Module 8 Identification of Mycobacterium tuberculosis




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Module 8

Identification of Mycobacterium tuberculosis





Purpose

To provide participants with the knowledge and skills to identify Mycobacterium tuberculosis.

Prerequisite

modules

Modules 2 and 7

Module time

1 hour 10 minutes

Learning objectives

At the end of this module, participants will be able to:

  • evaluate growth characteristics for species identification;

  • perform and interpret the PNB growth inhibition test;

  • perform and interpret the niacin production test;

  • perform and interpret the nitrate reduction test;

  • perform and interpret the catalase test:

    • heat-labile;

  • understand the principle of the immunochromatographic test.


Module overview

Step

Time

Activity/method

Content

Resources
needed


1

5 min

Presentation

Introduction to module

Slides 1–3

2

9 min

Presentation

MTB identification

Slides 4–9

3

16 min

Presentation

Cultural tests and microscopy

Slides 10–21

4

25 min

Presentation

Biochemical tests

Slides 22–36

5

8 min

Presentation

Identification - immunochromatographic test

Slides 37-40

6

2 min

Discussion

True/false exercise

Slide 41

7

2 min

Presentation

Take home messages

Slide 42

8

3 min

Self-evaluation

Self-assessment

Slide 43


Materials and equipment checklist

  • PowerPoint slides or transparencies

  • Overhead projector or computer with LCD projector

  • Flipchart


Practical exercise in the laboratory

  • Several culture tubes with colonies of: M. tuberculosis, non-tuberculous mycobacteria (NTM).

  • Reagents for niacin, nitrate reduction, catalase tests.

  • Immunochromatographic test

  • Reagents for Ziehl–Neelsen (ZN) staining.

  • Gown, gloves and appropriate respirators for the participants

Teaching guide

Slide number

Teaching points

1

Module 8: Identification of Mycobacterium tuberculosis

DISPLAY this slide before you begin the module. Make sure that participants are aware of the transition into a new module.



2



Learning objectives

STATE the objectives on the slide.



3

Flipchart



Content outline

WRITE the content outline on a flipchart before starting this module.

REFER to the flipchart frequently so that participants are sure of where they are in the module.

EXPLAIN that the topics listed are those that will be covered in this module.



4

M. tuberculosis identification

EXPLAIN that M. tuberculosis identification might be either phenotypic (biochemical or immunochromatographic) or genotypic. Phenotypic identification relies on the appearance of colonies and on biochemical and growth tests that underline the main characteristics of the species belonging to the TB complex. Immunochromatographic identification is based on detection of MPB 64 Ag – predominant protein Ag, secreted by MTB complex strains. Genotypic identification is based on molecular biology techniques and identifies mycobacteria species by analysing their DNA with specific probes (with or without amplification) or by sequencing the DNA and comparing the results with a database.



5

Phenotypic approach: biochemical tests

EXPLAIN the advantages and disadvantages of the phenotypic approach.

STATE that this approach has the following advantages:


  • It is cheap (especially when reagents are “home-made”).

  • It can distinguish between members of the MTB complex.

  • It can give reliable results for some NTM.

ADD that there are also disadvantages:

  • It is labour-intensive.

  • Turn-around times are long because of the long time needed for mycobacterial growth.

  • Technical expertise is required for interpretation of test results.

  • Limited to solid cultures (tests require large quantities of TB bacilli to be reliable)

New commercial immunochromatographic methods are available: they are fast (<1h) and easy to perform on liquid and solid cultures

6

Genotypic approach

EXPLAIN the advantages and disadvantages of the genotypic approach.

STATE that this approach has the following advantages:


  • It is fast.

  • It can distinguish between members of the MTB complex.

  • It will identify NTM.

ADD that there are also some disadvantages:

  • It is expensive.

  • It requires dedicated equipment.

  • It requires technical expertise.

  • Traditional methods are still required for culture and DST.

STATE that this module deals with the phenotypic identification of M. tuberculosis.

