Tap chi GAN MAT V I £ T NAM s^ 28-2014 5
Phat hif n cac dot bien khang thuoc cua virus viem gan B tir cac benh nhan b| viem gan B man if mien B^c Vift Nam
Detection of anti-HBV drug-resistant mutations from patients with chronic hepatitis B in Northern Vietnam
Nguyen Nghiim Luat'. Nguyin Him Quyin', Dang Vdn Tdn', VU Manh Hdng', Nguyen Thi Thu Hucmg', Vd Ngpc Lan' vd Pham Hung Vdn^
TOM TAT
Muc tieu nghiin cuu: muc tieu cua nghien ctiu nay la phat hien cac dot bien khang thudc chong HBV tren gen polymerase (?) cua virus viem gan B (HBV) tu nhirng benh nhan bi nhiem HBV man 6 Mien Bac Viet Nam.
Chdt li0u vd phuang phdp nghien cuu: tong cpng 198 mau huyet thanh tir cac benh nhan viem gan man gom 106 nam va 92 nii, tuoi tir 18 den 64, dupc phan tich bang phuoTig phap PCR va giai trinh tu de xac dinh cac dot bien kh^ng thuoc chong HBV.
Kit qud: ve kieu gen cua cac mau nghien cim, chi CO 2 kieu gen duac phat hien, do la kieu gen B chiam uu thS (145/ 198, 73,23% ) va kilu gen C (53/198, 26,77%). Ty le bpnh nhan nhilm HBV CO cdc dot bien khang thudc la 82/198 (41,41%). Trong so dpt bien khang thuoc cua
HBV duoc phat hien, chung tdi chi thay co 5 loai dot bian la M204I (3/198, 1,52%), AI81T (1/198, 0,5%), S85I/F/A (7/198, 3,5%), L80I (1/198,0,5%) vaV207M/L(62/198, 31,31%) cd kha nang khang vdi cac thudc chdng HBV nhu Lamivudine, Adefovir, Entecavir, Telbivudine va Emtiicitabine. Ngoai cac ddt bien nay, chting tdi cdn phat hien duac mdt sd dot bien la cd kha nang khang thudc voi tan suat thap nhu A222T (4/198, 2,02%), T259S (2/198, 1%), H267Q (2/198, 1%), N124H (1/198, 0,5%), T128A (1/198, 0,5%),...
Kit luan: cac dot bien khang thudc ciia HBV duac phat hien d Mien Bac Viet Nam chiem ty le rat thap vdi hau het la cac dpi bien don, ciing phat hien dupc mpt sd dot bien la; tuy nhien, chua phat hien dupc dot bien nao cd lien quan den khang Tenofovir.
Objectives: the objective of this study was to detect drug-resistant mutations on polymerase gene (?) of the hepatitis B virus (HBV) from pa- tients with chronic hepatits B in Northern Vietnam.
Material and methods: a total of 198 serum samples from patients with chronic hepatitis B including 106 male and 92 female, aged 18 to 64, were analyzed by PCR and sequencing to identify anti- HBV drug resistant mutations.
Results: among the genotypes of the samples, there were only 2 genotypes detected: genotype
B was predominant (145/198, 73.23%), and genotype C (53/198, 26.77%). The proportion of patients with HBV resistance mutations was 82/198 (41.41%). Among of patients with de- tectable HBV mutations, we found only 5 mu- tations which were M204I (3/198, 1.52%), A181T (1/198, 0.5%), S85I/F/A (7/198, 3.5%), L801 (1/198, 0.5%) and V207M/L (62/198, 31.31 %) that may resist to anti-HBV drugs such as Lamivudine, Adefovir, Entecavir, Telbivu- dine, and Emtricitabine. In addition to these mu- tations, we also discovered some novel 'B4nh vi4n Da khoa MEDLATEC. Hd Npi vd 'Cdng ty Nam Khoa, Thanh phd Ho Chi Minh
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Tap chi GAN MAT VIET NAM so 28-2014 mutations that may resist to drugs with low fre- quencies, such as A222T (4/198,2.02%), T259S (2/198, 1%), H267Q (2/198, 1%), N124H (1/198, 0.5%), T128A (1/198, 0.5%). No any Tenofovir resistant mutations were determined in this study.
Conclusion: drug-resistant mutants of HBV in Northern Vietnam was detected with a very low frequency, most of it were single mutations, also found some novel mutations; however, have not detected any mutations related to resistance to Tenofovir.
l.DATVANDE:
Nhilm virus viem gan B (hepatitis B virus:
HBV) la mdt van de sue khde toan cdu anh hudng din han 350 trieu ngudi tren thi gidi.
