DENGUE

PATHOGENESIS AND DIAGNOSIS

BY DR. AASHISH CHOUDHARY

MODERATOR PROF. SHOBHA BROOR

Dengue virus
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

WHY IS DENGUE AN EMERGING / RE EMERGING DISEASE ?

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 dengue is 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

Dengue in India

Trends in India

Q
Is Aedes aegypti a nervous feeder ? WHAT MAKES IT AN EFFICIENT EPIDEMIC VECTOR ??

Aedes aegypti ( tiger mosquito )

Transmission of Dengue Virus by Aedes aegypti

Mosquito feeds / acquires virus Extrinsic incubation period Mosquito refeeds / transmits virus Intrinsic incubation period

Viremia
0 5 8 12 16 20

Viremia
24 28

Illness Human #1

DAYS
Human #2

Illness

CLINICAL FEATURES
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 who have severe disease during epidemic transmission include :

-strain and serotype of the infecting virus

- immune status of host -age of host

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 start during 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 2448 hrs. ( saddle back fever ) A second rash maculopapular / scarlatiniform, may appear at the time of defervescence, 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

DENGUE HEMORRHAGIC FEVER (DHF)

Principally a disease of children under the age of 15 yrs. Infants <9 months of age ( transplacental transfer of antibody) Acute phase sudden onset fever with chills , facial flushing , headache , anorexia/ vomiting , epigastric pain as in DF Critical stage is at the time of defervescence ; as the fever remits in 2-7 days , manifeststions of plasma leakage appear hemorrharic manifestations petechiae , purpura , epistaxis , GI bleeding Scattered petechiae commonly on extremities Hepatomegaly

DSS DHF with shock- circulatory failure

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

 1.

2. 3. 4. 5. 6. 7.

The induction of vascular permeability and shock depends on : PRESENCE OF ENHANCING NON-NEUTRALISING ANTIBODIES-antibody elicited by previous heterologous dengue infection - transplacental maternal antibody may be present in infants < 9 months age AGE susceptibility to DHF/DSS drops considerably after 12 years of age NUTRITIONAL STATUS malnutrition is protective SEX females more affected than males SEQUENCE OF INFECTION for eg. Serotype 1 followed by serotype 2 RACE Caucasians more affected than blacks INFECTING SEROTYPE serotype 2 is more dangerous than others

Petechial Rash of Dengue Fever

DHF/DSS - bedside clues

The clinician should record the temperature and perform 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?

Q
WHAT CONVERTS A CLASSICAL FEBRILE DENGUE FEVER INTO DHF/DSS ?

Increased Probability of DHF

Hyperendemicity

Increased circulation of viruses Increased probability of occurrence of virulent strains

Increased probability of secondary infection Increased probability of immune enhancement

Increased probability of DHF

Gubler & Trent, 1994

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

Homologous Antibodies Form Non-infectious Complexes

Dengue 1 virus Neutralizing antibody to Dengue 1 virus Non-neutralizing antibody Complex formed by neutralizing antibody and virus

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

Heterologous Antibodies Form Infectious Complexes

Dengue 2 virus Non-neutralizing antibody to Dengue 1 virus Complex formed by non-neutralizing antibody and virus

Hypothesis on Pathogenesis of DHF (Part 3)

Antibody-dependent enhancement is the process in which certain strains of dengue virus, complexed with non-neutralizing antibodies, can enter a greater proportion of cells of the mononuclear lineage, thus increasing virus production

Heterologous Complexes Enter More Monocytes, Where Virus Replicates

Dengue 2 virus Non-neutralizing antibody Complex formed by non-neutralizing antibody and Dengue 2 virus

Hypothesis on Pathogenesis of DHF (Part 4)

Infected monocytes release vasoactive mediators, resulting in increased vascular permeability and hemorrhagic manifestations that characterize DHF and DSS

LAB. DIAGNOSIS
A. 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 inoculation 2. Mosquito cell culture C6/36 clone of A. albopictus cells 3. Baby mice (1-3 day old) - intracerebral inoculation 4. Mamalian cell culture : LLC-MK2 cells

C. D.

VIRUS IDENTIFICATION
1. Indirect fluorescent-antibody technique (IFA)

MOLECULAR TECHNIQUES
1. RT-PCR

2. Hybridization probes 3. Immunohistochemistry using enzyme conjugates

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

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 even longer) , 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. and tert. Inf. , and reciprocal antibody titres increase rapidly in the first few days , reaching > 5,120 or 10,240 ; these high titres fall below 1,280 in about 30-40 days Hence , a titre > 1,280 in an ac. phase / early convalesc. phase serum is presumptive diagnosis of current infection Major disadvantage lack of specificity ; unreliable for identifying infecting serotype

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 not persistent.

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 of detectable 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

VIRUS ISOLATION
MOSQUITO INOCULATION - intra thoracic inoculation of adult mosquitoes - most sensitive isolation method for Dengue viruses esp.in patients with severe hemorrhagic ds , or in fatal cases -species used Aedes aegypti, A. albopictus Toxorhynchities amboinensis , T. splendens - virus detected by DFA in brain , salivary glands MOSQUITO CELL CULTURE - most widely used cell line C6/36 Clone of A. albopictus cells - rapid , economical but not as sens. as mosquito inoculation - method of choice for routine virologic surveillance BABY MICE ( 3-4 days old) INOCULATION- intracerebral MAMALIAN CELL CULTURE primarily in LLC-MK2 cells

Q
WHAT SAMPLE TO COLLECT AND WHEN ??

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-capped vial 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

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

Q
CAN WE DIFFERENCIATE PRIMARY INFECTION FROM SECONDARY INFECTION ?

DIFFERENCIATION BETWEEN PRIMARY AND SECODARY INFECTIONS HI is the conventional test used IgG AVIDITY via ELISA to which a urea incubation step is done -in prim. inf. the specific IgG antibody response begins with low avidity IgG, which gradually evolve to high avidity antibodies -in sec. Inf. , the rapid antibody response is chara cterized by production of high avidity antibodies.

Risk Factors Reported for DHF

Virus strain Pre-existing anti-dengue antibody previous infection maternal antibodies in infants Host genetics Age  Higher risk in secondary infections  Higher risk in locations with two or more serotypes circulating simultaneously at high levels (hyperendemic transmission)

Viral Risk Factors 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

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 containers Aerosol 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 sources like T.v., Radio, Cinema slides, etc. 6. COMMUNITY PARTICIPATION Sensitilizing and involving the community for detection of Aedes breeding places and their elimination

Features of candidate dengue vaccines

Live attenuated

Chimeric virus

DNA

Inactivated

Subunit recomb

No. of antigens In vivo replication Immune response Memory T and B cells Protection in animals Status of developme

10 Yes Best Best Yes Phase I, II

2 Yes Best Best Yes Phase I

1 to many No Excellent Excellent Yes Preclinical

Several No Excellent Fair Yes Preclinical

Mainly 1 No Poor Fair Yes Animal studies