phase serum, collected 50 days after symptom onset , panel BBecause the patient seemed to have acquired the infection during her African research expedition, where she had had extensive contact with rodents and bats, other persons who also come in contact with bats or rodents, such as field biologists, local residents, or ecotourists, might be at risk for infection. This potential public health threat prompted us to develop diagnostic assays for the rapid detection of Sosuga virus.N gene and tested it on all available serum and blood samples from the patient. This test showed that the patient’s viremia peaked early in the course of the infection (cycle threshold 29.5 on day 3 after symptom onset), coinciding with the period of high fever and diverse irregularities in blood parameters  occurred 11 days after symptom onset. As expected, IgM levels later decreased (titer >400), and IgG levels remained high 50 days after symptom onset.Second, we developed a new ELISA specific for Sosuga virus by using the virus recombinant nucleocapsid protein produced and purified from  patient . AlthougIn addition, the new ELISA was tested for potential cross-reactivity with some common paramyxoviruses, including mumps and measles viruses. No cross-reactivity was detected on the ELISA plates when control serum from patients with high levels of IgG against mumps and measles viruses was used, a desired feature in a new diagnostic assay because most persons have IgG to these viruses as a result of vaccination or natural infection.A severe disease affected a wildlife biologist shortly after her return from rural Africa to the United States. Because of the disease characteristics  and travel history, a viral hemorrhagic fever was suspected, and clinical samples were rushed to CDC for investigation of a possible high-risk virus. After molecular and serologic diagnostic assays ruled out several well-known human pathogens  as the cause of the patient’s illness, a next-generation sequence approach was followed to detect a possible new infectious agent.The combination of next-generation sequencing and metagenomic analysis identified a novel paramyxovirus; the virus genome was completely characterized by use of standard sequencing techniques. The complete virus sequence clearly indicated a relationship with other rubula-like viruses isolated from bats. Moreover, the novel virus was isolated from acute-phase serum samples by infecting suckling mice and propagating the virus in cell culture. The specific molecular and serologic diagnostic assays that we developed will facilitate rapid identification of this novel infectious agent should new cases occur. We used these assays to retrospectively investigate all available clinical samples from the patient, and the results revealed periods of viremia and seroconversion.Henipavirus) are known to cause severe disease in humans (Although the exact source of the patient’s infection remains unknown, the sequence similarity with bat-derived rubula-like viruses is highly suggestive. In recent years, a large number of diverse paramyxoviruses have been detected in bats (


Anas platyrhynchos) recently infected with H5N2 or H7N3 viruses were introduced into a room housing other mallards plus chickens, blackbirds, rats and pigeons, and transmission was assessed by monitoring virus shedding (ducks) or seroconversion (other species) over the following 4 weeks. Additional animals of each species were directly inoculated with virus to characterize the effect of a known exposure. In both barnyard experiments, virus accumulated to high titers in the shared water pool. The H5N2 virus was transmitted from infected ducks to other ducks and chickens in the room either directly or through environmental contamination, but not to rats or blackbirds. Ducks infected with the H7N2 virus transmitted directly or indirectly to all other species present. Chickens and blackbirds directly inoculated with these viruses shed significant amounts of virus and seroconverted; rats and pigeons developed antiviral antibodies, but, except for one pigeon, failed to shed virus.Waterfowl and shorebirds harbor and shed all hemagglutinin and neuraminidase subtypes of influenza A viruses and interact in nature with a broad range of other avian and mammalian species to which they might transmit such viruses. Estimating the efficiency and importance of such cross-species transmission using epidemiological approaches is difficult. We therefore addressed this question by studying transmission of low pathogenic H5 and H7 viruses from infected ducks to other common animals in a quasi-natural laboratory environment designed to mimic a common barnyard. Mallards ( Gaining a more detailed understanding of the transmission potential of different avian influenza viruses (AIVs) among co-habitating species will enhance our