literature review of dengue fever

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• Published: 24 February 2023

Epidemiology, biology, pathogenesis, clinical manifestations, and diagnosis of dengue virus infection, and its trend in Ethiopia: a comprehensive literature review

• Biruk Zerfu   ORCID: orcid.org/0000-0002-2406-5971 1 , 2 ,
• Tesfu Kassa 2 &
• Mengistu Legesse 2  

Tropical Medicine and Health volume  51 , Article number:  11 ( 2023 ) Cite this article

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Dengue fever is a dengue virus infection, emerging rapidly and posing public health threat worldwide, primarily in tropical and subtropical countries. Nearly half of the world's population is now at risk of contracting the dengue virus, including new countries with no previous history-like Ethiopia. However, little is known about the epidemiology and impact of the disease in different countries. This is especially true in countries, where cases have recently begun to be reported. This review aims to summarize epidemiology, biology, pathogenesis, clinical manifestations, and diagnosis of dengue virus infection and its trend in Ethiopia. It may help countries, where dengue fever is not yet on the public health list-like Ethiopia to alert healthcare workers to consider the disease for diagnosis and treatment. The review retrieved and incorporated 139 published and organizational reports showing approximately 390 million new infections. About 100 million of these infections develop the clinical features of dengue, and thousands of people die annually from severe dengue fever in 129 countries. It is caused by being bitten by a dengue virus-infected female mosquito, primarily Aedes aegypti and, lesser, Ae. albopictus . Dengue virus is a member of the Flavivirus genus of the Flaviviridae family and has four independent but antigen-related single-stranded positive-sense RNA virus serotypes. The infection is usually asymptomatic but causes illnesses ranging from mild febrile illness to fatal dengue hemorrhagic fever or shock syndrome. Diagnosis can be by detecting the virus genome using nucleic acids amplification tests or testing NS1 antigen and/or anti-dengue antibodies from serum, plasma, circulating blood cells, or other tissues. Dengue cases and outbreaks have increased in recent decades, with a significant public health impact. Ethiopia has had nearly annual outbreaks since 2013, devastating an already fragmented health system and economy. Standardization of medication, population-level screening for early diagnosis and prompt treatment, and minimization of mosquito bites reduce overall infection and mortality rates.

Introduction

Dengue fever (DF) is a rapidly emerging acute febrile disease, with potentially fatal complications, of public health concern worldwide, mainly in tropical and subtropical regions [ 1 , 2 ]. The term ‘dengue fever’ was derived from the Swahili word ‘Ka-dinga pepo’ synonymous with ‘cramp-like seizure’. In 1787, Benjamin Rush coined the term as “break-bone fever” because of the symptoms of myalgia and arthralgia involvement during epidemic reports in Philadelphia in 1780 [ 3 ]. DF epidemics were first recognized clinically in the 1780s in Asia, Africa, and North America at about the same time [ 4 ]. Dengue virus (DENV) that is known to cause the full range of DF diseases worldwide comprises four independent but antigen-related serotypes (DENV1-4) and belongs to the genus flavivirus of the family  Flaviviridae [ 5 ]. The virus is a positive sense single-stranded encapsulated RNA virus and comprises virus-encoded three structural proteins, namely, nucleocapsid or core (C) protein, membrane-associated (M) protein and envelop (E) glycoprotein and seven non-structural (NS) proteins [ 3 ]. It is transmitted to humans by the bite of infected female Aedes mosquitoes [ 5 , 6 ].

The DF disease ranges from mild to undifferentiated fever illness to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [ 7 , 8 ]. For management of cases, in 2009 WHO revised and classified DF disease into uncomplicated DF and severe DF, though the 1997 WHO classification that classified DF into undifferentiated fever, DF and DHF is still in use [ 8 , 9 ]. The main clinical manifestations of each categories would be continuous high fever lasting 2–7 days, haemorrhage—manifested by petechiae, epistaxis, positive tourniquet test or thrombocytopenia, and plasma leakage shocks—manifested by hemoconcentration (hematocrit above 20%), pleural effusion and ascites [ 3 ]. In regions where DF case is found as endemic and/or epidemic, particularly in tropical and subtropical countries including in Ethiopia, patients with febrile illness compliance are main public disease, commonly sought medical attention [ 10 ]. In Ethiopia and in Africa as a whole, these febrile illnesses would likely caused by various infectious pathogens, complicating control and response programs to epidemic and pandemic diseases, such as malaria, Ebola and COVID-19 [ 11 ]. The manifestations of the febrile illnesses are the manifestations of DF and other arboviruses illness [ 12 , 13 , 14 ]. However, DF and other arboviral diseases are unknown or low, probably due to misdiagnosed or undiagnosed febrile patients [ 13 ]. In East African countries, even though few data were available so far, DF is notably recognized as epidemic-prone public health threat disease [ 15 ]. In countries where DF and other arbovirus diseases case potentially exist, accurate and multiplex diagnoses need to be employed for patient care and epidemiological surveillance for epidemic preparedness and response within the countries and at the regions at large [ 16 ].

The existing surveillance system is inadequate to detect new cases and the dynamics of the virus in a timely manner because of inadequate human, financial, material and technical resources to equip laboratory services and epidemiological capacity. Similarly, the diagnosis, diagnostic methods and health service systems variability of countries hinder the reliable estimations of burden and distributions of emerging diseases like DF disease [ 17 ]. These are the reasons why the 2015 United Nations plan through its sustainable development goals (SDGs) to end emerging or re-emerging neglected tropical diseases epidemics by 2030, which encompasses Arboviral diseases including DF, seems far from the target achievement. In Ethiopia, various DF outbreaks have occurred every year among undifferentiated febrile patients in different areas of the country, including in Afar region, in Somali region, and in Dire Dewa city administration [ 18 , 19 , 20 ]. Since 2013 during the first identified outbreak [ 21 ]. In addition, a serological survey has identified DF disease, revealing DF is spreading and emerging disease in the country [ 22 ]. However, the country has been hampered by challenges of lack of clinical experience to suspect and manage potential DF cases and by the absence of adequate laboratory diagnostic capabilities. As a result, DF cases possibly remain undiagnosed as mild or asymptomatic, misdiagnosed with other clinical manifestations resembling malaria, bacterial, or other viral infections or remaining unidentified febrile illness [ 13 , 23 ]. Therefore, DF cases, including epidemics, emerge and pose a significant unforeseen and none-responded challenge to the country due to weak health system, low experience of health service providers, and economic instability [ 24 ]. State-of-the-art reviews tend to address current status of DENV and DF dynamics reflection would be crucial for consideration of the emerging DF cases for diagnosis and management in countries like Ethiopia, where DF cases have not yet considered as a public health important disease. This review summarizes the current knowledge of epidemiology, biology, pathogenesis, clinical manifestations and diagnosis of dengue virus infection and its trend in Ethiopia.

Epidemiology of DENV infection

The DF was first identified and named in 1789 by Benjamin Rush, who coined the term “break-bone fever” because of the symptoms of myalgia and arthralgia. DF epidemics were occurred and recognized simultaneously in Asia, Africa, and North America in the 1780s [ 4 ]. The disease is endemic in many countries in WHO regions of Africa, the Americas, the Eastern Mediterranean, Asia, Australia and the Western Pacific [ 1 , 25 , 26 ], though the Americas, South–East Asia and Western Pacific regions are the most seriously affected, with Asia representing ~ 70% of the global burden of DF disease [ 5 ]. The lack of a unified and coordinated effort at the regional levels to initiate population-based epidemiological surveillance with clear operational goals leads to differences in burden reports within regions [ 27 ]. Since 1940, the risk of contracting the DENV infection has increased by over 30-fold and has widespread dramatically as population movements during World War II spread around the world [ 4 ]. In the late 1990s, DF was the second most important mosquito-borne disease after malaria, with approximately 40 million DF cases and hundreds of thousands of DHF cases each year [ 4 ]. Currently, a half of the world populations are at risk of contracting DF disease. Annually, an estimated 390 million DF infections, of which about 100 million manifest clinical features and nearly a thousand cases develop fatal DHF/DSS, predominantly in the tropical and subtropical regions including the Americas, Asia, Australia, and Africa [ 1 , 7 , 28 ]. The spreading and distribution of DF cases are attributed to the factors such as globalization, population and urbanization growth, variations in climate and environmental factors, lack of sanitary services, ineffective mosquito control, and increasing DENV surveillances and case reports. The factors may contribute to the increases mosquito populations and susceptibility to circulating serotypes and creating favourable temperature, precipitation, and humidity for the reproduction and feeding patterns of the mosquito populations and for the DENV incubation period [ 5 ]. Besides, in recent years, the losses of enzootic amplification requirements and adaptation for replication at higher temperatures of the vector have made DENV cause the largest and the most extensive epidemic in tropical urban areas [ 29 ]. Endemic and epidemic DF transmission cycles are augmented with significant morbidity, mortality, and economic costs, particularly in developing countries [ 2 ].

Since 2000, a sharp rise in DF incidence, the spread of cases to new countries, and the urban-to-rural spread risk of about half of the world’s population [ 5 , 17 ]. The DENV's limit of infectivity has now reached 129 countries with good evidence of DF cases and outbreaks, including 36 countries previously classified as dengue-free by WHO and/or the US CDC [ 17 ]. Annually, an estimated 390 million DENV infections could have occurred, from that estimated 67–136 million cases can manifest clinical features and thousands of case develops into severe/deaths most frequently in the population of countries in tropical and subtropical regions of the world. The tendency of increase may be due to globalization, the growth of the population and urbanization, variations in climate and environmental factors, the lack of sanitary services, ineffective mosquito control and increasing DENV surveillances and case reports [ 5 ]

Over the past 20 years, the number of DF cases and deaths reported to WHO has increased by more than eightfold and fourfold, respectively. The reported cases increased from 505,430 in 2000 to more than 2.4 million in 2010 and to more than 5.2 million cases in 2019, where the reported deaths increased from 960 in 2000 to 4032 in 2015 [ 5 ]. Moreover, the cases of DF have shifted from mostly affecting children 40–50 years ago to affect all age groups [ 26 ]. During the 2020–2021 years, the total number of reported cases and deaths contrarily seemed to decrease. However, the data were not completed yet as well as the COVID-19 pandemic that hampered case reporting in several countries [ 5 ]. Even though the DF case and deaths reports have been growing over wide geographical locations and in all ages of populations, the current global distribution remains highly uncertain [ 17 ].

In Africa, the epidemiology of DF is poorly characterized, even though the vector mosquitoes present a high burden in the neighbouring Middle East and in Sub-Saharan Africa as well all serotypes of DENV circulate in 19 countries of the continent [ 4 ]. In the region of Sub-Saharan Africa, DENV infection seems a significant burden for public health, with an estimated burden of about 25% (21–29%) by IgG, 10% (9–11%) by IgM and 14% (12–16%) by viral RNA tests [ 30 ]. Moreover, many countries of the region, including Burkina Faso in 2016 and 2017, Côte d’Ivoire in 2017, Cape Verde in 2009, and Egypt in 2015 have experienced of DF outbreaks that were reported to Africa CDC [ 31 ].

DENV infection in Ethiopia

DF is understudied in Ethiopia, but the country has a sharply increased number of DF outbreak cases, continued transmission, and increased viral infection [ 32 ]. These would be reasons to recommend Ethiopia has high potential risk factors for DENV transmission and the following findings would further substantiate the recommendation. First; Ae. Aegypti , the vector transmitting DENV, have been extraordinarily identified at indoor and outdoor levels [ 33 ]. From immature stages collected from discarded containers and other artificial water containers found around houses and peri-domestic areas, about 50–84% of them were morphologically identified as Ae. aegypti [ 34 , 35 ]. Second, DF has been potentially misdiagnosed in Africa including in Ethiopia as malaria, because over-diagnosis of malaria in areas of low transmission has been well documented and overestimating of malaria (≈ 75%) by clinical diagnosis [ 36 ]. Reporting of viruses that are similar to DENV by having same transmitting vector and being in the same member of genus Flavivirus was started in Ethiopia during the yellow fever (YF) outbreak in 1960–62 in southern Ethiopia [ 37 ]. Flavivirus including Zika virus (ZIKV), West Nile virus (WNV), Chikungunya virus (CHIKV), Wesselsbron, Talaguine and Sindbis viruses were serologically identified in human populations and in wild animals in Southern and Western Ethiopia [ 38 ]. Despite potential serological cross-reactivity due to the broad antigenic cross-reactivity of antibodies and clinical presentation similarities with causing febrile illness to these Flavivirus infections [ 39 ], DENV infection was not identified and reported until the last decade in Ethiopia.

In Ethiopia, DENV infection outbreak was first reported in 2013 during DF related outbreak that occurred in Dire Dawa city administration from eastern part of the country. During the outbreak, 12,000 DF-related suspected cases were registered, from which 88 of the cases were confirmed by ELISA and RT-PCR and 50 of the cases were found positive for DENV infection, specifically for DENV-2 serotype [ 21 , 33 ]. The next year, other many outbreaks were identified in a year round fashion in Dire Dawa city administration, in Godey Town of the Somali Region, and in the Adar district of the Afar region [ 18 , 19 ]. In the Somali region, during the same months of three series of years, which were in January, February and March of 2014, 2015 and 2016, DF-related outbreaks with a total of 440 cases occurred [ 40 ]. In May 2017, DF-related outbreak from Kabridahar Town in the Somali region was reported with a total of 101 cases, including five with severe DF and one death [ 19 ]. Similarly, the epidemiological evidence reveals that DF-related outbreaks were recorded from 2017 to 2021 in the Somali region and Dire Dewa city administration [ 19 , 41 , 42 , 43 ]. Furthermore, from Jan 01 to Feb 04, 2021, Ethiopian health officials reported DF-related outbreaks of 160 confirmed DENV infection cases in Warder Woreda of Dolo Zone and 47 suspected DF cases in Dolo Ado Woreda of Liban Zone of the Somali Region which are areas that had an experience of past DF outbreaks in 2017 and 2018 [ 43 ]. Contributing factors to the outbreaks include the weakened nutritional status of the community due to prolonged drought, population displacement, poor household water handling, living with ill people and lack formal education [ 19 , 41 ].

Similarly, few serological studies reported DENV infections from different localities of the country. From northwest Ethiopia, in Gonder referral hospital, 7.5% current (acute) and 13.0% past DENV infections [ 44 ] and in Metema and Humera, 19% current and 21% previous DENV infections were reported from acute febrile patients [ 22 ]. In southern Ethiopia, in the Borena zone of the Oromia region, 22.9% against anti-DENV IgG and 7.9% against IgM of DENV infection were reported [ 45 ]. From the confirmed DF infections, the responsible serotypes for the outbreaks were DENV1-3 serotypes [ 33 , 42 , 44 ]. Even though DENV infection would be transmitted all year round in Ethiopia, the risk of contracting it in the country is the most significant during and immediately after the rainy season, which runs from June to August [ 43 ]. The close contact with DF patients, non-uses of bed nets, and the presence of stagnant water around the village were identified as risk factors for contracting DF in Ethiopia [ 18 ].

Biology of DENV

Etiologic taxonomy and transmissions of denv.

DENV is a positive sense single-stranded encapsulated RNA virus and consists of three structural proteins and functional seven none-structural (NS) proteins [ 3 ]. It belongs to the genus flavivirus of the family  Flaviviridae [ 5 ]. The family Flaviviridae comprises various genera, including the genus flavivirus that contains viruses such as DENV, yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Zika virus (ZIKV), genus Hepacivirus that contains hepatitis C virus (HCV), genus Pestivirus and many others like the newly proposed genus consisting of G Barker Virus (GBV) isolates [ 46 , 47 , 48 ]. Although members of the family Flaviviridae share similarities in morphology, genome organization, and replication, each genus differs in antigenic and biological properties. The genus flavivirus has distinct characteristics including most of the viruses in the genus are arthropods (mosquito or tick) borne and share closely related genomic organization and sequence homology, leading to antigenic cross-reactivity among members [ 47 ].

DENV is a roughly spherical virus particle with a diameter of approximately 50 nm, and based on cross-reactivity assays, the virus consists of four independent antigen-related serotypes (DENV1-4) [ 5 , 49 ]. Epidemiologically, like in genetic diversity, transmission dynamics, and epidemic potential, all serotypes are almost similar, except the DENV-4 serotype which is genetically quite distinct [ 50 ]. The DENV exhibits two distinct morphological forms, namely, the intracellular immature virion and mature virion forms. The mature virion is distinguished by two virus-encoded membrane-associated E and M proteins, forming a relatively smooth surface. However, the intracellular immature virion has E protein and a precursor membrane (prM) protein which will be cleaved proteolytically into the M protein during maturation. The immature virion spikes the surface in an asymmetric, upright manner by undergoing extensive rearrangement of intracellular virus-encoded surface proteins upon acidification during maturation in the infected cells [ 51 , 52 ]. However, partially mature/immature forms may sometimes be released from infected cells [ 52 ]. All serotypes have similar geographic distributions and host/vector associations as well as all are known to cause the entire spectrum of DF diseases; considering reasons to be a single species even if they have quite distinct genetically and antigenically [ 53 ]. In addition to the four worldwide identified DENVs, a fifth serotype (DENV-5) has been detected from a suspected patient in Malaysia in 2007 using isolation and genetic sequence analysis and announced in 2013 [ 54 ].

