U'NG DUNG KY THUAT SINH HOC PHAN TU" (semi-nested PCR/Sequencing) XAC DINH CAC TYP VI RUT DU'ONG RUOT GAY BENH TAY CHAN MIENG d NGU'QI NAM 2009-2010 TLT CAC MAU LAM SANG
Nguyin Thj Hien Thanh, Dao Thi Hai Anh, Le Thj Quynh Mai Vien Ve Sinh Dich ti Trung Uffng, Hit noi
TOM
T A T .16 trong so 30 b?nh nhan m i c benh tay chan mieng nam 2009-2010 a mot so tinh Viet Nam du'gc xac djnh la do vi rut du-ong ruot b i n g phu'ang phap phan lap tren te bao va phu-ong phap sinh hoc phan Xis (semi-nested PCR/Seq). K i t qua phan lap vi rut tren t l bao cho ty le thap dat 23,3%; K i t qua phat hi§n vi riit tif cac m i u lam sang bang phu'ang phap sinh hpc phan tu' (semi-nested PCR/Seq) da phat hien du'p'c vi rut a ty le r l t cao dat 81,8%. K i t qua nghien CLPU da xac djnh dirac phu-ang phap sinh hoc phan tLK (semi-nested PCR/Seq) co dp nhay va do dac hieu r i t cao, c6 the ap dung de chan doan dich sam do vi rut duang rupt tCr cac m i u lam sang. Benh tay chan mieng nam 2009 la do vi rut Coxsackie A16 va nam 2010 la do vi rut duang rupt typ71/C4. Benh xay ra vao mua he, tap trung a tre du'ai 5 tuoi, tre trai m§c 2 lan cao han tre gai.
Tip khoa : Sinh hoc phan tir (semi-nested PCR/Seq), benh tay chan mieng, vi rut du-ang ruot
I. DAT VAN DE
Vi nit duong ruot thuong dugc xac dinh bang phuong phap phan lap vi riit tren te bao va djnh typ bang phuong phap trung hoa vi lugng voi cac khang huyet thanh dac hieu typ, bang phuong phap nay phai mat khoang 28 ngay moi CO dugc ket qua. Phuong phap sinh hgc phan tii' CO do nhay va do dac hieu cao, thoi gian cho ket qua nhanh (trong vong 2 ngay).
Chiing toi da ling diing ky thuat sinh hgc phan tir b;in to (snPCR/Seq) de xac djnh cac typ vi riit duong ruot gay djch benh tay chan mieng nam 2008 tii' cac mau nuoc noi te bao [1]. Bang phuong phap nay ciing phai mat it nhat 2 tuan moi c6 dugc ket qua vi phai dgi phan lap vi rut tren te bao. De dap irng thoi gian chan doan djch som, chiing toi tien hanh
nghien ciru li'ng dung ky thuat sinh hgc phan tii ban t6 (semi-nested PCR/Sequencing) xac djnh cac typ VRDR tir cac mau lam sang.
II. PHI/OTVG PHAP NGHIEN ClTU 9 mau phan, 18 mau djch ngoay hgng va 3 mau djch not phong cua 30 benh nhan mac benh tay chan mieng nam 2009-2010 dugc thu thap de phan lap vi rut tren te bao va lam phan irng snPCR/Seq true tiep tir cac mau lam sang
Cac cap moi dac hieu vi nit duong ruot Kit tach chilt ARN. hang Qiagen; Kit tinh khiet san pham PCR. hang Promega; sinh pham tong hgp san pham PCR, BigDye Termi- nator hang Promega;
Sinh pham khac, hang Roche bao gom muoi dem 5 x RT buffer, 10 x PCR buffer +
MgCI, Buffer, 20 mM dNTP, DTT (0.1M; En- zyme; RNasin RNase Inhibitor, Super Script II RT, Taq Polymerase, FastStart Taq.
Phan lap va djnh typ vi rut tren te bao bang each xir ly benh pham va gay nhiem 0,2 ml benh phan vao te bao RD, cac mau gay buy hoai te bao dugc djnh typ vi riit bang each trung hoa voi cac khang huyet thanh dac hieu vi rut duong ruot (theo thuong qui cua to chuc y te the gioi)
- Phuxmgphdp semi-nested RT-PCR/Seq.:
Bay mau nuoc noi te bao va 11 mau lam sang ciia benh nhan nam 2010 dugc tach chiet RNA bang kit Qiagen theo thuong qui ciia hang.
