1. Dr. R.Jayaprada
DENGUE

2. INTRODUCTION
 Dengue fever is one of the most important emerging disease of
the tropical and sub tropical regions, affecting urban and pre
urban areas.
 Dengue is the biggest Arbovirus problem in the world today with
over 2 million cases per year. Dengue is found in SE
Asia, Africa and the Caribbean and S America.
 Dengue fever (UK: /ˈd ɛŋɡeɪ/, US: /ˈd ɛŋɡiˈ also known as
/),
break bone fever. It some times causes severe joint and
muscle pain that feels like bone are breaking.
 Dengue is also called as water poison by Chinese.
 The Arbovirus are also called as Arthropod borne
viruses, represent an ecological grounding of viruses with
complex transmission cycles involving Arthropods
 These viruses have diverse physical and chemical properties
and are classified in several virus families.
 Dengue infection is caused by Arbovirus .
 Dengue is an arthropod borne flavivirus .

3. HISTORY
 The name dengue is derived from word ―Swahili ka
dinga pepo”, meaning sudden seizure by a demon.
 Earliest known documentation of symptoms of dengue
like illness was described in chinese encyclopedia
during 265 A.D.
 This disease was first described by Benjamin Rush in
Philadelphia, Pennsylvania in 1780.
 Rouss coined the term ―break bone fever‖.
 Dengue virus was first isolated in Japan in 1943 by
inoculation of serum of patients into suckling mice.
 The virus was isolated by Sabin 1944 from serum
samples of US soldiers.

4. EPIDEMIOLOGY
 Dengue virus infection is the most common arthropod-
borne disease worldwide with an increasing incidence in
the tropical regions of Asia, Africa, and Central and South
America.
 First epidemic of clinical dengue like illness was recorded
in Chennai in 1780.
 First virologically proved epidemic of DF in India occurred
in Calcutta & Eastern coast of India in 1963-64.
 First major epidemic of DHF occurred in Manila,
Philippines in 1953-54 followed by global spread.
 By 1997 most of the countries have experienced large out
breaks of the disease, currently DF / DHF is endemic in
Bangladesh , India, Indonesia, Maldives, Srilanka,
Thailand approximately 1.3 billion people are leaving in

5. BURDEN OF DISEASE IN S.E.ASIA
 CATEGORY-A
(INDONESIA,MYANMAR,AND THAILAND)
 CATEGORY-B
(INDIA,BANGALADESH,MALDIVES,AND
SRILANKA)
 CATEGORY-C (BHUTAN, NEPAL)
 CATEGORY-D (DPR KOREA)

6. EPIDEMIOLOGY IN INDIA
 The very first report of DF existed in India way back
in1946.
 Initial epidemic was reported in Eastern coast of India in
1963-64 spread northwards & reached Delhi in 1967
and Kanpur in 1968 (DV-4). In 1969, DV-2 & DV-4 were
isolated in Kanpur epidemic. In 1970 epidemic, it was
completely replaced by DV-2 in Kanpur & adjoining
Hardoi city.
 In 1966 epidemic, DV-3 was isolated from patients &
mosquitoes. In 1968, all 4 types of DV are isolated.
 Outbreaks occurred in Rajasthan –DV-1,DV-3,
Madhyapradesh—DV-3, Gujarat—DV-2, Haryana—DV-2.
 DV-2 was the predominant serotype circulating in North
India.
 Dv-2 has also been reported from South India –in Kerala

7. EPIDEMIOLOGY IN INDIA
 DV-3 has been isolated during epidemics at Vellore in
1966, at Kolkata in 1983 and in 1990,at jalore
city, Rajasthan in 1985, at Gwalior in 2003 and 2004 & at
Tirupur ,Tamilnadu in 2010.
 Emergence of DV-4 has been reported in Andhrapradesh
and Pune, Maharastra.
 At Delhi till 2003, the predominant serotype was DV-2
,but in 2003 all the 4 types were found to co-
circulate, thus changing Delhi into a hyper endemic
region. Then it is followed by complete predominance of
DV-3 in 2005.
 During 2004 epidemic of DHF/DSS , there predominance
of DV-3 replacing DV-2.
 In 2007-2009, there predominance of DV-1 replacing DV-

