DENGUE FEVER

The virus aetiology of dengue fever was not documented until 1943–44, when Japanese and American scientists simultaneously isolated the viruses from soldiers in the Pacific and Asian theatres during World War II. Albert Sabin isolated dengue viruses from soldiers who became ill in Calcutta (India), New Guinea, and Hawaii. The viruses from India, Hawaii, and one strain from New Guinea were antigenically similar, whereas three others from New Guinea were different. These viruses were called dengue 1 (DENV-1) and dengue 2 (DENV-2) and designated as prototype viruses (DENV-1, Hawaii, and DENV-2, New Guinea C). The Japanese virus, isolated by Susumu Hotta, was later shown to be DENV-1. Two more serotypes, called dengue 3 (DENV-3) and dengue 4 (DENV-4), were subsequently isolated by William McD. Hammon and his colleagues from children with haemorrhagic disease during an epidemic in Manila, Philippines, in 1956. Although thousands of dengue viruses have been isolated from different parts of the world since that time, all fit antigenically into the four-serotype classification.

Many early workers suspected that dengue viruses were transmitted by mosquitoes, but actual transmission was first documented by H. Graham in 1903. In 1906, T. L. Bancroft demonstrated transmission by Aedes aegypti, later known to be the principal urban mosquito vector of dengue viruses. Subsequent studies in the Philippines, Indonesia, Japan, and the Pacific showed that Aedes albopictus and Aedes polynesiensis also were efficient secondary vectors for dengue viruses.

During and following World War II, Aedes aegypti greatly expanded its distribution in Asia, becoming the dominant day-biting mosquito in most Asian cities. Multiple dengue virus serotypes were also disseminated widely at that time. A dramatic increase in urbanization in the post-war years created ideal conditions for increased transmission of urban mosquito-borne diseases. These changes, plus an increased movement of people within and among countries of the region via airplane, resulted in increased movement of dengue viruses between population centres, increased frequency of epidemic activity, the development of hyperendemicity (co-circulation of multiple serotypes), and the emergence of epidemic dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) in many countries of Southeast Asia during the 1960s.

By 1975, DHF/DSS was a leading cause of hospitalization and death among children in the region. During the 1980s and 1990s, epidemic DHF/DSS continued to expand geographically in Asia. In the 1970s DHF/DSS moved into the Pacific Islands after an absence of 25 years. In the Americas, where Aedes aegypti had been eradicated from many countries as a result of efforts to control yellow fever, increased epidemic dengue fever closely followed the reinfestation of countries by this mosquito in the 1970s, 1980s, and 1990s.

Classification

Dengue viruses belong to the family Flaviviridae, genus Flavivirus. There are four serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. They belong to a larger, heterogeneous group of viruses called arboviruses. This is an ecological classification, one which implies that transmission between vertebrate hosts, including humans, is dependent on hematophagous arthropod vectors.

As are other flaviviruses, dengue viruses are comprised of a single-stranded RNA genome surrounded by an icosahedral nucleocapsid. The latter is covered by a lipid envelope, which is derived from the host cell membrane from which the virus buds. The complete virion is about 50 nm in diameter. The mature virion contains three structural proteins as follows: the nucleocapsid core protein (C), a membrane associated protein (M), and the envelope protein (E). Functional domains responsible for virus neutralization, hemagglutination, fusion, and interaction with virus receptors are associated with the E protein. Epitope mapping has demonstrated three to four major antigenic sites. Antigenically, the four dengue viruses make up a unique complex within the genus Flavivirus. Although the four dengue serotypes are antigenically distinct, there is evidence that serologic subcomplexes may exist within the group. For example, a close genetic relationship has been demonstrated between DENV-1 and DENV-3 and between DENV-2 and DENV-4.

Host

There are only three known natural hosts for dengue viruses: Aedes mosquitoes, humans, and lower primates. Viremia in humans may last 2–12 days (average, 4–5 days) with titres ranging from undetectable to more than 108 mosquito infectious doses 50(MID₅₀) ml. Experimental evidence shows that several species of lower primates (chimpanzees, gibbons, and macaques) become infected and develop viremia titres high enough to infect mosquitoes, but do not develop illness. Viremia levels in lower primates are more transient, often lasting only 1–2 days if detectable, with titres seldom reaching 106 MID₅₀ ml.

Dengue viruses are known to cause clinical illness and disease only in humans.  Mosquitoes only of species of the genus Aedes appear to be natural hosts for dengue viruses. Species of the subgenus Stegomyia are the most important vectors in terms of human transmission, and include Ae. (S.) aegypti, the principal urban vector worldwide, Ae. (S.) albopictus (Asia, the Pacific, Americas, Africa, and Europe), Ae. (S.) scutellaris spp. (Pacific), and Ae. (S.) africanus, and Ae. (S.) luteocephalus (Africa).

Transmission

Most dengue virus transmission is by the bite of an infective mosquito vector. Any of the four serotypes may cause high levels of viremia in humans (≥10⁸ MID₅₀ ml) that lasts an average of 4–5 days (range, 2–12 days). If a competent mosquito vector takes a blood meal from a person during this viraemic phase, virus is ingested with the blood meal and infects the cells of the mosquito mesenteron. After 8–12 days, depending on ambient temperature, the virus, and the mosquito, the virus will disseminate and infect other tissues, including the mosquito salivary glands. When the mosquito takes a subsequent blood meal, virus is injected into the person along with the salivary fluids. Dengue virus infection has no apparent effect on the mosquito, which is infected for life.

