Iridoviridae are large, icosahedral, dsDNA viruses that are subdivided into 5 genera. Frog virus 3 (FV3) is the type species of the genus Ranavirus and the best studied iridovirus at the molecular level. Typically, antibodies directed against a virus act to neutralize the virus and limit infection. Antibody dependent enhancement occurs when viral antibodies enhance infectivity of the virus rather than neutralize it.Viruses included in the family Here we show that anti-FV3 serum present at the time of FV3 infection enhances infectivity of the virus in two non-immune teleost cell lines. We found that antibody dependent enhancement of FV3 was dependent on the Fc portion of anti-FV3 antibodies but not related to complement. Furthermore, the presence of anti-FV3 serum during an FV3 infection in a non-immune mammalian cell line resulted in neutralization of the virus. Our results suggest that a cell surface receptor specific to teleost cell lines is responsible for the enhancement.This report represents the first evidence of antibody dependent enhancement in iridoviruses. The data suggests that anti-FV3 serum can either neutralize or enhance viral infection and that enhancement is related to a novel antibody dependent enhancement pathway found in teleosts that is Fc dependent. Following a viral infection an immune response is elicited by the host, which includes both an innate and adaptive response. During the adaptive immune response, antibodies are produced that are designed to recognize and neutralize a pathogen. Typically, viral antibodies neutralize a virus by preventing the attachment of specific cell surface receptors with viral glycoproteins, while also activating the complement system. However, not all antibodies serve to reduce infectivity. Antibody dependent enhancement (ADE) occurs when viral antibodies enhance infectivity of a virus by promoting the attachment of viral particles to cells. Virus specific antibodies bind to viral particles to form complexes that can bypass normal routes of viral attachment and entry. The virus+antibody complex allows for increased viral entry or infection of cells that would not normally become infected. Virus+antibody complexes therefore result in a more efficient infection than with virus alone.There are several mechanisms of how ADE can occur. The most common mechanism of ADE is Fc receptor (FcR)-dependent . In FcR-in vitro study suggested that the mechanism behind ADE in dengue virus was FcR-dependent [ADE can result in increased viral pathogenesis because it enhances a virus's ability to bind to cells. It therefore can result in increased severity of disease. This was first shown with dengue virus where a second infection resulted in an increased number of infected cells and higher levels of virus production ,6. An inependent -9. Denguependent -9.Iridoviridae use ADE as a mechanism of infection. Viruses in the family Iridoviridae are large (~120-200 nm), icosahedral viruses that contain a linear, double-stranded DNA genome. Iridovirus infections appear to be restricted to invertebrates  and poikilothermic vertebrates  [Ranavirus) as a model virus, we propose to investigate whether ADE occurs in viruses of the family Iridoviridae, specifically in the Ranavirus genus.While ADE has been demonstrated for many RNA viruses, only a few DNA virus families, including poxviruses  and herptivirus) . AlthougIn order to investigate whether ADE occurs during an FV3 infection, FV3 was pre-incubated with either rabbit anti-FV3 serum (FV3+anti-FV3 serum) or rabbit pre-immune serum (FV3+pre-immune serum). The FV3+anti-FV3 serum and FV3+pre-immune serum complexes were added to BF-2 (teleost fibroblast) or FHM  cells. Two hours post-infection, BF-2 and FHM cells were overlaid with agarose and 48 hours later the number of plaques produced by the virus were counted and compared to the number of plaques from an FV3 only control infection. All experiments were repeated in at least 3 independent trials and mean results are shown Figure . The addFV3 replicates in a variety of cell types including cells of mammalian origin . In ordeIn order to determine if the ADE was specific to anti-FV3 serum, we challenged cells with non-specific rabbit serum to act as a competitive inhibitor of anti-FV3 serum. FV3+anti-FV3 serum (50 ng) or FV3+pre-immune serum (50 ng) complexes were allowed to form and were then added to cells along with increasing amounts  of non-specific rabbit serum. Infected cells were incubated for 2 hours and overlaid with agarose. Forty-eight hours later the plaques were counted. The addition of increasing amounts of non-specific competitive serum resulted in a reduction in ADE  was pre-incubated with rabbit anti-FV3 serum or rabbit pre-immune serum followed by the addition of FV3. FV3+anti-FV3 serum/(+/-)protein A or FV3+pre-immune serum/(+/-)protein A complexes were added to cells and plaques were counted 48 hours later. The addition of protein A to anti-FV3 serum completely abolished the ADE in BF-2 cells Figure . Note thTo determine whether FV3 ADE was complement-dependent, anti-FV3 serum and pre-immune serum were heat-inactivated to inactivate complement, or incubated with either EGTA or zymosan A. EGTA is a chelator that inhibits the classical complement pathway, while zymosan A disrupts the alternative complement pathway. Treated anti-FV3 serum or pre-immune serum was incubated with FV3 before addition to BF-2 cells. Forty-eight hours post-overlay plaques were counted. Infection by the FV3+anti-FV3 serum complexes treated with heat-inactivation, EGTA, or zymosan A did not reduce the ADE of FV3 infectivity as compared to the untreated FV3+anti-FV3 serum control Figure . RegardlThe ability of anti-FV3 serum to neutralize an infection in BGMK cells  and enhance infection in BF-2 and FHM cells  suggests that teleost cells may contain an Fc-binding component absent from BGMK cells. Since the pre-immune control serum was able to act as a competitive inhibitor Figure . No bandRanavirus genus.While ADE has been previously reported for some large DNA viruses, including herpesviruses -13 and pIn this study, anti-FV3 serum demonstrated the ability to either neutralize or enhance an FV3 infection depending on the cell line. Although the infection was less efficient in mammalian cells, FV3 exhibited the ability to enter the cell and spread as was revealed by the presence of numerous plaques post-infection. The addition of anti-FV3 serum to an FV3 infection in BGMK cells dramatically reduced plaque number and size demonstrating the ability of the anti-FV3 serum to neutralize the infection in a mammalian cell line. However, the opposite effect was seen in teleost (BF-2 and FHM) cell lines. ADE often occurs with neutralizing antibodies at sub-neutralizing concentrations and differences in the interaction between virus and antibody can lead to either neutralization or enhancement of a viral infection . Furtherin vivo, other closely related  ranavir protein -20. FV3 in vitro -23; therin vitro ,25. Howein vitro . Amphibiin vitro -29. HoweCommon mechanisms behind ADE can be dependent on either complement or FcRs. Our results suggest that complement pathways  do not play a role in the enhancement of FV3 infection by anti-FV3 serum. However, protein A eliminated any enhancement of the anti-FV3 serum suggesting the mechanism behind FV3 ADE to be FcR-dependent. Many virus families, including other DNA viruses, enhance viral infection through the binding of the Fc region of anti-viral antibodies to FcR on the surface of cells of the immune system -37. Howein vitro and in vivo currently remains elusive. For instance, ADE of dengue viruses has been well documented and extensively characterized in vitro, but in vivo studies remain unclear and controversial [in vitro studies using live fish and frogs. Ranavirus infections are spreading rapidly worldwide, however, the reasons behind this rapid spread are currently unknown and are most likely complex. While FV3 ADE has yet to be demonstrated in vivo, a strong second infection of FV3 may explain the increased severity and prevalence of ranavirus infections. Therefore, ADE may represent a potential hypothesis for the recent emergence and increased severity of ranavirus infections.Iridovirus infections of increased pathogenicity have been recently observed in several wild and cultivated fish and amphibian species ,47,48. Soversial -60. It win vitro. The anti-FV3 serum used in this study both enhanced and neutralized a viral infection depending on cell type and concentration. The mechanism behind enhancement was found to be independent of complement but dependent upon the Fc region of anti-FV3 antibodies. The addition of protein A to either anti-FV3 serum or teleost cells completely abolished ADE. This result was surprisingly because two non-immune cell lines most likely lacking FcR were used during this experiment. Our results suggests that the Fc region of anti-FV3 antibodies may promote viral entry through novel Fc binding activity on teleost cells.This study demonstrates for the first time that FV3, an iridovirus, utilizes ADE to increase infection 2 in Eagle's Minimal Essential medium with Earle's balanced salts  and 2 mM L-glutamine supplemented with 10% fetal bovine serum (FBS), 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin). Baby green monkey kidney (BGMK) cells were obtained from ATCC and were maintained in Dulbecco's modified Eagle's medium  supplemented with 7% FBS, 2 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 g/mL) at 37°C with 5% CO2. We have previously characterized an FV3 infection in BGMK cells [2.Bluegill fry (BF-2) cells were obtained from American Type Culture Collection  and were grown at 28°C in 5% COMK cells . FatheadFV3 (~50 PFU) was mixed with either rabbit anti-FV3 serum or control rabbit pre-immune serum  for a final volume of 100 μL in media and was incubated for 1 hour at 4°C. The FV3+anti-FV3 serum or FV3+pre-serum complexes were then added to BF-2, FHM, or BGMK cells grown to 90% confluence in 6-well plates. BF-2 and BGMK cells were overlaid with 2% agarose 2 hours post-infection. Forty-eight hours post-overlay cells were either stained with crystal violet (0.05%) or underwent indirect immunofluorescence and plaques were counted. FHM cells were incubated for 24 hours and indirect immunofluorescence was carried out and plaques were counted.Rabbit anti-FV3 serum  or rabbit pre-immune serum  were mixed with FV3 (~50 PFU) in a final volume of 100 μL in EMEM and were incubated for 1 hour at 4°C. Pre-immune serum was added to BF-2 cells grown to 90% confluence in 6-well dishes for final concentrations of 0 ng/μL, 0.1 ng/μL, 0.5 ng/μL, and 1 ng/μL. FV3+anti-FV3 serum or FV3+pre-immune serum complexes were added to BF-2 cells containing pre-immune serum and were incubated for 2 hours. Cells were overlaid with 2% agarose and 48 hours post-overlay crystal violet (0.05%) was added to cells and plaques were counted.Rabbit anti-FV3 serum  or rabbit pre-immune serum  were incubated with protein A  or EGTA (0.05 M) for 30 minutes at room temperature, zymosan A  for 1 hour at 37°C, or were heat-inactivated at 56°C for 30 minutes. Approximately 50 PFU of FV3 was added and the FV3+anti-FV3 serum or FV3+pre-immune serum complexes were brought up to a final volume of 100 μL with serum-free EMEM. An ADE plaque assay in BF-2 cells was then performed. Protein A (300 μg/mL) was incubated with BF-2 cells for 30 minutes at room temperature. Cells were washed several times with PBS and 50 PFU of FV3 previously incubated with 0-150 ng anti-FV3 serum or pre-immune serum for one hour at 4°C were added to the cells. An ADE plaque assay using 0 ng, 10 ng, 50 ng, 100 ng, and 150 ng of rabbit anti-FV3 serum or control rabbit pre-immune serum was then performed.