8. AETIOLOGY
 Flavivirus (type of Arbovirus)
 Transmitted from Aedes aegypti and Aedes albopictus
mosquitoes
 Four Serotypes (Dengue 1-4). These serotypes are
genetically quite similar, but different enough to
represent different virulence (Chamers, Liang, Droll, et.
al., 2003).
 DV virus is a positive strand encapsulated RNA virus &
composed of 3 structural protein genes , which encode
Nucleocapsid /core (c) protein,
Membrane associated protein (M),
Enveloped protein and 7 non structural proteins.

9. MORPHOLOGY & ANTIGENIC STRUCTURE
 Dengue virion are spherical particles approximately 40 nm
in diameter.
 contains a single plus strand of RNA. Surrounded by a
lipid bilayer.
 Mature virions are composed of 6% RNA, 9%
carbohydrate, and 17% lipid.
 Because of the lipid envelope, flaviviruses are readily
inactivated by organic solvents and detergents.
 Genome codes for 3 structural proteins and 7
nonstructural proteins (NS) in the following order :
 Nucleocapsid /Core (C)- Membrane (M)- Envelope (E)-
Nonstructural proteins-
NS1,NS2a, NS2b, NS3, NS4a, NS4b,NS5.
 These proteins are derived Co-translational/posttranslational
processing of a single long precursor polypeptide or polyprotein

10. BASIC DENGUE GENOME

11. MORPHOLOGY & ANTIGENIC STRUCTURE
 Pre M protein (prm, a premembrane protein) is present
in the intracellular nascent virions and is cleaved to M
protein found in mature extracellular virions.
 C protein is responsible for the group reactivity &
generation of complement fixing antibodies.
 The E protein is the major surface glycoprotein of the
viral particle probably interacts with viral receptors, and
mediates virus-cell membrane fusion.
 E protein is responsible for many biological activities like
Haemagglutination, Viral Neutralization, Viral binding
with cellular receptors, Membrane fusion & Virion
assembly.
 Antibodies that neutralize virus infectivity usually
recognize this E protein and mutations in E can affect
virulence.

12.  Among Nonstructural proteins, NS1 appears to be
highly conserved in all serotypes of Dengue (also in
Flavivirus).
 NS1 is synthesized in the rough ER and may remain
intracellular/ transported to plasma membrane/
secreted out of the cell.
 NS1 assist in virus morphogenesis.
 NS1 has the immunological importance since infected
cells expressing protein on the surface become the
targets
for immunocytolysis.
Antibodies against NS1 Ag are found to crossreact
against some self antigens.
Antibodies against NS1 Ag participates in the
pathogenesis of DHF.
 Viral Replication in cytoplasm

14. DENGUE VIRUS & ITS SEROTYPES
 Has 4 distinct serotypes
 DEN-1—First isolated from Hawaii in 1944
 DEN-2--First isolated from New Guinea in 1944
 DEN-3 &DEN-4--First isolated from Philippines in
1956.
 Speciation was done by Albert Sabin in 1944.
 Each serotype has different genotypes.
 Of the four serotypes, DENV-2, with six genotypes,
exhibits the most genetic diversity.
 DENV-2 is a predominant serotype causing
dengue fever and dengue haemorrhagic fever
outbreaks.

15. DENGUE VIRUS & ITS SEROTYPES
 Indian DV-1 isolates are divided into 4 lineages
 I—It is imported from Singapore.
 II –Evolving insitu.
 III –Oldest & extinct lineage.
 IV –African lineage.
• Each serotype provides specific lifetime immunity,
and short-term cross-immunity
• All serotypes can cause severe and fatal disease
• Genetic variation within serotypes
• Some genetic variants within each serotype
appear to be more virulent or have greater
epidemic potential

16. VIRUS CLASSIFICATION
 GROUP : IV
 FAMILY :Flaviviridae
 Genus :Flavivirus
 Species :Dengue Virus

17. ABOUT THE VECTOR
 The spread of dengue is attributed to expanding
geographic distribution of the four dengue viruses and
of their mosquito vectors, the most important of which
are the predominantly urban species Aedes aegypti
and Aedes Albopictus.
 A rapid rise in urban populations is bringing ever
greater numbers of people into contact with this
vector, especially in areas that are favorable for
mosquito breeding, e.g. where household water
storage is common and where solid waste disposal
services are inadequate.

