THANH TI^U HOAT BQNG KH, CN VA MT
0ng lung ky Ihual PCR
TRONG C H X N D O A N LAO PHdl M (-), LAO NGOAl P H 6 | , L A O K H A N G THUdC
TAI B E N H V I E N L A O V A B e N H P H 6 | C A N T H d
BS CKINeuyin Thi Thanh Nhdn vd ThS BS DS Hodne Lone Benh vien Lao vd B?nh phdi Cdn Tha
1 3
1. D a t vS'n d§
Vi khuan lao Mycobacterium tuberculosis la thu pham gay nen benh lao d ngifdi. Theo Td chd'c Y te The gidi (TCYTTG) ifdc linh cd 9,4 trieu benh nhdn lao mdi va hdn 1,8 trieu benh nhan lao id" vong. Trong nhffng nam gan day, sif Ian rdng vd dich te hdi chiJng suy giam mien dich m^c phai (AIDS) tren thd gidi da dnh hifdng nang ne ddi vdi benh lao d cdc nifdc dang phat tridn cung nhif d cac nifdc phat Iridn. nguydn nhan ndy da 1dm ISng sd ngifdi mac lao vd chdt do benh lao, nguy hidm hdn la ngifdi nhiem HIV mac benh lao rd't dd phdt sinh ra vi khu^n lao khdng tiiud'c. Tinh hinh lao/HIV va lao da khdng thud'c dang Id van dd nghidm trgng tiren todn cau de dga sif thanh cdng trong cdng tdc chdng lao.
De han chd sif gia tdng ty le mdc bdnh lao mdi, lao da khdng thudc do Mycobacterium tuberculosis gdy ra, viec phdt hien sdm cdc benh lao mdi va lao da khdng thudc dd qudn IJ vd dieu tri kip thdi la viec ra't can thidt cho Chu'dng trinh chdng lao qudc gia (CTCLQG).
Hien nay, trong chan dodn lao thifdng difa vao kdt qua soi dam trifc tidp tim vi khuan lao vd nudi ea'y vi khuan lao tif cdc m§u benh ph^m nhif (ddm, dich nao t i y , dich mdng phdi...) u-ong cac mSu sinh thidt, phifdng phap soi tnfc tidp b i n g kinh hidn vi cho bidt kdt qud nhanh tiiidu dd nhay, do dac hieu vd phu'dng phdp nudi ca'y cd dd nhay vd do ddc hidu cao hdn vd la tieu chuan vang trong viec ch^n dodn benh n h d n J a s - j a f e a J a ^ H B i B ^ ^^^
cham sau 8 tuln. ' ^ ^ l
IDCCAN THd
THANH TUU HOAT BQNG KH, CN VA MT
Do dd, viec phdt tridn mdt quy trinh ky thuat mdi trong chan doan benh lao vCfa nhanh vita nhay cam vifa dac hieu cao la nhu cau can thie't tai Benh vien Lao va Benh Phoithanhphd'CanThd.
2. Muc tidu nghien cd'u:
L/ng dung ky thuat PCR trong chan doim lao phoi M (-) vd lao ngodi phdi; u'ng dung ky Ihual giai trinh tif gen trong chan doan lao khdng thud'c va so sanh phu'dng phdp PCR vdi cac phifdng phdp thdng thifdng khae trong chan doan lao phdi M (-) vd lao ngodi phdi.
3. Phifdng phap nnhicMi cvCu:
- Thie't ke' nghien cifu: Nghien cifu md hi cat ngang.
- Cachchgn man: chgn loan bg so'benh nhan lao phdi M (-), lao ngoai phdi va lao tai Iri de'n khdm tai Benh vien Lao & Benh phdi tif 08/2008 den thdng 09/2011.
- Bie'n so' nghien cifu:
* Dac diem nghien cd'u xa hgi ciia ddi lifdng nghien ciJu-
-t- Tudi: Tinh iheo sd tuoi. chia lam 04 nhdm:
< 20; 20 - 40; 41 - 60 va > 60.
