PREVELENCE STUDY OF HEPATITIS C

VIRUS INFECTION (PCR BASED)

AMONG GENERAL PUBLIC

A RESEARCH REPORT IS SUBMITTED AS A PART OF REQUIREMENT

FOR THE DEGREE IN MASTER OF SCIENCE INMEDICAL
LABORATORY TECHNOLOGY

BY

MUHAMMAD JAMAL KHAN GADI

Regd. No.17-AUST-PC17-929

DEPARTMENT OF MEDICAL LABORATORY SCIENCESFRONTIER

INSTITUTE OF MEDICAL SCIENCES ABOTTABAD

ABOTTABAD UNIVERSITY OF SCIENCE &

TECHNOLOGY, ABBOTABAD

i
ii
 ACKNOWLEDGMENT

First of all, it is my humble obligation to express my fervent gratitude for the

clemency of ALMIGHTY ALLAH, the most compassionate, the most merciful, who
bestowed me with the fortitude, health and wisdom to accomplish this task. Numerous
blessing for the prophet Muhammad (peace be upon Him) who is forever torch of
guidance and knowledge for humanity as a whole.

Thanks to my teachers whose sincere efforts helped me to complete project

successfully.

Special thanks to Managing Director FIMS Respected Farmanullah Sb. & Sir
Muhammad Zuhaib sb. for encouraging attitude and subjective guidance, precious
criticism throughout this work and the tips which he provided and enabled me to
complete this project.

Muhammad Jamal Khan Gadi

Reg No.17-aust-pc17-929

iii
 CERTIFICATE

It is certified that the research work described here is the original work of Mr.
Muhammad Jamal Khan Gadi (reg.no.17-aust-pc17-929) and has been carried out
under my direct supervision. It is further certified that the material included in this
research have not been used in part or full in any manuscript already submitted or in
the process of submission from any other institution.

Supervisor

MUHAMMAD ZOHAIB

Department of Medical Lab Technology

Frontier Institute of Medical Sciences/

Abottabad University of Science & Technology

HOD SAIFULLAH KHAN

Department of Medical Lab Technology

Frontier Institute of Medical Sciences/

Abottabad University of Science & Technology

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 DECLARTION

I hereby declare that the work presented in the following research report is my own
effort except, where otherwise acknowledged and that the research report is my own
composition.

The research report is submitted to the department of Medical Laboratory

Technology, Frontier Institute of Medical Sciences Abbottabad by myself to fulfill the
research project course requirement.

I am to verify that all the data collection was collected by me and the data collection
is for only academic purpose.

Muhammad Jamal Khan Gadi

Reg No.17-aust-pc17-929

v
 DEDICATION

This work is dedicated especially

to My Parents,

My Family Members

For their endless support and encouragement, who have been a rock of stability
throughout my life for their struggle and prays for my success.

vi
TABLE OF CONTENTS
1. Abstract 1
2. Introduction 2
3. Materials used . 3
4. Test Principal 4
5. RNA isolation procedure 5
6. Scheme of Super Extract Viral RNA Minikit 9
7. Troubleshooting Viral RNA Isolation 11
8. General Instruction on Handling RNA 12
9. Real time PCR introduction, Advantages 13
10. Preventive Measures to Avoid 14
11. Safety Measures 14
12. PCR Master Mix Preparation 16
13. Flow chart master mixing 17
14. Quantification in PCR 18
15. Interpretation of results 19
16. Performance Characteristics 20
17. Troubleshooting in PCR Results Analysis 23
18. Results 26
19. Conclusion 29
20. Discussion 30
21. References 31
22. Evaluation performa 33

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 LIST OF ABBREVIATIONS
CDNA: Complementary DNA

HCV: Hepatitis C Virus

ICT: Immune-Chromatographic Test

PCR: Polymerase Chain Reaction

ALP: Alkaline Phosphatase

ALT ALANINE TRANSAMINASE

ELISA ENZYME LINKED IMMUNO SORBANT ASSAY

IU/ul INTERNATIONAL UNIT /MICROLITER

UDG URACIL DNA GLYCOSYLASE

QS QUANTITATIVE STANDARD

IC INTERNAL CONTROL

NTC NON TEMPLATE CONTROL

BIOAMP BIOMED AMPLIFICATION KIT

QIA QIAGEN

FDA FOOD & DRUG ADMINISTRATION

CE EUROPION CERTIFICATION

IVD INVITRO DIAGNOSTICS

RT-PCR REAL TIME PCR

LOD LIMIT OF DETECTION

TM TRADE MARK

ROTOR GENE PCR INSTRUMENT (THERMOCYCLER)

SYSTAAQ MANUFACTURER COMPANY PCR KITS

WHO WORLD HEALTH ORGANIZATION

viii
 ABSTRACT

HEPATITIS C INFORMATION INTRODUCTION

Background:

Hepatitis C virus infection is one of the major blood borne infections worldwide.
HCV carriers may develop chronic Hepatitis leading to the liver cirrhosis and
hepatocellular carcinoma (HCC). We have performed this research in order to
determine accurately the prevalence and risk factor of HCV infection among general
population.

