Mycobacterium tuberculosis

Useful For

Diagnosis and monitoring of respiratory and non-respiratory tuberculosis.



  • Epidemiological risk groups: foreign-born individuals, particularly those from countries with high TB incidence, Aboriginal Canadians, the elderly (particularly elderly males) and close contacts of infectious TB cases are at increased risk.
  • Symptoms: the classic symptom of pulmonary TB disease is a chronic cough of at least 3 week’s duration. This cough is initially dry but after several weeks to months will become productive. Fever and night sweats are common but may be absent in the young and the elderly. Hemoptysis, anorexia, weight loss, chest pain and other symptoms are generally manifestations of more advanced disease.
  • Signs: the most common physical finding in pulmonary TB is totally normal examination, even in relatively advanced cases. Bronchial breathing, rales or crepitations will be found in more advanced cases. It is important to examine for signs of extrapulmonary disease, such as lymphadenopathy, pleural effusion, and abdominal or bone involvement, as these may be present concomitantly, particularly in HIV-infected individuals.


Testing Algorithm

Reporting Name Available separately Always performed
AFB microscopic NO YES
AFB culture NO YES
susceptibility testing

*M. tuberculosis complex PCR is performed on all initial AFB-positive respiratory specimens.


Special Instructions and Forms

Every specimen submitted must be accompanied by a carefully completed requisition form providing the patient’s demographic data, the physician’s name and contact details, the date and time the specimen was collected, and the specimen type and site.
As much as possible, specimens collected for initial diagnosis should be obtained before the initiation of anti-tuberculosis therapy.


Method Name

Auramine fluorescent microscopy

Liquid and solid media culture


Drug susceptibility testing

Polymerase chain reaction (PCR) with fluorescent dye-labeled oligonucleotide probe detection.


Reporting Name

AFB Microscopy


TB Culture







Specimen Required


Sputum (X3)

5-10 ml of sputum specimen should be collected at least 1 hour apart; specimen may be collected on the same day.

Container/Tube: Sterile, leak-proof, laboratory approved container.



Induced sputum. For best results, an ultrasonic nebulizer should be used that can administer 5 – 6 ml per minute over 15 minutes. With this virtually all patients will produce sputum, and a single sputum induction will have equivalent or better yield than fibreoptic bronchoscopy. Sputum induction has been performed successfully in children as young as 2 years of age. Indicate on the requisition that the sputum was induced, because the resulting specimen usually appears watery.


Bronchoscopy. This can be used to confirm the diagnosis of TB when spontaneous sputum and induced sputum are unavailable, or all samples are smear-negative. Bronchoscopy entails risk and discomfort for the patient, is expensive and can contribute to nosocomial spread of TB. If bronchoscopy is performed, post-bronchoscopy sputum should be sent for AFB testing, as this has a yield similar to bronchial washings and lavage.



Gastric aspirate. The primary indication is for children who cannot expectorate sputum, or for the same reason elderly demented patients. The child needs to have nothing to eat or drink for 6 hours before the test. When the individual wakes, a nasogastric tube is intriduced to the stomach, and the contents are aspirated. If nothing is obtained, small quantities (20 – 50 ml) of sterile water can be instilled and aspirated. The fluid has to be adjusted to neutral pH within 4 hours of collection because acid is detrimental to mycobacteria. If sample cannot be processed within 4 hours, it should be placed in a container with 100 mg of sodium carbonate. This technique is uncomfortable and unpleasant for patients.


Urine: First morning urine collected at midstream, on three consecutive days (do not pool or batch specimens).



Nonrespiratory Tuberculosis


Specimen depends on the anatomic site of involvement. Tissue biopsy yields positive findings more often than fluid aspiration. Both are superior to swabs (swabs will be rejected).


Container/Tube: Sterile, leak-proof, laboratory approved container.


Biopsy: submitted fresh or in a small amount of sterile saline. Histopathologic examination requires the specimen to be placed in formalin, destroying mycobacteria and prevents attempts to culture.



CSF (TB meningitis): Serial sampling of CSF for AFB smear and culture may increase yield. The sensitivity may be increased by using the last CSF tube collected and obtaining a large sample volume (10 – 15 ml).


Special Instructions

Monitoring Response to treatment
All patients with smear- and culture-positive sputum should have repeat sputum examinations performed at the end of the second month of treatment. If culture remains positive, repeat after 4 months of treatment. In order to report treatment outcome as “cure”, there must be a negative culture at the completion of treatment. More frequent monitoring is recommended if the clinical and radiographic response is unfavorable. Treatment failure is defined as positive cultures after ≥4 months of treatment or two positive sputum cultures in different months during the last 3 months of treatment, even if the final culture is negative.


Discontinuation of airborne isolation in suspect TB cases
Three successive samples of sputum (spontaneous or induced) are negative on smear, unless TB is still strongly suspected, cultures are pending, and no other diagnosis has been made. 5-10 ml of sputum specimen should be collected at least 1 hour apart; specimen may be collected on the same day.


