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Subject: Mycobacteria (AFB, TB) Culture
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Mycobacteria may cause acute and chronic infections. Infections may be localized or systemic, and there is significant overlap with signs and symptoms of fungal and other bacterial infections. Isolation of mycobacteria requires special culture techniques.
Mycobacteria are usually acquired via the respiratory route, and the lower respiratory tract is the site of most serious mycobacterial infections. M. tuberculosis is the most common pathogen associated with these infections. Other mycobacterial species, including other species in the M. tuberculosis complex and M. avium complex (MAC), may cause chronic pulmonary infections.
Organisms may disseminate from the site of primary infection to cause localized or systemic infection. Virtually all organ systems may be involved. The CNS, bone, and urinary tract are common sites of extrapulmonary infection. Mycobacteria may be isolated from stool, most commonly in HIV-infected patients, but the role of mycobacteria as a cause of GI infection has been questioned.
Superficial mycobacterial infections, such as “swimming pool granuloma” caused by M. marinum and wound infections caused by rapidly growing mycobacteria, may be caused by direct inoculation of environmental non–M. tuberculosis species.
Mycobacterial culture is used to detect mycobacterial pathogens and to provide isolates for susceptibility testing and further characterization.
Collect specimens using procedures that minimize the contamination with the patient's endogenous flora.
Because routine bacterial, fungal, and other types of infections may be in the differential diagnosis when mycobacterial infection is suspected, ensure that a sufficient volume of infected material is collected to ensure that all testing can be performed.
For the diagnosis of TB, a minimum of three sputum specimens should be submitted for culture. Patients must be carefully instructed in the proper technique for sputum collection.
Early-morning specimens are preferred because of pooling of secretions at night. A minimum of 5–10 mL of sputum should be submitted for each specimen.
Collection of sputum induced by inhalation of nebulized hypertonic saline or BAL specimens improve detection of pulmonary TB.
Twenty-four–hour sputum collections should not be submitted.
First-morning gastric aspirates may be collected for patients unable to produce sputum, like small children and the frail elderly.
Up to five, first-morning urine specimens should be submitted for patients with suspected renal TB.
Lysis–centrifugation, biphasic, and automated mycobacterial culture techniques are optimal for blood and bone marrow specimens submitted for detection of systemic mycobacterial disease.
Specimens should be transported to the laboratory as soon as possible in sterile containers with tight-fitting lids.
If same-day AFB stain results are needed, the specimen should arrive in the laboratory early enough in the day to allow enough time for specimen processing (decontamination and concentration) and smear interpretation.
Specimens for mycobacterial culture should not be collected using swabs.
Patients with tuberculosis, and specimens collected from them, are a significant risk for health care–acquired infection. Appropriate safety precautions must be followed through all aspects of TB diagnosis.
AFB smears should be performed on all specimens submitted for mycobacterial culture. See ACID-FAST BACILLUS (AFB) SMEAR.
Large-volume liquid specimens should be concentrated, usually by centrifugation, and specimens likely to be contaminated by endogenous flora should be decontaminated and concentrated prior to medium inoculation.
Specimens are inoculated into liquid (e.g., Middlebrook 7H9) media and at least one type of solid media. Special media may be required for fastidious mycobacterial pathogens, like M. haemophilum, or special incubation temperature for agents causing superficial infection, like M. marinum.
Most cultures are incubated at 37°C; cultures from skin or superficial lesions should also be incubated at 30–32°C to improve isolation of mycobacteria that are common pathogens at these sites, such as M. marinum and M. haemophilum. Cultures are incubated in 3–10% CO2. Broth media may be monitored on automated platforms, allowing earlier detection time and providing organisms for identification using molecular genetic tests. Clear, agar-based solid media, like Middlebrook media, provide sensitive isolation of M. tuberculosis, early detection of “microcolonies,” and preliminary, presumptive identification by microcolony morphology. Selective media, which contain a variety of antibiotic agents, may be inoculated for specimens when heavy contamination is likely. Because pathogenic mycobacteria may be inhibited by selective media, nonselective media must always be included. If M. haemophilum infection is suspected, media supplemented with blood, hemin (X factor strip), or ferric ammonium citrate should be inoculated. Inoculation of parallel cultures, with one culture exposed to light during the early growth phase, and one incubated in the dark only, can be used to determine characteristics of pigment formation.
