BANGALORE
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION1 / nAME OF THE CANDIDATE AND ADDRESS (IN BLOCK LETTERS) / DR. ASMABEGAUM BIRADAR.
POST GRADUATE STUDENT,
DEPT OF MICROBIOLOGY,
KIMS, HUBLI –580 022.
2 / nAME OF THE INSTITUTION / KARNATAKA INSTITUTE OF
MEDICAL SCIENCES, HUBLI -580 022.
3 / cOURSE OF STUDY AND SUBJECT / M. D. MICROBIOLOGY.
4 / DATE OF ADMISSION TO COURSE / 25TH MAY 2010.
5 / tITLE OF THE TOPIC / Diagnosis Of Dengue Virus Infection By Immuno-Chromatoghraphic (IC) Card Test For NS1 (Non-Structural Protein 1) Antigen Detection And MAC-ELISA For IgM Antibody.
6 / BRIEF RESUME OF THE INTENDED WORK:
7 / 6.1 NEED FOR THE STUDY
Dengue fever is an acute febrile arbo-viral disease affecting the tropical and subtropical region of the world.
The diagnostic methods currently available are viral culture, viral RNA detection by reverse transcriptase polymerase chain reaction (RT-PCR) and serological tests such as an immunoglobulin M (IgM) capture enzyme- linked immunosorbent assay (MAC-ELISA). Viral isolation by cell culture and subsequent detection by immunofluorescence, though gold standard,2 cannot be used as a routine diagnostic procedure due to the requirement of a highly trained staff, the need of a sophisticated equipment as well as the cost factor. The MAC-ELISA, which is a commonly used assay, has a low sensitivity in the first four days of illness.3
NS1 (non-structural protein 1) antigen is a highly conserved glycoprotein that is essential for the viability of dengue virus and is produced both in membrane-associated and secretory forms by the virus. Enzyme-linked immunosorbent assays (ELISA) directed against NS1 antigen have demonstrated its presence at high concentrations in the sera of dengue infected patients during the early clinical phase of the disease.4 The detection of secretory NS1 antigen represents a new approach to the diagnosis of acute dengue virus infection.
In this study an attempt will be made to evaluate Immuno-chromatoghraphic card test for detection of NS1 antigen for early diagnosis of dengue virus infection in a tertiary care hospital.1
6.2. REVIEW OF LITERATURE :
1. Dussarat P, Labeau G, Louis P, Nunes MR, Rodrigues SG, Storck-Hermann C, Cesaire R, Morvan J, Flamand M, Baril L. reported that:
A diagnostic strategy combining the dengue NS1ag test for acute phase sera and immunoglobulin M capture ELISA for early convalescent phase sera increased
sensitivity from 88.7% to 91.9% . Thus NS1 kit antigen could be used for first
line testing for acute dengue virus infection in clinical diagnostic laboratories.4
2. Shamala devi sekaran, Ew cheng lan, Kantesh Basalingappa Mahesawarappa, Ramapraba Appanna and Geetha subramaniam. summarised their study as follows:
The ELISA is a highly specific and sensitive assay for the detection of dengue NS1antigen. The assay was able to detect NS1 antigen in convalescent sera up until day 10 of infection, whereas viral RNA was undetectable via PCR by day 7. However, as it is most effective during the acute phase of the disease, this kit should be used together with the IgM to increase the sensitivity of detection, especially in areas with higher prevalence of secondary dengue virus infections.5
3. Seok Mui Wang and Shamala Devi Sekaran. evaluated, Commercial SD dengue virus NS1 antigen capture enzyme-linked immunosorbent assay kit for early diagnosis of dengue virus infection and observed that:
The overall sensitivity and specificity of the standard diagnostic dengue NS1ag ELISA in the detection of confirmed dengue virus sera are 76.76% and 98.31% respectively. They have tested a total serum sample of 399 for detection of dengue virus NS1 (non -structural protein 1) antigen and compared the results with real-time reverse transcriptase polymerase chain reaction (RT-PCR), an in house IgM MAC-ELISA and a hemagglutination inhibition (HI) assay.6
