BaillieÁre's Clinical Haematology

Vol. 13, No. 2, pp. 261±276, 2000

doi:10.1053/beha.2000.0073, available online at http://www.idealibrary.com on

Haematology in dengue and dengue

haemorrhagic fever

Tanomsri Srichaikul MD, MSc

Emeritus Professor of Medicine, Mahidol University. Senior Consultant, Hematology Division,
Pramongkutklau Medical College, Bangkok
Vichaiyuth Hospital, 114/4 Sretsiri Road, Bangkok, Thailand

Suchitra Nimmannitya MD, MPH

Senior Consultant
Queen Sirikit National Institute of Child Health, Rajvithee Road, Bangkok, Thailand

Dengue fever (DF) and dengue haemorrhagic fever (DHF) are caused by the dengue virus. The
major pathophysiological hallmark that distinguishes DHF from DF is plasma leakage as a result
of increased vascular permeability. Following this leakage, hypovolaemic shock occurs as a
consequence of a critical plasma volume loss. Constant haematological abnormalities occurring
in DHF and frequently include bone marrow suppression, leucopenia and thrombocytopenia.
An enhanced immune response of the host to a secondary DV infection is a feature of DHF and
leads to many consequences. These are immune complex formation, complement activation,
increased histamine release and a massive release of many cytokines into the circulation, leading
to shock, vasculopathy, thrombopathy and disseminated intravascular coagulation (DIC).
The mechanisms underlying the bleeding in DHF are multiple. These are vasculopathy,
thrombopathy and DIC. Thrombopathy consists of thrombocytopenia and platelet dysfunction.
DIC is prominent in patients with shock. The most severe DIC and massive bleeding are the
result of prolonged shock and cause a fatal outcome. The mechanisms of thrombopathy and DIC
and the proper management of DHF are reviewed and discussed.

Key words: dengue fever; dengue haemorrhagic fever; vasculopathy; plasma leakage;
cytokines; thrombopathy; DIC.

Dengue illness caused by any of the four dengue serotypes (DEN1±4) is currently the
most important mosquito-borne viral disease in the tropical areas of the world.
Dengue haemorrhagic fever (DHF), the most severe form, has emerged to become
one of the major public health problems. The ®rst major epidemics in Southeast Asia
occurred in the mid 1950s. A global pandemic has intensi®ed during the past 18 years
with expanding geographical distribution, increased epidemic frequency and the
emergence of DHF in new areas. It has been estimated that 20 million cases of dengue
infection occur annually, with approximately 300 000 to 500 000 of these being DHF.
With case fatality rates varying from 1 to 5%, several thousand deaths occur each year,
mostly among children in tropical Asia.1
Unlike DF, known for over 200 years as a mild non-fatal disease, DHF has the potential
to develop into a fatal dengue-shock syndrome (DSS). The major pathophysiological
1521±6926/00/020261+16 $35.00/00 *
c 2000 Harcourt Publishers Ltd.
262 T. Srichaikul and S. Nimmannitya

hallmarks that determine disease severity and distinguish DHF from DF and other
viral haemorrhagic fevers are plasma leakage as a result of increased vascular permea-
bility and abnormal haemostasis.2 Hypovolaemic shock occurs as a consequence of, and
subsequent to, a critical plasma volume loss. Abnormal haemostasis including increased
capillary fragility (positive tourniquet test and a tendency to bruise), thrombocytopenia,
impaired platelet function and, in the most severe form, disseminated intravascular
coagulation (DIC), contribute to varying degrees of haemorrhagic diathesis.1±3
For the past four decades extensive research studies in various ®elds have contributed
a great deal to better understanding of the pathophysiology and pathogenesis of DHF.
Although the precise pathogenetic mechanism has not been de®ned, all available data
strongly suggest the involvement of immunological mechanisms. The association of
DHF/DSS with secondary dengue infection rather than the ®rst (primary) infection led
Halstead (1977) to study and describe antibody-dependent enhancement (ADE) of
dengue virus infection in macrophages or mononuclear cells.4 It is proposed that an
increase in the number of dengue virus-infected monocytes by the enhancing activity of
cross-reacting antibody from previous infection was responsible for the pathogenesis of
DHF. Complement activation, with profound depression of C3 and C5 levels, is a
constant ®nding and is thought to play an important role.5,6 Kurane and Ennis proposed
that, in addition to ADE and activation of complement, activation of T-lymphocytes also
plays an important role.5,6 Activation of monocytes and T-lymphocytes induces the
production of cytokines and chemical mediators. These authors hypothesized that a
rapid increase in the levels of the potential mediators, such as TNF-a, IL-2, IL-6, IFN-g,
PAF, C3a, C5a and histamine, and the synergistic e€ects of these mediators, induce
malfunction of vascular endothelial cells which leads to plasma leakage and shock,
and derangements of the coagulation system which may lead to haemorrhagic
manifestations.7,8

