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Dengue virus (DENV) belongs to the Flavivirus genus and is transmitted by mosquitoes, including Aedes albopictus and Ae. aegypti. There are four serotypes of DENV (DEVN 1–4), which can cause a spectrum of outcomes ranging from subclinical to death. Four serotypes (DENV 1–4) are circulating in tropical and ...
Keywords : Dengue Virus, epidemiology, vaccine, viral pathogenesis, innate immunity
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- Published: 09 December 2015
Tackling dengue fever: Current status and challenges
- Taoufik Nedjadi 1 ,
- Sherif El-Kafrawy 2 ,
- Sayed S. Sohrab 2 ,
- Philippe Desprès 3 ,
- Ghazi Damanhouri 1 &
- Esam Azhar 2
Virology Journal volume 12 , Article number: 212 ( 2015 ) Cite this article
According to recent statistics, 96 million apparent dengue infections were estimated worldwide in 2010. This figure is by far greater than the WHO prediction which indicates the rapid spread of this disease posing a growing threat to the economy and a major challenge to clinicians and health care services across the globe particularly in the affected areas.
This article aims at bringing to light the current epidemiological and clinical status of the dengue fever. The relationship between genetic mutations, single nucleotide polymorphism (SNP) and the pathophysiology of disease progression will be put into perspective. It will also highlight the recent advances in dengue vaccine development.
Thus far, a significant progress has been made in unraveling the risk factors and understanding the molecular pathogenesis associated with the disease. However, further insights in molecular features of the disease and the development of animal models will enormously help improving the therapeutic interventions and potentially contribute to finding new preventive measures for population at risk.
Dengue fever is a major cause of illness and death worldwide. The disease is caused by dengue virus which gets transmitted to humans by the bites of infected mosquitoes, Aedes (Ae.) aegypti and Ae. albopictus [ 1 ]. The disease represents a global health issue as it is endemic in around 100 countries, most of which are in tropical and sub-tropical areas. Over the last decades, the incidence rate and the geographic distribution of dengue have rapidly increased (almost 30-fold). Data from the World Health Organization (WHO) estimates up to 100 million cases of dengue fever each year [ 2 ]. However, a recent published work by Bhatt et al . (2013) suggested that the burden of dengue is far more than the WHO estimation and indicated that 390 million infections of dengue virus could have happened every year [ 3 ]. Changes in dengue epidemiology and the increase in incidence rates (with and without co-morbidities) have led the WHO to propose a new dengue classification system according to disease severity (Fig. 1 ) [ 2 ].
WHO dengue case classification ( Adopted from; Dengue Guidelines for diagnosis, treatment, prevention and control, New edn. Geneva: WHO; 2009 )
Etiology and mode of transmission
Dengue fever is caused by infection with dengue virus (DENV). The DENV is a vector-borne virus transmitted to humans primarily by bites from two mosquito species, Ae. aegypti or Ae. albopictus . DENV is a single positive-stranded RNA virus belonging to Flavivirus genus of the Flaviviridae family and has 4 major serotypes (DENV 1–4) that are antigenically distinct from each other. Each DENV serotype is phylogenetically distinct suggesting that each serotype could be considered a separate virus [ 4 ]. Three dengue serotypes out of four (DENV 1–3) have been found in Middle Eastern countries including Saudi Arabia and Yemen. Interestingly, DENV-1 strain isolated in Saudi Arabia exhibited a high genetic similarity with DENV-1 strain isolated from Asian population, suggesting a widespread of the Asian genotype, probably through Asian pilgrims [ 5 , 6 ]. A recently published article has unveiled a new serotype (DENV-5), to be added to the existing ones [ 7 ]. This discovery is still controversial and little-known enough to conclude how the 5 th dengue serotype might add to the burden associated with dengue infection.
Mosquitoes transmit the virus by feeding on blood of infected persons. At first, the virus infects and replicates in the mid-gut epithelium of the mosquito and then spreads to other organs until it reaches the salivary glands after 10–14 days where it can be inoculated to another person during subsequent blood meal. Vertical transmission of DENV in mosquitoes, i.e. from mosquito to larvae has been reported by a number of research groups. In India, Angel & Joshi (2008) reported the detection of dengue virus by indirect fluorescence antibody test (IFAT) in laboratory reared mosquitoes originating from larvae collected from urban and rural areas [ 8 ]. A similar study was conducted in Brazil by Martins et al. (2012) and confirmed the isolation of DENV-type 3 in Ae. albopictus larvae and DENV-type 2 in Ae. aegypti larvae [ 9 ]. Similar findings were also reported in Mexico [ 10 ] and Indonesia [ 11 ]. On the other hand, mother-to-infant transmission of dengue virus via cord blood or breast milk remains controversial [ 12 – 14 ].
Based on the results from several studies, the WHO has launched a new dengue classification. This classification divides dengue cases into a) cases with/without warning signs and b) severe dengue cases [ 2 ]. However, it is important to note that numerous research groups have debated the rational of this classification as it does not fit their unique local settings. The criteria for dengue case classification are presented in Fig. 1 .
Clinically, dengue infection has a broad spectrum of features. The vast majority of cases are asymptomatic and passes unnoticed. Typically, the symptoms start to be prominent after an incubation period of 3–10 days [ 15 ]. The severity of the clinical manifestations varies from mild symptoms to severe life threatening symptoms in the case of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [ 16 ]. Predicting the progression of the mild signs to a severe DHF/DSS remains a challenge due to non-specificity of clinical presentation and the incomplete understanding of pathophysiology of the disease and its underlying molecular mechanisms.
Dengue with warning signs
The early signs of the disease are non-specific. According to the WHO classification (2009), DF is characterized by febrile episode (≥40 °C for 2–7 days) frequently associated with rash, nausea, vomiting, and headache. Although the disease affects people of all ages from infancy through to adulthood [ 17 ], epidemiological data showed that children tend to tolerate this phase of illness better than adults [ 18 ]. The persistence of the aforementioned symptoms and appearance of other symptoms, such as abdominal pain, mucosal bleed, and lethargy and restlessness can be seen 3–7 days later. Laboratory analysis of mild dengue fever cases usually shows abnormal leukocyte counts and moderate elevation of the hepatic amino-transferase enzyme activity [ 19 ]. The emergence of these symptoms is a warning sign for disease progression to severe form (DHF/DSS) if therapeutic intervention is not undertaken. At this stage clinical intervention and continuous surveillance are imperative to prevent vascular leakage, especially in an endemic area.
This form of dengue infection can be attributed to any of the four known serotypes DENV 1–4. The likelihood of developing DHF/DSS is high in patients who have experienced dengue infection in the past with heterogeneous serotype [ 20 ]. About 5–10 % of patients progress to develop a severe DHF/DSS which can be fatal unless treated promptly [ 21 ]. This form develops at a late stage of DF, where patients may go through defervescence phase characterized by a sudden drop of body’s temperature. This phase is also distinguished by severe bleeding, particularly bleeding from the gastrointestinal tract (black, tarry stool), and thrombocytopenia (<50,000/mm3), which may affect up to 50 % of DHF cases [ 22 ]. Interestingly, there was an observed negative correlation between the severity of DHF and the level of platelets in the blood. The exact mechanism of this correlation has yet to be delineated. The drop of platelet counts and the loss of their functionality lead to a vascular fragility increasing the risk of hemorrhage and plasma leakage [ 23 ]. It has been suggested that during acute phase of the infection DENV replicates quickly in platelets, as this is very critical for virus survival and dissemination [ 24 , 25 ].
The existence of other symptoms such as retro-orbital pain, maculopapular rash, petechiae, or bleeding from the nose or gums will help making definitive diagnosis for DF [ 25 ]. Evidence of plasma leakage in various body cavities such as the pleural cavity and the peritoneal cavity, associated with profuse perspiration, adynamia, and sometimes fainting are signs of rapid progression to shock. Subsidence in systolic pressure and hypotension may result in profound shock, known as dengue shock syndrome (DSS). The duration of DSS for a long time might predispose to further complications such as massive bleeding, disseminated intravascular coagulopathy (DIC), respiratory failure, multi-organ failure, and infrequently encephalopathy leading to death [ 26 , 27 ]. It has been proposed that case fatality related to DHF may reach 15 % of all cases, however, proper medical care and symptomatic management can reduce mortality rate to less than 1 % [ 28 ].
An early and accurate laboratory diagnosis of dengue infection is of paramount importance in the management of the disease. It has been estimated that the number of misdiagnosed dengue cases could reach a record ratio of 50 % of all cases, mainly due to a large disparity of dengue signs and symptoms which overlap with the symptoms of other viral infections, especially for persons living in or traveling to endemic areas of tropical infectious diseases. Dengue fever should be distinguished from other illnesses which share similar symptoms such as chikungunya, Mayaro fever, Ross River fever, West Nile fever, Zika fever, yellow fever and viral hemorrhagic fevers [ 4 ]. Until the antiviral vaccine becomes available, the prevention of severe cases and cut-down of the economic burden of the disease rely enormously on early and accurate diagnosis. The latter is made possible through the availability of several diagnostic laboratory and virological tests.
The onset of later stage symptoms of the illness can be overwhelming and more pathognomonic. Nonetheless, based on WHO classification schemes, the appearance of leukopenia in patients with febrile illness is a major consideration in making diagnosis of dengue infection [ 29 ]. Overall, there is an urgent need to reduce dengue morbidity and mortality by improving the diagnosis and molecular analysis of emerging dengue virus. Thus far, two diagnostic modalities have been applied to detect the disease at an early stage. The first one is a direct method targeting the acute phase of dengue disease, which is based upon detection of genomic RNA by RT-qPCR or soluble NS1 by antigen capture in blood samples from viremic patients. The second is the indirect method that relies on serological tests to detect dengue-related immunoglobulins par Mac-ELISA for the capture of specific IgM or indirect ELISA for the capture of anti-DEN IgGs [ 30 – 34 ].
Genetic alteration/susceptibility to dengue infection
Several risk factors have been associated with dengue infection and its progression to severe DHF/DSS forms. Recent advances in molecular biology have revealed that the genetic makeup of the three elements of dengue infection (the virus, the vector, and the host) plays a primordial role in the pathogenesis of the disease and could potentially contribute to the DHF progression [ 19 , 24 , 35 ]. Hence, an in-depth analysis of genetic variability including polymorphism and mutations could be beneficial in identifying the possible factors and mechanisms of disease development [ 36 ]. The list of host’s genetic factors that confer susceptibility or resistance to dengue infection is summarized in Table 1 .
Like most arboviruses, DENV infect different organs of the mosquito, including the salivary glands and the central nervous system. Mosquito infection elicit behavioral changes including increase of the probing time which lead to host interruption that might lead to wider spread of the virus [ 37 ]. It has been demonstrated that DENV infection induced the expression of cathepsin-B, a putative cystatin, and a hypothetical ankyrin repeat-containing protein genes [ 38 ]. The latter could alter the efficiency of virus replication in the salivary gland. This study has shown that modulation of OBP10 and OBP22 genes expression as well as DENV infection-responsive odorant-binding protein genes increase the time length for initiation of probing before a successful blood meal, resulting in changes in the host seeking behavior of the mosquito. Comparative analysis of the salivary gland transcriptomes of native and DENV-infected Ae. aegypti identified a number of differentially expressed genes related to sugar/protein digestion enzymes, immunity related genes and blood meal acquisition enzymes that might have an impact on the efficiency of viral replication or mosquito feeding behavior. This study showed that DENV infection alter the expression of key host-seeking genes in the mosquito’s main olfactory organs and the antennae [ 38 ].
Recent updates have indicated that resistance of Ae. aegypti to conventional insecticides is related to different mechanisms, one of which is associated with genetic abnormalities within the vector’s genome. Single point mutation in the voltage-gated sodium channel gene at position 1534 ( F1534C ) resulting in phenylalanine to cysteine substitution in Ae. aegypti confers resistance to permethrin. This mutation is widespread in this vector in Southeast Asia and Latin America [ 39 , 40 ]. It has also been reported that a single amino acid substitution Valine to Glycine at position 1016 in domain II, segment 6 of the voltage-gated sodium channel gene was associated with less sensibility of Ae. aegypti to deltamethrin in Thailand [ 41 ].
Human susceptibility to dengue disease
Numerous multi-disciplinary studies confirmed that race, young age, virus strain, female sex and high body-mass index correlate well with increased burden of dengue infection. The observation that people of African background are less likely to develop DHF/DSS compared to their Caucasian counterpart has led to the suggestion that host genetic variability has a major impact on the clinical manifestations of dengue infection [ 42 , 43 ]. Thus, a closer consideration of human genes regulating the severity of dengue infection, especially genes associated with the immune response, might help in controlling disease spread and improve the acute symptoms of the infection. A number of studies have investigated the relationship between the host genetic polymorphisms and DENV infection (Table 1 ).
A single nucleotide polymorphism (SNP) in the promoter of CD209/DC-SIGN was associated to increased risk of developing dengue fever [ 44 ]. Association studies have successfully identified a link between polymorphisms in the human major-histocompatibility-complex (HLA) class I/II genes and non-HLA host genetic factors and severity of dengue disease [ 45 – 47 ]. Polymorphisms of the TAP1 and TAP2 genes could be directly associated with the risk of developing dengue disease among the primary-infected individuals [ 48 ]. Both TAP1 and TAP2 are located within the MHC class II region and homozygosity of the TAP1 at position 1333 and 1637 and for TAP2 at position 2379, respectively, was found to protect against developing severe forms of dengue [ 46 ].
In an independent study [ 49 ], the authors showed that single nucleotide polymorphism of the oligoadenylate synthetase genes ( OAS1, 2 and 3 ), of the OAS/RNase L antiviral immune system, enhance susceptibility to clinical outcomes of dengue infection. An association between the severity of the disease and other genes including human leukocyte antigen class I and class II genes, tumor necrosis factor-alpha, FcGRIIA , vitamin D receptor, transporters associated with antigen presentation, and JAK1 has also been proposed [ 50 ]. The importance of Vit-D in DENV pathogenesis was concluded from newly-gathered data showing that Vit-D impairs DENV replication and polymorphism of Vit-D gene increases the expression of both CD209/DC-SIGN and FcGRIIA receptors that enhance DENV entry in the target cells [ 51 , 52 ].
In another study [ 53 ], the authors have successfully applied genome-wide association study (GWAS) approach to identify loci that confer susceptibility to severe forms of dengue disease. The investigators used samples from 2008 children affected with severe dengue infection against 2018 population control cases in Vietnam. The data showed that SNPs at two loci, MICB and PLCE1 , significantly increased the likelihood of developing DSS in children. This finding was further validated in an independent cohort of 1737 cases and 2934 controls [ 53 ]. A SNP in the MICB gene coding for the MHC class I polypeptide-related sequence B, an inducible activating ligand for the NKG2D type II receptor of immune cells could alter the protective role of natural killer and CD8 + T cells in the host responsiveness to DENV at the early stage of infection [ 54 , 55 ]. On the other hand, PLCE1 plays a primordial role in maintaining intact vascular endothelial cell barrier function, hence, polymorphism of the PLCE1 gene may lead to blood vessels leakage and circulatory hypovolemia during DSS [ 56 ].
Other host candidate genes have also been associated with early onset dengue disease. Among these genes, there were receptors/attachment factors for DENV linked to immune system and inflammatory response. The chemokines CXCL10, CXCL11 and its respective chemokine receptor CXCR3 were reported as biomarkers for severe form of dengue infection [ 57 ]. These results are in agreement with recent emerging data indicating strong association between CXCL10, CXCL11 and CXCR3 and vascular permeability [ 58 ]. The three genes are components of the NF-kB pathway and are involved in the pathogenesis of SARS and West Nile virus encephalitis [ 59 , 60 ]. Cerney et al . (2014) interrogated the effect of DENV on the first point of human contact which is skin cells. The authors demonstrated an increase expression of IFN-β, STAT-1 and CCL5 in a susceptible population of skin dendritic cells (DC) which may facilitate the spread of DENV in the blood [ 61 ]. This process depends enormously on vector-derived salivary factors inoculated on the skin cells [ 62 ].
Current status of dengue vaccine development
Till-date, there is no effective, commercially available, therapy/vaccine for dengue virus. Numerous groups have already made intensive efforts and made good progress to develop a safe, affordable and effective vaccine against all serotypes for global public health [ 63 – 69 ]. Vaccines which are being developed use various approaches such as live attenuated viruses, inactivated viruses, subunit vaccines, DNA vaccines, and chimeric viruses using yellow fever vaccine and attenuated dengue viruses as backbones (Table 2 ).
Live attenuated yellow fever 17D/DENV chimeric vaccine
Currently, only one tetravalent vaccine against dengue virus, developed by Sanofi-Pasteur (France) has reached phase III clinical trial and is expected to be launched in 2015. This vaccine is based on the production of four chimeric live dengue-yellow fever viruses in which the yellow fever (YF) 17D vaccine sequences encoding the envelope proteins prM and E genes were substituted by the prM and E genes from DV of serotype 1, 2, 3, or 4 in a molecular clone of YF-17D [ 69 ]. This vaccine was produced and tested over 6000 people using four dengue virus isolates from Indonesia and Thailand. This candidate vaccine was found to be attenuated and stable in animal models with respect to plaque size and yellow fever virus neurotropism [ 70 ]. Results of the clinical trials showed no adverse effects except moderate injection site pain, headache, and myalgia. Another randomized, controlled trial was launched using a total of 4002 Thai school children to investigate the efficacy of a recombinant, tetravalent vaccine for dengue virus and only 134 dengue cases were reported [ 71 ]. Phase I trial of the vaccine in the Philippines showed that the seropositivity increased gradually (53, 72 & 92 %) after 1–3 vaccinations against all four serotypes as compared to control group. The most promising results were observed in children 2–5 years old who exhibited high levels of reactivity of 91, 100, 96, 100 % for DENV 1–4; respectively [ 72 ].
Another placebo-controlled trial was conducted on 10,275 children from Vietnam (vaccine, n = 6851 Vs placebo, n = 3424) to determine the clinical efficacy and safety of CYD-TDV. The results demonstrated virologically-confirmed cases in 47 % of the vaccine group as compared to the control group (53 %). The efficacy was achieved in up to 56.5 % (95 % CI 43.8–66.4). These findings indicated that the vaccine is highly efficacious with good safety profile when three injections were given to children with age group 2–14 years at 0, 6 and 12 months intervals [ 73 ]. The data emerging from another randomized phase II trial in India indicated that the vaccine has no serious adverse events and the immunogenicity and safety of CYD-TDV were satisfactory [ 74 ]. A pilot study carried out in five Latin American countries where more than 20,000 children aged 9–16 were recruited to receive either the CYD-TDV vaccine or placebo. The results on efficacy (60.8 %) and safety profiles were consistent with the previous findings [ 74 , 75 ]. Interestingly, the vaccine efficacy (80.3 %) against hospitalization for dengue was promising and represented a step forward to developing an effective dengue vaccine [ 75 ].
Live attenuated DENV delta-30 mutation and intertypic DENV chimeric vaccines
Other candidate dengue vaccines have been developed in USA by the Johns Hopkins University and National Institute of Allergy and Infectious Diseases (NIAID) and have reached advanced clinical trials [ 65 ]. Four live-attenuated DENV/delta-30 were generated each containing 30 nucleotides deletion of the 3’-untranslated region of genomic RNA (delta-30). These vaccines efficiently impaired viral growth in human liver carcinoma cells [ 76 ]. To improve the attenuation of DENV-2/delta-30 and DENV-3/delta-30, chimeric DENV were developed by substitution of the prM-E gene region of DENV-4/delta-30 virus with the prM-E genes of DENV-2 and DENV-3 [ 72 , 77 ]. The results from phase I clinical trial showed that all four live-attenuated DENV/delta-30 are safe and immunogenic with minor side effects such as faint rash and transient leucopenia only after higher dose [ 78 , 79 ].
Dengue virus serotype-1 antigen was expressed in a vector based on pediatric live-attenuated Schwarz measles vaccine (MV) by using the envelope domain III (EDIII) fused with the ectodomain of the membrane protein (ectoM). After immunization, long-term production of DENV-1 serotype-specific neutralizing antibodies was observed in measles virus susceptible mice [ 80 ]. A new strategy was evaluated based on single minimal tetravalent DENV antigen expression using viral vector derived from pediatric live-attenuated measles vaccine (MV). A recombinant MV vaccine construct was developed using envelope domain III (EDIII) and ectodomain of the membrane protein. The neutralizing antibodies were induced against all four serotypes of dengue virus after two injections in mice susceptible to MV infection. A strong memory neutralizing response was observed against all four serotypes in immunized mice after inoculation with live DENV from each serotype [ 81 ].
Dengue prM-E DNA vaccine
A naked DNA-based candidate vaccine against DENV has been developed by the Naval Medical Research Center [ 67 , 82 , 83 ]. The genes encoding prM and E of DENV were cloned into a shuttle vector under the transcriptional control of human cytomegalovirus (CMV) promoter. The results of phase I clinical trial showed no adverse effects except mild injection site pain, swelling, and fatigue. After second dose, strong IgM and IgG antibody response was observed which favors the safety profile of this vaccine. To get a better immunogenicity profile, a vaccine based on lipid adjuvant Vaxfectin (Vical Incorporated, San Diego, USA), was developed and the results demonstrated good protection profile against DENV compared to DNA alone [ 84 ]. Based on this technology, different groups have developed other candidate vaccines and achieved good protection in mouse models using envelope glycoproteins prM and E, the non-structural protein NS1 and the helicase/protease NS3 as vaccine antigens [ 85 – 87 ].
Purified inactivated vaccine (PIV)
The first purified inactivated vaccine was developed with aluminum hydroxide (alum) adjuvant and tested in mice and rhesus macaques in the mid-1990s, by Walter Reed Army Institute of Research against dengue 2 serotype and good virus protection was reported after two doses [ 88 , 89 ]. Using similar technology, second generation Japanese encephalitis (JE) PIV vaccine was developed [ 90 , 91 ]. Currently, a new JE vaccine (Ixiaro; Novartis Vaccines) has been approved for use in many countries, including the USA [ 92 ]. Another dengue vaccine (dengue 1 PIV), recombinant subunit dengue E glycoprotein antigen (r80E) was also developed and has entered phase I clinical trial [ 93 – 95 ]. The Centers for Disease Control and Prevention (USA) have also developed a live-attenuated vaccine named DENVax, which was found to be highly immunogenic in both children and adults and has currently entered phase I clinical trial in the United States [ 96 , 97 ]. Recently, a novel third generation approach is being used to develop a vaccine containing recombinant subunit E domain III ( ED3 ) and the results of laboratory tests have shown the development of potent neutralizing antibodies in a mouse model [ 98 – 100 ]. Using the same technology, a tetravalent vaccine was developed and expressed in Pichia pastoris by splicing and using flexible pentaglycyl linkers of the four EDIII. The observed results showed that this antigen elicit specific antibodies against all four DENV serotypes in BALB/c mice [ 101 ].
Lessons from animal models
Animal models are very useful for vaccine test development. The lack of animal models significantly hampered the development and efficacy testing of dengue vaccine. Currently only rhesus macaques and Aotus monkeys are being used for testing the vaccine before clinical trials are initiated [ 62 ]. The D1ME100 vaccine was evaluated in both Aotus monkeys and rhesus monkeys, and found to be immunogenic with 80–95 % protection against dengue infection [ 102 , 103 ]. Porter et al. (2012) demonstrated that injection of non-human primate with three doses on day 1, 28 and 84, with tetravalent dengue DNA vaccine Vaxfectin-adjuvanted, was more efficient against live dengue-2 virus compared to control animals. This finding support initiation of Vaxfectin-adjuvanted phase I clinical trial [ 84 ].
