Download name and designation( in block letters)

SYNOPSIS FOR PG DISSERTATION
FOR MD/MS,
UNDER RAJIV GANDHI
UNIVERSITY OF HEALTH SCIENCES,
BENGALURU
DR. ARUN KAUSHIK.R
DEPARTMENT OF MICROBIOLOGY
KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES
Banashankari II Stage,
Bangalore, Karnataka 560070
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
4th BLOCK, JAYANAGAR, BANGALORE, KARNATKA
ANNEXURE-11
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
NAME OF THE
CANDIDATE
DR. ARUN KAUSHIK. R
POST GRADUATE STUDENT,
DEPT OF MICROBIOLOGY,
AND ADDRESS
(IN BLOCK LETTERS)
KEMPEGOWDA INSTITUTE OF MEDICAL
SCIENCES
Banashankari II Stage, Bangalore, Karnataka
2.
NAME OF THE
INSTITUTION
KEMPEGOWDA INSTITUTE OF MEDICAL
SCIENCES
Banashankari II Stage, Bangalore, Karnataka
3.
COURSE OF THE
STUDY AND SUBJECT
4.
DATE OF ADMISSION
M.D MICROBIOLOGY
31st MAY – 2012
TO COURSE
5.
TITLE OF TOPIC:
“SEROLOGICAL CORRELATION OF CLINICALLY DIAGNOSED DENGUE
VIRUS INFECTION BY IMMUNOCHROMATOGRAPHY, ELISA TEST AND
PLATELET COUNT”
6.
BRIEF RESUME OF THE INTENDED STUDY:
6.1) NEED FOR THE STUDY:
•
Dengue infection is a rapidly spreading mosquito-borne viral illness caused by
flavivirus and transmitted by Aedes mosquitoes.1 The disease is endemic in India
with increasing prevalence and has a case fatality rate of 0.65%.
•
Symptomatic patients have febrile illness and symptoms which overlap with other
viral infections like chikungunya, Japanese encephalitis etc. Complications of
dengue fever (DF) like dengue haemorrhagic fever (DHF), dengue shock
syndrome (DSS) which are more common during secondary infection are major
international public health concerns. An early and rapid laboratory confirmation of
infection is important for management.1,2
•
Newer rapid diagnostic techniques like immunochromatography, which do not
require specialized equipment and training, are being developed. Results will be
available from this test within 10 minutes. They can be used for early detection of
NS1 antigen, IgM, IgG antibodies and to differentiate primary and secondary
infection. The results from these newer tests need to be correlated with
conventional tests like ELISA and with platelet counts.1-6
•
This study is undertaken to correlate the sensitivity, specificity and reproducibility
of Immunochromatography test with ELISA test and with platelet counts.
6.2) REVIEW OF LITERATURE:
•
Dengue viral infection is common mosquito borne febrile illness in developing
countries, and is endemic in India. The high prevalence rate and case fatality rate with
complications like dengue haemorrhagic fever (DHF), dengue shock syndrome (DSS)
make early diagnosis in acute phase of disease and management essential.1,2
•
Symptomatic Dengue infection patients have a broad spectrum of symptoms which
overlap with other viral hemorrhagic fever and bacterial infections. Patients with an
acute febrile illness of 2-7 days duration with two or more of the following
manifestations – headache, retro-orbital pain, arthralgia , myalgia, rash, haemorrhagic
manifestations, thrombocytopenia, leucopenia have to be evaluated by laboratory
investigation for confirmation of infection.1,2
•
Accurate diagnosis of infection can be done by isolating viral copies using culture as
definitive diagnostic test in the acute phase of the disease. Detection of Dengue RNA
in the serum using specific oligonucleotide primers by Reverse Transcriptase PCR,
Real time PCR, Nucleic acid sequence based amplification(NASBA) can also be used
for accurate diagnosis.
