Chlamydia trachomatis (CT) and Neisseria gonorrheae (NG) are commonly observed in men who have sex with men (MSM). There is increasing evidence that such infections at extra-genital sites are also common in women. In both sexes, these infections are largely overlooked as they are not routinely tested for in regular care. Testing based on sexual behavior or symptoms would only detect half of these extra-genital infections. This paper elucidates the differences and similarities between women and MSM, regarding the epidemiology of extra-genital CT and NG. It discusses the clinical and public health impact of untested extra-genital infections, how this may impact management strategies, and thereby identifies key research areas.Anorectal and pharyngeal infections with Mycoplasma genitalium.Extra-genital CT is as common in women as it is in MSM; NG in women is as common at their extra-genital sites as it is at their genital sites. The substantial numbers of extra-genital CT and NG being missed in women and MSM indicate a need to test and treat more patients and perhaps different choices in treatment and partner management strategies. Doing so will likely contribute to reduced morbidity and transmission in both sexes. However, in our opinion, it is clear that there are several knowledge gaps in understanding the clinical and public health impact of extra-genital CT and NG. Key research areas that need to be addressed concern associated morbidity , ‘the best’ management strategies, including testing and treatment for extra-genital CT, extra-genital treatment resistance, transmission probabilities between partners and between anatomic sites in a woman, and impact on transmission of other infections. Data are also lacking on cost-effectiveness of pharyngeal testing, and of NG testing and anorectal CT testing in women. Gaps in the management of extra-genital CT and NG may also apply for other STIs, such Current management strategies, including testing, to address extra-genital CT and NG in both sexes are suboptimal. Comparative data on several identified key themes in women and MSM are lacking and urgently needed to guide better management of extra-genital infections. Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are sexually transmissible bacteria that may result in serious complications such as pelvic inflammatory disease, infertility and ectopic pregnancy in women, and epididymitis in men and play an important role in enhancing HIV transmission [It is a continuous challenge to control the spread of sexually transmitted infections (STIs). smission , 2. The smission . In Eurosmission .The use of highly sensitive and specific laboratory assays, i.e. nucleic acid amplification tests (NAAT), has revealed the frequent presence of CT and NG at extra-genital sites. Infections at extra-genital sites are common in both men who have sex with men (MSM) and women. In MSM, this led to specific control guidelines, including expanded testing –9. In woThis paper addresses the state of the art in extra-genital NG and CT epidemiology, elucidating the differences and similarities in women versus MSM, argues how this may impact control strategies, and identifies the knowledge gaps to address these issues to guide testing.To provide an overview on state-of-the-art on the prevalence and anatomic site distribution of CT and NG infections in women, we have reviewed the literature focusing on including studies (in English) in women describing anorectal and/or pharyngeal CT and NG infections detected using NAATs. We conducted a Medline search (last update 8 June 2015) using the terms:  and . These terms were used in combination with  or  or . Additionally, relevant results on anorectal CT that could not directly be retrieved from these papers were kindly provided by the authors of these studies. We compared results to findings in several key papers in MSM from different countries and using NAAT. Because there is an abundant literature on extra-genital infections in MSM a literature search was deemed not necessary.Prevalence, which is defined here as the proportion of the study population that tested positive, has so far for women only been assessed in clinic-based populations. The observed prevalence ranges of extra-genital CT as detected by NAAT are notably similar between women –32 and MMost studies on anorectal CT  serovars comprised 2–16 % of CT positive anorectal samples and were also found in the pharynx and urethra , 41. In The prevalence of extra-genital NG reflects the well known disparity between MSM and women , a substantial part have a concurrent infection at the anorectal site: between 33 % (i.e. ) and 83 In contrast, MSM with anorectal infections usually do not have a concurrent infection at the genital site. In MSM anorectal infections are mostly single site infections, i.e. up to 91 % for CT and up to 70 % for NG –39, 43. Pharyngeal CT prevalence is low in MSM and in women . The presence of anorectal infections in women without anal sex and the high rate of concurrence with infections at the genital site, fuelled the belief that anorectal infections in women are largely the result of self-infection. The anatomical proximity of genital and anorectal site makes this plausible. However, self-infection has not been confirmed by rigorous data. Also, the observed high rates of concurring genital and anorectal infections in women is predictable given the likelihood that women who have anal sex will also have vaginal sex . PossiblThe vast majority of extra-genital CT and NG are asymptomatic, i.e. without oral or anorectal symptoms reported , 2. EvenGenerally, treatments recommended for extra-genital STI do not differ from that of genital STI. For CT however, the recommended first choice treatments may differ between anorectal and genital infection. For genital CT infection azithromycin 1-g single dose is advised. For anorectal CT, azithromycin or doxycycline 100mg twice daily for seven days are considered equal first line treatments in the US. Other countries  now have doxycycline as first choice in anorectal CT –9. TheseRoutine extra-genital testing for CT and NG is possible using NAAT and minimally invasive sampling methods. However, extra-genital infections are still not routinely tested for in MSM and testing is even less common in women. As these infections are mostly asymptomatic, they are frequently overlooked in health care. Extra-genital infections, in women especially anorectal CT, are common and testing based on exposure or symptoms misses over half of these extra-genital infections. More testing in women and in MSM would identify many more extra-genital infections. But, what are the implications for clinical practice of testing more? Untreated infections might continue to cause complications and potentially drive further transmission. Still, what are the public health gains of testing more? Should control strategies be different for MSM and women? Some answers to these questions will provide us with more insight into what is needed to guide extra-genital testing in MSM and in women.A substantial proportion of infected individuals do not attend a testing facility for testing or retesting, and are obviously missed, hence remain untreated . Even soOf patients who have an anorectal infection, women frequently have a concurrent infection at the genital site while MSM frequently do not have a concurrent genital infection. This implies that adding a extra-genital test to routine genital testing would reveal much more additional infected individual MSM than it would for women, even when the prevalence is similar . This also implies that to detect large part of extra-genital infections in women we could just test women with genital infections. A strategy to achieve this, without having to ask the women to come back for extra-genital testing, could be to directly take a extra-genital sample at the routine genital STI testing visit, but to only test the extra-genital specimen from women who tested positive for their genital infection. In such case, fewer extra-genital laboratory tests would have to be performed compared to routine testing of both specimens in women, perhaps reducing costs. Yet, such phased testing strategy would also demand more complex laboratory logistics and increase time to return test results. Using this strategy implies there is a reason to detect the extra-genital infection in a women who will be treated because of her genital infection (different treatment regimen or different partner management needed?). Also, such phased testing strategy would not detect the single site extra-genital infections that still will remain undiagnosed and untreated. Another testing strategy potentially reducing costs is to test pooled multiple site samples. Such approach precludes feedback on the anatomic site(s) where the infection(s) occurs precluding treatment guidance, and its effectiveness needs to be further explored.More information on the clinical and public health impact is needed to decide what would be the optimal cost-effective algorithm for testing. This clinical and public health impact of leaving extra-genital infections untested may be different between the two sexes. This is because the co-occurrence of infections at multiple anatomic sites differs between MSM and women and may impact the need for additional management strategies. The clinical and public health impact also is dependent on the (yet unknown) role of extra-genital infections in morbidity and transmission.Further insight on these issues is essential to know how the additional costs of testing extra-genital samples and managing more identified positive cases balances the costs avoided by reducing spread and morbidity due to extra-genital testing.If treatment for genital infections is also adequate for extra-genital infections, then part of the extra-genital CT and NG would be ‘inadvertently treated’ by treating the infection at the genital site . This would most frequently apply to anorectal infections in women, as these usually co-occur with a genital infection, reducing the negative consequences (in terms of morbidity and transmission) of leaving anorectal CT and NG infections in women untested. However, this scenario may not be true for anorectal CT. There are reports suggesting that doxycycline is a better treatment for anorectal CT than is azithromycin –63. If aIn MSM, an additional contributing factor to increasing the adverse consequences of an untested extra-genital CT, is the occurrence of LGV. LGV prevalence is low, and there are marked differences in geographic distribution, but it requires additional and different testing and treatment.The introduction of NG treatment resistant strains poses a challenge for its management . So far,For CT, there is yet no evidence of homotypic anti-microbial resistance . Still, Management strategies such as re-testing, partner notification and expedited or accelerated partner treatment have been shown to be highly effective strategies to prevent relatively high numbers of CT and NG . PartnerIt also is apparent that by not testing, a single extra-genital infection would always be missed and none of the treatment, retest or partner notification strategies would be employed. Therefore, by testing and treating at genital sites only, essential control opportunities in the management of both female and MSM patients and their partners are lost.Some studies in MSM linked anorectal NG to symptomatic proctitis, which is an inflammatory syndrome of the distal 10–12 cm of the rectum. Some