DfNG DUNG KYTHUAT REALTIME PCR OEXACejNH 3 NHOM VI KHUAN RICKETTSIA
GAY BENH SdT CAP TINH d NGl/dl
Le Thj Hpi', Vu Ngoc Lien', Tran Mai Hoa', Nguyen Vu TVung', Nguyen Van Kinh'.
Muc tieu: Xay dung phuang phap Realtime PCR dd xac dinh cac vi khuan O. tsutsugamushi gay benh sot mo, vi khuan R. typhi gay benh sot dich te va nhom vi khuan Rickettsia spp. gay benh sdt phat ban. Doi tucmg vd phuomg phap: Dung phuang phap tach chiet PBMC, ADN, Reahime PCR. Plasmit ADN duoc su dung Iam chung ducmg dd tdi uu phuotig phap Realtime PCR. Cac mau benh pham duac thu ttr 40 benh nhan s6t c4p tinh nghi ngd nhilm vi khuan rickettsia. Kit qud: Phuang phap Reahime PCR xac dinh dugc 3 nhom vi khuan rickettsia (nhom O. tsutsug- amushi, nhom R. typhi va nhom Rickettsia spp.), cho ket qua nhanh, thuc hien true tiep tu mau benh pham mau. Vdi so lugng mau 40 benh nhan sot cap tinh chua ro nguyen nhan, da xac dinh dugc 23% do vi khuan Rickettsia, trong do O. tsutsugamushi va sdt do R. typhi chiem ty Ie 12,5% va 7,5%. Ket lugn: Ky thuat Realtime PCR xac dinh dugc 3 nhom vi khuan rickettsia gdy benh sot cap tinh {O. tsutsugamushi, R. typhi va nhdm vi khuan Rickettsia spp.), co dp nhay cao. Ky thuat nay cho ket qua nhanh, chinh xac, thuc hien dugc true tiep tu benh pham mau. Tai cac benh vien nen trien khai ky thuat Realtime PCR de xac dinh Rickettsia.
Tir khoa: Rickettsia, sdt mo, sot do bg chet chugt truy8n, s6t phat ban.
DAT VAN OE
Cac vi khuan thudc hg Rickettsiaceae, gom hai chi RIckedsia va Orientia la cae vi khuan gram am, co kha nSng gay benh d ngu'di va ddng vatf^J. DuS vao dQ" lieu di truyen va dnh khang nguyen, cac Rid<ettslaeeae dddc chia lam 3 nhdm: (i) nhdm gay benh sdt djch te do R. typhi; (ii) nhdm gay benh sdt phat ban do eac loai Rickettsia spp;
(lii) nhdm gay benh sdt md do 0. tsutsugamushf^.
Cac ket qua dieu tra cho thay cac vi khuan thupe hp Rickettsiaceae cd mat va gay benh d hau het cae ehau lycP]. Tai Viet nam, cac vi khuan hp Rickettsiaceae eung da duHc ghi nhan cd mat va gay benh kha pho bieni^^PJ.
Tuy nhien trong sudt thdi gian qua, do nhutig han che cua
"Bfnh vifn Bfnh nhift dai Trung uang Ngi> a h f p b i i : 10/10/2015.
Ngiy p h i n bifo xong: 15/10/2015.
N g i y duyf I d i n g : 30/10/2015.
Ngirdi chju ( r i c h nbifm n^i d u n g khoa h f c : Le Thi Hpi. Khoa Xet nghifm, Bfnh vifn nhift dai Thing utmg
Difnlho^i 0986161979. E-mail Iehot2003@gmailcom
cac ky thuat vi sinh nen viec chan doan benh d cac tuyen dieu tri chu yeu du^ vao bieu hien lam sang. Hien nay ky thuat smh hpc phan tu", cu the la PCR du'dc khuyen cao la mpt xet nghiem cd dp nhay va do dac hieu cao. Phu'dng phap nay dap utig yeu cau chan doan nhanh va dang dupe utig dung tai cac phdng xet nghiem tren the gidi. Vi vay chiing tdi tien hanh nghien cutj nay v ^ mue dieh: LTng dung ky thuat Realtime PCR de xac djnh chan doan 3 nhdm vi khuan rickettsia gay benh sdt cap b'nh d ngUdi.
DOI Tl/ONG, PHUONG PHAP NGHIEN CUtJ 061 tu'dng: (i) Plasmit chuan du'dc cung cap tij" Phdng thi nghiem Rickettsia, Tnjng tam nghien cuU y sinh Naval - My lam chutig du'dng, de phat hien 3 nhom vi khuan rick- ettsia gay benh sdt cap tinh. (ii) 40 mau benh pham mau (4ml/mau) cua 40 benh nhan sdt cap b'nh nghi ngd do rickettsia. Benh pham dUpc chdng dong bang EDTA.
Phu'dng phap: Day la nghien CUIJ litig dung, du'dc thdc hien ta\ lOioa Xet nghiem, Benh vien Benh nhiet ddi Trung u'dng.
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« I Phu'dng phap t i e n hanh:
1. Thiet ke cae cap moi va Taqman probe: Cae cap moi va mau dd Taqman probe dddc thiet ke de nhan eac doan gen: (i) Gen ma hoa protein mang ngoai cua O. tet/t- sugamushi cd trpng ludng phan t d 47 kDa; (ii) Gen ma hoa protein Ompecua R. typhiva (iii) Gen ma hoa protein mang ngoai ehung cho 2 nhdm R. typhiva Rickettsia spp.
CO trpng lu'dng phan tu" 17kDa. Mdi va Taqman probe ciia 3 gen du'dc trinh bay d bang 1.
Bang 1 : Trinh td cac cap moi va probe cua cac gen 47kDa, 17kDa va OmpB
Ten
Pnmer 47F Primer 47R Probe 47
Pnmer 17F Primer 17R Probe 17 OmpBF OmpBR OmpB probe
Trinh t v (5" - 3') AACTGATiTTATTCAAAaAATGCrGCr TATGCCTGAGTAAGATACRTGAATRGAATT 6FAM - TGGGTAGCTTTGGTGGACCGATGTTTAATCT - TAMRA
GGGCGGTATGAAYAAACAAG CCTACACCTACTCCVACAAG
FAM - CCGAATTGAGAACCAAGTAATGC • TAMRA TGGTATTACrGCrCAACAAGCr CAGTAAAGTCTATTGATCCTACACC FAM-CGCGATCGTTAATAGCAGCACCA6CATTATCGCG - B H Q - 1
2. Xd ly mau benh pham va tach ldp te bao mau ddn nhan ngoai vi (PBMC) theo phu'dng phap gradient trong IUdng:
Hut 4ml dung djch Ficoll vao dng Falcon 15mi. Sau do nho tii tif vao dng Falcon toan bo 4ml mau benh pham.
Ly tam 1800 vong trong 20 phut d nhiet dp phong.
Hut Idp tren eung ehu^ huyet tu'dng ra dng eppen- dorf. Hut het Idp tiep theo ehu^ PBMC re ong Falcon 15ml. Loai bo ldp du'di cung ehiis hong cau.
3. Tach chiet ADN tdng sd: 200p] PBI^C du'dc vortex va chuan bj tach AND. Tach chiet DNA du'dc thUc hien theo hu'dng dan tren bp kit QIAamp DNA Mini Kit (Qia- gen, Du'c). Cac mau AND tach chiet ti^ benh pham dupe sir dung lam khuon cho phan utig Realtime PCR.
4. ThUc hien phan utig Realtime PCR
Qua trinh chuan bj mastermix du'dc tien hanh theo cdng thu'c sau:
Master Mix Pnmer F Primer R Probe MgCI2 H20 Template
12,5|J|
0,75 Ml 0,75^1 0,5 Ml 1,5 Ml 4^1 5 Ml Chu trinh ehay
Bien b'nh liic dau Bien tinh GSn moi
Nhiet do 9 5 ^ gs^C eo^c
Thdi gian 10' 20s 30s
40 chu ky
KET QUA
Cac plasmid chuan dddc sd dung lam chutig du'dng dupc cung cap bdi Tmng tam nghien cuU Y hoe Navai, My.
