Dengue

Prologue Introduction Classification Epidemiology

Virus Vector Transmission

Immunology Pathogenesis Clinical Features

Lab diagnosis Nonspecific Immunology NAT Virus isolation

Prevention & future

Dengue Dengue - Swahili “Ka-Dinga pepo”

Sudden cramp like disease caused by an evil spirit Dandy Fever, Breakbone Fever, Breakheart Fever Infectious thrombocytopenic purpura Mosquito borne Flavivirus infection Tropical and subtropical areas Earliest record - Chinese encyclopedia of disease

symptoms and remedies 265-420 AD

Classification WHO Classification 2009

Uncomplicated Severe

WHO Classification 1997 Undifferentiated fever Dengue Fever (DF) Dengue Hemorrhagic Fever (DHF)

Gd I – Easy bruising, + Tourniquet test, fever Gd II – Spontaneous bleeding Gd III – Shock Gd IV – Severe Shock (Unrecordable pulse and BP)

Epidemiology Second to malaria in tropical areas Threatens 2.5 bn (40%) population Disease burden – 1600 DALYs/million population Sporadic – Endemic – Hyperendemic – Epidemic –

Pandemic ??? Major public health problem No approved vaccines Ineffective antiviral therapy

Epidemiology 1799 – Egypt and Indonesia, 1780 – US Fewer epidemics till 1940s First epidemic DHF – 1954 Manila Increased epidemicity by 1980-90s

worldwide, all serotypes, DEN2 1998 – Dengue pandemic, DEN3

56 countries worldwide 2005 – South America, DEN3 DF 100 m, DHF 50 m, Deaths 50000 Case Fatality Rate - 0.5-3.5%

Dengue - SEAR

Dengue - SEAR

Dengue - SEAR

1963 – Kolkata epidemic80 regular outbreaks in many parts of India except North East

1967, 70, 82, 88, 92 – DEN1, DEN2 1996 – Major outbreak, DEN2 2009 – Delhi and other areas, DEN3 Ongoing

Dengue - India

Dengue Virus Spherical (Diameter 40-50 nm) Lipid enveloped Positive strand RNA Virus - 1940s, serotypes – 1956 4 serotypes: DEN1 - DEN4 10.7 kb RNA genome 10,233 nucleotides and 3411 amino acids

Dengue Virus

3 structured proteins 7 nonstructural proteins

Replication and polypeptide processingImplicated in severe disease

NS1: 55 kDa glycoproteinEssential for the viability of virusMembrane associated/secretory form

Dengue Vector Aedes (Stegomyia) A. aegypti – Epidemic vector A. albopictus, polynesiensis, scutellaris complex,

niveus, tabu Eggs Bite

Transmission Man-Mosquito-Man Both act as reservoirs Lifetime infectivity of mosquito Intrapartum, percutaneous,mucocutaneous Blood transfusion, Organ transplant Vertical transmission IP: 3-14 (4-7) days

PathogenesisVIRAL ENTRY & REPLICATION

DV protein binds to Langerhans cells membrane protein C-type lectins (DC-SIGN, mannose receptor, CLEC5A) DC-SIGN/CD209 is nonspecific mannose binding receptor

for foreign material on dendritic cell and macrophages DV replicates in membrane bound vesicles of ER Immature virus is transported to Golgi apparatus Mature virus buds and releases by exocytosis DV infects monocytes, macrophages, CD4+, CD8+ T cells

PathogenesisHOST RESPONSE

Innate and adaptive immune systems T cells attack DV infected cells Infected cells produce IFN Ab against DV

Neutralizing - Phagocytosis Non neutralizing – Further replication

PathogenesisIMMUNE ENHANCEMENT HYPOTHESIS

Antibody dependent enhancement (Kurane and Ennis)Antiviral Ab - ↑Viral entry - ↑ Infectivity - ↑T cell activation -

↑cytokines/mediators – Severe diseaseCD4+ cells produce IFNY, upregulation of FcY receptors Endothelial dysfunction - Vasoactive mediators -

↑permeability - Hypovoluemia, Shock Coagulation disorders - Quantitative/Qualitative platelet

defects - BT↑ - 100000/mm3

High viral load in blood, bone marrow, liver is associated with severe disease

Immunology of Dengue Day 0-4: Viral Ag, No Ab Day 5-7: IgM (30-90 days)/IgG (60 years) Primary infection:

Serotype specific AbSerotype cross reactive AbHigh IgM and low IgG

Secondary infection:Ag-Ab is bound & internalized but not neutralizedHigher risk of DHFHigher IgG and lower IgM

4 serotypes can infect in a lifetime

Host susceptibility Genetic polymorphisms

increasing risk TNF α Mannan binding lectin CTLA4 TGF β DC-SIGN HLA G6PD deficiency

Genetic polymorphisms decreasing risk Vit D receptor FcyR

Incubation peroid

Clinical Features Asymptomatic 80% Uncomplicated fever Severe illness

5% Life threatening

Diabetes Asthma

Febrile phase 2-7 days Fever (Biphasic) Headache Mucosal bleed Muscle ache Joint pain Vomiting Rash (Measles like) Diarrhoea

Clinical Features Critical phase

1-2 days Hypotension Pleural effusion Ascites Severe bleed (GIT) Organ dysfunction

Recovery phase

2-3 days Altered consciousness Seizures Itching Bradycardia

Clinical Features (DF)CASE DEFINITION OF DENGUE FEVER (WHO 2006)

Suspected - Acute febrile illness + >2 ofheadache, retro orbital pain, myalgia, arthralgia, rash, hemorrhagic manifestations and leucopenia

Probable - Clinical description and serology reciprocal HI Ab titre > 1280, comparable IgG ELISA

or positive IgM Ab test Confirmed Reportable - Any probable or confirmed case

Clinical Features (DHF)CASE DEFINITION OF DHF (WHO 2006)

Acute onset high fever for 2-7 days Hemorrhagic manifestations with at least a positive

tourniquet test Platelet <100 x 109 per litre Hemoconcentration (rising PCV >20%) or Evidence of plasma leakage- ascites, pleural effusion, low

serum protein

Laboratory criteriaLABORATORY CRITERIA FOR DENGUE FEVER (WHO 2006)

Isolation of the DV from serum or autopsy samples or Fourfold or greater rise in reciprocal IgG or IgM Ab titres

to DV Ags in paired sera or DV Ag in autopsy tissue, serum or CSF by

immunochemistry, IFA or ELISA or Detection of DV genomic sequences in autopsy tissue,

serum or CSF by PCR

Differential diagnosis Chickungunya West Nile, JE Influenza Measles Rubella Typhoid Meningococcemia Leptospirosis Rickettsial infections Malaria

Specimens Blood, serum, plasma Washed leucocytes CSF Saliva Homogenized/minced autopsy tissues Homogenized pooled mosquitoes Wet ice - - Thiomersal or sodium azide <24 hrs: 4-80C, -700C for longer duration

Acute phase sample Convalescent phase sample

Lab Diagnosis Biochemistry Hematology Serological tests

Antigen detection – NS1Antibody detection – IgM, IgG

Molecular detection Viral isolation

Non specific parameters Neutropenia Lymphocytosis (Atypical lymphocytes) Thrombocytopenia Raised PCV (Hallmark of DHF) Hypoproteinemia Microscopic haematuria Elevated liver enzymes Abnormal coagulogram

Antigen detection Immunochromatography (ICT) NS1 capture ELISA Immunofluorescence assay Microsphere based immunoassay Dot blot assay Immunochemistry Immunoperoxidase Avidin-biotin enzyme assays

Antibody detection Immunochromatography (ICT) ELISA Haemagglutination inhibition (HI) Complement Fixation (CFT) Neutralisation (NT) Microsphere based immunoassay Indirect immunofluorescent Ab test Microneutralisation assay Dot Blot assay Western Blot assay

ICT Lateral flow immunochromatography Most frequently used Sensitive and specific Simple, rapid, easy Can be used for mass screening

ELISA MAC ELISA NS1 serotype specific IgG ELISA Pathozyme IgM/Pathozyme IgG Platelia Dengue NS1 Ag ELISA – mAb IgG avidity ELISA Capture ultramicro ELISA Dot ELISA AuBioDOT IgM capture

