DENGUE

Dengue is the most widely distributed mosquito-borne viral infection of humans and is emerging as a major burden on global public health. A recent study has estimated that 100 million clinically apparent dengue infections occur worldwide each year, with a further 300 million asymptomatic infections (13) (Figure 1). Typically dengue manifests as a relatively mild self-limited febrile illness characterized by fever, headache, muscle and joint pains, and skin rash. However the clinical spectrum also includes severe and occasionally life threatening disease. Potential complications include an unusual plasma leakage syndrome that may result in hypovolaemic shock, a coagulopathy sometimes accompanied by severe bleeding, and organ impairment (142).

Virology

Infection can be caused by any one of the four antigenically distinct but closely related dengue viral serotypes (DENV-1, 2, 3, and 4) that together constitute one subgroup of the genus Flavivirus, family Flaviviridae. The Flavivirus genus includes approximately 80 other viruses, many of which are also arthropod-borne human pathogens, among them Japanese encephalitis virus, West Nile virus and yellow fever virus.

The dengue virus is a single stranded, positive-sense RNA virus with a genome of approximately 11 kilobases. The mature virion consists of a nucleocapsid core comprising the capsid protein and the RNA genome enclosed within a lipid bilayer made up of the envelope and membrane structural proteins. The outer diameter of the spherical virus particles is about 50 nm (83). Recently immature viral particles have been described and intra-cellular viral processing has been characterized using cryo-electron microscopy (188). Conformational changes occur during maturation of the virus and are pH (189) and temperature dependent (190). Seven non-structural proteins are expressed in infected cells, but do not form part of the mature virion; the contribution of these proteins, in particular non-structural protein 1 (NS1), to pathogenesis is a major focus of current research efforts (111). NS1 is a glycoprotein with a molecular weight of 46-55 kDa that exists as a stable homodimer on the surface membrane of infected cells, and as a soluble hexamer in plasma, forming a barrel shape around a lipid core (53). Recent work has defined the assembly and antibody binding of NS1using cryo-electron microscopy (39). Soluble NS1 has been identified in plasma, sometimes in very high concentrations, from the first day of fever and for up to 14 days afterwards (38), and sNS1 levels in the acute phase have been found to correlate with dengue severity (93). NS1 and/or anti-NS1 antibodies have also been implicated in the pathogenesis of the vascular leakage and coagulopathy, although the evidence arises primarily from in-vitro studies or animal models (6, 7).

Humans are the primary vertebrate hosts in the life cycle of the virus, and other vertebrate species are generally not susceptible to infection. Aedes mosquitoes (principally Ae. Aegypti and Ae. Albopictus) are responsible for person-to-person transmission of all serotypes. Ae. Aegypti, the most important vector, is a highly domesticated, day-biting mosquito that is adapted to living in peri-urban and urban environments where humans are the main feeding target. It is found around the globe, mainly in tropical and sub-tropical latitudes, and is usually limited to altitudes below about 1000 metres. The mosquitoes acquire DENV when they bite infected individuals with significant plasma viraemia (116). The virus then replicates in the mosquito's mid-gut before disseminating widely, including to the saliva, after 7-12 days, following which onward transmission can occur when the mosquito takes its next blood feed (87). In recent years considerable global spread in dengue virus activity has been noted, largely reflecting expansion in the geographic distribution of the mosquito vectors. The recent spread of the vector Aedes Albopictus to new areas of the world including southern Europe, America and Africa may explain the rise in dengue transmission in these areas (164, 169).

All four viral serotypes can cause severe and fatal disease. Infection with one serotype is thought to elicit specific life-long immunity to that serotype but not to provide long-term cross-protective immunity to the remaining serotypes. Therefore persons living in a dengue-endemic area can experience several dengue infections during their lifetime. There is strong epidemiological evidence to indicate that second (or subsequent) infections with a different serotype can be associated with a more severe clinical disease phenotype (58, 163). In addition there is evidence to suggest that certain serotypes, particularly DENV-2 and DENV-3, are more virulent than others (43, 65, 117).

Within each serotype there is also considerable genetic variation and certain genotypes are associated with increased disease severity. Thus, introduction of the Southeast Asian DEN-2 genotype into Cuba in the early 1980's coincided with documentation of the first cases of dengue haemorrhagic fever (DHF) seen in the Americas (125), and spread of this strain through the region was associated with further outbreaks of severe disease. In contrast, no cases of dengue haemorrhagic fever occurred during an epidemic caused by the American genotype of DEN-2 in Peru in 1995, even though the majority of infections were secondary (170).

Epidemiology

Epidemics of dengue like disease have been reported at varying intervals over the last 200 years from different locations across Asia, Africa and North America. Contemporary reports of these early epidemics describe self limited, febrile illnesses associated with rash, vomiting, and prominent muscle and joint pains (130). Although more severe and sometimes fatal presentations were occasionally documented, the general impression of dengue fever (DF) as a disease of minor clinical significance persisted until the 1950's when a serious clinical syndrome with a high mortality began to emerge among children in Southeast Asia. This syndrome, characterized by vascular leakage and bleeding, was initially thought to be a new disease. However in the 1960's the syndrome was identified as a severe manifestation of dengue infection and was given the name dengue haemorrhagic fever (56, 60), although more recently the terminology for classification of dengue has been revised.

