Seminar

Dengue
Scott B Halstead
Lancet 2007; 370: 164452 Supportive Research and Development, Pediatric Dengue Vaccine Initiative, Internal Vaccine Institute, Seoul, South Korea (S B Halstead MD) Correspondence to: Dr Scott B Halstead, 5824 Edson Lane, Rockville, MD 20852, USA halsteads@erols.com

The four dengue viruses are transmitted in tropical countries that circle the globe. All can cause syndromes that are self-limited or severe. The common severe syndromedengue haemorrhagic fever/dengue shock syndrome (DHF/DSS)is characterised by sudden vascular permeability generated by cytokines released when T cells attack dengue-infected cells. Dengue 1 virus became prevalent in Hawaii where it was transmitted by Aedes albopictus, producing a classic virgin soil epidemic, with clinical disease seen largely in adults. In Cuba and Singapore, sequential dengue infections at long intervals produced unusually severe disease in adults. Evidence suggests that enhancing and cross-reactive neutralising antibodies regulate dengue epidemics and disease severity. Classic DHF/DSS arises during initial dengue infections in infants with low circulating amounts of maternal dengue antibodies, an observation that precludes an exclusive causal role for secondary T-cell responses. Here, I review and discuss data on clinical diagnosis and pathophysiology of vascular permeability and coagulopathy, parenteral treatment of DHF/DSS, and new laboratory tests. Infection with one or more dengue viruses imperils an estimated 25 billion people living in tropical and subtropical countries, mostly in large and small cities. About 50100 million individuals are infected every year, and in some years as many as 500 000 people have been admitted to hospital.1 Genetic studies of sylvatic dengue strains provide evidence that the four dengue viruses evolved from a common ancestor in subhuman primate populations and that, around 500 years ago, all viruses emerged separately into a human urban transmission cycle.2 Infection of human beings with any dengue virus can result in two well dened syndromes (dengue fever and dengue haemorrhagic fever/dengue shock syndrome [DHF/DSS]), a range of intermediate responses, or no clinical response at all. DHF/DSS is characterised by rapid onset of capillary leakage accompanied by thrombocytopenia, altered haemostasis, and damage to the liver indicated by increases in aspartate aminotransferase and alanine aminotransferase.3 Dengue syndromes arise on a severity continuum: for example, cases of dengue fever without clearcut evidence of vascular permeability are generally accompanied by thrombocytopenia, rises in liver enzymes, and subthreshold vascular permeability.46 Use of the term haemorrhagic fever instead of the more descriptive phrase dengue vasculopathy has led doctors and patients alike to anticipate bleeding as the greatest threat to fatal outcome. Rather, uids lost into tissue spaces when not replaced promptly can lead to shock, which if prolonged leads to complications such as gastrointestinal bleeding. Dengue shock can be subtle, arising in patients who are fully alert, and is accompanied by increased peripheral vascular resistance and raised diastolic blood pressure. Bleeding, sometimes severe, can accompany dengue fever, usually in adults.7 These subtleties and complications have given rise to diagnostic diculties encountered by clinicians, particularly when applying WHO case denitions to identify dengue syndromes.812 Such problems can be avoided with ultrasound studies. Thickening of the gallbladder wall could be a precursor to clinically signicant vascular permeability in children with dengue.13 Furthermore, ultrasound examinations of the abdomen and thorax can reliably detect uid accumulation in serosal cavities.14 This low-cost diagnostic method coupled with careful examination for physical signs of vascular collapse should greatly enhance reliability of the diagnosis of DHF/DSS.15 Research into dengue has grown exponentially, generating several specialised reviews.1618 This Seminar will include topics of importance to clinicians in the areas of epidemiology, pathogenesis, pathophysiology, treatment, and diagnosis of dengue infections and disease.