Successful induction of immune response was obtained in mice and rhesus monkeys to the vaccines developed using dengue 4 prM-E, dengue 1 prM-E-nonstructural (NS)1 , and dengue 2 NS3 antigens, and PIV adjuvanted with alum [ 85 , 86 ]. Centers for Disease Control and Prevention (Fort Collins, CO), Hawaii biotech, and Simmons developed different vaccines that showed good immunogenicity in animal models [ 104 ]. Similarly, the psoralen/UV inactivation dengue vaccine was found to be more immunogenic and protective against dengue serotype 1 virus in Aotus monkeys [ 105 ].
Thus far, there are no antiviral drugs available to treat dengue fever; therefore the community will continue to depend on the control of the mosquito vector as the main route to prevent the spread of disease. Alternative approaches have been utilized against flaviviruses by targeting and inhibiting virus entry and the essential elements used in virus replication, nonstructural proteins, RNA polymerase, and proteases. The most important target elements include NS3 helicase nucleoside triphosphatase (NTPase/RNA 5’ triphosphatase (RTPase), NS5 methyl transferase/RNA-dependent RNA polymerase, and NS3/NS2B protease [ 106 – 108 ].
RNA interference (RNAi) technology is also being used to impair virus replication against respiratory syncytial virus, hepatitis viruses, influenza virus, poliovirus and HIV [ 109 , 110 ]. Low molecular weight phenolic compounds such as flavonoids and phytochemicals isolated from plants were previously tested and are being used for anti-dengue therapy [ 111 , 112 ]. An anti-viral inhibitory effect ranging from 50–75 % against DENV replication was observed when methanolic extracts of Momordica charantia and Andrographis paniculata were used in cultured primate cells [ 113 ].
Several attempts have been made in the past to tackle dengue through elimination of Ae. Aegypti. The most successful experiences were related to vector control programs adopted in Cuba and Singapore. The programs were based on intensive insecticidal treatment and reduction of the availability of Aedes larval habitats [ 18 , 114 ]. Unfortunately, lack of sustainability of these stringent measures led to reappearance of dengue outbreaks.
Recently, a novel form of biological control of dengue transmission has been developed and is currently being applied. This is based on the development of genetically modified (GM) mosquitoes infected with a bacterium known as Wolbachia to combat dengue infection. This bacterium blocks replication of the virus inside the mosquito and prevents its transmission to humans [ 115 ]. In 2012, 10 million GM male mosquitoes were released in the wild to decrease the number of Aedes mosquitoes and reduce the rate of dengue transmission. A closer monitoring of the insects revealed that over 85 % of the eggs were Wolbachia-positive which indicated that GM-mosquitoes were overriding wild-mosquitoes resulting in decreased virus transmission [ 116 ]. In an initiative to eradicate dengue fever, scientists from Australia, are leading Eliminate Dengue (ED) program which involves community engagement as a key component in this program. Since the program kicked off in 2011, millions of Wolbachia mosquitoes were released across the North Queensland city—Australia. Based on the promising results obtained from local trial, Eliminate Dengue became an international research program across countries affected by dengue including Australia, Vietnam, Indonesia, Brazil and Colombia [ 117 , 118 ].
Targets of antiviral therapy
Dengue infection can be prevented by alternative approaches. The first one includes blocking virus entry into cells which is mediated by the viral envelope glycoprotein E via receptor-mediated endocytosis [ 119 ]. Dendritic cells, monocytes, and macrophages are the main targets of DENV infectious entry. The second approach involves blocking virus attachment to specific cellular receptors expressed on immune cells, liver cells, and endothelial cells.
Fusion and glycosidase inhibitors
Small molecules and peptides targeting the hydrophobic pocket of the envelope E glycoprotein are characterized as inhibitors of virus entry. Nicholson et al. (2011) explored the inhibitory effects of DN59 and 1OAN1, peptide entry inhibitors. The authors demonstrated that DN59 and 1OAN1 can effectively block antibody dependent enhancement (ADE) in-vitro suggesting that entry inhibitors are potential candidates to prevent development of DHF/DSS [ 120 ]. Two other compounds have also been shown to qualify as potent inhibitors of dengue virus infection are imino-sugars deoxynojirimycin and castanospermine [ 121 ]. These compounds are natural alkaloids derived from the black bean and act as inhibitors against all 4 dengue serotypes by disrupting the folding pathways of the envelope glycoproteins prM and E [ 122 ].
Various types of carbohydrate-binding agents, isolated from different organisms, have been shown to have anti-viral activities. Three plant lectins, Hippeastrum hybrid agglutinin, Galanthus nivalis agglutinin and Urtica dioica agglutinin isolated from amaryllis, snowdrop and stinging nettle respectively were found to be potent inhibitors of DENV-2 infection by inhibiting viral replication [ 123 ].
Heparan sulfate (HS) is a putative receptor for DENV which interacts with domain III of the E-protein. Virus entry can be blocked by targeting the E-protein-HS interaction with soluble GAGs and other highly charged HS [ 124 ]. Fucoidan was isolated from marine algae and showed antiviral activity against DENV-2 in BHK cells [ 125 ]. Similarly, carrageenan and DL galactan, sulfated polysaccharides from red seaweeds, exhibited strong antiviral activity against DENV-2 and DENV-3 but a very weak activity against DENV-4 and DENV-1. Furthermore, two α -D-glucans were isolated from a Chinese herb and demonstrated high anti-DENV-2 activities in BHK cells [ 112 , 126 ].
Dengue fever represents a real economic burden especially in affected countries. Extensive efforts are needed to tackle disease spread and reduce the mortality rates and the associated healthcare cost. There is a need for more scientific research which we believe is a key route to provide further insight in the pathogenesis of dengue infection and help understanding the underlying molecular mechanisms associated with progression to the severe forms of the disease (DHF/DSS). This will be a step forward to develop an adequate preventive vaccine and effective treatment.
- Dengue hemorrhagic fever
Dengue shock syndrome
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This Project is funded by the King Abdulaziz City for Science and Technology (KACST) under grant number (ات-33-26) . The authors are also grateful to the kind cooperation provided by the Deanship of Scientific Research (DSR), King Abdulaziz University.
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Taoufik Nedjadi & Ghazi Damanhouri
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
Sherif El-Kafrawy, Sayed S. Sohrab & Esam Azhar
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TN Participated in the review design, coordination and helped to draft the manuscript. SK and SS Participated in the review design and helped to draft the manuscript. PD Participated in the article revision. GD and EA Participated in the review design. Read and approved the final manuscript.
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Nedjadi, T., El-Kafrawy, S., Sohrab, S.S. et al. Tackling dengue fever: Current status and challenges. Virol J 12 , 212 (2015). https://doi.org/10.1186/s12985-015-0444-8
Received : 12 May 2015
Accepted : 01 December 2015
Published : 09 December 2015
DOI : https://doi.org/10.1186/s12985-015-0444-8
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- Published: 18 August 2016
- Maria G. Guzman 1 ,
- Duane J. Gubler 2 ,
- Alienys Izquierdo 1 ,
- Eric Martinez 1 &
- Scott B. Halstead 3
Nature Reviews Disease Primers volume 2 , Article number: 16055 ( 2016 ) Cite this article
- Dengue virus
- Viral epidemiology
- Viral host response
- Viral infection
- Viral pathogenesis
Dengue is widespread throughout the tropics and local spatial variation in dengue virus transmission is strongly influenced by rainfall, temperature, urbanization and distribution of the principal mosquito vector Aedes aegypti . Currently, endemic dengue virus transmission is reported in the Eastern Mediterranean, American, South-East Asian, Western Pacific and African regions, whereas sporadic local transmission has been reported in Europe and the United States as the result of virus introduction to areas where Ae. aegypti and Aedes albopictus , a secondary vector, occur. The global burden of the disease is not well known, but its epidemiological patterns are alarming for both human health and the global economy. Dengue has been identified as a disease of the future owing to trends toward increased urbanization, scarce water supplies and, possibly, environmental change. According to the WHO, dengue control is technically feasible with coordinated international technical and financial support for national programmes. This Primer provides a general overview on dengue, covering epidemiology, control, disease mechanisms, diagnosis, treatment and research priorities.
Dengue is currently one of the world's most important neglected tropical diseases 1 and its incidence has increased >30-fold in recent decades alongside the geographical expansion of the Aedes vector mosquitoes and dengue viruses (DENVs) 2 , 3 . Transmission of DENVs occurs in Eastern Mediterranean, American, South-East Asian, Western Pacific and African regions, with new cases occurring and spreading to non-endemic areas in the United States and Europe. Dengue epidemics impose high costs to health services, to families and to the economic systems of affected countries 1 .
The term ‘dengue viruses’ groups four genetically and antigenically related viruses that are known as serotypes 1–4, each of them grouped into genotypes. Infection by any of the four serotypes can result in a range of clinical manifestations for which the timing or sequence of infections can be an important determinant of disease severity and course 4 . Dengue illness is clinically classified as either dengue with or without warning signs or severe dengue 2 ( Fig. 1 ). This classification was launched by the WHO in 2009 for the purpose of improving clinical management. The assessment of warning signs is designed to permit the early identification of patients with more-severe disease manifestations who require supportive therapy. Dengue illness can also be divided into three separate phases: the acute (febrile) phase, the critical (plasma leakage) phase and the convalescent or reabsorption phase ( Box 1 ). The 2009 classification replaced the previous 1997 WHO system that addressed and underscored the two pathological phenomena associated with the disease: plasma leakage and abnormal haemostasis. Under this classification, patients were designated as having either dengue fever — a nonspecific febrile illness and the most common manifestation of DENV infection — or dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS) — a combination of plasma leakage and coagulopathy, sometimes accompanied by bleeding that can lead to a rapid fall in blood pressure and consequently to circulatory shock and organ impairment 3 ( Box 1 ).
The criteria for dengue with or without warning signs and for severe dengue. Dashed arrows indicate that not all patients progress to severe dengue. * Important when there is no sign of plasma leakage. ‡ Requires strict observation and medical intervention. ALT, alanine aminotransferase; AST, aspartate aminotransferase. From Ref. 2 . Adapted with permission, from WHO/TDR, Dengue Guidelines for Diagnosis, Treatment, Prevention and Control — New Edition, World Health Organization and Special Programme for Research and Training in Tropical Diseases, WHO Press, Figure 1.4, 2009.
This Primer provides special emphasis on the epidemiology, diagnosis, clinical management, pathogenic mechanisms and control of dengue. Research priorities are also discussed.
Box 1: Key clinical terms
Characterized by high fever that is driven by high viral loads (viraemia)
Characterized by plasma leakage into the abdominal and pleural cavities, which becomes evident at the end of the febrile (acute) stage of illness (days 3–6)
Warning signs that announce shock are usually present
Involves both cessation of plasma leakage and reabsorption of leaked fluids
Dengue or dengue fever
A nonspecific febrile illness that is characterized by fever and the presence of two or more other symptoms, such as headache, rash, retro-orbital or ocular pain and myalgia
Most patients have a satisfactory resolution without signs of severity or warning signs (referred to as dengue without warning signs according to the 2009 WHO classification)
Dengue haemorrhagic fever
Characterized by increased vascular permeability, plasma leakage, bleeding, thrombocytopaenia and fever (according to the 1997 WHO classification)
The term and concept are not included in the revised 2009 WHO classification nor are they recommended for triage of patients because it is not necessarily associated with severity, among other reasons
Dengue with warning signs
At the end of the febrile period, some patients have signs or symptoms that are suggestive of important fluid loss associated with capillary leakage (for example, severe abdominal pain), announcing the imminence of shock and indicating that fluid replacement is urgently required (according to the 2009 WHO classification)
Circulatory shock or respiratory distress associated with severe plasma leakage, severe bleeding or severe organ involvement (frequently myocarditis, encephalitis and severe hepatitis) with or without shock or bleeding (according to the 2009 WHO classification)
DENVs are maintained in an endemic–epidemic cycle involving humans and mosquitoes in crowded tropical urban centres. These viruses are fully adapted to humans, and the highly domesticated principal vector mosquito Aedes aegypti emerged long ago from sylvatic cycles involving non-human primates and canopy-dwelling Aedes mosquitoes in the rainforests of Asia and Africa 4 . Although these cycles still exist, their public health importance is uncertain. Ae. aegypti was introduced to the Americas during the slave trade in the 1600s and spread worldwide as the shipping industry expanded. This species lives in intimate association with and feeds on humans, rests in their homes and lays its eggs in man-made water containers. The average female mosquito lives for approximately 1 week, but some females can live for ≥2 weeks 5 .
The female mosquito becomes infected when she takes a blood meal during the acute febrile and viraemic phase of illness. During the extrinsic incubation period, the virus first infects midgut cells and then disseminates to replicate in numerous mosquito tissues, ultimately infecting the salivary glands 5–12 (generally 8–10) days later, a process that is influenced by ambient temperature, the viral strain and the competence of the mosquito. Once the salivary glands are infected, the mosquito is infective and can transmit the virus to another person during blood-feeding 4 . The mosquito remains infective for life and can infect every person it subsequently feeds on or probes while trying to feed. The time from infection to onset of illness (the intrinsic incubation period) in humans ranges from 3 to 14 days, with an average of 4–7 days 4 , 6 ( Fig. 2 ). Vertical transmission can occur when the infected female mosquito transmits the virus through the eggs to her offspring, but the epidemiological importance of this mode of transmission is uncertain 4 .
An Aedes aegypti mosquito can become infected by feeding on a person in the viraemic phase of infection. During the extrinsic phase of the cycle, dengue viruses first infect mosquito midgut cells and other tissues before disseminating to the salivary glands. An infected mosquito can then transmit the dengue virus to several humans as it feeds or attempts to feed. Once infected, it takes an average of 4–7 days for the onset of symptoms and for a person to become capable of transmitting dengue virus to a new mosquito. Both symptomatic and asymptomatic individuals can transmit dengue virus to mosquitoes.
Global burden of disease
Although DENVs achieved distribution throughout the tropics in the eighteenth and nineteenth centuries, during the twentieth and twenty-first centuries, globalization enabled their more-rapid spread and the introduction of multiple viral serotypes into permissive areas, resulting in most tropical regions becoming hyperendemic (that is, with multiple viral serotypes co-circulating). This rapid spread began with a pandemic of dengue in South-East Asia in the 1950s that was associated with regional economic and urban growth after World War II. Epidemic activity dramatically accelerated in the 1970s and 1980s, leading to a global geographical expansion of viruses and mosquito vectors, and the consequent widespread DENV transmission across the tropics and subtropical areas 4 , 7 ( Fig. 3 ). This geographical expansion resulted in increased frequency and magnitude of epidemics and increased frequency of severe disease ( Fig. 4 ). The principal drivers of this twentieth century pandemic were global trends, such as human population growth, urbanization, modern transportation, global trade and the absence of effective mosquito control in endemic countries 4 , 7 – 11 . The geographical spread and increased epidemic activity in the 1970s coincided with the jet airplane becoming a principal mode of human travel 4 , 8 . This development led to more-frequent epidemics followed by clinically silent or undetected transmission during inter-epidemic periods. Large cities tend to be hyperendemic, with co-circulation of all four serotypes. Epidemics might occur when herd immunity to one of the four serotypes wanes and/or when a new epidemic strain of virus emerges or is introduced. Although not documented, an increase or change in vector competence of the mosquito population might also influence epidemic transmission.
The global evidence consensus, risk and burden of dengue is shown with evidence consensus on complete absence (dark green) through to complete presence (dark red) of dengue. Adapted from Ref. 10 , Nature Publishing Group.
The number of locations that reported dengue between 1960 and 2012 are shown. An increasing trend is apparent in the number of locations reporting dengue, with higher figures in American and Asian regions. The figure shows the number of occurrence points by date recorded in mapping exercises; it is neither a prevalence nor an incidence metric, but an audit of data sources per year. Adapted from Ref. 301 , Nature Publishing Group.
Recent best estimates of dengue disease burden suggest that over half of the world's population (3.6 billion people) live in areas that place them at risk of DENV infection 12 , with 390 million overall DENV infections, 96 million symptomatic infections 10 , 2 million cases of severe disease 12 and 21,000 deaths per year 13 . The highest incidence of DENV infection occurs in Asia where children between 5 and 15 years of age are primarily infected, followed by the American tropics where the modal age of infection is 19–40 years, depending on the country 8 . Dengue rates in Africa are unknown 14 because many outbreaks and cases might be misattributed to malaria 15 . During the past 40 years, a steady increase in dengue epidemic activity in Africa has been noted, as well as in the isolated islands of the Pacific and Indian Oceans 4 , 8 , 14 .
As with disease burden, the economic consequences of dengue are not well studied. However, estimates of disability-adjusted life years have shown dengue to be in the same order of magnitude or higher as most of the major infectious diseases, such as upper respiratory infections and hepatitis B virus infection 16 . Recent estimates of direct and indirect costs resulting from DENV infections are considerable, averaging US$2.2 billion per year in the Americas between 2000 and 2007, US$1.2 billion in South-East Asia per year between 2001 and 2010 and US$76 million in Africa per year 17 , 18 . A recent study estimates the annual global cost of dengue at US$8.9 billion 19 . These are conservative estimates and are subject to many uncertainties.
The viral genome . DENVs belong to the genus Flavivirus of the Flaviviridae family. The four serotypes are enveloped, spherical viral particles with a diameter of approximately 500 Å 20 . The genome of each serotype comprises approximately 11 kb of positive-sense, single-stranded RNA that encodes ten proteins. The three structural proteins encoded by the genome are the membrane (M) protein, envelope (E) protein and capsid (C) protein; the non-structural (NS) proteins are NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5.
Structure and function of the E protein and M protein . The delivery of the DENV genome into the host cytoplasm is a multistep process that begins with fusion of the viral membrane with the host plasma membrane, followed by endocytosis of the virus into an endosome and then pH-dependent fusion of the viral and endosomal membranes ( Fig. 5 ). The inside of the virus particle is formed by RNA complexed with capsid proteins and is surrounded by a lipid bilayer membrane that contains externally anchored M protein and E protein, which together orchestrate host–viral interactions during entry. The E protein is arranged on the surface of DENVs as 90 tightly packed monomers that lie flat against the membrane and facilitates viral entry into host cells by binding to cellular receptors and mediating the fusion of viral and cellular membranes 21 , 22 . Of its three domains (DI–DIII), DIII of the E protein is responsible for binding to host receptors and several mutations have been identified in this domain that affect receptor binding 21 – 23 . The hinge that connects DI to DII is highly flexible and is used to manoeuver DII in the low pH environment of the endosome, leading to the exposure of the DII fusion loop. This fusion loop then interacts with the endosomal membrane to facilitate fusion of the virus with the endosomal membrane and release of viral RNA into the host cell 24 . Upon release from infected cells, new DENV particles can either contain processed surface M protein, and therefore be infective or ‘mature’, or retain the uncleaved precursor form of the M (prM) protein on its surface and thereby remain in an ‘immature’ form. Some viral particles can have a mixture of M protein and prM protein on their surface and these particles may or may not be infective.
Mature viral particles attach to host cells by the binding of the envelope (E) protein to unknown receptors. Viral entry is achieved through receptor-mediated endocytosis. Inside the host cell, pH-dependent rearrangement of the E protein facilitates fusion of the viral and endosomal membranes to release the nucleocapsid, which disassembles to release the capped viral genomic RNA. The genomic RNA is then translated into a long polyprotein, which is autocatalytically cleaved by the non-structural 2B (NS2B) or NS3 viral protease and host proteases into individual proteins. The released NS proteins are targeted to the site of replication on endoplasmic reticulum (ER)-derived vesicle packets to initiate transcription. NS1 is initially expressed in association with the ER; the monomer is modified by the addition of high mannose carbohydrate (CHO) moieties, resulting in membrane association. A subset of dengue virus (DENV) NS1 acquires glycosyl-phosphatidylinositol (GPI). Both membrane-bound NS1 and GPI-anchored NS1 are trafficked to the cell surface via an unknown pathway, where they have been shown to associate with lipids such as cholesterol. Some of the cell surface-associated NS1 can be previously secreted NS1 that has bound directly to cell surface glycosaminoglycans (GAGs). Some NS1 can also be secreted from host cells. Meanwhile, the precursor form of the membrane (M) protein (prM) and the E protein are embedded into the ER membrane and enclose the newly formed nucleocapsid as it buds into the ER lumen to form an immature particle. This particle is trafficked via the secretory pathway, in which the low pH of the trans -Golgi network causes substantial rearrangement of the prM and E proteins that permits the cleavage of prM by furin protease to form the mature M protein. The virion is released from the host cell with the release of the pr peptide (dashed arrow). A subset of viral particles are released with prM still intact and are unable to infect new host cells. + and – signs indicate positive-sense and negative-sense RNA, respectively. C, capsid; DC-SIGN, dendritic cell-specific ICAM3-grabbing non-integrin; UTR, untranslated region. Adapted from Ref. 302 , Nature Publishing Group.
Structure and function of the NS proteins . The NS proteins are involved in viral replication and packaging, processes that are closely linked to host endoplasmic reticulum (ER) and secretory pathway function ( Fig. 5 ). NS1 is a 46 kDa glycoprotein that exists in three forms: the ER-resident form; the membrane-anchored form; and the secreted form. NS1 is initially synthesized as a soluble monomer and becomes associated with the membrane after dimerization in the lumen of the ER 25 . The crystal structure of NS1 has recently been determined and revealed exposed hydrophobic domains in the dimer that probably mediate this membrane association 26 . Intracellular NS1 participates in early viral RNA replication and is found in virus-induced vesicular compartments that house the viral replication complexes 27 . NS1 is also transported to the cell surface, where it either remains associated with the cell membrane or is secreted (sNS1) as a soluble, lipid-associated hexameric species. sNS1 can be detected in the blood of infected patients from the first day of symptoms and circulates at levels in the ng per ml to mg per ml range during the acute phase of infection 28 , and blood levels of sNS1 correlate with peak viraemia and disease severity in secondary DENV infection 28 . Several studies have suggested that sNS1 is a key mediator of dengue pathogenesis. For instance, highly purified recombinant NS1 (rNS1) devoid of bacterial endotoxin activity directly activates mouse macrophages and human peripheral blood mononuclear cells via Toll-like receptor 4 (TLR4), leading to the induction and release of pro-inflammatory cytokines and chemokines. In addition, in in vitro and in vivo models of vascular leakage, exposure to NS1 resulted in the disruption of endothelial cell monolayer integrity 29 , 30 . The key features of NS1 and of the other DENV NS proteins are detailed in Table 1 .
Locus of DENV infection in vivo
Overview of infection . During mosquito feeding, DENV is inoculated into the dermis and epidermis, and some virus is also injected directly into the bloodstream. In the skin, this delivery results in the infection of macrophages, dendritic cells and Langerhans cells. These infected cells can migrate from the initial site of infection to lymph nodes, which triggers the recruitment of monocytes and macrophages that then become subsequent targets of DENV infection. As a result, the number and variety of cells infected with DENV increases and the infection can become disseminated throughout the lymphatic system with the infection of cells of the mononuclear lineage, including blood-derived monocytes, myeloid dendritic cells and splenic and liver macrophages 31 , 32 .
Determination of infected cells in vivo. The identification of sites of DENV infection in vivo is problematic because many of the stains that are used to visualize dengue viral antigens do not discriminate between intracellular antigens that have been phagocytosed and those that are indicative of active viral invasion and replication. For this reason, to identify sites of DENV infection, it is crucial to use probes for DENV-specific RNA, negative-sense RNA or for NS proteins, which are produced at sites of viral replication or assembly. The few studies that have been published using these markers have detected DENV infection in monocytes that are present in the blood and macrophages from the liver, skin, spleen and thymus 33 , 34 . In addition, sites of infection can also be identified through the detection of viral particles. Peripheral blood monocytes from patients with dengue have been found to harbour DENVs 35 , and immature skin dendritic cells support the growth of DENVs 36 .
Although dengue viral particles or antigens have been shown to localize to neurons, microglia and endothelial cells in the human central nervous system (CNS) 37 , no evidence of DENV replication was found in the CNS when a sensitive viral RNA amplification method was applied to samples from patients who had died as a result of DENV infection 38 . Moreover, no DENV antigens were detected in CNS tissue from 13 children who had died from DHF/DSS 33 .
