Prevalence of hepatitis C virus mutants resistant to protease inhibitors among Polish HCV genotype 1-infected patients - Epidemiological Review

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ABSTRACT

$,0 7KHDLPRIWKLVVWXG\ZDVWRHYDOXDWHSUHYDOHQFHRIKHSDWLWLV&YLUXV+&9KDUERXULQJPXWDWLRQVDVVRFL-DWHGZLWKGHFUHDVHGVXVFHSWLELOLW\WRSURWHDVHLQKLELWRUV%RFHSUHYLU7HODSUHYLUDPRQJ3ROLVKXQWUHDWHGSDWLHQWV infected with HCV genotype 1.

0$7(5,$/$1'0(7+2'3RSXODWLRQVHTXHQFLQJZDVXVHGVHTXHQFLQJGDWDZHUHLQWHUSUHWHGE\ZHEEDVHG JHQRSKHQRDOJRULWKP$WRWDORIVHUXPVDPSOHVZHUHREWDLQHGIURPSDWLHQWVLQIHFWHGZLWK+&9JHQRW\SH 1, admitting Outpatient Clinics of Hospital of Infectious Diseases, Warsaw.

5(68/766HTXHQFLQJDQDO\VLVRIWKH16SURWHDVHFDWDO\WLFGRPDLQZDVVXFFHVVIXOLQRXWRIVXEMHFWV,Q VHYHQW\WKUHHRXWRIVDPSOHVZLOGW\SH+&9ZDVGHWHFWHGLQVDPSOHVPXWDWLRQVDVVRFL-DWHGZLWKFOLQLFDOO\REVHUYHG%RFHSUHYLU7HODSUHYLUGHFUHDVHGVXVFHSWLELOLW\ZHUHGHWHFWHG

6800$5<$1'&21&/86,2162EWDLQHGUHVXOWVGRFXPHQWWKHSUHVHQFHRI+&9VWUDLQVKDUERXULQJSURWHDVH LQKLELWRUV3,VUHVLVWDQFHDVVRFLDWHGPXWDWLRQVDPRQJ3ROLVKWKHUDS\QDwYHSDWLHQWV7KHGHWHUPLQHGSUHYDOHQFH of drug resistant HCV variants is 14.1%. Further and continuous surveillance is necessary to estimate how pre-existing and emerging drug resistance mutations influence clinical outcome in triple-therapy experienced patients.

.H\ZRUGV: hepatitis C Virus, Boceprevir, Telaprevir, drug resistant mutants

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Chronic hepatitis C (CHC) is one of the major SUREOHPVRISXEOLFKHDOWKVHUYLFHVZRUOGZLGH0RUH WKDQPLOOLRQSHRSOHDUHLQIHFWHGZLWKKHSDWLWLV& YLUXV+&9DFFRUGLQJWR:+2HVWLPDWLRQVHDFK\HDU 3 million persons are newly infected (1). Spontane-ous recovery from acute HCV infection is rare, and GHSHQGVRQKRVWDQGYLUDOIDFWRUVDURXQGRI+&9 LQIHFWHGLQGLYLGXDOVZLOOGHYHORS&+&8QWUHDWHG HCV infection may lead to chronic liver disease, cir-rhosis or hepatocellular carcinoma, and is a leading FDXVHRIOLYHUWUDQVSODQWDWLRQ8QWLOWKH\HDU VWDQGDUGWKHUDS\VFKHPHZDVEDVHGRQFRPELQDWLRQRI SHJ\ODWHG,)1Į3HJ,)1ĮDQGULEDYLULQ5%9 DQG WKH OHQJWK RI WUHDWPHQW EHWZHHQ  ZHHNV GHSHQGLQJRQYLUXVJHQRW\SHEDVHOLQHYLUDHPLDUDSLG and early viral response to therapy (4). However, only

DERXWRISDWLHQWVLQIHFWHGZLWK+&9JHQRW\SH RUDFKLHYHVXVWDLQHGYLUDOUHVSRQVH695RULQ WKHFDVHRIJHQRW\SHDQG$GGLWLRQDOO\WUHDWPHQW ZLWK3HJ,)1ĮDQG5%9FDXVHVQXPHURXVDGYHUVHVLGH effects, like fever, anemia and depression (5). Clinical REVHUYDWLRQVFRPELQHGZLWKH[SHULHQFHREWDLQHGGXULQJ HIV infection treatment allowed to design new classes of anti-HCV drugs.

