This document provides an overview of dengue virus, the disease it causes, epidemiology, pathogenesis, clinical presentation, diagnosis and prevention. It discusses the four serotypes of dengue virus and its mosquito vectors. It describes how the virus is transmitted between humans and mosquitoes. Clinical features of both dengue fever and dengue hemorrhagic fever are outlined. The document reviews various laboratory diagnostic methods including antigen detection, antibody detection, molecular detection and virus isolation. Prevention and management of dengue focuses on mosquito control and supportive care.

2. Prologue
 Introduction
 Classification
 Epidemiology
 Virus
 Vector
 Transmission
 Immunology
 Pathogenesis
 Clinical Features
 Lab diagnosis
 Nonspecific
 Immunology
 NAT
 Virus isolation
 Prevention & future

3. 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

4. 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)

5. 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

6. 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%

8. Dengue - SEAR

9. Dengue - SEAR

10. Dengue - SEAR

11.  1963 – Kolkata epidemic
80 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

12. 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

13. Dengue Virus
 3 structured proteins
 7 nonstructural proteins
Replication and polypeptide processing
Implicated in severe disease
 NS1: 55 kDa glycoprotein
Essential for the viability of virus
Membrane associated/secretory form

14. Dengue Vector
 Aedes (Stegomyia)
 A. aegypti – Epidemic vector
 A. albopictus, polynesiensis, scutellaris complex,
niveus, tabu
 Eggs
 Bite

15. 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

16. Pathogenesis
VIRAL 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

17. Pathogenesis
HOST 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

18. Pathogenesis
IMMUNE ENHANCEMENT HYPOTHESIS
Antibody dependent enhancement (Kurane and Ennis)
Antiviral Ab - ↑Viral entry - ↑ Infectivity - ↑T cell activation
-↑cytokines/mediators – Severe disease
CD4+ 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

19. Immunology of Dengue
 Day 0-4: Viral Ag, No Ab
 Day 5-7: IgM (30-90 days)/IgG (60 years)
 Primary infection:
Serotype specific Ab
Serotype cross reactive Ab
High IgM and low IgG
 Secondary infection:
Ag-Ab is bound & internalized but not neutralized
Higher risk of DHF
Higher IgG and lower IgM
 4 serotypes can infect in a lifetime

20. 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

21. Incubation peroid

22. 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

23. 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

24. Clinical Features (DF)
CASE DEFINITION OF DENGUE FEVER (WHO 2006)
 Suspected - Acute febrile illness + >2 of
headache, 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

25. 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

26. Laboratory criteria
LABORATORY 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

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

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

29. Lab Diagnosis
 Biochemistry
 Hematology
 Serological tests
Antigen detection – NS1
Antibody detection – IgM, IgG
 Molecular detection
 Viral isolation

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

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

32. 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

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

34. 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

35. 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

36. 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

37. 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

38. 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)

39. 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)

40. Viral isolation
 Mammalian cells - Vero
 Mosquito cell lines – A. albopictus C6/36
Aedes pseudoscutellaris AP61
 Newborn mice (intracerebral)
 Adult mosquitoes (intrathoracic)
A. aegypti, albopictus
Toxorhynchites splendens, amboinensis
 Lengthy, laborious, low sensitivity, expensive

41. Diagnostic decision
 Choice of test
Rapidity, sensitivity, specificity,
Suitability at a particular stage of disease
Ease of execution, standardization and
automation
 Combination of tests
 Quest for the new Gold Standard

42. 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 ?

43. References1. Chaturvedi UC, Agarwal R, Elbishbishi EA, Mustafa AS. Cytokine Cascade in Dengue Haemorrhagic Fever:
Implications for Pathogenesis. FEMS Immunol Med Microbiol 2000; 28: 183-188.
2. Berry N, Chakravarti A, Gur R, Mathur MD. Serological investigation of a febrile outbreak in Delhi, India, using
a rapid immunochromatographic test. J Clin Microbiol 1998; 36: 2795-6.
3. Tripathi BK, Gupta B, Sinha RS, Prasad S, Sharma DK: Experience in adult population in dengue outbreak in
Delhi. J Assoc Physicians India 1998, 46: 273-76.
4. Kumar M, Pasha S T, Mittal V, Rawat D S, Arya C , Agarwal N, Bhattacharaya D, Lal S, Rai A. Unusual
emergence of guate 98 like molecular subtype of Den -3 during 2003 dengue outbreak in Delhi, dengue
Bull.2004; 28: 101-167.
5. Gubler, DJ 1998. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev. 11:480–496.
6. Smith AW, Chen LH, Massad E, Wilson ME. Threat of dengue to blood safety in dengue-endemic countries.
Emerg Infect Dis 2009; 15:8-11.
7. Kurane I, Takasaki T: Dengue fever and dengue hemorrhagic fever: challenges of controlling an enemy still at
large. Rev Med Virol 2001, 11:301-311.
8. Messer WB, Gubler DJ, Harris E, Sivananthan K, de Silva AM: Emergence and global spread of a dengue
serotype 3, subtype III virus. Emerg Infect Dis 2003, 9:800-9.
9. Guzman MG, Kouri G. Dengue: An update. Lancet Infect Dis 2002; 2:33-42.
10. WHO (1997) Dengue hemorrhagic fever: diagnosis, treatment, prevention and control, 2nd edition. Geneva:
World Health Organization.
11. Broor S, Dar L, Sengupta S, Chakaraborty M, Wali JP, Biswas A, Kabra SK, Jain Y, Seth P: Recent dengue
epidemic in Delhi, India. In Factors in the emergence of arbovirus diseases.: Elsevier; 1997: P123-7.
12. Chouhan GS, Rodrigues FM, Shaikh BH, Khangaro SS, Mathur KN, et al. Clinical & virological study of
dengue fever outbreak in Rajasthan.1985. Indian J Med Res 1990; 91: 414-8.
13. Monath TP. Dengue: The risk to developed and developing countries. Proctl Natl Acad Sci USA 1994; 91:
2395-400.
14. Morens DM, Rigau-Perez JG, Lopez-Correa RH,et al.: Dengue in Puerto Rico, 1977: public health response
to characterize and control an epidemic of multiple serotypes. Am J Trop Med Hyg 1986, 35: 197-211.

44. 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.

45. Thank You