Cyprinus carpio L.) a prominent example. Cyprinid herpesvirus 3 (CyHV-3) has been proposed as a biological control (biocontrol) agent for invasive carp in Australia. Safety and efficacy are critical factors in assessing the suitability of biocontrol agents, and extensive host-specificity testing suggests that CyHV-3 is safe. Efficacy depends on the relationship between virus transmissibility and virulence. Based on observations from natural outbreaks, as well as the biology of virus-host interactions, we hypothesize that (i) close contact between carp provides the most efficient transmission of virus, (ii) transmission occurs at regular aggregations of carp that favour recrudescence of latent virus, and (iii) the initially high virulence of CyHV-3 will decline following its release in Australia. We also suggest that the evolution of carp resistance to CyHV-3 will likely necessitate the future release of progressively more virulent strains of CyHV-3, and/or an additional broad-scale measure(s) to complement the effect of the virus. If the release of CyHV-3 does go ahead, longitudinal studies are required to track the evolution of a virus-host relationship from its inception, and particularly the complex interplay between transmission, virulence and host resistance.Biological invasions are a major threat to global biodiversity. Australia has experienced many invasive species, with the common carp ( Cyprinus carpio L.), which now comprise up to 90% of the fish biomass in parts of the Murray-Darling Basin in south-eastern Australia [Along with climate change and habitat destruction, biological invasions represent one of the great human threats to global biodiversity . Australustralia . These iustralia .Unsurprisingly, the ecological burden imposed by carp has stimulated research into potential control measures. Key among these is the proposed release (in 2018) of cyprinid herpesvirus 3 (CyHV-3) – a double-stranded DNA virus first recognized in the late 1990s  – as a bNeoarius graeffei; eel-tailed catfish, Tandanus tandanus; [As it is critical that a biocontrol virus only affects the target host species, the species-specificity of the virus is obviously the most important safety requirement. Extensive host-specificity testing, involving a wide taxonomic range of animals, has been conducted for CyHV-3. Crucially, there is no evidence that CyHV-3 causes clinical, or histological, signs of disease in any non-target species . Nor is there evidence that the virus even replicates in any of the 14 species of fish, two species each of amphibians and reptiles, and single species of bird, mammal and invertebrate tested . Importaandanus; ) were amandanus;  occurrinandanus; , host-juandanus; , suggestandanus; .C. carpio in Australia will gradually decline, mirroring what has happened with MYXV [Biocontrol viruses need to be both readily transmissible and highly virulent, requirements that are complicated by a possible evolutionary trade-off between these two variables . It is tith MYXV , althougBiocontrol viruses will very likely select for the evolution of host resistance, which has been demonstrated in both MYXV and RHDV . It is tIt is also theoretically possible that the use of an imperfectly immunizing vaccine will lead to the evolution of progressively more virulent CyHV-3 strains. A modified-live vaccine has been employed in intensive carp aquaculture for about a decade in some countries . An inteTo use CyHV-3 as a biocontrol virus for carp in Australia it is critically important to determine the relationship between virulence and transmissibility. Although there are no published mortality figures for the years following a natural outbreak of CyHV-3, observations in wild and farmed populations of carp where CyHV-3 is endemic suggest that, following an initial high mortality, there is a rapid trend toward lower mortalities –28. AlthWhatever the nature of the relationship between transmissibility and virulence, the release of CyHV-3 undoubtedly represents a unique and exciting natural experiment that will provide the first opportunity to track, in real time, the co-evolution of both the biocontrol virus and the targeted host. Such a study could lead to major fundamental advances in our understanding of both pathogen evolution and the biological control of invasive pest species.


Lepeophtheirus salmonis) rhabdoviruses (LsRV) have been identified. The prevalence of these viruses was measured along the Norwegian coast and found to be close to 100%, and with the present lack of suitable cell lines to propagate these viruses, it is challenging to obtain material to study their host impact and infection routes. Thus, virus free lice strains were established from virus infected lice carrying one or both LsRVs by treating them with N protein dsRNA twice during development. The viral replication of the N protein was specifically down-regulated following introduction of virus-specific dsRNA, and virus-free lice strains were maintained for several generations. A preliminary study on infection routes suggested that the LsRV-No9 is maternally transmitted, and that the virus transmits from males to females horizontally. The ability to produce virus free strains allows for further studies on transmission modes and how these viruses influences on the L.salmonis interaction with its salmonid host. Moreover, this study provides a general fundament for future studies on how vertically transmitted rhabdoviruses influence the biology of their arthropod hosts.Rhabdoviruses are a family of enveloped negative-sense single-stranded RNA viruses infecting a variety of hosts. Recently, two vertically transmitted salmon louse ( The transgenic plants showed anti-viral activities, however, it was later that the requirement for double stranded RNA (dsRNA) to activate the RNAi pathway was reported3. Now it is clear that RNAi is a conserved mechanism in eukaryotic cells that can silence the gene expression of both viral and endogenous genes. Hence, it has been postulated to be a promising therapeutic method for viral diseases such as HIV, hepatitis B and the Ebola virus6.RNA interference (RNAi), sequence-specific post-transcriptional gene silencing, was initially discovered in transgenic tobacco plants expressing untranslated sense or antisense RNAs of a viral coat-protein gene8, this has been more promising in invertebrates due to the systemic nature of RNAi in many of these species. Trials in farmed shrimps have shown that muscular injection of dsRNA can be used therapeutically against yellow head virus and infectious myonecrosis virus, at least to decrease the viral load and increase survival rate of infected animals11. In insects such as the honeybee, oral treatment with dsRNA simultaneously with virus inoculation has been shown to induce resistance to the israeli acute paralysis virus (IAPV)12, and even large scale application of dsRNA against this virus has been attempted13. Also in mosquitos such as the Aedes aegypti, a vector for many human diseases like the zika and dengue virus, trials have been performed. Genetically manipulated A. aegypti expressing inverted-repeat dengue virus RNA sequences showed an increased protection against the virus15, and recently it was shown that RNAi can be induced by soaking larvae in a dsRNA solution16, important for using the method as a viral control measure.As the major challenge regarding the use of RNAi as antiviral treatment in mammals has been the delivery methodLepeophtheirus salmonis Krøyer, 1838) is a marine ectoparasitic copepod on salmonid fish, representing a severe problem for the salmon farming industry due to antiparasitic treatment resistance and the consecutive impact on wild salmonid fish20. Its life cycle consists of eight developmental stages each separated by a molt22. The two initial instars, nauplius I and II, are planktonic, whereas the next instar, the copepodid, detects and attaches to the epidermis or gill of the host fish. Here the louse passes through two chalimus and two pre-adult stages before the final molt to adult. The louse feeds on mucus, skin, and blood24; hence, lice infestation can increase the susceptibility to other pathogens as it disturbs the osmotic balance and stress the fish27. Thereby, RNAi has been used to study the function of various salmon louse genes by soaking of nauplius I larva or by injections of the larger pre-adult and adult stages32, in order to identify potential targets for new treatments and countermeasures.Salmon louse  and virus free (LsVF) salmon louse strains from a common virus infected origin by using RNA interference. Prior to this, the presence of the LsRVs in lice from or near Norwegian salmon farms was analysed, as to be able to make strains reflecting the wild population of salmon lice.Recently, two vertically transmitted salmon louse rhabdoviruses, LsRV-No9 and No127, have been identifiedThe presence of LsRV-No9 and No127 in salmon louse sampled from farmed and wild hosts was examined along the Norwegian coast in order to identify the distribution and co-occurrence of these viruses. Both viruses were present at all locations tested, and most lice carried both viruses (65%). Single infections of LsRV-No9 was observed in 25% of the lice, while 6% was positive for the No127 strain only . This knockdown success is not altogether very different from that in experiment 1 and 2. This demonstrates the importance of collecting early egg strings to establish the virus free F1 generation, before the viral level increases above the threshold for vertical transmission. This also demonstrates that it is possible to simultaneously knockdown both viruses sufficiently to produce virus free strains from a double infected origin. Interestingly, in experiment 4b, dsRNA treatment of the nauplius cohort originating from LsVF3 F0 females apparently cured all treated individuals, and the LsVF4b strain remained negative for three generations  were taken into the lab and confirmed to be positive for both LsRVs. Eggs were incubated and lice were subjected to dsRNA treatment as in previous experiments. However, in this third experiment the LsVly Table . RNAi ef2) were cohabitated with virus infected pre-adult females (LsV2) on naïve fish and later sampled