Dengue: a Continuing Global Threat

EVALUATING DIAGNOSTICS

Dengue: a continuing global threat

Maria G. Guzman*, Scott B. Halstead‡, Harvey Artsob§, Philippe Buchy||, Jeremy Farrar¶, Duane J. Gubler#, Elizabeth Hunsperger**, Axel Kroeger‡‡, Harold S. Margolis**, Eric Martínez*, Michael B. Nathan§§, Jose Luis Pelegrino*, Cameron Simmons¶, Sutee Yoksan¶¶ and Rosanna W. Peeling‡‡,## Abstract | Dengue fever and dengue haemorrhagic fever are important arthropod-borne viral diseases. Each year, there are ~50 million dengue infections and ~500,000 individuals are hospitalized with dengue haemorrhagic fever, mainly in Southeast Asia, the Pacific and the Americas. Illness is produced by any of the four dengue virus serotypes. A global strategy aimed at increasing the capacity for surveillance and outbreak response, changing behaviours and reducing the disease burden using integrated vector management in conjunction with early and accurate diagnosis has been advocated. Antiviral drugs and vaccines that are currently under development could also make an important contribution to dengue control in the future.

Dengue is the most important arthropod-borne viral countries in Asia and the Americas, the burden of den- infection of humans. Worldwide, an estimated 2.5 bil- gue is approximately 1,300 disability-adjusted life years lion people are at risk of infection, approximately 975 (DALYs) per million population, which is similar to the million of whom live in urban areas in tropical and disease burden of other childhood and tropical diseases, sub-tropical countries in Southeast Asia, the Pacific and including tuberculosis, in these regions10. the Americas1. Transmission also occurs in Africa and The geographical areas in which dengue transmission the Eastern Mediterranean, and rural communities are occurs have expanded in recent years (FIG. 1), and all four increasingly being affected. It is estimated that more than dengue virus serotypes (DENV-1–4) are now circulating 50 million infections occur each year, including 500,000 in Asia, Africa and the Americas, a dramatically differ- hospitalizations for dengue haemorrhagic fever, mainly ent scenario from that which prevailed 20 or 30 years among children, with the case fatality rate exceeding 5% ago (FIG. 2). The molecular epidemiology of these sero- in some areas1–4. types has been studied in an attempt to understand their The annual average number of dengue fever/den- evolutionary relationships11. gue haemorrhagic fever (DF/DHF) cases reported to This Review will provide an update on our under- the World Health Organization (WHO) has increased standing of the pathogenesis of this successful pathogen, dramatically in recent years. For the period 2000–2004, how we diagnose and control infection and the progress ‡‡ UNICEF/UNDP/World Bank/ the annual average was 925,896 cases, almost dou- that has been made in vaccine development. WHO Special Programme for ble the figure of 479,848 cases that was reported for Research and Training in the period 1990–1999. In 2001, a record 69 countries Dengue virus pathogenesis Tropical Diseases (TDR), World Health Organization, reported dengue activity to WHO and in 2002, the Dengue viruses belong to the genus flavivirus within the CH-1211 Geneva 27, Region of the Americas alone reported more than Flaviviridae family. DENV-1–4 evolved in non-human Switzerland. 1 million cases. Although there is poor surveillance and primates from a common ancestor and each entered ##London School of Hygiene no official reporting of dengue to WHO from coun- the urban cycle independently an estimated 500–1,000 and Tropical Medicine, 12 Dept of Clinical Research, tries in the African and Eastern Mediterranean regions, years ago . The virion comprises a spherical particle, Keppel Street, in 2005–2006 outbreaks of suspected dengue were 40–50 nm in diameter, with a lipopolysaccharide enve- London WC1E 7HT, UK. recorded in Pakistan, Saudi Arabia, Yemen, Sudan and lope. The positive single-strand RNA genome (FIG. 3), Correspondence to R.W.P. Madagascar1–4, and a large outbreak of dengue involving which is approximately 11 kb in length, has a single open e-mail: Rosanna.Peeling@ >17,000 cases was documented in the Cape Verde islands reading frame that encodes three structural proteins — lshtm.ac.uk 5 Copyright © WHO, on behalf in 2009 .Travellers from endemic areas might serve as the capsid (C), membrane (M) and envelope (E) glyco- 6–9 of TDR (WHO/TDR) 2010. vehicles for further spread . Dengue epidemics can proteins — and seven non-structural proteins (NS1, doi:10.1038/nrmicro2460 have a significant economic and health toll. In endemic NS2A, NS2B, NS3, NS4A, NS4B and NS5). Important

