Module 8

Identification of M. tuberculosis

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Learning objectives

At the end of this module you will be able to:

evaluate growth characteristics for species identification; perform and interpret the PNB growth inhibition test; perform and interpret the niacin test; perform and interpret the nitrate reduction test; perform and interpret the catalase test

– heat-labile; Understand the principle of the immunochromatographic

test.

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Content outline

• Definitive M. tuberculosis identification• Cultural tests:

– absence of growth on LJ containing PNB

• Biochemical tests:– niacin– nitrate reduction– catalase

• Immunochromatographic method

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M. tuberculosis identification

Da eliminare?

PositiveAFB culture

PositiveAFB culture

Morphology

Phenotypic Approach

Biochemicaland growth tests

(Slow)

GenotypicApproach

DNA probes

DNA Sequencing

with amplification

w/o amplification

Molecular biology

Immunochromatography(fast)

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Phenotypic approach: biochemical tests

Advantages:

• Inexpensive • Will identify members

of MTB complex• Gives reliable results

for some MOTT

Disadvantages:

•Labour-intensive•Long turn-around times•Technical expertise required• Limited to solid cultures

Immunochromatography: fast (<1 h), applicable to solid and liquid cultures

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Genotypic approach

Advantages:

• Fast• Able to distinguish

between members of MTB complex

• Identifies NTM• Fewer biosafety

concerns

Disadvantages:

• Expensive• Requires dedicated

equipment• Requires technical

expertise• Traditional methods

still required for culture and DST

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Tubercle bacilli identification chart without niacin test

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Acid fast bacilli

Colonies visible

< 7 days

PigmentationYES

NO

10d – 8 wks

PNB

Heat stable

NO

catalase

Thermo labile

+

= tubercle bacilli+ few MOTT

= tubercle bacilli+ MOTT

= tubercle bacilli+ MOTT

NO tubercle bacilli= MOTT

-

= tubercle bacilli Nitrate reductase+

M.bovisM. africanum

-M.tuberculosis

M. africanum

Tubercle bacilli identification chart with niacin test

Acid fast bacilli

Colonies visible

< 7 days

PigmentationYES

NO

10d – 8 wks

PNB

niacin

+

= tubercle bacilli+ few MOTT

= tubercle bacilli+ MOTT

= tubercle bacilli+ MOTT

NO tubercle bacilli= MOTT

-

CatalaseM.bovisM. africanum

+ few MOTT

M.tuberculosisM. africanum

+

-thermolabile

M.bovisM. africanum

thermoresistantNO tubercle bacilli

= MOTT8

Phenotypic identification

Has to consider all the characteristics, including:

• Morphology (colonies/bacilli)

• Cultural tests

• Biochemical tests

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Cultural tests

• Rate of growth

• Growth temperature

• Pigment production

• Colony morphology

• Selective inhibition (inoculation on PNB)

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Rate of growth

• Rapid-grower: isolated in less than a week– not TB

• Slow-grower: usually 3 weeks, up to 6 weeks– could be TB

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Growth temperature

• Incubation: 36 ± 1 °C

M. tuberculosis does not grow at lower or higher temperatures.

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Pigment production

Non-chromogen TB

Chromogens non-TB

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Appearance of colonies

M. tuberculosis

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Appearance of colonies

M. bovis

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Appearance of colonies

M. chelonae M. phlei

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ZN staining

Morphology “cord-like”

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ZN staining

Morphology:

“striped”

“dispersed”

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ZN staining

Morphology:

“sea urchin”

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Growth on medium containing p-nitrobenzoic acid (PNB)

Procedure

• 1 LJ with PNB at 37 °C

• 1 LJ without PNB at 37 °C

Examine at 28 days

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Growth on medium containing p-nitrobenzoic acid (PNB) – interpretation

• Abundant growth on both slopes: mycobacterial strain other than tubercle bacilli.

• Abundant growth on control tube and little or no growth on PNB medium: MTB complex strain.

• No growth on either slope: non-interpretable test, to be repeated.

M. tuberculosis fails to grow on PNB medium21

Biochemical tests• Niacin production• Nitrate reduction

• Catalase negative at 68 °C

Always use pure cultures, otherwise they will yield false results.

Test should be performed in the BSC – aerosols are produced.

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Niacin production

• Metabolite accumulating in medium:– culture requirements– extraction.

• Differentiates M. tuberculosis from other species.

• Rarely positive in other mycobacteria.

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Niacin test – procedure

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Niacin test – interpretation

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Always check expiry date of strips to avoid false-negatives!

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Niacin test

Advantages:• Reliable• Easy to perform• Faster than PNB

Disadvantages:• Toxic reagents (banned)• Paper strips (expensive)

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Nitrate reduction test

• M. tuberculosis reduces nitrates to nitrites.• Several species may reduce nitrates.• 4 hours.• Cultures tested:

– 4 weeks old– abundant growth.