Tong hgp cDNA bang each trgn 5 pi ARN tir mau lam sang dugc tach chiet, lOOpM cua moi dNTP, 2pl ciia x5 dem RT (x5 RT buffer), 0,0 IM dithiothreitol, Ipmol moi loai primer (primer AN32; GTYTGCCA, AN 33;
GAYTGCCA, AN 34: CCRTCRTA, AN 35; RC- TYTGCCA), 20 UI ARNsin va 100 UI enzymp phien ma ngugc Superscript II (tong the tich la 1 Opl). Trgn dIu va ii tai 22°C 10 phut: 45°C - 45 phut, va 9 5 ^ 5 phut;
T6ng hgp san pham PCRl bao gom 10 pi cDNA, 5pl dem PCR xlO (xlO PCR buf- fer). 200pM m6i loai dNTP, 50pmol moi loai primer S0224: GCIATGYTIGGIACICAYRT va S0222: CICCIGGIGGIAYRWACAT, va 2,5 UI enzyme Taq DNA polymerase. Trgn deu va chay 40 chu ky tai 95°C 30 giay, 42°C 30 giay, 60^0 - 45 giay;
T6ng hgp san pham PCR2 bao g6m 01
pi san pham PCRl, 5pl dem Taq fastStart xlO (xlO fastStart Taq buffer), 200pM moi loai dNTP, 40pmol m6i loai primer AN 89: CCAG- CACTGACAGCAGYNGARAYNGG" va AN 88:
TACTGGACCACCTGGNGGNAYRWACATb. va 2,5 UI fastStart Taq DNA polymerase. Iron deu va ii 95°C - 6 phut, sau do chay 40 chu ky tai 95"C 30 giay, 6 0 ^ 20 giay, 72°C 15 giay. Chay dien di xac djnh san pham PCR2 (khoang 350-400bp).
Tinh khilt san pham PCR2 bang kit Wizard SV gel and PCR clean up System (Pro- mega). Phan irng sequencing dugc thuc hien voi BigDye Terminator va primer sequencing la AN 88F. Chay tren may dieu khien nhiet ciia chuong trinh Sequencing tai 01 phiit 96°C;
25 chu ky cua 96''C - 10 giay, 50°C 05 giay, 60°C - 4 phiit. Tinh sach san pham sequencing bang SephadexTM G-50 Fine (Amersham Bio- sciences). Phan tich san pham gen bang may Sequencer ABI 3 130. Cac trinh tir quang pho dugc lap dat bang phan mem Sequencer. Su sap dat trinh tu dugc thuc hien bang chuong trinh Sequencer 4.2.2, chuong trinh Genetic Max va NCBI nucleotide - nucleotide BLAST. Trinh tu san pham PCR2 dugc so sanh voi trinh tu viing VPl ciia cac chiing VRDR chuan, bao gom it lihat 03 dai dien ciia moi serotyp da biet. Ket qiia CO su dong nhat ve nucleotide voi it nhat ba chiing chuan >75% dugc ghi nhan la ciing serotyp.
Cac chiing EV71 dugc ve cay di truyen dl xac djnh kieu gen bang phan mem MEGA4
Tap chi Y hpc du phong, Tap XXI, so 3 (121)
37III. KET QUA
Bang 1. Thoi gian lay mlu benh pham va kit qua phan lap vi rut tren td bao:
Loai mau benh pham
Thoi gian lay mlu k l tir ngay mic
So mau phan lap
N
Sd gay buy hoai t§ bao
(CPE) N(+)
S6 mlu lam So (+) VRDR snRT-PCR bang snRT-PCR
(N) N (+)
Miu phan
<= 5 ngay Khong ro
</= 4 ngay 5 ngay
3 7 10
1
2 3 2 0
0 0 g 0
0 0 9
Mau djch 0
ngoay hpng
Djch not Tong so m a u phan lap
Khong ro
</=3 ngay
6 3 30
0 0 7 (23,3%)
0 2 11
0 0 9 (81,8%)
30 mau benh pham dugc gay nhiem vao te bao RD. Ket qua eo 7 mau gay buy hoai te bao, chiem ty le 23,3%; Ty le phan lap dugc vi rut o cac mau phan cao hon d djch ngoay hgng (5/10 va 2/17 theo trinh tu), khong phan lap dugc vi rut tir djch not phong.