The hepatitis C virus (HCV) is a small, enveloped, single-stranded, positive sense

RNA virus in the family Flaviviridae. HCV mainly replicates within hepatocytes in
the liver, although there are clear evidence for replication in lymphocytes or
monocytes. Circulating HCV particles bind to receptors on the surfaces of hepatocytes
and subsequently enter the cells. Two putative HCV receptors are CD81 and human
scavenger receptor class B1 (SR-BI). However, these receptors are found throughout
the body. The identification of hepatocyte-specific cofactors that determine observed
HCV liver tropism are currently under investigation. Once inside the hepatocyte,
HCV utilizes the intracellular machinery necessary to accomplish its own replication.
[1] Specifically, the HCV genome is translated to produce a single protein of around
3011 amino acids. This "polyprotein" is then proteolytically processed by viral and
cellular proteases to produce three structural (virion-associated) and seven
nonstructural (NS) proteins. Alternatively, a frame shift may occur in the Core region
to produce an Alternate Reading Frame Protein (ARFP). HCV encodes two proteases,
the NS2 cysteine aurotease and the NS3-4A serine protease. The NS proteins then
recuit the viral genome into an RNA replication complex, which is associated with
rearranged cylasmic membranes. RNA replication takes places via the viral RNA-
dependent RNA polymerase of NS5B, which produces a negative-strand RNA
intermediate. The negative strand RNA then serves as a template for the production of
new positive-strand viral genomes. Nascent genomes can then be translated, further
replicated, or packaged within new virus particles. New virus particles presumably
bud into the secretory pathway and are released at the cell surface. Based on genetic
differences between HCV isolates, the hepatitis C virus species is classified
into six genotypes with several subtypes within each genotype. Subtypes are further

1
 broken down into quasispecies based on their genetic diversity. The preponderance
and distribution of HCV genotypes varies globally. For example, in North America,
genotype 1a predominates followed by 1b, 2a, 2b, and 3a. In Europe, genotype 1b is
predominant followed by 2a, 2b, 2c and 3a. Genotypes 4 and 5 are found almost
exclusively in Africa. Genotype is clinically important in determining potential
response to interferon-based therapy and the required duration of such therapy.
Genotypes 1 and 4 are less responsive to interferon-based treatment than are the other
genotypes.

Aims and objectives: because of the importance of hepatitis infection and its adverse
effect on the society, we accessed the commonness of HCV contamination among the
patients in NISTHAR Medical Hospital during April 2019 to May 2019 .To estimate
the prevalence and spectrum of Hepatitis C virus (HCV) infection in general
population .

Material and method: A total of 50 blood sample were collected randomly from apparently
healthy people in southern Punjab detailed information for each participant was recoded .All
the samples were tested for anti HCV antibodies and all the sero positive samples were
further tested for HCV RNA by polymerase chain reaction (PCR) . Blood samples were
collected randomly from individuals visiting different clinical laboratories in Punjab. Serum
was separated and processed by nested PCR qualitative assay for the detection of HCV RNA.
The samples were categorized into different age groups on the basis of pre-test questionnaires
in order to record the age-wise differences regarding the prevalence of active HCV. Data
were analyzed statistically using Chi-Square test.

Results: HCV infection was detected in 31/50 cases. overall prevalence of the
infection was 62% among male and female. One HCV infected subject was also
positive for hepatitis B surface antigen (HBsAg). However no cases showed HIV
seropositivity. overall people with the mean age of 42.16 year were included. The
youngest patient was 17 and the oldest was 70.40 % of total patients were male.

Conclusion: the study showed a high seroprevalence of anti HCV antibodies in a

general and apparently healthy population of southern Punjab Pakistan. Drug
injection, blood transfusion, and needle stuck were the factors more strongly
2
associated with HCV infection.

Key words: HCV infection, prevalence, general population, Multan.

Test Principle

Pathogen diagnosis by the polymerase chain reaction (PCR) is based on the

amplification of specific regions of the pathogen genome. In real-time PCR the
amplified product is detected via fluorescent dyes. These are usually linked to
oligonucleotide probes which bind specifically to the amplified product. Monitoring

3
the fluorescence intensities during the PCR run (i.e. in real-time) allows the detection
and quantitation of the accumulating product without having to re-open the reaction
tubes after the PCR run

Material and Methods

Present study was conducted at PCR laboratory, department of pathology, Nisthar

hospital. Total 50 serum sample were collected from patient visiting central
laboratory. Sera screening was done for anti HCV antibodies with the help of
immune-chromatographic tests by using kits. The positive samples were subjected to
further analysis.

All patients were seropositive for anti-HCV by enzyme immunoassay. Of the 50

patient 31 were calculated positive, about 17 patients have high ALT value and 19
patients were non-detected for HCV RNA has Nil ALT value.

HCV RNA was extracted from serum sample by RNA extraction kit. The amplified
cDNA was used for qualitative analysis of HCV the PCR reaction was carried out by
using an outer sense and anti-sense primers. The PCR positive samples were screened
for the nine different strains.in order to amplify a segment of DNA using PCR, the
sample is first heated so the DNA denatures, or separate into two pieces of single
stranded DNA. An enzyme called “taq polymerase” synthesizes-builds-two new
strands of DNA, using the original strand as a template.