HBV cd bd gen ban ch4t la DNA nen c6 kha nang tich hap vao bp gen cua tl bao gan ngudi, dan den cac vi xda nhiem sac thi, vl lau dai cd the dan den cac bien chiing nang nhu suy gan, xa gan hoac ung thu bilu md tl bao gan (Hepa- tocellular carcinoma: HCC). Ci Viet Nam, HBV duoc biet den nhu tac nhan benh nguyen chinh gay ra HCC [7]. HBVcd thi lay truyin tiJ me sang con khi sinh, qua dudng tinh dtic, do dimg chung kim tiem hoac sam tren da. Trong nhirng nam gan day chua cd bao cao nao ve cac dot bien khang thudc tren gen polymerase ciia HBV lay nhiem d khu vuc mien Bac Viet Nam dupc cdng bd. Vi vay, muc tieu cua nghien curu nay phan tich cac dpt bien khang thudc tren gen P cua virus viem gan B tir nhiing benh nhan bi nhiem HBV man d Mien Bac Viet Nam.
2. CHAT LIEU VA PHlTOfNG PHAP NGHIEN ClTU
2.1. Benh nhdn. Mau huyet thanh cila 198 benh nhan gdm 106 nam va 92 nii, tuoi tu 16 - 64, duoc xac dinh la viem gan man va khang thudc vdi cac tieu chuan: HBsAg (+) tinh trong han sau thang, HBcAb (+) tinh va HBcAb IgM (-) tinh, tai luong HBV DNA > 10^ copies/mL huylt thanh, tai lupfng HBV DNA tang trong khi vin dang sir dung lien tuc mpt loai thudc khang virus viem gan B. Cac mau huyet thanh nghien cuu dupc thu thap trong thdi gian 4 nam (5/2010-5/2014).
2.2. Tach chiet, tinh sach HBV DNA: HBV DNA duac tach chiet va tinh sach bang he thong may tu dpng ciia Roche.
2.3. Khich dai gen P bdng PCR: gen P duac khuech dai nhd PCR, sau do san pham PCR lai dugc tinh sach.
2.4. Gidi trinh tu-gen P cua HBV DNA:
trinh tu gen P cua HBV cua tung benh nhan duac giai trinh tu tren He thdng giai trinh tu gen ABI 3130 XL Genetic Analyzer vdi Cyle Se- quencing Kits.
2.5. Phan tich dot bien cua gen P cUa HBV:
cac dot bien khang thudc tren gen P cua HBV tur cac benh nhan dugc xac dinh chinh xac bing each gijri phan tich va dich thanh trat ti^ cac acid amin (codon) tai Ngan hang gen thi gidi (Gen- Bank), tuang ung vdi kieu gen B hoac C ciia HBV d tung benh nhan.
3. KET QUA
5.7. VS kieu gen: chi phat hien dupc 2 kilu gen la kieu gen B va kilu gen C, trong dd kilu gen B chilm mi thi (145/198, 73,23%)) vakilu gen C h han nhilu (53/198, 26,77%)).
Bdng 1. Cdc kieu gen ctia HBV duac phdt hien a 198 benh nhdn viem gan B man (n = 198)
S6TT 1 2
Kieu gen B C
So luong 145 53
T y l e % 73,23 26,77 3.2. Ve cac dot biin khang thuoc:
Cac dot bien tren gen polymerase cua HBV da duoc phat hien cr 82 trong s6 198 (41,41%)
Tap chi GAN MAT VIET NAM so 28-2014 mau huySt thanh cua benh nhan nhi&n HBV M204I (3/198, 1,52%), A181T (1/198, 0,5%), man. Trong s6 dot bign kh4ng thu6c ciia HBV, S85I/F/A (7/198, 3,5%), L80I (1/198,0,5%) va chung toi chi phat hien dugc 5 loai dot bian la V207M/L (62/198, 31,31%) (Bang 2).
Bang 2. Cdc dot bien khdng thuoc cua HBVdifocphdt Men a benh nhdn viem gan Brru2n (n= 198)
So TT 1 2 3 4 5
Cac dpt biSn tren gen P
M204I A181T S85I/F/A
L80I V207M/L
T6ng
S6 lupng 3 1 7 1 62 74
Ty le % 1,52
0,5 3,5 0,5 31,31 37,37
Su lien quan vol khang thuoc Khang Lamivudine, Entecavir, Telbivudine va Emtricitabine
Khang Lamivudine va Adefovir Adefovir
Khang Lamivudine, Adefovir, Telbivudine Khang Lamivudine
Cdc dot biln tren cd kha nang khang lai cac thuoc chdng virus Lamivudine, Adefovir, Ente- cavir, Telbivudine va Emtricitabine.