1,2
These tests can be employed for serotyping of the causative
strain, mainly used in epidemiological analysis of the disease. In the laboratory
diagnosis of the infection is done by detection of NS1 viral antigen and Dengue
specific IgM and IgG antibodies in the acute phase serum.1,2,6
•
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 NS1 viral antigen can be detected. The NS1 gene product
is a glycoprotein produced by the virus essential for its replication and viability. This
protein is secreted by virus infected mammalian cells and can be detected as early as
day 1 and declines by 5-6 days. Studies done for evaluation of the sensitivity of the
early diagnosis of NS1 antigen in the serum showed a high sensitivity compared with
the RT PCR and serves as an early marker for the disease.6 Antibody response seen
with IgM antibodies which are detectable by 3–5 days after the onset of illness, rise
quickly by about two weeks and decline to undetectable levels after 2–3 months. The
IgG antibodies are detectable at low level by the end of the first week, increase
subsequently and remain for many years. Because of the late appearance of IgM
antibody, i.e. after five days of onset of fever, serological tests based on this antibody
done during the first five days of clinical illness are usually negative.1-3 Hence the
diagnosis based on antigen detection helps in early detection of the disease even on
the day1 of the disease.
•
In the secondary dengue infection (when the host has previously been infected by
dengue virus), antibody titres rise rapidly. IgG antibodies are detectable at high levels,
in the initial phase; persist from several months to life long. IgM antibody levels are
significantly lower or can be undetectable. Hence, a ratio of IgM/IgG is commonly
used to differentiate between primary and secondary dengue infections.1-3
•
Recently many commercial rapid serological test-kits for detection of NS1 Antigen
and anti-dengue IgM and IgG antibodies have become available. They are rapid, easy
to perform, less time consuming (results within 10 minutes) and do not require
specialised training. They can be done in peripheral setups where the sample load is
low and there is lack of trained staff.
•
As the accuracy of most of these tests is uncertain they need to be validated against a
standard test like ELISA. Studies done by Vaughn DW et al, Sang CT et al and others
have determined that around 99% sensitivity and a high specificity can be achieved in
the diagnosis of dengue infection using rapid immunochromatographic techniques
when compared with ELISA, EIA and HA.3,5 But rapid tests can yield false positive
results due to cross-reaction with other flaviviruses, malaria parasite, leptospires and
patients of immune disorders. Studies done show that the rapid tests can be used for
identifying the presence of primary and secondary dengue infections.
•
Standard haematological parameters such as platelet count, haematocrit and leucocyte
count are important and are part of the biological diagnosis of dengue infection.
Thrombocytopenia, a drop in platelet count below 100 000 per μl1,2,11 is usually
observed between the third and eighth day of illness often before or simultaneously
with changes in haematocrit and leucocute count, followed by other haematological
changes. Haemoconcentration with an increase in the haematocrit of 20% or more and
leucopenia with reduced neutrophils are usually seen during increased vascular
permeability and plasma leakage.1,2 Their correlation with the acute phase of the
illness needs to be tested.
6.3 ) OBJECTIVES OF THE STUDY
1. Serological confirmation of dengue infection in febrile patients who are clinically
symptomatic by using immunochromatographic test (Rapid Diagnostic Tests) and
ELISA to detect the presence of NS1 Ag, IgM and IgG antibodies in blood
samples.
2. To assess and correlate the sensitivity, specificity and diagnostic capability of the
immunochromatographic test and ELISA in diagnosing dengue infection.
3. To correlate the hematological findings (platelet count and leucocyte count) with
the serological diagnosis of dengue infection.
4.
To determine if the patient is suffering from primary or secondary dengue
infection using ELISA.
7.
MATERIALS AND METHODS
7.1) SOURCE OF THE DATA
•
PERIOD OF STUDY: 1 year 6 months
•
STUDY SAMPLES AND SAMPLE SIZE: Testing blood samples from clinically
symptomatic Dengue infection patients as per WHO criteria in Kempegowda
Institute of Medical Sciences, Bangalore. 60 blood samples from such study
subjects will be tested using immunochromatography and ELISA.
