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Seminars in Diagnostic Pathology (2007) 24, 227-236
The pathology of dengue hemorrhagic fever
Anthony S-Y. Leong, MBBS, MD, FRCPA, FRCPath, FCAP, FASCP, FHKAMed (Pathol),
Hon FHKCPath, Hon FRCPT,a K. Thong Wong, MBBS, FRCPath,b
Trishe Y-M. Leong, MBBS (hons), FRCPA, FCAP,c
Puay Hoon Tan, MBBS, FAMS, MD, FRCPath, FRCPA,d
Pongsak Wannakrairot, MD, FRCPTe
a
From the Hunter Area Pathology Service and University of Newcastle, Newcastle, Australia;
Department of Pathology, University of Malaya, Selangor, Malaysia;
c
Victoria Cytology Service, Melbourne, Australia;
d
Department of Pathology, Singapore General Hospital, Singapore; and the
e
Department of Pathology, Chulalongkorn University, Bangkok, Thailand.
b
KEYWORDS
Dengue hemorrhagic
fever;
Dengue virus;
Inflammatory
cytokines;
Dendritic cells;
Pathology;
Hepatitis;
Apoptosis;
Immunology
An estimated 2.5 billion people are at risk of dengue infection, and of the 100 million cases of dengue fever
per year, up to 500,000 develop dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), the
life-threatening forms of the infection. The large majority of DHF/DSS occurs as the result of a secondary
infection with a different serotype of the virus. While not completely understood, there is evidence that the
target cells include dendritic reticulum cells, monocytes, lymphocytes, hepatocytes, and vascular endothelial
cells. Viral replication appears to occur in dendritic cells, monocytes, and possibly circulating lymphoid
cells, and damage to these and other target cells occurs through immune-mediated mechanisms related to
cross-reacting antibodies and cytokines released by dendritic cells, monocytes, and vascular endothelium.
There is evidence of a concomitant cellular activation as well as immune suppression during the infection.
The activation of memory T cells results in cascades of inflammatory cytokines, including tumor necrosis
factor-␣, interleukins (IL-2, IL-6, and IL-8), and other chemical mediators that increase vascular endothelial
permeability or trigger death of target cells through apoptosis. Pathological studies in humans are uncommon, and a suitable animal model of DHF/DSS does not exist. The current treatment of DHF/DSS is
symptomatic, and prevention is through vector control. As such, there is a great impetus for the development
of vaccines and novel therapeutic molecules to impede viral replication in infected cells or counteract the
effects of specific inflammatory mediators on target cells. The role of genetics in relation to resistance to
DHF/DSS also requires clarification.
© 2007 Elsevier Inc. All rights reserved.
The global problem
The first record of a disease clinically resembling dengue
fever (df) can be found in a Chinese medical encyclopedia
Address reprint requests and correspondence: Anthony S-Y Leong,
MD, Hunter Area Pathology Service, Locked Bag 1, HRMC, Newcastle,
NSW 2310, Australia.
E-mail: [email protected].
0740-2570/$ -see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1053/j.semdp.2007.07.002
dated 992.1 With the expansion of the world shipping trade
in the 18th and 19th centuries, both the viruses and their
principal mosquito vector, Aedes aegypti, were spread to
new geographic regions, particularly along the tropical trading routes and shipping ports. Because spread was slow and
via sailing ships, long intervals of 10 to 40 years separated
epidemics of the disease. The expansion of dengue was
closely related to development and economic growth in
tropical countries and the mode of the viral transmission.
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Seminars in Diagnostic Pathology, Vol 24, No 4, November 2007
Economic growth is associated with transmigration of large
numbers of the population into cities and the development
of new cities; this urbanization resulted in a concomitant
increase in breeding sites for the mosquito. Accelerated
modern travel, particularly by airplanes, allows the introduction of the virus from an endemic area to a denguereceptive area where both the vector and susceptible population exist. The virus is usually carried in an infected but
asymptomatic person during the incubation period of the
disease, a method of transmission that often accounts for
explosive outbreaks of df.
