DAC DIEM PHAN Tl> CUA CAC CHUNG VI KHUAN LAO KHANG RIFAMPICIN PHAN LAP TAI VIET NAM NAM 2008-2009
Vu Thi Kim Lien, Tran Thi Hai Au, Nguyen Thj Thu Thai*, Nguyen Thai Son**.
Vien Ve sinh Dich te Trung utrng, Ha Npi
* Dai hoc V khoa Thai Nguyen
** Hpc vien Qucin Y, Hd Ndi TOM TAT
Trong ting s6 126 chung vi khuan lao phan lap lit mien Bic, Trung, Nam cua Viet Nam nam 2008-2009 da diroc si> dung de kiem tra dot bien trong vung 81 bp hotspot cua gen rpoB lien quan den khang rifampicin Ket qua cho thay khong co dot bien diroc tim thay tren 21 chiing nhay. Trong si 58/59 chung khang rifampicin dirge tim thay dot bien tren gen rpoB, voi vi tri dot bien thirong gap la codon 531 (52,54%), 526 (27,12%) va 516 (11,86%). Co 16 kieu dot bien lien quan khang thudc tai viing 81 bp, trong do kieu Ser531 Leu phi bien nhat. Giam sat cac chiing lao khang rifampicin la van de quan trong nhat trong chien liroc dieu trj ddi VON benh nhan lao a Viet Nam.
Tir khoa: Mycobacterium tuberculosis, khang rifampicin; vi tri dot bien, giai trinh tir ADN [. DAT VAN DE
Viet Nam dung thu 12 trong so 22 quoc gia co benh nhan nhiem lao cao tren the gidi.
Mac du, chuong trinh chdng lao quoc gia dat duoc nhung thanh tuu nhat djnh, nhung cac benh nhan lao khang thudc va khang da thudc van tiep tuc tang. Trong cac loai khang thudc ciia vi khuan lao. khang rifampicin (RTF) duoc quan tam nhat vi la dau hieu ciia khang da thudc d vi khuan lao. Theo dinh nghTa cua WHO [1], vi khuan lao khang da thudc (MDR- TB) la nhung chiing vi khuan khang ddng thdi hai thudc chii luc dieu tri lao la isoniazid va ri- fampicin, gan day xuat hien nhung cluing sieu khang (XDR-TB) la nhung cluing vi khuan khang da thudc ddng mot kem khang it nhlt 3 thudc chdng lao ddng hai. Theo Chuong trinh Chong lao Quoc gia, mdi ngay cd gan 400 ngudi mac benh lao, trong do hon 75%
thudc lira tudi lao ddng, ly le tir vong do lao la 26/1 00.000 dan. 1 lien nay. nguy co nhiem lao hang nam d nude ta udc tinh la 1,5% (d cac tinh phia Nam la 2%, d cac tinh phia Bac la
1%). Tinh hinh khang thudc ciia vi khuim lao tai Viet Nam dang la mot van de dang lo ngai, ty le khang thudc tien phat la 30,7% vao loai cao tren the gidi, ty le khang da thudc chung la 4%, trong dd ty le da khang thudc thir phat la 19.3%, ty le da khang thudc tien phat la 2,7%. U'dc tinh den 2015. sd ca tir vong do lao khoang 14.000 don vi. Phat hien nhanh nhung cluing vi khuan lao khang thudc la vo ciing quan trong nham kiem soat benh cd hieu qua va nham ngan chan sir lay truyen ciia cac chiing lao khang thudc. Qua trinh xet nghiem ciia cac phong thi nghiem thdng thudng gom nhieu budc nhir phan lap chiing. nudi cay va thu khang sinh dd, qua trinh nay cd the ddi hdi thdi gian tir 2-3 thang. Cac ky thuat sinh Tac gia: Vu Thj Kim Lien
Dia chi: Vien Ve sinh djch tl Trung uong. Sd 1 Ycrsin.
