Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer The ability of retroviruses to integrate into the host cell chromosome also raises the possibility of insertional mutagenesis and oncogene activation. Both these phenomena are well known in the interactions of certain types of wild-type retroviruses with their hosts. However, until recently they had not been observed in replication defective retroviral vector-mediated gene transfer, either in animal models or in clinical trials. This has meant the potential disadvantages of retroviral mediated gene therapy have, until recently, been seen as largely, if not entirely, hypothetical. The recent clinical trial of γc mediated gene therapy for X-linked severe combined immunodeficiency (X-SCID) has proven the potential of retroviral mediated gene transfer for the treatment of inherited metabolic disease. However, it has also illustrated the potential dangers involved, with 2 out of 10 patients developing T cell leukemia as a consequence of the treatment. A considered review of retroviral induced pathogenesis suggests these events were qualitatively, if not quantitatively, predictable. In addition, it is clear that the probability of such events can be greatly reduced by relatively simple vector modifications, such as the use of self-inactivating vectors and vectors derived from non-oncogenic retroviruses. However, these approaches remain to be fully developed and validated. This review also suggests that, in all likelihood, there are no other major retroviral pathogenetic mechanisms that are of general relevance to replication defective retroviral vectors. These are important conclusions as they suggest that, by careful design and engineering of retroviral vectors, we can continue to use this gene transfer technology with confidence. Retroviruses are viruses that are found throughout the animal kingdom, including in chickens, mice, cats, sheep, goats, cattle, primates, fish and humans. The first retro viruses were identified as cell free oncogenic factors in chickens. Subsequently, many of the oncogenic retroviruses have been shown to be replication defective forms that have substituted a part of their normal viral gene complement with an oncogene sequence . Replicagag gene. In addition, the virion contains two identical copies of a genomic RNA molecule , a tRNA primer for reverse transcription as well as small amounts of the products of the viral pol gene. The virion may also include a range of other host cell derived proteins although it is unclear whether these represent a random assortment of proteins that are coincidently incorporated into the virion or whether they play some role in the viral life cycle. Both possibilities are probably true, certainly HIV-1 is known to incorporate into its virion a number of host cell proteins that play a vital role in its life cycle [The retroviral virion is a spherical particle of about 80–100 nm in diameter. It is enclosed by a lipid bilayer derived from the host cell plasma membrane into which one of the retroviral gene products, the envelope protein, is inserted. The virion has considerable internal structure that is mainly comprised of the products of the viral fe cycle ,10.gag, pol and env, the complex retroviruses encode a number of other proteins that are involved in regulating viral replication or the host cells response to the virus. For example, HIV-1 has six gene sequences in addition to the minimal retroviral complement of gag, pol and env. Two of these, tat and rev, encode proteins that regulate expression of the viral genome, while the other four, vpu, vif, vpr and nef, encode proteins that play multiple roles in enhancing viral replication.While the simple retroviruses have only three genes, It is the unique nature of the retroviral life cycle, combined with the simplicity and advantageous arrangement of the retroviral genome, which has made retroviruses so attractive as vectors for gene therapy ,12. The cis  and trans (protein coding sequences) functions  vectors. With these vectors the resulting provirus contains no active retroviral derived transcriptional promoter or enhancer elements [The genetic structure of the virus and the existence of the proviral form make it easy to manipulate retroviruses to make replication defective vectors for transfer of heterologous gene sequences. The proviral form, being DNA, can be readily isolated in standard plasmid cloning vectors and so made amenable to molecular manipulation. The genetic structure of the virus is such that the viral ons Fig.  are largems Fig. . As the elements ,14.transduction, to distinguish it from the process of infection with replication competent viruses. It is theoretically possible that with most, if not all, recombinant vector systems, that recombination of the constituent parts of the system with each other, or with cellular sequences, can regenerate a replication competent retrovirus (RCR) [The use of a replication-defective retroviral vector to transfer gene sequences into target cells has been termed us (RCR) ,16. HoweRetroviruses have historically been most intensively studied in animals that are either the subject of scientific experimentation , or are of commercial significance , where they cause a number of commercially significant diseases. Indeed, the first retroviruses to be described were the oncogenic retroviruses Avian leukosis virus (ALV), and Rous sarcoma virus (RSV), which are both found in chickens. A large number of oncogenic retroviruses have now been described. These tend to cause malignant disease in a very high proportion of infected hosts. In addition, the complex retroviruses human T-cell leukemia virus (HTLV) and bovine leukemia virus (BLV) can cause leukemia in their hosts, although they do so in only a small percentage of infected individuals.The lentiviruses are also overtly pathogenic and have been shown to be the causative agent of several slow progressive diseases in animals including arthritis and encephalitis in goats, leukemia in cattle, anaemia in horses, and immunodeficiency in cats, cattle, primates and humans. The AIDS epidemic means that the lentivirus HIV-1 is now the most intensively studied retrovirus ever-incredibly, given the relative genetic simplicity of the retroviruses, there appears to be much still to learn about many aspects of HIV-1. There are also a number of viruses that cause central nervous system (CNS) pathology. For some of these, such as HIV and HTLV, CNS disease is a secondary pathology, while others are more specific in their effects. Similarly, while ALV and RSV are best known as oncogenic viruses, they are also associated with wasting syndromes.The archetypal retroviral pathogen is the oncogenic retrovirus. Some of these are replication defective retroviruses that carry and express an oncogene sequence-indeed it was these retroviruses that largely allowed the concept of oncogenes to be first defined. These viruses induce cancers with relatively short latency periods. In addition, there are a large number of non-defective retroviruses that are oncogenic. These generally induce cancers after longer latency periods. HTLV and BLV and related viruses form a separate class of complex retroviruses that cause leukemia in a small percentage of infected individuals after very long latency periods. Retroviruses have also been associated with sarcomas in fish but these viruses have not been studied in great detail.These have been described in a number of species, but have been most extensively studied in the laboratory mouse. These are replication defective, simple retroviruses in which part of the normal viral genome has been replaced with a cDNA copy of a cellular oncogene. The viral oncogene sequence often contains mutations that make the protein it encodes act in a dominant manner. The capture of a cellular oncogene by a retrovirus is an extremely rare event, the major significance of these viruses in scientific terms is that they led to the discovery of cellular oncogenes. These viruses depend on the presence of a replication competent helper virus in order to replicate and they induce cancers with relatively short latency periods. The existence of a latency period suggests that oncogene expression is, in itself, not enough to cause malignant disease, but that additional genetic events are required. The majority of the cancers caused by these retroviruses are found in the haematopoietic system although sarcomas are also common. They are also able to transform the phenotype of cells grown in culture, principally by causing cells to lose their contact inhibition. The type of malignant event caused by any one virus is determined by the nature of the oncogene expressed by the virus and by the nature of the enhancer sequences present in the long terminal repeat which control the tissue specific expression of the oncogene.Replication defective vectors obviously also have the same potential to capture oncogenes. However, the mechanism of oncogene capture by retroviruses, and its extreme rarity, means it is probably not of major relevance when considering the risk factors associated with the use of retroviral vectors for gene therapy.via a number of different mechanisms. In contrast to the oncogene carrying retroviruses, these viruses are associated with much longer latency periods. This is a reflection of the relatively low probability that proviral insertion will result in activation of an oncogene, in combination with the requirement for other genetic changes before a cancer eventuates. Although proviral integration can also result in gene inactivation, inactivation of tumour suppressor genes does not appear to be a mechanism associated with any known instances of retroviral induced malignancy.Non-defective, replication competent retroviruses are also associated with malignant diseases. These viruses do not carry oncogene sequences. Although first discovered in the chicken they have been most extensively studied in the laboratory mouse. These viruses induce cancer by activating cellular oncogenes The principal routes of oncogene activation are transcriptional promotion from one of the viral LTRs, and activation of endogenous cellular promoters by the strong transcriptional enhancer elements present in the viral LTRs. In the former case the provirus must obviously integrate in the sense orientation and upstream of the relevant coding sequence. Transcription can be from either LTR , and mayNot all non-defective simple retroviruses are overtly oncogenic and the oncogenic, non-defective simple retroviruses show a spectrum of tissue specificity and oncogenic potential. Analysis of the oncogenic potential of different retroviruses has clearly shown that the major determinant of both the overall oncogenic potential of the virus, and the cell specificity of the type of cancer that results, is the viral long terminal repeat -27. Moreenv gene sequences. For example, the SU protein (p55) of the polycythemic strain of Friend virus binds to, and activates, the erythropoietin receptor resulting in massive erythroid proliferation and splenomegaly [sag gene of Murine Mammary Tumour Viruses (MMTV) induces an immune response by interacting with the T-cell receptor [via the antigen