ability to develop accurate models for disease spread, develop control strategies and, in some cases, assess risk of transmission to humans. Influenza A viruses are a common concern among many animal species including, birds, horses, pigs, sea mammals and humans, as the effects can range from asymptomatic to severe respiratory distress leading to death. While AIVs are maintained in wild water birds, they occasionally spread to other animals and humans and can lead to public health concerns, as currently is the case for highly pathogenic H5N1 viruses. All 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of influenza A virus are found in wild waterfowl, gulls, and shorebirds Most strains of AIV are designated low pathogenic (LP) and cause minimal illness in chickens, as well as in wild waterfowl and shorebirds, but infection results in high levels of virus shedding, efficient spread among susceptible hosts, and perpetuation of the agent. Other AIV strains are classified as highly pathogenic (HP) and are restricted to members of the H5 and H7 subtype. HPAIV classification comes from the ability to cause severe morbidity and mortality in domestic fowl and more recently has caused mortality in wild waterfowl, mammals, and humans Among water birds, mallards are of great interest due to their widespread distribution, reservoir for subtypes H1–H12, and ability to shed large amounts of virus with minimal pathology and disease The relative roles of direct contact versus environmental contamination in the transmission of AIVs remains poorly understood; both mechanisms likely occur based on experimental and field studies Common concerns with transmission of AIV from waterfowl to other species are evident when you observe interactions of multiple species, both domestic and wild, present within a single small farm, virtually anywhere in the world. Transmission to species such as rodents would not likely result in disease spread, but could be exploited to monitor disease incursion via serosurveillance; rats and mice are found in abundance on small farms and are of concern in their ability to move freely from outside into enclosures, and their propensity to eat and drink from common containers of poultry feed. Although rats are not considered reservoir hosts for influenza viruses, both laboratory and cotton rats have been shown to replicate unadapted avian and human influenza A viruses It is clear that wild and domestic ducks harbor and shed influenza A viruses and recurrently interact in nature with a broad range of other avian and mammalian species to which they might transmit such viruses. Estimating the efficiency and importance of such multispecies transmission using epidemiological approaches is difficult. We therefore addressed this question by studying transmission of LP H5 and H7 viruses from infected ducks to other common animals in a quasi-natural laboratory environment designed to mimic a common barnyard.All experiments were approved by the Institutional Animal Care and Use Committee of Colorado State University, Fort Collins, Colorado, USA, under approval number 09-168A.Mallard ducks and chickens were purchased from local producers at 2–4 months and 3–4 weeks of age respectively. Red-winged blackbirds and pigeons were captured locally. Sprague Dawley rats, 6–8 weeks of age were obtained from Charles River Laboratories. All animals tested negative for group-specific antibodies to influenza A virus by ELISA and strain specific antibodies  by hemagglutination inhibition (HAI) assay prior to infection.The viruses used in this study were A/Mallard/MN/346250/00 (H5N2) and A/Ruddy turnstone/ReedsBeachNJ/00 (H7N3). Both viruses were propagated to passage three in 10 day old specific pathogen free embryonated chicken eggs . Eggs were incubated at 37 C and allantoic fluid was harvested 48 hours after inoculation, aliquoted, and stored at −80 C until use. Both viruses were titrated by plaque assay. Briefly, plaque assays were performed on monolayers of MDCK cells  in 6-well plates. The monolayers were washed with PBS, inoculated with sample, incubated 60 minutes at 37 C, then overlaid with 0.5% agarose in minimal essential medium containing 0.5% bovine albumin, antibiotics and TPCK trypsin (1 µg/ml). Following a 48 hour incubation at 37 C, a second overlay containing neutral red (33 mg/L) was applied and plaques were visualized and counted 4–6 hours later.The challenge virus for each experiment was used to determine subtype-specific antibodies using the hemagglutination-inhibition assay Two independent experiments were conducted using different influenza viruses. In the first experiment, the barnyard contained 8 ducks, 8 chickens, 8 rats and 10 blackbirds. The second experiment consisted of 8 ducks, 8 chickens, 6 pigeons, 5 blackbirds and 7 rats. In both experiments, animals were allowed to