Dien thoai: 0904184282 Email: [email protected]
16 Tap chi Y hoc dir phong, Tap XX, so 6 (1 14)
hoc phan tu thuc hien tren cac mau benh pham ho hap co thd cho ket qua nhanh hon. Khang RIF chii vcu xay ra tai viing 81 bp hot-spot tren gen rpoB. Kieu va tan xuat ciia nhung dot bien nay thay doi theo cac vi tri dja ly cua tirng quoc gia | 1, 2, 3]. Phuong phap sinh hoc phan tu thudng duoc sir dung de phat hien nhanh vi khuan lao khang RIF va INH nhu: PCR- RFI.P. PCR-SSCP. ADN microarrays. 1NNO- I.IPA... tuy nhien. phuong phap giai trinh tir ADN co the xiic dinh chinh xac duoc cac dot
bien cu va dot bien moi xuat hien.
Nham gop phan phat hien nhanh cac chiing vi khuan lao khang thuoc, nhdm nghien ciru dii tien hanh phan tich, xac dinh cac vj tri dot bien lien quan den khang rifampicin ciia vi khuan lao. Cac cluing nghien ciru duoc giai trinh tu doan 528 bp chira viing 8 1 bp tren gen rpoB. chi ra tan suat dot bien hay xay ra nhat tai vi tri codon nao tren gen rpoB.
II. Pill ONG PHAP NGHIEN CI V 2.1. Doi tuong ngliicTi ciru
I 26 cluing \ i khuan lao nhay. khang don
\a khang da thuoc dupe thu thap nam 2008- 2009 tir cac loai benh pham cua benh nhan lap nlur: derm, djch mang phoi, djch rira phe quan, dich mang bung, dich mang phoi tren ca 3 mien Bac. Trung. Nam cua Viet Nam (gom benh vien 103. 108. Vien Lao Trung uong.
Benh \ ien Pham Ngoc Thach - TP. HCM.
Benh vien Trung uong Hue). Trong do gom 56 chung midn Nam, 30 chung mien 'Trung. 40 chung mi6n Bac. Ngoai ra. trong nghien ciru co su dung chung chuan quoc te H37Kv lam doi cluing.
2.2. Phuong phap nghien ciru:
2.2.1. I'll irong plnip tiich chih ADS ciia vi kliiidn lao
Lam chet te bao vi khuan lao o 80"C 30 phut, cho 20j.il ly so/y m ii qua dem 37"C. Sau do sir dung bp sinh pham QIAamp DNA cua
hang QIAGEN, cac budc tien hanh tach chict ADN theo hudng dan cua nha san xiuit.
2.2.2. Phuong plidp PCR:
Khuech dai ADN tren gen rpoB: San pham PCR cd chieu dai 528 bp, chua viing 81 bp khang rifampicin ciia gen rpoB vdi cap ADN mdi dac hieu: VSF1: 5'-ACCGACGA- CATCGACCACTT-3"; VSR.1: 5 - GGC GGT CAG GTA CAC GAT CT-3\
Hon hop mix cho phan ling PCR gom:
20()uM ciia mdi loai dNTP; 5 mM MgCI2:
0.2pmol ciia moi loai ADN mdi; 1U Taq ADN polymerase (Invitrogen); 1.x dem PCR (20m\l Tris-HCl. pll 8.4. 50mM KCI ); 50ng -I50ng ADN khudn. Dieu kien phan irng PCR: 5 phut/ 95"C. 35 chu ky (95"C/ 30giay:
64"C 45 giay: 72"C/45 giay), 7 phut o 72"C.
2.2.3. Plurtfng phap tach ddng gen:
Hinh 1. Te bao kha bien mang plasmid chira ADN la tao khuan lac trang San pham PCR lien quan khang rifam- picin cua cac cluing khang da, sicu khang sau khi tinh sach dupe dua vao plasmid pGl \1*-'.
(kit A 1360- Promega). Tiep tuc dupe bien nap vao te bao kha bien DH 5a. phan lap tren mdi trudng LB Agar cd bd sung X-gal. IPTG. am- picilin. Nudi khuan lac trang qua dem trong mdi trudng LB long cd ampicilin, tach chiet bang kit Plasmid purification (Bionecr). Su thanh cdng ciia plasmid tai td hop dupe kiem tra bang phuong phap PCR va phuong phap thuy phan sir dung enzym gidi han EcoRI.