+ Gidi: nam va nif
* Kdt qua chan doan lao phdi M (-) va lao ngoai phdi:
+ Ke't qua xel nghiem soi infc tie'p cac man benh pham
+ Ke't qua cay cac m l u benh pham + Ke't qua giai phau benh sinh thidt md viem lao
* Danh gia ke't qua lao khdng thud'c bang giai trinh tif gen'
- Cac ky thuat su" dung:
+ Soi tifdi dam (AFB): benh phd'm (2ml) difdc phe't mong tren lam kinh cd difdng kinh 2cm x 1cm du'dc cd' dinh bang each hd nhanh qua lifa 3 Ian rdi nhudm theo phifdng phdp Zielh-Neelsen
+ Nudi cay benh pham: 10ml benh pham du'dc lay tif benh nhan nghi lao. Cho benh ph£m (ddm) vao d'ng nghiem. Sau dd them mdt the lich NaOH - NaCl (0,25M NaOH, 25mM Na - citra.
15mM N-acetyl-cysten) bang vdi thd tich benh ph^m. Ddnh tan ddm bang may vortex trong 5 giay. Dd trdn mdy l^c 20 phut. Cho them vao d'ng nghiem mdt lifdng ntfdc ca't vd triing. Ly tdm 15 phut vdi td'c do 2.600 l/phut. Bo phan nifdc ndi guT lai can lang. Tidp Iheo la'y phan can ca'y len mdi tru'dng Lowcnslcin-.lensen, de lii a'm 37"C va doc ke'l qua sau 30, 60, 90 ngay.
+ Phan ifng khue'ch dai gen (PCR):
- Ly trich ADN ciia te' bao vi khuan lao bang phifdng phdp Boom.
- Phan u'ng khue'ch dai gen du'dc ihifc hien vdi PCR mix ed L-;ic doan mdi (primers) Ptl8 va INS2 cho doan gen 1S6110 ddc hieu ciia Mycobacterium tuberculosis.
- Kd't qua ciia phan ifng khuech dai gen difdc dgc tren gel agarose 2'/< cd ethidlum bromide vd difdc chup phim ky thuat sdde lifu cd sd khoa hgc.
+ Tinh khiel san pham PCR: san pham PCR lao khdng thud'c se difdc tmh khie't bang hda cha't va ca't giu" cho de'n khi difdc giai trinh lif ADN
+ Giai trinh lif gen: Kit va may gidi trinh ADN se giup tim ra doan gen quyet dinh cho sif khdng thud'c cua vi khuan lao dd'i vdi rifampicln hoac isoniazid.
+ Sinh thidt vd dgc lieu ban giai phau benh:
mang phdi ciia nhifng benh nhan nghi lao mang phdi se difdc sinh Ihidl va gdi de'n phong giai phau benh di lam lieu ban va dgc ket qua.
+ Sinh hda va te' bao: Cac mau benh ciia lao ngoai phdi nhif dich mang nao, dich mdng phdi, dich mang bung .. difdc xac dinh cac thanh phan sinh hda va te' bao cd Irong dich bang may smh hda va huye't hgc.
4. Ke't qua va ban luan 4.1. Bdc diem mdu nghien cihi:
Nghien cdu tie'n hanh 473 mdu benh pham chan dodn benh lao bang ea 3 phifdng phdp:
soi tnfc tie'p tim BK bdng phifdng phdp nhudm Ziehl - Neelsen; c^y cdc mau benh phim bang phu'dng phap Loweinstein va lam PCR Kdt quS cho thd'y:
TAP CHI KHOA HOC C A N THO 1 3
THANH TUU HOAT PQNG KH, CN VA MT
- Da sdld lao phdi M (-) 57.5%, lao phdi M (+) tdi Iri 26,6% vd lao ngodi phdi Id 15,9%
- T^ Id bdnh nhan nam cao hdn nff chid'm l^ Id 71,7%.
- Chiem da sdld bdnh nhan trong tudi lao ddng iCf chiem ly le 73,4%.
- Cd mdi lidn quan giifa nhdm tudi va kdt qud soi dam trifc tidp, (P < 0,001) ket qud soi (+) cd xu hifdng tdng d i n ddn 60l.
