logy in dengue and dengue

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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 first major epidemics in Southeast Asia occurred in the mid 1950s. A global pandemic has intensified 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. Baillie`re’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 8

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Page 1: logy in Dengue and Dengue

Haematology in dengue and denguehaemorrhagic fever

Tanomsri Srichaikul MD, MSc

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

Suchitra Nimmannitya MD, MPH

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

Dengue fever (DF) and dengue haemorrhagic fever (DHF) are caused by the dengue virus. Themajor pathophysiological hallmark that distinguishes DHF fromDF is plasma leakage as a resultof increased vascular permeability. Following this leakage, hypovolaemic shock occurs as aconsequence of a critical plasma volume loss. Constant haematological abnormalities occurringin 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 andleads to many consequences. These are immune complex formation, complement activation,increased histamine release and amassive release of many cytokines into the circulation, leadingto 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 theresult of prolonged shock and cause a fatal outcome. Themechanisms of thrombopathy andDICand 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 themost important mosquito-borne viral disease in the tropical areas of the world.Dengue haemorrhagic fever (DHF), the most severe form, has emerged to becomeone of the major public health problems. The ®rst major epidemics in Southeast Asiaoccurred in the mid 1950s. A global pandemic has intensi®ed during the past 18 yearswith expanding geographical distribution, increased epidemic frequency and theemergence of DHF in new areas. It has been estimated that 20 million cases of dengueinfection occur annually, with approximately 300000 to 500000 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

UnlikeDF, known forover 200 years as amild non-fatal disease,DHFhas the potentialto develop into a fatal dengue-shock syndrome (DSS). The major pathophysiological

1521±6926/00/020261+16 $35.00/00 *c 2000 Harcourt Publishers Ltd.

BaillieÁ re's Clinical HaematologyVol. 13, No. 2, pp. 261±276, 2000doi:10.1053/beha.2000.0073, available online at http://www.idealibrary.com on

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hallmarks that determine disease severity and distinguish DHF from DF and otherviral haemorrhagic fevers are plasma leakage as a result of increased vascular permea-bility and abnormal haemostasis.2 Hypovolaemic shock occurs as a consequence of, andsubsequent to, a critical plasma volume loss. Abnormal haemostasis including increasedcapillary fragility (positive tourniquet test and a tendency to bruise), thrombocytopenia,impaired platelet function and, in the most severe form, disseminated intravascularcoagulation (DIC), contribute to varying degrees of haemorrhagic diathesis.1±3

For the past four decades extensive research studies in various ®elds have contributeda great deal to better understanding of the pathophysiology and pathogenesis of DHF.Although the precise pathogenetic mechanism has not been de®ned, all available datastrongly suggest the involvement of immunological mechanisms. The association ofDHF/DSS with secondary dengue infection rather than the ®rst (primary) infection ledHalstead (1977) to study and describe antibody-dependent enhancement (ADE) ofdengue virus infection in macrophages or mononuclear cells.4 It is proposed that anincrease in the number of dengue virus-infectedmonocytes by the enhancing activity ofcross-reacting antibody from previous infection was responsible for the pathogenesis ofDHF. Complement activation, with profound depression of C3 and C5 levels, is aconstant ®nding and is thought to play an important role.5,6 Kurane and Ennis proposedthat, in addition to ADE and activation of complement, activation of T-lymphocytes alsoplays an important role.5,6 Activation of monocytes and T-lymphocytes induces theproduction of cytokines and chemical mediators. These authors hypothesized that arapid 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, inducemalfunction of vascular endothelial cells which leads to plasma leakage and shock,and derangements of the coagulation system which may lead to haemorrhagicmanifestations.7,8

CLINICAL PRESENTATIONS

Dengue fever

The clinical features of DF are age-dependent. Infants and children infectedwith denguevirus for the ®rst time usually develop a simple febrile illness. Dengue fever is mostcommon in adults and older children and may be either benign or present as the classicincapacitating disease with severe muscle, joint, and bone pain (break-bone fever).Erythematous/maculopapular skin rashes, occasionally with petechiae, are common, asare gastrointestinal symptoms. Rarely, massive haemorrhage, mostly from pre-existinglesions in the gastrointestinal tract may result in death. Leukopenia is common, andmoderate thrombocytopenia is occasionally observed. The clinical presentations of DFvary 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 tropicalareas where dengue is endemic, classic DF is infrequently found among indigenousadults.1

Dengue haemorrhagic fever

Unlike DF, DHF occurs mainly in children and its clinical features are ratherdistinctive. Typically DHF is characterized by four major clinical manifestationspresented below in order of their appearance and frequency.1,2

. High continuous fever for 2 to 7 days in most cases;

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. Haemorrhagic diathesis, most frequently presenting as skin petechiae (including apositive tourniquet test);

. Hepatomegaly; and

. Circulatory disturbances (presenting as shock in severe cases).

