cac ky thuat chan doan lao tai bv pham ngoc thach
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CC PHNG PHP XT NGHIM VI SINH
CHN ON VI KHUN LAOANG P DNG TI BV.PNT
KHOA VI SINH BV.PHM NGC THCH
ISO 15189 - 2007 1
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CC YU CU CHO MT XT NGHIM
VI SINH CHN ON
Nhanh nhy v c hiu n gin D thc hin
R tin An ton cho mi trng Khng c hi
ISO 15189 - 2007 2
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WHO
-
Khuy
n co v
cc k
thu
t ch
n on lao, lao
a khng
Soi knh
Knh hin vi nh sng thng
Kinh hin vi hunh quang thng
thngKnh hin vi hunh quang n LED
Nuicy, nhdanh
Khng sinh Thuc chng lao hng 1
Thuc chng lao hng 2
Sinhhcphnt
ISO 15189 - 2007 3
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QU TRNH PHT TRIN CA CC K
THUT CHN ON VK LAO
ISO 15189 - 2007 4
1880s Phng php nhumZiehl-Neelsen
1900s Mantoux test (tuberculin)
1920s Purified Protein Derivative (PPD)1930s Lowenstein-Jensen
1940s Dubos agar, Ogawa
1950s Middlebrook 7H9
1990s Khuych i a xt Nucleic
2000s ELISPOT, QuantiFERON
20XX Hain, Xpert &?????
Soi TT
Min dch
Nui cy
SHPT
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SOI KNH HIN VI
Tm s hin din ca AFB trong mu bnh phm Phng php soi trc tip : Lm tiu bn trc tip t mu
bnh phm
Phng php soi thun nht: Lm tiu bn t mu bnhphm c x l.
Nhum Ziehl Neelsen : S = 45-60% , Sp = 99%
Nhum Auramin : S = 45 80% , Sp > 90%
Bnh phm p dng: TT C CC LOI BNH PHM
ISO 15189 - 2007 5
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SOI KNH HIN VI (
tt
)
u im:
n gin, r tin, Cho kt qu trong vng 2 gi Pht hin c ngun ly
Gip theo di kt qu iu tr .
K thut n gin.
Nhc im:
nhy thp. Mu bnh phm phi c t > 5000 vi khun / 1ml , mi c kt qu dng tnh.
Khng phn bit c MTB vi NTM
Khng phn bit c VK sng hay cht; VK khng thuc.
ISO 15189 - 2007 6
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SOI KNH HIN VI (tt)
Nghin cu so snh nhy ca soi trc tip v thun nht tbnh phm m: khng c s khc bit v NHY gia 2phng php
Khng c bng chng thuyt phc cho thy x l m bngho cht, hoc li tm cho kt qu tt hn lm tiu bn trctip nn vic s dng cc k thut ny trong chng trnhchng lao khng c khuyn co.
ISO 15189 - 2007 7
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c kt qu (KQ KHQ c quy i sang Z-N)
Kt qu Z-NKt qu trn phiu yu cu
0 AFB 0 AFB/ 100 qt0 - AFB
Ghi sc th 1-9 AFB/ 100 qt AFB 4 AFB
1+ 10-99/ 100 qt AFB 1+
2+ 1- 10 AFB/ 1 qt AFB 2+
3+ >10 AFB/ 1 qt AFB 3+
ISO 15189 - 2007 8
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Nuicyvnhdanh
ISO 15189 - 2007 9
u imChn on xc nh MTB nhy cao, tng s BN pht hin (30%-50% so vi STT)Chn on gii on sm (trc khi thnh ngun ly chnh)Phn lp c chng VK lm KS,Nghin cu...