®-X Phalloidin (dilution 1/40) (Invitrogen) for one hour at room temperature. Finally, cells were incubated for 2 minutes in the nucleic acid stain DAPI (Invitrogen) diluted to 300 nM in PBS. Immunofluorescence was detected using a Leica DM6000 B fluorescent microscope . Images were assembled using Adobe Photoshop CS4 .Cells were fixed for 10 minutes in 3.7% paraformaldehyde in phosphate buffer saline (PBS). Following several washes, cells were incubated in block buffer  (w/v), 50 mM Tris HCl (pH 7.4), 150 mM NaCl, 0.5% NP-40 (v/v)) overnight at 4°C. Cells were incubated with rabbit anti-FV3 serum (dilution: 1/1000) for one hour at room temperature. Cells were then incubated in FITC-conjugated goat anti-rabbit immunoglobulin G (IgG) (dilution: 1/100)  and Texas Red® 20, 3 mM KCl) containing 5% non-fat milk powder. The membrane was incubated without primary serum, rabbit pre-immune serum (dilution 1:1000), rabbit pre-immune serum pre-incubated with 300 μg/mL protein A for 30 minutes at room temperature (dilution 1:1000), or a second unrelated rabbit pre-immune serum (dilution 1:1000) for 1 hour shaking at room temperature. The membrane was washed several times then incubated for 1 hour shaking at room temperature in peroxidase-conjugated AffiniPure F(ab')2 fragment goat anti-rabbit IgG (Jackson ImmunoResearchInc.) diluted 1/10,000. The membrane was washed several times and proteins were detected by applying Chemiluminescence Reagent Plus  to the membrane as per the manufacture's protocol. The images were then viewed using a Genius2 Bio Imaging System .BGMK, BF-2, and FHM cells grown to 100% confluence in a 6-well dish were scraped into the media and centrifuged at 10,000 × g for 5 minutes. The supernatant was removed and the cells were re-suspended in Laemmli reducing buffer . Cell lyThe authors declare that they have no competing interests.HEE performed the research and helped to draft the manuscript. EP helped to perform the research. CRB conceived the study and participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript.ADE occurs during an FV3 infection. ADE occurs during an FV3 infection in (A) BF-2 and (B) FHM cells. Original plaque numbers and standard error from three individual experiments are shown.Click here for fileRabbit anti-FV3 serum neutralizes an FV3 infection in BGMK cells. Original plaque numbers and standard error from three individual experiments are shown.Click here for fileAddition of rabbit pre-immune serum to BF-2 cells inhibits ADE. Original plaque numbers and standard error from three individual experiments are shown.Click here for fileADE in FV3 is Fc-dependent. Protein A incubated with either (A) anti-FV3 serum or (B) BF-2 cells abolished ADE in BF-2 cells. Original plaque numbers and standard error from three individual experiments are shown.Click here for fileADE in FV3 is complement independent. Treatment of anti-FV3 serum with (A) heat inactivation, (B) EGTA, and (C) zymosan A did not affect ADE. Original plaque numbers and standard error from three individual experiments are shown.Click here for file


Although a sizeable budget is available for HIV/AIDS control in Thailand, there will never be enough resources to implement every programme for all target groups at full scale. As such, there is a need to prioritize HIV/AIDS programmes. However, as of yet, there is no evidence on the criteria that should guide the priority setting of HIV/AIDS programmes in Thailand, including their relative importance. Also, it is not clear whether different stakeholders share similar preferences.Criteria for priority setting of HIV/AIDS interventions in Thailand were identified in group discussions with policy makers, people living with HIV/AIDS (PLWHA), and community members ). On the basis of these, discrete choice experiments were designed and administered among 28 policy makers, 74 PLWHA, and 50 VHVs.In order of importance, policy makers expressed a preference for interventions that are highly effective, that are preventive of nature (as compared to care and treatment), that are based on strong scientific evidence, that target high risk groups , and that target both genders (rather than only men or women). PLWHA and VHVs had similar preferences but the former group expressed a strong preference for care and treatment for AIDS patients.The study has identified criteria for priority setting of HIV/AIDS interventions in Thailand, and revealed that different stakeholders have different preferences vis-à-vis these criteria. This could be used for a broad ranking of interventions, and as such as a basis for more detailed priority setting, taking into account also qualitative criteria. While the number of new HIV positive cases in Thailand decreases -3, HIV/AAlthough a sizeable budget for implementation of this plan is available - approximately US$193 million in 2008 derived from both the Thai government and the International Monetary Fund [A number of criteria can guide this priority setting process . First, It is clear that relying on a single criterion, e.g. efficiency, falls short to capture the important moral and ethical notions, and is unlikely to be acceptable for most policy makers -21 incluHowever, as of yet, there is no evidence on the criteria that should guide the priority setting of HIV/AIDS programmes in Thailand, including their relative importance. Also, it is not clear whether different stakeholders share similar preferences. It is against this background that this paper elicits preferences on the relative importance of criteria for priority setting of HIV/AIDS programmes in Thailand from policy makers, PLWHA and community members. The study uses discrete choice experiments (DCE) to elicit explicit preferences in HIV/AIDS area, and is the first study to do so in this area. The technique allows the assessment of the relative importance of different criteria that influence choice, in this case the priority setting of health interventions in HIV/AIDS control. The technique has shown promising results in a number of other disease areas in low-income settings -25. The Discrete choice experiments are a quantitative methodology for evaluating the relative importance of the different product attributes that influence consumer choice behavior . In suchWe employed DCE to determine the relative importance of criteria for priority setting, according to various stakeholders. In a DCE, respondents choose their preferred option from sets of hypothetical scenarios, each consisting of a bundle of criteria that describe the scenario in question. The criteria are constant in each scenario, but the levels that describe each criterion may vary across scenarios. Analysis of the options chosen by respondents in each scenario reveals the extent to which each criterion is important to the decision at hand ,28. RunnIn this study, we chose to explore the views of policy makers in comparison with two other groups of stakeholders, i.e. PLWHA, and community members represented by village health volunteers (VHVs).The policy makers were represented by 28 national - and province level decision makers strongly involved in health resource allocation decisions in Thailand specifically on HIV/AIDS. As a first step in the selection process, members of the National AIDS Committee were asked to participate. As a second step, they were asked to nominate other decision-makers meeting the above criterion. A total of 30 decision makers were invited, and 28 agreed to participate in the study. They were predominantly male 71.4%), and all being higher educated (bachelor degree or more)  with a minority being higher educated.The community members were represented by VHVs - these are community members who have been trained by public health providers in order to provide basic health care delivery including first aid and necessary health information to members of the village they reside in. In the selection process, we invited 100 VHVs in a semi-urban district of Samutprakan province, and out of these, 50 agreed to participate. They were predominantly female (88%), with a minority being higher educated.To define the criteria in DCE, group discussions were organized with each group of stakeholders including six representatives of that group. As an initial step, two HIV/AIDS interventions were presented. Then participants were asked to decide which intervention should be funded and reasons for the choices were discussed. The discussion was then broadened to discuss general reasons, or criteria, to fund HIV/AIDS interventions, and finally agreement was reached on a comprehensive set of criteria. Resulting criteria and associated levels from the three group discussions were compared. The final selection of criteria and levels included those that were identified by two or more discussion groups. This resulted in identification of one criterion at four levels, two criteria at three levels, and two criteria at two levels Table .1 × 32 × 22), a limited number is chosen on the basis of a fractional experimental designs catalogue produced by Hahn and Shapiro [The DCE was designed on the following principles. To avoid information overload from a full factorial of 144 possible scenarios based on identified criteria and levels  are the coefficients of the model indicating the probability of selection relative to the reference criterion level, and ε is the unobservable error term. To control for differences in attractiveness of DCE scenarios, dummies were added for scenarios to equation (1).where P is the probability of an intervention being selected by the respondents, βMarginal effects reflect the change in the probability of selection of an intervention. These were computed by taking the average difference in predicted probability of P with and without the variable, while holding the distribution of the other variables at their sample value, and then taking the sample mean of these differences.2[The relative contributions were calculated to signify the contribution of one criterion to the variation in preferences explained by the regression model and therefore describe the relative importance of the various criteria in the choice of interventions. This relative importance depends on the variation in the levels that are chosen for each of the attributes. Variation explained by the model is based on Efron R2,(2)i indicates the observed choice and 2of the above model minus Efron's R2of the model where the criterion is held constant at its sample mean. This procedure shows the contribution of criteria irrespective of the number of levels they have.where yTo test whether the decision makings on choice selections vary between perspectives, the likelihood ratio test was analyzed. At