18. MOSQUITO VECTORS
 All known vectors belong to genus Aedes
 Vector competence and vectorial capacity of different
species vary
 Different species
 Different geographic populations of the same species
 No correlation between clinical features of subsequent
disease
 Subgenus Stegomyia contains the most important vectors
of dengue viruses
 Ae. aegypti, Ae. albopictus and Ae. polynesiensis
 Ae. aegypti
 African origin
 Not found in Hong Kong
 Most important vectors worldwide
 Linked with human activities such as uncontrolled
urbanization, deterioration of urban environment and decreasing
standard of sanitation

19. VECTOR
 Lays egg singly, and eggs are cigar shaped.
 Female mosquito acts as vector.
 They do not fly over long distance-
<100mts(110yards), this factor facilitates its
eradication.

20. LIFE CYCLE OF AEDES MOSQUITO
1-2 days
Pupae Eggs
Larvae
4-5 days
2-3
days

22. AEDES AEGYPTI
• The most common
epidemic vector of dengue
in the world is the Aedes
aegypti mosquito. It can be
identified by the white
bands or scale patterns on
its legs and thorax. Dengue
transmitted by infected
female mosquito
• Primarily a daytime feeder
• Lives around human
habitation
• Lays eggs and produces
larvae preferentially in
artificial containers

24. AE. ALBOPICTUS
 Asian species
 South-East Asia, China, Japan, Indonesia, islands in the Indian
Ocean, Hawaii
 Spreading to the United States, South America, Africa, the Pacific
and south of europe
 Originally a forest mosquito feeding on a variety of animals
and breeding in tree holes
 Become adapted to human environment
 Natural containers such as tree holes, plant axils, cut
bamboo stumps and opened coconuts
 Outdoor artificial containers such as water storage barrels
and trash receptacles.

25. AE. ALBOPICTUS
 Can persist as far north as Beijing or Chicago (average
isotherm of 0ºC)
 Optimal growth at 25 °C to 30°C
 Eggs can resist desiccation for several months
 10 days for egg-larva-pupa-adult cycle
 Ae. albopictus females known to survive for up to 122 days
(daily mortalities 8-15%)
 Density much influenced by rainfall
 Feed outdoors during daytime
 Peak at 8-9 a.m. & 5-6 p.m.
 Multiple bites per feed
 Active maximum dispersal range of females about 400 to 600m
 Passive dispersal less important

26. DISTRIBUTION OF DENGUE
Present in most tropical and sub-tropical (less humid) climates
Africa
Southeast Asia and China
India
Middle East
Caribbean and Central and South America
Australia and the South and Central Pacific
Some parts of the U.S., namely Texas and Hawaii

27. Dengue is found anywhere that Aedes aegypti can live, and that means any
tropical and sub-tropical climate. This includes Central & South America, parts
of Africa, South Asia, and countries in the Pacific like Australia, New
Zealand, Fiji, etc. It is also present in parts of North America, though so far the
only outbreaks have been recorded in Hawaii and Texas (Stephenson, 2005).
With the impending threat of mosquito evolution and global
warming, however, Ae. aegypti could vastly increase its range.

28. DENGUE INFECTION
 Dengue virus (DENV) infects 50 million (WHO) to
100 million (NIH) people annually.
 Forty per cent of the world’s
population, predominately in the tropics and sub-
tropics, is at risk for contracting dengue virus.
 DENV infection can cause dengue fever, dengue
hemorrhagic fever, dengue shock syndrome, and
death.

