Y HQC VlgT NAM T H A N G S - SO D A C BIET/2013
DANH GIA s a BQ HIEU QUA CUA PHiniNG PHAP REALTIME PCR MULTIPLEX TRONG XAC DjNH N H I I M MYCOBACTERIUM TUBERCULOSIS
TAI BENH VIEN HOU NGHI VIET TIEP HAI PHONG
Bui Thj Tuy^t Anh*, Do Ngoc Hai*, Ph^m Thj Nguy?t*
TOM TAT
Oat van de - Chan doan lao nhanh va chinh xac la van de can thiet trong dieu trj lao.
Realtime PCR Multiplex du'oc trien khai tai benh vien Hihj nghi Viet Tiep Hai Phong la mot cong cu hillj hieu trong xac djnh Mycobacterium Wberculosis
Phu'cfng phap - PCR djnh lu'tfng thdi gian that (realtime PCR) su* dung Taqman probe va 2 cap moi khac nhau trong cung mpt phan litig de xacdjnh dong thdi ISSUOva gen quy djnh 16S rRNA cua M. tuberculosis
Ket qua - Tong so 352 benh pham du'dc chl dmh lam PCR lao, gom cac benh pham dUdng ho hap va cac benh pham khong thuoc du'dng ho hap. 39 tru'dng hop du'dc xac dinh du'dng tinh d cac nong do khac nhau bang phu'dng phap realtime PCR Multiplex. Chi 2 trong so 21 ca diiWng tlnh ke tren la du'dc xac dinh du'dng tfnh bang nhuom soi.
Ban luan - Realtime PCR Multiplex co hieu qua trong chan doan xac dmh /Mycobacterium tubera/losis, ciing vdi nhuom soi gop phan tang cu'dng chan doan lao
SUMMARY
Background- Dignoslng M.tuberculosis fast and reliable is necesary. Multiplex Realtime PCR has been being developed In VietTlep Hospital Is useful diagnosis.
Methods- Realtime PCR using Taqman probe and two difference primes to recognize both iseno and 16S rRNA gene of
*Benh vien Wet Tiep
Mycobacterium tuberculosis.
Results- There are 352 specimens indicated diagnosis M. tuberculosis by realtime PCR. Kinds of specimens is diversity, respiratory and non respiratory. There are 39 possitive at ifferent M.
/^£/te/ry/o5ffi concentrations. 21 in 39 these cases are stained and observed by microscope and fluorescent microscope. Result is only 2 positive in 21.
Conclusions- Multiplex realtime PCR is effective in diagnosis M. tuberculosis
\. OAT VAN of
Benh lao do vi khuan M. tuberculosis gay ra d^n nay van la mot trong 3 benh truyen nhilm gay tir vong cao nhat the giai, tap trung a cac nuac dang phat trien. Viet Nam diing thir 12 trong so 22 nuac c6 ty Ie nhilm Iao cao nhat the gioi, hang nam co khoang 200 000 nguofi nhiem Iao cac the, trong do cd ca nhiing truang hop nhilm Iao khang thudc.
Cac phuang phap chan doan Iao: xet nghiem vi sinh (nhupm soi), nuoi cay, cac test thii duai da (Mantoux,...), chup X-quang deu CO van de ve do nhay va dp dac hieu, rieng nuoi cay thi chinh xac nhung thai gian keo dai khoang 4-8 tuan mai co kSt qua.
Vai sir phat trien ciia cong nghe sinh hpc, PCR da duac ung dimg vao trong chin doan Iao vai hy vpng la tang dp nh^y, dp dac hieu va thoi gian tra ket qua nhanh. Reahime PCR Muhiplex xac djnh M. tuberculosis da duac triln khai tai benh vien HiJu nghi Viet Tiep Hai Phong. Trong nghien cijru nay
CHUVeN B 6 : HQI NGHI KHKT CAC TINH VUNG OUYBN HAI BAU ny u n i nu n
chiing toi se phan tich va dua ra nhCtag dinh gid sa t)0 ve lii?u qua cua phuong phdp de gop phan dua iin cdi nhin day du va lihdch quan ve mi rimfc aim ciia PCR uong chan doan lao.
II. OOl TUQNG VA PHUONG PHAP NGHIEN CCfU 2.1. Bii tufng nghien ciiu
Mau b?nh phdm dugc chi djnh ldm PCR lao tai b$nh vi$n HOu nghj Viet Tiep Hai Phong trong khodng th&i gian tir thdng 07/2012 iin hit thdng 12/2012.
