Download rajiv gandhi university of health sciences ,bangalore

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES ,BANGALORE-41,
KARNATAKA
PROFORMA FOR REGISTRATION OF SUBJECT FOR PROJECT
1. NAME OF THE CANDIDATE
ANUJA.M
2. ADDRESS
POST GRADUATE (M.Sc.M.L.T.)
DEPARTMENT OF MICROBIOLOGY,
ST. JOHN’S MEDICAL COLLEGE HOSPITAL,
BANGALORE-34
3. NAME OF INSTITUTION
ST.JOHN’S MEDICAL COLLEGE,BANGALORE
4. COURSE OF STUDY
1ST YEAR M.Sc.M.L.T.
5. SUBJECT
MICROBIOLOGY
6. DATE OF
COURSE
7. TITLE
ADMISSION
TO 5 SEPTEMBER 2011
OCCURENCE OF
HEPATATIS C VIRUS
ANTIBODIES IN HEMODIALYSIS PATIENTS
8.1NEED FOR STUDY
Hepatitis C virus is estimated to infect 170 million people and 3% of the world
population and creates a huge disease burden from chronic progressive liver diseases. It has a
major role in hepatic carcinoma and is the most common indication of liver
transplantation.1Paients with end stage renal disease (ESRD) treated with hemodialysis are at a
higher risk of acquiring HCV infection. A number of risk factors have been identified for HCV
infection among dialysis patients, which include number of blood transfusions, duration of end
stage kidney disease, mode of dialysis, and the concurrent prevalence of HCV infection in the
dialysis unit.2 The frequent sharing of facilities over a prolonged period may result in
accumulated risk.3This study helps to detect the occurrence of HCV antibodies in hemodialysis
patients. This study also helps to segregate positive patients from negative for the use of
hemodialysis equipment.
8.2 REVIEW OF LITERATURE
Hepatitis C virus was discovered in 1989 and was then called as non A non B virus.It can
lead to hepatic carcinoma. The infection has an incubation period of 8 weeks. There is a high rate
of chronicity following acute infection.1
Hepatitis C virus is positive stranded RNA virus coming under the family of Flaviviridae.1
PROPERTIES OF VIRUS
MORHOLOGY
It is a small 40-60 nm virus with a lipid envelope .It is difficult to be visualized by electron
microscopy.
GENOME
Single stranded RNA viral genome comprising approximately 9500 nucleotides. The Nterminal encodes the basic nucleocapsid (c).This is followed by 2 glycoprotein domains, the
envelope E 1 and second envelope Nonstructural -1(NS1/E2).Downstream to this region are the
non structural genes NS2,NS3,NS4,NS5 respectively .The non structural genes encodes for
enzymes like helicase , polymerase etc. which are needed for viral replication.
PHYSICOCHEMICAL PROPERTIES
Density of HCV is difficult to measure accurately because of its binding to various factors
in plasma .Beta lipoprotein decreases the density of HCV.HCV is inactivated by exposure to
chloroform, ether, and other organic solvents and by detergents. It is inactivated by dry heat
treatment at 800c or by wet heat treatment at 600c.
REPLICATION OF HCV GENOME
HCV replicates its RNA genome through the production of a minus strand replication
intermediate. The minus strand copy becomes the template for the generation of positive strand
copies.
HCV TRANSLATION
The synthesis of HCV proteins occurs through translation and cotranslational or
subsequent proteolytic cleavage of large potential polyprotein encoded by open reading frame.
VIRUS ENTRY, UNCOATING, ASSEMBLY AND RELEASE
CELLULAR ENTRY
The identity of the receptor for HCV remains uncertain, although it is believed that
tetraspanin (CD81), scavenger receptor B,and LDL receptor have significant role in viral entry4.
PROTEINS OF HCV
The genome of HCV is thought to encode at least ten proteins of which 3 are
structural (core,E1,E2) and six are non structural. It also has a core protein. Non structural proteins
are encoded by NS2,NS3,NS4A,NS4B,NS5A,NS5B which produces
proteases,
1
helicases,NTPase,RNA- dependent RNA polymerase.