29. TRANSMISSION OF VIRUSES

30. REPLICATION AND TRANSMISSION
1.Mosquitoes transmit Vector
dengue to human
dendritic cells Humidity:
Extrinsic
2. Dengue targets areas Incubation Rainfall & Temp.
with high WBC counts Period:
(liver, spleen, lymph 1-2 weeks
nodes, bone marrow,and
glands)
3. Dengue enters
WBCs & lymphatic Incubation
Tissue. Period:
4.Dengue virus enters the 3-14 days
circulation.
Viraemia & Fever: 5-7 days

31.  1.The virus is inoculated into humans with the mosquito saliva.
 2.The virus localizes and replicates in various target organs, for
example, local lymph nodes and the liver.
 3.The virus is then released from these tissues and spreads
through the blood to infect white blood cells and other lymphatic
tissues.
 4.The virus is then released from these tissues and circulates in
the blood.
 5.The mosquito ingests blood containing the virus.
 6.The virus replicates in the mosquito mid gut, the ovaries, nerve
tissue and fat body. It then escapes into the body cavity, and later
infects the salivary glands.
 7.The virus replicates in the salivary glands and when the
mosquito bites another human, the cycle continues.

32. TRANSMISSION CYCLE
 The transmission cycle of dengue virus by the
mosquito Aedes aegypti begins with a dengue-
infected person. This person will have virus
circulating in the blood—a Viraemia that lasts for
about five days.
 During the viremic period, an uninfected female
Aedes aegypti mosquito bites the person and
ingests blood that contains dengue virus. Although
there is some evidence of transovarial transmission
of dengue virus in Aedes aegypti, usually
mosquitoes are only infected by biting a viremic
person.
 Then, within the mosquito, the virus replicates
during an extrinsic incubation period of eight to

33. VIRAL PATHOGENESIS
 The mosquito then bites a susceptible person and
transmits the virus to him or her, as well as to every
other susceptible person the mosquito bites for the
rest of its lifetime.
 The virus then replicates in the second person and
produces symptoms. The symptoms begin to appear
an average of four to seven days after the mosquito
bite—this is the intrinsic incubation period, within
humans. While the intrinsic incubation period
averages from four to seven days, it can range from
three to 14 days.
 The viremia begins slightly before the onset of
symptoms. Symptoms caused by dengue infection
may last three to 10 days, with an average of five
days, after the onset of symptoms—so the illness
persists several days after the viremia has ended.

34. TRANSMISSION CYCLE CONTD

35. VIRAL PATHOGENESIS
 Bite of the Aedes mosquito
Virus replicates at the site of the bite&
lymph nodes
Enters the blood stream (Primary Viraemia)
Reaches the RES (virus replicates preferentially in
the cells of Mononuclear phagocytic lineage)
Blood steam (secondary Viraemia)--------Organs
Leakage of plasma caused by increased capillary
permeability is the major abnormality in DHF &DSS.

36. ENTRY INTO THE CELL
Dengue infection
Endosome entry & pH change
E protein conformational change
Release of viral RNA into cell
Replication & further infection

37. VIRAL REPLICATION

38. PATHOGENESIS OF DHF
 1. A widely accepted hypothesis is the ―secondary
infection/Immune enhancement hypothesis‖. This is
also referred as Antibody dependent Enhancement
hypothesis.
 2. Another hypothesis assumes that epidemics of
DHF are due to emergence of more virulent
genotypes by selection pressure.
 Phenotypic expression in virus genome may include
 a) Increased virulence.
 b) Increased levels of Viraemia.
 C) Increased epidemic potential.

39. DHF/DSS – EPIDEMIOLOGY
 Early reports
 1897 Northern Australia
 1928 Greece
 1935 Taiwan
 1950 Thailand
 mid-1980s Southern China and Hainan Island
 Asian DHF/DSS epidemics
 Multiple types of dengue viruses simultaneously or
sequentially endemic
 Secondary-type antibody responses observed
 Only during secondary dengue infections

40.  Infection parity and enhancing antibodies
 Secondary-type dengue infections
 Primary in infants born to dengue-immune
mothers
 Antigens shared between first and second
infecting serotypes
 Shift the spectrum towards more severe disease

41.  Serum antibodies developed can neutralize dengue virus
of that same serotype (homologous)
 Pre-existing heterologous antibodies form complexes but
no neutralization.

