S6 1-2012 T»P CHi P H O N G C H 6 N G BgNH S 6 T RET VA CAC B$NH K^ SINH TRCING 45
NGHlfeN c i r u C O CAU KY SINH T R U N G S 6 T R E T TAI NINH T H U . ^ BANG KY T H U ^ T PCR (2009-2010)
L6 Dih Bio', Trmnig Viin Hjnh', Naiiazawa' S., Maeno' Y., Trin VSn Cir' Nguyin Tlij Tliuong', Nguyin Tlij Tliu Huyin', Nguyin Thi Tri', Kawal' S. VO Quylt Thing.
'Vi?nS4t rtt, KST-CT Tir
^Tniimg d^i hoc t6ng hpp Nagoya, Vi^n Y hoc nhi^t ddri, Nh§t B^n
^ Tnr&ng dai hgc Y khoa Fujita, Nagoya, Nh§t Bin
'Tnmg ttoi phdng ch6ng s6t rit, KJ sinh tntag, C6n trCing Ninh Thujn ' Thrtmg dai hpc Y khoa Dokkyo, Nhf t bin
Tdm tdt:
Chiing toi da dp dung ky thudt phdn ling chuSi polymerase (PCR) di khdo sdt thdnh phdn, ca cdu lodi ky sinh trimg sdt ret (KSTSR) trin ngu&i & tinh Ninh Thudn giai dogn
2009-2010. Cdc doi moi dugc sii dgng Id mdi ddc hieu doi vdi 4 lodi KSTSR nguai:
P.falciparum, P. vivax, P. malariae vd P. ovale vd mdi xdc dinh P. knowlesi Dogn ADN khudn dac thil cho tiing lodi Id gen ghi md 18 dim vi nhd ARN ribosome (18ssu-rRNA). Kit qud cho Ihiy ci JK tin tgi cita 3 lodi KSTSR nguai Id P.falciparum chiim 63,12%, P.vivax chiim 29,79%, P.malariae chiem 4,96% vd P. knowlesi chiim 2,12% ky sinh trSn ngudi.
1. D.^T VAN Bt
Xac dinh chinh xac timg loai KSTSR gay b$nh trSn nguoi la mOt khoi diu quan trpng cho vi^c lira chpn phac do dieu tri phu hop.
Phat hien KST SR bang kinh hien vi do Laveran phat minh tlJr nam 1880 d6n nay vin thuc t^ va dang tin cSy. Tuy nhiSn, bat Ipi cua ky thuSt nay la khSng th^ phdt hien, phan biet day dli vk chinh xdc thanh phan cac loai KSTSR tren m6t b$nh nhSn, dac biet nhiing trucmg hpp c6 mat dp nhilm KST thip hoac nhiem phoi hop hai hay nhieu lodi, trong do chi c6 mot loai tr^i ban ve s6 lupng.
Ky thudt PCR c6 the phdn tich dupe tdt cd cdc mau thu lupm tir thirc dia [10,11] va phdt hidn dupe 4 lodi KST SR [10] trong nguW vd KSTSR khi P.knowlesi [3]. Doan ADN dich elm nhdn bdn trong ky thudt PCR nay Id gen ghi mS 18 don vi nho ARN ribosome (18 small subunit ribosomal RNA -18ssu-rRNA). Doan gen ndy da dupe xdc dinh trinh tu vd ddc hi^u cho moi loai trong 4 loai KSTRS tr6n ngu6ri [5] vd trSn khi [3]. Sir khdc nhau ve trinh tir oligonucleotide cua 5 loai cho ph6p thiet k^ nhihig doi m6i dac hieu loai phii hpp cho ky thu4t PCR.
Muc tiSu cua nghiSn cim ndy Id:
Xic djnh thdnh phdn, cff cdu KSTSR tfi tinh Ninh Thufn 2009-2010.