LGV strains cause a severe proctocolitis . In womeHypothetically, anorectal infections in women could have an impact on the reproductive outcomes. This would be if an anorectal infection could be spread to the genital site. A recent study estimated that, when self-infection from the anorectal site plays a role, a less adequate treatment would sustain CT in a woman . Thus anPharyngeal and anorectal CT and NG are probably capable of being transmitted to genital sites of a partner , 78. In In the absence of better data on the transmission potential of extra-genital infections, any extra-genital STI that remains untreated should be considered transmissible. Given that there is a large number of extra-genital CT and NG currently missed by the current standard of care , the transmission potential of such infections may be huge in women and in MSM. Infections at extra-genital sites may impact the total STI burden in the population by spread between sexual partners and even between a woman’s anatomic sites. A recent mathematical study in MSM suggest that oral sex has an important role in sustaining NG in MSM by providing a pool of untreated asymptomatic infection . If extrData on factors facilitating transmission are yet unavailable. It is for example unknown whether anorectal and pharyngeal bacterial load is associated with an increased risk for transmission, although such associations may be biologically plausible. The CT and NG load detected on pharyngeal or anorectal swabs load is generally lower than on genital swabs , 80–82. Missing extra-genital infections may also impact the transmission of other STI. In MSM, it has been shown that anorectal CT and NG facilitate HIV transmission –85. AnalMycoplasma genitalium (MG), Herpes Simplex Virus, Human Papilloma Virus (HPV) and Trichomonas vaginalis (TV) can be found at the anorectal site in a woman. Presence at the anorectal site may be correlated to their presence in the vagina, such as has been shown for MG, TV and HPV [As we have started to gain awareness of the occurrence of extra-genital CT and NG in women, we have been learning about other extra-genital STI as well. We know that  and HPV , 89–91. Extra-genital CT and NG, and perhaps other STI such as MG, may form a high potential for avoidable transmission and to some extent for avoidable morbidity in MSM and in women. Extra-genital CT and NG can be successfully detected using NAAT on simple to obtain self-taken samples. Both men and women are willing to test, and the so far hidden extra-genital STI epidemic can be revealed. Strategies can be taken to increase the reach of the testing and reduce costs by using internet based programs, e-health strategies and home-collection. Still several of the tests  and strategies (home-collection) are not FDA cleared. It is likely that, at least in the US, the needed paradigm shift from a genital-centered approach to testing (MSM and others at risk) to include a broader sampling frame, will not become routine until FDA clearance for extra-genital testing is obtained.Testing should not solely be guided by indication of symptoms or behavioral exposure as the large majority of extra-genital STI are asymptomatic and over half of the extra-genital infections occur in the absence of (report of) such symptoms or behavioural exposures in both women and MSM. The majority of extra-genital infections in MSM are single site infections that are not being ‘inadvertently managed’ by treating urethral infections. Based on the current available data routine universal testing of all MSM instead of only testing MSM who report symptoms or behavioural exposure is therefore an imperative. To date, there is no evidence that the public health impact, in terms of transmission, of anorectal or pharyngeal infections is any different for women than it is for MSM. However, in women, the clinical implications of extra-genital infections, and gains by testing these, present uncertainties. In women, whether or not to implement routine testing would depend on the increment achieved in terms of avoided morbidity and further spread. For some less prevalent STI, such as NG in some countries, the increment is likely smaller in women than MSM due to the more infrequent occurrence of these infections in women. For more prevalent STI, such as CT and perhaps MG, the increment would depend on the number extra-genital infections that could be adequately managed (by treatment and partner management) above the number of infections that are already adequately managed by standard care for the genital infection. We lack essential information in several key area’s (summarized in Table Future studies should address extra-genital and genital morbidity caused by extra-genital infections. Randomized controlled trials are needed to determine whether treatment efficacy for extra-genital infections differs from that of genital infections. The full spectrum including potential for further transmission of extra-genital infections to genital sites and related morbidity has not yet been evaluated in women. Prospective studies in couples or modeling studies could reveal more insight on what are the transmission probabilities between partners and between anatomic sites, and the associated factors. Morbidity and transmission may depend on the extent the bacteria is able to replicate or presents in high loads, which is unknown in MSM and in women. Studies using appropriate laboratory techniques, such as quantitative PCR for measuring bacterial load, and culture to detect viable bacteria and antibiotic resistance, to address these aspects are needed. These will greatly aid our understanding of extra-genital infections and the differences presented by these infections in MSM and women. Finally, cost-effectiveness of extra-genital testing has been evaluated for HIV infected MSM and was shown cost-effective in case of anorectal CT and NG , 88. WhiHidden extra-genital infections might fuel the transmission of CT and NG and other STI and thereby increase the overall STI burden. This may be very important in the context of HIV and anorectal infection. Health care providers should be aware of the common occurrence of extra-genital infections. They should be doing more and urgently need a better understanding, especially in women, to obtain the highest gains of the increased CT and NG testing and treating efforts. Increased testing is an imperative for MSM. There is much to be learned to see if the same is true for women, and possibly for some heterosexual men as well.Ethical approval was not required for this paper as it is a debate paper and not original research involving humans.


A duck tembusu virus (DTMUV) was isolated from the brain of a Cherry Valley duckling that showed neurological signs by using a specific-pathogen-free chicken embryo. The isolate was named GX2013G (GenBank accession no. KM275941). The strain GX2013G was identified with reverse transcription-PCR (RT-PCR), and the amplicon was sequenced. The genome that was obtained is 10,990 nucleotides in length and contains a single open reading frame encoding a putative polyprotein of 3,425 amino acids. This study will advance the understanding of the epidemiology and molecular characteristics of tembusu virus (TMUV) in Guangxi and further studies of the mechanisms of virus replication and pathogenesis. Tembusu virus (TMUV) was isolated and identified for the first time in 2010 in the east of China , Zhejian6–In 2013, a flock of ducklings showed neurological signs and loss of appetite in a Cherry Valley duck farm in Guangxi. Encephaledema was observed, and the brains were collected to isolate viruses by using specific-pathogen-free (SPF) chicken embryos and identified by reverse transcription-PCR (RT-PCR). A strain of TMUV was isolated, and other pathogens which cause similar symptoms were ruled out –14. Thir–Compared to genome sequences of previously isolated TMUVs from different species of ducks, geese, and chickens in various areas of China, there were 96.5% to 97.5% homology at the nucleotide level and 99.7% homology with GX2013H isolated in Guangxi (By predicting the potential glycosylation sites, we found 13 glycosylation sites in six viral proteins: 2 glycosylation sites in prM, 1 in E, 3 in NS1, 1 in NS2A, 3 in NS4B, and 3 in NS5. The numbers and positions of glycosylation sites in each protein were the same as GX2013H.In conclusion, this study of the whole-genome sequence of TMUV provides further information about the epidemiology and evolution of TMUV and may help elucidate mechanisms of virus replication and pathogenesis.KM275941.The complete genome sequence of the duck tembusu virus isolate has been deposited in GenBank under the accession no.


The term arbovirus denotes viruses that are transmitted by arthropods, such as ticks, mosquitoes, and other biting arthropods. The infection of these vectors produces a certain set of evolutionary pressures on the virus; involving migration from the midgut, where the blood meal containing the virus is processed, to the salivary glands, in order to transmit the virus to the next host. During this process the virus is subject to numerous bottlenecks, stochastic events that significantly reduce the number of viral particles that are able to infect the next stage. This article reviews the latest research on the bottlenecks that occur in arboviruses and the way in which these affect the evolution and fitness of these viruses. In particular we focus on the latest research on three important arboviruses, West Nile virus, Venezuelan equine encephalitis virus and Chikungunya viruses and compare the differing effects of the mosquito bottlenecks on these viruses as well as other evolutionary pressures that affect their evolution and transmission. Some arboviruses can also be transmitted vertically via infected eggs, or venereally, or via nonreplicative viremia to co-feeding vectors. However, for the most part, these modes of transmission are thought to be insufficiently efficient to perpetuate circulation in the absence of some degree of horizontal transmission.Arboviruses are arthropod-borne viruses that are transmitted between vertebrate amplifying hosts by mosquitoes, ticks and other biting arthropods that serve as vectors ,2. TheirAlphavirus, one of two genera in the family Togaviridae); the flaviviruses ; the bunyaviruses, nairoviruses and phleboviruses (three of five genera in the family Bunyaviridae); the orbiviruses (one of nine genera in the family Reoviridae); the vesiculoviruses (one of six genera in the family Rhabdoviridae) and the thogotoviruses (one of four genera in the family Orthomyxoviridae) [African swine fever virus (Asfarviridae: Asfarvirus), suggesting that the genetic plasticity and mutant swarm characteristics of RNA viruses are critical to maintaining the alternating vertebrate-invertebrate host cycle in nature . Traditionally, it has been hypothesized that arboviruses are constrained genetically due to the “trade-off” in fitness that they must make when adapting simultaneously for infection and replication in both kinds of hosts. This view has been partially supported by experimental and retrospective studies of host range changes [Arboviruses do not comprise a taxon because arthropod-borne transmission has evolved convergently several times, as indicated by the presence of arboviruses in several different RNA viral taxa, all of which also include non-arthropod-borne members: the alphaviruses (genus viridae) . Only on changes ,5. HowevIn addition to their taxonomic