as adults. After 35 days of cohabitation, three out of seven males had become marginally positive for the LsRV-No9 and eight out of 14 lice slightly positive at 56 days of cohabitation , Drosophila immigrans, and speckled wood butterflies (Pararge aegeria) have been shown to mainly transmit maternally, with only lower rates of paternal transmission seen46. The presence of highly positive females producing offspring that were either negative or slightly positive for the LsRVs indicates that the infection needs to develop beyond a threshold for vertical transfer in females before entering the eggs at some point during oocyte development. At any point in time, an adult female carry at least four batches of eggs in different phases of development. One batch are developing embryos within external egg strings, one batch of eggs undergoing vitelogenesis in the genital segment, one batch developing in the oviduct  and at least one batch of future oocytes developing within the large tubular syncytium that make up the ovary47. At 10 °C, each batch require around 9–10 days before moving to the next stage of development48. Thus, the oocytes giving rise to the egg strings collected at 56 days were formed in the ovary roughly 20–30 days earlier, at a point in time where females tested negative for the virus. Previously, high levels of both LsRV transcripts have been detected in the salmon louse ovaries33. Altogether, this suggests that the LsRV-No9 virus transmits to eggs in an early phase of oocyte development.To study transmission of LsRV-No9 from males to females, pre-adult II LsVFive Fig. , hence iing Fig. . In contL. salmonis by subjecting the lice to two dsRNA treatments during development. As the degree of horizontal transmission of LsRV-No9 through the salmon louse gut seems to be limited, the main mode of transmission is most likely vertically as previously suggested33. A low horizontal transfer rate may have been vital for the present success of RNAi mediated viral treatment in salmon louse since variability in the degree of knock-down and virus proliferation could potentially have produced a significant horizontal infection pressure increasing the virus level above the threshold for vertical transfer. The present study indicates that LsRV-No9 is, although to a lesser extent, horizontally transmitted with male sperm. This emphasizes the need to sample the first egg string before the females become re-infected by virus positive males. The fact that multiple infections appear to be common advocates the need to study transmission mechanisms in lice strains carrying several viruses. Moreover, since the LsRVs seem to be omnipresent at sites with extensive farming of salmon, further research on how these rhabdoviruses influence louse biology and the interaction between the lice and its salmonid host are of importance. As no cell lines for LsRVs cultivation are presently known, the method reported herein provide a vital fundament for further research on vertically transmitted RNA viruses in sea lice. In principle, this study also demonstrates in more general terms that it is possible to remove vertically transmitted virus from strains of arthropods by means of dsRNA treatment. This allows for experimental designs comparing virus infected and virus free strains from a common origin, reducing the genetic variability within the system observed.The present study shows that it is possible to simultaneously remove two vertically transmitted rhabdoviruses from a strain of Salmo salar) according to Hamre et al.49. The salmon were hand fed on a commercial diet, and reared in sea water with a salinity of 34.5 g/kg and a temperature of 10 °C. Eggs, nauplii and copepodids were kept in seawater from the same supply. Nauplii were obtained from hatching eggs, and kept in single wells in a flow through system49.Laboratory strains of salmon lice were maintained on farmed Atlantic salmon , Alta (LsAlta) and the fjord of Oslo (LsOslo). Moreover, two to ten lice taken from or near salmon farms in Varanger, Tana, Alta, Senja (Laksfjord), Bodø (Skjerstadfjorden), the coast of Helgeland, Bjugn, Agdenes, Romsdalen, Gulen, Hardanger, and Dirdal (Høgsfjorden) were analyzed for the presence of the two LsRVs.http://www.fdu.no/fdu/).All experimental procedures were performed in accordance with national legislation for animal welfare, and approved by the governmental Norwegian Animal Research Authority  according to the Trizol reagent protocol described by Invitrogen. Samples were either frozen at −80 °C until use, or cDNA synthesis was done directly. For real time RT-PCR, cDNA synthesis was carried out using the AffinityScript qPCR cDNA Synthesis Kit (Stratagene) according to the supplier recommendations, adding 200 ng total RNA. The cDNA samples were diluted 3 times and stored at −20 °C until use. For PCR, the qScript cDNA SuperMix (Quanta Bioscience) was used, applying 1 µg total RNA.Lice for RNA purification were stored in RNAlater (Qiagen). Total RNA from nauplius, copepodids and chalimus stages was isolated with a combined Tri reagent (Sigma Aldrich) and RNeasy (Qiagen) method, as previously described1/slope − 1) × 100 51. The salmon louse elongation factor 1 alfa (eEF1α) was used to normalize the data52, and always included in the run. When the relative differences in threshold cycle between the viral genes and the reference gene (ΔCT) and expression relative to controls (ΔΔCT) were calculated, they were transformed by the equation 2−ΔΔCT 53. If only CT values were displayed, the reference gene were always run and evaluated to be stably expressed. For detection of viral RNA in pre-adult and adult animals, one animal per sample was purified, while 10–50 animals were pooled in one sample for the copepodid and chalimus stages.For the detection of viral N protein RNA, real time RT-PCR was performed with 1x SYBR Select Master mix (Life Technologies), 500 nM gene specific primers Table  and 2 µl30, with primers listed in Table RNAi was performed as previously describedTo analyze the specificity of the knock-down, nauplius I from three females positive for both viruses were each divided into five groups. One group were not added any dsRNA (untreated) while the rest of the groups were added control dsRNA, N protein dsRNA from the No9 strain, N protein dsRNA from the No127 strain, or N protein dsRNA from both No9 and No127 strain. Samples were taken at 0, 4 and 8 days after soaking for analyzing the viral N protein RNA level.49. Each female and her offspring were tested for presence of virus to select the F0 parents for the virus free strain (LsVF), parents for the virus infected strain (LsV) were selected from the control fragment group. Altogether, four experiments were carried out, and strains established in each of the experiments were named according to the experiment number (n), LsVFn and LsVn  and hosted on 6 fish/strain. One fish from each group were sampled at 8 days post infestation (dpi), 25 dpi and 55 dpi (LsVF1a). To test whether an additional RNAi treatment at the pre-adult stage can improve the LsRV N protein knock-down in adults, a group of LsVF1 named LsVF1b were established by administering a second round of RNAi to 15 female and 12 male pre-adult lice, and putting them on naïve fish (n = 3). Surviving lice were sampled again at the adult stage (55 dpi) and their egg strings were placed in separate incubators. Copepodids were checked for presence of virus, and the F1 generation was founded based on the offspring from one F0 virus negative female and followed for two more generations.Experiment 1: LsVF2 and LsVF2 were established from the LsGulen strain and the F0 generation was hosted on 3 fish/strain. At 26 dpi, all lice were sampled and given a second round of RNAi. Subsequently, for each of the two strains, four naïve fish were infested with ten F0 females and seven F0 males and allowed to develop into adults. At 58 dpi, the lice were sampled and the first egg string collected. The F1 copepodids were analyzed for the presence of viral N protein. The LsV2 and LsVF2 strains were further propagated for three generations.Experiment 2: LsV3 and LsV3 were based on lice from the Hardanger fjord hosting both the LsRV-No9 and No127 virus strains. In this experiment both viruses were knocked down simultaneously to create the LsVF3 strain. The F0 generation of each strain was hosted on three fish respectively, and following dsRNA injection at the pre-adult I stage, ten female and male lice were put back on 3 fish/strain, and then sampled at 75 dpi when the lice carried their third egg string.Experiment 3: LsVF3 was slightly positive, two new virus free strains were made: LsVF4a based on LsV3, and LsVF4b based on LsVF3. Also note that the LsV4 F0 was based on the LsV3 F1 generation. For all three strains, ten female and eight male lice per fish were added to three fish after a second RNAi treatment at the pre-adult stage. Further, the first egg string produced by the females was sampled at 55 dpi, and the LsVF4b strain was followed  for three generations.Experiment 4: Since the F1 generation of LsVF2 and LsVF2 were used to infest two groups of naïve fish. Ten LsV2 female lice were put together with ten LsVF2 males on one fish in a single fish tank, while ten LsVF2 female lice were put on a second fish together with ten LsV2 males. After 35 days, when the females had become egg bearing adults (63 dpi), the lice were sampled and tested for virus.To study the degree of horizontal transmission of LsRV-No9, pre-adult II females and adult males from the F2 generation of LsV2 and LsVF2 F3 generation, with an equal experimental design, only this time adding eight females and eight males to each of three fish for each combination. Adult lice were sampled after 56 days (81 dpi), and the level of viral RNA was analyzed. Egg strings were collected and incubated in flow-trough incubators, and the copepodids were sampled and tested for virus by real time RT-PCR.A second transmission experiment was carried out using the LsVThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.We carefully reviewed the ethical standards of the journal and we hereby certify that the procedures used with the investigated species comply fully with those standards.