The DENV serotypes can be undergone to several subtypes/ lineages or genotypes based on several changes in the viral genome [ 49 , 55 ]. DENT-1 was previously classified into five (IV) genotypes based on the sequence of the E gene. Of these, genotypes I, IV, and V appeared to predominate, while genotypes II and III appeared to be dormant [ 49 , 56 ]. DENV-1 was classified recently into three genotypes (I, II, and III) by whole-genome sequencing [ 57 , 58 ]. However, the role of different genotypes in triggering outbreaks is poorly studied [ 58 ]. DENV-2 consists of 6 genotypes, including Asian/American, Asian I, Asian II, Cosmopolitan, American, and Sylvatic, and 15 other subpopulations/ lineages. These variations are occurred when few codons in envelope genes confer antigenicity and lineage diversity to American strains of the Asian/American genotypes, codons from NS genes of DENV-2 confer lineages diversity to the Asian I, Cosmopolitan, and Silvatic genotypes [ 59 ]. DENV-3 consists of five genotypes ((I, II, III, IV and V) of which genotype III formed three heterogeneous subpopulations (III-a, III-b, and III-c) [ 60 , 61 ]. Subpopulations of genotypes I, II, and III-a include Asians, and III-c and IV includes Americans strains. The III-b subpopulation contains mainly American strains and a minority of South Asians, but many genotype III strains and genotype V strains contain Asians and Americans [ 61 , 62 ]. DENV-4 consists of five genotypes (I, IIA, IIB, III, and Sylvatic) [ 60 , 63 ]. Genotype I was found in the Philippines and genotype IIA is common in Southeast Asia and China. Genotype IIB has been isolated in Southeast Asia and the Pacific Islands, and genotype III has been reported only in Thailand [ 63 , 64 ]. The genotype and serotype diversity are primarily due to the high rate of genetic mutation driven by error-prone RNA-dependent RNA polymerases; the RNA polymerase lacks proofreading activity and generates mutation approximately per round of DENV genome replication [ 49 ]. On the other hand, a serotype can provide lifelong immunity to the infected person only against that specific serotype [ 65 , 66 ]. Hence, the diversities in the genotypes and serotypes are major challenges in the development of a tetravalent vaccine.

Transmissions of DENV infection to humans can be known to undergo two types of transmission cycles, called urban and enzootic cycles [ 49 ]. The urban transmission cycle to humans occurs from domestic/peri-domestic habitats by female mosquitoes mainly of the species  Ae. aegypti  and, to a lesser extent,  Ae. albopictus [ 5 , 59 ]. The mosquitoes are infected when they bite a person infected with the virus, consequently, the infected mosquitoes can, therefore, extend the virus to other people through bites. These mosquitoes prefer to bite people during both the day and night and live indoors and outdoors near people [ 67 ]. Unlike many flavivirus DENVs are confined to their natural vertebrate host range that use primates as their amplification and reservoir hosts. The DENV transmission that occurs as an enzootic cycle would be in non-human primates of sylvatic habitats and arboreal mosquitoes, such as Ae. taylori  and  Ae. fucifer [ 59 ]. The urban transmission cycles are an endemic or epidemic cycle that takes place between human reservoir hosts and the mosquitoes, where larval maturation occurs around domestic water containers. DENV cannot be spread directly from person to person; however, infected humans are known to carry the infection from one country to another or from one area to another during the stage when the virus circulates and reproduces in the blood system [ 5 ].

Structural components of DENV

The three virus-encoded structural proteins of DENV are C protein (12 kDa), M protein (8 kDa) which is cleaved from PrM protein (21 kDa) and E protein (53 kDa), whereas the seven NS proteins are NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [ 68 ]. The virion particle is organized as virus-encoded outer protein layer, host cell-derived lipid bilayer envelope membrane, and an inner spherical nucleocapsid core [ 69 ]. On the outer surfaces, 180 copies of each E and M/PrM protein are anchored into the lipid bilayer membrane and spanned through the membrane to form an icosahedral arrangement. In the mature DENV and flavivirus , the 180 copies of 395 residues of E monomer proteins are organized into 90 head-to-tail homodimers on the outer layer. Each E monomer protein is composed of three domains that are arranged as central domain I connecting immunoglobulin-like domain III to a dimerization domain II [ 70 ], a membrane-proximal stem, and a transmembrane anchor [ 71 ]. Domain III is supposed to interact with host cell receptors for entry and key epitope to bind neutralizing antibodies [ 55 ]. The membrane-proximal stem has two predicted amphipathic helices of hydrophilic and hydrophobic parts lie against the viral membrane and span to the length of domain II during fusion. At the tip of the domain II, there is a hydrophobic fusion loop which is exposed after the removal of the pr peptide from prM and is important for interaction and fusion with host membranes [ 71 ].

The prM protein is cleaved at position 91 by furin or furin-like protease to produce the pr peptide and M protein. The M protein contains an N-terminal loop (the first 20 residues), α-helical domain (MH), and two transmembrane spans (MT1 and MT2). The MH domain which is a highly conserved residue located 20-38aa downstream from the prM cleavage site can regulate prM cleavage during viral particle maturation and host cell entry [ 72 ]. The cleavage of the ‘pr’ peptide from prM is occurred to keep the M protein remains transmembrane under the protein E shell in the mature particle during maturation [ 68 ]. The lipid bilayer envelope membrane, which encloses the central spherical core of the virion, is taken from the host endoplasmic reticulum (ER) membrane during the maturation process [ 73 ].

During reconstructions by cryoelectron microscopy and fitting of the known structure of E glycoprotein, the viral surface envelope exhibits an icosahedral scaffold of 90 E glycoprotein dimers [ 69 ]. The conformational changes take place on the two surface proteins by different environmental levels of pH, confer on the surfaces unique structural features of the immature and mature DENV forms [ 74 ]. In the immature form, the PrM and E form 90 heterodimers that extend as 60 trimeric spikes on the surface of the particle (Fig.  1 A), wherein the mature particles the E protein form 90 homodimers of 30 raft-shaped groups of three E protein dimers that lie flat against the viral surface to form a ‘smooth’ herringbone-like pattern (Fig.  2 D). During transition through the trans-Golgi network (TGN), predominantly conformational changes happened in the E protein due to pH level change determines to occur the structural transition from spiky immature to smooth shape mature [ 73 ]. Using the knowledge of E protein mass and comparing the size with other viruses, it was suggested that DENV may have three E subunits per icosahedral asymmetric unit [ 69 ]. The three E-monomers present in each icosahedral asymmetric unit indicate that the DENV virion lacks true T = 3 symmetry; this may contribute to its presence in three chemically distinct environments and may play different roles when present in different stages of the disease [ 66 ].

Structure of the dengue virion and conformations of the E protein

Schematic representation of the DENV genome: A ORFs showing structural proteins (C-prM-E) and NS proteins (NS1–NS2AB–NS3–NS4AB–NS5) and 5′ and 3′ UTRs. Positions of complementary sequences are indicated by solid lines for 5′–3′CS and dashed lines for 5′–3′UAR. B Predicted secondary structure of the 5′ UTR of the genome. Structural elements of the 5′ terminal region include stem loop A (SLA), stem loop B (SLB), oligo (U) track spacer, translation initiator AUG, capsid region hairpin (cHP), and 5′ CS element. C Predicted representation of RNA elements at the 3′UTR of the genome. Predicted secondary structures of three defined domains are shown: domain I (variable region, VR), domain II (dumbbell structure, DB1 and DB2), and domain III (conserved sequences CS1 and 3′SL). In addition, the respective positions and sequences of the conserved elements corresponding to RCS2, CS2, 3′CS and 3′UAR are indicated [ 76 ]

The central core of the virus is nucleocapsid, consisting the RNA genome molecule merged with C protein to form a central spherical core [ 66 ]. The NS proteins are functional proteins for a sequence motif characteristic, including viral serine protease, RNA helicase, and RNA-dependent RNA polymerase. They involve in controlling, coordinating and regulating various intracellular processes of viral life cycles [ 47 , 75 ].

Genome of DENV

The DENV comprises of about 11 kb long single-stranded positive-sense RNA molecule genome. The genome has single open reading frame (ORF) flanked by 5′- and 3′-terminal untranslated regions (UTRs) (Fig.  2 A). The ORF encoded for a single of about 3390 residues of large polyproteins [ 53 ]. The 5′ and 3′ UTRs are non-coding regions used as RNA genome maintenance. The UTRs containing conserved sequences are cis-acting elements that regulate the processes of genome amplification, translation, and packaging by altering stability, localization, and translational efficiency [ 25 , 50 , 55 , 76 ].

The 5′ UTR is an upstream region of ORF which is a short and highly structured sequences region of between 95 and 101 nucleotides of the four serotypes. The region has two structural domains of distinct functions during RNA synthesis. The domains are separated by a short oligo(U) sequence that functions as a spacer to enhance viral RNA synthesis. The first domain is about 70 nucleotides sequence, predicted to fold to large stem-loop A (SLA) which is proposed to act as a promoter for the viral RNA-dependent RNA polymerase (NS5). SLA shows portions of three helical structure regions (S1, S2, and S3), a side stem-loop (SSL), and a top loop (TL) which are essential structures recognized by viral RNA-dependent RNA polymerase (NS5) during viral RNA synthesis. The S1 and S2 regions represent one of the most conserved elements, but the sequence and structure of the S3 and side stem-loop show the most variation in flaviviruses . The second domain of 5′ UTR is a 16-nucleotide-long sequence which is predicted to form a short stem-loop B (SLB). The SLB is identified as a 5′ upstream AUG region (5′UAR), which is a complementary region to the counterpart 3′UAR present at the 3′ ends of the genome. The downstream of 5′ UTR is positioned by ~ 100 nucleotides long coding sequence for C that contains a highly conserved in all four DENV serotypes 5′ complementary sequences (5′CS) to 3′CS, a stable capsid region hairpin (cHP) and RNA element that modulates DENV replication in mosquito and mammalian cells. The 5′ CS, which is an 11 nucleotides long (134-UCAAUAUGCUG), mediates long-range RNA–RNA interactions between the ends of the RNA for the genome cyclization. The cyclization of the viral RNA occurs when the 5′UAR and 5′CS hybridize with the counterparts in the 3′ UTR, which is a process required for transferring the viral polymerase from the 5′ SLA to the 3′ ends to initiate genome replication (Fig.  2 B) [ 53 , 76 , 77 , 78 , 79 , 80 , 81 ].

The 3′UTR is a relatively long nucleotide sequence region that comprises about 470, 450, 430, and 385 (shortest) nucleotides sequence for DENV-1, -2, -3 and -4, respectively. The 3`UTR lacks a poly (A) tail (polyadenylation), which is a crucial tail for stimulation and stabilization of cellular mRNAs translational initiation but ends with a conserved 3′ stem-loop (3′SL). The 3′ UTRs of DENV and ZIKV have 3 major domains. Domain I: A stem-loop (SL) domain that immediately follows the stop codon of NS5 and a highly variable region inside the 3′ UTR. In the DENV serotypes, SL has a significant sequence and length variation that may vary from less than 50 nucleotides to greater than 120 nucleotides between the serotypes. Domain II: A dumbbell (DB) characteristic shape and duplicate in tandem. This domain has conserved CS2 and its repeated CS2 (RCS2) sequences. Domain III: The most conserved region of the 3′UTR, containing a CS1 element which is followed by a terminal 3′SL structure. CS1 is a structure involved in long-range RNA–RNA interaction between the end of the viral genome during cyclization (Fig.  2 C) [ 53 , 76 , 81 ].

Life cycle of DENV

DENV infection and replication require step-by-step processes in host immune cells using cellular machinery. The DENV targets immune cells, including dendrite cells (DC), skin Langerhans cell, B cells, T cells, monocytes, macrophages, lymphocytes and liver cells to infect [ 82 , 83 ]. The life cycle of the infection occurs in a subsequent fashion, as detailed below and depicted in Fig.  3 .

Step-by-step processes of dengue virus entry in the host cell and its life cycle

Attachment and cell entry

DENV attaches to the surface of the immune cells and enters the cells by a process known as endocytosis. During the binding to the immune cells, receptors on the surface of the cells trigger the virus to be taken in. The cognate receptor molecules such as glycosaminoglycans (GAG), lipopolysaccharide-binding protein in association with CD14 molecules, heparan sulfate and lectin-like receptors such as dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) are crucial for DENV infection. FC receptor involves in the mechanism referred to as antibody-dependent enhancement (ADE) dengue infection. During infection, E protein binds to the relevant receptors and triggers receptor-mediated endocytosis via clathrin-coated vesicles, sac-like structures, called endosomes [ 55 , 84 , 85 ]. DENV lands on the cell surface and either rolls over various surface receptors or migrates diffusively as a virus-receptor complex toward pre-existing clathrin-coated cavities during the process [ 86 ]. Alternatively, the entry of DENV into target cells can also occur independent of clathrin, caveolae and lipid rafts, depending on a non-classical endocytic pathway, for example, via dynamin [ 87 ].

After internalization, the virus particles are delivered to Rab5-positive early endosomes and mature into Rab7-positive late endosomes, where membrane fusion exclusively occurs [ 86 ]. The endosome’s interior pH can be decreased by proton pumps which trigger the virus to change the protein E conformation to form spike-like structures. As a postulate, the acidic pH in the endosomes triggers E homodimers dissociation, leading domain II to project outward and exposing the hydrophobic fusion loop peptide to the target endosome membrane [ 73 ]. The hydrophobic residues of the fusion loop penetrate the membrane of the endosome. Domain III also shifts and folds back toward the fusion loop peptide into a hairpin-like conformation [ 88 ]. Subsequently, the membrane-proximal stems span the length of domain II. Finally, the stems are aligned across the entire length of domain II to bring the TM anchor and the fusion loop together, completing membrane merger and pore formation (Fig.  3 ). The pore helps the virus to release the nucleocapsid into the cytoplasm [ 66 , 89 ].

RNA uncoating and translation

The viral translation and replication to be undertaken, the nucleocapsid needs to be broken and leave the C protein aside to release the viral RNA into the cytoplasm. The C protein is a highly basic protein that binds to viral RNA with high affinity but low specificity. Uncoating of the C protein from the viral genome occurs by an unexplained mechanism. The non-degradative steps of ubiquitination, however, are thought to function in genome uncoating [ 90 ]. In support of this thought, some studies suggested that inhibition of ubiquitination blocks the uncoating of the DENV genome, by demonstrating that the inhibition of the ubiquitin E1 activating enzyme stabilizes the viral genome by retaining it in endosomes or nucleocapsids during infection [ 2 , 91 ]. The uncoated viral genome translocation to the rough endoplasmic reticulum (ER) occurs through still exactly unknown mechanisms, though believed that constant alteration of cytoskeletal machinery facilitates the translocation [ 92 ].

Translation of the genome RNA occurs at the surface of the ER membrane to produce viral proteins, subsequently initiating the critical step of genome RNA synthesis and amplification [ 93 ]. The members of the Flavivirus genome translation initiation are canonical cap-dependent. The genome RNA contains, like cellular messenger RNAs (mRNA), an m7GpppN-cap structure at the 5′ end though unlikely to lack a 3′ poly-A tail [ 81 , 94 ]. To mediate genomic mRNA cyclization that is occurred to stimulate and stabilize translation initiation, the Flavivirus genome does not have a poly (A) tail binding to a poly (A) binding protein (PABP) to interact with the cap-bind eukaryotic translation initiation factor 4 complex (eIF4F). However, the eIF4F which is a crucial regulator of the cellular translation initiation complex for the cap-dependent translation-like DENV recognizes and binds to the m 7 GpppN cap structure at the 5′ end of mRNA and bridges it to the 40S ribosomal subunit to translate into a single polyprotein [ 95 ]. DENV also has relied alternatively on cap-independent cellular translation initiation to enable viral protein synthesis [ 95 ]. During the inhibition of cap-dependent translation by targeting the cap-binding protein eIF4E, the DENV replication and translation are unaffected [ 96 ]. However, the internal ribosome entry site (IRES), a region in the mRNAs that allow the internal initiation of translation, has not been identified for the Flavivirus [ 97 ]. Therefore, a study suggested that cap-independent translation appears to be regulated by both 5′ and 3′UTRs [ 93 ]. The ribosomal subunit and associated factors would recruit viral mRNA and scan 5′UTR until getting the AUG starts codon. The AUG selection may be assisted by a secondary structure element called cHP, located at 14 nucleotides downstream of the start codon and stops the 40S ribosomal subunit to ensure correct start codon selection [ 98 ].