Nepal

Hawaii Bhutan Sudan Hong Kong SAR Macao SAR Galapagos Islands

Madagascar – La Reunion Easter Island

Geographical extension of dengue, 2000–2007 Risk of dengue transmission

Figure 1 | countries and areas at risk of dengue transmission, 2007. Data from WHO. Nature Reviews | Microbiology

biological properties of dengue viruses, including recep- that takes approximately 10 days and is most rapid at tor binding, haemagglutination of erythrocytes and the high ambient temperatures14. Mosquito bites after the induction of neutralizing antibodies and the protective extrinsic incubation period result in infection, which immune response, are associated with the E glycoprotein. might be promoted by mosquito salivary proteins15. Each DENV shares around 65% of the genome, which is In the skin, dengue viruses infect immature dendritic approximately the same degree of genetic relatedness as cells through the non-specific receptor dendritic cell- West Nile virus shares with Japanese encephalitis virus. specific ICAM3-grabbing non-integrin (DC-SIGN)16. Despite these differences, each serotype causes nearly Infected dendritic cells mature and migrate to local or identical syndromes in humans and circulates in the regional lymph nodes where they present viral antigens same ecological niche13. to T cells, initiating the cellular and humoral immune The mosquito vectors, principally Aedes aegypti, responses. There is also evidence of abundant replica- become infected when they feed on humans during tion of DENVs in liver parenchymal cells and in mac- the usual five-day period of viraemia. The virus passes rophages in lymph nodes, liver and spleen, as well as in from the mosquito intestinal tract to the salivary peripheral blood monocytes17. Both in vitro and in vivo, glands after an extrinsic incubation period, a process macrophages and monocytes participate in antibody- dependent enhancement (ADE)18–20. ADE occurs when mononuclear phagocytes are infected through their Fc Author addresses receptors by immune complexes that form between *Instituto de Medicina Tropical, ‘Pedro Kouri’, PO Box 601, Marianao 13, Ciucad de la DENVs and non-neutralizing antibodies. These non- Habana, Cuba. neutralizing antibodies result from previous heterotypic ‡Pediatric Dengue Vaccine Initiative, Kwanak PO Box 14, Seoul, Korea 151-600. dengue infections or from low concentrations of dengue § Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public antibodies of maternal origin in infant sera21. The co- Health Agency of Canada, 1015 Arlington Street ,Winnipeg, Mannitoba R3E 3R2, Canada. circulation of four DENV serotypes in a given population ||Institut Pasteur in Cambodia, Unit 5, Monivong Boulevard, PO Box 983, Phnom Penh, might be augmented by the ADE phenomenon22. Cambodia. ¶Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford DENVs produce several syndromes that are condi- University Clinical Research Unit, Ho Chi Minh City, Viet Nam. tioned by age and immunological status. During initial #Duke–NUS Graduate Medical School Singapore, 8 College Road, Singapore 169857. dengue infections, most children experience subclinical **Dengue Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease infection or mild undifferentiated febrile syndromes. Control and Prevention, 1324 Calle Cañada, San Juan 00920-3860, Puerto Rico. During secondary dengue infections the pathophysiology ‡‡ UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in of the disease changes dramatically, particularly sequen- Tropical Diseases (TDR), World Health Organization, CH-1211 Geneva 27, Switzerland. tial infections in which infection with DENV-1 is followed §§ World Health Organization, Department of Neglected Tropical Diseases, Geneva, by infection with DENV-2 or DENV-3, or infection with Switzerland. DENV-3 is followed by infection with DENV-223–25. Such ¶¶Center for Vaccine Development, Mahidol University Institute of Science & infections can result in an acute vascular permeability Technology for Development, 25/25 Phutthamonthon 4 Road, Salaya, Nakhon Pathom 73170, Thailand. syndrome known as dengue shock syndrome (DSS). The severity of DSS is age-dependent, with vascular leakage