2. NGUYEN LIEU VA PHUONG PHAP 2.1. NguySn ]i§u:
2.1.1 Mdu mdu
Mlu mdu c6 KST dupe thu thdp tai Ninh Thudn trong 2 nam 2009 va 2010
46 TAP CHl P H 6 N G C H 6 N G B $ N H SOT R 6 T VA CAC BgNH K t SINH TRpNG S6 1-2012
21.2 Hoa chdt:
Cdc hoa chit diing de tdch, tinh khi^t ADN mua ciia hdng Qiagen, Hilden, Germany.
• H6a chdt dimg cho phdn img PCR:
- dATP, dTTP, dCTP, dGTP (TAKARA, Japan).
- Taq Polymerase: Vifn SR, KST-CT TW sdn xuat.
- Cdc loai Primer (moi): PLUI, PLUS, PLU6, FALl, FAL2, VIVl, VIV2, MALI, MAL2, OVAl, 0VA2, Pmk8, Pmk9: (Tmimg dai hpc y khoa Fujita, Nagoya, Nh|t bdn t6ng hpp).
2.2 Phinmg phdp;
2.2.1 Phuffng phdp Ihu thgp mdu mdu:
- Sir dung k^ thu^t x^t nghiem lam mdu nhupm giSm sa, soi kinh phdt hi$n cdc trudng hpp c6 KSTSR (di^u tra cdt ngang tai cdc di^m nghiSn ciru d cdc huy?n thu$c tinh Ninh Thu$n).
- Phuong phdp thu thap mau tir dau ngdn tay de ddng, khong ddi h6i nhilu dyng cy phric tap vd eung phii hpp v6i phuong phdp phan tich PCR. Dyng CU chi can kim trich mdu dau ng6n tay, b6ng, c6n sdt trimg. Phuong phdp ndy tien hdnh tai th\rc dja de ddng vd bdo qudn don gidn.
Thu thap mau theo phuong phdp ndy thudng ket hpp dong thiri vdi idy lam gipt ddy va gipt mong. Thi tich mdu nho len giiy thdm Whatman 3MM 30 :]l-50 Dl. Do diiu ki$n kho khdn d thuc dia va do nhiing chuyen di dai ngdy (1 thdng), vao vimg sau, vimg xa chung toi bdo quan cac mau nhu sau:
a/ De tdch rcri rieng timg mau de trdnh t^p nhilm chSo.
b/ Diiu kien thyc dja dl lau kho, viec bao qudn mau ddi hdi de kho hodn todn mdi trdnh dupe vi khuan va nam moc phdt trien. Khi de khd ty nhien d dieu ki?n thuc dia phai tranh cdc lo^i cdn thing den an (rudi, muoi, kien,...).
2.2.2 Phuffngphdp tdch linh khiet ADN:
ADN dupe tinh khiet bang bp sinh pham QIAamp DNA blood mini kit (Qiagen, Hilden, Germany). Quy trinh tinh khilt dupe thyc hi?n theo hudng dan cda hdng san xult.
2.2.3. Phdn Ong PCR:
- Phdn ting PCR xdc dinh 4 loai KSTSR tren ngudi vd P. knowlesi:
Phdn ling nhan bdn dupe tiln hdnh tren mdy GeneAmp- PCR System 9700, Applied Biosystems, USA. Trong tdt cd cdc phdn ilng cd ting the tich 20 DI chila nong do cdc chat Tris-HCl 10 mM, pH 8.3, 0.1 mg/ml gelatine, 50 mM KCl, 250 nM dNTPs (dATP, dCTP, dGTP, dlTP), 125 nM moi moi xudi vd m6i ngupc.
Phdn irng PCR dl nhan ban gen djc hi$u ciia Plasmodium gpi Id Nestl. Hai mdi Plu5 X Plu6 sit dyng cho phdn iing Nestl ddi vdi 4 loai KSTSR tren ngudi, Plui x Plu5 ddi vdi
S6 1-2012 TAP CHl P H 6 N G CHONG BgNH S 6 T RfeT VA CAC BgNH KY SINH TRUNG 47
KSTSR khi P. knowlesi cd trinh ty oligonucleotide nhu bdng 1. Phdn ilng PCR de nhdn bdn gen d^c hi^u cho timg lodi P.falciparum, P.vivax, P.malariae, P.ovale, P. knowlesi dupe gpi la Nest 2. Phdn img Nest 2 dupe thyc hi$n bdng ky thudt PCR da moi (multiplex PCR). Trinh ty oligonicleotide dac hi?u cho timg lodi nhu trong bdng 1. Thdnh phan phdn ilng Nest2 nhu Nestl, chi khac ADN khudn dupe ily tii sdn phim Nestl. Dieu ki?n phdn ilng nhu Nestl, chi khdc budc 5 dupe lap lai 30 lln.
