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Hindawi Publishing CorporationISRN Infectious DiseasesVolume 2013, Article ID 571646, 6 pageshttp://dx.doi.org/10.5402/2013/571646

Review ArticlePathogenesis of Dengue Haemorrhagic Fever and Its Impact onCaseManagement

Kolitha H. Sellahewa

Department of Medicine, Melaka Manipal Medical College, Jalan Batu Hampar, Bukit Baru, 75150 Melaka, Malaysia

Correspondence should be addressed to Kolitha H. Sellahewa; [email protected]

Received 5 September 2012; Accepted 30 September 2012

Academic Editors: R. Bologna, R. Favory, and K. Sawanyawisuth

Copyright © 2013 Kolitha H. Sellahewa. is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Plasma leakage and intrinsic coagulopathy are the pathological hall marks in dengue haemorrhagic fever (DHF). Viral virulence,infection enhancing antibodies, cytokines and chemical mediators in the setting of intense immune activation are the key playersimplicated in the pathogenesis of DHF; the exact nature of which is yet to be fully understood.e pathophysiological changes theattended clinical features of plasma leakage necessitate recognition of changing physiological parameters for the early recognitionof plasma leakage and appropriate �uid therapy. On the other hand, the changes in the haematological indices resulting fromcoagulopathy can tempt the clinician to initiate other modalities of therapy. A clearer understanding of the pathogenesis ofDHF and the appreciation that both of these fundamental pathological changes share common pathogenic mechanisms wouldfacilitate the appropriateness of management decisions and the early recognition of severe disease. us, thrombocytopaenia,reduced �brinogen, and prolonged partial thromboplastin time early in the disease course connoted severe disease and attendedplasma leakage rather than clinical bleeding. e detection of plasma cytokine pro�le by a multiple bead immunoassay could alsocomplement clinical parameters in predicting severe disease early in the disease course. us, MIP-𝛽𝛽 indicates good prognosiswhile IFN-𝛾𝛾 portends severe disease.

1. Introduction

Infection by any one of the four serotypes of dengue virus(DENV) remains asymptomatic in the vast majority. Clinicalspectrum among symptomatic infection ranges from undif-ferentiated fever (viral syndrome), dengue fever (DF), anddengue haemorrhagic fever (DHF) to the expanded denguesyndrome with isolated organopathy (unusual manifesta-tions). DF can be without haemorrhage or have unusualhaemorrhage,whileDHF can bewithout shock orwith shock,that is, dengue shock syndrome [1].

e WHO criteria for the clinical diagnosis of DHFrequires the presence of acute and continuous fever of2 to 7 days, haemorrhagic manifestations associated withthrombocytopenia (100,000 cells/c.mm or less) and haemo-concentration (haematocrit >20% from baseline of patientor population of same age). Haemorrhagic manifestationscould be mucosal and or skin or even a positive tourniquettest which is the commonest. Hepatomegaly occurs at some

stage of DHF and oen precedes plasma leakage and hence avaluable early predictor of plasma leakage [1].

DHF is most commonly seen in children with secondarydengue infection but has been documented in primaryinfection with DENV-1 and DENV-3, as well as in infants.ese infants had acquired maternal dengue antibody andsubsequently experienced a dengue infection [2]. Greaterbaseline vascular permeability among children could also bea contributor for more severe disease among children thanamong adults [3]. Epidemiological and serological studiesdone both in ailand and Cuba support the importance ofsecondary dengue infections as a risk factor for DHF. Sincethe �rst observations by Halstead et al. in 1�70, DHF hasbeen present in situations where more than one serotypecirculates [4, 5]. e disease burden and a resurgence ofrecurrent epidemics of DHF are attributable to social dynam-ics and a variety of epidemiological factors such as a highvector density, a high virus circulation, and a population atrisk of secondary infection by virtue of previous exposure

2 ISRN Infectious Diseases

[6]. Besides secondary infection, chronic diseases such asbronchial asthma and diabetes have been suggested as riskfactors for DHF. Also, whites have higher risk of developingDHF than blacks. DENV-2 virus is known to replicate tohigher concentration in the peripheral blood cells of whitescompared with those of blacks [6].

