CASE PRESENTATION

By
Dr Raksha Bhat

Moderated by
Dr Shalini Shenoy
 OVERVIEW
 CASE
 Background
 Transmission
 Virus morphology
 Viral replication and pathogenesis
 Host immune response
 OVERVIEW

 Clinical features & complications

 Treatment
 Epidemiology
 LABORATORY DIAGNOSIS
 Control measures
 Vaccine development
 Areas of concern
CASE:

 A 40 year old male, resident of Mangalore

with presenting complaints of
 Fever – since 6 days
 Severe headache – since 4 days
 Body ache – since 3 days
 Loss of appetite- since 3 days
History of presenting illness :

 Fever – high degree, sudden onset, intermittent,

no chills and rigors, not responding to oral
antipyretics
 Associated with severe body ache, headache
and loss of appetite past 3 to 4 days
 No cough, cold
 No urinary or bowel complaints.
 No history of rash or bleeding from any site
.
Past history :
 Known diabetic since 1 year and on oral
hypoglycemic drugs
 No other significant co morbidities
 History of typhoid fever 15 years back
 No similar fever episodes in the past

Personal history:
 No history of tobacco or alcohol abuse
 Non smoker
 Non vegeterian diet
 Family history:
 Six members, no similar complaints
among them
 No significant history

Socioeconomic and occupational

history:
 Shop keeper by occupation
 Lower middle class
Treatment History:
 Treated on outpatient basis with the
following medications in a private clinic:
 Tab Paracetamol
 Tab Azithral

 The following investigations were asked

for – Hemoglobin, TC, DC, Platelet count,
urine culture
 The patient did not respond to treatment
and the results of the investigations were
as follows:

 Haemoglobin: 9.2 g/dl

 Total count: 4600 cells/mm3
 Direct count: N-73, L-25, M- 02
 Platelet Count: 48,000 cells/ μL
 Urine culture : No growth
Clinical examination:
 GENERAL PHYSICAL EXAMINATION:

 Patient was conscious, oriented

 Pallor: Present
 Icterus: Absent
 Cyanosis: Absent
 Clubbing: Absent
 Lymphedanopathy: Absent
 Edema: Absent
Clinical examination continued…
 Weight: 56 kgs

 Blood pressure: 112/78 mm of Hg right arm

 Pulse: 98 beats/ min, regular rhythm

 Temperature: 102˚F

 Conjunctival congestion present

 No rash, no petechial heamorrhages

 Tourniquet test: Negative

 SYSTEMIC EXAMINATION:

 CVS: Normal heart sounds

 RS: 18 breath/ min, lung fields clear, no
abnormal findings
 Per abdomen: Liver- normal size, mild
splenomegaly present
 CNS: No abnormal findings
Course in the hospital:
 The patient was admitted in view of
high fever and low platelet count.

 He was started on intravenous fluids

NS 20ml/kg/hr and the following
investigations were asked for:
On the day of admission:

Investigation Report Normal range

Haemoglobin 8.6 12-16
Total WBC count 5000 4000-10000
Direct N 78 50-70
count M 01 0-10
E 00 0-10
L 21 20-40
Hematocrit 25.3 40-55
ESR 6 0-10
Platelet Count 30000 150000-4000000
Blood Group B POSITIVE -
Investigation Report Normal range
Serum creatinine 0.6 0.4-1.4
Serum Na+ 149 136-149
Serum K+ 3.0 3.5-5.3

Total bilirubin 0.2 0.2-1.2

Direct bilirubin 0.12 Up to 0.3
Serum protein 3.2 6.0-8.3
Serum albumin 2.2 3.2-5.5
Serum globulin 1.6 1.8-3.4
AST 56 5-40.0
ALT 34 5-40.0
Alkaline 37 40-129
phosphotase
Investigation Report
Peripheral RBCs Microcytic hypochromic
smear polychromasia

WBCs High normal number

Smear for malaria parasite Negative

Filaria Negative
Dengue Ig M ELISA POSITIVE >53.76 Panbio
units ( >11 positive)

Widal Test Negative

Ig M Leptospirosis Negative
Prothrombin time 14.8
Prothrombin time control 14.9
INR 0.99
 Blood culture showed no growth after 48 hours
of incubation
 Chest Xray : clear, normal lung fields
 USG Abdomen: Mild splenomegaly, no other
abnormal findings