Epidemiology
Dengue viruses continue to expand their range, as shown by movement of dengue virus type 1 (DENV-1) into Hawaii in 200102 (gure 1). This outbreak of dengue was the rst in Hawaii since World War II ended.19 Over a period of nearly 1 year, 122 serologically reported cases of dengue fever spread on Maui, Oahu, and Kauai, with disease noted predominantly in adults or young adults.20 The epidemic was unique in that virus was transmitted by Aedes albopictus mosquitoes. This inecient vector produces a slow-moving outbreak by contrast to the sharp epidemics associated with Aedes aegypti (gure 2).21 Aected individuals lived in fairly isolated homes surrounded by abundant vegetation that provided a breeding habitat for A albopictus.19 The epidemic was initiated by two variant DENV-1 viruses imported by visitors from Tahiti, an island then in the throes of a severe outbreak of DHF/DSS in children.20,22 During the past several decades, all four dengue viruses had been introduced sequentially into Tahiti, establishing conditions prerequisite to secondary dengue infections.23
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Search strategy and selection criteria I searched PubMed with the terms: dengue; dengue fever; dengue hemorrhagic fever; viral hemorrhagic fevers; aviviruses; immune response; and dengue viruses AND antibody response, T cell response, cytokine response, and endothelium.

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Areas with recent dengue transmission Areas infested with Aedes aegypti

Figure 1: Approximate global distribution of dengue and Aedes aegypti in 2005 Reprinted with permission of the US Centers for Disease Control and Prevention.

The Hawaii outbreak illustrates textbook descriptions of dengue in virgin populationsclassic cases of dengue fever in adults. Adults have been implicated in two other island dengue outbreaks, in Cuba and Singapore. These outbreaks illustrate a dierent feature of dengue immunobiology. DENV-1 was introduced to Cuba in 1977, encountering a largely susceptible population. Based on ndings of serological surveys, more than 44% of the population was infected.24 Only mild disease was reported, the most severe cases being of dengue fever. 4 years later, an Asian genotype called dengue virus type 2 (DENV-2) was brought into the country, leading to hundreds of thousands of overt cases of dengue fever along with an estimated 10 000 incidences of DHF/DSS.21 Individuals aged up to 55 years had the same rates of infection with DENV-1 in 1977 and DENV-2 in 1981.25 During the 1981 epidemic, severe dengue and deaths were recorded in both children and adults, with children being most highly aected.26 After a long period of successful mosquito control without any dengue transmission, in 1997, Asian genotype DENV-2 was reintroduced into Santiago, Cuba. DHF/DSS was noted only in adultsspecically, those who previously had been infected with DENV-1 in 1977.27 A similar occurrence arose 3 years later when dengue virus type 3 (DENV-3) was brought into Havana.28 Endemic dengue in Singapore in 2006 bears remarkable similarities to the infection in Cuba.29 In both Singapore and Cuba, dengue transmission rates fell during long periods of eective mosquito control.27,30 Soon after the end of World War II, several dengue viruses were endemic
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in Singapore, producing DHF/DSS in children with only occasional mild dengue illnesses in adults.31,32 In 1973, an intensive, island-wide control programme for A aegypti was begun.33 Cases of DHF/DSS in children steadily declined then disappeared, with ndings of serological surveys showing that successive cohorts of children born after the mosquito-control programmes were almost entirely non-immune to dengue.34 These children have now reached young adulthood. Despite the best eorts of the vector-control programme, small pockets of mosquitoes still persist.30 These mosquitoes can transmit dengue viruses from infected visitors and between Singaporean children and adults. Many of the Singaporean people infected are dengue-naive and, as expected, recognisable cases of dengue fever arise, mainly in adults. However, some adults are immune to one or more dengue viruses owing to dengue infections acquired before 1973 when dengue viruses were highly endemic. Similar to the situation in Cuba, infections in people who are part-immune to dengue lead to a mixture of cases of dengue fever and DHF/DSS, almost exclusively in adults.35 In Cuba, when DENV-1 and DENV-2 outbreaks arose 20 years apart, disease was especially severe and the ratio in adults of dengue fever to DHF/DSS was 24:1.27 From published descriptions, this ratio could be close to that reported in Singapore. In Cuba, disease and case-fatality rates were much higher in individuals who acquired DENV-2 infections 20 years apart than in those infected with the same virus type 4 years apart.27 As described above, long-interval secondary dengue infections can account for the mystifyingly severe
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Figure 2: Image of Aedes aegypti mosquito Reprinted with permission of the US Centers for Disease Control and Prevention.