,QQHZGUXJVRI16$SURWHDVHLQKLELWRUV FODVV3,VERFHSUHYLU%2&DQGWHODSUHYLU739 for therapy of HCV genotype 1-infected (G1) patients, ZHUHDSSURYHGE\)RRGDQG'UXJ$GPLQLVWUDWLRQDQG VRRQDIWHUWKDWE\(XURSHDQ0HGLFLQHV$JHQF\&OLQL-cal trials documented that addition of BOC or TPV to VWDQGDUGWKHUDS\VFKHPHLQFUHDVHG695UDWLRWRDERXW LQSUHYLRXVO\XQWUHDWHG*LQIHFWHGSDWLHQWV &RPELQDWLRQRIGLIIHUHQWDQWLYLUDOGUXJVLVQHFHVVDU\ EHFDXVHRIUDSLGVHOHFWLRQRIGUXJUHVLVWDQFHYDULDQWV

3LRWU=ąEHN-RODQWD2SRND.HJOHUHWDO

 No 3

GXULQJ3,VPRQRWKHUDS\*HQRPLFDQDO\VLVRI51$ HCV revealed that strains containing mutations related WRUHGXFHGVXVFHSWLELOLW\WR3,VDUHSUHH[LVWLQJPLQRULW\ YDULDQWVSUHVHQWEHIRUHLQLWLDWLRQRIWKHUDS\UDWKHUWKDQ 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 UDWHRI+&9DQGODFNRISURRIUHDGLQJDELOLW\RIYLUDO RNA-dependent RNA polymerase cause an accumu-ODWLRQRIGLIIHUHQWVXESRSXODWLRQVLQLQIHFWHGSDWLHQW - quasispecies (11).

Several mutations in NS3 and NS4 coding regions, DIIHFWLQJ+&9VXVFHSWLELOLW\WR739DQGRUWR%2& KDYHEHHQLGHQWLILHGXQWLOQRZ7KHPRVWFOLQLFDOO\LP-portant are V36A, T54A, R155K and A156V/S which result in high resistance of HCV to approved and WHVWHGSURWHDVHLQKLELWRUV7KHSUHVHQFHRIGUXJ UHVLVWDQFHDVVRFLDWHGVXEVWLWXWLRQVFDQOLPLWXVDJHRI ILUVWDSSURYHG+&9SURWHDVHLQKLELWRUV

The aim of this study was to evaluate prevalence RISULPDU\GUXJUHVLVWDQFHWRSURWHDVHLQKLELWRUVDPRQJ QDwYHKHSDWLWLV&*LQIHFWHGSDWLHQWVLQ3RODQG