Over the next 20 years the incidence of dengue haemorrhagic fever increased rapidly in Southeast Asia, eventually becoming hyperendemic in most Asian cities, with major epidemics superimposed on the regular background transmission pattern at 3 to 5 year intervals.

The geographic distribution of the dengue viruses and their mosquito vectors has continued to expand and severe disease associated with dengue has emerged as a significant problem in the Pacific region and the Americas as well as in Asia (51, 99). Reports of dengue outbreaks in Africa are also increasing (33), and seroprevalence studies indicate that all 4 dengue serotypes are circulating in many locations across the African continent (42, 45, 47). However, reports of large epidemics and cases of severe dengue are rare. This may be due to under-reporting or failure to recognize the severe cases as part of the dengue spectrum, or may reflect the existence of protective genetic host factors in African populations, or low vector competence and reduced transmission efficiency (4). The eastern Mediterranean area has also seen an increase in dengue in the last three decades, and recent outbreaks in Yemen, Saudi Arabia and Pakistan. In 2012 the first European outbreak of dengue since the 1920's occurred in Madeira, Portugal, with over 2000 cases documented and 120 hospitalizations (155). At the present time, some 2.5 billion people living in tropical and sub-tropical areas of the world are at risk for infection, and an estimated 100 million clinically apparent dengue infections occur annually. Dengue is also a leading cause of hospitalization and death for children in many of the more than 100 tropical countries where the disease is now common (13). Important risk factors that are considered to influence disease severity include the serotype and strain of the infecting virus as mentioned above, as well as the age, immune status, and genetic predisposition of the patient. The age distribution for severe dengue among dengue-infected patients is bimodal. Infants commonly present at a peak age of approximately 7 months with primary antibody responses; they are always born to mothers who are dengue immune and the age at presentation appears to be related to the level of antibody acquired from the mother (25, 81, 143). Young children aged between 1 and 5 years rarely develop severe disease but a second peak occurs among older children and young adults and is almost invariably associated with secondary antibody responses. In hyperendemic situations, with repeated exposure to multiple serotypes from early childhood, the peak age for severe dengue is around 10 - 12 years. However in environments with lower endemicity adults may be the predominant group affected during an outbreak, depending on previous exposure and immunity (54). Plasma leakage is more likely to occur, and to be severe, in children than in adults (55, 61), probably because the microvasculature is intrinsically more permeable in the young and matures with age (45). Women also have a lower threshold for vascular leak than men and, although infection rates between the sexes are similar in endemic areas, a preponderance of females has been noted among those with dengue shock syndrome and among fatal cases (57). A large retrospective study of 130,000 hospitalized dengue patients in Vietnam identified female sex and age less than 5 years, as risk factors for poorer outcomes (5).

Ethnicity has been highlighted as another possible risk factor for severe dengue; black individuals were less likely to experience dengue hemorrhagic fever than white individuals during well-documented epidemics in Cuba (18, 47). Similarly nutritional status has been identified as a possible risk factor, with well-nourished children having a higher risk for severe disease than children with malnutrition (114, 134). Elderly patients and those with co-morbidities including diabetes and hypertension have also been found to be at an increased risk of severe dengue (119).

Various genetic markers for dengue hemorrhagic fever have been examined. Several groups have identified specific HLA alleles associated with both susceptibility and resistance to severe dengue disease (94, 147). In addition various polymorphic non-HLA host genetic factors that might influence susceptibility have been investigated; variations in the vitamin D receptor and Fc gamma receptor II genes conferred some protection from dengue hemorrhagic fever, but no associations were found with polymorphisms in the mannose binding lectin, interleukin-1, and interleukin-1 receptor antagonist genes and disease severity (95). Other genetic association studies have shown the potential role of ICAM-3 grabbing nonintegrin (DC-SIGN1, encoded by CD209), which is a major dengue dendritic cell receptor. A strong association between the promoter variant of CD209 (G allele variant of DCSIGN1-336) was found to be protective against severe disease (133). A genome-wide association study in Vietnam has identified two distinct loci, MICB and PLCE1 that were associated with severe disease (80), and a further study confirmed these loci were also associated with less severe forms of dengue, as well as with dengue in infants (174).

Clinical Manifestations

Dengue virus infection can cause a wide variety of clinical manifestations ranging from asymptomatic infection to life-threatening hypovolaemic shock. Previously, symptomatic disease was separated into two main clinical syndromes: dengue fever, a non-specific febrile illness; and dengue hemorrhagic fever, an illness characterized by increased vascular permeability and altered haemostasis resulting respectively in capillary leak syndrome and hemorrhage. However, recognizing that DENV infection causes a spectrum of disease manifestations rather than two separate clinical syndromes, the World Health Organization (WHO) published revised guidelines for case classification in 2009. The new WHO system classifies patients as having dengue, dengue with warning signs, or severe dengue (175) (Table 1). This set of 'warning signs' were intended to help clinicians identify patients who may develop complications during the critical phase of the illness (Figure 2).They were derived mainly from a study of 2000 patients with dengue in Asia and Latin America, but as only a small number of patients progressed to severe disease during the study, further work is ongoing to validate these findings (3).