7

Tubercle bacilli identification chart without niacin test

EXPLAIN the identification chart shown on the slide.

FOLLOW all the arrows and EXPLAIN each result.

ADD that every test shown in the chart will be explained in the slides that follow.



8

Tubercle bacilli identification chart with niacin test

STATE that, if niacin is used for the identification, a positive result identifies M. tuberculosis; a negative result is seen with M. bovis and with atypical mycobacteria.



9

Phenotypic identification

EXPLAIN that traditional identification is based on cultural and biochemical tests. When biochemical tests are used in combination with the growth characteristics of M. tuberculosis colonies (discussed in Module 7), it is possible to precisely identify MTBC in more than 95% of cases.



10

Cultural tests

LIST the characteristics of growth to be observed while performing cultural tests.



11

Rate of growth

EXPLAIN that mycobacteria are divided into rapid- and slow-growers.

READ the definitions of rapid-growers and slow-growers and STRESS that MTBC is a slow grower.

ADD that the time required for primary growth is not used as identification criterium



12

Growth temperature

STATE that M. tuberculosis grows at 36 ± 1 °C. Higher or lower temperatures may inhibit the growth.



13

Pigment production

POINT OUT that M. tuberculosis is a non-chromogen – that is, its colonies are colourless.

ADD that some non-tuberculous mycobacteria may be photochromogens, producing pigment only after exposure to light, or scotochromogens, which produce pigment in the dark. If pigmented colonies grow, they should be considered as contaminants.


14

Appearance of colonies

DESCRIBE the colonies of M. tuberculosis shown on the slide.



15

Appearance of colonies

DESCRIBE the colonies of M. bovis shown on the slide.



16

Appearance of colonies

DESCRIBE the colonies of M. chelonae (smooth, in contrast to M. tuberculosis colonies) and of M. phlei (orange) shown on the slide.



17

ZN staining

STRESS that any appearance-related identification is merely presumptive.

EXPLAIN that the picture in the slide is of a ZN-stained M. tuberculosis colony: it resembles a rope (cord), hence the use of “cord-like” to describe its morphology.


18

ZN staining

DESCRIBE the mycobacteria shown in the pictures. The first two exhibits a “striped” morphology and is related to M. kansasii and M. gordonae, while the third is described as “dispersed” and is related to M. avium.



19

ZN staining

DESCRIBE the “sea-urchin” morphology of the mycobacteria shown in the picture, related to M. xenopi.



20

Growth on medium containing p-nitrobenzoic acid (PNB)

EXPLAIN that different mycobacteria have different susceptibilities to chemicals that can be added to the medium, such as p-nitrobenzoic acid (PNB).

DESCRIBE the procedure: one LJ with PNB and one with no PNB are inoculated, incubated at 37 °C and examined at 28 days.


21

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

EXPLAIN the interpretation of the results given on the slide.

STRESS that M. tuberculosis does not grow on LJ with PNB.


22

Biochemical tests

LIST the main biochemical tests used to identify M. tuberculosis

UNDERLINE the importance of the sentences in the two boxes.


23

Niacin production

EXPLAIN that niacin is a metabolic product of M. tuberculosis; it is released slowly in the medium, which is why an old culture (at least 3 weeks old and with a good growth) is needed and the medium is “scraped” or punctured for its extraction. A positive niacin test has a high positive predictive value as very few other mycobacteria (even within the MTB complex) produce niacin.



24

Niacin test - procedure

EXPLAIN the procedure for performing the niacin test with commercial paper strips:



  1. Add 2 ml of distilled water to the 3-week-old culture tubes.

  2. “Scrape or puncture” the medium with a stick or a loop.

  3. Leave tubes in a slanted position for at least 30 minutes to let the water soak the scraped medium and extract the niacin.

  4. Transfer 1 ml of water to a tube.

  5. Place a paper strip in each tube.

25

Niacin test – interpretation

EXPLAIN that a reaction is considered negative when no colour develops, and positive when a yellow colour develops within 15 minutes at the bottom of the tube.