DENV can also infect the liver, resulting in apoptosis of hepatocytes 39 , and when apoptotic DENV-infected hepatocytes are engulfed by Kupffer cells, they form Councilman bodies — the classic histopathological finding in the livers of those with dengue and yellow fever. Results from studies that attempted to culture DENVs in explanted human tissue, primary human cells and human cell lines suggest that DENV1 is unable to replicate in mature Kupffer cells. By contrast, primary hepatocytes and hepatocyte cell lines were successfully infected but underwent apoptosis shortly after 40 , 41 . Although DENV infection imparts considerable damage to the liver, infection of this organ might not make a considerable contribution to the distribution or maintenance of acute infection 33 .
Host receptors of DENV entry . DENVs are capable of infecting many different cell types in vitro , including epithelial cells, endothelial cells, hepatocytes, muscle cells, dendritic cells, monocytes, bone marrow cells and mast cells. Despite the ability to infect these cells with DENVs in a laboratory setting, their roles in dengue pathogenesis and the cellular receptors involved in their infection remain unknown. Although candidate receptors — such as heparan sulfate, dendritic cell-specific ICAM3-grabbing non-integrin, macrophage mannose receptor 1, heat shock protein 70 (HSP70) and HSP90 — have been described for in vitro systems, their contribution to infection in humans is not established 42 .
Infection and response
Four main factors control DENV disease response along a response continuum: immune status, virus strain, genetic status and age ( Fig. 6 ).
Four main factors control dengue virus (DENV) disease response along a response continuum: immune status, virus strain, genetic status and age. Individuals at risk of a secondary infection are at a higher risk of severe disease through the antibody-dependent enhancement phenomenon, with a higher production of virus. Genetic dengue susceptibility favours disease severity. For example, white individuals have been found to have a higher chance of developing severe disease than black individuals. Younger age is associated with disease severity in individuals experiencing a secondary infection. The association of each serotype and genotype to disease severity, epidemic potential and efficiency transmission could be influenced by the differences among them, but also by other conditions such as host immunity, the ability of the vector to become infected and to disseminate the virus to humans, among others. FcγR, Fcγ receptor.
Immune status . Infection with any DENV serotype results in long-term homotypic immunity (that is, immunity against the serotype causing the infection) with a short period of heterotypic immunity (that is, immunity against another serotype) 43 , 44 . Only a small fraction of circulating antibodies in monotypic-immune individuals (that is, those only immune to one viral serotype) neutralize homologous DENV. Polyclonal antibodies are directed against several epitopes; some are directed against quaternary epitopes located at the hinge region between DI and DII of the E protein on the surface of intact virions 45 , 46 . Immediately after an individual's first DENV infection, antibodies might neutralize heterotypic DENVs in in vitro assays. Over the next few months, antibodies become increasingly specific to the DENV serotype that is causing the infection 47 . These in vitro changes in neutralizing antibody specificity correlate with in vivo observations. Monotypic-immune humans demonstrate an initial short period of cross-protection against infection with heterotypic DENVs (approximately 2 months) and a longer period of protection against severe disease (approximately 2 years) caused by heterotypic DENV infections 47 . Recently, a class of strong broadly cross-neutralizing antibodies was recovered and characterized 48 , 49 . Whether these antibodies are preferentially selected during second heterotypic DENV infections and contribute to pan-DENV protection are unknown. Neutralizing antibodies circulate for a lifetime and are thought to explain the observed long-lasting protection against re-infection with homotypic DENVs 50 , 51 .
The majority of circulating antibodies are non-neutralizing and are directed against various antigens on the E protein and the prM protein. In the absence of blocking by type-specific neutralizing antibodies, non-neutralizing antibodies usually enhance the entry of any DENV into Fc receptor-bearing cells. This phenomenon is called antibody-dependent enhancement (ADE) 52 , 53 and makes DENVs unique among human viral infections, in that pre-infection partial immunity to one or more DENV (so-called sensitization) upgrades disease severity. As such, severe dengue and DHF/DSS occur most often in individuals with monotypic immunity during a second heterotypic DENV infection. Severe cases also occur (infrequently) in primary infections. By contrast, third and fourth DENV infections are typically mild or asymptomatic 54 , 55 .
In dengue-endemic countries, DHF/DSS also occurs during primary infections in infants born to mothers who are immune to DENV. These mothers circulate antibodies from two or more lifetime DENV infections that occurred before pregnancy 56 – 58 . As discussed above, these individuals develop broad neutralizing antibodies and are usually protected against severe disease with heterotypic DENV strains 55 , 59 . Epidemiological data from many countries indicate that these multitypic antibodies protect infants from dengue illness for several months after birth 60 , 61 . Then, the concentration of these neutralizing maternal antibodies eventually falls below a protective threshold, with most antibodies having a half-life of approximately 40 days. For a short period, if the infant is infected with DENV, severe disease mediated by ADE may occur owing to the presence of maternal non-neutralizing antibodies.
In addition to serotype-specific immunity, the interval between an individual's first DENV infection and subsequent infections might be an important determinant of disease severity. For example, in a comparison of groups of identical age, DHF occurred at an eightfold higher rate in those whose secondary DENV2 infections were separated by 20 years than those whose secondary infection was 4 years after their first. A possible reason why severe disease rates increase with longer intervals between infections might be related to the steady decline in heterotypic neutralization of DENV2 by DENV1 antibodies 62 . A second phenomenon sometimes observed in dengue is that infection sequences that were previously benign can suddenly become pathogenic. For example, in Tahiti in 2001, DHF occurred in children 4–13 years of age who contracted DENV1 infections, even though they had previously been infected with DENV2 4–5 years earlier 63 . By contrast, during the 1980 Rayong (Thailand) epidemic, no severe disease accompanied secondary DENV1 infections, even though they comprised 37.5% of all secondary DENV infections 64 , indicating that, although infection sequence is important, a secondary infection with DENV1 does not necessarily cause severe disease.
The relative contribution of antibodies versus T cells to protection against dengue is not well understood, although there is growing evidence that both components are required to prevent infection, overt disease and severe disease 65 – 67 . For example, recent studies on naturally infected humans and infection of humanized mice indicate that T cells contribute to protection against severe disease associated with heterotypic secondary DENV infections 68 . Effective CD8 + T cell immunity is largely mediated by epitopes on NS proteins, their respective contribution varying between different DENV serotypes 69 .
Viral serotypes . Although all four DENV serotypes are transmitted by Aedes mosquitoes and, in principle, cause the same clinical manifestations and show similar patterns of systemic dissemination, there are some biological differences between them 60 , 70 . Indeed, associations between particular serotypes or genotypes and disease severity, epidemic potential and the efficiency of transmission have been described, but these associations could be influenced by factors other than intrinsic viral characteristics, such as host immunity, the ability of the mosquito vector to become infected and to transmit the virus to humans, and the conditions (which are not well known) that support the displacement of one genotype by another 71 – 73 .
The hypothesis that some DENVs have greater ‘virulence’ and epidemic potential than others was introduced in the 1970s 61 , 74 . The DENV polyprotein demonstrates 30% divergence between the four serotypes, and several genotypes within each serotype show different geographical distributions 71 . Some data indicate that genetic changes in DENVs might directly affect transmission potential in mosquitoes or disease expression in infected humans. For example, some studies have shown that DENV2 of Asian origin replicates to higher titres in human dendritic cells, infects Ae. aegypti mosquitoes more efficiently and is transmitted at a higher rate than American DENV2 strains 75 , 76 . In addition, some strains of DENV3 replicate at a higher rate in mosquitoes than other DENV3 strains, leading to the capacity to displace established DENV3 strains 68 , 77 . Similarly, when DENV4 was introduced to Puerto Rico, it caused three major epidemics in 1982, 1986 and 1998; the latter two epidemics were each associated with a clade (that is, a monophyletic group of the same genotype) change in the circulating virus 78 , 79 . Similar clade changes associated with endemic and epidemic transmission have been observed with DENV2 in the South Pacific 74 , 80 and DENV3 in Sri Lanka 81 , 82 . To date, most of the genetic changes associated with epidemic potential have resulted in amino acid changes in NS proteins.
Several pathogenesis studies have been performed on patients with dengue who were clinically classified as having either dengue fever or DHF/DSS 3 . Although both dengue fever and DHF/DSS can be associated with any serotype, some sequences of infection have been associated with severe disease at a higher frequency than others 83 . In addition, some serotypes may be associated with DHF/DSS during a secondary infection but result predominantly in mild or asymptomatic infections during primary infections. Furthermore, there is no evidence that severe dengue regularly accompanies primary infections of susceptible individuals. In this context, DENVs are not inherently ‘virulent’ but are instead conditionally virulent. That condition is usually the presence of pre-infection circulating DENV antibodies, as discussed above 73 . For example, in the very ‘clean’ epidemiological setting of the Santiago de Cuba DENV2 epidemic of 1997, susceptible individuals of all ages who were infected by the DENV2 Asian genotype primarily developed subclinical disease. By contrast, individuals who contracted a secondary infection with DENV2 after first having a DENV1 infection almost always experienced overt disease (the overt to subclinical disease ratio was nearly 1). Similar observations have been reported for primary DENV4 infections 84 .
Of interest is the possibility that, during the course of epidemics, there is a rapid selection of DENV neutralization escape mutants or of mutations that affect the ability of certain DENVs to cause infections that involve different degrees of ADE 85 . In support of this possibility, in three Cuban epidemics, month-to-month increases were observed in the proportion of severe secondary DENV cases compared with mild cases as well as in case fatality rates. For example, in the 1997 DENV2 outbreak in Santiago de Cuba, there was an emergence of severe disease that was accompanied by a stable amino acid switch in NS1 (Ref. 86 ). Somewhat similar increases in severity of secondary DENV2 infections have been described in Taiwan and Nicaragua; these were associated with several mutations, including changes in the structure of the DENV 3′ untranslated region (UTR) 87 , 88 . Although the precise mechanisms of the rapid acquisition of increased fitness of DENVs are unknown, suspicions increasingly point to improved viral survival during interactions with the human innate immune system 89 .
Host genetics . As with most infectious diseases, host factors, many of which are measured by proxy genetic markers, can control the outcome of infection through mechanisms that are not yet fully understood. Some host factors that affect the outcome of DENV infection have been well documented. For example, an unidentified gene that is present in black individuals moderates the clinical severity of secondary DENV infections, and studies have shown that the rates of DHF/DSS were lower in infected black patients than in white patients with the same secondary DENV2 infection experience 90 , 91 . Table 2 includes some human leukocyte antigen (HLA) and non-HLA genes that are linked with increased susceptibility or resistance to dengue 92 – 102 .
Patient age and sex . Age affects dengue disease expression in a contradictory manner. The disease severity that accompanies a first DENV infection is directly related to age. In susceptible young children, first DENV infections are usually occult or mild, whereas adults experiencing a first DENV infection often develop dengue fever. More complicated outcomes are observed in the elderly or in those with chronic diseases, such as diabetes mellitus, chronic obstructive pulmonary disease or cardiovascular disease 2 . Bleeding phenomena are common in both the elderly and those with comorbidities. Menorrhagia has been observed in adult women during primary DENV infections and gastrointestinal bleeding has been observed in individuals with peptic ulcer disease. The risk of progressing to DHF/DSS in sensitized individuals varies inversely with age. When an entire population (people ≥3 years of age) was exposed to identical rates of secondary DENV2 infection, DHF rates were more than fivefold higher in children than in adults 103 . This observation can probably be explained by intrinsic host susceptibility to vascular permeability that accompanies a secondary DENV infection 104 , 105 . Indeed, healthy children have been reported to have a higher capillary fragility than adults. The greater density and surface area of growing microvessels in childhood could be the reason for why children have this higher microvascular permeability 106 , 107 . The outcome of secondary DENV infections is also controlled by sex, with girls >4 years of age having higher rates of DSS than boys of any age 59 , 86 .
Acute DENV infections are expressed along a continuum from inapparent to undifferentiated fever, to an acute febrile viral exanthema and finally to a complex of physiological abnormalities that affects multiple systems, including the liver, blood coagulation, complement, haematopoiesis and the vascular systems.
Vascular permeability . Dengue vascular permeability syndrome, which was historically known as DHF/DSS, includes the abnormalities that affect the vascular system. Several studies have shown that there is a range of capillary permeability and plasma leakage affecting most individuals with overt dengue illnesses, rather than distinct pathological mechanisms underlying DHF/DSS. For example, endothelial cell damage by infection or extensive cell death does not seem to be responsible for the increase in vascular permeability associated with dengue 33 , 108 . Indeed, patients who are recovering from DHF regain normal endothelial function relatively quickly, implying that whatever causes vascular permeability is more reversible than endothelial damage, and might include one or more soluble mediator 109 . In addition, increased microvascular permeability has been reported in patients with DHF/DSS at or around the time they experience defervescence (a decrease in increased temperature) 109 , 110 , indicating that vascular permeability is not directly correlated with peak viraemia that usually occurs on the first day after the onset of fever. A mild increase in microvascular permeability has been observed in infected volunteers experiencing dengue fever, indicating that dengue disease severity occurs across a continuum 111 , 112 .
Recent studies on myeloid cells, both in vitro and in mice, indicate that NS1 induces vascular leakage and activation of TLR4, resulting in the production of inflammatory cytokines 29 , 30 . These studies suggest that circulating DENV NS1 triggers endothelial barrier dysfunction, which causes increased permeability of human endothelial cells in vitro . These findings open a new window of opportunity for dengue drug and vaccine development 29 , 30 .
Thrombocytopaenia . Thrombocytopaenia results from transient bone marrow suppression and increased peripheral destruction of platelets during the febrile and early convalescent phases of the disease, and results in platelet counts as low as 5,000 per ml (compared with approximately 200,000 platelets per ml in healthy individuals) 113 , 114 . Whereas thrombocytopaenia occurs commonly across a wide range of infectious diseases, severe thrombocytopaenia accompanies clinically significant vascular permeability during acute DENV infections. Remarkably, in the bone marrow, early suppression of the production of all blood cell types occurs during the early febrile phase of DENV infection 115 , 116 . A possible explanation for this suppression comes from studies on lymphocytic choriomeningitis virus infection. Here, in infected laboratory animals, bone marrow suppression was mediated by interferon-α (IFNα) production 117 . Towards the end of the febrile period of a DENV infection, the bone marrow cells recover to normal density and diversity, leaving only a residual megakaryocyte arrest that can be observed in autopsy studies 118 .
Coagulopathy . The impaired haemostasis that accompanies DHF/DSS involves a series of alterations in the coagulation system that disrupts the regulation of clot formation. For example, an increase in activated partial thromboplastin time (APTT; which measures time to clot formation) and a reduction in the level of fibrinogen (a factor that promotes clot formation) are fairly consistent findings in DHF/DSS 119 – 121 . The evidence that haemorrhage in dengue is caused by classic disseminated intravascular coagulation is under debate 120 . The concentration of procoagulant markers is increased in some DHF/DSS cases, but this increase is usually mild and is accompanied by a considerable reduction in the concentration of anticoagulant proteins.
Factors that might contribute to these alterations include secreted viral effectors. Recent reports have shown that NS1 binds to thrombin in vivo to form NS1–thrombin complexes. In addition, in vitro , rNS1 inhibits prothrombin activation and prolongs APTT in human platelet-deficient plasma 122 . Release of heparan sulfate or chondroitin sulfate (molecules similar in structure to heparin that mimic its function as an anticoagulant), which are possibly sheared off by NS1 from the glycocalyx, can also contribute to altered haemostasis 112 .
In most patients, coagulopathy is relatively minor and resolves within a few days. In some children with severe shock, these minor derangements are compounded by the effects of prolonged hypotension and tissue hypoxia. Major bleeding occurs by erythrocyte extravasation in the gastrointestinal tract.
Complement activation . The complement system becomes activated to control DENV infection, and this activation contributes to pathogenesis through interaction with the coagulation system. In classic studies on complement in children with DHF/DSS, temporal and peak production of complement split products correlated with blood fibrinogen levels and thrombocytopaenia 123 . Complement activation has been described in detail in a 6-month-old infant with DSS during a primary DENV infection 124 . Most studies on complement activation in dengue have centred on patients with secondary DENV infections and have led to the conclusion that complement activation was mediated via the classical pathway by circulating immune complexes. By contrast, studies on complement activation in infants experiencing a primary DENV infection have implicated the alternative pathway in activating complement during infection. Indeed, in children experiencing a secondary DENV infection, NS1 might be responsible for activating complement by the alternative pathway 122 .
Liver enlargement . Liver enlargement and dysfunction are common during DENV infection, with liver enlargement having a significantly stronger association with DHF/DSS than with dengue fever (55% compared with 18%; P < 0.01) 125 . For instance, one study in Thailand showed that hepatomegaly was observed in a high percentage of all children admitted with serologically confirmed severe dengue 126 – 128 . Liver enlargement seems to occur for two reasons: generalized oedema due to vascular permeability and an inflammatory response that occurs after infection of hepatocytes by DENVs. However, almost no cellular inflammatory response was observed in livers from patients who died as a result of DENV infection, reinforcing the observation that liver damage might be caused by apoptosis, as discussed above.
Despite the prevalence of liver enlargement, jaundice in dengue illness, even in DSS, is rare 125 , 129 . By contrast, changes in liver enzyme levels, which are markers of liver dysfunction, are common. Aspartate aminotransferase and alanine aminotransferase blood levels are increased in 60–90% of children with DHF. The increase in the levels of the transaminases was shown to be mild to moderate in one study, but a small group of patients (7–10%) had transaminase levels that were tenfold higher than the upper limit of normal. In this study, co-infection with hepatitis B virus or hepatitis C virus was not related to liver enzyme changes, and liver enzyme levels were significantly higher in patients with prolonged shock 130 . Finally, this study also demonstrated that the levels of serum bilirubin, alkaline phosphatase and gamma-glutamyl transpeptidase were raised in 7%, 16% and 83% of patients, respectively.
Models of vascular permeability in dengue . Many researchers attribute vascular permeability to a lethal ‘cytokine storm’ caused by overactive T cells, heterologous T cell responses or defective T cell responses (original antigenic sin) 131 – 133 . Other hypotheses to explain this phenomenon posit that circulating immune complexes activate complement or that immune responses to DENV proteins cross-react with host systems to generate short-lived immune or autoimmune DHF/DSS 134 – 136 . However, none of these explanations can account for why infants who have never previously experienced DENV infection develop severe dengue disease. Alternatively, capillary leakage might be caused by factors produced in target cells that are directly related to DENV infection 137 – 142 . However, this hypothesis does not fit with the observation that, although target cells are infected throughout the course of infection, the onset of vascular permeability is delayed to the time of defervescence.
The onset of thrombocytopaenia, altered haemostasis, complement activation, liver damage and detectable vascular permeability before defervescence suggest that DHF/DSS might result from a factor or factors that circulate throughout the acute illness, which exceeds a threshold at defervescence. DENV virions and NS1 circulate throughout the acute phase. As discussed above, NS1 interacts with the complement system, can extend APTT and several lines of evidence support the notion that NS1 contributes to vascular permeability. Indeed, APTT values are the strongest correlate of vascular permeability in patients with dengue illness 143 . Moreover, it has been known for several decades that antibodies against DENV NS1 protect against lethal dengue disease in animal models 144 . Perhaps then, terminal T cell-mediated cytolysis of DENV-infected cells releases a bolus of cell-bound NS1 to surpass a threshold resulting in vascular permeability 145 . Finally, the mechanisms that underlie peak vascular permeability during defervescence in dengue remain unclear, but intensified research efforts might uncover these in the future.
Diagnosis, screening and prevention
Signs and symptoms.
Dengue is a dynamic illness, despite its short duration (no more than 1 week in nearly 90% of cases). Its clinical expression can change as the days go by and can also worsen suddenly. Dengue illness can evolve into three phases: the acute febrile phase — observed in most of the patients — and the critical and the recovery (convalescent) phases 2 ( Box 1 ).
Fever occurs during the acute febrile stage and is generally the first clinical manifestation of illness with a variable intensity. It is associated with headache and vomiting, as well as body pains. In children, fever is frequently the only clinical manifestation or is associated with rash and/or unspecific digestive symptoms. The pharynx can become reddened, but other signs and symptoms of the respiratory system are not frequent or clinically significant. Slight abdominal pain and diarrhoea can occur; diarrhoea more frequently occurs in patients who are <2 years of age and in adults. In general, compared with children, adolescents and adults show a ‘flu-like syndrome’ (including malaise, headache and body pains) with more prominent digestive symptoms than respiratory symptoms, if any. During the febrile stage, leukocyte counts are usually decreased. Petechiae (small spots on the skin caused by broken capillaries) or ecchymosis (large subcutaneous bleeding spots) can be present, with or without thrombocytopaenia. After 2–5 days, these symptoms can be followed by rapid clinical deterioration. Most patients with dengue recover after defervescence; however, the clinical state of some patients worsens when the fever drops. Thus, the period during which the fever subsides indicates the beginning of the critical phase.
The critical phase coincides with the leakage of plasma that can lead to shock, which is characterized by coldness in the teguments, weak pulse, delayed capillary filling, tachycardia, oliguria and hypotension. Shock is caused by low blood volume (hypovolaemia). At the beginning, not all clinical signs of shock are observed, and, in this setting, shock can be detected by a narrowing of the differential arterial tension or pulse pressure (a difference of ≤20 mmHg between the maximum or systolic arterial tension and the minimum or diastolic arterial tension). At this stage, patients usually have a flushed face, a warm trunk, cold and clammy extremities, diaphoresis (sweating), slow venous filling, restlessness, irritability, pain in the upper and middle abdomen and decreased urinary output. In addition, patients might also exhibit signs of impaired haemostasis, including scattered petechiae on the forehead and extremities, spontaneous ecchymoses, easy bruising and bleeding at venipuncture sites, and circumoral and peripheral cyanosis (blue skin discolouration). Gastrointestinal bleeding occurs in <10% of patients and usually follows a period of uncorrected hypotensive shock. Patients with shock also experience rapid and potentially laboured breathing, a weak pulse and have a rapid heartbeat that sounds ‘thready’. Finally, their livers are usually firm, tender and can become enlarged to 4–6 cm below the costal margin, the haematocrit level is increased and the platelets — which were decreasing progressively — reach their lowest count. In those who recover, this critical phase lasts for 24–36 hours and is followed by a rapid convalescence.
Convalescence can involve complications, such as encephalopathy, bradycardia, ventricular extarsystoles and, rarely, myocarditis and encephalitis.
According to the 2009 WHO clinical classification, a patient can have dengue with or without warning signs or severe dengue ( Fig. 1 ), highlighting that severity is considered as the second step of the same disease. In other words, dengue can be considered to be a single disease entity that is both systemic and dynamic.
Detection of viraemia . DENV viraemia is detectable 24–48 hours before fever onset and continues for 5–6 days ( Fig. 7 ). During this period, infective virus, its specific RNA and the NS1 protein can be detected in patient blood, serum and plasma, and also in tissues from fatal cases 146 . Virological, molecular and serological methods are used to confirm DENV infection for epidemiological surveillance and clinical diagnosis.
Viraemia, non-structural 1 (NS1) antigen and antibodies change over time; thus, different diagnostic tests will be appropriate depending on the stage of infection. ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription. Adapted from Ref. 153 , Nature Publishing Group.
Anti-DENV IgM antibody detection is the most widely used test in routine practice 147 . Anti-DENV IgM titres in sera from patients in the acute phase of disease are measured to serologically confirm infection, whereas patients in convalescence are identified through IgM and IgG seroconversion by comparing antibody titres in paired acute and convalescent sera 146 , 148 . For patients who are suspected of having dengue, a presumptive diagnosis can be made by the detection of anti-IgM antibodies in samples collected at day 6 of acute symptoms. Commercial kits for IgM or IgG detection in enzyme-linked immunosorbent assay (ELISA) and less-sensitive rapid test formats are available 149 , 150 .
