British Society for Microbial Technology

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British Society for Microbial Technology

• The laboratory diagnosis of tuberculosis
• 25 years of progress

D A Mitchison St Georges, University of London
With assistance from FINDdiagnostics
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Diagnostic testing at different levels of health
system
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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

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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
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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.

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Genetic systems
Sensitivity
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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

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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.

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Reserve drugs
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MGIT 960 Reserve Critical Concentrations
1Rusch-Gerdes S et al. JCM 200644688-92. 2Rodri
gues C et al. IJTLD 2008121449-55. 3Kruuner A
et al. JCM 200644811-8.
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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

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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

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What is drug resistance?
Defined from distribution of MICs on wild
strains
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Studies of early bactericidal activity define
the therapeutic margin
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Can high drug dosage still have an effect on
resistant strains?