During the polyprotein translation, signaling and stopping translates sequence direct back and forth translocation across the ER membrane and co- and post-translationally cleaved the polyprotein by viral and cellular proteases to produce the viral proteins. Specifically, cleavage of the polyprotein at conserved sites by the viral serine protease (NS2B/NS3), or by a host-derived signalase to cleave pr/M by furin or furin-like protease after assembly [ 47 ]. The three structural proteins occupy the N-terminal of the polyprotein and are arranged in the order of highly basic C protein (100 amino acids), followed by the prM protein (166aa) which proteolysis into M protein (75aa) during maturation, and then E protein (495aa) [ 47 , 75 ]. In the meantime, the Flavivirus manipulate host cell gene expression at the translational level to facilitate the production of viral proteins and create a replication-friendly cellular state [ 93 ].

RNA replication

The Flaviviridae family viruses have an identical structural arrangement and positive sense single-stranded RNA genome. The viral genome is an mRNA template for the replication of viruses into complete intermediate negative-sense single-stranded RNA upon entry into the host cell [ 47 ]. The positive-sense RNA viruses possesses a limited number of viral replication proteins, consisting of 3–10 genes; however, they extensively use host proteins, membranes, lipids, and metabolites during the life cycle process, including replication [ 99 ]. The positive sense RNA viruses induce massive rearrangements of intracellular membranes to create ultra-structural microenvironments derived either from the ER, mitochondria, Golgi apparatus, plasma membranes, or other organelles for virus RNA replication. These microenvironments are organelle-like membranous structures that favour replication by coordinating various steps of the viral life cycle through spatial separation of the replicating RNA from the ribosome and assembly to the C protein. Moreover, besides increasing the concentration of components required for efficient replication and assembly by reducing the diffusion space, these organelle-like structures also protect viral RNA from cellular nucleases and innate immunity-triggering pattern recognition receptors [ 100 ]. The flavivirus- induced membraneous microenvironment compartment of the RNA replicate is a vesicle packet (VP), created by ER invagination. The VPs are forms of a continuous membranous network in the ER, where functional viral replication complexes (VRCs) are assembled and to create isolated compartments to be hidden from the viral double-stranded RNA (dsRNA)-activated innate cellular immunity [ 101 ]. In the VPs, the VRCs function like a molecular factory by coordinating viral RNA replication through accommodating most of the viral NS proteins, such as NS3, NS4B, and NS5, viral replication intermediate dsRNA, and the host factors [ 93 , 102 , 103 , 104 ].

The DENV RNA serves as a template for replication and actively provides regulated signals that act as promoters, enhancers, and silencers of RNA replication. The elements of the regulate signals for RNA replications are located within the 5′ and 3′ UTRs and in the viral coding sequences [ 76 ]. The RNA-dependent RNA polymerase of the NS5 protein initiates the viral RNA synthesis by binding to SLA located in the 5′ UTR of the viral RNA [ 105 ]. In flavivirus , the presence of the complementary sequence 5′–3′ CS and the cyclization sequence 5′–3′ UAR between the 5′ and 3′ base pairs are essential for viral RNA synthesis. To reach the replication initiation sites at the 3′ ends of the RNA molecule, the SLA-bound polymerase exploits the long-range 5′–3′ RNA–RNA interactions within the template mediated by genome 5′–3′CS and 5′–3′UAR hybridizations of the long RNA molecules [ 76 ]. The change in a hybridization of complementary sequences leading to RNA cyclization exposes the 3′ end of the viral genome, which is a template during the initiation of negative-sense RNA synthesis [ 77 , 78 , 106 ]. The RNA-dependent RNA polymerases, along with the viral protease/helicase NS3, other viral NS proteins, and presumably host factors, catalyze the enzymatic reaction process to synthesize a negative sense RNA that will serve as a template for the amplification of positive sense genomic RNA [ 107 ]. The progeny viral RNA synthesis occurs by asymmetric, semi-conservative replication on the replicative form (RF) templates or dsRNA recycling [ 108 ], because the viral replications are the production of 10–100-fold more positive-sense progeny RNA than intermediate negative-sense RNA. As the viral RNA was required to be recruited to the replication compartment for replication, the newly synthesized positive-strand RNA is required to be released from the compartment after the optimum RNA is synthesized.

Virion assemblage and liberation

Virion assembly involves encapsulation, envelopment and acquisition of a lipid envelope containing glycoproteins by budding across the intracellular membrane. The NS2A protein recruits viral genome RNA, structural proteins and proteases to virion assembly sites and orchestrates nucleocapsid and virus formation. The 3′UTR end of the viral genome that serves as a recruitment signal for packaging is linked to the cytoplasmic loop of NS2A, allowing NS2A to recruit nascent RNA from the VRC to the virion assembly site. NS2A also recruits the C-prM-E polyprotein and the NS2B–NS3 protease to the virion assembly site through interactions with prM, E, and NS3, resulting in coordinated C-prM-E cleavage [ 109 ].

In members of the genus Flavivirus , nascent RNA is assembled into virions by encapsulation, after which the envelope buds into the ER lumen. Primarily mature C proteins are encapsulated into the viral RNA to form nucleocapsids, which are subsequently loaded with prM and E proteins and conquer a lipid bilayer envelope containing glycoproteins by budding across the intracellular membrane to form virions. In DENV VPs, the opening into the cytosol allows access for metabolites [such as nucleoside triphosphates (NTPs)] to the VRC and for newly replicated viral RNA to exit the VP for translation or assembly of virus particles. The replicated DENV genomes released through the VP pore can be used directly for packaging into virion particles and buds through the ER membrane in near the VP [ 100 ]. The close proximity of the replication and assembly sites to the VP may reveal DENV RNA selectivity for encapsulation by shifting the balance from RNA translation to genome encapsulation. This transient regulation is C protein accumulates to sequester viral RNA for replication DENV life cycle, whereas released viral RNA is preferably used for translation during early timepoints after infection when low levels of structural proteins are present [ 100 ]. The formed virus particles are transported to cytoplasmic vesicles via the secretory pathway before being released by exocytosis (Fig.  3 ). The initial immature virion, containing 60 prM and E heterotrimers in an icosahedral arrangement on the surface virion, migrates through the Golgi network, where the acidic environment triggers cleavage of prM by the cellular furin protease, leading to infectious mature virions production [ 110 , 111 ].

Pathogenesis of DENV infection

Pathogenesis of DENV infection is complex and not fully understood though the spectrum of the pathogen severity of all serotypes ranges from mild DF to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [ 112 ]. The pathogenesis is attributed to a complex interaction of the virus, host genes, and immune responses of the host [ 113 ]. Because, the clinical features and severity of DF occur during the existence of factors like being a neonate or young child, female, high body mass index, genetic polymorphisms and previous infection with DENV-1 if the patient contracts DENV-2 or DENV-3, co-morbidities, such as diabetes and asthma disease [ 9 , 25 ]. During severe DF cases, the DENV induces blood coagulation abnormality and plasma leakage and increases vascular fragility to lead to DHF. Furthermore, the virus increases capillary permeability to cause a body fluid loss that results in a hypovolemic shock DSS and multiple organ failures [ 114 ]. Hence, the patho-physiological features of severe DF may be due to plasma leakage and abnormal hemostasis. The plasma loss in DF and its complication outcome have been known for the past 10 years, but the mechanism of the expression demonstrated by the virus remains unclear [ 115 ]. On the other hand, the DENV infection severity peaks after the virus has been cleared by the host immune system, not during the viral load is at peak [ 116 ]. It is an important finding to confirm that the host immune response plays a crucial role in the pathogenesis of DENV infection.

The host organs and tissues’ tropism of DENV are considered major determinants in the pathogenesis; however, the absence of adequate tropism assays and animal models has hampered the understanding of replication tropism in the tissues cell for pathogenesis [ 117 ]. The presence of DENV (−) sense RNA or NS3/NS5 proteins in specific cells of tissue may indicate DENV replication, as these antigens are present when DENV replicates, whereas detection of other DENV antigens (E, prM, C, (+)-sense RNA) may indicate no active replication of DENV in cells, as they do not permit replication of DENV rather cells may non-specifically take up viral RNA and other antigens from the surroundings [ 112 ]. In vitro and autopsy studies, after mosquito bites, cells of the skin are infected and deliver DENV to the draining lymph nodes through lymph in which resident macrophages and different unknown cells are infected and deliver the virus to the lymphatic and vascular system, leading to the infection of bone marrow and spleen [ 112 ]. Then, Peyer’s patches and lymph nodes are infected or may acquire DENV immediately from draining lymph nodes or through bone marrow and spleen and numerous non-lymphoid organs such as stomach, thymus, lung, brain, gastrointestinal tract, liver, kidney, and heart are likely infected [ 112 ]. It is clear that the immune system cells and endothelial cell (EC) lining of blood vessels play a vital role for DENV tropism and severe pathogenesis. The infection of the DENV to host cells such as macrophages, hepatocytes, and EC influences the hemostatic and immune responses to the virus, representing a considerable risk factor for severe illness development. Infected cells die in particular via apoptosis and, to a lesser extent via necrosis; necrosis releases toxic products, which activate coagulation and fibrinolytic systems. On the other hand, the hemopoiesis process is depressed, resulting in to decrease in thrombogenicity in the blood depending on the extension of the infection to the bone marrow stromal cells and levels of IL6, IL8, IL10, and IL18. The high viral load blood, viral tropism for the EC and platelet dysfunction by severe thrombocytopenia result in a high capillary fragility to cause DHF and clinically manifested as petechiae, easy bruising, and gastrointestinal mucosal bleeding [ 118 ]. The DENV infection stimulates the development of specific antibodies and cellular immune responses despite the immune response aggravating the pathogenesis. Studies identified that when IgM antibodies produced against DENV can cross-react with EC, platelets, and plasmin to result in a cycle of amplification for higher vascular permeability and coagulopathy, the improved IgG antibodies bind heterologous viruses during secondary infection of different serotypes and improved antigen-presenting cells (APCs) infection to contribute to the highest viral load viremia in some patients during secondary infection. The viral load overestimates both low and high avidity cross-reactive T cells as in some haplotypes of HLA; cross-reactive T cells delay the virus clearance, producing high levels of pro-inflammatory cytokines and other mediators. The high levels of soluble factors induce changes in the CE leading to coagulopathy and attributing plasma loss to DSS (Fig.  4 ).

Model for the pathogenesis of DF, DHF, and DSS. Black arrows—processes leading to the indicated event; colored boxes with white centers–pathological events. Each event will ultimately affect the EC or the hemostatic system (purple arrows)

Most primary infections usually present as an asymptomatic or mild febrile illness but also cause hemorrhagic fever in some patients, particularly babies born to a DENV-immune mother. Subsequent infection with different serotypes can lead to severe clinical manifestations, such as DHF and DSS [ 119 , 120 ]. The DENV-infected person can produce lifelong immunity against only the infecting serotype; however, the product provides only temporary and partial immune responses against other serotypes [ 66 ]. The severity of DENV increases during secondary infection of different serotypes due to the weakly neutralizing antibodies from the first infection binding to the second serotype and enhancing antibody-dependent enhancement (ADE) infection by Fc-receptor-bearing immune cells, such as monocytes and macrophages [ 85 ]. The cross-reactivity of the primary DENV infection antibodies forms a combination with a second infecting serotype to form infectious immune complexes that enter Fc-receptor-bearing cells, ensuing an extended number of infected cells and increased viral output in line with the infected cell [ 119 ]. The synergistic actions of viral serotypes and various host factors, including ADE, memory cross-reactive T cells, anti-DENV NS1 antibodies, and autoimmunity play a vital role in the severe manifestations of DF in humans [ 113 ]. The severities of DF are likely to be multi-factorial, though the mechanisms leading to severe are yet to be understood.

Clinical manifestations of DENV infection

DENV and other flavivirus can cause serious diseases ranging from febrile illness to fatal hemorrhagic, neurologic, and gastrointestinal symptoms [ 121 ]. DENV infections, following an incubation period of 4–10 days after being bitten by an infected mosquito, may have clinical features that last 2–7 days including asymptomatic or may lead to undifferentiated fever, DF or DHF with plasma leakage that may lead to hypovolaemic shock, DSS [ 9 ]. Symptomatic DENV infections were classified into DF, DHF and DSS according to the 1997 WHO classification guidelines until 2009 [ 8 ]. However, in the 2000s, the DF expert groups agreed that DF is a fundamental disease that has different clinical manifestations and often has unpredictable clinical features and outcomes; DF cases reclassification into severity levels has strong potential for practical utility in clinicians’ decisions about, where and how intensively a patient should be monitored and treated [ 9 ]. Furthermore, the 1997 classification of DHF and DSS case definitions was too difficult to apply in resource-limited settings, and too specific and failed to identify significant proportion of severe DF cases, including cases of hepatic failure and encephalitis [ 122 ]. Consequently, in 2009 WHO categorized DF as non-severe and severe DF based on a set of clinical and/or laboratory parameters [ 9 ].

Furthermore, the 2009 classification had split a large non-severe DF patient group for practical purposes into two categories: DF without warning signs (D − W) and DF with warning signs (D + W). Criteria for probable DF (D − W) would be living in or travelling to DF endemic area and showing fever and two of the following clinical symptoms: nausea (vomiting), skin rash, soreness, positive tourniquet test, leucopenia or any of the warning signs. The D + W patients, which require strict observation and medical intervention, can show all clinical symptoms of D-W, and abdominal pain or tenderness, persistent vomiting, clinical fluid retention, mucosal bleeding, lethargy/restlessness, liver enlargement > 2 cm, and/or laboratory findings with an increase in hematocrit (HCT) associated with a rapid decrease in platelet count. The criteria for DHF are all symptoms of DF and associated with hemorrhagic manifestations (positive tourniquet test or spontaneous bleeding), thrombocytopenia, and signs of increased vascular permeability that increase hemoconcentration or fluid effusion in the chest or abdominal cavity (Fig.  5 ). As the DHF severity extends, plasma leakage occurred to leads DSS and fluid accumulation with respiratory distress, severe bleeding based on clinician assessment, or severe organ involvement including liver (AST or ALT ≥ 1000), CNS (impaired consciousness), heart and other organs [ 9 ]. However, the 2009 WHO classification guidelines have been criticized for being too comprehensive, allowing several different methods of diagnostic criteria for severe DF and using non-specific warning signs as diagnostic criteria for DF. Besides, the guidelines have failed to have defined clinical criteria for the diagnosis of severe DF apart from providing laboratory breakpoints for transaminase levels, and determination of severity is subject to individual clinical judgment [ 122 ]. The 2009 WHO scheme is effective in identifying severe cases [ 123 ], but when the 1997 guidelines were followed, patients tended to fall into lower severity grades [ 124 ]. The 1997 classification appeared to identify truly severe cases, whereas the 2009 guidelines were more useful in recognizing a wide range of severe clinical manifestations.

Suggested dengue case classification and levels of severity

The clinical features of DF frequently depend on the age of the patient. Infants and young children may have an undifferentiated febrile disease, often with a maculopapular rash. Older children and adults may have mild febrile symptoms or classic debilitating disease with rapid onset of fever, severe headache, retro-orbital pain, myalgia, arthralgia, and gastrointestinal discomfort, often with a skin rash and sometimes minor bleeding in the form of petechiae, nosebleeds, gastrointestinal bleeding and bleeding gums. In addition, in those with signs of bleeding, usually leucopenia and occasionally thrombocytopenia can be observed in DF [ 8 , 125 , 126 ]. In Children, DHF commonly presents with a sudden temperature rise accompanied by facial flush and other non-specific constitutional symptoms resembling DF, such as anorexia, vomiting, headache, and muscle or bone and joint pain. DSS is a shock and deterioration occurs suddenly after a fever of 2–7 days or shortly after defervescence (during the return of fever to normal), whereas DSS is a rapid, weak pulse (≤ 20 mmHg) or hypotension accompanied by cold skin and dizziness in the early stages of shock. Hence, for patients who do not receive prompt and appropriate treatment, a period of profound shock can occur, in which pulse and blood pressure are undetectable, leading to death within 12–36 h after the onset of shock [ 8 , 9 ].

Diagnosis and control of DENV infection

DF should be considered in a patient typically present with acute onset of fever, headache, body aches and sometimes rash spreading from the trunk and who lives in or recently traveled to a disease-endemic area in the 2 weeks before symptom onset [ 122 ]. The diagnosis can be performed by detecting the virus, viral nucleic acids, antigens, anti-DENV antibodies, or combinations of these techniques [ 5 ]. In the early stages of the disease (seven or less than 7 days after onset of illness), DENV infection can be diagnosed from serum, plasma, circulating blood cells, or from other tissues by detecting viral RNA with nucleic acids amplification tests, NS1 protein using some commercial tests and viral isolation in mammalian or mosquito cell culture to further genotyping and lineage for virus characterization [ 122 ]. DENV is thermally labile; RNA detection and isolation of the virus are highly dependent on well-preserved specimens for accurate diagnosis results [ 127 ]. The samples awaiting shipment to the laboratory should be stored in a refrigerator or freezer. That is, for storage up to 24 h, samples should be stored at 4–8 °C and for long-term storage samples should be frozen in a − 70 °C refrigerator or liquid nitrogen container [ 9 ].

