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

Peripheral health centre

Proportion of patients

TB tests

Peripheral centre 60% None

Microscopy centre 35% Microscopy

Referral laboratory 10% Culture, DST

Reference laboratory 5% Reference methods

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

• 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)

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

Fluorescence v. Bright field microscopy

Fluorescence: Introduced in 1940s.5x more rapid than Bright fieldBUT: Mercury vapour bulb: Expensive. Limited life. Gradual decline.

LED illumination introduced during past 5 years

Find/Zeus collaboration

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

Equipment cost

Cost specimen

(£)

Sm +

Cult +

Sm –

Cult +

Specifity

Hain TBDR+ Moderate 48 98% 100%

Gene Xpert (Cepherd)

High

(£100,000)

40 99% 87% 97%

LAMP

(Eiken)

Low Low 98% 49% 99%

Sensitivity

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

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.

Reserve drugs

Fluoroquinolones Ethionamide

Moxifloxacin Prothionamide

Levofloxacin Cycloserine

Injectables PAS

Streptomycin Linezolid

Amikacin (Kanna) Amoxicillin/clavulanate

Capreomycin

Ethambutol

Pyrazinamide

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.

DrugStudy

1Study 2 Study 3

Amikacin 1.0 1.0 1.0

Kanamycin ND 2.5 ND

Capreomycin 2.5 2.5 1.25

Ethionamide 5.0 5.0 ND

Proteonamide 2.5 ND 2.5, 5.0

Ofloxacin 2.0 2.0 1.0

Moxifloxacin ND 1.0 0.125

Levofloxacin ND ND ND

Rifabutin 0.5 ND 0.5

PAS ND 4.0 ND

Linezolid 1.0 ND ND

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 DSTThin-layer agar plate (TLA) method

7H11 thin layer plates made selectiveEach plate with up to 6 strains in quadrants

Control: no drugPNB (p-nitrobenzoate): TB inhibited.INH 0.2 µg/mlRIF 2.0 µg/mlSM 2.0 µg/mlPZA 2,000 µg/ml nicotinamideetc

What is drug resistance?

Defined from distribution of MICs on ‘wild’ strains

EBA titrations of INH, RMP & SM

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.5 1 1.5 2 2.5 3 3.5

Log drug concentration

Sta

nd

ard

EB

A

INH

RMP 1

RMP 2

SM

9

19

38

75

150

300600

600

150

1.5 g

0.38 g

Studies of early bactericidal activity define the ‘therapeutic’ margin

Can high drug dosage still have an effect on resistant strains?

Isoniazid Mutants katG – high MIC

inhA – low MIC

Early clinical trial

Guinea-pig study

Quinolones Mutants Mainly in gyrA – low MIC