P<0.01). The sequential sampling provided the flow of rTSPY CN over six observations in 30 months and wide-spread variation of rTSPY CN was detected. Although a clear trend of the direction of change was not identifiable, the highly dynamic changes of individual rTSPY CN in aging bulls were observed here for the first time. In summary we have observed a highly variable rTSPY CN in bulls over a short period of time. Our results suggest the importance of further long term studies of the dynamics of rTSPY CN variablility.The Y-chromosomal TSPY gene is one of the highest copy number mammalian protein coding gene and represents a unique biological model to study various aspects of genomic copy number variations. This study investigated the age-related copy number variability of the bovine TSPY gene, a new and unstudied aspect of the biology of TSPY that has been shown to vary among cattle breeds, individual bulls and somatic tissues. The subjects of this prospective 30-month long study were 25 Holstein bulls, sampled every six months. Real-time quantitative PCR was used to determine the relative TSPY copy number (rTSPY CN) and telomere length in the DNA samples extracted from blood. Twenty bulls showed an altered rTSPY CN after 30 months, although only 9 bulls showed a significant change (4 significant increase while 5 significant decrease,  While copy number variations (CNVs) occur throughout the mammalian genome, the Y-chromosome is particularly interesting. Its’ appeal lies in the fact that its’ structure is rich in multicopy genes and repeats that are prone to be involved in intra-chromosomal rearrangements, thus frequently leading to CNVs . AlthougTSPY is among the most studied Y-chromosomal gene in cattle and extensive copy number variation was described among 14 different breeds, as well among individual bulls of the same breed . FurtherWe hypothesize that an individual’s TSPY CN changes over time, just as the burden of common autosomal CNVs was observed in large studies of aging populations . This stThe subjects of this prospective 30-month long study were 25 Holstein bulls of similar age maintained in the same artificial insemination centre under the same housing and feeding conditions. At the outset, these animals were young bulls entering an AI program and were not selected for research purposes. Blood samples from fifteen animals were sampled every 6 months providing 6 observations  and the additional ten bulls were collected at the beginning and end of the study . Sampling was done by veterinarians as part of routine animal health check and mandatory sampling for CFIA (Canadian Food Inspection Agency) testing, according to the Canadian Council on Animal Care and University of Guelph’s Animal Care Committee guidelines. DNA was extracted from blood samples using the standard phenol chloroform extraction and precipitation method as previously described [T method [°C for 2 min, followed by 50 cycles of 98°C for 5s, 60°C for 5s and 72°C for 10s. Melting curve was obtained between 72°C and 95°C with measurements taken every 0.2°C. All samples were run in triplicates. Primer efficiency was calculated from standard curves created for both sets of primers according to the equation E = 10−1 / slope. The rTSPY values were tested for normality with D’Agostino and Pearson, Shapiro-Wilk and Kolgomorov-Smirnov tests, then Student’s t-test was used to for statistical comparisons.Real-time quantitative PCR (qPCR) was used to determine the relative TSPY copy number (rTSPY CN) in the samples. rTSPY CN was calculated after normalization against the single copy SRY and a Calibrator sample according to the efficiency corrected ΔΔCT method . In ordeT method ,9 and liP value of < 0.01 was used to establish statistical significance.Relative telomere length were determined by the standard qPCR protocol against the single copy reference gene ZAR-1 and the Calibrator ,15. qPCRrTSPY CN was determined for all bulls at T0 and revealed extensive variation among the individual bulls . SeventeP<0.01). Of those 9 bulls, 4 (44.4%) showed a significant increase (P<0.01) while 5 (55.6%) showed a significant decrease (P<0.01) in rTSPY CN over time. Out of the 16 remaining animals, rTSPY CN of 5 bulls did not change at all in 30 months when the two end-point observations (T30-T0) are compared.The overall change in rTSPY CN during the 30-month sampling period was calculated for all 25 bulls by comparing T30 vs. T0 Figs  and 2. TTo get a detailed view of rTSPY CN variability over time, qPCR was performed on samples from 15 bulls for all six sampling time point over the 30-month period. Analysis of the rTSPY CN of these bulls revealed a highly variable rTSPY CN that nearly doubled the starting copy number (at T0) in some animals. Most animals showed an increase or decrease of rTSPY CN within six months (two consecutive observations) and 11 changed significantly. A clear trend in the direction of rTSPY CN change was not identifiable .P<0.01) in 11 bulls when compared with the calibrator  over time and we observed minimal variability between the start and end points (T30 vs T0). Thirteen bulls (87%) displayed no significant difference in RTL, while 2 bulls showed a slight, but significant increase (P<0.01). One of these two bulls showed an increased rTSPY CN (T30-T0), while the other did not.The TSPY gene is situated on the mammalian Y-chromosome and represents a unique biological model to study various aspects of genomic copy number variations, such as its dynamics in aging. Not only TSPY is one of the highest copy number protein coding gene in the mammalian genome, but its extensive individual variation was also observed . The subAlthough positive selection pressure for evolutionary CN amplification of TSPY in various species suggests male-specific function, its exact cellular role is still mainly unknown ,20. TSPYIn summary we have observed a highly variable rTSPY CN in bulls over a short period of time. Our results suggest the importance of further long term studies of the dynamics of rTSPY CN variablility and its potential association with prolongation of male fertility.S1 Fig*marks samples with significantly different rTSPY CN as compared to the calibrator sample (CALI) P<0.01.(TIF)Click here for additional data file.S2 FigP<0.01.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Successful breast conserving cancer surgeries come along with tumor free resection margins and account for cosmetic outcome. Positive margins increase the likelihood of tumor recurrence. Intra-operative fluorescence molecular imaging (IFMI) aims to focus surgery on malignant tissue thus substantially lowering the presence of positive margins as compared with standard techniques of breast conservation (ST). A goal of this paper is to assess the incremental number of surgeries and costs of IFMI vs. ST.We developed a decision analytical model and applied it for an early evaluation approach. Given uncertainty we considered that IFMI might reduce the proportion of positive margins found by ST from all to none and this proportion is assumed to be reduced to 10% for the base case. Inputs included data from the literature and a range of effect estimates. For the costs of IFMI, respective cost components were added to those of ST.The base case reduction lowered number of surgeries  by 0.22  and changed costs  by €-663 . A tornado diagram identified the Diagnosis Related Group (DRG) costs, the proportion of positive margins of ST, the staff time saving factor and the duration of frozen section analysis (FSA) as important determinants of this cost.These early results indicate that IFMI may be more effective than ST and through the reduction of positive margins it is possible to save follow-up surgeries–indicating further health risk–and to save costs through this margin reduction and the avoidance of FSA. Breast cancer is the most common cause of cancer deaths in women in Germany. 30.8% of all cancer incidence in women in 2012 were caused by the disease . The corresponding results regarding the costs are €4,695 for IFMI and €5,358 for ST, resulting in incremental costs of €-663  by linear interpolation. Results of the whole spectrum of relative risks calculated by linear interpolation are shown in Figs The most important cost drivers of the intervention are shown in the tornado diagram . BesidesRegarding sensitivity analyses compared to the base case, increasing the DRG costs leads to a downward shift of the incremental costs, the slope becomes steeper and uncertainty increases. The opposite direction of the effects can be seen when the DRG costs are decreased Fig A in .Furthermore, setting the staff time saving factor for waiting times of FSA to unity leads to a downwards shift of the incremental costs while uncertainty increases Fig C in –on the oHigher costs of Bevacizumab-IRDye800CW of €800 lead to an upward shift of the incremental costs and the confidence intervals Fig H in . In the The first case of structural sensitivity analysis describes the situation in which both re-excision of negative margins and no re-excision of positive margins are possible. In the second case further surgeries do not depend on the type of margins after the surgery. The cost scenario of the first case worsens the costliness of IFMI vs ST while the cost scenario of the second case improves it Fig J in . In the In our base case IFMI saves 0.22 surgeries per person scheduled to receive breast conserving therapy. The more the proportion of positive margins was reduced by IFMI the more surgeries could be avoided. While future trials will show stronger evidence regarding the effect of IFMI, we developed a model framework to analyze possible results at a very early stage. Results of a phase I study were used as a base case, rendering a first possible order of magnitude of the effects of IFMI on number of surgeries and costs. In order to address uncertainty, the whole range of possible margin reductions was investigated. By considering up to three operations per person to finally achieve negative margins the model also covers a wide range. For more detail, sensitivity analyses revealed the most important determinants of results, for example, the DRG costs. These influential variables indicate need for future consideration both in patient management as well as in data collection, for more accurate analysis. In structural sensitivity analysis it was shown that consideration of re-excisions for negative margins and no re-excisions for positive margins reduced incremental surgeries by about a quarter as compared to the base case.One key result, the incremental costs of IFMI vs. ST are negative for the base case, i.e. the IFMI intervention is less expensive than the strategy without IFMI, but significant only to a slightly higher level than 5%. Within the intervention, the DRG costs, the proportion of positive margins of ST, the staff time saving factor and the duration of FSA have the highest cost impact. Most of the sensitivity analyses showed significant negative incremental costs for relative risks below 0.33. Furthermore, higher costs of the molecular agent and a lower proportion of positive margins within ST were tested in a sensitivity analysis. The change in the slope for the latter indicated that the potential impact of a reduction in the share of positive margins through the application of IFMI has diminished.In the model, costs of IFMI have been assumed using data of a clinical trial. If IFMI will be applied within a daily clinical practice, costs would most likely be reduced through the higher rate of breast cancer surgeries. It is likely that e.g. costs of the contrast agent could be reduced as higher volume can be ordered from pharmaceutical companies.To reflect the additional costs of IFMI versus ST, some additions to the DRGs have been implemented in the model. Financing IFMI for daily usage in hospitals in Germany would thus most likely require a submission to the New Methods of Diagnosis and Treatment (“Neue Untersuchungs- und Behandlungsmethoden” or NUB) procedure. By this procedure, hospitals can negotiate extra reimbursement for new technologies of which the costs would reach beyond the current level of DRG reimbursement , 51. AccTo improve quality we referred to the checklist of Philips et al. . The strBecause of short term effects being most relevant a decision tree structure seemed adequate. Focusing on the surgical event, integration of the natural course of breast cancer by using a Markov-model did not seem helpful. Furthermore, the linear character of the results made it possible to construct a graph for the whole spectrum of relative risks, thus allowing for interpolation and a flexible focus of the reader on areas of results considered to be relevant.Some limitations regarding our study exist. The setting is restricted to the German context, e.g. costs of breast cancer cases are taken from the German DRG system. A direct transfer to other countries is not recommended without close consideration of the cost assumptions though the model easily allows for parameter adaptation to other contexts . Within Another restriction is that our analysis has focused on cost consequences and on number of surgeries while the impact on quality of life and thus quality-adjusted life time could not reasonably be included at this early stage.Beyond, there are more possible consequences of IFMI which are difficult to quantify. For example, reducing surgery may increase availability of time slots in operating rooms and reduce waiting times. Or, patients who can avoid multiple operations might even enjoy better prognosis due to earlier treatment while this would require evidence from future studies. Effects on final positive margins would be another issue which is difficult to address due to the lack of evidence regarding IFMI. Furthermore, false positive readings of IFMI can lead to the excision of healthy tissue or adverse reactions to the contrast agent might occur. Another complicated modeling strategy would be considering hospitals in rural areas, in which surgical efficiency is less compared to hospitals of urban areas. Another limitation, this study could not consider whether cases exist where applying the IFMI-technology could lead to more tissue removed than needed.Cost effectiveness strongly depends on staff time which can be saved by IFMI. Taking the base case relative risk of 0.33, IFMI would begin to save costs significantly, if about 2/3 of costs of surgery staff for FSA would be saved; the exact value was found between 0.66 and 0.68 depending on run of the probabilistic model. Otherwise, it would be more difficult or even impossible to save costs. For the base case a conservative assumption has been made, however, an accurate estimate would require an own representative survey of the workflow during breast surgery.Bevacizumab-IRDye800CW plays an important role within the surgical costs of IFMI. This drug can be applied for other cancer types, and optical imaging is not restricted solely to breast cancer , 55. BeiAnother area of future application of IFMI is that it seems essential in a surgical field in which re-operations are not possible or very difficult. This is especially the case for patient groups who incur a high risk of complications or even mortality when undergoing surgery .The aim of IFMI is to improve quality of life as a consequence of avoided surgeries. In this early-stage analysis, we were able to indicate ranges for the amount of surgeries saved, and the cost impacts linked to that. The model quantifies the reduction of number of surgeries for patients, an importantly beneficial effect, depending upon the reduction of the share in positive margins. Results also indicate that IFMI might lead to cost savings, especially if waiting times for the results of frozen section analysis can be saved. Key cost drivers were identified of which reduction can be considered in the further development of IFMI strategies.S1 Fig(DOC)Click here for additional data file.