Nhcim
Phc tp, Gi thnh cao; chun b mi trng, x l bnh phm
Thi gian ko di (VK mc)Trang thit b c chngi hi k nng ca KTViu kin ATSH
Hn ch
Vic kh tp cng git ch mt phn vi khun lao
LOD: 100 bacilli/ml sputumCy lng nhy hn 10%, nhanh hn vi tun, nhng c t l ngoi nhim cao hn Cyc v i hi iu kin ATSH cao hnnh danh ch xc nh MTB v NTMCc k thut nh danh: SVHH, min dch, SHPT
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Phng php nui cyC 2 phng php:
Nui cy trn mi trng c Cy LJ/ OGAWA
Nui cy trn mi trng lng Cy MGIT
c coi nh tiu chun vng v c th nh danhc VK lao vi c hiu > 98%
Bnh phm p dng: TT C CC LOI BNH PHM
ISO 15189 - 2007 10
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QUY TRNH XN LAO TI PXN
ISO 15189 - 2007 11
Mu bnh phm X l mu
Nui cyTB/NTM
6-8 tun
Nhum soi
m tnh
Dng tnh
nh danh
KS
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Nui cy trn mi trng c
Nguyn tc: VK lao c kh nng mc trn mt s mi trng c bit to
thnh cc khun lc trn b mt mi trng c th quan stbng mt thng . Thi gian (+) trung bnh 3 - 6 tun ; (-) 8
tun Bnh phm lm sng gi ti PXN cy tm VK lao nu
khng m bo v trng th s c kh tp loi b cc VKthng thng- cc loi VK mc nhanh.
ISO 15189 - 2007 12
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c v ghi nhn KQ cy
c Bo co KQ
Cckhun lc mc dy khp mt
mitrng(>500khun lc) 4+
Mc nhiu > 200 khun lc,
nhng khng y khp mt mi
trng
3+
100 ~ 200khun lc 2+
20 ~ 100khun lc 1+
< 20khun lc Ghis khun lc m c
Khngmc m tnh
Ngoi nhim Ngoi nhim
ISO 15189 - 2007 13
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Nui cy trn mi trng lngBACTEC MGIT 960
960 v tr t tup
Thi gian nui cy: (+) 7- 21 ngy
(-) 6 tun p dng cho tt c cc loi bnh phm lm
sng tr mu v nc tiu
ISO 15189 - 2007 14
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Cy m tnh
t hoc khng pht quang
Cy dng tnh
Pht quang mnh
V tr nhy cm
gia
B mt
Nguyn l
ISO 15189 - 2007 15
FF F
F
FO2FO2
F
F
F F
CO2
O2
O2
O2
O2O2
FF FO2
FO2FO2FO2
FO2
F
FO2 FO2
CO2O2
O2
O2 O2
O2
O2
O2O2
CO2
CO2
O2
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Cy vo mi trng
ISO 15189 - 2007 16
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c kt qu
ISO 15189 - 2007 17
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ISO 15189 - 2007 18
M. TB, NTM Yu t thng
NTM Ngoi nhim tp khun
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Mt s phiu KQ nui cy
ISO 15189 - 2007 19
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MA = MOTT = NTM
T l 7% Nhm sinh trng nhanh (M. fortuitum, M. chelonae
)
Nhm sinh trng chm (MAC , M. kansasii , M.marinum )
Nhm sinh trng chm khc (M. simiae complex, M.xenopi . )
Nhm sinh trng kh (M. haemophilium,
M. ulcerans)
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Thu thp mu bnh phm
Mu thu thp trong l v trng, c np vn cht Lu tr lnh nu thi gian chuyn mu n phng XN >
1 gi
Khng cn mi trng vn chuyn hay cht bo qun
Mu bnh phm h hp : thu thp 3 mu sng sm trong3 ngy khc nhau
Mu dch th, pxe, m : ht hay ct ch khng dnggc ly mu. Mu m c th ngm trong t nc mui
v trng trnh b kh.