binding site itself, a large proportion of the T-cell population (up to 10%) is stimulated. This, in turn, stimulates B-cells, the initial cellular target for infecting MMTV, allowing enhanced viral replication and the subsequent infection of mammary epithelial cells, the eventual site of tumour formation. Although Sag is a major determinant of the oncogenic potential of MMTV it should be noted that in the final analysis malignancy is due to oncogene activation.In contrast to oncogene activation, the oncogenic potential of some retroviruses maps to the nomegaly . Howeverreceptor . This dogag, pol and env genes common to all retroviruses, have two genes that encode regulatory proteins. HTLV causes adult T-cell leukemia, often after a very long latency period . Only a small percentage of infected individuals (about 1% for HTLV) develop cancer. Although the mechanism of disease induction is unclear it is certainly related to the clonal proliferation of infected cells in vivo. Although viral gene expression does not appear to be necessary for maintenance of the disease, evidence suggests that one of the regulatory proteins, Tax, is important in inducing the initial T cell proliferation.How HTLV  and BLV Given the recent development of vectors from lentiviruses, including HIV, it is worth noting that despite intense scientific scrutiny, examples of insertional mutagenesis or gene activation resulting from infection with these viruses have not been documented. However, in the case of HIV-1 the limited lifespan of most infected cells means that this observation must be interpreted with caution.via the provision of promoter or enhancer sequences, but especially the latter, will be the major risk factor for disease induction. In addition, selection of the retroviral envelope used for vector pseudotyping could also potentially play a role as could inadvertent transfer and expression of other retroviral proteins, at least for vectors developed from particular retroviruses, such as Friend virus.In terms of replication defective retroviral vectors, the study of oncogenic retroviruses suggests that oncogene activation, via infection of the epithelial cells of the blood-brain barrier. After these become infected they release virus directly into the CNS where it infects microglial cells, resulting in a spongiform encephalopathy. The SU (env) protein has been shown to be a major determinant of the neuropathogenesis of Cas-Br-E MLV [Several retroviruses cause CNS disease. Some of these, such as the murine retroviruses Cas-Br-E MLV  and FMCFBr-E MLV  and otheBr-E MLV , analogoHTLV causes CNS disease in only a small percentage (about 1%) of infected individuals after a latent period that can be as short as two, or as long as thirty years . The devin vitro and a number of the cytokines induced by HIV infection of monocytes and macrophages also have the capacity to damage neural tissue, either directly or indirectly [Most individuals infected with HIV have virus within the CNS and the route of infection is thought to be transmigration of infected macrophages across the blood-brain barrier. As well as allowing opportunistic infections within the CNS there is a specific condition, AIDS dementia complex (ADC), which is a direct result of HIV infection of the CNS . Within directly .All of the retroviruses that cause CNS disease would appear to do so as a consequence of their active replication. In the case of HIV there is direct evidence for this-treatment of patients with antiretrovirals can significantly decrease the severity of CNS disease . Howevergag gene product, Pr60 Gag, which is not processed normally [The AIDS epidemic has brought a substantial focus to bear on the retroviruses that cause immunodeficiencies in general, and the subset of these that are lentiviruses in particular. Simple retroviruses that cause immune deficiencies in mice , cats 4 and primnormally , is requnormally .via expression of an altered SU (Env) protein. This protein is incapable of causing resistance to superinfection [In cats the simple retroviruses that induce immunodeficiency do so nfection ; as a confection  and immuThe lentiviruses that have been associated with immune deficiency are FIV, SIV and HIV. All appear to share a common pathogenetic mechanism where virus infection of, and replication in, T-cells directly causes cell death, T-cell depletion and immunodeficiency . Cell deAgain, the pathogenetic mechanisms involved with these retroviruses do not have major relevance to replication defective retroviral vectors. However, the pathogenetic mechanisms involved in the murine and feline immunodeficiencies caused by simple retroviruses do reiterate the point that expression of certain retroviral gene products can induce serious pathogenetic states and that this fact may have some relevance to vector design.Apart from the lentiviruses mentioned above that result in immunodeficiency, there are a number of other lentiviral-associated diseases including those caused by caprine arthritis encephalitis virus (CAEV) , equine For all these viruses pathology appears to be intimately linked to viral replication. Therefore, the pathological mechanisms involved are not of direct relevance to replication defective retroviral vectors.Retroviral infection has also been shown to be the cause of wasting and osteopetrosis in birds  and anaeper se did not appear to have an overt pathogenetic affect, rather a T-cell lymphoma eventuated [From the known mechanisms of retroviral pathogenesis discussed above the most obvious pathogenic potential of retroviral vectors is (i) the production of a replication competent virus, and (ii) insertional mutagenesis, specifically oncogene activation. Clearly, the production of replication competent virus not only creates the potential of pathogenetic disease, but will also greatly increase the probability of insertional mutagenesis. In fact, in the one instance where a vector contaminated with a replication competent virus was administered to animals viral replication entuated , presumagag, env and other retroviral genes also has the potential of inducing a pathogenetic state this would appear to depend on the specific retroviral gene sequence in question and to not be of general significance. Even so, minimizing the inadvertent transfer of retroviral gene sequences should clearly be an objective when developing retroviral vectors, not only because of this issue but also because it will have a bearing on the likelihood of replication competent virus being produced and of an endogenous retrovirus being activated.While the inadvertent transfer of In addition, even though oncogene capture by retroviruses is an extremely rare event, the very significant pathogenic potential of the viruses that result means that it should also be taken into consideration during the development of retroviral vector systems.For various reasons, not least of which has been the problem of achieving positive experimental outcomes, only the issue of reducing the probability of replication competent virus arising has been systematically addressed during the development of retroviral vector technology. Indeed, great care has been taken in the development of retroviral vector systems to minimise the chance of producing replication competent retroviruses ,64. Howetrans function plasmids), either with themselves or with endogenous viral sequences in the cell lines used for virus production [cis sequences present in the vector; (iv) the development of SIN vectors; (v) the minimisation of homology between the separate plasmids that make up the system; and (vi) the use of cell lines that do not contain endogenous retroviral sequences with homology to the vector system [via any of these mechanisms appears to be remote. While this doesn't negate the need for appropriate quality control procedures, especially as there is still the remote probability of inadvertent activation of an endogenous retrovirus from the cell line used for virus production, it means that the major safety issue faced by those wishing to use retroviral vectors is that of insertional mutagenesis and oncogene activation.The generation of replication competent virus has, from the very beginning, been seen as the major safety issue for retroviral vectors and this has led to a prolonged effort to develop means of minimising the probability of it arising. There are two principal ways in which replication competent virus can be produced. The first of these is through recombination of the constituent parts of the vector system .The minimization of the inadvertent transfer of retroviral genes to target cells is clearly a worthwhile objective as some of these genes have direct pathogenic potential and they may also influence the probability of endogenous retroviral sequences in the target cell being activated. Generally, the principles applied to the design of vector systems in order to minimize the probability of RCR being produced will also minimize the probability of inadvertent retroviral gene transfer. However, as the production of RCR requires multiple recombination events more effort should be made to analyse the rate of transfer and expression of individual retroviral gene sequences by vector systems. It is clear that the rate of individual gene transfer is much higher than the rate of RCR generation and can occur at a significant frequency even in highly evolved systems where RCR cannot be detected . This suThe mechanism of oncogene capture appears to be dependent on the generation of a chimeric retroviral-oncogene transcript (69, 70). This suggests that the risk of oncogene capture will be related to the efficiency of termination/polyadenylation of the proviral transcript and that this should be considered and assessed in the process of vector development, especially as retroviral polyadenylation sequences are often relatively inefficient, perhaps reflecting the necessity for the polyadenylation signal to be inactive in the context of the 5' LTR. However, in transient virus production systems, where the transfected vector plasmid presumably remains either entirely, or almost entirely extrachromosomal, this mechanism would appear to preclude the probability of oncogene capture. In the case of stable producer cell lines there is clearly an argument for categorizing the integration site of the vector sequence and discarding any clones where this is in a known or suspected oncogene.The adverse events that have been observed in animal experiments and clinical trials reinforce the conclusions discussed above, that replication competent virus  and inseIn terms of the vector technology used on the two occasions where oncogene activation has been observed the following comments can be made:via splicing. MoMLV is a strongly oncogenic, non-defective virus that causes B-and T-cell lymphomas and leukemias in mice. As with other non-defective oncogenic retroviruses the primary determinant of its pathological properties is the long terminal repeat enhancer. MoMLV has been shown to induce oncogenesis via activation of any one of a number of different cellular genes .1) The vector is derived from MoMLV and uses the LTR sequence to drive the transgene 2) The vector LTR is used to control transcription