Bdng 1: Trinh ty oligonucleotide m6i.
Ki€u phan umg
Plasmodium genus, Nest 1 Plasmodium P.know. Nest 1 P.falciparum Nest 2 P.vivax
Nest 2 P.malariae Nest 2 P.ovale
Nest 2 P. knowlesi
Nest 2
Ky hieu
PLu5 PLu6 PLul PLu5 FALl FAL2
vrvi
VIV2 MALI MAL2 OVAl 0VA2 PmkS Pmk9
Trinh ty
5'-CCT GTT GTT GCC TTA AAC TTC-3' 5'-TTA AAA TTG TTG CAG TTA AAA CG-3' 5'-TCA AAG ATT AAG CCA TGC AAG TGA-3' 5'-CCT GTT GTT GCC TTA AAC TCC-3'
5'- TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT -3' 5'-ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC-3' 5'-CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC-3' S'-ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA-3' 5'-ATA ACA TAG TTG TAC GTT AAG AAT AAC CGC-3' 5'-AAA ATT CCC ATG CAT AAA AAA TTA TAC AAA-3' 5'-ATC TCT TTT GCT ATT TTT TAG TAT TGG AGA-3' 5'-GGA AAA GGA CAC ATT AAT TGT ATC CTA GTG-3' 5 '-GTTAGCGAGAGCCACAAAAAAGCGAATT-3' 5 '- ACTCAAAGTAACAAAATCTTC CATA - 3'
Kfch thudc san pham PCR
~1,.2 kb
1670 bp
205 bp
120 bp
144 bp
786 bp
153 bp
DiSu kidn tdi uu cua phan ling Nested 1 la: 95*0 - 5 phut tiip theo la 25 chu ky gdm 94''C - 1 phiit, 58°C - 1 phiit, 72°C - 2 phut. Chu kJ- cudi cimg 72°C - 8 phiit.
Thanh phin phdn irng Nested 2 nhu Nested 1, chi khdc ADN khudn dupe ily tit sdn pham Nested 1. Diiu kien phdn ling nhu sau: 95°C - 5 phiit tiep theo id 30 ehu ky gom 94°C - 1 phut, 58°C -1 phiit, 72°C - 2 phut. Chu ky cu6i cimg 72°C - 8 phiit.
2.2.4. Phan tich kit qud:
Sdn phim PCR dupe phan tich bang ky thiMit di?n di agarose gel. Sd lupng bdn sao cua sdn phim PCR cd thi nhin thiy dupe U-8n agarose gel 2% chila Ethidium Bromide 30ng/nil.
- Kich thudc san phim PCR ddc hi|u P.falciparum : 205 base pairs (bp).
48 TAP CHl P H 6 N G C H 6 N G BgNH SOT RgT VACAC BgNH K-? SINH T R O N G S 6 t ^
- Kich thudc sdn phim PCR dac hi?u P.vivax : 120 bp.
- Kich thudc sdn phim PCR dac hi§u P.malariae : 144 bp.
- Kich thudc sdn phim PCR dac hi^u P.ovale : 800 bp.
- Kich thudc sdn phim PCR cua P.knowlesi : 153 bp
San phim PCR cua P. knowlesi dupe tinh khilt bang bd sinh phim QIAEX" II gel Extraction kit (Qiagen, Hilden, Germany). Quy trinh tinh khilt theo hudng din cda hang sdn xult. Sdn phim tinh khilt dupe giri din Trudng d?i hpc y khoa Fujita, Nagoya, Nhjt Bdn dl gidi tnnh ty. Gidi trinh ty dupe thyc hi^n trSn mdy ABI-CEl.