Ngoai cdc dot bien neu tren, chiing tdi cdn Bdng 3. Cdc dpt bien la cda HBV duac phdt
phat hien dugc mdt sd dot bien la tren gen HBV polymerse nhu A222T (4/198, 2,02%), T259S (2/198,1%),H267Q (2/198,1%),N124H (1/198, 0,5%), T128A (1/198, 0,5%),... (Bang 3).
hiin a benh nhdn viem gan B man (n = 198) SoTT
1 2 3 4 5 6 7 8 9
Dot biSn T70P L91I V103I SI 06 A N124H T128A Q130P Y135S N139K Tong
S6 luong
2 1
Ty le % 0,5 0,5 0,5 0,5 0,5 0,5 0,5 1,0 0,5
S6TT 10 11 12 13 14 15 16 17 18
Dot bien XI631 V187I L2201 A222T S223A 1224V T259S H267Q M271L
So lugmg 1 2 1 4 1 I 2 1 1 24
Ty le % 0,5 1,0 0,5 2.0 0,5 0,5 1,5 0,5 0,5 12,12 Cdc dpt biln la nay chi dupc xuat hien vdi
t4n suat thdp va c6 thi c6 kha nang khang thudc d nhirng muc dp nhat dinh.
4. BAN LUAN
4.1. Ve kieu gen: virus HBV gdm 10 kilu gen (dugc xep loai hi A-J ). Sir phd biln cua
8 Tap chi GAN MAT VIET NAM so 28-2014 kieu gen HBV khac nhau tiiy thudc vao su phan bo dia ly. Kieu gen A cd phd bien d Hoa Ky, Bac Au, Trung Phi va My La tinh. Kilu gen B va C chiem uu the trong khu vuc chau A, trong khi kieu gen D pho bien d vung Dia Trung Hai va cac nudc Tay A. Su phan bd cua kieu gen E va F da dugc bao cao d Tay Phi va Hoa Ky, trong khi dd kieu gen G chi dugc phat hien d Phap va Hoa Ky. Su hien dien ciia kieu gen H dugc thay d Tnmg My, kieu gen I va kieu gen J dugc phat hien d Nhat.
Trong nghien cum ctia chiing tdi, chi cd 2 loai kieu gen cua HBV dugc phat hien, trong dd, kilu gen B chilm uu thi (145/198,73,23%) so vdi kilu gen C (53/198,26,77%). Theo H6 Tin Dat va cdng sir, 2007 [2], ty le kilu gen B ctia HBV lay nhiem d Mien Nam la 63% va kieu gen C la 37,7%. Ket qua nghien ciiu cua chiing tdi tuang ddi phti hgp vdi nhieu bao cao dugc cdng bd ve sir phan bd dia ly ciia HBV kieu gen, theo dd ca hai kieu gen B va C la cac kieu gen pho bien nhat d Chau A [7].
4.2. Ve cdc dot Men khdng thudc cua HBV:
cac dot bien d vung gen P ciia HBV cd y nghia quan trpng ve mat lam sang vi chiing cd lien quan den su khang thudc chong virus d benh nhan nhiem HBV. Trong nhiing nam gan day, de dieu tri chdng virus HBV, mpt sd loai thudc tuang tu nucleoside /nucleotide gdm Lamivu- dine (LAM hoac 3TC), Adefovir (ADV), Ente- cavir (ETV), Telbivudine (LdT), Emtricitabine (FTC) va Tenofovir (TDF) da dupc Cue Quan
Bdng 4. So sdnh cdc dot biin khdng thudc cua HB V a binh nhdn viim gan B man a Mien Bac vd Mien Nam Viit Nam
ly Thuc phSm va Dugc phSm Hoa Ky (Food and Drug Administration: FDA) phe duy^t.
Cac dot biln khang Lamivudine chinh gdm mdt dot biln d codon (acidamin) M240V/I trong kilu (motif) YMDD (Tyrosine- Methionie-As- partate-Aspartate) ciia gen polymerase vi +/- L180M [8]. Cac dot biln khang Adefovir chinh d cac vi tri N236T +/- A181V [5]. Cac ddt biln khang Entecavir chinh d cac vi tri L180M +M204V +/- Tl 84G, +/- S202I, +/- M250V [9].
Cac dot bien khang Telbivudine cd lien quan den dot biln M204I [6, 7]. Cac dot biln khang Emtricitabine thudng di kem vai khang lamu- vudine nhu L180M, V173L va M204I [7].
Trong nghien ciiii nay, cac dot bien trSn gen polymerase ciia HBV da dugc phat hien d 41,41%) trong tdng sd mau huyet thanh cua benh nhan nhiem HBV man. Sd dot bien khang thudc cua HBV dugc phat hien gdm 5 loai dpt biln la M204I (1,52%), A181T (0,5%), S85I/F/A (3,5%), L80I (0,5%) va V207M/L (62/198,31,31 %) (Bang 2). Cac dot biln ndy c6 kha nang khang lai cac thudc chdng vims Lamivudine, Adefovir, Entecavir, Telbivudine va Emtricitabine. Ve cac dot biln nay, k6t qua nghien ciiu ciia Hd Tan Dat va cdng su, 2007 [2] cho th4y d Miln Nam Viet Nam, cac da dpt biln cua HBV xuat hien nhilu ban (Bang 4).