cases of dengue seen in adult Singaporeans.35,36 Cuban workers have sought to understand why the severity of secondary DENV-2 infections rose as the interval from rst to second infections widened. DENV-2, which caused the 1981 and 1997 epidemics in Cuba, belongs to the same genotype that has been associated with DHF/DSS elsewhere in the Americas.37,38 By analysis of a stored collection of serum samples, the researchers noted progressive loss of cross-reactive neutralising antibodies to DENV-2 in people as time after infection with DENV-1 increased.39 Findings of related studies in Thailand and Peru showed that heterotypic DENV-2 neutralising antibodies in serum samples gathered before illness were correlated with mild second DENV-2 infections.40,41 Co-circulation of four dierent dengue viruses in the same habitat for many years generates a complex of antibody-mediated occurrences, such as cross-protection and infection enhancement, which are of interest to mathematical modellers. Consensus suggests that infection enhancement contributes to the pattern of variable-sized outbreaks observed.42 Researchers in Bangkok did phylogenetic analysis of viruses, clade replacement, and mathematical modelling of the Bangkok dataset and reported that the noted 810-year epidemic oscillations of DENV-1 and dengue virus type 4 (DENV-4) could be attributed to moderate levels of cross-immunity between these two virus types.43 Adopting a theoretical approach, which combined ecological and immunological mechanisms, another group concluded that Bangkok dengue epidemiological patterns were consistent with a period of short-lived cross-immunity and were not dependent on heterogeneity of virus virulence or antibody-dependent enhancement.44 An explanation frequently given for the occurrence of severe dengue disease is that the infecting virus is virulent, whereas viruses associated only with dengue fever are non-virulent. Virulence is best understood as a ratio of severe outcomes to the total number of individuals infected with an organism. The closer the ratio is to 1, the more virulent the virus. For example, rabies is a classic virulent
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virus with nearly all infections resulting in death. The American genotype of DENV-2a virus that has not been isolated from DHF casesis described as non-virulent on the basis of laboratory observations, which show that changes in the secondary structure of the virus in the 3untranslated region of its genome reduce the ability of the virus to grow in cell cultures and mosquitoes, compared with the Asian genotype of DENV-2, which is more readily transmitted by vector mosquitoes and is associated with outbreaks of DHF/DSS.45,46 In nature, American genotype DENV-2 has been predicted to be poorly transmitted by mosquitoes and dengue disease in human beings to be mild.46 Predicted behaviour of this virus in mosquitoes does not accord with actual observations. In 1995, a large epidemic of American genotype DENV-2 arose in the Amazonian city of Iquitos, Peru.47 This virus was probably transported up the Amazon River to Peru, a process accompanied undoubtedly by silent infections in human beings and implying ecient transmission from people to mosquitoes to people by bites from geographically dispersed and genetically distinct populations of A aegypti. A parallel systematic eort failed to associate disease severity with changes in the 3 untranslated region of any of 61 dengue viruses of all four types gathered over a 30-year period in Bangkok.48 The observation that American genotype DENV-2 bears an unusual epitope that permits substantial cross-neutralisation by antibodies to DENV-1 could account for their failure to produce severe disease in people who are immune to DENV-1.41 Regulation of disease severity by heterotypic antibodies accords with evidence cited above that circulation of dengue viruses can be controlled by the presence or absence of heterotypic antibodies.