- Cd mdi quan he giCfa nhdm tudi vdi ke't qud ca'y ddm (P = 0,002) nhdm > 601 cd kdt qud ca'y ddm tha'p nha't, nhdm tudi tif 20 - 60t chie'm 86,53% cd kdt qud cd'y ddm difdng tinh.
- Cd md'i quan he giila n h d m tudi vdi kdt qud PCR, ttf 20t trd Idn PCR (+) chidm 32,6%
(P = 0.026)
- Trong 473 m§u benh phd'm tiii ty Id PCR difdng tinh d m l u benh pham dich rijfa phd qudn cao chie'm 44% va mau benh pham dam 34,6%.
- Ddi vdi kdt qud cd'y chdng tdi 1dm ty Id difdng tinh la 24,94%.
- Dd'i vdi ke't qua soi chdng tdi Idm ty le difdng tinh la 26,63% so vdi tdc gid Nggc Lan Id 36,8%.
4.2. Ung dung PCR trong chan dodn lao phSi M (-) vd lao ngodi phdi:
4.2.1. Lao phdi M(-):
' Thifc hien 272 mSu bdnh pham dam trong dd: 100% am tinh vdi ke't qud soi tnfc tie'p, ke't qud cd'y difdng tinh chidm 24,94% trong khi dd phifdng phdp PCR cho kdt qua difdng tinh 31,92%.
- Kdt qud phifdng phdp PCR trong nghien ciJu Id 31,92% so vdi kdt qua phifdng phdp soi trifc tidp (0%) vd phifdng phdp nudi ca'y Id 24,94%.
Nhif vay, kdt qua PCR luon difdng tinh cao so vdi phifdng phdp nudi ea'y vi cd mdt sd' lifdng vi khuan Mycobacterium tuberculosis bi ehe'l di trong qud trinh xvt I^ m l u tnfdc khi cay va da Idm han chd sif xdc dinh Mycobacterium tuberculosis d phifdng phdp nudi ca'y. Trong khi do ADN khdng bi phd hiiy trong qud trinh xu" If mSu tiTfdc khi thifc hien ky thuat ly tdm. vi vay ket qua PCR khdng bi anh hifdng. Ngoai ra, cdn cd mot sd mau benh pham cd chu'a nhffng vi khuan lao da chdt.
- Cd 34 tnfdng hdp nudi cd'y difdng tinh nhiftig PCR (-) cd Ihd ddy Id nhffng chung Mycobacterium tuberculosis thidu trinh Iff IS6110 trong bd gen vi cd khodng 2% chung Mycobacterium tuberculosis ciia ngifdi Vidt Nam khdng cd trinh ttf ndy trong bO gen.
- Cd 54 truOng hdp soi trifc tidp dm linh PCR (-) nhifng cay (+) sd Iffdng vi khuan mgc tren moi tnfdng nudi cay ciia nhffng U-ffdng hdp nay rat it Iff 1 ddn 2. Day Id cho sal sdl cua mau benh ph^m xdy ra khi cd 1 sd Iffdng r^t it vi khuan Mycobacterium tuberculosis. Didu nay 1dm cho mSu ndy c6 chOng vi khu^n cdn mau kia thi khdng, hay do the tich mSu ddm khi Idm ddn phffdng phdp PCR khdng dii thd tich. Ddi vdi phifdng phdp nudi cd'y thi loan bd thd tich mlu benh pham sau khi xff 1^ tnfdc cd'y dffdc si3' dung hdt eho nudi ca'y, trong khi dd dd'i vdi phifdng phdp PCR chi sff dung 1 phffdng thffc ly trich ADN (khodng 20ml) dd thffc hien phan ffng.
