1. Virology , Pathogenesis and
Laboratory Diagnosis of
Dengue
Dr. Nandita Pal
Professor & HOD,
Dept. of Microbiology
Malda Medical College &
Hospital, Malda
2. Dengue - Introduction
• The most prevalent mosquito-borne viral
disease in human
• The spectrum of infection
– Asymptomatic infection
– Clinically apparent disease
– Severe disease
– May result in death
3. Dengue - Introduction
• Symptomatic infections can present as:
– A mild non-localizing fever syndrome
or
– Classic dengue fever characterized by
• Intense headache, retro-orbital pain
• Severe myalgias and arthralgias
• Rash
• Nausea
• And vomiting
4. • Dengue hemorrhagic fever (DHF)
characterised by
– Abrupt onset of abnormally low platelet counts
– Leakage of plasma into the pleural and abdominal
cavities
– Hemorrhagic symptoms
– Dengue shock syndrome (DSS): DHF with
evidence of systemic hypoperfusion)
• In a small percentage of cases: hepatitis,
encephalitis or multiorgan failure
Dengue - Introduction Continued
5. Epidemiology
• Globally around 3.4 billion people, are at
risk for DENV infection (WHO Bulletin 17th
March 2023: Brady,O.J., et al., 2012).
• Total number of infections (apparent and
in apparent) likely reach 390 million per
year (WHO Bulletin 17th March 2023 :Bhatt
et al., 2013)
6. Age and Sex Distribution of the Dengue positive cases
0
5
10
15
20
25
30
35
40
45
0-10 11-20 21-30 31-40 41-50 51-60 61-70 >70
Dengue IgM ELISA Male Dengue IgM ELISA Female
Dengue NS-1 ELISA Male Dengue NS-1 Female
7. Dengue virus serotyping (2016-17)
46 samples (fever<8 days) that were NS1 and/or IgM positive were selected for
dengue virus serotyping.
Dengue viral RNA could be detected in 27 samples (58.70%).
74% (20) samples had monotypic infection with DENV-2.
DENV-3, was found in 15% (4 cases) and Den virus1 was found in 3% of the
samples.
Two cases were found to have dual infection with DENV-1,-3 and DENV-2,-3.
DENV-4 was not found.
74%
15%
3% 4% 4% DENV-2
DENV-3
DENV-1
DENV-1 & 3
COINFECTION
DENV- 2&3
COINFECTION
8. Den 1 Den 2 Den 3 Den 4 Co Infections Total
NS1 positive 5 5 62 10 9
(Den1 + Den 3 =5 cases,
Den2+Den3 = 3 cases,
Den3 + Den4 = 1 case)
91
IgM positive 1 0 0 0 1 (Den2+Den3) 2
Total 6 5 62 10 10 93
n = 185
Dengue serotypes in and around
Kolkata –April 2021-February 2022
10. Dengue Virus
• The agent of dengue, dengue viruses, is categorized
under the genus Flavivirus, Family Flaviviridae.
• Spherical in shape with diameter 50 nm approx.
CDC. Dr. Fred Murphy; Sylvia Whitfield. Public
Health Image Library image ID# 10228
11. • This Viral envelope- lipid bilayer surrounds the
nucleocapsid and throughout, two types of viral
proteins are present.
– envelope (E) protein and
– membrane (M) protein.
• These two proteins are responsible for controlling
the entry of virus into the human cell.
Dengue Virus
This virus Nucleocapsid is
composed of the following:
1. Viralgenome
2. Capsid protein(C)
12. Genomic Structure
• A positive-stranded 11 kb genomic RNA
• The RNA is composed of
– Three structural protein genes that encode
• A nucleocapsid or core (C) protein,
• A membrane-associated (M) protein,
• An envelope (E) protein and
– Seven non-structural (NS) protein genes:
• NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5
• The NS proteins are assumed to be involved in viral
replication and viral assembly
13. Dengue Serotypes
• There are four dengue virus serotypes –
– DENV-1
– DENV-2
– DENV-3
– DENV- 4
• Each serotype is capable of causing the full spectrum
of clinical disease.
• The fifth variant DENV-5, not yet reported in India
– has been circulating among non-human primates in the
forests of South East Asia with occasional spillover into
humans.
