dengue pathogenesis and diagnosis

8/6/2019 Dengue Pathogenesis and Diagnosis

1/45

DENGUEPATHOGENESIS AND

DIAGNOSIS

BY

DR. AASHISH CHOUDHARY

MODERATOR PROF. SHOBHA BROOR


2/45

RNA virus , virion is spherical,40-50 nm in diameter

Family Flaviviridae

Genus -Flavivirus

Lipid enveloped

Icosahedral (cubical) symmetry

+ve sense, ss RNA; the genome- a

single linear 11 kb molecule

Has 4 serotypes ( DEN 1,2,3 and

4) an arbovirus

Causes Dengue fever, DHF/DSS ;

transmitted by Aedes mosquitoes

Dengue virus


3/45

Q

WHY IS DENGUE AN EMERGING /

RE EMERGING DISEASE ?


4/45

EMERGENCE OF DENGUE AS A PUBLIC

HEALTH PROBLEM

Unprecedented global population growth

Unplanned and uncontrolled urbanisation esp. in tropical

developing countries

Lack of effective mosquito control in areas where dengueis endemic

Increased air travel transport of the virus between

populations of the world

Decay in public health infrastructures in most countries in

the past 30 years


5/45


6/45


7/45

Dengue in India


8/45

Trends in India


9/45

Q

Is Aedes aegypti a nervous feeder ?

WHAT MAKES IT AN EFFICIENT

EPIDEMIC VECTOR ??


10/45

Aedes aegypti

( tiger mosquito )


11/45

Transmission of Dengue Virus

byAedes aegypti

Viremia Viremia

Extrinsic

incubation

period

DAYS0 5 8 12 16 20 24 28

Human #1 Human #2

Illness

Mosquito feeds /

acquires virus

Mosquito refeeds /

transmits virus

Intrinsic

incubation

period

Illness


12/45

CLINICALFEATURES Spectrum of illness ranging from inapparent / asymptomatic or mild

febrile illness to severe and fatal hemorrhagic disease due to shock Infection with any of the 4 serotypes will cause a similar clinical

syndrome i.e. classical DF . In rare cases , second infection with a

serotype of dengue virus different from that involved in the primary

infection leads to DHF/DSS Incubation period 3 to 7 days (varies from 2 to 14 days)

In endemic areas , the illness is often clinically non-specific , esp. in

children

Important risk factors influencing the proportion of patients whohave severe disease during epidemic transmission include :

-strain and serotype of the infecting virus- immune status of host

-age of host


13/45

CLASSIC DENGUE FEVER

Primarily a disease of older children/adults; all ages and both sexes susceptible

Can occur epidemically or endemically; epidemics may be explosive and startduring the rainy season

Onset sudden high fever (may rise to 102-105 C) + chills , intense

headache(frontal) ,severe myalgia, retro-orbital pain, joint pains, back-pain

(hence the colloquial designation break-bone fever )

Other features anorexia

/vomiting, constipation, altered taste sens.

Often there is a macular rash on the first day, as well as adenopathy and palatal

vesicles and scleral injection

The fever is typically (but not inevitably) followed by a remission of a few to 24-

48 hrs. ( saddle back fever )

A second rash maculopapular

/scarlatiniform, may appear at the time ofdefervescence, and lasts about 2-3 days; begins on trunk spreading to limbs,face

and may be asso. with pruritis/hyperaesthesia

Fever lasts for about 5 days ( range 2-7 days); ; recovery is usually complete,

although convalescence may be protracted


14/45

DENGUE HEMORRHAGIC FEVER(DHF)

Principally a disease of children under the age of 15 yrs.

Infants


15/45

HEMATOLOGICAL PARAMETERS IN DHF/DSS

1. Thrombocytopenia 100,000/mm3 or less

2. Hemoconcentration rise in hematocrit by >= 20 % of baseline

GRADING OF SEVERITY OF DHF / DSS

Grade 1 fever , constitutional symp. , the only

hemorrhagic manifestation is a +ve tourniquet test

Grade 2 - grade 1 + spontaneous bleeding into skin /

other sites

Grade 3 circulatory failure rapid and weak pulse ,narrowing of pulse pressure( 20 mm of Hg or less)

Grade 4 profound shock , with unrecordable blood

pressure


16/45

The induction ofvascular permeability and shockdepends on :

1. PRESENCE OF ENHANCING NON-NEUTRALISING

ANTIBODIES-antibody elicited by previous heterologous

dengue infection

- transplacental maternal antibody may be

present in infants < 9 months age

2. AGE susceptibility to DHF/

DSS drops considerably after12 years of age

3. NUTRITIONAL STATUS malnutrition is protective

4. SEX females more affected than males

5. SEQUENCE OF INFECTION

for eg. Serotype 1 followed by serotype 2

6. RACE Caucasians more affected than blacks

7. INFECTING SEROTYPE serotype 2 is more dangerous than others


17/45

Petechial Rash of DengueF

ever


18/45

DHF/DSS - bedside clues

The clinician should record the temperature andperform a tourniquet test and look for petechiae

Tourniquet test : > 20 petechiae/ 2.5cm.(1inch) square

All suspected cases of fever with bleeding should be

investigated thoroughly for low platelet count In case of shock, tests should be done for detection

of small fluid in the abdomen or in the chest?