2.2.4. Phuong phap gidi trinh tir ADS:
Chay phan ling PCR gan BigDyc: ADN 'ap chi V hoc dir phong. Tap XX. so 6 (l 14) 17
khudn la san phim PCR d6i voi nhung chiing mamid va dua vao may giai trinh ty tu dong nhay va chiing khang don, plasmid doi voi ABI avant 3100. Xac djnh vi tri dot bien ciia chung sieu khang va da khang. Sau do tinh cac chiing nhay. khang don, khang da va sieu sach san phim PCR bang kit DyeF.x 2.0 Spin khang bang cac chuong trinh Cl.USTA!. X, (QTAGI N). bidn tinh ADN trong HiDi 1 or- Bioedit. DN A Star
110 120 130 140 150 160 170 180 190
OCA .,CCTTG.-T».-C,-TCCGffiCG^ -T 'CAT GG ACCAOAACAACCCOC
Hinh 2: Kit qua giai trinh ADN chung khang da mien Trung TB05-259 tren gen rpoB III. KKT QUA VA BAN LUAN don, 45 chiing khang da, 15 cluing sieu
khang. Trong so 106 cluing khang thuoc co 59 Dac tinh khang thuoc ciia 127 cluing
... . ,„ , . , . . . i i chiing (55.66%) khang rifampicin, 47 chiing
nghien cu'u: 20 chung nhay khang sinh; 1 " o r a
chung chuan qu6c te H37Rv; con lai 106 (44,34%) khang voi cac loai thudc khac nhu chiing khang thuoc gom: 46 chiing khang streptomycin, isoniazid (Bang 1).
Bang 1. Dac tinh khang thuoc cua chung vi khuan lao Chung vi khuan lao
Tong so Loai khang thudc Mien Bac Mien Nam Mien Trung Tong so
Khong khang (nhay) 10 10 20
H 3 4 5 12
S 12 9 10 31
H S 4 4
R 3 3
HSR/ HSRE 15 15 11 41
HSRE Ofx Th Eto Cs Km PAS 15 15
Tdng so 40 56 30 126
A Streptomycin. H: Isoniazid. E: Ethambutol, R: Rifampicin. Ofx : Ofloxacin , Th : Thioaceta- /.one. Eto:Ethionamide. Cs:Cyc loserine. Km:Kanamycin. PAS:P-aminosalicylic acid.
IS Tap chi Y hoc du phong, Tap XX, so 6(114)
Ket qua giai trinh tir doan 528 bp tren gen rpoB cua 1 27 chung: 20 chung nhay va I chiing chuan H37Rv khong xuat hien dot bien:
46 chung khang don co 20 chiing (45.65%) xuat hien dot bien: 45 chiing khang da co 42 chung (93.3%) phat hien dot bien; 15 chiing sieu khang mien Nam trong do co 14 (93,3%) xuat hien dot bien va 1 chiing sieu khang (11306-2 1 7) (6,7%) khong thay dot bien.
Khi phan tich ket qua giai trinh tu viing 81 bp tren gen rpoB theo ba mien Bac Trung Nam ciia 106 chung khang thuoc cho thay: 46 chung khang don co 3 chung khang don vdi rifampicin co dot bien tai codon 526 va 531:
43 cluing khang voi isoniazid hoac streptomy- cin khong thay dot bien tai codon 5 1 6, 526.
53 I. Trong tong so 59 cluing khang rifampicin phat hien dot bien a 58 cluing chiem 98.3%.
l y le dot bien trong vung "Hot spot mutation"
ciia 58 cluing khang rifampicin thuong gap nhat tai vi tri codon 53 I co cac kieu dot bien sau: S5311.; S53II-; S531Q: S531W chiem 3 1 59 (52.54%), sau do lan krol la codon 526 115261.; H526R; 11526 1: H526Y: H526S 1I526D chiem 16/59 (27.12 %); codon 516 D516V chiem 7/59 (11,86%) va mot s6 v tri codon khac nhu L530M; S522I.: L533P L530Stop: N519Stop chiem 1/59 (1.69%).
Tren viing 81 bp xuat hien 7 \ i tri dot bien.
kieu dot bien thuong gap nhat la Ser 53 1 Leu (S53 1 L). Ty le va so luong dot bien cac chiing khang thuoc theo ba mien the hien o bang 2.