diversity, arboviruses are ecologically highly diverse, utilizing vectors in four different orders of insects  and two families of ticks (Ixodidae and Argasidae) as vectors . VertebrIn evolutionary biology, a bottleneck is a stochastic or random event that decreases the number of viable replicating organisms in a population to a much smaller number, therefore reducing the effective population size. Following a bottleneck, one of two options will occur: the population will either go to extinction or recover to a larger effective population size. The genetic diversity of the population will be reduced regardless of the outcome . This iset al. [Bottlenecks occur in all types of organisms and their populations including humans , the Euret al.  have shoCulex taeniopus and an enzootic VEEV strain associated with this mosquito showed that almost the whole midgut was infected with the virus [C. taeniopus, showed that the number of infected midgut cells was determined by the amount of virus ingested by the mosquito [For arboviruses, the presence and effect of bottlenecks is especially important as the mosquito vector subjects the virus to several anatomical bottlenecks (reviewed in ). As arbhe virus . These fmosquito . FurtherIxodes scapularis, POWV is subject to a high degree of purifying selection and bottlenecks that probably occur when the ticks moult to the next stage [For tick-borne viruses, the only study of diversity was performed using Powassan virus (POWV), and this showed that POWV populations isolated from ticks contain little viral genetic variation. This suggests that, upon infection of xt stage . FurtherGiven the evidence presented above, it is likely that all arboviral infections are affected to some degree by bottlenecks. However, the severity and effect of these bottlenecks on the viral diversity is only just beginning to be uncovered and further experiments are required to determine the evolutionary pressures that such bottlenecks impose on arboviruses.i.e., making the virus a hyper-mutator, also decreases the infectivity of CHIKV [Arboviral transmission cycles are complex and the viruses encounter different environments during their transmission cycles. Infection of the vertebrate host requires the virus to evade both innate and adaptive immune responses as well as to replicate to high enough titers to facilitate transmission to the next arthropod vector. It has long been postulated that the diversity present within the viral populations facilitates evasion of the immune system long enough for transmission. Certainly, viruses that have reduced diversity are no longer able to replicate as well within the host ,29,30. Eof CHIKV , reducinDrosophila and is reviewed elsewhere [Drosophila genes, and homologues of the important genes Dicer-2 (Dcr-2), R2D2 and Argonaute-2 (Ago-2) have been found in Anopheles gambiae, C. pipiens and Aedes aegypti [Unlike vertebrates where the immune response is well understood, we are only just beginning to understand the immune responses associated with arbovirus vectors such as the mosquito and tick. Most of the work on insect immune systems has been done with lsewhere . For moslsewhere . Most of aegypti ,35.An. gambiae, the virus reaches the salivary glands in 9 days. If the virus is co-infected with double-stranded ONNV RNA that stimulates the siRNA response, this leads to a slower infection with the virus increasing the time required to disseminate and reach the salivary glands. However, when ONNV is co-infected with siRNAs that inhibit the RNAi response, the virus spreads more rapidly and achieves higher mosquito titers, even leading to increased mosquito mortality when An. gambiae is infected with ONNV [A. aegypti cell cultures or in vivo mosquitoes results in increased infection, dissemination and high viral titers and increased mortality [In general, arboviruses infect mosquito cells without causing significant damage, with little evidence of the cytopathic effects (CPE), usually seen in vertebrate cells. This allows mosquitoes to remain infectious for their lifetime, compared to vertebrates that typically clear the infection from their systems or succumb to infection. Recent evidence suggests that this long-term vector infection is the result of suppression of the virus via siRNA responses. When the alphavirus O’nyong-nyong (ONNV) is used to experimentally infect ith ONNV ,36. Thisith ONNV . Upon inortality . Thus thGiven that arboviruses are subject to antiviral pressures from the mosquito as well, the presence of viral diversity appears to be more important in the mosquito vector than was previously recognized. Recent work examining the effect of the RNAi response on the production of siRNAs from the virus has shown that there are areas of the viral genome that disproportionately produce the viral RNAs (viRNAs) that trigger the immune response. Studies with SINV show that these viRNAs are generated from both the positive and negative-strand of the genome, though mainly from the positive strand ,40. ThesArboviral genetic diversity is therefore an essential strategy for the virus to ensure survival and represents a new and exciting avenue of virus evolution research. More work is needed to examine the interactions between the RNAi response and viral diversity. Additionally, the impact of the repeated bottlenecks described above needs to be investigated to give a complete picture of the complex interplay between the mosquito and the virus that facilitates viral transmission without adverse effects to the mosquito.versus vertebrate hosts and can be virus‑specific, but that arbovirus fitness is not severely limited by alternating between vertebrate and invertebrate hosts.Arboviruses are unique in that they obligately cycle between vertebrates and invertebrates, unlike most RNA viruses that use just one or several closely related host species. The need to replicate in two different host types imposes special selective constraints on arboviruses and shapes their evolution. Invertebrate and vertebrate hosts are believed to present conflicting challenges that limit adaptation to either host alone by imposing fitness costs where adaptations are antagonistic . As evidAlthough recent work has focused on defining the viral genetic determinants of emergence and host range changes , knoAlphavirus. Of these VEEV has been of historic interest since the 1930s due to its periodic reemergence as an epidemic virus and the ability of the virus to spread rapidly throughout Central and South America, and even to the U.S., during epidemic outbreaks [The three viruses studied below are the most well studied arboviruses in terms of their evolution and transmission. West Nile virus has been studied in detail since its first identification in the U.S. in 1999. Since then, there have been numerous studies on its evolution in both avian and mosquito hosts and it remains the best understood flavivirus in terms of how the virus and the hosts interact. CHIKV and VEEV both belong to the genus utbreaks . ChikungWest Nile virus, has been well studied since its arrival in New York in 1999 , and theStudies of WNV have shown that it, like all RNA viruses, exhibits diversity within infected hosts. Evidence for the presence of diversity has been identified in both the mosquito vector and the avian host. However, experimental quantification of the amount of variation suggests that there is more diversity in vectors compared to avian hosts . ExperimC. pipiens mosquitoes do not appear to exhibit bottlenecks, showing no reductions in the number of viral variants between the midgut, the hemocoel and the salivary glands. Instead, the amount of diversity decreases over time in all vector tissues and organs over a 21-day extrinsic incubation period. This decrease appears to be stochastic, with no one variant consistently being selected [C. pipiens mosquitoes and WNV. Further study of other systems is needed to assess the relative changes in temporal versus anatomical reduction in variants in different systems.For WNV two experiments were conducted, the first showed that upon infection of a mixture of five variants in roughly equal quantities (much like the VEEV work described below) the selected . HoweverC. taeniopus [The presence of bottlenecks in VEEV during mosquito infection has been studied using subtype IE strains and the associated enzootic vector aeniopus   to cohorts of mosquitoes, they all present in the infected mosquito midguts. However, when the hemocoel was tested for the presence of the marked viruses there was a significant reduction in the number of marked clones, suggesting the presence of a bottleneck upon virus escape from the midgut into the hemocoel. Effective population size measures estimated that ≈50 infectious virus particles escaped from the midgut into the hemocoel. However, when mosquitoes were fed with lower VEEV titers (105 pfu/mL) the number of marked viruses detected in the midgut decreased, indicating the presence of a bottleneck upon midgut entry. Midgut infection is therefore dependent on the initial concentration of ingested virus, and in natural transmission cycles probably represents a significant bottleneck as most rodents have viremia titers lower than 104 pfu/mL during infection with VEEV IE [5 pfu/mL) titer there was still a bottleneck associated with escape from the midgut, and the number of particles calculated to escape was approximately two. Following injection of the virus via the intrathoracic route to artificially bypass midgut infection, evidence of a third bottleneck was detected when VEEV infects the salivary glands from the hemocoel. Although transmission experiments were conducted, only three transmission events were documented, so it was not possible to determine a consistent bottleneck associated with vertebrate infection. Therefore it appears that for VEEV, the mosquito is the major source of bottlenecks, and these are associated with the anatomical barriers in mosquitoes, unlike WNV where the reduction in viral genetic diversity is associated with time.A series of eight viruses marked in the nsP2 gene with synonymous mutations was created, all with equal mosquito fitness  VEEV IE . At the Chikungunya virus is a re-emerging alphavirus that causes febrile disease with arthritis and has recently expanded from Africa to Indian Ocean Islands, Asia, Europe and the Americas in vivo study with VEEV that detected significant fitness increases after serial single-host passages but no genome-wide consensus changes [The precedent for CHIKV studies came from an  changes , suggest changes . By comp changes . In paraRecent studies with CHIKV  employinIn vitro studies have generally showed that arboviruses are subject to fitness declines following repeated bottlenecks . Population bottlenecks in vivo during vector infection have been shown using experimentally barcoded arboviral populations and deep sequencing of wt viral populations analysed in different compartments at several stages of vector infection and transmission. However, somewhat surprisingly, although these bottlenecks are apparent in several arbovirus-vector systems, there is little or no evidence that they affect fitness or even genetic stability in experimental settings or in nature. Key questions requiring further study include: (1) How do arboviruses recover their genetic diversity, which has been shown experimentally with high fidelity mutants to be critical for fitness and transmission [Since the pioneering work of Muller  the potesmission , after p