CLINICAL PRESENTATIONS
Dengue fever
The clinical features of DF are age-dependent. Infants and children infected with dengue
virus for the ®rst time usually develop a simple febrile illness. Dengue fever is most
common in adults and older children and may be either benign or present as the classic
incapacitating disease with severe muscle, joint, and bone pain (break-bone fever).
Erythematous/maculopapular skin rashes, occasionally with petechiae, are common, as
are gastrointestinal symptoms. Rarely, massive haemorrhage, mostly from pre-existing
lesions in the gastrointestinal tract may result in death. Leukopenia is common, and
moderate thrombocytopenia is occasionally observed. The clinical presentations of DF
vary from location to location and from epidemic to epidemic. It is almost impossible,
therefore, to make a clinical diagnosis of DF, particularly in isolated cases. In tropical
areas where dengue is endemic, classic DF is infrequently found among indigenous
adults.1

Dengue haemorrhagic fever

Unlike DF, DHF occurs mainly in children and its clinical features are rather
distinctive. Typically DHF is characterized by four major clinical manifestations
presented below in order of their appearance and frequency.1,2
. High continuous fever for 2 to 7 days in most cases;
 Dengue and dengue haemorrhagic fever 263

. Haemorrhagic diathesis, most frequently presenting as skin petechiae (including a

positive tourniquet test);
. Hepatomegaly; and
. Circulatory disturbances (presenting as shock in severe cases).

Thrombocytopenia and haemoconcentration (an increase in the haematocrit of 20% or

more) representing the pathophysiological hallmarks of abnormal haemostasis and
plasma leakage, respectively, are constant ®ndings.
The course of DHF is rather stereotypical. An early and accurate clinical diagnosis
could be made based on the above-mentioned four major manifestations together with
the presence of thrombocytopenia and haemoconcentration.2 The World Health
Organization (WHO) has adopted these to be WHO criteria.1 The disease severity is
categorized according to the clinical evidence of shock into grade I and II non-shock
cases; and grade III and IV shock cases. The grade II patient is distinguished from the
grade I patient by the presence of spontaneous bleeding in the former. The grade IV
patients are those who experience profound shock with undetectable pulse and/or
blood pressure, regardless of the presence of a bleeding diathesis.1,2
During the acute febrile phase, which usually lasts 2 to 7 days, DHF resembles DF in
many respects, but a maculopapular rash and myalgia/arthralgia are less frequent in
DHF.1 Haemorrhagic manifestations, which are invariably present in DHF, are usually
mild and frequently consist of tiny, scattered, petechial haemorrhages on the skin or
occasionally in the buccal cavity and subconjunctivae, and a tendency to bruise. Early in
the febrile phase, a positive tourniquet test is frequently observed. Bleeding from the
nose, gums and gastrointestinal tract are less common, but may be severe. Massive
gastrointestinal haemorrhage may occur and is usually found later in association with
prolonged shock. Haematuria is extremely rare.
The liver is often enlarged and palpable a few days after the onset of fever. It is
usually soft and tender, but jaundice is not observed. Splenomegaly is rarely noted in
small infants. Generalized lymphadenopathy is observed in about half of the cases.1
The critical stage is reached by the end of the febrile phase of illness. Accompanying
or shortly after a rapid drop in temperature, varying degrees of circulatory disturbance
develop. The patient is often sweating, restless and has cold extremities. In mild DHF
cases, (WHO grades I and II) the changes in vital signs are minimal and transient.
Patients recover spontaneously or shortly after a brief period of treatment. The onset
of shock is acute and generally occurs at the time of defervescence, which is on or after
the third day of illness (the shortest duration of fever is 2 days). The temperature is
often subnormal, the skin is cold and clammy, and the pulse becomes rapid and weak.
The pulse pressure is narrow ( 4 20 mmHg), with a characteristically high diastolic
pressure (e.g. 100/90, 110/90 mmHg) in the early stage of shock. It is noteworthy that
patients who are in shock usually remain conscious almost to the terminal stage. The
course of shock is short but life-threatening. If the proper management is not given,
the patient deteriorates rapidly into the stage of profound shock, and the pulse and/or
blood pressure become undetectable (WHO grade IV). Patients who do not receive
treatment usually die within 12 to 24 hours after shock ensues. Prolonged shock is
often complicated with metabolic acidosis that may precipitate the occurrence of DIC,
or enhance the ongoing DIC to the point that massive bleeding may occur. The most
common site of severe bleeding is in the gastrointestinal tract, and the bleeding usually
presents as haematemesis (with dark-coloured blood) and/or melena. Occasionally the
bleeding may be concealed, leading to a more complicated course if not recognized.
The least common haemorrhagic manifestation is intracranial bleeding, but these
264 T. Srichaikul and S. Nimmannitya

