Download prevalence of hepatitis c virus mutants resistant to protease

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Outpatient Clinic of Infectious Diseases, Warsaw, Poland
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ABSTRACT
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infected with HCV genotype 1.
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1, admitting Outpatient Clinics of Hospital of Infectious Diseases, Warsaw.
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of drug resistant HCV variants is 14.1%. Further and continuous surveillance is necessary to estimate how preexisting and emerging drug resistance mutations influence clinical outcome in triple-therapy experienced patients.
.H\ZRUGV: hepatitis C Virus, Boceprevir, Telaprevir, drug resistant mutants
INTRODUCTION
Chronic hepatitis C (CHC) is one of the major
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3 million persons are newly infected (1). Spontaneous recovery from acute HCV infection is rare, and
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HCV infection may lead to chronic liver disease, cirrhosis or hepatocellular carcinoma, and is a leading
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and early viral response to therapy (4). However, only
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effects, like fever, anemia and depression (5). Clinical
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HIV infection treatment allowed to design new classes
of anti-HCV drugs.
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for therapy of HCV genotype 1-infected (G1) patients,
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VRRQDIWHUWKDWE\(XURSHDQ0HGLFLQHV$JHQF\&OLQLcal trials documented that addition of BOC or TPV to
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HCV revealed that strains containing mutations related
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as a result of spontaneous de novo replication during
treatment (9, 10). High genetic diversity of HCV may
lead towards appearance of primary or emerging drug
resistance mutations. Additionally intense replication
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RNA-dependent RNA polymerase cause an accumuODWLRQ RI GLIIHUHQW VXESRSXODWLRQV LQ LQIHFWHG SDWLHQW
- quasispecies (11).
Several mutations in NS3 and NS4 coding regions,
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result in high resistance of HCV to approved and
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The aim of this study was to evaluate prevalence
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MATERIAL AND METHOD
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hepatitis C patients admitting Outpatient Clinics of
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clinics treats the patients from different regions of
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RealTime HCV test - the limit of detection (LOD) was
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was done using Versant HCV Genotype LiPa Assay
(Siemens, Germany). HCV RNA was isolated from
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Acid Kit, Roche, Switzerland). RT-PCR was performed
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according to following thermal profile: reverse tranVFULSWLRQDWo&IRUPLQGHQDWXUDWLRQVWHSDWoC
IRUPLQWKHQF\FOHVDWoC for 15 sec, 55oC for
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for 5 min. Amplification and sequencing of HCV NS3
region were carried out with oligonucleotide primers
synthesized according to Bartels DJ, et al. (9). Used
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in agarose gel electrophoresis. Sequencing reaction was
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for 10 sec, 50oC for 5 sec and 60oC for 55 sec. The
sequencing was performed using 3100-Avant Genetic
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was used for the interpretation of the results, fold change
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RESULTS
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DISCUSSION
Development of new antiviral drugs directly affecting viral pathogens’ life cycles was a great success of
pharmacological industry. Introduction of new agents
allowed efficient replication control of clinically
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recognised – generation, selection and transmission
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resistance to used drugs. Implementation of protease
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scheme improves SVR ratio. However, there is a risk
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genetic variants of HCV with drug resistance. The aim
of this study was to evaluate prevalence of primary
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No 3
Hepatitis C virus mutants resistant to protease inhibitors
Presented results document naturally occurring
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determined prevalence of drug resistant HCV variDQWVLVLQWHVWHGVDPSOHV7KHQXPEHURIWHVWHG
samples and treatment of the patients from the different
regions of Poland ensure fair representativeness of the
presented data.
Determined prevalence of genetic variants with
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to those presented in Vicenti et al. study (13). However,
there are studies documenting lower percentage of drug
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of drug resistance strains, geographic location.
Existence of fairly high prevalence of HCV strains
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of viral failure during future treatment. Recommended
antiviral schemes of chronic hepatitis C therapy with
BOC and TPV suggest usage of PIs for short time - such
therapy regime prevents mutants’ accumulation.
The necessity of routine HCV drug resistance testing is still discussed. The main reasons for this are conIOLFWLQJGDWDDERXWFOLQLFDOVLJQLILFDQFHRISUHH[LVWLQJ
drug resistance mutations and the fact that HCV genetic
material is not archived in infected cells, like in the case
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of patients with viral failure with the same therapy regiPHQZKHQGUXJUHVLVWDQFHVWUDLQVZLOOEHUHSODFHGE\
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helpful in some groups of patients i.e. non-responders,
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experts (HCV Drug Development Advisory Group)
in guidelines specifying different issues of HCV drug
resistance monitoring (14).
413
REFERENCES
$QRQ *OREDO VXUYHLOODQFH DQG FRQWURO RI KHSDWLWLV &
5HSRUWRID:+2&RQVXOWDWLRQRUJDQL]HGLQFROODERUDtion with the Viral Hepatitis Prevention Board. J Viral
+HSDW
%RZHQ '*:DONHU &0$GDSWLYH LPPXQH UHVSRQVHV
in acute and chronic hepatitis C virus infection. Nature
±
3. Verna EC, Brown RS Jr. Hepatitis C virus and liver
WUDQVSODQWDWLRQ6HPLQ/LYHU'LV
4. Berg T. Tailored treatment for hepatitis C. Clin Liver Dis
±
5. Fried M, Hadziyannis S. Treatment of chronic hepatitis
& LQIHFWLRQ ZLWK SHJLQWHUIHURQV SOXV ULEDYLULQ 6HPLQ
/LYHU'LV6XSSO±
6. Poordad F, McCone J Jr, Bacon BR et al. Boceprevir for
Untreated Chronic HCV Genotype 1 Infection. N Engl J
0HG
-DFREVRQ,00F+XWFKLVRQ-*'XVKHLNR*HWDO7HODprevir for previously untreated chronic hepatitis C virus
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6DUUD]LQ & =HX]HP 6 5HVLVWDQFH WR GLUHFW DQWLYLUDO
agents in patients with hepatitis C virus infection. GasWURHQWHURORJ\±
9. Bartels DJ, Zhou Y, Zhang EZ, Marcial et al. Natural
prevalence of hepatitis C virus variants with decreased
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QDLYHVXEMHFWV-,QIHFW'LV
10. Kuntzen T, Timm J, Berical A et al. Naturally occurring dominant resistance mutations to hepatitis C virus
SURWHDVH DQG SRO\PHUDVH LQKLELWRUV LQ WUHDWPHQWQDLYH
SDWLHQWV+HSDWRORJ\±
11. Le Guillou-Guillemette H, Vallet S, Gaudy-Graffin C
et al. Genetic diversity of the hepatitis C virus: impact
and issues in the antiviral therapy. World J Gastroenterol
+DOIRQ 3 /RFDUQLQL 6 +HSDWLWLV & YLUXV UHVLVWDQFH WR
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13. Vicenti I, Rosi A, Saladini F et al. Naturally occurring
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resistance-related mutations in HCV genotype 1-inSUMMARY AND CONCLUSION
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14. Kwong AD, Najera I, Bechtel J et al. Sequence and Phe1. The results of presented studies document the exnotypic Analysis for Resistance Monitoring in Hepatitis
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C Virus Drug Development: Recommendations From the
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patients. BOC and TPV resistant HCV mutants were
detected in 14% of tested samples.
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further and continuous surveillance is necessary
to estimate how pre-existing and emerging drug
resistance mutations influence clinical outcome in
triple-therapy experienced patients.
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