Ae. aegypti is a highly competent epidemic vector of dengue viruses. It lives in close association with humans because of its preference to lay eggs in artificial water holding containers in the domestic environment, and to rest inside houses and feed on humans rather than other vertebrates. It has a nearly undetectable bite and is very restless in the sense that the slightest movement will make it interrupt feeding and fly away. It is not uncommon, therefore, to have a single mosquito bite several persons in the same room or general vicinity over a short period of time. If the mosquito is infective, all of the persons bitten may become infected.

In addition to transmitting the virus to humans or lower primates, the female mosquito may also transmit the virus vertically to her offspring through her eggs. Although the implications of vertical transmission are not fully understood, it is thought to be an important mechanism in the natural maintenance cycles of dengue viruses, especially in rural and forest settings. The primary site of replication of dengue viruses after injection into humans by the feeding mosquito is believed to be dendritic cells. Other tissues from which these viruses have been isolated include phagocytic monocytes, liver, lungs, kidneys, lymph nodes, stomach, intestine, and brain, but it is not known to what extent the virus replicates in these tissues. Pathological changes similar to those observed in yellow fever, with focal central necrosis, have been observed in the liver of some patients who died of dengue virus infection. There is some evidence that the viruses also replicate in endothelial cells and possibly in bone marrow cells. Encephalopathy has been documented in dengue infection but whether dengue viruses cross the blood–brain barrier and replicate in the central nervous system is still open to question. Dengue viruses have been transmitted by blood transfusion and organ transplantation.

Seasonality and intensity of transmission

Dengue transmission usually occurs during the rainy season when the temperature and humidity are conducive for build-up of the vector population breeding in secondary habitats as well as for longer mosquito survival.

In arid zones where rainfall is scanty during the dry season, high vector population builds up in man-made storage containers. Ambient temperature, besides hastening the life-cycle of Ae. aegypti and resulting in the production of small-size mosquitoes, also reduces the extrinsic incubation period of the virus as well. Small-size females are forced to take more blood meals to obtain the protein needed for egg production. This has the effect of increasing the number of infected individuals and hastening the build-up of the epidemic during the dry season.

A number of factors that contribute to initiation and maintenance of an epidemic include: (i) the strain of the virus, which may influence the magnitude and duration of the viraemia in humans; (ii) the density, behaviour and vectorial capacity of the vector population; (iii) the susceptibility of the human population (both genetic factors and pre-existing immune profile); and (iv) the introduction of the virus into a receptive community.

Pathogenicity

Two pathogenetic mechanisms are associated with severe dengue infection. Classical DHF/DSS is characterized by a vascular leak syndrome which, if not corrected, may rapidly lead to hypovolemia, shock, and death. The underlying pathogenetic mechanism for this syndrome is thought to be an immune enhancement phenomenon whereby the infecting virus complexes with non-neutralizing dengue antibody, thus enhancing infection of mononuclear phagocytes.

The latter produce vasoactive mediators, which are responsible for increased vascular permeability. Loss of plasma from the vascular compartment may range from mild and transient to severe and prolonged, the latter often resulting in irreversible shock and death. Although classical DSS is most commonly associated with secondary dengue infections, it has also been documented in primary infections, which suggest that sub neutralizing levels of homologous antibody or other immune factors may also cause immune enhancement.

The most consistent feature associated with the emergence of DHF/DSS in an area is the development of hyperendemicity. This increases the probability of secondary infection, which is thought to be associated with DHF/DSS. However, hyperendemicity is also associated with increased transmission and movement of viruses between population centres, which increases the probability of genetic change and introduction of virus strains that have greater epidemic potential or virulence.

Patients infected with dengue viruses may also experience severe and uncontrolled bleeding, usually from the upper gastrointestinal (GI) tract. This severe haemorrhagic disease may be associated with multiple organ failure, and is more difficult to manage than classical DHF/DSS. The underlying pathogenetic mechanism for this type of bleeding is clearly different from that of the vascular leak syndrome, and involves disseminated intravascular coagulation and thrombocytopenia.

A third type of severe and fatal dengue infection, which may or may not involve overt haemorrhagic disease, is encephalopathy. Although many patients with this syndrome present clinically as viral encephalitis, conclusive evidence that dengue viruses infect the central nervous system has not yet been documented. Available data suggest that neurologic symptoms may be secondary to cerebral haemorrhage, oedema, or other indirect effects of dengue virus infection.

Clinical features

Dengue virus infection may be asymptomatic or may cause undifferentiated febrile illness (viral syndrome), dengue fever (DF), or dengue haemorrhagic fever (DHF) including dengue shock syndrome (DSS). Infection with one dengue serotype gives lifelong immunity to that particular serotype, but there is only short-term cross-protection for the other serotypes. The clinical manifestation depends on the virus strain and host factors such as age, immune status, etc.