Farmácia Popular Program (Popular Pharmacy Program). To analyze the costs of public pharmaceutical services compared to Aqui Tem Farmácia Popular Program (Farmácia Popular is available here) with the full costs of medicine provision by the Municipal Health Department of Rio de Janeiro. The comparison comprised 25 medicines supplied by both the municipal pharmaceutical service and Aqui Tem Farmácia Popular Program. Calculating the cost per pharmaceutical unit of each medicine included expenditure by Municipal Health Department of Rio de Janeiro with procurement (price), logistics, and local dispensation. The reference price of medicines paid by Aqui Tem Farmácia Popular was taken from the Brazilian Ministry of Health standard in force in 2012. Comparisons included full reference price; reference price minus 10.0% copayment by users; and maximum reference paid by the Ministry of Health (minus copayment and taxes). Simulations were carried out of the differences between the costs of Municipal Health Department of Rio de Janeiro with the common medicines and those potentially incurred based on the reference price of Aqui Tem Farmácia Popular. Comparison between prices paid by Aqui Tem Farmácia Popular Program for 20 medicines, regardless of reference prices. The potential costs incurred by Health Department if expenditure of its consumption pattern were based on the reference prices of Aqui Tem Farmácia Popular would be R$124,170,777.76, considering the best scenario of payment by the Brazilian Ministry of Health . The Municipal Health Department of Rio de Janeiro spent R$28,526,526.57 with 25 medicines of the common list in 2012; 58.7% accounted for direct procurement costs. The estimated costs of the Health Department were generally lower than the reference prices of the Farmácia Popular Program indicates that some reference prices could be reviewed aiming at their reduction. The difference in costs between public provision by Municipal Health Department of Rio de Janeiro and  Farmácia Popular Program (PFPB) by the Brazilian Ministry of Health (MH) in 2004Difficulties in access to medicines due to their high share in private household spending in BrazilThe program expanded into arrangements involving the public and private sectors. It has its own network of pharmacies and a public-private partnership with the retail pharmaceutical sector, both of them with or without copayment by users.Aqui Tem Farmácia Popular, increased over 750% in 2006-2013 and was responsible for the program’s geographical spread,,The number of pharmacies accredited by the private division of PFPB, called The Ministry of Health’s direct disbursement to the accredited retail network is based on a reference price (RP) per medicine. This price is valid for the entire country and comprises, in addition to the procurement price, costs related to logistics, dispensation and maintenance of pharmacies, insurance and taxes.Tribunal de Contas da União  raised the debate on the differences in prices found in public tenders compared to those in the Program. The audit identified a huge discrepancy between the RP paid by ATFP and the prices of 13 medicines purchased by the public sector in April 2010. For four of them, the difference between the RP and the average procurement price exceeded 1,000%The costs of the ATFP Program are subject to questioning within the public model for provision of access. An audit by New medicine supply arrangements in Brazil are important strategies to back policies aimed at expanding access. However, they lack data to support analyses of funding sustainability and more efficient use of public resources.Farmácia Popular Program.This study aimed to analyze the costs of public pharmaceutical services compared to A comparative study of costs (in reais – R$) of the two models of pharmaceutical services (PS), the ATFP Program and public services, in Rio de Janeiro, Southeastern Brazil, in 2012.Secretaria Municipal de Saúde do Rio de Janeiro  has a recognized track record of efficiency in the procurement of medicines,,Estratégia de Saúde da Família  reached around 40.0%, marked by recent expansion arising from the creation of new units (Clínicas da Família – Family Clinics) managed by social organizations (SO) and including pharmacies within their structureComponente Básico da Assistência Farmacêutica , and 971/2012Aqui Tem Farmácia Popular.The comparative analysis involved 25 medicines common to public municipal pharmaceutical services and the ATFP Program, according to the lists contained in Ordinances 1555/2013Calculating the cost per pharmaceutical unit (PU) of each medicine included SMS-RJ costs with procurement, logistics  and local dispensation. Administrative costs involved in procurement or loss and misplacement were not computed.Procurement costs were obtained from the price registration minutes of SMS-RJ bidding processes, published in the Municipal Official Gazette, following the methodology used by Silva and CaetanoThe source of logistics costs was the SMS-RJ outsourcing contract for this activity. The amount paid to the company was prorated to all medicines, considering the following specific adjustments: (i) monthly consumption of each medicine in PU; (ii) storage and transport space , used to monitor and evaluate the management contracts of those organizations. The computation included wages and benefit values and other labor costs such as provision, Government Severance Indemnity Fund for Employees, Instituto Nacional do Seguro Social , Programa de Integração Social  and Programa de Formação do Patrimônio do Servidor (Public Servant Savings Program).Staff costs were based on a specific SMS-RJ census of September 2012, which identified the number of personnel dedicated to pharmacy, separated by professional category – pharmaceutical and support staff  – and employment relationship . Civil servant salaries were determined from the municipal administration salary scale related to the average monthly remuneration values of the corresponding levels, including hazard pay, three-year service bonus and transportation allowance. The salaries of professionals hired by SO were obtained from the SMS-RJ internal system , material , building management expenses and other administrative costs. The SMS-RJ Family Clinics follow a standard structure and operation model. Amounts and values estimated for a FC were arbitrarily extrapolated to 201 basic units. Furniture and equipment costs were based on the lowest unit purchase price recorded in the 2012 OS INFO system and depreciated according to the Regulatory Instructions of the Brazilian Internal Revenue Service2) was used for energy, water and sewage, cleaning/maintenance, security and building maintenance; quantity of equipment for air conditioning and refrigeration; and number of network access points in pharmacies and number of logins to access Internet service providers and electronic medical record systems, respectively.An apportionment method was applied to the administrative costs of service contracts and maintenance, which cannot be individually measured by service item. Physical area = cost of medicine A in dispensation stageCdisp = Total dispensation costsTCMed A = Annual consumption of medicine A in 2012Annual consEstimate of the final cost per PU to each medicine of the SMS-RJ corresponded to the sum of the cost components of procurement, logistics and dispensation for each one of the 25 medicines.The RP of medicines in the common list paid by ATFP was obtained from Ordinance 971, in force in 2012Comparisons between the RP and the estimated cost per PU of SMS-RJ were based on the percentage variation between both of them. Three situations were considered: full RP, disregarding copayment percentages; RP minus 10.0% copayment by users; and maximum RP paid by MH minus taxes, that is, minus 4.27% taxes on the gross revenue of private pharmacies in 2011Based on the annual consumption of SMS-RJ, simulations were performed on the differences between SMS costs with each medication of the common list and those potentially incurred based on the RP of ATFP.