Abnormal haemostasis and plasma leakage are the mainpathophysiological hall marks in DHF. Even though morethan half a century has elapsed since plasma leakage was �rstidenti�ed its precise mechanism remains elusive. e mainfactor implicated in the development of DHF rather thanthe relatively innocuous DF in dengue infection is secondarydengue infection but other factors like viral virulence andhost characteristics are also important. Severe disease isthe result of a complex interaction between the virus andthe immune response evoked by the host with secondaryinfection [7].

2. Plasma Leakage in DHF

2.1. Pathophysiology. Plasma leakage is speci�c to the pleuraland peritoneal surfaces. In DHF there is no vasculitis andhence no injury to the vessel walls, and plasma leakage resultsfrom cytokine mediated increase in vascular permeability.e ensuing movement of albumin and the resultant reduc-tion of intravascular oncotic pressure facilitate further loss of�uid from the intravascular compartment. e basic Starlingprinciple still holds true in explaining microvascular ultra�l-tration based on the balance of the oncotic and hydrostaticpressures. However the glycocalyx, which is a gelatinouslayer lining the vascular endothelium is also implicated incontrolling �uid movement by the adherence of albuminmolecules in to its matrix, damage of which, leads to loss ofalbumin into the extravascular compartment [8–11].

2.2. Immunopathogenesis. e immune system is implicatedin the pathogenesis ofDHFowing to the increased propensityto develop DHF with secondary dengue infection.e innateimmune mechanisms comprising the complement pathwayand NK cells as well as humoral and cell-mediated immunemechanisms launched in response to antigenic stimulationare involved in the clinical manifestations. Complementactivation as well as vascular permeability may be in�uencedby viral products like NS1. Different immune mechanisms inthe form of antibody enhanced viral replication leading to anexaggerated cytokine response impacts vascular permeability[12–14].

Infection with one dengue serotype elicits immunity tothat serotype but does not provide long-term cross-protectiveimmunity to the remaining serotypes. Subsequent infectionwith a different serotype results in the binding of the newvirus to cross reactive nonneutralising antibody from theprevious infection facilitating the uptake by mononuclearphagocytes enabling ampli�ed viral replication.e resultingincrease in viral load then drives an immunopathogeniccascade and the resultant exaggerated cytokine response leadsto a transient increase in microvascularpermeability. eprecise way in which microvascular permeability is alteredis not clear but is more likely to be a functional change

rather than structural damage, as dengue shock is rapidlyrecoverable, and no in�ammation is evident in the leakingsurfaces [15–19]. Adding to the complexity of the under-lying immunopathogenic mechanisms resulting in changesin vascular permeability is the proposal of an alternativemechanismwhereby the rapidmobilisation of serotype cross-reactivememory T cells trigger the release of biologicalmedi-ators. Some of the other factors implicated in this orchestra-tion include viral virulence, molecularmimicry, and immunecomplex and/or complement mediated dysregulation, andgenetic predisposition, all of which have been shown to corre-late with disease severity. However, as yet no mechanism hasbeen identi�ed that links any of these established immuno-logical derangements with a de�nitive effect on microvascu-lar structure or function consistent with the observed alter-ation in permeability. In addition, most of the immunologicalabnormalities so far identi�ed do not differ substantially fromthose seen in other infections without an apparent effect onpermeability.

Neutralising antibodies are key factors in the aetiopatho-genesis of the disease. However, the cellular immuneresponse is also important. It has been demonstrated thatmemory dengue T lymphocyte response aer a primaryinfection includes both serotype-speci�c and serotype-cross-reactive T lymphocytes [20]. NS3 protein seems to be themajor target for CD4+ and CD8+ T cells.

Cytokines that may induce plasma leakage such as inter-feron g, interleukin (IL) 2, and tumour necrosis factor (TNF)𝛼𝛼 are increased in DHF cases [20, 21]. Also, interferon 𝛾𝛾enhances uptake of dengue particles by target cells throughincreasing Fc cell receptors [22].Other cytokines such as IL-6,IL-8, and IL-10 are also increased. A protein of 22–25 kDa hasbeen associated with the pathogenesis of DHF.is cytotoxicfactor able to induce increased capillary permeability in miceis capable of reproducing in mice all the pathological lesionsthat are seen in human beings, and has been detected in seraof DHF patients [23].