In the latter part of the 20th century, globalization and
rapid urbanization of many developing tropical countries
produced increased transmission and hyperendemicity of
the disease. Today, dengue is the most frequent arbovirus
infection with more than 100 million cases annually
throughout the world, including up to 0.5 million cases of
dengue hemorrhagic fever (DHF) and 24,000 deaths.2,3 An
estimated 2.5 billion people are at risk for the infection in
the subtropical and tropical regions of the world,4 and recent
years have witnessed unprecedented global dengue epidemics with large numbers of fatalities5; such statistics make df
one of the most important arbovirus diseases in humans
today.
The magnitude of the global problem is compounded
by the fact that there is currently no specific treatment
or vaccines for the disease, and control is dependent
largely on public health measures directed against the
vectors.
Presentation, pathogenesis, and pathology
The Dengue virus (DV) is a positive-sense, singlestranded RNA surrounded by an icosahedral nucleocapsid. The virus is composed of three structural proteins
[the capsid (C), premembrane (prM), and envelope (E)
proteins] and seven nonstructural (NS1-7) proteins. With
infection, the DV is endocytosed and acidic pH triggers a
conformational change of the E protein. This allows
fusion of the virus envelope to the endosomal membrane
and release of the viral genome into the cytoplasm, where
it is translated into viral proteins that replicate in the host
endoplasmic reticulum. The newly synthesized viral genomes are packaged as viral core, envelope, and membrane proteins into immature virions before being secreted from the cell.6 The virus belongs to the family of
Flaviviridae and the genus Flavivirus, which includes
yellow fever, West Nile, Japanese encephalitis, and St.
Louis encephalitis viruses. There are four distinct serotypes of DV (DEN1-4), and infection with any one of the
serotypes confers lifelong immunity to that serotype.
Whereas DVs exhibit 65% to 70% sequence homology,
serotype cross-reactive immunity is only transient and
wanes after 6 months so that the host remains susceptible
to the remaining three heterologous dengue serotypes.7
Presentation
DV infection results in protean manifestations ranging
from a mild nonspecific or subclinical febrile illness to DHF
or dengue shock syndrome (DSS). df uncomplicated has an
incubation period of 5 to 9 days. The clinical features are
age-dependent. Infants and young adults may only have
simple fever with a maculopapular rash. In older children
and adults, df is characterized by the sudden onset of high
fever of about 40°C, chills, severe headache that is mostly
frontal or retro-ocular, skin rash, general malaise, and severe
muscle ache in the lumbar region, legs, and joints. There is loss
of appetite with nausea and vomiting, and photophobia with
puffiness of the eyelids and cervical lymphadenopathy may
be present. The fever lasts for 2 to 4 days, and after an afebrile
period of about 1 day, a second febrile period may follow,
producing the typical “saddle back” temperature curve. At
this stage, an itchy maculopapular rash develops, often
spreading from the extremities to the trunk to involve the
entire body, except for the face. The palms and soles may
also be red. Convalescence can take several weeks or
months, but there is virtually no mortality associated.8 The
triad of fever, rash, and arthralgia requires separation from
other nonarbovirus infections, such as measles, rubella,
Epstein–Barr virus, cytomegalovirus, and protozoan infections such as toxoplasmosis. Due to the wide clinical variation, it is not possible to make a definite diagnosis on
clinical findings alone. A positive tourniquet test (ⱖ10
petechiae/inch2 read 1 minute after release of 5 minutes
of pressure midway between systolic and diastolic) and
leukopenia (WBC ⱕ5000 cells/mm3) in a febrile patient is
highly predictive but still nonspecific for df. Real-time polymerase chain reaction (RT-PCR) to detect the virus remains
the gold standard, although this test is not available in many
developing nations.