I was very lucky to have stumbled into curiosity-driven scientific research for a career, because I suspect that I have always had a poorly developed herd instinct. In my case, when I set up my first research lab as a new primary investigator in 1980, I elected to study a family of viruses that infect only rabbits. Why rabbits, you ask? I had become fascinated by events that had happened a half-century earlier, when a rabbit poxvirus called myxoma virus had been deliberately released into the Australian outback in a failed attempt to wipe out the hoards of feral rabbits that had proliferated out of control and devastated the ecological balance of the countryside. This release failed to reduce rabbit populations as planned but it did prove another point that later turned out to be significant: myxoma virus was completely harmless to all other vertebrates, including humans. So, it was pretty much guaranteed that the research in my lab would never be relevant to any human disease. But fortunately for me, I received research funding to investigate how this virus causes disease only in this one particular host species.After several years of investigating the molecular features of this virus, we discovered that the virus genome encoded a large family of proteins with remarkable similarity to elements of the host immune system. In due course, the field began to appreciate that many viruses capable of selectively infecting vertebrates have co-evolved to counteract or evade the immune defenses of their hosts. Brett Finlay and I coined the term “anti-immunology” to describe an amazingly diverse family of pathogen-derived proteins that evolved to control the vertebrate immune system. Over time, my lab began genetically deleting viral genes from myxoma virus one at a time, in order to observe the effects of the various targeted gene knockouts on the virus, but then two unexpected discoveries began to drag my career more into the world of translational research.The first discovery, made with Alexandra Lucas, revealed that some anti-host viral proteins were powerful inhibitors of the host immune responses to the virus. These viral proteins could be purified individually and used as drugs to inhibit the same immune cells when they become hyper-activated during inflammatory diseases like atherosclerosis. This led to the formation of a new startup biotech company (Viron Therapeutics), and introduced me into the world of applied translational research and clinical trials. Although it is still not a given that viral proteins will end up being approved and licensed for clinical use, I am now an advocate of virus-derived proteins as a new source of drugs to treat immune-based diseases that have no known connection with viruses.The second discovery, beginning about a decade ago, occurred when we discovered that myxoma virus also grows in many classes of human cancer cells, but not in normal tissues (unless you are a rabbit). In fact, when my longtime collaborator Peter Forsyth injected it into human gliomas transplanted into the brains of test immunodeficient mice, the virus grew selectively within the transplanted human brain tumors just like it does in the internal tissues of myxoma-infected rabbits! Since it is totally nonpathogenic for humans, this discovery has convinced us to develop myxoma virus as a new potential therapeutic for a variety of human cancers. I have now formed a partnership with a biotech company that specializes in virotherapy for cancer (DNAtrix), and our first clinical trial goal will be to improve the outcomes for cancer patients receiving bone marrow transplants. I am very excited about this new approach against cancer in general, but only time will tell whether using live oncolytic viruses to treat cancer will become a licensed clinical tool for oncologists in the future.I think my own take-home message is that the results of true fundamental research still remain virtually impossible to predict, despite what pundits or politicians might have you believe. In fact, I continue to passionately support curiosity-based research for its own sake of expanding the boundaries of knowledge, not just to fulfill a pre-approved agenda. To me, the single most important justification for fundamental research in biology remains this: Mother Nature is mysterious and magnificent but some of Her secrets can still be revealed if we only allow curious minds to ask the right questions. But with the growing din of anti-science sentiments, those of us who have been lucky enough to pursue fundamental research as a career now more than ever need to speak up. If we want the next generation of scientists to lead the way to the transformative discoveries of the future, we all need to articulate more clearly to nonscientists why, in our modern world, basic research matters more than ever.


Diabetes mellitus (DM) is a major health problem with severe complications and a significant impact on quality of life. It constitutes an enormous burden of disease due to high prevalence, severe co-morbidities and high costs for society. This study is the first comprehensive study on the direct and indirect costs of DM (type 1 and type 2) and associated complications in Poland.In order to estimate the direct medical costs of DM and its complications, including the costs of medical consultation, hospitalisation, rehabilitation, drugs and medical equipment, data from the National Health Fund were used. Indirect costs on loss of productivity due to diabetes and its complications were based on data obtained from the ZUS  and from GUS . Attributable risk methodology was used to assess the burden of DM complications.A continuous increase of the direct costs of diabetes has been observed since the year 2005. In the analysed time period (2005–2009) the direct costs of medical services for both types of DM doubled. DM is a cause of significant sickness absence and incapacity for work and therefore is associated with a growing productivity decline in Poland. The highest direct costs and indirect costs are associated with treatment of diabetes-related complications. Direct costs of hospital complication treatment were EUR 332 million, which exceeded by more than five times the direct costs of hospital treatment of diabetes per se, which in the same year amounted to EUR 58.5 million. The indirect costs of diabetes-related complications were higher by 41 % compared with indirect costs related to DM itself. Total costs of health care services for DM and its complications amounted to EUR 654 million, which constitutes a 2.8 % of total health care costs in Poland. Total DM cost in Poland in 2009 amounted EURO 1.5 billion.DM is causing a growing economic burden on the health care system and on Polish society in terms of health care and productivity losses. Most of the total cost of diabetes are indirect costs caused by productivity losses. Both direct and indirect costs are driven by the cost of diabetes complications. Diabetes mellitus (DM) is a widespread clinical and public health problem in many countries in the world, especially when we take into account its high prevalence, increasing morbidity and impact on health care systems in terms of costs. An estimation of the influence of DM on health care expenditures is complicated because of the many aspects of the issue. Total costs can be divided into medical and non-medical or direct and indirect costs. In addition to costs associated with DM treatment , treatment of immediate and distant future complications caused by DM should be taken into consideration. People suffering from the disease are at high risk of serious cardio- or cerebrovascular problems, neuropathy and/or microvascular complications , which lead to a rise in the total costs of the illness and significantly lower the quality of life. Increased risk of disability, inability to work or premature death is important as well. Loss of productivity caused directly by DM as well as by DM-related complications constitutes a substantial economic burden.The economic impact of DM on a health care system depends, in particular, on the type of illness. According to the classification recommended by the WHO, there are two main types of DM: type 1 DM and type 2 DM. Although these separate conditions have similar long-term consequences, they affect different age groups and require different treatment strategies. Type 1 DM, previously called insulin dependent, usually develops in childhood and adolescence as a result of impairment of pancreatic islets β-cells, whereas type 2 DM results from insulin resistance—an inappropriate response of body tissue to insulin—and usually develops in adulthood. It is estimated that more than 90 % of cases are type 1 or 2 DM . This hiA number of pharmacoeconomic or cost-of-illness analyses related to DM have been performed recently. However, reported costs vary significantly depending on the data source used, type of assessment and scope of the analysis. Some cost estimates are made on the basis of the existing population data on health care use, disability and mortality, while others are performed using the data of patients with DM at an individual level. Methodologies differ in terms of methods of calculating the direct and indirect costs, costs attributable to DM, and in the way in which the results are presented. Furthermore, a number of investigations are concentrated on one type of DM only. Therefore, the results of different studies either cannot be compared or the comparison is problematic and unreliable.An analysis performed in Spain , conductSo far, three studies of costs related to DM have been conducted in Poland. In the cost of DM type 2 in Poland study (CODIP) , direct Severe criticism has been raised against cost of illness studies , pointinThe goal of this article is to present the results of the first Polish top-down study that aimed to assess the total costs of DM in Poland, including both direct and indirect costs of DM and its complications.There is a need of a comprehensive and systematic study of the DM-related costs in Poland, as well as discussion on proper DM prevention and management. The number of people living with DM in Poland is expected to increase by 28 % by 2030 as a result of the obesity epidemic, the ageing of the population and other factors such as lifestyle changes and models of consumption. There is great public concern about the obesity epidemic among children in Poland. In the future, these children will face a substantial risk of DM, and this will result in an increasing social and economic burden. Another rationale for our study is to estimate the structure of DM-related costs and to compare them with the results of analyses conducted in other countries. Our results can serve as a benchmark for future studies on the effectiveness of DM management and efficiency of health resources allocation in the health care system and in the public finance sector.Ischaemic heart diseases: angina pectoris (I20), acute myocardial infarction (I21), chronic ischaemic heart disease (I25)Other forms of heart disease: heart failure (I50)Cerebrovascular diseases: intracerebral haemorrhage (I61), cerebral infarction (I63), stroke, not specified as haemorrhage or infarction (I64)Renal failure (N17–N19)Retinal disorders in diseases classified elsewhere (H36)Visual disturbances and blindness: visual impairment including blindness (H54).This study is a prevalence-based top-down cost of illness study, which analysed the direct and indirect costs of DM and DM-related complications. We defined prevalence as all