Pathogenesis
For more than two decades, eorts to identify the mechanisms that underlie the sudden onset of vascular permeability and of haemorrhaging have focused on the role of the T-cell immune response. Research has been centred almost exclusively on secondary dengue infections, thereby ignoring the important immunological subgroupcases of DHF in infantsthat accompanies primary dengue infections.49 During symptomatic secondary dengue infections, high concentrations of interferon are recorded 12 days after onset of fever.50 At the end of the febrile period and coincident with onset of vascular permeability, high concentrations of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon are noted.51 Amounts of soluble tumour necrosis factor receptors, soluble CD8, soluble interleukin 2 receptors, interleukin 10, detectable circulating tumour necrosis factor , and macrophage migration inhibition factor are all correlated with severity of dengue disease.52,53 Raised concentrations of soluble tumour necrosis factor receptor 7554 or tumour necrosis factor have been reported repeatedly in DHF cases.5558 Very high concentrations of CCL2, a protein that reduces
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tight junctions of vascular endothelium cells, have been seen in DHF/DSS patients.59 Production of many of these cytokines or cellular factors can be induced in vitro by incubation of antigen-responsive human T lymphocytes with dengue-infected cells, usually monocytes.5961 Dominant epitopes that interact with antigen-responsive T lymphocytes are peptides of the dengue non-structural protein NS3.62 In an early review, Rothman and Ennis concluded that because the number of cells presenting the dengue viral antigen to T lymphocytes is increased during a secondary infection, a striking and more rapid rise in amount of T-lymphocyte activation would be expected.63 A growing trend has been to attribute severe dengue disease to T-cell immunopathology. So-called pathological outcomes are postulated when a dengue infection generates T-cell responses to heterologous dengue antigenic sequences.17,64 Variant peptides or altered peptide ligands induce dierent activation signals in antigen-specic T lymphocytes and, thereby, modulate specic eector functions of CD4+ and CD8+ T cells.6467 A situation analogous to so-called original antigenic sin has been recorded in T-cell responses. During secondary DENV infection, expansion of pre-existing lower avidity memory T cells takes precedence over that of naive T cells, with higher avidity for the new DENV serotype.68 CD8+ T cells generated during infection bind weakly to MHC tetramers presenting epitopes of the infecting virus but strongly to other epitopes, presumably from previously encountered viruses. A high frequency of these T cells shows an apoptotic phenotype and seems destined to die before adequately controlling the infection. Low avidity T cells that dominate the response to secondary DENV infections are less than optimally ecient at elimination of DENV-infected cells.69 These hypotheses are at variance with the biological reality that heterologous T-cell responses are not needed to produce DHF in infants. Exactly the same severe vascular permeability clinical syndrome and the same concentrations of cytokines in blood are produced during primary dengue immune responses in infants (with enhanced primary dengue infections) as are in children with augmented secondary dengue infections.7074 Antibodies, predominantly of the IgG1 subclass, are the only immunological substances known to be transferred from mother to fetus.75 The fact that infants from southeast Asia fail to develop clinical dengue illnesses until around 6 months of age accords with the presence of broadly reactive dengue neutralising antibodies in their mothers serum samples and the protection aorded by passively transferred dengue antibodies.76,77 DHF in both groups is linked by a model in which enhancing antibodies lead to an increased infected cell mass; T-cell and cytokine responses are proportionate to this antigenic stimulus. Evidence suggests that antibody-dependent enhancement results from idiosyncratic Fc-receptor signalling. When mouse macrophages were infected by complexes of Ross River virus with enhancing antibodies, during the ensuing