5′ UTR links between the virus, complement activation and the NS2A NS4A development of DHF/DSS50. Alternative hypotheses CM E NS1 NS3 NS5 of dengue pathogenesis include the suggestions that secondary T-cell responses are blunted because stim- 3′ UTR NS2B NS4B ulation of T-cell memory results in the production of Figure 2 | The dengue virus genome. The single open reading frame encodes three heterotypic CD4+ and CD8+ cells that have a dimin- structural proteins (the capsid (C), membrane (M) and envelope (E) glycoproteins) and Nature Reviews | Microbiology ished capacity to kill but nonetheless release inflam- seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and N55). matory cytokines that contribute to disease severity51; that severe disease is caused by DENVs of increased virulence52; and the suggestion that cross-reactivity being most severe in young children, a phenomenon that between NS1 and human platelets and endothelial cells is thought to be related to the intrinsic integrity of the raises antibodies that damage these cells53. capillaries26,27. In adults, primary infections with each One working hypothesis of dengue pathogenesis that of the four DENV serotypes, particularly with DENV-1 is consistent with the available evidence is that severe dis- and -3, often results in DF. Some outbreaks of primary ease in infants with primary infections and in older indi- DENV-2 infections have been predominantly subclini- viduals with secondary infections is the result of ADE cal24. Nonetheless, dengue infections in adults are often of infection of mononuclear phagocytes. Infection by accompanied by a tendency for bleeding that can lead to an antibody–virus complex suppresses innate immune severe haemorrhages. responses, increasing intracellular infection and gen- Dengue infections can be life-threatening when erating inflammatory cytokines and chemokines that, they occur in individuals with asthma, diabetes and collectively, result in enhanced disease. Liver infection other chronic diseases28–30. Host factors that increase and a pathogenic role for NS1 add to the complexity. In the risk of severe dengue disease include female sex, patients with DF, IFN production and activated natural several human leukocyte antigen (HLA) class I alleles, killer cells can limit disease severity. a promoter variant of the DC-SIGN receptor gene, a single-nucleotide polymorphism in the tumour necrosis Clinical signs and immunological response factor (TNF) gene and AB blood group31–36. Host factors Dengue-associated deaths are usually linked to DHF/ that reduce the risk of severe disease during a second DSS. Even though no vaccines or drugs are available, dengue infection include race, second or third degree severe disease can be successfully managed by careful malnutrition, and polymorphisms in the Fcγ receptor monitoring of the warning signs and early initiation of and vitamin D receptor genes37–42. Secondary dengue aggressive intravenous rehydration therapy. During the infections in adults can produce the classical DSS or early febrile stage (the symptoms of which include fever, severe disease complicated by haemorrhages. The sever- malaise, headache, body pains and rash), clinicians can- ity of secondary dengue infections has been observed not predict which patients will progress to severe disease. to increase from month-to-month during island out- Later, during defervescence, symptoms such as bleeding, breaks43; the longer the interval between the first and thrombocytopenia of <100,000 platelets mm−3, ascites, second infection the more severe is the accompanying pleural effusion, haematocrit >20% and clinical warn- disease44,45. Tertiary dengue infections can cause severe ing signs, such as severe and continuous abdominal pain, disease, but only rarely25. restlessness and/or somnolence, persistent vomiting and In vitro studies demonstrate that the infection of a sudden reduction in temperature (from fever to sub- human monocytes and mature dendritic cells results in normal temperature) associated with profuse perspira- increased virus replication as a result of the suppression tion, adynamia (loss of strength or vigor) and sometimes of the interferon system45. Type I interferon-associated fainting, can be indicative of plasma extravasation and the genes are less abundantly activated in peripheral blood imminence of shock. At this point, patients should receive mononuclear cells taken from patients with severe den- fluid replacement (crystalloids) to avoid haemodynamic gue disease compared with milder disease46. Subsequently, instability, narrowness of blood pressure and hypotension. the increased number of infected cells present targets Early resuscitation can prevent other complications, such for CD4+ and CD8+ T cells, resulting in large quanti- as massive haemorrhage, disseminated intravascular coag- ties of interleukin (IL)-10, IL-2, interferon (IFN)-γ and ulation, multiple organ failure, and respiratory failure due TNF that, singly or in combination, might contribute to non-cardiogenic pulmonary oedema54–57. Treatment of to endothelial damage and altered haemostasis. Virions uncomplicated dengue cases is only supportive, including released from infected cells might also directly damage plenty of oral fluids during the febrile period and paraceta- endothelial cells and the uptake of the non-structural mol (acetaminophen), the daily dosage of which should protein NS1 by hepatocytes might promote viral infection not be exceeded to prevent intoxication mainly related to of the liver47–49. During DHF, the complement cascade is liver function. When dengue shock becomes prolonged also activated and the levels of the complement activa- or recurrent, intravenous fluids should be given carefully tion products C3a and C5a correlate with the severity according to age and dosage to prevent fluid overload as of illness49. Soluble and membrane-associated NS1 have this can result in pulmonary oedema. been demonstrated to activate human complement. The Recent publications have suggested that the WHO levels of the terminal SC5b–9 complement complex and syndromic case definition of DHF/DSS should be evalu- plasma NS1 correlated with disease severity, suggesting ated for clinical utility58–62. A prospective multi-centre