The incubation period may be as short as 3 days and as long as 14 days, but most often is 4–7 days. The majority of patients present with mild, nonspecific febrile illness, or with classical dengue fever. The latter is generally observed in older children and adults, and is characterized by sudden onset of fever, frontal headache, retroocular pain, and myalgias. Rash, joint pains, nausea and vomiting, and lymphadenopathy are common. The acute illness, which lasts for 3–7 days, is usually benign and self-limiting, but it can be very debilitating, and convalescence may be prolonged for several weeks.

Undifferentiated fever

Infants, children and adults who have been infected with dengue virus, especially for the first time (i.e. primary dengue infection), may develop a simple fever indistinguishable from other viral infections. Maculopapular rashes may accompany the fever or may appear during defervescence. Upper respiratory and gastrointestinal symptoms are common.

Dengue fever

Dengue fever (DF) is most common in older children, adolescents and adults. It is generally an acute febrile illness, and sometimes biphasic fever with severe headache, myalgias, arthralgias, rashes, leucopenia and thrombocytopenia may also be observed. Although DF may be benign, it could be an incapacitating disease with severe headache, muscle and joint and bone pains (break-bone fever), particularly in adults. Occasionally unusual haemorrhage such as gastrointestinal bleeding, hypermenorrhoea and massive epistaxis occur. In dengue endemic areas, outbreaks of DF seldom occur among local people.

After an average intrinsic incubation period of 4–6 days (range 3–14 days), various non-specific, constitutional symptoms and headache, backache and general malaise may develop. Typically, the onset of DF is sudden with a sharp rise in temperature and is frequently associated with a flushed face and headache. Occasionally, chills accompany the sudden rise in temperature. Thereafter, there may be retro-orbital pain on eye movement or eye pressure, photophobia, backache, and pain in the muscles and joints/bones. The other common symptoms include anorexia and altered taste sensation, constipation, colicky pain and abdominal tenderness, dragging pains in the inguinal region, sore throat and general depression. These symptoms usually persist from several days to a few weeks. It is noteworthy that these symptoms and signs of DF vary markedly in frequency and severity.

• Fever: The body temperature is usually between 39 °C and 40 °C, and the fever may be biphasic, lasting 5–7 days in the majority of cases.
• Rash: Diffuse flushing or fleeting eruptions may be observed on the face, neck and chest during the first two to three days, and a conspicuous rash that may be maculopapular or rubelliform appears on approximately the third or fourth day. Towards the end of the febrile period or immediately after defervescence, the generalized rash fades and localized clusters of petechiae may appear over the dorsum of the feet, on the legs, and on the hands and arms. This convalescent rash is characterized by confluent petechiae surrounding scattered pale, round areas of normal skin. Skin itching may be observed.
• Haemorrhagic manifestations: Skin haemorrhage may be present as a positive tourniquet test and/or petechiae. Other bleeding such as massive epistaxis, hypermenorrhoea and gastrointestinal bleeding rarely occur in DF, complicated with thrombocytopenia.
• Course: The relative duration and severity of DF illness varies between individuals in a given epidemic, as well as from one epidemic to another. Convalescence may be short and uneventful but may also often be prolonged. In adults, it sometimes lasts for several weeks and may be accompanied by pronounced asthenia and depression. Bradycardia is common during convalescence. Haemorrhagic complications, such as epistaxis, gingival bleeding, gastrointestinal bleeding, haematuria and hypermenorrhoea, are unusual in DF. Although rare, such severe bleeding (DF with unusual haemorrhage) is an important cause of death in DF.

Dengue fever with haemorrhagic manifestations must be differentiated from dengue haemorrhagic fever.

Clinical laboratory findings

In dengue endemic areas, positive tourniquet test and leukopenia (WBC ≤ 5000 cells/mm³) help in making early diagnosis of dengue infection with a positive predictive value of 70%–80%.

The laboratory findings during an acute DF episode of illness are as follows:

• Total WBC is usually normal at the onset of fever; then leucopenia develops with decreasing neutrophils and lasts throughout the febrile period.
• Platelet counts are usually normal, as are other components of the blood clotting mechanism. Mild thrombocytopenia (100000 to 150000 cells/mm³) is common and about half of all DF patients have platelet count below 100000 cells/mm³; but severe thrombocytopenia (<50000 cells/mm3) is rare.
• Mild haematocrit rise (≈10%) may be found as a consequence of dehydration associated with high fever, vomiting, anorexia and poor oral intake.
• Serum biochemistry is usually normal but liver enzymes and aspartate amino transferase (AST) levels may be elevated.

Dengue haemorrhagic fever and dengue shock syndrome

Dengue haemorrhagic fever (DHF) is more common in children less than 15 years of age in hyperendemic areas, in association with repeated dengue infections. However, the incidence of DHF in adults is increasing. DHF is characterized by the acute onset of high fever and is associated with signs and symptoms similar to DF in the early febrile phase. There are common haemorrhagic diatheses such as positive tourniquet test (TT), petechiae, easy bruising and/or GI haemorrhage in severe cases. By the end of the febrile phase, there is a tendency to develop hypovolemic shock (dengue shock syndrome) due to plasma leakage.

