Download 6 Brief Resume of the intended work 6.1 Need for the study Dengue

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A. Brief Resume of the intended work
6.1
Need for the study
Dengue is one of the most serious mosquito borne viral infection mainly affecting
tropical and subtropical countries of the world. 1
Since there is no immunoprophylactic or specific antiviral therapy available, timely
and rapid diagnosis plays a vital role in patient management and implementation of
control measures. 2
Early and rapid diagnostic tool, management and confinement of dengue cases are
critical to halt the dengue virus transmission. 3
WHO recommends rapid and accurate dengue diagnosis for (i) epidemiological
surveillance (ii) clinical management (iii) Research (iv) vaccine trials. 4
This proposed study is an attempt to detect NS1 antigen by Capture ELISA vis-avis rapid immunochromatography test for early detection of dengue virus infection.
6.2
Review of Literature
Dengue virus is a positive-strand RNA virus of the Flaviviridae family with 4
distinct serotypes (DV1-4), and is transmitted to humans by several species of
the Aedes mosquito.1 All four serotypes of dengue viruses are known to be able
to infect humans and also have been documented to cause severe fatal diseases.5
It is a major cause of morbidity throughout the tropical and subtropical regions
of the world. 1 The number of dengue cases has increased over the last three to
five years, with recurring epidemics. 4 Dengue is endemic in many parts of India
and epidemics are frequently reported from various parts of India.6
Dengue fever (DF) and its severe forms—dengue haemorrhagic fevers (DHF)
and dengue shock syndrome (DSS)—has become major international public
health concerns.4 Early diagnosis of dengue shock syndrome (DSS) is
particularly important, as patients may die within 12 to 24 hours if appropriate
treatment is not administered. 3
Dengue viraemia in a patient is short, typically occurs 2–3 days prior to the onset
of fever and lasts for four to seven days of illness. During this period the dengue
virus, its nucleic acid and circulating viral antigen can be detected.4
Among the nonstructural proteins, NS1 is a highly conserved glycoprotein which
appears essential for virus replication, although no precise function has yet been
assigned to it. During acute dengue virus infection, NS1 is found associated with
intracellular organelles or is transported through the cellular secretory pathway to
the cell surface.2 NS1 is of diagnostic and pathological importance.4
Laboratory diagnosis of dengue can be made following different research lines,
which are: virus isolation, detection of viral genome, detection of viral antigens,
and serological studies. Serology is the most widely used tool in the laboratory
routine.7
Though virus isolation and characterization are considered as the gold standard
of laboratory diagnosis for acute dengue virus infection, it is expensive and takes
at least 6-10 days for the virus to replicate. Detection of viral genomic sequence
by RT-PCR is also an expensive method and not widely available in most
hospital diagnostic laboratories. 2
NS1 Antigen detection is a recent alternative to specific IgM antibody detection.8
An enzyme-linked immunosorbent assay specific to dengue virus non-structural
1 (NS1) protein has been developed for the detection of dengue NS1 antigen
during the acute phase of disease in patients experiencing primary and secondary
infections. 2
Studies done by Young et al. 2000, Alcon et al. 2002 demonstrated high
circulating level of NS1 in the acute phase of dengue by antigen capture
ELISA.9,10 Antigen detection of non-structural dengue antigens may be of benefit
for an early-stage rapid diagnosis of infection due to its long half life in the
blood. 11
A study done by Hang et al. in 2009, showed sensitivity of Platelia NS1 ELISA
assay and NS1 lateral flow rapid test to be 82% and 72% respectively.12
According to a study done by A Shrivastava et al., 2011, at New Delhi, 26% of
samples were positive for NS1 antigen by capture ELISA and 16% were positive
by immunochromatography.2
6.3
Objectives of the study
1. Detection
of
NS1
antigen
by
Capture
ELISA
and
Immunochromatographic test and comparison of results between the two
techniques.
7
B. Materials and methods
7.1
Source of data
The study will be conducted on patients with clinical suspicion of dengue virus
infection admitted in Rajarajeswari medical college and hospital, Bangalore after
obtaining informed consent.
7.2
Methods of collection of data ( including sampling procedure, if any)
A study will be conducted on 100 purposive single serum samples collected from
clinically suspected cases of dengue virus infection at Rajarajeswari medical
college, Bangalore.
 The duration of study will be of 12 months i.e. from December 2011 to
November 2012.
 The serum samples will be subjected to detection of
1. NS1 antigen by capture ELISA technique
2. NS1
antigen, anti-dengue
IgM
and
IgG antibodies by
immunochromatographic method.
Inclusion criteria
Patients of all age groups with fever who are clinically suspected of dengue virus
infection as per WHO criteria.13
Exclusion criteria
i.
Patients with fever due to a proven etiology other than dengue virus
infection (Enteric fever, Malaria, HIV infection, Hepatitis B)
Plan for data analysis
The data collected will be tabulated and analyzed statistically using appropriate
methods.