4.2.2. Lao mdng ndo:
- Trong 10 trffdng hdp lao mdng nao cd 3 tnfdng hdp cd tdn thffdng phdi vd 7 trffdng hdp c6n lai Id lao mdng nao ddn thuan khdng cd tdn thffdng d phdi. 10 trffdng hdp ndy khdng cd ton thffdng khdng cd trffdng hdp ndo hdn me hay bidn chd'ng nhff net. Td't cd cdc benh nhdn ndy deu dffdc Idm chge dich ndo tiiy gffi 1dm PCR, c3'y tren moi trffdng Loweinstein vd nhudm Zlehl - Neelsen,
- Phffdng phdp ca'y dffdc xem Id tidu chuan c h i n dodn benh. Trong 10 tnfdng hdp trdn thi kdt qua ca'y la 20%.
- Kdt qua PCR (+) 10 ca lao mdng nao Id IQ%.
- Kdt qua PCR dich nao tdy ciia BV Lao vd Benh Phdi Can Thd cho kdt qud tiia'p Id do mgt sd didu kidn: mau bdnh phim (dich nao liy) lay sd Iffdng the tich khdng nhidu. Sau khi soi vd cay cdn phai di chuyen ddn Trffdng DH Y Dffdc Can Thd de lam PCR do dd sd Iffdng mau benh phim it qud nen ke't qud tha'p.
Tuy vay so sdnh 3 phffdng phdp trdn soi, cd'y va PCR tiii phffdng phdp PCR cd tinh ffu vidt trong chan dodn lao ngodi phdi hdn phffdng phdp soi vd cd'y cd tiidi gian trd Idi kdt qua nhanh sau 24h
THANH T\JU HOAT S Q N G KH, CN VA MT
vd ne'u tidn hdnh lam xdl nghiem: ea^y, soi, PCR cCing mdt dia didm vd cilng thdi gian thi kdt qua PCR se cao hdn rd't nhidu.
4.2.3. Dfch ri^a phi'qudn:
Trong 25 tnfdng hdp lao phdi M (-); soi ddm tnfc tidp dm tinh lidn tuc 6 m§u ddm va trdn phidn phim Xquang cd tdn thffdng nghi lao phdi eho tie'n hanh:
- Ndi soi phd qudn: Id'y dich rffa phd qudn de soi tnfc tidp nhudm Ziehl - Neelsen, cay tren mdi trffdng Loweinstein va Idm PCR, kdl qua 2/25 (+) t^ Id 8%.
- Ca'y tim vi trffng lao BK (trong dich rffa phe qudn) Id 16%.
- PCR dich phd quan: kdt qud 44% vd PCR (+) 82,7%. Ld'y dich rffa phd qudn Idm PCR, cd'y, soi trffc tidp cho ke't qua ifu viet hdn ho khae ld'y ddm ddn thuan 1dm PCR soi vd cd'y.
4.2.4. Lao mdng phdi
Trong nhffng trffdng hdp TDMP nghi ngd do lao: cd 14 tnfdng hdp TDMP kem cd tdn thffdng nhu md phdi vd 26 tnfdng hdp TDMP ddn thuan khdng cd tdn thffdng nhu md phdi, Ta't cd cdc bdnh nhdn ndy ddu dffdc Idm:
- Chge dd mdng phdi Idm xel nghidm PCR, c^y trdn m o i trffdng Loweinstein va nhudm Ziehl - Neelsen.
- Sinh thie't mang phdi: gffi mSu md mang phdi de'n khoa gidi ph§u benh Benh vien da khoa trung ffdng Idm xdl nghiem vd trd Idi kd't qua sau 7 ngdy - 10 ngay.
- Soi trifc Hep tim BK trong dich mang phdi cho ke't qua dffdng tinh rat thap va ddi hoi phdi cd trdn 1000 vi khuan/ Iml mdi eho ke't qua dffdng tinh.
Trong nghien cffu nhffng ca TDMP ddu ld'y dich soi, nhudm Zlehl - Neelsen kdt qud 100% dm.
- Cd 1/40 trffdng hdp cd^y cd ke't qua (+) t^ le 2,5%. Nhff vdy, kdt qua ca'y dich mdng phdi thffdng cho ty le thd'p va phai mat thdi gian ddi cd kdt qud trd Idi (8 tuan).