For patients with suspected DF disease, serum specimens during acute phases (≤ 7 days after onset of illness) would be collected and diagnosed by detecting the viral RNA sequence by reverse transcription–polymerase chain reaction (RT–PCR) or NS1 protein and/or anti-DENV antibodies by enzyme-linked immunosorbent assays (ELISA) or rapid point-of-care tests [ 128 ]. IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) is used for the qualitative detection of DENV IgM antibodies starting 4–5 days after onset of symptoms and is also reliably detectable for approximately 12 weeks. The MAC-ELISA is based on capturing human IgM antibodies on a micro-titer plate using anti-human–IgM antibody followed by the addition of DENV antigens derived from the envelope proteins of the four serotypes. Plaque Reduction Neutralization Tests (PRNTs) that detect specific neutralizing antibodies against DENV and other flavivirus are performed on IgM positive patients to determine the cause of infection or to rule out other flavivirus, such as ZIKV, YFV and, in some cases, to determine the infecting DENV serotypes [ 5 , 122 ]. Moreover, the recent development of ELISA and dot blot assays targeting the E/M and NS1 antigens has demonstrated that high levels of these antigens in the form of immune complexes are detected in patients with both primary and secondary dengue infections up to 9 days after the onset of illness. The NS1 glycoprotein is produced by all flavivirus , secreted by mammalian cells, and generates a very strong humoral response, so that detection of NS1 allows early diagnosis of dengue virus infection, although serotypes are not differentiated [ 9 ]. The nested RT-PCR protocol was developed using universal dengue primers targeting the C/prM region of the genome for the initial reverse transcription and amplification step, followed by a nested PCR amplification for identification of the infecting serotype-specific qualitatively [ 129 ]. Moreover, the combination of the four serotype-specific oligonucleotide primers in a single reaction tube which utilizes one-step multiplex RT-PCR was an interesting alternative to the nested RT-PCR [ 130 ]. The advancement of RT-PCR into real-time (rRT-PCR) by incorporating dyes and probes (SYBR green and TaqMan) in a single step is capable of providing quantitative data [ 131 ]. The presence of the virus by rRT-PCR or NS1 antigen in a single diagnostic sample is considered laboratory-confirmed dengue in patients with compatible clinical and travel histories [ 122 ]. For patient illness of more than 4 days after the onset of fever, DF can be diagnosed by testing serum for IgM antibodies produced against DENV using MAC-ELISA, whereas for patients presenting within the first week after fever, testing for DENV should include detection of rRT-PCR or NS1 and IgM [ 122 ].

After the acute phase of infection has subsided or after 7 days of fever onset, detection of IgM antibodies is the preferred method of diagnosis using ELISA and hemagglutination inhibition (HI), although NS1 has been reported positive up to 12 days after fever onset [ 9 , 67 ]. If the DENV infection occurred in a person who had no previous flavivirus infections or had not been vaccinated against flavivirus, such as ZIKV, YFV, JEV, and TBE, patients would develop a primary antibody response which slowly increases for a limited time long [ 128 ]. The IgM isotype is the primary emerging antibody and detection rate in serum is increased as follows: by days 3–5 after the disease onset, it is detected in 50% of patients, by day 5 detection increased to 80% of patients and by day 10–99% of patients [ 9 ]. During DENV infection occurs in a place, where other potentially cross-reactive flavivirus such as ZIKV, WNV, YFV, and JEV are not a risk, a single serum sample IgM test result strongly suggests a recent DENV infection and should be presumed confirmatory for DF [ 67 ]. The IgM level peaks at about 2 weeks after disease onset and declines to undetectable levels after 2–3 months. Contrarily, IgG started to be detected at low titers in serum, usually at the end of the first week of onset, and slowly increases thereafter and is detected after months and even years [ 9 ].

During secondary DENV infection or after vaccination or infection with a non-dengue flavivirus , the IgG isotype antibody titers rise rapidly; the predominant antibody isotype is detected in secondary infection with high levels in an acute phase and lasts 10 months and sometimes lifelong [ 128 ]. During the convalescent phase, IgM antibodies can be reliably detected but negative for viral RNA or NS1 test [ 122 ]. The IgM levels during early convalescence are significantly lower within secondary than primary infections and may be undetectable depending on the tests used. Hence, IgM detection is a reliable serological diagnostic test target in primary DENV infections [ 132 ]. IgM/IgG antibody ratios and HI tests are used to distinguish between primary and secondary dengue infections [ 133 ]. IgM and IgG anti-DENV antibodies detection are useful to confirm recent or past infection, because where IgM can be formed about 1 week after infection and reaches their peak 2–4 weeks after the onset of disease, the formation time of IgG level is longer than that of IgM but IgG will stay in the body for many years [ 5 ]. The presence of IgM indicates a recent infection; the presence of IgG indicates a previous DENV infection. Similarly, during the clinical course, the IgG/IgM ratio plays an important role in differentiating DENV infection. A ratio of 1.10 or higher is found the optimal cutoff point for differentiating secondary from primary DENV infection [ 134 ].

DENV can be isolated from serum, plasma, and peripheral blood mononuclear cells and obtained from tissue autopsies (e.g., liver, lung, lymph nodes, thymus, or bone marrow), although specimens for isolation should be collected early in the infection process and during the viremia stage (usually before the 5th day) [ 112 ]. Cell culture is a widely used method to isolate DENV as a golden standard for DENV infection diagnosis, using the mosquito cell lines C6/36 (cloned from Ae . albopictus ) or AP61 (a cell line from Ae. pseudoscutellaris ) and rarely and mammalian cell cultures, such as Vero, LLCMK2, and BHK21. Consequently, the viral RNA genome sequencing is performed for the genotyping of the serotypes and to characterize the molecular epidemiology of DENV infections [ 135 , 136 ].

Rapid diagnostic tests (RDTs) for detecting NS1 protein antigen, IgM, IgG, and IgA antibodies have been developed by many commercial companies and are widely used due to their ease of use and rapid results. In many DENV endemic settings and areas with limited laboratory diagnostic resources, RDTs provide opportunities for point-of-care diagnosis, as well as secondary infection or convalescent timepoints after recent infections [ 137 ]. The IgM-based RDT format alone is not sensitive enough for acute DF diagnosis. On the other hand, the IgG-based RDT format is not recommended for diagnosing of acute DF, because IgG antibodies persist for lifelong and are more likely to be misdiagnosed as false-positive. The NS1 antigen-based RDTs are important part of modern point-of-care diagnostics, but are sensitive only in the early stages of infection and are not suitable for single use in epidemic settings, where late clinical manifestations may occur [ 138 ].

Careful medical detection and monitoring of patients with DF can significantly reduce mortality from severe dengue [ 31 ]. Currently, there is no specific treatment for DENV infection. Symptoms of muscle pain, fatigue, and fever can be relieved and reduced by treatment with acetaminophen or pain relievers, such as acetaminophen. Non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and aspirin, are not recommended, because these anti-inflammatory drugs have a blood losing effect and blood anticoagulants that can worsen the prognosis of diseases with a risk of hemorrhage. For severe DF, medical care by doctors and nurses familiar with the effects and course of the disease can reduce mortality from more than 20% to less than 1% by maintaining patients' fluid volume, essential for the management of severe DF [ 5 , 8 , 9 ]. Currently, Dengvaxia ®  is the only DENV vaccine approved and in use in the United States for children ages 9 to 16 with laboratory-confirmed evidence of previous DENV infection and living in areas, where DF is common [ 67 ]. Control of DF/DHF relies primarily on the use of insect repellents, wearing long sleeves and long trousers, and mosquito repellent inside and outside the home. According to the Global Vector Control Response (GVCR) noted, epidemiological surveillance with case detection and control, and entomological surveillance and control would be pillars to prevent and control DENV infections [ 139 ].

Limitations of the review

This review covers the most relevant aspects of DF global epidemiology, transmission, etiology, morphology, life cycle, genome, pathogenesis, clinical features, and diagnosis. Although this review article discusses DENV infection in detail, it has limitations. Only 139 research articles and organizations' report documents are covered, and the detailed aspect of DENV prevention and treatment were barely covered due to the absence of proven curative drugs. Only the DENV scenario observed in Ethiopia was detailed; other countries were not emphasized in-depth due to the time and length limitations of the article. This article also lacks information on the detailed genome segment-based DENV diagnostic method. It also does not include information on failures and progress in developing preventive vaccines against DENV.

DENV infection is a complex systemic disease with severe medical and economic consequences. To fully understand the impact on patients and the general public, assessing the full spectrum of the DF disease burden would be substantial. Considering Ethiopia is a representative developing country, it has been experiencing at least one DF epidemic every year since 2013, severely depleting the country's economy and health system. The high vector burden and rapid increase in seroprevalence of both current and past DENV infections in febrile individuals suggest that mosquito-borne DENV infection plays an important role in the spread of the disease. Globalization, population growth, and urbanization, lack of sanitation facilities, change in climate and environmental factors ineffective mosquito control, and increased DENV surveillance are factors behind the increase in DF worldwide. In addition, major risk factors for individuals to contracting DENV and developing DF include poor nutrition, persistent drought, population displacement, poor water handling, living with the ill, and lack of formal education. Appropriate precautions are recommended to minimize the risk of DENV infection.

Efforts are being made to expand surveillance coverage, but achieving the goal of eliminating DENV by 2030 will require ensuring prevention and control practices in all sectors of healthcare organizations. However, there is still a long way to go before this global health burden is reduced and DF is eliminated. In the meantime, human struggles to achieve this without preventive vaccines and efforts must be made to utilize effective preventive vaccines.

Availability of data and materials

Not applicable.

Abbreviations

Antibody-dependent enhancement

Antigen presenting cells

Centres for Disease Control and Prevention

Chikungunya virus

Capsid region hairpin

Complementary sequence

Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin

Dengue virus

Dengue fever

Dengue hemorrhagic fever

Double-stranded RNA

Dengue shock syndrome

DF without warning signs

Eukaryotic translation initiation factor 4 complex

Enzyme-linked immunosorbent assays

Glycosaminoglycans

G Barker virus

Hepatitis C virus

Internal ribosome entry site

Japanese encephalitis virus

IgM antibody capture enzyme-linked immunosorbent assay

Poly (A) tail binding to a poly (A) binding protein

Real-time reverse transcription–polymerase chain reaction

Reverse transcription–polymerase chain reaction

Large stem-loop A

Short stem-loop B

Tick-borne encephalitis virus

Trans-Golgi network

Untranslated region

Vesicle packet

West Nile virus

Yellow fever virus

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Biruk Zerfu

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Biruk Zerfu, Tesfu Kassa & Mengistu Legesse

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Zerfu, B., Kassa, T. & Legesse, M. Epidemiology, biology, pathogenesis, clinical manifestations, and diagnosis of dengue virus infection, and its trend in Ethiopia: a comprehensive literature review. Trop Med Health 51 , 11 (2023). https://doi.org/10.1186/s41182-023-00504-0

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• Review Article
• Published: December 2010

Dengue: a continuing global threat

• Maria G. Guzman 1 ,
• Scott B. Halstead 2 ,
• Harvey Artsob 3 ,
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• Jeremy Farrar 5 ,
• Duane J. Gubler 6 ,
• Elizabeth Hunsperger 7 ,
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• Harold S. Margolis 7 ,
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• Rosanna W. Peeling 8 , 11  

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• Dengue virus
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Dengue fever and dengue haemorrhagic fever are important arthropod-borne viral diseases. Each year, there are ∼ 50 million dengue infections and ∼ 500,000 individuals are hospitalized with dengue haemorrhagic fever, mainly in Southeast Asia, the Pacific and the Americas. Illness is produced by any of the four dengue virus serotypes. A global strategy aimed at increasing the capacity for surveillance and outbreak response, changing behaviours and reducing the disease burden using integrated vector management in conjunction with early and accurate diagnosis has been advocated. Antiviral drugs and vaccines that are currently under development could also make an important contribution to dengue control in the future.

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Dengue is the most important arthropod-borne viral infection of humans. Worldwide, an estimated 2.5 billion people are at risk of infection, approximately 975 million of whom live in urban areas in tropical and sub-tropical countries in Southeast Asia, the Pacific and the Americas 1 . Transmission also occurs in Africa and the Eastern Mediterranean, and rural communities are increasingly being affected. It is estimated that more than 50 million infections occur each year, including 500,000 hospitalizations for dengue haemorrhagic fever, mainly among children, with the case fatality rate exceeding 5% in some areas 1 , 2 , 3 , 4 .

The annual average number of dengue fever/dengue haemorrhagic fever (DF/DHF) cases reported to the World Health Organization (WHO) has increased dramatically in recent years. For the period 2000–2004, the annual average was 925,896 cases, almost double the figure of 479,848 cases that was reported for the period 1990–1999. In 2001, a record 69 countries reported dengue activity to WHO and in 2002, the Region of the Americas alone reported more than 1 million cases. Although there is poor surveillance and no official reporting of dengue to WHO from countries in the African and Eastern Mediterranean regions, in 2005–2006 outbreaks of suspected dengue were recorded in Pakistan, Saudi Arabia, Yemen, Sudan and Madagascar 1 , 2 , 3 , 4 , and a large outbreak of dengue involving >17,000 cases was documented in the Cape Verde islands in 2009 5 .Travellers from endemic areas might serve as vehicles for further spread 6 , 7 , 8 , 9 . Dengue epidemics can have a significant economic and health toll. In endemic countries in Asia and the Americas, the burden of dengue is approximately 1,300 disability-adjusted life years (DALYs) per million population, which is similar to the disease burden of other childhood and tropical diseases, including tuberculosis, in these regions 10 .

The geographical areas in which dengue transmission occurs have expanded in recent years ( Fig. 1 ), and all four dengue virus serotypes (DENV-1–4) are now circulating in Asia, Africa and the Americas, a dramatically different scenario from that which prevailed 20 or 30 years ago ( Fig. 2 ). The molecular epidemiology of these serotypes has been studied in an attempt to understand their evolutionary relationships 11 .

Data from WHO.

The single open reading frame encodes three structural proteins (the capsid (C), membrane (M) and envelope (E) glycoproteins) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and N55).

This Review will provide an update on our understanding of the pathogenesis of this successful pathogen, how we diagnose and control infection and the progress that has been made in vaccine development.

Dengue virus pathogenesis

Dengue viruses belong to the genus flavivirus within the Flaviviridae family. DENV-1–4 evolved in non-human primates from a common ancestor and each entered the urban cycle independently an estimated 500–1,000 years ago 12 . The virion comprises a spherical particle, 40–50 nm in diameter, with a lipopolysaccharide envelope. The positive single-strand RNA genome ( Fig. 3 ), which is approximately 11 kb in length, has a single open reading frame that encodes three structural proteins — the capsid (C), membrane (M) and envelope (E) glycoproteins — and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Important biological properties of dengue viruses, including receptor binding, haemagglutination of erythrocytes and the induction of neutralizing antibodies and the protective immune response, are associated with the E glycoprotein. Each DENV shares around 65% of the genome, which is approximately the same degree of genetic relatedness as West Nile virus shares with Japanese encephalitis virus. Despite these differences, each serotype causes nearly identical syndromes in humans and circulates in the same ecological niche 13 .

The figure shows the distribution in 1970 ( a ) and 2004 ( b ). Reproduced with permission from Ref. 141 .

The mosquito vectors, principally Aedes aegypti , become infected when they feed on humans during the usual five-day period of viraemia. The virus passes from the mosquito intestinal tract to the salivary glands after an extrinsic incubation period, a process that takes approximately 10 days and is most rapid at high ambient temperatures 14 . Mosquito bites after the extrinsic incubation period result in infection, which might be promoted by mosquito salivary proteins 15 . In the skin, dengue viruses infect immature dendritic cells through the non-specific receptor dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN) 16 . Infected dendritic cells mature and migrate to local or regional lymph nodes where they present viral antigens to T cells, initiating the cellular and humoral immune responses. There is also evidence of abundant replication of DENVs in liver parenchymal cells and in macrophages in lymph nodes, liver and spleen, as well as in peripheral blood monocytes 17 . Both in vitro and in vivo , macrophages and monocytes participate in antibody-dependent enhancement (ADE) 18 , 19 , 20 . ADE occurs when mononuclear phagocytes are infected through their Fc receptors by immune complexes that form between DENVs and non-neutralizing antibodies. These non-neutralizing antibodies result from previous heterotypic dengue infections or from low concentrations of dengue antibodies of maternal origin in infant sera 21 . The co-circulation of four DENV serotypes in a given population might be augmented by the ADE phenomenon 22 .

