Cac Ky Thuat Chan Doan Lao Tai Bv Pham Ngoc Thach

7/25/2019 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

Cac Ky Thuat Chan Doan Lao Tai Bv Pham Ngoc Thach

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