The envelope proteins (E1, E2) are likely to form the principal target of antibody
mediated neutralization of virus infectivity.5
HCV GENETIC VARIABILITY
HCV displays genomic diversity with different genotypes (clades) predominantly in
different parts of the world .The virus undergoes sequence variation during chronic infection. This
complex viral population in a host is referred as ‘quasi species’. This mixed viral population can
modulate the characteristics of the wild type virus and mutation rate, in the genes coding
glycoprotein account for the antigenic drift and the continued selection of neutralizing
antibodies.5
CLINICAL AND PATHOLOGICAL ASPECTS
Hepatitis C virus is usually clinically mild, with only minimal to moderate elevation
of liver enzymes.Hospitalisation is unusual and jaundice occurs in less than 25% of patient’s
.Despite the mild nature of the disease, 70-90% of cases progress to chronic liver disease. Most
patients are asymptomatic, but histologic evaluation often reveals evidence of chronic active
hepatitis. Many patients (20-50%) develop cirrhosis and are at high risk for hepatocellular
carcinoma (5-20%).End stage liver disease associated with HCV is the most frequent indication
for adult liver transplantation.6
EPIDEMOLOGICAL AND CLINICAL FEATURES OF HEPATITIS C VIRUS
Incubation period
15-160 days
Principal age distribution
Adults
Seasonal incidence
Throughout year
Route of infection
Predominantly parenteral
Occurrence of virus in blood
Months to years
Fever
Less common
Duration of ALT elevation
1-6 months
Immunoglobulin
Normal to slightly elevated
Complication
Chronicity in 70-90
Immunity
Low(probably)
6
EPIDEMIOLOGY
Common route of transmission is the parenteral route of transmission which include
injecting Drug Users (IDU’S), transfusion recipients, transplant recipients, hemodialysis patients,
and health care workers. HIV/HCV coinfection may increase the risk for sexual transmission of
HCV and this may be a function of the higher HCV titre which is measured in the blood of the
HIV coinfected patients.Tatooing and acupuncture may also be responsible for percutaneous
exposure1
The prevalence of anti–HCV antibodies among hemodialysis patients is consistently higher
than in general population indicating increased risk for acquiring HCV infection among
hemodialysis patients. Using third generation ELISA ,prevalence of
anti–HCV antibodies
among dialysis patients was found to be 42% in France ,75% in Moldavia and 49% in Syria.There
is a wide variation in the prevalence of HCV infection among dialysis units and countries as
shown by Dialysis Outcomes and Practice Pattern studies.The mean HCV facility prevalence was
13.5%and varied among countries from 2.6%-22.9%2. However, the data on the prevalence of
anti HCV among Indian hemodialysis patients is scanty. Salunkhe et al in 1992 reported 45%,
Chadher et al in 1993 reported 12.1%, Sumathi et al in 1993 reported 37.5%, Agarwal et al in
1999 reported 42%, and Jaiswal et al in a study from 1992- 2000 reported prevalence of 30%.7
Several reports have suggested cross infection of HCV in dialysis patients who shared
hemodialysis machines.2
LAB DIAGNOSIS
It includes serodiagnosis method and molecular diagnostic method.HCV circulates in low titre in
infected serum and is rapidly degraded at room temperature,.There is currently no vaccine
available and superinfection can occur. Further anti HCV test cannot distinguish current and past
infection8.
SERODIAGNOSIS
Antibody reactivity can be detected to both structural and non structural proteins of
HCV.Recombinant proteins or peptides containing these antigenic regions are used for serological
test for antibody to HCV.Different generations of ELISA detecting antibodies against NS4 , core
protein, NS3 and NS5 is available. The detection of antibody is useful to measure present or past
infections irrespective of the actual infectivity of the patient. Patients who have cleared the virus
may gradually loss their antibodies and consequently the antibody screening will not detect all
past infections. 9While ELISA detect antibodies to specific HCV antigens in a standard ELISA
plate, recombinant immunoblot assays (RIBA’S) detect antibodies on a strip that is read visually
.The limited sensitivity of the first and second generation anti HCV test lead to the development
of third generation anti HCV test, which have incorporated multiple recombinant antigens from
different parts of the HCV genome to overcome the limitations of previous test.9
GENERATIONS OF ELISA
FIRST GENERATION ELISA
This ELISA detected antibodies against c100-3 antigen which is a non structural protein encoded
by NS4 region of HCV genome.
