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 10th 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.
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Diagnostic
Test
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≤ 7 Days After Symptom Onset
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> 7 Days Post Symptom Onset
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Specimen
Types
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Molecular Tests
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ü
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Serum, plasma, whole blood, cerebrospinal fluid*
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Dengue Virus Antigen Detection (NS1)
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ü
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Serum
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Serologic Tests
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ü
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ü
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Serum,
cerebrospinal fluid*
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Tissue Tests
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ü
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ü
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Fixed tissue
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* 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.
