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Abstract
Hepatitis C is a major global public health problem and is
one of the main causes of cirrhosis and Hepatocellular
carcinoma. About 200 million people; 3% of the world’s
population are infected with hepatitis C virus and 3 to 4 million
persons are newly infected each year with a global 170 million
chronic carriers at risk of developing liver cirrhosis and/or liver
cancer. At least 85% of infected persons become chronically
infected. Blood bank and community-based surveys conducted
in Egypt have reported sero-prevalence rates of HCV to be as
high as 40% in some parts of the country. Blood transfusion is
thought to be the main route of transmission.
While hepatocytes are the major site of viral replication,
the virus can also replicate at the extrahepatic sites including
PBMC. This is reflected by a higher frequency of extrahepatic
complications and diseases associated with chronic HCV
infection, including mixed cryoglobulinaemia, cutaneous
vasculitis, glomerulo-nephritis, neuropathy and lymphoproliferafive
disorders in HCV-infected persons. There is strong
support from recent studies that hepatocytes are the major site
of viral replication, the virus can also replicate at the
extrahepatic sites including PBMCs.
Patients with detectable negative-strand HCV RNA in
PBMCs had lower IFN sustained response rates compared to
those without detectable negative-strand HCV RNA in PBMCs.
Low-level replication of HCV in PBMCs may lead to
Summary
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reactivation of HCV after termination of therapy.
Because of the frequency and the severity of the disease,
many tests have been developed for diagnosis of hepatitis C
virus. They include non specific tests e.g. liver transaminases
and auto antibodies detection; specific tests which include the
antibody detection (serological) methods as ELISA and RIBA
tests, detection of the HCV RNA itself through the use of NATs
and finally, genotype testing. While traditional HCV ”viral
load” assays quantify HCV RNA in the serum, they are unable
to distinguish positive- from negative-strand HCV RNA and
thus have limited utility in assessing extrahepatic replication.
The use of ”strand-specific” RT-PCR to detect the
replicative intermediate negative-strand HCV RNA is often
cited as evidence for viral replication and the use of nested RTPCR
(in which the products of a first round of PCR were
reamplified using a second, nested set of primers) gave
extreme- sensitivity and increased specificity.
This study was done on 30 HCV infected patients who had
sero-positive HCV results (using 3rd generation ELISA technique)
including 21 patients positive for HCV RNA in the serum and 9
patients negative for HCV RNA in the serum by Real time
PCR. Blood samples were collected from the Outpatient of Internal
Medicine Hospital of Ain Shams University. All cases were
subjected to full history taking and complete general and local
examination.Twenty healthy controls matched age and sex also
included in this study.
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All cases were also subjected to HCV positive- and
negative-stranded RNA detection in PBMCs samples by using
thermal cycler (Gene Amp PCR System 9700) for conventional
RT-PCR qualitative assay, also nested PCR was performed.
A statistical study was done among the results and this study
revealed that: the HCV-RNA positive-strand was detected in
PBMCs from 20 out of 30 sero-positive patients (66.7%) and also
from 20 out of 21 serum HCV-RNA positive patients (95.2%).
There is highly statistically significant association (p<0.01)
between HCV-Ab positive cases and the presence of HCV-RNA
positive-strand and also between serum HCV-RNA positive cases
and the presence of HCV-RNA positive strand in PBMCs. The
HCV-RNA negative-strand was detected in PBMCs from 7 out of
30 sero-positive patients (23.3%) and this association was
statistically significant (p<0.05). Also HCV-RNA negative-strand
was detected in PBMCs from 7 out of 21 serum HCV-RNA
positive patients (33.3%), so there is highly statistically significant
association (p<0.01) between HCV-RNA positive serum and
HCV-RNA negative-strand in PBMCs. There was a high
statistically significant association between positive- and negativestrand
HCV-RNA (p<0.01). No positive results for both positiveand
negative-strands HCV- RNA were detected in PBMCs of the 9
patients with negative serum HCV-RNA nor in the controls.
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from this study and other studies we concluded that PBMCs
act as possible site for extrahepatic replication and may represent
a reservoir for HCV which cause reinfection of hepatocytes