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nh danh NTM
Hnh thi vi khun, sc t Thi gian sinh trng
Sinh ha
Hain test
DNA Sequence hay PCR - ELISA
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DST - NTM
Mi tng quan gia DST in vitro v p ng lm sng :hn ch Ngng khng ca nhiu loi NTM khng c ngha
lm sng (xc nh ngng cho tng loi)
lp li km Cn trng khi s dng DST v nhiubnh NTM khng
p ng iu tr khi da trn kt qu DST in vitro Khng lm DST cho NTM nhm sinh trng nhanh trn
mi trng agar v kt qu mu thun DST trong mi trng broth
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iu tr NTM
ISO 15189 - 2007 24
iu tr theo c th
Phi hp thucThi gian iu tr ???Theo di iu tr ??? n khi cy (-)
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Mt s vd v iu tr NTM
M. kansasii: rifampin, isoniazid, ethambutol,ethionamide, streptomycin, and clarithromycin
M. fortuitum : macrolides v quinolones, doxycycline vminocycline, sulfonamides . Mt s nghin cu mc
nhy cm amikacin (100%), ciprofloxacin & ofloxacin(100%), sulfonamides (100%), cefox-itin (50%),imipenem (100%), clarithromycin (80%), doxy-cycline(50%)
M. chelonae: tobramycin (l00%), clarithromycin (l00%),linezolid (90%), imipenem (60%), amikacin (50%), clo-fazimine, doxycycline (25%), and ciprofloxacin (20%)
*Nhm mc nhanh: Khngtnhin vi cc thuc khng lao
ISO 15189 - 2007 25
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Khngsinh
ISO 15189 - 2007 26
u im
Chn on xc nh lao khng thucL tiu chun vngCc k thut: KS gin tip Kiu hnh: KS trn mi trng c; mi trng lng Cc thuc: Hng 1: H, S, R, E, Z
Hng 2: Of, Km, Cm, Am
Nhcim
Thi gian di: 42 ngyPhi c chng thun
p dng Mu cy dng tnh MTB
Hn ch: Mt s thuc chng lao hng 2 hin khng thc hin v KQ khng chnh xc
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KQ KS
ISO 15189 - 2007 27
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Khng thc hin KS
ISO 15189 - 2007 28
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Skhng cho gia cc thuc chng lao
Cc thuc c chung lin ha hc:Nhm I- thuc tng hp: Isoniazid, Ethionamid,
Thiacetazon, Kanamycin, Capreomycin
* EthionamidThiacetazon: -CS.NH2* IsoniazidEthionamid: -NHNH2 (khng H nng
thp)
Nhm II- khng cho mt chiu
* Kanamycin Capreomycin
* Kanamycin Steptomycin
ISO 15189 - 2007 29
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WHO- Khuyn co v cc kthut chn on lao, lao a khng
Sinhhcphnt
ISO 15189 - 2007 30
u im
Nhanh,Chun haKhng i hi iu kin ATSH cao so vi KSLPAs c WHO chng thc 2008
Nhc im LPAs ch c thc hin vi mu soi dng (V1)
Hn ch LPAs ch p dng c ti cc PXN tuyn trung ng
Xpertc WHO chng thc 2011, nhanh, n gin, thc hin c vi musoi m,khngi hi iu kinATSH cao, ph hp vi cc PXN tuyn di
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K thut MTBDR plus( Hain test)
Hain test da trn k thut lai vi on d c hiu gn trnthanh giy, cho php nh danh VK lao MTB complex v tnh khng i vi R v H
Bnh phm: t mu cy dng hoc tt c cc mu bnh phmtr :
Mu m
Mu
nc tiu
phn,
ISO 15189 - 2007 31
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ISO 15189 - 2007 32
RIF-RES INH-RES MDR
SENSITIVITY 93.2% 92.7% 89%
SPECIFICITY 100% 100% 100%
PPV 100% 100% 100%
NPV 93% 92.8% 89.8%
Performance of the Hain MTBDR plus assay to
predict drug resistance
(n=111, Culture)
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ISO 15189 - 2007 33
RIF-RES INH-RES MDR
SENSITIVITY 93.5% 88.7% 86.7%
SPECIFICITY 96.7% 97.8% 96.7%
PPV 94.7% 97.9% 94.2%
NPV 95.9% 88.2% 92.2%
Performance of the Hain MTBDR plus assay to