freely range inside a room within an ABSL3 facility. The room had dimensions of 12 (width)×18 (length)×12 (height) feet and basic illumination was provided through a skylight in the roof. The barnyard rooms contained a plastic children's swimming pool  and two large bowls that contained commercial duck and chicken layer feed . Three s6 PFU/0.5 ml of the respective virus. After four hours, the four inoculated ducks were returned to the barnyard where all animals were free to move about the room and interact. All eight ducks (4 inoculated and 4 non-inoculated) were sampled on days 0–7 by collecting oral and cloacal swabs into 2 ml BA-1 medium  supplemented with antibiotics , refrigerated and tested within 2 days for virus titer by plaque assay on MDCK cells; titers are reported as PFU/ml. The only barnyard animals to have daily swab samples collected for virus titration were ducks as we wished to minimize the stress on remaining barnyard species and use seroconversion as the determination of virus infection. All animals in the barnyard room were bled on days 0, 14, 21 and 28, and those sera were tested for seroconversion using HAI and ELISA.On day 0 of each experiment, four ducks were removed from the barnyard room, placed in a separate room, and inoculated orally, intranasally and ocularly with 10in vitro experiment in which pool water from a room containing non-infected ducks was spiked with the H5 or H7 viruses, maintained at room temperature and tested by plaque assay, as described above, at intervals up to 42 days.Two to three water samples were collected daily before the addition of new water. Samples were collected by skimming the top of the pool with a tube, sediment by running the tube along the bottom of the pool to collect sample, and, if present, taking a sample of splashed water from the floor near the pool. To assist in interpreting pool water virus titers, we conducted an 6 PFU in 0.1 ml. Once daily on days 0–7, oropharyngeal and cloacal swabs were collected from the birds, and oral swabs from rats; these samples were processed as described above for duck samples. Sera were collected on days 0, 14, 21, and 28, and tested for anti-influenza antibodies by ELISA and for challenge virus-specific antibodies by HAI.For both experiments, groups of each of the animals in the barnyard except ducks were housed in cages in a separate room and directly inoculated with virus to determine the effects of known exposure. Chickens, pigeons, blackbirds and rats were inoculated intranasally with 106 PFU/ml of H5N2 or H7N3 virus and placed at room temperature. A tube of the same water not spiked with virus served as the negative control and was collected and tested for virus. Samples were collected once daily on days 0 through 7 then weekly for 6 weeks. One ml aliquots were collected at each time point and stored at −80 until all samples were collected. Samples were then tested for virus titer utilizing the plaque assay.Water was collected from a pool that had non infected ducks swimming in it for 24 hours prior to water collection. This was done to mimic the natural state of the water from the barnyard study where water would also contain feces and food particles. Pool water was then placed in a 50 ml conical tube and spiked with either 1×10Clinical signs of disease were not observed in any of the birds or rats in the barnyard environments nor among those caged and directly inoculated with either virus. Animals in the barnyard were observed several times daily. The ducks and chickens tended to cluster and move about in their own groups. Blackbirds and pigeons spent much of their time perched above the floor, but were frequently observed walking on the floor or perched on the side of the pool. All of the birds and rats were observed drinking from the pool and eating out of common feed bowls on the floor. The rats were almost never seen out of their houses during daylight, but were confirmed by video to be exceptionally active in running around the room and through the pool of water during the dark .In vitro testing of H5N2 virus stability in pool water demonstrated a steady decline in virus titer with viable virus detected out to day 35 (Virus was shed by all four inoculated ducks and transmitted to all four contact ducks either through direct contact or environmental contamination of the floor and shared pool . As woulo day 35 .In the direct inoculation of control animals experiment, 83% of chickens and 100% of blackbirds shed detectable virus orally on days 1 through 5 and 1 through 6 respectively ; small aIn order to determine infection rates of all animals exposed in the barnyard or directly inoculated, sera were collected on days 0, 14, 21 and 28 post-inoculation or exposure.For the contact chickens in the barnyard we observed seroconversion rates in chickens of 63% by HAI and 100% by