 Platelet transfusion was started, platelet count

which was monitored on a daily basis

 Tab Paracetamol 500 mg QID was given orally

for fever and body temperature was monitored

 Patient was observed for signs and symptoms

and monitored to avoid any drop in the platelet
count, development of bleeding signs or shock.
Investigation DAY 1 8/8 DAY 2 9/8 DAY 3 10/8 DAY 4 11/8 DAY 5 12/8

Hemoglobin 8.6 8.8 9.6 10.2 10.4

Total Count 5000 6700 8000 6700 6800

Direct 78 45
Count 21 52 [Not investigated]

01 02
00 01
Platelet 30000 31000 40000 67000 90000
Count
Investigation DAY 6 13/8 DAY 7 14/8 DAY 8 15/8 DAY 9 16/8

Hemoglobin 11.2 11.4 12.3 11.9

Total Count [Not investigated]

Direct
Count

[Not investigated]

Platelet 92000 148000 172000 205000

Count
Platel Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9
et
Transf
usion
Numbe 4 4 2 2 3 2 2 0 0
r of
units

PLATELET COUNT
250000

200000

150000

100000

50000

0
DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9
 FEVER
100.5

100

99.5

99

98.5

98

97.5
DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9
 The patient responded well to treatment
and symptomatically improved.
 Platelet count, hematocrit improved and
body temperature reached normal

 The patient was discharged on Day 9 after

complete recovery with the diagnosis of :

‘DENGUE FEVER’
Follow up:
 The patient visited the outpatient
department a week later, he had no fresh
complaints.
 Follow up investigations were done:
Investigation Report Normal range
Haemoglobin 11.7 12-16
Haematocrit 41.3 40-55
Total WBC Count 5000 4000-10000
Direct N 67 50-70
Count L 28 20-40
M 01 0-10
E 00 0-10
 Background:
 Arthropod borne flavi virus infection

 Variable symptoms ranging from:

Dengue
Asymptomatic Dengue Haemorrhagic
infection fever fever/ Dengue
Shock Syndrome

 Transmitted by Aedes aegypti and Aedes albopictus

mosquitoes

 Four Serotypes (DEN 1, DEN 2, DEN 3 & DEN 4)

Transmission:
Human –mosquito- human cycle

Vector: Aedes aegypti and Aedes

albopictus

Incubation period: 4 - 7 days.

Extrinsic incubation : 8-12 days

The mosquito remains infected for

the remainder of its life i. e days or
a few weeks.
 Morphology:
 Family: Flavivirideae
 Genus: Flavivirus
 Single, positive-stranded RNA surrounded by
an icosahedral core
 2 proteins – M & E
 E Protein structure:

3 DOMAINS- I, II & III

•Domain II – has fusion loops which are
responsible for membrane fusion once dengue
enters the cell
•Important step in the pathogenesis
Pathogenesis and viral replication:
Dengue infection

Endosome entry & pH change

E protein conformational change

Release of viral RNA into cell

Replication & further infection

 VIRUS RELEASE IN THE CELL
Host immune response:
 Both humoral cellular immune response
play a role
2 aspects:
 FIRST INFECTION
 SECOND INFECTION
FIRST INFECTION
 Humoral response increases the antibody
serum neutralizing levels, which prevents
dengue from ever fusing with the cell
membrane

 Cellular response involves dendritic cells

which migrate to the lymph nodes and
activate T cells

 Memory cells develop antibodies to fight off

future infection of same serotype
SECOND INFECTION

 Antibody Dependent Enhancement

occurs when infected with a second
serotype
 Ig G antibodies- form a complex with the
virus which binds to Fc receptor and help in
cell entry
 Abs attach to the new strain, cross-react
with it and increase the virus-binding
efficiency
This triggers the immune system to release
excessive amounts of proinflammatory
cytokines.