2.2, Tdch chiit acid nucleic
Vai cdc benh phdm Id dam, tinh djch hay dich khap g6i co dp nhdy nhat cao vd nhieu protein thi can duac XLT ly bdng dfm phosphat va dung dich mucolytic ket hpp vai N-Acetyl-L-Cystein.
Benh phdm duac ly tdm a 6500 vong/
phut, trong khodng 15-20 phut de thu cdn ti bdo va vi khuan. Tdch chiet theo phuang phap mo phong ciia Chomczynski va Sacchi (1987): 100-200 |jl benh phdm dugc cho vdo 900 pl Trizol pH8 (Invitrogen) de pha thdnh TB vd bien tinh protein. Acid nucleic sau do duac tiia bdng isopropanol voi chat tra tua co mdu hong. Tua dupc lam kho vd hoa trong dung dich TE IX (Tris EDTA), bdo quan a - 20°C cho dSn khi sir dung.
2.3. Phin img realtime PCR Multiples Phdn img dupc ttin hdnh a chu ky nhijt;
95°C trong 5 phut, va 40 chu ky lap lai vdi chi dp nhi?t 95°C-15 gidy, 55°C-1 phdt va 72°C- 20 giay.
Phdn ling realtime PCR Multiplex;la phdn ling realtime PCR sir dung 2 cjp moi hon hpp de huong tai 2 dich khdc nhau: trinh tu chta 1S6//0 vd gen quy dinh 16S rRNA, thay vi chi six dung mpt cdp 'moi hay chi huong t6i mpt dich dan le nhu PCR thong thuang. Trinh tu moi vd mau do nhu sau:
Trinh tu moi vd mau do ii xdc dinh trinh tu ddc hi?u tren gen quy dinh 16S rRNA nhir sau: 16Sf: 5'-GTG CGA GCG TTG TCC GGA AT-3'
16Sr: 5'-GCC CGT ATC GCC CGC ACG CTC ACA-3'
16Sp: 5'-TAA AGA GCT COT AGO TGG TTT GTC GCG TTG T-3'
Trinh tu moi vd mau do de xdc dinh trinh ; tu dac hieu ndm tren trinh tu chen lS(S//0:
ISeUOf: 5'-AGC CCG TCC CGC CGA TCT-3'
1S61 lOr: 5'-GCG TAG GCG TCG GTG ACA AA-3'
1S61 lOp: 5'-TCG ACC TGG GCAGGG TTC GCC TAC -3'.
III. KET QuA
Dac diem phdn bo cda benh nhdn theo d^c trung khoa, phong trong benh vien Bdng 1: Phan bo benh nhan chi dinh ldm PCR iao
STT 1 2
3
KHOA Than kinh Hoi su'c npi
Npi 2
Am tinh 84 59
54
Du'dng tinh
1 4
18
Dac diem benh pham Djch nao tiiy
Kiem tra lai: 3
Dich nao tiiy, djch mang phoi, djch mang bung, tam bong ngoay hpng...
Kiem tra lai: 3
Djch mang phoi, djch Ichcfp goi, dcim Kiem tra lai: 4 d6m, 8 dich manq ohoi 134
Y HQC VI j T NAM T H A N G 5 - S6 PAC BlfeT/2013
4 5
>
6 7 8 9 10
Lay Tim mach
N o l l
Npi 3
Noi 4 I5ng ngUc
Phong kham va cac khoa
khac
38 20 17 10 5 4 14
9 2 1 Khong
1 1 3
Dich nao tiiy, co ca djch mang phoi, idm Kiem tra lai: IDNT
Ddm, djch mang phoi, djch mang ngoai tim Kiem tra lai: l D 6 m
Dich 6 bung, doi khi co dcJm Kiem tra lai: Idich 6 bunq, Idcfm Djch mang phdi, d6m
Kiem tra lai:Iddm, 1 dich manq phoi Khonq CO
Djch mang phoi, djch mang ngoai tim Kiem tra lai: 1 dich manq phoi
Oa dang: ©dm, tinh djch, nUdc tieu, djch l<hdp goi, DMP...
Kiem tra lai: 2
Nhu vay trong 6 thang cuoi nam 2012, CO 352 benh pham Iam realtime PCR Iao tir tat ca cac khoa khac nhau trong benh vien.