Thrombocytopenia and haemoconcentration (an increase in the haematocrit of 20% ormore) representing the pathophysiological hallmarks of abnormal haemostasis andplasma leakage, respectively, are constant ®ndings.

The course of DHF is rather stereotypical. An early and accurate clinical diagnosiscould be made based on the above-mentioned four major manifestations together withthe presence of thrombocytopenia and haemoconcentration.2 The World HealthOrganization (WHO) has adopted these to be WHO criteria.1 The disease severity iscategorized according to the clinical evidence of shock into grade I and II non-shockcases; and grade III and IV shock cases. The grade II patient is distinguished from thegrade I patient by the presence of spontaneous bleeding in the former. The grade IVpatients are those who experience profound shock with undetectable pulse and/orblood 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 inmany respects, but a maculopapular rash and myalgia/arthralgia are less frequent inDHF.1 Haemorrhagic manifestations, which are invariably present in DHF, are usuallymild and frequently consist of tiny, scattered, petechial haemorrhages on the skin oroccasionally in the buccal cavity and subconjunctivae, and a tendency to bruise. Early inthe febrile phase, a positive tourniquet test is frequently observed. Bleeding from thenose, gums and gastrointestinal tract are less common, but may be severe. Massivegastrointestinal haemorrhage may occur and is usually found later in association withprolonged shock. Haematuria is extremely rare.

The liver is often enlarged and palpable a few days after the onset of fever. It isusually soft and tender, but jaundice is not observed. Splenomegaly is rarely noted insmall 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. Accompanyingor shortly after a rapid drop in temperature, varying degrees of circulatory disturbancedevelop. The patient is often sweating, restless and has cold extremities. In mild DHFcases, (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 onsetof shock is acute and generally occurs at the time of defervescence, which is on or afterthe third day of illness (the shortest duration of fever is 2 days). The temperature isoften 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 diastolicpressure (e.g. 100/90, 110/90 mmHg) in the early stage of shock. It is noteworthy thatpatients who are in shock usually remain conscious almost to the terminal stage. Thecourse 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/orblood pressure become undetectable (WHO grade IV). Patients who do not receivetreatment usually die within 12 to 24 hours after shock ensues. Prolonged shock isoften 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 mostcommon site of severe bleeding is in the gastrointestinal tract, and the bleeding usuallypresents as haematemesis (with dark-coloured blood) and/or melena. Occasionally thebleeding may be concealed, leading to a more complicated course if not recognized.The least common haemorrhagic manifestation is intracranial bleeding, but these

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patients have the highest fatality rates. Convulsions and coma may occur in patientswith DIC and bleeding into the brain.9

The pathogenetic mechanisms that lead to plasma leakage and abnormal haemostasisare self-limiting. The critical period of plasma leakage and shock rarely last longer than48 hours. In most cases, early and e�ective replacement of lost plasmawith isotonic saltsolution, plasma or plasma expander results in a rapid and uneventful recovery. Bloodtransfusion is indicated in patients with severe bleeding and occasionally bloodcomponents may be needed. With early diagnosis and proper treatment of shock thecase fatality rate of DHF has been markedly reduced. The causes of death are stillmainly 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 occurselectively into the pleural and abdominal cavities. Pericardial e�usion, if there is any, israther minimal. Chest radiographs demonstrate pleural e�usion which correlates wellwith 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 studiessuggest that DSS results from hypovolaemia due to plasma loss accompanied by anincrease in peripheral resistance.2,10

It is likely that the increase in vascular permeability leading to plasma leakageresults from a functional change caused by short-lived pharmacological mediator(s) andproducts of the immune mechanism, rather than from structural destruction ofendothelial 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 recoverywith 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 andhaemorrhage.11

Two observations have been important in understanding the mechanisms of vascularchanges, plasma leakage and shock. First, DHF patients have no generalized oedemawhen 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 afterdefervescence, which is after the tourniquet test becomes positive and the petechiae®rst appear. These observations suggest that the vascular changes (vasculopathy) thatlead to increased vascular permeability and plasma leakage probably occur at venulesin the thoracic and abdominal cavities while those changes related to haemorrhage inthe form of leakage of erythrocytes are probably con®ned to capillaries.12 The capillaryfragility change may be a direct e�ect of the virus as it appears early in the ®rst fewdays of illness during the viraemic phase.3,4

Among possible mediators, it has been recently shown that C3a and C5a areelevated, and that both their levels and duration of elevation correlated well with theoccurrence of shock and disease severity.13 Among the cytokines and chemicalmediators, including tumour necrosis factor, interleukin-1 (IL-1), IL-2 and IL-6, IFN-g,

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

Haemorrhagic diathesis

Haemorrhagic manifestations, which are invariably present in DHF, are usually mildand are most commonly found as scattered tiny petechiae on the skin and occasionallyon the submucosa. A positive tourniquet test, which indicates increased capillaryfragility, and bruises at venepuncture sites, are the most common ®ndings that appearearly. Epistaxis is the next most common manifestation and may be severe. Massivebleeding that requires blood transfusion is less common and usually occurs after theonset of shock.14 Gastrointestinal bleeding in the form of haematemesis and/ormelaena is the most common type of severe bleeding. In those who died afterprolonged shock, bleeding in various organs (gastrointestinal tract, heart, lungs, liverand brain) have been observed.9,11 Some pre-existing host factors may contribute togastric 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 tostudy and understand the mechanism of bleeding in order to improve the managementof a patient with bleeding.