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infection interferon transcription factors, STAT1, and NFB complexes were suppressed instead of activated, as seen in cells infected with virus only.78 Similarly, DENV-2 antibody-dependent enhancement of infection of a human macrophage cell line suppressed STAT1 phosphorylation, decreased nitric oxide production, and increased interleukin 10 and virion production compared with cells infected with virus only.79 Mirroring these in-vitro data, DHF patients circulated reduced nitric oxide and enhanced interleukin 10 concentrations in blood. In a separate study, the abundance of type I interferon gene transcripts in peripheral-blood mononuclear cells from six Vietnamese DSS patients was lower in than in cells from eight nonshock DHF cases.80 Correlation of size of dengue-infected cell mass with disease severity has been shown many times by high titres of circulating virus in early illness blood samples or by persisting high concentrations in blood of dengue viral RNA and dengue non-structural protein 1 (NS1).8185 Some researchers have assumed that, because random serum samples can be used to quantify viral load in chronic HIV infections, a similar strategy holds in dengue.86 In an acute, self-limited viral infection, peak viraemia provides the best quantitative estimate of cellular infection, but this event is transient, happening early in infection.81 Dengue NS1 protein production also parallels cellular dengue infection.83 The correlation of disease severity with markers of immune activation (eg, interleukins 6 and 8, tumour necrosis factor , interferons n1 and , soluble tumour necrosis factor receptor, complement components 3a and 5a) together with altered platelet, dendritic-cell, monocyte, and T-cell functions has been interpreted by some researchers to suggest that immune responses to components of dengue viruses could contribute to autoimmune processes that result in DHF/DSS. Antibodies directed against DENV NS1 cross-react with human platelets and endothelial cells.87 Serum samples from DHF patients show higher binding activity to platelets than do those of people with dengue feverthe isotype of platelet autoantibodies being IgM and not IgG.88 The autoimmune hypothesis posits that endothelial dysfunction is the result of cross-reactivity between anti-DENV NS1 to host proteins and endothelial cells.89,90 After binding to endothelial cells, anti-DENV NS1 induce cells to undergo apoptosis mediated by nitric oxide.91,92 Further, these antibodies induced inammatory endothelial cell activation of interleukins 6 and 8 and CCL2.93 NS1 might activate complement by the alternative pathway,94 a mechanism that could explain why complement activation has been recorded in infants with DHF during primary infections.95 The autoimmune NS1 hypothesis is contradicted by the kinetics. The autoimmune NS1 hypothesis is contradicted by the durability of NS1 antibodies contrasted with the transient nature of vascular permeability and haemostatic disorders. Further, although the kinetics of
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antibody production in primary and secondary infections dier strikingly, the same dierences are not seen in the evolution of pathophysiological features of DHF in infants and children. Considerable attention has been paid to the possibility that dengue virus infection of endothelial cells underlies vascular permeability. Unquestionably, dengue viruses can infect endothelial cells in vitro.96100 This experimental approach seems unprotable because, when studied carefully, no evidence exists of dengue infection of endothelial cells in vivo.101 On the other hand, uninfected endothelial cell monolayers have been rendered permeable by treatment with cytokines generated in various ways, including by antibody-mediated dengue virus infection of monocytes,102,103 release of cytotoxic factors from dengue-infected monocytes, macrophages, or dendritic cells,104107 or endogenous interleukin 1 produced by peripheral-blood mononuclear cells stimulated with interferon and tumour necrosis factor.108,109