4. Kumarswamy V,Wahab AH, Chua SK, Hssan Z, Chem YK, Mohamad m, Chua KB. Reported,
The dengue NS1 antigen-capture ELISA gave an overall sensitivity of 93.4% and a specificity of 100%. The sensitivity was significantly higher in acute primary dengue (97.3%) than in acute secondary dengue (70%). The positive predictive value of the dengue NS1 antigen-capture ELISA was 100% and negative antigen predictive value was 97.3%. These result indicate that the commercial dengue NS1 antigen-capture ELISA may be superior to virus isolation and RT-PCR for the laboratory diagnosis of acute dengue infection based on a single serum sample.7
5. Vianney Tricou , Hang TT Vu , Nhu VN Quynh , Chau VV Nguyen , Hien T Tran, Jeremy Farrar, Bridget Wills, and Cameron P Simmons. Evaluated:
NS1 Ag Strip and Dengue (NS1/IgM/IgG) lateral flow rapid tests in a panel of plasma samples from 245 Vietnamese patients with RT-PCR confirmed dengue and 47 with other febrile illnesses. The NS1 rapid tests had similar diagnostic sensitivities (respectively 61.6% and 62.4%) in confirmed dengue cases but were 100% specific. When IgM / IgG results from the SD Dengue Duo were included in the test interpretation, the sensitivity improved significantly from 62.4% with NS1 alone to 75.5% when NS1 and IgM was positive, 83.7% when NS1 and IgM / IgG was positive. NS1 assays were significantly more sensitive for primary than secondary dengue. NS1 positivity was associated with the underlying viraemia as NS1-positive samples had a significantly higher viraemia than NS1-negative samples.
These data suggest that the NS1 test components of the assays are highly specific. The IgM parameter improved overall test sensitivity without compromising specificity. The Dengue lateral flow rapid test deserves further prospective evaluation in dengue endemic settings.8
6.3 Objectives of Study :
1. To detect NS1 (non-structural protein1) antigen by rapid immuno-chromatographic (IC) card test.
2. To detect dengue specific IgM by immunoglobulin M capture enzyme immunosorbent assay (MAC-ELISA)
3. Comparison of two diagnostic tests for early/rapid diagnosis of dengue infection.
4. To know the sero-prevalence of dengue infection in North Karnataka.
MATERIALS AND METHOD
7.1 Source of Data :
Patients attending Karnataka Institute of Medical Sciences Hospital Hubli, suffering from fever clinically suspicious of Dengue infection. Serum samples collected from these patients are used as study material.
7.2 Methods of collection of data :
(a).Design of study: Cross sectional study.
(b).Size of the study sample: The total serum samples received in department of Microbiology Karnataka Institute Medical Sciences Hubli for investigation of dengue infection are 200-300 in a year. As ours is a time bound study only one hundred patients with acute febrile illness, clinically diagnosed as having dengue fever as per the WHO criteria between December 2010 to November 2011 are included in the study.
(c). Inclusion criteria:
Serum samples of patients with acute onset of fever of 2 to 7 days, clinically suspicious of dengue virus infection
Controls: A total of 40 samples are collected as control.
· Twenty samples are from patients with fever due to a proven aetiology
other than dengue infection.
· Twenty samples from healthy individuals.
(d). Exclusion criteria:
· Patients with history of prolonged fever of more than 10 days.
· Patients with any other proven febrile illnesses like Malaria, Typhoid etc.
are excluded.
(e).Method:
Study group
Sample:
By using standard aseptic precautions 5 to 8 ml of blood is collected from each patient and distributed in two aliquots.
· First portion- 2 to 3ml is collected in a tube containing EDTA and used for
1. Hb%.
2. Total count and differential count.
3. Peripheral smear.
4. Haematocrit.
5. Platelet count.
· Second portion- 3 to 5ml collected in a plain tube. (Without anticoagulant.)