The World Health Organization (WHO) distinguishes
DHF/DSS from df with hemorrhage, which is considered a
mild disease.9 DHF/DSS is associated with poorer outcomes
and a mortality rate that approaches 5%.10 The classical
features of DHF are high fever, increased vascular permeability with hemorrhagic manifestations, thrombocytopenia
(platelet count ⱕ100,000/mm3), and hemoconcentration or
signs of plasma leakage. DSS is defined as DHF plus either
hypotension for age or narrow pulse pressure in the presence of clinical signs of shock. The increased capillary permeability is generally not accompanied by morphological
damage to the capillary endothelium, and there are altered
numbers and functions of leukocytes, increased hematocrit,
and thrombocytopenia. Raised levels of aspartate-amino-transferase (AST) and alanine amino-transferase (ALT) have been
observed in 98% and 37% of patients, respectively, indicating
that liver dysfunction is a very common occurrence in DHF.11
As dengue spreads worldwide, there is evidence that the
four key criteria of severe disease employed in the WHO
grading system (shock, plasma leakage, marked thrombocytopenia, and internal hemorrhage) may not be universally
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Leong et al
Dengue Hemorrhagic Fever
applicable, and many severe cases (including those that
involve shock and fatality) may be missed, especially as the
criteria were based on initial observations in children with
the disease in Southeast Asia.12
Other less common manifestations include neurological
symptoms from cerebral edema and encephalopathy from
hepatic failure, complications of disseminated intravascular
coagulopathy, diastolic dysfunction, abdominal compartment syndrome, adult respiratory distress syndrome,13 acute
renal failure,14 postinfectious fatigue syndrome,15 intracranial hemorrhage,16 fulminant hepatitis,17 acute abdomen,18
hemophagocytic syndrome and dyserythropoiesis,19 and
myocarditis.20
Pathogenesis
The pathogenesis of DHF/DSS is poorly understood and
has been the subject of much research interest in recent
years. Although df is a self-limiting disease in the great
majority of cases, the problem becomes a significant one as
the large numbers of infected persons result in about half a
million cases per year of potentially life-threatening DHF/
DSS. Up to 90% of DHF/DSS cases occur in secondary
heterologous DV infection; the remaining are primary infections, usually in infants between 6 and 12 months of age.
Clearly, secondary infection with a heterologous dengue
serotype is a major risk factor. To date, several immunological aspects of DV infection have been studied in detail.
These relate to the target cells of the virus and their immunological effects and the effects of antibody-mediated
mechanisms in heterologous secondary infections.
Antibody-enhanced viral duplication
After introduction of the dengue virus through the bite of
an infected Aedes mosquito, local viral duplication is
thought to take place in target epidermal dendritic cells that
are up to 10 times more permissive to dengue infection than
monocytes or macrophages. A C-type lectin expressed by
the dendritic cells can bind to the dengue envelope (E)
protein and probably serves as a coreceptor for viral entry.21,22 Migration of interdigitating dendritic cells to regional lymph nodes also allows transfer of the virus to T
cells. IgG antibodies and enhancement via IgM and complement C3 receptors have been implicated in dendritic cell
infection.23,24 The infection of CD14-positive dendritic
cells as well as bone marrow dendritic cells leads to the
production of TNF-␣, IFN-␣, and IL-10 and inefficient
maturation of infected dendritic cells, which undergo apoptosis. The dendritic cells display impaired ability to upregulate cell surface expression of costimulatory, maturation, and
major histocompatibility complex molecules, resulting in reduced T cell stimulatory capacity. There is also an impaired
ability to stimulate allogenic T cells, which is accompanied
by further enhanced IL-10 production.25,26 These findings
suggest a possible immune evasion strategy of the virus by
impairing antigen-presenting cell function through matura-
229
tion blockade and induction of apoptosis.27 Natural killer
cells, which comprise another arm of the innate immune
system, may also play a role. These cells can clear virusinfected cells either by direct cytotoxicity or via antibodydependent cell-mediated cytotoxicity. Dengue immune serum has been shown to mediate antibody-dependent cellmediated cytotoxic lysis of DV-infected cell lines.28
Research into the effects of primary DV infection has
been hampered by the absence of a true animal model of the
disease. Nonhuman primates display viremia but not DHF.