patients have the highest fatality rates. Convulsions and coma may occur in patients
with DIC and bleeding into the brain.9
The pathogenetic mechanisms that lead to plasma leakage and abnormal haemostasis
are self-limiting. The critical period of plasma leakage and shock rarely last longer than
48 hours. In most cases, early and e€ective replacement of lost plasma with isotonic salt
solution, plasma or plasma expander results in a rapid and uneventful recovery. Blood
transfusion is indicated in patients with severe bleeding and occasionally blood
components may be needed. With early diagnosis and proper treatment of shock the
case fatality rate of DHF has been markedly reduced. The causes of death are still
mainly prolonged shock and massive bleeding.

PATHOPHYSIOLOGY AND PATHOGENESIS

Vascular change and increased vascular permeability
The most prominent feature of DHF is plasma leakage which appears to occur
selectively into the pleural and abdominal cavities. Pericardial e€usion, if there is any, is
rather minimal. Chest radiographs demonstrate pleural e€usion which correlates well
with disease severity.2 Other evidence of plasma leakage include a rising haematocrit/
haemoconcentration and hypoproteinaemia/hypoalbuminaemia.2 Although the patho-
genesis of shock is not well understood, clinical observations and laboratory studies
suggest that DSS results from hypovolaemia due to plasma loss accompanied by an
increase in peripheral resistance.2,10
It is likely that the increase in vascular permeability leading to plasma leakage
results from a functional change caused by short-lived pharmacological mediator(s) and
products of the immune mechanism, rather than from structural destruction of
endothelial cells. Evidence for this conclusion includes:
. Rapid onset of plasma leakage with sudden elevations in haematocrit;
. Short duration of leakage/shock for 24 to 48 hours;
. Rapid recovery with proper treatment (24±28 hours) with uneventful convalescence;
. No sequelae;
. No in¯ammatory vascular changes found at autopsy11; and
. No severe pathological changes in major organs other than serous e€usion and
haemorrhage.11
Two observations have been important in understanding the mechanisms of vascular
changes, plasma leakage and shock. First, DHF patients have no generalized oedema
when they present with shock. This supports selective leakage into serous spaces.2
Second, the timing of leakage is from the end of the febrile phase to 24±48 hours after
defervescence, which is after the tourniquet test becomes positive and the petechiae
®rst appear. These observations suggest that the vascular changes (vasculopathy) that
lead to increased vascular permeability and plasma leakage probably occur at venules
in the thoracic and abdominal cavities while those changes related to haemorrhage in
the form of leakage of erythrocytes are probably con®ned to capillaries.12 The capillary
fragility change may be a direct e€ect of the virus as it appears early in the ®rst few
days of illness during the viraemic phase.3,4
Among possible mediators, it has been recently shown that C3a and C5a are
elevated, and that both their levels and duration of elevation correlated well with the
occurrence of shock and disease severity.13 Among the cytokines and chemical
mediators, including tumour necrosis factor, interleukin-1 (IL-1), IL-2 and IL-6, IFN-g,
 Dengue and dengue haemorrhagic fever 265

all except, IL-1 have been found to be elevated in DHF.7,8 Although the results of
various studies support the role of these cytokines and chemical mediators, they are
still far from being conclusive. Further studies in search of mediators responsible for
plasma leakage are urgently necessary.

