TB Methodologies Dr. John G. Magee Regional Reference Centre for Mycobacteriology Health Protection...

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


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)



In-House Assays:

Single PCR; Semi-Nested PCR; Nested PCR…... and now

Real-Time PCR


Amplification

control (58C)Target (62C)

Negative

control

-dF/dT

Temp (°C)


Diagnostic and Reference Mycobacteriology

Microscopy for AFB, if done well, remains a cheap, simple, fast and effective diagnostic technique


Isolation


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


Isolation: Liquid Media circa 1993


Continuous Automated Mycobacterial Liquid Culture systems


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


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


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



“Gene-Probes” can confirm M.tuberculosis complex in under 2 hours



“Genetic probes” are NOT amplification procedures -

They can identify a limited number of common species:

M.tuberculosis complexM.avium complexM.aviumM.intracellulareM.kansasiiM.gordonae


Characterisation of mycolic acids by HPLC

Detection of unique fragments of genomic DNA

16S rRNA sequencing equipmentdatabase variances

Identification of Mycobacteria

[ ]


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


Continuous Automated Mycobacterial Liquid Culture Systems


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



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)


HIP fingerprinting results

1353

603

310


SpoligotypingSpacer Oligonucleotide Typing

Less discriminatory than IS6110 typing … BUT...

Faster turnaround time

Digital results, facilitating comparisons

Does not require viable cultures


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)


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


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

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