45. ANTIBODY DEPENDENT ENHANCEMENT HYPOTHESIS
 ADE is mainly due to a)Enhancing antibodies.
 b) DC-SIGN.
 Dengue may be caused by any of the 4 viral
serotypes.
 Infection with one serotype confers lifelong
protective immunity against particular serotype only.
 1.These antibodies are non neutralizing, but cross
reactive for other serotypes.
 2.These cross reactive antibodies are directed
against some M’ protein epitopes &non structural
epitopes (p’ly NS1 &NS3) of other serotypes.
 3. When the person gets infected with different
serotype, a more severe infection results because of
these heterologous antibodies.

46. ADE CONTD
 4. These non neutralizing but cross reactive antibodies
complexes with the virus  Fails to neutralize its
infectivity.
 5.The Fc portion of the Ab in these complexes binds to Fcγ
R bearing permissive cells, usually mononuclear
phagocytic cells.
 6.Virus enters the cells by binding to the specific receptors
on the cell.
 7.Heterologous Ab and Fcγ are enhancing the infection by
acting as co-receptor.
 8. When once virus enters the cell it is free to replicate
as it was not neutralized.
 9. Virus can enter a great number of target cells
Increased virus production in the presence of enhancing
Ab’s.

47. DC-SIGN
 Another receptor was identified which mediates the
dengue infection in dendritic cells is DC-
SIGN=Dendritic Cell-Specific ICAM-3 Grabbing Non
integrin.
 Normally dendritic cells capture the antigen and
disseminates the Ag after DC-SIGN interaction with
the glycosylated protein Ag.
 Mosquito modify the E’ protein of flaviviruses by
glycosylating them and injects them into dermis
Local dendritic cells are infected with virus  carries
to the regional lymph nodes.

48. DHF/DSS EPIDEMIOLOGY
 Age
 Greatest susceptibility to shock is 8 to 10 years
 ? Capillaries of of children more prone to cytokine-mediated
increased permeability
 Sex
 Shock cases and deaths more frequently in female than in
male children
 ? Immune responses of females more competent
 ? Capillary bed of females more prone to increased capillary
permeability
 Nutritional status
 Moderate to severe protein-calorie malnutrition
reduces risk to DHF/DSS in dengue infected children
 Malnutrition suppresses cellular immune responses
 Preceding host conditions
 Peptic ulcer and menstrual periods risk factors for
severe bleeding

49. ROLE OF CELLULAR IMMUNE RESPONSE
 Following primary infection, serotype specific &
serotype cross reactive memory T’ cells are
generated.
 NS3 has multiple T’ cell epitopes and most T’ cells
cross react with these NS3 epitopes.
 In response to a second infection with a different
(cross reacting ) serotype, memory T’ cells get
activated quickly & show poor binding capacity 
resulting in Inefficient killing of target cells &
inadequate immune response.
 This phenomenon is known as original antigenic
sin.
 Because of this non optimal T’cell

50. ROLE OF CYTOKINES
 Proinflammatory cytokines (TNF-α, IFN-γ, IL-8, IL-12 )
 Anti-inflammatory cytokines (IL-10).
 IFN-γ can up regulate Fcγ receptors on the cells of monocytic
lineage and augments the viral infection.
 TNF-α prolongs the dendritic cell survival by up regulating the
anti apoptotic factors.
 A cytokine ,recently identified is a protein of 22-23 Kda,named
as Human Cytotoxic factor (hCF).
 Virus infected macrophages produce
free radicals+ Reactive O2 intermediates
Induces CD4 cells
hCF TNF-α, IFN-γ Histamine
increases the
IL-8, IL-12
vascular permeability-------------- SEVERE DISEASE.

51. SUPPRESSION OF INNATE IMMUNE RESPONSE
 NS4b protein of dengue virus blocks the JAK-STAT
pathway of IFN signaling results in failure of IFN
signaling  Suppression of innate immune
response.