a lower in secondary dengue infections, false-negative test results for dengue-specific IgM have been reported during secondary infections55,66,67. Following a dengue infection, IgG can be lifelong, which complicates the serodiagnosis of past, recent and current infections65,67. IgA and IgE responses have also been documented but the utility of detecting these immunoglobulins as markers for dengue 68 DEN-1 serodiagnosis requires further study . DEN-2 In areas where two or more flaviviruses are circulat- DEN-1 DEN-1 ing, multiple and sequential flavivirus infections make DEN-2 DEN-2 DEN-3 differential diagnosis difficult owing to the presence of DEN-4 pre-existing antibodies and the phenomenon of original antigenic sin (during sequential flavivirus infections, B-cell clones responding to the first infection synthe- size antibodies with higher affinity for the first infecting virus than for the second infecting virus)69. b Laboratory diagnosis of dengue infection Laboratory confirmation of dengue infection is crucial as the broad spectrum of clinical presentations, ranging from mild febrile illness to several severe syndromes, can make accurate diagnosis difficult. Among the methods DEN-1 available for dengue diagnosis, virus isolation provides DEN-1 DEN-1 DEN-2 the most specific test result. However, facilities that can DEN-2 DEN-1 DEN-1 DEN-2 DEN-3 DEN-1 DEN-3 DEN-2 support viral culture are not always available. The detec- DEN-2 DEN-4 DEN-1 DEN-2 DEN-4 DEN-3 DEN-3 DEN-2 tion of the viral genome or viral antigens also provides DEN-3 DEN-4 DEN-4 DEN-4 DEN-1 DEN-3 evidence of infection. DEN-2 DEN-1 DEN-4 Seroconversion of IgM or IgG antibodies is the stand- DEN-3 DEN-2 DEN-4 DEN-3 ard for serologically confirming a dengue infection. The DEN-4 presence of IgM or high levels of IgG in acute serum collected from a suspected dengue case suggests a probable dengue infection54,55. BOX 1 shows the laboratory criteria Figure 3 | The change in distribution of dengue serotypes. The figure shows the for confirmed and probable dengue infections. distribution in 1970 (a) and 2004 (b). Reproduced with permissionNature Re fromviews REF. | Micr 141obiolog. y Virus isolation The Aedes albopictus mosquito C6/36 cell line is the study in several Latin-American and Southeast asian method of choice for DENV isolation, although other countries is planned that will provide standardized mosquito (such as Aedes pseudoscutellaris AP61) and descriptions of dengue clinical presentations in the mammalian (including Vero cells, LLC-MK2 cells and context of the current WHO case definitions. BHK21 cells) cell lines can also be used70,71. Sera that The acquired immune response to dengue infection have been collected from suspected dengue cases in the consists of the production of antibodies that are primarily first 3–5 days of fever (the viraemic phase) can be used directed against the virus envelope proteins. The response for virus isolation. After an incubation period permitting varies depending on whether it is a primary or secondary virus replication, viral identification is performed using infection63,64. A primary antibody response is seen in indi- dengue-specific monoclonal antibodies in immunofluo- viduals who are not immune to dengue and a secondary rescence and PCR assays63,64,72,73. Serum is often used for immune response is observed in patients who have had a virus isolation but plasma, leukocytes, whole blood and previous dengue infection (FIG. 4). A primary infection is tissues obtained at autopsy can also be used63,74,75. characterized by a slow and low-titre antibody response. Immunoglobulin (Ig)M antibodies are the first isotype Serological testing to appear, by day 3–5 of illness in 50% of hospitalized Serological assays are most commonly used for diagno- patients and by day 6–10 of illness in 93–99% of cases. The sis of dengue infection as they are relatively inexpensive IgM levels peak ~2 weeks after the onset of fever and then and easy to perform compared with culture or nucleic generally decline to undetectable levels over the next 2–3 acid-based methods. When a dengue infection occurs months54,55,65. Dengue-specific IgG is detectable at low titre in individuals who have experienced a previous dengue at the end of the first week of illness and slowly increases. infection, a secondary immune response occurs, which By contrast, during a secondary infection, high levels of generates high levels of IgG through the stimulation of IgG antibodies that crossreact with many flaviviruses are memory B cells from the previous infection as well as detectable even in the acute phase and rise dramatically an IgM response to the current infection. Because high over the following 2 weeks65. The kinetics of the IgM levels of IgG compete with IgM for antigen binding, an response are more variable; as IgM levels are significantly IgM capture assay can be used.