© software was used to estimate costs and comparisons.ExcelThe study was approved by the Ethics Committees of the Institute of Social Medicine – Rio de Janeiro State University  and of SMS-RJ (Opinion 350A/2012).SMS-RJ spent R$28,526,526.57 on medications of the common list in 2012, 58.7% of which with the direct procurement of products . The aveThere was significant variation in the percentage share of cost components, especially for some specific products . For mosLogistics costs predominated in three products: ethinylestradiol + levonorgestrel (72.4%); propranolol (54.8%), and glibenclamide (52.1%). Ipratropium spray and carbidopa + levodopa showed proportionally higher dispensation costs than the others.Comparing the costs of SMS-RJ and the RP of ATFP Program, municipal costs were generally lower, regardless of the RP considered . The difCaptopril and enalapril showed percentage differences above 1,000%, with a 14 times lower cost in the municipal context, regardless of the comparison scenario with ATFP. Five other medicines also stood out favorably in the municipal dispensation, with costs at least five times lower than ATFP, even after discounting copayment and taxes: alendronate sodium, losartan, glibenclamide, atenolol and ipratropium solution .Simulations of potential costs incurred by SMS-RJ if its medicine consumption pattern were paid according to the RP of ATFP showed values of R$124,170,777.76, considering the best payment scenario by MH . SMS-RJ would spend 3.4 times more than estimated with the entire common list if it used the RP of ATFP .Comparison between the reference price paid to establishments accredited by the Brazilian Ministry of Health in the ATFP Program and the costs of public supply of common list medications by SMS-RJ, calculated per pharmaceutical unit, in general showed differences in favor of the municipal public service, which were 3.5 times greater, on average. The cost of municipal provision was lower in 20 of the 25 items in common with the ATFP Program. Considering the demand of each drug consumed in 2012 by SMS-RJ, the simulation showed that if the municipality had acquired them for the lowest RP, it would incur over R$95 million in the overall cost for the same 25 products.The ATFP Program offers medicines fully covered by CBAF for use in primary health care. Nationwide expenditure with ATFP totaled R$1,293,874,112.05 in 2012, of which approximately R$261 million were used exclusively to pay pharmacies established in Rio de Janeiro, RJFor most products, procurement costs were the highest compared to the other components investigated. A common feature of medicines with procurement costs above 70.0% of the total cost per PU was low volume purchases.The contraceptives levonorgestrel and estradiol + norethisterone are acquired centrally by MH and passed on to the municipality, with low volume purchases by SMS. A similar condition occurs with insulin, which is only purchased by the municipality when the MH supply is disrupted. Beclomethasone and budesonide, indicated for treatment of asthma and rhinitis, have little market competition due to the low number of products and manufacturers in the country and the absence of generics, and were only incorporated into SMS-RJ procurement in 2011. Benserazide + levodopa had a single manufacturer until 2012, which classified it as a unique product, hindering price negotiations.,,,Low volume purchases and less bargaining power contribute to higher prices and inefficient medicine purchases in SUSStocks improve the level of services by deploying resources needed for the production process and encouraging economies of scale in procurement, protecting against price increases and demand uncertaintySuch elements account for the more prevalent logistics costs of medicines transferred by MH for local distribution, such as ethinyl estradiol + levonorgestrel and NPH and regular insulin, and therefore tend to occupy a lot of storage space and/or demand significant human resources for frequent monthly transfers.Ipratropium bromide spray and carbidopa + levodopa showed proportionally higher dispensation costs compared to the others, with no clear justification observed. This is probably due to the low costs of the other components, increasing dispensation proportionately. Low volume purchases, extremely specific use instructions, and relatively low consumption demand support this hypothesis.The differences identified in the comparisons were more favorable to costs at municipal level: SMS-RJ cost estimates resulted in prices below those of ATFP for 20 medicines. The RP paid by MH to private pharmacies is more than four times the price paid by SMS-RJ, considering procurement, logistics and dispensation costs, in eight products. These differences remain relevant even in the best payment scenario .Câmara de Regulação do Mercado de Medicamentos (Medicine Market Regulation Chamber), information on market revenue and retail trade volume of the medicines, and the average discount on the factory price of the respective medicinesThe more advantageous results of SMS persist when using the median rather than the average of the percentage differences. The RP for medicines featured in the ATFP list are established considering the factory price approved by The identified differences intensify the debate raised by the 2011 TCU report. Aggregating the other cost components produced important variations. The percentage difference of 2,507.0% between the median of the municipal cost and the RP of ATFP for the medicine captopril in the abovementioned document fell to 1,325.6% in the most favorable scenario for MH. A similar reduction trend occurred with enalapril: from 1,937.0% to 1,325.6%.Despite the comparative reductions, the RP paid by MH are still 14 times higher than prices paid by SMS. These angiotensin-converting enzyme inhibitors, recommended for treating systemic high blood pressure and congestive heart failureA similar investigation carried out by CarraroAlthough the percentage differences are systematically favorable to estimated municipal costs per PU in both studies, there was a greater disparity in SMS-RJ (3.5 to 28.6 times lower). City size, volume purchases, epidemiological profile and local health system structure, including pharmaceutical services, may have contributed to these findings.Simulations based on medicine consumption in SMS-RJ in 2012 and expenditures estimated by the lowest RP of ATFP also showed savings in municipal provision, with the difference in overall costs exceeding R$95 million if prices of the ministry program had been used. Items with a RP lower than the estimated municipal cost have low consumption and, analyzed in an integrated way, have a small impact and do not reverse the municipal advantage. The savings correspond to more than three times the amount of federal transfers  from CBAF to SMS-RJ.,There are other debates related the concurrence of the two pharmaceutical services provision models: the size and overlapping of lists of medicines available from SUS units and ATFP accredited retail pharmacies, which lead to service duplication; lack of use of public sector purchasing power; concentration of the Brazilian Ministry of Health as the key player in supplying medicines for basic care, which is primarily provided at municipal levelThe advantages of municipal provision found in this study cannot, however, be generalized to other local arrangements and realities. The estimated costs concern a single location. The city of Rio de Janeiro and SMS-RJ have singularities that are not necessarily nor frequently reproduced in Brazilian areas where approximately 80.0% of Brazilian municipalities have up to 30,000 inhabitants. The size of the population and the health service network favor the volume of purchases and increase the city’s power of negotiation. Coupled with the relatively well-structured management of pharmaceutical services, they can potentially reduce costs and maximize efficiency. Finally, other unobserved factors such as the complex logistics in a continental-size country and the different sales taxation schemes of states and municipalities also have repercussions.The study was limited to a single year, providing a static picture in time. It may not have picked up some relevant cost elements of the municipal pharmaceutical chain that might have emerged in a longer study period, such as seasonality, prescription profile changes, etc.Administrative costs directly involved in the procurement process and those from loss and misplacement were not included. The former are difficult to be individualized in the pharmaceutical services management chain, especially because the human resources involved in the administrative process of medicine procurement also engage in other procurement activities. This prevents time estimates or the definition of an apportionment criterion capable of identifying this element. Moreover, in general there is only one bidding process per year. The literature on losses is scarce and national statistics are unavailable. Medicine procurement in SMS-RJ is by electronic trading with price registration, with on-demand delivery based on consumption, enabling smaller stocks and minimizing losses from product expiry. Misplacements are covered by insurance provided in the logistics contract, which was included in the specific component costs. The health units have a technical accountability structure and dispensation rules that allow control and reduce misplacement.This study reinforces the importance of costs in analyzing SUS policies, in particular those related to pharmaceutical services and the provision of medicines by the government. The pharmaceutical services model of the ATFP Program is based on the logic of medicine consumption as a promoter of access, with no emphasis on matters related to costs compared to large public medicine purchasers, who must consider their financial sustainability. The difference in costs between the public provision of SMS-RJ and ATFP indicates that some of the reference prices could be reduced, when compared to prices paid by SMS, which are systematically more favorable.