Ty le phan lap dugc vi riit ciing lien quan nhieu den thoi gian lay mau. Cac mau phan thu thap trong vong 5 ngay ke tir ngay mac co ty le phan lap cao (2/3) chiem 66,6%; Djch ngoay hgng CO ty le phan lap dugc vi rut thap hon nhieu (2/17) chiem 11,7% mac dii mau dugc
thu thap trong vong 4 ngay ke tir ngay mac tuong doi cao, cac mau thu thap ngay thii 5 ke til' ngay mac hoac khong co thong tin ve nga\
mac da khong phan lap dugc vi nit. Djch not phong khong phat hien dugc vi nit mac dii thoi gian lay mau som trong vong 3 ngay ke tir ngay mac.
Ty le phat hien vi riit bang ky thuat di truyin phan tur ban to (semi-nested PCR/Seq) tai viing gen VPl tir 11 mau lam sang cho ket qua rat cao (9/11), chilm ty le 81,8% .
Bang 2. K§t qua xac djnh typ vl rut diFong ruot blng giai trinh tu> gen (sequencing)
Nguon gdc N mlu vi rut (+)
Ket qua xac djnh typ vi rut dirong ruot bing phu'ang phap Sequencing
EV71 CA16 CA10 CBS E19 EVS
Nuoc noi te bao Mlu lam sang Tong
7
9
16
2
4
6 (37,5%)
3
3 6 (37,5%)
1
0
1
1
0
1
0
1
1
0
1
1
Ket qua giai trinh tu gen 16 chiing vi nit duong ruot da xac djnh dugc 6 chiing la vi rut duong ruot typ71; 6 chimg la vi nit Cox- sackie A16; bon chung con lai la vi nit Cox- sackieAlO, vi rut Coxsackie B5, vi nit Echo
19 va mot chiing la vi riit duong ruot chua xac djnh. Tir ket qua nghien ciru da cho thay vi riit EV71 va vi nit Cosackie luu hanh va gay benh tay chan mieng la chinh chiem 37,5% cho moi loai vi riit.
Bang 3. Kit qua xac djnh can nguyen vi rut dirong ruot gay b#nh theo nam
Nam
(+) vi rut duong
rupt
Kit qua xac djnh typ vi rut duong ruot bling phirong phap Sequengcing
EV71 CA16 CA10 CBS E19 Chua xac dinh 2009
2010 Tong
18
12 30
6
10 16
1
5 6
3
3 6
1
0 1
1
0 1
0
1 1
0
1 1
Nam 2009, vi nit Coxsackie A16 gay benh tay chan mieng la chinh, chilm ty le 50%
trong t6ng s6 cac vi nit phan lap dugc; vi nit EV71, Coxsackie AlO va Coxsackie B5 cung phan lap dugc nhung mang tinh roi rac.
Nam 2010, vi rut EV71 phan lap dugc la chinh, sau do la vi nit Coxsackie A16 (3/10) chilm ty le 50% va 30% theo trinh tu. Ngoai ra con xac djnh dugc mot vi rut echo 19 va mot vi rut chua xac dinh.
Tap chi Y hpc du phong, Tap XXI, so 3 (121) 39
Bang 4. Kit qua xac djnh can nguyen vi rut duong rupt theo vung dja ly nam 2009-2010
Tinh Ha Giang Quang Ninh
Nam 09-2010
2009
N(+) 3 1
EV71 0 0
CA16 3 1
CA10 0 0
CBS 0 0
ECH019 0 0
EVS 0 0 2009
Lang Son
2010 3 3/C4
Ha Noi Hai Phong Quang Tri Bac Giang Bac Can Tong
2009 2009 2009 2010 2010
1 1 2 1 3 16
0 1/C5
0 1/C5 1/04 6
1 0 0 0 0 6
0 0 1 0 0 1
0 0 1 0 0 1
0 0 0 0 1 1
0 0 0 0 1 1
Ket qua bang 4 cho thay can nguyen vi va Bac Can la do vi nit EV71; Rieng tai Quang nit duong ruot gay benh tay chan mieng tai tri la mot tinh mien trung lai do vi nit Coxsa- Ha Giang, Ha Ngi va Quang Ninh la do vi nit ckieAlO va vi rut CoxsackieB5 gay "benh tay CoxsackieA16; tai Lang Son co sir luu hanh chan mieng. Ngoai ra eon xac djnh dugc vi gay benh ciia ca vi rut CoxsackieA16 va vi rut riit echo 19 cung lien quan den benh tay chan duong ruot typ 71; Tai Hai Phong, Bac Giang mieng tai tinh Bac Can