ELISA . None ofin vitro for stability, and behaved similarly to the H5N2 virus with viable virus detected out to day 42 (Virus was shed by all four inoculated ducks and transmitted to all four contact ducks . Virus wo day 42 .Virus isolation was also performed on all control animals directly inoculated with H7N3 virus, including blackbirds, pigeons, chickens and rats. Virus was isolated from oropharygeal swabs at various time points from 83% of chickens, 16% of pigeons, and 100% of blackbirds , but wasTransmission rates in the barnyard were assessed serologically on days 0, 14, 21 and 28 post inoculation. Only 25% of virus-inoculated ducks seroconverted based on HAI results but 100% were positive by ELISA, correlating with the fact that 100% shed virus to varying degrees in the experiment . Of the For the directly inoculated chickens, rats, blackbirds, and pigeons we observed seroconversion rates of 67%, 80%, 100%, and 0% by HAI respectively, and 100%, 100%, 0%, and 33% by ELISA respectively . As withIn both barnyard experiments, introduction of recently-infected mallards was followed rapidly by infection and shedding of virus by contact ducks, and accumulation of substantial quantities of virus in water from the shared pool. Based on detection of seroconversion, ducks infected with either virus efficiently transmitted the virus to other species either through direct contact, which would be most likely with the contact ducks, or through contamination of the environment such as the pool and floor water where high virus titers were recovered in titers high enough to infect the other species. The H7N3 virus was transmitted to a large fraction of other animals in the room, including blackbirds, pigeons and rats, but transmission of the H5N2 virus to blackbirds and rats was not detected. This apparent difference in cross-species transmission may reflect, in part, differences in transmissibility between the two viruses, but it is more likely that transmission of the H7 virus was enhanced due to its higher magnitude and duration of shedding, and higher levels of accumulation in the shared water source compared to that of the H5 virus. As anticipated, neither of the two viruses induced noticeable signs of disease in any of the exposed animals, including those directly inoculated with virus.The high titers of virus that accumulated in water of the shared barnyard pools undoubtedly were in excess of what might typically be expected in natural situations involving wild mallards, but may not be altogether unrealistic for low pathogenic AIVs in small bodies of water. Moreover, it seems likely that such titers may occur in ponds associated with high density domestic duck production facilities, although studies attempting to measure virus titers in such situations are lacking with ducks. The presence of environmental AIV in water habitats of turkeys supports the need for increased environmental sampling along with avian surveillance studies Rats were included in the barnyard transmission experiments because they or other rodents are inevitably present on small farms, sometimes in large numbers, are in direct contact with ducks and poultry and are able to move freely among enclosures. Live markets in Asia, where H5N1 influenza is prevalent, are an additional setting where large numbers of rats live in close contact with ducks and chickens Sero-surveys in natural settings Both LPAIVs tested efficiently spread from ducks to chickens within the shared environment, and a majority of chickens directly inoculated with these viruses shed them at reasonable levels from the intestinal tract; we did not test whether chicken-to-chicken transmission occurred. Transmission of LPAIV from ducks to chickens, if accompanied by mutations in the hemagglutinin gene encoding the HA-1∶HA-2 cleavage site, could lead to generation of a HPAIV and subsequent outbreak in poultry. It is not known whether either of these viruses replicating in chickens, blackbirds, or pigeons might evolve and adapt to those hosts, allowing the new host to better transmit the virus or whether the virus would encounter a dead end in the new host.Small farms, live and wet markets, and many poultry shows provide abundant opportunities for interactions among free ranging and domestic species which may result in transmission and perpetuation of AIVs, particularly when ducks are involved. The studies reported here indicate that introduction of ducks infected with LPAIV into a room designed to mimic a typical barnyard resulted in efficient dissemination of virus to a number of other species, including other birds and rodents. This model system should be extended to investigate more refined questions, such as transmission from passerines to ducks or chickens, multiround transmission, and transmission involving additional viruses, including H5N1 AIV.