Co mau benh pham ciia ducmg ho hap nhu dom, dich mang phoi, tam bong ngoay hong..., va cac benh pham khong phai cua duong ho hap nhu tinh dich, nuac ti6u, dich nSo tliy, dich khap goi, dich mang ngoai tim, dich 6 bung, hach, mu hach, dich ap xe.
Trong do benh pham la djch nao tuy va dich mang phoi chiem so lupng nhieu hon ca. Co 39/ 352 truong hpp benh pham dupc xac djnh duong tinh. Khoa Npi 2 co ty Ie benh phSm duong tinh cao nhSt, chiem 24% tong so benh phSm gui Iam. Ben canh do c6 den 26/ 352 benh prnn la phai kiem tra Iai do khi chay Realtime PCR CO bieu hien phan iing bi uc chS.
Trong phan iing realtime PCR chiing toi su dung a day ngoai probe gan tin hieu FAM de cho tin hieu benh pham duang tinh thi chiing toi con su dung probe mang tin hieu HEX Iam chung npi, de giam cac truong hap am tinh gia do phan ung bi uc che. Phan ung realtime PCR la mot phan ung rat nhay do do benh pham, cGng nhu nhung thao tac de tien hanh phan ung can rat chinh xac, de toi thieu hoa cac sai so.
Dp nhgy vd dp dgc hiiu ciia pitdn irng Realtime Multiplex PCR
Dp nhay cua phan ung
Trong tong so 352 truang hop dupc chi dinh Iam Iao trong 6 thang cuoi nam 2012, co 39 benh pham dupc xac dinh duang tinh.
Bang 2: Ty le benh pham duang tinh a cac mirc do khdc nhau Mii'c d o du'dng t i n h
DifOng tlnh dudi ngUSng ( < 3 0 0 copies/ mil
10^ coDies/ mi
; 10' cooies/ mi
^O^ippies/ ml
Sd lu'dng b§nh pham 23
4 5 3
Tv le {%) 58.97%
10.25%
12.82%
7.69%
CHUVfeN oi: HQI NGH| KHKT CAC TINH VUNG DUVfeN HAI BAC BQ LAN JHJJ NHAT
Nhu v^y tren 50% so truong hg'p duang tinh I^ nh&ng trudng hpp duong tinh yeu, v6i chu ky ngu&ng dao dpng khoang tir chu ky 36-40, so lupng vi khuin c6 the tir 10 den <
300 copies/ ml. Cdc trudng hpp duong tinh CO tin hi?u huynh quang vupft qua tin hi?u nen so Iugng vi khuan dao dpng tCr 10^ den 10* copies/ml. Vi phan Idn cdc b?nh nhan lao CO bieu hi?n va tri?u chiing r6 rang it khi co t^ B?nh vi?n Hihi nghj Vipt Ti?p Hdi Phong, ben canh d6 neu b?nh pham nhupm soi duong tinh r6 rang thi cung c6 the sg khong giii Iam realtime PCR lao, chinh vi v^y ty le benh pham duong tinh chi co khodng tren 10% (39/352), va trong s6 do thi hon mia la cac trucmg hpp duong tinh y^u.
Khi so sdnh cac ket qud duong tinh nay vai cdc ket qua nhupm soi true ti^p do khoa Vi sinh benh vien hiiu nghj Viet Tiep cung cap, chiing toi thay co sir sai khac Idn vk dp nhay giua 2 phuang phap nay. Trong s6 39 trudng hpp duang tinh k^ tren, chi co 21 tnrdng hcrp duprc giii nhupm soi ben vi sinh.
K^t qua nhupm soi duong tinh vdi chi 2 trong so 21 trudng hpp da dupc xac dinh la duang tinh bdng realtime PCR lao.
Trong 2 trudng hpp co k6t qua duong tinh vdi cd nhupm soi va PCR thi mpt trudng hop benh pham la dich phe quan, n6ng dp vi khuin dupc xac djnh la 10^ copies/ ml.
Trudng hap duang tinh thii 2 la tren b?nh nhan HIV va mau ddm dup'c giri nhupm soi, mau PCR la djch nao tuy. Nhu vay co \hk thay ve co ban nhupm soi thudng chi cho kit qud duong tinh vdi nhijng trudng h(;yp Immg vi khudn trong benh phdm nhilu.