Dang Mai Anh Tu4n va cpng sii, 2010 [ 1 ] ciing cho thdy d Miln Nam Viet Nam co nhilu da dot bien han, dieu nay cd nghTa la cac chting HBV cd kha nang khang thudc manh han cac chiing HBV d Miln Bdc Viet Nam.
MEDIC (n=122) MED- LATEC (n=198)
Loai dot bien Soluong
Ti le % So luong
Ti le % M2041/V
17 13,9%
3 1,52%
A181T 0
1 0,5%
S851/F/A 0
7 3,5%
L801 0
1 0,5%
V207M 0
62 37.37%
M2041/V +L180M 58
0 0 0
L180M + M204V
+V173L 1 47,6%
0 0
L180M +M204V
+V2071 1 0.8 0,8
0 0
M2041 +V2071 1 0,8 0 0
Tap chi GAN MAT VIET NAM so 28-2014 9 Cho den nay, chung tdi chua phdt hien dugc
dot bien tiao cd Hen quan den khdng thuoc Teno- fovir d b?nh nhan viem gan B man. Theo Kitri- nos KM va cong su, 2014 [3], nhdm nghiSn cuu ciia ho ciing khdng phdt hien dugc dot bien nao cd lien quan d i n khang thudc Tenofovu d benh nhan viem gan B man.
Ngoai cdc dot b i l n neu tren, chiing tdi cdn phdt hien dugc mpt so dot b i l n la tren gen HBV polymerse vdi tdn sudt thdp nhuA222T (4/198, 2,02%), T259S (2/198, 1%), H267Q (2/198, 1%), N124H (1/198, 0,5%), T128A (1/198, 0,5%), ...(Bdng 3). Cdc dpt bien la nay da dugc mpt sd tai li6u qudc te cdng bd. Tuy nhien, cho den nay, d Viet Nam chua cd tdi lieu ndo cong bd v l nhihig dot bien la nay. Khd nang khang thudc cua cdc dot bien la nay hien cdn dang dugc tiSp tuc nghien cuu.
Phdt hien sdm cdc dot bien khang thudc cua HBV cd t h i giup cac bac sT quyet dinh lua chpn thudc dieu tri khdng virus va quan ly cdc benh nhan viSm gan B man hieu qud ban.
TM lif u t h a m khao
1. Dang Mai Anh TuSn, Vo Diic Xuyen An, Pham Hiing Van. Tim hiiu dpt biln khang Adefovir va Lamivudine tren HBV tach chiet tir huyet thanh benh nhan viem gan B man tinh. Yhpc Thanh phd Hd Chi Minh 2010; 14(1): 287-293.
2. Ho Tdn Dat, Pham Thu Thuy, Nguyin Bao Toan, Nguyen Thi Kilu Oanh, Nguyen Thanh Tong
Xac dinh kieu gen va cac dot bien khang thuoc ciia sieu vi viem gan B bang ky thuat giai trinh tu chuSi. Yhpc ThdnhphdHd ChiMinh 2007; 11(1):
153-158.
3. Kitrinos KM, Corsa A, Liu Y, et al. No detectable resistance to tenofovir disoproxil fumarate after 6 years of therapy in patients with chronic hepatitis B. Hepatology 2014 Feb; 59(2): 434-442.
4. Lai CL, Gane E, Liaw YF, et al. Telbivudine ver- sus lamivudine in patients with chronic hepatitis B. NEnglJMedlOOl; 357(25): 2576-2588.
5. Schildgen O, Sirma H, Funk A, et al. Variant of hepatitis B virus with primary resistance to ade- fovir. 7^ E n g / M e ^ 2006; 354(17): 1807-1812.
6. Strasfeld L, Chou S. Antiviral drug resistance:
mechanisms and clinical implications. Infect Dis Clin North Am 2010; 24(2): 413-437.
7. Suppiah J, Zain RM, and Saat Z. Drug-resistance associated mutations in polymerase (?) gene of hepatitis B virus isolated from Malaysian HBV carriers./fepa/Mo« 2014; 14(1): el3173-13189.
8. Tacke F, Shirvani-Dastgerdi E. Impact of dmg-re- sistance polymerase mutations on the replication of HBeAg-positive and HBeAg-negative hepatitis B virus strains in vitro. Hepat Mon 2012; 12(6):
357-60.
9. Tenney DJ, Rose RE, Baldick CJ, et al. Long-term monitoring shows hepatitis B virus resistance to entecavir in nucleoside-naive patients is rare through 5 years of therapy. Hepatology 2009; 49 (5): 503-1514.