individual flaviviruses as no crossreactivity was observed

-PCR NS1

ELISA in sera collected from individuals infected with dengue or IgG y Japanese encephalitis virus85. Similarly, it has been dem- by RT by onstrated that IgG specific for the NS5 protein can poten- Viraemia tially discriminate between infections caused by West Nile, dengue and St Louis encephalitis viruses86. Finally, IgM dengue-specific IgG was shown to have high specificity in

al load detected detected load al an assay using a recombinant polypeptide located in the 87 Vir 046142150 Antibodies quantified b N-terminal region of the envelope protein . IgG assays Acute illness Days are also useful for sero-epidemiological studies to identify past dengue infection. Figure 4 | Dengue virus, antigenNa turande Reantibodyviews | Micr responsesobiology used in diagnosis. Ig, immunoglobulin; NS, non-structural. IgM:IgG ratio. A dengue virus E and M protein-specific IgM:IgG ratio can be used to distinguish primary from MAC-ELISA. The Armed Forces Research Institute of secondary dengue virus infections. IgM capture and IgG Medical Sciences (AFRIMS) developed an IgM anti- capture ELISAs are the most common assays for this body-capture enzyme-linked immunosorbent assay purpose. According to this method, a dengue infection is (MAC-ELISA) for dengue in regions where dengue defined as a primary infection if the IgM:IgG OD ratio is and Japanese encephalitis virus co-circulate65. Today, greater than 1.2 (using patient sera at 1:100 dilution) or many groups have developed their own in-house 1.4 (using patient sera at 1:20 dilution), or as a secondary MAC-ELISAs. Dengue-specific IgM in the test serum is infection if the ratio is less than 1.2 or 1.4 (REFS 88,89). detected by first capturing all IgM using human-specific However, in a recent publication the authors indicated IgM bound to a solid phase. The assay uses a mixture that the IgM:IgG ratio varies depending on whether of four dengue antigens (usually derived from dengue the patient has a serologically non-classical or classical virus-infected cell culture supernatants or infected dengue infection, and redefined the ratios84. Hence the suckling mouse brain preparations)76. Compared to the cut-off for the IgM:IgG ratio is not well defined. haemagglutination inhibition assay as the gold standard, MAC-ELISA shows a sensitivity and specificity of 90% Neutralization assays. The plaque reduction neutrali- and 98%, respectively, in samples collected after 5 days zation technique (PRNT) and the micro-neutralization of fever55. In addition to serum, dengue-specific IgM can assay are used to define the infecting serotypes following be detected in whole blood on filter paper (sensitivity a primary infection. These tests are mainly for research 98.1% and specificity 98.5%)77,78 and in saliva (sensitivity and vaccine studies90–94. 90.3% and specificity 92.0%)79, but not in urine68. More than 50 commercial kits are available with variable sen- Nucleic acid amplification tests sitivity and specificity65,80–82. False-positive results due to Many nucleic acid amplification tests (NAATs) have dengue-specific IgG and crossreactivity with other fla- been developed for the diagnosis of dengue infection. viviruses is a limitation of the MAC-ELISA, mainly in Some techniques are quantitative and others can be used regions where multiple flaviviruses co-circulate. Some for serotyping. However, none has been commercialised tests also show non-specific reactivity in sera from to date and quality assurance materials are not widely patients with malaria and leptospirosis82. available to ensure the quality of the results.