A recent study has demonstrated the plasma cytokinepro�le in dengue fever from a Bra�ilian population whichwas detected by a multiplex bead immunoassay. MIP-𝛽𝛽was indicated as a good prognostic marker which is incontrast to IFN-𝛾𝛾 that was associated with severe disease.Both cytokines serve to discriminate mild from severe cases.It has also been shown that during the course of denguedifferent cytokine pro�les may be present and vary accordingto determined clinical manifestations. e cytokine pro�lesidenti�ed by bead arraymultiplex systemmay favour an earlyidenti�cation of patients with the worst prognosis and maycontribute to the establishment of more directed therapeuticprocedures than the present ones [24].

Complement activation as a result of immune com-plexes (virus-antibody) or immune activation and cytokineproduction could also be involved in the mechanism ofplasma leakage. Certain complement fragments such as C3aand C5a are known to enhance permeability. NS1 antigenin dengue virus has been shown to regulate complementactivation and hence could play a role in the pathogenesisof DHF [12, 13, 25–27]. Clearly immunopathogenic mech-anisms are involved in plasma leakage and coagulopathy.

ISRN Infectious Diseases 3

However alternate immune pathways are also implicated in aprotective role adding to the complexity and intricacy of thepathogenesis of DHF. Activated NK cells release granzyme A,which has cytolytic functions. MIP-1𝛽𝛽 produced by humanmonocytes and dendritic cells as well as activated NK cellsand lymphocytes is chemoattractant for NK cells, recruitingthem to in�ammatory sites. ese mechanisms could play aprotective role in the immunopathology of DHF by the earlyand efficient clearance of DENV by direct or indirect NKfunctions thereby limiting viral replication and its attendedcascading cytokine mediated plasma leakage. NK cells havebeen associated with mild dengue [28, 29].

In summarymonocytes,macrophages, and dendritic cellsare the major targets for DENV.

During secondary infection with a different DENVserotype cross-reactive nonneutralising antibodies bind toDENV and facilitate uptake via Fc receptors resulting inenhanced viral replication. e resultant higher viral antigenload leads to an exaggerated activation of cross-reactivedengue speci�c T cells. �iological mediators released bythe activated T cells as well as virus infected cells alongwith complement activation by viral proteins, and immunecomplexes are implicated in increasing vascular permeabilityand coagulopathy.

ese biological mediators in�uence clinical outcomesto a variable extent. us IL-1𝛽𝛽, IFN-𝛾𝛾, IL-4, IL-6, IL-13, IL-7, and GM-CSF are associated with severe clinicalmanifestations whileMIP-1𝛽𝛽 is elevated in patients withmilddengue. Marked thrombocytopaenia is evident in patientswith elevated IL-1𝛽𝛽, IL-8, TNF-𝛼𝛼, andMIP-1, while increasedlevels of MIP-1 and GM-CSF correlated with hypotension[24].

2.3. Haemorrhagic Manifestations in DHF. e pathogenesisof bleeding in DHF is unclear even though well-recognisedcoagulation disturbances do exist. e clinical haemorrhagicmanifestations range from a mere positive tourniquet test,skin petechiae and ecchymoses to epistaxis, and gumbleedingto severe gastrointestinal haemorrhages.rombocytopaeniais a consistent �nding, while prolonged partial thrombo-plastin time and reduced �brinogen concentration are theother abnormal haemostatic indices evident from early in thedisease course. ese haematological abnormalities seem tocorrelate betterwith the timing and severity of plasma leakagerather than the clinical haemorrhagic manifestations [30].

ese recent �ndings raise the possibility for commonpathogenic mechanisms responsible for both plasma leakageand abnormalities in the haemostatic indices.e true natureof the intrinsic coagulopathy evident early in the diseasecourse and in mild forms of dengue can be confounded bythe advent of hypovolemic shock and hypoxia in DHF withsevere plasma leakage with less than optimal correction.

