1. British Society for Microbial Technology The laboratory diagnosis of tuberculosis 25 years of progress D A Mitchison St George’s, University of London With assistance from FINDdiagnostics

2. Diagnostic testing at different levels of health system Peripheral health centre Proportion of patients TB tests Peripheral centre60%None Microscopy centre35%Microscopy Referral laboratory10%Culture, DST Reference laboratory5%Reference methods

3. Sputum: 25 years ago (1985) 1.Poor countries: Microscopy alone 2.Richer countries. Microscopy, LJ culture, DST 3.Advanced countries. Microscopy, Liquid culture, ID, DST

4. Direct smears Culture on LJ slopes (3-6 weeks) Identification as M. tuberculosis (Chemical; PNB, niacin, catalase) Drug susceptibility tests (DSTs) (Rifampicin screen) Sputum bacteriology UK (1985)

5. FIND and Carl Zeiss Micro Imaging GmbH have co- developed a fluorescent LED microscope based on the proven Primo Star platform. FIND/Zeiss microscope offers superior optics, reflected light illumination, easy switch from brightfield to fluorescent light

6. Direct smears Fluorescence v. Bright field microscopy Fluorescence: Introduced in 1940s. 5x more rapid than Bright field BUT: Mercury vapour bulb: Expensive. Limited life. Gradual decline. LED illumination introduced during past 5 years Find/Zeus collaboration

7. Culture: solid v. liquid Solid: LJ slopes. 7H11 slopes or plates. Liquid: Early attempts high contamination. 1971 Selective medium paper (Mitchison et al J Med Microbiol 1971; 5: 165) Penta used in Bactec machine Automated liquid systems v. solid media Sensitive. Rapid. Contamination. NTMs v. TB.

8. Genetic systems Equipment cost Cost specimen (£) Sm + Cult + Sm – Cult + Specifity Hain TBDR+Moderate4898%100% Gene Xpert (Cepherd) High (£100,000) 4099%87%97% LAMP (Eiken) Low 98%49%99% Sensitivity

9. Culture, identification & DSTs HAIN MDTBDR plus PCR & Line probe based 1. Identifies as TB complex. 2.DSTs for RIF & INH (95%) Can be used directly on sputum avoiding culture

10. What to do about MDR TB? (MDR = Resistance to INH & RIF) Genetic tests for reserve drugs not adequate yet. Therefore cultures in liquid or on solid medium necessary as well as genetic techniques.

11. Reserve drugs FluoroquinolonesEthionamide MoxifloxacinProthionamide LevofloxacinCycloserine InjectablesPAS StreptomycinLinezolid Amikacin (Kanna)Amoxicillin/clavulanate Capreomycin Ethambutol Pyrazinamide

12. MGIT 960 Reserve Critical Concentrations 1 Rusch-Gerdes S et al. JCM 2006;44:688-92. 2 Rodrigues C et al. IJTLD; 2008;12:1449-55. 3 Kruuner A et al. JCM 2006;44:811-8. Drug Study 1 Study 2Study 3 Amikacin1.0 KanamycinND2.5ND Capreomycin2.5 1.25 Ethionamide5.0 ND Proteonamide2.5ND2.5, 5.0 Ofloxacin2.0 1.0 MoxifloxacinND1.00.125 LevofloxacinND Rifabutin0.5ND0.5 PASND4.0ND Linezolid1.0ND

13. DSTs Phenotypic Classic on LJ slopes or 7H11 plates. Takes 7 weeks +. MGIT or other automated liquid tests. Microcolony methods Liquid medium: Mods. Sensitive, time consuming, ?dangerous Solid medium: Thin layer agar (TLA): Quicker. Less dangerous

14. Phenotype DST Thin-layer agar plate (TLA) method 7H11 thin layer plates made selective Each plate with up to 6 strains in quadrants Control: no drug PNB (p-nitrobenzoate): TB inhibited. INH 0.2 µg/ml RIF 2.0 µg/ml SM 2.0 µg/ml PZA 2,000 µg/ml nicotinamide etc

15. What is drug resistance? Defined from distribution of MICs on ‘wild’ strains

16. Studies of early bactericidal activity define the ‘therapeutic’ margin

17. Can high drug dosage still have an effect on resistant strains? IsoniazidMutants katG – high MIC inhA – low MIC Early clinical trial Guinea-pig study QuinolonesMutantsMainly in gyrA – low MIC