HAEMATOLOGICAL DISORDERS

Peripheral blood

Leucopenia is a common ®nding in both DF and DHF. Initially, the leucocyte countsmay 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 totalleucocytes and neutrophils. Simultaneously, a relative lymphocytosis is noted with thepresence of atypical lymphocytes. The leucopenia usually reaches a nadir shortly beforeor at the time the temperature drops and the leucocyte count returns to normal 2±3days after defervescence. The numbers of atypical lymphocytes in DHF, varying from15 to 20%, are clearly greater than in patients with DF.15,16 These cells may representactivated B- and T-lymphocytes.17 Moderate to marked reduction in the number ofplatelets usually follows the reduction in leucocytes and reaches a nadir on the day ofdefervescence.2 Giant platelets have been observed in blood smears of DHF patients,suggesting an increased platelet turnover.12 The platelet counts usually drop rapidly tobelow 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 plateletcounts correlate well with disease severity.2,18,19

Bone marrow changes

The haemopoietic suppression is a well known phenomenon during dengue virusinfection. The degree of suppression is similar in both DF and DHF. In experimentalstudies of humans infected with dengue virus, neutropenia developed on the secondday of clinical symptoms.20 Therefore, the suppression of haemopoiesis probably beginsaround 4±5 days after the innoculation of virus by the bites of an infected mosquito,

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during the incubation period (5±8 days).21 This suppression lasted approximately 7±10days and ended in the acute febrile phase approximately 2±3 days before the onset ofshock or subsidence of fever. In various studies11,22±26 the bone marrow of patientswith DHF at the early phase of the acute febrile illness showed marked hypocellularitywith a decrease in all the haemopoietic cell types including megakaryocytes, erythro-blasts and myeloid precursors. In vitro studies have shown that the colony formingunits for granulocytes and macrophages (CFU-GM) were markedly decreased oralmost absent. The colonies were also abnormally small or appeared as a cluster ofcells.27 The decrease in granulopoiesis sometimes was accompanied by a dispro-portionate reduction in cells after the myelocytic stage.23 Following the haemopoieticsuppression, recovery of haemopoiesis occurred a few days before the onset of shockor subsidence of fever. The bone marrow then appeared hypercellular, with anincrease in the number of megakaryocytes, erythroblasts and myeloid precursors.Despite the increased or normal number of megakaryocytes, some of these cellsshowed signs of degeneration as manifested by nuclear fragmentation and/or vacuol-ization and naked nuclei.23,27 Haemophagocytosis of young and mature erythroid andmyeloid 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 thenumber 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 maximumreduction of these cells was seen on the day of shock or subsidence of fever. In therecovery phase of the disease, the number of leucocytes, neutrophils and plateletsgradually returned to normal within 7 days. The time sequence of the onset ofneutropenia and thrombocytopenia following the bone marrow suppression indicatedthat neutropenia and thrombocytopenia were the result of the haemopoietic suppres-sion. The haemophagocytosis observed in the phase of bone marrow recovery11,23 maybe another mechanism inducing further leucopenia and thrombocytopenia. Ine�ectivethrombopoiesis may also contribute to the thrombocytopenia. The peripheraldestruction 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 mainfactors. These are: (1) direct injury to haemopoietic progenitor cells by infection ofthese 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 wasdemonstrated 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 viruscaused inhibition of the proliferation of erythroid burst-forming units (BFU-E) andgranulocyte-macrophage colony-forming units (CFU-GM). In 1997, Murgur et al29

demonstrated in vitro that DEN3 could infect the cord blood mononuclear cells. Thisinfection caused suppression of progenitor cell growth in cultures. The degree ofhaemopoietic suppression was correlated with the clinical spectrum of dengueinfection. 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 Russaet al.30,31 They demonstrated that the infection of stromal cells byDV2 caused inhibitionof the growth of early haemopoietic progenitor cells in Dexter cultures. During theinfection of stromal cells by DV, many cytokines were released into the culturesupernatant. These cytokines weremacrophage in¯ammatory protein-1 alpha (MIP-1a),

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IL6 and IL-8.32±34 MIP-1a is capable of inhibiting the growth of early haemopoieticprogenitor cells. Simultaneously, there was a decrease in stem cell factor35 leading to adecreased 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 ofdengue 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 thesecytokines correlated well with the clinical severity of dengue infection.37±40 The timeof bone marrow suppression also corresponded with the increased level of cytokinesin the blood.41±44

In summary, the bone marrow suppression in DF and DHF appears to result fromthe direct infection of haemopoietic progenitor cells and bone marrow stromal cells bydengue virus as well as the release of various haematodepressive cytokines during thedengue virus infection. The haemopoietic suppression, leads to granulocytopenia andthrombocytopenia. The haemopoietic suppression occurs transiently and recoversrapidly by the end of the febrile period. Following this recovery the number ofneutrophils and platelets continues to be low until the day of shock or subsidence offever and then gradually returns to normal within 7 days in the convalescent period ofthe disease.