3. K £ T Q U A VA BAN LU^^N
Trong 2 ndm 2009 vd 2010, chiing tdi da tiln hdnh 2 dpt diiu tra cdt ngang tsii 2 huy^n: Ninh Son vd Bdc Ai. Tong so ddn dupe xdt nghiem Id 1974 ngudi. S6 ngudi mang kf sinh trimg s6t rit Id 115. 7^ 1^ ngudi nhiem ky sinh trimg 5,82%.
Trong ting so 115 miu mdu b^nh nhdn cd KSTSR, dupe phdn tich bing ky thujt PCR, so lln xuit hi^n KST Id 141. Cd 26 mlu nhiem ph6i hpp. Kit qud phdn tich thu dupe thi hi^n trong bdng 2.
Bdng 2: Thdnh phin, co ciu iodi vd ti le nhiem phoi hpp tai Ninh Thu|n
^ \ ^ Lodi K y t h u d t ~ \ PCR
Tdng sd .
Nhiem don P. falciparum
89 (63,12%)
P. vivax 42 (29,79%)
P. malariae 7 (4,96%)
P. knowlesi 3 (2,12%)
Nhilm ph6i hpp 26/115 (22,6%) 115 (141 lln xuit hi^n KST)
Bdng 2 cho thay ket qud phdn tich cde miu mdu nhilm KST tai Ninh Thudn, dd phdt hien dupe 3 lodi KSTSR trdn ngudi. Ty If nhilm dem chilm 77,4%, trong dd nhiem don do P.falciparum chilm 63,12%, nhiem don P. vivax Id 29,79%, ?. malariae 4,96%. DJc bift phft hi?n dupe 3 truimg hpp ngudi nhiem ky sinh trimg khi P. knowlesi (chilm 2,12%). Hinh dnh dien di sdn phim PCR cdc bfnh nhdn nhilm don P.falciparum va P. vivax, P. knowlesi iai/c the hien tren hinh 1, hinh 2
Hinh I: dnh difn di nhiing bfnh nhdn nhilm iariP. falciparum vd P. vivax
S6 1-2012 TAP CHi P H 6 N G C H 6 N G BENH SQT RET VA CAC BENH K^ SINH TRONG 49
L I M (-)
//////( 2. anh ilicii di san iiliani PCR ^:K- hcnli nhan nliicin /' kiunylcsi L Ladder - Thang do Irviig luang plian iir 5(1 bp (+): Chinig duang P. knowk'.si
(-): Chirng dm
8. 15. 18: Cik mau benh nhan nhiem KST P.knowlesi
De khang dinh chae chan cac mau 8. 15. 18 nhiem KST kJii P. kiumle.si. chung toi da gui cac mau na}' sang dai hoc '\' Fujita Nhat Ban giai trinh tu.
Ket qua giai Irinh ttr da kliang dinli cac mau 8. 15. 18 chinh xac la P. knowlesi (hinh 3, hinh 4 va hinh 5)
Hinh 3: Hinh anh giai trinh tu nucleotid tren gen 18ssu-rRNA tur san phim PCR mlu 8
50 TAP CHl P H 6 N G CHISNG BgNH S O T RgT VA CAC BgNH KY SINH T R O N G S 6 1-2012
CATATATGTTTTCTOCTTTATOTCiCCiCATCCTCTACCTATTAAGTGTTAATTAAATTAAAG
A ' j B Applied
# v . O Biosystems
CATATATGTTTTCTGCTTTATGTGCGCATCCTCTACCTATTAAGTGTTAATTAAATTAAAGOG TTCTTTTTAAAATCTTCTATAACTAATAAAAAAATACGGAAGATTTTGTTACTTTGAGTAAA
Hinh 4: H i n h anh giai trinh tir tren gen 18ssu-rRNA tit sdn p h a m P C R m a u 15 CATATATGTTTTCTGCTTTATGTGCGCATCCTCTACCTATTAAGTGTTAATTAAATTAAAGGG TTCTTTTTAAAATCTTCTATAACTAATAAAAAAATACGGAAGATTTTGTTACTTTGAGTAAA
# v . O Biosystems
9giH« G - » 4 A , M 2 T « 3 a C 1 M A , v S 4 3 M T S t M CRLMK OMO'MW
Hinh 5: Hinh dnh gidi trinh tv nucleotid tren gen 18ssu-rRNA tir sdn phdm PCR mdu 18 CATATATGTTTTCTGCTTTATGTGCGCATCCTCTACCTATTAAGTGTTAATTAAATTAAAGGGT TTCTTTTTAAAATCTTCTATAACTAATAAAAAAATACGOAAGATTTTGTTACTTTGAGTAAA Ti le v l thdnh phan lodi KSTSR dupe t h i hifn trfn hmh 6.