Discontinuation of airborne isolation in confirmed TB cases

Smear-negative, culture-positive respiratory TB: After 2 weeks of appropriate therapy, as long as there is clinical evidence of improvement.

Smear-positive TB: Three consecutive sputum smears are negative. Each 5-10 ml should be collected on the same day, at least 1 hour apart. In patients who are no longer able to spontaneously produce sputum specimen, sputum induction is useful and appropriate. More invasive testing, i.e. bronchoscopy, is not recommended for this purpose.

MDR-TB: must remain in airborne isolation for the duration of hospital stay or until three consecutive sputum cultures are negative after 6 weeks’ incubation.

XDR-TB: must remain in airborne isolation for the duration of hospital stay or until three consecutive sputum cultures are negative after 6 weeks’ incubation.


Transport Temperature

Specimen Room temperature Refrigerated Frozen
Respiratory NO YES NO
Nonrespiratory (excl. CSF) NO YES NO



Reject Due To

24-hour collection ACCEPT
Gastric aspirate without neutralization received at PHL >4 hours post collection REJECT
Transport > 7 days REJECT
Pooled urine REJECT
Pooled sputum REJECT

Useful For

Diagnosis and monitoring of respiratory and non-respiratory tuberculosis.


Clinical Information

Delayed laboratory confirmation of TB leads to delays in initiation of therapy, potentially inappropriate therapy, and missed opportunities to prevent transmission.


Tuberculosis involving any site may produce symptoms and findings that are not specifically related to the organ or tissue involved but, rather, are systemic in nature. Of the systemic effects, fever is the most easily quantified. The frequency with which fever has been observed in patients with tuberculosis varies from approximately 37 to 80%. The most common hematologic manifestations of tuberculosis are increases in the peripheral blood leukocyte count and anemia, each of which occurs in approximately 10% of patients with apparently localized tuberculosis. Anemia is common when the infection is disseminated. In some instances, anemia or pancytopenia may result from direct involvement of the bone marrow and, thus, be a local, rather than a remote, effect. In many patients tuberculosis is associated with other serious disorders. These include HIV infection, alcoholism, chronic renal failure, diabetes mellitus, neoplastic diseases, and drug abuse, to name but a few. The signs and symptoms of these diseases and their complications can easily obscure or modify those of tuberculosis and result in considerable delays in diagnosis or misdiagnoses for extended periods of time, especially in patients with HIV infection.


For pulmonary tuberculosis cough is the most common symptom. Early in the course of the illness it may be nonproductive, but subsequently, as inflammation and tissue necrosis ensue, sputum is usually produced and is key to most diagnostic methods. Hemoptysis may rarely be a presenting symptom but usually is the result of previous disease and does not necessarily indicate active tuberculosis. Dyspnea is unusual unless there is extensive disease. Tuberculosis may however cause severe respiratory failure. Physical findings in pulmonary tuberculosis are not generally helpful in defining the disease. Rales may be heard in the area of involvement as well as bronchial breath sounds if there is lung consolidation.


Extrapulmonary tuberculosis usually presents more of a diagnostic problem than pulmonary tuberculosis. The combination of small numbers of bacilli and inaccessible sites causes bacteriologic confirmation of a diagnosis to be more difficult, and invasive procedures are frequently required to establish a diagnosis. Extrapulmonary tuberculosis in HIV-infected patients presents a challenge: Because of the frequency of extrapulmonary tuberculosis among HIV-infected patients, diagnostic specimens from any suspected site of disease should be examined for mycobacteria. Moreover, cultures of blood and bone marrow may reveal M. tuberculosis in patients who do not have an obvious localized site of disease but who are being evaluated because of fever. Disseminated tuberculosis occurs because of the inadequacy of host defenses in containing tuberculous infection. Multi-organ involvement is probably much more common than is recognized because, generally, once M. tuberculosis is identified in any specimen, other sites are not evaluated. Because of the multisystem involvement in disseminated tuberculosis, the clinical manifestations are protean. The presenting symptoms and signs are generally nonspecific and are dominated by systemic effects, particularly fever, weight loss, night sweats, anorexia, and weakness. A productive cough is common because most patients with disseminated disease also have pulmonary involvement. Headache and mental status changes are less frequent and are usually associated with meningeal involvement. Physical findings likewise are variable. Fever, wasting, hepatomegaly, pulmonary findings, lymphadenopathy, and splenomegaly occur in descending order of frequency. A finding that is strongly suggestive of disseminated tuberculosis is the choroidal tubercle, a granuloma located in the choroid of the retina. Tuberculous lymphadenitis usually presents as painless swelling of one or more lymph nodes. Intrathoracic adenopathy may compress bronchi, causing atelectasis leading to lung infection and perhaps bronchiectasis. This manifestation is particularly common in children. Needle biopsy or surgical resection of the node may be needed to obtain diagnostic material if the chest radiograph is normal and the sputum smear and culture are negative. In patients with genitourinary tuberculosis, local symptoms predominate and systemic symptoms are less common. Dysuria, hematuria, and frequent urination are common, and flank pain may also be noted. However, the symptoms may be subtle and often there is advanced destruction of the kidneys by the time a diagnosis is established. The finding of pyuria in acidic urine with no routine bacterial organisms isolated from a urine culture should prompt an evaluation for tuberculosis by culturing the urine for mycobacteria. The suspicion of genitourinary tuberculosis should be heightened by the presence of abnormalities on the chest film.


