Module 8
Identification of
M. tuberculosis
1
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
Molecular
biology
Positive
AFB culture
Morphology
Phenotypic Approach
Biochemical
and growth tests
(Slow)
Da eliminare?
Genotypic
Approach
DNA probes
with amplification
Immunochromatography
(fast)
w/o amplification
DNA Sequencing
4
Phenotypic approach:
biochemical tests
Advantages:
Disadvantages:
• Inexpensive
• Will identify members
of MTB complex
• Gives reliable results
for some MOTT
•Labour-intensive
•Long turn-around times
•Technical expertise
required
• Limited to solid cultures
Immunochromatography: fast (<1 h), applicable to
solid and liquid cultures
5
Genotypic approach
Advantages:
Disadvantages:
• Fast
• Able to distinguish
between members of
MTB complex
• Identifies NTM
• Fewer biosafety
concerns
• 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
Acid fast bacilli
Colonies visible
< 7 days
10d – 8 wks
= tubercle bacilli
+ MOTT
NO tubercle bacilli
= MOTT
YES
Pigmentation
NO
+
NO
= tubercle bacilli
+ MOTT
PNB
= tubercle bacilli
+ few MOTT
catalase
Thermo labile
= tubercle bacilli
+
Heat stable
Nitrate reductase
M.tuberculosis
M. africanum
-
M.bovis
M. africanum
7
Tubercle bacilli identification chart
with niacin test
Acid fast bacilli
Colonies visible
10d – 8 wks
< 7 days
= tubercle bacilli
+ MOTT
NO tubercle bacilli
= MOTT
YES
Pigmentation
NO
+
= tubercle bacilli
+ MOTT
PNB
= tubercle bacilli
+ few MOTT
niacin
M.bovis
M. africanum
+ few MOTT
+
M.tuberculosis
M. africanum
thermolabile
Catalase
thermoresistant
M.bovis
M. africanum
NO tubercle bacilli
= MOTT
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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
Chromogens
TB
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
Examine at 28 days
• 1 LJ without PNB at 37 °C
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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 medium
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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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Nitrate reduction test –
results and interpretation
• 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+
POSITIVE
purplish red = 5+
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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.
32
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
34
Catalase test at 68 °C, pH 7.0 –
results and interpretation
• 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 catalasenegative, 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.
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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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Why the immunochromatographic
test for low-income countries?
• 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.
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What is the
immunochromatographic test?
• 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%
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How does the test work? (1)
• 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.
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How does the test work? (2)
• 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.
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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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Module review: take-home messages

•
•
•
•
•
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
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Self-assessment
• 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?
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