Download - TB Methodologies
TB Methodologies
Dr. John G. Magee
Regional Reference Centre for MycobacteriologyHealth 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
Detection Smear /Culture Molecular amplification of unique fragments
Identification Phenotypic tests •“Gene probes” • PCR• Sequencing
Susceptibilitytesting
Phenotypic expression
Detection of gene mutations
Fingerprinting(Typing)
Numericaltaxonomy
•RFLP … HIP•Spoligotyping•VNTR/MIRU
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Diagnostic and Reference Mycobacteriology
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
Commercial Assays:
Roche Amplicor (Cobas & “Manual”)
Abbott LCx (LCR)
GenProbe Direct (TMA)
BD ProbeTec ET (SDA)
Direct detection using molecular biological tests
Regional Centre for Mycobacteriology, Newcastle upon Tyne
In-House Assays:
Single PCR; Semi-Nested PCR; Nested PCR…... and now
Real-Time PCR
Direct detection using molecular biological tests
Amplification
control (58C)Target (62C)
Negative
control
-dF/dT
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
Sample 6492 - M.tuberculosis present
of 330 laboratories 31.8% failed to isolate M.tuberculosis
of 176 UK laboratories 39.2% failed
UK NEQAS Distribution 1601 - Mycobacterium culture
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
0
20
40
60
80
100
120
140
Nu
mb
er
of
isola
tes
1 2 3 4 5 6 >6
Weeks after inoculation
LJ
CAMLiC
Regional Centre for Mycobacteriology, Newcastle upon Tyne
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
Surety of Competence
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 also- de Boer et al. J Clin Microbiol 2002; 40; 4004
They found that labs processing <3000 samples per annum showed agreater risk of cross-contamination
Identification of mycobacteria
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 complexM.avium complexM.aviumM.intracellulareM.kansasiiM.gordonae
Regional Centre for Mycobacteriology, Newcastle upon Tyne
Characterisation of mycolic acids by HPLC
Detection of unique fragments of genomic DNA
16S rRNA sequencing equipmentdatabase variances
Identification of Mycobacteria
[ ]
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 resistanceMolecular detection of drug resistance
DrugPutative
resistancegene
Resistant strainswith mutation (%)
Isoniazid
Rifampicin
Pyrazinamide
Ethambutol
katG
inhA
ahpC
kasA
22-64
20-34
10
14
pncA
90-98
embB 48-62
rpoB
72-96
Riska et al. Int J Tuberc Lung Dis 2000; 4(2):S4-10
UK 1994-1999 Initial isolates showing resistance to any drug;Isoniazid & MDR resistance (%),
0500
10001500200025003000350040004500
1994 1995 1996 1997 1998 1999
Year
Nu
mb
er o
f is
olat
es
0
1
2
3
4
5
6
7
Per
cen
tage
Res
ista
nt
N % Isoniazid resistant % MDRN
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
SpoligotypingSpacer 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 - MIRUVariable 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)
0
10
20
30
40
50
60
70
80
<=1 day 2 days 3-4 days 5-6 days 7-8 days 9-10 days >10days
Time
No
of s
ampl
es
Regional Centre for Mycobacteriology, Birmingham