The presence of preceding warning signs such as persistent vomiting, abdominal pain, lethargy or restlessness, or irritability and oliguria are important for intervention to prevent shock. Abnormal haemostasis and plasma leakage are the main pathophysiological hallmarks of DHF. Thrombocytopenia and rising haematocrit/haemoconcentration are constant findings before the subsidence of fever/ onset of shock. DHF occurs most commonly in children with secondary dengue infection. It has also been documented in primary infections with DENV-1 and DENV-3 as well as in infants.

Typical cases of DHF are characterized by high fever, haemorrhagic phenomena, hepatomegaly, and often circulatory disturbance and shock. Moderate to marked thrombocytopenia with concurrent haemoconcentration/rising haematocrit are constant and distinctive laboratory findings are seen. The major pathophysiological changes that determine the severity of DHF and differentiate it from DF and other viral haemorrhagic fevers are abnormal haemostasis and leakage of plasma selectively in pleural and abdominal cavities.

The clinical course of DHF begins with a sudden rise in temperature accompanied by facial flush and other symptoms resembling dengue fever, such as anorexia, vomiting, headache, and muscle or joint pains. Some DHF patients complain of sore throat and an injected pharynx may be found on examination. Epigastric discomfort, tenderness at the right sub-costal margin, and generalized abdominal pain are common. The temperature is typically high and, in most cases, continues as such for 2–7 days before falling to a normal or subnormal level. Occasionally the temperature may be as high as 40 °C, and febrile convulsions may occur. A bi-phasic fever pattern may be observed.

The haemorrhagic form of disease, DHF/DSS, is most commonly observed in children under the age of 15 years, but it also occurs in adults in areas of lower endemicity. It is characterized by acute onset of fever and a variety of nonspecific signs and symptoms that may last 2–7 days. During this stage of illness, DHF/DSS is difficult to distinguish from many other viral, bacterial, and protozoal infections. In children, upper respiratory symptoms caused by concurrent infection with other viruses or bacteria are not uncommon. The differential diagnosis should include other haemorrhagic fevers, hepatitis, leptospirosis, typhoid, malaria, measles, influenza, etc.

The critical stage in DHF/DSS occurs when the fever subsides to or below normal. At that time, the patient’s condition may deteriorate rapidly with signs of circulatory failure, neurologic manifestations, shock and death if proper management is not implemented. Skin haemorrhages such as petechiae, easy bruising, bleeding at the sites of venepuncture, and purpura/ecchymoses are the most common haemorrhagic manifestations; GI haemorrhage may occur, usually after, but in some cases before, onset of shock.

A positive tourniquet test (≥10 spots/square inch), the most common haemorrhagic phenomenon, could be observed in the early febrile phase. Easy bruising and bleeding at venipuncture sites are present in most cases. Fine petechiae scattered on the extremities, axillae, and face and soft palate may be seen during the early febrile phase. A confluent petechial rash with small, round areas of normal skin is seen in convalescence, as in dengue fever. A maculopapular or rubelliform rash may be observed early or late in the disease. Epistaxis and gum bleeding are less common. Mild gastrointestinal haemorrhage is occasionally observed, however, this could be severe in pre-existing peptic ulcer disease. Haematuria is rare. The liver is usually palpable early in the febrile phase, varying from just palpable to 2–4 cm below the right costal margin. Liver size is not correlated with disease severity, but hepatomegaly is more frequent in shock cases. The liver is tender, but jaundice is not usually observed. It should be noted that the incidence of hepatomegaly is observer dependent. Splenomegaly has been observed in infants under twelve months and by radiology examination. A lateral decubitus chest X-ray demonstrating pleural effusion, mostly on the right side, is a constant finding. The extent of pleural effusion is positively correlated with disease severity. Ultrasound could be used to detect pleural effusion and ascites. Gall bladder oedema has been found to precede plasma leakage. The critical phase of DHF, i.e. the period of plasma leakage, begins around the transition from the febrile to the afebrile phase. Evidence of plasma leakage, pleural effusion and ascites may, however, not be detectable by physical examination in the early phase of plasma leakage or mild cases of DHF. A rising haematocrit, e.g. 10% to 15% above baseline, is the earliest evidence. Significant loss of plasma leads to hypovolemic shock. Even in these shock cases, prior to intravenous fluid therapy, pleural effusion and ascites may not be detected clinically. Plasma leakage will be detected as the disease progresses or after fluid therapy. Radiographic and ultrasound evidence of plasma leakage precedes clinical detection. A right lateral decubitus chest radiograph increases the sensitivity to detect pleural effusion. Gall bladder wall oedema is associated with plasma leakage and may precede the clinical detection. A significantly decreased serum albumin >0.5 gm/dl from baseline or <3.5 gm% is indirect evidence of plasma leakage.