14. Dengue Serotypes
• These serotypes can co-exist in the endemic
areas
• The immunity to one serotype does not afford
protection to an infection caused by a
heterotypic serotype.
16. • It is not fully understood why
– Most patients resolve DENV infections
quickly and without complications, whereas
– Others experience a potentially fatal
vascular leak syndrome or severe
hemorrhages
Dengue - Pathogenesis
17. • Primary DENV infection confers:
– Long-lasting immunity to the infecting serotype an
– Partial immunity to subsequent infection with
other serotypes
• Yield of primary infection:
– Cross reactive antibodies
– Cross reactive memory T cells
• Secondary infection with a heterologous serotype is
a risk factor to develop severe forms of the disease
Theories of DHF/DSS pathogenesis
18. • Higher incidence of DHF/DSS upon secondary
infections is due to Antibody-dependent
enhancement (ADE) (Halstead, 2007)
• Antibodies from a primary infection are cross-
reactive with other DENV serotypes, but do
not neutralize the infection.
Theories ….. ADE
19. • These antibodies mediate increased uptake of
opsonized virus particles into Fc-g receptor-
bearing cells (i.e., DCs, monocytes, and
macrophages)
• This results in:
– increased viral replication and
– immune activation accompanied by enhanced
cytokine release (Halstead, 2007)
Theories of DHF/DSS pathogenesis
21. • An analogous mechanism -at the level of activated T
cells, designated “original antigenic sin” (mongkolsapaya
et al., 2003)
• This model argues for
– A reactivation of cross-reactive memory T cells
specific for the primary DENV infection induce
increased cytokine secretion
– Higher virus titres due to delayed viral clearance,
and
– Apoptosis of both infected and uninfected
bystander cells
Theories …… original antigenic sin
22. • In both models
– Cytokines have pro-inflammatory effects on
vascular endothelial cells
– This leads to leaky junctions and increased
capillary permeability (Pang, Cardosa, & Guzman,
Eliana G. Acosta et al,2007)
Theories ………
23. • In fact, elevated levels of numerous cytokines
have been observed in the sera of infected
patients during the course of DENV infection
– high concentrations of IFN-g, TNF-a, and IL-10
(Chakravarti & Kumaria, 2006; Nguyen et al., 2005; Perez et al., 2004), and
– elevated levels of IL-6 in children with DSS (Juffrie et
al., 2001).
Theories …….
24. Some observations
• Severe dengue also occurs during primary
DENV infection of infants born to DENV-
immune mothers
• Previously infected children or adults and
infants born to DENV-immune mothers have
in common a single immune risk factor –
DENV reactive IgG antibodies
25. • These hypotheses cannot explain severe
courses of disease after primary DENV
infection
• Most secondary infections do not result in
severe disease, suggesting there are other
important factors involved
Gaps in understanding
26. • PATHOGENESIS: (Costa et al., 2013; Martina et al., 2009;
Whitehorn & Simmons, 2011)
– the activation of the complement system,
– virus virulence
– and, most importantly, host genetic factors
• RISK FACTORS
– Young age
– Female gender,
– Virus strain and
– Genetic variants of
• the human major histocompatibility complex class I-related
sequence B and
• phospholipase C epsilon 1 genes
Probable Additional Factors
27. The co-circulation of multiple virus serotypes in a community
(hyperendemicity) is the most important risk factor for the
occurrence of dengue hemorrhagic fever
D J Gubler, John A. Burns School of Medicine, Honolulu, HI, USA
28. Increased transmission of multiple dengue serotypes
raises the iceberg further out of the water, and increases
the probability that severe disease will occur
D J Gubler, John A. Burns School of Medicine, Honolulu, HI, USA
30. • Serological test – currently the most widely applied
method in routine diagnosis
• NS1 Antigen Detection (ELISA)
• IgM-captured enzyme-linked immunosorbent assay
(MAC ELISA)
• IgG ELISA
• Rapid test – not recommended
• Haemagglutination-inhibition
• Neutralization
• RT-PCR and Real time RT PCR
• Virus Isolation and Culture
Laboratory Confirmation of Dengue Virus
31. Comparative merits of direct and indirect laboratory
methods for the diagnosis of dengue infections
• Opportunity refers to the fact that antibody testing is
usually the most practical diagnostic option available
33. NVBDCP Recommended Tests
• ELISA Based NS1 Ag Detection Tests from 1st Day
onwards (since 2010).