Platelet alteration

There are two major changes observed in DHF: thrombocytopenia and plateletdysfunction.

Thrombocytopenia

A platelet count less than 100 � 109/l may be occasionally observed in DF but is aconstant feature of DHF.1,2 Mean platelet nadir for patients with DHF categorized bygrade according to the World Health Organization (WHO) was successively lower forgrades I, II, and III/IV.1,4 The degree of thrombocytopenia is well correlated with theclinical 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 only15% of the shock cases with platelets lower than 50 � 109/l experienced severebleeding.2 Very severe thrombocytopenia, less than 10 � 109/l, could be seen in severecases. The degree of thrombocytopenia was also correlated with the activation ofcomplement and plasma kinin system.45,46 In the study of Srichaikul et al19, a plateletcount less than 10 � 109/l could be observed at 2 days before the onset of shock orsubsidence of fever. In the convalescent period, the platelet count rose by the secondafebrile day andwas normal in all patients by 7 days after subsidence of fever19 (Figure 2).

The pathogenesis of thrombocytopenia in DF and DHF involves two majormechanisms. These are decreased production and increased peripheral destruction orincreased utilization. Decreased production has already been discussed in the section onbone marrow suppression. The mechanisms inducing peripheral destruction orincreased utilization are more important and play a major role in the induction ofthrombocytopenia in DHF. This mechanism was suggested by the observations thatduring the 2 days before shock or defervescence, rapidly progressive thrombocytopeniaoccurred along with the normal or increased number of megakaryocytes in the bonemarrow. Strong evidence supporting peripheral destruction of platelets inDHFpatientscame from a study by Mitrakul et al in 1977.47 In this study it was demonstrated that

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during the acute phase of DHF, survival of platelets was markedly decreased due todestruction mainly in the liver and to a lesser extent in the spleen. Subsequently,immune-mediated injury was demonstrated to be the underlyingmechanism of plateletdestruction. By using a direct immuno¯uorescence technique, Phanichyakarn et al48

demonstrated C3 on the surface of platelets from 11 of 13 patients with DHF. Inaddition, more C3 was found on the surface of platelets in the shock than in the non-shock group. Themore sophisticated work of Boonpuchnaving et al in 197949, using thesame technique, demonstrated dengue antigen, human immunoglobulin and C3 on thesurface of platelets in 48% of patients with DHF. A correlation between the amount ofimmunoglobulin andC3with the degree of thrombocytopenia and the dayof illnesswasalso demonstrated in this study. Lateron, itwas shown that the immunedestructionwasmediated by interaction between dengue antibody and dengue antigen which waspresent on the platelet surface.50,51 C3dg, the activated form of C3, was demonstratedon the surface of platelets, and the amount of the C3dg positively correlated with thedecrease in the number of circulating platelets.52 The latter observation suggests that

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

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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 andendothelial cells having DV antigen and suggested that some injury to vascularendothelial cells by DV may allow the blood circulating in the vessel to interact withsubendothelial collagen and lead to the promotion of platelet aggregation and lysis ofplatelets resulting in thrombocytopenia. Finally, the other important mechanism ofthrombocytopenia in DHF is the consumption of platelets during the process ofconsumptive coagulopathy which occurred in most DHF patients. The supportingevidence for this latter mechanism was the correlation between the lowest plasma®brinogen and platelet nadir, and the lowest plasma ®brinogen and serum C3 level inpatients with DHF and shock. These ®ndings suggested that complement activation,coagulation and peripheral destruction of platelets are related. Complement activationis known to be capable of initiating coagulation.53

Platelet dysfunction

Most patients who exhibit petechiae during the early febrile phase in DHF havenormal platelet counts. This raised the question of whether there is an acquiredplatelet dysfunction early in DHF before thrombocytopenia occurs. A functionalabnormality 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 theconvalescent period. Srichaikul et al in 198954 demonstrated a decrease in plateletaggregation after stimulation with 5 mmMADP in 35 patients with DHF studied duringthe febrile phase or early convalescent period. The characteristic abnormality wasseverely depressed primary aggregation and absent secondary aggregation. These

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

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functional abnormalities were found in both shock and non-shock patients. Themajority of the patients had normal platelet aggregation responses to ADP whenstudied 2±3 weeks later. Plasma levels of platelet factor 4 and beta thromboglobulinwere 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 ofshock or subsidence of fever. Srichaikul et al interpreted their results as indicating thatplatelets circulating during the critical period of plasma leakage were being activatedby some mechanism and became exhausted. The activated platelets were then unableto respond to in vitro stimulation because of prior in vivo degranulation. Thishypothesis was supported by Chanthraksri et al in 199055 who demonstrated thatplatelets from healthy dengue-immune donors, when incubated with dengue virus,release platelet dense granule contents including ATP, show enhanced ATP release inresponse to subminimal ADP challenge and become hyporesponsive to ADPstimulation later on.