Several promising murine models of DV infections have
recently been developed, including interferon-␣/␤ and ␥
receptor-deficient mice that developed encephalitis and
nonobese diabetic/severe combined immunodeficent mice
reconstituted with human hematopoietic stem cells with
skin erythema.29,30
Not surprisingly, because of the severity and mortality
associated with DHF immunological reactions, secondary
heterologous DV infections have been extensively studied.
Antibody-dependent enhancement is one mechanism that
has been proposed to explain the severity of DHF/DSS. This
mechanism evokes the binding of preexisting dengue antibodies at nonneutralizing conditions to heterologous DV to
enable viral entry into FcRII-bearing target cells.31 The
target cells are predominantly cells of the reticuloendothelial system of spleen, liver, and bone marrow, including
monocytes, lymphocytes, Kupffer cells, and alveolar
macrophages.32 The enhanced viral entry into such cells
produces an increased viral burden in the host. Anti-E
antibodies enhanced infection via FcRII, whereas antiprM
antibodies enhanced infection of both FcRII- and nonFcRII-bearing cells.33 Some studies have demonstrated an
association between viral burden and disease severity,34,35
with the ability of preinfection plasma to enhance infection
of monocytes corresponding to disease severity.36 However,
a more recent study contradicts those findings by showing
no association between the ability of preillness plasma to
enhanced infection in vitro and subsequent dengue viremia
or disease severity in secondary dengue-2 or dengue-3 virus
infections.37
In the small percentage of DHF cases that manifest
during primary DV infection, usually in infants, it has been
postulated that maternal transmission of nonneutralizing
DV antibodies result in the same phenomenon as described
above in adults with secondary infections.38
Although the viral envelope glycoprotein E is responsible for viral attachment and entry, and is the antigenic target
for neutralizing antibodies, there are also several viral nonstructural proteins that are involved in viral replication and
have other effects in vivo. The dengue nonstructural protein
NS1 has both a secreted and cell-associated form. Secreted
NS1 levels have been found to be associated with viremia
levels in secondary dengue-2 infection.39 Anti-NS1 antibodies appear to have both a protective as well as pathogenic
role in DV infection. In animal models, anti-NS1 antibodies
protect against lethal flavivirus challenge,40,41 but these
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antibodies to NS1 have also been suggested to have a direct
role in the pathogenesis of vascular leakage. Sera from acute
dengue-infected individuals are able to bind to human
umbilical cord endothelial cells, and this process can be
blocked by the addition of recombinant NS1.42 Furthermore, the treatment of such umbilical cord cells with murine
antidengue NS1 antibodies, but not Japanese encephalitis
NS1 antibodies, induced the production of IL-6, IL-8, and
monocyte chemo-attractant protein type 1, an inflammatory
response that was abrogated in the presence of recombinant
dengue NS1.43 Anti-NS1 antibodies have also been implicated to cause damage to endothelial cells by inducing nitric
oxide-mediated apoptosis.42 Antibodies to NS1 persist long
after dengue infection has resolved but do not cause pathology. Furthermore, the persistence and kinetics of development of anti-NS1 antibodies during and after secondary DV
infection do not correlate with the timing of plasma leakage,
raising questions as to the clinical relevance of this in vitro
evidence for molecular mimicry.