Haemorrhagic diathesis
Haemorrhagic manifestations, which are invariably present in DHF, are usually mild
and are most commonly found as scattered tiny petechiae on the skin and occasionally
on the submucosa. A positive tourniquet test, which indicates increased capillary
fragility, and bruises at venepuncture sites, are the most common ®ndings that appear
early. Epistaxis is the next most common manifestation and may be severe. Massive
bleeding that requires blood transfusion is less common and usually occurs after the
onset of shock.14 Gastrointestinal bleeding in the form of haematemesis and/or
melaena is the most common type of severe bleeding. In those who died after
prolonged shock, bleeding in various organs (gastrointestinal tract, heart, lungs, liver
and brain) have been observed.9,11 Some pre-existing host factors may contribute to
gastric bleeding (haematemesis) early in the course of illness. Examples of such pre-
existing factors are gastritis caused by aspirin ingestion or pre-existing peptic ulcer.14
As severe bleeding remains the major cause of death in DHF/DSS, it is important to
study and understand the mechanism of bleeding in order to improve the management
of a patient with bleeding.

HAEMATOLOGICAL DISORDERS
Peripheral blood
Leucopenia is a common ®nding in both DF and DHF. Initially, the leucocyte counts
may be normal, or slightly increased, predominantly due to an increase in neutrophils.
Towards the end of the febrile phase there are reductions in the number of total
leucocytes and neutrophils. Simultaneously, a relative lymphocytosis is noted with the
presence of atypical lymphocytes. The leucopenia usually reaches a nadir shortly before
or at the time the temperature drops and the leucocyte count returns to normal 2±3
days after defervescence. The numbers of atypical lymphocytes in DHF, varying from
15 to 20%, are clearly greater than in patients with DF.15,16 These cells may represent
activated B- and T-lymphocytes.17 Moderate to marked reduction in the number of
platelets usually follows the reduction in leucocytes and reaches a nadir on the day of
defervescence.2 Giant platelets have been observed in blood smears of DHF patients,
suggesting an increased platelet turnover.12 The platelet counts usually drop rapidly to
below 100  109/l shortly before defervescence and before the onset of shock in DHF.
Platelet counts usually remain low for 3±5 days in most cases. The levels of platelet
counts correlate well with disease severity.2,18,19

Bone marrow changes

The haemopoietic suppression is a well known phenomenon during dengue virus
infection. The degree of suppression is similar in both DF and DHF. In experimental
studies of humans infected with dengue virus, neutropenia developed on the second
day of clinical symptoms.20 Therefore, the suppression of haemopoiesis probably begins
around 4±5 days after the innoculation of virus by the bites of an infected mosquito,
266 T. Srichaikul and S. Nimmannitya