Srichaikul et al54 also demonstrated that platelets from 5 convalescent DHF patientsstimulated by ADP underwent greater aggregation when mixed with autologous acutephase platelet-poor plasma that had been stored at ÿ708C than when mixed with

Shock or defervescence

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Figure 3. Platelet aggregation, BTG PF4 in 12 DHF patients.K � control value. Reproduced from Srichaikulet al (1989), Southeast Asian Journal of Tropical Medicine and Public Health 20: 19±25) with permission.

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fresh convalescent platelet-poor plasma. These ®ndings suggest that acute plasmacontained substances capable of triggering platelet aggregation. The pro-aggregatingagent in acute-phase plasma was not identi®ed, but it may be dengue virus or denguevirus coated with antibody and complement. Scott et al56 could isolate dengue virusfrom platelet-rich plasma in two of 50 patients with dengue infection. Immunecomplexes with or without complement were also demonstrated on the surface ofplatelets during the acute febrile and early convalescent periods of DHF.49 Plateletshave 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 partialthromboplastin time and prothrombin time in 60 and 30% of cases respectively.47 Assayof 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 moderateincrease in FDP were observed in all grades of the disease.47,59±63 The degree ofclotting abnormalities correlated well with the degree of thrombocytopenia and theclinical severity, particularly with the degree of shock and bleeding.19,60 Reduction ofantithrombin III and a2 antiplasminogen were also demonstrated in grade 2 DHFpatients.62 In addition, a slight increase in the level of D-dimer was recently found inDHF patients (Uthaisang E, personal communication, 1999). All of the above ®ndingsclearly demonstrate the occurrence of increased intravascular coagulation in patientswith 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 casesfavoured the process of consumptive coagulopathy. Furthermore, a correlationbetween coagulopathy and liver impairment was not observed.2,60 There was alsoevidence of increased ®brinolysis secondary to disseminated intravascular coagulationas indicated by the decreased a2 antiplasminogen and increased FDP and D-dimerlevels during the actue phase. However, the degree of hyper®brinolysis was rathermild 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 havingsevere 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 andadrenal gland in two adult DHF patients who died because of severe bleeding andintractable shock.19 Furthermore, platelet ®brin thrombi were found in the brain ofgrade 2 DHF patients who died of CNS complications (Hemsrichart V, personalcommunication, 1985). Second, there was increased consumption of 125I-®brinogeninjected into grade 2, 3, 4 DHF patients60 (Figure 4). The half life of 125I-®brinogencorrelated well with the haemostatic changes which were compatible with DIC. Acorrelation between the above abnormal ®ndings and severity of shock was alsoobserved in this study. Increased intravascular coagulation as indicated by rapidconsumption of 125I-®brinogen was demonstrated in 82 and 56% of the shock and non-shock groups, respectively. However, these abnormalities were only mild to moderateand rapidly returned to normal after the recovery from shock. None of the patientsdied and anticoagulant therapy was not required in these patients.

In conclusion, increased intravascular coagulation de®nitely occurs inDHF patients. Itis 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

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proper treatment of, and rapid recovery from, shock. However, DIC could becomesevere and clinically signi®cant in patients with severe intractable shock, particularly incases with prolonged acidosis or fulminant hepatic failure. In these circumstances, avicious circle involving DIC, bleeding and shock would occur. Without e�ectivemanagement to stop this vicious circle, patients would die from bleeding or shock ormultiple organ failure (Figure 5). Therefore, the early and propermanagement of shock

Figure 4. Plasma disappearance curve and mean T12of the groups of control, febrile subjects, and DHF

patients with grade II and grades III and IV. The number in parentheses indicates the number of subjectsstudied in each group. The P value of DHF grade II, DHF grade III and IV versus control � 0.005. Reproducedfrom Srichaikul et al (1989), Southeast Asian Journal of Tropical Medicine and Public Health 20: 19±25) withpermission.

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is very important in order to prevent this vicious circle. The management of patientswith prolonged shock, severe DIC and bleeding remains a challenging problem. In ourexperience, 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 ofthe four dengue serotypes (DEN1±4). The major pathophysiological hallmarks thatdistinguish DHF from DF are plasma leakage as a result of increased vascularpermeability followed by hypovolaemia and shock in severe DHF.