The aim of this study is to evaluate the efficacy of Dynesys® posterior dynamic stabilization (PDS) in the treatment of L4–S1 degenerative diseases and to assess the influence of postoperative motion on lumbar degeneration.Included in this retrospective study were patients with L4–S1 degenerative disease who underwent fusion or PDS from September 2010 to September 2014. Clinical outcomes were assessed by preoperative and postoperative visual analog scale (VAS) and Oswestry Disability Index (ODI). Preoperative and postoperative X-rays assessed range of motion (ROM) of the non-surgical and surgical levels and whole lumbar. MRI assessed degeneration of non-surgical levels.P < 0.05). However, there was a significant difference in a 6-month follow-up ODI between the two groups (P < 0.05). X-rays showed PDS patients partially maintained surgical level ROM and non-surgical level ROM increased less than in the fusion group. MRI showed adjacent segment degeneration (ASD) in both groups, and patients whose preoperative L3–4 Pfirrmann classification was higher than grade 2 had more ASD than lower than grade 2.A total of 56 consecutive patients were divided into two groups: group A, PDS, and group B, fusion. Patient demographics and baseline characteristics were similar in the two groups. In both groups, there was a significant difference between preoperative and postoperative VAS and ODI scores (PDS can maintain surgical level ROM and had less influence on whole and non-surgical level ROM. Following PDS, patients recovered faster and had a better lumbar function. It may be a better choice for multi-level lumbar degenerative diseases. Traditional posterior lumbar fusion is the primary procedure for multi-level lumbar degenerative disease . Its purBecause of improved understanding of lumbar motion, surgeons now consider how to treat lumbar degenerative disease while maintaining ROM. New materials and techniques have led to the development of many non-fusion techniques and devices: nucleus replacement, artificial disc, interspinous process devices, and posterior dynamic stabilization. Lumbar non-fusion techniques treat the disease while maintaining ROM –10. A syFew articles have reported on the efficacy of lumbar ROM and degeneration following decompression and different levels of PDS fixation –19. A syOur study focuses on L4–5 and L5–S1, the most common degenerative lumbar levels. We evaluated the change in ROM before and after L4–S1 PDS  or active hepatitis; and mental illness, drug abuse, and/or alcoholism.Inclusion criteria were ages 18–65 years, clinical symptoms consistent with lumbar degenerative disease , radiographic evidence of L4–5 and L5–S1 disc degenerative disease , history of L4–S1 decompression and fusion or Dynesys® stabilization, no symptoms caused by non-surgical levels, and at least had 2 years follow-up. Exclusion criteria were cauda equina syndrome, severe osteoporosis, dual-energy X-ray absorptiometry  All operations were performed by one experienced surgical group (primary surgeon QZ). In the PDS group, a midline incision and Wiltse approach was used according to the manufacturer’s instructions . Pedicle screws were inserted and the cord-rod constructs were installed. Dynesys® screws were fixed in such a way as to avoid influencing facet joint motion at the surgical and adjacent levels. Decompressions were performed at the levels indicated by imaging and patients’ symptoms. In the fusion group, all patients underwent L4–S1 transforaminal lumbar interbody fusion (TLIF).The visual analog scale (VAS) and Oswestry Disability Index (ODI) were performed preoperatively as well as postoperatively at 3 months, 1 year, and 2 years. X-rays and magnetic resonance imaging (MRI) were done preoperatively; both X-rays and MRI were done at 2 years postoperatively. The CT scan was done in fusion group at 6 months postoperatively, in order to confirm the interbody fused. If not, the CT scan would be done at 24 months.All radiological data was measured in the picture archiving and communication system (PACS) by two spinal surgeons. If there was dissent, the final decision was made by the corresponding author (QZ). To ensure consistency of the measurement results, the second measurement was done 4 weeks after the first one.Measured preoperatively and at 2 years postoperatively were the lumbar sagittal Cobb angle; surgical segment sagittal Cobb angle; flexion, extension, and total ROM; surgical segment flexion, extension, and total ROM; non-surgical segment ROM; and non-surgical segment interspace disc height.Sagittal Cobb angle: the angle between the superior endplates of L1 and S1 in the neutral lateral position.Surgical segment sagittal Cobb angle: the angle between the superior endplates of L4 and S1 in the neutral lateral position.flexion and extension angles were calculated by measuring the angles between the superior endplates of L1 and S1 in flexion and extension. Flexion ROM: The difference between the flexion and neutral angle. Extension ROM: The difference between the extension and neutral angle. Total ROM: The difference between the flexion and extension angle.Lumbar flexion, extension, and total ROM: First, surgical flexion and extension angles were calculated by measuring the angles between the superior endplates of L4 and S1 in flexion and extension. Surgical segment flexion ROM: The difference between the surgical flexion and neutral angle. Surgical segment extension ROM: The difference between the surgical extension and neutral angle. Surgical segment total ROM: The difference between the surgical flexion and extension angle.Surgical segment flexion, extension, and total ROM: First, L3–4 flexion and extension angles were calculated by measuring the angles between the superior endplate of L4 and the inferior endplate of L3 in flexion and extension. The difference between the flexion and extension angles was the non-surgical segment L3–4 ROM. The same procedure was followed for L1–2 and L2–3.Non-surgical segment ROM at L1–2, L2–3, and L3–4: First, L1–2 and L2–3.Non-surgical segment interspace disc height: The average of the anterior and posterior L3–4 segment disc heights. The same procedure was followed for Non-surgical segment disc degeneration: group 1, preoperative MRI Pfirrmann classification of L3–4 of ≤1.5; group 2, ≥2.Adjacent segment degeneration (ASD): Radiographic ASD (ASDeg) was defined as follows: (1) L3–4 disc height reduction more than 3 mm measured on the lateral X-ray; (2) L3 vertebral spillage of more than 3 mm compared with the preoperative on the lateral X-ray or the ROM of L3–4 segment was more than 15°; and (3) MRI evidence of aggravation of L3–4 degeneration, including L3–4 Pfirrmann grade increasing, disc herniation, or new stenosis. And the clinical ASD (ASDis) was defined as follows: (1) At least, the L3–4 segment had one of ASDeg and also had new clinical symptoms such as low back pain or lower extremity radiating pain.The following definitions were used:t test was used to assess patient age, body mass index (BMI), and follow-up time. Chi-square/Fisher’s test was used to assess gender. Independent sample t test was used to assess patient VAS and ODI. Independent sample t test was used to assess sagittal Cobb angle; surgical segment sagittal Cobb angle; flexion, extension, and total ROM; surgical segment flexion, extension, and total ROM; non-surgical segment ROM; and interspace disc height. A probability (P) value of <0.05 was considered statistically significant.All data were analyzed using SPSS 19.0 statistical software . All data are presented as the mean ± standard deviation (SD). In both treatment groups, 2 in group A compared to 25.03 kg/m2 in group B. Patient demographics and baseline characteristics for group A are shown in Table A total of 56 patients met inclusion and exclusion criteria. There were 27 patients in group A (PDS) and 29 patients in group B (fusion). Mean patient age, follow-up time, and BMI were similar in the two treatment groups. Mean patient age was 48.28 years in group A compared to 50.10 years in group B. Mean follow-up time was 28.78 months in group A compared to 29.90 months in group B. Mean BMI was 24.64 kg/mP < 0.05). However, in both groups, there was no significant difference in pain relief between the postoperative and final follow-up. In addition, in the two treatment groups, there was no significant difference in preoperative, postoperative, or follow-up VAS values (P > 0.05). However, there was a significant difference in ODI values between the two groups at 6 months follow-up (P < 0.05), but no difference preoperatively or at 2 years follow-up (P > 0.05) . However, the PDS group maintained ROM postoperatively. Comparing the two groups, there was a significant difference in postoperative whole lumbar ROM and flexion ROM (P < 0.05). Postoperative X-ray showed surgical segment ROM decreased in the fusion group, and there was a significant difference between preoperative and postoperative surgical segment ROM at 2 years follow-up (P < 0.05). However, the PDS group partly maintained surgical segment ROM postoperatively. Comparing the two groups, there was a significant difference in postoperative surgical segment extension ROM, flexion ROM, and whole lumbar ROM (P < 0.05) (Table X-rays showed 5) Table .Table 3SNon-surgical segment ROM increased at all levels, and ROM at L3–4 mostly increased in both groups. There was a significant difference in ROM preoperatively and postoperatively in the fusion group (P < 0.05), but there was no difference in the PDS group (P > 0.05).Disc height also decreased at L3–4 in both groups. There was a significant difference in disc height preoperatively and postoperatively in the fusion group (P < 0.05), but there was no difference in the PDS group (P > 0.05)  ROM 2 years postoperatively. Flexion ROM (at 33% preoperatively) decreased more than extension ROM (at 70% preoperatively). However, in the fusion group, there was almost no ROM at the surgical level following surgery. Because of the loss of ROM at the surgical level, whole lumbar ROM was also affected. Whole lumbar ROM was nearly the same preoperatively as in the PDS group, but was 70% in the fusion group; flexion ROM, which was 63% preoperatively, obviously decreased.Our results showed PDS can maintain 36% of whole lumbar ROM decreased 21% following dynamic stabilization and 40% following fusion. After fusion, ROM at the surgical level was almost lost in the fusion group. The authors concluded that dynamic stabilization maintains ROM better than fusion. While the non-surgical level partly compensated for ROM, whole lumbar ROM still decreased. However, because dynamic stabilization can partly maintain ROM at the surgical level, whole lumbar ROM is less affected. Yu et al. used the midline approach, in which the para-spinal muscles were dissected from the spinous processes and facet joint, which damaged muscle function. In our study, we used the Wiltse approach, in which para-spinal muscles were split to expose the screw insert points, which protected para-spinal muscle function in order to better maintain ROM.Yang et al.  comparedBoth types of surgery affect ROM at the surgical level, but compared to fusion surgery which involves rigid fixation and fusion, dynamic stabilization results in less loss of ROM. To accommodate ROM, non-surgical levels will compensate for the loss of ROM, especially at the adjacent levels. At postoperative follow-up of L4–S1 stabilization in both groups, ROM at the non-surgical levels increased. ROM at L3–4, which compensated most for lost ROM, increased more than ROM at L1–2 and L2–3. However, ROM at L3–4 in the PDS group increased 21% less than ROM in the fusion group, which increased 37%. ROM at L1–2 and L2–3 did not increase. Wang et al.  showed RSimilar to extremity joints such as the knees, the spinal joints will degenerate faster if exercise frequency and intensity are increased. Consequently, degeneration of the discs and ligaments of the adjacent levels was accelerated. Our results showed disc heights at L3–4 decreased 18.1% in the fusion group and 5% in the PDS group; however, disc heights at L1–2 and L2–3 did not decrease after surgery. These results are consistent with the increase in ROM. It is known that decrease in disc space height is a signal of disc degeneration. We concluded that PDS maintained ROM at the surgical level; therefore, ROM at the non-surgical level increased less than that following fusion, which resulted in slower degeneration than that seen following fusion.MRI showed that disc degeneration at L3–4 was worse postoperatively than preoperatively. In patients whose preoperative Pfirrmann classification was worse than grade 2, disc degeneration was faster than in the other group after both PDS and fusion surgery. In a meta-analysis by Xia et al. , 26.6% oThere are some limitations of the Dynesys® dynamic stabilization system. In the PDS group, the postoperative surgical sagittal Cobb angle was just 70% of the preoperative angle, but in the fusion group, the surgical sagittal Cobb angle was almost the same as the preoperative angle. However, in both groups, the lumbar sagittal Cobb angle maintained well postoperatively. We believe the Dynesys® system can offer great support for the spinal posterior column, but there is a lack of support for the anterior and middle columns. After the discectomy, support for the anterior and middle columns declined. Therefore, the surgical level sagittal Cobb angle decreased. The length of spacer should be measured precisely during surgery, because a long spacer may cause surgical level kyphosis.In conclusion, our results showed PDS can maintain surgical level ROM and had less influence on whole lumbar and non-surgical level ROM. The patients recovered faster and had a better lumbar function. For multi-level lumbar degenerative diseases, PDS could reduce trauma and decrease fusion segments. It may be a better choice in the clinic.