IgG ELISA. An ELISA for dengue-specific IgG detection Reverse transcriptase PCR (RT-PCR). Many dengue can be used to confirm a dengue infection in paired sera. RT-PCR assays have been described in the past 10 years. It is also widely used to classify primary or secondary These in-house assays target different genes and use dif- infections53,54,63,64. Some protocols use serum dilutions ferent amplification procedures. The most commonly to titre dengue-specific IgG83 and others use the ratio of used NAATs are based on a single RT-PCR assay95,96, a IgM to IgG66,84. The assay uses the same dengue antigens nested RT-PCR assay96 or a one-step multiplex RT-PCR as MAC-ELISA and it correlates with results from the assay97. The nested PCR reaction involves an initial haemagglutination inhibition assay. In general, an IgG reverse transcription and amplification step using den- ELISA lacks specificity within the flavivirus serocomplex gue primers that target a conserved region of the virus groups, however it has been demonstrated that the IgG genome followed by a second amplification step that is response to the prM membrane glycoprotein is specific to serotype specific. The products of these reactions are separated by electrophoresis on an agarose gel, which Box 1 | Laboratory diagnosis of a dengue virus infection allows the dengue serotypes to be differentiated on the basis of size. The sensitivity of RT-PCR assays in com- confirmed dengue infection Probable dengue infection parison to virus isolation in mosquito cell culture varies • Virus isolation • IgM positive between 25% and 79%98. • Genome detection • Elevated IgG titre (that is, 1,280 or greater • Antigen detection by haemagglutination inhibition test) Real-time RT-PCR. The real-time RT-PCR assay is a • IgM or IgG seroconversion one-step assay that allows virus titre to be quantified in approximately 1.5 hours. The detection of the amplified

Box 2 | The global strategy for dengue prevention and control IgM and IgG responses. NS1 antigen detection kits are now commercially available. As yet, these kits do not • Vector control, based on the principles of integrated vector management differentiate between the different DENV serotypes. • Active disease surveillance based on a comprehensive health information system Additional independent studies are needed to confirm • Emergency preparedness the performance of these kits and to further validate • Capacity building and training the diagnostic and prognostic significance of NS1 and 107–109 • Vector control research NS1-specific antibody detection . Dengue control and prevention strategies A global strategy for dengue prevention and control was target by fluorescent probes replaces the need for post- promulgated more than 10 years ago and comprises five amplification electrophoresis. Many real-time RT-PCR major elements (BOX 2). assays have been developed that are either ‘singleplex’, Efforts have since been made to focus on three detecting one single serotype per reaction, or ‘multiplex’, fundamental aspects: surveillance for planning and identifying all four serotypes from a single sample99–101. response, reducing the disease burden and changing One advantage of this assay is the ability to determine behaviours to improve vector control110. The 2002 viral titre early in dengue illness, which is believed to be World Health Assembly Resolution urged greater com- an important predictor of disease severity102. mitment among Member States and WHO to imple- ment this strategy111. Of particular significance is the Nucleic acid-sequence based amplification assay 2005 revision of the International Health Regulations112, (NASBA). The NASBA assay is an isothermal RNA- which includes mention of DF (and yellow fever) as an specific amplification assay that has been adapted for example of a health ‘event that may constitute a pub- dengue virus. Its performance is comparable to that of lic health emergency of international concern’ and other NAATs103. which, under such circumstances, should be notified to WHO. Antigen detection In recent years several new, improved or validated Dengue antigens can be detected in tissues such as liver, tools and strategies for dengue control and prevention spleen and lymph nodes as well as tissues from fatal cases have been developed and are available to public health (slides from paraffin-embedded, fresh or frozen tissues) practitioners and clinicians (BOX 3). using an enzyme and a colorimetric substrate with antibodies that target dengue-specific antigens104–106. Vector control. To reduce or prevent dengue virus transmission there is currently no alternative to vector con- NS1 antigen and antibody detection. NS1 is a glyco- trol. Most endemic countries have a vector control protein produced by all flaviviruses and is essential for component in their dengue control and prevention pro- viral replication and viability. Because this protein is grammes but its delivery by public health practitioners secreted into the bloodstream, many tests have been is frequently insufficient, ineffective or both. developed to diagnose DENV infections using NS1. Given its behaviour and generally close association These tests include antigen-capture ELISA, lateral flow with humans, the principal vector A. aegypti requires the antigen detection and measurement of NS1-specific use of a combination of vector-control methods, nota- bly environmental management methods and chemical control methods based on the application of larvicides Box 3 | Tools and resources for dengue control and prevention and adulticide space sprays113. Chemical controls typi- cally must be added to water stored for domestic use, • Rapid commercial diagnostic tests in use in endemic countries including drinking water. The active ingredients of • Pocket Book of Hospital Care for Children (inclusion of dengue in the management of four larvicides have been assessed by the International fever)137 Programme on Chemical Safety (IPCS) to determine 138 • An audiovisual guide and transcript for health care workers responding to outbreaks their safety for use as mosquito larvicides in drink- • Guidelines for planning social mobilization and communication139 ing water at dosages that are effective against Aedes • Global strategic framework for integrated vector management140 spp. larvae. Since the early 1970s the organophosphate • TDR–Wellcome Trust CD-ROM. Topics in International Health Series: dengue temephos has been widely used, but increasing levels of 114,115 • Entomological survey to identify the most productive container habitats of the resistance , householders’ rejection of the treatment vector(s)116 of their drinking water, and difficulties in achieving high • Seven insecticide products evaluated by WHO as mosquito larvicides (five insect and regular levels of coverage are important technical growth regulators and two bacterial larvicides), four of which are approved for use in and operational constraints. drinking water and three for space spray applications to control mosquitoes Biological control agents, including larvivorous fish • Advances in the development and operational deployment of DengueNet (http:// and copepods, have had a demonstrable role in control- 116,117 apps.who.int/globalatlas/default.asp) for global dengue surveillance ling A. aegypti , but operational difficulties — partic- • International Health Regulations 2005 (REF. 112): voluntary compliance in effect ularly the lack of facilities and expertise in mass rearing, and the need to frequently re-introduce these agents into • Planning Social Mobilization and Communication for Dengue Fever Prevention and Control: A Step-by-Step Guide139 some container habitats — have largely precluded their widespread use.