Reverse transcription PCR (RT-PCR), real-time RT-PCR, DENV isolation in mosquito cell lines and by mosquito inoculation facilitate confirmation and identification of the agent virologically. Although virus isolation and identification is highly specific, it has a relatively low sensitivity and is resource-consuming and time-consuming. By contrast, DENV RNA detection provides a rapid, sensitive and specific method for virological diagnostic confirmation. NS1 protein detection provides a window of opportunity for early aetiological diagnosis. The sensitivity and specificity of DENV NS1 detection depend on the infecting serotype, the timing of sample collection and the parity of DENV infection (primary versus secondary), as well as the format of the test 151 , 152 .
Box 2 shows the interpretation of dengue tests and Table 3 summarizes the WHO recommended diagnostic tests according to laboratory surveillance level 2 , 153 . Whereas all of these methods can be used to establish aetiological diagnosis, bedside rapid tests for antigen, antibody or simultaneous antigen and antibody detection are preferable if they are of satisfactory sensitivity and specificity.
The recent introduction and extension of two new arboviruses in dengue-endemic countries of the American region — chikungunya (an alphavirus detected at the end of 2013 in the Caribbean island of St Martin) and Zika (a flavivirus detected in May 2015 in Brazil) — impose new challenges for the diagnosis of dengue and the arbovirus in general. The diagnosis of any of these viruses is based on RNA and/or IgM detection. However, the duration of viraemia is different between these infections, antibody cross-reaction is observed between DENV and Zika virus (which belong to the same viral family: Flaviviridae ) and commercial, adequately evaluated kits for serology are needed for chikungunya virus and Zika virus infections. To face this emergency, the network of Arbovirus National Laboratories of the American region (RELDA; formerly the Dengue Laboratory Network of the Americas) conducted by the Pan American Health Organization (PAHO), recommended a new diagnostic algorithm for DENV, Zika and chikungunya viruses. In the first step, DENV, chikungunya and Zika viral RNA is detected by real-time PCR in acute samples. If available, IgM serology on serum samples collected from individuals with clinically suspected DENV infection or Zika virus infection should be tested by IgM Capture ELISA to both DENV and Zika virus. If positive to both viruses, a secondary flavivirus infection should be considered 154 .
Considering this new epidemiological situation, it is expected that dengue and flavivirus diagnostic guidelines will change with new algorithms according to the epidemiological situation and more-sensitive and specific as well as better evaluated commercial kits for serology.
Identification of patients at risk of severe disease . As DENV infections can result in severe and life-threatening illness, identifying which patients are at risk of an outcome that requires supportive interventions is important. Differentiating this group from the thousands of mild cases during outbreaks is a major medical challenge; simple and inexpensive strategies are urgently needed. The 2009 WHO classification system for the identification of patients at risk of severe disease is summarized in Fig. 1 (Ref. 155 ).
Biomarkers for dengue prognosis that are under evaluation include a high level of viraemia and NS1 protein, the level of microparticles that are produced as a consequence of apoptotic cell death and cellular activation, the level of some immune-response mediators, such as IL-1 receptor-like 1 (IL1RL1; also known as ST2), tumour necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL), and some biochemical alterations, but to date none have been approved for routine practice 156 , 157 . However, as part as the routine laboratory follow-up of a suspected dengue case, a full blood count should be done at the first patient visit. A decreasing white blood cell count makes a diagnosis of dengue very likely, whereas plasma leakage is suggested by a rapid decrease in platelet count, mainly if it is associated with a rising haematocrit level. Fluid accumulation, which can be detected by X-ray or ultrasonography, is a conclusive warning sign of plasma leakage. In addition, laboratory findings during the critical stage of illness that vary according to the severity of vascular permeability include prolonged bleeding time, increased APTT, thrombocytopaenia, increased levels of liver enzymes, activated complement with high levels of C3a and C5a, fibrin split products and low levels of fibrinogen. Chest X-ray is the best method for detecting pleural effusions, and abdominal sonograms can detect gallbladder wall thickening and ascites 2 , 158 . Patients should also be closely monitored for signs of shock.
Daily monitoring of the clinical warning signs to detect early progression from mild to severe illness remains the most useful method to prevent fatal disease 2 , and serial ultrasonographic studies could be better than existing markers, such as the haematocrit level, to identify patients who are at risk of developing severe dengue who merit intensive monitoring 159 . Clinical algorithms have also been proposed for both dengue case identification and dengue prognosis, but none are in routine use 160 – 162 .
Classification systems . There are somewhat competing views in the field as to the optimal approach for the clinical classification of patients with dengue and the identification of warning signs of severe disease, and several reviews and position papers regarding the usefulness of the 2009 WHO system compared with the 1997 WHO system have been published 163 , 164 . Prospective clinical studies developed in Asian and Latin-American countries have concluded that the 2009 WHO dengue classification system may be better at detecting severe DENV infection cases compared to the previous WHO classification system 165 – 167 . Others have argued that the revised 2009 WHO classification has a high sensitivity for identifying severe dengue and is easy to apply 168 ; some consider the 2009 system to be promising from both research and clinical perspectives 169 . Indeed, the 2009 classification system has greater discriminatory power for detecting patients who are at risk of progression to severe disease and those who need hospitalization than the 1997 classification 170 . Furthermore, the 2009 system is simple to use for triage and case management according to disease severity, even in primary care settings 171 , and for disease surveillance. It also reflects the natural course of dengue illness from mild to severe disease and covers all clinical manifestations 172 . A formal expert consensus was reached in La Habana, Cuba, in 2013 with dengue experts from the Americas 173 , where a decrease in disease lethality after the introduction of the revised classification was evident 174 .
That said, through the analysis of retrospective data, some investigators have found that warning signs are not as useful in adults as they are in children 175 , and have argued that the current recommended predictors of severe dengue are, therefore, limited 176 . Others have put forward that there is a need for a more precise definition of warning signs to enable optimal triaging for accurate identification of patients who require hospitalization 177 . In addition to these critiques, one study described that both the 1997 and the 2009 WHO classification systems show high sensitivity but lack specificity 178 , and that the 2009 system requires refined definitions of severe bleeding and organ impairment to improve its clinical relevance 179 . A major ongoing clinical study, coordinated by one of the three large European Union-funded consortia that are currently working on dengue research themes, might address some of these issues 180 . Finally, since the introduction of the revised criteria, a high number of patients have been admitted to hospital or placed under clinical observation during dengue epidemics. This increase is probably owing more to traditional hospital-based methods of managing patients with dengue than to the 2009 WHO classification system, a conclusion that is supported by the fact that this increase in clinical intervention can be alleviated through the participation of trained primary care health units, which the WHO is trying to facilitate 181 .
Although the 2009 WHO classification is more applicable to clinical and epidemiological purposes than the 1997 classification, debate continues regarding its usefulness for pathogenesis research 182 . In particular, some have argued that the dengue fever, DHF and DSS classifications were more capable of correctly identifying cases of plasma leakage than the 2009 system, and that this identification served as a useful predictor of disease severity that was directly related to the main underlying model of pathogenesis. However, in a separate study, the same authors concluded that the 1997 system misclassified a substantial proportion of patients 183 . Specifically, only 68% of patients who were in need of clinical intervention were classified as having DHF and, therefore, in using this system, it could be inferred that 32% of severe cases would be missed. One of these studies has been analysed by a group of experts 184 , who concluded that the revised classification reflects clinical severity in real time, which is something that clinicians have wanted for some time, and with its simplified structure will facilitate effective triage and patient management and also allow collection of improved comparative surveillance data.
Box 2: Interpretation of dengue diagnostic tests
Dengue virus infection is confirmed if any of the following test results are produced:
RT-PCR or real-time RT-PCR are positive
Positive virus culture
Positive NS1 test
IgM seroconversion *
A presumptive dengue diagnosis is made if either of the following test results are produced:
Positive IgM in a single serum sample *
High titre of IgG in a single serum sample tested by haemagluttination inhibition assay or ELISA (≥1,280) *
ELISA, enzyme-linked immunosorbent assay; NS1, non-structural 1 protein; RT, reverse transcription. * The recent introduction and expansion of Zika virus infection into dengue-endemic countries impose a further diagnostic challenge. IgM antibodies in patients with secondary dengue virus infection can cross-react with Zika virus. For this reason, the Pan American Health Organization recommends IgM detection to both dengue virus and Zika virus antigens. Samples that are IgM positive against dengue virus or Zika virus antigens individually are considered a primary infection of dengue virus or Zika virus, respectively, whereas samples that are positive against both viruses are considered a presumptive flavivirus infection. Similarly, specific IgG cross-reaction can be observed with Zika virus antigens. For a more specific serological diagnosis, neutralization assays to both viruses are recommended 154 .
The mechanisms of protective dengue immunity are not well understood. Neutralizing antibodies against viruses serve as the most commonly used correlate of protection. As discussed above, antibodies produced during an infection provide lifelong protection to the homologous virus but short-lived protection against the other three serotypes 185 . Most neutralizing antibodies recognize the E protein, and high DENV neutralizing antibody titres in mice and monkeys have been correlated with protection 186 . However, there is no proof that protection is always associated with neutralizing antibodies, as evidenced by the absence of protection against DENV2 in some vaccinated individuals who have appreciable levels of circulating neutralizing antibody 187 , 188 . Different antibody responses, such as antibody-dependent cell-mediated cytotoxicity and complement fixation, might also correlate with antibody-mediated protection against DENVs 189 , 190 . Moreover, T cell-mediated functions might correlate with protection in vivo . In general, CD4 + T cells can control viral infection through various mechanisms, including the production of antiviral and inflammatory cytokines, cytotoxic killing of infected cells, the enhancement of CD8 + T cell and B cell responses and the promotion of immune memory responses. Similarly, CD8 + T cells also act through the production of pro-inflammatory cytokines, such as TNF and IFNγ, and can be directly cytotoxic to viral infected cells 191 , 192 .
The development of safe and fully protective dengue vaccines faces several challenges: ideally, the vaccine should protect against the four serotypes; long-term protection is required otherwise an individual might become susceptible to breakthrough infection and enhanced disease owing to waning and non-protective immunity (that is, a dengue vaccine could lead to DHF/DSS through ADE if immunity is not sustained or is partial); there is no animal model that exactly replicates human dengue disease; although DENV neutralizing antibodies protect in some circumstances, the full correlates of protection are not known; and vaccine candidates need to be evaluated in the context of changing patterns of transmission intensity and circulating viruses.
Several DENV vaccines are currently under development, including some in phase III safety and efficacy testing. One that has completed phase III efficacy testing is under registration in several countries ( Table 4 ). These vaccines are outlined below.
Live attenuated vaccines . Live attenuated vaccines have numerous advantages, including the ability to induce an immune response that mimics the response to natural infection, the induction of robust B cell and T cell responses and the ability to confer lifelong immune memory. Live attenuated vaccines can be produced at relatively low cost and might be effective after one dose 186 . Early dengue vaccine efforts focused on passaging wild-type DENV strains through various types of primary cells or cell lines, including primary dog kidney (PDK) and African green monkey kidney (GMK) cells. Passaging of DENV in vitro renders it less virulent in humans and was investigated in two series of work.
In the first series, vaccine strains from each serotype obtained by passage through PDK cells or primary GMK cells were selected and tested in monovalent, bivalent, trivalent and tetravalent vaccinations in Thai adults 193 . Of the tetravalent recipients, only one of ten seroconverted to all four serotypes, and neutralizing antibody responses were directed primarily against DENV3. Subsequently, several tetravalent vaccine formulations were tested and the dominant neutralizing antibody response was still against DENV3 (Refs 194 , 195 ). Following on from these studies, the DENV3 vaccine strain was re-derived genetically, grown in Vero cells and tested in volunteers 196 . All recipients had adverse reactions and the trial was halted 186 .
In the second series, different formulations of the tetravalent vaccine were tested in monkeys and flavivirus-naive adults and children 197 , 198 . The formulations were improved to reduce the reactogenicity and increase the immunogenicity 199 , 200 . These new formulations were safe and moderately effective, and the authors recommend that studies in a larger number of adults and then in children are warranted 186 .
Another attenuation strategy is the targeted mutagenesis of 3′ UTR regions of DENV RNA 201 . The viral 3′ UTR is approximately 450 nucleotides long and comprises four defined domains: domain A; domains A2 and A3, which seem to work as enhancers for viral RNA replication; and domain A4 and the 3′ stem loop, which are essential elements for viral replication 202 . The deletion was created by the removal of nucleotides 172–143 from the 3′ UTR. This deletion, designated Δ30, has been shown to attenuate DENV1 and DENV4 in rhesus monkeys and to inhibit dissemination of DENVs in mosquitoes 203 , 204 . Monovalent and tetravalent preparations have been given to human volunteers and produced good immune responses 205 . A phase I trial investigated a single dose of four different formulations of a live tetravalent vaccine in flavivirus-naive volunteers. The vaccines were well tolerated, produced no severe adverse events and only one dose induced a good neutralizing antibody response in 75–90% of the individuals 206 . One of these tetravalent DENV vaccines was licensed to several vaccine developers 207 and entered large-scale phase III efficacy trials in Brazil following a small human challenge trial conducted in the United States. A single dose of the dengue vaccine TV003 fully protected 21 vaccinated volunteers against infection in a virus challenge study, whereas 20 unvaccinated controls all developed an infection 208 .
In addition, a candidate tetravalent dengue vaccine (called CYD-TDV) has been developed, via the insertion of the prM and E genes of the four DENV serotypes into the genetic backbone of the 17D yellow fever vaccine virus 209 . Two ChimeriVax phase III trials were conducted in >30,000 children in five Asian and five American countries. Overall efficacy in the Asian trial was 56.5% and 60.8% in the American trial 187 , 188 . In addition, a reduction in severe complications was reported with a vaccine efficacy of >80% against DHF. These vaccines seem to boost immune responses and protect individuals who have had one previous DENV infection and are, therefore, at risk of ADE. However, these vaccines failed to protect seronegative individuals against clinical infection with all four DENV serotypes, and a group of young vaccinated children had higher rates of hospitalized breakthrough DENV infections than controls 210 . Children who were ≤5 years of age when vaccinated experienced a DENV disease resulting in hospitalization at five times the rate of controls. The aetiology of disease in placebo and vaccinated children that results in hospitalization during a DENV infection, while clinically similar, are of different origin. The implications of the observed mixture of DENV protection and enhanced disease in CYD vaccinees is under study 211 . CYD-TDV seems to protect people who have been infected once and, accordingly, are at risk of severe disease. But, conversely, it puts people who were susceptible to a first infection at risk of severe disease. Even so, the vaccine is approved in Mexico, the Philippines and Brazil.
Another vaccine construct has been developed by substituting the prM and E genes of DENV2 PDK-53 with those of wild-type DENV1, DENV3 or DENV4 (Ref. 212 ). Three different formulations of these tetravalent vaccine (DENVax) were tested in monkeys, and all vaccinated monkeys developed neutralizing antibodies against all four serotypes after one or two doses 213 . On the basis of these results, phase I and phase II trials were carried out to evaluate different vaccination regimens, formulations and alternative routes of immunization 214 . The vaccine was well tolerated in children and adults 1.5–45 years of age, irrespective of prior dengue exposure; mild injection-site symptoms were the most common adverse events. DENVax induced a neutralizing antibody response and seroconversion to the four DENVs, as well as cross-reactive T cell-mediated responses that could be necessary for a broad protection against dengue illness 215 . Currently, phase III trials of the vaccine have been initiated in several Asian countries.
Following on from live attenuated vaccines, another generation of vaccine candidates, including subunit vaccines, inactivated vaccines, DNA vaccines and viral vector vaccines, is being launched.
Subunit vaccines . The advantages of protein vaccines compared with live attenuated vaccines are that they are safe, the induction of a balanced immune response to the four DENV serotypes should be feasible and the immunization schedule can be accelerated, reducing the risk of incomplete immunity and the potential for ADE. However, these vaccines require the use of adjuvants and multiple doses to achieve optimal immunogenicity, and they may not be as efficient as live attenuated vaccines at inducing long-lasting immunity 186 .
The protein target of subunit vaccine development for dengue has been the E glycoprotein, as the majority of neutralizing epitopes on the DENV virion are located in this protein. Recombinant E protein has been produced using Escherichia coli , baculovirus and insect cells, yeast and mammalian cells 216 – 219 . Truncated recombinant E protein subunits (80E) of each serotype were obtained in a Drosophila melanogaster Schneider 2 cell expression system and were found to induce neutralizing antibody responses in mice and in non-human primates 220 . A phase I trial of the DENV1-80E vaccine candidate has been completed 221 and a phase I trial of a tetravalent formulation began in 2012 (Ref. 222 ). The subunit vaccine might be an important component in a prime–boost vaccine regimen.
Domain III-capsid (DIII-C) is a novel candidate vaccine containing viral fragments that might potentially induce neutralizing antibodies and cell-mediated immunity. DIII-C has been evaluated in Balb/c mice and Vervet monkeys 223 , 224 . In animal models, DIII-C has been shown to induce a serotype-specific immune response in terms of both antiviral antibodies and cellular immune response with partial protective efficacy 225 . This candidate is at an advanced stage of preclinical development.
Inactivated vaccines . Vaccination with inactivated vaccines ideally should induce a balanced immune response without the viral interference (wherein the replication of one virus can inhibit the generation of a balanced immune response against all four serotypes as it can interfere with the replication of the other serotypes) that can occur with live attenuated vaccines. In addition, there is no risk of viral replication or reversion to wild-type virus. Inactivated vaccines are less effective in inducing long-lasting immunity than live attenuated vaccines, so multiple doses and adjuvants are needed for optimal immunogenicity in unprimed individuals. A dengue inactivated vaccine might be useful as part of a heterologous prime–boost vaccine regimen 186 .
A dengue purified formalin-inactivated vaccine (DPIV) is being developed and has been shown to be immunogenic in rhesus macaques. A phase I trial began in 2011, and two phase I trials of a tetravalent candidate began in 2012 in a dengue-primed population and in a non-endemic area 186 .
DNA vaccines . DNA vaccination results in antigen expression by both major histocompatibility complex (MHC) class I and MHC class II, leading to the activation of CD4 + and CD8 + T cells, as well as an antibody response. In addition, DNA vaccines are non-replicating and, therefore, safer than live attenuated vaccines, with low reactogenicity. Other advantages include low cost, ease of production and temperature stability 186 . Most of the DNA vaccine-based approaches in dengue have focused on eliciting immune responses to the prM protein and the E protein in mice and monkeys 186 . DNA vaccines based on the NS1 protein have also been tested in mice 226 , 227 , and another DNA vaccine, based on the expression of DENV1 prM, E and NS1 proteins, induced better protection than a DNA vaccine without NS1 (Ref. 228 ). Further advances in DNA vaccination may lead to a successful DENV vaccine.
Viral vector vaccines . Several viral vector platforms, including vaccinia virus, adenovirus and alphavirus vectors, have been explored as delivery vehicles for DENV antigens. Viral vector dengue vaccine candidates are focused on eliciting and evaluating anti-E protein antibody responses. No viral vector vaccine has advanced to clinical phase I testing 186 .
Vector control-based prevention
As the twentieth century dengue pandemic expanded over the past 40 years, prevention and control of the disease relied solely on mosquito control, as there was no licensed vaccine. However, as evidenced by the increasing global disease burden and expanding geographical distribution of both the viruses and the mosquito vectors, it is clear that mosquito control, as used in most countries, has failed to control dengue 229 – 231 ( Fig. 3 ). The reasons for this failure are complex and a detailed discussion is beyond the scope of this Primer. Briefly, after the successes of the American hemispheric Ae. aegypti and global malaria eradication programmes in the 1950s and 1960s 232 , there was widespread complacency about vector-borne diseases in general, and dengue in particular 229 . Moreover, dengue was not considered a major public health problem by policy makers who controlled budgets because epidemics were intermittent and mortality was low. This led to a redirection of resources and a lack of commitment to dengue control on the part of permissive countries and to deteriorating public health infrastructure 233 . Finally, limitations placed on the use of effective insecticides, such as dichlorodiphenyltrichloroethane (DDT), and the improper use of mosquito control tools that were available were contributing factors to the failure.
There were two countries that, temporarily, were exceptions to this general failure: Singapore and Cuba. Singapore was one of the first countries in Asia to experience DHF in the 1960s 234 . A highly successful Ae. aegypti control programme, which prevented epidemic dengue in Singapore for nearly 20 years, was implemented in 1968. The programme had three main pillars: legislation that levied fines on individuals whose premises were found to be infested by Ae. aegypti mosquitoes; larval source reduction and control; and community outreach and education 235 . Although this programme is still functioning and effectively controlling Ae. aegypti , it has failed to prevent the re-emergence of epidemic DENV transmission in the past 20 years 236 . The reasons for this re-emergence are not fully understood, but are thought to be a combination of low herd immunity, high frequency introduction of DENVs from neighbouring endemic or epidemic countries that had not controlled the disease and a highly dense human population 236 , 237 .
The Cuban programme was initiated in 1981 during the first large epidemic of DHF in the Americas 237 , 238 . This programme was based on the same three pillars used in Singapore, but added a fourth pillar: extensive use of space spraying of pyrethroid and organophosphate insecticides to kill adult mosquitoes using ultra-low volume and thermal fogging machines 238 . Epidemic dengue was controlled in Cuba for almost 30 years, but this programme also ultimately failed because of economic problems and, as with Singapore, the introduction of DENVs from neighbouring endemic or epidemic countries that had not controlled dengue have occurred.
There are several important lessons to be learned from these experiences. First, sustainable dengue control cannot be achieved by individual countries or communities when they are surrounded by areas with endemic or epidemic dengue. Thus, effective sustainable programmes must be developed on a regional basis as clearly demonstrated by the American hemispheric eradication programme 231 , 239 . Second, sustainable control requires long-term commitment by endemic countries. Those countries must use their own resources instead of relying on international agencies whose funds might not be relied on with certainty 229 , 231 . Last, to be effective, mosquito control tools must be used properly by trained personnel 240 . Otherwise, dengue control efforts become a waste of time and money.
Fortunately, the future for dengue control using vector control looks brighter as there are numerous new tools in the development pipeline. A new organization, the Partnership for Dengue Control, has recently been formed to facilitate an integrated approach to dengue control 241 . As a global alliance of partners and stakeholders interested in controlling dengue, it will bring together the leading expertise in dengue and public health to design new strategies for dengue control by integrating new and existing mosquito control tools with vaccination. Recent expert consensus workshops have reviewed currently available mosquito control methods as well as those in the development pipeline that might become available in the next 5 years. Briefly, the reviews concluded that, for currently available tools, targeted indoor residual spraying with synthetic pyrethroid insecticides combined with larval control were the most likely to provide effective Ae. aegypti control, provided the methods were used properly. The new tools in the pipeline include new non-pyrethroid residual insecticides that can be used for the control of dengue as well as new uses for those insecticides. Thus, in addition to targeted indoor and outdoor spraying, these compounds, which may have a residual activity of ≥6 months, can also be used as spatial repellents, to treat curtains and other materials hanging in mosquito resting areas and in lethal ovitraps (devices that mimic natural mosquito breeding sites).
Other tools in the pipeline include biological ( Wolbachia ) and genetic (sterile males) control 241 – 243 . A strain of Wolbachia , a natural bacterial parasite of insects, has been adapted to Ae. aegypti . When infected, the female Ae. aegypti has a reduced lifespan and has increased resistance to infection with DENVs, both of which can decrease transmission. When released into a natural population of Ae. aegypti , the Wolbachia spreads via normal mating, ultimately infecting most individuals in that population. A major advantage of this method is that it provides sustainable control. The sterile male method uses a dominant lethal gene carried by male Ae. aegypti , which are released into the natural mosquito population. When the males carrying the lethal gene mate with wild-type female Ae. aegypti , the progeny die as larvae, therefore, reducing the population. The advantage of this method is rapid reduction of the mosquito population, but the disadvantage is that it is not sustainable. Both of these approaches are in advanced field trials in several countries in Asia, South America and Central America, with promising results 241 .
Unfortunately, none of these mosquito control methods are likely to be completely effective in controlling dengue if used alone, but if used in an integrated control programme with other synergistic mosquito control tools and vaccines, effective control might be achieved 241 .
Central health policy-making institutions of each country should have programmes aimed at avoiding dengue-related fatalities. Permanent capacity building is necessary 244 to ensure the adequate classification and supportive care of patients 245 . Moreover, judicious fluid management during the critical phase coupled with continuous monitoring 246 , reorganization of sanitary services during epidemics 247 and dengue research are all vital to improve outcomes for patients 248 . A very comprehensive review on case treatment and management can be found in the WHO Dengue Guidelines for Diagnosis, Treatment, Prevention and Control 2 . In general, more histopathological and virological studies are needed to define the causes and pathogenesis of all of the complications that can accompany dengue illness.
No specific treatment for dengue is currently available; consequently, patients are provided with supportive care. As management decisions need to be made before a confirmed dengue diagnosis by serology or other tests, clinicians must rely on clinical and epidemiological data to decide whether to classify a patient as a suspected dengue case 249 . In the absence of an effective antiviral drug, the prescription of bed rest and abundant liquids by mouth can be pivotal in determining the outcomes of patients. Indeed, fluid intake during the 24 hours before being seen by a clinician has been significantly associated with decreased risk for hospitalization of patients with dengue fever 250 . Analgesic and antipyretic drugs, such as paracetamol (neither aspirin nor NSAIDs should be taken), can be prescribed at the usual dosage for children and adults. Bed nets or repellents to avoid mosquito bites should be used to prevent other cases at home and in the neighbourhood.
Fluid therapy is key to dengue management and is applied based on disease severity. In mild dengue, oral fluid therapy can be as effective as intravenous fluid replacement and there is no need for hospitalization. Nevertheless, the requirement for hospital admission must be made according to the analysis of each clinical case, and hospitalization might be required, for instance, when a patient has diabetes mellitus or other comorbidities or characteristics, such as in the case of pregnant women, newborns or the elderly 251 , 252 . Old age and comorbidities, such as cardiovascular disease, stroke, respiratory disease and renal disease, might contribute to the development of severe dengue 253 .
Capillary leakage and shock . Capillary leakage becomes evident at the end of the febrile stage (days 3–6) and increases during the next 24–48 hours. At defervescence, the identification of warning signs ( Fig. 1 ) has a greater discriminatory power for detecting patients who are at risk of progression to severe disease and those who need hospitalization 254 , as the 2009 WHO guidelines recommend 2 , 163 . Families also must be educated to identify the so-called warning signs of dengue shock when fever subsides 171 and, when available, patients should be monitored clinically for markers of severe disease risk. Warning signs are the clinical heralds that announce the imminence of shock. In this situation, the fluid lost from the circulatory system owing to capillary leakage must be replaced by immediately initiating intravenous fluid therapy, of which crystalloid solutions (volume expanding fluid replacements such as lactate Ringer solution) or physiological (normal) saline solution are recommended 255 . Therapy with fluid support should be continued, according to the clinical situation of the patient and their fluid balance. As a rule, most patients with dengue recover if they are stratified and managed according to WHO recommendations 167 , 173 , which are guidelines that are supported by recognized experts 9 .
Some patients have a poor clinical evolution and present with pronounced frank hypotension, mental confusion and worsening of the other signs 256 . In these cases, intravenous administration of crystalloid solutions should be continued according to the WHO recommendations 257 , 258 . If appropriate management is continued, most patients will recover and go on to require maintenance fluid therapy, which is calculated according to the clinical situation of the patient. For those who do not recover, colloids (volume expanding solutions that contain human albumin, gelatin or starch) can be administered as an alternative to crystalloid solutions.
Impaired haemostasis . The haemorrhages that can accompany dengue illness are multifactorial. Although haemorrhages can worsen outcomes for patients who experience DSS, they are not necessarily associated with capillary leakage and can occur at any moment of the critical phase or during the convalescent phase 259 . Nevertheless, haemorrhages are frequent complications of prolonged shock rather than a general dengue complication (such as disseminated intravascular coagulation) and, therefore, the best way to prevent major haemorrhages is to prevent shock or treat it quickly and appropriately. Risk factors for severe haemorrhage include the duration of shock and low-to-normal levels of haematocrit at shock onset, whereas platelet count is not predictive of bleeding and does not correlate with its severity 260 . A sudden decrease in the levels of haematocrit and haemoglobin without an improvement of the patient is a sign of silent haemorrhage. Bleeding most commonly occurs in the gastrointestinal system where it can manifest as haematemesis (the vomiting of blood) and/or melaena (black, tar-like faeces). Treatment of gastrointestinal bleeding involves transfusion with packed red blood cells. Intracranial or lung bleeding can also occur and these are associated with a poor prognosis that can sometimes involve multiple organ system failure 261 . Finally, thrombocytopaenia is frequently observed in the course of dengue illness, but there is currently no evidence to support the practice of prophylactic platelet transfusions, which are costly and sometimes harmful 262 .
Other complications of severe dengue . Cardiac, hepatic, respiratory and neurological complications can all accompany dengue illness. DENV infection has been shown to cause cardiac disease with clinical manifestations ranging from a mild increase of disease biomarkers to myocarditis and/or pericarditis, and patients with severe dengue might show evidence of systolic and diastolic cardiac impairment that predominantly affects the septum and right ventricular walls 263 . Patients with dengue and clinical cardiovascular manifestations have been tested for abnormalities in the biomarkers troponin I and the amino-terminal fragment of brain natriuretic peptide, and these patients have also been studied using echocardiography and cardiac MRI 264 . These studies have shown that DENV infection can induce the destruction of cardiac fibres, the absence of myocyte nuclei and the loss of striations 265 . Dengue myocarditis or myocardiopathy can occur alone, can be associated with other organ dysfunctions or can be a complication of DSS. As such, cardiac function should be carefully monitored and, where appropriate, the patient should be admitted to an intensive care unit and given inotrope therapy (such as dopamine and dobutamine), according to local guidelines 266 .
The capillary leakage involved in severe dengue can have consequences for the respiratory system. These can include pleural effusion and respiratory distress associated with pulmonary oedema, which can be worsened by over-hydration during fluid therapy 267 . To avoid respiratory distress, during fluid therapy, the balance of fluids must be monitored 246 , oxygen therapy is mandatory and in some cases mechanical ventilation should be initiated.
In cases of acute dengue hepatitis, patients might require liver support and a hepatic pre-coma regimen (in attempt to avoid hepatic encephalopathy). In addition, patients with dengue hepatitis might also require management of other associated complications, such as bleeding (using transfusion of blood rather than platelets or fresh frozen plasma). Metabolic complications, such as acidosis, hypoglycaemia and hypocalcaemia, could be important components of dengue severity, although not necessarily associated with severe liver dysfunction. Pancreatitis is an infrequent complication of DENV infection 268 , 269 .
A major unmet need in dengue management is a safe and effective antiviral drug 270 . Although some antiviral candidates have been tested in randomized controlled trials, the results have been poor 271 . In addition, a trial of chloroquine — which has had in vitro antiviral effects against DENV infection and has additional anti-inflammatory properties — did not show any significant effect on the duration of viraemia or on the clinical course of illness 272 .
An alternative approach to using antivirals to treat dengue is to modulate the immune system, as cytokines have been implicated in contributing to capillary leakage. Although corticosteroids can suppress the immune system and decrease inflammation, a study in patients with dengue demonstrated no effect of steroid use on clinical signs or symptoms. Similarly, statins (3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors) were unable to produce anti-inflammatory or endothelial-stabilizing effects in those with dengue 273 . Modern bioinformatics and high-throughput screening approaches are being used to try to identify new antiviral molecules and drugs that could stabilize the endothelium and, therefore, prevent or reverse capillary leakage in dengue illness.
Quality of life
Effects of acute illness.
Patients with dengue usually visit the physician with a main complaint of an unexplained high fever and malaise, sometimes with rash, petechiae or other skin and mucosal bleeding and vomiting. As a rule, these patients cannot attend school or go to work for ≥1 week. Dengue (also named ‘breakbone disease’) is frequently a very painful disease and mainly produces abdominal pain, backache, headache and myalgias 274 . By contrast, myositis (muscle inflammation) and muscle weakness are distinctly uncommon manifestations of DENV infection. Patients with dengue who have myositis have high levels of creatine phosphokinase and could have respiratory muscle involvement, which sometimes requires mechanical ventilation 275 . Guillain–Barré syndrome should be considered in patients who present with myalgias and general weakness or progressive limb weakness after the acute illness 276 .
Long-term effects of infection
Although fever and acute illness usually resolve within 1 week (mainly in children but also in some adults), most adult patients take longer to recover. In some patients, some dengue symptoms last for 6 months after the acute phase. The most frequent symptoms that are present at 6 months after acute illness are weakness (27.6% of patients), headache (14.8% of patients) and arthralgias (10.6% of patients). One study showed that these symptoms had an irregular appearance, were inconsistent and were mostly related to physical or mental exertion 277 . The persistence of some dengue symptoms has even been demonstrated 2 years after the acute illness 278 . Liver enzymes can normalize 1 month after acute illness 279 or continue to be raised for a much longer duration as a marker of liver cytolysis 280 . Fatigue post-DENV infection can result in clinical disease beyond the acute phase of infection, which may be explained by the immune alterations that are triggered by the DENV 281 . Some immunocompetent patients who had experienced severe dengue among other infectious diseases have had an increase in lymphocyte count preceding clinical deterioration that included interstitial pneumonitis, airway obstruction, CNS disturbances and systemic capillary leak syndrome, all of which were thought to represent immunopathological tissue damage 282 .
Neurological, renal and haematological outcomes
Neurological features have had an increased frequency in patients with dengue who have been admitted to hospital. These can be categorized into dengue encephalopathy (which is caused by hepatic failure or metabolic disorders), encephalitis (which is caused by direct virus invasion), neuromuscular complications (such as Guillain–Barré syndrome or transient muscle dysfunctions) and neuro-ophthalmic manifestations. Other neurological symptoms, such as transitory post-dengue Parkinsonism have been reported following serologically confirmed DENV infection. Transient and permanent visual disturbances have also been reported 283 . Impaired vision caused by dengue-associated ocular inflammation is an emerging ophthalmic condition and often involves the posterior segment (dengue-related maculopathy). The prognosis for these patients is variable and, although they usually regain good vision, they can retain persistent partially altered vision (which is defined as partially altered vision even up to 2 years after the clinical resolution of the acute disease) 284 . The aetiology and mechanisms of these complications deserve careful study.
Renal manifestations are uncommon and include acute kidney injury, proteinuria, glomerulonephritis and haemolytic uraemic syndrome 285 .
Among haematological alterations, haemophagocytic lymphohistiocytosis (an inflammatory condition characterized by increased proliferation of activated lymphocytes and macrophages that are capable of phagocytosing red blood cells leading to severe anaemia) has been recognized as a dengue-associated condition 286 .
There have been major advances in vaccines, specific antiviral drug development and vector control activities in the past 10 years. Used effectively, these new tools will provide new opportunities to control the disease. New molecular diagnostic and antigen detection tests and a better understanding of pathogenic mechanisms will enable earlier diagnosis and more-effective clinical management. In addition, a better understanding of virus–vector interactions and the dynamics of DENV transmission along with the new vector control tools that are being developed should lead to more-effective prevention and control of the disease.
Several vaccine candidates are currently in clinical trials, with live attenuated vaccines being the most successful developed to date 287 . The impact of the vaccination should be estimated by also considering vaccine coverage, the epidemiological situation of dengue, genetic polymorphisms within populations, dengue immunity of the target population and vector control activities 288 . The introduction of a licensed dengue vaccine into national programmes represents a challenge. According to the epidemiological situation, countries should decide whether high-risk groups or the total population should be vaccinated, and in which age groups and regions where the vaccine should be introduced. Mathematical models could become a very useful tool for the understanding of population-wide dengue strategies 289 .
As of April 2016, Sanofi Pasteur completed phase III testing and received approval from the WHO's Scientific Advisory Group of Experts on Immunization. The company has licensed their three-dose tetravalent live attenuated CYD vaccine (Dengvaxia) in five countries (Brazil, El Salvador, Mexico, Paraguay and the Philippines) for use in individuals 9–45 years of age, 70% of whom should be circulating DENV antibodies 188 , 210 , 290 , 291 .
Despite these advances, dengue research is still a priority. In 2006, the Special Programme for Research and Training in Tropical Diseases and the WHO convened a Dengue Scientific Working Group with experts from 20 countries who reviewed existing knowledge on dengue and established the research priorities, which were organized into four main streams: research related to reducing dengue fatality rates and disease severity; research on transmission control through improved vector management; research related to primary and secondary prevention; and health policy research aimed at contributing to an adequate public health response. The objective was to provide information for policy makers and foster the development of more cost-effective strategies to reverse the epidemiological trend of dengue. Unfortunately, owing to the current complex global dengue epidemiology, after almost 10 years, this approach is yet to stop the spread and emergence of dengue 292 , 293 .
Today, there are many promising areas for research. Interdisciplinary studies of the interactions between the virus, the human and the mosquito vector are required for a better understanding of the illness pathogenesis, DENV transmission dynamics, vaccine and drug development, and better diagnostic tools. The integration of clinical and epidemiological data with basic research, genomic studies and the application of advanced technology (such as ‘omics’, nanotechnology, biosensors and molecular modelling, among others) should be supported. No less important are studies to define the social, environmental and other risk factors for DENV transmission. Box 3 summarizes the main research priorities and topics.
Recognizing the importance of dengue, several international initiatives are ongoing. As a result of these efforts, the WHO published the Global Strategy for Dengue Prevention and Control in 2012 with three main targets: to reduce mortality to 50% by 2020; to reduce morbidity to 25% by 2020; and to better study disease burden. This strategy relies on five technical elements: diagnosis and case management; integrated surveillance and outbreak preparedness; sustainable vector control; future vaccine implementation; and research 13 .
At present, some of the WHO efforts are to identify early warning signs of outbreaks, to implement a strategy for integrated vector management and to analyse issues related to dengue vaccine and vaccination 2 , 294 – 298 . Other international initiatives include the Dengue Vaccine Initiative, the Partnership for Dengue Control and the multi-country research projects International Research Consortium on Dengue Risk Assessment, Management and Surveillance, Dengue Research Framework for Resisting Epidemics in Europe, and Innovative Tools and Strategies for Surveillance and Control of Dengue, which are supported by the European commission, among others 241 , 299 , 300 .
Overall, the international multi-sector response, as outlined in the 2012 WHO Global Strategy 13 and the Partnership for Dengue Control, will be crucial to reversing the global dengue trend as well as addressing other emerging vector-borne diseases, such as chikungunya, Zika and yellow fever virus infections. This strategy should include improved vector control through strategies such as community-based interventions to reduce breeding sites, an increase in the scope and breadth of dengue surveillance, improved case management to reduce the case fatality rate, a vaccination strategy and, potentially, novel entomological approaches to reduce transmission by altering mosquito ecology or genetics.
Box 3: Dengue research priorities topics
Reduce disease severity and fatality rates
Evaluate the 2009 WHO classification in clinical practice
Identify and validate prognostic markers (warning signs and risk factors) for severe disease
Prepare guidelines for clinical management of severe cases
Study dengue and comorbidities, pregnancy and in the elderly
Develop new diagnostic methods for early clinical diagnosis and clinical differentiation to chikungunya virus and Zika virus infections, tests for dengue confirmatory diagnosis, evaluation of commercial kits and quality assurance for diagnosis
Implement training programmes in case management at the different levels of the health care system
Define the molecular mechanisms of dengue and severe dengue with special emphasis on plasma leakage, bleeding and dengue in infants
Investigate the mechanisms of virus–human and virus–mosquito cell interactions, including host genetic associations with dengue virus (DENV)
Study the protective and pathogenic immune responses in DENV infection and the mechanisms of antibody-dependent enhancement, as well as the relationship with Zika immunity
Investigate the association between genetic changes in DENV and phenotypic expression
Study of vector biology and ecology
Develop and evaluate new vector control tools
Introduce and evaluate a strategy for integrated vector management
Implement training programmes in vector control to strengthen capacity
Study temporal, virological and immunological variables associated with transmission dynamics in different epidemiological settings
Apply mathematical modelling and geographical information systems for evaluating transmission dynamics, predicting transmission patterns and developing control measures
Define and evaluate better vector indices for surveillance and epidemic responses
Improve methods to study insecticide resistance
Develop early warning indicators of dengue outbreaks and epidemics and effective response systems
Develop new vaccine candidates via research on various areas
Apply mathematical modelling to estimate the effect of vaccination and the interaction between vaccination and vector control
Define indicators for post-vaccination surveillance
Integrate vaccine introduction with effective mosquito control
Health policy research
Define the burden and cost of illness, including in Africa
Identify strategies for multi-sectorial and multi-country collaboration for addressing dengue, integrating all factors and efforts to diminish transmission
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Institute of Tropical Medicine ‘Pedro Kouri’, PAHO/WHO Collaborating Center for the Study of Dengue and its Vector, Autopista Novia del Mediodia, Km 6 1/2, Havana, 11400, Cuba
Maria G. Guzman, Alienys Izquierdo & Eric Martinez
Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore
Duane J. Gubler
Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
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Introduction (M.G.G.); Epidemiology (D.J.G.); Mechanisms/pathophysiology (S.B.H.); Diagnosis, screening and prevention (A.I., D.J.G., E.M. and M.G.G.); Management (E.M.); Quality of life (E.M.); Outlook (M.G.G.); Overview of Primer (M.G.G.).
Correspondence to Maria G. Guzman .
D.J.G. is a patent holder on the Takeda vaccine and a stock holder in the company. He has consulted for Takeda and Sanofi, and was on the Scientific Advisory Board of Novartis Institute for Tropical Diseases from 2003 to 2012. All other authors declare no competing interests.
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Guzman, M., Gubler, D., Izquierdo, A. et al. Dengue infection. Nat Rev Dis Primers 2 , 16055 (2016). https://doi.org/10.1038/nrdp.2016.55
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- 1 Department of Microbiology and Molecular Genetics, Center for Vaccine Research, University of Pittsburgh, PA 15261, USA. [email protected] <[email protected]>
- PMID: 20513545
- PMCID: PMC7115719
- DOI: 10.1016/j.cll.2009.10.007
Dengue is the most prevalent arthropod-borne virus affecting humans today. The virus group consists of 4 serotypes that manifest with similar symptoms. Dengue causes a spectrum of disease, ranging from a mild febrile illness to a life-threatening dengue hemorrhagic fever. Breeding sites for the mosquitoes that transmit dengue virus have proliferated, partly because of population growth and uncontrolled urbanization in tropical and subtropical countries. Successful vector control programs have also been eliminated, often because of lack of governmental funding. Dengue viruses have evolved rapidly as they have spread worldwide, and genotypes associated with increased virulence have spread across Asia and the Americas. This article describes the virology, epidemiology, clinical manifestations and outcomes, and treatments/vaccines associated with dengue infection.
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- Aedes / virology
- Dengue / diagnosis*
- Dengue / epidemiology
- Dengue / etiology
- Dengue Vaccines / immunology
- Dengue Virus / immunology
- Dengue Virus / isolation & purification*
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Dengue virus in humans and mosquitoes and their molecular characteristics in northeastern Thailand 2016-2018
Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing
Affiliation Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
Roles Methodology, Supervision
Affiliations Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, HPV & EBV and Carcinogenesis Research Group, Khon Kaen University, Khon Kaen, Thailand
Roles Methodology, Supervision, Writing – review & editing
Roles Supervision, Writing – review & editing
Affiliation Mahidol-Osaka Center for Infectious Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
Roles Funding acquisition, Supervision
Affiliations Mahidol-Osaka Center for Infectious Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
Roles Funding acquisition, Methodology, Supervision, Writing – review & editing
Roles Formal analysis
Affiliations Program in Bioinformatics and Computational Biology, Graduate School, Chulalongkorn University, Bangkok, Thailand, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Roles Formal analysis, Supervision
Affiliation Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Roles Project administration
Affiliation Department of Disease Control, Office of Disease Prevention and Control, Region 7 Khon Kaen, Ministry of Public Health, Khon Kaen, Thailand
Roles Funding acquisition, Project administration, Writing – review & editing
Affiliation Faculty of Science and Technology, Norwegian University of Life Sciences, Ås, Norway
Roles Funding acquisition, Writing – review & editing
Affiliations Public Health & Malaria Control, PT Freeport Indonesia/International SOS, Kuala Kencana, Papua, Indonesia, Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand
Affiliation MRC Tropical Epidemiology Group, London School of Hygiene and Tropical Medicine, London, United Kingdom
Roles Conceptualization, Data curation, Funding acquisition, Project administration, Supervision, Writing – review & editing
- Patcharaporn Nonyong,
- Tipaya Ekalaksananan,
- Supranee Phanthanawiboon,
- Sirinart Aromseree,
- Juthamas Phadungsombat,
- Emi E. Nakayama,
- Tatsuo Shioda,
- Vorthon Sawaswong,
- Sunchai Payungporn,
- Published: September 14, 2021
- Reader Comments
Dengue is hyperendemic in most Southeast Asian countries including Thailand, where all four dengue virus serotypes (DENV-1 to -4) have circulated over different periods and regions. Despite dengue cases being annually reported in all regions of Thailand, there is limited data on the relationship of epidemic DENV infection between humans and mosquitoes, and about the dynamics of DENV during outbreaks in the northeastern region. The present study was conducted in this region to investigate the molecular epidemiology of DENV and explore the relationships of DENV infection in humans and in mosquitoes during 2016–2018. A total of 292 dengue suspected patients from 11 hospitals and 902 individual mosquitoes (at patient’s houses and neighboring houses) were recruited and investigated for DENV serotypes infection using PCR. A total of 103 patients and 149 individual mosquitoes were DENV -positive. Among patients, the predominant DENV serotypes in 2016 and 2018 were DENV-4 (74%) and DENV-3 (53%) respectively, whereas in 2017, DENV-1, -3 and -4 had similar prevalence (38%). Additionally, only 19% of DENV infections in humans and mosquitoes at surrounding houses were serotypically matched, while 81% of infections were serotypically mismatched, suggesting that mosquitoes outside the residence may be an important factor of endemic dengue transmission. Phylogenetic analyses based on envelope gene sequences showed the genotype I of both DENV-1 and DENV-4, and co-circulation of the Cosmopolitan and Asian I genotypes of DENV-2. These strains were closely related to concurrent strains in other parts of Thailand and also similar to strains in previous epidemiological profiles in Thailand and elsewhere in Southeast Asia. These findings highlight genomic data of DENV in this region and suggest that people’s movement in urban environments may result in mosquitoes far away from the residential area being key determinants of DENV epidemic dynamics.
Citation: Nonyong P, Ekalaksananan T, Phanthanawiboon S, Aromseree S, Phadungsombat J, Nakayama EE, et al. (2021) Dengue virus in humans and mosquitoes and their molecular characteristics in northeastern Thailand 2016-2018. PLoS ONE 16(9): e0257460. https://doi.org/10.1371/journal.pone.0257460
Editor: Baochuan Lin, Defense Threat Reduction Agency, UNITED STATES
Received: April 7, 2021; Accepted: September 1, 2021; Published: September 14, 2021
Copyright: © 2021 Nonyong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the Research Council of Norway (project no. 250443); the Invitation Research Grant, Faculty of Medicine, Khon Kaen University, Thailand, grant number IN62113; the Japan Agency for Medical Research and Development (AMED) JP19fm0108003 and 20wm0225010h0101.
Competing interests: The authors declare no competing interests exist.