Bang 5. Su phan bd theo tuoi va gioi
Do tuoi
1 tuoi 2 tuoi 3tu6i 4 tuoi 9 tuoi 32 tuoi
N(+) VRBR 5 5 2 2 1 1
EV7 3 2 0 1 1 0
A16 1 3 1 1 0 0
Vi riit khac 1 0 1 0 0 1
Ket qua bang 6 cho thay benh gap chii yeu o tre duoi 5 tuoi (15/16) va tap trung chinh a tre trong vong 4 tuoi (14/16). Khong co sir khac biet theo tuoi vS s6 tre mac do vi nit EV71 va CoxsackieA 16 (6/6). Tre trai mic benh cao gap 2 lan so voi tre gai (2,2:1,0)
1 9 3 7
29
91
9 9 34
91
53 33
51
76 94
90
43
39
9V 100
14
31
99
93
37
69
9 8
100
9 / 8 0
9 9
100 51
6 3
99
2A
2 5
79 100
6 1
93 59
88
5 2
1
y ^ ^
• 1 0 0 0 4 . L S . N I H E
• 1 0 0 0 5 . L S . N I H E
• 1 0 0 0 2 . B K . N I H b 5 5 9 S A / N M / 0 5 / C 4
H 2 5 - C H N - 0 0 / C 4
' ^ 1 8 T A / N M / 0 5 / C 4 ^
K O R - E V 7 1 - 1 3 / C 3 2 M / A U S / 3 / 9 9 / C 2 0 3 7 5 0 - M A A - 9 7 / C 2 1 8 1 4 - Y a m a g a t a - 0 1 / C 2 6 7 1 - Y a m a g a t a - 9 8 / C 2 9 8 3 - Y a m a g a t a - 9 9 / C 2 1 1 1 6 - Y a m a g a t a - 0 0 / C 2 1 M / A U S / 1 2 / 0 0 / C 1 S B 9 4 6 5 - S A R - 0 3 / C 1 S 1 8 1 9 1 / S A R / 0 2 / C 1
/ ^ 3 9 . N T h . N I H E ^
• 0 9 2 1 . H P . N I H E
• 1 0 0 1 2 . B G . N I N E 0 8 1 4 . N D . N I H E
0 8 2 1 . N B . N I H E 0 8 3 4 . H u . N I H E 1 0 0 1 V / V N M / 0 5 / C 5 . 2 1 0 8 9 T A / N M / 0 5 / C 5 . 2 0 7 5 3 . H P . N I H E 0 8 6 5 . H P . N I H E 0 8 1 7 . N B . N I H E
I
S 1 1 0 0 3 1 - S A R - 0 3 / B 6 M a l 2 . 1 N I H E / B 5
• 5 5 3 6 / S I N / 0 0 / B 4
• S 2 1 0 8 2 / S A R / 0 0 / B 4 - 7 3 8 - Y a f n a g a t a - 0 0 / B 4
• 1 0 8 4 - Y a m a g a t a - 0 0 / B 4
• M Y 1 6 / I / S A R / 9 7 / B 3
• 0 8 9 9 - M A A - 9 7 / B 3 - 4 3 5 0 / 8 1 N / 9 8 / B 3 - 2 6 0 4 - A U S - 7 4 / B 1 - 2 2 3 2 - N Y - / y / B 1 - 2 2 2 2 - U S A - 8 8 / B 2 - 7 6 3 3 - P A . 8 7 / B 2 - 7 6 7 3 - U S A - 8 7 / B 2
Hinh1. Cay di truySn cac chung vi rut EV71 nam 2009-2010
Tap chi Y hpc dir phong. Tap XXI, so 3 (121) 41
Phan tich gen cac chiing vi rut duong ruot typ71 phan lap dugc trong cac nam 2009- 2010 bang ve cay di truyen so sanh voi cac chiing chuan cho thay vi riit EV71 luu hanh gay benh 6 cac tinh dong bang (Hai Phong va Bac Giang) thugc nhom gen C5, vi nit EV71 luu hanh gay benh o cac tinh mien nui ( Bac Can va Lang Son) thugc nhom gen C4
IV. BAN LUAN
Ty le phan lap dugc vi rut duong ruot lien quan nhieu den loai mau va thoi gian thu thap mau, benh pham thu thap cang som thi kha nang xae djnh dugc can nguyen VRDR cang cao. Benh pham phan co kha nang phan lap dugc VRDR cao nhat la do vi nit duong ruot thuong dao thai theo phan voi so lugng cao trong thoi gian dai (trong vong 14 ngay ke tir ngay phat benh); Benh pham djch ngoay hgng phan lap dugc a ty le thap nhung lai co kha nang phat hien dugc axit nucleic vi nit o ty le cao neu lay mau dung thoi gian qui djnh (trong vong 4 ngay ke tii' ngay co not phong).