4
 Results: to the best of available knowledge this is my first work carried out in MPhil
microbiology. All the patients were assessed using real time PCR. The total number
of patients tested by ELIZA were 70.out of these 50 positive cases were Considered
for PCR. According to the results announced by RT-PCR it was cleared that

62 %( 31) samples were having HCV-RNA positive. The ALT is highly specific for
liver whereas AST and ALP were considered less significant for this analysis, it
means patients had prognosis of liver disease or no cirrhosis. According to SPSS data
editor software results the ALT level of mean is 2.46 this means that genotype most
common in Pakistan is more elevated. Demographic data of the participants is shown
in table given.

Principle and procedure for HCV RNA ISOLATION (EXTRACTION)

The Super Extract Viral RNA mini kit procedure comprises the following steps:

 Lyses of the infection molecule

 Adjustment of the coupling conditions, trailed by the exchange of the example into
the RNA restricting Super Extract Spin Column
 Binding of the viral RNA to the layer of the Super Extract Spin Column
 Washing of the layer and disposal of contaminants and ethanol. Elution of profoundly
unadulterated viral RNA from the film
 Repeated wash steps ensure that defilements and compound inhibitors are
productively expelled and high decontaminated RNA is eluted in Elution Buffer or
RNase free water. This manual contains 6 conventions.
Sampling and Storage of Starting Material:

Best outcomes are gotten utilizing crisply removed examples. For whatever length of
time that the examples are not stun iced with fluid nitrogen or is hatched with RNase
inhibitors or denaturing reagents, the RNA isn't verified. In this way it is fundamental,
that examples are promptly streak solidified resulting to the reaping by utilizing fluid
nitrogen and are put away at - 80°C. RNA contained in such profound solidified
examples is steady for a considerable length of time. RNA refinement ought to be
prepared as quickly as time permits. Tests can likewise be put away in RXV Buffer for
1h at room temperature, medium-term at 4°C, and for long haul stockpiling at – 80°C.
Capacity under profound solidified conditions is suggested.

Serum, plasma, pee, cerebrospinal liquid or other cell free body liquids, just as cell
culture supernatants, swabs, and feces tests can be put away on ice for 1 - 2 hours, for

brief timeframe (up to 24h) examples might be put away at - 20°C. For long haul
stockpiling, we suggest freezing tests at – 80°C. Numerous defrosting and freezing
5
before disengaging the viral RNA ought to be stayed away from.

Serum and Plasma (and other cell free body liquids).

Following centrifugation, plasma or serum from blood treated with anticoagulants like
EDTA or citrate, however not with heparin, can be put away at 2–8°C for as long as 6
hours. For long haul stockpiling, freezing at – 20°C to – 80°C in aliquots is prescribed.
Continued freezing and defrosting cycles must be stayed away from in light of the fact
that denaturation and precipitation of proteins bring about a reduction of the infection
titer and accordingly diminish the yield of the removed viral RNA. Happening
cryoprecipitates can be pelleted by quickly centrifuging (6.800 x g for 3 min). The
cleared supernatant ought to be evacuated, without upsetting the pellet, and handled
right away. This progression won't diminish viral titers.
RNA Isolation Procedure

Lysis
Tests are lysed under denaturing conditions at raised temperatures. Because of the solid
denaturing lyses conditions within the sight of Proteinase K and RXV Buffer cells are
immediately broken and R Nases are inactivated at the same time. The viral RNA is verified.
The expansion of Carrier RNA is vital for the upgrade of viral RNA recuperation so an
exceptionally modest number of viral RNA particles will likewise be cleaned. Bearer RNA
likewise settles nucleic acids in tests with exceptionally little nucleic corrosive fixations.

Binding Viral RNA

After adding Binding Solution to optimize the binding of viral RNA to the Super
Extract Spin Column membrane, the lysate will be applied onto the Super Extract
Spin Column and the viral RNA is bound to the surface of the Super Extract spin
column membrane as the lysate is drawn through by centrifugation.

6
Removing Residual Contaminants
Contaminants are proficiently washed away utilizing WS Buffer R1 and R2, while the viral
RNA stays bound to the film of the Super Extract Spin Column.

Elution

High caliber viral RNA is eluted from the film utilizing Elution Buffer (or R Nase Free
Water). The eluted RNA is prepared to use in various ensuing applications.

Important Indications Preparing RNA

While getting ready viral RNA, work rapidly during the manual strides of the technique.
The RXV Buffer of the Super Extract Viral RNA smaller than expected pack
disentangles viral RNA confinement by consolidating proficient lyses of the beginning
material and the inactivation of exogenous and endogenous R Nases. Outrageous
consideration ought to be taken to maintain a strategic distance from defilements with R
Nases when taking care of Elution Buffer.