Cae plasmid nay dUdc pha loang tii nong dp 10^ copies den lO'copies de tdi du dieu kien phan dng Realtime PCR cho 3 gen moi. Ket qua Realtime PCR d nong do 10' copies tren ca 3 gen deu khong phat hien du'dc. NhUng c(
nong dp 10^ va IO"* copies ca 3 gen ma hoa protein mang ngoai 0. tsutsugamu^i (trpng lu'dng phan tir 47kDa), gen ma hoa protein mang ngoai R. typhi va Rickettsia spp (trpng lu'dng phan tir 17kDa) va gen ompB cua R. tyf^i deu du'dc phat hien va cd ti'n hieu huynh quang d cac chu ky 34 vdi nong dp plasmid la 102 copy va chu ky 28 vcfi ndng dp la 10" copy (Hinh 1, 2, 3).
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Hinh 1 : Ket qua Realtime PCR gen ma hoa protein mang ngoai O. tsutsugamushi
(trong li^dng phan til' 47kDa) PCI - 10": plasmid co nong dp ID* copy; PCI - llf.
plasmid CO nong dp W copy; NTC: ciiutig am
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Hinh 2: Ket qua Realtime PCR gen ma hoa R. typhiva Rickettsia spp (trong lu'dng phan tuT 17kDa) PCI - 10": plasmid cd nong dp 10" copy; PCI - 10^:
plasmid co nong dp 10^ copy; NTC; chutig am.
Hinh 3: Ket qua Realtime PCR doi vdi gen ompB cua R. typhi
PCI - 1 0 " : plasmid c6 nong dp 10" copy; PCI -10^:
plasmid ed nong dp 10^ copy; NTC: chiitig am.
Cac ket qua tren cho thay cac dieu kien phan utig Re- altime PCR da dddc tdi Uu hoa, vdi dp nhay cho 3 gen d nong dp plasmid la 10^ copy. Sau khi da tdi uli phUdng phap, chung toi tien hanh phan uTig Realtime PCR cho 40 mau benh pham dUdc thu thap tai Benh vien Benh nhiet ddi Trung ddng, d nhutig benh nhan sdt cao chUa ro nguyen nhan, trong do cd benh nhan cd vet ddt dien hinh ciia sot md ( 0 . tsutsugamushi). NhiJYig benh nhan nay deu da dUdc sang Ipc cac can nguyen gay sdt thudng gap nhu" cae vi rut sot xuat huyet, cum va mot sd benh nhiem trung. Ket qua cho thay 9/40 mau benh pham dUdng tfnh vdi vi khuan Rickettsia (chiem 23%). Cu the 5 mau du'dng tf'nh vdi 0. tsutsugamushi{12.5%), 3 mau du'dng tfnh vdi R. typhi (7.5%) va 1 mau dUdng tinh vdi Rickettsia spp (2.5%). Ket qua xet nghiem 8 mau benh pham cd chan doan nghi ngd nhiem Rickettsia dupc trinh bay d hinh 4,
5 va 6. Cac benh nhan so 1, sd 3 va sd 4 tren lam sang cd cac vet dot d cac vj trf khae nhau va deu du'dng b'nh vdi 0. tsutsugamushi {Hmh 4).
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Hinh 4: Ket qua Realtime PCR cua gen ma hoa cho protein mang ngoai vi khuan O. tsutsugamushi co trong lUcfng phan tit 47 kDa tren 8 b^nh nhan
Hinh 5 la ket qua cua benh nhan sd 2 va 7, di/dng tinh vdi gen ma hoa protein mang ngoai eiia vi khuan co trpng lu'dng 17 kDa, day la gen chung eho R. typhi va Rickettsia spp.
Hinh 5: Ket qua Realtime PCR cua gen ma hoa cho protein mang ngoai nhom vi khuan Rickettsia spp. co trong lu'dng phan tu' 17 kDa
Oe xac djnh vi khuan R. typhi, chung toi thu'c hien xet nghiem Realtime PCR vdi gen ompB. Ket qua eho thay benh nhan sd 2 dUPng ti'nh vdi gen ompB (Hinh 6).
Hinh 6: Ket qua Realtime PCR vdi gen ompB tren benh nhan sd 2
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BAN LUAN
Benh Rickettsia dieu til khong kho khan do vi khuan nay van nhay cam vdi cac khang sinh ttiong thu'dng. Tuy nhien, neu chan doan diam tre, cd tiie de iai nhutig bien chutig nang ne, hoac cd tfie gay tii vongt^^. Nhiem vi khuan ho Rickettsiaceae tiiu'dng tiieu chutig khdng dien hinh, dii cd bieu hien sdt keo dai khd chan doant^l Do vay, viec chan doan can nguyen gay benh sdm, chfnh xac cd y nghTa idn trong dieu h i .
