TB Methodologies
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Transcript TB Methodologies
TB Methodologies
Dr. John G. Magee
Regional Reference Centre for Mycobacteriology
Health Protection Agency Regional Laboratory,
Newcastle upon Tyne
Annual report, tuberculosis cases reported in 2000,
England, Wales and Northern Ireland
Of 6323 cases ONLY 3350 (53%) were culture confirmed
Of 3729 with pulmonary disease:
ONLY 2249 (60%) were culture confirmed
ONLY 2513 had a smear result!
ONLY 1406 (56%) were smear positive
DoH - Getting ahead of the curve “Tuberculosis Action Plan”
The HPA will work with reference laboratories and NHS microbiologists
to improve the speed and consistency of laboratory diagnosis by:
providing high quality diagnostic services through a network of suitably
equipped and experienced laboratories
standardising methods
establishing quality assurance/performance monitoring programmes
covering
.. liquid culture for all specimens
.. molecular confirmation
.. unique typing designation
DoH - Getting ahead of the curve “Tuberculosis Action Plan”
STANDARDS EXPECTED
smear turnaround time - 1 working day
all clinical samples to have access to automated liquid culture
performed in experienced centres with large throughput and
dedicated facilities and staff
all isolates referred to regional mycobacteriology centre for
identification & susceptibility testing
Diagnostic and Reference Mycobacteriology
Detection
Smear /Culture
Molecular amplification
of unique fragments
Identification
Phenotypic tests
•“Gene probes”
• PCR
• Sequencing
Susceptibility
testing
Phenotypic
expression
Detection of gene
mutations
Fingerprinting
(Typing)
Numerical
taxonomy
•RFLP … HIP
•Spoligotyping
•VNTR/MIRU
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Direct detection using molecular biological tests
The problems are have something to do with low organism numbers
and much to do with extraction of DNA from clinical samples
Direct detection using molecular biological tests
Commercial Assays:
Roche Amplicor (Cobas & “Manual”)
Abbott LCx
(LCR)
GenProbe Direct (TMA)
BD ProbeTec ET (SDA)
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Direct detection using molecular biological tests
In-House Assays:
Single PCR; Semi-Nested PCR; Nested PCR…... and now
Real-Time PCR
Amplification
control (58C)
Target (62C)
-dF/dT
Negative
control
Temp (°C)
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Diagnostic and Reference Mycobacteriology
Microscopy for AFB, if done well, remains a cheap, simple, fast
and effective diagnostic technique
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Isolation
Regional Centre for Mycobacteriology, Newcastle upon Tyne
UK NEQAS Distribution 1601 Mycobacterium culture
Sample 6492 - M.tuberculosis present
of 330 laboratories 31.8% failed to isolate M.tuberculosis
of 176 UK laboratories 39.2% failed
Sample 6491 - M.tuberculosis present
of 331 laboratories 5.1% failed to isolate M.tuberculosis
of 176 UK laboratories 8.5% failed
Isolation: Liquid Media circa 1983
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Isolation: Liquid Media
circa 1993
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Continuous Automated Mycobacterial
Liquid Culture systems
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Cumulative weekly totals of Mtb complex
isolates by CAMLiC and LJ slope culture
Number of isolates
140
CAMLiC
120
100
80
60
40
LJ
20
0
1
2
3
4
5
6
>6
Weeks after inoculation
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Surety of Competence
Detection of Acid-Fast Bacilli: 20 smears per week
1000 per year
Mycobacterial Culture: 25 samples per week
1250 per year
Susceptibility testing: 20 isolates per week
1000 per year
Regional Centre for Mycobacteriology, Newcastle upon Tyne
UK NEQAS Distribution 1601 Mycobacterium culture
Sample 6490 - M.tuberculosis NOT present
8/331 laboratories (2.4%) isolated a mycobacterium!
In the last 4 negative samples there were 11,4, 2 & 10 false positives
False positivity is probably due to laboratory cross contamination.