during the incubation period (5±8 days).21 This suppression lasted approximately 7±10
days and ended in the acute febrile phase approximately 2±3 days before the onset of
shock or subsidence of fever. In various studies11,22±26 the bone marrow of patients
with DHF at the early phase of the acute febrile illness showed marked hypocellularity
with a decrease in all the haemopoietic cell types including megakaryocytes, erythro-
blasts and myeloid precursors. In vitro studies have shown that the colony forming
units for granulocytes and macrophages (CFU-GM) were markedly decreased or
almost absent. The colonies were also abnormally small or appeared as a cluster of
cells.27 The decrease in granulopoiesis sometimes was accompanied by a dispro-
portionate reduction in cells after the myelocytic stage.23 Following the haemopoietic
suppression, recovery of haemopoiesis occurred a few days before the onset of shock
or subsidence of fever. The bone marrow then appeared hypercellular, with an
increase in the number of megakaryocytes, erythroblasts and myeloid precursors.
Despite the increased or normal number of megakaryocytes, some of these cells
showed signs of degeneration as manifested by nuclear fragmentation and/or vacuol-
ization and naked nuclei.23,27 Haemophagocytosis of young and mature erythroid and
myeloid cells as well as lymphocytes and platelets was also observed.11,23
During the early period of bone marrow suppression, there were no changes in the
number of white blood cells and platelets in the peripheral blood. Following haemo-
poietic suppression for 7±10 days, the number of leucocytes, including neutrophils,
lymphocytes and platelets started to decline below the normal level. The maximum
reduction of these cells was seen on the day of shock or subsidence of fever. In the
recovery phase of the disease, the number of leucocytes, neutrophils and platelets
gradually returned to normal within 7 days. The time sequence of the onset of
neutropenia and thrombocytopenia following the bone marrow suppression indicated
that neutropenia and thrombocytopenia were the result of the haemopoietic suppres-
sion. The haemophagocytosis observed in the phase of bone marrow recovery11,23 may
be another mechanism inducing further leucopenia and thrombocytopenia. Ine€ective
thrombopoiesis may also contribute to the thrombocytopenia. The peripheral
destruction of platelets by various mechanisms has been reported by many investi-
gators, and this issue will be discussed later.
The pathogenesis of bone marrow suppression in DHF probably involves three main
factors. These are: (1) direct injury to haemopoietic progenitor cells by infection of
these cells by dengue virus, (2) infection of stromal cells by dengue virus, and (3)
changes in marrow regulators.
The direct injury by dengue virus to the haemopoietic progenitor cells was
demonstrated by Nakoa et al in 1989.28 They showed that dengue virus type 4 (DEN4)
could replicate in normal bone marrow mononuclear cells. The replication of the virus
caused inhibition of the proliferation of erythroid burst-forming units (BFU-E) and
granulocyte-macrophage colony-forming units (CFU-GM). In 1997, Murgur et al29
demonstrated in vitro that DEN3 could infect the cord blood mononuclear cells. This
infection caused suppression of progenitor cell growth in cultures. The degree of
haemopoietic suppression was correlated with the clinical spectrum of dengue
infection. The most severe suppression was observed in dengue shock syndrome
(DSS), followed by DHF and DF respectively.
The injury to bone marrow stromal cells by dengue virus was observed by La Russa
et al.30,31 They demonstrated that the infection of stromal cells by DV2 caused inhibition
of the growth of early haemopoietic progenitor cells in Dexter cultures. During the
infection of stromal cells by DV, many cytokines were released into the culture
supernatant. These cytokines were macrophage in¯ammatory protein-1 alpha (MIP-1a),
 Dengue and dengue haemorrhagic fever 267

IL6 and IL-8.32±34 MIP-1a is capable of inhibiting the growth of early haemopoietic
progenitor cells. Simultaneously, there was a decrease in stem cell factor35 leading to a
decreased growth of haemopoietic stem cells/early progenitor cells in culture.35,36
Recently, it has been shown that cytokines, some of which could suppress haemo-
poiesis, were released into the circulation during the early acute febrile phase of
dengue infection. These cytokines included tumour necrosis factor (TNF-a), inter-
leukins (IL-2, IL-6, IL-8) and interferons (INF-a and INF-g). The levels of these
cytokines correlated well with the clinical severity of dengue infection.37±40 The time
of bone marrow suppression also corresponded with the increased level of cytokines
in the blood.41±44
In summary, the bone marrow suppression in DF and DHF appears to result from
the direct infection of haemopoietic progenitor cells and bone marrow stromal cells by
dengue virus as well as the release of various haematodepressive cytokines during the
dengue virus infection. The haemopoietic suppression, leads to granulocytopenia and
thrombocytopenia. The haemopoietic suppression occurs transiently and recovers
rapidly by the end of the febrile period. Following this recovery the number of
neutrophils and platelets continues to be low until the day of shock or subsidence of
fever and then gradually returns to normal within 7 days in the convalescent period of
the disease.

Platelet alteration
There are two major changes observed in DHF: thrombocytopenia and platelet
dysfunction.