Storing Samples

Solidified Serum or plasma tests must not be defrosted more than ones. Rehashed freeze
defrosting prompts denaturation and precipitation of proteins, bringing about decreased
viral titers and in this manner diminished yields of viral nucleic acids Furthermore
cryoprecipitate shaped during freeze defrosting will stop up the Super Extract Spin Column
layer.

Adding Carrier RNA

Transporter RNA fills two needs. Right off the bat, it upgrades the authoritative of viral acids
to the Super Extract turn section layer, particularly if there are not very many objective atoms
in the example. Also, the expansion of a lot of Carrier RNA decreases the opportunity of viral
RNA corruption in the uncommon occasion that R Nase particles are not denaturated by the
chaotropic salt and cleansers in the RXV Buffer. On the off chance that Carrier RNA isn't
added to the RXV Buffer, this may prompt decreased viral RNA recuperation. The utilization
of an inward control is prescribed when utilizing the Super Extract Viral RNA little pack in
blend with symptomatic intensification frameworks. Inner Control RNA and reconstituted
Carrier RNA ought to be added to the RXV Buffer and blended completely by rearranging
the cylinder multiple times. To abstain from frothing, don't vortex.

7
Eluting Viral RNA

For downstream applications, that require little gazing volumes, utilizing viral RNA
eluted in 40 µl Elution Buffer may expand measure affectability. The volume of eluate
recuperated might be up to 5 µl not exactly the volume of elution cushion applied to the
Super Extract Spin Column. The volume of eluate recouped relies upon the idea of the
example.

Extraction Control

Internal Controls (IC) from the PCR assay provider can be used as extraction controls
if the fragments are longer than 100 bp. In this case they have to be added after
finalization of the lysis step. Alternatively it can be mixed with the Carrier RNA.

Attention: don´t add directly these Internal Controls to the sample!

Important points before starting a protocol.

 Always change pipet tips between liquid transfer. To avoid cross-

contaminations, we recommend the use of aerosol-barrier pipet tips.
 All centrifugation steps are carried out at room temperature.
 When working with chemicals, always wear a suitable lab coat, disposable
gloves, and protective goggles.
 Discard gloves if they become contaminated.
 Do not combine components of different kits unless the lot numbers are
identical.
 Avoid microbial contamination of the kit reagents.
 To minimize the risk of infections from potentially infectious material, we
recommend working under laminar air-flow until the samples are lysed.
 This kit should only be used by trained personal.
 Microcentrifuge ( 11.000 rpm)
 Thermomixer (65°C-80°C)
 ddH2O
 Ethanol (96-100%)

8
The following notes are valid for all protocols:

The RNA can also be eluted with a lower (but not lower than 40 µl) or a
Note: higher volume of Elution Buffer (depends on the expected yield or needed
concentration of RNA).

After extraction place the Elution Tube on ice. For a long time
Important
storage place the nucleic acids at –20°C or –80°C.

Troubleshooting in Viral RNA Isolation Process

Problem/ probable cause Comments and suggestions

Clogged Super Extract
Spin Column
After lysis spin lysate to pellet debris and continue
Insufficient disruption or
with the protocol using the supernatant. Increase g
homogenization of the
force and/ or centrifugation time. Reduce the amount
starting
of starting material. All centrifugation steps should
material.
be conducted at room temperature.
Little or no viral RNA
eluted
Incomplete removal of cell Make sure that the cell culture medium is complete
culture medium. removed after the cell harvest.
Insufficient mixing of the Mix sample sufficient by pipetting up and down with
sample with Binding Solution prior to transfer the sample onto the
Binding Solution Super Extract Spin Column.
Prolong the incubation time with preheated Elution
Incomplete elution.
Buffer to 5-10 min or repeat elution step once again.
RNA degraded
The RNA purification protocol should be performed
quickly (see also “General notes on handling RNA”,
Inappropriate handling of page 22).
the starting
material. Cell pellets stored at - 80°C for later processing
should be immediately frozen after cell harvest by
liquid nitrogen treatment.
Viral RNA does not
perform well in
downstream-applications

9
(e.g. RT
PCR)

Ethanol carryover during Increase g-force or centrifugation time when drying

elution. the Super Extract Spin Column.
Ensure that WS Buffer R1 and R2 are at room
temperature.
Salt carryover during
Checkup WS Buffer R1 and R2 for salt precipitates.
elution.
If there are any precipitates solve these precipitates
by careful warming.
Appendix
Instructions on handling RNA

RNA is far less stable than DNA. It is very sensitive to degradation by endogenous
RNases in the biological material and exogenous RNases which are permanently
present everywhere in the lab. To achieve satisfactory qualitative and quantitative
results in RNA preparations, contaminations with exogenous R Nases has to be
reduced as much as possible. Avoid handling bacterial cultures, cell cultures or other
biological sources of R Nases in the same lab where the RNA purification is to be
carried out. All glassware should be treated before use to ensure that it is R Nase-free.
Glassware should be cleaned with detergent, thoroughly rinsed and oven baked at
240C for four or more hours before use. Autoclaving alone will not completely
inactivate many R Nases. Oven baking will both inactivate R Nases and ensure that
no other nucleic acids (such as Plasmid DNA) are present on the surface of the
glassware. You can also clean glassware with 0.1% DEPC (diethyl pyrocarbonate).
The glassware have to stand 12 hours at 37C and then autoclave or heat to 100C for
15 min to remove residual DEPC.