Haematological changes which are constantly found in DHF and frequently in DFinclude bone marrow suppression, leucopenia and thrombocytopenia. In DHF, but notin DF, there is an enhanced immune response in a host with a secondary infection, andthis results in the formation of circulating immune complexes, complement activation,increased histamine release and massive release of many cytokines into the blood. Allof 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 skinto massive bleeding. Mechanisms of bleeding are multifactorial, namely, vasculopathy,thrombopathy and DIC. Thrombopathy consisting of thrombocytopenia and plateletdysfunction is caused by bone marrow suppression (thrombocytopenia), immuneinjury, infection of platelets by DV and, ®nally, DIC.

DIC of a mild degree occurs in 56% of non-shock patients but becomes moreprominent in patients with shock. The most severe DIC and massive bleeding arealways accompanied by prolonged intractable shock and cause fatality. Mechanisms of

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

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DIC are multifactorial, namely, vascular endothelial cell damage, hyperactivity ofplatelets and, ®nally, microcirculatory stasis caused mostly by shock. In general, thedegree of DIC is mild to moderate and can be self-limiting following recovery fromshock. The most important aspect of the management of DHF patients is therefore totreat shock early and e�ectively in order to prevent the vicious circle produced bysevere DIC with massive bleeding and intractable shock. The management of patientswith prolonged shock, severe DIC and bleeding remains a challenging problem.

REFERENCES

* 1. Anon. Dengue hemorrhagic fever: diagnosis and control. Geneva: World Health Organization, 1997.* 2. Nimmannitya S. Clinical spectrum and management of dengue hemorrhagic fever. Southeast Asian Journal

of Tropical Medicine and Public Health 1987; 18: 392±398.* 3. Halstead SB. Dengue haemorrhagic fever, haematologic aspect. Seminars in Haematology 1982; 19:

116±131.4. Halstead SB. Dengue hemorrhage fever; challenges to molecular biology. Science 1988; 239: 476±481.5. Bokisch VA, Top FH Jr, Russell PK et al. The potential pathogenic role of complement in dengue

hemorrhagic shock syndrome. New England Journal of Medicine 1973; 289: 996±1000.6. Suvatte V. Immunological aspects of dengue hemorrhagic fever: studies in Thailand. Southeast Asian

Journal of Tropical Medicine and Public Health 1987; 18: 312±315.* 7. Kurane T & Ennis FA. Immunopathogenesis of dengue virus infections. In Gubler DJ & Kuno G (eds)

Dengue and Dengue Hemorrhagic Fever, pp 273±290. Wallingford, UK: CAB International, 1997.8. Kurane I, Innis BL, Nimmannitya S et al. Human immune response to dengue viruses. Southeast Asian

Journal of Tropical Medicine and Public Health 1990; 21: 658±662.9. Nimmannitya S, Thisyakorn U & Hemsrichart U. Dengue hemorrhage fever with unusual manifestation.

Southeast Asian Journal of Tropical Medicine and Public Health 1987; 18: 398±406.10. Pongpanich B. Hemodynamic changes in shock associated with dengue hemorrhage fever. Southeast Asian

Journal of Tropical Medicine and Public Health 1987; 18: 326±330.*11. Bhamarapravati N, Tuchinda P & Boonpuknavik V. Pathology of Thai hemorrhagic fever: a study of 100

autopsy cases. Annals of Tropical Medicine and Parasitology 1967; 61: 500±510.12. Bhamarapravati N. Hemostatic defect in dengue hemorrhagic fever. Reviews of Infectious Disease 11,S: 4:

5826±5829.13. Malasit P, Mongkotsapaya J, Kalayanarooj S & Nimmanitya S. Complement and dengue hemorrhagic

fever/dengue shock syndrome. Southeast Asian Journal of Tropical Medicine and Public Health 1987; 18:316±320.

14. Tsai JT, Kuo CH, Chen PC & Changeheng CS. Upper gastrointestinal bleeding in dengue fever. AmericanJournal of Gastroenterology 1991; 86: 33±35.

15. Suwatte V & Longsaman M. Diagnostic value of bu�y coat preparation in dengue hemorrhagic fever.Southeast Asian Journal of Tropical Medicine and Public Health 1997; 10: 7±12.

16. Thisyakorn U, Nimmannitya S, Ningsanond V & Sookarun S. Atypical lymphocyte in dengue hemor-rhage fever. Its value in diagnosis. Southeast Asian Journal of Tropical Medicine and Public Health 1984; 15:32±36.

17. Boonpucknavig S, Lohachitranond C & Nimmannitya S. The pattern and nature of the lymphocytepopulation response in dengue hemorrhagic fever. American Journal of Tropical Medicine and Hygiene1979; 28: 885±889.

18. Mitrakul C. Bleeding problems in dengue hemorrhagic fever: platelet and coagulation changes. SoutheastAsian Journal of Tropical Medicine and Public Health 1987; 18: 407±412.