patients suffering from DM and alive on 31 December 2009. The resources used for DM per se were identified based on DM being the first diagnosis for resource consumption. Additionally, we estimated the costs of DM complications. DM-related complications included in the analysis were identified based on the ICD-10 codes of complications included in the German CoDiM Study [The research was based on the data acquired from the National Health Fund (NFZ), ZUS , and from GUS .The direct costs assessed in the research included: medical care  and drug reimbursement calculated from the payer perspective. Due to the marginal share of co-payments in the public health care system the costs calculated from the payer perspective are a good estimate of social cost.Intangible costs have been omitted. The scope of the analysis does not include the costs associated with indirect costs of informal care, which can account for up to 30 % of the indirect costs in DM treatment . The cosP stands for the prevalence rate of DM among hospital patients, and R is a relative risk of suffering from a given chronic complication among people with DM [A calculation of direct costs of DM-related complications was also carried out on the basis of the data acquired from NFZ. These data cover complete information about all patients treated in hospitals in 2009. The data were extracted by NFZ in two stages. First, patients with a diagnosis of primary and co-morbid DM  were selected. Then, episodes containing primary or DM-related co-morbidity were identified in these patients. The direct costs of DM complications were calculated using aetiological fractions (EF), to estimate what share of co-morbidity costs is attributable to DM. The EF is calculated based on the following:mentclasspt{minimaThe methodology applied is a source of possible systematic error in our estimates. When calculating relative risks and prevalence rates values we used panel data of hospital patients as a sample. This sample is not representative of the entire population with overrepresentation of patients with severe DM; therefore there is a risk that the estimated variables are overestimated. This possible bias can thus lead to overestimation of the indirect costs share in the total costs. Moreover, because of this limitation in methodology, the calculated values of EFs are not easily comparable with the results of other epidemiological studies.The indirect costs assessed in the research included the costs of productivity loss due to work absence or inability to work due to DM and DM complications. The indirect costs were estimated using the data from ZUS to GUS. In the case of the indirect costs of DM-related complications, similarly to the approach applied in the direct cost estimations, we used EF to estimate what share of the total indirect cost of chosen co-morbidities can be attributed to DM. ZUS reports the first diagnosis in the ICD-10 coding; therefore it was impossible to directly identify patients who were unable to work because of DM-related complications.While estimating the indirect costs, the human capital method was used. The costs of lost productivity due to sickness absence were calculated with the use of the ZUS data (i.e. the number of days taken off for sickness caused by type 1 and 2 DM) and the average daily gross wage in the economy (EUR 34.06 in 2009). The analysis of indirect costs also involved lost productivity related to work incapacity. These costs are incurred because of inactivity in the labour market and the collection of social security benefits and were calculated on the basis of the ZUS data concerning the volume of social security benefits transferred, along with the GUS data on the average gross salary in the economy.Lost productivity caused by premature death was not included in the analysis of indirect costs because of the lack of data.In our study we applied point estimates of EF for DM complications. The variables used to calculate EF are subject to uncertainty; therefore we provided upper and lower limits of the direct and indirect costs resulting from adjusting the relative risks and prevalence rate. We applied sensitivity analysis of ±10 % to relative risks. With regard to the prevalence rate, we used the results of an epidemiological study conducted in the Halemba district in Poland (6.54 %)  and the Figure In the DM services analysis, care of patients treated within the PHC network was not included because of the method of reporting by the NFZ. Estimation of the PHC costs was, however, possible for 2009. In 2009, care for patients with DM was granted a higher financing level. General practitioners treating DM patients receive a yearly lump sum assigned to an individual patient that is three times higher than that for a ‘regular’ patient. The data from the performance of the contracts with the PHC doctors show that the total number of patients with DM who were treated in POZ in 2009 was 1,584,716. Thus, we can estimate the PHC costs of DM treatment in 2009 to be EUR 70.4 million.The total costs of health care services for type 1 and type 2 DM in 2009 amounted to EUR 163 million, and the largest individual factors are the costs of PHC and hospital treatment, which constitute 82.8 % of the total costs.On the basis of the available data there is no possibility of classifying the costs of drugs according to the type of DM. Therefore, the costs were analysed for both DM types. The total costs of drug reimbursement for DM in 2009 amounted to EUR 159 million. The distribution of the costs for the years 2005–2009 demonstrated that the total expenditures for drugs have increased by 25.7 % since 2005.The available data do not allow for a separate analysis of the drug reimbursement and PHC costs for each type of DM. Therefore the total direct costs were calculated together for type 1 and type 2 DM  are presented in Table In 2009, the direct costs of hospital complication treatment were EUR 332 million, which exceeded by more than five times the direct costs of hospital treatment of DM per se, which in the same year amounted to EUR 58.5 million. The biggest share in the costs of DM-related complications goes to heart diseases (84.4 %).The total indirect costs of type 1 and type 2 DM consist of the following fractional costs: the costs of lost productivity because of sickness absence and the costs of lost productivity due to work incapacity.The costs of lost productivity because of sickness absence in the case of type 1 DM were EUR 12.8 million and in the case of type 2 DM were EUR 32.1 million. The analysis of absenteeism revealed a growing trend of indirect costs related to both types of DM in the period 2006–2009. A 74 % growth in the costs was observed for both types of DM; however, a more rapid increase in the costs was observed for type 2 DM patients (88.6 vs. 53.7 %). This correlated with an increase in the number of sickness absence days by 23.2 % for type 1 DM and 51.2 % for type 2 DM (Fig. Lost productivity due to work incapacity in the case of type 1 and type 2 DM was respectively EUR 223.6 million and EUR 79.5 million.In 2009, the total indirect costs of DM exceeded EUR 349 million, of which around 13 % was associated with loss of productivity because of sickness absence, whereas the remaining 87 % with lost productivity was attributed to work incapacity.The indirect costs of complications include the costs of lost productivity caused by sickness absence and costs of lost productivity related to work incapacity. These costs were EUR 53.8 million and EUR 438.05 million respectively. The structure of indirect costs related to DM-related complications was then similar to the corresponding cost structure of DM itself and demonstrated that 89 % of the costs were associated with productivity losses due to incapacity for work, while only 11 % with capacity lost because of sickness absence days.In both the costs of lost productivity due to sickness absence and the costs of lost productivity related to work incapacity, the prevalence of heart disease complications was respectively 91.5 and 83.9 %.Overall, the indirect costs of DM-related complications were higher by 41 % compared with the indirect costs related to DM itself.The total costs of DM in 2009 amounted to EUR 1.5 billion Table . In the A sensitivity analysis Tables , 5 reveaThe analysis shows that a 10 % increase in relative risks results in a 3 and 3.4 % increase in the direct and indirect costs respectively. The values of the direct and indirect costs calculated for lower limits of relative risks are 2.3 and 4.4 % lower in comparison with the model values. The figures in Tables From a social perspective, the total annual cost of DM in Poland is about EUR 1.5 billion. The total costs of health care services for DM and its complications amount to EUR 654 million, which constitutes 2.8 % of the total health care costs in Poland. Since 2005 a systematic increase in direct costs of DM treatment has been observed. In the case of type 1 DM such a substantial increase in the costs was caused by a more than 90 % rise in the average cost of medical services (Table This is the first time that the attributable risk approach has been used for estimating the direct and indirect cost of DM complication in Poland. The Polish DM COI studies mentioned above have estimated the cost of DM complications directly from the study sample without adjusting for attributable risk. Due to a major methodology discrepancy the results are barely comparable with the previous studies conducted in Poland, although it can be observed that indirect costs constitute a high fraction of the total costs regardless of the methodology used.The fact that both the total health care cost and the cost per person diagnosed with DM were found to be lower than in the UK  is not sMost of the previous studies in Poland were based on epidemiological data and surveys conducted on a representative sample of the Polish population. This results from low data availability, dispersion or a lack of data. Our top-down study of DM costs is the first of its type in Poland. It is based on reliable source data, and although some data could not be obtained, the results provide an insight into the structure of DM costs while demonstrating the scale of the problem. Such measurements are the first step in the proper DM control, and this study can be a starting point for further research in this field in Poland to provide a full picture of the DM burden. An improvement of data availability and quality is needed to monitor the real costs of the disease. We should indicate a number of difficulties connected with conducting COI studies in Poland. These are primarily low data availability, dispersion or lack of data: difficulty in identification of costs related to the most frequent complications, lack of legal regulations and underdevelopment of the information infrastructure of the health care system.The study has certain limitations that should be mentioned. It is likely that the results underestimate the real costs associated with DM and DM-related complications. One reason is not including the loss of productivity associated with mortality. This cost can account for a substantial part of the total DM cost. A recent DM COI study reported that it can amount to as much as 46 % of the total indirect DM burden . The stuIn conclusion, our study shows that DM imposes a major economic burden in Poland. Therefore there is a need to introduce an integrated system of DM management that would enable containing future DM costs.