Storage of RNA

Purified RNA can be stored -80°C and is stable for months and years.

10
PCR, RT PCR, Real Time PCR Molecular Diagnosis of HEPATITIS
C Virus Outline

 Introduction to PCR
 Reverse Transcriptase PCR
 Real Time PCR
 Implementation in HEPATITIS C virus

Diagnosis History of PCR

 Kary Mullis, 1983, thinking about a simple method of determining a specific

nucleotide from along a stretch of DNA developed in conjunction with Cetus.
 He realized you could amplify a target, by annealing primers specific to the
sequence and using DNA polymerase to replicate that sequence. Importantly
he realized this could be done again and again, cycle after cycle, causing the
target to be amplified exponentially – a polymerase chain reaction.
 Method was published in Science 1985, with more detail published in 1987.
 What is PCR? PCR (polymerase chain reaction) is a process used to make
many copies of selectregions of DNA. What is PCR PROCESS?
 Basic Ingredients Target DNA, 2 Primers, Taq Polymerase PCR Buffer.
 Steps Heat to denature (94C) – Cool to anneal primers (54C) Heat to extend
using TAQ (72C)
 Cycle steps for multiple copies & exponential increase in copy number. The
exponential amplification of the gene in PCR INTRODUCTION TO PCR
Reverse Transcriptase PCR Reverse Transcriptase PCR (RT PCR) Viewing
the results of PCR
 Before the PCR product is used in further applications, it has to be checked if :
1. There is a product formed.
2. The product is of the right size
3. Only one band is formed. Limitations of conventional PCR Technique
 Time consuming, non-automated
 Poor precision, size-based discrimination only
 Low resolution – about 10 fold. (Real-Time PCR can detect as little as a two-
fold change!)

11
  Results are not expressed as numbers Ethidium bromide for staining is not
very quantitative
 Low sensitivity, not necessarily related to amount of input DNA
 Short dynamic range < 2 logs
 Post PCR processing
REAL TIME PCR
Assay that monitors the accumulation of a DNA product from a PCR reaction in
real time
1. Uses fluorescence-based chemistries
2. Data collection during the early exponential phase of PCR WHY REAL
 TIME PCR? Benefits over traditional PCR assays Sensitivity (as little a single
copy) Enhanced dynamic range of detection (7-8 magnitudes)
 Increased reproducibility
 Permits rapid target as low as 15- 30 min
 Cost efficiency Can be performed outside the lab Molecular Beacon Assays
Each method has advantages and disadvantages One method may be more
appropriate for an application over another ƒReal-Time Instrumentation is
equipped to handleall chemistries

Preventive Measures to avoid PCR Contamination

The BioAmp RT Mix contains a heat-labile form of Uracil DNA Glycosylase (UDG).
UDG and dUTP in the BioAmp RT Mix prevents the reamplification of carryover
PCR products between reactions. dUTP ensures that the amplified DNA will contain
uracil, while UDG removes uracil residues from single- or double-stranded DNA.
UDG destroys previously formed dU-containing HCV PCR product at 37°C for 15
mins before the reverse transcription of HCV RNA. The UDG is then inactivated at
50°C, thereby allowing cDNA sysnthesis of HCV RNA.

Safety and Environment

 Please refer to the Material Safety Data Sheet (MSDS) and product labeling
for information on potentially hazardous components.
 Specimen should always be handled as potentially hazardous.
 Use of protective equipment is necessary: gloves and safety spectacles when
manipulating dangerous or infectious agents.

12
  Waste should be handled according to the institution’s waste disposal
guidelines.

Also observe all federal, state, and local environment regulations.

Precautions

 Use sterile pipette tips with filters.

 In order to avoid DNA/RNA contamination, a maximum physical separation
between pre- and post-amplification steps is recommended: separate rooms,
separate pipettes and other lab material, separate lab coats and gloves (and
their stock) are minimum precautions for good laboratory practice. The
reagents should be isolated from any source of contaminating DNA/RNA,
especially
amplified DNA products. Also avoid microbial contamination of reagents.
 Avoid any return from the post-amplification room to the pre-amplification
room.
 Store and extract positive material (specimens, controls and amplicons)
separately
from all other reagents and add it to the reaction mix in a separated facility.
 Thaw all components thoroughly at room temperature before starting an assay.
 When thawed, mix the components and centrifuge briefly.
 Work quickly on ice or in the Cooling Block (72/96 well loading block).

13
PCR Mix Preparation

Each component must be added, and supplied in the right amount. Too much or too
little sample or reagents might result in a specific amplification or even in no
amplification at all.

BioAmp RT Mix 10 µl 120 µl

BioAmp RT Enzyme 2 µl 24 µl

BioAmp Enhancer 1 µl 12 µl

Total Mastermix Volume = 13 µl 156 µl

Purified Sample
Containing Internal 12 µl Each
Control =

14
FLOW CHART MASTER MIX PREPARATION

First of all make calculations according to your samples, positive and negative
controls. Plan your plate setup according to parameters.