On primary infection with DV, antibodies are generated
against NS1 and the viral envelope protein E. Serotypespecific and serotype cross-reactive neutralizing antibodies
are directed against the E protein. Enhancing antibodies can
affect the severity of the disease as long as 20 years after the
primary infection, especially during dengue-2 and dengue-3
infection following a primary infection with dengue-1 virus.44 However, all serotypes have been shown to be capable of causing severe disease,45 and milder disease is associated with lower viral loads and high levels of preexisting
heterotype neutralizing antibodies during the secondary infection. This is not necessarily always true; in the case of
secondary dengue-3 virus infection, higher levels of these
cross-reactive memory humoral immune responses appeared
to be beneficial as demonstrated by reduced viremia levels and
decreased disease severity, but the same did not hold true for
secondary dengue-1 or dengue-2 virus infection.46
Immunological mechanisms
The observation that onset of plasma leakage occurs up
to several days following significant reduction or clearance
of viremia suggests that, instead of the proposed antibodydependent enhancement due to viral burden, an immunemediated mechanism may be responsible for the extreme
capillary permeability that is characteristic of DHF/DSS.
Furthermore, despite high viral loads in secondary infections, progression to more severe forms of the infection
occurs only in ⬍5% of cases. Serotype-specific and crossreactive T cells have been detected in the peripheral blood
of individuals with acute DV infections. It is suggested that
these serotype cross-reacting T cells show low affinity for
the infecting virus and a higher affinity for another virus
serotype (presumably from a previous infection), a phenomenon called the “original antigenic sin.”47 However, definite
identification of the primary infecting DV serotype is difficult because neutralizing antibodies can be highly serotype
cross-reactive after secondary infection, a caveat that questions
the validity of this hypothesis.27 Importantly, it has been dem-
onstrated that cross-reactive dengue-specific T cells induce
high levels of cytokine production that may lead to increased
vascular permeability.48
CD4 and CD8 T cell responses after primary DV infection have shown interesting differences. CD4⫹ T cells
produced greater amounts of IFN-␥ to homologous DV
antigens, but the ratio of TNF-␣ to IFN-␥ was higher after
stimulation with heterologous serotype antigens or CD4⫹ T
cell epitopes.49 CD8⫹ T cells have shown partial agonist
responses in vitro in which a heterologous serotype variant
could sensitize target cells for lysis but not cytokine production or proliferation.
Several studies have suggested a possible role for “immunodominant” variants of dengue epitopes in which the
sequence of DV serotypes may influence the risk of DHF/
DSS. Human leukocyte antigen (HLA) A2-restricted CD8⫹
T cell responses in primary dengue-immune individuals
have shown both quantitative as well as qualitative differences in their cytokine responses to variant dengue epitopes,
suggesting that previous infection as well as the sequence of
heterologous DV infections may affect subsequent clinical
outcome.50 For some epitopes, a single dengue variant was
able to elicit the highest response in all donors, regardless of
infecting serotype, suggesting that certain epitopes may be
immunodominant. A recently identified dengue HLA-A11restricted NS3 T cell epitope has also been found to be presented by HLA A24, an unexpected finding because these
alleles belong to different HLA superfamilies.48 Previous infection history to unrelated viruses can also lead to immunopathology, as shown in human and animal models.51
The studies performed so far appear to show a concomitant cellular activation as well as immune suppression
during acute DV infection. T cell proliferation to mitogens
is impaired during the acute infection, and this appears to be
caused by a defect in the antigen-presenting cell.52 There is
an impairment of the normal plasmacytoid dendritic cell
response in children who subsequently develop DHF. This
blunted response is thought to result in inadequate control of
viremia with the subsequent enhanced activation of crossreactive memory T lymphocytes resulting in DHF.53 Overall numbers of CD4 and CD8 T cells, natural killer cells,
and ␥␦ T cells have been found to be decreased in DHF
compared with df, but despite their reduced numbers, CD8
and natural killer T cells expressed higher levels of the
activation marker CD69 in patients with DHF compared
with uncomplicated df. It has been shown that low affinity