Dot bien nhieu codon dong thoi (tir 2 codon tro len). hau het chi thay o cac chiing khang da thuoc.
Bang 2. So lirong va kieu dot bien trong viing "Hot spot mutation"
Codon 516 Codon 526 Codon 531 Codon khac
Sieu khang (n= 15) 3 D516V
(20%) 3 (H526D; H526R;
H526T) (20%) 8 (S531L; S531F;
S531Q) (53,33%) L530M (6,67%) Mien
Nam Ba khang (n = 15) 0 3 (H526Y; H526R;
H526S) (20%) 9 (S531L; S531W) (60%) S522L
(6,67%) Don khang (n= 16) 0 H526L (6,25%) 2 (S531L) (12,5%)
Mien Da khang (n = 15) 4 (D516V)
(26,67%) H526R (6,67%) 6 (S531L) (40%) L533P (6,67%) Trung
Don khang(n = 15) 0 0 0 L530Stop
(6,67%)
X8 (H526L; H526Y;
Da khang ( n = 15) 0 H526D; H526R) 6 (S531L) (40%)
Mien (53,33%)
Bic
Don khang (n = =15) 0 0 0 N519Stop
(6,67%)
Tap chi Y hoc dir phong. Tap XX, so 6 (114) 19
Trong 59 chung khang thudc rifampicin co 45 chung (76.27%) vi khuan lao mang mot codon dot bien la dot bien sai nghia; 3 chiing mang mot dot bien mat nghTa hoac dong nghia; 8 chung (13,56%) mang hai codon dot bidn. chii ygu la dot bien sai nghia va 3 chiing (5.08%) mang ba codon dot bien chii yen tao dot bien sai nghTa.
Ngoai ra, khi th6ng ke kit qua nghien ciiu ngoai viing "Hot spot mutation", tren doan 528 bp tren gen rpoB ciia 106 cluing khang thu6c cho thay t\ le dot bien cao nhat tai vj tri codon 490 tai ba mien Bac Trung Nam lii (Q490H; Q490R: Q490Stop) chiem 13/106 (12.26%) va xuat hien dot bien tai cac vi tri codon tiich biet khac v di ty le thap:
* Mien Nam: Sieu khang: M479I;
1-505 V: H588K. Da khang: Q490R; l572F.Don khang: 3 (Q490H); T491Stop; 2(l-5()5Stop);
F506T.
* Mien Trung: Da khang: I460V;
N462S; F504K; 2(R542C); G570S; K588G.
Don khang: Q482Stop: 3(O490H): 2 (T491S- top)
* Mien Bac: Da khang: V456A; T480A;
T'5631. Don khang: R476L: 6(Q490H; Q490S- top): 2 (T49IR); I 5()5Stop.
Trong 46 chiing khang don cd 3 chiing khang don vdi riTampicin va 43 chiing khang don vdi streptomycin hoac isoniazid. Ket qua giai trinh tu 3 chiing khang don vdi rifampicin cho th;i\ ch ung 11306-128 co dot bien tai vi tri codon 526. 2 chiing TB06-I27, TB06-208 ciing xuat hien dot bien tai codon 531. Con lai 43 cluing khang don vdi cac thudc khac cd 17 cluing xuat hien dot bien. cac dot bien nav thudng xuat hien ngoai viing 81 bp, hoan toan khdng xay ra dot bien tai vi tri codon 531.526.
516 nhu cac cluing khang da hay sieu khang thudc. Cite dot bien ciia cac chiing khang don vdi khang sinh khac thudng xuat hien dot bien ngoai viing "Hot spot mutation" 81 bp gap d cac vi tri 482: 490; 491; 505; 506; 476.
Dac biet dot bien tai vi tri codon 490 (Q490H/R/Stop) xuit hien 13 cluing trong tdng sd 106 cluing gdm cac cluing khang don.
khang da va sieu khang. Vi tri dot bien nav cung duoc phat hien qua mot nghien ciru ciia Thd NhT Ky nam 2006 |4|. Phuong phap giai trinh tir cd the phat hien chinh xac dot bien cii thudng xuat hien va dot bien mdi, d;i\ chinh la uu the cua phuong phap giai trinh tu hon so vdi cite phuong phap khac. vi dot bien tai vi tri codon 490 hay cac vi tri codon khac ngoai viing 81 bp se khdng xac dinh duoc bang cac phuong phap sinh hoc phan tu khac nhu INNO-I.iPA Rif.TB kit; PCR-RFLP; MAS- PCR...