Pathophysiology and management

The current notion of typical microvascular ultraltration asserts that intrinsic permeability is regulated by the endothelial surface glycocalyx as much as by endothelial cells themselves.110 This highly anionic proteoglycan matrix is located on the luminal surface of all vascular endothelial beds, anchored in the plasma membrane of endothelial cells. The glycocalyx has proved dicult to visualise by conventional histopathological techniques. It forms an electrostatic barrier, repelling negatively charged plasma proteins from the endothelial cell surface, thus eectively restricting access to underlying cellular transport mechanisms. Preliminary evidence from children with DSS indicates that molecular size and charge characteristics determine which molecules are preferentially lost from the circulation and suggests that a transient disturbance in the function of the endothelial glycocalyx layer could arise during dengue infections.111 Endothelial cells themselves are dislodged into circulation.112 How is the glycocalyx layer damaged? Possibilities include dengue virus, one of the dengue non-structural proteins, or one of the components of the immune response to infection that might interact directly with the glycocalyx layer in such a way as to alter temporarily the characteristics of the bre matrix. Heparan sulphate, an important constituent of the structure to which dengue virus can adhere, might have a role in this process.113 In the few studies of dengue-infected patients in which endothelial architecture has been examined, absence of detectable abnormalities at the cellular level or evidence of viral infection despite profound disturbances in permeability lends support to the idea that the pathological process takes place elsewhere.101,114 New techniques to visualise the surface glycocalyx layer are becoming available and could prove interesting in this context.110 Thrombocytopenia is almost always present in patients with dengue, with several mechanisms thought to be entailed. Early bone-marrow suppression combined with
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increased peripheral destruction of platelets during the febrile and early convalescent phase of dengue disease can lead to profound thrombocytopenia, with platelet nadirs as low as 5000 cells per L recorded in some cases.115,116 However, during the recovery period, platelet numbers rise promptly as production increases in the now hypercellular marrow. In the absence of substantial bleeding (ie, bleeding sucient to warrant consideration of blood transfusion), no evidence exists to show that prophylactic platelet transfusions improve outcome, but they confer a very denite risk of acute and long-term complications.117 Important changes in blood pressure take place as DSS worsens, including enhanced peripheral vascular resistance with diminished cardiac output and normal or low central venous pressure.118 Shock is not due to congestive heart failure but to venous pooling. With increasing cardiovascular compromise, diastolic pressure rises towards the systolic and pulse pressure narrows. Finally, decompensation happens and both pressures disappear abruptly. Successful management of severe dengue relies on meticulous regulation of parenteral uids and colloid during the period of increased vascular leakage, together with proactive management of major bleeding should this situation develop.3 The doctor should remember that all uid administered will be reabsorbed and uid overload could result. Careful consideration of the risks associated with any intervention is also essential if informed and rational decisions are to be made. Methodical clinical research can provide invaluable data on which to base management guidelines for the future and to design specic interventions to counteract or prevent leakage, haemorrhage, or both.111,119122 Findings of a double-blind randomised comparison of three uids for initial resuscitation of 383 Vietnamese children with DSS showed that Ringers lactate was sucient to resuscitate infants with moderately severe disease.122 However, should the disease proceed to severe shock, administration of dextran 70 or 6% hydoxyethyl starch will stabilise vascular volume and blood pressure in most cases. In view of adverse reactions associated with use of dextran, starch might be preferable for this group, although dextran 1 can be given immediately before dextran 40 or 70, acting as a hapten inhibitor blocking molecules of dextran 40 or 70 from forming toxic immune complexes.123 The coagulopathy associated with dengue infections is well described but unfortunately underlying mechanisms still remain unclear. Severe bleeding happens only rarely in children (almost invariably in association with profound shock) and thrombotic complications are not seen. An increase in activated partial thromboplastin time and a reduction in brinogen concentrations are fairly consistent ndings, together with thrombocytopenia, and these abnormalities correlate with overall severity.124,125 However, evidence for classic disseminated intravascular coagulation in most patients is not convincing.125,126 Concentrations of procoagulant markers are raised to some degree (usually mild), with substantial reductions in amounts of
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anticoagulant proteins, but ndings with respect to the brinolytic pathway are conicting. In general, data indicate enhanced brinolytic activity, and this occurrence could suggest a direct interaction between virus and plasminogen, one of the key proteins in this pathway. Several groups have noted the presence of plasminogen cross-reactive antibodies during and after dengue infection. Release of heparan sulphate or chondroitin sulfate (molecules similar in structure to heparin that can mimic its function as an anticoagulant) from the glycocalyx might also contribute to the overall picture.111 In most patients with dengue fever, the coagulopathy is fairly minor and resolves within a few days as the virus is cleared. However, in some children, usually those with severe shock, these minor derangements are compounded by the eects of prolonged hypotension and tissue hypoxia, and major haemorrhage takes place, usually from the gastrointestinal tract. In these patients, true disseminated intravascular coagulation probably develops. As yet, little information is available about the coagulopathy in adults, but bleeding seems to be more prominent in this group, sometimes without shock. Systematic investigation of dengue disease in adults could prove valuable.

Plaque reduction neutralisation test Haemagglutination inhibition IgM and IgG ELISA Rapid tests Virus isolation Molecular techniques Dengue antigen capture ELISA

Shock Haemorrhage

Dengue antibodies

Mosquito bite Fever Viraemia

4 Day of illness

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Figure 3: Course of dengue infection and timings of diagnosis Published with permission of Timothy Endy, Syracuse University, Syracuse, NY, USA.