memory T cells occur at higher levels than cytotoxic T cells,
the latter being lost through apoptosis,47 and DV epitopespecific T cell responses are associated with disease severity.47 Dengue-specific T cells are increased in DHF, but
peripheral blood mononuclear cells are unable to produce
IFN-␥ in response to dengue epitope stimulation,48 reflecting the reduced ability of monocytes and dendritic cells to
present antigen adequately. Alternatively, antigen-induced
cell death may ensue after the in vitro stimulation of re-
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Dengue Hemorrhagic Fever
cently activated CD8 T cells,52 a finding that has been
directly demonstrated.48,54
The short-lived nature of plasma leakage syndrome seen
in DHF is another point to support a functional rather than
destructive effect of DV infection on endothelial cells. In
vitro studies of human endothelial cell lines infected with
DVs can produce pro-inflammatory mediators such as IL-6
and IL-8 RANTES (regulated on activation; normal T cell
expressed and secreted), alter intercellular cell adhesion
molecule type 1 surface expression and actin cytoskeleton
structure, and increase permeability to small molecules.55,56
These effects could be partially reversed by neutralizing
antibodies directed against IL-8.55 Also, the infection of
human umbilical cord endothelial cells with DV in the
presence of antidengue immune serum induced the formation of activated complement via both classical and alternative pathways with complement activation appearing to
be mediated by dengue NS1 protein.57 Serum from patients
with acute dengue infection induced the activation and
apoptosis of cultured endothelial cells that could be partially
reversed by anti-TNF-␣ monoclonal antibodies, further supporting the role of inflammatory mediators for plasma leak-
A
C
231
age.58 A recent murine model for DV-induced lethal vascular permeability has also demonstrated the importance of
TNF-␣ as a key mediator of DV induced in mice.59
The presence of IgM, ␤-1-globulin, and fibrinogen has
been demonstrated in the cutaneous vessels of patients with
DHF with dengue antigen in perivascular mononuclear
cells, indicating that the skin rashes associated with DHF
are immune-mediated.60
Cytokine cascades, complement, and other mediators
Although incomplete, the current evidence suggests that,
after massive activation of memory T cells, a cytokine
cascade that targets vascular endothelial cells is primarily
responsible for the critical leakage of fluid and protein in
DHF/DSS. Such cytokines, including IFN-␥, TNF-␣, IL-2,
IL-6, IL1-␤, and IL-8, are released in high concentrations
mostly by T cells, monocytes/macrophages, and endothelial
cells and are demonstrable in the serum of patients with
DHF/DSS. More recent studies have confirmed the presence
of elevated levels of IFN-␥, TNF-␣, and IL-10 in patients
from Vietnam, India, and Cuba.61-63 Such cytokines have
the potential to induce the release and production of other
B
D
Figure 1 Organs from a 2-year-old Thai boy who died from dengue hemorrhagic fever. (A) The eviscerated intestines were markedly
edematous and focally hemorrhagic, and there was a hemoperitoneum of 300 mL. (B) The kidneys were edematous with focal hemorrhage
and hemorrhage into the calyces and renal pelvis. (C) The liver was swollen with focal hemorrhage. (D) Both lungs were heavy and beefy
in consistency and hemorrhagic.
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Seminars in Diagnostic Pathology, Vol 24, No 4, November 2007
cytokines so that a complex interactive network results in
further increases in the levels of cytokines and other chemical mediators in a cascade, often with synergistic effects on
vascular permeability. IFN-␥ is also able to upgrade the
expression of Fc␥ receptors on monocytes and macrophages, further facilitating viral replication. Although this
cascade of cytokine activation and production stimulated by
memory T cells is an attractive proposition in adult DHF/
DSS, it fails to explain the occurrence of this severe form of
dengue infection in the primary infection of infants born of
dengue-immune mothers.
Complement has been suggested to have a role in the
immunopathogenesis of DHF/DSS, although its cause remains unknown. Large amounts of dengue NS1, complement anaphylatoxin C5a, and the terminal complement
complex SC5b-9 were found in pleural fluids of patients
with DHF/DSS.57 Other mediators, such as histamine, tissue
plasminogen activator, and macrophage inhibitory factor,
have also been found in this disease.64,65
Together with the endothelial permeability seen in DHF,
there is a marked thrombocytopenia. This has also been seen
in df, although the drop in platelet numbers is less marked.