Ket qua phan lap dugc vi rut trong djch ngoay hgng thap la do hau hgng chi la noi cu trii tam thoi ciia vi riit ducmg ruot nen nong do vi rut 6 day rat thap. Ket qua xac dinh dugc 81.8% mau duong tinh voi vi rut duong ruot tir cac mau djch ngoay hgng lam sang da cho thay ky thuat di truyen phan tir ban to (semi-nested PCR/Seq) CO do nhay va do dac hieu rat cao, ket qua co tinh thuyet phuc de su dung cho cong tac xac dinh djch benh do vi rut duong ruot gay ra [2].
Ket qua xac djnh typ vi nit duong ruot cho thay co sir dong luu hanh va gay benh tay chan mieng o nhieu typ vi nit duong ruot khac nhau (bang 3) va luu hanh cung khac nhau theo
ie AlO, va vi rut Coxsackie B5 nhung voi ty le thap chilm 16,6% cua moi loai vi rut. Nam 2010 thi vi riit EV7! lai gay benh tay chan mieng la chinh, chiSm ty le 50% trong tong so cac vi riit phan lap dugc, sau do la vi riit Coxsackie A16 chiSm 30%, ngoai ra con phan lap dugc vi rut Echo 19. Tuoi mic tap trung chinh o tre duoi 5 tuoi, tre trai mac cao hon tre gai 2 lan.
Cac ket qua nghien ciru ve benh tay chan mieng tren the gioi cung cho thav vi riit Cox- sackie Alb la nguyen nhan pho bien nhat gav benh tay chan mieng a tre em. Vi riit duong ruot typ71 gay benh tay chan mieng khong pho bien nhung thuong eo bi§u hien lam sang tram trgng, ty le tu vong cao va doi khi ciing gay ra cac vu djch Ion voi cac kieu gen khac nhau a moi nuoc [3,4,5]. Hien nay vi riit duong ruot typ 71 dugc To chirc Y te The gioi khuyen cao la loai vi nit nguy hiem, tat ca cac nuoc tren the gioi can phai de phong vi vi riit co the gay ra dich Ion va co ty le tir vong cao o tre em duoi 5 tuoi.
Ket qua nghien cuu ve kieu gen cua sau chiing vi nit duong ruot typ 71 xac djnh trong nam 2009-2010 cho thay co bon chimg vi nit thugc kieu gen C4 giong cac vi nit duong rupt typ 71 luu hanh gay dich benh ta\ chan mieng 6' Trung Quoc nam 2000. Bon chiing vi riil thugc kieu gen C4 xac djnh dugc 6 tinh Bac Can va tinh Lang Son co bien gioi voi Trung Quoc va trong nhirng nam gan day Trung Quoc lien tuc xay ra djch benh tay chan mieng ma can nguyen do EV71/C4 nen co the day la nhirng chiing vi rut gay benh bj xam nhap vao tir Trung Quoc. Hai chimg v i nit thugc kieu gen C5 xac djnh dugc 6 tinh Hai Phong va Bac Gi- ang thi giong cac chiing vi nit HV71 gay benh
V. KET LUAN
Tir ket qua nghien ciru cho thay:
1. Ty le phan lap vi nit lien quan nhieu den loai mau
- MIU phan CO ty le phan lap cao, chiSm 50% trong tong s6 mau phan thu thap dugc
- Djch ngoay hgng co ty le phan lap thap, chiem 20% trong tong so mau djch ngoay hgng thu thap dugc
Khong phan lap dugc vi rut tir mau djch not phong
2. Ky thuat di truyen phan tu ban to (semi-nested PCR/Seq) c6 do nhay va do dac hieu rat cao voi ty le phat hien dugc vi nit la 81,8%. Chi phat hien dugc vi nit tii' cac mau lam sang co thoi gian lay mau chuan (Benh pham djch ngoay hgng lay trong vong 4 ngay ke tir ngay phat benh)
Khong phat hien dugc vi nit tir cac mau djch not phong
3. Benh tay chan mieng nam 2009 la do vi rut Coxsackie A16 va nam 2010 la do vi riit EV71 gay benh la chinh. Benh xay ra o ca cac tinh dong bang va mien niii, tuoi mac chii yeu o tre em duoi 5 tu6i, tre trai mac cao gap 2 lan tre gai
4. Vi nit EV71 luu hanh gay benh nam 2009 thugc nhom gen C5 gi6ng voi vi nit EV71 luu hanh gay benh o Viet Nam trong cac nam 2005-2008
Vi rut EV71 luu hanh gay benh nam 2010 thugc nhom gen C4 gidng cac chung vi riit EV71 gay benh o Trung Qu6c nam 2008 TAI LIEU T H A M K H A O