52. ROLE OF AUTOIMMUNITY
 AntiNS1 Ab’s and AntiprM Ab’s acts as Antiplatelet
&anti endothelial Ab’s due to cross reactions.
 Anti endothelial Ab’s induce endothelial apoptosis in
a caspase dependent manner  damage to
vascular endothelium Increases the vascular
permeability.
 Antiplatelet Ab’s cause complement mediated
lysis
 Hemorrhagic manifestations.

53. REEMERGENCE OF DF/DHF
 Unprecedented human population growth
 Unplanned and uncontrolled urbanization
 Inadequate waste management and water
supply
 Increased distribution and densities of vector
mosquitoes
 Lack of effective mosquito control
 Increased movement and spread of dengue
viruses

54. CLINICAL PRESENTATION OF DENGUE
 There are actually four dengue clinical
syndromes: (WHO CLASSIFICATION 1997).
1. Undifferentiated fever;
2. Classic dengue fever;
3. Dengue hemorrhagic fever, or DHF; and
4. Dengue shock syndrome, or DSS.
 Dengue shock syndrome is actually a severe
form of DHF.
 According to WHO classification (WHO
2009),
 Dengue is classified as
 1.Uncomplicated ,2.Severe.

55. CLINICAL PRESENTATION OF DENGUE
 Clinical Case Definition for Dengue Fever
 Classical Dengue fever or Break bone fever is an acute febrile
viral disease frequently presenting with headaches, bone or joint
pain, muscular pains,rash,and leucopenia
 Clinical Case Definition for Dengue Hemorrhagic Fever
 4 Necessary Criteria:
1. Fever, or recent history of acute fever
2. Hemorrhagic manifestations
3. Low platelet count (100,000/mm3 or less)
4. Objective evidence of “leaky capillaries:”
• elevated hematocrit (20% or more over baseline)
• low albumin
• pleural or other effusions

56. CLINICAL PRESENTATION OF DENGUE
 Clinical Case Definition for Dengue Shock Syndrome
• 4 criteria for DHF
 +
• Evidence of circulatory failure manifested indirectly by
all of the following:
• Rapid and weak pulse
• Narrow pulse pressure (< 20 mm Hg) OR
hypotension for age
• Cold, clammy skin and altered mental status
• Frank shock is direct evidence of circulatory failure

57. HEMORRHAGIC MANIFESTATIONS OF DENGUE
 1.Skinhemorrhages:
petechiae, purpura, ecchymoses petechiae
 Gingival bleeding
 Nasal bleeding / Epistaxis.
 2.Gastrointestinalbleeding:
Hematemesis, melena, hematochezia
 Hematuria
 Increased menstrual flow
purpura

58.  Signs and Symptoms of
Encephalitis/Encephalopathy
Associated with Acute Dengue
Infection
• Decreased level of consciousness:
lethargy, confusion, coma
• Seizures
• Nuchal rigidity
• Paresis

59. FOUR GRADES OF DHF
 Grade 1
 Fever and nonspecific constitutional symptoms
 Positive tourniquet test is only hemorrhagic manifestation
 Grade 2
 Grade 1 manifestations + spontaneous bleeding
 Grade 3
 Signs of circulatory failure (rapid/weak pulse, narrow pulse
pressure, hypotension, cold/clammy skin)
 Grade 4
 Profound shock (undetectable pulse and BP)

60. DENGUE GRADATION

61. DANGER SIGNS IN DENGUE HEMORRHAGIC FEVER
• Abdominal pain - intense and sustained
• Persistent vomiting
• Abrupt change from fever to hypothermia, with
sweating and prostration
• Restlessness or somnolence
• All of these are signs of impending shock and
should alert clinicians that the patient needs
close observation and fluids.

63. Ecchymosis
NASAL HEMORRHAGE

64. DIAGNOSIS D.H.F
 WHO Criteria For DHF :
 Fever, Minor or Major Hemorrhagic
manifestations.
 Thrombocytopenia ( < 100000 / mn3)
 Objective evidence of increased capillary
permeability (hematocrit increased > 20%) X-
ray pleural effusion
 Hypoalbuminemia.
 DSS : Above mentioned criteria plus hypo
tension and narrow pulse pressure ( < 20 mm
of Hg)

65. DENGUE DIAGNOSIS

66. LABORATORY DIAGNOSIS OF DENGUE
 I. Direct examination
 —Electron Microscopy
 ---Detection of dengue viral Ag by
Immunohistochemistry staining/
Immunofluorecence.
 ---Antigen capture ELISA.
 ---Nucleic acid detection techniques.
 II. Virus Isolation
 III. Viral identification
 IV. Serologic tests.