MATERIAL AND METHOD

$WRWDORIVHUXPVDPSOHVREWDLQHGIURPFKURQLF hepatitis C patients admitting Outpatient Clinics of +RVSLWDO RI ,QIHFWLRXV 'LVHDVHV ZHUH DQDO\]HG WKH clinics treats the patients from different regions of 3RODQG0HDQDJHRISDWLHQWVZDVUDQJLQJIURP WR\HDUVRXWRIWKHPZHUHZRPHQ 3DWLHQWVLQFOXGHGLQWKHVWXG\ZHUH*LQIHFWHG RIWKHPZLWKVXEW\SHE0HDQYLUDOORDGZDVORJ ,8POUDQJLQJIURPWRORJ,8PO1RQHRI WKHSDWLHQWVKDGHYHUEHHQWUHDWHGZLWKVWDQGDUG3HJ ,)15%9 WKHUDS\ DQGRU ZLWK SURWHDVH LQKLELWRUV +&9YLUDOORDGWHVWLQJZDVSHUIRUPHGXVLQJPVS PUW V\VWHP $EERWW 0ROHFXODU ,/ 86$ ZLWK RealTime HCV test - the limit of detection (LOD) was ORJ,8POXSSHUOLPLWRITXDQWLWDWLRQZDVORJ ,8PO$EERWW0ROHFXODU,/86$+&9JHQRW\SLQJ was done using Versant HCV Genotype LiPa Assay (Siemens, Germany). HCV RNA was isolated from VHUXPE\VSLQFROXPQPHWKRG+LJK3XUH9LUDO1XFOHLF Acid Kit, Roche, Switzerland). RT-PCR was performed LQ7KHUPDO&\FOHU/LIH7HFKQRORJLHV1<86$ according to following thermal profile: reverse tran-VFULSWLRQDWo&IRUPLQGHQDWXUDWLRQVWHSDWo_C

IRUPLQWKHQF\FOHVDWo_{C for 15 sec, 55}o_{C for}

VHFDQGo&IRUVHFILQDOHORQJDWLRQVWHSDWo_C

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

SULPHUVHQDEOHGDPSOLILFDWLRQEDVHSDLUVIUDJPHQW RI16$UHJLRQTXDQWLW\RI3&5SURGXFWZDVDQDO\]HG in agarose gel electrophoresis. Sequencing reaction was FDUULHGRXWLQIROORZLQJFRQGLWLRQVF\FOHVDWo_C

for 10 sec, 50o_{C for 5 sec and 60}o_{C for 55 sec. The}

sequencing was performed using 3100-Avant Genetic $QDO\]HU/LIH7HFKQRORJLHV1<86$WKHREWDLQHG VHTXHQFHVZHUHDOLJQHGE\6HT6FDSHYHUVRIWZDUH /LIH7HFKQRORJLHV1<86$)LQDOO\JHQRSKHQR SURJUDPODWHVWYHUVLRQDYDLODEOHDWWKHWLPHRIDQDO\VLV was used for the interpretation of the results, fold change FXWRIIYDOXHZDVVHWWR RESULTS 3RSXODWLRQEDVHGVHTXHQFLQJRIWKH16SURWHDVH FDWDO\WLFGRPDLQZDVVXFFHVVIXOLQRIVXEMHFWV,Q FDVHRIVDPSOHVWKHUHZDVQRDPSOLILFDWLRQRUREWDLQHG VHTXHQFHVZHUHRISRRUTXDOLW\6HYHQW\WKUHH RXWRIVDPSOHVZHUHZLOGW\SH+&9QRGUXJUHVLV-WDQFHPXWDWLRQVZHUHGHWHFWHGDFFRUGLQJWRJHQRSKHQR DOJRULWKP,QVDPSOHVPXWDWLRQVDVVRFLDWHG ZLWKFOLQLFDOO\REVHUYHG3,VGHFUHDVHGVXVFHSWLELOLW\ ZHUHGHWHFWHG)LYHRXWRIWKHVHPXWDQWVKDU-ERXUHGWKH'(VXEVWLWXWLRQVWUDLQVKDG $7PXWDWLRQERWKRIWKHPUHVSRQVLEOHIRUUHVLVWDQFH WR%2&VDPSOHVFRQWDLQHGWKH76PXWD-WLRQUHVXOWLQJLQSRVVLEOHUHVLVWDQFHWR739,QWKHFDVH RIUHPDLQLQJVWUDLQVFDUULHG5+DQG  9$ VXEVWLWXWLRQV HIIHFWLQJ LQ UHGXFHG VXVFHSWLELOLW\WRERWK3,V1RQHRIWKHVHVWUDLQVFDUULHG PRUHWKDQRQHGUXJUHVLVWDQFHDVVRFLDWHGVXEVWLWXWLRQ