19. Srichaikul T, Punyagupta S, Nitiyanant P & Alkarawong K. Disseminated intravascular coagulation inadults dengue hemorrhagic fever: report of three cases. Southeast Asian Journal of Tropical Medicine andPublic Health 1975; 6: 106±114.

20. Simmon JS, St John JH & Raynold FHK. Experimental studies of dengue. Phillipine Journal of Science 1931;44: 12±47.

*21. La Russa VF & Innis BI. Mechanisms of dengue virus induced bone marrow suppression. BaillieÁres ClinicalHaematology 1995; 8: 249±270.

22. Bierman HR & Nelson ER. Hematodepressive virus disease of Thailand. Annals of Internal Medicine 1965;62: 867±884.

274 T. Srichaikul and S. Nimmannitya

Page 15: logy in Dengue and Dengue

23. Na Nakorn S, Suingdumrong A, Pootrakul S & Bhamarapravati N. Bone marrow studies in Thaihemorrhagic fever. Bulletin of the World Health Organisation 1966; 35: 54±55.

24. Nelson ER, Bierman HR & Chulajata R. Hematologic phagocytosis in postmortem bone marrow ofdengue hemorrhagic fever. American Journal of the Medical Sciences 1966; 252: 68±74.

25. Nelson ER, Bierman HR & Chulajata R. Hematologic ®ndings in the 1960 hemorrhagic fever epidemic(dengue in Thailand). American Journal of Tropical Medicine and Hygiene 1964; 13: 642±649.

26. Kho LK, Wulur H & Himawan T. Blood and bone marrow in dengue hemorrhagic fever. PaediatricaIndonesiana 1972; 12: 31±39.

27. Lin SF, Liu HW, Chang CS et al. Hematological aspects of dengue fever. Kaohsiung Journal of MedicalSciences 1989; 5: 12±16.

28. Nakao S, Lai CJ & Young NS. Dengue virus, a ¯avi virus, propagates in human bone marrow progenitorsand hematopoietic cell lines. Blood 1989; 74: 1235±1240.

29. Murgue B, Cassar O, Guigon M & Chungue E. Dengue virus inhibit human hematopoietic progenitorgrowth in vitro. Journal of Infectious Disease 1997; 175: 1497±1501.

30. La Russa VF, Putnak JR & Knight RD. Dengue virus infection of stromal cells in Dexter cultures ofhuman marrow [abstract]. Experimental Hematology 1991; 19: 479.

31. La Russa VF, Cutting MA, Kanshal S et al. Generation of stromal cell colonies from CD34� cells[abstract]. Blood 1993; 82: 98a.

32. Maze R, Sherry B, Kwon BS et al. Myelosuppressive e�ect in vivo of puri®ed recombinant murinemacrophage in¯ammatory protein-1 alpha. Journal of Immunology 1992; 149: 1001±1009.

33. Oppenheim J, Zacharie C, Kukuaica N & Matsuskinna K. Properties of the novel proin¯ammatorysupergene `intercrine' cytokine family. Annual Review of Immunology 1991; 9: 617±648.

34. Dunlop D,Wright E, Lorimore S et al. Demonstration of stem cell inhibition and myeloprotective e�ectsof SCI/rh MIP 1a in vivo. Blood 1992; 79: 2221±2225.

35. Williams DE, Devries P, Namen AE et al. The steel factor. Developmental Biology 1992; 151: 368±376.36. Williams DE & Lyman SD. Characterization of the gene product of the steel locus. Progress in Growth

Factor Research 1991; 3: 235±242.*37. Green S, Vanghn DW, Kalayanarooj S et al. Early immune activation in acute dengue illness is related to

development of plasma leakage and disease severity. Journal of Infectious Disease 1999; 179: 755±762.38. Laur F, Murgue B, Deparis X et al. Plasma levels of tumour necrosis factor alpha and transforming

growth factor beta-1 in children with dengue-2 virus infection in French Polynesia. Transactions of theRoyal Society of Tropical Medicine and Hygiene 1998; 92: 654±656.

*39. Avirutnan P, Malasit P, Seliger B et al. Dengue virus infection of human endothelial cells leads tochemokine production, complement activation and apoptosis. Journal of Immunology 1998; 161:6338±6346.

40. Raghupathy R, Chaturvedi UC, Al-Sayer H et al. Elevated levels of IL-8 in dengue hemorrhagic fever.Journal of Medical Virology 1998; 56: 280±285.

41. Kurane I, Innis BL, Nimmannitya S et al. Activation of T lymphocytes in dengue virus infections. Highlevels of soluble interleukin 2 receptor, soluble CD8, interleukin 2, and interferon-g in sera of childrenwith dengue. Journal of Clinical Investigation 1991; 88: 1473±1480.

42. Kurane I, Innis BL, Nimmannitya S et al. High levels of interferon alpha in the sera of children withdengue virus infection. American Journal of Tropical Medicine and Hygiene 1993; 48: 222±229.