Identifying the regions where wild animal populations could transmit the Ebola virus should help with efforts to prepare at-risk areas for future outbreaks. Related research article Pigott DM, Golding N, Mylne A, Huang Z, Henry AJ, Weiss DJ, Brady OJ, Kraemer MU, Smith DL, Moyes CL, Bhatt S, Gething PW, Horby PW, Bogoch II, Brownstein JS, Mekaru SR, Tatem AJ, Khan K, Hay SI. Mapping the zoonotic niche of Ebola virus disease in Africa. eLife3:e04395. doi: 10.7554/eLife.04395Image Large regions of West and Central Africa (red) have the right environmental conditions for Ebola infection in wild animalsThe scale of the current Ebola outbreak, the first ever to hit West Africa and the first to spread to urban areas, is unprecedented. Already more people have fallen ill and died than in all previously recorded outbreaks in Africa combined. The on-going outbreak now spans five West African countries  and was recently declared a Public Health Emergency of International Concern by the World Health Organization. Health services are being overwhelmed, and the long-term social and economic consequences of the epidemic seem daunting.eLife, Simon Hay of the University of Oxford and co-workers in the UK, US, Canada and Sweden have mapped regions that are potentially at the most risk of a future Ebola outbreak (The extent of the epidemic and the devastation it has caused has clearly, and rightly, dominated both the scientific discourse and media reporting. Yet, questions remain. How did the virus arrive in West Africa, a region that had no history of reported cases in humans? Where does the virus circulate between outbreaks? And where might future outbreaks strike? Now, in outbreak .It has been known for years that several non-human species, notably primates and fruit bats, can be infected with the Ebola virus and that wild animals are the likely source of infection for outbreaks in humans. There have been 23 known human outbreaks of Ebola virus disease since its discovery in 1976 , but theEidolon helvum;Fruit bats, on the other hand, do not appear to fall ill when infected with Ebola. As such, bats are considered the most likely candidates for the reservoir species in which the virus lingers between outbreaks in humans. Antibodies against the Ebola virus have been found not only in three bat species in Central Africa , but alsTo predict where people might be at risk of Ebola outbreaks, it is important to know where the virus can circulate in wild animal populations, and this is exactly what Hay and co-workers—including David Pigott and Nick Golding of Oxford University as joint first authors—set out to do. After collecting information on all the reported Ebola cases in humans with a suspected origin in other species, and all reports of Ebola infections in non-human species from the last 40 years, they mapped the data across Africa. Hay and co-workers then matched the locations of these reports with a variety of environmental factors such as climate and vegetation, as well as with estimates for the distribution of fruit bats. The information obtained was used to plot a risk map of the potential for Ebola virus outbreaks in Africa.The new map reveals that large swathes of Central and West Africa appear to have the right environmental conditions for Ebola infection to occur in non-human species.Furthermore, when the analysis was repeated without using data about the on-going outbreak, an area deemed to be at risk was still within 5 kilometres of the village thought to the origin of the current outbreak.This ‘at-risk area’ spans 22 countries and is inhabited by 22 million people. It should be stressed that this is not necessarily the size of the population at risk of getting infected with Ebola virus; rather, it represents the population that lives within the area estimated to be suitable for transmission in animals. Once an outbreak has started, transmission from human-to-human could easily spread the virus away from the source. This is an important issue given that the Democratic Republic of Congo—where most outbreaks so far have occurred—is currently home to more than 60 million people.Given the large population found to be at risk of Ebola infection from other species, human outbreaks are rare. At the same time, when transmission does occur, a combination of factors are clearly able to cause an outbreak to grow and become as large as the one currently observed. These factors include: poorly prepared health care systems, a slow international response, and a lack of trust or even hostility towards control measures and care. Traditional beliefs and funeral practices that involve touching the body of the deceased can also contribute to the spread, as can the fact that there are now more people living in, and travelling between, the at-risk areas than ever before.During the current emergency, caring for the sick and stopping the spread of the virus are clearly the most important and immediate tasks. Once the epidemic is over, however, it will be paramount to reassess the extent of the area at risk of outbreaks. Health care systems in these regions will then need to be strengthened and made capable of curbing any further outbreaks at the source. The area found to be at risk is home to some of the most under-resourced health care systems in the world—with the five countries afflicted by the current outbreak ranking among the world's worst both in terms of maternal mortality and human development . Further


The purpose of this study is to understand the course of costs over a 2-year period in a cohort of recently diagnosed fibromyalgia (FM) patients receiving different treatment strategies. Following the diagnosis, patients were randomly assigned to a multidisciplinary programme (MD), aerobic exercise (AE) or usual care (UC) without being aware of alternative interventions. Time between diagnosis and start of treatment varied between patients. Resource utilisation, health care costs and costs for patients and families were collected through cost diaries. Mixed linear model analyses (MLM) examined the course of costs over time. Linear regression was used to explore predictors of health care costs in the post-intervention period. Two hundred three participants, 90 % women, mean (SD) age 41.7 (9.8) years, were included in the cohort. Intervention costs per patient varied from €864 to 1392 for MD and were €121 for AE. Health care costs (excluding intervention costs) decreased after diagnosis, but before the intervention in each group, and increased again afterwards to the level close to the diagnostic phase. In contrast, patient and family costs slightly increased over time in all groups without initial decrease immediately after diagnosis. Annualised health care costs post-intervention varied between €1872 and 2310 per patient and were predicted by worse functioning and high health care costs at diagnosis. In patients with FM, health care costs decreased following the diagnosis by a rheumatologist. Offering patients a specific intervention after diagnosis incurred substantial costs while having only marginal effects on costs. Fibromyalgia (FM) is characterised by chronic widespread musculoskeletal pain, often accompanied by other clinical manifestations such as fatigue and stiffness, but also cognitive dysfunction or mood disorders , 2. FM tVarious interventions have been studied, among which multi-modal non-pharmacological programmes as well as pharmacological therapies. However, the effectiveness of these interventions showed conflicting results , 13. In Also, beneficial effects of a diagnosis in itself have been suggested but results of research, all conducted in claims databases, are conflicting; two studies showed that a diagnosis of FM increases costs, primarily attributable to an increased use of medication , 21, whiThe clinical burden of FM and the gaps in available evidence-based treatment recommendations , 25 justPrevious analyses of a pragmatic trial among patients with recently diagnosed FM patients showed no consistent differences in relation to health outcomes between those that received a partially individualised multidisciplinary intervention with aftercare (MD), those receiving aerobic exercise (AE) and those receiving care as usual (UC) . The preA two-year cost-analyses of data of an observational study in which a pragmatic trial was embedded.n = 108), AE (n = 47) or UC (n = 48). Patients assigned to MD or AE were asked again to consent to participate in the proposed intervention without being aware of the alternative intervention. The study was approved by the Medical Ethical Committees of the participating medical centres. The precise procedures in this pragmatic trial, registration number ISRCTN32542621, have been published elsewhere [A cohort of 203 consecutive patients that were recently (<3 months) diagnosed with FM according to the American College of Rheumatology criteria  at one oThe MD intervention was a two-phased group programme of 1 year. Phase l consisted of a 12-week course  with two therapy sessions of 1.5 h per day. Sociotherapy and physiotherapy were given twice per week; psychotherapy and creative arts therapy were given once per week. Phase II was an aftercare programme that was provided over the course of the remaining year and consisted of five group meetings. In addition, a maximum of seven individual therapy sessions with one of the therapists could be scheduled if considered necessary by the therapist and/or the patient. The AE intervention was a 12-week group course given twice a week by a trained physiotherapist in a community gym, following recommendations for exercise . The UC Demographic characteristics  and health status were assessed by means of patient-reported questionnaires at entry in the observational study. Health status comprised symptom duration, and the impact of FM, measured by the Fibromyalgia Impact Questionnaire (FIQ), that consists of 10 items on health in the past week; physical functioning, numbers of days feel good, number of days missed work, interference of symptoms with ability to activities, pain, fatigue, unrefreshed sleep, stiffness, anxiety and depression . Each itSelf-reported FM-related health care resource use and costs for patients and their families served as a basis for the cost analysis and were collected by means of 12 cost diaries over a total study period of 2 years. In each diary, patients had to indicate the number of visits to general practitioners (GPs), medical specialists, physiotherapists and other paramedical therapists such as psychologists; the prescribed medication taken; the kind of assistive devices purchased; and the number of hours professional home help per week received during the 2 months prior to measurement . Next, pHealth care costs for each category were calculated by multiplying the number of each resource used with its unit cost, derived from the Dutch Cost Manual or the Pharmacotherapeutic compass. If true costs were not available, market prices or tariffs were used –32. Patiwww.cbs.nl) [The time input from all health care providers in the MD or AE group sessions were the basis for the calculation of the costs of the interventions. Total costs were calculated by summing the product of each hour of work by the gross salary per hour for each professional, augmented with 39 % charges for social security. As recommended, 45 % charges for overhead, including for use of accommodation, were added . Finally.cbs.nl) . All cosTo understand the course of resource utilisation and costs per patient over time, we distinguished four clinical meaningful periods; the diagnostic phase representing the 2 months before referral to the rheumatologists and diagnosis of FM (diagnosis), the period after the diagnosis but before the intervention (pre-intervention), Phase I of MD, and AE (intervention) and the period after the intervention (post-intervention). As the start of the MD and AE programme varied in time among individual patients, the average number of questionnaires available for the period after the diagnosis but before the start of the MD or AE programmes varied from 1 to 4, and after the programmes from 6 to 9, while the main intervention period itself was always represented by one questionnaire. For the UC group, the number of questionnaires for each period was assigned after matching each subject randomly to a participant of the MD or AE group , and in order to achieve complete data, missing data were imputed using a non-parametric regression forest method .First, mixed linear model analyses (MLM), with a random intercept, a random slope and an unstructured correlation structure, were used to assess differences between the intervention groups in