There are five closely related mycobacteria grouped in the M. tuberculosis complex: M. tuberculosis, M. bovis, M. africanum, M. microti, and M. canetti. Mycobacterium tuberculosis is transmitted through the airborne route and there are no known animal reservoirs. Mycobacterium bovis may penetrate the gastrointestinal mucosa or invade the lymphatic tissue of the oropharynx when ingested in milk containing large numbers of organisms. Airborne transmission of both M. bovis and M. africanum can also occur. Mycobacterium bovis BCG is a live-attenuated strain of M. bovis and is widely used as a vaccine for tuberculosis. It may also be used as an agent to enhance immunity against transitional-cell carcinoma of the bladder. When used in this manner, adverse reactions such as dissemination may be encountered, and in such cases M. bovis BCG may be cultured from nonurinary tract system specimens, i.e., blood, sputum, bone marrow, etc.


Tuberculosis is spread from person to person through the air by droplet nuclei, particles 1 to 5 µm in diameter that contain M. tuberculosis complex. Droplet nuclei are produced when persons with pulmonary or laryngeal tuberculosis cough, sneeze, speak, or sing. They also may be produced by aerosol treatments, sputum induction, aerosolization during bronchoscopy, and through manipulation of lesions or processing of tissue or secretions in the hospital or laboratory.


Tuberculosis remains one of the deadliest diseases in the world. The World Health Organization (WHO) estimates that each year more than 8,000,000 new cases of tuberculosis occur and approximately 3,000,000 persons die from the disease. In Canada 1,577 new active and re-treatment cases were reported to the Canadian Tuberculosis Reporting System (CTBRS) in 2010, corresponding to an incidence rate of 4.6 per 100,000 population. The incidence in 25 – 34 year olds accounted for 18% of the total (6.0 / 100,000), whereas the incidence in those > 74 years old was 9.6 / 100,000. Of all TB cases in 2010 66% were from foreign-born individuals, 21% among Canadian-born Aboriginal people and 12% of cases were among Canadian-born non-Aboriginal people. For 2010, pulmonary TB constituted 64% of all reported cases.




AFB Microscopic
The threshold of detection of AFB in concentrated specimens using flourochrome stain is 5,000 – 10,000 bacteria/ml of sputum. The specificity of the AFB smear is high for mycobacteria, but it is important to remember that all nontuberculous mycobacteria (NTM) will be AFB positive. Other organisms, such as Nocardia and Actinomycetes, can be weakly acid-fast, but these are rare. Therefore, a positive AFB smear almost always indicates the presence of mycobacteria, but not necessarily M. tuberculosis.


Number of bacteria seen on microscopy and laboratory interpretation

Flourochrome (250X

Laboratory Report

0 in 30 fields Negative
1-2 per 30 fields Report exact number
1-9 per 10 field 1+
1-9 per field 2+
10 – 90 per field 3+


POSITIVE: M. tuberculosis complex DNA detected.
NEGATIVE: M. tuberculosis complex DNA NOT detected. A negative PCR result does not exclude the presence of tuberculosis disease.


AFB culture
As few as 10 – 100 viable bacteria can be detected by culture.

Solid and liquid cultures are incubated for 8 weeks before being reported as NEGATIVE.



American Thoracic Society. Diagnostic Standards and Classification of Tuberculosis in Adults and Children. Am J Repir Crit Care Med. 2000; 161:1376-1395. Pfyffer, G, and F Palicova. 2011. Mycobacterium: general Characteristics, Laboratory Detection, and Staining Procedures, p 472 – 502, In Versalovic, J., Carroll, K. C., Funke, G., Jorgensen, J, H., Landry, M. L., and Warnock, D, W. Manual of Clinical Microbiology, 10th ed., vol. 2. ASM Press, American Society for Microbiology, Washington, DC.


Menzies D., and Khan, K. Diagnosis of tuberculosis infection and disease. In Long, R., and Ellis, E. Eds. Canadian Tuberculosis Standards 6th Ed. Public Health Agency of Canada, 2007.


Public Health Agency of Canada. Tuberculosis in Canada 2010. Public Health Agency of Canada, Ottawa, ON. 2012.



Analytic Time

Maximum Laboratory Time

Specimen Retention

AFB Microscopic


Mon – Fri 24h 24h 1 month


Mon – Sun 24h 24h 1 month


Mon – Sun 24h 48h 1 month


Mon – Fri 8 weeks 10 weeks 1 month

Method Description

AFB Microscopic: Auramine fluorescent microscopy

TB PCR: Realtime TaqMan PCR with probe confirmation

Culture: 1) BACTTEC MGIT Liquid Media, 2) LJ Solid Media


Performing Laboratory Location

Newfoundland & Labrador Public Health Laboratory
100 Forest Road
St. John’s, NL A1A 3Z9


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