19/45

Q WHAT CONVERTS A CLASSICAL

FEBRILE DENGUE FEVER INTO

DHF/DSS ?


20/45

Increased Probability of DHF

Hyperendemicity

Increased circulationof viruses

Increased probabilityof secondary infection

Increased probability of

occurrence of virulent strains

Increased probability of

immune enhancement

Increased probability of DHF

Gubler & Trent, 1994


21/45

Hypothesis on Pathogenesis

of DHF

(Part 1)

Persons who have experienced a dengue

infection develop serum antibodies that can

neutralize the dengue virus of that same

(homologous) serotype


22/45

Neutralizing antibody to Dengue 1 virus

Dengue 1 virus

Homologous Antibodies Form

Non-infectious Complexes

Non-neutralizing antibody

Complex formed by neutralizing antibody and virus


23/45


of DHF

(Part 2)

In a subsequent infection, the pre-existing

heterologous antibodies form complexes

with the new infecting virus serotype, but

do not neutralize the new virus


24/45

Non-neutralizing antibody to Dengue 1 virus

Dengue 2 virus

Heterologous Antibodies Form

Infectious Complexes

Complex formed by non-neutralizing antibody

and virus


25/45


of DHF

(Part 3) Antibody-dependent enhancement

is the process in which certain

strains of dengue virus, complexedwith non-neutralizing antibodies,

can enter a greater proportion of

cells of the mononuclear lineage,thus increasing virus production


26/45

Heterologous Complexes Enter More

Monocytes, Where Virus Replicates

Non-neutralizing antibody

Dengue 2 virus

Complex formed by non-neutralizing

antibody and Dengue 2 virus


27/45


of DHF

(Part 4) Infected monocytes release vasoactive

mediators, resulting in increased vascular

permeability and hemorrhagicmanifestations that characterize DHF and

DSS


28/45


29/45


30/45


31/45

LAB. DIAGNOSISA. SEROLOGY

1. IgM capture ELISA

2. Hemeagglutination-Inhibition (HI)

3. Neutalization test (NT)

4. Complement Fixation (CF) test

5. IgG avidity test

B. VIRUS ISOLATION

1. Intra-thoracic mosquito inoculation2. Mosquito cell culture C6/36 clone ofA. albopictus cells

3. Baby mice (1-3 day old) - intracerebral inoculation

4. Mamalian cell culture : LLC-MK2 cells

C. VIRUS IDENTIFICATION

1. Indirect fluorescent-antibody technique (IFA)

D. MOLECULAR TECHNIQUES1. RT-PCR

2. Hybridization probes

3. Immunohistochemistry using enzyme conjugates


32/45

IgM capture ELISA

Most widely used serological test

Simple , rapid , doesnt require sophisticated equipment

Most patients develop anti-dengue IgM by day 5 ; these wane to

undetectable levels by 60 days

Is produced in both primary and secondary dengue infections

Advantage over HI - a single, properly timed acute phase serum

sample has a sensitivity comparable to the paired serum samples

reqd. for HI

Disadvantage owing to persistence of IgM for 1-3 months , a

positive result does not always mean current infection


33/45

Hemeagglutination inhibition (HI)

A frequently used test ; reliable if properly done

Is sensitive , easy to perform , requires only minimal equipment.

Since HI antibodies persist for long periods ( upto 48 yrs. Or evenlonger) , the test is ideal for seroepidemiologic studies

HI antibodies are detectable by day 5 or 6 of illness , convalescent

titres are at or below 640 in primary infections

By contrast , there is an immediate anamnestic response in sec. andtert. Inf. , and reciprocal antibody titres increase rapidly in the firstfew days , reaching > 5,120 or 10,240 ; these high titres fall below1,280 in about 30-40 days

Hence , a titre > 1,280 in an ac. phase / early convalesc. phase serumis presumptive diagnosis of current infection

Major disadvantage lack of specificity ; unreliable for identifyinginfecting serotype


34/45

Complement fixation(CFT)

Also used widely

CF antibodies appear later than HI antibodies , are more specific in

primary infections , and usually persist for for short periods

Diagnostically valueable test because of this late rise in CF Abs ;

some patients thus show a diagnostic rise of titres by CF but have

only stable Ab by HI or ELISA Greater specificity in prim. Inf. since CF responses are monotypic

whereas HI responses are broadly heterotypic

NOT specific in sec. Inf.