The flaviviruses are small single-stranded RNA viruses that are typically transmitted by mosquito or tick vectors. These “arboviruses” are found around the world and account for a significant number of cases of human disease. The flaviviruses cause diseases ranging from mild or sub-clinical infections to lethal hemorrhagic fever or encephalitis. In many cases, survivors of neurologic flavivirus infections suffer long-term debilitating sequelae. Much like the emergence of West Nile virus in the United States in 1999, the recent emergence of Zika virus in the Americas has significantly increased the awareness of mosquito-borne viruses. The diseases caused by several flaviviruses have been recognized for decades, if not centuries. However, there is still a lot that is unknown about the flaviviruses as the recent experience with Zika virus has taught us. The objective of this review is to provide a general overview and some historical perspective on several flaviviruses that cause significant human disease. In addition, available medical countermeasures and significant gaps in our understanding of flavivirus biology are also discussed. The intent of this review is to provide a broad-brush overview of the flaviviruses, some of their historical highlights and to identify significant gaps in our understanding of these very interesting viruses. The diversity in arthropod-vectors, reservoir species and diseases caused in humans is unlike any other virus family. The recent outbreaks of Zika, yellow fever and Usutu viruses should highlight the potential impact of the flaviviruses to human health while the continuing challenges of dengue, Japanese encephalitis and tick-borne encephalitis reminds us of the number of people at risk for infection with one of these viruses.Flaviviridae; genus Flavivirus; type species yellow fever virus, Asibi strain) that are associated with infection of mammals, there are two main groupings of viruses: those transmitted by ticks and those transmitted by mosquitoes. The tick-borne flaviviruses are a closely related, monophyletic group consisting of a single “serocomplex”, despite distinct differences in the disease caused by representative viruses. The mosquito-borne viruses are far more diverse, consisting of the Japanese encephalitis virus (JEV) serocomplex, yellow fever virus (YFV) and members of the four dengue virus (DENV) serotypes, among many others. Evaluations of phylogenetic divergence times indicate the origins of the flaviviruses go back around 100,000 years to a common ancestor with the split between mosquito- and tick-borne flaviviruses around 40,000 years ago [Flaviviridae in 1984 based on differences in structure, gene sequence and replication strategy [Togaviridae. In addition, several viruses have been identified as “non-vectored” flaviviruses and a number of insect-specific flaviviruses (ISFs) have also been discovered [In the current classification of flaviviruses  and generally associated with neurotropic viruses, and those transmitted by Aedes spp. mosquitoes and more closely associated with viscerotropic or hemorrhagic disease in humans. The recent discovery that Zika virus (ZIKV), a virus transmitted by Aedes aegypti, can cause severe neurological disease in developing fetuses may stimulate a re-evaluation of the impact of infection by the closely related DENV or YFV on pregnant women. The tick-borne flaviviruses cause of number of significant diseases in humans that are typically associated with neurological symptoms although hemorrhagic manifestations have been documented following infection with some of these viruses. The diversity of arthropod vectors, disease characteristics and the wide geographic distribution of the flaviviruses makes these viruses especially interesting, particularly if one considers that most people throughout the world live in a flavivirus endemic region. The relative ease with which some of these viruses can be introduced into new environments should also raise concerns and highlight the need for extensive additional research on these viruses, both in the lab and in the field.A number of flaviviruses are considered major human pathogens  and the As far back as the mid-1600s the “Black Death” or “Blood Vomit” (Xekik in Mayan) was a known disease that afflicted people primarily in port cities throughout the Caribbean and in the Americas. Outbreaks of this disease were documented in cities in Europe, particularly along the Mediterranean coast where the disease was thought to be imported from Africa. Although evidence of a yellow fever (YF)-like disease was reported in Hispaniola in 1495 . OutbreaIn 1897 the Yellow Fever Commission was established to investigate the origins of YF in Cuba. Previous work by J.C. Nott and Carlos Finlay had suggested that mosquitoes may be a means of transmitting the disease YF between people . MembersBacillus icteroides or Leptospira icteroides) was the transmissible element causing YF as some studies identified bacteria in cultures taken from YF patients. It was not until 1928 when Stokes and colleagues, in a seminal series of studies, identified a “filterable” agent as the transmissible component of YF infection that the concept of a “yellow fever virus” was understood [The concept of “viruses” was not understood at the time of the Yellow Fever Commission. Instead, the prevailing hypothesis was that a bacterium  were transmitted in the same manner was recognized in the 1800s; Dr. William Smart noted “…there were those who attributed its (dengue) diffusion to a widely spread so-called ‘epidemic constitution of the atmosphere’, such as was at the same period maintained to be the sole cause of epidemic yellow fever” . It was o vector ,25,26,27o vector . In whatf dengue .Using “cross-immunity” and “dermal neutralization” tests in addition to “intracerebral neutralization” tests with sera from convalescent human volunteers, Sabin and Schlesinger demonstrated that there were at least two different “immunological types” of dengue virus . SubsequPeople at risk for dengue disease inhabit tropical and subtropical regions around the world with an estimated 40% of the global population at risk for DENV infection and 390 million cases annually . DENV inThere have been a number of studies evaluating the role of antibody dependent enhancement (ADE) and its role in development of severe dengue disease. The prevailing hypothesis is that the presence of low-affinity, cross-reactive, non-neutralizing antibodies from a primary DENV infection  will enhance DENV infection with a second serotype (heterotypic). The premise is that once the non-neutralizing antibodies bind virus, they then bind a cell presenting an Fc receptor to facilitate virus entry into that cell. The fact that the virus is not “neutralized” in the antibody-virion complex allows the virus to release viral RNA and leads to a productive infection. The occurrence of ADE may be exacerbated by the occurrence of “original antigenic sin” wherein the response of T cells to secondary DENV infection may increase the potential for severe disease . For morEpidemics of encephalitis had been noted in Japan as far back as 1871. In 1924, an outbreak of encephalitis that affected 6000 people and killed 60% of those affected, gave rise to a disease called Japanese B summer encephalitis . The ageThere are an estimated three billion people who live within areas of 24 countries impacted by JEV . The annCulex spp. mosquitoes in an enzootic cycle that includes pigs and birds [JEV is transmitted by nd birds ,61. Pigsnd birds . The invInfection with JEV causes an acute non-specific febrile illness that consists of rapid onset with headache, myalgia, diarrhea and vomiting. In some patients, the disease can be complicated by neurological signs including opisthotonus, acute flaccid paralysis, convulsions, mental confusion, mask-like facies and cogwheel rigidity . Severe Ixodes persulcatus was identified as the vector for TBE [Ix. ricinus tick. Over the course of outbreak investigations, a third variant of TBEV was identified and termed the Siberian subtype of TBEV. This virus, transmitted by Ix. persulcatus, caused a disease that was of intermediate severity between far-eastern TBEV and its European relative. Genetic analysis of far-eastern TBEV, Siberian and central European encephalitis virus demonstrated that these viruses were closely related not only serologically and in the clinical disease they caused, but also genetically [Tick-borne encephalitis (TBE) was first recognized in 1932 as a severe neurological disease that occurred in forest workers in the far eastern Soviet Union (now Russia). In 1936 the Soviet Union established an exploratory expedition to determine the source and cause of this disease. As a result of this expedition,  for TBE . In 1937 for TBE . A simil for TBE . Western for TBE . Westernetically ,69. The etically .Peromyscus leucopus, did not develop disease when infected with POWV and did not develop a sufficient viremia to directly infect feeding ticks [The TBEV are maintained in a life cycle that includes their tick hosts and small rodents upon which the ticks feed. While TBEV can be maintained in tick-populations by trans-stadial and trans-ovarial transmission , horizonng ticks . Infection with TBEV-FE can cause a very severe disease following an uneventful prodrome. The disease manifests with a rapid onset, high fever, myalgia and neurological indications including headache, photophobia and clinical evidence of encephalitis or meningitis with complications including flaccid motor neuron paralysis, ascending paralysis or hemiparesis ,75. The Unlike TBEV-FE infections, the disease caused by TBEV-Eu can be relatively mild with a number of infections resulting in subclinical infections . In thosInfection by TBEV-Sib results in a disease that is described as intermediate between those caused by TBEV-FE and TBEV-Eu. However, a unique characteristic of this virus is that it has been associated with chronic infection in both humans and non-human primates, a complication not typically described for TBEV-FE or TBEV-Eu ,80,81,82Since the initial discovery of TBEV, a number of related viruses causing human disease have been identified including Omsk hemorrhagic fever virus (OHFV), Kyasanur forest disease virus (KFDV), Alkhumra hemorrhagic fever virus (AHFV) and POWV. OHFV is found in a small region near Novosibirsk in Russia , KFDV isIn August 1999, the introduction of West Nile virus (WNV) into the USA  significConcurrent with the spread of WNV in the United States was the recognition of an increase in cases of POWV or Deer Tick virus (DTV) infection , perhapsIn 2001, Usutu virus (USUV) was identified in Austria , the firThe expansion of ZIKV into the Americas has again highlighted the importance of surveillance and of disease reporting and recognition. For many months, clinicians in Brazil were reporting increases in the occurrence of microcephaly in newborns and a potential correlation with ZIKV infection. These reports were met with a healthy dose of skepticism. Now we have come to realize that there is a correlation between microcephaly and ZIKV infection and that the impact of ZIKV infection is much different from other flaviviruses. A more extensive review of ZIKV is presented elsewhere in this Special Issue.10 neutralization index of >0.7 (or a dilution titer of 1:10) [There are a number of highly efficacious vaccines for protection against flavivirus infection, including, perhaps, the best vaccine ever produced. In 1937, Max Theiler reported production of the YFV 17D vaccine . This vaof 1:10) . While tof 1:10) ,108, butof 1:10) . While tof 1:10) , vacciniof 1:10)  (Clinicaof 1:10) ,112, andof 1:10)  , and a vaccine composed of the live SA-14-14-2 virus itself that is available in China and other Asian countries [The first JE vaccine produced and introduced in Japan in 1954 was a formalin-inactivated vaccine using mouse brain homogenates from JEV-Nakayama infected mice ,115. Sinountries . An inacountries . A recenountries .