NEQAS refer us to Breese at al. Arch Pathol Lab Med 2001, 125(9): 1213
BUT see alsode Boer et al. J Clin Microbiol 2002; 40; 4004
They found that labs processing <3000 samples per annum showed a
greater risk of cross-contamination
Identification of mycobacteria
M.tuberculosis
M.bovis
M.avium
M.intracellulare
M.malmoense
M.haemophilum
M.xenopi
M.gastri
M.terrae
Niacin
phosphatase
+
+
catalase Resistance to Resistance to
inah
inah
(45mm)
HydroxylHydroxyl- (1 mg/l) (5 mg/l)
amine (125) amine (500)
+
V
+
+
+
+
+
V
+
+
+
+
+
+
+
+
+
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Identification of mycobacteria
“Gene-Probes” can confirm M.tuberculosis complex in under 2 hours
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Identification of mycobacteria
“Genetic probes” are NOT amplification procedures -
They can identify a limited number of common species:
M.tuberculosis complex
M.avium complex
M.avium
M.intracellulare
M.kansasii
M.gordonae
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Identification of Mycobacteria
Characterisation of mycolic acids by HPLC
Detection of unique fragments of genomic DNA
16S rRNA sequencing
[
equipment
database variances
]
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Susceptibility testing of mycobacteria
In the U.K. susceptibility testing of mycobacteria is
performed by one of two methods:
The
Resistance Ratio Method comparing MICs of test and
control strains
The Radiometric or Proportional Method using the Bactec
460 TB
The
Resistance Ratio Method is reliable & reproducible
but laborious and relatively slow
The
Radiometric Method is faster but suffers from
problems inherent in the Bactec 460
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Continuous Automated Mycobacterial
Liquid Culture Systems
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Molecular detection of drug resistance
Drug
Putative
resistance
gene
Resistant strains
with mutation (%)
Isoniazid
katG
inhA
ahpC
kasA
22-64
20-34
10
14
Rifampicin
rpoB
90-98
Pyrazinamide
pncA
72-96
Ethambutol
embB
48-62
Riska et al. Int J Tuberc Lung Dis 2000; 4(2):S4-10
4500
4000
3500
3000
2500
2000
1500
1000
500
0
7
6
5
4
3
2
1
Percentage Resistant
Number of isolates
UK 1994-1999
Initial isolates showing resistance to any drug;
Isoniazid & MDR resistance (%),
0
1994
1995
1996
1997
1998
1999
Year
N N
% Isoniazid resistant
% MDR
Regional Centre for Mycobacteriology, Newcastle upon Tyne
DoH - Getting ahead of the curve “Tuberculosis Action Plan”
Develop and implement protocols for DNA fingerprinting
taking customers needs into account.
Establish a central database …
linking fingerprinting and epidemiological data
MTBC Strain Typing Methods
IS6110-based fingerprinting
Most discriminatory method
Slowest method (3-6 weeks)
Difficult to compare large numbers of patterns
Restriction Fragment Length Polymorphism, RFLP
PCR-RFLP
Hemi-nested Inverse PCR (HIP)
Regional Centre for Mycobacteriology, Newcastle upon Tyne
HIP fingerprinting results
1353
603
310
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Spoligotyping
Spacer Oligonucleotide Typing
Less discriminatory than IS6110 typing … BUT...
Faster turnaround time
Digital results, facilitating comparisons
Does not require viable cultures
Regional Centre for Mycobacteriology, Newcastle upon Tyne
VNTR - MIRU
Variable Number of Tandem Repeats
Measures variability in 6 loci
Mycobacterial Interspersed Repetitive Units
Measures variability in 12 loci
PCR-based = rapid turnaround
Digital results, facilitates comparisons
Highly discriminatory
Does not require viable cultures
High throughput (automated sequence analysers)
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Future Genotyping Strategy
Primary typing will be high throughput, automated,
PCR-based i.e. MIRU/VNTR
Secondary typing by IS6110 RFLP when needed for
discrimination
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Time taken for turnaround of final reports
(from date sent by RCM to date final report reaching clinicians)
80
70
60
50
40
30
20
10
0
<=1 day
2 days
3-4 days
5-6 days
7-8 days
9-10 days
>10days
Time
Regional Centre for Mycobacteriology, Birmingham