67. LABORATORY DIAGNOSIS OF DENGUE
 Specimens:
 1. Blood collected during first 3-5 days of illness for
virus isolation, NAAT (RT-PCR).
 2.CSF
 3.Serum

68. DIRECT EXAMINATION
 Antigen capture ELISA:
 This test detects the DEN virus in mosquitoes
&acute phase blood samples.
 NS1 Ag ELISA : This detects DEN virus in the
infected patients as early as 1st day of post onset of
symptoms (DPO) to 18th DPO.
 NS1Ag assay is useful for differentiation between
flaviviruses because of specificity of the assay.
 NS1Ag of dengue is detected by
 1.Fluorescent antibody assay.
 2.Immunoperoxidase staining.
 3.Avidin-biotin enzyme assay.
 NS1Ag assay is 99-100% specific & is more
sensitive in the early phase of the primary dengue

69. LABORATORY DIAGNOSIS OF DENGUE CONTD
 Nucleic acid detection techniques:
 1.RT-PCR
 2.Nested RT-PCR
 3. Genomic sequencing -NASBA
 4.RT-LAMP method (Reverse
Transcriptase Loop mediated
Isothermal Amplification).
 Quicker, more reliable means of
diagnosis
 NASBA method (RNA-specific
amplification assay)
 RT-PCR method to provide most
accuracy, uses 5’-3’ nuclease
oligonucleotide probe (which may not

70. VIRUS ISOLATION
 Old ―Gold Standard‖
 Cell Culture (mammals &
mosquitoes) -Indirect
Immunofluorescence
 Useful to study basic
virology, epidemiology &
pathogenesis. Impractical for
rapid diagnosis &
treatment.
 Cell lines include:
Primary duck/chick embroyo.
Primary monkey & Hamster
kidney cells, Vero cells,
BHK-21
 Mosquito cell lines are
C6/36 Aedes albopictus,
AP61 Aedes

71. SEROLOGY-ANTIBODY DETECTION
 A)ELISA 1. Mac ELISA: (IgM Antibody Capture
ELISA)
 2. IgG ELISA
 3. IgM/ IgG ratio
 4.IgG antibody Avidity assay
 B) Indirect Immunofluorescence
 C) Haemagglutination Inhibition test
 D)Neutralization tests –1.Plaque Reduction
Neutralization tests2. Micro Neutralization test

72. SEROLOGY-ANTIBODY DETECTION
 Mac ELISA: (IgM Antibody Capture ELISA)
 Principle: Assay is based on capturing human IgM
Antibody on a micro titer using antihuman- IgM Ab
followed by addition of dengue virus specific Ag
(DEN1-4).
 Antigens used for this purpose are derived from
envelope protein of the virus.
 Limitations:
 1.Crossreactivity between other circulating
flavivirus.
 2. Some cases of secondary infection will produce
false negative results in Mac ELISA as the levels of
IgM Ab’s is low during secondary infection.

73. ELISA
 B)IgG ELISA: It is used to detect past dengue
infection.
 It utilizes envelope protein of the virus as the
antigen.
 Primary v/s Secondary infections can be determined
using a simple algorithm—
 Samples with a negative IgG in the acute phase
and a positive IgG in the convalescent phase 
Primary infection.
 Samples with a positive IgG in the acute phase and
a four fold rise in titer in the convalescent phase (
with at least 1 week interval between 2 samples).
Secondary infection.