DENVs produce several syndromes that are conditioned by age and immunological status. During initial dengue infections, most children experience subclinical infection or mild undifferentiated febrile syndromes. During secondary dengue infections the pathophysiology of the disease changes dramatically, particularly sequential infections in which infection with DENV-1 is followed by infection with DENV-2 or DENV-3, or infection with DENV-3 is followed by infection with DENV-2 23 , 24 , 25 . Such infections can result in an acute vascular permeability syndrome known as dengue shock syndrome (DSS). The severity of DSS is age-dependent, with vascular leakage being most severe in young children, a phenomenon that is thought to be related to the intrinsic integrity of the capillaries 26 , 27 . In adults, primary infections with each of the four DENV serotypes, particularly with DENV-1 and -3, often results in DF. Some outbreaks of primary DENV-2 infections have been predominantly subclinical 24 . Nonetheless, dengue infections in adults are often accompanied by a tendency for bleeding that can lead to severe haemorrhages.

Dengue infections can be life-threatening when they occur in individuals with asthma, diabetes and other chronic diseases 28 , 29 , 30 . Host factors that increase the risk of severe dengue disease include female sex, several human leukocyte antigen (HLA) class I alleles, a promoter variant of the DC-SIGN receptor gene, a single-nucleotide polymorphism in the tumour necrosis factor (TNF) gene and AB blood group 31 , 32 , 33 , 34 , 35 , 36 . Host factors that reduce the risk of severe disease during a second dengue infection include race, second or third degree malnutrition, and polymorphisms in the Fcγ receptor and vitamin D receptor genes 37 , 38 , 39 , 40 , 41 , 42 . Secondary dengue infections in adults can produce the classical DSS or severe disease complicated by haemorrhages. The severity of secondary dengue infections has been observed to increase from month-to-month during island outbreaks 43 ; the longer the interval between the first and second infection the more severe is the accompanying disease 44 , 45 . Tertiary dengue infections can cause severe disease, but only rarely 25 .

In vitro studies demonstrate that the infection of human monocytes and mature dendritic cells results in increased virus replication as a result of the suppression of the interferon system 45 . Type I interferon-associated genes are less abundantly activated in peripheral blood mononuclear cells taken from patients with severe dengue disease compared with milder disease 46 . Subsequently, the increased number of infected cells present targets for CD4 + and CD8 + T cells, resulting in large quantities of interleukin (IL)-10, IL-2, interferon (IFN)-γ and TNF that, singly or in combination, might contribute to endothelial damage and altered haemostasis. Virions released from infected cells might also directly damage endothelial cells and the uptake of the non-structural protein NS1 by hepatocytes might promote viral infection of the liver 47 , 48 , 49 . During DHF, the complement cascade is also activated and the levels of the complement activation products C3a and C5a correlate with the severity of illness 49 . Soluble and membrane-associated NS1 have been demonstrated to activate human complement. The levels of the terminal SC5b–9 complement complex and plasma NS1 correlated with disease severity, suggesting links between the virus, complement activation and the development of DHF/DSS 50 . Alternative hypotheses of dengue pathogenesis include the suggestions that secondary T-cell responses are blunted because stimulation of T-cell memory results in the production of heterotypic CD4 + and CD8 + cells that have a diminished capacity to kill but nonetheless release inflammatory cytokines that contribute to disease severity 51 ; that severe disease is caused by DENVs of increased virulence 52 ; and the suggestion that cross-reactivity between NS1 and human platelets and endothelial cells raises antibodies that damage these cells 53 .

One working hypothesis of dengue pathogenesis that is consistent with the available evidence is that severe disease in infants with primary infections and in older individuals with secondary infections is the result of ADE of infection of mononuclear phagocytes. Infection by an antibody–virus complex suppresses innate immune responses, increasing intracellular infection and generating inflammatory cytokines and chemokines that, collectively, result in enhanced disease. Liver infection and a pathogenic role for NS1 add to the complexity. In patients with DF, IFN production and activated natural killer cells can limit disease severity.

Clinical signs and immunological response

Dengue-associated deaths are usually linked to DHF/DSS. Even though no vaccines or drugs are available, severe disease can be successfully managed by careful monitoring of the warning signs and early initiation of aggressive intravenous rehydration therapy. During the early febrile stage (the symptoms of which include fever, malaise, headache, body pains and rash), clinicians cannot predict which patients will progress to severe disease. Later, during defervescence, symptoms such as bleeding, thrombocytopenia of <100,000 platelets mm −3 , ascites, pleural effusion, haematocrit >20% and clinical warning signs, such as severe and continuous abdominal pain, restlessness and/or somnolence, persistent vomiting and a sudden reduction in temperature (from fever to subnormal temperature) associated with profuse perspiration, adynamia (loss of strength or vigor) and sometimes fainting, can be indicative of plasma extravasation and the imminence of shock. At this point, patients should receive fluid replacement (crystalloids) to avoid haemodynamic instability, narrowness of blood pressure and hypotension. Early resuscitation can prevent other complications, such as massive haemorrhage, disseminated intravascular coagulation, multiple organ failure, and respiratory failure due to non-cardiogenic pulmonary oedema 54 , 55 , 56 , 57 . Treatment of uncomplicated dengue cases is only supportive, including plenty of oral fluids during the febrile period and paracetamol (acetaminophen), the daily dosage of which should not be exceeded to prevent intoxication mainly related to liver function. When dengue shock becomes prolonged or recurrent, intravenous fluids should be given carefully according to age and dosage to prevent fluid overload as this can result in pulmonary oedema.

Recent publications have suggested that the WHO syndromic case definition of DHF/DSS should be evaluated for clinical utility 58 , 59 , 60 , 61 , 62 . A prospective multi-centre study in several Latin-American and Southeast asian countries is planned that will provide standardized descriptions of dengue clinical presentations in the context of the current WHO case definitions.

The acquired immune response to dengue infection consists of the production of antibodies that are primarily directed against the virus envelope proteins. The response varies depending on whether it is a primary or secondary infection 63 , 64 . A primary antibody response is seen in individuals who are not immune to dengue and a secondary immune response is observed in patients who have had a previous dengue infection ( Fig. 4 ). A primary infection is characterized by a slow and low-titre antibody response. Immunoglobulin (Ig)M antibodies are the first isotype to appear, by day 3–5 of illness in 50% of hospitalized patients and by day 6–10 of illness in 93–99% of cases. The IgM levels peak ∼ 2 weeks after the onset of fever and then generally decline to undetectable levels over the next 2–3 months 54 , 55 , 65 . Dengue-specific IgG is detectable at low titre at the end of the first week of illness and slowly increases. By contrast, during a secondary infection, high levels of IgG antibodies that crossreact with many flaviviruses are detectable even in the acute phase and rise dramatically over the following 2 weeks 65 . The kinetics of the IgM response are more variable; as IgM levels are significantly lower in secondary dengue infections, false-negative test results for dengue-specific IgM have been reported during secondary infections 55 , 66 , 67 . Following a dengue infection, IgG can be lifelong, which complicates the serodiagnosis of past, recent and current infections 65 , 67 . IgA and IgE responses have also been documented but the utility of detecting these immunoglobulins as markers for dengue serodiagnosis requires further study 68 .

Ig, immunoglobulin; NS, non-structural.

In areas where two or more flaviviruses are circulating, multiple and sequential flavivirus infections make differential diagnosis difficult owing to the presence of pre-existing antibodies and the phenomenon of original antigenic sin (during sequential flavivirus infections, B-cell clones responding to the first infection synthesize antibodies with higher affinity for the first infecting virus than for the second infecting virus) 69 .

Laboratory diagnosis of dengue infection

Laboratory confirmation of dengue infection is crucial as the broad spectrum of clinical presentations, ranging from mild febrile illness to several severe syndromes, can make accurate diagnosis difficult. Among the methods available for dengue diagnosis, virus isolation provides the most specific test result. However, facilities that can support viral culture are not always available. The detection of the viral genome or viral antigens also provides evidence of infection.

Seroconversion of IgM or IgG antibodies is the standard for serologically confirming a dengue infection. The presence of IgM or high levels of IgG in acute serum collected from a suspected dengue case suggests a probable dengue infection 54 , 55 . Box 1 shows the laboratory criteria for confirmed and probable dengue infections.

Virus isolation

The Aedes albopictus mosquito C6/36 cell line is the method of choice for DENV isolation, although other mosquito (such as Aedes pseudoscutellaris AP61) and mammalian (including Vero cells, LLC-MK2 cells and BHK21 cells) cell lines can also be used 70 , 71 . Sera that have been collected from suspected dengue cases in the first 3–5 days of fever (the viraemic phase) can be used for virus isolation. After an incubation period permitting virus replication, viral identification is performed using dengue-specific monoclonal antibodies in immunofluorescence and PCR assays 63 , 64 , 72 , 73 . Serum is often used for virus isolation but plasma, leukocytes, whole blood and tissues obtained at autopsy can also be used 63 , 74 , 75 .

Serological testing

Serological assays are most commonly used for diagnosis of dengue infection as they are relatively inexpensive and easy to perform compared with culture or nucleic acid-based methods. When a dengue infection occurs in individuals who have experienced a previous dengue infection, a secondary immune response occurs, which generates high levels of IgG through the stimulation of memory B cells from the previous infection as well as an IgM response to the current infection. Because high levels of IgG compete with IgM for antigen binding, an IgM capture assay can be used.

MAC-ELISA. The Armed Forces Research Institute of Medical Sciences (AFRIMS) developed an IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) for dengue in regions where dengue and Japanese encephalitis virus co-circulate 65 . Today, many groups have developed their own in-house MAC-ELISAs. Dengue-specific IgM in the test serum is detected by first capturing all IgM using human-specific IgM bound to a solid phase. The assay uses a mixture of four dengue antigens (usually derived from dengue virus-infected cell culture supernatants or infected suckling mouse brain preparations) 76 . Compared to the haemagglutination inhibition assay as the gold standard, MAC-ELISA shows a sensitivity and specificity of 90% and 98%, respectively, in samples collected after 5 days of fever 55 . In addition to serum, dengue-specific IgM can be detected in whole blood on filter paper (sensitivity 98.1% and specificity 98.5%) 77 , 78 and in saliva (sensitivity 90.3% and specificity 92.0%) 79 , but not in urine 68 . More than 50 commercial kits are available with variable sensitivity and specificity 65 , 80 , 81 , 82 . False-positive results due to dengue-specific IgG and crossreactivity with other flaviviruses is a limitation of the MAC-ELISA, mainly in regions where multiple flaviviruses co-circulate. Some tests also show non-specific reactivity in sera from patients with malaria and leptospirosis 82 .

IgG ELISA. An ELISA for dengue-specific IgG detection can be used to confirm a dengue infection in paired sera. It is also widely used to classify primary or secondary infections 53 , 54 , 63 , 64 . Some protocols use serum dilutions to titre dengue-specific IgG 83 and others use the ratio of IgM to IgG 66 , 84 . The assay uses the same dengue antigens as MAC-ELISA and it correlates with results from the haemagglutination inhibition assay. In general, an IgG ELISA lacks specificity within the flavivirus serocomplex groups, however it has been demonstrated that the IgG response to the prM membrane glycoprotein is specific to individual flaviviruses as no crossreactivity was observed in sera collected from individuals infected with dengue or Japanese encephalitis virus 85 . Similarly, it has been demonstrated that IgG specific for the NS5 protein can potentially discriminate between infections caused by West Nile, dengue and St Louis encephalitis viruses 86 . Finally, dengue-specific IgG was shown to have high specificity in an assay using a recombinant polypeptide located in the N-terminal region of the envelope protein 87 . IgG assays are also useful for sero-epidemiological studies to identify past dengue infection.

IgM:IgG ratio. A dengue virus E and M protein-specific IgM:IgG ratio can be used to distinguish primary from secondary dengue virus infections. IgM capture and IgG capture ELISAs are the most common assays for this purpose. According to this method, a dengue infection is defined as a primary infection if the IgM:IgG OD ratio is greater than 1.2 (using patient sera at 1:100 dilution) or 1.4 (using patient sera at 1:20 dilution), or as a secondary infection if the ratio is less than 1.2 or 1.4 (Refs 88 , 89 ). However, in a recent publication the authors indicated that the IgM:IgG ratio varies depending on whether the patient has a serologically non-classical or classical dengue infection, and redefined the ratios 84 . Hence the cut-off for the IgM:IgG ratio is not well defined.

Neutralization assays. The plaque reduction neutralization technique (PRNT) and the micro-neutralization assay are used to define the infecting serotypes following a primary infection. These tests are mainly for research and vaccine studies 90 , 91 , 92 , 93 , 94 .

Nucleic acid amplification tests

Many nucleic acid amplification tests (NAATs) have been developed for the diagnosis of dengue infection. Some techniques are quantitative and others can be used for serotyping. However, none has been commercialised to date and quality assurance materials are not widely available to ensure the quality of the results.

Reverse transcriptase PCR (RT-PCR). Many dengue RT-PCR assays have been described in the past 10 years. These in-house assays target different genes and use different amplification procedures. The most commonly used NAATs are based on a single RT-PCR assay 95 , 96 , a nested RT-PCR assay 96 or a one-step multiplex RT-PCR assay 97 . The nested PCR reaction involves an initial reverse transcription and amplification step using dengue primers that target a conserved region of the virus genome followed by a second amplification step that is serotype specific. The products of these reactions are separated by electrophoresis on an agarose gel, which allows the dengue serotypes to be differentiated on the basis of size. The sensitivity of RT-PCR assays in comparison to virus isolation in mosquito cell culture varies between 25% and 79% 98 .

Real-time RT-PCR. The real-time RT-PCR assay is a one-step assay that allows virus titre to be quantified in approximately 1.5 hours. The detection of the amplified target by fluorescent probes replaces the need for post-amplification electrophoresis. Many real-time RT-PCR assays have been developed that are either 'singleplex', detecting one single serotype per reaction, or 'multiplex', identifying all four serotypes from a single sample 99 , 100 , 101 . One advantage of this assay is the ability to determine viral titre early in dengue illness, which is believed to be an important predictor of disease severity 102 .

Nucleic acid-sequence based amplification assay (NASBA). The NASBA assay is an isothermal RNA-specific amplification assay that has been adapted for dengue virus. Its performance is comparable to that of other NAATs 103 .

Antigen detection

Dengue antigens can be detected in tissues such as liver, spleen and lymph nodes as well as tissues from fatal cases (slides from paraffin-embedded, fresh or frozen tissues) using an enzyme and a colorimetric substrate with antibodies that target dengue-specific antigens 104 , 105 , 106 .

NS1 antigen and antibody detection. NS1 is a glycoprotein produced by all flaviviruses and is essential for viral replication and viability. Because this protein is secreted into the bloodstream, many tests have been developed to diagnose DENV infections using NS1. These tests include antigen-capture ELISA, lateral flow antigen detection and measurement of NS1-specific IgM and IgG responses. NS1 antigen detection kits are now commercially available. As yet, these kits do not differentiate between the different DENV serotypes. Additional independent studies are needed to confirm the performance of these kits and to further validate the diagnostic and prognostic significance of NS1 and NS1-specific antibody detection 107 , 108 , 109 .

Dengue control and prevention strategies

A global strategy for dengue prevention and control was promulgated more than 10 years ago and comprises five major elements ( Box 2 ).

Efforts have since been made to focus on three fundamental aspects: surveillance for planning and response, reducing the disease burden and changing behaviours to improve vector control 110 . The 2002 World Health Assembly Resolution urged greater commitment among Member States and WHO to implement this strategy 111 . Of particular significance is the 2005 revision of the International Health Regulations 112 , which includes mention of DF (and yellow fever) as an example of a health 'event that may constitute a public health emergency of international concern' and which, under such circumstances, should be notified to WHO.

In recent years several new, improved or validated tools and strategies for dengue control and prevention have been developed and are available to public health practitioners and clinicians ( Box 3 ).

Vector control. To reduce or prevent dengue virus transmission there is currently no alternative to vector control. Most endemic countries have a vector control component in their dengue control and prevention programmes but its delivery by public health practitioners is frequently insufficient, ineffective or both.

Given its behaviour and generally close association with humans, the principal vector A. aegypti requires the use of a combination of vector-control methods, notably environmental management methods and chemical control methods based on the application of larvicides and adulticide space sprays 113 . Chemical controls typically must be added to water stored for domestic use, including drinking water. The active ingredients of four larvicides have been assessed by the International Programme on Chemical Safety (IPCS) to determine their safety for use as mosquito larvicides in drinking water at dosages that are effective against Aedes spp. larvae. Since the early 1970s the organophosphate temephos has been widely used, but increasing levels of resistance 114 , 115 , householders' rejection of the treatment of their drinking water, and difficulties in achieving high and regular levels of coverage are important technical and operational constraints.

Biological control agents, including larvivorous fish and copepods, have had a demonstrable role in controlling A. aegypti 116 , 117 , but operational difficulties — particularly the lack of facilities and expertise in mass rearing, and the need to frequently re-introduce these agents into some container habitats — have largely precluded their widespread use.

