Download The laboratory diagnosis of tuberculosis

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 TB tests
patients
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
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)
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 field
BUT: 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
(£)
Sensitivity
Sm +
Sm –
Cult +
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%
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
Moxifloxacin
Levofloxacin
Injectables
Streptomycin
Amikacin (Kanna)
Capreomycin
Ethambutol
Pyrazinamide
Ethionamide
Prothionamide
Cycloserine
PAS
Linezolid
Amoxicillin/clavulanate
MGIT 960 Reserve Critical Concentrations
Drug
Study
1
Study 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
1Rusch-Gerdes
S et al. JCM 2006;44:688-92.
C et al. IJTLD; 2008;12:1449-55.
3Kruuner A et al. JCM 2006;44:811-8.
2Rodrigues
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 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
What is drug resistance?
Defined from distribution of MICs on ‘wild’ strains
Studies of early bactericidal activity
define the ‘therapeutic’ margin
EBA titrations of INH, RMP & SM
0.6
300
600
0.5
150
0.4
Standard EBA
INH
RMP 1
RMP 2
SM
75
0.3
600
38
0.2
1.5 g
19
0.1
0
150
0.38 g
-0.1
9
-0.2
0.5
1
1.5
2
2.5
Log drug concentration
3
3.5
Can high drug dosage still have an effect on
resistant strains?
Isoniazid
Mutants
katG – high
MIC
inhA – low
MIC
Quinolones Mutants Mainly in
gyrA – low
MIC
Early clinical
trial
Guinea-pig study