74. ELISA
 C)IgM/ IgG ratio :
 IgM Capture ELISA & IgG Capture ELISA are
commonly used for this purpose.
 Dengue infection is primary –If IgM/ IgG OD ratio is
greater than 1.2 (using patient’s sera at 1/100
dilution) or 1.4 (using patient’s sera at 1/20 dilution).
 Dengue infection is secondary –if ratio is < 1.2/1.4.
 But this algorithm has to be standardized as the ratio
varies between laboratories.
 D).IgG antibody Avidity assay :
 This test is done on acute phase serum samples.
 It is used to discriminate between primary &
secondary infection.
 This assay is useful in patient’s hospitalized with

75. INDIRECT IMMUNOFLUORESCENCE
 IgM Ab’s are detected by Immunofluorescence within
few days of illness.
 Four fold rise in IgG titer is diagnostic of recent
infection,
 In some circumstances single stable elevated titer
(>1:128) –indicates recent infection.

76. HAEMAGGLUTINATION INHIBITION TEST
 It is based the ability of dengue antigens to
agglutinate R.B.C’s of gander or trypsinized human O
RBC.
 Anti dengue Ab’s in sera inhibit this agglutination &
potency is measured in HI test.
 Optimally it requires 2 samples—tested at 1:10
dilution& further at 2 fold dilution to end point.
 A four fold rise between acute & convalescent sera is
diagnostic of recent infection.

77. NEUTRALIZATION TEST
 1.Plaque Reduction Neutralization test
 2. Micro Neutralization test
 Plaque Reduction Neutralization test(PRNT) :
 PRNT is used to determine the infecting serotype in
the convalescent sera.
 PRNT is a biological assay based on the principle of
interaction of the virus and antibody so that virus no
longer able to infect/replicate in cell culture.
 Micro Neutralization test:
 This assay based on same principle.
 This test uses a calorimetric measurement of the
virus induced cell lysis to determine the endpoint
instead of counting the number of plaques/well.

78. OTHER PROCEDURES
 V. Dot Blot assay
 VI. Dipstick assay
 VII. Microsphere based immunoassays (MIA): It is
based on the covalent bonding of Ag/ Ab to
microspheres or beads. Detection methods include
lasers to elicit fluorescence of varying lengths.
 Future developments include :
 IX. Molecular profiles using Mass spectrometry.
 X. Microarray Analysis.

79. TREATMENT
 No specific therapy
 Supportive measures:
adequate hydration
acetaminophen (if no liver dysfunction)
avoid ASA and NSAIDs
 DHF or DHF w/ shock:
IV fluid resuscitation and hospitalization
blood or platelet transfusion as needed

80. VACCINATION
 No current dengue vaccine
 Estimated availability in 5-10 years
 Vaccine development is problematic as the
vaccine must provide immunity to all 4
serotypes
 Lack of dengue animal model
 Live attenuated tetravalent vaccines under
phase 2 trials
 New approaches include infectious clone
DNA and naked DNA vaccines

81. CHIMERIVAX-DENGUE
 ChimeriVax-Dengue
- Tetravalent
- Uses yellow fever
vaccine as base
- 92% of monkeys
passed ―virulent
virus challenge‖
The most promising vaccine to
date is being worked on in
the U.S. and it uses the
same base as the yellow
fever virus vaccine, but
replaces the premembrane
and Virion envelope genes
for yellow fever with the ones
for each dengue serotype.
Current studies are largely
successful because the
genomes remain stable even
after 20 passages through

82. CHIMERIVAX-DENGUE
 Tetravalent vaccine
 ChimeriVax-Dengue?
 20% seroconversion
rate
 More research
necessary!

83. PREVENTION
Personal:
 clothing to reduce exposed skin
 insect repellent especially in early morning,
late afternoon. Bed netting is of little utility.
Environmental:
 reduced vector breeding sites
 solid waste management
 public education

84. PREVENTION
Biological:
 Target larval stage of Aedes in large water
storage containers
 Larvivorous fish (Gambusia), endotoxin
producing bacteria (Bacillus), copepod
crustaceans (mesocyclops)
Chemical:
 Insecticide treatment of water containers
 Space spraying (thermal fogs)

85. PUBLIC HEALTH STRATEGIES
 Vector Control
 Surveillance
 Preparation for outbreaks
 Research

89. Programs to Minimize the Impact of
Epidemics
•Education of the medical community
•Implementation of emergency contingency
plan
•Education of the general population