Sir,et al. reported that the level of reactive oxygen molecules (ROMs) are hugely raised in malignant effusions as opposed to no or modest rise in nonmalignant effusions . ROM in serum and pleura fluid differentiated the two categories with a 100% sensitivity and specificity . We would like to raise the following concerns:We read with great excitement your September 2010 publication titled “Reactive oxygen metabolites can be used to differentiate malignant and non-malignant pleural effusions”.[At the time the various criteria  did not achieve 100% accuracy in distinguishing transudative and exudative effusions, here we have a single test that 100% sensitive and specific in differentiating the etiology of exudates.The Introduction and Discussion sections downplay the importance of ROM in inflammatory conditions and highlight their association with malignancy. As a matter of fact, ROM  and also called free oxygen radicals are abundantly produced by polymorphs and other phagocytes to enhance their bactericidal action. Moreover, death in pneumonia was attributed to imbalance (excess) of ROM and lung injury. SignificChest in 2005, the mean and distribution of ROM were the same in malignant and inflammatory effusions.[Chest. Nitrous oxide (NO) is not a ROM but is closely related and enhances their action. NO levels in pleural fluid also failed to differentiate inflammatory and malignant effusions as it was raised in both.[We agree with the authors that ROM levels are generally raised in malignant pleural effusions. Studies showed that the elevated levels of ROM differentiated exudative and transudative effusions with excellent sensitivity and specificity. They also contradict the authors’ findings that the ROM level distinguishes malignant and inflammatory effusions. In the best of these studies, published in ffusions. However, in both.The method used by the authors to estimate ROM may throw a light on the discrepancy of results. ROMs are unstable products at room temperature and immediate freezing is mandatory. The storage temperature varies according to the duration of storage. Immediate chilling at –4°C is needed for a few hours storage, but much colder snap freezing is required for long storage. In the publication in your journal we read “pleural fluid, blood samples were kept at room temperature for 30–60 minutes and centrifuged for 10–15 min. Thereafter, fluids were separated from the shaped elements using an automatic pipette. The sera and pleural fluid were kept in the deep freezer at –35°C….” Deep freezers take some time to freeze samples. The considerable delay may explain why inflammatory samples, which are highly cellular and may contain bacteria, had low levels of ROM. The degradation of ROM  is enhanced by contact with high levels of organic matter. These methods compare very unfavorably with other studies where pleural fluid samples were analyzed immediately or lung samples were snap frozen in liquid nitrogen and stored at –80°C.6


Aims. To examine all relevant literature on the economic costs of osteoarthritis in the UK, and to compare such costs globally. Methods. A search of MEDLINE was performed. The search was expanded beyond peer-reviewed journals into publications by the department of health, national orthopaedic associations, national authorities and registries, and arthritis charities. Results. No UK studies were identified in the literature search. 3 European, 6 North American, and 2 Asian studies were reviewed. Significant variation in direct and indirect costs were seen in these studies. Costs for topical and oral NSAIDs were estimated to be £19.2 million and £25.65 million, respectively. Cost of hip and knee replacements was estimated to exceed £850 million, arthroscopic surgery for osteoarthritis was estimated to be £1.34 million. Indirect costs from OA caused a loss of economic production over £3.2 billion, £43 million was spent on community services and £215 million on social services for osteoarthritis. Conclusions. While estimates of economic costs can be made using information from non-published data, there remains a lack of original research looking at the direct or indirect costs of osteoarthritis in the UK. Differing methodology in calculating costs from overseas studies makes direct comparison with the UK difficult. Bone and Joint Decade (2000–2010) , anWithin the envelope of musculoskeletal disorders, Osteoarthritis represents a complex musculoskeletal disorder with multiple genetic, constitutional, and biomechanical risk factors. It represents the most common form of joint disease and disability in older people and ranks amongst the top 5 causes of disability . The economic costs of osteoarthritis can be broken down into direct costs and indirect costs. Direct costs represent the pharmacological/nonpharmacological treatments, including surgery, as well as use of hospital resources and management of complications arising from the treatment of osteoarthritis. Indirect costs represent loss of time from work, decreased productivity because of pain, care-giver time, premature mortality, and disability compensation/benefits. These costs are summarised in A third category sometimes considered is that of intangible costs. These are defined as the pain and suffering experienced by the patient as a result of the disease; the reduction in the patient's quality of life. They remain an area of controversy, with only a few studies making the attempt to estimate them . The aim of this paper is to examine all relevant literature on the economic costs of Osteoarthritis in the UK, and to see what comparisons can be made regarding such costs