Environmental management is generally considered are being tested120 as well as controlled release larvicides to be an essential component of dengue prevention and that provide several months of control following a single control, particularly when targeting the most productive application to targeted containers. container habitats of the vector118. Source reduction, ‘clean- Products for personal and household protection have up’ campaigns, regular container emptying and cleaning a huge potential for household pest control. Generally (targeting not only households but also public spaces speaking, these commercial products tend to be used such as cemeteries, green areas and schools), installation by consumers not so much in response to any perceived of water supply systems, solid waste management and public health concerns, but to alleviate the nuisance of urban planning all fall under the rubric of environmental biting mosquitoes and in some settings households are management. However, huge investments in infrastruc- prepared to spend substantial amounts of money on ture are needed to increase access to safe and reliable water these products121. supplies and solid waste disposal systems. In addition to With the increased political recognition of dengue as overall health gains, such provision would clearly have a a public health problem and commitment to prevention major impact on vector ecology, although the relation- and control, better organized control services using new ship is complex. For instance, cost recovery mechanisms, tools and partnership strategies, based on the principles such as the introduction of metered water, might actually of integrated vector management, are likely to have a encourage the household collection and storage of roof major impact on dengue transmission2. catchment rainwater, which can be harvested at no cost. Although not studied carefully, the construction of com- Vaccine development. As a result of the failure of vector munity water distribution services to rural townships and control, the continuing spread and increasing intensity villages might be contributing to the rural spread of den- of dengue has renewed interest and investment in dengue in Southeast Asia and elsewhere by facilitating domes- gue vaccine development, making a safe, effective and tic water storage. When decisions on such infrastructure affordable tetravalent dengue vaccine a global public development are being made, the views of Ministers of health priority122. Dengue vaccine development has been Public Health and municipal health departments are sel- in progress for several decades, however the complex dom voiced loudly, even when the economic and public pathology of the illness, the need to control four virus health burden of diseases linked to water and sanitation serotypes simultaneously and insufficient investment by are recognized, including those associated with dengue. vaccine developers have hampered progress122. Most efforts in vector control are centred at the house- The observation that DHF/DSS is associated with hold and community levels, but with few exceptions, the DENV secondary infection poses a special challenge achievements to date have been largely unspectacular to the development of a dengue vaccine, leading to a and there have been difficulties in scaling up from the requirement that such vaccines should induce a robust project level119. Nevertheless, such community-based immune response against the four serotypes in naive as interventions are widely seen as the most promising way well as previously immune individuals. Animal models of improving delivery and achieving long-term control of are only partially useful for vaccine evaluation. The poor the vector through behaviour change. Towards this end, understanding of the mechanisms involved in inducing a TDR/WHO guide for planning social mobilization protective immunity against dengue infection poses and communication for dengue fever prevention and additional challenges123. Finally, cases of DHF/DSS have control has been developed113. Additionally, new ‘con- recently been documented 20 or more years after pri- sumer-friendly’ tools such as window curtains and water mary dengue infection, which adds a new dimension to container covers treated with long-lasting insecticide the problem25,44.