MAC ELISA Most widely used Simple, rapid, requires minimal equipment Specificity is not very high Unequivocal diagnosis – 4 fold rise Not a confirmatory test when single sample used Cannot be used to identify serotypes Useful tool for surveillance of DF, DHF and DSS

HI Frequently used Sensitive, easy and reliable Detectable by day 5-6 of fever Titers >1280 considered significant HI Ab persist for at least 48 years Suitable for seroepidemiologic studies Not suitable for secondary infections Not specific

CFT Not widely used - Difficult Complement is consumed during Ag-Ab reactions Detectable earlier than HI Ab More specific in primary infections Not specific for secondary infections Not valuable for seroepidemiologic studies

NT Most sensitive and specific Serum dilution plaque reduction Suitable for primary infections NT Ab persist for at least 48 years Suitable for seroepidemiologic studies Not suitable for secondary infections Demanding (time, effort and cost)

Molecular detection RT-PCR qRT-PCR Nested PCR Multiplex PCR NASBA TMA bDNA RT LAMP

Nucleic Acid Hybridization Self sustained sequence

replication (3SR) Strand displacement

amplification (SDA)

Viral isolation Mammalian cells - Vero Mosquito cell lines – A. albopictus C6/36

Aedes pseudoscutellaris AP61 Newborn mice (intracerebral) Adult mosquitoes (intrathoracic)

A. aegypti, albopictusToxorhynchites splendens, amboinensis

Lengthy, laborious, low sensitivity, expensive

Diagnostic decision Choice of test

Rapidity, sensitivity, specificity,Suitability at a particular stage of diseaseEase of execution, standardization and

automation Combination of tests Quest for the new Gold Standard

Management & Prevention Supportive

Rehydration Antipyretics Blood transfusion Whole blood Packed RBCs Platelets FFP

Mosquito reduction Habitat reduction Prevention of bites Anti-dengue drug ? New vaccine ?

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References15. Halstead SB, Nimmannitya S, Cohen SN: Observations related to pathogenesis of dengue hemorrhagic fever. Yale J Biol Med 1970, 42: 311-328.16. WHO: Strengthening implementation of global strategy for dengue fever/ dengue hemorrhagic fever, prevention and control, report on informal consolation, WHO; 1999.17. Gubler D J, Sather G E. Laboratory diagnosis of dengue and dengue hemorrhagic fever. In: Homma A, Cunha J F, editors. Proceedings of the International Symposium on Yellow Fever and Dengue. 1988. pp. 291–322.18. World health organization.1997, Dengue hemorrhagic fever: diagnosis, treatment, prevention and control, second edition. World health organization Geneva, Switzerland.19. Vaughn DW, Kalayanarooj S, Innis BL, Nimmannitya S et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 2000, 181: 2-9.20. Groen, J., P. Koraka, J. Velzing and A. D. M. E. Osterhaus. 2000. Evaluation of of dengue virus-specific immunoglobulin M and G antibodies. Clin. Diagn. Lab. Immunol. 7: 867–871.21. Mantke, O.D.,K,Lemmer, S.S.Biel, J.et al.2004. Quality control assessment for the serological diagnosis of dengue virus infection. J.Clin. Virol. 2004; 29: 105-112.22. Lanciotti, R. S., C. H. Calisher, D. J. Gubler, G. J. Chang, and A. V. Vorndam. 1992. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J. Clin. Microbiol. 30: 545–551.23. Henchal, E. A., J. M. McCown, M. C. Seguin, M. K. Gentry, and W. E.Brandt. 1983. Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay. Am. J. Trop. Med. Hyg. 32: 164–169.24. Harris, E., T. G. Roberts, L. Smith, J. Selle, L. D. Kramer, S. Valle, E. Sandoval, and A. Balmaseda. 1998. Typing of dengue viruses in clinical specimens and mosquitoes by single-tube multiplex reverse transcriptase PCR. J. Clin. Microbiol. 36: 2634–2639.25. Notomi T et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 2000, 28: E63.26. Kurane, I., and F. A. Ennis. 1997. Immunopathogenesis of dengue virus infections, p. 273-290. In D. J. Gubler, and G. Kuno (ed.), Dengue and dengue hemorrhagic fever. CAB International, London, United Kingdom.

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