Gram and AFB staining is performed on growth from positive AFB cultures; further testing for identification and susceptibility, as appropriate, is performed.
Growth rate and pigment formation, including photoreactivity, are used to initially characterize non–M. tuberculosis mycobacterial species (NMTB) and help to determine the panel of tests required for full identification. Rapidly growing mycobacterial species yield mature colonies within 10 days after subculture.
Newer technologies for definitive identification of isolates have replaced biochemical and phenotypic testing in many laboratories. The NAP (p-nitro-acetylamino-hydroxypropiophenone) test may be used to rapidly identify M. tuberculosis. Nucleic acid probes are available for identification of M. tuberculosis complex, M. avium complex (MAC), Mycobacterium kansasii, and M. gordonae. Nucleic acid sequencing technology is emerging as an important tool for identification of mycobacteria in reference laboratories.
Cultures are incubated for 6–8 weeks. Specimens with a positive AFB smear or direct molecular test result should be incubated for an additional 4 weeks before signing out as negative.
In positive cultures, several additional weeks may be required for isolation, identification, susceptibility testing, and further characterization, as needed.
Antimicrobial susceptibility testing should be performed on initial isolates, if M. tuberculosis, as well as isolates from cultures, positive longer than 3 months after initiation of therapy. Susceptibility testing should also be performed on most NMTB isolates.
The primary panel for TB isolates include isoniazid, rifampin, ethambutol, and pyrazinamide. For isolates resistant to rifampin or any other two drugs from the primary panel, a secondary panel is performed, including amikacin; capreomycin; cycloserine; ethionamide; kanamycin; PAS; and streptomycin at a low level and a high level.
Various species-specific drug panels are used for significant NMTB isolates.
Using optimal growth, identification, and susceptibility testing systems, complete identification and susceptibility testing should be completed for the majority of M. tuberculosis isolates within 4 weeks of submission of specimen to the lab.
Expected results: No growth.
Positive: Growth of M. tuberculosis in culture is usually very specific for mycobacterial infection. Because of their wide distribution in the environment, cultures positive for NMTB species must be interpreted carefully, taking into consideration factors such as the species, number of positive cultures, and the patient's clinical presentation.
M. gordonae (tap water bacillus) is often isolated from patient specimens, but is rarely associated with disease; its growth is most likely caused by contamination of the specimen, or transient contamination of the patient, with organisms from external water sources.
Negative: The post-test probability of mycobacterial infection is significantly diminished if cultures are negative, but additional cultures and specimens collected from different patient sites may be needed in patients with continued suspicion for mycobacterial disease in spite of initial negative cultures.
Three or more specimens, and specimens from different sites, may be required for sensitive detection; invasive collection techniques may be needed. The final results of testing may not be available for up to 2 months after collection; decisions regarding empirical therapy and management may be required before culture results are available.
The following mycobacterial species are most commonly associated with human disease:
M. tuberculosis complex: M. tuberculosis, M. africanum (rare), M. bovis, including BCG, and M. microti (rare)—pulmonary and other localized infections and systemic disease
M. avium complex (MAC)—systemic infection in immunocompromised patients, like patients with AIDS or chronic pulmonary disease
M. kansasii—pulmonary disease
Rapid growers: M. fortuitum, M. chelonae, M. abscessus—wound infections, localized and systemic infection
M. scrofulaceum—cervical lymphadenitis
M. marinum, M. ulcerans, M. haemophilum—skin and superficial infections
M. genavense—disseminated and GI disease in immunocompromised patients