Thaw PCR Master Mix (Buffer) and water prior to master mix preparation.

Add PCR Water

Vortex RCR master
Mix before adding

Add PCR Master

Mix (Buffer)

Vortex Primer and probe

Add Primer (F), Primer (R) and Probe

After adding PCR master Mix (Buffer), water, primer and probes vortex eppendorf tube for one
second and then centrifuge for five seconds.

Centrifuge enzyme mix

(Do not vortex enzymes)

After adding enzyme centrifuge

Add Enzymeeppendorf
Mix tube for five seconds.

Cut strip tubes according to parameters

Then add 5 ul PCR water in NTC tubes row wise and cap the tubes in mater mix room.
Dispense 20ul of master mix
in each PCR stripIntubes
RNA extraction room Vortex
column wise Samples and positive control before addition
ncubate for 3 min. and centrifuge at 5.900 x g
(8.000 rpm) for 1 min.

Dispense 5ul of samples

and positive controls in
each PCR strip tubes row
wise and cap respective

15
Quantification

The Quantitation Standards HCV QS 1 – 4 are pre-extracted standards and do

not require any extraction. They can be use either with internal control or
without internal control. To generate a standard curve on the Rotor-Gene TM, all
four Quantitation standards should be used and defined in the menu window
Edit Samples as standards with the specified concentrations in IU/ μl.
The Quantitation Standards are defined as IU/μl. The following equation has to
be applied to convert the values determined using the standard curve into IU/ml
of sample material: Result (IU/ml) = Result (IU/μl) x Elution Volume (μl)
Sample Volume (ml)

Programming of Thermocycler

For the detection of HCV RNA, create a temperature profile on your Rotor-Gene™
according to the following six steps.

A. Setting of General Assay Parameters

B. Reverse Transcription of the RNA

C. Initial Activation of the Hot Start Enzyme

D. Amplification of the cDNA

E. Adjustment of the Fluorescence Channel Sensitivity

F. Starting of the Rotor-Gene™ Run

All specifications refer to the Rotor-Gene latest software version. Please find further
information on programming the Rotor-Gene™ in the Rotor-Gene™ Manual, latest
Version.

16
First, enter the PCR reaction volume in the menu window 25 µl. Temperature profile
will be as below.

37 15 min -

50 30 min -

95 3 min -

95 15 sec 50

60 60 sec*

* Acquiring fluorescence at 60 degree on cycling A on FAM channel and cycling B

on JOE channel. The detection range of the fluorescence channels has to be
determined according to the fluorescence intensities in the PCR tubes. This
adjustment is done in the menu window Auto Gain Calibration Setup (activation in
menu window New Experiment Wizard under Calibrate). Please set the calibration
temperature to the annealing temperature of the amplification programmed.

The gain values determined the channel calibration are saved automatically and are
listed in the menu window of the programming procedure.

Result Interpretation

Data analysis is performed with the Rotor Gene TM software according to the
manufacturer’s instructions (Rotor-GeneTM Manual, latest version). The following
results are possible:

1. A signal is detected in fluorescence channel Cycling A.FAM / Green and in

channel Cycling A.JOE/Yellow. The result of the analysis is positive: The
sample contains HCV RNA.
2. 2. In fluorescence channel Cycling A.FAM / Green no exponential phase
signal is detected whereas a signal is detected in fluorescence channel Cycling
A.JOE/Yellow. In the sample no HCV RNA is detected. It can be considered
negative.

17
Analytical Specificity

Cross-Contamination Frequency

The potential cross-contamination frequency was determined by testing replicates of a

high titer HCV genotype 1 positive specimen and replicates of an HCV negative
specimen. HCV positive samples (1 x 106 copies/ml) were alternated with HCV
negative samples using a “checkerboard” pattern. Forty-five (45) replicates each of
HCV negative and HCV positive samples were tested in each of five runs, for a total
of 225 replicates of the HCV positive samples and 225 replicates of the HCV negative
samples. No false results were obtained; two negative samples were invalid.
Combined results across all runs yielded a cross contamination frequency of 0%
(0/223). interference were observed in the SYSTAAQ HCV Real Time RT-PCR
Assay.
Potentially Interfering Substance - Endogenous Substances

Potentially interfering endogenous substances were tested by adding these substances

to HCV negative specimens and specimens spiked with HCV genotype 1 at 57.7
IU/ml (300 copies/ml). The concentrations of potentially interfering endogenous
substances were tested according to CLSI/NCCLS Document EP7-P (20). The
following endogenous substances were tested: 500 mg/dL hemoglobin, 60 mg/dL
bilirubin (conjugated), 60 mg/dL bilirubin (unconjugated), 3,000 mg/dL triglycerides,
and 8 g/dL protein. None of the endogenous substances tested interfered with the
sensitivity and specificity of the SYSTAAQ HCV Real Time RT-PCR Assay. -

HCV Real Time RT-PCR Assay. - Other Potentially Interfering Substances

The effect of other potentially interfering substances was determined by testing HCV
negative specimens and specimens spiked with HCV genotype 1 at 57.7 IU/ml (300
copies/ml). The disease categories tested were: myeloma IgG (n=12) positive
specimens, anti-nuclear antibody positive specimens (n=10), anti-double-stranded
DNA positive specimens (n=6), rheumatoid factor positive specimens (n=19), and
specimens from subjects with systemic lupus erythematous (n=10). With the
exception of a subset of the myeloma specimens, none of the tested samples from
subjects with HCV-like disease states interfered with the performance of the HCV
Real Time RT-PCR Assay.