- Sinh thie't mdng phdi vd ly le dffdng tinh Id 12,5%. Thdi gian trd Idi ket qua 7 ngdy ddn 10 ngay, c6 nhidu tnfdng hdp phai sinh thidt mang phdi 2-3 l l n mdi cho kdt qud dffdng tinh.
- PCR dieh m a n g phdi: chi cho k e t qud dffdng tinh 2,5%, trd Idi kdt qud 24 gid.
4.3. Kit qud gidi trinh tif gen cua lao da khdng thuoc:
Ta't ca 126 benh nhdn lao tdi tri: (tdi phdt vd Mi bai ciia 2 phdc dd 2SHRZ/6HE vd 2SHERZE/
REHZ/R3E3H3) ddu dffdc soi ddm dffdng tinh.
Sau dd cay ddm vd lam PCR cho thd'y:
- 21,4% khdng gidi trinh dffdc, khong cd nghia Id edc man benh pham nay khdng khdng vdi RH hay R ddn thuan md do doan ADN khdng dii hay do ky Ihudt ld'y man ehffa tdt, hay sd Iffdng the tich mSu ehffa du.
- 18,3% cd (khdng vdi RH 15,9% vd 2,4%
khdng R ddn thuan) dffdc chan dodn Id lao da khdng thud'c.
Nhff thd tinh hinh lao da khdng thud'c dd'i vdi phac dd tai tri tai TP. Can Thd tha'p so vdi kd't qua dieu tri lao da khdng thud'c l l n 3 vd thdi gian trd Idi kdt qud 24h, Do dd cd thd ffng dung phffdng phdp PCR trong chin dodn lao da khdng thudc.
4.4. So sdnh cdc phUOng phdp chan dodn:
soi trUc tiip, cdy vM PCR
Thffc hidn phffdng phdp soi, nudi ca'y vd Idm PCR cho 473 mSu bdnh pham dffdc chan doan Id lao phdi M (-) la 272 mSu. lao ngodi phdi Id 75 mau va lao phdi tai tri la 126 m l u , nhdn thd'y:
* Ddi vdi lao phdi M (-):
- Phffdng phdp PCR va soi tnfc tidp ddu cho ke't qua trong thdi gian ngan 24 gid.
- Ty le dffdng tinh eua phffdng phap PCR cao (31,61%) hdn phffdng phdp soi tnfe tie'p.
- Dffa vao tidu chuan vang ciia chan dodn vi trung la nudi cd'y, thdi gian tra Idi kdt qua Id 8 luan:
- Ty le dffdng tinh cua PCR Id 31,61% eao hdn phffdng phdp nudi ca'y la 23,89%.
* Dd'i vdi lao ngodi phdi:
- Phffdng phdp PCR vd phffdng phdp soi trffc tidp deu cho kdt qua ff-ong thdi gian ngan 24 gid va phffdng phdp nudi cd'y cho kdl qua 8 tuin:
- Ty le dffdng tinh cua phffdng phdp PCR Id 56,5%, cay la 45% va soi tnfc tidp la 1,6%.
TAP CHI KHOA HOC C A N THO 1 5
THANH TIJU HOAT PQNG KH, CN VA MT
* Ddi vdi cdc mSu benh phim lao phdi M (-) vd lao ngoai phdi:
- Kdt qud soi trffc tidp (+) vd kdt qud PCR (+) la 2,1%.
- Kdt qud soi tnfc tidp (-) va PCR (+) 2,38%.
- Kiidng cd mdi lidn he giffa kdt qud soi trffc lidp va PCR vdi p = 0,55.
So sdnh kdl qud cd'y PCR cua mau bdnh pham lao phdi M(-):
- Cd'y (+) vd PCR (+): 43,66%
- Cd'y (-) vd PCR (+): 26,44%
vd cd mdi quan hd giffa ke't qud ca'y vd PCR vdi p = 0,007 kdt qua cay (+) thi PCR (+) cao hdn ke't qud ca'y (-) vd ke'l qud ca'y (+) cho kd't qud PCR (+) cao hdn 2,19 lln cd'y (-).