26

Always check expiry date of strips to avoid

false-negatives!

WARN participants of the consequences of improper storage of reagents. The two tests shown on the slide were performed on the same strain, one with a valid strip and one with an expired strip. The expired strip gave a false-negative result. Remind that the colour change should be in the liquid not on the strip



27

Niacin test

EXPLAIN the advantages and disadvantages of the niacin test, STATING the points in the slide.



28

Nitrate reduction test

EXPLAIN that M. tuberculosis reduces nitrates to nitrites, but shares this characteristic with other mycobacteria.

HIGHLIGHT that this test requires 4 hours. To avoid the risk of false-negatives, culture tubes should be 3–4 weeks old and show abundant growth.


29

Nitrate reduction test

REMIND participants that preparation of the necessary reagents is described in Annex 8.1.

DESCRIBE the steps of the procedure.


30

Nitrate reduction test – controls

REMIND participants of the importance of testing both a positive and a negative control and INDICATE which control should be used.



31

Nitrate reduction test – results and interpretation

EXPLAIN that the nitrate reaction is considered positive when a pink colour develops. The intensity of colour should be compared with a series of standards and only 3+ to 5+ should be considered positive.

If no colour develops, either the reaction is negative or it is positive but reduction has gone beyond the nitrite stage. To discriminate between the two possibilities, add zinc: if no colour develops, the reaction is positive; this should be confirmed by repeating the test.

STRESS that the standard must be added every time the nitrate reduction test is performed.



32

Catalase test: principle

EXPLAIN that catalase is an intracellular enzyme that produces water and bubbles of oxygen from hydrogen peroxide. Almost all mycobacteria have this enzyme, but different characteristics in different mycobacteria may help in species identification.

ADD that some isoniazid-resistant M. tuberculosis and M. bovis strains lack the enzyme.


33

Catalase test

EXPLAIN that M. tuberculosis loses catalase activity at 68 °C, while other mycobacteria have thermoresistant catalases.



34

Catalase test at 68 °C, pH 7.0

REMIND participants that the time and temperature of incubation are critical for this test!

Care should be taken to avoid shaking of the test-tubes: the Tween 80 may form bubbles, giving false-positive results!


35

Catalase test at 68 °C, pH7.0 – results and interpretation

STATE the points on the slide.



36

Catalase tests – controls

STATE the points on the slide.



37

Why the immunochromatographic test for low-income countries?

STATE the points on the slide.

STRESS that the immunochromatographic test can be performed from liquid or solid cultures.


38

What is the immunochromatographic test?

STATE the points on the slide.

REMEMBER that the immunochromatographic test cannot differentiate among tubercle bacilli


39

How does the test work? (1)

EXPLAIN that the positive culture sample applied in the sample well flow laterally through the membrane

The Ab/colloidal gold conjugate binds to MPB Ag in the sample. The complex then flows further and binds to the MABs on the solid phase in the test zone, producing a red band (positive for MTBC).

In the absence of MPB 64, no positive reaction (red band) is seen in this area.

The liquid continues to migrate along the membrane and produces a red band in the control zone (showing normal reagent activity).


40

How does the test work? (2)

The slide shows a positive and a negative test. EXPLAIN the meaning of the different bands.



41

Discussion



True and false exercise

Participants should be provided with two cards (green and red) which they should use to state whether the messages are true (green) or false (red).

True: 2, 3

False: 1


42

Module review: take-home messages

LIST the characteristic of M. tuberculosis that allow its identification.



43

Self-assessment


The participants should evaluate what they have learned from this module by answering the questions.

ANSWER any questions the participants may have




Practical activity in the laboratory (BSL3 facility)

  1. Ensure that all participants are wearing appropriate personal protective equipment. Ask them to examine several culture tubes positive for mycobacteria and describe the colony morphology.

  2. Participants should carry out a niacin test (or another biochemical test) assisted by the supervisors.

  3. If available, participants should carry out a immunochromatographic test and interpret the results.




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