of the transgene. In the case of the X-SCID trial there is a strong selective pressure for gene corrected cells and accordingly there will clearly be an equally strong selection for transduced T-cell clones in which the LTR is active.3) The PHSC is notoriously difficult to transduce with oncogenic retroviral vectors and the protocol used was designed to enhance transduction by using multiple cytokines to stimulate division of PHSC. This is likely to induce many genes involved in regulating cell growth. As retroviruses preferentially integrate into active gene sequences, this would increase the number of growth regulating genes accessible as targets for provirus integration and hence promiscuous, unregulated activation. Specifically, LMO2, the oncogene activated in the X-SCID trial, is normally expressed in primitive haematopoietic cells (the target for gene transfer) but not in mature cells (80). Therefore, it will be accessible for proviral integration during the transduction process and its continuing expression in maturing T cells generated from gene corrected precursors is biologically inappropriate.The problems that occurred in this X-SCID trial, their broader relevance and possible answers, have all been reviewed from a number of aspects ,73,77. HGiven what is known about retroviral mediated insertional mutagenesis it is surprising that more attention has not been paid to the technology used in many of the retroviral mediated gene therapy animal studies and human trials. With hindsight, it seems that the technologies used were selected on the basis of efficacy, not safety, that is achieving adequate gene expression took preference over consideration and assessment of insertional mutagenesis. However, given the technical difficulties involved in developing a workable protocol this is not surprising, and it is a pre-occupation that was, and is, shared by all gene therapy researchers.Possible technological approaches that would appear to provide answers to these issues include:1) The use of self-inactivating (SIN) vectors would make a major difference in that the provirus would lack all U3 enhancer sequences, negating the ability of the LTR to activate cellular genes. The vector should also not contain active splice signals. However, given the ability of SIN vectors to be repaired at a significant rate during virus production (see below) careful selection of the retrovirus used to build the vector backbone is also important if this risk is to be minimised. Clearly the construction of vectors from non-oncogenic retroviruses and the development of more effective (i.e. less prone to LTR repair) SIN vectors is warranted. If SIN vectors are to be used the transgene must be expressed from an internal promoter which must also be presumed to have the potential for oncogene activation. Therefore it would be preferable to use a promoter without highly active enhancer elements. In addition, the wisdom of incorporating matrix/scaffold attachment regions into vectors to increase expression may also be contraindicated as these sequences have long-range enhancer like properties (81). If high levels of gene product are required, consideration should be given to other means to enhance transgene expression, such as codon-optimisation of coding sequences.trans-activate adjacent promoters. The different integration specificities of lentiviral  and oncogenic (promoter adjacent in active gene sequences) retroviruses and vectors [2) Vectors should be developed from non-oncogenic retroviruses. The recent development of vectors from HIV-1 and other lentiviruses for unrelated reasons (predominantly their ability to transduce non-cycling cells) means that this has already happened. The Tat dependence of the HIV-1 LTR may also provide an extra measure of safety as long as Tat is not transferred along with the vector. However, the enhancing properties of the HIV-1 LTR in the presence and absence of Tat needs to be carefully defined in order to test the assumption that the HIV-1 LTR lacks the ability to  vectors  also giv3) The incorporation of strong transcription termination/polyadenylation signals and gene isolator sequences (83) may provide another means to reduce the possibility of adjacent genes being activated. These sequences should also reduce the probability of oncogene capture in virus producer cells. However, the incorporation of insulator sequences appears to lead to a significant loss of vector titre (84).4) When the transgene plays a role in regulating cell growth, extra consideration should be given to using the relevant control signals from the gene in question to regulate expression of the transgene.5) Although the PHSC is theoretically a very attractive target for gene transfer it is extremely difficult to transduce with retroviral vectors derived from oncogenic viruses such as MoMLV. Although efficient transduction of human PHSC can now be achieved this requires exposure to multiple cytokines over a relatively long culture period. The potential of new retroviral vectors derived from lentiviruses (85) and spumaviruses (86) to transduce PHSC with shorter exposure to less cytokines needs to be fully explored.+ cells were infused into these patients. Therefore, it would appear the patient is exposed to a much greater number of transduced cells than is theoretically necessary to produce the desired result. In other words, the process of generating gene corrected T cell clones by transduction of CD34+ cells is very inefficient.6) In general the limitations of vectors should be taken into account when designing gene therapy protocols. For example, in the case of X-SCID, it may be just as efficacious to target a more committed T-cell precursor that can be transduced more easily, and without biological manipulation using multiple cytokines. Alternatively, if the PHSC is to be targeted as highly enriched a PHSC population as possible should be used in order to expose the patient to the minimum number of transduction events compatible with the desired outcome. In the two X-SCID patients who developed T cell leukemia, molecular analysis of samples collected before the appearance of malignant disease showed the presence of >50 γc transduced T cell clones. Approximately 14 to 20 million transduced CD34both LTRs of the provirus. As the transcriptional enhancer elements in the 3' LTR are redundant in the context of a retroviral vector construct they can theoretically be deleted without affecting vector performance. After transduction of the target cell both LTRs are deleted and are transcriptionally silent. Although this requires that an internal promoter is used to control expression of the transgene, and makes it more difficult to generate high titre stable packaging cell lines, the advantages of the approach are obvious. However, SIN vectors have not been widely used in the case of oncogenic retroviral vectors, principally because viral titres were low, because of high rates of repair of the SIN deletion [With hindsight SIN vectors ,87,88 nodeletion ,89 and bdeletion . Subsequdeletion  but thisIn contrast, SIN vectors have been widely adopted in the lentiviral vector field ,65,92,93The most important determinant of the safety of retroviral vectors remains ensuring they are free of replication competent retrovirus of any sort. Clearly, the technologies available for the production of vector virions would appear able to preclude the production of replication competent virus by recombination of the constituent parts of the vector system (i.e. vector and helper plasmids) with a very high degree of certainty. However, production of replication competent virus from the cell lines used for virus production remains a theoretical possibility and more work needs to be done on generic assays for replication competent retroviruses.gag, env (SU) and other retroviral gene sequences would appear to of relevance and needs to be specifically addressed in the development of vector systems.Apart from the issue of replication competent virus, analysis of the pathologies associated with retroviruses, and the results of the X-SCID trial, demonstrate that careful attention must be paid to the ability of sequences in retroviral vectors to activate transcription of genes adjacent to proviral integration sites. Although the use of SIN vectors will greatly reduce the risk of such events, given the predilection of current SIN vectors to be repaired during virus production these vectors need to be further developed, especially for vectors derived from strongly oncogenic viruses. In addition, inadvertent transfer to, and expression in, transduced cells of As both oncogenic (MoMLV derived) and lentiviral (HIV-1 derived) vectors have been shown to preferentially integrate into transcribed sequences it would appear logical that the likelihood of proviral integration near cellular genes involved in the positive regulation of cell growth would be increased in actively growing cell populations. This suggests that the use of transduction protocols that target non-cycling cells, or cells that are subjected to the minimum of stimulatory signals as is compatible with efficient gene transfer, would be greatly advantageous in terms of minimising the risk of malignant events after the stimulatory signals are removed.With hindsight, the observation of malignant events induced by replication defective MoMLV retroviral vectors is not surprising although the frequency of these events in the X-SCID gene therapy trail certainly was. The concern is that these events will now cause a significant backlash against the use of all retroviral vectors while the real message is that we need to make better use of the knowledge we now have in terms of designing vectors and gene therapy protocols. Clearly, the known oncogenic potential of MoMLV and its relationship to viral sequences has, for one reason or another, been largely ignored to date. Indeed, most of the retroviral vectors used in trials to date are based on MoMLV and contain an intact 5' LTR. While the historic reasons for this are obvious we now need to evaluate and adopt more appropriate technologies as rapidly as possible.et al [There are several obvious conclusions to be drawn from the X-SCID trial and the results of Li et al . The firet al ,86,95-97Regulatory authorities also have a role to play. Clinical trials are based on extensive preclinical experimentation and animal trials that take many years to complete. Clearly, the particular vector system that has been used to develop a protocol may no longer be the best to use ten years later when clinical trials become a reality. The question is, then, how can the regulation of clinical trials be made flexible enough to allow the introduction of new and improved vector technology late in the process?In conclusion, retroviral mediated gene transfer remains an extremely attractive option for gene therapy when the stable and permanent genetic modification of the target cell is optimal. However, we must take greater care, and utilise more resources, for the pro-active, rather than reactive, refinement and testing of the basic technology that is used for gene therapy and for the adoption of improved vector systems if adverse events are to be minimised.