Thrombocytopenia
A platelet count less than 100  109/l may be occasionally observed in DF but is a
constant feature of DHF.1,2 Mean platelet nadir for patients with DHF categorized by
grade according to the World Health Organization (WHO) was successively lower for
grades I, II, and III/IV.1,4 The degree of thrombocytopenia is well correlated with the
clinical severity of dengue haemorrhagic fever (Figure 1). In shock cases (grades III±IV)
the mean nadir platelet count was about 20  109/l.2 It is of interest to note that only
15% of the shock cases with platelets lower than 50  109/l experienced severe
bleeding.2 Very severe thrombocytopenia, less than 10  109/l, could be seen in severe
cases. The degree of thrombocytopenia was also correlated with the activation of
complement and plasma kinin system.45,46 In the study of Srichaikul et al19, a platelet
count less than 10  109/l could be observed at 2 days before the onset of shock or
subsidence of fever. In the convalescent period, the platelet count rose by the second
afebrile day and was normal in all patients by 7 days after subsidence of fever19 (Figure 2).
The pathogenesis of thrombocytopenia in DF and DHF involves two major
mechanisms. These are decreased production and increased peripheral destruction or
increased utilization. Decreased production has already been discussed in the section on
bone marrow suppression. The mechanisms inducing peripheral destruction or
increased utilization are more important and play a major role in the induction of
thrombocytopenia in DHF. This mechanism was suggested by the observations that
during the 2 days before shock or defervescence, rapidly progressive thrombocytopenia
occurred along with the normal or increased number of megakaryocytes in the bone
marrow. Strong evidence supporting peripheral destruction of platelets in DHF patients
came from a study by Mitrakul et al in 1977.47 In this study it was demonstrated that
268 T. Srichaikul and S. Nimmannitya

Figure 1. Nadir platelet counts and severity in dengue haemorrhagic fever. Reproduced from Nimmannitya
et al (1987), Southeast Asian Journal of Tropical Medicine and Public Health 18: 392±398) with permission.

during the acute phase of DHF, survival of platelets was markedly decreased due to
destruction mainly in the liver and to a lesser extent in the spleen. Subsequently,
immune-mediated injury was demonstrated to be the underlying mechanism of platelet
destruction. By using a direct immuno¯uorescence technique, Phanichyakarn et al48
demonstrated C3 on the surface of platelets from 11 of 13 patients with DHF. In
addition, more C3 was found on the surface of platelets in the shock than in the non-
shock group. The more sophisticated work of Boonpuchnaving et al in 197949, using the
same technique, demonstrated dengue antigen, human immunoglobulin and C3 on the
surface of platelets in 48% of patients with DHF. A correlation between the amount of
immunoglobulin and C3 with the degree of thrombocytopenia and the day of illness was
also demonstrated in this study. Later on, it was shown that the immune destruction was
mediated by interaction between dengue antibody and dengue antigen which was
present on the platelet surface.50,51 C3dg, the activated form of C3, was demonstrated
on the surface of platelets, and the amount of the C3dg positively correlated with the
decrease in the number of circulating platelets.52 The latter observation suggests that
 Dengue and dengue haemorrhagic fever 269

Figure 2. Serial platelet count in 35 DHF patients. Number above each column ˆ number of cases studied;
K ˆ control value. Reproduced from Srichaikul et al (1989), Southeast Asian Journal of Tropical Medicine and
Public Health 20: 19±25) with permission.

complement plays a role in the immune destruction of platelets in DHF patients.

Funahara et al in 198750 also demonstrated an interaction in vitro between platelets and
endothelial cells having DV antigen and suggested that some injury to vascular
endothelial cells by DV may allow the blood circulating in the vessel to interact with
subendothelial collagen and lead to the promotion of platelet aggregation and lysis of
platelets resulting in thrombocytopenia. Finally, the other important mechanism of
thrombocytopenia in DHF is the consumption of platelets during the process of
consumptive coagulopathy which occurred in most DHF patients. The supporting
evidence for this latter mechanism was the correlation between the lowest plasma
®brinogen and platelet nadir, and the lowest plasma ®brinogen and serum C3 level in
patients with DHF and shock. These ®ndings suggested that complement activation,
coagulation and peripheral destruction of platelets are related. Complement activation
is known to be capable of initiating coagulation.53

Platelet dysfunction
Most patients who exhibit petechiae during the early febrile phase in DHF have
normal platelet counts. This raised the question of whether there is an acquired
platelet dysfunction early in DHF before thrombocytopenia occurs. A functional
abnormality of platelets in DHF patients was ®rst observed by Mitrakul et al in 197747
who demonstrated the absence of ADP release by platelets in DHF patients during the
convalescent period. Srichaikul et al in 198954 demonstrated a decrease in platelet
aggregation after stimulation with 5 mmM ADP in 35 patients with DHF studied during
the febrile phase or early convalescent period. The characteristic abnormality was
severely depressed primary aggregation and absent secondary aggregation. These
270 T. Srichaikul and S. Nimmannitya