In mild cases of DHF, all signs and symptoms abate after the fever subsides. Fever lysis may be accompanied by sweating and mild changes in pulse rate and blood pressure. These changes reflect mild and transient circulatory disturbances as a result of mild degrees of plasma leakage. Patients usually recover either spontaneously or after fluid and electrolyte therapy. In moderate to severe cases, the patient’s condition deteriorates a few days after the onset of fever. There are warning signs such as persistent vomiting, abdominal pain, refusal of oral intake, lethargy or restlessness or irritability, postural hypotension and oliguria. Near the end of the febrile phase, by the time or shortly after the temperature drops or between 3–7 days after the fever onset, there are signs of circulatory failure: the skin becomes cool, blotchy and congested, circum-oral cyanosis is frequently observed, and the pulse becomes weak and rapid. Although some patients may appear lethargic, usually they become restless and then rapidly go into a critical stage of shock. Acute abdominal pain is a frequent complaint before the onset of shock. The shock is characterized by a rapid and weak pulse with narrowing of the pulse pressure ≤20 mmHg with an increased diastolic pressure, e.g. 100/90 mmHg, or hypotension. Signs of reduced tissue perfusion are: delayed capillary refill (>3 seconds), cold clammy skin and restlessness. Patients in shock are in danger of dying if no prompt and appropriate treatment is given. Patients may pass into a stage of profound shock with blood pressure and/or pulse becoming imperceptible (Grade 4 DHF). It is noteworthy that most patients remain conscious almost to the terminal stage. Shock is reversible and of short duration if timely and adequate treatment with volume-replacement is given.

Without treatment, the patient may die within 12 to 24 hours. Patients with prolonged or uncorrected shock may give rise to a more complicated course with metabolic acidosis and electrolyte imbalance, multiorgan failure and severe bleeding from various organs. Hepatic and renal failure are commonly observed in prolonged shock. Encephalopathy may occur in association with multiorgan failure, metabolic and electrolyte disturbances. Intracranial haemorrhage is rare and may be a late event. Patients with prolonged or uncorrected shock have a poor prognosis and high mortality.

Pathogenesis and pathophysiology

DHF occurs in a small proportion of dengue patients. Although DHF may occur in patients experiencing dengue virus infection for the first time, most DHF cases occur in patients with a secondary infection.The association between occurrence of DHF/DSS and secondary dengue infections implicates the immune system in the pathogenesis of DHF. Both the innate immunity such as the complement system and NK cells as well as the adaptive immunity including humoral and cell mediated immunity are involved in this process. Enhancement of immune activation, particularly during a secondary infection, leads to exaggerated cytokine response resulting in changes in vascular permeability. In addition, viral products such as NS1 may play a role in regulating complement activation and vascular permeability.

The hallmark of DHF is the increased vascular permeability resulting in plasma leakage, contracted intravascular volume, and shock in severe cases. The leakage is unique in that there is selective leakage of plasma in the pleural and peritoneal cavities and the period of leakage is short (24–48 hours). Rapid recovery of shock without sequelae and the absence of inflammation in the pleura and peritoneum indicate functional changes in vascular integrity rather than structural damage of the endothelium as the underlying mechanism.

Various cytokines with permeability enhancing effect have been implicated in the pathogenesis of DHF. However, the relative importance of these cytokines in DHF is still unknown. Studies have shown that the pattern of cytokine response may be related to the pattern of cross-recognition of dengue-specific T-cells. Cross-reactive T-cells appear to be functionally deficit in their cytolytic activity but express enhanced cytokine production including TNF-a, IFN-g and chemokines.

Of note, TNF-a has been implicated in some severe manifestations including haemorrhage in some animal models. Increase in vascular permeability can also be mediated by the activation of the complement system. Elevated levels of complement fragments have been documented in DHF.

Some complement fragments such as C3a and C5a are known to have permeability enhancing effects. In recent studies, the NS1 antigen of dengue virus has been shown to regulate complement activation and may play a role in the pathogenesis of DHF.

Higher levels of viral load in DHF patients in comparison with DF patients have been demonstrated in many studies. The levels of viral protein, NS1, were also higher in DHF patients. The degrees of viral load correlate with measurements of disease severity such as the amount of pleural effusions and thrombocytopenia, suggesting that viral burden may be a key determinant of disease severity.