1. Nguyin Thj Hi6n Thanh, VQ Hong Nga, Trkn Thj Nguyin Hoa, Dao Thj Hai Anh.
Benh tay chan mieng o nguoi nam 2008 do
virut duong ruot typ 71 va virut coxsackie A16. Tap chi Y hgc du phong 2010 tap XX s6 6(114),tr.46-54
2. W. Allan Nix, M. Steven Oberste,* and Mark A. Pallansch. Sensitive, Seminested PCR Amplification of VPl Sequences for Direct Identification of All Enterovirus Se- rotypes from Original Clinical Specimens.
JCM, 2006. p 2689-2704
3. Brown BA, Oberste MS, Alexander JP Jr.
Kennett ML, Pallansch MA, et al. Molecu- lar epidemiology and evolution of enterovi- rus 71 strains isolated from 1970 to 1998.
Journal of Virol 1999; p.9969 75.
4. Hosoya, Mitsuaki; Kawasaki, Yukihi- ko; Sato, Masatoki; Honzumi, Ken; Kato, Asako; Hiroshima, Toy omasa; Ishiko, Hi- roaki; Suzuki, Hitoshi. Genetic Diversity of Enterovirus 71 Associated With Hand, Foot and Mouth Disease Epidemics in Ja- pan from 1983 to 2003. Pediatric Infectious Disease Journal. August 2006; 25(8): p.
691-694.
5. Cardosa Mj, Perera D, Brown BA, et al.
Molecular epidemiology of human entero- virus 71 strains and recent outbreaks in the Asia-Pacific region: Comparativeanalysis of the VPl and VP4 genes. Emerg Infect Dis2003;9. p. 461-8.
6. Phan Van Tu,* Nguyen Thi Thanh Thao,*
David Perera,i" Khanh Huu Truong,J Nguyen Thi Kim Tien,* Tang Chi Thuong,J Ooi Mong How,§ Mary Jane Cardosa,t and Peter Charles McMinn. Epidemiologic and Viroiogical Investigation of Hand, Foot, and Mouth Disease, Southern Vietnam, 2005.
EID Journal Home > Volume 13, Number Il-November2007. p. 1-7
Tap chi Y hpc dir phong. Tap XXI, so 3 (121)
43APPLICATION OF MOLECULAR METHOD (SEMI-NESTED PCR AND SEQUENCING) IN TYPING ENTEROVIRUSES WHICH CAUSE HUMAN HAND-FOOT-AND-MOUTH DISEASE (HFMD) FROM CLINICAL SPECIMENS IN 2009 2010.
Nguyen Thi Hien Thanh, Dao Thi Hai Anh, Le Thi Quynh Mai National Institute of Hygiene & Epidemiology, Hanoi
Isolation of virus on cell culture and molecular methods (Semi-nested PCR/ Seq), enteroviruses were identified to be responsible for HFMD in 16 out of 30 patients from 2009 to 2010 in several Vietnam provinces. Virus isolation in cell culture gave rise to a low rate of virus identification as 23.3%. Virus deter- mination from clinical samples by semi-nested PCR/ Seq achieved a high rate of virus identifi- cation as 81.8%. The study result revealed that the molecular method of semi-nested PCR/ Seq
had a high level of sensitivity and specificity, which can be applicable to early diagnosis of enterovirus outbreak from clinical samples.
HFMD in 2009 was caused by Coxsacki- evirus Alb and in 2010 was caused by Entero- virus 71/ C4. The disease occurred in summer, focusing on children below 5. Boys acquired twice more than girls did.
Key words: Molecular method (Semi- nested PCR/ Seq), hand-foot-and-mouth dis- ease, enteroviruses.