SECOND GENERATION ELISA
Multi antigen ELISA included antigen to the core region (c22-3) and one or more further non
structural regions NS3(c33) , NS4(C100-3), or NS5.11
THIRD GENERATION ELISA
This ELISA is more sensitive and detect seroconversion significantly earlier.It omitted the orginal
5 -1 -1 antigens and included the NS5 antigen.
RAPID IMMUNOCHROMATOGRAPHIC METHOD
Adoption of the ELISA technique by embedding it in a nitrocellulose membrane of a test strip
allows rapid detection of antibodies in serum samples. The presence of these specific proteins is
indicated by the development of coloured band on strips .It is simple, cheap, rapid and reliable.1
GOLD STANDARD FOR THE DIAGOSIS OF HCV INFECTION
The gold standard for the diagnosis of HCV infection is the nucleic acid amplification technique
like PCR , and branched chain DNA assay. They can detect the viremia.1PCR can detect HCV
RNA within 1- 3 weeks of exposure and prior to appearance of Anti HCV antibodies or elevation
in ALT level2.
PREVENTION
The centre for disease control and prevention(CDC) recently issued guidelines to control hepatitis
C in hemodialysis centres.They recommended that all patients should be monitored monthly for
aspartate aminotransferase and alanine aminotransferase levels to detect non A non B hepatitis.
They also suggested conducting serological screening for anti HCV among hemodialysis patients
in order to assess prevalence of HCV and management of hepatitis C patients. 10The use of
dedicated machines along with strict enforcement of universal precautions is sufficient to avoid
nosocomial infection.11
8.3OBJECTIVE OF THE STUDY
The objective of this study is to determine the occurrence of antibodies to HCV by ELISA among
hemodialysis patients
9 MATERILAS AND METHODS
9.1 SOURCE OF DATA
A total of 100 serum samples collected from the patients undergoing hemodialysis in St.Johns
Medical College,Hospital(SJMCH) will be studied starting from january 2011.
9.2 MATERIALS
Serum samples from patients undergoing hemodialysis.
9.3 INCLUSION CRITERIA
Serum samples from patients undergoing hemodialysis.
9.4 EXCLUSION CRITERIA
Serum samples of patients who are not undergoing hemodialysis.
9.5 METHODS
100 serum samples collected from the patients undergoing hemodialysis will be analyzed for
antibodies to HCV by ELISA.A proportion (about 25) of serum samples will be tested using rapid
immunochromatographic technique.
ELISA
A third generation using ortho HCV 3.0 ELISA test.
It is a qualitative ELISA for the detection of antibodies to Hepatitis C virus (anti HCV) in human
serum or plasma.
It uses micro wells coated with recombinant Hepatitis C virus encoded antigen as the solid phase.
PRINCIPLE
Antibody or antigen which becomes bound to the solid phase can be detected by complementary
antibody or antigen which is labeled with an enzyme capable of acting on a chromogenic
substrate. The presence of antigen or antibody can be detected by the development of coloured
end product on applying an enzyme substrate. In this test, three recombinant HCV encoded
antigens are used which are c22-3, c200, NS5.
C22-3 is encoded by the putative core region of the HCV genome and it encodes for RNA binding
nucleocapsid protein.In many cases antibodies to C22-3 develop sooner following HCV
infection.HCV recombinant proteins C200 is encoded by the putative NS3 NS4 region of the HCV
genome which codes for nonstructural region of genome.C33-c is encoded by the putative
NS3,which codes for viral helicase,which is an enzyme involved in the unwinding of RNA during
replication of the viral genome by RNA dependent RNA polymerases.This antibody also develop
sooner.
HCV recombinant protein NS5 is encoded by the putative NS5 region of the HCV genome which
codes for viral polymerase, an enzyme involved in replication of HCV .Although antibody
responses to NS5 region encoded antigen, the addition NS5 to C22-3 andC200 recombinant
proteins in ortho HCV 3.0 ELISA test system affords antibody detection to a greater number of
HCV encoded epitopes.
REQUIREMENTS
1.REAGENTS
(a)HCV encoded antigen (Recombinant C 22 -3, c200, and NS5) coated microwell plates (96
wells each).
(b)Conjugate-Antibody to Human IgG (murine monoclonal)-Anti human IgG heavy chains
(murine monoclonal )conjugated to horseradish peroxidase with bovine protein stabilizers.