The autonomic nervous system (ANS) plays an important role in the genesis and maintenance of atrial fibrillation (AF), but quantification of its electrophysiologic effects is extremely complex and difficult. Aim of the study was to evaluate the capability of linear and non-linear indexes to capture the fine changing dynamics of atrial signals and local atrial period (LAP) series during adrenergic activation induced by isoproterenol (a sympathomimetic drug) infusion.Nine patients with paroxysmal or persistent AF (aged 60 ± 6) underwent electrophysiological study in which isoproterenol was administered to patients. Atrial electrograms were acquired during i) sinus rhythm (SR); ii) sinus rhythm during isoproterenol (SRISO) administration; iii) atrial fibrillation (AF) and iv) atrial fibrillation during isoproterenol (AFISO) administration. The level of organization between two electrograms was assessed by the synchronization index (S), whereas the degree of recurrence of a pattern in a signal was defined by the regularity index (R). In addition, the level of predictability (LP) and regularity of LAP series were computed.SR = 0.75 ± 0.07 RSRISO = 0.69 ± 0.10, p < 0.0001; RAF = 0.31 ± 0.08 RAFISO = 0.26 ± 0.09, p < 0.0001; LPSR = 99.99 ± 0.001 LPSRISO = 99.97 ± 0.03, p < 0.0001; LPAF = 69.46 ± 21.55 LPAFISO = 55 ± 24.75; p < 0.0001). Electrograms analysis shows R index reductions both in SR (RSR = 0.49 ± 0.08 RSRISO = 0.46 ± 0.09 p < 0.0001) and in AF (RAF = 0.29 ± 0.09 RAFISO = 0.28 ± 0.08 n.s.).LAP series analysis shows a reduction of both LP and R index during isoproterenol infusion in SR and AF (RThe proposed parameters succeeded in discriminating the subtle changes due to isoproterenol infusion during both the rhythms especially when considering LAP series analysis. The reduced value of analyzed parameters after isoproterenol administration could reflect an important pro-arrhythmic influence of adrenergic activation on favoring maintenance of AF. Atrial Fibrillation (AF) results from multiple, rapidly changing and spatially disorganized activation wavelets sweeping across the surface of the atria . Among fIn normal hearts, both vagal and sympathetic mechanisms have been associated with paroxysmal atrial fibrillation (PAF) initiation. Most of PAF episodes observed in patients with structural heart disease are triggered by sympathetic activation and vagal withdrawal .Spectral analysis of heart rate variability before PAF episodes has further clarified the pro-arrhythmic role of the autonomic nervous system ,10. BettEven if AF has been classically described as a random process, a few studies have recently documented, using various signal processing methods, the existence of some determinism underlying AF. Linear analysis techniques documented relationships between intra-atrial recordings using both time-domain methods  and specNine patients  selected to sustain a left atrial ablation with encirclement of the pulmonary veins by transeptal approach were included in the study. All subjects were suffering from atrial fibrillation (AF) and were non responsive to anti-arrhythmic therapy . Paroxysmal and persistent AF episode were present in, respectively, 5 and 4 subjects. A history of AF was present for an interval ranging from 2 months to 10 years. The mean left ventricular ejection fraction was > 40% in all patients; the mean left atrial diameter was 37 ± 3 mm in 7 patients and 51 ± 8 mm in 2. Structural heart disease was present in 4 patients. Reported symptoms included palpitations (6 subjects), fatigue after effort (9 subjects) and syncope (2 subjects). Arterial hypertension was the most common comorbidity in our study group (4 patients). All the patients were in anti-arrhythmic drug wash-out at the time of the study. Flecainide, propafenone, metoprolol, cordarone and methyldigoxin were ceased ≥ 5 half-lives before ablation. Transoesophageal echocardiography was performed the day before the procedure to exclude atrial thrombus.The Medical Ethical Committee approved this study and all subjects gave their written consent.We investigated the effect of adrenergic activation induced by infusion of isoproterenol on atrial electrical activity. The electrophysiological procedure was performed in the Electophysiology Laboratory of the "Istituto Clinico Sant'Ambrogio" of Milan, Italy. Intracavitary electrocardiograms were recorded during the ablation procedure in which arrhythmic foci inside the pulmonary veins of the left atrium were electrically isolated.The research project protocol included an intracavitary recording of multiple atrial electrograms during sinus rhythm and after induction of atrial fibrillation. In both experimental conditions, the recording was repeated during intravenous infusion of isoproterenol (0.01–0.02 mcg/kg/min) tiered to determine a 30% increase of heart rate. The four clinical experimental conditions were defined as sinus rhythm (SR), sinus rhythm during isoproterenol administration (SRISO), atrial fibrillation (AF) and atrial fibrillation during isoproterenol administration (AFISO). Details on the four epochs of the study can be found in Figure In all the patients, AF inducibility was obtained at twice diastolic threshold by burst atrial pacing (5'-second bursts at an output of 20 mA) from the mid coronary sinus beginning at a cycle length of 250 ms and reducing by 10 ms intervals until atrial refractoriness. All the nine patients were inducible. AF was considered inducible if it persisted for more than 1 minute. If AF terminated after less than 1 minute, induction was repeated until a maximum of 3 times. If AF became sustained (lasting > 10 minutes), ablation was performed after external DC cardioversion. All our patients underwent the procedure in spontaneous SR.The duration of registration was an important parameter for the reliability of the analysis, because an insufficient number of atrial potentials (less than 250 – 300) could give errors in the estimation of conditioned probability. Therefore at least 5 minutes (range 5 – 8 minutes) of sinus rhythm and 90 seconds (range 60 – 120 seconds) of atrial fibrillation were registered.St Jude catheter (length 95 cm, 7 F, interelectrode spacing 2 – 10 mm), a deflectable decapolar catheter with a distal ring configuration, Lasso-Cordis Biosense Webster catheter (length 115 cm, 7 F, interelectrode spacing 2 – 5 mm) and 4 mm distal electrode catheter, Medtronic Sprinklr, with irrigated tip (for ablation, length 115 cm, 7 F, interelectrode spacing 2 – 5 mm). The St Jude catheter was in contact with the right atrial wall and inserted in the coronary sinus below the left atrium. The Lasso-Cordis Biosense Webster catheter and the Medtronic Sprinklr catheter were positioned in the superior pulmonary veins at the inside of the atrium.The electrophysiological study was carried out using a deflectable 20 pole For the purpose of this study, one surface ECG tracing and nine intracavitary atrial electrograms were stored on digital memory for subsequent off line analysis. In all patients, electrograms labeled 2 – 3 – 4 – 5 corresponded, respectively, to the superior, middle, middle inferior and inferior wall of the right atrium; electrogram 6 to coronary sinus ostium; electrograms 7 – 8 – 9 indirectly corresponded to the inferior and the left wall of the left atrium whereas electrogram 10 to the left superior pulmonary vein.St Jude catheter, electrogram 10 with Lasso-Cordis Biosense Webster catheter.Electrograms 2 – 9 were recorded with 20 pole CE) may be used to estimate a regularity index, defined as the degree of recurrence of a pattern in a signal. CE represents the amount of information carried by the most recent sample x(i) of a normalized realization of x when its past L - 1 samples are known. CE is defined as [Conditional entropy (fined as :p(xL - 1) represents the probability of the pattern xL - 1 (i - 1) of length L - 1 (xL - 1 (i - 1) = {x(i - 1),..., x(i - L + 1)}) and p(x(i) / xL - 1) the conditional probability of the sample x(i) given the pattern xL - 1. In (1) the first summation is extended to all the possible xL - 1 patterns, the second one is extended to all the different Lth samples of the pattern xL (i) (xL (i) = {x(i), xL - 1 (i - 1)}).where CE is maximum if x is complex and unpredictable and it reaches zero as soon as a new sample can be exactly predicted from the previous L -1 ones.CE over short data series can cause an unreliable estimate of CE (CÊ) : when the conditioning pattern xL - 1 (i - 1) is found only once in the series x (i.e. p(x(i) / xL - 1) = 1), CÊ decreases to zero with L. As a consequence both periodic and completely unpredictable signals exhibit CÊ equal to zero when L increases. Therefore the corrected conditional entropy (CCE) must be introduced to perform a reliable measure over short data series:Using CCE(L) = CÊ(L) + perc(L)·Ê(1)     (2)perc(L) is the percentage of length L patterns found only one time in the data set and Ê(1) is the estimate of Shannon entropy of the process x. perc(L)·Ê(1) represents the corrective term that compensates the null information associated to the pattern found only once and it increases with L, while CÊ(L) decreases with L.where CCE is the best estimate of CE and it's taken as an index of complexity: the larger the index, the less predictable the processes. The CCE is normalized by the Shannon entropy of the process in order to derive an index independent of the different probability distribution of the processes, thus obtaining:The minimum value of the An index of regularity (the opposite of complexity) may be defined as:Rx = 1 - min(NCCE(L))     (4)Rx tends to zero if x is a fully unpredictable process, it tends to one if x is a periodic signal and it assumes intermediate values for those processes that can be partially predicted by the knowledge of the past samples [ samples .The cross-conditional