Shock or defervescence

1000 Shock (8)

800 Non-shock (4)
Platelet 108
BTG IU per

600
400
200

200
100
Platelet 108
PF4 IU per

120
80
40

100
aggregation in 108/ml

80
% Platelet

60

40

20

−1 0 +1 +2 +3 +14 +21
Day of illness
Figure 3. Platelet aggregation, BTG PF4 in 12 DHF patients. K ˆ control value. Reproduced from Srichaikul
et al (1989), Southeast Asian Journal of Tropical Medicine and Public Health 20: 19±25) with permission.

functional abnormalities were found in both shock and non-shock patients. The
majority of the patients had normal platelet aggregation responses to ADP when
studied 2±3 weeks later. Plasma levels of platelet factor 4 and beta thromboglobulin
were increased above the levels of controls in all the patients with platelet dysfunction
(Figure 3). Levels of these proteins returned to normal 2±3 days after the onset of
shock or subsidence of fever. Srichaikul et al interpreted their results as indicating that
platelets circulating during the critical period of plasma leakage were being activated
by some mechanism and became exhausted. The activated platelets were then unable
to respond to in vitro stimulation because of prior in vivo degranulation. This
hypothesis was supported by Chanthraksri et al in 199055 who demonstrated that
platelets from healthy dengue-immune donors, when incubated with dengue virus,
release platelet dense granule contents including ATP, show enhanced ATP release in
response to subminimal ADP challenge and become hyporesponsive to ADP
stimulation later on.
Srichaikul et al54 also demonstrated that platelets from 5 convalescent DHF patients
stimulated by ADP underwent greater aggregation when mixed with autologous acute
phase platelet-poor plasma that had been stored at ÿ708C than when mixed with
 Dengue and dengue haemorrhagic fever 271

fresh convalescent platelet-poor plasma. These ®ndings suggest that acute plasma
contained substances capable of triggering platelet aggregation. The pro-aggregating
agent in acute-phase plasma was not identi®ed, but it may be dengue virus or dengue
virus coated with antibody and complement. Scott et al56 could isolate dengue virus
from platelet-rich plasma in two of 50 patients with dengue infection. Immune
complexes with or without complement were also demonstrated on the surface of
platelets during the acute febrile and early convalescent periods of DHF.49 Platelets
have receptors for complement57 and type II FC g receptors.58

Coagulopathy and disseminated intravascular coagulation

Coagulopathy in DHF has been studied for more than 30 years by many investigators.
During the acute febrile stage, plasma clotting tests revealed a prolonged partial
thromboplastin time and prothrombin time in 60 and 30% of cases respectively.47 Assay
of coagulation factors revealed a mild to moderate reduction of factors II, V, VII, VIII,
IX, X and XII.59 A constant decrease in ®brinogen, along with a mild to moderate
increase in FDP were observed in all grades of the disease.47,59±63 The degree of
clotting abnormalities correlated well with the degree of thrombocytopenia and the
clinical severity, particularly with the degree of shock and bleeding.19,60 Reduction of
antithrombin III and a2 antiplasminogen were also demonstrated in grade 2 DHF
patients.62 In addition, a slight increase in the level of D-dimer was recently found in
DHF patients (Uthaisang E, personal communication, 1999). All of the above ®ndings
clearly demonstrate the occurrence of increased intravascular coagulation in patients
with DHF. A prothrombin complex de®ciency was also suggested in some cases.61
However, the constant reduction of F XII and antithrombin III in the majority of cases
favoured the process of consumptive coagulopathy. Furthermore, a correlation
between coagulopathy and liver impairment was not observed.2,60 There was also
evidence of increased ®brinolysis secondary to disseminated intravascular coagulation
as indicated by the decreased a2 antiplasminogen and increased FDP and D-dimer
levels during the actue phase. However, the degree of hyper®brinolysis was rather
mild since the euglobulin clot lysis time was normal in almost all DHF patients.9,61,63
On the other hand, the hyper®brinolysis could become severe in fatal cases having
severe DIC with intractable shock and severe bleeding.19
De®nitive evidence of DIC in DHF patients came from two major observations.
First, generalized ®brin thrombi were present in the lung, bone marrow, kidney and
adrenal gland in two adult DHF patients who died because of severe bleeding and
intractable shock.19 Furthermore, platelet ®brin thrombi were found in the brain of
grade 2 DHF patients who died of CNS complications (Hemsrichart V, personal
communication, 1985). Second, there was increased consumption of 125I-®brinogen
injected into grade 2, 3, 4 DHF patients60 (Figure 4). The half life of 125I-®brinogen
correlated well with the haemostatic changes which were compatible with DIC. A
correlation between the above abnormal ®ndings and severity of shock was also
observed in this study. Increased intravascular coagulation as indicated by rapid
consumption of 125I-®brinogen was demonstrated in 82 and 56% of the shock and non-
shock groups, respectively. However, these abnormalities were only mild to moderate
and rapidly returned to normal after the recovery from shock. None of the patients
died and anticoagulant therapy was not required in these patients.
In conclusion, increased intravascular coagulation de®nitely occurs in DHF patients. It
is likely to be a major mechanism contributing to severe bleeding in DHF. In general,
the degree of DIC is only mild to moderate and DIC could cease spontaneously after
272 T. Srichaikul and S. Nimmannitya