(c)Specimen diluents-Phosphate buffered saline with bovine protein stabilizer
(d)OPD-Ortho phenylene diammine dihydro chloride tablets.
(e)Substrate buffer- Citrate phosphate buffer with 0.02% hydrogen peroxide.
(f)Positive control: photo chemically treated human serum of plasma containing anti
non-reactive for HBsAg and antibodies to HIV 1and HIV 2.
HCV and
(g)Negative control; - Human serum of plasma nonreactive for HBsAg and antibodies to HIV1,
HIV 2 and anti- HCV.
2. Multichannel micro pipettes-50 µl and 200µl
3. Single channel micropipette -200µl and 300µl.
4. Serological pipettes.
5. Multi channel aspirator-washer device
6.Dual wavelength microwell reader reading at 490 nm with a reference filter of 620
nm.
7. Incubator 37±1 0c
8. Distilled or deionized water
9. 5.25 % sodium hypochrorite
10.4N sulphuric acid.
11. Distilled or deionized water
12. 22 X wash buffer concentrate.
13. Variable speed microwell plate shaker.
PREPARATION OF SUBSTRATE
Add 1 tablet of OPD to 6 ml of the substrate diluent.
TEST PROCEDURE
1. Bring kit components to room temperature prior to beginning of procedure.
2. Determine the total number of wells needed for the assay. In addition to specimens
substrate blank, 3 negative controls, 2 positive controls must be included on each plate or
partial plate.
3. Pepare a record (plate map) identifying the placement of the controls and the specimens
in the microwell.
Arrange the assay control/caliberator wells so that well 1A is the substrate blank. From
well 1A arrange all control or calibrator in a row (horizontal or vertical) as follows.
Well 1A-substrate blank
Negative control
Negative control
Negative control
Positive control
Positive control
(A) Add specimens, calibrators or controls to the micro well as follow
1. Add 200 µl of specimen diluents to all wells except 1A.
2. Add 20 µl of control ,20µl of specimens to the appropriate wells.
3. Incubate at 37 0c.±10c for 60±5 min
4. With an aspirator–washer device,aspirate and wash all wells three to five
buffer.
times with wash
5. Add 100µl of conjugate(ready to use) to all wells.
6.Incubate at 370c for I hour.
7.Aspirate and wash all wells three to five time with wash buffer.
8. Add 100 µl of the substrate solution to all wells.
9. Incubate at room temperature in the dark for 30 min.
10. Add 50µl of 4 N sulphuric acid to all wells including 1A.Gently tap the plate or use a
microwell shaker to mix the contents.
11.Read the microwell strip plate at a wavelength of 490 nm and 620 nm.
Microwell strip plates must be read within 60 min following the addition of 4 N sulphuric acid.
INTERPRETATION OF RESULTS

Cut off value is calculated by adding 0.6 to the mean values of all the negative
controls.

Specimens with absorbance values less than cutoff value should be considered non
reactive and specimens with cutoff value greater than cutoff value will be taken as
positive.
SD BIOLINE HCV 3.0 RAPID TEST PROCEDURE
EXPLANATION OF TEST
HCV genes for expression of recombinant antigens such as core, NS3, NS4, and
NS5regions of the HCV genome are constructed in E.coli.These recombinant materials
were used as capture materials and coated on a membrane of an immunochromatographic
(rapid) test.The use of multiple recombinant proteins avoid non specific cross reactivity
and increases the sensitivity of the HCV antibody test.
The SD BIOLINE HCV test is an immunochromatographic test for the qualitative
detection of antibodies in serum, plasma, and whole blood. The SD BIOLINE HCV test
contains a membrane strip, which is precoated with recombinant HCV capture antigen
(core NS3, NS4 and NS5) on the test band region. The protein A colloid gold conjugate and
serum samples moves along the membrane chromatographically to the test region (T) and
forms a visible line as the antigen-antibody protein A gold particle complex forms with
high sensitivity and specificity. Both the test and and control line in the result window are
not visible before applying any sample. The control line is used for procedural control and
should always appear if the test procedure is performed correctly.
MATERIALS PROVIDED
1. SD BIOLINE HCV test device individually foil pouched with desiccant.
2. Capillary pipette
3. Assay diluents
4. Package insert
SPECIMEN USED
serum
TEST PROCEDURE
1) Remove the test device from the foil pouch, place it on a flat, dry surface.