Diagnosis
Diagnosis of dengue falls into two stages: stage I, fever and viraemia accompanied by NS1 antigens in blood; and stage II, the early post-febrile period lasting a few weeks when IgM and IgG antibodies are in excess. During primary infection, viraemia more or less coincides with fever (gure 3). However, during a secondary infection, the duration of viraemia can be 2 or 3 days, whereas presence of NS1 antigens in blood lasts somewhat longer. The diagnostic importance of the two stages of dengue infection should be understood by the doctor. Usually, detection of newly formed antibodies (IgM) is not possible until after viraemia ends or after fever subsides. Dengue syndromes dier in presentation. In dengue fever, because of the acute onset of fever and malaise, patients generally seek medical attention within the rst 2 days of fever. At this stage, diagnosis is possible only by detecting virion, RNA, or dengue proteins in blood. Serological diagnosis will not be positive until defervescence (ie, abatement of fever; gure 3). If a serological test is ordered the patient must be asked to return for a second blood sample once fever has ended. In individuals with DHF/DSS, vascular permeability is recognised usually at defervescence, at which time the IgM-capture serological test should be positive but tests to detect virions, dengue RNA, or dengue proteins could be negative. Commercial dengue serological tests in most countries are not subject to quality control. In a study in which one serum sample from a series of patients was tested, a commercial dengue IgM-capture ELISA test had a high rate of false-positive results.127 A seemingly attractive alternative is use of the rapid immunochromatographic assay to detect viral antigens. Some commercial tests are of low sensitivity.128
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Because of the desire by doctors and patients for a diagnosis during the febrile period, attempts are being made to optimise and simplify PCR. With genus-specic and serotype-specic nested NS3 primers, one group was able to identify infecting virus in 80% of dengue patients; nearly all positive serum samples were obtained within 5 days of onset of fever.129 Alternatively, dengue RNA can be detected rapidly with primers for all four dengue viruses in a one-step TaqMan real-time reverse transcriptase-PCR.130 An inexpensive, rapid, sensitive, and specic test is needed to diagnose dengue during the febrile stage. One such test is marketed by BioRad (Hemel Hempstead, UK) and uses a dengue group-specic NS1 monoclonal antibody in an ELISA format to detect dengue NS1 antigen in blood. This test conrmed 85% of PCR-positive serum samples.131 Sensitivity was enhanced when NS1 antigen-capture was combined with a test to detect NS1 antibodies. Kinetics of the appearance of NS1 antigens in blood (peak titres on days 610 after fever) have been studied in a group of patients with DENV-1 infection.132 Articial NS1 receptors implanted on a reuseable microchip can capture and identify NS1 nearly instantaneously, oering possible bedside diagnosis of ongoing dengue virus infection.133 NS1 can be used in an ELISA-based test to replace the detection of viral antigens, making possible diagnosis of specic causes during primary infections and accurate characterisation of primary and secondary dengue infections.134 Finally, a word of warning about the use of blood adsorbed to lter paper for diagnostic tests. A substantial amount of IgM-dengue antibodies are lost on adsorption, reducing the diagnostic accuracy and sensitivity of this technique.135 However, IgG antibodies are not adsorbed to lter paper, permitting their use for seroepidemiological studies of dengue-antibody prevalence or for detection of IgG responses during secondary dengue infections.