DISCUSSION

Development of new antiviral drugs directly affect-ing viral pathogens’ life cycles was a great success of pharmacological industry. Introduction of new agents allowed efficient replication control of clinically LPSRUWDQWEORRGERUQYLUXVHV+,9+%9DQGQRZD-days, HCV. Soon after the introduction of these drugs, VLPLODUO\WREDFWHULDOLQIHFWLRQVDQHZSUREOHPZDV recognised – generation, selection and transmission RIYLUDOJHQHWLFYDULDQWVZLWKORZHUHGVXVFHSWLELOLW\RU resistance to used drugs. Implementation of protease LQKLELWRUV WR FKURQLF KHSDWLWLV & VWDQGDUG WUHDWPHQW scheme improves SVR ratio. However, there is a risk RIWKHUDS\IDLOXUHFDXVHGE\WUDQVPLWWHGRUVHOHFWHG genetic variants of HCV with drug resistance. The aim of this study was to evaluate prevalence of primary GUXJUHVLVWDQFHWRSURWHDVHLQKLELWRUVDPRQJ+HSDWLWLV &JHQRW\SHLQIHFWHGWKHUDS\QDwYHSDWLHQWV

Hepatitis C virus mutants resistant to protease inhibitors ₄₁₃

No 3

Presented results document naturally occurring +&9 VWUDLQV KDUERXULQJ GUXJ UHVLVWDQFHDVVRFLDWHG PXWDWLRQVDPRQJ3ROLVKWKHUDS\QDwYHSDWLHQWV7KH determined prevalence of drug resistant HCV vari-DQWVLVLQWHVWHGVDPSOHV7KHQXPEHURIWHVWHG 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 ORZHUHGVXVFHSWLELOLW\RUUHVLVWDQFHWRWKH3,VLVVLPLODU to those presented in Vicenti et al. study (13). However, there are studies documenting lower percentage of drug UHVLVWDQFH+&9VWUDLQV2EVHUYHGGLVFUHSDQFLHV FRXOGEHDUHVXOWRIGLIIHUHQFHVLQWKHJURXSVRISDWLHQWV WHVWHGWKHLUQXPEHUGXUDWLRQRILQIHFWLRQWUDQVPLVVLRQ of drug resistance strains, geographic location.

Existence of fairly high prevalence of HCV strains QDWXUDOO\UHVLVWDQWWRSURWHDVHLQKLELWRUVLVDULVNIDFWRU 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 test-ing is still discussed. The main reasons for this are con-IOLFWLQJGDWDDERXWFOLQLFDOVLJQLILFDQFHRISUHH[LVWLQJ drug resistance mutations and the fact that HCV genetic material is not archived in infected cells, like in the case RI+%9RU+,97KLVVLWXDWLRQHQDEOHVWKHUHWUHDWPHQW of patients with viral failure with the same therapy regi-PHQZKHQGUXJUHVLVWDQFHVWUDLQVZLOOEHUHSODFHGE\ ZLOGW\SHSRSXODWLRQ+RZHYHUVXFKWHVWLQJFRXOGEH helpful in some groups of patients i.e. non-responders, UHODSVHUVRUSDWLHQWVZLWKXQIDYRXUDEOH,/ESRO\PRU-SKLVPZKLFKZDVWDNHQLQWRDFFRXQWE\+&9'5$* experts (HCV Drug Development Advisory Group) in guidelines specifying different issues of HCV drug resistance monitoring (14).

SUMMARY AND CONCLUSION

1. The results of presented studies document the ex-LVWHQFHRI+&9VWUDLQVKDUERXULQJGUXJUHVLVWDQW DVVRFLDWHGPXWDWLRQVDPRQJ3ROLVKWKHUDS\QDwYH patients. BOC and TPV resistant HCV mutants were detected in 14% of tested samples.

&XUUHQWO\ EHFDXVH RI WKH ODFN RI VXIILFLHQW GDWD 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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