Clinical laboratory findings of DHF

• The white blood cell (WBC) count may be normal or with predominant neutrophils in the early febrile phase. Thereafter, there is a drop in the total number of white blood cells and neutrophils, reaching a nadir towards the end of the febrile phase. The change in total white cell count (≤5000 cells/mm3)63 and ratio of neutrophils to lymphocyte (neutrophils<lymphocytes) is useful to predict the critical period of plasma leakage. This finding precedes thrombocytopenia or rising haematocrit. A relative lymphocytosis with increased atypical lymphocytes is commonly observed by the end of the febrile phase and into convalescence. These changes are also seen in DF.
• The platelet counts are normal during the early febrile phase. A mild decrease could be observed thereafter. A sudden drop in platelet count to below 100 000 occurs by the end of the febrile phase before the onset of shock or subsidence of fever. The level of platelet count is correlated with severity of DHF. In addition, there is impairment of platelet function. These changes are of short duration and return to normal during convalescence.
• The haematocrit is normal in the early febrile phase. A slight increase may be due to high fever, anorexia and vomiting. A sudden rise in haematocrit is observed simultaneously or shortly after the drop in platelet count. Haemoconcentration or rising haematocrit by 20% from the baseline, e.g. from haematocrit of 35% to ≥42% is objective evidence of leakage of plasma.
• Thrombocytopenia and haemoconcentration are constant findings in DHF. A drop in platelet count to below 100000 cells/mm3 is usually found between the 3rd and 10^th days of illness. A rise in haematocrit occurs in all DHF cases, particularly in shock cases. Haemoconcentration with haematocrit increases by 20% or more is objective evidence of plasma leakage. It should be noted that the level of haematocrit may be affected by early volume replacement and by bleeding.
• Other common findings are hypoproteinaemia/albuminemia (as a consequence of plasma leakage), hyponatremia, and mildly elevated serum aspartate aminotransferase levels (≤200 U/L) with the ratio of AST:ALT>2.
• A transient mild albuminuria is sometimes observed.
• Occult blood is often found in the stool.
• In most cases, assays of coagulation and fibrinolytic factors show reductions in fibrinogen, prothrombin, factor VIII, factor XII, and antithrombin III. A reduction in antiplasmin (plasmin inhibitor) has been noted in some cases. In severe cases with marked liver dysfunction, reduction is observed in the vitamin K-dependent prothrombin co-factors, such as factors V, VII, IX and X.
• Partial thromboplastin time and prothrombin time are prolonged in about half and one third of DHF cases respectively. Thrombin time is also prolonged in severe cases.
• Hyponatremia is frequently observed in DHF and is more severe in shock.
• Hypocalcemia (corrected for hypoalbuminemia) has been observed in all cases of DHF, the level is lower in Grade 3 and 4.
• Metabolic acidosis is frequently found in cases with prolonged shock. Blood urea nitrogen is elevated in prolonged shock.

The World Health Organization (WHO) has defined strict criteria for diagnosis of DHF/DSS, with four major clinical manifestations: high fever, haemorrhagic manifestations, haemoconcentration, and circulatory failure. WHO has classified DHF/DSS into four grades according to severity of illness: grades I and II represent the milder form of DHF and grades III and IV represent the more severe form, DSS. Thrombocytopenia and haemoconcentration are constant features. However, there is some disagreement with the WHO case definition in that some patients may present with severe and uncontrollable upper GI bleeding with shock and death in the absence of haemoconcentration or other evidence of the vascular leak syndrome. These patients by the WHO criteria cannot be categorized as having DHF/DSS. In addition, hepatomegaly may not be a constant feature in all epidemics of DHF/DSS. The WHO is currently re-evaluating the case definitions for dengue fever, DHF and DSS.

LABORATORY DIAGNOSIS

Efficient and accurate diagnosis of dengue is of primary importance for clinical care (i.e., early detection of severe cases, case confirmation and differential diagnosis with other infectious diseases), surveillance activities and outbreak control.

Laboratory diagnosis methods for confirming dengue virus infection may involve detection of the virus, viral nucleic acid, antigens or antibodies, or a combination of these techniques. After the onset of illness, the virus can be detected in serum, plasma, circulating blood cells and other tissues for 4–5 days. During the early stages of the disease, virus isolation, nucleic acid or antigen detection can be used to diagnose the infection. At the end of the acute phase of infection, serology is the method of choice for diagnosis.

The following laboratory tests are available to diagnose dengue fever and DHF:

• Virus isolation- serotypic/genotypic characterization
• Viral nucleic acid detection
• Viral antigen detection
• Immunological response-based tests-IgM and IgG antibody assays
• Analysis for haematological parameters

Patients with symptoms consistent with dengue can be tested with both molecular and serologic diagnostic tests during the first 7 days of illness. After the first 7 days of illness, test only with serologic diagnostic tests.

Diagnostic Test	≤ 7 Days After Symptom Onset	> 7 Days Post Symptom Onset	Specimen Types
Molecular Tests	ü	_	Serum, plasma, whole blood, cerebrospinal fluid*
Dengue Virus Antigen Detection (NS1)	ü	_	Serum
Serologic Tests	ü	ü	Serum, cerebrospinal fluid*
Tissue Tests	ü	ü	Fixed tissue

* Testing cerebrospinal fluid is recommended in suspect patients with central nervous system clinical manifestations such as encephalopathy and aseptic meningitis.

Acute Phase: Initial 1-7 days after symptom onset

• The initial 1-7 days after symptom onset are referred to as the acute phase of dengue.
• During this period, dengue virus is typically present in blood or blood-derived fluids such as serum or plasma.
• Dengue virus RNA can be detected with molecular tests.
• The non-structural protein NS1 is a dengue virus protein that also can be detected using some commercial tests.
• A negative result from a molecular or NS1 test is not conclusive. For symptomatic patients during the first 1-7 days of illness, any serum sample should be tested by a NAAT or NS1 test and an IgM antibody test. Performing both molecular and IgM antibody (or NS1 and IgM antibody) tests can detect more cases than performing just one test during this time period, and usually allows diagnosis with a single sample.