entropy is introduced to define an index of synchronization, related to the repetition of a complex pattern involving two signals.x given a pattern y is defined as [Given two normalized signals, the cross-conditional entropy of fined as :p(yL - 1) represents the probability of the pattern yL - 1 (i) and p(x(i) / yL - 1) the conditional probability of the sample x(i) given the pattern yL - 1.where CEx/y represents the amount of information carried by the most recent sample of the signal x when L - 1 past samples of y are known. Over short data series, this definition suffers from the same limitations as conditional entropy, so analogously corrective terms and normalization are introduced. The uncoupling function (UF) is defined as:UFx,y(L) = min(NCCEy/x(L), NCCEx/y(L))     (6)L pattern, UF chooses as input the signal that can be the best predictor of the other one. Besides, the joint pattern does not take into account past samples of the output signal to prevent to have a high coupling strength only because one signal has a large index of regularity.in order to measure the amount of information carried by one signal that can't be derived from the knowledge of past samples of the other signal. In this way both causal directions are tested and it is taken the one that leads to the best prediction. For every length UF is taken as an index of uncoupling between x and y, therefore an index of synchronization (the opposite of uncoupling) can be defined as:The minimum of Sx,y = 1 - min(UFx,y (L))     (7)Sx,y tends to zero if the two processes are uncoupled, it tends to one if they are perfectly synchronized and it assumes intermediate values when the two signals are able to exchange a certain amount of information [and it quantifies the maximum amount of information exchanged between the two signals. ormation .x(n) can be modeled as the output of an autoregressive model of p orderA discrete time series n is the discrete-time index, the ak are the model coefficients and w(n) is a Gaussian white noise process of variance  feeding the model. The actual sample differs from its model prediction, thus generating the prediction errorwhere LP) may be defined as followsAn index of the level of predictability (LP = (1 - σe / σx)     (10)σe is the standard deviation of e(n) and σx is the standard deviation of the process x. LP measures the percentage of power which may be predicted by the autoregressive model. In the case of a purely random signal (σe is quite close to σx) LP tends to zero, while in the case of a linearly predictable signal (σe tends to zero) the index tends to one and it assumes intermediate values for those processes that may be partially predicted from the model.where All signals were appropriately recorded and digitized to a 1000 Hz sampling rate at 16-bit resolution. All bipolar electrograms were band-pass filtered (40 – 250 Hz) to remove baseline shift and high frequency noise. In order to cancel the possible effects of ventricular interference , the averaged ventricular interference complex was computed and subtracted from each atrial signal [After detecting time-instants of local atrial depolarization using a derivative / threshold algorithm, the detected depolarizations were visually scored and missed / erroneous detections were corrected by an expert operator using an interactive software. Then, the local atrial period (LAP) series were derived as the sequence of temporal distances between two consecutive local atrial activations  the pro.In analogy with previous studies ,23, afteConsidering the number of recorded signals and patients, we analyzed 79 recordings in SR and in SRISO and 77 recordings in AF and in AFISO. Twelve recordings were disregarded for the low quality of electrograms. For each recording, atrial signals were divided into six-second segments and then analyzed. Regularity index was estimated for each six-second segment in each recording site and for each patient, while synchronization index was estimated for each pair of close recording sites .t-test for paired data, comparing each rhythm before and after isoproterenol administration, and between organized (SR) and not organized rhythm (AF).The statistical analysis was carried out using Student's Figure Concerning the synchronization index (S), a significant decrease was observed only when comparing sinus rhythm to atrial fibrillation Table . EvaluatNCCE function. The regularity (R = 1 - min(NCCE)) decreases visibly passing from sinus rhythm to atrial fibrillation. A decrease in both SR and AF after isoproterenol administration is also observed. The mean values showed in Table LAP series were analyzed using the level of predictability and the regularity index. Figure Figure The autonomic nervous system plays an important role in the genesis and maintenance of atrial fibrillation, but characterization and quantification of its pro-arrhytmic effects are extremely complex and therefore difficult to define. Aim of this study was to evaluate the capability of linear and non-linear parameters to capture the fine changing in the dynamics of atrial signals and LAP series during adrenergic activation induced by the injection of a sympathomimetic drug. The existence of determinism and of an underlining order during AF has recently been shown. In particular both linear -16 and nConcerning results obtained from atrial signals, both synchronization and regularity indexes showed a marked reduction passing from SR to AF well in keeping with previous findings  that doc


Psychiatric sleep research has attempted to identify diagnostically sensitive and specific sleep patterns associated with particular disorders. Both schizophrenia and alcoholism are typically characterized by a severe sleep disturbance associated with decreased amounts of slow wave sleep, the physiologically significant, refreshing part of the sleep. Antisocial behaviour with severe aggression, on the contrary, has been reported to associate with increased deep sleep reflecting either specific brain pathology or a delay in the normal development of sleep patterns. The authors are not aware of previous sleep studies in patients with both schizophrenia and antisocial personality disorder.The aim of the present case-study was to characterize the sleep architecture of a violent, medication-free and detoxified female offender with schizophrenia, alcoholism and features of antisocial personality disorder using polysomnography. The controls consisted of three healthy, age-matched women with no history of physical violence. The offender's sleep architecture was otherwise very typical for patients with schizophrenia and/or alcoholism, but an extremely high amount of deep sleep was observed in her sleep recording.The finding strengthens the view that severe aggression is related to an abnormal sleep pattern with increased deep sleep. The authors were able to observe this phenomenon in an antisocially behaving, violent female offender with schizophrenia and alcohol dependence, the latter disorders previously reported to be associated with low levels of slow wave sleep. New studies are, however, needed to confirm and explain this preliminary finding. Female violent behaviour has been studied less than that of men. This is partly because women commit fewer crimes than men , but alsHuman sleep consists of two main components: rapid eye movement (REM) sleep, and non-REM sleep, the latter divided into stages 1–4 (S1–S4). Stage 3 sleep (S3) and stage 4 sleep (S4) in non-REM sleep are defined as slow-wave sleep (SWS), also called delta sleep or deep sleep. In normal sleep, REM and non-REM sleep periods alternate cyclically. Though the exact functions of the different sleep stages are unknown, it is generally accepted that SWS is the physiologically significant, refreshing part of sleep. Feelings of unwellness, either somatic or psychiatric, are frequently associated with decreased SWS.Serious sleep disturbances are associated both with schizophrenia and alcoholism. Typical findings in both disorders are long sleep latency, reduced sleep length, long periods of waking after sleep onset, abnormalities in REM parameters as well as decreased SWS -12. The The aim of the present study was to characterize the sleep architecture in a severely violent, medication-free, detoxified woman with schizophrenia, alcoholism and a history of attempted homicide. She is the first subject in a larger study concerning sleep structure in women with this type of comorbidity.The patient was a 23-year-old woman charged with attempted manslaughter. She was ordered by the court to undergo a pre-trial forensic psychiatric examination which took place in a maximum security state mental hospital. The trial records and all available background information were reviewed. Both of her parents were alcoholics and she was taken to protective custody under the age of one year. Her grandmother suffered from schizophrenia and her mother had been in a psychiatric hospital after a suicide attempt. The patient used to shoplift before the age of 15 and later she stole money from time to time. She also worked as a prostitute to earn money. She started to abuse alcohol at the age of 15. She had several boyfriends but has never married and has no children. Her personality was noticed to change before the age of 17. She started to have obsessive-compulsive behaviour, paranoid thoughts as well as depressive symptoms. She tried to kill herself by hanging and by several drug intoxications. She brutally killed her own pet. She was never hospitalized before the forensic mental examination but she irregularly visited an outpatient clinic. Despite psychiatric treatment, she impulsively tried to kill her male friend with whom she was drinking. During the psychiatric examination the diagnoses – schizophrenia paranoid type, alcohol dependence and features of antisocial personality disorder – were made by a senior forensic psychiatrist using the structured clinical interview for DSM-IV, SCID I and II ,17. NeitThe control group consisted of three 23-year-old female students without criminal records or a history of physical violence. They were healthy with no signs of somatic, psychiatric, or neurological disorders. As part of a psychiatric interview, the SCID-non-patient version  was fillWritten consent was obtained from all participants after the study procedure had been fully explained to them. The study was approved by the local human ethics committee.Polysomnography (PSG) was recorded over two consecutive nights but only the second night was considered for the study. The study patient slept in a single room within the department, while controls slept in the hospital guest room. All participants were allowed to sleep as long as they wanted to. Recordings took place on an ambulatory basis; the participant had a portable recording device (Embla, Flaga hf, Reykjavik, Iceland) that was connected to the recording electrodes. The recordings were performed using the standard Rechtschaffen-Kales method . The higA sleep diary was used for one week during the study period to ensure a normal sleep-wake rhythm and to exclude the effects of daytime naps. The participant filled in the time of retiring to bed, estimated time of falling asleep and time of awakening in the morning for each consecutive night as well as daytime naps.The patient's PSG recording with reduced sleep length, long sleep latency, and a high number of awakenings after sleep onset as well as reduced sleep efficiency Table  is very The finding concerning SWS is completely contrary to previous sleep reports in patients with schizophrenia as well as with alcohol dependence. The patient had abstained from alcohol for several months before the PSG, and this raises the question if the result could be explained with a withdrawal state. However, reduced SWS is also associated also with alcohol withdrawal and the phenomenon has been reported to be extremely long lasting . Even afMRI disclosed no post-traumatic signs in the brain substance of the patient. Waking EEG was also within normal limits. Minor head injuries (concussions) are, however, frequent events among people with alcoholism. Kaufman et al.  demonstrThe patient had a history of violent acts and her lifestyle was described to be antisocial and unstable and she was diagnosed as having features of antisocial personality disorder in addition to schizophrenia and alcohol dependence. The authors are not aware of previous sleep studies in patients with both schizophrenia and antisocial personality disorder. In a PSG study among habitually violent male offenders with antisocial personality disorder as a primary diagnosis, increased amounts of SWS and S4 sleep were observed as compared with age-matched healthy men , which iAll patients with schizophrenia should not be considered to be violent, although there are minor subgroups of schizophrenic patients among whom the risk for violence is remarkably high. It has been estimated that this increase in risk could be associated with the paranoid form of schizophrenia, coexisting substance abuse and antisocial behaviour - the disIncreased deep sleep has been associated with antisocial behaviour with severe aggression. The authors were able to observe this phenomenon in an antisocially behaving, violent female offender with schizophrenia and alcohol dependence, the latter disorders previously reported to be related to low levels of SWS. New studies are, however, needed to confirm and explain this preliminary finding.The author(s) declare that they have no competing interests.This manuscript was prepared by a multidisciplinary team consisting of:NL, generated the idea for this case report, interviewed the controls, scored the sleep recordings, and prepared the manuscript together with the team.PT, had a substantial contribution in theoretical background and processing of the present study as expert in sleep researchPT, reviewed all material concerning the patient as expert in forensic psychiatryES, participated in processing of the manuscript as expert in forensic psychiatryHP, participated in the processing of the manuscript as expert in forensic psychiatry and female violenceME had a central role in planning the study design as well as in formulating the theoretical background of the present study. He also allocated financial resources to this project and helped solving practical problems of the study projectMV, supervised and participated with great impact in all stages of this manuscriptThe pre-publication history for this paper can be accessed here:


Dengue is the most important arthropod-borne viral disease of public health significance. Compared with nine reporting countries in the 1950s, today the geographic distribution includes more than 100 countries worldwide. Many of these had not reported dengue for 20 or more years and several have no known history of the disease. The World Health Organization estimates that more than 2.5 billion people are at risk of dengue infection. First recognised in the 1950s, it has become a leading cause of child mortality in several Asian and South American countries.This paper reviews the changing epidemiology of the disease, focusing on host and societal factors and drawing on national and regional journals as well as international publications. It does not include vaccine and vector issues. We have selected areas where the literature raises challenges to prevailing views and those that are key for improved service delivery in poor countries.Shifts in modal age, rural spread, and social and biological determinants of race- and sex-related susceptibility have major implications for health services. Behavioural risk factors, individual determinants of outcome and leading indicators of severe illness are poorly understood, compromising effectiveness of control programmes. Early detection and case management practices were noted as a critical factor for survival. Inadequacy of sound statistical methods compromised conclusions on case fatality or disease-specific mortality rates, especially since the data were often based on hospitalised patients who actively sought care in tertiary centres.Well-targeted operational research, such as population-based epidemiological studies with clear operational objectives, is urgently needed to make progress in control and prevention. Aedes aegypti and Aedes albopictus mosquito species. Recovery from infection by one serotype provides lifelong immunity against that serotype but confers only partial and transient protection against subsequent infection by the other three. First recognised in the 1950s, it has become a leading cause of child mortality in several Asian and South American countries.Dengue is the most important arthropod-borne viral disease of public health significance. Compared to nine reporting countries in the 1950s, today the geographic distribution includes more than 100 countries worldwide. Many of these had not reported dengue for 20 or more years and several have no known history of the disease. The World Health Organization (WHO) estimates that more than 2.5 billion people are at risk of dengue infection. Most will have asymptomatic infections. The disease manifestations range from an influenza-like disease known as dengue fever (DF) to a severe, sometimes fatal disease characterised by haemorrhage and shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is on the increase. Dengue fever and dengue haemorrhagic fever/dengue shock syndrome are caused by the four viral serotypes transmitted from viraemic to susceptible humans mainly by bites of The average number of DF/DHF cases reported to WHO per year has risen from 908 between 1950 and 1959 to 514,139 between 1990 and 1999. The real figure is estimated to be closer to 50 million cases a year causing 24,000 deaths. Of an estimated 500,000 cases of DHF/DSS requiring hospitalisation each year, roughly 5% die according to WHO statistics. Regional distribution of dengue and its serotypes are described elsewhere [Half the world's population lives in countries endemic for dengue, underscoring the urgency to find solutions for dengue control. The consequence of simple DF is loss of workdays for communities dependent on wage labour. The consequence of severe illness is high mortality rates, since tertiary level care required for DHF/DSS management is beyond the reach of most of the persons at risk.This paper reviews the changing epidemiology of the disease, focusing on host and societal factors and drawing on national and regional journals as well as international publications. It does not include vaccine and vector issues. Although each one of the issues taken up below merits an independent, in-depth treatment, we have selected only those issues where the literature raises challenges to prevailing views and therefore require further research, particularly given that most of these issues are key for improved service delivery in poor countries.Dengue infection can cause a spectrum of illness ranging from mild, undifferentiated fever to illness up to 7 days' duration with high fever, severe headache, retro-orbital pain, arthralgia and rash, but rarely causing death. Dengue Haemorrhagic Fever (DHF), a deadly complication, includes haemorrhagic tendencies, thrombocytopenia and plasma leakage. Dengue Shock Syndrome (DSS) includes all the above criteria plus circulatory failure, hypotension for age and low pulse pressure. DHF and DSS are potentially deadly but patients with early diagnosis and appropriate therapy can recover with no sequelae. Case management for DF is symptomatic and supportive. DHF requires continuous monitoring of vital signs and urine output. DSS is a medical emergency that requires intensive care unit hospitalisation .The increase in dengue mortality is considered to be a reflection of the increase in the proportion of DF patients who develop DHF/DSS. The pathogenesis of DHF/DSS is widely considered to be antibody-dependent enhancement in secondary infection with a virus of different serotype . Evidencet al., and Libraty et al. [Alternative or additional factors associated with severe illness, such as high viraemia titres, have also been suggested . So far,y et al. ,12. On tViral virulence , immunolThe evidence from different studies also shows that the pathogenesis of DHF/DSS may be multi-factorial and understanding remains incomplete.Demographic, economic, behavioural and social factors are often keys for effective communicable disease control and underpin successful public health programmes. Despite promising indications in the literature, these factors have remained poorly understood in the case of dengue. Furthermore, recent field evidence raises some questions regarding widely accepted characteristics of dengue that need review and confirmation.DF is typically acknowledged to be a childhood disease and is an important cause of paediatric hospitalisation in southeast Asia. There is, however, evidence of increasing incidence of DHF among older age groups. Since the early 1980s, several studies in both Latin America and southeast Asia have reported a higher association of DHF with older ages. The earliest studies were by Guzmán (1981) in Cuba and Rigau-Pérez in Puerto Rico ,19. LateThree studies in Asia using surveillance data report increasing age of infected patients. In Singapore, surveillance data showed a shift in peak dengue mortality from paediatric ages (1973–1977) to adults in 1982, since which year more than 50% of the deaths occurred in patients older than 15 years. From 1990–96, the highest age-specific morbidity rates were in the 15 to 34 year age groups . In IndoHospital-based studies have similarly reported increasing infection rates among adults, mentioning that it is contrary to the popular belief that dengue is a paediatric disease ,28. The Race-related susceptibility to dengue has been observed in a few studies and merits further investigation. In a retrospective seroepidemiologic study Guzmán reported that blacks and whites were equally infected with DEN-1 and DEN-2 viruses during the Cuban epidemics of 1977 and 1981, while severe dengue disease was observed less frequently in dengue-infected black persons than whites ,6.A study in Haiti observed that despite virologic pre-conditions hypothesised to be precursors for DHF (i.e. the evidence of previous infection by DEN virus types 1, 2 and 4), local children did not develop severe illness . The autGenetic polymorphism in cytokine profiles and coagulation proteins has been proposed as a factor protecting persons of African origin . EvidencIn Asia, two studies report racial differences in disease incidence. A 15-year study of the epidemiology of dengue reports a significantly higher incidence of DHF among Chinese compared to Malaysian males . This fiUnderstanding male-female differences in infection rates and severity of disease is important for public health control programmes. A few hospital-based studies and surveillance data show a male-female difference in infection rates and in severity of disease. Three independent studies from epidemics in India and Singapore found nearly twice the number of male patients compared to females  -35. In hStudies in South America generally report that both sexes are equally affected ,38 althoOf significance are two studies in Asia by Kabra and Shekhar where severe illness and CFR were consistently higher among females despite higher incidence in males ,40. HalsNo studies suggest gender bias in home care and male preferences in health care seeking, still prevalent in many Asian and other traditional societies. It is widely recognised that in many of the Asian communities, lower disease incidence in women may be a statistical artefact related to lower reporting and care-seeking for women from traditional practitioners who do not report to public surveillance systems. By the same token, women are less likely to be taken for care at a hospital when ill or are taken at late stages of disease, when no other options are available. Determining sex differences, both in infection and severity of disease, requires well-designed and targeted studies to capture both biological and social factors that drive disease patterns in a community.Historically, DF/DHF has been reported as occurring predominantly among urban populations where density of dwellings and short flying distance of the vector create the right conditions for transmission. However, the literature shows that dengue transmission and, in some cases, outbreaks occur in rural settings in both Asia and Latin America. In the WHO Western Pacific region, WHO has confirmed that disease spread into rural areas from where it had not been reported previously .Aedes aegypti, both in rural and urban areas during an outbreak in Gujarat in 1988 and 1989 [Rural epidemics occurred as early as 1976 in Indonesia, and in 1994 the outbreak in Laos began in a remote, rural district of Nasaithong ,44. Todaand 1989 . Increasand 1989 .Aedes aegypti mosquito has not been seen in Hawaii since it was supposedly eradicated by pesticide spraying in 1943 [In industrialised settings, the Centers for Disease Control and Prevention (CDC) reported an outbreak of DF among residents of the rural towns of Hana and Nahiku in Hawaii in 2001. The outbreak was historically unusual because infection occurred among residents who have no history of recent travel and the  in 1943 .Increased transport contact, mobility and spread of peri-urbanisation have been the most frequently cited reasons for spread of dengue to rural areas . While sThe incidence and, in particular, epidemics of dengue have been commonly associated with the rainy season, and the El Niño phenomenon has been incriminated in the increases of certain vector-borne diseases, including dengue ,50.Despite the number of studies, convincing data or models supporting these hypotheses are scarce. The relationship between temperature, rainfall and vector-borne disease are increasingly seen as oversimplifications. A study modelling DF transmission and seasonal temperature on data from Puerto Rico from 1988 to 1992 revealed weak relationships between monthly mean temperature and incidence of DF . The stuP. falciparum transmission in the past century or during the period of reported malaria resurgence. Others have questioned models linking global temperatures and disease incidence, stating that, historically, climate has rarely been the principal determinant of vector-borne disease prevalence. Neither does the literature provide an adequate evidence base establishing the impact of climate change on vector-borne disease [More recently, long-term meteorological trends were studied in four high-altitude sites in East Africa, where increases in malaria have been reported in the past two decades . They di disease ,54. The  disease  and denget al. (2003) studied dengue transmission on the Mexico-USA border and found higher rates in the Mexican city compared to the American one [Social and economic factors play an essential role in the incidence and prevalence of DF and DHF. Air conditioning, screens and safe water supplies in wealthier countries help prevention and better health services reduce or eliminate mortality from DHF. Unplanned urbanisation and inadequate resources for vector control are factors that promote transmission and are characteristic of poor rather than richer countries. Reiter ican one .However, some anomalies persist despite the rich/poor divide in disease incidence. Despite energetic control programs in the wealthier endemic countries of southeast Asia such as Singapore, Malaysia and parts of China (eg. Hong Kong), dengue continues to be a problem. Malaysia reports some of the highest numbers of cases during epidemics compared to other countries in the region. In some of these cases, particular traditional practices, such as rainwater storage on roofs, expose them to higher risk.The major epidemic in Puerto Rico in 1977 serves as a reminder that advanced public health capacities and economic development may not guarantee protection against massive epidemics [On an individual level, evidence points to greater susceptibility among well-nourished or middle-class communities rather than malnourished and poorer patients commonly associated with other tropical diseases.A case-control study of serologically-confirmed DHF patients, other infectious diseases patients and healthy children in the Children's Hospital in Bangkok showed that malnutrition amongst DHF patients was significantly lower . In IndiMiddle classes have been specifically noted as the proportionally predominant group during the epidemic in Dhaka Bangladesh  and uppeFew studies specifically measure and test socio-economic determinants of exposure at community levels. Heukelbach in Fortaleza, Brazil did examine socio-economic variables but their study did not show an association with DF . Since aOn a macro level, the impact of dengue on the economy is likely to be increasingly similar to that of malaria. Prevalent in communities characterised by subsistence or daily wage labour, a week's illness can be catastrophic for poor families. As a primarily paediatric disease in the past, the active labour force or the family wage earners were less affected. Now, as the modal age of illness and incidence increases, losses in productivity and earning capacity may be expected. The economic lesson from malaria was learnt late and when the resurgence was already in full swing. Dengue fever risks the same fate.et al. undertook a cost analysis of the epidemic of DF/DHF in Puerto Rico in 1977 using upper and lower