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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 l.jpg
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 l.jpg
Sputum: 25 years ago (1985)

  • Poor countries: Microscopy alone

  • Richer countries. Microscopy, LJ culture , DST

  • Advanced countries. Microscopy, Liquid culture, ID, DST


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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)


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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 l.jpg
Direct smears 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

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 l.jpg
Culture: solid v. liquid 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

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 l.jpg
Genetic systems 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

Sensitivity


Culture identification dsts l.jpg
Culture, identification & DSTs 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

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 l.jpg

What to do about MDR TB? 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(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.


Reserve drugs l.jpg
Reserve drugs 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


Mgit 960 reserve critical concentrations l.jpg
MGIT 960 Reserve Critical Concentrations 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

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.


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DSTs 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

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 l.jpg
Phenotype DST 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 lightThin-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 l.jpg

What is drug resistance? 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

Defined from distribution of MICs on ‘wild’ strains


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Studies of early bactericidal activity 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

define the ‘therapeutic’ margin