2) Using capillary pipette add 10µl of serum or plasma or blood specimen upto the
black line into the sample well.
3) Add 4 drops (about 120 µl) of the assay diluents into the sample well.
4) Interpret the results in 5- 20 min.
INTERPRETATION OF RESULTS

A colour band will appear in the left section of the result window .This is the
control line.(C)

Colour band will appear in the right section of the result window is the test
line.
POSITIVE RESULT
The presence of 2 colour bands(T and C bands) in the result window , no matter which
band appears first, indicate positive result.
NEGATIVE RESULT
The presence single colour band in the control region indicates negative result.
INVALID TEST
If the control band C is not visible in the result window after performing the test
Result is considered invalid.
10. Does the study require any investigation or interventions to be conducted
or other human beings or animals if so describe briefly.
No
11. Has ethical clearance been obtained from your institution in case of 10?
No
on patients
REFERENCES
1.
Peter Simmonds and david multimer .Hepatitis C virus .In: Brian
W.J.Mahy and Volker T.M .editors , Topley & Wilson’s Virology,10th
edn.Washington :ASM Press.Vol.2,p.1196
2.
S.Jasuja, A.K.Gupta , R.Chowdary et al .Prevalence and association of
Hepatitis C viremia in hemodialysis patient at a tertiary care hospital ,India
Journalof Nephrology ;2009 april vol 19 issue 2 ,p.62
3.
Peter M.schneerger , Ingrid keur etal .HCV infection in dialysis centres in
Netherlands.J. clin microbiol;june 1998 p1711-1715
4.
Stantly M Lemon ,Cristopher M Walker,Mirian J.Alter and Min Kyung
Yi;Hepatitis C.Virus .Fields virology edi by David M Knipe ,Peter M Howley 5 th
edi Vol 1.p.1263
5.
J Gracia ,Valdecases ,C.Bernal ,F.Garcia ,N Leyva,S.Cerzo
.Epidemiological factors involved in hepatits C virus infection in patients with
renal disease ,Nephrology Dialysis Transplant 1995 supl 6 p.82 vol 10
6.
Geo. F Brooks, Karen C Carroll,Janets s. Butel,Stephen A morsi.Hepatatis
C virus,Jawetz melniks and adelberg medical microbiology 22 edition p 468,
475,481.
7. A.K Reddy, KVD murthy, V.lakshmi.Prevalence of HCV infection in patients in
hemodialysis ;survey by anibody and core antigen detection ;Indian journal of
medical microbiology 2005, 23:106-110
8.Pinto dos Santos ,A.Loureiro ,M.Cendoroglo Neto and B.J.G.Pereira .Impact of
dialysis room and reuse strategies on the incidence of Hepatitis C virus infection in
hemodialysis units.Nephrolog y Dialysis Transplantation 1996 vol 11 :2017
9.Svetlozar N.Natar , M D Johnson , Y.N.Lau et al:Serological and virological
profile of Hepatits C virus in renal transplant candidates.American Journal of
Kidney disease 1998 sup.vol .31 p-921
10.F.Fabrizi ,G.Lunghi ,L.Raffale Nephrology Dialysis Transplant 1997 vol 12 :298303.Serological survey of control of Hepatitis C in hemodialysis patients : third
generation assays and analysis of costs
11.A.Blumberg ,C.Zehnder , J.J Burckardh:Prevention of HCV infection in
hemodialysis units.a prospective study.Nephrology Dialysis Transplant
1995(10)230-233
SIGNATURE OF THE
CANDIDATE
NAME AND DESIGNATION
OF THE GUIDE
Dr SRIKANTH N.S
PROFESSOR
DEPT.OF MICROBIOLOGY
ST.JOHN S MEDICAL
COLLEGE BANGALORE 560034
REMARKS OF THE GUIDE
SIGNATURE OF THE GUIDE
HEAD OF THE
DEPARTMENT
Dr S.MURALIDHARAN
PROFFESOR AND HEAD
DEPT.OF MICROBIOLOGY
ST.JOHNS MEDICAL
COLLEGE , BANGALORE 560034
SIGNATURE OF THE HEAD
OF THE DEPARTMENT
REMARKOF THE DEAN OF
THE INSTITUTION
SIGNATURE OF THE
DEAN