•
CONTROLS: 18 positive controls and 18 negative controls provided by the
manufacturer along with the kits will be tested for the efficiency of the kits.
•
STUDY DESIGN : Comparative study
•
SAMPLING DESIGN : Purposive sampling method
7.2) METHOD OF COLLECTION OF DATA
•
Study will be conducted from routine blood samples collected from patients from
Department of Medicine, Paediatrics who are clinically symptomatic with dengue
infection at the time of first contact with the hospital and stored with suitable
precautions.
•
The blood samples collected will be used for performing immunochromatography
and ELISA tests.
•
The information regarding complete blood count will be obtained and used for
correlation with the diagnosis.
7.3) METHOD OF PERFORMING THE STUDY
•
The blood samples from clinically symptomatic patients, collected by
venipuncture under sterile precautions, is allowed to clot at room temperature (2025oC) and centrifuged according to the CLSI procedures. The serum separated will
be refrigerated (2-8oC).
•
Above serum will be subjected to rapid immunochromatographic test using SD
BIO LINE Dengue Duo kits. 100μl of serum added to sample well and appearance
of coloured bands at ‘T’ and ‘C’ levels are observed. Coloured bands at ‘T’ and
‘C’ indicates positive test for presence of NS1 Ag and coloured bands at ‘C’, ‘M’,
‘G’ indicates IgM and IgG antibodies. The results will be tabulated.
•
Above serum will be subjected to ELISA test using DENGUE EARLY ELISA kit
for NS1 antigen, DENGUE IgM CAPTURE ELISA and DENGUE IgG
CAPTURE ELISA kits from pan bio for dengue specific antibodies. 100μl of
serum sample and the control samples provided, will be introduced into respective
micro wells coated with anti-NS1 antibody. 100μl of HRP conjugated Anti-NS1
Mab and 100μl of TMB chromogen will be added with incubation and washing
between each step. Absorbance read at a wavelength of 450nm using a
spectrophotometer. The index values and pan bio units will be calculated and the
results will be tabulated.
•
Haematological data (Complete blood count) obtained using Analyzer and smear
will be correlated with the laboratory diagnosis of dengue infection.
•
The IgM/IgG ratio will be determined from ELISA values to determine if the
patient is suffering from primary (>1.2) or secondary (<1.2) dengue virus
infection.
7.4) INCLUSION CRITERIA
•
Patients with febrile illness who are clinically symptomatic for dengue infection of
all ages and both sex are included in the study
7.4) EXCLUSION CRITERIA
•
Patients who fail to give consent for the serological diagnosis.
•
Patients with autoimmune diseases
•
Serum obtained from patients which are icteric or exhibiting hemolysis, showing
lipaemia, microbial growth.
7.5) DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR
INTERVENTIONS TO BE CONDUCTED IN PATIENTS OR OTHER
HUMANS OR ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY?
Yes. The study requires use of patients’ blood for obtaining serum for the tests. No
interventions, experiments on patients, other humans, animals are involved in my study.
7.6) HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR
INSTITUTION :
Yes. The certificate obtained from KIMS Institutional Ethics committee, KIMS,
Bangalore is enclosed.
8.
LIST OF REFERENCES
1. World Health Organization. Comprehensive Guidelines for Prevention and Control
of Dengue and Dengue Haemorrhagic Fever, Revised and expanded edition. 2011
World Health Organization, Region of South-East Asia.
2. World Health Organization. Dengue Guidelines for diagnosis, treatment,
prevention and control. 2009 World Health Organization, Geneva, Switzerland
3. Vaughn DW, Nisalak A, Kalayanarooj S, Solomon T, Dung GM, Cuzzubbo A,
et al. Evaluation of a Rapid Immunochromatographic Test for Diagnosis of
Dengue Virus Infection. Journal Of Clinical Microbiology 1998;36(1):234-8
4. Xu H, Di B, Pan Y, Qiu L, Wang Y, Hao W, et al. Serotype 1-Specific
Monoclonal Antibody-Based Antigen Capture Immunoassay for Detection of
Circulating Nonstructural Protein NS1: Implications for Early Diagnosis and
Serotyping of Dengue Virus Infections. Journal Of Clinical Microbiology
2006;44(8):2872-8
5. Sang CT, Hoon LS, Cuzzubbo A and Devine P. Clinical Evaluation of a Rapid
Immunochromatographic Test for the Diagnosis of Dengue Virus Infection.
