Development of drug resistance in M. tb and detection tests Cali, Colombia - March 28, 2007

Transcription

1 Development of drug resistance in M. tb and detection tests Cali, Colombia - March 28, 2007

2 Hugo David Applied Microbiology : Probability distribution of drug-resistant resistant mutants in unselected populations of M. tuberculosis

3 Within wild-type M. tuberculosis populations, small populations of mutants are found to be resistant to anti-tb drugs: 1.84 x 10-8 and 3.5 x 10-6 res to INH 2.2 x and 1.2 x 10-8 res to RIF 1.0 x 10-7 and 3.1 x 10-5 res to EMB 2.9 x 10-8 and 3.8 x 10-6 res to SM

4 Do the math for MDR: A mutant resistant to both INH and RIF would be really, really, really, really rare! 4 x and 4.2 x 10-14

5 Do the math for MDR: A mutant resistant to both INH and RIF would be really, really, really, really rare! 4 x and 4.2 x Canetti: a cavity lesion contains roughly 10 8 bacteria! ARRD :687 :

6 Clinician Specialty TB / Infection Control * Point of Care Time *FAST TRACK the highest priority specimens

7 Florida MDR TB ( ): Chart Review [N=81]: Cured Community Care [N=31] 48% A.G. Holley Hospital [N=39] 79% Narita et al CHEST 120:343 : (2001) 348(2001)

8 Bactec 460TB and MGIT 960 growth detection and DST systems Bactec 460 TB system shorter TAT semi-automated radioactive requires needles special gas mix MGIT 960 system fully automated, walk-away non-radiometric no need for needles (inoculation and testing) no need for manual loading of vials no need to establish reading schedules

9 Testing concentrations: Drug 460TB MGIT 7H10 7H11 INH 0.1/ / / /1.0 RIF EMB 2.5/ / SM 2/6 1/4 2/10 2/10 PZA N/A N/A

10 RIF resistance yes or no? rpob analysis Clinical course Egg based AST Agar based AST Radiometric / Non-r.

11 The Dynamic C..! Care Cure Cash

12 The Dynamic C..! Care Cure Cash Crash?

13 reativity!

14 Amalio Telenti Lancet 1993 (13 March); Telenti et al: Detection of rifampicin-resistant resistant mutations in M. tuberculosis

15 Alignment of E. coli and M.tb rpob.. The E. coli sequence edited according to Jin [1988]

16 Molecular testing: Drug RIF PZA INH INH-ETH INH INH Gene rpob >96% pnca 97% % mutations katg 40-60% inha 15-43% ahpc 10% kasa unknown

17 rpob analysis Codon 526 (CAC) encodes histidine in susceptible strain replaced with (GAC) aspartate in resistant strain.

18 Challenge!

19 It is easier with DNA line probes...

20 INNO-LiPA Rif. TB Innogenetics,, Belgium GenoType MTBDR Hain Lifescience,, Germany

21 Further DST solution Use of molecular assays in combination with broth-based based systems

22 Latest development

23 GenoType MTB-DR (Hain( Lifescience) INH resistant strains are often lacking their catalase peroxidase activity (Middlebrook et al., 1954) Cloning of the katg gene encoding catalase peroxidase that activates INH identified mutations which result in high degree of INH resistance (Zhang et al., 1992) Mutations resulting in an amino acid change within the 81-bp core region of the RNA- polymerase β-subunit (rpob)) gene are found in more than 96% of RIF resistant MTB strains (Telenti et al., )

24 Hain Lifescience

25 GenoType MTB-DR (Hain( Lifescience)-II Overall TAT within hours vs. 14 to 18 days Automated Sensitivity for MTBC is comparable to Amplicor or MTD: 135/143 (94.4%) Successful amplification: katg: 135/143 (94.4%) rpob: 130/143 (90.9%) JCM Dec :

26 GenoType MTB-DR (Hain( Lifescience)-III Detection of drug resistance*: katg: 48/57** (84.2%) High-level INH r rpob: 25***/26 (96.1%) RIF resistance * Determined by Radiometric BACTEC ** With sequencing 49 strains with katg mutation [98%] *** 3 susceptible strains with rpob mutation (sequencing & MTB-DR) *** JCM Dec :

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28 If you think health is expensive, try disease!

29 CHEST 131: (2007) XDR detected by agar proportion method Genotyping: W-Beijing MDR detected by broth-based method week 8 katg, rpob, and pnca assay from growth positive broth week 7 week 4 Direct katg, rpob, and pnca assays on a clinical specimen week 2 week 3 day 2 week 1 Clinical suspicion of MDR or XDR ± Beijing-W genotype

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31 Alexander Bell invented telephone: 1876

32 Microscopy - LJ - Broth-based

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34 Phone camera organizer Transformation

35 Detection Molecular Identification Drug resistance Genotyping

36 About changes... Said is not heard Heard is not understood Understood is not implemented Implemented is not retained

37 hink old!!!

38 Let s s be bold: Move from AFB microscopy to molecular detection of DR, MDR, or XDR TB in sputum samples without first growing the tubercle bacilli!!!

39 Obrigadoh! Gracias! Tesekkür ederim! Spacibo! Merci! Grazie! Danke! Thank you!