Serotypes of Dengue in Pushpagiri - Yengage

PREVALENT
SEROTYPES OF
CIRCULATING DENGUE VIRUS AND ITS
ASSOCIATION WITH THE SEVERITY OF
DISEASE IN A TERTIARY CARE
CENTRE,
1
KERALA
Kavitha R Nair
Reg No.119/Jan 2014
PhD Scholar(External part time)
Kavitha R Nair
Reg No:119/Jan 2014
External Part Time PhD Scholar
Yenepoya University.
Official address
Tutor
Department of Microbiology
Pushpagiri Institute of Medical Sciences and Research Centre
Tiruvalla - 689101
Kerala.
Email: [email protected].
Mobile: +919539049477
2
Name of the Guide
Dr Vidya Pai MD
Professor and Head
Department of Microbiology
Name of the Co-Guide
Dr Seema K Oommen MD,DNB
Professor and Head
Department of Microbiology,
Yenepoya Medical College and
Pushpagiri Institute of Medical
Research Centre
Sciences and Research Centre,
Yenepoya University
Tiruvalla-689101,
Mangalore
Kerala,
Karnataka
India
India
3
COMMENTS AND ACTION TAKEN
Comments
Action taken
1
Mistakes should be avoided
in the data presented in the
slides
Mistakes have been
corrected
2
Review of literature can be
shown in a couple of slides.
Added more reviews
4
INTRODUCTION
5
VIROLOGY
 Family Four
 The

OF DENGUE
Flaviviridae, Genus – Flavivirus, RNA virus
serotypes:DEN-1, DEN-2, DEN-3, DEN-4
mature virion contains three structural proteins:
C, the nucleocapsid or core protein; M, a membraneassociated protein; and E, the envelope protein
 Seven
are nonstructural proteins: NS1, NS2A,
NS2B, NS3, NS4A, NS4B, and NS5.
( Field’s virology Vol.2 6th ed )
6
MODE OF TRANSMISSION
Bite of infected Aedes mosquitoes ,mainly Aedes
aegypti and Ae.albopticus
SIGNS AND SYMPTOMS OF DENGUE
Malaise and influenza like symptoms
 Myalgia, Cytopenia, Rash, Elevated liver enzymes

COMPLICATIONS
 Dengue
hemorrhagic fever (DHF)
 Dengue shock syndrome (DSS)
( WHO bulletin.,2012)
7
MAJOR DIAGNOSTIC MARKERS FOR DENGUE
INFECTION
(Rosanna W. Peeling et al. Evaluation of diagnostic tests: dengue
Nature Reviews .,Dec 2010)
8
PRESENT STATUS
9
INTERNATIONAL

-
Endemic in over 100 countries worldwide
causes nearly 100 million cases of DF,
500,000 cases of DHF and
24,000 deaths each year
( WHO Dengue Bulletin.,2011)
10
COMPARISON OF DENGUE CASES INDIA
2015&2016-STATEWISE
2015
2016
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
Delhi
Source:NVBDCP
Andhra Pradesh
Assam
Gujarat
Haryana
Karnataka
Kerala
Madhya Pradesh
Maharashtra
Orissa
Punjab
Tamil Nadu
Uttar Pradesh
West Bengal
11
COMPARISON OF DENGUE CASES KERALA
2015&2016-MONTHWISE
1400
1200
1000
800
600
2015
2016
400
200
0
december
november
october
september
august
july
june
may
april
march
february
january
12
SOCIAL
13
RELEVANCE
 Dengue
is the most rapidly spreading mosquitoborne viral disease in the world.
 Dengue
infection has a wide clinical spectrum that
includes both severe and mild manifestations
 The
number and frequency of outbreaks increased
and dengue is almost endemic in India.
 At
present, all the four serotypes are seen in
circulation, but the predominant serotype keeps
changing.
14
CONTD …..
Dengue morbidity and mortality can be reduced by:
-
Implementing early case detection
-
Implementing improved outbreak prediction and
detection through coordinated epidemiological and
entomological surveillance
-
Promoting integrated vector management
-
Managing severe cases with appropriate treatment
15
REPORTED SEROTYPES IN INDIA
Author and article
Year &Prevalent serotype
Nivedita Gupta et al.,2012.Dengue in 1956-2005
India
DENV-2
2005 onwards
DENV-2 & DENV-3-N.India
DENV-3 – S.India
Bharaj at al.,2008. Concurrent
infections by all four dengue virus
serotypes during an outbreak of
dengue in 2006 in Delhi, India
2006
DENV-3
DENV 1 & 3 (co infection)
N.Pradeep Kumar et al.,2013.
Genetic characterization of dengue
viruses prevalent in Kerala State,
India
2008-2010
DENV-3 & DENV-2
16
DENV-2 &3 (co infection)
Author and article
Ahmed NH et al.,2015. Dengue Fever
Outbreak in Delhi, North India: A ClinicoEpidemiological Study.
Nazia Afreen et al., 2014. Molecular
Investigation of 2013 Dengue Fever
Outbreak from Delhi, India
Nazia Afreen et al.,2016 .Evolutionary
Analysis of Dengue Serotype 2 Viruses
Using Phylogenetic and Bayesian Methods
from New Delhi, India
Year &Prevalent
serotype
2010
DENV 1
2013
DENV-2
DENV 1&2
(co infection)
2011-2014
DENV1,DENV2,
DENV3
17
AIM & OBJECTIVES
18
AIM
The aim of the present study is to detect the
prevalent serotypes of circulating dengue virus
and its association with severity of disease in a
tertiary care centre, Kerala
19
OBJECTIVES:1. Detection of the prevalent serotypes of the
circulating dengue virus with the annual
seasonal variation in a tertiary care centre .
2. To compare the dengue serology with the
molecular methods
3. To characterise the prevalent genotypes in the
locality by molecular methods
4. To classify primary and secondary dengue
based on the serological test.
.
20
5. To document the haematological
parameters and the clinical outcomes.
6. To associate the serotypes with the clinical
outcome
21
MATERIALS & METHODS

Research Design: Prospective

Study Setting: Microbiology laboratory of a tertiary
care teaching medical college.

