Content

1. Scope

2. Definitions and abbreviations

3. Personnel qualifications

3.1 Medical fitness

3.2 Education and training

4. Procedure

4.1 Principle

4.2 Samples

4.3 Equipment and materials

4.4 Reagents and solutions

4.5 Process of identification of M. tuberculosis

4.6 Interpretation and identification

5. Related documents

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1. Scope

This SOP describes the differentiation of tubercle bacilli from other mycobacteria and the phenotypic identification of Mycobacterium tuberculosis based on biochemical tests.

The vast majority of strains phenotypically identified as M. tuberculosis are genuine M. tuberculosis strains, although phenotypic traits may not always differentiate between M. africanum strains and M. tuberculosis . However, this has no major impact on patient management and follow-up.

For precise epidemiological studies in a geographical region where M. africanum or M. bovis is frequent or for a study of patients from such regions, a link should be established for reference purposes with a laboratory that carries out genotypic identification of tubercle bacilli.

Identification based on biochemical tests has severe limitations: tests are time-consuming, can be performed only on solid cultures, and require large quantities of TB bacilli if they are to give reliable results.

By contrast, immunochromatographic assays enable rapid identification of tubercle bacilli from either solid or liquid cultures and minimize the handling of large quantities of tubercle bacilli that is required in biochemical tests. WHO recommends the use of immunochromatographic tests for the rapid identification of cultures of TB bacilli. These tests are commercially available; if used, the manufacturer’s instructions must be followed.

2. *Definitions and abbreviations

PNB: p-nitrobenzoate.

NA: not applicable

3. Personnel qualifications

1. Medical fitness

In accordance with national laws and practices, arrangements should be made for appropriate health surveillance of TB laboratory workers:

• before enrolment in the TB laboratory;

• at regular intervals thereafter, annually or bi-annually;

• after any biohazard incident;

• at the onset of TB symptoms.

Ideally, individual medical records shall be kept for up to 10 years following the end of occupational exposure.

Laboratory workers should be educated about the symptoms of TB and provided with ready access to free medical care if symptoms arise.

Confidential HIV counselling and testing should be offered to laboratory workers. Options for reassignment of HIV-positive or immuno-suppressed individuals away from the high-risk areas of the TB laboratory should be considered.

All cases of disease or death identified in accordance with national laws and/or practice as resulting from occupational exposure to biological agents shall be notified to the competent authority.

2. Education and training

Basic education and training must be given on the following topics:

• potential risks to health (symptoms of TB disease and transmission);

• precautions to be taken to minimize aerosol formation and prevent exposure;

• hygiene requirements;

• wearing and use of protective equipment and clothing;

• handling of potentially infectious materials;

• laboratory design, including airflow conditions;

• prevention of incidents and steps to be taken by workers in the case of incidents (biohazard incidents, chemical, electrical and fire hazards);

• good laboratory practice;

• organization of work flow;

• procedures;

• importance of laboratory results for patient management;

• importance of laboratory results for the national TB programme.

The training shall be:

• given before a staff member takes up his/her post;

• strictly supervised;

• adapted to take account of new or changed conditions; and

• repeated periodically, preferably every year.

4. Procedure

4.1 Principle

The process involves the phenotypic identification of cultures of acid-fast bacilli grown on solid media. Identification charts are based on the combination of observed colony morphology, inability to grow on a culture medium containing PNB, and results of biochemical tests specific for detection of niacin, heat-labile catalase and nitrate reductase.

4.2 Samples

• Cultures grown on solid media. These could be primary cultures if growth is abundant.

• Subcultures, e.g. control tubes used for drug susceptibility testing.

Identification must be carried out on pure cultures only.

4.3 Equipment and materials

NA

4.4 Reagents and solutions

NA

4.5 Tests of identification of M. tuberculosis

4.5.1 Observation of colonies: morphology and growth rate

The following characteristics of isolated colonies may yield a tentative identification of M. tuberculosis :

• formation of visible colonies 10–28 days after incubation at 37 °C;

• greyish white or buff, dry, cauliflower-like colonies with no late production of pigment (up to 28 days).

4.5.2 Interpretation of the growth test on PNB

Growth is inhibited on Löwenstein–Jensen medium containing PNB (500 μg/ml).

4.5.3 Interpretation of the catalase test

The catalase activity is not heat-stable.

4.5.4 Interpretation of the nitrate reductase test

The nitrate reductase test is positive.

4.5.5 Interpretation of the niacin test

The niacin test is positive.

4.6 Interpretation and identification

4.6.1 Identification of M. tuberculosis

Two charts (see below) can be followed:

• Niacin test not used

Criteria 4.5.1 to 4.5.4 defined above must be met for the specific identification of M. tuberculosis .

• Niacin test used

Criteria 4.5.1 and 4.5.5 defined above allow the specific identification of more than 95% of M. tuberculosis strains.

Note: The niacin test should be performed using commercially available strips. The conventional method requiring the use of cyanogen bromide, an extremely hazardous chemical, is banned in most countries and should be discontinued.

The vast majority of tubercle bacilli are M. tuberculosis . However, if more precise identification is required, strains should be referred to laboratories that perform genotypic tests.

Identification chart without niacin test

Identification chart with niacin test

5. Related documents

Collins C, Grange J, Yates M. Organization and practice in tuberculosis bacteriology . London, Butterworths, 1985.

de Kantor IN, Laszlo A. (1997). Tuberculosis laboratory procedures for developing countries. In: Gangadarham P, Jenkins PA, eds. Mycobacteria: basic aspects . Vol. 1 . New York, Chapman and Hall, 1997.

Hasegawa N et al. (2002). New simple and rapid test for culture confirmation of Mycobacterium tuberculosis complex: a multicenter study. Journal of Clinical Microbiology , 2002, 40(3):908–912.

Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory . Atlanta, GA, United States Department of Health and Human Services, Centers for Disease Control, 1985.

Leao S et al. Practical handbook for the phenotypic and genotypic identification of mycobacteria. Brugge, Belgium, Van den Broele, 2005.

Vincent V, Gutiérrez MC. Mycobacterium : laboratory characteristics of slowly growing mycobacteria. In: Manual of clinical microbiology . Washington, DC, American Society for Microbiology, 2007:573–588.

Laboratory services in tuberculosis control. Part III: Culture . Geneva, World Health Organization, 1998.