Figure 4. Plasma disappearance curve and mean T1 of the groups of control, febrile subjects, and DHF
2
patients with grade II and grades III and IV. The number in parentheses indicates the number of subjects
studied in each group. The P value of DHF grade II, DHF grade III and IV versus control ˆ 0.005. Reproduced
from Srichaikul et al (1989), Southeast Asian Journal of Tropical Medicine and Public Health 20: 19±25) with
permission.

proper treatment of, and rapid recovery from, shock. However, DIC could become
severe and clinically signi®cant in patients with severe intractable shock, particularly in
cases with prolonged acidosis or fulminant hepatic failure. In these circumstances, a
vicious circle involving DIC, bleeding and shock would occur. Without e€ective
management to stop this vicious circle, patients would die from bleeding or shock or
multiple organ failure (Figure 5). Therefore, the early and proper management of shock
 Dengue and dengue haemorrhagic fever 273

Figure 5. Fatal DHF in a patient with shock, severe DIC and bleeding.

is very important in order to prevent this vicious circle. The management of patients
with prolonged shock, severe DIC and bleeding remains a challenging problem. In our
experience, the use of heparin or exchange transfusion may be bene®cial in a few cases.
However, further well-controlled studies of these forms of therapy are needed.

SUMMARY

Dengue fever (DF) and dengue haemorrhagic fever (DHF) are both caused by any of
the four dengue serotypes (DEN1±4). The major pathophysiological hallmarks that
distinguish DHF from DF are plasma leakage as a result of increased vascular
permeability followed by hypovolaemia and shock in severe DHF.
Haematological changes which are constantly found in DHF and frequently in DF
include bone marrow suppression, leucopenia and thrombocytopenia. In DHF, but not
in DF, there is an enhanced immune response in a host with a secondary infection, and
this results in the formation of circulating immune complexes, complement activation,
increased histamine release and massive release of many cytokines into the blood. All
of these events lead to several pathological consequences, namely, vasculopathy, shock,
thrombopathy and disseminated intravascular coagulation (DIC).
Bleeding manifestations in DHF vary in severity, ranging from petechiae on the skin
to massive bleeding. Mechanisms of bleeding are multifactorial, namely, vasculopathy,
thrombopathy and DIC. Thrombopathy consisting of thrombocytopenia and platelet
dysfunction is caused by bone marrow suppression (thrombocytopenia), immune
injury, infection of platelets by DV and, ®nally, DIC.
DIC of a mild degree occurs in 56% of non-shock patients but becomes more
prominent in patients with shock. The most severe DIC and massive bleeding are
always accompanied by prolonged intractable shock and cause fatality. Mechanisms of
 274 T. Srichaikul and S. Nimmannitya

DIC are multifactorial, namely, vascular endothelial cell damage, hyperactivity of

platelets and, ®nally, microcirculatory stasis caused mostly by shock. In general, the
degree of DIC is mild to moderate and can be self-limiting following recovery from
shock. The most important aspect of the management of DHF patients is therefore to
treat shock early and e€ectively in order to prevent the vicious circle produced by
severe DIC with massive bleeding and intractable shock. The management of patients
with prolonged shock, severe DIC and bleeding remains a challenging problem.

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