It has been suggested that there is transient suppression of
hematopoiesis, although megakaryocytes have not been
shown to be infected with the virus.66 Other suggestions for
the thrombocytopenia include binding of the virus to platelets in the presence of virus-specific antibody, antivirus
antibodies that cross-react with human platelets, and antiplatelet antibodies, the latter in a mouse model.67
The elevation of serum liver enzymes is a frequent finding in DV infection, and infrequently midzonal necrosis is
demonstrable in the liver and fulminant hepatitis may occur.
Hepatocytes are therefore a likely target for the virus. In
vitro infection of HepG2 cells results in apoptosis mediated
by TNF-related apoptosis-inducing ligand and other chemokines, such as IL-8, RANTES, and monocyte chemoattractant proteins, with evidence that these actions may be mediated by NS5 protein.68
Other mediators, such as histamine, tissue plasminogen
activator, and macrophage migration inhibitory factor, have
also been implicated in the pathogenesis of the severe forms
of dengue infection.69
volume with augmented endocapillary and mesangial cellularity.
Children who die from DHF have entensive edema of
viscera with leakage of blood and focal hemorrhage (Figure
1). In a study of five fatal cases of DHF in Vietnamese
children, severe hepatitis was observed with midzonal necrosis and micro-vesicular steatosis, although in one patient
the liver appeared normal. The necrotic areas showed apoptosis by the TUNEL technique, with destruction of both
hepatocytes and Kupffer cells, and there was no recruitment
of polymorphonuclear cells or lymphocytes.71 Another
study of nine fatal cases with fulminant hepatitis confirmed
the presence of DV cDNA by reverse transcriptase in situ
PCR in more than 80% of the hepatocytes and in many
Kupffer cells. Five livers showed massive hepatocyte necrosis or apoptosis with no accompanying involvement of
bile ducts (Figure 2). There was rare bile canalicular cholestasis, and micro-vesicular steatosis was common. The
pauci-cellular areas of massive hepatic necrosis showed
only rare Kupffer cells to be positive for TNF-␣ and IL-272
compared with the upregulation of these and many other
cytokines seen in the livers of fatal cases of hepatitis C.
Thrombotic microangiopathy may be observed in the glomeruli (Figure 3), most likely the result of disseminated
intravascular coagulopathy from hemoconcentration.14
Localization of DV antigen and RNA by immunohistochemistry and in situ hybridization confirmed the presence
of viral antigens in Kupffer cells, lymphoid cells in the
splenic red and white pulp, renal tubular epithelium, vascular endothelium of the liver and lung, monocytes in the
liver, spleen, and lung, and peripheral blood monocytes and
lymphocytes (Figure 4 A and B), although high levels of
Pathology
There are a limited number of morphological studies in
DHF/DSS. In BALB/c mice inoculated with dengue-2 obtained from human serum, focal alterations were found in
the liver, kidney, lung, and cerebellum. The presence of the
virus in these organs was confirmed by ultrastructural and
immunolocalization techniques in mosquito cell cultures of
the infected tissues.70 Hepatocytes were ballooned; portal
and centrilobular veins were congested; lungs were focally
hemorrhagic with vascular congestion and focal alveolitis;
cerebellar tissue displayed focal neuronal compaction and
perivascular edema; and there was increased glomerular
Figure 2 Liver biopsy from a 13-year-old Chinese boy from
Singapore who presented with dengue hemorrhagic fever and
fulminant hepatitis. The needle core biopsy showed extensive
pauci-cellular necrosis concentrated in the midzone, but in areas
tending to involve the entire lobule, the portal tract elements were
not involved (arrows). The nuclear debris represented apoptotic
bodies.
