Investigation of samples for the presence of Mycobacterium sp.
The genus Mycobacterium is a member of the family Mycobacteriaceae and consists of over 100 species and 10 subspecies of which a few have been reclassified to other species within the family.
TB is caused by members of the Mycobacterium tuberculosis complex (MTBC); in humans this is predominantly Mycobacterium tuberculosis, though less often by other members of the complex such as Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canettii or Mycobacterium caprae.
Strains of the live vaccine Mycobacterium bovis BCG, which are also used intra-vesically in the treatment of bladder cancer, can occasionally cause disease in patients who are immunocompromised. The two other species currently in the complex, Mycobacterium microti and Mycobacterium pinnipedii are almost exclusively associated with different mammalian hosts, but a few cases have been reported in patients who are immunocompromised.
Non-tuberculous mycobacteria (NTM) are also increasingly encountered as a cause of disease in humans. Not all persons infected with tubercle bacilli develop disease, and not all those that are infected become infectious to others. Overt disease may develop months or years after the initial exposure. Patients with evidence of potential acquisition of MTBC, for example, on the basis of a positive tuberculin skin test and/or an interferon gamma blood test, but with no symptoms of active disease nor positive sample for MTBC may have latent TB infection (LTBI). Disease due to tuberculosis may occur in virtually any organ of the body but is most common in the lungs and infection is characterized by caseating granuloma formation.
Collect specimens before antimicrobial therapy where possible.
Some antimicrobials may also have significant anti-mycobacterial activity, notably the fluoroquinolones such as ciprofloxacin, levofloxacin or moxifloxacin, and the macrolides such as clarithromycin or azithromycin.
White topped sterile container
Samples received in formalin will not be processed.
The following specimen types may be sent:
- Pleural fluid
- Bronchoalveolar Lavage
- Blood (contained in x2 Lithium heparin Green topped tubes)
- Bone marrow
- Skin and tissue biopsies
- Post-mortem specimens
- Sterile site body fluids (CSF, pleural fluids etc)
- Gastric washings
For initial diagnosis of mycobacterial infection all specimens to be fresh (less than 1 day old) to minimize contamination, and taken, before anti-tubercular treatment is started.
Purulent specimens are best, 2 to 3 samples of >5 ml collected 8-24 hours apart and one being an early morning sample, due to them having the greatest yield.
Induced sputum preferred over gastric washings, the reason why they are used is due to small children swallowing their respiratory secretions rather than cough them up. Early morning sample before breakfast, on 3 days, greater than 5 ml, delivered promptly to the laboratory due to avoided acidic deterioration of organism, microscopy on this type of sample can be misleading as other acid fast bacilli can be present in the stomach.
Pleural or pericardial fluids
Pleural or pericardial fluids are not sensitive samples for the detection of mycobacterium tuberculosis and concurrent pleural or pericardial biopsy taken with fluid is more useful. Negative result does not rule out a diagnosis.
Urine should be collected on three consecutive days. The sample should be the first void of the day and collected into a white topped sterile universal.
Skin, bone and tissue
Collect aseptically and placed in sterile container without preservatives sterile distilled water added to sample to prevent desiccation.
TB and M. avium may be isolated from stool samples especially in patients that are immunocompromised such as with HIV and AIDS, but NTM can be isolated from healthy individuals may represent colonisation only. If found there is probably this may be due to ingestion of respiratory secreations rather than intestinal disease. This type of sample is heavily contaminated with other bacteria so this type of samples not recommended to be used.
Pus and pus swab
Pus is the sample of choice, over a swab due to the mycobacterium sticking to the swab rather than transferring to the culture medium.
Clear saliva or nasal discharge is not recommended.
Bronchoalveolar lavage/bronchial washings
These may be sent if spontaneous or induced sputum is unavailable or if such specimens are AFB smear negative.
Note: Contamination of the bronchoscope with tap water, which may contain environmental Mycobacterium species, should be avoided. Minimum sample size is preferably 5mL.
Sterile site body fluids
CSF, pleural fluid, etc. Collect aseptically as much (for example >6mL in adults) CSF sample as possible into a CE Marked leak proof container in a sealed plastic bag. If only a small volume is available after initial lumbar puncture, and the findings of cell counts and protein suggest TB meningitis, a second procedure should be considered to obtain a larger volume to improve chances of achieving positive cultures.
It should be noted that pleural or pericardial fluids are not very sensitive samples for the detection of M. tuberculosis, and that a concurrent pleural or pericardial biopsy taken with the fluid is more useful. A negative result on these fluids does not rule out the diagnosis.
Skin, bone, and tissue including postmortem samples
Specimens should be collected aseptically and placed in a CE Marked leak proof container without preservatives in a sealed plastic bag. A caseous portion should be selected if possible: most organisms will be found in the periphery of a caseous lesion. Tissue biopsy specimens received in formalin are unacceptable and will not be processed.
As large a sample of bone marrow as possible should be aspirated and added directly to the culture medium in accordance with the manufacturer’s instructions.
Note: EDTA, even in trace amounts, inhibits the growth of some Mycobacterium species and so is not acceptable, but is accepted in lithium heprin tubes (green topped) need to be at least 6ml of blood.
See above for individual sample types
Limitations and restrictions
Send to laboratory as quickly as possible. If delays likely, refrigerate at 2-8⁰C
- AFB Smear result: 1 working day
- Mycobacterium tuberculosis complex PCR: 1 working day
- Culture: 2 – 12 weeks
The Regional Centre for Mycobacteria, Freeman Hospital, Freeman Road, High Heaton, Newcastle Upon Tyne, Tyne and Wear, NE7 7DN