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Nitrate reduction test

Reagents • Preparation: see Annex 8.1.

Procedure

1. Add 0.2 ml sterile saline to a screw-cap tube.

2. Use a sterile loop/spade to emulsify 2 loopfuls/spadefuls of a 4-week-old culture in the saline.

3. Add 2 ml of the NaNO3 substrate.

4. Shake well and incubate upright in a 37 °C water bath for 3 hours, then remove.

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Nitrate reduction test – controls

• Negative control: extract from an uninoculated tube of medium

• Positive control: extract from a culture of M. tuberculosis H37Rv.

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• Negative: no colour. • Positive: red colour, varying from pink to very

deep crimson:

faint pink = +/-clear pink = 1+deep pink = 2+red = 3+deep red = 4+purplish red = 5+

Nitrate reduction test – results and interpretation

POSITIVE

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Catalase test: principle

• Intracellular, soluble enzyme.

• Release of O2 and production of bubbles.

• Virtually all mycobacteria possess catalase enzymes, except for rare isoniazid-resistant mutants of M. tuberculosis and M. bovis.

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Catalase test

• 68 °C test at pH7: strains of M. tuberculosis lose catalase activity.

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Catalase test at 68°C, pH 7.0

• All mycobacteria produce catalase, usually thermoresistant but M.tuberculosis produces thermolabile catalase– 1 tube: incubation at 68 °C for 20 minutes– 1 tube: incubation at room temperature

for 20 minutes

– detection by H2O2

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• If the unheated tube forms oxygen bubbles and the heated tube does not, the test strain produces heat-labile catalase and is likely to belong to the M. tuberculosis complex.

• If both heated and unheated tubes form oxygen bubbles, the test strain produces heat-stable catalase and is unlikely to belong to the M. tuberculosis complex.

• If neither the heated nor the unheated tube forms oxygen bubbles, the test has failed or the strain may be catalase-negative, such as rare isoniazid-resistant strains of M. tuberculosis. Repeat the test with a larger quantity of bacilli to eliminate the possibility of a false-negative test.

Catalase test at 68 °C, pH 7.0 –results and interpretation

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Catalase tests – controls

68 °C tests

• Positive control, no bubble formation after heating: previously identified M. tuberculosis strain or an MTB reference strain from a culture collection.

• Negative control, bubble formation after heating: M. terrae grown on LJ.

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• Alternative to molecular methods.

• Species ID by immunochromatographic method from positive culture (NOT from specimens).

• Rapid (15 minutes).

• User-friendly.

• Quality assured.

• Reproducible.

• ID on culture grown on liquid medium (protocol can be adapted to colony identification).

• Performance, cost, turn-around time compare well with other fast molecular methods.

Why the immunochromatographic test for low-income countries?

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• Simple, rapid immunochromatographic method.• Detects MPB 64 Ag – predominant protein Ag, secreted

by MTB complex strains.• Simple lateral flow speciation test.• No sample processing/instrumentation required.• Storage and cryopreservation do not interfere with

detection.• Sensitivity of tubercle bacilli detection – 100%

What is the immunochromatographic test?

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• The test strip consists of: - sample pad

– reagent pad– nitrocellulose membrane– absorbent pad.

• Antibodies are immobilized on nitrocellulose membrane as capture reagent (anti-MBP64) or control (anti-IgG) lines.

• Secondary Ab, which recognizes another epitope of Ag-MBP64 conjugated with colloidal gold particles, is used for Ag capture and detection on the membrane in a sandwich type.

How does the test work? (1)

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• Negative, only one reddish-purple band appears in the control window.

• Positive, in addition to the control band, a clear distinguishable reddish-purple band also appears in the test window.

How does the test work? (2)

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True and false exercise

1. M. tuberculosis can be identified only by the morphology of the colonies.

2. Expired reagents affect the results of identification tests.

3. The immunochromatographic test allows fast identification of TB complex from positive cultures.

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Characteristics of M. tuberculosis

• AFB • Slow growth rate• Growth temperature 35–37 °C only • Typical morphology• No pigmentation

Additional• No growth on LJ medium containing p-nitrobenzoic acid• Niacin test: positive• Catalase test: negative at 68 °C • Nitrate test: positive

Module review: take-home messages

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• What are the advantages and disadvantages of phenotypic and genotypic identification?

• Why should a complete set of standards be used for comparison during each nitrate reduction test?

• If all mycobacteria have the catalase enzyme, why is this test useful in species identification?

• List the culture tests for Mycobacteria species identification.

• What are the main characteristics that allow identification of M. tuberculosis?

Self-assessment

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