43. Hober D, Poli L, Roblin B et al. Serum levels of tumour necrosis factor-a (TNF-2), interleukin 6 (IL-6)and interleukin-1b (IL-1b) in dengue infected patients. American Journal of Tropical Medicine and Hygiene1993; 48: 324±331.

44. Yadar M, Kamath KR, Iyngkaran N & Sinniah M. Dengue hemorrhagic fever and dengue shocksyndrome: are they tumour necrosis factor-mediated disorders? FEMS Microbiology and Immunology 1991;89: 45±50.

*45. Memoranda. Pathogeneic mechanisms in dengue hemorrhagic fever. Report of an internationalcollaboration study. Bulletin of World Health Organization 1973; 48: 117±133.

46. Edelman R, Nimmannitya S, Colman RW et al. Evaluation of the plasma kinin system in denguehemorrhagic fever. Journal of Laboratory and Clinical Medicine 1975; 86: 410±421.

47. Mitrakul C, Poshyachinda M, Futrakul P et al. Hemostatic and platelet kinetic studies in denguehemorrhagic fever. American Journal of Tropical Medicine and Hygiene 1977; 26: 975±984.

48. Phanichyakarn P, Israngkura P, Krisarin C et al. Studies on dengue haemorrhagic fever IV. Fluorescentstaining of the immune complexed on platelets. Journal of the Medical Association of Thailand 1977; 60:307±311.

49. Boonpucknavig S, Vattiviroj O, Bunnag C et al. Demonstration of dengue antibody complexes on thesurface of platelets from patients with dengue hemorrhagic fever. American Journal of Tropical Medicineand Hygiene 1979; 28: 881±884.

Dengue and dengue haemorrhagic fever 275

Page 16: logy in Dengue and Dengue

50. Funahara Y, Ogawa K, Nobuya F & Yoshinobu O. Three possible triggers to induce thrombocytopenia indengue virus infection. Southeast Asian Journal of Tropical Medicine and Public Health 1987; 18: 351±355.

51. Wang S, He R, Patarapotikul J et al. Antibody enhanced binding of dengue-2 virus to human platelets.Virology 1995; 213: 254±257.

52. Malasit P, Mongkolsapaya J, Kalayanarooj S & Nimmannitya S. Surface associated complement fragment(C3g) on platelets from patients with dengue infection [abstract]. Southeast Asian Journal of TropicalMedicine and Public Health 1990; 21: 705.

53. Zimmernan TS & Muller-Eberhard HJ. Blood coagulation initiation by a complement-mediated pathway.Journal of Experimental Medicine 1971; 134: 1601±1607.

54. Srichaikul T, Nimmannitya S, Sripaisarin T et al. Platelet function during the acute phase of denguehemorrhagic fever. Southeast Asian Journal of Tropical Medicine and Public Health 1989; 20: 19±25.

55. Chantharaksri U, Laoharoengpanya N, Malasit P & Innis BL. Time dependent e�ects of dengue virus onplatelets: a cyclooxygenase dependent mechanism [abstract]. Southeast Asian Journal of Tropical Medicineand Public Health 1990; 21: 706.

56. Scott RM, Nisalak A, Cheamudom U et al. A preliminary report on the isolation of viruses from theplatelets and leukocytes of dengue patients. Asian Journal of Infectious Diseases 1978; 2: 95±97.

57. Lambris JD. The multifunctional role of C3, the third component of complement. Immunology Today1988; 12: 387±393.

58. Fanger MW, Shen L, Graziano RF & Guyra PM. Cytotoxicity mediated by human Fc receptors for IgG.Immunology Today 1989; 10: 92±99.

59. Weiss HJ & Halstead SB. Studies of hemostasis in Thai hemorrhagic fever. Journal of Pediatrics 1965; 66:918±926.

60. Srichaikul T, Nimmannitya S, Artchararit N et al. Fibrinogen metabolism and disseminated intravascularcoagulation in dengue hemorrhagic fever. American Journal of Tropical Medicine and Hygiene 1977; 26:525±532.

61. Isaranggura P, Pongpanich B, Pintadit P et al. Hemostatic derangement in dengue hemorrhagic fever.Southeast Asian Journal of Tropical Medicine and Public Health 1987; 18: 331±339.

62. Funahara Y, Sumarmo Shirahata A & Dharma R. Dengue hemorrhagic fever characterized by acutedisseminated intravascular coagulation with increased vascular permeability. Southeast Asian Journal ofTropical Medicine and Public Health 1987; 18: 346±350.

63. Suvatte V, Pongpipat D, Tuchinda S et al. Studies on serum complement C3 and ®brinogen degradationproduct in Thai hemorrhagic fever. Journal of the Medical Association of Thailand 1973; 56: 24±32.

276 T. Srichaikul and S. Nimmannitya