Dengue, caused by the dengue virus (DENV), is one of the most important re-emerging arboviral diseases in the tropical and subtropical regions of the world, including Southeast Asia. The virus is transmitted between humans primarily by the mosquitoes Aedes aegypti , and Ae . albopictus being a secondary vector [ 1 ]. The disease is endemic in more than 100 countries, with more than 2.5 billion people at risk [ 2 ]. There are an estimated 390 million DENV infections annually, of which 96 million are symptomatic, ranging from mild dengue fever, with or without warning signs, to severe dengue with plasma leakage that may lead to shock, bleeding, and/or organ impairment [ 3 ]. Increasing numbers of dengue cases, and the propagation of all four serotypes of DENV, are facilitated by uncontrolled urbanization, suboptimal management of water and solid waste, gaps in vector control, and rapid population movement, especially travel [ 4 , 5 ]. In the absence of specific antiviral drugs and an effective vaccine, virus surveillance for early warning and integrated vector management are the primary options for the prevention and control of dengue outbreaks [ 6 , 7 ]. Dengue prevention and control programs in Thailand are mainly based on hospital case reporting, conducted jointly by the hospital and the Offices of Disease Prevention and Control (ODPC), Ministry of Public Health. The dengue surveillance team responds when a case is reported by a hospital within 24 hours of notice in order to prevent transmission by spraying insecticides within 100 meters of the patient’s house [ 8 ].
DENV is a single-stranded, positive-sense RNA virus within the Flaviviridae family, and has four distinct serotypes: DENV-1 to -4 [ 9 ]. The viral genome has length approximately 11 kb which contains a single open reading frame (ORF) encoding three structural proteins (C, prM/M, E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) [ 10 ]. Within each serotype, genotypes can be phylogenetically classified on the basis of their E gene; DENV-1 includes six genotypes (I, II, III (sylvatic), IV, V, and VI); DENV 2 includes six genotypes (Asian I, Asian II, Asian/American, American, Cosmopolitan, and sylvatic); DENV-3 includes five genotypes (I, II, III, IV, and V); DENV-4 includes five genotypes (I, IIA, IIB, III, and sylvatic) [ 2 , 11 ]. Previous studies have suggested that the transmission of the various serotypes is cyclic, with distinct serotypes periodically re-emerging to dominate, and the introduction of new serotypes or genotypes leading to new epidemics or outbreaks [ 11 – 15 ]. However, the circulating patterns of viruses during infection in human hosts and mosquito vectors are still unclear.
In Thailand, the first case of dengue disease was reported in 1949 and the first major outbreak of severe dengue (dengue hemorrhagic fever, DHF) was documented in Bangkok (central region) in 1958 [ 2 , 11 ]. Dengue affects inhabitants throughout the country and has become a major public health problem annually [ 2 , 16 ]. Although the dengue situation in northeastern Thailand has become critical in recent years, with both rising case numbers and increasing disease severity, there is no information available on the DENV genotype in this region.
In the present study, we performed molecular characterization of DENV detected in humans and Aedes mosquitoes in order to explore, for the first time, the epidemiological relationship between humans and mosquitoes and molecular characteristics of circulating DENV strains in northeastern Thailand.
Materials and methods
Study site, recruitment, and sample collection.
Plasma and mosquito samples obtained from a prospective, hospital-based, case-control study in northeastern Thailand [ 8 ] were used. The study was conducted in four provinces in northeastern Thailand from June 2016 to December 2018 ( Fig 1 ). Eleven district hospitals in these provinces were included: Mancha Khiri (16° 12’ N 102° 29’ E), Chum Phae (16° 34’ N 102° 5’ E), Ban Phai (16° 3’ N 102° 43’ E), Ban Haet (16° 12’ N 102° 46’ E), and Mueang Khon Kaen (16° 25’ N 102° 49’ E) district hospitals in Khon Kaen Province; Selaphum (16° 2’ N 103° 59’ E), Phon Thong (16° 18’ N 103° 57’ E), and Thawat Buri (16° 1’ N 103° 45’ E) district hospitals in Roi Et Province; Kamalasai (16° 18’ N 103° 36’ E) and Kuchinarai (16° 30’ N 104° 1’ E) district hospitals in Kalasin Province; and Chiang Yuen (16° 25’ N 103° 3’ E) district hospital in Maha Sarakham Province.
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Sample collections were done in four provinces of northeastern Thailand which are highlighted in green, brown, blue, and yellow. The locations of study districts are marked on the map in gray. The map was created using QGIS 3.16 software.
The methodology of sample collection was previously described [ 8 ]. Briefly, plasma samples were collected from patients presenting with dengue-like symptoms, with potential dengue infections based on the presence of fever (> 38°C), no recent travel history during the previous 7 days, and being older than five years of age.
For the entomological investigation, adult Aedes mosquitoes were collected from the patient’s household and four additional neighboring houses within a 100-meter radius of the patient’s house using portable Prokopack aspirators [ 17 ]. The collection was conducted for 15 minutes indoors (mainly in bedrooms and living rooms) and 15 minutes outdoors near the house (primarily around human-made articles, backyard/patio, vegetation, etc). Mosquito sex and species ( Ae . aegypti or Ae . albopictus ) were identified under a stereomicroscope using morphological keys [ 18 , 19 ]. Aedes mosquitoes from the patient’s house and neighboring houses were designated as being from one combined collection cluster.
Confirmation of dengue virus infection in humans
During the study period, we obtained 292 plasma samples from suspected dengue patients ( S1 Fig ). The preliminary screening for DENV in the patient’s plasma was performed using the SD BIOLINE Dengue Duo kit (Standard Diagnostics, Suwon, Korea), according to the manufacturer’s instructions. Laboratory-confirmed DENV detection and serotyping were carried out in the laboratory at Khon Kaen University, Khon Kaen province, Thailand. Briefly, viral RNA was extracted from 140 μl of each plasma sample using QIAamp Viral RNA Mini Kits following the manufacturer’s instructions (Qiagen, Germany), and DENV detection and serotyping were performed using qRT-PCR as described by Shu et al. [ 20 ].
DENV detection in adult mosquitoes
A total of 192 female Aedes mosquito pools containing 902 Aedes mosquitoes (1–32 mosquitoes per pool) were processed for DENV identification by qRT-PCR ( S1 Fig ). These 192 pools originated from different collection clusters. Mosquito abdomens were separated from the head-thorax and pooled. These pooled and individual head-thorax samples were triturated, using sterile pestles and 1.5 ml Eppendorf tubes (Eppendorf AG, Hamburg, Germany) in 500 μl of Leibovitz’s L-15 medium (Gibco, Thermo Fisher Scientific, USA). The resulting suspension was clarified by centrifugation 800×g at 4°C for 5 minutes. Next, 140 μl of each sample was transferred to Eppendorf tubes for RNA extraction, and the remaining suspensions were stored at −80°C. Viral RNA extraction was performed by using QIAamp Viral RNA Mini Kits following the manufacturer’s instructions (Qiagen, Germany). Mosquito samples were subjected to two rounds of PCR for DENV detection and serotyping using the primer set of Lanciotti et al. [ 21 ] with minor modifications to perform it on real-time PCR. The viral RNA was reverse-transcribed into cDNA by specific D2 primer synthesized using SuperScript III first-strand synthesis system (Invitrogen, USA) according to the manufacturer’s instructions and cDNA was stored at -20°C until used. The first round of PCR aimed to screen DENV infection from mosquito pooled samples using SYBR green-based real-time and the primer set of Lanciotti et al. [ 21 ]. All individual mosquito specimens from each PCR-positive pool were subjected to the second round of PCR for DENV serotyping by SYBR green-based real-time PCR using the primer set of Lanciotti et al. [ 21 ]. The PCR reaction comprised pre-denaturation at 95°C for 2 minutes followed by 35 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and a final period of 72°C for 42 seconds. The PCR amplification used an Applied Biosystems® 7500 Real-Time PCR machine (Applied Biosystems, CA, USA). Positive control and negative control (without cDNA template) were included with each amplification reaction. The resulting data was analyzed using the software provided by Applied Biosystems based on the Tm and Ct amplification plot values. The positive results were re-confirmed by visualization on 2% agarose gel electrophoresis staining with ethidium bromide.
Serotype matching between dengue patients and mosquitoes
To evaluate the relationship of DENV infection between patients and mosquitoes in the same residential area, DENV serotypes from 75 patients’ plasma were matched with those from mosquitoes collected from the collection clusters around each patient’s residence ( S1 Fig ). Where the patient’s plasma had at least one serotype in common with the mosquito pool, a match was assigned. On the other hand, if there was no DENV serotype in common between the patient’s plasma sample and the mosquito pool, or if the latter was negative for DENV, this was assigned as a serotype mismatch.
DENV envelope gene sequencing
Full-length E gene PCR products were directly amplified from patient plasma and mosquito pooled samples to avoid genetic changes due to the culture process. The viral RNA was extracted from the samples using QIAamp Viral RNA Mini kit (Qiagen, Germany) according to the manufacturer’s instructions. cDNA fragments covering the E gene of DENV were synthesized by specific primers of each serotype as previously described [ 22 ] using SuperScript III first-strand synthesis system (Invitrogen, USA) according to the manufacturer’s instructions and stored at -20°C until use. Two overlapping fragments (F and L fragments) of the E gene were amplified primers as previously described [ 22 ], using PrimeStar GXL DNA polymerase (Takara, Japan) according to the manufacturer’s instructions. The PCR amplification cycles were performed with a step of initial DNA denaturation at 98°C for 5 minutes followed by 35 cycles of denaturation at 98°C for 10 seconds and annealing at 55°C for 15 seconds and extension at 68°C for 90 seconds. The specific amplicons were purified from 0.8% agarose gel by gel cutting and extraction using the QIAquick gel extraction kit (Qiagen, Germany). Purified amplicons of each sample were subjected to Sanger sequencing by using cycle sequencing reactions and dye terminator methodologies of Macrogen company (Macrogen, Seoul, Korea) using four overlapping primers for each serotype as previously described [ 22 ].
The phylogenetic analysis based on the nucleotide sequence of the envelope gene-encoding region of the DENV was constructed and used to elucidate the origins of disease outbreaks. The E gene sequences obtained in this study were aligned with other DENV sequences from those previously isolated in Thailand and neighboring countries and from other regions of the world available in the GenBank database (www.ncbi.nlm.nih.gov) using ClustalW in Bioedit. Maximum-likelihood trees were constructed in MEGA X using 35 reference sequences for DENV-1, 65 sequences for DENV-2, and 40 sequences for DENV-4 ( S2 Table ) with the best-fit nucleotide substitution model (TN93+G). Bootstrap values were done with 1000 replications. Genotypic classification of DENV-1, -2, and -4 followed Goncalvez et al. [ 23 ], Twiddy et al. [ 24 ] and AbuBakar et al. [ 25 ] respectively.
The full-length E gene sequences obtained in this study were deposited in GenBank and granted accession numbers MT524489 to MT524513 ( Table 1 ).
The human samples in the present study were obtained from a dengue case-control study [ 8 ]. The present study was approved by the Khon Kaen University Ethics Committee for Human Research (KKUEC, no. HE611454). Informed consent was obtained in writing from all participants before sample collection.
DENV serotype distribution in human samples
A total of 103 of 292 plasma samples were positive for DENV ( S1 Fig ). Serotype identification revealed all four DENV serotypes circulated in these samples. Of the 103 DENV positive samples, single DENV serotypes indicating mono-infection were detected in 79 (77%) samples, while multiple serotypes indicating co-infection were found in 24 (23%) samples. The predominant DENV serotypes in 2016 and 2018 were DENV-4 (74%) and DENV-3 (53%), respectively, whereas in 2017 DENV-1, -3, and DENV-4 were found with equal prevalence ( Table 2 ).
DENV serotype distribution in mosquito samples
A total of 902 female Aedes individual mosquitoes (contained in 192 pools) were captured during the study period ( Table 2 ). The predominant mosquito species was Ae . aegypti (98.8%) followed by Ae . albopictus (1.2%) ( Table 2 ). One hundred forty-nine (16.5%) of the 902 female mosquitoes were positive for DENV. Serotyping of individual mosquito head-thoraces revealed that the predominant DENV serotypes were DENV-3 (73%) in 2016, DENV-2 and DENV-3 (38% of each serotype) in 2017, and DENV-2 (94%) in 2018 ( Table 2 ). Of the 149 DENV positive individual mosquito samples, single DENV serotypes indicating mono-infection were detected in 105 (70%) samples, while multiple serotypes indicating co-infection were found in 44 (30%) samples.
Relationships of DENV infection between dengue patients and mosquitoes from the same residence area
Out of 103 DENV-positive patients, mosquito collection was done only in 75 patient’s residential areas. DENV serotypes in plasma of those patients were compared with DENV serotypes from the infected mosquitoes from their residences. Of the 75 plasma samples, 14 (19%) matched a serotype from the DENV positive mosquitoes samples ( Fig 2 ). Among 61 (81%) mismatched samples, 10 (13%) had a different DENV serotype in the corresponding mosquito samples, while 51 (68%) corresponded to DENV negative mosquito samples. The molecular characteristics of 24 pairs out of 75 paired samples are presented in S3 Table .
In 14 households (19%), the serotype in patients and mosquitoes matched each other, i.e. were the same (blue); in 10 households (13%), the serotypes in patients and mosquitoes were the different (orange); and in 51 households (68%) patients were DENV-positive whereas mosquitoes were DENV-negative (grey). More details of the molecular characteristics of DENV-positive patients and mosquitoes are provided in S3 Table .
Sequencing of the E gene was successful for 25 samples, 21 from patients, and 4 from mosquito pools. They contained eight DENV-1, six DENV-2, and 11 DENV-4 sequences ( Table 1 ).
Phylogenetic analysis of DENV-1 following Goncavez’s classification [ 23 ] showed that all eight DENV-1 strains were genotype I ( Fig 3 ), and that all eight were from the same clade. Seven strains were closely related to the DENV-1 genotype I isolated from Taiwan in 2015, the other (D1H/4405-08/17) being closely related to DENV-1 isolated between 2016 and 2018 from other Asian countries including Singapore in 2016, China in 2017–18 and Myanmar in 2017 ( Fig 3 ).
Maximum-likelihood tree of the E gene sequences of DENV-1 was generated in the MEGA X program using the TN93 + G model with 1000 bootstrap replications. Bootstrap support values exceeding 80% are shown on branch nodes. The virus names of each sequence retrieved from the NCBI database are labeled as follows: GenBank accession number/country/isolated year. Samples collected in the present study are in red bold font. Annotation on the right denotes DENV genotype.
Of the six DENV-2 strains, two were classified as Asian I and four as Cosmopolitan genotypes [ 24 ] ( Fig 4 ). In turn, the Asian I genotype was clustered into two clades. The Asian I strain D2H/4005-02/16 was closely related to the DENV-2 Thai strain isolated in 2013, while the other (D2H/4603-11/18) was closely related to one isolated in Thailand in 2017, and in China in 2015 ( Fig 4 ). The four Cosmopolitan strains also clustered into two distinct clades. Three Cosmopolitan strains (D2H/4603-10/18, D2H/9918-02/18, and D2H/9918-09/18) were closely related to ones isolated in Thailand and China during 2016–2017, whereas the remaining strain (D2H/4405-26/18) was closely related to DENV-2 isolated from Singapore in 2016 ( Fig 4 ).
Maximum-likelihood tree of the E gene sequences of DENV-2 was generated in the MEGA X program using the TN93 + G model with 1000 bootstrap replications. Bootstrap support values exceeding 80% are shown on branch nodes. The virus names of each sequence retrieved from the NCBI database are labeled as follows: GenBank accession number/country/isolated year. Samples collected in the present study are in bold red font. Annotation on the right denotes DENV genotype.
Phylogenetic analysis of DENV-4 following AbuBakar et al. [ 25 ] revealed that all 11 DENV 4 strains obtained in this study belonged to genotype I ( Fig 5 ). However, they were clustered into four distinct clades ( Fig 5 ). Seven strains from 2016 were closely related to one isolated in Taiwan in 2015 and in Thailand in 2017, whereas two strains collected during 2016 (D4H/4507-15/16 and D4H/4507-18/16) were closely related to one isolated in Taiwan in 2013. The two remaining strains were closely related to ones found in Thailand and neighboring countries, e.g. Singapore in 2014 and China in 2015 to 2016 ( Fig 5 ).
Maximum-likelihood tree of the E gene sequences of DENV-4 was generated in the MEGA X program using the TN93 + G model with 1000 bootstrap replications. Bootstrap support values exceeding 80% are shown on branch nodes. The virus names of each sequence retrieved from the NCBI database are labeled as follows: GenBank accession number/country/isolated year. Samples collected in the present study are in red bold font. Annotation on the right denotes DENV genotype.
Recent evidence indicates that the dengue outbreak situation in northeastern Thailand has become critical, causing annually peaks of the disease associated with increasing morbidity and mortality humans [ 2 ]. So far, there is limited information on the molecular characteristics of DENV in this region. The present study is the first to describe the relationship of DENV infection between humans and mosquitoes, and molecular characteristics of DENV in northeastern Thailand.
Approximately one third (35.3%) of dengue-suspected patients recruited in this study had a confirmed DENV infection, indicating a high burden of dengue in 2016 and 2018 ( Table 2 ). All four DENV serotypes were found co-circulating during the three years of the study. These findings are similar to those from other regions of Thailand during the same period [ 11 , 16 ]. The most prevalent serotypes in 2016 and 2018 were DENV-4 and DENV-3 respectively, whereas, DENV-1, -3, -4 were equally prevalent in 2017. With the lack of molecular data on DENV in northeastern Thailand, we were not able to compare this serotype profile to those of previous years. During the three years (June 2016 to December 2018) of our study, Thailand faced dengue outbreaks in 2016 and 2018 in several provinces [ 26 , 27 ]. Fewer dengue cases in 2017 were also reported by national surveillance (The Office of Disease Prevention and Control Region, ODPC). In addition, a previous report also showed that, prior to our study, during 2013–2015, the yearly distribution of DENV serotypes in Thailand included a high prevalence of DENV-2 [ 27 ]. This could have resulted in fewer DENV-2 human cases in 2017 due to recently acquired prior immunity. However, DENV-2 is still present in the collected mosquitoes at the same time, it is common to find all serotypes circulating in vectors or hosts, given the hyperendemicity of DENV in Thailand. The virus can also be directly transmitted to the next vector generation by vertical transmission. Similarly, a study conducted in India observed that the DENV-2 disappeared in 2016 after predominating between 2011–2015 [ 28 ].
Dengue virus detection in mosquitoes during an outbreak has been suggested as a potential tool for early warnin and designing effective vector control strategies [ 29 , 30 ]. We collected Aedes mosquitoes from houses of suspected dengue patients in northeastern Thailand, the most prevalent species was Ae . aegypti (98.8%) followed by Ae . albopictus (1.2%) ( Table 2 ). Our study found 16.5% (149/902) of mosquitoes were DENV-positive. Similarly, in southern Thailand, 16% of field-caught Aedes aegypti and 36.2% of Aedes albopictus were infected with DENV during the early rainy season of 2005 [ 31 ]. Several studies have described DENV infection in field-caught Aedes mosquitoes, with various infection rate including 10.8% in Brazil [ 32 ], 12.7% and 62% in Colombia [ 33 , 34 ], 2.8% in Philippines [ 35 ]. These findings suggested that the infection rate of virus in field-caught mosquitoes is highly variable, possibly depending on the design and conditions of each study [ 36 , 37 ]. Serotyping results illustrated that all four serotypes were circulating in the mosquitoes in this region. DENV-3 and DENV-2 were predominant in 2016 and 2018 respectively, while both serotypes were most prevalent in 2017.
Moreover, we found co-infection of different DENV serotypes: 23% and 30% in human and mosquito samples respectively. This finding is consistent with several studies which reported the concurrence of DENV infection in the same patient with multiple serotypes at different percentages ranging from 3% to 43% [ 38 – 41 ]. Similar observations of co-infection with multiple DENV serotypes have been reported in other countries and also in Thailand [ 31 , 42 , 43 ]. All four serotypes circulating in the region have been found in combinations within single patients [ 41 ]. Several factors might be involved in co-infection including: i) the multiple feeding behavior of Ae . aegypti , which feeds more than once during its genotropic cycle [ 31 , 41 , 42 ], ii) the high attack rates of cases during dengue epidemics which may result in many infections with multiple serotypes in humans, and also provide opportunities for mosquitoes to become infected with multiple serotypes [ 42 ], iii) transovarial transmission of dengue viruses in mosquitoes [ 31 ], iv) asymptomatic cases as a source of infection in mosquitoes [ 42 ].
Results from the present study, show the difference of serotype distribution in hosts and vectors by year, and suggest that mosquitoes may serve as a maintenance mode for the virus in the environment. When the immunity level in human population to a certain serotype decreases, a burst of infections may follow [ 32 ]. Moreover, our results are consistent with previous studies which showed specific DENV serotypes building up in mosquitoes, then becoming predominant in humans in the subsequent year [ 32 , 33 ].
Although several studies have evaluated the presence of DENV-infected Aedes mosquitoes in all four regions of Thailand including central [ 44 – 49 ], southern [ 31 ], northern [ 47 , 50 ], and northeastern [ 51 ], our study is the first to target the residential area of DENV-infected patients within the onset of symptoms in northeastern Thailand. The DENV serotypes of patient samples were compared with mosquito samples collected from the patient’s residential area within a 100-meter radius. About one fifth (19%) of the patients had a serotype matching the serotype in the mosquitoes collected, while remaining 81% were serotype mismatched, consistsing of 13% with a different serotype and another 68% whose corresponding mosquitoes were DENV negative ( Fig 2 ). This result suggests that transmission outside the residence may be important. Since most of the cases were children and adults ( S4 Table ) who have spent day time outside their residence (e.g., school, worksite/office), it is possible that they were exposed there to infectious mosquitoes. This finding is consistent with previous studies conducted in Brazil and Philippines that found differences in DENV serotypes between patients and mosquitoes in the same home [ 32 , 35 ]. Previous reports suggest that DENV infection often occurs elsewhere, other than the home, e.g. workplace, school or college, temple or farm field [ 9 , 35 , 52 ]. Numerous factors may influence these results including: i) asymptomatic infections with different DENV serotypes in members of the same household, ii) transovarial transmission of DENV in the household involving serotypes to which the residents have recent prior immunity [ 35 ]. It is essential to note that spending time in an area or work environment during the day that is infested with Aedes mosquitoes may increase the risk of dengue infection. This supports the idea that DENV transmission is probably driven by the movement of infected humans [ 35 ].
The phylogenetic analysis revealed that all eight DENV-1 strains were classified as genotype I ( Fig 3 ), which mainly circulates in Asian countries especially Thailand [ 11 , 16 , 53 – 56 ]. Interestingly, these DENV-1 strains were not grouped within the same clade as Thai strains isolated during the same period in northern [ 11 ] and southern [ 16 ] regions. Rather, those other strains were closely related to strains from other Asian countries during 2015–2018, such as a Taiwanese strain isolated from a traveler who returned from Thailand in 2015, a Singaporean strain in 2016, a Myanmar strain in 2017, and Chinese strains in 2017–2018. This indicates that DENV-1 circulating strains in northeastern Thailand are likely to have been imported from other Asian countries ( Fig 3 ).
We observed DENV-2 Asian I and Cosmopolitan genotypes co-circulating in this region ( Fig 4 ), as they commonly do elsewhere in Asia, especially Southeast Asia (SEA) [ 11 , 14 , 16 , 57 – 59 ]. The Asian I strains were clustered into different 2 clades. Interestingly, the strain D2H/4005-02/16 was closely related to the Taiwanese strain isolated from two travelers returning from Thailand in 2010 [ 60 ] and 2013 [ 61 ], suggesting they were likely to have circulated for at least 3–4 years in northeastern Thailand. Moreover, the remaining strain D2H/4603-11/18 was closely related to one isolated in Guangzhou (China) during 2019, suggesting they have a common ancestor. Therefore, northeastern Thailand seems to be a potential source of dengue transmission to other parts of Asian countries ( Fig 4 ). The four Cosmopolitan strains were clustered into two clades. Three strains (D2H/4603-10/18, D2H/9918-02/18, and D2H/9918-09/18) were closely related to ones isolated during the same period in the central and northern regions of the country, and in other Asian countries such as China and Singapore ( Fig 4 ). This demonstrates that the DENV-2 northeastern strains were not only transferred from other parts of Thailand in the same period but also were persistent in this region ( Fig 4 ).