Serum sample is separated by centrifugation, and used for
1. Detection of NS1 (non-structural protein1) antigen by rapid Immuno-chromatographic (IC) card test.
2. Detection of specific IgM antibody by immunoglobulin M (IgM) capture enzyme- linked immunosorbent assay (MAC ELISA).
Control group
2-3ml serum samples obtained from each individual is subjected to both NS1 Ag IC card test and IgM MAC-ELISA to find out any possibility of false positive reaction.
· Comparison of two tests is done by using appropriate statistical methods like Z-test.
7.3 Does the study require any investigation to be conducted on patients (or) animals specify.
Blood samples routinely received for diagnosis of dengue virus infection are used as
Study samples. No additional invasive procedure involved.
7.4 Has ethical clearances been obtained from Ethical Committee of your institution in case of 7.3?
‘Yes’, ethical clearance has been obtained from Ethical Committee of Karnataka Institute of Medical Sciences, HUBLI.
8 / LIST OF REFERENCES :
1. S. Datta, C Wattal. Dengue NS1 antigen detection: A useful tool in early diagnosis of dengue virus infection. Indian J Med Microbiol 2010; 28(2):107-10.
2. Chakravarthi A, Kumaria R, Batra VV, Verma V. Improved detection of dengue virus serotypes from serum samples – Evaluation of single – tube multiplex RT – PCR with cell culture. Dengue Bulletin 2006; 30:133-40.
3. Alcon S, Talarmin A, Debruyene M, Falconar A, Duebel V, Flamand M. Enzyme linked Immunosorbent Assay specific to Dengue Virus Type 1 NS1 (non- structural Protein1) antigen. 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; 40:376-81.
4. Dussart P, Labeau B, Lagathu G, Louis P, Nunes MRT, Rodrigues SG, et al. Evaluation of an enzyme Immunoassay for detection of dengue virus NS1
antigen human serum. Clin Vaccine Immunol 2006; 13:1185-9.
5. Sekaran SD, Lan EC, Mahesawarappa KB, Appanna R, Subramanium G. Evaluation of a dengue NS1 capture Elisa assay for the rapid detection of dengue. J Infect Developing Countires 2007; 1:182-8
6. Seok Mui Wang and Shamala Devi Sekaran. Evaluation of a Commercial SD Dengue Virus NS1 Antigen Capture Enzyme-Linked Immunosorbent Assay Kit for Early Diagnosis of Dengue Virus Infection. J Clin Microbiol 2010;48( 8 ); 2793-2797.
7. Kumarswamy V,Wahab AH, Chua SK, Hssan Z, Chem YK, Mohamad m, Chua KB. Evaluation of a commercial dengue NS1 antigen-capture ELISA for laboratory diagnosis of acute dengue virus infection. J Virol Methods. 2007 mar: 140 (1-2): 75-9.
8. Vianney Tricou , Hang TT Vu , Nhu VN Quynh , Chau VV Nguyen , Hien T Tran , Jeremy Farrar, Bridget Wills, and Cameron P Simmons. Comparision of two dengue NS1 rapid test for sensitivity specificity and relationship to viremia and antibody responses. BMC infect dis 2010;10: 142.
9 / SIGNATURE OF THE CANDIDATE:
10 / REMARKS OF THE GUIDE:
11 /
NAME AND DESIGNATION OF
11.1 GUIDE
/DR.SHOBHA D. NADAGIR MD
PROFESSOR & HEAD, DEPARTMENT OF MICROBIOLOGY KIMS, HUBLI.11.2 SIGNATURE:
11.3 CO-GUIDE: / DR.RAJENDRA V. NAIDU. MD
PROFESSOR DEPARTMENT OF PAEDIATRICS, KIMS, HUBLI.
11.4 SIGNATURE:
11.5 HEAD OF THE DEPARTMENT: /
DR.SHOBHA D. NADAGIR MD
PROFESSOR & HEAD, DEPARTMENT OF MICORBIOLOGY,KIMS, HUBLI
11.6 SIGNATURE:12 / 12.1 REMARKS OF THE CHAIRMAN & PRINCIPAL:
12.2 SIGNATURE:
10