Following the bite of an infected mosquito there is a 3-15 day incubation period (commonly 5-7days), after which symptoms begin abruptly and typically follow 3 phases – a febrile phase, a critical phase and a spontaneous recovery phase. Increased vascular permeability and altered haemostasis are the most frequent complications encountered during the critical phase, but a number of other severe manifestations involving specific organs are increasingly being recognized and will be described separately.

Febrile phase

This phase commences with sudden onset of high fever and non-specific constitutional symptoms. Anorexia, nausea and vomiting are prominent features. Adults are often more symptomatic than children, complaining of headache, retro-orbital pain, and severe myalgia and arthralgia. Other non-specific symptoms may include upper respiratory symptoms, diarrhea or constipation, and dysuria. On examination, lymphadenopathy is common, and conjunctival suffusion and an evanescent macular rash may be noted. Hepatomegaly is often present, but palpable splenomegaly is unusual. The high fever persists for about 5-7 days and usually terminates abruptly. A maculopapular rash may appear at around this time particularly in older children and adults. Sometimes this convalescent rash may be extremely florid, with an intensely erythematous appearance interspersed with islands of pale skin. The rash fades after a few days, although desquamation and pruritus may follow. Adults often complain of extreme tiredness for weeks after recovery and convalescence may be complicated by depression.

The differential diagnosis of dengue includes other arboviral infections, as well as measles, rubella, enterovirus infections, adenovirus infections, and influenza. Other differential diagnoses that should be considered depending on local disease prevalence include typhoid, malaria, leptospirosis, hepatitis A, rickettsial diseases, and bacterial sepsis. Certain laboratory tests may be helpful. Patients with dengue are usually leucopenic during the first few days, but may go on to develop a lymphocytosis, often including many atypical lymphocytes. Some degree of thrombocytopenia is common and hepatic transaminases are often moderately raised.

Critical Phase

The critical phase occurs around the time of defervescence, usually between days 3–7 of illness, when the temperature drops to 37.5–38^oC or less. While most patients start to improve at this time, a small proportion develop complications, in particular a vasculopathy characterized by endothelial dysfunction and plasma leakage, manifested by a rising haematocrit and development of serosal effusions. Ultrasound studies have demonstrated that pleural effusions, ascites, and gall bladder wall oedema are often present in the critical phase and are associated with disease severity (12, 31, 139), but subclinical fluid accumulation can be detected as early as day 2-3 of fever (146). It is likely that a slow plasma leak occurs during the febrile phase, balanced by increased lymphatic return acting together with compensatory cardiovascular, renal and adrenal mechanisms to maintain normal plasma volume. However, as leakage progresses during the transition to the critical phase these processes result in peripheral vasoconstriction and a rise in the diastolic pressure towards the systolic pressure so that the pulse pressure narrows. When the pulse pressure narrows to ≤ 20 mm Hg the patient is classified as having dengue shock syndrome (DSS) (176). A rapid weak pulse, impaired peripheral perfusion and lethargy or restlessness are usually apparent by this stage. Interestingly however, some patients do not develop a significant tachycardia and the severity of plasma leakage may be underestimated if there is failure to monitor the blood pressure and peripheral perfusion carefully during the critical time around defervescence. If fluid resuscitation is not instituted promptly when the pulse pressure narrows the ongoing intravascular depletion of plasma becomes critical, the systolic pressure falls rapidly, and irreversible shock and death may follow.

Haemorrhagic manifestations can occur at any stage of the disease, in both uncomplicated and severe dengue, but are usually limited to presence of skin petechiae or development of bruising at sites of minor trauma. Mucosal bleeding, e.g. epistaxis, gum or gastrointestinal bleeding, is not common but may occasionally be severe. Menorrhagia is noted sometimes in post-pubertal girls, and life threatening uterine haemorrhage has been reported in pregnant women (9, 149). Intracranial haemorrhage is a very rare complication. In children severe mucosal bleeding tends to occur in those with profound or prolonged shock, and is often accompanied by metabolic acidosis and laboratory evidence of disseminated intravascular coagulation. Rarely, massive gastrointestinal bleeding causing haemorrhagic shock (as distinct from shock due to plasma leakage) has been reported in adults, and may occasionally be fatal (27, 160).

Until or unless shock develops, clinical identification of increased vascular permeability is difficult but the degree of haemoconcentration as judged by serial haematocrit measurements can be a useful marker of the severity of vascular leakage (105). Platelet counts fall over the illness course reaching a nadir around the time of maximal vascular leakage (158). Profound thrombocytopenia with counts below 20,000 platelets/mm³ is common in severe disease (17), although rarely associated with spontaneous bleeding despite the fact that the remaining platelets are also dysfunctional (107, 145). Lower platelet counts have also been found to correlate more closely with the severity of vascular leakage rather than bleeding (182). An increase in the activated partial thromboplastin time and a reduction in fibrinogen levels are the two most consistent coagulation abnormalities detected, but the true nature of the coagulopathy remains to be fully characterized (82, 100, 179). Plasma protein concentrations, particularly albumin concentrations, may be low in dengue, but severe hypoproteinaemia is usually a feature of dengue shock syndrome (180). Evidence of renal compromise, markedly raised liver enzymes, and metabolic acidosis may be seen in those with profound shock and are poor prognostic signs.

Recovery Phase

The increased vascular permeability and abnormal haemostasis are transient and usually resolve within 48 – 72 hours of becoming clinically apparent. Spontaneous reabsorption of the excess fluid from the interstitial compartment begins, usually around day 6-8 of illness, and progresses rapidly. With appropriate fluid management the outcome is generally good and convalescence is usually short and uneventful. An erythematous rash with patches of normal skin or 'islands of white' may become visible in older children and adolescents during recovery. In experienced hands and with meticulous attention to detail, mortality rates of <1 - 5% are achievable even for patients with established dengue shock syndrome managed in endemic areas. However, fluid management in infants, the elderly, and those with underlying diseases is not straightforward, and death rates are correspondingly higher in these groups. In many cases volume overload is a significant contributor to the adverse outcome. Rapid improvement in the platelet count is usually apparent during the second week of illness, along with other laboratory parameters and the general clinical improvement.

Other organ involvement

In addition to the major clinical presentations discussed above, patients may develop severe organ involvement including cardiac, hepatic or neurological syndromes, and these are included in the definition of severe dengue in the new WHO classification. Such presentations are rare however, and represent only a tiny proportion of the total disease burden (52).

A number of cardiac manifestations ranging from myocardial dysfunction to arrhythmias, and occasionally fulminant myocarditis, are now recognized to occur (186). Myocardial impairment of varying degrees has been described using several different techniques, and in some cases likely contributes to the haemodynamic instability observed during the critical phase (106, 185).

Mild to moderate hepatomegaly with a rise in a transaminase levels is frequently seen in dengue patients, with more marked derangements usually associated with more severe disease (159, 178).

Fulminant liver failure, sometimes associated with severe haemorrhage and/or encephalopathy, occurs infrequently and mortality tends to be high (112). A possible association with aspirin and/or traditional medicine consumption has led some clinicians to suspect a Reye like syndrome, and occasionally increased ammonia levels have been documented. In view of this, and the possible effects on platelets and haemostatic competence, aspirin is contraindicated in patients with dengue.

Patients with major neurological involvement usually present with coma and convulsions after several days of a non-specific febrile illness, most often indicating an encephalopathy rather than a direct neurotropic effect of the virus. Biochemical derangements, hypoxia, cerebral oedema and intracranial haemorrhage may all contribute to this picture (23). However, there is now increasing evidence that a true encephalitis with direct viral invasion of the central nervous system does occur, albeit infrequently (20, 97, 144). Given the similarities between dengue and Japanese encephalitis viruses this neurotropism is not unexpected. Spinal cord involvement and peripheral neuropathy have also been reported occasionally (74, 136).

Laboratory Diagnosis

During the febrile stage of the illness virus isolation, using mosquito inoculation or cell culture techniques, was traditionally considered the optimal method for laboratory diagnosis of dengue. However since these techniques are time consuming and require specialist laboratory facilities, detection of viral RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), has now become the preferred method. Viral RNA can be detected up to about day 5-6 of illness, rarely day 8, but as the viraemia falls this method becomes less sensitive (167). Several different PCR based techniques are available which now permit identification of the serotype, and quantification of the level of plasma viraemia, with good sensitivity, specificity and limit of detection, especially for real-time RT-PCR (64, 76, 88). Multiplex real-time RT-PCR that allows detection of DENV serotypes in a single reaction has also been developed (69).

Serological diagnosis is complicated by the existence of cross-reactive antigenic determinants shared by dengue and other members of the Flavivirus family. Capture ELISA techniques are now the most commonly employed method for serological diagnosis, but in view of this cross-reactivity, ideally tests for other locally prevalent flaviviruses should be performed in parallel with dengue virus serology. In regions where Japanese encephalitis virus (JEV) co-circulates with dengue, quantitative techniques that measure the relative levels of anti-dengue and anti-Japanese encephalitis virus IgM and IgG can differentiate between the two infections (70). Although IgM can be detected as early as 3-5 days after illness onset, paired samples demonstrating seroconversion are required to make a definitive diagnosis. The pattern of antibody responses can be helpful in differentiating between primary and secondary infections; an early IgM response detectable during the febrile period or within 2-3 days of defervescence with a slower IgG response rising over several weeks suggests a primary infection, while a rapid and marked rise in IgG levels during the first two weeks of illness in association with a less marked rise in IgM suggests a secondary infection. A number of methods using different cutoffs for the IgM/IgG ratio are suggested, but since these methods do not take into account the evolving nature of the immune response over time, interpretation is difficult. Other serological tests such as haemagglutination inhibition, complement fixation and plaque reduction neutralization tests can also be used to help differentiate primary from secondary antibody responses.

Several serological tests have been developed into commercial kits intended to assist with rapid diagnosis (162, 184). In addition commercial NS1 ELISA kits have been developed, which are highly specific in early disease, although sensitivity is variable depending on the viral serotype and/or the presence of a concurrent humoral immune response (62, 157). Rapid tests combining NS1 detection with an antibody test are also available, which can extend the window of detection (44).

Pathogenesis

The pathogenic mechanisms underlying the characteristic clinical features of severe dengue remain poorly understood. Although a number of animal models for severe dengue exist, the relationships to human clinical disease are uncertain, and thus the findings from studies using these models must be interpreted with caution. Dengue viruses have been shown to infect and replicate in a wide range of cells of endothelial and epithelial derivation in vitro, but few data are available to establish the major sites of dengue virus replication in humans. Peripheral blood mononuclear cells do support infection, and dengue virus antigen has also been detected in cells of the macrophage-monocyte lineage in the lymphoid organs, lung and liver from patients dying from dengue (11, 75, 129). Infection of dendritic cells was observed in a skin biopsy from a dengue vaccine recipient who experienced a dengue-like illness (183). However, there is no evidence that the dengue virus infects endothelial cells in vivo and only minor non-specific changes have been demonstrated in histopathological studies of the microvasculature (11).

A complex interaction between host and virus seems to determine the intensity of viral replication, and it is thought that this influences disease severity. Most researchers have found higher levels of viraemia, viral RNA, and/or free circulating NS1 in patients with dengue hemorrhagic fever as compared to dengue fever (91, 163, 167). Based on well established epidemiological evidence indicating that dengue hemorrhagic fever/severe dengue occurs predominantly in patients experiencing a second infection with a dengue virus serotype different from that encountered during their first infection or in infants with transmitted maternal antibody (58), and on in vitro studies showing that dengue infection of mononuclear cells is enhanced in the presence of cross-reactive but non-neutralizing antibodies (59), the theory of "antibody-dependent enhancement (ADE) of infection" was originally proposed to explain the pathogenesis of dengue hemorrhagic fever. The antibody-dependent enhancement hypothesis suggests that heterotypic non-neutralizing antibodies bind to the virus introduced in the second infection, and form antibody-virus complexes that bind to the Fc receptors of monocytes and macrophages, favoring uptake and productive infection in these cells, and leading to an increase in viral load in the early phase of the infection. Viral antigens are then presented by the infected cells, resulting in priming and stimulation of T lymphocytes. Cytokine release and activation of other immune cells follows, and an immunopathogenic cascade is set in motion (19, 35, 141).

Rapid mobilization of serotype cross-reactive memory T cells has been suggested as an alternative mechanism to trigger the inflammatory cascade and release vasoactive molecules. In support of this theory relatively higher frequencies of cross-reactive CD8+ T cells have been found in association with more severe disease during secondary infections (109). Cells of low affinity for the infecting virus but higher affinity for other, probably previously encountered, serotypes may be less effective at clearing the infection resulting in a higher viraemia. Further work has demonstrated the T cell response was most marked to NS3 protein, with high cytokine and low CD107a (a marker of cell degranulation) predominating (36).

However, although a range of markers of immune activation have been found in association with severe disease during secondary infections (10, 48), these response patterns do not differ significantly from those occurring in other viral infections which do not progress to a shock syndrome. Secondly, as yet no specific pathway has been identified linking these immunopathogenic mechanisms with the profound derangements in microvascular permeability and haemostatic control that are seen. In recent years considerable advances have been made towards understanding the mechanisms underlying microvascular permeability, with the current concept that intrinsic permeability is regulated by the endothelial surface glycocalyx as much as by endothelial cells themselves (105). This highly anionic proteoglycan matrix is located on the luminal surface of the vascular endothelium, anchored in the plasma membrane of endothelial cells, and forms an electrostatic barrier repelling negatively charged plasma proteins away from the endothelial surface and thus effectively restricting access to underlying cellular transport mechanisms. Preliminary evidence from children with dengue shock syndrome suggests that structural or functional changes in this layer may be important in pathogenesis (180), through the binding of heparan sulfate by the DENV E protein and the NS1 antigen (6, 26). Circulating levels of free heparan sulfate are raised in plasma of dengue patients and can be detected early in the disease (151), but further efforts to link aspects of the inflammatory response to effector mechanisms at the vascular endothelial level are needed.

Complement dysregulation has been suggested to play a role in the pathogenesis through the generation of anaphylatoxins and the terminal complement complex SC5b-9 by soluble and membrane-associated NS1 (7, 16). High levels of NS1 and SC5b-9 in the plasma of dengue patients correlated with severity and were also found in large amounts in pleural fluid of dengue shock syndrome patients along with the anaphylatoxin C5a. Other possible pathogenic mechanisms for which there is supporting data include differences in viral virulence (89, 124, 125). molecular mimicry (30, 41, 102), virus-specific plasmablast responses (46), and immune complexes (121, 167, 168). It seems likely that a complex interaction of many of these factors, together with host genetic factors, contributes to the overall disease phenotype. However, since the severe manifestations occur relatively late in the disease course, often when the infecting virus is no longer detectable in plasma, immune mediated mechanisms are thought to play a significant role in pathogenesis.