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Dengue Hemorrhagic Fever
233
vesicles in cutaneous vascular endothelium has suggested
that the presence of viral antigen in these cells represents
endocytosis and not infection.73
Genetic predisposition and resistance
Figure 3 A 46-year-old Chinese woman with dengue hemorrhagic fever. Her hemoglobin was 3 g/dL, platelets 64,000/dL, and
hematocrit 26.4%. Peripheral blood flim showed no evidence of
fragmented cells. Thrombotic microangiopathy was evident in the
renal biopsy (arrows, methanamine silver).
viral RNA were only found in the antigen-bearing cells of
the spleen and peripheral blood supporting viral replication
in these latter cells (Figure 4C and D).32 Viral antigen was
present in the endothelium of the organs studied, but there
was no evidence of viral replication. The presence of increased numbers of ultrastructural vacuoles and pinocytic
The patient’s genetic background appears to be a critical
factor in determining progression to DHF/DSS. Outbreaks
in Cuba have shown a reduced risk of people of Negroid
race for DHF/DSS compared with those of Caucasoid
race,74 an observation coinciding with the low reported
incidence of dengue disease in African and Black Caribbean
populations. Although dengue has been documented in 19
African nations and the virus repeatedly isolated, only sporadic cases of dengue have been reported, and mainly in
nonindigenous populations. Even when outbreaks have
been reported in Africa and the Seychelles, the clinical
manifestations have been very mild.75 There is a clear
absence of DHF/DSS despite hyperendemic dengue virus
transmission in the Haiti,76 this despite the annual infection
rate in Port-au-Prince being about 30% higher than that of
Yangoon, Myanmar, where DHF/DSS rates and fatalities
are high.
Although polymorphic genes have been suggested as
possible contenders to account for the genetic susceptibility,
the HLA region is an obvious candidate as it encodes
several proteins involved in the immune response, including
complement and TNF-␣. Certain HLA types have been both
A
B
C
D
Figure 4 Avidin– biotin peroxidase staining with antidengue polyclonal antibodies for three serotypes (hyperimmune mouse ascitic fluid; kindly
provided by R.E. Shope. WHO Centre for Tropical Diseases, University of Texas Medical Branch, Galveston, TX). (a) Staining was seen in
Kupffer cells. (b) The antigen was present in the white pulp of the spleen as well as in scattered larger monocytes in the red pulp. (c) In situ
hybridization showed viral genome in macrophages and stimulated lymphoid cells of the splenic red pulp and peripheral blood monocytes (d).32
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Seminars in Diagnostic Pathology, Vol 24, No 4, November 2007
positively and negatively associated with DHF. For example, variations in HLA-A locus were significantly associated
with susceptibility to DHF/DSS77; HLA-DR04 has the reverse association.78 Genetic background may also be related
to the proliferation of low-affinity T cells, or the persistence of cytokine production by these cells, or both factors
together.
Conclusions
Current information on the immunopathogenesis of DHF/
DSS is still fragmentary. The severe forms of the DV
infection occur as secondary infections. The target cells
appear to be dendritic cells, monocytes, hepatocytes, T
lymphocytes, and possibly vascular endothelial cells. The
disease results from two major immune mechanisms that
involve the production of nonneutralizing enhancing antibodies that cross-react between the serotypes of DV enhancing viral entry into dendritic cells and monocytes to increase
the viral load and produce inefficient maturation of the
infected cells. The other component of the immune response
involves the massive activation of memory T cells sensitized in a previous infection. This activation results in the
proliferation and release of pro-inflammatory cytokines and
a cytokine cascade that targets the susceptible cells causing
cell death through apoptosis and is responsible for the fluid
and protein leakage and the liver damage characteristic of
DHF/DSS. Research is hampered by the absence of a suitable animal model for DHF/DSS, and detailed pathological
studies in humans are few. Current treatment is largely
symptomatic, and prevention is through vector control,
making it imperative that greater understanding of the
pathogenesis be achieved with the view of developing novel
molecules to inhibit viral replication and to stem the damaging effects of immune mediators. There is also impetus
for vaccine development, and a greater understanding of the
relation of resistance to the severe forms of the infection and
genetics is needed.
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