Mot sd nghien ciru trudc day cho thay hon 95% cac cluing khang riTampicin xay ra dot bien trong viing "Hot spot region muta- tion" 81 bp ciia gen rpoB, tir codon 507-533 dupe biet nhu lii viing de xac djnh khang ri- fampicine. Sir dot bien trong codon 516. 526.
531 ciia gen rpoB thudng ket hop vdi khang rifampicine d mire do cao, dieu nay hoan toan phii hop vdi ket qua nghien ciru thu duoc, dot bien tai ba codon na\ ludn gan lien vdi cac chiing da khang va sieu khang (Bang 2) |3.
5] [6]. Hau het cac cluing khang da thudc va sieu khang deu phat hien dot bien trong viing
"Hot spot mutation" 81 bp nhir cac nghien cuu trudc da cdng bd [2. 4. 3]. Trong so 42/45 chiing khang da xuat hien dot bien chii yeu tai vj tri 53 1; 526 va cd 4 cluing xuat hien dot bien tai vj tri 516. Cd 14/15 chiing sieu khang xuat hien dot bien tai vj tri 531; 526: 516. 1 cluing sieu khang khdng phat hien dot bien cd the dot bien ngoai viing 528 bp duoc giai trinh tu. Trong sd 59 cluing khang rifam- picin cd 58 chung chiem 98,3% xuat hien dot bien. thirdng gap nhat tai vj tri codon 531 (52,54%); sau dd lan lupt la codon 526 (27,12 %): codon 516 (11.86%); ngoai ra con cd cac vi tri dot bien khac nhu 530; 522: 533:
519. Vj tri dot bien 522; 533 thudng dupe 20 Tap chi Y hpc dir phong. Tap XX. so 6(114)
phat hien tai cac nghien ciiu khac, vi tri dot bien 519 duy nhat duoc phat hien bdi tac gia Saeed Zaker Bostanabad Iran nam 2007:
nhung dac biet trong nghien ciiu nay chiing toi phat hien duoc vi tri dot bien 530, la vj tri chua thay tac gia nao trong nude va tren the gioi cong bo. Day co the la diem khac biet trong nghien ciiu tren cac chiing vi khuan lao co nguon gdc tir Vict Nam. Tuy nhien. trong nghien ciiu nay cluing tdi khdng phat hien duoc cac dot bien tai cac vj tri nhu 513: 516;
507... ma cac tac gia khac xac djnh duoc.
Cd the do dac diem phan tir ciia cac chung vi khuan lao phan bd theo viing dja ly. thdi gian thu thap khac nhau. Tan suat dot bien ciia ket qua nghien cuu thu duoc phu hop vdi mot so nghien ciru trong va ngoai nude trudc day nhu;: lac gia M. Caws tai thanh phd Hd Chi Minh nam 2006: 53 1 (43%); 526 (31%); 516 (15%); l ac gia Amina Abdelaal Ai Cap 2009: 531 (45%); 526 (30%); 516 (20%); Klin llae Cho I lan Qudc 2009: 531 (64.6%); 526 (1 1.1%); 516 (6,6%).
Dii-e biet, trong nghien ciru ciia cluing tdi phat hien mot cluing khang da thudc thu thap tai mien Bac (11502-133) mat doan TGAGC- CAATTCA gom 12 nucleotit tir codon 5 I I den codon 515 dim den dot bien mat nghTa. kieu dot bien cluing nay dac biet khdng thay xuat hien a cac nghien ciru khac. vi vay can tiep tuc nghien ciru them ddi vdi cluing \ i khuan lao nay.