All 11 DENV-4 strains were clustered into genotype I, which commonly circulates and was recently found in Thailand [ 11 , 16 , 62 ] ( Fig 5 ). Although all strains were grouped into this single genotype, they were clustered into 3 different clades ( Fig 5 ). Nine strains were closely related to ones isolated in Thailand, Taiwan, and China during 2015–2016, with the remaining two (D4H/4507-15 and D4H/4507-18) being closely related to one isolated in Taiwan in 2013 from a traveler who returned from Thailand ( Fig 5 ). This demonstrates that DENV-4 strains circulating in this region were not only transferred from other regions of Thailand in the same period but also imported from Southeast Asian countries ( Fig 5 ). Interestingly, all seven DENV-4 northeastern strains collected during 2016 were closely related to the LC410202-03/Thailand/2017 strain which was isolated from the central region of Thailand in 2017 [ 11 ]. These results suggest that the northeastern region is a locus of active circulation of DENV genotypes, with the potential to export them not only to other regions of the country but also other countries around the world especially SEA through the movement of infected persons and mosquitoes [ 10 ].
Our study has some limitations, in particular in terms of generalizability (extrapolation). First, we defined inconsistent serotyping results as a mismatch, which included both the occurrence of different genotypes, and an infection linked to negative sample. Since negative was assigned as mismatched, it could increase the chance of mismatch due to the low titer of DENV in mosquitoes. Second, our mosquito collection focused on households, but transmission could occur in other locations where patients spend their daytime such as schools, work offices, and community centers [ 8 ]. Last, we were unable to sequence any DENV-3 viruses. Since our study performed E gene sequencing directly from the sample without virus isolation, to avoid working on passaged viruses, this gap might be attributed to low viral titers which were insufficient for conventional PCR amplification. Shorter fragments of PCR products have been suggested to be more suitable for sequencing [ 10 ], and this is being considered for our future studies.
In conclusion, our study provides the first data on molecular characteristics of DENV in northeastern Thailand during 2016 to 2018. Our data confirmed the hyperendemicity of all four DENV serotypes in northeastern Thailand. The low proportion of matches between DENV serotypes in patients and mosquitoes from the patients’ residence areas suggests that non-residential transmission may be important. Our phylogenetic analysis revealed that the virus circulating in this region shared high homology with the virus from other regions of Thailand and Southeast Asian countries in the same period, as well as with persisting strains that originated in Thailand and other southeast Asian countries. These findings indicated that human movement has an important role in infection and disease transmission in the region. Taken together, our findings highlight that vector control, and early warning based on DENV detection in vectors, are important strategies for the prevention of dengue epidemics in northeastern Thailand. Continuous molecular surveillance of DENV in northeastern Thailand to observe the virus replacement and also better understand dengue transmission dynamics in this region is required.
S1 fig. the diagram of the number of the samples used in this study..
S1 Table. Primer used for envelope (E) gene fragment amplification and sequencing in the present study.
S2 Table. Published sequences used in this study for phylogenetic analysis.
(accessed from GenBank, the National Centre for Biotechnology Information https://www.ncbi.nlm.nih.gov/genbank/ ).
S3 Table. Molecular characteristic of dengue virus between patient and adult mosquitoes collected from patient’s resident area.
The molecular characteristics of 24 pairs of DENV positive samples were shown in this table. Gray row label represents serotype matched between patients and mosquitoes from their resident area.
S4 Table. Age of 75 dengue patients who have mosquitoes inside the resident area.
We would like to thank the attending physicians, nurses, and clinical laboratory staff at eight district hospitals in Khon Kaen, Roi Et, Kalasin, and Mahasarakham provinces for their kind assistance in recruiting potential participants and logistic support. We would like to acknowledge the entomology team, Office of Disease Prevention and Control, Region 7, Khon Kaen for mosquito collections. The officers of Mahidol-Osaka Center for Infectious Diseases (MOCID), Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand are thanked for their technical support. We are grateful to all members of Assoc. Prof. Sunchai Payungporn lab group (SP lab), Chulalongkorn University for their kind assistance and support in data analysis of DENV sequence. Our special thanks to Thawaree Nakpook, Le Thi Bao Chi and Benedicte Fustec for their valuable suggestions and assistance in the laboratory.
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- v.12(7); 2018 Jul
Dengue infection in India: A systematic review and meta-analysis
1 Department of Epidemiology, National Institute of Epidemiology, Chennai, Tamil Nadu, India
Manoj V. Murhekar
2 School of Public Health, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
Krishnendu sukumaran, anandan anandaselvasankar.
3 Campbell Collaboration, New Delhi, India
Sanjay M. Mehendale
4 Division of Epidemiology and Communicable Diseases, Indian Council of Medical Research, New Delhi, India
All relevant data are within the paper and its Supporting Information files.
Dengue is the most extensively spread mosquito-borne disease; endemic in more than 100 countries. Information about dengue disease burden, its prevalence, incidence and geographic distribution is critical in planning appropriate control measures against dengue fever. We conducted a systematic review and meta-analysis of dengue fever in India
We searched for studies published until 2017 reporting the incidence, the prevalence or case fatality of dengue in India. Our primary outcomes were (a) prevalence of laboratory confirmed dengue infection among clinically suspected patients, (b) seroprevalence in the general population and (c) case fatality ratio among laboratory confirmed dengue patients. We used binomial–normal mixed effects regression model to estimate the pooled proportion of dengue infections. Forest plots were used to display pooled estimates. The metafor package of R software was used to conduct meta-analysis.
Of the 2285 identified articles on dengue, we included 233 in the analysis wherein 180 reported prevalence of laboratory confirmed dengue infection, seven reported seroprevalence as evidenced by IgG or neutralizing antibodies against dengue and 77 reported case fatality. The overall estimate of the prevalence of laboratory confirmed dengue infection among clinically suspected patients was 38.3% (95% CI: 34.8%–41.8%). The pooled estimate of dengue seroprevalence in the general population and CFR among laboratory confirmed patients was 56.9% (95% CI: 37.5–74.4) and 2.6% (95% CI: 2–3.4) respectively. There was significant heterogeneity in reported outcomes (p-values<0.001).
Identified gaps in the understanding of dengue epidemiology in India emphasize the need to initiate community-based cohort studies representing different geographic regions to generate reliable estimates of age-specific incidence of dengue and studies to generate dengue seroprevalence data in the country.
Dengue fever, an extensively spread mosquito-borne disease, is endemic in more than 100 countries. Information about dengue disease burden, its prevalence and incidence and geographic distribution is necessary to guide in planning appropriate control measures including the dengue vaccine that has recently been licensed in a few countries. We performed a systematic review and meta-analysis of published studies in India on dengue. The overall estimate of the prevalence of laboratory confirmed dengue infection based on testing of more than 200,000 clinically suspected patients from 180 Indian studies was 38.3%. The pooled estimate of dengue seroprevalence in the general population and CFR among laboratory confirmed dengue patients was 56.9% and 2.6% respectively. There were no community-based studies reporting incidence of dengue. Our review also identified certain knowledge gaps about dengue epidemiology in the country. Identified gaps in the understanding of dengue epidemiology in India emphasize the need to initiate community-based cohort studies representing different geographic regions to generate reliable estimates of age-specific incidence of dengue and studies to generate dengue seroprevalence data in the country.
Dengue is the most extensively spread mosquito-borne disease, transmitted by infected mosquitoes of Aedes species. Dengue infection in humans results from four dengue virus serotypes (DEN-1, DEN-2, DEN-3, and DEN-4) of Flavivirus genus. As per the WHO 1997 classification, symptomatic dengue virus infection has been classified into dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The revised WHO classification of 2009 categorizes dengue patients according to different levels of severity as dengue without warning signs, dengue with warning signs (abdominal pain, persistent vomiting, fluid accumulation, mucosal bleeding, lethargy, liver enlargement, increasing haematocrit with decreasing platelets) and severe dengue [ 1 , 2 , 3 ]. Dengue fever is endemic in more than 100 countries with most cases reported from the Americas, South-East Asia and Western Pacific regions of WHO [ 1 ]. In India, dengue is endemic in almost all states and is the leading cause of hospitalization. Dengue fever had a predominant urban distribution a few decades earlier, but is now also reported from peri-urban as well as rural areas [ 4 , 5 ]. Surveillance for dengue fever in India is conducted through a network of more than 600 sentinel hospitals under the National Vector Borne Disease Control Program (NVBDCP) [ 6 ], Integrated Disease Surveillance Program (IDSP) [ 7 ] and a network of 52 Virus Research and Diagnostic Laboratories (VRDL) established by Department of Health Research [ 8 ]. In 2010, an estimated 33 million cases had occurred in the country [ 9 ]. During 2016, the NVBDCP reported more than 100,000 laboratory confirmed cases of dengue [ 6 ]. It is therefore possible that dengue disease burden is grossly under-estimated in India.
High dengue disease burden and frequent outbreaks result in a serious drain on country’s economy and stress on the health systems. In India, case detection, case management, and vector control are the main strategies for prevention and control of dengue virus transmission [ 6 ]. A new dengue vaccine is now available and several vaccines are in the process of development [ 10 , 11 , 12 ]. Information about dengue disease burden, its prevalence, incidence and geographic distribution is necessary in decisions on appropriate utilization of existing and emerging prevention and control strategies. With this background, we conducted a systematic review and meta-analysis to estimate the disease burden of dengue fever in India. We also reviewed serotype distribution of dengue viruses in circulation, and estimated case fatality ratios as well as proportion of secondary infections.
Search strategy and selection criteria
This systematic review is registered in PROSPERO (Reg. No. CRD 42017065625). We searched Medline (PubMed), Cochrane Central, WHOLIS, Scopus, Science Direct, Ovid, Google Scholar, POPLINE, Cost-Effectiveness Analysis (CEA) Registry and Paediatric Economic Database Evaluation (PEDE) databases for articles published up to 2017. The main search terms included incidence, prevalence, number of reported cases, mortality, disease burden, cost of illness, or economic burden of dengue in India. The complete search strategy is described in S1 Appendix . Back referencing of included studies in bibliography was also done to identify additional studies.
The search results were initially imported to Zotero software (Version 220.127.116.11) and duplicate records were removed. During title screening, we examined relevant studies from various databases. Our inclusion criterion was studies reporting dengue infection in India, not restricted to setting, design, purpose and population. Titles thus selected were subjected to abstract screening. Studies were considered eligible for further examination in full text if their abstracts reported incidence, prevalence, number of reported cases, mortality or the burden of dengue fever anywhere in India. Studies reporting complications of dengue, serotype details of dengue virus as well as seroprevalence of dengue were also included. Using a pre-designed data extraction form, two reviewers extracted details from selected studies independently. The data, which differed between the reviewers, were resolved by consensus. Information about the year of publication, study setting (hospital/laboratory based, or community-based), study location, study period, laboratory investigations, number of suspected patients tested and positives, age distribution of cases, and details of dengue serotypes were abstracted ( S1 Dataset ).
The primary outcome measures of interest were (a) prevalence (proportion) of laboratory confirmed dengue infection among clinically suspected patients in hospital/laboratory based or community-based studies, (b) seroprevalence of dengue in the general population and (c) case fatality ratio among laboratory confirmed dengue patients. The diagnosis of acute dengue infection among the clinically suspected patients was based on any of the following laboratory criteria: (a) detection of non-structural protein-1 (NS1) antigen, (b) Immunoglobulin M (IgM) antibodies against dengue virus (c) haemagglutination inhibition (HI) antibodies against dengue virus, (d) Real-time polymerase chain reaction (RT-PCR) positivity or (e) virus isolation. Seroprevalence of dengue was based on detection of IgG or neutralizing antibodies against dengue virus. Studies providing prevalence (proportion) of laboratory confirmed dengue infection among clinically suspected patients were classified into (a) hospital/laboratory-based surveillance studies and (b) outbreak investigations or hospital/laboratory-based surveillance studies when the outbreak was ongoing in the area, as mentioned in the original research paper. Studies regarding outbreak investigations considered an increase in number of reported cases of febrile illness in a geographical area, as the criteria for defining an outbreak. The outbreak investigations included one or more of the following activities: active search for case-patients in the community, calculation of attack rates for suspected case-patients, confirmation of aetiology and entomological investigations. For the case fatality ratio, the numerator included reported number of deaths due to dengue and denominator as laboratory confirmed dengue patients.
Our secondary outcomes of interest were the following: (a) proportion of primary and secondary infections among the laboratory confirmed dengue patients. This classification was made based on the information about dengue serology provided in the paper. Primary dengue infection was defined as acute infection, as indicated by qualitative detection of NS1 antigen, and/or IgM or HI antibodies or RT-PCR positivity and absence of IgG antibodies against dengue virus. A case of acute infection as defined above, in presence of IgG antibodies, was considered as secondary dengue infection [ 2 , 13 , 14 ]. Some of the studies used the ratio of IgG to IgM antibodies as the criteria for differentiating primary and secondary infections [ 14 ]; (b) distribution of predominant and co-circulating dengue virus serotypes; (c) proportion of severe dengue infections based on WHO 1997 or WHO 2009 criteria [ 1 , 2 ]. The category of severe dengue infection included patients with DHF and DSS as per the WHO 1997 classification as well as severe dengue infections classified as per the WHO 2009 classification and (d) cost of illness, which included reported direct and indirect costs associated with dengue hospitalization.
Risk of bias
The risk of bias was assessed using a modified Joanna Briggs Institute (JBI) appraisal checklist for studies reporting prevalence data [ 15 ] and essential items listed in the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist [ 16 ]. The criteria for assessing bias primarily included methods for selecting participants, methods for laboratory testing, and outcome variables (Supplementary file S2 Appendix ).
We conducted quantitative synthesis to derive meta-estimates of primary and secondary outcomes (severity of disease and primary/ secondary infections) and qualitative synthesis to describe the serotype distribution and economic burden due to dengue. We followed Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines [ 17 ]. For each study, primary outcomes (prevalence of acute infection, seroprevalence and CFR) were summarized as proportion and their 95% confidence intervals were computed. We used logit and inverse logit transformations for variance stabilization of proportions [ 18 ]. Binomial–Normal mixed effects regression model was used to estimate the pooled proportion of dengue infections. Forest plots were used to display pooled estimates. Heterogeneity was tested using likelihood ratio test. Funnel plots with logit prevalence on x-axis and standard errors on y-axis and Egger’s test were used to evaluate publication bias. Independent variables potentially associated with the prevalence of laboratory confirmed dengue were included as fixed-effects in univariate and multivariate binomial meta-regression models. P <0.05 was considered statistically significant. Sensitivity analysis was carried out by leaving out one study at a time in the order of publication to check for consistency of pooled estimates. Analyses were performed in the R statistical programming language using the ‘metafor’ package [ 19 , 20 ].
Characteristics of included studies
The search strategy initially identified 2,285 articles from different databases. After removal of duplicates, 1,259 articles were considered for title and abstract screening. Seven hundred and forty-six articles were excluded for reasons provided in Fig 1 . Thus, 513 articles were found to be eligible for full-text review. After the review of full-text articles, 233 studies were included for the analysis [ 21 – 253 ]. The details of the studies included in the review are provided in the PRISMA flowchart ( Fig 1 ). None of the studies reported incidence of dengue fever.
Prevalence (proportion) of laboratory confirmed dengue fever.
Of the 233 studies included in the analysis, 180 provided information about proportion of laboratory confirmed dengue cases among clinically suspected patients [ 21 – 200 ]. This included 154 studies conducted in hospital or laboratory setting [ 21 – 174 ] and 26 studies reporting outbreak investigations [ 175 – 200 ]. Of the 154 studies conducted in hospital/ laboratory setting, 40 were conducted when an outbreak was ongoing in the area [ 135 – 74 ]. The diagnosis of acute dengue infection was based on a single assay in 86 studies (IgM antibodies = 68, RT-PCR = 11, HI antibodies = 4, virus isolation = 2, detection of NS1 antigen = 1) and more than one assay in 95 studies.
Case definitions used : Of the 154 studies conducted in hospital settings, WHO or NVBDCP case definitions were used by 39 and 2 studies respectively. The remaining studies used case definitions such as acute febrile illness/acute undifferentiated illness (n = 20), and clinically suspected dengue fever (n = 93). Similarly, of the 26 reported outbreaks, investigators used WHO or NVBDCP case definitions in 7 and 2 settings respectively, whereas acute febrile illness and clinically suspected dengue fever case definitions were used in 5 and 12 settings respectively.
Place and time distribution of studies : Of the 154 studies conducted in hospital setting, 75, 41, 27 and 7 were from north, south, east and western Indian states respectively, whereas 3 studies were from north-eastern states. One study reported data from VRDL network, covering multiple regions in India [ 65 ]. Of the 26 outbreaks, most (10, 38.5%) were reported from Southern states, followed by 9 (34.6%) in the north, 4 (15.4%) in the east, and 3 (11.5%) in the north-eastern Indian states. Most (65, 42.2%) studies conducted in hospital settings were between 2011–2017, while 48 (31.2%) were conducted between 2006–2010 and 41 (26.6%) were conducted before 2006. Eighteen (69.2%) of the 26 outbreaks were reported after 2000.
Of the 180 studies which reported proportion of dengue cases, 74 studies (30%) provided the details of laboratory confirmed cases by month with most (n = 60, 81%) reporting higher dengue positivity between August and November months.
Age distribution of dengue cases : The age distribution of laboratory confirmed dengue patients was available from 52 out of 180 studies. The pooled median age of laboratory confirmed dengue cases in these studies was 22 years ( Fig 2 ). Fifteen (28.8%) studies reported the median age of dengue cases below 15 years.
Estimates of prevalence (proportion) : The overall estimate of the prevalence of laboratory confirmed dengue infection in the random effects model based on testing of 213,285 clinically suspected patients from 180 studies was 38.3% (95% CI: 34.8%–41.8%) ( Fig 3 ). There was a significant heterogeneity in the prevalence reported by the 180 studies (LRT p<0.001). The prevalence of laboratory confirmed dengue infection was higher in studies reporting outbreaks or hospital-based surveillance studies during outbreaks (47.3%, 95% CI: 40.9–53.8) as compared to hospital-based surveillance studies (33.6%, 95% CI: 29.9–37.5) ( S1A and S1B Fig ). The attack rates of suspected dengue case patients were available in 8 out of the 26 outbreak investigations reports. The attack rates ranged between 1.9% and 19.5%.
Error bars indicate 95% confidence intervals. Diamonds show the pooled estimates with 95% confidence intervals based on random effects (RE) model.
In the univariate mixed effect meta-regression model, odds of laboratory confirmation were higher in case of outbreaks or hospital-based studies conducted during outbreaks (OR = 1.8, 95% CI: 1.3–2.4). Studies which used WHO/ NVBDCP case definitions for enrolment of patients also had higher odds of detecting laboratory confirmed dengue compared to studies which used acute febrile illness/ clinically suspected dengue cases as case definitions. Compared to studies conducted before 2006–10, studies conducted between 2011 and 2017 had higher odds of identifying laboratory confirmed patients (OR = 1.33, 95% CI: 0.93–1.9). The odds of laboratory confirmation did not differ by region ( Table 1 ). In the multivariate meta-regression model constructed by including all covariates, case definition (WHO/NVBDCP), type of study (hospital-based surveillance studies conducted during outbreaks or outbreaks) and period of study (prior to 2005 and 2011–2017) were associated with higher odds of dengue cases being laboratory confirmed.
Ref—Reference category; CI—Confidence interval; P*–P value.
Seroprevalence of dengue among healthy individuals
We included 7 studies reporting seroprevalence of dengue based on detection of IgG (n = 5), neutralizing antibodies (n = 1) or HI antibodies (n = 1) against dengue in the analysis [ 201 – 207 ]. These studies, conducted in 12 Indian states [Andaman and Nicobar islands (n = 1), Andhra Pradesh (n = 2), Tamil Nadu (n = 3), Delhi (n = 4), West Bengal (n = 1), and Maharashtra (n = 1)], surveyed 6,551 individuals. The study population surveyed in these studies included healthy children (n = 2), general population (n = 3), blood donors (n = 1) and neighbourhood contacts of dengue confirmed cases (n = 1). The overall seroprevalence of dengue fever based on these studies was 56.9% (95% CI: 37.5–74.4) ( Fig 4 ). The age-specific prevalence of IgG antibodies was available in three studies [ 201 , 204 , 206 ]. There was a significant heterogeneity in the seroprevalence reported by the seven studies (LRT p<0.001). In the 3 studies which provided age specific seroprevalence, by the age of 9 years, 47.6% -73.4% children were reported to have developed IgG or neutralizing antibodies against dengue ( Table 2 ).
Figure in square bracket indicate reference
Case fatality ratios (CFR)
Seventy-seven studies provided information about case fatality ratios; most of them (n = 72, 93.5%) were conducted after 2000. The reported CFRs in these studies ranged from 0% to 25%. There was a significant heterogeneity in the CFRs reported by the 74 studies (LRT p<0.001). Twenty (25.9%) studies reported CFR of 2% or more. Three studies [ 30 , 239 , 195 ] which affected overall meta-estimates due to small denominator and hence were excluded from analysis. The pooled estimate of CFR was 2.6% (95% CI: 2.0–3.4) ( Fig 5 ).
Primary and secondary dengue infection.
A total of 49 studies provided data which enabled classification of laboratory confirmed dengue into primary and secondary dengue infections. The number of patients with acute dengue infections in these studies ranged between 13 and 1752. Only two studies estimated the proportion of secondary infection based on IgG to IgM ratio [ 174 , 237 ]. The prevalence of secondary dengue infection was <10% in 6 studies, 10–25% in 9 studies, 26–50% in 12 studies, 51–75% in 17 studies and >75% in 5 studies. The overall proportion of secondary dengue infection among laboratory confirmed patients was 42.9% (95%CI: 33.7–52.6) ( Fig 6 ).
Proportion of severe cases
Information about severity of dengue was available in 49 studies. Most studies (n = 46, 93.9%) used the WHO 1997 classification while 3 studies used the WHO 2009 classification for dengue severity. The reported proportion of severe dengue cases among laboratory confirmed patients ranged between 1.4% and 97.4%. The overall proportion of severe dengue among laboratory confirmed studies in the random effects model was 28.9% (95% CI: 22.2–36.6) ( Fig 7 ).
Serotypes of dengue virus
Information about dengue serotypes was available in 51 studies. These studies were conducted in 19 Indian states; with a regional distribution of north (n = 28), south (n = 13), east (n = 4), northeast (n = 4), and west (n = 2). Thirty-eight (75%) of the 51 studies reported circulation of more than one serotype. The predominant serotypes reported in these studies were DEN-2 and DEN-1 in the northern region, DEN-2 and DEN-3 in the southern region, and DEN-1 and DEN-2 in the eastern and the western regions. In the four studies reported from the north-eastern region, the predominant serotypes was DEN-3 followed by DEN-1 and DEN-2 serotypes ( Table 3 ).
Key for coloured cell: Blue—One circulating serotype, Yellow—two co-circulating serotypes, Green—three co- circulating serotypes, Orange—four co- circulating serotypes. Numbers mentioned in the cell indicate predominant serotypes, in descending order.
Direct and Indirect cost analysis : An estimate of direct and indirect costs was reported in three studies. The average direct cost per case of dengue ranged between USD 23.5 and USD 161 and the indirect cost was around USD 25 whereas the average cost of hospitalization ranged between USD 186 and USD 432.2 [range 249 -252]. The cost of dengue treatment in the private health sector was two to four times higher than that in the public sector hospitals [ 249 , 253 ].