Clinical and Diagnostic Laboratory Immunology 1998;5(3):407-9
6. Sharmin R, Tabassum S, Jahan M, Nessa A, Mamun KZ. Evaluation of an
immunochromatographic test for early and rapid detection of dengue virus
infection in the context of Bangladesh. Dengue Bulletin. December 2011;35:84-93
7. Shankar SG, Dhananjeyan KJ, Paramasivan R, Thenmozhi V, Tyagi BK, Vennison
SJ. Evaluation and use of NS1 IgM antibody detection for acute dengue virus
diagnosis: report from an outbreak investigation. Clinical Microbiology and
Infection 2012;18(1):8-10
8. Shrivastava A, Dash PK, Tripathi NK, Sahni AK, Gopalan N, Lakshmana Rao PV.
Evaluation of a commercial Dengue NS1 enzyme-linked immunosorbant assay for
early diagnosis of dengue infection. Indian J Med Microbiol 2011;29:51-5
9. Blacksell SD, Bell D, Kelley J, Mammen MP, Gibbons RV, Jarman RG, et al.
Prospective Study To Determine Accuracy of Rapid Serological Assays for
Diagnosis of Acute Dengue Virus Infection in Laos. Clinical And Vaccine
Immunology 2007;14(11):1458-64
10. Prince HE, Yeh C, Nixon ML. Utility of IgM/IgG Ratio and IgG Avidity for
Distinguishing Primary and Secondary Dengue Virus Infections Using Sera
Collected More than 30 Days after Disease Onset. Clinical And Vaccine
Immunology 2011;18(11):1951-6
11. Kulkarni RD, Patil SS, Ajantha GS, Upadhya AK, Kalabhavi AS, Shubhada RM
et al. Association of platelet count and serological markers of dengue infection importance of NS1 antigen. Indian J Med Microbiol 2011; 29(4): 359-62
9.
SIGNATURE OF
CANDIDATE
10.
REMARKS OF THE
GUIDE:
Recommended
11.
NAME AND
DESIGNATION( IN
BLOCK LETTERS) OF
DR. K.L. RAVIKUMAR
11.1 GUIDE
11.2 SIGNATURE
PROFESSOR AND HEAD,
DEPARTMENT OF MICROBIOLOGY,
KEMPEGOWDA INSTITUTE OF MEDICAL
SCIENCES
Banashankari II Stage, Bangalore, Karnataka
11.3 CO-GUIDE ( if any )
DR. RAJEEV. H
ASSOCIATE PROFESSOR
DEPARTMENT OF MEDICINE
KEMPEGOWDA INSTITUTE OF MEDICAL
SCIENCES
Banashankari II Stage, Bangalore, Karnataka
11.4 SIGNATURE
11.5 HEAD OF THE
DEPARTMENT
DR. K.L. RAVIKUMAR
PROFESSOR AND HEAD,
DEPARTMENT OF MICROBIOLOGY,
KEMPEGOWDA INSTITUTE OF MEDICAL
SCIENCES
Banashankari II Stage, Bangalore, Karnataka
11.6 SIGNATURE
12
12.1 REMARKS OF THE
CHAIRMAN &
PRINCIPAL
12.2 PRINCIPAL OF THE
INSTITUTTION
12.3 SIGNATURE
DR. M.K. SUDARSHAN
PRINCIPAL AND DEAN,
KEMPEGOWDA INSTITUTE OF MEDICAL
SCIENCES
Banashankari II Stage, Bangalore, Karnataka