the end, as a validity check, the resulting rank ordering of attributes derived from DCE exercise was compared (presented by the relative contributions) to those derived from a simple rank ordering.This study was approved by Institute for the Development of Human Research Protections (IHRP), Ministry of Public Health, Thailand. All participants provided their written informed consent for the discussion and the interview.Table Second, PLWHA had similar preferences as policy makers with some exceptions. Most notably, PLWHA expressed a strong preference for treatment or care for AIDS patients, and the probability of selection of these interventions is 24.5% higher than treatment and care for HIV-infectious people. Moreover, they expressed a strong preference for targeting both genders rather than one gender only. Third, VHVs preferences cohered largely with that of policy makers.2, and Hosmer-Lemeshow chi-square. The likelihood ratio test presents that the preferences on the criteria of each group of stakeholders are significantly different.The different models for policy makers, PLWHA and VHVs demonstrate a good fit as indicated by the pseudo R2 indicates the overall importance of criteria  are well reflected through decisions made by policy makers. This study also highlights the differences in preferences between PLWHA and the other stakeholder groups. The preferences of the former for care and treatment may reflect self-interests, whereas the preferences of the latter may reflect preferences for the society at large.Thirdly, our findings show overlap between the ranking of criteria resulting from DCE and as obtained from simple ranking for policy makers, but less so for other stakeholders. This may indicate validity and hence usefulness of DCE for (well-educated) policy makers, but possibly less so for other (less-educated) stakeholders.Recently, a number of empirical priority setting studies have included the views of different stakeholders, such as patients and community members, besides those of policy makers . InclusiThe DCE in this study only includes the criteria that were found to overlap from the focus group discussions. The rationale for doing so was to accommodate comparability of study findings  on the one hand, while maintaining the number of criteria to a manageable number  on the other hand. However, this choice may have led to the omission of important criteria for some groups of stakeholders, and may have reduced the validity of study findings. Next studies should seek to strike a balance between comparability and validity.Our study findings are based on small sample sizes , and should therefore be interpreted with caution. This also indicates the explorative character of our study. A proper sample size calculation is difficult in the absence a prior information on the variances on the responses - we based our sample sizes on previous similar studies, e.g. in Ghana  and NepaIntervention utility can be calculated by assuming a main effects additive utility model on the basis of a linear combination of the weights of each level of all criteria . This utThe present study findings, and associated rank ordering of HIV/AIDS interventions, can be considered as general principles to prioritization of HIV/AIDS interventions in Thailand. Since the DCE design only involves a set of criteria amendable to quantification, it ignores a range of non-quantifiable considerations - e.g. ethical, political, and social concerns ,35. As sThis exploratory study has shown the feasibility of eliciting explicit preferences on the criteria for prioritization of HIV/AIDS interventions in Thailand. Further studies should refine methodological aspects, and interpret the findings in terms of the prioritization of interventions.The authors declare that they have no competing interests.SY and RB conceptualized the study. SY carried out the data collection, and drafted the manuscript. RB, ST and YT participated in the study design, made substantial contributions to the data interpretation and writing of the paper. XK participated in data analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/10/197/prepubThe DCE questionnaire. The questionnaire presents the DCE questions and explanatory notes used in the survey (10 pages)Click here for file


Somatolactin (Sl) is a fish specific adenohypophyseal peptide hormone related to growth hormone (Gh). Some species, including salmonids, possess two forms: Sl alpha and Sl beta. The somatolactin receptor (slr) is closely related to the growth hormone receptor (ghr). Sl has been ascribed many physiological functions, including a role in sexual maturation. In order to clarify the role of Sl in the sexual maturation of female Atlantic salmon , the full length cDNAs of slr, Sl alpha and Sl beta were cloned and their expression was studied throughout a seasonal reproductive cycle using real-time quantitative PCR (RTqPCR).Atlantic salmon Sl alpha, Sl beta and slr cDNAs were cloned using a PCR approach. Gene expression of Sl alpha, SL beta and slr was studied using RTqPCR over a 17 month period encompassing pre-vitellogenesis, vitellogenesis, ovulation and post ovulation in salmon females. Histological examination of ovarian samples allowed for the classification according to the degree of follicle maturation into oil drop, primary, secondary or tertiary yolk stage.The mature peptide sequences of Sl alpha, Sl beta and slr are highly similar to previously cloned salmonid forms and contained the typical motifs. Phylogenetic analysis of Atlantic salmon Sl alpha and Sl beta shows that these peptides group into the two Sl clades present in some fish species. The Atlantic salmon slr grouped with salmonid slr amongst so-called type I ghr. An increase in pituitary Sl alpha and Sl beta transcripts before and during spawning, with a decrease post-ovulation, and a constant expression level of ovarian slr were observed. There was also a transient increase in Sl alpha and Sl beta in May prior to transfer from seawater to fresh water and ensuing fasting.The up-regulation of Sl alpha and Sl beta during vitellogenesis and spawning, with a subsequent decrease post-ovulation, supports a role for Sl during gonadal growth and spawning. Sl could also be involved in calcium/phosphate mobilization associated with vitellogenesis or have a role in energy homeostasis associated with lipolysis during fasting. The up-regulation of both Sl alpha and Sl beta prior to fasting and freshwater transfer, suggests a role for Sl linked to reproduction that may be independent of the maturation induced fasting. The slr primer pair and probe were based on those used to study slr in masu salmon  form a distinct clade amongst other recently cloned non-salmonid type II ghr. Both black sea bream type I and type II ghr have been characterized in ligand binding studies as ghr, not slr, with the use of heterologous  Prl and Sl [A phylogenetic analysis of putative fish slr and ghr Fig.  reveals l and Sl . Native l and Sl . This hal and Sl . Howeverl and Sl  has estal and Sl , in contThe Salmonidae family underwent an additional whole-genome duplication event (4R) about 25–100 million years ago . This gain vitro and in vivo studies are needed to distinctly characterize both types of receptors in their binding, signaling and biological actions.Salmon slr appears to have reduced affinity for Gh, perhaps due to the lack of conserved site B involved in Gh binding, as found for sheep and rat . Salmon ci can reproduce [In salmonids, most studies link Sl to sexual maturation, as plasma Sl levels increase during sexual maturation -11 and seproduce .slr transcripts became downregulated as maturation advanced during the autumn and spring preceding ovulation. Furthermore, tissue screens have shown a relatively high expression level of slr transcripts in the gonads of 1 year old coho salmon parr, lesser only to that of liver, visceral fat and muscle [A recent study on previtellogenic oocyte maturation in coho salmon  found lid muscle  and in islr transcripts was found between oocyte oil-drop stage, vitellogenesis, spawning and post ovulation , with an ensuing down-regulation to baseline levels during ovarian post ovulation in December. The fact that Slα and Slβ transcripts reach their highest levels at the time of spawning (November) and then drop drastically to baseline levels post ovulation suggests a direct role for Slα and Slβ in the final stages of ovarian growth and spawning.In the present study, no change in ovarian tion Fig .. This lSlα and Slβ during September, October and November  stimulates hepatic vitellogenin production and mobilizes calcium and phosphate from the environment and/or internal stores to the salmonid oocytes [Another possible explanation for the observed changes in pituitary ber Fig. , could b oocytes .2 also correlates with Sl [Slα correlates positively with E2 during post ovulation while Slβ correlates positively with E2 in August when the GSI increases from 2.8% to 7.9%, and also in November during spawning and post ovulation. During the final months of oocyte maturation, Slα and Slβ correlate with both gonad weight and GSI. The largest increase in Slα and Slβ coincides with the sharpest increase in GSI starting in August, which is also the time when calcium-containing vitellogenin is rapidly incorporated into the oocytes. It is also tempting to speculate that the transient peak in Sl observed in February could signal the start of the Ca mobilization that occurs around this time (i.e. onset of exogenous vitellogenesis). The putative role of Sl in calcium mobilization could be in part mediated by PTHrP which incidentally is produced in the vicinity of somatolactotrophs [2 [2-stimulated vitellogenesis [slr expression is high in the gills (but not in the kidney) of Atlantic salmon  coincides with the shift from somatic to gonadal growth that has also previously observed in maturing coho and Atlantic salmon [On the other hand, it has long been suggested  that Sl c salmon ,8. Sl hac salmon  and likec salmon ,58-60. TSlα and Slβ transcripts takes place  and nutritional status (voluntary anorexia) during the anadromous spawning migration. However, there is no evidence in salmonids that Sl has a direct freshwater-adapting role [In May, another important transient increase in pituitary ace Fig. , prior ting role ,8,11,20 Slα and Slβ and ovarian slr transcripts and stage of gonadal development/time during a reproductive cycle. Hence it was not designed to precisely distinguish between different possible roles associated with reproductive development in salmon such as body colour transformations, feed deprivation and anorexia, gonadal growth, spawning and associated metabolic changes. More studies are therefore needed to further clarify the specific roles of Sl during sexual maturation, to elucidate specific functions related to gonadal growth and development, lipolysis, calcium and phosphate metabolisms and body coloration.The current study is based on correlations between expression of pituitary Slα during gonadal growth and down-regulation during post ovulation, and shows for the first time that Slβ expression also changes before, during and after spawning. The increases of both Slα and Slβ during late vitellogenesis and spawning, and downregulation to baseline levels after ovulation strongly suggests an important role for Sl either directly or indirectly related to reproductive functions in salmon. Among these functions, Sl could be involved in plasma calcium and/or phosphate regulation, lipid metabolism or the regulation of maturationally induced fasting as well as have a direct effect on gonadal development and spawning.In summary, the aim of the study has been to sequence and phylogenetically characterize Atlantic salmon Slα, Slβ and slr, and to elucidate their potential role in sexual maturation of female Atlantic salmon. The study confirms an increased mRNA expression of The authors declare that they have no competing interests.SB carried out the cloning, phylogenetic analysis, gene expression studies and drafted the manuscript. EA carried out the design of the study, sampling and gene expression studies. GLT carried out the design of the study, fund-raising and sampling. BThB carried out the design of the study and fund-raising. All authors helped to draft the manuscript and read and approved the final version.


The evaluation of academic research performance is nowadays a priority issue. Bibliometric indicators such as the number of publications, total citation counts and h-index are an indispensable tool in this task but their inherent association with the size of the research output may result in rewarding high production when evaluating institutions of disparate sizes. The aim of this study is to propose an indicator that may facilitate the comparison of institutions of disparate sizes.h) of an institution over the h-index anticipated for that institution on average, given the number of publications (N) it produces i.e. α and β denote the intercept and the slope, respectively, of the line describing the dependence of the h-index on the number of publications in log10 scale). MII values higher than 1 indicate that an institution performs better than the average, in terms of its h-index. Data on scientific papers published during 2002–2006 and within 36 medical fields for 219 Academic Medical Institutions from 16 European countries were used to estimate α and β and to calculate the MII of their total and field-specific production.The Modified Impact Index (MII) was defined as the ratio of the observed h-index . The MII was positively associated with the average number of citations/publication , the h-index , the number of publications with ≥ 100 citations  but not with the number of publications . It was the most highly associated indicator with the share of country-specific government budget appropriations or outlays for research and development as % of GDP in 2004 (r = 0.229) followed by the average number of citations/publication (r = 0.153) whereas the corresponding correlation coefficient for the h-index was close to 0 (r = 0.029). MII was calculated for first 10 top-ranked European universities in life sciences and biomedicine, as provided by Times Higher Education ranking system, and their total and field-specific performance was compared.From our biomedical research data, the slope The MII should complement the use of h-index when comparing the research output of institutions of disparate sizes. It has a conceptual interpretation and, with the data provided here, can be computed for the total research output as well as for field-specific publication sets of institutions in biomedicine. As a result, it combines information on both the number of papers and the number of citations. However, due to its inherent association with the size of the research output it may result in rewarding institutions with high production [Bibliometric indices are an indispensable tool in evaluating the research output of individuals and institutions. Recently, novel indicators have been proposed with the aim to overcome deficiencies of the "traditional" bibliometric indices  and to combine more efficiently information on both the quantity and the quality of the research output -4. H-indoduction . Thus, w5 papers, the dependence of the h-index on the size of the set is characterised by a "universal" growth rate [Recenlty, it has been shown that when evaluating sets of publications ranging from several hundreds to 10wth rate . This wawth rate  as well wth rate  have proIn the present study we aim to extend the interpretation of the h-index by proposing a size-corrected, h-index based indicator (Modified Impact Index – MII). The concept of this index is to assess whether the h-index of an institution deviates from the average h-index, as estimated for a particular number of publications. MII shares all the merits of the impact index. Additionally, we will show that it has a more informative numerical interpretation and, with the data that we will provide in the following sections, it may be used also in the case of smaller publication sets. We will illustrate the use of this index in biomedical research and explore its application within specific biomedical disciplines.α and slope β of the line describing the dependence of h-index on the number of publications  were obtained through least-squares estimation.The Academic Medical Institutions located in 16 European countries  were identified from the database of medical schools provided by the Institute for International Medical Education [h: h-index and N: number of publications. As Molinari and Molinari have shown in their paper [β of 0.4 estimated when accumulating data on h-index over time is similar to the slope of the regression line obtained from cross-sectional data . Thus, we used the latter approach and estimated the impact index of papers published within 2002–2006 using the slope β obtained from our data on 219 institutions.The impact index of each institution was calculated as ir paper , the sloTo illustrate our findings, we used the rankings provided by Times Higher Education to select top-ranked European universities in life sciences and biomedicine .When the h-index of each institution was plotted against the corresponding number of papers from 36 medical fields on a log-log plot, the resulting points were fitted by a regression line Figure :(1)hi and Ni the h-index and the number of publications of the ith institution, respectively, α and β the intercept and the slope of the regression line and εi the ith residual. The estimated α and β were 0.207 and 0.445, respectively. The parameter β = 0.445 governing the dependence of h-index on the number of publications in biomedical research was found to be similar to that estimated in other disciplines (≈0.4). The number of publications ranged from 102 to 104 papers, with the exception of one institution with very low number of publications. The exclusion of this institution did not alter the estimated slope. Our estimate for β in biomedical sciences was consistent among different countries  between the observed log10 hi  expresses the deviation εi of the observed h-index of the ith institution from the average estimate for the number of publications it produces. In the original scale, this difference is transformed into the ratio h-index is higher than that estimated by the regression model based on the number of publications. Thus, a value higher than 1 indicates that the particular institution performs better in terms of h-index than it would be expected for the number of publications it produces. Similarly, a value lower than 1 indicates that the particular institution performs worse in terms of h-index than it would be expected for the number of publications it produces. The ratio 10 scale:The fitted regression line of equation 1) provides the average h-index for a particular number of publications. Thus, points above the regression line correspond to institutions with h-index higher than the average. Similarly, points below the regression line correspond to institutions with h-index lower than the average. The difference log providesMolinari  multipliVar(MII) was estimated to be equal to 0.013475.In the original scale We explored the validity of the MII by examining its association with other indices. The MII was positively associated with the average number of citations/publication , the h-index , the number of publications with ≥ 100 citations  but not with the number of publications . We further examined whether the country-specific modified impact indices  correlated with the share of government budget appropriations or outlays for research and development (GBAORD) as % of GDP in 2004 (GBAORD are a way of measuring government support to R&D activities) . The MIIβ may not be necessarily equal to 0.445 as the range of the evaluated sets of publications lies on a much lower scale. The evaluated sets of publications per field for the years 2002–2006 ranged from less than 10 papers to several hundreds (on average up to 500 papers) as opposed to the range of 102–104 papers for all 36 subjects. Some fields were characterized by a small range in the number of publications (e.g. Anatomy with range over 219 institutions up to 76 papers) and others reached thousands .When evaluating the MII or the impact index of an institution within a specific medical field, the value of the slope α and β were estimated  . Howeveroduction  and the oduction  gives moh.The proposed index deals with the fact that the inherent association of the h-index with the size of the research output may result in rewarding high production when evaluating institutions of disparate sizes. By definition, the h-index cannot exceed the number of publications. Thus, as noted by Glanzel  "it putsβ that characterises the dependence of h-index on the number of publications was approximately 0.4 and similar to that estimated in other disciplines . These Molinari  who haveβ the MII provides the same ranking as the impact index proposed by Molinari and Molinari [β provided here can be used to calculate the impact index of institutions in biomedical research and within specific biomedical disciplines. Both indices have the advantage that they can be well estimated by using a representative subset of the publications rather than the total set of publications produced by an institution [The proposed index correlated with the share of government budget appropriations or outlays for research and development as % of GDP in 2004 (r = 0.229) whereas the corresponding correlation coefficient for the h-index was close to 0. Additionally, it was positively associated with the average number of citations/publication, the h-index and the number of highly cited papers. Furthermore, for a given α s and β s were based on data from European Medical Institutions. In order to assess whether these estimates can be used to calculate the MII for non-European institutions too, we performed a preliminary analysis to check whether the slope based on data from top-ranked US universities is similar to that obtained from the top-ranked European ones. We observed that these slopes were similar unless universities with number of publications outside the evaluated range were included (e.g. Harvard and Johns Hopkins). Thus, we advocate that the estimates provided here can be used to calculate the MII for non-European institutions, as long as their number of