ANTIMICROBIAL THERAPY

At present no effective antimicrobial therapy exists for the treatment of dengue infections, but several different classes of viral inhibitor are under consideration, including experimental agents that inhibit viral binding and entry, intracellular multiplication and maturation, or secretion of viral particles (90, 150, 171). However the work is hampered by lack of a suitable animal model for dengue with most studies being carried out in cell culture systems that bear little resemblance to natural infections. Primates and rodents can be infected experimentally and do develop detectable viraemia but do not usually demonstrate the clinical features seen during human infection; however the use of immune deficient and humanized mice has shown some promise in better representing human pathology (152, 191). Several drugs already licensed for human use have been investigated. Ribavirin, a drug with significant in vivo activity against some RNA viruses, had little effect on viraemia in a blinded placebo-controlled study of ribavirin pre-treatment of rhesus monkeys subsequently infected with dengue (101). Pegylated recombinant alpha interferon 2a was shown to lower viraemia levels significantly and improve viral clearance as compared to placebo, when given as a single dose one day after onset of experimental DEN-2 viraemia in rhesus monkeys, but evidence from human studies is lacking (1). Another drug with potential antiviral properties is chloroquine; apart from its well-known anti-malarial effects, chloroquine exerts inhibitory effects on several viruses, such as retroviruses, coronaviruses and flaviviruses (135). However a randomized controlled trial of chloroquine in early dengue showed no clinical benefit, change in viraemia or NS1 clearance times (156). Two more recent randomized and blinded human trials assessing the safety and efficacy of other antiviral agents were also disappointing; neither balapiravir in Vietnam (115), nor celgosivir in Singapore (115), demonstrated any clinical benefit in patients with confirmed dengue, or altered virological or host inflammatory kinetics.

The search for effective dengue therapeutics continues, and with several novel treatments under investigation plus improved animal models and human infection models in development, this is a rapidly changing field (172).

ADJUNCTIVE THERAPY

The only treatment currently available for symptomatic dengue infections is supportive, with a particular focus on careful fluid management. No specific pharmacological therapy has been shown to be beneficial but the options investigated to date have been limited. Carabazochrome, an agent thought to influence haemostasis and vascular function, did not influence the severity of plasma leakage in a randomized controlled trial (154). Corticosteroid therapy, often employed for the treatment of dengue hemorrhagic fever in the early days, showed no convincing benefit on mortality from shock in several small randomized controlled trials during the 1970's and 1980's (148, 153). More recently a randomized, blinded, placebo-controlled trial of oral prednisolone in early dengue infection, with the primary goal of assessing safety during the phase of active viral replication, failed to show any effect on clinical outcome (151). Although the trial did not show evidence of harm with early prednisolone use compared to placebo, there was also no evidence of efficacy in preventing dengue shock syndrome. Currently, there is an ongoing study of statin therapy in early dengue, and it is speculated that the anti-viral and endothelial stabilizing properties of lovastatin could modulate disease severity (103, 173).

The basic principles of fluid management are described below. Paracetamol (10-15 mg/kg/4-6 hourly) is the preferred antipyretic agent for control of fever. Aspirin and non-steroidal anti-inflammatory drugs should not be administered because of their anti-platelet effects and, in the case of aspirin, because of the possible association with Reye's syndrome and severe hepatic involvement.

Parenteral fluid therapy

During the first few days of illness oral fluids should be encouraged to offset losses from high fever and vomiting. Although patients with dengue without shock may require small volumes of parenteral fluid, particularly if vomiting is severe, the primary indication for parenteral fluid therapy is in patients with significant vascular leakage leading to cardiovascular compromise. Prompt restoration of an adequate circulating plasma volume is essential, followed by fluid therapy to support the circulation at a level just sufficient to maintain critical organ perfusion until vascular permeability reverts to normal. Attempts to restore the plasma volume to normal are likely to precipitate significant fluid overload and to contribute to mortality, especially in health-care settings without access to advanced respiratory support.

The current WHO management guidelines recommend use of crystalloid solutions in the first instance, followed by plasma or colloid solutions for dengue shock syndrome patients with profound or unresponsive shock (175). Formal research studies broadly support this approach, although there are some indications that early intervention with colloid solutions may improve outcome in those with severe shock (37, 113, 181). The majority of children with shock respond to 10-20 ml/kg/hr of a crystalloid solution given for 1-2 hours, followed by a schedule reducing to maintenance levels over 6-8 hours. However, children have a relatively high requirement for fluid, and it is important for adult patients, the obese, the elderly, or those with underlying diseases, that the volume of fluid given is carefully adjusted to suit the individual circumstances and thus limit the likelihood of precipitating fluid overload. Only isotonic crystalloid solutions, which distribute primarily within the extracellular fluid compartment and thus provide the greatest intravascular support, should be used. Intravenous fluid should be stopped as soon as the patient stabilizes, ideally before the reabsorption phase of the illness begins, in most cases within 24 - 48 hours of the onset of shock.