Sensitivity
and
specificity
of
capture
ELISA
v/s
immunochromatographic method will be compared.
7.3
Does the study require any investigation or intervention to be conducted on
patients or other humans or animals? if so please describe briefly
Yes. 5 ml of blood will be collected by venipuncture under strict aseptic
precautions from clinically suspected cases of dengue virus infection after
obtaining informed consent. Serum will then be separated by centrifugation and
subjected to detection of the serological markers of dengue virus infection. There
will be no financial liability on the patients included in the study.
No animal intervention / investigation will be done.
7.4
Has ethical clearance been obtained from your institution in case of 7.3?
Yes. Ethical clearance has been obtained from Institutional Ethics Committee,
Rajarajeswari Medical College and Hospital, Bangalore.
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C. LIST OF REFERENCE
1. Atul Garg, Jaya Garg, Rao YK, Upadhyay GC, Suman Sakhuja. Prevalence of dengue
among clinically suspected febrile episodes at a teaching hospital in North India.
Journal of Infectious Diseases and Immunity 2011;3(5):85-9
2. Shrivastava A, Dash PK, Tripathi NK, Sahni AK, Gopalan N, Lakshmana Rao PV.
Evaluation of a commercial Dengue NS1 enzyme-linked immunosorbent assay for early
diagnosis of dengue infection. Indian J Med Microbiol 2011;29(1):51-5
3. Tan YY, Sekaran SD, Wang SM, Ahmed F, Hossain A, Sil BK. Development of
ASSURE dengue IgA rapid test for the detection of anti-dengue IgA from dengue
infected patients. J Global Infect Dis 2011;3(3):233-40
4. Comprehensive Guidelines for Prevention and Control of Dengue and Dengue
Haemorrhagic Fever. Revised and expanded edition. New Delhi: SEARO, World
Health Organization, 2011.
5. Mustafa B, Asmah Hani AW, Chem YK, Mariam M, Khairul AH, Abdul Rasid K, Chua
KB,Epidemiological and Clinical Features of Dengue Versus other Acute Febrile
Illnesses Amongst Patients Seen at Government Polyclinics. Med J Malaysia 2010;
65(4):293-98
6. Ukey PM, Bondade SA, Paunipagar PV, Powar RM, Akulwar SL. Study of
seroprevalence of dengue fever in central India. Indian J Community Med
2010;35(4):517-9
7. Dutra NR, de Paula MB, de Oliveira MD, de Oliveira LL, de Paula SO. The laboratorial
diagnosis of dengue: Applications and implications. J Global Infect Dis 2009;1(1):3844
8.
Singh M, Majumdar M, Singh G, Goyal K, Preet K, Sarwal A, et al. NS1 antigen as an
early diagnostic marker in dengue: report from India. Diagn Microbiol Infect Dis
2010;68(1):50-4
9. Young PR, Hilditch PA, Bletchly C, Halloran W. An antigen capture enzyme-linked
immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of
infected patients. J Clin Microbiol 2000;38(3):1053-7.
10. Alcon S, Talarmin A, Debruyne M, Falconar A, Deubel V, Flamand M. Enzyme-linked
immunosorbent assay specific to dengue virus type 1 nonstructural protein NS1 reveals
circulation of the antigen in the blood during the acute phase of disease in patients
experiencing primary or secondary infections. J Clin Microbiol 2002;40(2):376-81.
11. Das D, Mongkolaungkoon S, Suresh MR. Super induction of dengue virus NS1 protein
in E. coli. Protein Expr Purif 2009;66(1):66-72.
12. Ty Hang V, Minh Nguyet N, The Trung D, Tricou V, Yoksan S, Minh Dung N, et al.
Diagnostic accuracy of NS1 ELISA And Lateral Flow Rapid Tests for Dengue
Sensitivity, Specificity and Relationship to Viraemia and Antibody Responses. PLoS
Negl Trop Dis 2009[cited 2011 Nov 9];3(1):360+.Available from:
http://www.plosntds.org/.
13. Dengue guidelines for diagnosis, treatment, prevention and control. New Ed. Geneva:
World Health Organization,2009.
RAJARAJESWARI MEDICAL COLLEGE AND HOSPITAL
BANGALORE
INFORMED CONSENT
I, _________________________________________ aged _____________ years, unreservedly and in my full
senses give my consent to take part in the study which includes procedures like venipuncture.
These procedures have been explained to me in the language understood by me.
I hereby give my consent for the same.
Signature / LTI of the participant
Name:
Date:
RAJARAJESWARI MEDICAL COLLEGE AND HOSPITAL
BANGALORE
PARENTAL INFORMED CONSENT
(FOR CHILDREN BELOW 18 years of age)
I ___________________________parent of __________________________ unreservedly and in my full senses
give my consent to allow my child to take part in the study which includes procedures like venipuncture. These
procedures have been explained to me in the language understood by me and I hereby give my consent for the
same.
Signature / LTI of the parent
Name:
Date: