DENGUEDr. R.Jayaprada

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: /ˈdɛŋɡeɪ/, US: /ˈdɛŋɡiː/), 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 .

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.

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 endemic areas

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)

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 along with DV-3.

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-2 and DV-3.

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.

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

BASIC DENGUE GENOME

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.

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

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.  

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

VIRUS CLASSIFICATION GROUP : IV

FAMILY :Flaviviridae

Genus :Flavivirus

Species :Dengue Virus

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.

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

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.

LIFE CYCLE OF AEDES MOSQUITO

Pupae Larvae

Eggs

4-5 days2-3 days

1-2 days

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

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.

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

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

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.

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.

TRANSMISSION OF VIRUSES

REPLICATION AND TRANSMISSION 1.Mosquitoes transmit dengue to human

dendritic cells2. Dengue targets areaswith high WBC counts(liver, spleen, lymph nodes, bone marrow,andglands)

3. Dengue enters

WBCs & lymphatic

Tissue.

4.Dengue virus enters the

circulation.

Incubation Period: 3-14 days

Viraemia & Fever: 5-7 days

ExtrinsicIncubation Period: 1-2 weeks

VectorHumidity: Rainfall & Temp.

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.

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 twelve days.

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.

TRANSMISSION CYCLE CONTD

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.

ENTRY INTO THE CELL

Dengue infection

Endosome entry & pH change

E protein conformational change

Release of viral RNA into cell

Replication & further infection

VIRAL REPLICATION

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.

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

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

Serum antibodies developed can neutralize dengue virus of that same serotype (homologous)

Pre-existing heterologous antibodies form complexes but no neutralization.

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.

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.

10. Infected cells secrete vasoactive mediators Increased vascular permeability SHOCK

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.

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

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 activation,

infection persists and immune mediated tissue damage occurs.

Cytotoxic T’cells die and release Cytokines.

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.

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.

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.

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

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.

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

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

HEMORRHAGIC MANIFESTATIONS OF DENGUE

1.Skinhemorrhages:petechiae, purpura, ecchymoses

Gingival bleeding Nasal bleeding / Epistaxis. 2.Gastrointestinalbleeding:

Hematemesis, melena, hematochezia Hematuria Increased menstrual flow

purpura

petechiae

Signs and Symptoms of Encephalitis/Encephalopathy Associated with Acute Dengue Infection

• Decreased level of consciousness: lethargy, confusion, coma

• Seizures • Nuchal rigidity • Paresis

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)

DENGUE GRADATION

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.

NASAL HEMORRHAGEEcchymosis

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)

DENGUE DIAGNOSIS

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.

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

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 infection.

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 be able to distinguish among serotypes) – new “Gold Standard”

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 pseudoscutellaris.

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

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.

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.

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

dengue

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.

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.

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.

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.

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

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

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 cells.

CHIMERIVAX-DENGUE

Tetravalent vaccine ChimeriVax-Dengue? 20% seroconversion

rate

More research necessary!

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

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)

PUBLIC HEALTH STRATEGIES

Vector Control

Surveillance

Preparation for outbreaks

Research

Programs to Minimize the Impact of Epidemics

•Education of the medical community

•Implementation of emergency contingency plan

•Education of the general population

THANK YOU