Nhff vay phffdng phdp PCR ty le dffdng tinh cao hdn so vdi phffdng phdp ca'y vd sol trffc tie'p.
5. Kdt lufin va kidn nghi 5./. Ket luan:
- Yd phffoTng phap PCR.
Qua kdt qua da dat dffdc, phffdng phdp PCR thff nghiem bffdc dau ed the dffa vao ap dung trong chan dodn nhdm ho trd cdc phffdng phdp thffdng qui hien nay.
Trong 473 mau bdnh phim:
- Phffdng phdp PCR kdl qud (+) 31,92%
- Phffdng phdp soi trffc tie'p 26,63%.
- Phffdng phap cd'y kdl qua (+) 24,94%
Khdng cd mdi quan he giffa ke'l qud phffdng phdp soi trffc tidp va PCR vdi P = 0,55.
Cd mdi quan he giffa ke't qua cd'y vd phffdng phdp PCR vdi P = 0,007
Phffdng phdp PCR da cho kdt qua Iffdng dd'i phd hdp vdi cdc nghien cffu trffdc day.
Do dd phffdng phdp PCR cd the dffa vdo sff dung trong chan dodn lao phdi M (-), lao ngodi phdi va lao khdng thud'c
- Gdp ph^n cha'n doan b e n h lao - lao ngoai phdi.
Viec chan doan sdm bdnh lao dd dieu tri Id mdt dieu ra'l can thidt trong qudn ly vd dieu tii benh lao.
- Dd'i vdi benh lao phdi M (-): t^ Id PCR dffdng tinh 31,61%, ca'y 23,89%.
- Dd'i vdi lao ngodi phdi: ly le PCR dffdng linh 56,65, ca'y 45% vd soi 1,6%.
- Phffdng phdp soi trffc lldp eho kdt qud trong thdi gian 24 gid, phffdng phdp ea'y cho kdt qud trong thdi gian 8 t u I n vd phffdng phdp PCR bao gid cQng cho kdt qud cao hdn so vdi 2 kdl qud thffdng qui, Ngodi ra phffdng phdp soi tnfc tidp vd phffdng phdp edy ddu cd lien quan dd'n nhdm tudi: ddi vdi nhffng bdnh nhan > 60 tudi ihi kd't qud soi vd cdy ddu thdp. Trong khi dd PCR it dnh hffdng tudi ciia benh nhdn trong kdt qud c h i n dodn. Do dd phffdng phdp PCR rat cd gid tri trong chan dodn lao sdm, dac biet Id cac trffdng hdp lao mdng nao.
- G d p ph^n chin d o a n b^nh lao da khang thud^c
Lao da khdng thudc dang Id mdt ihdch thffc rat Idn ddi vdi nhan loai, phdt hien sdm va dieu tri kip thdi dd'i vdi lao khdng thudc Id muc ti6u hdng dIu tiong Chffdng trinh Chd'ng lao qudc gia.
Trong 126 man benh pham lao phdi tdi phat.
tha't b a l e d :
- Ty le khdng RH la 15.9%
- T y l e khdng R i d 2,4%
- Ty le lao khdng da Ihude Id 18,3%
Va thdi gian u-a Idi ket qua Id 24 gid vdi do ddc hieu Id 90%. Do dd: phffdng phdp PCR gdp phan tiong chan dodn lao da khang thudc nhanh vd chinh xac.
5.2. Kien nghi
- Dffa phffdng phap PCR vdo tidu chuin chan dodn lao phdi M (-), lao ngodi phdi va lao nhi tai Benh vien Lao & Benh phdi TP. Can Thd.
- Phffdng phdp PCR Id mot trong nhffng phffdng phdp c h i n dodn nhanh trong lidu chuIn chan dodn lao da khdng thudc tai Bdnh vien Lao vd Benh phdi TP. Cin Thd.
- Phffdng phdp PCR Id mot trong nhffng phffdng phdp chan doan nhanh tiong tieu chuan chan doan lao da khang thudc trong CTCLQG.
1 6 TAP CHI KHOA HOC C A N THO