Table 1 | Selected dengue vaccine candidates Vaccine approach Developer Status Live attenuated tetravalent chimeric YF–DEN vaccine Sanofi Pasteur Phase II Live attenuated tetravalent viral isolate vaccine WRAIR and GSK Phase II Live attenuated chimeric DEN2–DEN vaccine CDC and Inviragen Phase I Recombinant E subunit vaccine Merck Phase I Live attenuated tetravalent vaccine comprising 3′ deletion mutations US NIH LID and NIAID Phase I and DEN–DEN chimeras Subunit recombinant antigen (domain III) vaccine IPK/CIGB Preclinical Live attenuated chimeric YF–DEN vaccine Oswaldo Cruz Foundation Preclinical Tetravalent DNA vaccine US NMRC and GenPhar Preclinical Purified inactivated tetravalent vaccine WRAIR and GSK Preclinical CDC, Centers for Disease Control and Prevention; CIGB, Center for Genetic Engineering and Biotechnology; GSK, GlaxoSmithKline; IPK, Pedro Kouri Tropical Medicine Institute; NIAID, National Institute for Allergy and Infectious Diseases; US NIH LID, United States National Institutes of Health Laboratory of Infectious Diseases; US NMRC, United States Naval Medical Research Center; WRAIR, Walter Reed Army Institute of Research; YF, yellow fever.

The available data suggest that neutralizing antibodies attenuated dengue viruses as backbones129–134. TABLE 1 are the major contributors to protective immunity124,125, summarizes the most advanced vaccine candidates. however the role of the cellular immune response Significant progress in the development of dengue requires further study123. In this context, clinical tri- vaccine candidates has been achieved lately135,136. An als are crucial for vaccine development owing to the Acambis/Sanofi Pasteur yellow fever–dengue chimeric unique information they provide on immune responses vaccine is in advanced Phase II testing in children in and reactogenicity. Also, long-term observations of vac- Thailand and others are in Phase 1 or advanced preclini- cinated populations will be required to demonstrate the cal evaluation. It is expected that a licensed vaccine will absence of ADE or severe disease. be available in less than 10 years. The ideal dengue vaccine should be free of important reactogenicity, induce life-long protection against Conclusions infection with any of the four DENV serotypes and be Dengue is now a global threat and is endemic or epi- affordable126,127. Vaccine candidates should be evaluated demic in almost every country located in the tropics. in population-based efficacy trials in several at-risk While we wait for new tools such as vaccines, antiviral populations in different geographical settings includ- drugs and improved diagnostics, better use should be ing Asia and the Americas, which experience different made of the interventions that are currently available. patterns of dengue transmission intensity and dengue The challenge that awaits us in the near future will be virus circulation122. Vaccine developers are working how to scale up to deploy these new tools. with the Pediatric Dengue Vaccine Initiative (PDVI) to In recent years, several partnerships such as the establish suitable field sites. Developers are also working PDVI, the Innovative Vector Control Consortium, with the WHO Initiative for Vaccine Research (WHO/ the Asia-Pacific Dengue Prevention Partnership and IVR) to define the immunological correlates for protec- the European union’s DENFRAME and DENCO tion and clinical trial design. Because of the important projects have come into existence, receiving funding role of neutralizing antibodies as surrogates of protec- from the Bill and Melinda Gates Foundation, regional tion, the validation of neutralization tests is a priority128. Development Banks and the private sector. These part- Current approaches to vaccine development involve nerships are working with WHO and national govern- using live attenuated viruses, inactivated viruses, subu- ments to develop new tools and strategies to improve nit vaccines, DNA vaccines, cloned engineered viruses diagnostics and clinical treatments and to achieve a and chimeric viruses using yellow fever vaccine and successful vaccine.

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