1. Module 8 Identification of M. tuberculosis

2. 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.

3. Content outline Definitive M. tuberculosis identification
Cultural tests:
absence of growth on LJ containing PNB
Biochemical tests:
niacin
nitrate reduction
catalase
Immunochromatographic method

4. M. tuberculosis identification
Molecular biology
Positive
AFB culture
Morphology
Phenotypic Approach
Biochemical
and growth tests
(Slow)
Genotypic
Approach
DNA probes
DNA Sequencing
with amplification
w/o amplification
Da eliminare?
Immunochromatography
(fast)

5. 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

6. Genotypic approach Advantages: Disadvantages: 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

7. Tubercle bacilli identification chart without niacin test

8. Tubercle bacilli identification chart with niacin test8

9. Phenotypic identification
Has to consider all the characteristics, including:
Morphology (colonies/bacilli)
Cultural tests
Biochemical tests

10. Cultural tests Rate of growth Growth temperature Pigment production
Colony morphology
Selective inhibition (inoculation on PNB)

11. 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

12. Growth temperature Incubation: 36 ± 1 °C
M. tuberculosis does not grow at lower or higher temperatures.

13. Pigment production
Non-chromogen TB
Chromogens
non-TB

14. Appearance of colonies
M. tuberculosis

15. Appearance of colonies
M. bovis

16. Appearance of colonies
M. chelonae
M. phlei

17. ZN staining
Morphology
“cord-like”

18. ZN staining
Morphology:
“striped”
“dispersed”

19. ZN staining
Morphology:
“sea urchin”

20. 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

21. 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

22. 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.

23. Niacin production Metabolite accumulating in medium:
culture requirements
extraction.
Differentiates M. tuberculosis from other species.
Rarely positive in other mycobacteria.

24. Niacin test – procedure

25. Niacin test – interpretation

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

27. Niacin test Advantages: Disadvantages: Reliable
Easy to perform
Faster than PNB
Disadvantages:
Toxic reagents (banned)
Paper strips (expensive)

28. Nitrate reduction test
M. tuberculosis reduces nitrates to nitrites.
Several species may reduce nitrates.
4 hours.
Cultures tested:
4 weeks old
abundant growth.

29. Nitrate reduction test
Reagents
Preparation: see Annex 8.1.
Procedure
Add 0.2 ml sterile saline to a screw-cap tube.
Use a sterile loop/spade to emulsify 2 loopfuls/spadefuls of a 4-week-old culture in the saline.
Add 2 ml of the NaNO3 substrate.
Shake well and incubate upright in a 37 °C water bath for 3 hours, then remove.

30. Nitrate reduction test – controls
Negative control: extract from an uninoculated tube of medium
Positive control: extract from a culture of M. tuberculosis H37Rv.

31. 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+ purplish red = 5+
POSITIVE

32. 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.

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

34. 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

35. 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 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.

36. 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.

37. 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.

38. 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%

39. 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.

40. 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.

41. True and false exercise
M. tuberculosis can be identified only by the morphology of the colonies.
Expired reagents affect the results of identification tests.
The immunochromatographic test allows fast identification of TB complex from positive cultures.

42. 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

43. 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?