Hien nay nhiaj phdng tiif nghiem tren thegidl da phat trien cae phUdng phap xac djnh benh Ricketisia khac nhau. Trong dd tfeu chuan vang la phUdng phap mien dicti huynh quang gian tiep IFA. Tuy nhien phu'dng phap nay ddi hoi phai trang bi kfnh hien vi huynh quang cung nhU yeu cau ky thuat cao. Do vay phUdng phap nay kho utig dung rong rai tai eae benh vien d Viet Nam. Chan doan sdt md bang phUdng phap ELISA cOng dude phat trien. Tuy nhien, phUdng phap nay iai cd ban che do dap iitig mien djch khang tfie chi phat hien dUde 6 tuan tfid hai ciia benh va khong cd ngudng gia trj thap cua hieu gia khang the, hdn niJa ddi hoi phai lay mau 2 lan de tim dgng lu'c khang thet'i. Ngoai ra phu'dng phap ELISA thudng xuat hien phan LJtig cheo, dan den chan doan nham lln. Ben canh cac phu'dng phap xac dinh neu tren, phUdng phap nuoi cay cung du'pc thUc hien. Tuy nhien Rickettsiaceae la hp vi khuan ky sinh ndi bao bat buoc va rat kho nudi eay.
Ngoai ra do tfnh chat nguy hiem ciia vi khuan nay doi hoi phai thUe hien trong phdng xet nghiem an toan cap 3 trd len. Vi vay nen phUdng phap nay khdng the thUc hien du'dc d cac phdng xet nghiem thdng thudng.
PhUdng phap Realtime PCR da dUdc chutig minh cd
dp nhay va do dac hieu cao hdn so vdi phudng phap PCR tfiudng. Ky thuat nay ed utj diem cho ket qua nhanh, co the biet ket qua ngay sau moi ehu ky eua phan utig, dSc biet sir dung Taqman probe du'dc tfiiS ke dac hieu vdi gen dich. Tnjng nghien cifu nay ehung tdi da tdi UU hoa ky thuat Realtime PCR de xac djnh 3 nhdm vi khuan gay benh Rickettsia. Ket qua cho tfiay phu'dng phap Realtime PCR xae djnh du'dc cae vi khuan 0. tsutsugamushi gay benh sdt md, vi khuan R. typhi gay benh sdt dieh te va nhdm vi khuan Rickettsia spp. gay benh sdt phat ban. Ket qua nay tu'dng tu" vdi cae nghien cutj ciia Dong - Mm Kim™, Tasawan SinghsilarakPi va Ju jiangt^l. Vdi sd lUdng m l u la 40 benh nhan sdt cap ti'nh chu'a ro nguyen, ket qua xae djnh ban dau cho thay 23% dupc xac (finh do rickettsia, trong do 0. tsutsugamushi va sdt do R. typhi chiem ty le 12,5% va 7,5%. Day la can nguyen chiem ty le kha cao trong nhdm benh nhan sdt cap tfnh nhap vi§n chUa ro nguyen nhan.
KET LUAN
Ky thuat Realtime PCR xae djnh 3 nhom vi khuan rick- ettsia gay benh sot cap tinh (vi khuan 0. tsutsugamushi gay benh sdt md, vi khuan R. typhi gay benh sdt cap tinli do bp chet ehupt truyen va nhom vi khuan Rickettsia spp.
gay benh sdt phat ban) tru'c tiep tir mau benh pham co do nhay eao. Phu'dng phap nay cho ket qua nhanh va chinh xac, thdc hien trUc hep tii" mau benh pham. Ky thuat Re- altime PCR nham xac djnh cac tac nhan gay b&nh Rick- ettsia nen dddc trien khai tai eac benh vien, giup chan doan sdm va dinh hudng dieu trj. De cd ket luan ve t / le nhiem cae nhom vi khuan rickettsia can du'dc tien hanh tren sd lu'dng mau Idn hdn.
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TAI LIEU THAM KHAO
1. Allen L. Richards (2012). Worldw/ide detection and identification of new and old riekettsiae and rickettsial dis- eases. FEMS Immunol Med Microbiol. 64(1): 107 - 1 0 .