in the UK and other countries in North America, Europe, and Asia.A comprehensive review of the literature was performed using a computerised bibliographical search of MEDLINE databases from 1946 to 31st Dec 2011. English language articles were reviewed that contained the words “economic cost,” “direct cost,” or “indirect cost” in combination with “osteoarthritis” in either their title or abstract. To expand the review beyond only published studies, an internet search was made for publications from the UK Department of Health, the National Institute for Clinical Excellence, the UK National Joint Registry, Hospital Episode Statistics, and charities Arthritis Research (UK) and Arthritis Care, and all publications were reviewed for information on costs for osteoarthritis. Further internet searches were made for publications from the British Orthopaedic Association, the Royal College of Surgeons, the Royal College of Physicians, and the Royal College of General Practitioners. Publications from American Association of Orthopaedic Surgeons and other regional orthopaedic associations in Europe and Asia were also reviewed.Studies on prevalence of osteoarthritis in the United States have shown that osteoarthritis affects 13.9% of adults aged 25 and older, and 33.6% of those over the age of 65, with an approximate 27 million Americans of all ages suffering from disease . Much of the data available on osteoarthritis in the United States is derived from studies conducted in the 1960s and 1970s. The Framingham study  represen£4.7 billion) [£13.8 billion) [Lethbridge-çejku et al.  examinedbillion) . Less th£2 billion–£8 billion) is spent annually on job-related OA costs in the USA. Meanwhile, Kotlarz et al., in 2010, using evidence from the national health survey data from 1996–2005, looked at absenteeism as a result of osteoarthritis. This study, estimated the indirect cost of the absenteeism to be approximately US$10.3 billion [Buckwalter et al.  used dat£8183 annually for the former, and $2300 or £1449 for the latter) [£824) per month, which may explain the relatively higher costs reported. A survey done by Gupta et al, in ON, Canada in 2005, estimated that the indirect costs incurred by a patient aged over 55 with hip or knee arthritis may be much higher than previously estimated when compared to direct costs ($12 990 or  latter) . IndirecMarch and Bachmeier (1997) looked at the global cost of osteoarthritis and found the cost of osteoarthritis in the USA, Canada, UK, France, and Australia to account for between 1–2.5% of the gross national product (GNP) for those countries . £1260), with direct costs representing 86% (£1084) of the total cost. Indirect costs were much lower (14% or £176) and mainly involved domestic help.Loza et al., in their Spanish study, assessed the burden of knee and hip osteoarthritis by examining 1071 patients across all the provinces of Spain . The aveIn contrast to this, the COART study  in Franc£785) and indirect costs €1236 (£1039) per patient per year [In Italy, Leardini et al. examined the economics of osteoarthritis of the knee in 2004. They used a bottom-up method, utilising data collected from each patient, and reviewed patients across 29 medical institutes. They concluded that the direct costs came to €934 . The direct costs ranged from HK $4860–$11180 (£384–£883) and indirect costs HK$3300–$6640 (£261–£525) per person annually.In contrast to the Western literature, Woo et al., in their Hong Kong  study es£610–£730), around 2.8% and 3.3% of the annual household income [£775) per year.Xie et al. assessed indirect costs in Singapore for OA and noted that there were estimated at between US$1000–1200 £610–£70, aroundThe Arthritis Research Council (UK) estimated in 2002 that at least 4.4 million patients in the UK have X-ray evidence of moderate-to-severe osteoarthritis in their hands, while 550 000 have similar evidence of osteoarthritis in their knees, and 210 000 have evidence of this in their hips .Pye et al., in 2004, showed that almost 8.5 million people in the UK have X-ray evidence of osteoarthritis in their spine, with back pain being the most frequent symptom . While p£36 for a 12-minute consultation [The Royal College of General Practitioners estimated in 2006 that in the UK over 1 million adults consult their GP each year with symptoms of osteoarthritis . Anotherultation .During the year from 1999-2000, there were 114,500 hospital admissions related to osteoarthritis in the UK . The latSurprisingly, there are no published studies in the literature with regard to direct or indirect costs of osteoarthritis in the UK. Data, however, is instead only available from a variety of other sources. The National Institute of Clinical Excellence (NICE) recently published a costing report in 2008 with regard to implementing the guidelines for treatment of osteoarthritis . In the £8.5 million and £25 million for oral NSAIDs. The cost for topical NSAID prescriptions was anticipated to double, and the cost of oral treatment reduced by 10%, if the new guidelines are followed. Adjusting for inflation, in 2010 prices, this would equate to £19.2 million and £25.65 million, respectively.The cost of topical and oral nonsteroidal anti-inflammatories (NSAIDs) was estimated using prescribing data from 2005/06 . An esti£32–£70 per patient prescribed an NSAID in the UK. This equates to a total cost of £44.8–£98 million per year (£56.9–£124.4 million at 2010 prices) [The cost of iatrogenic events related to NSAID use is also substantial. NSAID-related iatrogenic events have been estimated to be between  prices) .