predict drug resistance
(n=198, sputum)
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Nguyn l ca reverse line blot
ISO 15189 - 2007 34
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Quy trnh
ISO 15189 - 2007 35
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Din gii kt qu
Hain test :- Pht hin vi khun lao
- Khng sinh : RIF v INH
ISO 15189 - 2007 36
MTB
RIF
INH
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Cc trng hp c bit
C th c vi khun lao nhng s lng t khng pht hin c
ISO 15189 - 2007 37
Nn cy chn on xc nh
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Kt qu
ISO 15189 - 2007 38
-Nu soi AFB (+): nhiu kh nng l MOTT: nn cy chn onxc nh
-Nu soi AFB (-) v nghi ng BN b lao: nn cy chn on xcnh
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Kt qu
ISO 15189 - 2007 39
C th do vi khun qu t
Nn cy v thc hin Hain t mu cy (+)
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Trng hp c bit
KS : nhy RIF v INH
Hain test : Nhy Rif v khng INH ( 3 ln u nh nhau)
L do : Hain t bin gen inhA khng INH nng thp nn KS khng pht hin c
t bin gen inhAt bin gen katG
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Khng INH nng thpt bin gen katG
Khng INH nng cao
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Thc hin Hain test
TRONG VNG 2NGY = KT QU
Khoa VS: Lm Hain vo 2 ngy th 2 v th 5
Tr KQ : Th 3 & th 6
Sp ti: C lm tip hay khng ???
ISO 15189 - 2007 42
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GeneXpert
- Khuych i acid nucleic (NAAT )- Cepheid Xpert MTB/RIF- Xc nh phn t MTB v khng RIF (rpoB)
u im :- Mu m AFB(+) v (-)- Cc loi bnh phm khc (hn ch s liu bng chng)- H thng kn v t ng hon tan, gn nh
- Kt qu ~ 2h- Khng i hi yu cu an ton sinh hc- Thao tc d, n gin
ISO 15189 - 2007 43
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Nhc im :
- i tng chn on lin quan ti PPV- Hiu chnh my hng nm ( gi v ni sn xut )
- Phng my lnh
- Gi thnh cn cao so vi soi , cy- Phn mm cha Vit ha
ISO 15189 - 2007 44
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Xpert MTB/RIF
ISO 15189 - 2007 45http://www.who.int/tb/features_archive/new_rapid_test/en/index.html
WHO-endorsed December, 2010
http://www.who.int/tb/features_archive/new_rapid_test/en/index.htmlhttp://www.who.int/tb/features_archive/new_rapid_test/en/index.html -
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ISO 15189 - 2007 46
XPERT MTB/RIF
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XPERT MTB/RIF
nhy c hiu
Cy (+) Soi (+) /cy (+) Soi (-)/cy(+) Khng lao3 mu m
n=x/y(%)
723/741(97.6)
566/567(99.8)
157/174(90.2)
604/616(98.1)
2 mu mn=x/y
(%)1423/1482
(96.0)1127/1134
(99.4)296/348(85.1)
1215/1232(98.6)
1 mu m
n=x/y(%)
675/732(92.2)
551/561(98.2)
124/171(72.5)
604/609(99.2
ISO 15189 - 2007 47
Catharina C Boehme et al. NEJM, 2010
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Catharina C Boehme et al. NEJM, 2010
ISO 15189 - 2007 48
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ISO 15189 - 2007 49
1. Bnh Thun2. Ninh Thun3. B Ra- Vng tu4. TP.HCM
5. ng Nai6. Ty Ninh
7. Long An
8. Bn Tre9. ng Thp
10. Tin Giang11. Sc Trng12. C Mau
13. Cn Th14. Bc Liu
15. Kin Giang16. An Giang
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Ngun: WHO Policy Framework for implementing
New Tuberculosis Diagnostics 2010
ISO 15189 - 2007 50
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CHN THNH CM
N S
CH THEO DI
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