Dp ddc hieu ciia phdn iing
Khi su dyng cac m6i dupc thilt kl rieng cho phdn iing realtime PCR xdc djnh vi khuan Iao ydi cdc mau benh pham am linh vdi vi khudn lao va duong tinh vdi cac vi
khuiri hay virus kh^c nhu vdi Hepatitis B virus,' Cytomegalovirus, Escherichia CQ^
Shigella sonnei deu cho cac tin hi^u huynh quang khong co chu ky ngudng va khong vuiyt qud chu ky nen. Nhu vay cd the so bp cho thay tinh ddc hieu ciia moi va mau do.
Trong so cdc trudng hpp duang tinh dup'c xdc dinh bdng realtime PCR Multiplex khong thdy cd trudng hpfp ndo cd su phan hSi tu cac khoa Iam sang la b?nh nhan duong tinh gid. Ben canh do do ddc dilm ciia b^nh pham Id rat hiem mau duang tinh, do dd ducmg tinh gid do dong nhiem tir mau np sang mau kia Id hdu nhu khong thi xay ra.
Mdc du vay, van d& nay chiing toi ciing da tiing g^p phdi khi Idm vdi ciing Iiic nhik benh phdm Iao HIV duong tinh manh trong dieu ki?n khong co tu an toan sinh hpc cdp II.
Khong he cd mau benh pham ndo nhupm soi vi sinh duong tinh ma Iam PCR am tinh, tuy nhien cung khdng the chdc chdn la ta tinh gia khong he xdy ra vdi phuang phap PCR Iao nay vi vi khuan lao la mpt trong nhiing lo^ vi khuan khd tdch chilt va xac dinh nhdt, la mpt thdch thiic vdi tdt ca cac hang hoa chat ciing nhu cdc kit tdch chiet va chan doan dang dupc su dyng tren the gidi.
IV. KET LUAN
Trong khuon kho nghien cuu nay ciia minh, chiing toi mudn giii gdm den mot dieu rdng chiing toi c6 gdng trong kha ndng cao nhat de dua den mpt phuang phdp chdn doan Iao nhanh va hi?u qua bdng Realtime PCR Multiplex, ben canh phuang phdp nhupm soi vi khudn Iao hien nay tai benh vien.
Vdi realtime PCR Multiplex hudng toi 2 dich la: Gen quy dinh I6S rRNA chi co 1 ban sao trong te bdo, nhung te bao ndo cung co va IS61W d$c hieu cho M.tuberculosis co nhieu ban sao trong Wt bdo, nhung 2%
136
Y HOC VlgT NAM T H A N G 5 - Sti P A C BlgT/2013
chiing Iao d Vi^t Nam thieu doan chen ndy.
Nhu vdy phircmg phdp nay da tinh tdi tinh viing mien, sao cho phii hpp vdi cac chiing lao d Viet Nam. Mdt khac phuang phap nay CO thi tiln hdnh vdi tdt cd cac b?nh pham khac nhau, ca benh phdm dudng h6 hap va benh phim khdng thupc dudng ho hdp vd gid thanh lai re hon nhieu, dieu nay da duprc nhilu bdi bdo khdng dinh Id uu the hem ciia realtime PCR so vdi he th6ng tu d^ng h6a nhu GenXpert.
TAI UEU THAM KHAO
1. G M Mulcahy, Z C Kaminski, E A Albanese, R Sood and M Pierce: 186/70- based PCR methods for detection of Mycobacterium tuberculosis. J. Clin.
Microbiol 1996, 34(5): 1348.
\. Melissa B. Miller, Elena B. Popowitch, Michael G. Backlund and Edward P. C.
Ager: Performance of Xpert MTB/RIF RUO Assay and IS 6U0 Real-Time PCR for Mycobacterium tuberculosis Detection in
Ciinical Samples. J. Clin. Microbiol. 2011, 49(10):3458. DOl
M Maher, M Glennon, G Martinazzo, E Turchetti, S Marcolini, T Smith and M T Dawson: Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples. J. Clin. Microbiol. 1996, 34(9):2307.
Sathish Sankar*, Suresh Kuppanan, Babu Balakrishnan, Balaji Nandagopal:
Analysis of sequence diversity among IS6110 sequence of Mycobacterium tuberculosis: possible implications for PCR based detection. Bioinformation (2011). Vo!
6(7).
Syivie Armand, Pascal Vanhuls, Guy Delcroix, Rene Courcoi and Nadine Lemaitre: Comparison of the Xpert MTB/RIF test witli an IS(5/70-Taqman Real- time PCR Assay for direct detection of Mycobacterium tuberculosis in respiratory and non respiratory specimens. J. Clin.
Microbiol. 20\\, 49(5): Mil.