limits of incidence [With regard to costs of care, few economic studies exist and most estimate economic loss to range in millions. Von Allmen ncidence . They esncidence . At 658 On a micro level, a detailed study on costs of care in 3 hospitals in Bangkok estimated direct adult patient costs at US$67. Including opportunity costs, this figure increased to US$161.49. The net hospital cost for each DHF patient was US$54.60 and the public sector cost of prevention and control of the outbreak was US$4.87 million. The total expenditure for DHF in 1994 was estimated to be at least US$12.56 million, of which 45% was borne by the patients .These figures are reminders that most of the countries subject to DF/DHF cannot realistically afford a US$5 million prevention and control budget for a single disease and that the monthly income of many families in these countries is less than the direct cost of US$70 a month.Much needs to be done in finding effective strategies for behaviour change. Since mothers are the first-line care-givers, this aspect is key, particularly for childhood diseases. KAP studies are rare and therefore little is known regarding knowledge and attitude of the exposed population towards dengue. However, the little that is known is encouraging.Straightforward community education to reduce breeding sites for mosquitoes performed better than chemical spraying in a controlled experiment in Mexico . HoweverTreatment-seeking behaviour and lay symptom assessment is the first step in the chain to early diagnosis and was found to have an impact on duration of illness in Thailand . In thatFinally, reducing mortality from DHF and strengthening its control and prevention clearly cannot be done by the population alone. In most circumstances, these are poor populations with other pressing agendas. The programme requires public sector leadership with strong intersectoral collaboration. The WHO has made important progress to determine ways and mechanisms through which to achieve collaboration between sectors and state policy directions for control.Two aspects present themselves for useful discussion in this area. One relates to wide variations in CFRs between countries, sub-national units and hospitals under similar virological conditions. The other relates to differential risks of severe illness and mortality between children and adults.The global case-fatality rate (CFR) for DHF/DSS has been declining in most of the endemic countries according to government statistics. The overall CFR in the southeast Asia region is now less than 1% . HoweverFrom 1995–2000, the CFR in the countries of WHO Western Pacific Region ranged from 0.06% in Singapore and 0.17% in Malaysia to 3.4% in Cambodia. Hong Kong reported no deaths . In VietDuring the 1998 epidemic in Cambodia the CFR in Kantha Bopha, a private, charitable hospital, was substantially lower (1.96%) than the national average (2.91%) . Inter-dSecondly, studies have postulated higher risk of DF/DHF morbidity and mortality among children compared to adults . RecentlIn addition to vector control, widely recognised as a preventive strategy of choice, key health sector response for reduction of mortality and morbidity lies primarily in two areas: early detection (including care-seeking behaviour change and better surveillance) and improved case management of patients. Mortality in excess of 1% may be considered the consequence of inadequate care, late diagnosis and delayed hospitalisation.A hospital-based study during the dengue outbreak in Delhi revealed that mortality could be very low in patients who came early to the hospital . Late prThe short interval between onset of haemorrhage and death, especially in young children, makes rapid medical intervention for DHF/DSS a critical factor for survival. For most communities at highest risk of disease, intensive care facilities are only available at distant capitals requiring motorised transport, usually beyond the reach of many. Early diagnosis and leading indicators for DHF/DSS can ensure the availability of travel time to transfer the patient for effective treatment. Case-control studies have shown that low-normal hematocrit count at time of shock is a significant risk factor for haemorrhage  and poteHowever, research into predictive factors for severe illness is neither abundant nor conclusive. Moreover, as Van Gorp concludes, low capacity and lack of resources at secondary levels of health services limit the operational use of many of these findings .At this time, the WHO classification of dengue diseases is often not feasible in many countries because of lack of trained health professionals, inadequate laboratories, and radiological support. Neither are facilities to detect DHF by using hematocrit and plasma leakage signs readily available in many tropical countries. As successful treatment of dengue depends on symptom recognition and careful fluid management, a simpler dengue disease classification scheme, realistic in poor, provincial conditions and better training of district-level personnel is needed.A few creative approaches to primary health care to improve quality of care and case management at primary health care levels have been reported in the literature. For example, encouraging results have been found in Vietnam where they reduced dengue mortality rates by 64% through innovative primary healthcare concepts, including paediatric priority training units for medical staff, health education for patient carers and promotion of outpatient treatment to avoid unnecessary admissions . ReductiThe effect of strengthened health systems is recognised by public health authorities including WHO but is missing operational research and policies to put them into effect.Unreliable statistics are an extremely serious weakness from many perspectives. Estimates of DHF/DSS CFR from surveillance data are consistently lower than those from single sample study data suggesting under-reporting or misclassification of deaths. Inadequate knowledge of case definitions among district health personnel compromise complete reporting even within the public health service system. Inappropriate denominators further add to the confusion in estimating prevalence and incidences.Reporting deviations can lead to seriously misleading CFRs in countries where reliable estimates are urgently needed for effective resource programming. In Laos, for instance, 8197 DHF cases and 24 deaths were registered by the WHO in 1996, compared to 2563 cases and 23 deaths registered by the Institute of Malariology, Parasitology and Entomology (IMPE) for a CFR of that is 3 times higher than WHO statistics , Most naAn evaluation of the dengue reporting system in Bandung, Indonesia  found that only 31% of hospitalised DHF/DSS cases were reported to the Municipal Health Authorities . In PuerBased on the above studies, a conservative estimate would be that a third of the total cases are captured by surveillance systems, indicating that the global incidence rate could be around 1.5 million cases of DHF on an average year rather than the 0.5 million estimated by WHO.While complete surveillance data may be an unrealistic option in many the affected countries, sentinel surveillance and sample surveys using reliable methodologies could be undertaken to provide more accurate estimates of the disease burden and fill in the gaps. Occasional sample surveys of the private sector could help better estimate the bias in disease burden.On 18 May 2002, the WHO General Assembly confirmed dengue fever as a matter of international public health priority through a resolution to strengthen dengue control and research.Today, changing characteristics of the disease deserve serious research attention. Shifts in modal age, rural spread, social and biological determinants of race- and sex-related susceptibility have major implications for health service planning and control strategies. Behavioural risk factors, individual determinants of outcome and leading indicators of severe illness are poorly understood, compromising the effectiveness of control programmes. Early detection and case management practices have been noted as a critical factor for survival. Yet well-targeted operational research in these areas is rare. Population-based epidemiological studies with clear operational objectives should be launched as concerted efforts at regional levels.A major weakness is the inadequacy of sound statistical methods in some of the reviewed studies. Samples are exceedingly small in many cases, selection methods are often inadequately described or are self selecting, tests of significance are frequently not undertaken or not reported and denominators are not clearly described. Conclusions therefore do not have the full benefit of objective statistical analyses, reducing the scientific strength of the results. Furthermore, conclusions regarding case fatality or disease-specific mortality rates are hard to draw since they are frequently based on hospitalised patients who had actively sought care in tertiary centres. However, a systematic approach and a clear international research agenda can quickly bring forward the frontiers of knowledge. Better understanding of the above will not only feed into operational policy for dengue control, but also provide fertile terrain for vaccine application strategies in the future.Today, dengue control and prevention requires thinking outside the tropical disease box. Many of the affected countries are some of the poorest. Approaches that are realistic for their infrastructure need to be urgently developed.The author(s) declare that they have no competing interests.Debarati Guha-Sapir set out the plan of the paper, its focus areas and main messages. Barbara Schimmer carried out the literature search, summarised the studies and their results. She helped D. Guha-Sapir with the writing of the text.