18
Nonclinical Specimen Studies- Anticoagulant Studies

HCV negative specimens and specimens spiked with HCV genotype 1 at 57.7 IU/ml
(300 copies/ml) were collected in serum separator tubes (SST PLUS, plastic), K2
EDTA (PLUS, plastic), K2 EDTA (PPT), sodium citrate (glass, 4%), ACD-solution A
(glass) and sodium heparin (PLUS, plastic 60 USP units) tubes. None of the
anticoagulants tested affected the sensitivity and specificity of the SYSTAAQ HCV
Real Time RT-PCR Assay. - Specimen Storage Studies Samples from the
anticoagulant study (see above) were used to evaluate the effect of storing whole
blood samples for up to 24 hours at room temperature and the subsequently
processed specimens (serum and plasma) for up to 48 hours at 2°C to 8°C. No adverse
effects on sensitivity or specificity of the SYSTAAQ HCV Real Time RT-PCR Assay
were observed for the whole blood or processed specimens under the storage
conditions tested. Whole blood collected in the tested tube types can be stored at room
temperature for up to 24 hours and subsequently processed serum and plasma
specimens can be stored at 2°C to 8°C for up to 48 hours prior to testing in the
SYSTAAQ HCV Real Time RT-PCR Assay. - Multiple Freeze-Thaw Cycles
Studies The effects of one, two, and three freeze-thaw cycles on processed specimens
were tested in HCV negative specimens and specimens spiked with HCV genotype 1
at 57.7 IU/ml (300 copies/ml). Up to three freeze-thaw cycles of HCV negative and
HCV positive processed specimens had no effect on the performance of the
SYSTAAQ HCV Real Time RT-PCR Assay.

Reproducibility

Reproducibility testing was performed at three laboratories (A, B, C) to obtain

measures of repeatability and reproducibility during the clinical trial; two of the sites
were outside laboratories and one was in-house. Testing was also conducted in-house
during the preclinical phase (D). In the clinical testing, the three sites were provided
with six identical panels of eight samples containing 0 to 9,615 IU/ml (0 to 50,000
copies/ml) genotype 1 or 0 to 577 IU/ml (0 to 3,000 copies/ml) genotype 2b in serum
or plasma. In the pre-clinical phase testing, six member serum or plasma panels for
genotype 1 at 0 to 57.7 IU/ml (0 to 300 copies/ml) and genotype 2b at 0 to 69.2 IU/ml
(0 to 360 copies/ml) were tested. At sites A, B, and C, each of two operators
performed two days of testing with each of three kit lots for a total of six days of
testing. At Site D, three operators tested the genotype 1 panel with each of three kit
19
lots on six separate days. Similarly, Site D tested the genotype 2b panel
with each of three kit lots on each of five separate days. Reproducibility testing at or
near.
Linear Range

The linear range (analytical measurement) of SYSTAAQ HCV Real Time RT-PCR
wasdetermined by analyzing a dilution series of quantitation standards ranging from
1.0×109 to 1.0×10-2 IU/ml. This dilution series was calibrated against WHO
international standards. Each dilution was tested in replicates on consecutive days and
thus determined linear range up to 1.0×109 IU/ml.

Troubleshooting

 No signal with positive controls / HCV QS (1-4) in fluorescence channel

Cycling A.FAM /Green:
 The selected fluorescence channel for PCR data analysis does not comply with
the protocol.
 For data analysis select the fluorescence channel A.FAM / Green for the
analytical SYSTAAQ HCV Real Time RT-PCR.
 Incorrect programming of the temperature profile of the Rotor-Gene™.
 Compare the temperature profile with the protocol.
 Incorrect configuration of the PCR reaction.
 Check your work steps by means of the pipetting scheme and repeat the PCR,
if necessary.
 The storage conditions for one or more kit components did not comply with
the instructions given in 2. Storage or the Reagents had expired.
 Please check the storage conditions and the expiration date of the reagents and
use a new kit, if necessary. Weak or no signal in channel Cycling A.FAM /
Green:
 The PCR was inhibited.
 Make sure that you use a recommended isolation method (See 8.1 RNA
Isolation) and stick closely to the manufacturer's Instructions.
 Make sure that during the RNA isolation the recommended additional
Centrifugation step has been carried out before the elution in order to remove
any residual ethanol (see 8.1 RNA Isolation).