rombocytopaenia is initially due to bone marrowsuppression during the febrile viraemic phase of the illness.Progressive thrombocytopaenia with defervescence resultfrom immune mediated platelet destruction. Virus-antibodycomplexes have been detected on the platelet surface of DHFpatients suggesting a role for immune-mediated destruction

of platelets [31, 32]. Augmented platelet adhesiveness tovascular endothelial cells resulting from the release of highlevels of platelet-activating factor by monocytes with het-erologous secondary infection also contributes to the throm-bocytopaenia [33]. rombocytopaenia however correlatespoorly with bleeding manifestations. Spontaneous bleedingbeen uncommon evenwith counts below 100,000 cells/c.mm.It is strongly associated with the severity of vascular leakage.Counts below 100,000 cells/c.mm or a rapid drop in theplatelet count was associated with severe disease.

e role of the glycocalyx rather than the endothelial cellsper se in controlling ultra�ltration in the microvasculatureis increasingly recognised and in vivo animal studies haveshown the permeation of �brinogen to the endothelial surfacesimilar to albumin [11].

e low plasma �brinogen detected in DHF could thusbe a re�ection of loss into the interstitial spaces in the settingof increased vascular permeability. Heparan sulphate formsan integral part of the glycocalyx which when damaged bythe initial cytokine response in DHF gets liberated to thecirculation and acts like an anticoagulantwhich could explainthe prolonged APTT [34]. e disturbance in both theseimportant haemostatic indices are unlikely to cause sponta-neous bleeding.Haemorrhages are triggered by trauma in thissetting of coagulopathy.

Development of antibodies potentially cross-reactive toplasminogen could have a role in causing haemorrhage inDHF [35]. However different studies have shown con�ictingresults as some have demonstrated an activation of �brinol-ysis while others have shown an inhibition of the �brinolyticpathway in DHF [30].

2.4. Endothelial Cells in DHF. Precise knowledge on theextent to which DENV infects endothelial cells is lacking asfew studies have addressed the issue in the viraemic phase ofthe illness. Even though DENV has infected endothelial cellsin vitro it is doubtful whether it re�ects the effect in humaninfection as limited human autopsy studies have detectedonly the dengue antigen but not the genome in various celltypes ranging frommonocytes, liver sinusoidal cells, alveolarmacrophages, peripheral blood, and splenic lymphocytes.How important these �ndings are in the pathogenesis ofclinical features are uncertain as some studies have shownswelling of endothelial cells but not cell death or vasculitis[36], while others have detected apoptosis of endothelial cellsin lungs and intestinal mucosa in fatal DHF cases, but theextent of apoptosis has not been documented [36]. DENValters the endothelial cell surface protein production, itsexpression, and transcriptional activity.

Expression of ICAM-1 (intercellular adhesion molecule-1) and beta-integrin on micro vascular endothelium byDENV has been reported. DENV also affects the expressionof cytokine receptors. ese may contribute to the mecha-nisms involved in plasma leakage in DHF.

e role of DENV infected endothelial cells in thepathogenesis of coagulopathy in DHF is equally intriguing.ere is upregulation of tissue plasminogen, thrombomod-ulin, protease activated receptor-1, and tissue factor receptor,


while there is downregulation of tissue factor inhibitor andactivated protein C.

3. Clinical Implications

DHF cases have increased in the recent past andwill continueto increase in numbers in time to come as DHF is commonerin secondary dengue infection. e probability of secondarydengue infection in a given population is expected to increaseowing to the presumed high prevalence of previous exposureto clinical or asymptomatic dengue infection based on epi-demiological data particularly in dengue endemic regions inthe world. Despite the complexity of the immunopathogenicmechanism involved in severe disease, what is inexorableis that all patients with DHF have plasma leakage, themagnitude and progression of which will impact outcome.Dengue infection must be diagnosed early and in all suchpatients clinicians need to be alert and vigilant to identifyDHF patients early at the inception of plasma leakage beforeshock sets in. Appropriate interventions with �udicious �uidtherapy at this stage could offset adverse outcomes andensure a favourable outcome. Immunopathogenic mecha-nisms implicated in DHF could serve to meet the challengesof identifying in the febrile phase patients who could behaveas DHF during the disease course. In this context assay ofspeci�c biomarkers identi�ed in dengue could be useful.uswhile MIP-1 𝛽𝛽 indicates good prognosis, IFN-𝛾𝛾 portendssevere disease. Clinicians should also appreciate that bothplasma leakage and disturbances of haemostatic indices sharecommon immunopathogenic mechanisms. Disturbances inthe haemostatic indices should thus be correlated to theseverity of plasma leakage rather than the tendency forspontaneous clinical bleeding manifestations. Such consid-erations would serve to complement the accuracy of theprediction and identi�cation of patients with severe disease.Intelligent application of such knowledge in relation to thetemporal relation of the disease course will also facilitateinterventional decisionmaking and improve its accuracy andappropriateness. us, low plasma �brinogen and prolongedAPTT in the absence of shock early in the disease is to beexpected inDHF and interpreted as heralding plasma leakageand not DIC, and its magnitude gives an idea of the severityof leakage. On the contrary the same indices of coagulopathyshould have a different interpretation in the setting of shockowing to the confounding effects of hypovolemia and hypoxiaand even the probability of associated DIC in such a setting.

