dengue pathogenesis and diagnosis
TRANSCRIPT
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DENGUEPATHOGENESIS AND
DIAGNOSIS
BY
DR. AASHISH CHOUDHARY
MODERATOR PROF. SHOBHA BROOR
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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
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Q
WHY IS DENGUE AN EMERGING /
RE EMERGING DISEASE ?
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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
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Dengue in India
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Trends in India
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Q
Is Aedes aegypti a nervous feeder ?
WHAT MAKES IT AN EFFICIENT
EPIDEMIC VECTOR ??
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Aedes aegypti
( tiger mosquito )
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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
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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
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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
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DENGUE HEMORRHAGIC FEVER(DHF)
Principally a disease of children under the age of 15 yrs.
Infants
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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
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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
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Petechial Rash of DengueF
ever
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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?
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Q WHAT CONVERTS A CLASSICAL
FEBRILE DENGUE FEVER INTO
DHF/DSS ?
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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
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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
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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
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Hypothesis on Pathogenesis
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
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Non-neutralizing antibody to Dengue 1 virus
Dengue 2 virus
Heterologous Antibodies Form
Infectious Complexes
Complex formed by non-neutralizing antibody
and virus
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Hypothesis on Pathogenesis
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
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Heterologous Complexes Enter More
Monocytes, Where Virus Replicates
Non-neutralizing antibody
Dengue 2 virus
Complex formed by non-neutralizing
antibody and Dengue 2 virus
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Hypothesis on Pathogenesis
of DHF
(Part 4) Infected monocytes release vasoactive
mediators, resulting in increased vascular
permeability and hemorrhagicmanifestations that characterize DHF and
DSS
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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
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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
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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
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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.
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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
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Q
WHAT SAMPLE TO COLLECT AND
WHEN ??
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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
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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
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Q CAN WE DIFFERENCIATE PRIMARY
INFECTION FROM SECONDARY
INFECTION ?
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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.
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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)
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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
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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
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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