Economic impact of dengue on National Economy : Three macro-level studies addressed the economic impact of dengue faced by India [ 250 , 251 , 253 ]. It was estimated that the average total economic burden due to dengue in India was USD 27.4 million [ 251 ]. Another study estimated that the total direct medical cost of dengue in 2012 was USD 548 million [ 253 ]. The overall economic burden of dengue would be even higher if the cost borne by individual patients is combined with the society level cost of dengue prevention, vector control, disease control and its management, dengue surveillance as well as the cost of research and development [ 250 , 251 , 253 ].
Publication bias and sensitivity analysis
Funnel plots and Egger’s test revealed no publication bias in the estimates of dengue prevalence in hospital-based surveillance studies, hospital-based surveillance studies during outbreaks and outbreak investigations. CFR estimates, however, showed a significant publication bias, and studies with high prevalence were more likely to be published. In the sensitivity analysis, the estimated pooled proportions were found to be consistent for all study outcomes. ( S3 Appendix )
The present study has estimated the burden of dengue fever based on published literature from India spanning over five decades. Most of the published literature included in the analysis were hospital/ laboratory-based surveillance studies or reports of dengue outbreak investigations. Additionally the published data from VRDL network has been included in the analysis [ 65 , 96 ]. The data from the other two nationally representative surveillance platforms could not be used for the analysis because surveillance data from NVBDCP only reports the number of laboratory confirmed dengue cases, while the IDSP data is not available in the public domain.
There was no community-based epidemiological study reporting the incidence of dengue fever. Our analysis revealed that among the clinically suspected dengue fever patients, the estimated prevalence of laboratory-confirmed dengue infection was 38%. The burden of dengue was also variable in studies conducted in different settings. Our findings indicated that most of the laboratory confirmed dengue cases in India occurred in young adults. Dengue positivity was higher between the months of August and November, corresponding to monsoon and post-monsoon season in most states in India.
In the meta-regression, studies that had used WHO/NVBDCP case definitions and the hospital based studies conducted during outbreaks or studies reporting outbreaks were more likely to have laboratory confirmation of dengue. The odds of laboratory confirmation were also higher among studies conducted during the period of 2011 to 2017, as compared to studies conducted prior to the year 2000.
Information about seroprevalence of dengue in the general population is a useful indicator for measuring endemicity of dengue fever. The dengue vaccine (CYD-TDV) manufactured by Sanofi Pasteur has been introduced in two sub-national programs in Philippines and Brazil [ 254 ] and it has been suggested that vaccine acts by boosting the naturally acquired immunity [ 255 ]. WHO SAGE conditionally recommends the use of this vaccine for areas in which dengue is highly endemic as defined by seroprevalence in the population targeted for vaccination [ 12 , 256 ]. The results of the two vaccine trials and mathematical modelling suggest that optimal benefits of vaccination if seroprevalence in the age group targeted for vaccination was in the range of ≥70% [ 255 , 256 ]. In 2018, WHO revised the recommendation from population sero-prevalence criteria to pre-vaccination screening strategy [ 257 ]. The pooled estimate based on the seven studies conducted in India indicated a dengue seroprevalence of 57%. However, this estimated seroprevalence is not representative of the country, as these studies were conducted only in 12 Indian states, and some had used a convenience sampling method [ 201 ].
The computed pooled estimate of case fatality due to dengue in India was 2.6% with a high variability in the reported CFRs. The CFR estimated in our study was higher than the estimate of 1.14% (95% CI: 0.82–1.58) reported in the meta-analysis of 77 studies conducted globally; in the 69 studies which adopted WHO 1997 dengue case classification, the pooled CFR was 1.1% (0.8–1.6) while the pooled CFR for 8 studies which used the WHO 2009 case definition, the pooled CFR was 1.6% (95% CI: 0.64–4.0) [ 258 ]. Higher CFR observed in our analysis could be due to smaller sample sizes as 14 of the 35 studies that reported CFR of 2.6 or higher had a sample size of 100 or less, while in the remaining 21 studies the denominator ranging between 101 and 400. Also, we only considered laboratory confirmed dengue cases in the denominator for the calculation of CFR. As per the NVBDCP surveillance data, a total of 683,545 dengue cases and 2,576 deaths were reported in India during 2009–2017 giving a CFR of 0.38% [ 6 ]. The lower CFR estimates from NVBDCP data could probably be on account of under-reporting of deaths due to dengue, or inclusion of higher number of mild cases in the denominator [ 259 ]. As per the NVBDCP surveillance data, an average of 28,227 dengue cases and 154 deaths were reported annually during 2009–2012. The number of dengue cases reported increased thereafter, with an average of 100,690 cases per year during 2013–2017. However, the reported number of deaths did not increase proportionately. The information about severity of dengue cases is not available from NVBDCP surveillance data.
The published studies from India indicated circulation of all the four-dengue serotypes, with DEN-2 and DEN-3 being the more commonly reported serotypes. Two third of the studies reported circulation of more than one serotype. Co-circulation of multiple serotypes was particularly evident from the published studies in Delhi. More than two third (16/19) studies from Delhi reported circulation of more than one serotype; and most of the studies conducted in the last 10 years identified co-circulation of more than one serotype [ Table 3 ]. Our review also revealed that more than two-fifth of the laboratory confirmed infections were secondary dengue infections and nearly one-fourth of the cases were severe in nature. Circulation of numerous dengue serotypes is known to increase the probability of secondary infection, leading to a higher risk of severe dengue disease [ 260 ].
Our systematic review has certain limitations. First, our study included only peer-reviewed literature from selected databases and we excluded grey literature which may have provided additional data. Second, most of the studies on disease burden were hospital-based, with no community-based studies estimating incidence. Hospital-based studies do not provide any information about the community level transmission as hospitalization is a function of health-seeking behaviour of the population. In absence of the information about health seeking behaviour provided in these studies, we estimated the prevalence of dengue using number of patients tested in the hospitals as the denominator. Third, the hospital-based studies used varying case definitions and laboratory tests to confirm dengue infection. Fourth, information about the type of health facility (public or private), or residential status of patients (urban or rural), and age was not uniformly reported and hence we did not estimate the dengue prevalence by these variables.
In conclusion, the findings of our systematic review indicate that dengue continues to be an important public health problem in India, as evidenced by the high proportion of dengue positivity, severity and case fatality as well as co-circulation of multiple dengue virus serotypes. Our review also identified certain research gaps in the understanding on dengue epidemiology in the country. There is a need to initiate well planned community-based cohort studies representing different geographic regions of the country in order to generate reliable estimates of age-specific incidence of dengue fever in India. As such studies are cost intensive, a national level survey to estimate age-stratified dengue seroprevalence rates could be an alternative. Such estimates could be used to derive the relative proportions of primary and secondary infections using mathematical models [ 261 ]. Well planned studies in different geographic settings are also needed to generate reliable data about economic burden from India. Although the existing dengue surveillance platforms of NVBDCP, IDSP and VRDL are generating data about dengue disease burden, these systems could be strengthened to also generate data about dengue serotypes, severity, and primary and secondary infection from India.
S1 appendix, s2 appendix, s3 appendix, s1 checklist, funding statement.
The study was funded by the Department of Bio-technology, Govt of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Research shows maternal dengue immunity worsens birth defects caused by Zika virus
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Dengue Research Paper Example
Type of paper: Research Paper
Topic: Dengue Fever , Health , Medicine , World , Control , Vaccination , Disease , Prevention
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History of dengue
Dengue is described by the World Health Organization (n.d.) as a fast emerging pandemic viral disease with worldwide prevalence. The viral disease was initially traced to the epidemics in Java and in Egypt in 1779. Some literatures trace back the virus and its vectorsto the Chin Dynasty. It was hypothesized that the disease occured in the Western hemisphere in the midddle of the 17th century and believed to originate from tropical America. However, some researchers would point out that the accounts of dengue outbreaks were traced back during the 18th and 19th century in Southeast Asia.The dengue outbreaks in the tropical and subtropical areas across all continents were extensive during teh 19th and 20th centuries. The epidemics extend across other subcontinents and islands in the Caribean and South Pacific regions (Schlesinger, 1977). The global dengue epidemics was also caused by the expansion of the global shipping industry in the 18th and 19th centuries that enhanced the spread of the principal mosquito vector to new geographic areas.Moreover, the major epidemics with its severe and fatal form of dengue called the dengue haemorrhagic fever (DHF) happened first in the Southeast Asia.Gubler (2006) ascribed this epidemic as caused by the unplanned urbanization in tropical developing countries, globalization and the lack of effective mosquito control.Dengue has become one of the most serious infectious diseases that affect the tropical urban areas.The impact of dengue in the global community is significant, but there is some difficulty in ascertaining the actual data of the prevalence and incidence of the disease due to the inadequate disease surveillance, underreporting of its incidence and misdiagnosis.
The dengue epidemics can result in massive outbreaks, such as one that occured in Athens and Pireus where about 80% of the population were affected.The prevalence of dengue outbreaks is expected to expand further due to the dynamics of climate change, changes in socioeconomics, globalization, travel and the evolution of the dengue virus.The dengue virus currently has no cure. There is no vaccine available and the disease cannot be treated by any specific antiviral medication. This fact alone can contribute to the growing threat and incidence of dengue worldwide.The incidence of dengue is expected to increase 30 fold across geographic borders with expansions to new countries affecting both the urban and rural areas. The annual incidence of dengue infection is estimated to reach 50 million of cases. The historical patterns of spread of the dengue virus continue to grow. In 2000, the epidemic spread to new areas with increasing incidence to those regions already affected. In 2003 new countries reported incidence of the dengue virus for the first time.It was in 2005 when the high cases of fatal incidence of the dengue outbreak occured in Timor Leste.The fatality rate of dengue infection in India, Indonesia and Myanmar is about 3 to 5 percent and only 1 percent across the other regions. The highest on record of dengue cases was reported in 2007 that occured in Indonesia.Dengue outbreaks in America occured in a cyclical manner, usually every 3 to 5 years. The most commonly affected regions are the Caribbean and Central and South America. The highest reported cases of dengue outbreak was in 2002 in America due to the lack of vector surveillance and control measures (World Health Organization, 2009). The main cause of vector transmission for the dengue virus are the Aedes Aegypti and Aedes Albopictus.The Aedes Aegypti is known to be the main arthropod vector for the transmission of majority of dengue outbreaks.These mosquitoes are highly active on daylight and they predominantly spread across both tropics and subtropic regions.The transmission of the virus is made between people through the mosquito bite. Prevalent in Indonesia, Philippines and Pacific Islands are the transmission of the virus through the Aedes albopicus and Aedes polynesiensis as common vectors of dengue.The dengue outbreaks in the African region shows evidence that it is increasing in size and in frequency. However due to poor surveillance data, dengue reports are not reliable in this region. The reports on dengue outbreaks are often extracted from laboratory confirmation.The origin of the Aedes Aegypti as the primary vector of the dengue virus is traced back to Asia and Africa. They became widespread across regions and transcends geographic boundaries due to the commercial expansions and shipping vessels with humans as the breeding site of the virus. This process allows for the slow but persistent spread of the virus across coastal destinations. Currently, the Centers For Disease Control and Prevention (2014) reported that the global pandemic of dengue accounts to affect about 40% of the world's population with about 2.4 billions of individuals at risk of dengue transmission. The most serious form of dengue infection is dengue hemorrhagic fever (DHF) that accounts to 500,000 cases worldwide and about 22,000 cases of reported deaths annually with children as the most common victims.The dengue fever is now known to be the most important arthropod-borne viral disease in humans in terms of the mortality and morbidity rates (Kurstak, Marusyk, Murphy and Regenmortel, 1990).
The main focus of the Global Strategy for Dengue Prevention and Control is sustainable vector control. This appears to be the most effective preventive method against dengue morbidity and mortality rate by 2020 considering the lack of vaccines against the dengue virus. The World Health Organization advocates the strategic method of Integrated Vector Management which was described to be the most cost effective and rational process of dengue vector control (Murray, Quam and Smith, 2013).There is no vaccine or chemoprophylaxis that are available for dengue infection. This makes anyone living or travelling to dengue endemic areas highly susceptible to getting the condition. The Centers for Disease Control and Prevention advocates preventive measures like using insect repellent, wearing protective clothes with covering on the legs and arms and to find an accommodation with screened windows for travelers. The local community should also cover any standing water which are common breeding sites for mosquitos (Tomashek, Sharp and Margolis, 2015). The main goals and objectives of World Health Organization in its global strategy for dengue prevention and control includes reducing the dengue mortality rate by 50% and morbidity rate by 25% by 2020. According to the World Health Organization, the preventive measures against dengue mortality include the implementation of timely and appropriate clinical management of the condition.This process involves an early clinical diagnosis of the condition, hospital reorganization, intravenous rehydration, and clinical staff training. In the primary and secondary care levels for dengue, which are the stages where the patients are first evaluated, an efficient front line response is necessary in order to prevent hospital admissions. Because dengue is a global health problem, preventive measures across national and international levels must be implemented. The advocacy of the WHO is to implement programs with international collaboration and the harmonization of the regional efforts through strong leadership from the health care workers. A successful clinical outcome is possible with early diagnosis of the infections and through the implementation of an accurate differential diagnosis and a quick laboratory assessment and confirmation of the disease. Emphasized is the need for training of both the medical and non-medical staff who are involved in dengue control to have optimal dengue case management. As part of the dengue prevention program, a surveillance system for dengue should also become part of the national health information system (World Health Organization, 2012). The National Institute of Health (NIH) provides for the prevention program against dengue which includes the use of precautionary measures from being bitten by mosquitoes. It advocates the use of mosquito repellent and the wearing of protective clothing. The National Institute of Allergy and Infectious Disease (NIAID) of the National Institutes of Health (NIH) of the US Department of Health and Human Services are currently funding several dengue research projects that seek to discover the best prevention measure agaist the dengue infection and in the development of a new vaccine against dengue. The Dengue vaccine is currently on the third phase of its clinical trial in Brazil (National Institute of Health, 2016). The development of a vaccine is considered to be the most effective weapon against infectious diseases, such as dengue, according to Healthy People 2020 (2014). The advocacy of developing a vaccine against dengue is in line with the Healthy People 2020 objectives in the prevention of the spread of infectious diseases. Among the important prevention programs against dengue conducted by the World Health Organization include the strengthening of the epidemiological surveillance of dengue, enhancing laboratory networks, strengthening the vector monitoring and control, improve clinical management of patients and strengthen social communications (World Health Organization and Pan American Health Organization, 2014). The Healthy People 2020 advocates the prevention and treatment of infectious diseases such as dengue within the clinical and community levels using evidence based practices. Therawiwat, et al (2005) indicated that the levels of prevention for dengue and its worse form called the dengue hemorrhagic fever in the community should begin within the sub-district health level within villages that are at risk to dengue outbreaks. Community dengue prevention and control programs include long term and integrated community based mosquito control with the responsibility being shared between the central government and health ministries to the community members. Access to health care facilities are also an important preventive and control measure against dengue. Immediate diagnosis and medical intervention can prevent the morbidity and mortality rate from dengue. Since no vaccine and treatment is available to eradicate the disease, the World HealthOrganization and the CDC advocate environmental management and modification to prevent mosquitoes from gaining access to egg laying habitats. Other preventive measures include cleaning and covering domestic water storage or containers on a regular basis, use of insecticides, using personal household protection such as screening the doors and windows to prevent entrance of daylight biting mosquitoes, and enhancing the community participation for a sustainable vector control. The Healthy People 2020 objective includes the eradication of the burden of infectious diseases in the community and the health care settings and these preventive measures and control are currently the best approach against global dengue outbreaks.
Centers For Disease Control and Prevention (2014). Dengue epidemiology. Atlanta, GA: Centers For Disease Control and Prevention. Gubler, D.J. (2006). Dengue/dengue haemorrhagic fever: HIstory and current status. Novartis Found Symp. 277:3-16. Healthy People 2020 (2014). Easy Access Project Eases Immigrants into a New Life – A CDC Preventive Health and Health Services Block Grant Success Story. Healthy People. Retrieved from https://www.healthypeople.gov/2020/healthy-people-in-action/story/easy-access-project-eases-immigrants-new-life-%E2%80%93-cdc-preventive. Kurstak, E., Marusyk, R.G., Murphy, F.A. and Regenmortel, M.H.V. (1990). Virus variability, epidemiology and control. New York: Springer Science Business Media. Murray, N., Quam, M.B. and Smith, A. (2013). Epidemiology of dengue: past, present and future prospects. Clin Epidemiol. 5: 299–309. National Institute of Health (2016). Dengue Vaccine Enters Phase 3 Trial in Brazil. Retrieved from https://www.niaid.nih.gov/news/newsreleases/2016/Pages/DengueBrazilTrial.aspx. Schlesinger, R.W. (1977). Dengue viruses. New York: Springer-Verlag. Therawiwat, M. et al. (2005).Community-based approach for prevention and control of dengue hemorrhagic fever in Kanchanaburi Province, Thailand. Southeast Asian Journal Tropical Medicine Public Health. 36(6):1439-49. Tomashek, K.M., Sharp, T.M. and Margolis, H.S. (2015). Dengue. Centers for Disease Control and Prevention. Retrieved from http://wwwnc.cdc.gov/travel/yellowbook/2016/infectious-diseases-related-to-travel/dengue. World Health Organization (n.d.). Dengue. World Health Organization. Retrieved from http://www.who.int/denguecontrol/en/. World Health Organization (2009). Dengue guidelines for diagnosis, treatment, prevention and control. France: World Health Organization. World Health Organization (2012). Global strategy or dengue prevention and control 2012-2020. France: World Health Organization. World Health Organization and Pan American Health Organization (2014). State of the art in the prevention and control of dengue in the Americas. Washington, D.C: WHO.
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Five more residential areas to be part of Project Wolbachia to curb dengue
SINGAPORE – From early 2024, five more residential areas will be covered by a dengue-curbing programme that releases lab-grown male mosquitoes to suppress the population of disease-carrying mozzies.
Project Wolbachia, as the programme is called, will be expanded to Bukit Merah-Telok Blangah, Clementi-West Coast, Commonwealth, Holland and the Marine Parade-Mountbatten areas, after positive results from ongoing field studies.
The male Wolbachia bacteria-carrying Aedes aegypti mosquitoes will be released to those residential sites from the first quarter of 2024, covering 480,000 households, up from 350,000 currently.
This means that 35 per cent of all homes in Singapore – including HDB and landed estates – will be covered under the programme.
Announcing the expansion of the programme on Nov 21, Senior Parliamentary Secretary for Sustainability and the Environment Baey Yam Keng, who was speaking at the International Vector-Borne Diseases Conference, said: “In the last four years, residents in areas with at least one year of releases were up to 77 per cent less likely to be infected with dengue.”
Those areas include Tampines and Yishun. Thirteen residential areas have been covered under the programme.
The National Environment Agency (NEA) added that the five new locations were chosen based on historical dengue risk, the Aedes mosquito population, and the agency’s capacity for producing and releasing male Wolbachia-Aedes mosquitoes.
Associate Professor Ng Lee Ching, group director of NEA’s Environmental Health Institute, noted that as a sizeable number of dengue cases in study sites were isolated and not linked to clusters, infected individuals could have acquired dengue elsewhere rather than within the release sites.
But while the programme has led to the Aedes mosquito population falling by more than 90 per cent in some areas, it has been observed that Project Wolbachia’s impact on mosquito population and dengue varied across sites and years, added Mr Baey at the conference.
The three-day conference from Nov 21 to 23, held at the National University of Singapore (NUS), and led by the NUS Yong Loo Lin School of Medicine and the university’s Department of Biological Sciences, convenes renowned entomologists, vector biologists and virologists who will discuss developments in vector-borne diseases.
NEA is also using data analytics and artificial intelligence as a predictive tool to adjust the number of male mosquitoes at each site.
“This helps to optimise our deployment strategy and reduce the number of male mosquitoes needed to support dengue suppression in these areas,” said Mr Baey.
In his presentation on Singapore’s dengue control efforts, Dr Chong Chee Seng from the Environmental Health Institute said differences in the way buildings are designed and structured also affect the release of Wolbachia-Aedes mosquitoes.
NEA also plans to develop an additional source that can supply more Wolbachia-carrying mosquitoes, to increase the programme’s capacity as it expands. Currently, the mosquitoes are supplied by the agency and Verily Life Sciences.
NEA said it will engage the industry on this matter.
Currently, about seven million Wolbachia-Aedes mosquitoes are produced every week. With the expansion, 11 million mozzies will be produced.
Project Wolbachia started in 2016. Under the programme, non-biting male mosquitoes, infected with the Wolbachia bacteria, are released across the city to mate with uninfected females, producing eggs that do not hatch, to reduce reproduction of their kind.
As at May, more than one million residents have benefited from the programme.
While Project Wolbachia is a novel solution, it is not a silver bullet to prevent dengue outbreaks, said NEA.
“The presence of Aedes mosquitoes, ongoing circulation of multiple dengue virus serotypes, and low population immunity will continue to challenge dengue control efforts,” noted the agency, reminding residents to continue removing stagnant water and take steps to prevent getting bitten.
So far, more than 8,740 dengue cases have been recorded in 2023, around a quarter of the 32,325 cases recorded in the whole of 2022, which saw a major outbreak.
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- Notifiable diseases: causative agents reports for 2023
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NOIDs causative agents: week 46 (week ending 19 November 2023)
Updated 20 November 2023
© Crown copyright 2023
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Laboratories in England have a statutory duty to notify the UK Health Security Agency ( UKHSA ) of the identification of the following causative agents:
- Bacillus anthracis
- Bacillus cereus
- Bordetella pertussis
- Borrelia spp
- Brucella spp
- Burkholderia mallei
- Burkholderia pseudomallei
- Campylobacter spp
- Carbapenemase-producing Gram-negative bacteria
- Chikungunya virus
- Chlamydophila psittaci
- Clostridium botulinum
- Clostridium perfringens
- Clostridium tetani
- Corynebacterium diphtheriae
- Corynebacterium ulcerans
- Coxiella burnetii
- Crimean-Congo haemorrhagic fever virus
- Cryptosporidium spp
- Dengue virus
- Ebola virus
- Entamoeba histolytica
- Escherichia coli O 157
- Francisella tularensis
- Giardia lamblia
- Guanarito virus
- Haemophilus influenzae (invasive)
- Hanta virus
- Hepatitis A
- Hepatitis B
- Hepatitis C
- Hepatitis D
- Hepatitis E
- Influenza virus
- Junin virus
- Kyasanur Forest disease virus
- Lassa virus
- Legionella spp
- Leptospira interrogans
- Listeria monocytogenes
- Machupo virus
- Marburg virus
- Measles virus
- Mpox (monkeypox) virus
- Mumps virus
- Mycobacterium tuberculosis complex
- Neisseria meningitidis
- Omsk haemorrhagic fever virus
- Plasmodium falciparum
- Plasmodium knowlesi
- Plasmodium malariae
- Plasmodium ovale
- Plasmodium vivax
- Polio virus
- Rabies virus
- Rickettsia spp
- Rift Valley fever virus
- Rubella virus
- Sabia virus
- Salmonella spp
- SARS coronavirus
- Shigella spp
- Streptococcus group A (invasive)
- Streptococcus pneumoniae (invasive)
- Varicella zoster virus
- Variola virus
- Vibrio cholerae
- West Nile virus
- Yellow fever virus
- Yersinia pestis
Statutory notifications of causative agents, grouped by root organism
Totals for the current week compared to the previous 5.
Carbapenemase-producing Enterobacterales ( CPE )
For all Carbapenemase-producing Gram-negative organisms, the reports are de-duplicated by first mention of organism species and resistance mechanism by person in a rolling 52-week period.
Note: the numbers presented here do not include specimens that have been referred to the AMRHAI Reference Unit.
Other carbapenemase-producing Gram-negative organisms
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