Increases vascular permeability results which

causes the hemorrhaging characteristic of
dengue hemorrhagic fever
Sequence of events:
EXACT MECHANISM???
• Increased cytokine release
• Increased vascular permeability
• Plasma leakage
• Platelet aggregation
• Bleeding
• DIC & consumption of clotting factors
• Excessive Haemorrhage
• Shock
• Death
Clinical features:
Petechiae Sub conjunctival haemorrhage
Haemorrhagic Rash Dengue shock syndrome
Grading:
 GRADE I: Fever with other symptoms such as
vomiting, headache, muscle and joint pain: positive
tourniquet test is the only evidence of hemorrhaging
 GRADE II: Grade I symptoms + spontaneous bleeding
 GRADE III*: Failure of circulatory system, clammy
skin, rapid & weak pulse, restlessness

 GRADE IV*: Severe shock, no measurable blood

pressure or pulse

 *Considered Dengue Shock Syndrome (DSS)

Treatment:
 Treatment is symptomatic.
 Immediate replacement of plasma loss with
isotonic salt solution, plasma or plasma
expanders is required in cases of profound
shock.
 Electrolyte and acid-base balance must be
maintained.
 Blood loss must be replaced.

 Continued vigilance – Platelet count

Haematocrit
Bleeding manifestations
 Antivirals have no role
Epidemiology: Why Dengue is
important?
 Increasing incidence and recent outbreaks
 CDC Category A Infectious Disease
 Infects 50-100 million people every year
 About half the world lives in a “hot zone”
 Global warming has increased the range of
mosquitoes
 Previously un established serotypes are
establishing themselves in various countries
 Difficulty in diagnosis, creation of an effective
vaccine and therefore control
In India Continued…
30000 DEATH
108

25000 CASES

20000
169

15000 96
28292
80
10000 18860 70
15535
69 12561
5000 8693
5534
0
2007 2008 2009 2010 2011 2012

SOURCE: National Vector Borne Disease Control Programme Data

In Karnataka…
2500 DEAT
H
7
CASES
2000
8
1500
10
2285
1000
1764
1226
500
3 5
0 339 405
230
0
2007 2008 2009 2010 2011 2012
SOURCE: National Vector Borne Disease Control Programme Data
Role of the laboratory in diagnosis:
 Early case detection

 Novel diagnostic techniques – serological

and molecular methods have been developed
and many are in use.

 Has a potential role in management,

control and outbreak investigations

 There is a need for a specific and

inexpensive test
 Specifications for an ideal dengue
test
 Should distinguish between dengue and other
diseases with similar presentations eg: malaria,
leptospirosis, chikungunya
 Highly sensitive during the acute stage of
infection
 Rapid results
 Inexpensive
 Easy to use
 Stable at temperatures > 300C for field use
when necessary
Laboratory Diagnosis
 Currently available methods:
1. Virus detection
2. Viral RNA detection
3. Antigen detection
 NS-1 based assays
 Immuno histochemistry
4. Serological methods
 Ig M based assays
 Ig G based assays
Virus detection
 Old “Gold Standard”
 Cell culture – mosquito cell lines C6/36, AP61
or mammalian cell lines
 Intracerebral inoculation of suckling mice
 Whole blood or tissue(spleen or lymph node)
 Immunofluoroscence assays for serotype
identification using mAbs

 Useful to study basic virology,

epidemiology,
and pathogenesis

 Impractical for rapid diagnosis &

treatment
Dengue virus detection using immunofluoroscence
Viral RNA Detection
 NASBA (Nucleic Acid Sequence Based
Amplification) using tissue, whole blood
or sera in the acute phase

 Serotype specific regions of the dengue

genome can be identified by Nested PCR

 Improved sensitivity of detection

 RT - PCR
 Acute serum samples
 5’-3’ oligonucleotide
probe- Sequence
specificity
 Early laboratory
confirmation in a
significantly high
proportion of patients
 Advantages –
reliability and sensitivity
 CDC has developed a DC DENV-1-4
Real Time RT PCR Assay which was
approved by FDA in June 2012
 Detects DENV serotypes 1, 2, 3 or 4 in
clinical samples
 Not approved for blood bank screening
 Antigen detection
NS 1 based assays:

 NS 1- synthesised by all flavi virus and

secreted by infected mammalian cells
 Can be detected in the acute phase of both
primary and secondary dengue infection up
to 9 days
 Rapid immunochromatographic assays are in
use
Dengue Day 1 Test
 Serotpe specific mAb- based NS1 Ag-
capture ELISA has been developed which
shows good serotype specificity