Limited value in seroepidemiologic studies CF Abs are notpersistent.


35/45

Neutralization test(NT)

Is the most specific and sensitive serologic test for Dengue viruses

Commonest protocol serum dilution plaque reduction NT

Neutralizing Ab titres rise at par/ slower than HI or ELISA Ab

titres but more quickly than CF Ab titres and persist for > 48 yrs.

Can be used for seroepidemiologic studies

More sensitive , neutralizing are Abs are present in the absence ofdetectable HI Abs in some patients with past inf.

Since relatively monotypic Ab response is observed in properly

timed convalesc.- phase sera , NT can be used to identify the

serotype in primary infection

Major disadvantages expensive , time consuming , technically

difficult hence NOT routinely used


36/45


37/45

Q

WHAT SAMPLE TO COLLECT AND

WHEN ??


38/45

SAMPLE COLLECTION

GUIDELINES FOR CLINICIANS

IF FEVER IS OF < 5 DAYS DURATION

-only viral isolation possible at this stage

-send 3-5 ml. blood in a plain sterile screw-cappedvial ON ICE , IMMEDIATELY

IF FEVER IS OF > 5 DAYS DURATION

-send 3-5 ml. blood in a plain sterile screw-capped

vial for virus SEROLOGY

Requisition forms must include the following info;

-duration of fever

-if fever has subsided , no. of days since defervescence


39/45

NEWER TECHNIQUES

RT-PCR

-provides a rapid serotype-specific diagnosis- is sensitive simple, and reproducible if properly controlled

- should not be used as a substitute for viral isolation (the availability of viral

isolates for characterizing virus strain differences , since this info. is critical for

viral surveillance and pathogenesis studies.

HYBRIDIZATION PROBES

- detection of viral nucleic acids with cloned hybridization probes

IMMUNOHISTOCHEMISTRY

- detection of viral antigen using enzyme conjugates(peroxidase,phosphatase)with polyclonal/monoclonal Abs


40/45

Q CAN WE DIFFERENCIATE PRIMARY

INFECTION FROM SECONDARY

INFECTION ?


41/45

DIFFERENCIATION BETWEEN PRIMARY

AND SECODARY INFECTIONS

HI is the conventional test used

IgG AVIDITY via ELISA to which a urea

incubation step is done-inprim. inf. the specific IgG antibody

response begins with low avidity IgG, whichgradually evolve to high avidity antibodies

-in sec. Inf. , the rapid antibody response ischara cterized by production of high avidityantibodies.


42/45

RiskFactors Reported for DHF

Virus strain

Pre-existing anti-dengue antibody

previous infection

maternal antibodies in infants Host genetics

Age

Higher risk in secondary infectionsHigher risk in secondary infections

Higher risk in locations with two or more serotypes circulatingHigher risk in locations with two or more serotypes circulating

simultaneously at high levels (hyperendemic transmission)simultaneously at high levels (hyperendemic transmission)


43/45

Viral RiskFactors

for DHF

Pathogenesis Virus strain (genotype)

Epidemic potential: viremia level, infectivity

Virus serotype

DHF risk is greatest for DEN-2, followed by

DEN-3, DEN-4 and DEN-1


44/45

VECTOR CONTROL MEASURES

1.PERSONAL PROPHYLATIC MEASURES

Use of mosquito repellent creams, liquids, coils, mats etc.

Wearing of full sleeve shirts and full pants with socks

Use of bednets for sleeping infants and young children during day time to prevent mosquito bite

2. BIOLOGICAL CONTROL

Use of larvivorous fishes in ornamental tanks, fountains, etc.

Use of biocides

3. CHEMICAL CONTROL

Use of chemical larvicides like abate in big breeding containersAerosol space spray during day time

4. ENVIRONMENTAL MANAGEMENT & SOURCE REDUCTION METHODS

Detection & elimination of mosquito breeding sources

Management of roof tops, porticos and sunshades

Proper covering of stored water

Reliable water supply

Observation of weekly dry day

5. HEALTH EDUCATION

Impart knowledge to common people regarding the disease and vector through various media sourceslike T.v., Radio, Cinema slides, etc.

6. COMMUNITY PARTICIPATION

Sensitilizing and involving the community for detection ofAedes breeding places and their elimination


45/45

Features of candidate dengue vaccines

Live attenuated Chimeric virus DNA Inactivated Subunit recomb

No. of antigens 10 2 1 to many Several Mainly 1

In vivo replication Yes Yes No No No

Immune response Best Best Excellent Excellent Poor

Memory T and B cells Best Best Excellent Fair Fair

Protection in animals Yes Yes Yes Yes Yes

Status of developme Phase I, II Phase I Preclinical Preclinical Animal studies

dengue pathogenesis and diagnosis

Documents