Environmental management is generally considered to be an essential component of dengue prevention and control, particularly when targeting the most productive container habitats of the vector 118 . Source reduction, 'clean-up' campaigns, regular container emptying and cleaning (targeting not only households but also public spaces such as cemeteries, green areas and schools), installation of water supply systems, solid waste management and urban planning all fall under the rubric of environmental management. However, huge investments in infrastructure are needed to increase access to safe and reliable water supplies and solid waste disposal systems. In addition to overall health gains, such provision would clearly have a major impact on vector ecology, although the relationship is complex. For instance, cost recovery mechanisms, such as the introduction of metered water, might actually encourage the household collection and storage of roof catchment rainwater, which can be harvested at no cost. Although not studied carefully, the construction of community water distribution services to rural townships and villages might be contributing to the rural spread of dengue in Southeast Asia and elsewhere by facilitating domestic water storage. When decisions on such infrastructure development are being made, the views of Ministers of Public Health and municipal health departments are seldom voiced loudly, even when the economic and public health burden of diseases linked to water and sanitation are recognized, including those associated with dengue.

Most efforts in vector control are centred at the household and community levels, but with few exceptions, the achievements to date have been largely unspectacular and there have been difficulties in scaling up from the project level 119 . Nevertheless, such community-based interventions are widely seen as the most promising way of improving delivery and achieving long-term control of the vector through behaviour change. Towards this end, a TDR/WHO guide for planning social mobilization and communication for dengue fever prevention and control has been developed 113 . Additionally, new 'consumer-friendly' tools such as window curtains and water container covers treated with long-lasting insecticide are being tested 120 as well as controlled release larvicides that provide several months of control following a single application to targeted containers.

Products for personal and household protection have a huge potential for household pest control. Generally speaking, these commercial products tend to be used by consumers not so much in response to any perceived public health concerns, but to alleviate the nuisance of biting mosquitoes and in some settings households are prepared to spend substantial amounts of money on these products 121 .

With the increased political recognition of dengue as a public health problem and commitment to prevention and control, better organized control services using new tools and partnership strategies, based on the principles of integrated vector management, are likely to have a major impact on dengue transmission 2 .

Vaccine development. As a result of the failure of vector control, the continuing spread and increasing intensity of dengue has renewed interest and investment in dengue vaccine development, making a safe, effective and affordable tetravalent dengue vaccine a global public health priority 122 . Dengue vaccine development has been in progress for several decades, however the complex pathology of the illness, the need to control four virus serotypes simultaneously and insufficient investment by vaccine developers have hampered progress 122 .

The observation that DHF/DSS is associated with DENV secondary infection poses a special challenge to the development of a dengue vaccine, leading to a requirement that such vaccines should induce a robust immune response against the four serotypes in naive as well as previously immune individuals. Animal models are only partially useful for vaccine evaluation. The poor understanding of the mechanisms involved in inducing protective immunity against dengue infection poses additional challenges 123 . Finally, cases of DHF/DSS have recently been documented 20 or more years after primary dengue infection, which adds a new dimension to the problem 25 , 44 .

The available data suggest that neutralizing antibodies are the major contributors to protective immunity 124 , 125 , however the role of the cellular immune response requires further study 123 . In this context, clinical trials are crucial for vaccine development owing to the unique information they provide on immune responses and reactogenicity. Also, long-term observations of vaccinated populations will be required to demonstrate the absence of ADE or severe disease.

The ideal dengue vaccine should be free of important reactogenicity, induce life-long protection against infection with any of the four DENV serotypes and be affordable 126 , 127 . Vaccine candidates should be evaluated in population-based efficacy trials in several at-risk populations in different geographical settings including Asia and the Americas, which experience different patterns of dengue transmission intensity and dengue virus circulation 122 . Vaccine developers are working with the Pediatric Dengue Vaccine Initiative (PDVI) to establish suitable field sites. Developers are also working with the WHO Initiative for Vaccine Research (WHO/IVR) to define the immunological correlates for protection and clinical trial design. Because of the important role of neutralizing antibodies as surrogates of protection, the validation of neutralization tests is a priority 128 . Current approaches to vaccine development involve using live attenuated viruses, inactivated viruses, subunit vaccines, DNA vaccines, cloned engineered viruses and chimeric viruses using yellow fever vaccine and attenuated dengue viruses as backbones 129 , 130 , 131 , 132 , 133 , 134 . Table 1 summarizes the most advanced vaccine candidates.

Significant progress in the development of dengue vaccine candidates has been achieved lately 135 , 136 . An Acambis/Sanofi Pasteur yellow fever–dengue chimeric vaccine is in advanced Phase II testing in children in Thailand and others are in Phase 1 or advanced preclinical evaluation. It is expected that a licensed vaccine will be available in less than 10 years.

Conclusions

Dengue is now a global threat and is endemic or epidemic in almost every country located in the tropics. While we wait for new tools such as vaccines, antiviral drugs and improved diagnostics, better use should be made of the interventions that are currently available. The challenge that awaits us in the near future will be how to scale up to deploy these new tools.

In recent years, several partnerships such as the PDVI, the Innovative Vector Control Consortium, the Asia-Pacific Dengue Prevention Partnership and the European Union's DENFRAME and DENCO projects have come into existence, receiving funding from the Bill and Melinda Gates Foundation, regional Development Banks and the private sector. These partnerships are working with WHO and national governments to develop new tools and strategies to improve diagnostics and clinical treatments and to achieve a successful vaccine.

Box 1 | Laboratory diagnosis of a dengue virus infection

Confirmed dengue infection

Genome detection

IgM or IgG seroconversion

Probable dengue infection

IgM positive

Elevated IgG titre (that is, 1,280 or greater by haemagglutination inhibition test)

Box 2 | The global strategy for dengue prevention and control

Vector control, based on the principles of integrated vector management

Active disease surveillance based on a comprehensive health information system

Emergency preparedness

Capacity building and training

Vector control research

Box 3 | Tools and resources for dengue control and prevention

Rapid commercial diagnostic tests in use in endemic countries

Pocket Book of Hospital Care for Children (inclusion of dengue in the management of fever) 137

An audiovisual guide and transcript for health care workers responding to outbreaks 138

Guidelines for planning social mobilization and communication 139

Global strategic framework for integrated vector management 140

TDR–Wellcome Trust CD-ROM. Topics in International Health Series: dengue

Entomological survey to identify the most productive container habitats of the vector(s) 116

Seven insecticide products evaluated by WHO as mosquito larvicides (five insect growth regulators and two bacterial larvicides), four of which are approved for use in drinking water and three for space spray applications to control mosquitoes

Advances in the development and operational deployment of DengueNet ( http://apps.who.int/globalatlas/default.asp ) for global dengue surveillance

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Guzman, M., Halstead, S., Artsob, H. et al. Dengue: a continuing global threat. Nat Rev Microbiol 8 (Suppl 12), S7–S16 (2010). https://doi.org/10.1038/nrmicro2460

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AN LITERATURE REVIEW OF DENGUE FEVER: DENGUE HAEMORRHAGIC FEVER IS MORE DEADLY THAN DENGUE FEVER

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Dengue disease surveillance: an updated systematic literature review

S runge-ranzinger.

1 Special Programme for Research and Training in Tropical Diseases, World Health Organization, Geneva, Switzerland

2 Liverpool School of Tropical Medicine, Liverpool, UK

3 Institute of Public Health, University of Heidelberg, Heidelberg, Germany

To review the evidence for the application of tools for dengue outbreak prediction/detection and trend monitoring in passive and active disease surveillance systems in order to develop recommendations for endemic countries and identify important research needs.

This systematic literature review followed the protocol of a review from 2008, extending the systematic search from January 2007 to February 2013 on PubMed, EMBASE, CDSR, WHOLIS and Lilacs. Data reporting followed the PRISMA statement. The eligibility criteria comprised (i) population at risk of dengue, (ii) dengue disease surveillance, (iii) outcome of surveillance described and (iv) empirical data evaluated. The analysis classified studies based on the purpose of the surveillance programme. The main limitation of the review was expected publication bias.

A total of 1116 papers were identified of which 36 articles were included in the review. Four cohort-based prospective studies calculated expansion factors demonstrating remarkable levels of underreporting in the surveillance systems. Several studies demonstrated that enhancement methods such as laboratory support, sentinel-based reporting and staff motivation contributed to improvements in dengue reporting. Additional improvements for passive surveillance systems are possible by incorporating simple data forms/entry/electronic-based reporting; defining clear system objectives; performing data analysis at the lowest possible level (e.g. district); seeking regular data feedback. Six studies showed that serotype changes were positively correlated with the number of reported cases or with dengue incidence, with lag times of up to 6 months. Three studies found that data on internet searches and event-based surveillance correlated well with the epidemic curve derived from surveillance data.

Conclusions

Passive surveillance providing the baseline for outbreak alert should be strengthened and appropriate threshold levels for outbreak alerts investigated. Additional enhancement tools such as syndromic surveillance, laboratory support and motivation strategies can be added. Appropriate alert signals need to be identified and integrated into a risk assessment tool. Shifts in dengue serotypes/genotype or electronic event-based surveillance have also considerable potential as indicator in dengue surveillance. Further research on evidence-based response strategies and cost-effectiveness is needed.

Analyser les résultats de l'application d'outils pour la prédiction/détection des épidémies de dengue et la surveillance des tendances dans les systèmes de surveillance active et passive des maladies, afin d’élaborer des recommandations pour les pays endémiques et identifier les besoins importants de recherche.

Introduction

Dengue remains a major and growing public health threat worldwide. With the most recent study estimating that global infection rates of 390 million infections occur annually ( Bhatt et al. 2013 ), the need for improved dengue surveillance is evident. Dengue surveillance is essential for the detection of outbreaks and, in the longer term, to monitor disease trends. In order to trigger timely interventions, outbreak alerts are particularly important to mobilise vector control and to prime or reorganise healthcare delivery services in preparation for a surge in suspected cases. Although vector control tools can be effective in principle, implementation remains an issue and effective dengue routine prevention is rarely achieved, particularly in high-density urban communities ( Horstick et al. 2010 ; Pilger et al. 2010 ). Emergency vector control operations in response to dengue outbreaks are more typically applied, even though the efficacy of the most widespread method used, insecticide fogging or space-spraying, is dubious ( Esu et al. 2010 ). Surveillance for dengue can include different indicators and systems ( Harrington et al. 2013 ; Henning 2004 ; Stroup et al. 1989 ) to detect outbreaks and monitor trends. The authors' earlier systematic literature review ( Runge Ranzinger et al. 2008 ) analysed ‘the evidence on the structure, purpose and usefulness of dengue disease surveillance in dengue endemic countries’ and described a general lack of evidence for the usefulness of dengue disease surveillance for early outbreak detection, especially the lack of indicators/alert signals available to trigger response. A stepwise adaptation of surveillance systems after evaluation in combination with active surveillance was recommended. Since then, new articles in the field of dengue surveillance have been published, and new initiatives towards early detection of dengue outbreaks have been launched (WHO expert meeting on dengue outbreak detection and response, June 2012). Common to all reports and recommendations is an increased recognition of the need for dengue control to focus on surveillance, vector control and adequate case management.

The aims of this review were to strengthen the evidence base and (where required) adjust the recommendations of Runge Ranzinger et al . (2008) by incorporating new trends and tools and to identify and summarise advances or improvements made. As in the earlier review, dengue vector surveillance was not covered here, but has been reviewed independently elsewhere ( Bowman et al. 2014 , in press).

Methodology

This review followed the protocol ( York 2001 ) used in a previous systematic literature review ( Runge Ranzinger et al. 2008 ) on dengue disease surveillance, and the reporting guidelines set out in the PRISMA Statement for systematic reviews and meta-analyses ( Liberati et al. 2009 ). The eligibility criteria of the reviewed literature were as follows: (i) population at risk of dengue, (ii) dengue disease surveillance, (iii) outcome of surveillance described and (iv) empirical data evaluated. After the recent dengue outbreaks in France and Croatia (29 and 30), the population at risk in the search was extended to include the European region. Literature reported in English, German and Spanish was included although the search was conducted in English only. Studies focusing on risk mapping, transmission dynamics, forecasting or prediction methods were excluded (e.g. Buczak et al. 2012 ; Chen & Chang 2013 ; Racloz et al. 2012 ), as they did not directly study surveillance systems.

The literature search and analyses were developed and continued until 15th February 2013, with two persons working as data extractors. Search fields included Medical Subject Heading (MeSH) terms/subjects and free text, considering population, intervention and outcome. The terms ‘dengue’ and ‘severe dengue’ (dengue fever (DF), dengue h(a)emorrhagic fever (DHF) and dengue shock syndrome (DSS)), ‘surveillance’ (disease, clinical, active, passive, sentinel, epidemiologic, population), ‘communicable disease control’, ‘effectiveness’, ‘evaluation’, ‘disease notification’, ‘disease outbreaks’, ‘hospital and clinical laboratory information system’ were used. The search strategy was adapted according to the databases, consistent with the process undertaken during the primary review published in 2008.

The search strategy was applied to the following databases to locate peer-reviewed studies: The United States National Library of Medicine and the National Institutes of Health Medical Database (PubMed) (1966–2013), Excerpta Medica Database (EMBASE) (1983–2013), the Cochrane Database of Systematic Reviews (CDSR), the World Health Organization (WHO) library database (WHOLIS) and the Latin American and Caribbean Health Sciences Database (Lilacs) (1967–2013). The references cited by relevant literature, including grey literature, were also screened for further articles. Grey literature and unpublished studies were included if found relevant to the research question and if they fulfilled the inclusion and exclusion criteria.

All results were screened for duplication by author, title, journal and publication date, and then screened for relevance, based on the title and abstract only. The full text of all studies considered to be relevant was then reviewed for final assessment by two independent data extractors. Where necessary, consensus was achieved by discussion. Relevant information, including study bibliographic information, study design and objectives, levels of endemicity and population, components of the surveillance system (surveillance subjects, scope and method), resources spent on the system, delivery of the surveillance system (information flow, outbreak and/or case definition, additional relevant information), purpose of the surveillance system and outcome attributes, was extracted and tabulated in evidence tables (Table ​ (Table1 1 ).

Evidence tables

As many studies were descriptive or ‘ecological studies’ and therefore could not be ranked according to the ‘hierarchy of study designs’ ( York 2001 ), the National Health and Medical Research Council ( NHMRC 2000 ) evidence hierarchy ( Merlin et al. 2009 ) was used to group the studies according to study design. Only studies at evidence ‘level IV or level III-2 and III-3’ were included. Studies were grouped according to study types: models, time-series, case studies, ecological studies, evaluations, expert consensus, descriptive studies, prospective and retrospective cohorts.

No studies were excluded in the analysis for quality reasons if the eligibility criteria were met, and the limitations and possible biases in such studies are reported in the results section. The analysis grouped studies into four categories based on the purpose of the surveillance approach under investigation: (A) outbreak prediction/detection; (B) trend monitoring; (C) both outbreak prediction/detection and trend monitoring; and (D) low/non-endemic countries.

A total of 1116 studies, including duplicates, were identified during the electronic search as potentially relevant to the research question. After screening of titles and abstracts, 90 studies remained eligible. Full assessment of the text eliminated 54 further studies, leaving 36 studies included (Figure ​ (Figure1). 1 ). Data of the 36 studies were extracted to a table (Table ​ (Table1), 1 ), also assigning a unique identifier number for each study.

Flow chart of articles included and excluded.

When grouped according to purpose of the surveillance system studied and compared with the 2008 review, an increase in research interest in early outbreak detection was apparent, particularly in endemic countries: (A) outbreak prediction or detection (14 studies/previously 5); (B) monitoring dengue trends (4 studies/previously 6); (C) outbreak prediction and trend monitoring (9 studies/previously 7); and (D) non-endemic countries (9 studies/previously 6). Within each of these categories, key components essential for improving surveillance in endemic and non-endemic countries were identified. The detailed findings are summarised and presented in Table ​ Table1 1 .

Surveillance systems for outbreak detection and/or prediction (Groups A and C)

Most of these studies were from highly endemic settings and were intended to predict or detect outbreaks at an early stage.

Using electronic event-/search query-based surveillance for early detection of increased dengue activity

Two studies investigated the value of data quantifying the numbers of internet searches seeking dengue information in a number of countries (Bolivia, Brazil, India, Indonesia and Singapore; studies 1 and 2), by comparing with epidemiological data from the surveillance system using time-series analysis. The curve of the search queries over time was similar to the epidemic curve constructed from surveillance data, underlining the usefulness of this new and relatively simple approach. Study 13 used a real-time electronic approach based on Health Map in order to enhance timeliness and outbreak detection and to provide an added value for monitoring the ongoing spread of dengue.

Using the appearance of a new dengue serotype/genotype as an alert signal for dengue outbreaks

Six studies investigated serotype changes as a dengue outbreak signal (studies 3–8) using virus surveillance information, analysing laboratory data (genotyped or sequenced data) or hospital data (severity of cases) and examining how these correlated with the number of reported cases or dengue incidence. Five of these studies (3–7) also analysed whether a serotype shift or a clade replacement was positively associated with a subsequent epidemic. The sixth study investigated the correlation between population-wide serotype-specific data and an increase in cases (study 8) and showed that outbreaks occurred following the introduction of new serotypes in specific islands.