S6 1-2012 TAP CH( P H 6 N G C H 6 N G B $ N H S 6 T RET VA CAC B$NH KV SINH TRCING 51
Ll P malariae D P knowlesi
Hinh 8: Ti le vl co cdu KSTSR t?ii Ninh thu^n
So sanh ket qud ve thdnh phan, ca c^u lodi KSTSR cua Ninh Thudn vdi cdc tinh Lam Ding, Binh Phudc, Ddc Lie, Gia Lai thi thanh phdn vd co c4u lodi till Ninh Thu|in don gidn hon [1]. Trong khi cd bdn tinh tren, chiing tdi diu phdt hifn dupe sir tdn tai eua 4 loai KSTSR trdn ngudi, phdt hifn dupe nhidu trudng nhilm phoi hpp 3,4 lodi trdn cimg mdt bfnh nhdn tlli t^ Ninh Thu|in chi phdt hifn dupe 3 lodi KSTSR trfn ngudi va chi phdt hien dupe nhifm phdi hpp 2 loai.
3.2. T h a o lu§n
Cd mdt sd nghien ciiu ve di truyen quan the ngudi [6] bieu hien su nhiem benh bdi KST. Nhihig nghien cihi so sdnh vl di truyin cua quan the KST ty nhien cdn tuong dli it va chua cd thu nghiem ndo dupe tien hanh ve mdi lien quan di truyen KST ddi vdi di truyen qudn the ngudi vd vec to trong mpt vimg. Nhiing quan sdt vf lam sdng bfnh sot ret trong vimg sdt ret luu hanh va tir viec dp dung cac bien phdp dieu tri sdt ret the nao thay sir cd mat cua vdi loai trong 4 loai KSTSR trfn ngudi [4]. Bdn lodi KSTSR tren ngudi khdc nhau ve lam sang, bfnh hpc va cdch lan truyin ciing nhu ddp iing cua chiing voi thudc s6t ret. Vi thi cac chung trong cimg mpt loai cung cd nhiing dac tnmg khac nhau [7].
Phdn tich bdng ky thu4t PCR hifn nay dd chi ra rdng nhilm phdi hpp thudng Id nhilu hon nhilu so vdi ngudi ta nghi trudc ddy [8, 9, 10]. Trong mdt nghien cuu gdn ddy [12] da chi ra rdng mat dd nhiem KST vd bfnh hpc do nhilm phdi hpp cd sir khdc bift dang kl so vdi cimg lodi KST nhilm don. Rd rdng Id d diiu kifn lan truyin ty nhien, con ngudi, muoi ludn thay ddi, vi vay qudn thi KST cung phirc tap. Nghifn ciru vl thdnh phin vd co cdu lodi KST tai 2 huyfn sdt ret luu hanh tinh Ninh Thudn kit qud cho thdy cd su tdn cda 3 lodi KSTSR trdn ngudi P. falciparum, P. vivax, P. malariae, vd phat hifn dupe cd KST khi P.
knowlesi trdn ngudi. Trong dd P. falciparum chilm 63,12%, P. vivax chilm 29,79%, P.