2. Behzad Nadjm, Pham T Thuy, Van D. Trang, Le Dang Ha, Nguyen V Kinh and Heiman F. WerUieim (2014).
Scrub typhus in the nortfiern provinces of Vietnam, an ob- servational study of admissions to a national refen^l hos- pital. Trans R Soe Trop Med Hyg; 108(11): 739 - 740.
3. Daryl J. Kelly, Allen L Richards, Joseph Temenak, Daniel Strickman, and Gregory A. Dasch (2002). The Past and Present Threat of Rickeflsial Diseases to Military Med- icine and Internatfonal Public Health. Clinical Infectious Diseases 2002; 34(Suppl 4): 5145 - 69.
4. Dong-Min Kim, Geon Park, Hyong Sun Kim, Joo Young Lee, Ganesh Prasad Neupane, Stephen Graves, and John Stenos (2011). Comparison of Conventional, Nested, and Real-Time Quantitatfve PCR for Diagnosis of Scrub Ty- phus. J Clin Microbiol. 2011 Feb; 49(2): 607 - 612.
5. Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, Ray SC, Rikihisa Y, Rurangirwa FR (2001). Reorgani- zation of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: Unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with NeorickeOsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol. 51(Pt 6): 2145 - 6 5 .
6. Ju Jiang, Teik chye chan, Joseph J, Temenal, Gre- grory A, Dasch, Wei Mei Ching, and Allen L Richards (2004). Development of a quantitative Realtime Poly- merase Chain Reaction assay specific fororientia Tsutsug- amuehi. The American Society of Tropical Medicine and Hygiene. 70(4), 2004, pp. 351 - 356.
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Short report: detection of Orientia tsutsugamushi in clini- cal samples by quantitative real - time polymerase chain reaction. The American Society of Tropical Medicine and Hygiene. 72(5): 640 - 1 .
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9. Sugihiro Hamaguchi, Ngo Chi Cuong, Doan Thu Tra, Yen Hai Doan, Kenta Shimizu, Nguyen Quang Tuan, Lay - Myint Yoshida, Le Quynh Mai, Dang Due - Anh, Shuji Ando, Jiro Arikawa, Christopher M. Parry, Koya Ariyoshi, and Pham Thanh Thuy (2015). Clinical and Epidemiological Charac- tenstics of Scrub Typhus and Murine Typhus among Hospi- talized Patients with Acute Undifferentiated Fever in Northern Vletiiam. Am. J. Trop. Med. Hyg. 972 - 978.
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DEVELOPMENT OF REALHME PCR FOR DETECHON OF RICKETTSIA DISEASES I N HUMAN
Summary
Objectives: develop Realtime PCR assays with high sensitivity for detection of RickeOsia diseases including scrub typhus group (STG) caused by 0. tsutsugamushi, typhus group (TG) caused by R. typhi and spotted fever group (SFG) caused by Rickettsia spp. in clinical samples.
Objects and methods: The DNA plasmids were used as positive eonfrols for optimizing the Realtime PCR assays.
Qinical specimens were collected from 40 suspected cases infected with Rickettsia species. Methods: PBMC extinc- tion, total DNA extraction, Realtime PCR assays. Results and conclusions: The Realime PCR assays have approved highly sensitive, accurate and reliable for tfie detection of scrub typhus 0. tsutsugamushi, spoH:ed fever Rickettsia spp. group, typhus group R. typhi in clinical samples. In this study, we collected samples from 40 patients with
acute fever of unknown causes admited to the National Hospital of Tropical Diseases. The results showed that ttie prevalence of rickettsia infection was 23% in which scrub typhus caused by O.tsutsugamushi and typhus R. typhi group accounted for 12.5% and 7.5% respectively. The high prevalence of rickettsia infection was detected in pa- tients with fever of unknown origin. Therefore there is an urgent need to apply the Realtime PCR technique to de- tect rickettsia diseases in ttie hospitals which have infec- tious diseases department, in the institutes of preventive medicine in order to diagnose eariy stage of rickettsia in- fection. Early, accurate diagnosis is beneficial for patients and prevents complieatf ons occurring due to delays of di- agnosis and tfeatment.
Key words: Rickettsia, scrub typhus, typhus group, spotted fever group.
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