Examining these studies in detail, retrospective studies in Singapore (studies 3, 5) found that a serotype switch from DENV-2 to DENV-1 in 2004/2005 was associated with the 2005 epidemic. However, according to Schreiber (study 6), viral genome sequencing would not have been sufficient to predict this outbreak. A switch from DENV-1 back to DENV-2 in early 2007 was used as a warning sign and led to response actions that were believed to have reduced the impact of an outbreak 6 months later. A clade replacement within DENV-2 was also considered a contributing factor to the 2007 Singapore outbreak (study 3) and another outbreak at the end of 2010 (study 4). Similarly, three surveys in Surabaya (Indonesia) investigated prospectively the correlation of DENV type and disease incidence. Here, an increase in case numbers in 2010 was attributed to a genotype shift in DENV-1 from genotype IV to I between April and September 2009 (study 7). Retrospective analysis of serotype-specific surveillance data in the Pacific region (study 8) demonstrated that the rapid replacement of DENV-1 by DENV-4 in the region was associated with dengue outbreaks in 2008 and 2009 in Kiribati, New Caledonia, Samoa, Tonga and other islands.

Using syndromic surveillance to create alert signals for dengue outbreaks

Five studies investigated the value of syndromic surveillance for early outbreak detection. These included a comparison of community-based fever surveillance with surveillance of school absenteeism in Peru (study 9) and two studies in French Guiana (studies 10 and 12) that described the advantages of reporting dengue cases using a syndromic case definition compared with routine reporting. These two French Guiana studies and another in Madagascar (study 14) used sentinel sites and reported higher sensitivity and outbreak early warning capacity compared with the routine reporting systems (which were based on laboratory surveillance and passive case reporting). Studies 10 and 12 highlighted the need for maintaining the traditional surveillance and considering the increased potential for false alerts in syndromic surveillance systems.

The prospective study in Peru indicated that community door-to-door fever surveillance had higher sensitivity than school absenteeism records as an indicator for dengue (study 9); the community-based fever cohort captured twice as many cases as the school-based approach.

In French Guiana (study 10), the syndromic clinical surveillance in a military population and the routine laboratory reporting systems were found to be complementary: the syndromic approach detected an outbreak 3–4 weeks earlier and was six times more sensitive than laboratory-based surveillance, but the specificity was lower in the former. Further analysis (study 11) using CDC criteria ( CDC 2001 ) showed that the ideal reporting time was often not achieved due to barriers at data entry and that an increased risk of false alerts needed to be considered. However, all respondents perceived that this system detected outbreaks adequately and subsequent countrywide introduction of sentinel-based syndromic reporting in French Guiana identified 80 signals for confirmed cases and 64 for clinical cases and predicted three major epidemics (study 12). In Madagascar, a sentinel-based syndromic surveillance system for six diseases was evaluated: it detected ten outbreaks, five were confirmed and two of which were dengue (study 14).

Use of other sentinel site-based approaches to increase capacity for outbreak detection

Three studies analysed sentinels sites for early outbreak warning, either in the form of sentinel-based reporting and virus surveillance (Cambodia, study 20) or for non-endemic countries (studies 22, 23, see group D below). One study described an enhanced routine surveillance system in Puerto Rico by motivating public health staff, which resulted in an increase in reported dengue incidence three times above the incidence during the two most recent epidemics in 1994 and 1998 (study 21). In Cambodia, passive surveillance plus sentinel site surveillance including virus surveillance increased the sensitivity of detecting outbreaks (defined as numbers of cases exceeding two standard deviations [SD] above the mean) although the response was delayed, mainly due to inadequate financial management (study 24).

In Europe, ten new strains of dengue viruses were detected in travellers returning from Africa, and increased observation of dengue in travellers by surveillance networks (TropNetEurop) was correlated with outbreaks documented in national data (study 23).

Surveillance for describing endemic/epidemic trends (Group B and C)

These surveillance systems under investigation were mostly population-based and passive. Some included additional sentinel sites or virus surveillance but they were used only to monitor viral trends and were not applied to early warning.

Four cohort-based studies calculated the level of underreporting, either using capture–recapture approaches comparing two independent surveillance systems or by comparing cohort-based data with the national routine reporting. The expansion factor indicating the level of underreporting was calculated to be:

• 14–28 times in Nicaragua for a paediatric cohort (study 15)
• 8.7 times in Thailand (2.6 times for hospitalised cases) (study 16)
• 9.1 times in Cambodia (1.4 for hospitalised cases) (study 16, 18)
• 3.9–29 times in Cambodia following a capture–recapture analysis (study 17)
• 1.1–2.4 times in Cambodia following a capture–recapture analysis of hospitalised cases (study 17)

The results demonstrated remarkably high levels of underreporting in the surveillance systems, particularly for non-hospitalised cases. It was a common experience that a large proportion of the affected population was not captured by passive routine reporting (e.g. non-users of health services, users of private/traditional sectors or certain age groups (e.g. adults in Cambodia).

Four evaluations of routine dengue surveillance systems (studies 24–27) in 6 countries (Brazil, Bolivia, Cambodia, Indonesia, Maldives and Thailand) were conducted using a similar protocol for evaluations based on CDC Guidelines ( CDC 2001 ). Both trend monitoring and outbreak detection were evaluated. All evaluations found that a clear understanding of the objectives of the surveillance system by all stakeholders was crucial. The routine reporting systems – some of them with laboratory support – were perceived to be useful for trend monitoring and national planning but, as they did not apply appropriate thresholds/alert signals or include additional surveillance components, they had little capacity for early outbreak detection. In particular, reporting timeliness was perceived to be low, ranging from a few days for notification in the Maldives (study 27) to six to seven weeks until data analysis in Cambodia (study 24). Moreover, the responses were delayed, as shown in the Maldives, where no threshold for taking action was implemented, and in Cambodia, where lack of sufficient financial management and other constraints undermined any response to the alert signal of ‘increased transmission (above two SD) in low transmission season’. In Thailand, where the system relied exclusively on clinically confirmed cases, respondents felt that outbreak responses were delayed because decision-makers did not trust the data and feared false alerts (study 24). All evaluations reported that timeliness could have been increased by electronic reporting or simplified reporting forms and that data analysis should have been performed at the lowest possible level (e.g. every district, once per week), given that sufficient capacity was available.

Dengue surveillance in low/non-endemic countries (Group D)

The value or effectiveness of primarily laboratory-supported active dengue surveillance systems in non-endemic settings was described in several studies from Asia and Europe. Timeliness of the system and laboratory support were reported to be crucial elements.

Three European studies described the recent detection of dengue in France (study 30) and in Croatia (study 29) and imported dengue cases in Italy (study 28). In Croatia, the notification of returning travellers led to the detection of autochthonous cases, while survey-based investigations revealed additional cases (29).

An evaluation of routine reporting in Korea (study 31) reported a 2- to 15-day delay from disease onset to reporting, which was shortened when electronic reporting components were introduced.

Four studies from Taiwan (studies 32–36) demonstrated the effectiveness of linking routine reporting with strong laboratory support and active and syndromic reporting elements in monitoring epidemiological, virological and clinical trends. Airport fever screening (studies 32 and 36) detected around 45% of imported dengue cases, but any impact this might have had on subsequent autochthonous transmission could not be determined.

Key findings

A greater number of the studies included in the present study (19/36) were performed in Asia than in the Americas (8/36; previously 17/24), illustrating a shift in research attention to Asia from the Americas since the 2008 review, when 17 and 6 studies, respectively, of 24 were recorded. In the present study, four studies (4/36) had a global focus, one study was from Africa and three studies were from Europe, most likely reflecting the global spread of, and consequent interest in, dengue disease in these regions in recent years.

Tools for trend monitoring (Group B and C), and as baseline for ‘excess reporting’ for outbreak detection

The surveillance systems deployed for this purpose were mainly population-based and passive. Some included additional sentinel sites or virus surveillance, but in those cases, the data were used only to monitor viral trends and were not applied to early warning. Four cohort-based prospective studies calculated an expansion factor with a range between 1.1 and 2.6 for inpatients in Cambodia and Thailand, respectively, and between 3.9 and 29 in Cambodian, Nicaraguan and Thai cohorts for non-hospitalised cases.

The results demonstrate remarkable levels of underreporting in the surveillance systems, particularly for non-hospitalised cases. It was a common experience that a large proportion of the affected population was not captured by passive routine reporting (e.g. non-users of health services, users of private/traditional sectors or certain age groups, e.g. adults in Cambodia). However, while less than satisfactory, this does not mean that such a system is entirely inadequate, because as long as it is accurately reflecting the disease trend, it may still be used effectively as a baseline for detecting excess reporting (e.g. more than 2xSD above the mean of the previous 5 years) and thus outbreak detection. In the context of a public health system, it is not clear how sensitive surveillance data need to be (i.e. what is an acceptable level of under-reporting) in order to fulfil the dual purposes of reflecting disease trends accurately and providing a baseline for outbreak early alert. The studies reviewed here indicated that underreporting to a limited extent can be tolerated in high endemic settings, as long as the data are geographically representative and, ideally, laboratory confirmed as dengue. The calculation of an expansion factor enables a more accurate value for the national burden of disease, which is important for targeting public health measures and advocacy.

In the earlier systematic review ( Runge Ranzinger et al. 2008 ), the sensitivity of the DF/DHF/DSS case classification was considered to be too low (studies 20, 24–26), especially for DHF cases ( Bandyopadhyay et al. 2006 ). With the new WHO dengue case classification, described in the WHO dengue guidelines (World Health Organization and the Special Programme for Research and Training in Tropical Diseases (TDR) 2009) and the Handbook on Clinical Management of Dengue ( WHO 2012 ), this problem has been overcome, because the new WHO dengue case classification classifies according to disease severity, permitting more sensitive reporting of severe disease and allowing comparison of data across all regions ( Barniol et al. 2010 , Horstick et al. 2012 , Horstick et al. 2014 ) as described in study 27.

Four evaluations of routine dengue surveillance systems (studies 24–27) in 6 countries (Brazil, Bolivia, Cambodia, Indonesia, Maldives and Thailand) were conducted using similar protocols for trend monitoring and outbreak detection, based on the CDC Guidelines ( CDC 2001 ). However, all evaluations found that a clear understanding of the objectives of the surveillance system by all stakeholders was crucial. All routine reporting systems, with or without laboratory support, were perceived to be useful for trend monitoring and national planning. However, without the use of appropriate thresholds or alert signals or additional surveillance components to increase timeliness or sensitivity (e.g. as sentinel sites or syndromic surveillance components), they had little capacity for early outbreak detection. Improvements indicated by the evaluations were not exploited.

An appropriate alert signal with a defined threshold level (‘trigger’) for initiation of a response is crucial for any system. None of the reviewed studies investigated the specific threshold for excess reporting within a routine surveillance system. However, analysis of the included articles suggested that in general, an excess of reported cases (pattern recognition technique; Buehler et al. 2004 ) – identified through a population-based routine surveillance system – has potential for dengue outbreak prediction. Studies that evaluate sensitivity, specificity and positive predictive values of such a threshold are likely to be particularly valuable.

Throughout the studies, reporting time was slow, and without any threshold, responses were delayed while poor financial management and lack of trust in the data by decision-makers hindered further the delivery of adequate and timely response measures. Despite that, all evaluations reported that timeliness could have been increased by electronic reporting or the use of simplified reporting forms and that data analysis should have been performed at the lowest possible level (e.g. once per week in every district) if sufficient capacity was available.

In summary, the country evaluations consistently highlighted that immediate improvement is possible using a number of options, many of which are already available and easily implementable: (i) simplified data forms/data entry protocols/electronic-based reporting, (ii) clearly defined and easily understood system objectives, (iii) appropriate and regular/frequent data analysis at the lowest possible level (iv) and regular data feedback from top to bottom levels. As evidence becomes available, two additional components will be required to complete the model: (i) clearly defined and locally appropriate triggers for an outbreak response (no studies were found exploring the optimal sensitivity and specificity of such thresholds) and (ii) implementation of evidence-based response strategies.

Alert signals 1 (triggers/indicators/thresholds) for epidemic response (Group A, C and D)

Predicting outbreaks through the introduction or shift of a dengue sero-/genotype: six studies (studies 3–8) investigated serotype changes as a dengue outbreak signal demonstrated a positive correlation with the number of reported cases or dengue incidence, although the lag times could extend up to 6 months. However, viral genome sequencing alone would, according to Schreiber (study 6), not have been sufficient to predict an outbreak.

But these events are highly site-specific and are influenced by herd immunity, population size, co-circulation of additional dengue viruses and potentially numerous other factors. Moreover, only those countries with reliable serotype-/genotype-specific surveillance would be able to monitor changes in any patterns. Genotypic shifts were used as an early warning signal in Singapore prior to the 2007 epidemic and initiated an early response (study 3). Taking into consideration the possibility that publication bias (i.e. that only positive results are likely to be published) would have excluded additional studies where serotype shifts were not associated with subsequent outbreaks and that numerous potential confounding factors would have been possible in all studies, and it is not yet possible to draw any firm conclusions on the value of this as a measure in surveillance. Nonetheless, the sensitivity, specificity and positive predictive value of this parameter merit evaluation in prospective and comparative studies.

Predicting or detecting dengue outbreaks by syndromic surveillance data: Five studies investigated the value of syndromic surveillance for early outbreak detection. These included a comparison of community-based fever surveillance with school absenteeism in Peru (study 9), and two studies in French Guiana (studies 10 and 12) describing the advantages of reporting dengue cases using a syndromic case definition as compared to routine reporting. The prospective study in Peru indicated that community door-to-door fever surveillance had higher sensitivity than school absenteeism records. In French Guiana (study 10), the syndromic approach detected an outbreak 3–4 weeks earlier and was six times more sensitive than laboratory-based surveillance, but specificity was lower. However, in another study in French Guiana (11), the ideal reporting time of 60 min for a real-time syndromic surveillance approach was often not achieved due to barriers at data entry, while a risk of false alerts was expected, given the high sensitivity of the system. In Madagascar (14) and French Guiana (12), syndromic sentinel-based surveillance built on clinical syndromic case definitions showed promising results, increasing the sensitivity of dengue case detection in comparison with routine reporting and allowing the early detection of epidemic events.

Two studies investigated the value of data quantifying internet searches for dengue information carried out in a number of countries (Bolivia, Brazil, India, Indonesia and Singapore; studies 1 and 2). The curve of the search queries over time was similar to the epidemic curve constructed from surveillance data underlining the usefulness of this new and remarkably simple approach. Study 13 used a real-time electronic event-based approach based on Health Map to enhance timeliness, outbreak discovery and provide an added value for monitoring the ongoing spread of dengue.

A number of studies that were included in the earlier 2008 review also dealt with this topic; in summary, the following syndromic surveillance-based indicators were identified:

• Proportion of virologically confirmed cases (study 3, Rigau-Pérez & Clark 2005 )
• Malaria negative rate in fever patients in a malaria endemic areas ( Carme et al. 2003 , Talarmin et al. 2000 )
• Fever alerts ( Pirard et al. 1997 ; Kourı et al . 1998 )
• Clinical syndromic case definitions (study 10, 11, 12 and 14)
• School absenteeism (study 9)
• Google search queries or event-based surveillance (Study 1, 2 and 13)

Fever alert for the purpose of outbreak detection was not found to be useful in Cuba and Bolivia (Pirard et al. 1997 ; Kourı et al . 1998 ). None of the studies included in this update analysed syndromic surveillance based on laboratory parameters or the proportion of virologically confirmed cases. One study from Singapore (study 3) mentioned that during the 2007/2008 epidemic, the proportion of DENV-positive samples detected by PCR rose from 57.9% in January 2007 to 91.0% in July 2007 at the peak of transmission. A similar trend has been shown in Puerto Rico previously ( Rigau-Pérez & Clark 2005 ).

In summary, detection of increases in proportions of positive tested samples and quantification of electronic search queries are both promising approaches to dengue outbreak detection. They are inexpensive and offer near real-time data and their value for operational use should be considered and investigated. Syndromic surveillance based on a clinical case definition remains a complementary tool to national routine reporting.

Limitations

The main limitation of this review was its restriction to English, German and Spanish. However, as the bulk of literature accessible on electronic databases today is indexed in English by title and abstract, and no additional articles in other languages were found during the extensive search, the impact of a language bias is likely to be limited. While publication bias is a potential concern, by screening carefully the reference lists of assessed articles and grey literature, the bias has been reduced.

A ‘research hot spot’ in Singapore and Taiwan was identified: these two countries accounted for 10 of the total of 36 studies, potentially introducing some level of bias in the overall assessment of the published literature. Potential for bias also may have occurred with respect to the evidence demonstrating an association between newly introduced dengue serotypes and subsequent outbreaks (see below), because no studies reporting the absence of any association (i.e. new serotypes not followed by an increase in dengue; a phenomenon that is arguably, less likely to be published) were found.