Convalescent Phase: >7 days post symptom onset

• The period beyond 7 days following symptom onset is referred to as the convalescent phase of dengue.
• Patients with negative NAAT or NS1 test results and negative IgM antibody tests from the first 7 days of illness should have a convalescent sample tested for IgM antibody test.
• During the convalescent phase, IgM antibodies are usually present and can be reliably detected by an IgM antibody test.
• IgM antibodies against dengue virus can remain detectable for 3 months or longer after infection.
• Patients who have IgM antibodies against dengue virus detected in their serum specimen with an IgM antibody test and either: 1) have a negative NAAT or NS1 result in the acute phase specimen, or 2) without an acute phase specimen, are classified as having a presumptive, recent dengue virus infection.

Testing to differentiate dengue from other flaviviruses

Special considerations:

• Cross reactivity: Cross reactivity is a limitation of dengue serologic tests. Serologic tests to detect antibodies against other flaviviruses such Japanese encephalitis, St. Louis encephalitis, West Nile, yellow fever, and Zika viruses may cross react with dengue viruses. This limitation must be considered for patients who live in or have traveled to areas where other flaviviruses co-circulate. Therefore, a patient with other recent or past flavivirus infection(s) may be positive when tested to detect IgM antibodies against dengue virus. To more precisely determine the cause of infection in IgM positive patients, the IgM-positive specimens can be tested for specific neutralizing antibodies by plaque reduction neutralization test (PRNT) (against the four dengue virus serotypes and other flaviviruses; however, PRNT does not always conclusively distinguish specific flaviviruses.
• Areas with co-circulating flaviviruses: For people living in or traveling to an area with endemic or concurrently circulating dengue, Zika, and other flaviviruses, clinicians will need to order appropriate tests to best differentiate dengue virus from other flaviviruses, and may consult with state or local public health laboratories or CDC for guidance.
• Pregnant women: If the patient is pregnant and symptomatic and lives in or has travelled to an area with risk of Zika, test for Zika using NAAT in addition to dengue.

Current dengue diagnostic methods

Virus isolation

Specimens for virus isolation should be collected early in the course of the infection, during the period of viraemia (usually before day 5). Virus may be recovered from serum, plasma and peripheral blood mononuclear cells and attempts may be made from tissues collected at autopsy (e.g. liver, lung, lymph nodes, thymus, bone marrow). Because dengue virus is heat-labile, specimens awaiting transport to the laboratory should be kept in a refrigerator or packed in wet ice. For storage up to 24 hours, specimens should be kept at between +4 °C and +8 °C. For longer storage, specimens should be frozen at -70 °C in a deep-freezer or stored in a liquid nitrogen container. Storage even for short periods at –20 °C is not recommended.

Cell culture is the most widely used method for dengue virus isolation. The mosquito cell line C6/36 (cloned from Ae. albopictus) or AP61 (cell line from Ae. pseudoscutellaris) are the host cells of choice for routine isolation of dengue virus. Since not all wild type dengue viruses induce a cytopathic effect in mosquito cell lines, cell cultures must be screened for specific evidence of infection by an antigen detection immunofluorescence assay using serotype-specific monoclonal antibodies and flavivirus group-reactive or dengue complex-reactive monoclonal antibodies. Several mammalian cell cultures, such as Vero, LLCMK2, and BHK21, may also be used but are less efficient. Virus isolation followed by an immunofluorescence assay for confirmation generally requires 1–2 weeks and is possible only if the specimen is properly transported and stored to preserve the viability of the virus in it.

Nucleic acid detection

RNA is heat-labile and therefore specimens for nucleic acid detection must be handled and stored according to the procedures described for virus isolation.

RT-PCR

Compared to virus isolation, the sensitivity of the RT-PCR methods varies from 80% to 100% and depends on the region of the genome targeted by the primers, the approach used to amplify or detect the PCR products (e.g. one-step RT-PCR versus two-step RTPCR), and the method employed for subtyping (e.g. nested PCR, blot hybridization with specific DNA probes, restriction site-specific PCR, sequence analysis, etc.). To avoid false positive results due to non-specific amplification, it is important to target regions of the genome that are specific to dengue and not conserved among flavi- or other related viruses. False-positive results may also occur as a result of contamination by amplicons from previous amplifications.

Real-time RT-PCR

The real-time RT-PCR assay is a one-step assay system used to quantitate viral RNA and using primer pairs and probes that are specific to each dengue serotype. The use of a fluorescent probe enables the detection of the reaction products in real time, in a specialized PCR machine, without the need for electrophoresis. Many real-time RT-PCR assays have been developed employing TaqMan or SYBR Green technologies. The TaqMan real-time PCR is highly specific due to the sequence-specific hybridization of the probe. Nevertheless, primers and probes reported in publications may not be able to detect all dengue virus strains: the sensitivity of the primers and probes depends on their homology with the targeted gene sequence of the particular virus analysed.

Real-time RT-PCR assays are either “singleplex” (i.e. detecting only one serotype at a time) or “multiplex” (i.e. able to identify all four serotypes from a single sample). The multiplex assays have the advantage that a single reaction can determine all four serotypes without the potential for introduction of contamination during manipulation of the sample. However, the multiplex real-time RT-PCR assays, although faster, are currently less sensitive than nested RT-PCR assays. An advantage of this method is the ability to determine viral titre in a clinical sample, which may be used to study the pathogenesis of dengue disease.