the longitudinal course of health care costs and patient and family costs, with the baseline values of the dependent variables as covariates in the models.Next, mean between group differences of the direct costs in- and excluding intervention costs over the total 2-year period were tested using a non-parametric bootstrap method to obtain 95 % confidence intervals.Finally, to explore predictors of the health care costs in the period after the intervention a linear regression analysis on the square roots transformed costs was performed, entering as predictors age, symptom duration, educational level, FIQ-total score and health care costs in the diagnostic phase.Data imputation was performed using R2.10.1, and all other analyses were performed using IBM Statistics SPSS version 20.0.Baseline characteristics of the patients are presented in Table resource utilisation in the three groups is presented in the The detailed course of health care as well as patient and family Health care costs, excluding intervention costs, decreased statistically significantly after diagnosis but before the intervention in all groups  (not presented), reflecting a sooner increase of costs in the UC group . Patient and family costs increased in the three groups. MLM indicated no statistical significance between group differences.s Figure . During Patient and family costs increased in the three groups. MLM indicated no statistical significance between group differences.While the changes in costs incurred by the intervention were marginal and not statistically significant, the intervention costs were substantial; for MD €864 per person in phase I and in phase II, the costs varied between €86 and 528 per person depending on the number of individual contacts. Costs for AE were €121 per person. The resulting total direct costs per person (with and without programme costs) over the total observation period after the diagnostic phase are presented in Table Finally, the 2-monthly health care costs in the post-intervention period were predicted by a high impact of FM (FIQ) already at entry in the cohort and by higher health care costs before the intervention  along a randomised pragmatic trial. While the costs of the MD and AE intervention were substantial, their influence on health care costs and patient and family costs over time was similar.The major interest of the paper can be found in the course of health care utilisation and costs over time which decreased immediately after the diagnoses, before the MD or AE intervention was started. Since we previously failed to show any effects of the interventions on different aspects of health, including the 5-dimensional EuroQol (EQ-5D)  and FIQ The higher health care costs around the period of diagnosis likely reflect the high needs of patients to find help for their complaints. It is understandable that GPs first explore several diagnostic and treatment options in such periods and next refer patients, if complaints persist, to a rheumatologist for confirmation of the diagnosis and for a better treatment plan. In addition, the diagnosis itself includes several consultations and therefore costs are higher in this period as in FM, follow-up visits are limited. The decrease in costs after the diagnosis could partially be attributed to regression to the mean. Notwithstanding, it cannot be excluded that also the diagnosis itself reassures patients and reduces resource utilisation in the period short after diagnosis and before the start of the MD or AE programme , 35.The increase in patient and family costs, reflecting increased participation in health activities, and increased use of paid help and informal care over time is not surprising. In the usual care setting, patients receive information and education from the rheumatologist, the rheumatology nurse or the GP and they are encouraged to implement sports, to pace their tasks and activities and ask for support from relatives and friends , 37. OurOur results have far reaching consequences for clinical practice which is a strength of our study, as it suggests that care as usual in the Netherlands is as good and substantially cheaper compared to more complex interventions and might be appropriate for many patients.However, some limitations need to be addressed. First, we have used data from a cohort of patients participating in a trial and missing data occurred within the cohort. Missing data were carefully explored and were imputed. Of course, some level of uncertainty cannot be excluded but the advanced method that was used for the data imputation limits errors and contributes to valid data. Also, we did not include indirect costs in our study. Only 50 % of the patients were employed, and this small sample would limit the possibility for a reliable assessment of the course of costs due to productivity loss. Further, our study comprised a cohort of recently diagnosed patients but in the cohort, there was not necessarily a recent onset of FM. Likely, patients had been managed by their GPs during a longer period. Well-trained GPs in the Netherlands can diagnose and manage FM appropriately , but it In summary, our results show that after diagnosing FM, the use of health care resources decreases, and the slight increase afterwards is largely independent of the interventions offered. Given the absence of beneficial health effects but additional high intervention costs of MD and AE, such interventions cannot be recommended to all patients with FM. Notwithstanding, we cannot exclude that a subgroup of patients might have benefit from MD or AE; an appropriate selection of patients for interventions may result in larger effects and may contribute to cost-effectiveness. Future research should focus on improved selection of patients for specific health care innovations.


The goal of malaria elimination faces numerous challenges. New tools are required to support the scale up of interventions and improve national malaria programme capacity to conduct detailed surveillance. This study investigates the cost factors influencing the development and implementation of a spatial decision support system (SDSS) for malaria elimination in the two elimination provinces of Isabel and Temotu, Solomon Islands.Financial and economic costs to develop and implement a SDSS were estimated using the Solomon Islands programme’s financial records. Using an ingredients approach, verified by stakeholders and operational reports, total costs for each province were quantified. A budget impact sensitivity analysis was conducted to investigate the influence of variations in standard budgetary components on the costs and to identify potential cost savings.A total investment of US$ 96,046 (2012 constant dollars) was required to develop and implement the SDSS in two provinces . The single largest expense category was for computerized equipment totalling approximately US$ 30,085. Geographical reconnaissance was the most expensive phase of development and implementation, accounting for approximately 62% of total costs. Sensitivity analysis identified different cost factors between the provinces. Reduced equipment costs would deliver a budget saving of approximately 10% in Isabel Province. Combined travel costs represented the greatest influence on the total budget in the more remote Temotu Province.This study provides the first cost analysis of an operational surveillance tool used specifically for malaria elimination in the South-West Pacific. It is demonstrated that the costs of such a decision support system are driven by specialized equipment and travel expenses. Such factors should be closely scrutinized in future programme budgets to ensure maximum efficiencies are gained and available resources are allocated effectively.The online version of this article (doi:10.1186/1475-2875-13-325) contains supplementary material, which is available to authorized users. Malaria in the Pacific region has been an essential component of the global health agenda since the 1950s. More recently, considerable renewed interest has developed towards malaria elimination , with reThe management and control of this global disease has seen numerous significant achievements matched equally with disappointments –10. RegrSince 2008, the Solomon Islands’ National Vector Borne Disease Control Programme (NVBDCP) has embarked on a programme of aggressive malaria control. With a markedly low incidence of malaria transmission in the provinces of Temotu and Isabel, pilot malaria ‘elimination’ programmes commenced in these locations , 20, 22.A substantial literature exists on the cost dynamics of malaria programmes , particuThe present analysis examines the costs of the development and implementation of a SDSS for malaria elimination in two provinces of the Solomon Islands. Whilst this work does not provide either cost effectiveness or cost-benefit analyses, it does provide a clear picture of the investment required in developing this new tool for malaria elimination and provides the basis for future cost analyses. The analysis also assesses the degree to which variations in the design of the intervention influence programme costs. Other countries in the region with national policies of malaria elimination may potentially utilize this cost information in the planning and resource allocation process when developing their strategies and making operational decisions for malaria control and elimination.The Solomon Islands’ NVBDCP established the SDSS in mid-late 2008, with the goal of improving planning, implementation, and monitoring and evaluation of malaria interventions, as well as managing the more detailed malaria case data (to the household level) for malaria elimination. The programme’s design, technical specifications and applications are detailed elsewhere , 26, 27.Following an intensive planning process, the development and implementation of the SDSS involved training of local staff, the purchase of required equipment and materials, and the collection of geo-reference data at the household level . Concurrently, the development of a central-level database was undertaken, sharing common units and data structures where possible, while allowing the information to remain applicable to locally sensitive needs. Updates and modifications were undertaken to ensure maximum applicability of the SDSS to local needs , 27, 38.The SDSS was implemented in two different geographical locations – Isabel Province and Temotu Province  through to full operation (August 2013); from which time the SDSS was assumed to be fully operational in its intended capacity. The analysis was undertaken from a programme management perspective, which identified direct investment or administrative costs from stakeholders to support the development and implementation of the SDSS. These were considered additional costs outside of the routine expenses incurred by the NVBDCP for programme monitoring and reporting utilising the existing information system. The comparison (null) for this analysis was no SDSS, with the NVBDCP utilising the previously existing system. In an approach similar to other cost analyses of malaria interventions –42, an iDetailed retrospective direct programme cost data were collected from financial records of the key budgetary stakeholders. This included accounting costs recorded in transaction listings from the Solomon Islands Ministry of Health and Medical Services (MHMS), and from financial records of the Pacific Malaria Initiative Support Centre (PacMISC). When financial costs were unavailable for specific SDSS-related activities, MHMS or PacMISC records were sourced for unit costs values and quantities for various inputs.Costs were identified in either Australian Dollars (AUD) or Solomon Islands Dollars (SBD). These costs were converted to US Dollars using historical exchange rates , and werIn cases where costs were identified as having a mixed purpose (e.g. training for IRS and use of the SDSS for planning and reporting), a proportional allocation of each budgetary component was estimated based on the purpose and contribution specific to the SDSS. Proportional allocations were done through review by implementing stakeholders, and based on criteria including the main purpose or objective of the item or activity, the mix of staff and personnel involved in the activity, and the units and/or quantities of resources utilized specifically for the SDSS. Supporting documentation such as receipts, field reports and meeting/workshop minutes were used to support the proportional allocation to the overall cost for the SDSS development.Total costs were classified under two separate categories. First, costs were categorized into standard budgetary resource