publications falls within the evaluated range (102–104 papers for the 36 fields).The estimates of α s and β s and thus is not as straightforward as in the case of usual bibliometric indices. As mentioned before, the parameter β has a "universal" estimate of 0.4 independent of the discipline but dependent on the size of the publication set. As a result, the estimates for the α as a "universal" estimate of  0.4 independent of the discipline but dependent on the size of the publication set. As a result, the estimates for the a s and β s, as e.g. those provided here for biomedicine, can be applied to compute the MII of an institution as long as the number of its publications falls within the evaluated range (e.g. 102–104 papers in our case). Thus, it would not be safe to use them for outliers, i.e. for institutions with productivity outside the evaluated range.Bibliometric methods have been criticised due to technical and methodological problems generally encountered when they are employed to assess the research output of a university . Furthermore, the bibliometric indices currently used appear to be related to the size of research output and thus they probably tend to favour large institutions. The proposed index presents some clear advantages compared to existing bibliometric indices: it is not associated with the size of the publication output and thus can be used to compare institutions of disparate size, it has a conceptual interpretation (performance below or above the average) and can be computed by using a representative subset of the publications rather than the total set of publications produced by an institution. However, its computation requires estimates for the In conclusion, there is a growing demand for transparent and valid evaluation of universities but any ranking is bound to give rise to controversy. The assessment of medical research performance, in particular, is a challenging task. Peer-review, the currently thought gold standard of research evaluation is usually not feasible for large-scale evaluations. For large-scale evaluative purposes, we advocate the use of a combination of bibliometric indices that will include an indicator not associated with the size of the research output. The proposed modified impact index is such an indicator that has a conceptual interpretation and with the data provided here can be computed for large as well as for small field-specific publication sets in biomedicine.MII: Modified Impact IndexThe authors declare that they have no competing interests.VS oversaw the data collection and advised on the search strategy, analysed the data and co-wrote the first and subsequent drafts. AH conceived of the study, advised on the search strategy, oversaw data analysis and co-wrote the first and subsequent drafts. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/9/33/prepub


The behavior of adolescents puts them at an increased risk for HIV and other STIs, and their knowledge about HIV/AIDS is often inadequate. An understanding of how AIDS knowledge and sexual activity co-vary among Flemish secondary school students and of how education type, specifically, affects these students is limited. This study addresses the question of whether the effects of education type on HIV/AIDS knowledge and sexual activity are independent of the socio-demographic characteristics of the students.N = 11,872), were used. Data were analyzed using multilevel logistic and Poisson regression techniques.Data from the Flemish Educational Assessment survey, which collected data from a large representative sample of third- and fifth-grade high school students (There is an indication that type of education affects both an adolescent's sexual activity and his/her AIDS knowledge; these effects prove robust for differences in socio-economic backgrounds. Students in lower status education types are more likely to be sexually active and to have poorer AIDS knowledge. The relationship between AIDS knowledge and sexual activity is, however, more complex. Although students in education types with poorer AIDS knowledge are more sexually active, within each of these groups the sexually active have better AIDS knowledge than the non-sexually active. There is also evidence of active information seeking by sexually active students, which leads to improved AIDS knowledge.These findings are consistent with the literature on the role of the educational system in the reproduction of social inequalities. Students from lower status education types are at increased sexual risk compared to those from higher status types. There is also evidence of active information seeking by sexually active students, which leads to improved AIDS knowledge. In much of Europe, the big AIDS scare is over. People see AIDS as just another chronic disease and no longer see it as a deadly threat associated with "risky" sexual behavior. As the sense of threat diminishes, so does the salience of HIV/AIDS knowledge. A recent report  complainAdolescents and young adults are at an elevated risk for STIs (including HIV) because of their sexual behavior. They tend to practice serial monogamy and are at best inconsistent condom users -8. This Several studies have shown a relationship between educational attainment and HIV/AIDS knowledge ,9. PersoBourdieu's idea of the habitus refers to usually class-based internalized dispositions for behavior . Sexual Pelleriaux  found siThe Flemish secondary education system is characterized by a "waterfall" mechanism in which poorly performing students are steered towards ever "easier," and thus lower status, programs and education types. BSO students are therefore often labeled as educational failures ,25, whicHowever, poorer AIDS-knowledge in the lower status education types not only reflects the differences in school and youth cultures, but also differences in instruction in the different types. Students in lower status education types, and in vocational education in particular, may be exposed to substantially less and poorer quality sexual education than their colleagues in general education. Where the latter receive fairly formal and academic sexual education in their biology and/or social studies classes, such approach will be less successful for the former. Not only will there be, given the cultural backgrounds of the students, greater resistance against such topics, but the school culture, the learning styles of the students and their school fatigue makes instruction much less efficient. One therefore can expect that these differences in sexual education contribute to poorer reproductive health knowledge (including AIDS knowledge) in the lower status education types.The data used here are from the Flemish Educational Assessment survey, in which a representative sample (N = 11.872) of students in the third and fifth grades of secondary school  were interviewed in 85 randomly selected schools in Flanders during the 2004-2005 school year. The data collection was approved by the Ethics Committee of the Faculty of Political and Social Sciences of Ghent University.We used a subsample of 9,400 students (79.1%), with valid responses on all variables, for the multivariate analyses. The variables responsible for most missing observations were sexual activity (13.8% missing), HIV/AIDS knowledge (8.8%), and family socio-economic status (6.2%).The measure we used for HIV/AIDS knowledge was based on Zimet's  AdolesceEducation type consists of four categories: general (ASO), artistic (KSO), technical (TSO), and vocational secondary education (BSO). Control variables included gender, minority status (majority vs. minority), socio-economic status (SES), study motivation, and grade. We defined minority students as students of other than West European ancestry . A gender by minority status interaction term was included as most of the minority students were from cultures that have norms that are considerably more restrictive regarding sexuality for girls than for boys . Three SThe results in Table On average, the respondents answered about two thirds of the HIV/AIDS knowledge items correctly. Only 1.2% of the respondents scored all items correctly, while 13% had less than half the items correct, and 1.9% did not have a single correct answer. Although a substantial minority had poor HIV/AIDS knowledge, these students showed a decent knowledge of HIV/AIDS overall. The results in Figure p < 0.050) on overall AIDS knowledge and on the myths, transmission, risk behaviors, and blood transfusions dimensions than BSO students, while TSO students scored in between (p < 0.050). The differences among the education types were less pronounced with regard only to the perceived severity of AIDS, but there as well ASO and KSO students scored significantly higher than TSO and BSO students did. We also observed that AIDS knowledge improved with age. Fifth-grade students scored significantly (p < 0.001) higher on both the overall HIV/AIDS knowledge scale and on all five subscales than third-grade students did. Girls on average had better HIV/AIDS knowledge than boys did. On the overall scales and on four of the subscales  girls scored significantly higher than boys did. They only scored worse that boys (p < 0.001) regarding the dangers of blood transfusions. Minority students had significantly poorer HIV/AIDS knowledge than majority students did. For the overall AIDS knowledge scale as well as for all subscales except severity, minority students scored significantly (p < 0.001) lower than majority students. No significant gender-minority status interaction effects were observed. Family SES was also related to AIDS knowledge. High-SES students on average scored better on all AIDS knowledge variables than middle-SES students, who in turn scored better than low-SES students did. All pairwise differences were significant at p < 0.001, except for the one between high- and middle-SES students on the severity subscale (p = 0.025). Study motivation was weakly but significantly correlated with the overall AIDS knowledge scale  and two of the subscales: myths  and blood transfusions .HIV/AIDS knowledge clearly differs by education type Table . ASO and2[p = 0.000). There was a negative relation between the students' family SES and their sexual activity. Lower SES students were not only sexually active earlier but were also more often sexually active than higher SES students (χ2[p = 0.000).Table 2 = 895.1,p = 0.003). By the fifth grade, this difference had disappeared. However, the gender differences regarding sexual activity among minority status respondents were even more interesting. While there were no meaningful gender differences regarding sexual activity among majority students, clear gender differences emerged among minority students. While in the third grade 49.5% of the minority boys reported already being sexually active, only 14.8% of the girls reported the same (p = 0.000). In the fifth grade the proportion of sexually active minority boys versus girls had risen to 59.7% and 25.5%, respectively, (p = 0.000). These results clearly indicate the different standards for minority (mainly Muslim) boys versus girls. We also observed a small but significant negative correlation between study motivation and sexual activity .Overall, over the two grades there were a few differences regarding sexual activity based on gender or minority status (results not shown). Boys were somewhat more likely to start their sexual career early than girls were. For boys in the third grade, 22.2% reported being sexually active compared to only 18.8% of girls (s = 4.58) and 15.23 (s = 4.46) (pdiff = 0.000). Except for contagion, we found similar results for the various subscales (results not shown). This finding, however, is largely an artifact, due to the mix of third- and fifth-grade students, the latter not only being more sexually active but also having better knowledge about AIDS. When we split the sample by grade (Table p = 0.000) better AIDS knowledge  than non-sexually active ones. However, as the proportion of sexually active students was higher in the lower status education types, and in BSO in particular, and the latter also tended to have poorer AIDS knowledge, an overall negative association between sexual activity and AIDS knowledge emerged. This negative association is compensated for, only to an extent, by better knowledge about AIDs by sexually active students within each of the education types. The AIDS knowledge of sexually active BSO students, however, was still significantly lower (at p < 0.000) than that of the non-sexually active students in the other education types. The relationship between AIDS knowledge and sexual activity, therefore, is quite complex. On the one hand, there are groups with poorer AIDS knowledge that are more sexually active, but within each of these groups the sexually active have better AIDS knowledge than the others.Overall, sexually active respondents were observed to have better HIV/AIDS knowledge than non-sexually active ones. Their mean scores on the total AIDS knowledge scale were, respectively, 15.70 (BSO/ASO = 5.48). KSO and TSO students also remained more likely to be sexually active, but for them the effect was more moderate .Because cluster sampling methods were used to collect the data and the dependent variables were either dichotomous or counts, multilevel logistic and Poisson regression techniques were used for the multivariate analysis. The findings in Table The AIDS knowledge of students in KSO, TSO, or BSO also remained significantly poorer than that of ASO students, even after controlling for the other variables in the model. Again, BSO students performed worst; their total AIDS knowledge was on average only 85% of that of otherwise similar ASO students. For TSO and KSO students, total AIDS knowledge was only 93% and 91%, respectively. For the various subscales, a similar pattern was seen, except that the difference between KSO and ASO students was no longer significant for most of them, due to the small number of KSO students in the sample.That fifth-grade students were more sexually active and had better AIDS knowledge than those in the third grade does not come as a surprise. Girls were also more likely to be sexually active and to have better AIDS knowledge. Minority boys, but not girls, also had a significantly higher probability of being sexually active. Minority students also had significantly poorer AIDS knowledge than majority ones. On the subscales, this effect was significant only for the myths and transmission scales, but the effects on the other scales were in the same direction. No gender-minority status interaction effect was detected regarding AIDS knowledge.The socio-economic status of the students, controlling for the other variables, had little impact on whether or not they were sexually active. The socio-economic background of the students, however, had a more important effect on AIDS knowledge. The lower the SES of the student, the poorer his or her AIDS knowledge was. Study motivation was found to negatively affect not only the likelihood of being sexually active, but also AIDS knowledge.The type of education a Flemish adolescent attends affects not only the likelihood of being sexually active, but also his or her knowledge about AIDS. These effects prove robust when controlling for other socio-demographic characteristics of the respondents. Students in lower status education types and those in BSO in particular are more inclined to have sex early and tend to have poorer AIDS knowledge than ASO students do. However, the exact nature of the relationship between sexual activity and AIDS knowledge remains unclear as within each type of education sexually active students have better AIDS knowledge than non-sexually active ones, while the likelihood of being sexually active is highest among the groups with the poorest AIDS knowledge.HIV/AIDS knowledge may also be seen as a proxy for general reproductive health knowledge. The observation that youths who start their sexual careers earlier have, on average, poorer reproductive health knowledge than those who delay the onset may indicate that the former group is at increased risk, not only for HIV/AIDS, but also for other STIs and for unwanted pregnancy. On a more positive note, we find that within each type of education the sexually active students have better AIDS knowledge than non-active ones. This may indicate active information seeking on the part of sexually active students or may indicate an increased receptiveness in these students to reproductive health messages, since this information becomes more personally relevant. For students who are not sexually active, knowledge about reproductive health matters may be less pertinent.The analyses show that education type has a distinct effect on both the sexual behavior of the respondents and on their knowledge about AIDS. This effect of education type is not reducible to the differences in the backgrounds of the students. The education type reinforces influences from the students' backgrounds. BSO students therefore are at a double risk, from their background, as they are recruited disproportionally from minority and low-SES families, and from their school environment. This finding is consistent with the literature on the role of the education system in the reproduction of social inequalities -24. SchoA limitation of this study is that it is based on self-report and that a social desirability bias may affect the results for sexual activity. For instance, Muslim communities strongly curtail sexuality, especially for females, which may have lead to underreporting or non-response on this variable. Another limitation is the use of cross-sectional data. This makes it difficult to establish causality, especially with regard to the relation between AIDS knowledge and sexual activity, but also regarding whether the relationship with education type is due to self-selection or socialization. For instance, we have no information on the students' reproductive health knowledge in general and AIDS knowledge in particular prior to entering high school. One can expect this knowledge to be related to overall academic performance in elementary school and thus also to the tracking in the different education types. Both the knowledge and the tracking are also related to the background characteristics of the children, mainly SES and ethnicity. For the backgrounds of the students we controlled in the multivariate analysis, and to the extent that differences in knowledge levels and academic performance contribute to the tracking of students, they form constituent parts of the specific cultures that develop in the various education types.This study emphasizes that the type of education affects both the onset of sexual activity and AIDS knowledge, and that these effects prove robust for differences in socio-economic backgrounds of the students. Furthermore, the relationship between AIDS knowledge and sexual activity is quite complex and sexually active students may engage in active information seeking.Our results have implications for reproductive health education and campaigns. Flemish schools do not provide comprehensive reproductive health education but cover these issues in a range of courses. Our results suggest that schools need to develop a differentiated approach according to type of education and that they should give more attention to reproductive health education in lower status education types. Education and learning styles differ substantially among the education types. While ASO programs may present reproductive health knowledge in a more academic fashion , reproductive health classes in BSO need to be oriented more towards the everyday practices of their students. There is also evidence that the way reproductive health information is provided in schools is not popular, especially among minority teens with a Muslim background, which are disproportionally overrepresented in these lower educational tracks . FurtherThe authors declare that they have no competing interests.RVR collected the data, conducted all statistical analyses, wrote, and coordinated the paper. HB provided theoretical additions and other comments to the manuscript, and CVT provided statistical support. All authors commented on drafts, have read the revised manuscript, and have approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/30/prepub


Inpatient HIV/AIDS charges decreased from 1996–2000. This decrease was mainly attributable to treatment of HIV/AIDS patients with Highly Active Antiretroviral Therapy (HAART). This study aims to evaluate the trend in inpatient charges from 2000–2004.Rhode Island Hospital Discharge Data (HDD) from 2000 to 2004 was used. International Classification of Disease (ICD-9) diagnosis code 042–044 was used to identify HIV/AIDS admissions. The final study population included 1927 HIV/AIDS discharges. We used a multivariable linear regression model to examine the factors associated with inflation adjusted inpatient charges.We found a significant increase in inpatient charges from 2000–2004 after adjusting for length of stay (LOS), gender, age, race and point of entry for hospitalization. In addition to calendar year, LOS, gender and race were also associated with inpatient charges.HIV/AIDS inpatient charges increased after adjusting for inflation despite earlier studies that showed a decline. Our results have implications for uninsured, as well as insured HIV/AIDS patients who do not have a medical plan that covers their charges sufficiently. Future research should investigate what factors are contributing to rising inpatient charges among HIV/AIDS patients. Recent statistics show that an estimated 623,435 individuals in the United States (US) are living with HIV. HIV/AIDThe purpose of his study was to describe trends in inpatient charges in Rhode Island between 2000 and 2004 for hospital discharges where HIV/AIDS was the primary reason for admission. In addition, socioeconomic demographic and clinical variations in inpatient hospital charges among these discharges were examined.The Rhode Island Department of Health's Center for Health Data and Analysis collects Hospital Discharge Data (HDD) each year on inpatients from all 13 licensed acute care hospitals and the rehabilitation facility in Rhode Island from 2000–2004. The data reported to the Department of Health includes information on demographic data, clinical items, and hospital charges. The datInpatient charges (in dollars) were measured as the total charges of an admission to the hospital. Inpatient charges do not include professional fees, and non-covered charges. Non-covered charges are charges that patients or insurance providers are not required to pay. Emergency department charges incurred prior to admission are included in total charges. Inpatient charges were adjusted for inflation. Charges for subsequent years were deflated to 2000 dollars. This was done by taking into account the average annual percent growth in National Health Expenditures. This measure estimates the amount spent on all health services, supplies, health-related research, and construction activities in the United States during the calendar year.The independent variables included, LOS, and point of entry for hospitalization. LOS was measured in days. Point of entry for hospitalization had two categories: Emergency Room, and patients who were referred to the hospital through a physician, hospital clinic referral, or a facility referral. Specific data on income, education, and other components of SES were not available in the HDD data. Therefore, we utilized the point of entry for hospitalization variable as a proxy for socioeconomic status (SES). Previous studies have shown that patients referred through the emergency room are of lower SES when compared to patients with other types of hospital referrals  ,18. The Rhode Island HDD data from 2000–2004 were analyzed using the Statistical Analysis System . We performed a descriptive analysis of demographic and clinical characteristics by year, using one-way Analysis of Variance (ANOVA) and two-sample t-tests for continuous outcomes and Pearson's chi-square test of independence or Fisher's exact test for categorical outcomes. Fisher's exact test replaced Pearson's chi-square test when the expected cell count was less than 5 for a particular descriptive analysis. We used histograms and the Kolmogorov-Smirnov test to determine if inpatient charges and LOS followed a normal distribution. Due to the skewness of the data a natural log transformation was taken for LOS and inpatient charges. ANOVAs and t-tests were performed on the log transformed data which was subsequently back-transformed to generate the geometric means and standard errors for LOS and inpatient charges. Geometric means and standard deviations reported are likely to be smaller than normal calculations involving untransformed data. The reason for this is that the natural log transformation reduces the influence of extreme observations and outliers resulting in exponentiated geometric values that inherit the reduced influence of extreme observations. We used 2 multivariable linear regression models to compare the coefficients for different years across the models to understand how they change when LOS is controlled. The 2 models examined factors associated with inflation adjusted inpatient charges, with LOS excluded from the first model and included in the second model. Comparing the 2 models enabled us to examine how LOS mediates the effects of sociodemographic and clinical variables. The inflation adjusted inpatient charges were used as the outcome variable. The other independent variables in these models included year, age, race, gender, and point of entry for hospitalization. To meet the assumption of normality of the linear regression model, we performed a natural log transformation for inpatient charges and LOS. Regression coefficients were then exponentiated to obtain the estimated mean percentage increase in inpatient charges for each independent variable. We used a test of linear trend to examine if certain factors are related to each other linearly. All tests were two-sided with a type I error (α) of 0.05. The Bonferroni adjustment of α/k (k number of comparisons) for post-hoc ANOVA analysis was used to address the multiple comparisons problem.Among HIV/AIDS discharges with a primary diagnosis in Rhode Island from 2000–2004, 66.6% were male, 44.5% were white, 36.8% were black and 13.5% were Hispanic. The mean age of patients was approximately 42.7 ± 9.0 years. An estimated three quarters of the patients were admitted through the emergency room and the geometric mean for LOS was 4.7 ± 2.4 days. The inflation adjusted geometric mean inpatient charges over the 5 year time period was $11,480 ± 2.9.Inflation adjusted inpatient charges differed by point of entry for hospitalization in 2001, and gender from 2002–2004 (Table The test of trend showed a significantly increasing trend for inpatient charges among HIV/AIDS admissions from 2000–2004 (Table 2 = 0.0646) accounted for only 6.46% of the variance in log inpatient charges. This small R2 is primarily due to not having LOS in the model.In both multivariable models inpatient charges significantly increased over time based on the test of trend after adjusting for LOS, calendar year gender age, race and point of entry for hospitalization Table , 4. In t2 of 0.7473, showing that our model accounted for 74.73% of the variance in log inpatient charges.In the model that included LOS, compared to 2000, inpatient charges increased significantly in 2001 (11%), 2002 (31%), 2003 (45%) and 2004 (73%) respectively. LOS and other race were also found to be significantly associated with log inpatient charges in this model. For each 1 day increase in LOS, inpatient charges increased on average by 166%  after adjusting for other factors. Admissions that belonged to the other race category had a 19%  mean increase in inpatient charges compared to whites. Inpatient charges were significantly lower among blacks  and Asians  compared to whites. Males had a 10%  increase in inpatient charges compared to females in this model. The results of the multivariable model generated a RThis study found that mean inflation adjusted inpatient charges for HIV/AIDS patients in Rhode Island increased significantly from 2000 to 2004, after adjusting for LOS, gender, age, race, and point of entry of hospitalization. This increase in inpatient charges followed a significantly increasing linear trend from 2000–2004. These results indicate that HIV/AIDS charges are increasing at a faster than inflation. The variable LOS helped explain most of the variance in HIV/AIDS inpatient charges. Males had significantly higher inpatient charges than females in both multivariable models. Race was also a significant predictor of inpatient charges in the multivariable model that included LOS. However, this result was likely due to the few observations in the other race category. Some admissions in the other race category had higher than expected inpatient charges most likely enabling this finding to occur.Our findings are in contrast to the findings of studies conducted in the late 1990's, when HAART treatment led to a decrease in inpatient HIV/AIDS charges ,19,20. DIn addition to calendar year and LOS, race, and gender influenced inpatient HIV/AIDS charges. Contrary to other studies, we found that blacks and Asians had significantly lower inpatient charges than whites,21. AlsoThis study, like most studies, has limitations. The data we examined cannot account for multiple admissions. It is likely that there were multiple admissions by a single patient. Secondly, Rhode Island Hospital Discharge Data does not contain information on other key factors that are known to influence HIV/AIDS charges. For example, we are unable to determine the proportion of the HIV/AIDS charges that were attributable to medications, co-morbidities, physician charges, and other factors that may influence charges. We were unable to determine how inpatient charges differed by insurance status. This variable was excluded from the analyses based on the fact that more than 25% of the sample had missing insurance information. Another potential limitation is that a proportion of the uninsured HIV/AIDS admissions that were admitted may have decided to not receive hospital care due to lack of finances and thus they would not have the normal LOS and inpatient charges as others. Also, we are unable to attribute what percentage of these inpatient charges were due to other conditions that patients may have been treated for in addition to HIV/AIDS. Lastly, our results are not generalizable beyond Rhode Island. Based on this lack of generalizability we are unable to make inferences for the entire US based on our results.Despite these limitations our study has important implications for uninsured, as well as insured HIV/AIDS patients whose health care plan does not cover HIV/AIDS treatment adequately. As stated earlier a large portion of HIV/AIDS patients in the US are uninsured. Many of these individuals are of low SES and cannot afford to purchase an adequate health care plan. The increase in HIV/AIDS inpatient charges makes it more difficult for many of these individuals to pay for their treatment, resulting in lack of treatment options which can negatively impact morbidity and mortality rates.Charges for inpatient HIV/AIDS services seem to be rising faster than inflation. Our results suggest that LOS is associated with an increase in inpatient charges from 2000–2004 in Rhode Island however the level of LOS does not seem to fully account for this increase. Other factors seem to also be influencing this increase in inpatient charges over time. It is possible that charges for medical tests and procedures are increasing however we are unable to examine these variables due to the limitations of our data. Future research should focus on obtaining informative clinical data on these medical factors in order to determine their influence on inpatient charges over time. Also, it is essential to examine if these findings are consistent in the other parts of the US.The authors declare that they have no competing interests.All authors provided an intellectual contribution to the study. Study design: KEB, DNP; data analysis: KEB; manuscript preparation: KEB, DNP, AF; literature review: KEB, DNP; discussion: KEB, DNP, AF.The pre-publication history for this paper can be accessed here:


Tanzania is a wild orchid biodiversity hotspot and has a high prevalence of HIV/AIDS. The wild orchids in the study are endemic and protected by the Convention on International Trade in Endangered Species. Every year, however, between 2.2 and 4.1 million orchid plants consumed in Zambia are estimated as originating from Tanzania. This research examines the differences between HIV/AIDS wild edible orchid gatherers and non-HIV/AIDS gatherers with regards to the frequency of gathering, salience in naming the various orchids, gathering knowledge acquisition and perceptions regarding the current state of abundance of the edible species.Data was collected through interviews with 224 individuals in the Makete District of Tanzania close to the boarder of Zambia. Free-listings were conducted and Sutrup's Cultural Significance Index (CSI) constructed. The independent t-test was used to compare the differences in gathering frequencies between affected and non-affected gatherers. A multiple comparison of the 4 subgroups  in gathering frequencies was done with a one way ANOVA test and its post hoc test. To examine the difference between affected and non-affected gatherers difference in source of gathering knowledge, a chi square test was run.Disa, Satyrium, Habenaria, Eulophia and Roeperocharis. Ninety-seven percent of HIV/AIDS affected households state that orchid gathering is their primary economic activity compared to non-HIV/AIDS affected households at 9.7 percent. The HIV/AIDS affected gathered significantly more often than the non-affected. AIDS orphans, however, gathered most frequently. Gatherers perceive a decreasing trend of abundance of 6 of the 7 species. Gathering activities were mainly performed in age based peer groups. The results revealed a significant difference between affected and non-affected individuals in terms of their source of gathering knowledge.Forty two vernacular names of gathered orchid species were mentioned corresponding to 7 botanical species belongs to genera HIV/AIDS is related to increased reliance on the natural environment. This appears even more so for the most vulnerable, the AIDS orphaned children followed by HIV/AIDS widows. One impact of HIV/AIDS has been the creation of a large number of orphaned children. It is estimated that 12 million children in African countries are currently HIV/AIDS orphans and that the number of AIDS orphaned children under the age of 18 will increase to more than 14 million by 2015 .While gathering wild plants for both consumption and market sale is quite common among farming communities there is evidence that households affected by HIV/AIDS may utilise wild food plants as one of their survival strategies -7. Such Disa, Habenaria and Satyrium are the main ingredients of "chikanda" , including wild gathered orchids, requires not only that the interface of the ecological, biological and economic dimensions need to be taken into consideration, but that the socio-cultural dimension be taken into consideration as well . EnvironIt is now well known that the illegal trade in wild edible orchids contributes to their further endangerment yet little is understood about the gatherers themselves. Despite several scholars noting the devastating impact that HIV/AIDS has on most households and wild edible plants, no quantitative or in-depth studies have been conducted to examine differences between HIV/AIDS affected and non-HIV/AIDS affected households in terms of wild edible orchid gathering. Therefore, the aim of this study is to investigate how HIV/AIDS affected gatherers differ from non-HIV/AIDS affected gatherers. The differences investigated in this paper include gathering frequency, salience in naming gathered wild orchids, gathering knowledge acquisition and perceptions of changes in abundance. Several questions are answered in this paper: Do HIV/AIDS affected wild edible orchid gatherers gather more frequently than non-HIV/AIDS affected gatherers? Do HIV/AIDS affected orphans gather more frequently than HIV/AIDS affected adults? Is the source of wild orchid gathering knowledge different for the different groups in the study ? What are the most salient edible orchid species and what are the gatherer's perceptions regarding edible wild orchid abundance?Three villages were selected based on the communities having a prevalence of HIV/AIDS and practicing wild orchid gathering. The villages are located within Makete district in the southern highlands of Tanzania where wild orchids have a hospitable natural habitat. The Makete district, which is close to Zambia, is about 332 kilometres from the Iringa municipality, between 33°85' and 34°30' E and between 8°45 and 9°40' S  and 113 gatherers live in non-affected households (including 56 non-orphaned children).Field observations conducted to determine the types of orchids present and species diversity that confirm the diversity of orchids in the study area has been reduced due to gathering pressure is reported elsewhere in detail .Data used in this paper comes from a semi-structured in-depth questionnaire administered to 224 orchid tuber gatherers. The data selected for analysis includes the HIV-AIDS status, orphan status, wild orchids known, gathering patterns, perceived abundance of the species known to the informants, and source of orchid tuber gathering knowledge.Embedded within the larger questionnaire was a free listing task where informants were asked to list the edible orchids they know. Free listing is argued to be among the most successful initial techniques for delimiting a domain of knowledge, especially when there is a need of uncovering the most salient items ,18. It ithe cognitive salience index (CSI) was calculated using the formula S = F/(N mP)Estimation of F being the frequency of a term across the lists;N being the total number of lists (subjects);j being the rank of a term in an individual list.RThe mean position of a term (across the lists) is calculated as follows:j)/F, hence, the cognitive salience index is rewritten as:mP = , a one way ANOVA test (and its post hoc test) was run to test for statistical significance at the X2), was employed to determine if there was a significant difference (p < 0.05) in the source of gathering knowledge between the two major categories of gatherers (HIV/AIDS affected and non-HIV/AIDS affected).A chi square   mentioneis Table .Gathering households differ drastically with regard to their level of reliance on orchids Table . Ninety-While the number of wild orchids mentioned in free listing from individual gathers ranged from one to seven species, the results from the Student's t-test indicate that there is no significant difference in the number of wild orchids known between HIV/AIDS and non-HIV/AIDS affected gatherers. Nor is there any significant difference in the number of gatherers in the two kinds of households. We do find, however, a difference in the frequency of gathering per week between HIV/AIDS affected gatherers and non-affected gatherers Table .t (221) = 4.13, p = 0.00. That is, the mean number of days per week a HIV/AIDS affected individual gathers  is significantly more than a non-HIV/AIDS affected gatherer .Table Further analysis Table  using thThe in-depth interviews reveal five self-reported sources of wild orchid gathering knowledge: from parents (mother), other child gatherers, middlemen/brokers, guardians, and fellow adult gatherers Table . The majchi square test reveals that the source of gathering knowledge was significantly different for the two major categories of gatherers, HIV/AIDS and non-HIV/AIDS affected .Further analysis reveals that HIV/AIDS affected gatherers differ significantly from non-HIV/AIDS affected gatherers in terms of gathering knowledge acquisition. A Disa robusta N.E. had the highest CSI (0.51), followed by Satyrium atherstonei Rchb.f (0.37), while Eulophia schweinfrthii Kraenzl and Roeperocharis wentzeliana Kraenzl had the lowest CSI (0.0045). The majority of gatherers who knew a given species had the perception that the species abundance was decreasing except for Satyrium buchananii (Table ).The cognitive salience index and knower's perception of wild orchid abundance is shown in Table chb.f 0.3, while EHIV/AIDS orphans gather more frequently than any other category. HIV/AIDS affected adults follow in gathering frequency and lastly non-affected adults and non-affected children. There was no statistically significant difference between the number of gatherers in HIV/AIDS households and non-HIV/AIDS households, but the number of gatherers in both types of households is high. Taking the frequency of gathering into consideration and that multiple household members are engaged in gathering, the collection of edible orchid tubers appears to be an important activity for AIDS orphans and HIV/AIDS affected adults. There is a greater relative reliance on orchids among those affected by HIV/AIDS.The significant difference in gathering frequency observed between AIDS orphans and others illustrates the precarious position of AIDS orphans. These children probably have a deep need to obtain cash income and must make their own way in the world. Gathering orchid tubers requires only the input of their own labor for a relatively high return, given the market demand for the edible orchids. More than two million orchid tubers find their way illegally to the markets of Zambia annually. This dependence on wild gathered products was anticipated in high prevalence HIV/AIDS communities. Increased dependency on wild gathered products usually follows a reduction of agricultural production or shortage of labor to perform farm activities ,7. In suLocal users' perceptions are essential in ascertaining species numbers and health where quantitative data on previous management outcomes (indicators) are not available . PerceptBoth HIV/AIDS affected and non-HIV/AIDS affected gatherers have overwhelming numbers of women gatherers. Very few men participate in gathering wild orchid tubers in the communities studied as most men are interested in timber and hence leave gathering orchids to children and women. All women gatherers in the HIV/AIDS affected category are widows, while women from the non-HIV/AIDS affected category are from dual-headed households . Gender and age based division of labor in gathering is noted for many culture areas -28.Disa robusta Rendle had the highest CSI (0.51), followed by Satyrium atherstonei Rchb.f., Habenaria xanthochlora Schltr, Satyrium buchananni Schltr, Disa erubescence Rendle. The lowest CSI (0.0045) was observed in Eulophia scweinfurthii Kraenzl and Roeperocharis wentzeliana Kraenzl. Disa robusta Rendle and Satyrium atherstonei Rchb.f had not only the highest CSI, but the majority of gatherers also knew the species and perceived it to be decreasing. The two species with both the lowest CSI and percentage of gatherers who free listed the orchids where Eulophia scweinfurthii Kraenzl and Roeperocharis wentzeliana Kraenzl.A pattern emerges in the overall cognitive salience index of the seven gathered orchids. Disa erubescence Rendle and Disa robusta Rendle can no longer be easily found. As a result, orchid tubers considered to have inferior consumption quality (such as Satyrium buchananii Kranzl) are currently gathered. This finding agrees with what has been previously reported for Uganda [Irrespective of age, most gatherers in this study perceived that marketable wild orchid species are no longer found in close proximity to their communities, but are very far away from their villages, a typical indication of declining abundance. This finding corresponds with a study in Uganda, where the gathering of non-timber forest products was one of the factors for abundance decline of non-timber forest products . In the r Uganda , and ther Uganda . This isIt is our hope that in uncovering the differences in patterns between HIV/AIDS affected gatherers and non-affected gatherers we can ultimately make a contribution to understanding where future interventions might support people living with AIDS and the orchids.The authors declare that they have no competing interests.JFX-C collected the data in the three villages in the study area and analyzed the data under the supervision of LLP. JFX-C and LLP interpreted the data and drafted the framework for the paper and the discussion and conclusions. The writing of the article was a joint enterprise. All authors read and approved the final manuscript.