Duration of Study: 3 years
22
 Sample
size:
The sample size is calculated using the formula:
N =Z(1-α)2PQ
d2
Where the prevalence rate p is calculated from
previous studies d= allowable error is put as 5%, α
=5%
 Sample size (n) =340 NS1 positives
N. Pradeep Kumar et al.,2013
23
Inclusion criteria:
 All
consecutive serum specimens suspected of
Dengue within the first five days of onset of
fever with NS1 positives and only hospitalized
would be included in the study
A
paired serum after 3-4 days for those
samples which are only NS1 positives.
 Only
adult population is included
24
Exclusion criteria:
 Patient
serum specimens received after 5
days of onset of fever and NS1 negatives.
25
WORKPLAN
26
WORK
PLAN
Patient history, rain fall and
seasonal knowledge
Sample collection (Two aliquots)
Serological
test-ELISANS1,IgM,IgG
IgM/IgG
Ratio
Primary
NS1
positives
Secondary
NS1+IgM
positives
Repeat sample
IgM detection
RNA
extraction
-70°C
NS1 typing
PCR(DENV1,DENV2,DENV3,DENV4

Sequencing
Analysis of data
28
 All
serum samples subjected to NS1, IgM and IgG
Capture ELISA
Molecular analysis
 Reverse Transcriptase multiplex PCR
 Core-pre-Membrane (CprM) junction nucleotide is
the target of interest for the amplification
 Gel electrophoresis for dengue serotypes.
(Lancoitti et al.,1992)
o
All the amplified products gel are cut into blocks,
purified and will be subjected to sequencing
(Pradeep kumar et al.,2013)
29
PCR- Protocol
 Sample: Serum
 Reagents : Qiamp Viral RNA kit (as per kit protocol)
 Primers
Forward primer
 Dl 5'-TCAATATGCTGAAACGCGCGAGAAACCG-3'
Reverse primers
 TS1 5'-CGTCTCAGTGATCCGGGGG-3'
 TS2 5'-CGCCACAAGGGCCATGAACAG-3'
 TS3 5'-TAACATCATCATGAGACAGAGC-3'
 TS4 5'-CTCTGTTGTCTTAAACAAGAGA-3'
30
Lancoitti et al.,1992
STATISTICAL
ANALYSIS
 Data
will be analyzed by Sensitivity, specificity,
positive predictive value (PPV), negative
predictive value (NPV), chi-square, and
Cohen’s kappa values.
 The
significance of the association between
different serotype and disease severity on the
results of NS1 and IgM will be assessed using
generalized logistic regression by SPSS
windows software.
31
PROGRESS
o
OF WORK
A total of 304 NS1 positive serum samples
were collected during a period of 1 year from
31st Oct 2015 to 30th November 2016.
 Stored
at -70º C in duplicate.
 Patient
details recorded
 Performed
RNA extraction from 180 samples
using viral RNA extraction kit (Qiagen)
32
RESULT
33
MONTHWISE DISTRIBUTION OF DENGUE NS1
POSITIVE SAMPLES
100
91
90
80
65
70
60
50
50
40
28
8
8
Nov/16
Aug/16
Jul/16
Jun/16
May/16
Apr/16
Dec/15
Nov/15
0
4
Mar/16
1
4
Feb/16
10
12
9
9
Sep/16
15
Jan/16
20
Oct/16
30
34
PERCENTAGE DISTRIBUTION OF DENGUE
CASES , N=304
secondary
dengue
65(21%)
primary
dengue
239(79%)
35
RESULTS CONTD…..
 Out
of 304 cases 156 (51%) are males and
148 (49%) are females.
 Out
of 156 males,120(77%) cases were
diagnosed as primary dengue and 36 (23%)
were diagnosed as secondary dengue.
 Out
of 148 females, 117(79%) were having
primary dengue and 31 (21%) were
diagnosed as secondary dengue.
36
TIMELINE
37
Period of
Achievable target
Work
study
accomplished
6 Months -Literature review,
Yes
- Establishing protocols for
sample collection,
transport and testing,
-Procurement of kits,
reagents for RNA
extraction,
- Sample collection
processing (extraction) and
storage.
38
Period of
study
7-12
months
Achievable target
Work
accomplished
- Sample collection,
Yes
patient details,
transport,
processing and
storage,
- RNA extraction procurement of primers
and PCR reagents,
- Standardization of
conventional PCR
-Literature review
39
Period of
study
13-18
Months
Achievable target
-Sample processing and storage
.
-Testing of efficacy of ELISA
-Procurement of primers and PCR
reagents.
-Testing of samples by the PCR.
- Literature review
40
Period of
study
Achievable target
19-24 Months Sample processing and storage.
Testing of samples by the
methodologies developed.
Sequencing to be done. Reviewing
the literature, Publication
25-30 Months Testing of all samples by the
methodologies developed.
Comparison of results. Reviewing
the literature
41
Period of study
Achievable target
31-36 months
Statistical analysis,
Interpretation, rough draft
preparation.
37-42 months
Dissemination by publication,
Report writing.
42-48 months
Final Report writing.
42
PLAN OF WORK FOR THE NEXT SIX MONTHS
Period of study Achievable target
-Sample processing and storage
13-18 Months
.
-Testing of efficacy of ELISA
-Procurement of primers and
PCR reagents.
-Testing of samples by the PCR.
- Literature review
43
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