components utilized by the Solomon Islands’ Government. Where expenses were identified through an ‘imprest’ payment, the imprest budget and/or acquittal were used to identify amounts under each budgetary code. Secondly, costs were also categorized into start-up, geographical reconnaissance, or ongoing management expenses of the SDSS , with US$ 49,806 for Temotu Province and US$ 46,240 for Isabel Province. The single largest cost component was for equipment to implement the SDSS , accounting for over 30% of the total cost. Other significant cost categories included software licenses at approximately 14%, as well as expenses associated with travel , which combined totalled almost 40%. All costs by category and by province are detailed in Table The geographical reconnaissance phase of the SDSS development proved to be the most costly, with over 60% of total costs  associated with this activity. The ongoing management and support costs accounted for just under one-third of the overall costs over the five-year implementation period. Costs by phase of development and implementation are described below in Table A summary of cost categories, unit costs and values included in the sensitivity analysis are detailed in Table Reducing the number of supervisory support visits had a major impact in both provinces. The change could potentially cater for a 9.6% and 6.7% budget saving in total SDSS costs in Temotu and Isabel, respectively. In Temotu Province, changes in equipment costs demonstrated significant overall budget impacts with 4% overall budget savings with reduced unit costs. In light of the challenging logistical setting in Temotu Province, and its isolation and remoteness from the capital (i.e. Honiara), travel costs had a contributable influence on the budget, with a combined impact of approximately 11%. Fuel costs, salaries and changed discount rate all presented a budget impact of less than 2%.In Isabel Province, equipment costs were also found to be the most sensitive component, with approximately 9% savings potential achieved with reduced equipment costs. Software licenses and their annual renewal fees were found to have an almost 4% impact. Given the closer proximity of the Isabel Province to Honiara and the approach to undertake reconnaissance, variation in travel costs for programme staff had smaller impacts on the overall budget costs.This study presents the first cost analysis of the implementation of a SDSS designed to facilitate the elimination of malaria in the Pacific region. The current analysis estimates that the total cost of developing and implementing the SDSS is US$ 96,046. The largest cost components were equipment and travel expenses. The sensitivity analysis highlighted that cost savings depend on the characteristics of the province. In more remote areas, the intervention would be more affordable if changes in travel costs were achieved, while reduced equipment costs would deliver considerable budget savings more generally.Provincial-level costs are valuable information required for accurate programme planning within any context, but particularly in the Solomon Islands, where limited evidence of costs exists. This analysis demonstrates organizational structures and the subsequent allocation of equipment can have a considerable impact on the total budget required for malaria elimination activities. As the malaria elimination programme continues in the Solomon Islands and elsewhere, the roll out of any SDSS in other contexts would require careful consideration of the costs related to the computerized equipment, especially in larger provinces with a greater number of operational zones. Recent advances in the capacity and accessibility of technology, and the ubiquity of GIS-enabled portable devices has seen rapid changes in the market . In factIt is now widely accepted that geo-referenced case data are integral to inform malaria elimination , 37. An Ongoing management and support costs accounted for approximately 32% of the total cost. In countries heavily dependent on international aid for service delivery, such as the Solomon Islands, this poses policy questions regarding the financial feasibility of malaria elimination for both the international donor and the recipient. This applies not only for the SDSS, but potentially other information management tools .Whilst the cost analysis attempts to capture all costs relevant to the SDSS development and implementation, some irregular costs were part of the analysis. For example, a research vessel was hired as part of a broader baseline malaria survey in the outer islands of the Temotu Province and used by the SDSS mapping teams. The mapping teams were accommodated on board the research vessel, providing a significant cost saving under the accommodation line item. However, when costs were calculated based on an implementation model with standardized unit costs for accommodation, the cost of this proved to have a significant impact on the total budget (with a 20% reduction in the unit costs translating into a 4.2% budgetary saving). This becomes evident when comparing accommodation costs between Temotu and Isabel Province in the total cost summary.The applicability of the costs for geographical reconnaissance and the SDSS in other jurisdictions should be interpreted with some caution because geographical and topographical variations in provinces as well as infrastructure – including the availability of electricity, access to boats and vehicles, ports and airstrips – could significantly influence the total investment required. Moreover, the different approaches undertaken to conduct the reconnaissance in each of the two provinces presented difficulties for the costing analysis. Costs for certain categories may have been shared, or in contrast, overly allocated to the SDSS budget. For example, in Isabel Province, where the mapping officers worked closely with spraying teams, the fuel costs may have been under- or over-estimated because they were shared between the two activities. Whilst attempts were made to correctly apportion the costs where information exists, uncertainty inevitably exists. More generally, the availability and reliability of cost data in Solomon Islands also represents a limitation of this study. Inadequate financial infrastructure and limited financial management capacity may have introduced bias to the cost estimates. They are, nonetheless, the best estimates available. Efforts were made to clean the cost data. Within-country experience over a number of years provided the lead author an opportunity to gain an in-depth understanding of the operational requirements and familiarity with the financial systems, potentially improving the quality of the data.The SDSS is a new tool which has required investment in capacity building of local staff. This study does not include expenses associated with technical assistance, which is a limitation of the present analysis. This was due to the complexities of the design, delivery and support mechanisms for the development and implementation of the SDSS. Technical assistance expenses varied greatly between the provinces, with a greater focus on technical assistance support during the initial implementation of the SDSS. As systems were developed and local staff trained, an inherent capacity was built within the programme, with the need for international technical assistance  reducing significantly. This was evident on review of cost data, which indicated that stakeholder expenses associated with technical assistance in Isabel Province in 2010 were minimal. This in part may be due to the awareness and general increase in capacity of local staff following the experience in Temotu Province. Future cost analyses could investigate the role of capacity building and technical assistance in the development and implementation of the SDSS. For instance, the increased accessibility and reach of IT systems and equipment will no doubt improve the base capacity and operating efficiency to develop and implement a province-specific SDSS. On the other hand, as information systems are developed, IT requirements will increase . It is debatable whether this ‘ingredient’ contributes directly to the SDSS or whether it is a routine operational expense. For the purposes of this investigation, the latter has been assumed, and thus, IT systems were not included in the analysis. Future analysis of information systems should attempt to quantify the more complex costs associated with the SDSS development and implementation, including technical assistance.Long-term commitment to malaria elimination is required from donors, recipients and the international community , 54. TheFigure S2: Malaria cases, Santa Cruz Island, Temotu Province, 4th Quarter 2011. Figure S3: Distribution of all confirmed malaria cases, Isabel Province, 2011. Figure S4: IRS coverage map vs. Households, Santa Cruz Island, Temotu Province, 2010. Table S1: SDSS Development Phases. Table S2: Cost details, unit costs, and assumptions for SDSS. (DOCX 538 KB)Additional file 1: Figure S1: Solomon Islands, Isabel Province, Temotu Province.


Culex tritaeniorhynchus Giles mosquitoes from Ruili city, Dehong prefecture, Yunnan Province, in the People’s Republic of China.There have been four strains on Manzanilla virus (MANV) identified to date. Here, we identify a novel MANV strain (DHL10M107) isolated from The DHL10M107 L, M and S genes were sequenced at the nucleotide and deduced amino acid levels. The L, M and S gene sequences of DHL10M107 clustered with the MANV strains VN04-2108, TRVL3587, SA An 4165, and AV 782. DHL10M107 was most closely related to VN04-2108. Nucleotide homology ranged between 96 and 99% between DHL10M107 and VN04-2108. In terms of amino acid homology, all of the amino acid differences were in the L (96.3% homologous) and M (97.7% homologous) fragments.DHL10M107 is likely a MANV isolated from mosquitos in the Yunnan Province. This is the first reported isolation of MANV in mainland China.The online version of this article (doi:10.1186/s13104-015-1198-5) contains supplementary material, which is available to authorized users. Orthobunyavirus of family Bunyaviridae [Alouatta seniculus insularis) in Trinidad in 1954. More recently, in 2004 Bryant et al. isolated a MANV strain identified as the Cat Que virus (VN04-2108) from mosquitoes (Culex sp.) in Vietnam [Hyphanturgus ocularis) in 1959 [Progne subis) in the United States in 1964 [Manzanilla virus (MANV) belongs to the Simbu serogroup of the genus aviridae –3. MANV aviridae  from theFlavivirus, Family Flaviviridae), Dengue virus , Chikungunya virus , Sindbis virus , Getah virus , Batai virus (BATV) , Banna virus , Yunnan orbivirus , and several other viruses have been isolated from human patients and from mosquitoes collected in Yunnan Province [Yunnan Province is located in southwest China, adjacent to Myanmar, Laos and Vietnam. Ecologically, Yunnan Province supports the distribution of vector mosquitoes and the transmission of mosquito-borne viruses , 7. The Province –16. ThisCulex tritaeniorhynchus Giles and the remaining 107 were from the other nine species (25.18%). The mosquito samples were sorted by species into 15 pools and ground together. The supernatant was used to inoculate Baby hamster kidney (BHK-21) cells to isolate any viruses present as previously described [C. tritaeniorhynchus (DHL10M107) caused significant cytopathic effects (CPE) in BHK-21 cells, characterized by the cells shrinking, rounding, and eventually floating.In August 2010, mosquitoes were collected using the Kongfu Xiaoshuai trap  from cattle barns in the suburb of Ruili city, Dehong prefecture, Yunnan Province, People’s Republic of China. A total of 425 mosquitoes representing 10 species in four genera were collected. Of these, 318 (74.82%) were The viral RNA was extracted from the culture supernatant of the DHL10M107 isolate, using the QIAamp viral RNA mini kit  per the manufacturer’s instructions. cDNA was prepared with Ready-To-Go You-Prime First-Strand Beads™ , and amplified with MANV specific primers  and an Oya virus (OYV) strain (SC0806) isolated from mosquitos in Sichuan province, China , althougOrthobunyavirus strains (Table We then compared the homology of the open reading frame (ORF) and amino acid sequences in DHL10M107 to five other ns Table .Table 1NIn conclusion, the high degree of similarity in the whole genome sequence between DHL10M107 and the four MANV strains in GenBank, but not other viruses in the Simbu serogroup, confirmed it was a MANV strain. This is the first report of MANV in mainland China. Currently, the records pertaining to MANV in the International Committee on Taxonomy of Virus (ICTV)  excludes