Two key knowledge gaps were identified: none of the studies investigated whether the thresholds currently in use for triggering an outbreak response were at an appropriate level of sensitivity or geographical scale, and none indicated how outbreaks were distinguished from standard or ‘expected’ seasonal changes in transmission. Further research in this area remains of the highest priority and is strongly recommended.

Following the systematic review of the evidence of the value or potential of various tools or approaches in for dengue outbreak prediction or trend monitoring, the following conclusions can be drawn:

• Passive surveillance remains the backbone of disease monitoring, also providing the baseline for outbreak alert. All opportunities for improvement should be exploited to ensure that disease trends are accurately reflected. While underreporting could be tolerated to a certain extent, further research will be required to determine how much.
• The usefulness of the new dengue case classification for epidemiological use should be evaluated, as it is currently underway for its clinical use.
• Country evaluations of dengue surveillance systems should be conducted and published following CDC criteria.
• More research is necessary to identify appropriate thresholds of excess reporting that can be used to trigger an outbreak response; such studies must take into account both the geographical scale as well as the level of sensitivity.
• Appropriate additional alert signals need to be identified and tested and integrated risk assessment tools need to be developed.
• Additional well-designed and well-implemented enhancement tools (such as active surveillance components, laboratory support or motivation strategies) would strengthen surveillance.
• Shifts in dengue serotypes or genotype have considerable potential in dengue surveillance, and the value of these data merits evaluation in prospective and comparative studies. It is crucial that both negative and positive results be published to overcome publication bias in favour of positive associations.
• Syndromic surveillance approaches have potential as useful complementary tools offering increased timeliness and sensitivity but with an increased risk of false alerts. Further studies investigating laboratory parameters (e.g. the proportion of confirmed-to-requested laboratory tests) are also merited. Internet searches or electronic event-based surveillance strategies also show promise, although their operational usefulness remains to be demonstrated.
• Further research on evidence-based response strategies and cost-effectiveness is still needed.

Acknowledgments

The project was financially supported by a grant from the European Commission (Grant Number m281803) to the IDAMS network (International Research Consortium on Dengue Risk Assessment, Management and Surveillance) within the 7th Framework Programme of the European Commission and by the Special Programme for Research and Training in Tropical Diseases (TDR/WHO).

Alert signals for an unexpected increase in dengue case numbers (an ‘outbreak’) could include signals from within the dengue time series itself, or signals from external indicators associated with changes in dengue transmission. Other indicators (additional to the increase in cases) could be considered as indicators for a dengue outbreak. (Note: ‘Alert signals’ – also called alarm signals – are indicators that, at a defined threshold, are intended to ‘trigger’ a response).

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Dengue and severe dengue

• Dengue is a viral infection transmitted to humans through the bite of infected mosquitoes.
• About half of the world's population is now at risk of dengue with an estimated 100–400 million infections occurring each year.
• Dengue is found in tropical and sub-tropical climates worldwide, mostly in urban and semi-urban areas.
• While many dengue infections are asymptomatic or produce only mild illness, the virus can occasionally cause more severe cases, and even death.
• Prevention and control of dengue depend on vector control. There is no specific treatment for dengue/severe dengue, and early detection and access to proper medical care greatly lower fatality rates of severe dengue.

Dengue (break-bone fever) is a viral infection that spreads from mosquitoes to people. It is more common in tropical and subtropical climates.

Most people who get dengue will not have symptoms. But for those who do, the most common symptoms are high fever, headache, body aches, nausea, and rash. Most will get better in 1–2 weeks. Some people develop severe dengue and need care in a hospital. 

In severe cases, dengue can be fatal.  

You can lower your risk of dengue by avoiding mosquito bites especially during the day.

Dengue is treated with pain medicine as there is no specific treatment currently.

Most people with dengue have mild or no symptoms and will get better in 1–2 weeks. Rarely, dengue can be severe and lead to death.  

If symptoms occur, they usually begin 4–10 days after infection and last for 2–7 days. Symptoms may include:

• high fever (40°C/104°F)
• severe headache
• pain behind the eyes
• muscle and joint pains
• swollen glands
• rash. 

Individuals who are infected for the second time are at greater risk of severe dengue.

Severe dengue symptoms often come after the fever has gone away:

• severe abdominal pain
• persistent vomiting
• rapid breathing
• bleeding gums or nose 
• restlessness
• blood in vomit or stool
• being very thirsty
• pale and cold skin
• feeling weak.

People with these severe symptoms should get care right away. 

After recovery, people who have had dengue may feel tired for several weeks.

Diagnostics and treatment

There is no specific treatment for dengue. The focus is on treating pain symptoms. Most cases of dengue fever can be treated at home with pain medicine.

Acetaminophen (paracetamol) is often used to control pain. Non-steroidal anti-inflammatory drugs like ibuprofen and aspirin are avoided as they can increase the risk of bleeding.

For people with severe dengue, hospitalization is often needed.

Global burden

The incidence of dengue has grown dramatically around the world in recent decades, with cases reported to WHO increasing from 505 430 cases in 2000 to 5.2 million in 2019. A vast majority of cases are asymptomatic or mild and self-managed, and hence the actual numbers of dengue cases are under-reported. Many cases are also misdiagnosed as other febrile illnesses  (1) . 

The highest number of dengue cases was recorded in 2023, affecting over 80 countries in all regions of WHO. Since the beginning of 2023 ongoing transmission, combined with an unexpected spike in dengue cases, resulted in a historic high of over 6.5 million cases and more than 7300 dengue-related deaths reported.

Several factors are associated with the increasing risk of spread of the dengue epidemic: the changing distribution of the vectors (chiefly  Aedes aegypti and Aedes albopictus mosquitoes), especially in previously dengue naïve countries; the consequences of El Niño phenomena in 2023 and climate change leading to increasing temperatures and high rainfall and humidity; fragile health systems in the midst of the COVID-19 pandemic; and political and financial instabilities in countries facing complex humanitarian crises and high population movements.

One modelling estimate indicates 390 million dengue virus infections per year of which 96 million manifest clinically  (2) . Another study on the prevalence of dengue estimates that 3.9 billion people are at risk of infection with dengue viruses (3).

The disease is now endemic in more than 100 countries in the WHO Regions of Africa, the Americas, the Eastern Mediterranean, South-East Asia and the Western Pacific. The Americas, South-East Asia and Western Pacific regions are the most seriously affected, with Asia representing around 70% of the global disease burden.

Dengue is spreading to new areas in Europe, the Eastern Mediterranean and South America.

The largest number of dengue cases reported was in 2023. The WHO Region of the Americas reported 4.5 million cases, with 2300 deaths. A high number of cases were reported in Asia: Bangladesh (321 000), Malaysia (111 400), Thailand (150 000), and Viet Nam (369 000).

Transmission

Transmission through the mosquito bite

The dengue virus is transmitted to humans through the bites of infected female mosquitoes, primarily the  Aedes aegypti  mosquito. Other species within the Aedes genus can also act as vectors, but their contribution is normally secondary to  Aedes aegypti . However, in 2023, a surge in local transmission of dengue by Aedes albopictus (tiger mosquito) has been seen in Europe.

After feeding on a infected person, the virus replicates in the mosquito midgut before disseminating to secondary tissues, including the salivary glands. The time it takes from ingesting the virus to actual transmission to a new host is termed the extrinsic incubation period (EIP). The EIP takes about 8–12 days when the ambient temperature is between 25–28°C. Variations in the extrinsic incubation period are not only influenced by ambient temperature; several factors such as the magnitude of daily temperature fluctuations, virus genotype, and initial viral concentration   can also alter the time it takes for a mosquito to transmit the virus. Once infectious, the  mosquito can transmit the virus for the rest of its life .

Human-to-mosquito transmission

Mosquitoes can become infected by people who are viremic with the dengue virus. This can be someone who has a symptomatic dengue infection, someone who is yet to have a symptomatic infection (they are pre-symptomatic), and also someone who shows no signs of illness (they are asymptomatic).

Human-to-mosquito transmission can occur up to 2 days before someone shows symptoms of the illness, and up to 2 days after the fever has resolved.

The risk of mosquito infection is positively associated with high viremia and high fever in the patient; conversely, high levels of DENV-specific antibodies are associated with a decreased risk of mosquito infection. Most people are viremic for about 4–5 days, but viremia can last as long as 12 days.

Maternal transmission

The primary mode of transmission of the dengue virus between humans involves mosquito vectors. There is evidence however, of the possibility of maternal transmission (from a pregnant mother to her baby). At the same time, vertical transmission rates appear low, with the risk of vertical transmission seemingly linked to the timing of the dengue infection during the pregnancy. When a mother does have a dengue infection when she is pregnant, babies may suffer from pre-term birth, low birthweight, and fetal distress.

Other transmission modes

Rare cases of transmission via blood products, organ donation and transfusions have been recorded. Similarly, transovarial transmission of the virus within mosquitoes have also been recorded. 

Risk factors

Previous infection with DENV increases the risk of the individual developing severe dengue.

Urbanization (especially unplanned), is associated with dengue transmission through multiple social and environmental factors: population density, human mobility, access to reliable water source, water storage practice etc.

Community risks to dengue also depend on a population’s knowledge, attitude and practice towards dengue, as the exposure is closely related to behaviours such as water storage, plant keeping, and self-protection against mosquito bites.  Routine vector surveillance and control activities engaging community greatly enhances a community’s resilience. 

Vectors might adapt to new environments and climate. The interaction between dengue virus, the host and the environment is dynamic. Consequently, disease risks may change and shift with climate change in tropical and subtropical areas, in combination with increased urbanization and movement of populations.

Prevention and control

The mosquitoes that spread dengue are active during the day. 

Lower the risk of getting dengue by protecting yourself from mosquito bites by using: 

• clothes that cover as much of your body as possible;
• mosquito nets if sleeping during the day, ideally nets sprayed with insect repellent;
• window screens;
• mosquito repellents (containing DEET, Picaridin or IR3535); and
• coils and vaporizers.

Mosquito breeding can be prevented by:

• preventing mosquitoes from accessing egg-laying habitats by environmental management and modification;
• disposing of solid waste properly and removing artificial man-made habitats that can hold water;
• covering, emptying and cleaning domestic water storage containers on a weekly basis;
• applying appropriate insecticides to outdoor water storage containers.

If you get dengue, it’s important to:

• drink plenty of liquids;
• use acetaminophen (paracetamol) for pain;
• avoid non-steroidal anti-inflammatory drugs, like ibuprofen and aspirin; and
• watch for severe symptoms and contact your doctor as soon as possible if you notice any.

So far one vaccine (QDenga) has been approved and licensed in some countries. However, it is recommended only for the age group of 6 to 16 years in high transmission settings. Several additional vaccines are under evaluation.

WHO response

WHO responds to dengue in the following ways:

• supports countries in the confirmation of outbreaks through its collaborating network of laboratories;
• provides technical support and guidance to countries for the effective management of dengue outbreaks;
• supports countries in improving their reporting systems and capture the true burden of the disease;
• provides training on clinical management, diagnosis and vector control at the country and regional level with some of its collaborating centres;
• formulates evidence-based strategies and policies;
• support countries in the development of dengue prevention and control strategies and adopting the Global Vector Control Response (2017–2030) and the Global Arbovirus Initiative (2022–2025).
• reviews and recommends the development of new tools, including insecticide products and application technologies;
• gathers official records of dengue and severe dengue from over 100 Member States; and
• publishes guidelines and handbooks for surveillance, case management, diagnosis, dengue prevention and control for Member States.
• Waggoner, J.J., et al., Viremia and Clinical Presentation in Nicaraguan Patients Infected Wi1. Waggoner, J.J., et al., Viremia and Clinical Presentation in Nicaraguan Patients Infected With Zika Virus, Chikungunya Virus, and Dengue Virus. Clinical Infectious Diseases, 2016. 63(12): p. 1584-1590.
• Bhatt, S., et al., The global distribution and burden of dengue.  Nature , 2013. 496(7446): p. 504–507.
• Brady, O.J., et al., Refining the global spatial limits of dengue virus transmission by evidence-based consensus.  PLOS Neglected Tropical Diseases , 2012. 6(8): p. e1760.

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Dengue Infection During Pregnancy and Adverse Birth Outcomes: A Systematic Review and Meta-Analysis

Affiliations.

• 1 University Center for Research and Development, Chandigarh University, Mohali, India.
• 2 Medical Laboratories Techniques Department, AL-Mustaqbal University, Hillah, Iraq.
• 3 Division of Evidence Synthesis, Global Consortium of Public Health and Research, Datta Meghe Institute of Higher Education, Wardha, India.
• 4 Division of Evidence Synthesis, Global South Asia Infant Feeding Research Network (SAIFRN), Global Consortium of Public Health and Research, Datta Meghe Institute of Higher Education, Wardha, India.
• 5 Department of Chemistry and Biochemistry, School of Sciences, JAIN (Deemed to be University), Bangalore, India.
• 6 Department of Sciences, Vivekananda Global University, Jaipur, India.
• 7 Department of Medicine, NIMS University, Jaipur, India.
• 8 Chandigarh Pharmacy College, Chandigarh Group of College, Mohali, India.
• 9 Department of Chemistry, Raghu Engineering College, Visakhapatnam, India.
• 10 Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University, Dehradun, India.
• 11 Department of Biotechnology, Graphic Era (Deemed to be University), Dehradun, India.
• 12 Department of Allied Sciences, Graphic Era Hill University, Dehradun, India.
• 13 Department of Paediatrics, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pune, India.
• 14 Department of Public Health Dentistry, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Pune, India.
• 15 School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, India.
• 16 Center for Global Health Research, Saveetha Medical College and Hospital, Saveetha Institute of Medical and Technical Sciences, Chennai, India.
• 17 Evidence for Policy and Learning, Global Center for Evidence Synthesis, Chandigarh, India.
• PMID: 39245582
• DOI: 10.1002/rmv.2582

Dengue is a rapidly spreading mosquito-borne viral disease, posing significant public health challenges in tropical and subtropical regions. This systematic review and meta-analysis aimed to evaluate the relationship between maternal dengue virus infection and adverse birth outcomes. A literature search was conducted in PubMed, Embase, and web of science databases until April 2024. Observational studies examining the association between laboratory-confirmed maternal dengue infection and adverse birth outcomes such as preterm birth, low birth weight (LBW), small for gestational age (SGA), stillbirth, and postpartum haemorrhage were included. Data were extracted, and risk of bias was assessed using the Newcastle-Ottawa Scale. Random-effects meta-analysis models were used to pool data in R software (V 4.3). Twenty studies met the inclusion criteria. The pooled prevalence of preterm birth among dengue-affected pregnancies was 18.3% (95% CI: 12.6%-25.8%), with an OR of 1.21 (95% CI: 0.78-1.89). For LBW, the pooled prevalence was 17.1% (95% CI: 10.4%-26.6%), with an OR of 1.00 (95% CI: 0.69-1.41). SGA had a pooled prevalence of 11.2% (95% CI: 2.7%-36.9%) and an OR of 0.93 (95% CI: 0.41-2.14). The prevalence of stillbirth was 3.3% (95% CI: 1.6%-6.8%), with significant associations found in some studies (RR: 2.67; 95% CI: 1.09-6.57). Postpartum haemorrhage had an OR of 1.97 (95% CI: 0.53-2.69). While maternal dengue infection was associated with a higher prevalence of preterm birth and LBW, the associations were not statistically significant. Significant associations were observed for stillbirth in specific studies. Further research with standardized methodologies is needed to clarify these relationships and identify potential mechanisms.

Keywords: dengue viral infection; low birth weight; meta‐analysis; pregnancy; preterm birth; still birth.

© 2024 John Wiley & Sons Ltd.

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• Dengue and Severe Dengue, (World Health Organization, 2024), https://www.who.int/news‐room/fact‐sheets/detail/dengue‐and‐severe‐dengue .
• Dengue–Global Situation, (World Health Organization, 2024), https://www.who.int/emergencies/disease‐outbreak‐news/item/2024‐DON518 .
• E. S. Paixão, M. G. Teixeira, N. C. Maria da Conceição, and L. C. Rodrigues, “Dengue During Pregnancy and Adverse Fetal Outcomes: A Systematic Review and Meta‐Analysis,” Lancet Infectious Diseases 16, no. 7 (2016): 857–865, https://doi.org/10.1016/s1473‐3099(16)00088‐8 .
• J. Yang, A. A. Mosabbir, E. Raheem, W. Hu, and M. S. Hossain, “Demographic Characteristics, Clinical Symptoms, Biochemical Markers and Probability of Occurrence of Severe Dengue: A Multicenter Hospital‐Based Study in Bangladesh,” PLoS Neglected Tropical Diseases 17, no. 3 (2023): e0011161, https://doi.org/10.1371/journal.pntd.0011161 .
• V. H. Ferreira‐de‐Lima and T. N. Lima‐Camara, “Natural Vertical Transmission of Dengue Virus in Aedes aegypti and Aedes albopictus: A Systematic Review,” Parasites & Vectors 11 (2018): 1–8, https://doi.org/10.1186/s13071‐018‐2643‐9 .

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