20
 The storage conditions for one or more kit components did not comply with
the instructions given in 2. Storage or the Reagents had expired.
 Please check the storage conditions and the expiration date (see the kit label)
of the reagents and use a new kit, if necessary. Signals with the negative
controls in fluorescence channel Cycling A.FAM / Green of the analytical RT-
PCR.
 A contamination occurred during preparation of the PCR.
 Repeat the PCR with new reagents in replicates.
 If possible, close the PCR tubes directly after addition of the sample to be
tested.
 Strictly pipette the positive controls at last.
 Make sure that work space and instruments are decontaminated at regular
intervals.
 A contamination occurred during extraction.
 Repeat the extraction and PCR of the sample to be tested using new reagents.
 Make sure that work space and instruments are decontaminated at regular
intervals. If you have any further questions or if you encounter problems,
Please contact our Technical Service.

21
Product Use Limitations

 All reagents may exclusively be used in in-vitro diagnostics.

 The product is to be used by personnel specially instructed and trained in the
in-vitro diagnostics procedures (EN375) only.
 Strict compliance with the user manual is required for optimal PCR results.
 Attention should be paid to expiration dates printed on the box and labels of
all components. Do not use expired components.

22
 Total ALT Calculated

20
18
16
14
12
10

Total ALT Calculated

8
6
4
2
0

High Medium Low Nill

Statistics

ALT value PCR results

Valid 50 50
N
Missing 0 0

Mean 2.46 1.38

Std. Deviation 1.297 .490

Variance 1.682 .240

Minimum 1 1

Maximum 4 2

Sum 123 69

23
 35

30

25

20
total
detected
15

10

0
male female

Search No Gender Total No of Virus Detected Percentage

cases

1 Male 20 14 70 %

2 female 30 17 56.66 %

24
25
 CONCLUSION:

Real time PCR is powerful latest technique that gives the quantitative answers
difficult to obtain with end point PCR. Each step in real time PCR based assay is
controlled for proper analysis of the samples.

The prevalence of anti-hepatitis C virus is low in females as compared to males

during my period of study. The main factors which are associated with transmission
of infection were blood transfusion in association with surgical procedures also.

The HCV prevalence in our population is higher as compare to HBV infection in the
region. Blood safety measures are the essential step in limiting the transfer of HCV
infection.

The prevalence of HCV recorded in different age and gender groups show that its
frequency has increased with the increase in age. Children whether male or females
were the least infected on the other hand its prevalence is highest in the age group of
27-47.

Furthermore, the rate of infection in female population was almost 56.66% as

compare to males. Least incidence in females is because of low exposure to HCV risk
factors due to the male dominating society of area and also the estrogen hormone in
females in considered to play a role in the spontaneous clearance of HCV infection.

26
 DISCUSSION

Hepatitis C is rapidly emerging as a major health problem in developing countries

including Pakistan. The world health organization estimates that approximately 3% of
the world populations have been infected with HCV thus far; there are about 170
million patients with HCV in the world and 3 to 4 million are diagnose as new cases
every year. Prevalence of HCV may be different in different regions and the various
groups of the same community.

For the investigation of active HCV infection we relied on the highly sensitive
method of detection that is polymerase chain reaction. We collected 50 samples and
subjected them to the ICT and PCR. Almost all the individuals were actively infected
with HCV.

The age wise study revealed that young people of southern Punjab have less active
HCV infection as compared to elders because of their least exposure to some of the
high risk factor causing HCV such as barbers. The highest active HCV infection was
12% observed in the age group 37-47. These people had variable history of exposure
to HCV risk factor.

Our studies partially in agreement with one study which were conducted on the
prevalence of HCV in relation to the age group and the gender. The prevalence of
HCV in different age groups of both sexes was studied and it was found that
prevalence of HCV was maximum in males as compared to female our studies in
agreement with the fact that male population is more affected by HCV as compared to
female overall our study reveals that 62% of total patient under study are actively
infected with HCV.

27
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Engle J Med 1992, 327: 1899-1905.

Ohno T, Mizokami M, Wu RR, Saleh MG, Ohba KI, Orito E, Mukaide M, Williams
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Syed T, Jamal MM: Epidemiology of hepatitis C virus (HCV) infection. Int J Med
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Mackay IM. Real-Time PCR in the microbiology laboratory. Clin. Microbiol Infect.
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McHutchison JG, Gordon SC, Schiff ER, et al. Interferon alfa-2b alone or in
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Davis GL, Esteban-Mur R, Rustgi V, et al. Interferon alfa-2b alone or in combination

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Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin
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29
 EVALUATION PERFORMA
Prevalence study of Hepatitis C Virus Infection (PCR based) Among General
Public IN PAKISTAN.

(APRAIL 2019-MAY 2019)

LAB Reg No: Date:

Patient Name: Gender: Age:

Patient Clinical Findings:

1. Anti HCV antibody test  Yes  No

2. Raised ALT/inflamed liver  40 IU/m  > 40 IU/m
3. HCV RNA by PCR  Detected  Not Detected
4. Presence of significance coinfection.  Yes  No
5. Quantitative HCV RNA viral load.  High  Medium  Low

Evaluator Signature:

30