 Ag detection in secondary infection can

be compromised by the presence of pre
existing virus Ig G complexes
Immunohistochemistry
 Dengue Ags in tissue section can be
detected using labelled Abs with fluoroscent
dyes, enzymes or colloidal gold
Serological methods*
 Acquired immune response – Ig M, Ig G & Ig A
Abs specific for virus envolope protein

 Ig M based assays
 Ig G based assays

 Ig M – higher titre and more specific in primary

infection

 Ig G – higher in secondary infection

*Cross reactivity with other flavi viruses and

serotypes
 Ig M based assays:
 3- 5 days post onset in ~ 50% of
hospitalized patients
 90 % sensitivity & 98 % specificity
 Depends on quality of Ag used, varies
greatly between commercially available
products
 ELISA based use dengue E protein Ag from
all 4 serotypes
 Ig M may be present up to 3 months,
therefore may not diagnose current illness
 Rapid Ig M tests – quick and easy, can be
used bedside
 Recombinant Ags are used
 Results in 15- 90 minutes
 21-99 % sensitivity and 77-98% specificity
 High false positives
*ELISA - Sensitivity
*Rapid tests – easy and results in short
time
 Ig G based assays:
 For past dengue and current infections
 Paired sera to demonstrate sero conversion,
four fold increase in titre
 Multiple dilutions of each serum is tested to
determine end point titre
 Ig G avidity test can be used to differentiate
primary vs secondary infection
 INVESTIGATION ADVANTAGES LIMITATIONS

Virus isolation and •Confirmed identification •Requires acute sample

identification •Specific •Requires expertise and
•Identified serotypes appropriate facilities
•Takes more than a week
•Does not differentiate
primary and secondary
infection
•Expensive
RNA detection •Confirmed identification •Potential false positives due
•Sensitive & specific to contamination
•Identifies serotype and •Requires acute sample
genotype •Requires expertise and
•Results in 24-48 hours appropriate facilities
•Does not differentiate
primary and secondary
infection
 INVESTIGATION ADVANTAGES LIMITATIONS

Antigen detection

NS 1 assay •Confirmed infection • Not as sensitive as virus

•Easy isolation
•Less expensive

Immuno •Confirmed infection • Not as sensitive as virus

histochemistry isolation
•Requires expertise
 INVESTIGATION ADVANTAGES LIMITATIONS

Serological tests

Ig M or Ig G •Confirmed infection • Ig M levels can be low in

seroconversion •Easy to perform secondary infection
•Least expensive •Paired sera requirement

Ig M detection •Identifies probable dengue • Ig M levels is low in

( single sample) cases secondary infection
•Useful for surveillance ,
tracking outbreaks and
monitoring effectiveness of
intervention
 Control measures:
 Public health strategies to
decrease the incidence
and prevalence:
 Health education
 Mosquito nets or long
clothing
 Eliminate standing water
Vaccines
 Vaccine development for the dengue virus is
extremely difficult because of:
 Four serotypes
 Single-stranded RNA therefore mutations
can occur
 Limited understanding of the pathogenesis
and protective immune responses
• Tetravalent ChimeriVax-Dengue vaccine -
only a low percentage seroconvert for all
four serotypes
• Incomplete sero conversion may lead to
cross-reactivity
• Several dengue vaccine candidates are
progressing through clinical trials
 Areas of concern:
 Dengue is a disease with a complex case
presentation
 Annual incidence has increased over
decades
 Lack of complete understanding of dengue
virulence
 No IDEAL diagnostic test
 Development of vaccine
 References:
1. Medical Microbiology 17th edition by David Greenwood, Richard
Slack, John Peutherer Mike Barer

2. Principles and Practices of Infectious Diseases. Mandell, Bennett &

Dolin. 6th Edition
3. Harrison’s Principle of Internal Medicine 17th edition by Fauci et al

4. Peeling RW, Artsob H, Pelegrino JL, Buchy P, Cardosa MJ, et al.

(2010) Evaluation of diagnostic tests: dengue. Nat Rev Microbiol 8:
S30–38
5. www.cdc.gov. Atlanta, USA: Centers for Disease Control and
Prevention
6. www.nvbdcp.gov.in. India: National Vector Borne Disease Control
Programme Directorate General of Health Services Ministry of Health
& Family Welfare
THANK YOU