Similarly thrombocytopaenia is best used as a markerof severe disease particularly when it is <100,000 cells/c.mmor when there is a rapid drop. Its usefulness is as anindicator of prognosis during the disease course rather thana parameter for therapeutic interventions. Recognising thepoor correlation of thrombocytopaenia with bleeding shouldcaution the clinician against the futility albeit danger ofprophylactic platelet transfusions.

Clinicians should also bear in mind that cytokines playdifferent roles in the pathogenesis of DHF. Some beenstimulatory while others tend to downregulate the immuno-logical network.

Critical alterations in the cytokine balance with attendedadverse, rather than bene�cial outcomes could be expected ifcorticosteroids are used for immunosuppression when suchmanagement decisions are based on the super�cial considera-tion of immunological mechanisms as the underlying basis ofDHF pathogenesis. Even though cytokines are implicated inthe pathogenesis of increased vascular permeability absenceof in�ammation and the transient nature of altered perme-ability with a tendency for spontaneous cessation of plasmaleakage also raises the irrationality of using steroids and otheranti-in�ammatory agents.

4. Concluding Remarks

Plasma leakage and coagulopathy are the fundamentalpathological changes responsible for clinical manifestations,morbidity, and mortality in DHF. A complex interplaybetween immunological mechanisms with viral and hostfactors are implicated in the pathogenesis. Both humoral andcell-mediated immune mechanisms eventually result in therelease of cytokines responsible for changes in the selectivemicrovascular permeability and the resultant plasma leakage.Plasma leakage progresses either rapidly or slowly to ceasecompletely and predictably aer 24 to 48 hours of onset,raising the possibility of existence of underlying functionalchange rather than structural damage and in�ammation inthe vasculature. e in�uence of DEN� on endothelial cellsmay be direct or indirect via release of mediators frominfected or activated immune cells. Changes in the expressionof adhesion molecules, enzymes, and cytokine receptors onendothelial cells are implicated in increasing the vascularpermeability as well as activation of the coagulating system.

e two fundamental pathological attributes in DHF areplasma leakage and intrinsic coagulopathy.

e balance of hydrostatic and oncotic pressures isimportant in plasma leakage. However the glycocalyx alsoplays a crucial role in �uid �uxes. e permeation of �b-rinogen apart from albumin into its matrix, as well as therelease of heparan sulphate from its brush surface impactsboth plasma leakage and intrinsic coagulopathy. e recog-nition of the role of biological markers on these pathogenicmechanisms can have far reaching diagnostic and therapeuticimplications.

Clinicians should strive to predict severe disease beforethe advent of shock. Clinical predictors such as tender hep-atomegaly and tachycardia aer defervescence are exceed-ingly useful to suspect incipient plasma leakage. Technologi-cal advances and the availability of multiplex cytokine pro�lewould facilitate these efforts. It could also open up new vistasin developing interventions targeting speci�c cytokines toreduce plasma leakage. However the importance of diligentand accurate monitoring of heart rate, pulse pressure, urineoutput, and haematocrit for the early detection of plasmaleakage and ad�ustments to �uid therapy should not beoverlooked and constitute an essential and integral part ofcase management. Our understanding of the pathogenesis ofDHF and the availability of biological markers could serveto complement the clinician’s efforts. Prevention of immuneenhanced viral replication is another area to focus speci�c

ISRN Infectious Diseases 5

therapeutic interventions. Use of fresh frozen plasma for thispurpose is an exciting area of research [37].

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