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Conict of interest statement SBH is a director of the Supportive Research and Development Program of the Pediatric Dengue Vaccine Initiative, a programme of the International Vaccine Institute, Seoul, South Korea. The Pediatric Dengue Vaccine Initiative is supported by grants from the Gates and Rockefeller Foundations. References 1 Halstead SB. Pathogenesis of dengue: challenges to molecular biology. Science 1988; 239: 47681. 2 Wang E, Ni H, Xu R, et al. Evolutionary relationships of endemic/epidemic and sylvatic dengue viruses. J Virol 2000; 74: 322734. 3 WHO. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control, 2nd edn. Geneva: World Health Organization, 1997. 4 Kuo CH, Tai DI, Chang Chien CS, Lan CK, Chiou SS, Liaw YF. Liver biochemical tests and dengue fever. Am J Trop Med Hyg 1992; 47: 26570. 5 Kalayanarooj S, Vaughn DW, Nimmannitya S, et al. Early clinical and laboratory indicators of acute dengue illness. J Infect Dis 1997; 176: 31321. 6 Gamble J, Bethell D, Day NP, et al. Age-related changes in microvascular permeability: a signicant factor in the susceptibility of children to shock? Clin Sci (Lond) 2000; 98: 21116. 7 Tsai CJ, Kuo CH, Chen PC, Changcheng CS. Upper gastrointestinal bleeding in dengue fever. Am J Gastroenterol 1991; 86: 3335. 8 Deen JL, Harris E, Wills B, et al. The WHO dengue classication and case denitions: time for a reassessment. Lancet 2006; 368: 17073. 9 Rigau-Perez JG. Severe dengue: the need for new case denitions. Lancet Infect Dis 2006; 6: 297302. 10 Balasubramanian S, Janakiraman L, Kumar SS, Muralinath S, Shivbalan S. A reappraisal of the criteria to diagnose plasma leakage in dengue hemorrhagic fever. Indian Pediatr 2006; 43: 33439. 11 Balmaseda A, Hammond SN, Perez MA, et al. Short report: assessment of the World Health Organization scheme for classication of dengue severity in Nicaragua. Am J Trop Med Hyg 2005; 73: 105962. 12 Bandyopadhyay S, Lum LCS, Kroeger A. Classifying dengue: a review of the diculties in using the WHO case classication for dengue haemorrhagic fever. Trop Med Int Health 2006; 11: 123855. 13 Colbert JA, Gordon A, Roxelin R, et al. Ultrasound measurement of gallbladder wall thickening as a diagnostic test and prognostic indicator for severe dengue in pediatric patients. Pediatr Infect Dis J 2007; 26: 85052. 14 Srikiatkhachorn A, Krautrachue A, Ratanaprakarn W, et al. Natural history of plasma leakage in dengue hemorrhagic fever: a serial utrasonographic study. Pediatr Infect Dis J 2007; 26: 28390. 15 Halstead SB. Dengue: the case denition dilemmaa commentary. Pediatr Infect Dis J 2007; 26: 29192. 16 Fink J, Gu F, Vasudevan SG. Role of T cells, cytokines and antibody in dengue fever and dengue haemorrhagic fever. Rev Med Virol 2006; 16: 26375. 17 Green S, Rothman A. Immunopathological mechanisms in dengue and dengue hemorrhagic fever. Curr Opin Infect Dis 2006; 19: 42936. 18 Thomas S, Redfern JB, Lidbury BA, Mahalingam S. Antibody-dependent enhancement and vaccine development. Expert Rev Vaccines 2006; 5: 40912. 19 Hayes JM, Rigau-Perez JG, Reiter P, et al. Risk factors for infection during a dengue-1 outbreak in Maui, Hawaii, 2001. Trans R Soc Trop Med Hyg 2006; 199: 55966. 20 Eer PV, Pang L, Kitsutani P, et al. Dengue fever, Hawaii, 20012002. Emerg Infect Dis 2005; 11: 74249. 21 Kouri G, Guzman MG, Bravo J. Hemorrhagic dengue in Cuba: history of an epidemic. Bull Pan Am Health Organ 1986; 20: 2430. 22 Imrie A, Zhao Z, Bennett SN, Kitsutani P, Laille M, Eer P. Molecular epidemiology of dengue in the Pacic: introduction of two distinct strains of dengue virus type-1 into Hawaii. Ann Trop Med Parasitol 2006; 100: 32736. 23 Deparis X, Murgue B, Roche C, Cassar O, Chungue E. Changing clinical and biological manifestations of dengue during the dengue-2 epidemic in French Polynesia in 1996/97: description and analysis in a prospective study. Trop Med Int Health 1998; 3: 85965.

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