V. KET LUAN
Ket qua giai trinh tir doan 528 bp chira viing "Hot spot mutations" tren gen rpoB cua 127 chung vi khuan lao nhay va khang thudc d ca ba mien ciia Viet Nam cho thay cac cluing khdng khang thudc khdng xuat hien dot bien:
58 cluing phat hien dot bien trong tdng sd 59 cluing khang riTampicin chiem 98.3%. Cd 7 vj tri dot bien xay ra tren viing 81 bp gen rpoB ciia vi khuan lao, cac vj tri dot bien hay gap la
531 va 526 vdi tan suat dot bien tuong ung la 52.54% va 27,12 %, tiep theo la vj tri dot bien 516 chidm 11.86% va mot sd vi tri codon khac nhu 530; 522; 533; 519 chiem 1.69%. 13/106 chiing (12,26%) gdm ca khang don, khang da va sieu khang phat hien duoc dot bien lai codon 490 (Q490H/R/Stop). Cd 40 kieu dot bien tren viing 528 bp va 16 kieTi dot bien lien quan ddn khang rifampicin thudc viing 81 bp tren gen rpoB. trong do kieu dot bien Ser53 I - Leu la phd bien nhat.
TAI LIEU THAM KHAO
1. A. Telenti., N. Honore., C. Bernasconi.. J.
March.. A. Ortega., et al. (1997). "Genoty- pic Assessment of Isoniazid and Rifampin Resistance in Mycobacterium tuberculosis:
a Blind Study at Reference Laboratory Le- vel.'' clinical microbiology 35(3): 719-722.
2. Bartfai, Z., A. Somoskovi, C. Kodmon, N.
Szabo, E. Puskas, et al. (2001). "Molecu- lar characterization of rifampin-resistant isolates of Mycobacterium tuberculosis from Hungary by DNA sequencing and the line probe assay." .7 Clin Microbiol 39( 10):
3736-9.
3. S L Moghazeh.. X Pan., TArain., C K Sto- ver., J M Musser., et al. (1996 November).
"Comparative antimycobacterial activities of rifampin, rifapentine. and KRM-1648 against a collection of rifampin-resistant Mycobacterium tuberculosis isolates with known rpoB mutations." Anlimicroh Agents Chemother 40( 1 1): 2655-2657.
4. Ccngiz Cavusoglu., Ajda T'urhan., Pinar Akinci., and llknur Soyler. (2006 July).
"Ev aluation of the Genotype MTBDR As- say for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Isolates." J Clin Microbiol 44(7): 2338-2342.
5. Thomas Bodmer., Gabriela Ziircher., Paul Tap chi Y hpc dir phong, Tap XX. sd 6 (114) 21
Imboden.. and Amalio Telenti. (1995 ).
••Mutation position and type of substitution in the beta-subunit of the RNA polymerase influence in-vitro activity of rifamycins in rifampicin-resistant Mycobacterium tuber- culosis." Antimicrobial Chemotherapy 35:
345-348.
6. M. Caws.. Phan Minh Duy.. Dau Quang The. Nguyen Thi Ngoc Lan., Dai Viet Hoa.. et al. (2006). "Mutations Prevalent among Rifampin-and Isoniazid-Resistant- Mycobacteriumtuberculosislsolates from a Hospital in Vietnam, "journal of clinical microbiology 44: 2333-2337.
MOLECULAR CHARACTERIZATION OF RIFAMPICIN RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS ISOLATES IN VIET NAM, 2008 TO 2009.
Vu Thi Kim Lien, Tran Thj Hai Au, Nguyen Thi Thu Thai*, Nguyen Thai Son**
National Institute of Hygiene and Epidemiology, Hanoi
* Thai Nguyen Medical University
** Military Academy of Medicine, Hospital, Hanoi total 126 Mycobacterium tuberculosis
isolated from North, Central, and Southern of Viet Nam in 2008-2009 was used to examine the mutation in 81 bp hotspot region of rpoB gene which encoding rifampicin resistance.
The results showed that no mutation w as found on 21 drug sensitive strains. Among 58/59 (98.3%) rifampicin resistance strains have been found the mutation on rpoB gene, with high frequency was at codon 531 (52.54%);
526 (27.12 %); and 5 16(11.86%). There were 16 types of drug resistance mutation of 81 bp hotspot region, of which the most common type Ser531Leu. The data suggest that rifam- picin resistance in Mycobacterium tuberculo- sis were associated with the mutations in rpoB gene. Further study on rifampicin resistance is needed in order to set up treatment policy for tuberculosis patients in Vietnam.
s s Tap chi Y hpc du phong, Tap XX, so 6 (1 14)