Further research is needed to determine whether children with profound dengue shock syndrome benefit from first-line treatment with colloid solutions. However, current practice is to give patients already being treated for dengue shock syndrome who fail to respond to crystalloid infusions (no improvement in pulse pressure or pulse rate, persisting peripheral shutdown, a rising haematocrit) 10 ml/kg of a colloid solution over one hour. Some patients with severe vascular leakage experience repeated episodes of cardiovascular decompensation over the 24-36 hours after onset of dengue shock syndrome, and may require repeated supplementary treatment with small infusions of 5-10 ml/kg of colloid. The volume given on each occasion should be kept to a minimum. The selection of which colloid to use depends on local availability and personal familiarity, but an iso-oncotic medium molecular weight preparation that combines a good initial osmotic effect with reasonable intravascular persistence is probably the most appropriate choice (49). Hyper-oncotic solutions are best avoided in patients with hypovolaemic shock in view of the recognised association with acute renal failure (14, 42). Other concerns relating to the use of colloid solutions include the possibility of adverse effects on blood coagulation, particularly given the intrinsic coagulopathy associated with dengue, and the propensity of all colloids to cause allergic reactions (165). In many dengue-endemic countries hydroxyethyl starch (HES) solutions have become the standard colloid used for managing severe dengue, since dextran solutions are no longer available and gelatin based solutions have a high potential to cause allergic reactions and are perceived to be ineffective. However in the light of recent large-scale fluid intervention studies that indicate an increased risk for acute kidney injury in adults with severe sepsis treated with hydroxyethyl starch solutions, the use of any semisynthetic colloids for fluid resuscitation in critically ill patients has become controversial (138). Dengue shock syndrome is unique however, in that the population affected typically consists of young, previously healthy persons presenting with critical dysfunction of a single organ system; given the huge global burden of disease, and the limited access to ventilatory support in many dengue-endemic areas, resuscitation with crystalloids alone might well result in increased global morbidity and mortality secondary to fluid overload and respiratory failure. Formal evaluation of the indications for and usage of colloid therapy for dengue shock syndrome is warranted.

Monitoring

All patients hospitalized with dengue warning signs must be observed closely, including regular pulse and blood pressure monitoring, until they have been afebrile for at least 24 hours without antipyretics. The critical period for the development of shock is around the time of defervescence. Assessment of the degree of haemoconcentration using serial capillary haematocrit measurements is the simplest and most accessible method for monitoring the development and severity of the vascular leak syndrome. In the first few days of the illness the haematocrit should be checked daily or twice daily, but if it starts to rise rapidly or the patient's clinical condition deteriorates, it should be checked every few hours. Similarly, a platelet count should be performed on admission and then every one to two days, but if the patient develops significant mucosal bleeding or severe thrombocytopenia is documented (platelet count < 20-30,000 mm³), more frequent measurements are advisable.

Among patients with dengue shock syndrome frequent observation of pulse rate and pulse pressure (at least hourly), peripheral perfusion, mental state and urine output, together with serial haematocrit measurements are used to assess the response to treatment and guide decision making with respect to the need for additional fluid therapy. However it is important to recognize that it is the clinical response that matters, rather than normalization of the haematocrit. If the patient is warm, well perfused and has good urine output, then a relatively high but stable haematocrit is acceptable.

Management of haemorrhagic complications

In children clinically significant bleeding occurs only rarely, usually in association with profound and prolonged shock. However major mucosal bleeding (gastrointestinal, vaginal) may occur in adults without obvious vascular leakage. For most patients close inpatient observation with regular monitoring of the platelet count is all that is necessary, although nasal packing may be required for persistent epistaxis. Individuals with profound thrombocytopenia (<20,000 platelets/mm³) but without major bleeding should be managed expectantly with bed rest and protection from trauma. Platelet concentrates are not indicated and may be harmful; thrombocytopenia improves spontaneously within a few days, the half-life of transfused platelets is markedly reduced such that they are only effective for a few hours, and the patient may develop fluid overload (79, 83, 84, 98).

Major bleeding is almost always associated with very severe or prolonged shock, and is usually from the gastrointestinal tract. Contributing factors include profound thrombocytopenia, tissue hypoxia, metabolic acidosis and disseminated intravascular coagulation. Internal bleeding may not become apparent for many hours until the first melaena stool is passed. It should be considered in all those with dengue shock syndrome who fail to improve clinically after appropriate fluid resuscitation, particularly if the haematocrit is stable or falling and the abdomen is distended and tender. Transfusion should be undertaken with extreme care because of the risk of fluid overload in these circumstances; in the event that major internal bleeding is suspected in a patient with severe dengue shock syndrome, a small volume of fresh whole blood (5-10ml/kg) should be given over 1-2 hours and the response observed. Further small transfusions may be given subsequently if there is a good clinical response and significant bleeding is confirmed. Use of platelet concentrates, fresh frozen plasma and other blood products should be guided by the platelet count and coagulation profile. Two small studies indicate that recombinant activated factor VII may be helpful in controlling life threatening bleeding but further research is needed (28, 29)

Significant mucosal bleeding is more common in adults with dengue infection, often related to the presence of gastritis or peptic ulcer disease, and may contribute to shock (27, 160). More aggressive transfusion is often necessary although care is still required to avoid the pitfalls of fluid overload.