®  In the 1980s, a similar European vaccine was produced using the K23 strain of TBEV and is marketed as ENCEPUR®  [® and ENCEPUR® have similar formulations and both are highly immunogenic and clinically effective [®, nor ENCEPUR® are licensed for use outside of Europe despite extensive demonstration of safety and clinical efficacy. In addition to the European vaccines, Russian scientists have generated two vaccines for use against TBEV by utilizing either TBEV-FE strain Sofjin or strain 205 [The first vaccine for TBEV was an inactivated virus vaccine that was produced shortly after the virus was discovered in 1937 and was used to vaccinate workers during the course of outbreaks . VaccineGermany) . Both Euffective ,120. Neirain 205 .®) from Sanofi-Pasteur . The CYD-TDV has been through two phase 3 clinical trials and has been approved for use in individuals aged 9–45 years living in endemic areas of Mexico, Brazil, the Philippines, El Salvador and Costa Rica [The development of a vaccine for DENV has been an ongoing effort for several decades. Vaccine development is complicated by the need for the vaccine to be protective against all four DENV serotypes. The potential for enhanced disease during secondary DENV infections could be exacerbated if a vaccine is not fully protective against all four serotypes. At the current time, there are several vaccines that show promise, including CYD-TDV  developed by Takeda Vaccines (Singapore) is based on work initiated at CDC-Fort Collins a number of years ago. DENVax is similar to the CYD-TDV vaccine, except that it uses an attenuated DENV-2 virus backbone rather than YFV to generate chimeras for DENV-1, -3 and -4 and uses the authentic attenuated virus for DENV-2 ,129. TheThe TV003 vaccine (NIAID/Johns Hopkins)  is an admixture of four recombinant DENV that have mutations in the 3′-non-coding region leading to attenuation of the viruses . Early sIn addition to the above-mentioned vaccines, there are a number of inactivated, subunit or DNA vaccines currently in development or in clinical trials. For more comprehensive reviews on DENV vaccines and the issues related to vaccine development, please see articles by Thomas and Rothman , Thomas Major milestones in flavivirus research are highlighted in There are a number of significant gaps in our understanding of flaviviruses and the diseases they cause. Comparatively, more is known about DENV due to its broad distribution, significant health impact and the lower biocontainment level (BSL-2) required for safely performing research with this virus. The recent global spread of ZIKV has provided impetus and financial support for research with this interesting virus which has allowed for significant advances in our understanding of ZIKV structure, pathogenesis and for vaccine development in a short period of time. However, many questions remain regarding fundamental components of flavivirus biology.Given the divergent diseases caused by the different flaviviruses, it is probable that there is not a single receptor for all of the viruses, but rather a family or groups of structurally similar cell surface proteins that function as receptors for these viruses. Receptor proteins probably have different functions and cell expression patterns, given that there appears to be variable target cell types between different flaviviruses. For example, most of the mosquito-borne flaviviruses will infect both Vero and C6/36 cells, while tick-borne flaviviruses do not easily infect C6/36 cells and many replicate poorly in Vero cells. Previous work has suggested that highly glycosylated molecules, such as DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), may be receptors for flaviviruses. However, interactions with molecules such as DC-SIGN appear to be low affinity interactions rather than high affinity and are not the sole component required for viral entry ,139. FurWith the exception of dengue, research efforts toward understanding of the role of T cell mediated immunity during flavivirus infection in humans has only recently become of significant interest. In addition to extensive work with dengue virus, a number of studies with WNV and TBEV have evaluated T cell responses to wild-type virus infection. The majority of work with WNV has focused primarily in mouse models ,142. WhiThe animal models routinely used for evaluation of flavivirus pathogenesis and the testing of medical countermeasures are not generally representative of disease as it manifests in humans. Mouse models for neurotropic flaviviruses develop neurological disease, but the disease is typically monophasic and lethal, depending upon virus inoculum and mouse strain, within 7–14 days. Primate models for neurotropic flaviviruses are also limited as most do not develop disease when the virus is delivered peripherally . In the Our understanding of YFV-induced pathogenesis is limited by a lack of useful animal models. As established by Stokes et al. , the rheSimilar to the case for YFV, there are a limited number of animal models for DENV that are useful for studying human disease. The rhesus macaque may be a faithful model for mild cases of dengue in that they have a productive infection with reasonably high viremia, but do not have the clinical picture that is typically seen in humans. The laboratory mouse is also a poor model for DENV pathogenesis. Since the early 2000s, several modified mouse models have been developed to address specific questions of DENV pathogenesis. These include animals that have some component of their innate immune response, specifically interferon-related, removed or are humanized mouse models. While not necessarily accurately representing human disease, each of these models provides information regarding aspects of DENV-induced pathophysiology that might be applicable to humans. For comprehensive reviews of the existing models for DENV infection see Sarathy et al.  and ChanThe discovery that ZIKV can be transmitted sexually and that virus appears to persist in semen for some time ,158 was The discovery that ZIKV could be transmitted from mother to fetus and cause neurological disease in the fetus was an alarming discovery. The mechanism the virus uses to cross the placental barrier is still being evaluated, but this virus infects a number of cells in the placenta . PreviouTo date, there are no therapeutic options for the treatment of any flavivirus infection. Supportive care is the norm and has shown some success for the treatment of flavivirus infection, particularly yellow fever and DHF/DSS where fluid loss is a critical concern due to vascular leakage. The lack of treatment options is a significant problem as is evidenced by the continuing dilemma with DENV infections, the recent outbreaks of YFV in Angola and Brazil, and the continuing spread of ZIKV. The typical approach to drug screening focuses on direct antiviral effects in cultured or primary cells. In the case of most flavivirus infections, once symptoms are apparent the virus has been cleared from the blood (no viremia), is seeded in tissues and the host immune response is a significant contributor to the disease. In order to develop successful therapeutic approaches for treatment of flavivirus infections, a combination approach utilizing antivirals and host response directed countermeasures might be required.The objective of this review was to provide a broad overview of flaviviruses and the diseases they cause. A secondary objective was to heighten awareness of the risks of flavivirus induced disease, the potential for continued spread of these viruses and the relative lack of understanding regarding the mechanisms these viruses use to cause disease. Diseases caused by the flaviviruses have been recognized for more than 200 years, but there is a lot of work yet to do before we have a proper understanding of these very interesting viruses.


Epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2 were isolated from Florida white-tailed deer in 2015 and 2016, respectively, and their genomes were completely sequenced. To our knowledge, these are the first full genome sequences for EHDV-1 and -2 from Florida. Epizootic hemorrhagic disease virus (EHDV) is a nonenveloped double-stranded RNA virus within the family Reoviridae, genus Orbivirus (Odocoileus virginianus) in the United States  and neck  cells grown as monolayers at 37°C. After cytopathic effect became apparent, vRNA was extracted from virions in spent medium using a QIAamp viral RNA minikit (Qiagen), and a cDNA library was prepared using a NEBNext Ultra RNA library prep kit. Sequencing was performed using a version 3 600-cycle kit on a MiSeq platform (Illumina). De novo assembly of the paired-end reads was performed in SPAdes  and 2016 (OV215), respectively. After EHDV viral RNA (vRNA) was detected in the blood (OV202) and spleen (OV215) specimens by reverse transcription-PCR (RT-PCR) , aliquotTo date, these are the first cases of EHDV in white-tailed deer in Florida for which complete genome sequences have been determined. Future molecular epidemiological studies across a greater number of isolates are needed to determine whether EHDV strains circulating in white-tailed deer in Florida are unique or related to other regional strains.MF688826 to MF688835 and MF688816 to MF688825, respectively.The genome sequences for EHDV-1 isolated from OV202 and EHDV-2 isolated from OV215 have been deposited in GenBank under accession no.