malariae chilm 4,16%, P. knowlesi chilm 2,12%. Hifn nay mdt sd tde gid cho ring P.
knowlesi nhu Id lodi KSTSR thii 5 ky smh tren ngudi [3]. Ti If nhiem phdi hpp 2 lodi rk cao chilm 22,6%, chi phdt hien dupe nhiem phdi hpp 2 lodi P. falciparum vd P. vivax, hodc P.
vivax vd P. knowlesi. So sdnh vdi kit qud nghidn ciiu trudc ddy cua nhdm chiing tdi tai Lam Ddng, Ddc Lie, Gia Lai [1, 2] ti If nhilm phdi hpp thip hon (Ldm Ddng: 41%, Ddc Ldc:
52 TijlP CHl PH6NG CH6NG BgNH S O T RgT VA CAC BgNH KY SINH TRUNG S6 1-201
36,5%. Gia Lai: 54,7%). Tuy nhien t^ii 4 tinh Ldm Dong, Binh Phudc, Ddc Ldc, Gia Lai phd hifn dupe cd 4 lodi KSTSR tren ngudi. Ding thdi t^i 4 tinh ndy phdt hifn dupe nhilu trudn{
hpp trong mft bfnh nhdn nhiem dong thdi 3, 4 lodi KST. Qua s6 lifu phdn tich bdng ky thu^i PCR chung tdi thiy cd mdt diim chung Id ti If nhilm phii hpp rit cao. Dieu ndy cling chiing td rdng t§ii nhOng vimg ndy tin suit lan truyin cao vd lan truyin liin tyc, quanh ndm [11]. Ti If nhiem phii hpp tsii Ninh Thujn thip Id phd hpp do sau 5-10 ndm tich eye phdng ching SR, ti If ngudi nhiem KSTSR t?i tinh ndy dd gidm ddng kl.
4. KtT LU^N VA Bt X U A T 4.1. Ket luan:
Qua so lifu thu dupe bdng ky thudt PCR chiing tdi cd kit ludn sau:
- Thdnh phin KSTSR t?ii tinh Ninh Thujn giai d o ^ 2009-2010 cd mjt 3 lodi KSTSR tren ngudi P.falciparum, P. vivax, P.malariae vd KST khi P. knowlesi ky sinh tren ngudi
- Co ciu KSTSR P.falciparum chilm 63,12%, P.vivax Id 29,79%, P. malariae 4,96%.
Ddc bift phdt hifn dupe 3 truimg hpp ky sinh triing khi P. knowlesi (chilm 2,12%) ky sinh tren ngudi.
- Ti If nhiem phdi hpp trong vimg SR luu hdnh rit cao 22,6%.
4 . 2 . 9 e xuat:
Nen dp dung ky thu4t PCR dl khdo cdc lodi KSTSR khi ky sinh tren ngudi tai Vift Nam.
TAI LIEU THAM KHAO
1. Le Due Ddo, Biii Quang Phuc, Nguyen Vdn Tuin vd CS ( 2000), "Nghien cdu ca ciu ky sinh trimg sot ret d 3 tinh Lam Ddng, Binh Phudc, Ddc ldc bdng ky thu|it PCR ldng (Nested PCR)", Kyyeu cdng trinh nghien ciiu khoa hgc 1996-2000, Vi(n Sdt ret - Ky sinh triing - Cdn triing Trung uang, Nxb Y hpc,Hd Npi, Tr. 195-200.
2. Le Diic Ddo, Nguyin Vdn Tuin, Biii Quang Phuc, Nguyin Diic Giang, Truang Vdn Hanh (2002), "Xdc djnh co ciu ky sinh triing sot rft trong mdu ngdi vd phdn bift tdi phdt, tdi nhilm bdng ky thudt phdn irng chuoi Polymerase ldng t?i tinh Gia Lai", Dft dn hgp tdc Viet Nam- EC, Vifn Sdt rit - Ky sinh trimg - Cdn trimg Trung uong, Hd Npi.