• IgM Capture ELISA (MAC ELISA) Tests after 5th day of
illness.
• Networking of Laboratories:
– Apex Referral Laboratories (ARLs): Facilities for
serotyping –ICMR Virus Unit, NICED , Kolkata
– Sentinnel Surveillance Hospitals (SSHs)
34. Procedure for specimen collection
1. 3-5 ml of venous blood to be collected in screw
capped vials/vacutainers.
2. Appropriate labeling is most important
3. If the specimens cannot be analyzed or shipped to
laboratory within 24 hours, serum must be
separated, frozen and to be shipped in cold chain
4. For serology only, shipment within 24 hours may be
made in ambient temperature
NB: Whole blood sample not to be frozen
35. Sensitivity and Specificity
• Strongly influenced by the quality of the antigen
used and can vary greatly between commercially
available products.
• Many ELISAS use dengue E protein antigens from
all four dengue virus serotypes.
• This ensures that the assay is capable of identifying
any dengue infection regardless of the serotype.
36. • IgM ELISA (MAC ELISA):
Sensitivity and specificity depend on kit quality but are
generally > 95% and > 98% respectively
IgM circulates for up to three months or longer. So, its
presence might not be diagnostic of a current illness.
Clinical corroboration would be necessary.
• NS1Ag ELISA:
Sensitivity and specificity are usually > 97% and >
98% respectively
Sensitivity and Specificity…..2
37. • Rapid IgM-based dengue diagnostic tests have been
developed as a quick and easy method for use at point of
care or bedside.
• Usually have lower and variable sensitivity in
comparison to ELISA based Tests.
• False-positive results frequent in patients with malaria,
leptospiral infections, COVID, immune disorders such as
rheumatoid and lupus or previous dengue infections.
Hence, Rapid diagnostic tests are avoided.
Rapid diagnostic tests are preferably avoided
42. Serological Diagnostic Test Report
Acute Febrile Illness from patient samples collected from an outbreak
on 17.5.2022
Sl.No. Age Sex
Results of
Dengue NS
1 ELISA
(SERUM)
Cutoff Value
: >0.1636
Results of
Dengue IgM
ELISA
(SERUM )
Non-Reactive :
<9 Units
Reactive : >11
Units
Equivocal : 9 -
11 Units
Results of
Dengue IgG
ELISA
(SERUM )
Non-Reactive :
<9 Units
Reactive : >11
Units
Equivocal : 9 -
11 Units
Results of
Chikungunya
IgM ELISA
(SERUM )
Cutoff Value :
>0.270
Results of
Scrub Typhus
IgM[ELISA]
Cutoff Value :
>0.5
Leptospira
IgM
Cutoff Value :
>1.13
OB/22/28 43 F
Reactive
(2.9081)
Equivocal
(9.846)
Reactive
(16.04)
Non Reactive Non Reactive Non Reactive
OB/22/29 30 F
Non
Reactive
Non Reactive Non Reactive Non Reactive Non Reactive Non Reactive
OB/22/30 17 F
Reactive
(0.3213)
Non Reactive Non Reactive Non Reactive Non Reactive Non Reactive
OB/22/31 56 M
Non
Reactive
Non Reactive Non Reactive Non Reactive Non Reactive Non Reactive
Pls. note the low IgM and high early IgG (within day-5) in the first case.
43. Take home message
• The most prevalent mosquito-borne viral disease in human
• Immediately after the febrile phase(Dengue fever, DF) the disease may
progress to the more severe but less common forms, which include
dengue hemorrhagic fever (DHF)
• Serological test – currently the most widely applied method in routine
diagnosis
NS1 Antigen Detection (ELISA)
IgM-captured enzyme-linked immunosorbent assay
(MAC ELISA)
• IgM circulates for up to three months or longer. So, its presence might
not be diagnostic of a current illness.
• Clinical corroboration would be necessary.
