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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. Diagnostic testing at different levels of health system. Sputum: 25 years ago (1985).

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british society for microbial technology
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

sputum 25 years ago 1985
Sputum: 25 years ago (1985)
  • Poor countries: Microscopy alone
  • Richer countries. Microscopy, LJ culture , DST
  • Advanced countries. Microscopy, Liquid culture, ID, DST
slide4

Sputum bacteriology UK (1985)

  • Direct smears
  • Culture on LJ slopes (3-6 weeks)
  • Identification as M. tuberculosis
  • (Chemical; PNB, niacin, catalase)
  • Drug susceptibility tests (DSTs)
  • (Rifampicin screen)
slide5
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
direct smears
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

culture solid v liquid
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.

genetic systems
Genetic systems

Sensitivity

culture identification dsts
Culture, identification & DSTs

HAINMDTBDR plusPCR & Line probe based 1. Identifies as TB complex.

2.DSTs for RIF & INH (95%)

Can be used directly on sputum avoiding culture

what to do about mdr tb mdr resistance to inh rif

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.

mgit 960 reserve critical concentrations
MGIT 960 Reserve Critical Concentrations

1Rusch-Gerdes S et al. JCM 2006;44:688-92.

2Rodrigues C et al. IJTLD; 2008;12:1449-55.

3Kruuner A et al. JCM 2006;44:811-8.

slide13
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

phenotype dst thin layer agar plate tla method
Phenotype DSTThin-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

what is drug resistance

What is drug resistance?

Defined from distribution of MICs on ‘wild’ strains

slide16

Studies of early bactericidal activity

define the ‘therapeutic’ margin

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