3. Balbir Singh, Lee Kim Sung, Asmad Matusop, Anand Radhakrishnan, Sunita S G Shamsul, Janet Cox-Singh, Alan Thomas, David J Conway. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet 2004; (363): 1017-24 4. Brumpt, E., "The human parasites of the genus Plasmodium, in: Boyd M.F. Malariology:
A comprehensive survey of all aspects of this group of diseases from a global standpoint", Philadelphia, W.B. Saunders Copany 1949, pp.65-121.
S6 1-2012 TAP CHl P H 6 N G CH6NG BgNH S 6 T RET VA CAC BgNH KY SINH TRUNG 53
5. Goman, M., Mons, B., and Scaife, J. (1991), "The complete sequence otaP.malariae ssu- rRNA gene and its comparison to the other Plasmodials ssu-rRNA genes", M. Biochem.
Parasitol. (45), pp.281 - 288.
6. Hill S.V. et al (1991), "Common West African HLA antigens are associated with protection from severe malaria". Nature, (352), pp.595-600.
7. James, S.P.et al (1932), "A study of induced maUgnant tertian malaria". Transaction of the Royal Society of Tropical Medicine and Hygen, (25): pp. 1153-1186.
8. Scherf A. et al (1991), "Multiple infections and unusual distribution of block 2 of MSAl gene of Plasmodium falciparum detected in West African clinical isolates by polymerase cham reaction analysis". Molecular and Biochemical Parasitology, (44): pp.297-300.
9. Snewin V.A. et al (1991), "Polymorphism of alleles of the merozoite surface antigen MSAl and MSA2 in Plasmodium falciparum wild isolates from Colombia", Molecular and Biochemical Parasitology, (49): pp. 265-276,
10. Snounou, G. Viriyakosol, S., Jaira, W., Thai Thong, S., and Brown, K.N. (1993),
"Identification of the four human malaria species in fiel samples by the polymerase chain reaction of a high prevalence if mixed infection", Moi Biochem. Parasitol, (58), pp.283 - 292.
11. Snounou, G. Viriyakosol, S., Zhu, X.P., Jarra, W., Pinheiro, L., Rosario, V.E.D., Thai Thong, S., and and Brown, K.N. (1993), "High sensitivity of detection of Human malaria parasites by the use of nested polymerase chain leactio",. Moi. Biochem. Parasitol, (61), pp.315-320.
12. SnonouG. etal (1993), "The importance of sisitive detection of malaria parasites in the human and msect hosts in epidemiological studies, as shown by the analysis of field samples from Guinea Bissau", Transaction of the Royal Society of Tropical Medicine and Hygene, (87): pp. 649-653
COMPOSITION OF MALARIA PARASITES BASE ON PCR IN NINH THUAN PROVINCE
Le Due Dao', Truong Van Hanh', Nakazawa^ S., Maeno* Y., Tran Van Cu^
Nguyen Thi Thuong', Nguyen Thj Thu Huyen', Nguyin Thj Tra', Kawai^ S., Vu Quyet Thang et al.
' National institue of IVIalarioiogy Parasitology, Entomology, Hanoi, VN
^Nagoya University, Institute of Tropical Medicine, Japan ' Fujita Health University School of Medicine, Nagoya, Japan
* Ninh Thuan Center for Malaria Control
* Dokkyo Medical University, Japan
Abstract
The composition of human malaria parasites was studied by PCR (Polymerase Chain Reaction) in Ninh Thuan province within a period 2009-2010. The specific primers based on the four human malaria and P.knowlesi specific DNA sequences on the 18ssu-rRNR
54 TAP CHl P H 6 N G C H 0 N G BgNH SOT RgT VACAC BgNH KV SINH TRUNG SO 1-2012
genes. The results of PCR assay showed thai: Malaria parasite species composition in Ninh thuan province were abundant with presence of 3 human malaria parasite species:
P.falciparum was contained 63,12 %, P vivax 29,79 %, P. malariae 4,96 % and monkey malaria parasite P. knowlesi was infected in human 2,12%.
Cin bO phin bi{n PGS.TS. T? Thi Tinh Vifn s6t Tit KST-CT T l f