Isothermal amplification methods

The NASBA (nucleic acid sequence-based amplification) assay is an isothermal RNA specific amplification assay that does not require thermal cycling instrumentation. The initial stage is a reverse transcription in which the single-stranded RNA target is copied into a double-stranded DNA molecule that serves as a template for RNA transcription. Detection of the amplified RNA is accomplished either by electrochemiluminescence or in real-time with fluorescent-labelled molecular beacon probes. NASBA has been adapted to dengue virus detection with sensitivity near that of virus isolation in cell cultures and may be a useful method for studying dengue infections in field studies.

Detection of antigens

Until recently, detection of dengue antigens in acute-phase serum was rare in patients with secondary infections because such patients had pre-existing virus-IgG antibody immunocomplexes. New developments in ELISA and dot blot assays directed to the envelop/membrane (E/M) antigen and the non-structural protein 1 (NS1) demonstrated that high concentrations of these antigens in the form of immune complexes could be detected in patients with both primary and secondary dengue infections up to nine days after the onset of illness.

The NS1 glycoprotein is produced by all flaviviruses and is secreted from mammalian cells. NS1 produces a very strong humoral response. Many studies have been directed at using the detection of NS1 to make an early diagnosis of dengue virus infection. Commercial kits for the detection of NS1 antigen are now available, though they do not differentiate between dengue serotypes.

Serological tests

MAC-ELISA

For the IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) total IgM in patients’ sera is captured by anti-μ chain specific antibodies (specific to human IgM) coated onto a microplate. Dengue-specific antigens, from one to four serotypes (DEN-1, -2, -3, and -4), are bound to the captured anti-dengue IgM antibodies and are detected by monoclonal or polyclonal dengue antibodies directly or indirectly conjugated with an enzyme that will transform a non-coloured substrate into coloured products. The optical density is measured by spectrophotometer.

For detection of IgM, samples are taken within the appropriate time frame (five days or more after the onset of fever). Most of the antigens used for this assay are derived from the dengue virus envelope protein (usually virus-infected cell culture supernatants or suckling mouse brain preparations). MAC-ELISA has good sensitivity and specificity but only when used five or more days after the onset of fever. Different commercial kits (ELISA or rapid tests) are available but have variable sensitivity and specificity.

IgG ELISA

The IgG ELISA is used for the detection of recent or past dengue infections (if paired sera are collected within the correct time frame). This assay uses the same antigens as the MAC-ELISA. The use of E/M-specific capture IgG ELISA (GAC) allows detection of IgG antibodies over a period of 10 months after the infection. IgG antibodies are lifelong as measured by E/M antigen-coated indirect IgG ELISA, but a fourfold or greater increase in IgG antibodies in acute and convalescent paired sera can be used to document recent infections.

Haemagglutination-inhibition test

The haemagglutination-inhibition (HI) test (see Figure 4.4) is based on the ability of dengue antigens to agglutinate red blood cells (RBC) of ganders or trypsinized human “O” RBC. Anti-dengue antibodies in sera can inhibit this agglutination and the potency of this inhibition is measured in an HI test. Serum samples are treated with acetone or kaolin to remove non-specific inhibitors of haemagglutination, and then adsorbed with gander or trypsinized type “O” human RBC to remove non-specific agglutinins. Each batch of antigens and RBC is optimized. PH optima of each dengue haemagglutinin requires the use of multiple different pH buffers for each serotype. Optimally the HI test requires paired sera obtained upon hospital admission (acute) and discharge (convalescent) or paired sera with an interval of more than seven days. The assay does not discriminate between infections by closely related flaviviruses (e.g., between dengue virus and Japanese encephalitis virus or West Nile virus) nor between immunoglobulin isotypes.

Haematological tests

Platelets and haematocrit values are commonly measured during the acute stages of dengue infection.

A drop of the platelet counts below 100 000 per μL may be observed in dengue fever but it is a constant feature of dengue haemorrhagic fever. Thrombocytopaenia is usually observed in the period between day 3 and day 8 following the onset of illness.

Haemoconcentration, as estimated by an increase in haematocrit of 20% or more compared with convalescent values, is suggestive of hypovolaemia due to vascular permeability and plasma leakage.

Prevention and Control of Dengue

Currently, the only way to prevent dengue infection is to control the mosquito vector that transmits the virus. Unfortunately, our ability to control Ae. aegypti is limited. For more than 25 years, the recommended method of control was the use of ultralow volume (ULV) application of insecticides to kill adult mosquitoes. Field trials showed that this method was not effective in significantly reducing natural mosquito populations for any length of time. This supports epidemiologic observations that ULV has little or no impact on epidemic transmission of dengue viruses.

The only truly effective method of controlling Ae. aegypti is larval control, that is to eliminate or control the larval habitats where the mosquitoes lay their eggs. Most important larval habitats are found in the domestic environment, where most transmission occurs. To have sustainability of prevention and control programs, some responsibility for mosquito control should be transferred from government to citizen homeowners. For long-term sustainability, mosquito control programs must be community-based and integrated. Persons living in Ae. Aegypti infested communities have to be educated to accept responsibility for their own health destiny by helping government agencies control the vector mosquitoes, and thus prevent epidemic DF/DHF/DSS.