£26 000 in 2005/06 but expected to rise significantly to £10.5 million (£11.6 million in 2010) with implementation of the new guidance. The cost of proton pump inhibitor (PPIs) prescription for use with NSAID treatment was £1264, resulting in a cost £25 million for such treatment. It should be noted that NICE expected the cost of this to fall dramatically (by 19000 patients), with guidelines restricting the use of arthroscopic treatment to patients with “mechanical” symptoms such as locking or giving way. The new cost for arthroscopic treatment of osteoarthritis is calculated (£1264 × 1000 patients) at £1.26 million (£1.3 million in 2010). The 2005/06 Hospital Episode statistics stated that the total number of people aged 45 and over who received arthroscopic lavage and debridement for knee osteoarthritis was approximately 20 000. The national tariff for arthroscopies set by the Health Resource Group in 2008/09 was According to the 8th annual report of the NJR, published September 2011 , a 76 75While there are other causes for joint replacement surgery, osteoarthritis remains the most frequent cause for hip replacement (93% of primary hip replacements in 2010) and knee replacement (97% of primary knee replacements in 2010). £6000 for a primary total hip replacement and £6800 for a primary total knee replacement. The national tariff for 2010 was set at £5552 for an uncomplicated total hip replacement and £5198 for a similar total knee replacement. This leads to an estimated cost of £426 million for total hip replacements and £426 million for primary total knee replacements, giving a combined total cost for primary hip and knee replacements of £852 million in 2010. This represents a substantial increase in costs over the last 10 years, when compared to the expenditure of £405 million in 2000 for 44 000 hip and 35 000 knee replacements [£514 million in 2010, representing a 66% increase in the last 10 years. The costs of hip and knee replacements vary considerably from trust to trust in the UK with no set national price for implants, and the cost also being significantly dependant on length of hospital stay. The tariff reimbursement paid to the trust in one study  in 2005/acements . Even ad£3.2 billion; while at the same time, £43 million was spent on community services and £215 million spent on social services for osteoarthritis [Osteoarthritis has a significant negative impact on the UK economy with an estimated total cost of 1% of GNP . The Deprthritis . £2.41 billion paid to people claiming incapacity benefit due to arthritis and related conditions in 2001 [Arthritis remains the most common condition for people to receive the Disability Living Allowance (DLA), with  in 2001 . More th in 2001 . Only ar in 2001 . The most recent review of disability costs in the UK was done by Dame Carol Black  in her rThe review demonstrates that osteoarthritis represents an increasing economic burden to all countries, both from direct costs and indirect costs. Economic data on osteoarthritis has been made difficult because of problems defining the prevalence and incidence of the disease. There is only sparse literature available regarding economic costs in the east, but what is more surprising is the lack of clear costing studies in the west, especially in the UK. Direct costs may vary from country to country, which is to be expected given their different health systems, and even between institutions in a country. Significant variability is seen from these studies, making direct comparison difficult. Compounding the problem is the fact that the methodology used in estimating these costs can vary from study to study, and not all studies give a clear breakdown of the calculation of the direct costs involved . £723 per annum). Hence, the true economic burden of direct costs in osteoarthritis is likely to be significantly higher than most of these studies indicate.Furthermore, in the studies that provide a breakdown of the direct costs, few include the cost of alternative therapies in the treatment of osteoarthritis. There is evidence that nearly half (47%) of older patients in one American study  used an The significant variability in indirect costs from these studies is also a concern. This is likely to be due to the lack of a standardized method to estimate indirect costs–unfortunately, there remains at present no good evidence to support one preferred method over the others . Most stOur review of the literature suggests that while there are a large number of studies on economic costs of osteoarthritis, from multiple countries, the information available in the literature remains patchy and difficult to interpret. Some studies focus on the macroeconomic angle, looking at costs at a national scale or costs per capita while others focus on costs from the view of the individual patient with OA. Other studies are only specific for arthritis of a single joint. Even with studies compatible from this point of view, the varied methodology and lack of standardization of costing make it impossible to accurately compare economic costs, whether direct or indirect. These studies are summarized in Despite such difficulties, one conclusion does seem clear from these studies: that such costs are very substantial and are continuing to rise.The continuing lack of published data regarding direct and indirect OA costs in the UK, especially from the patient perspective, shows that more research into this area is vital. This will allow us to fully appreciate the healthcare burden of OA in the UK, as well as to make more accurate financial planning for the provision for healthcare services for the treatment of OA in the subsequent decade to come.