Management of other complications

Invasive procedures should generally be avoided in patients with severe dengue because of the thrombocytopenia and coagulopathy that is usually present. However, patients with dengue shock syndrome who develop severe fluid overload sometimes require haemodynamic monitoring, artificial ventilation, drainage of pleural effusions and ascites etc. Very occasionally inotropic support is required; in a large series of dengue shock syndrome patients managed over a 10 year period from a single hospital in Vietnam, only 4% required additional haemodynamic support, with the majority recovering with standard crystalloid resuscitation or following a single colloid infusion (86). Few hospitals in the tropics have sophisticated intensive care facilities where respiratory failure can be safely managed, so every effort must be made to ensure that severe overload does not occur. Fortunately, in the majority of cases fluid overload is not severe and can be successfully treated with oxygen, bed rest, and diuretic therapy once the patient is cardiovascularly stable. Diuresis proceeds rapidly as soon as the reabsorption phase of the illness commences.

Management of unusual manifestations such as dengue encephalopathy, myocarditis or fulminant hepatitis is similar to standard management for these disorders.

VACCINES

Development of a safe and effective vaccine is essential for long-term control of dengue. A combination vaccine that simultaneously induces sustained protective immunity against all four serotypes is needed to avoid creating a situation where vaccinees might be sensitized to develop severe disease in the future rather than being protected.

The first ever phase III dengue vaccine studies have recently been reported assessing a tetravalent live attenuated vaccine (CYD-TDV Sanofi-Pasteur) in approximately 30,000 participants across Asia (22) and Latin America (166). The vaccine was generally well tolerated and showed an overall efficacy of 56.5% in children aged 2-14 years in Asia and 64.7 % in children aged 9-16 years in Latin America. However, although the neutralizing antibodies to all 4 serotypes were demonstrated among vaccines participating in a substudy this did not translate into balanced protection against all 4-serotypes, with efficacy for DENV-2 of only 35% in the Asian study and 42.3% in Latin America. Long term follow-up to assess continuing safety and further work to improve our knowledge on the immunological correlates of disease will be key next steps. If, as is likely, this vaccine proceeds to full licensure in the near future, careful consideration will be needed to decide how to deploy it safely in different epidemiological situations.

Several other live attenuated vaccines have shown promise in early human trials (40, 118). There are some concerns about possible reversion to wild-type and/or recombination between vaccine virus and circulating wild-type flaviviruses allowing the emergence of recombinants with increased virulence (137). Long-term studies looking at the genetic stability of the vaccine viruses will be essential. Various strategies are also being explored to develop genetically engineered vaccines, among them recombinant subunit vaccines, chimeric vaccines, and DNA vaccines (24, 68). With the recent discovery of a new class of broadly neutralizing antibodies that is capable of potently neutralizing all four dengue serotypes, this has opened up a new avenue for the development of a future sub-unit vaccine targeting a single conserved epitope (32).

PREVENTION

Until a safe, balanced immunogenic vaccine is widely available, prevention of epidemics will continue to rely on eradication of Ae. Aegypti and other vectors by a) environmental management to eliminate potential breeding sites, and b) biological and chemical vector control. Well-organized and rapidly responsive entomological, clinical and virological surveillance systems are essential if vector control is to be effective. Biological control methods include treatment of water containers with larvivorous fish, bacteria that kill mosquito larvae but are not toxic to humans, or copepods that consume the larva while leaving the water potable. Programs using copepods of the genus Mesocyclops have proved to be successful in the short term in several countries (34, 78). However a very high level of community participation is required for such programs to be effective (77). Also, some groups remain concerned about "contamination" of their water supplies with live organisms. Another technology showing great promise for dengue control is release of biologic and/or genetically modified mosquitoes designed to invade and displace wild mosquito populations. The intracellular bacterium Wolbachia, when introduced into Aedes mosquitoes can influence the ability of the insects to transmit dengue virus, indirectly by reducing the mosquito life span and directly by reducing viral replication in the mosquito (104, 110). Studies investigating the feasibility of Wolbachia infected mosquitoes in wild mosquito populations in Australia and Vietnam are currently underway (67, 72). Genetically modified mosquitoes are also entering field trials, with promising results from engineered male Aedes mosquitoes in the Cayman Islands (63).

Chemical control of adult vectors involves use of residual insecticide sprays and both indoor and outdoor fogging techniques. Unfortunately many mosquito species have developed resistance to the commonly used insecticides following decades of use.

In the end it is likely that a combination of host protective strategies and vector control initiatives will be required if the spread of dengue is to be contained or reversed (127).

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Tables

Table 1. Criteria for Dengue Warning Signs, Taken from the 2009 WHO Classification

Criteria for Dengue
- Abdominal Pain or tenderness - Persistent vomiting - Clinical fluid accumulation - Mucosal bleeding - Lethargy/restlessness - Liver enlargement >2 cm - Laboratory increase in HCT concurrent with rapid decrease in platelet count
Criteria for Severe Dengue
- Shock (DSS) - Fluid accumulation with respiratory distress - Severe bleeding - Severe organ involvement: ---● Liver: AST or ALT > = 1000 ---● CNS: Impaired consciousness ---● Heart and other organs

Figure 1. Global Evidence Consensus, Risk and Burden of Dengue in 2010

Figure 2. Dengue Disease Phases and Potential Complications

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