TB NAAT, Smear, Culture, and Identification of Acid Fast Bacilli

Test Code

AFB1

Description

This test represents the full workup for acid fast bacilli and partially acid fast organisms from a clinical specimen, especially if there is unknown or potential risk of M. tuberculosis infection.

This algorithm includes the following components: nucleic acid amplification testing (NAAT), acid fast smear, culture, and appropriate identification. NAAT is a molecular test used to detect M. tuberculosis complex and rifampin resistance from a specimen. The AFB smear is a direct fluorescent stain from the specimen and can be used to quickly establish the presence of mycobacteria. The AFB culture is the inoculation of a clinical specimen onto multiple media types followed by monitoring for six weeks. Growth of any AFB (i.e. mycobacteria) or partially acid fast organisms (e.g. aerobic actinomycetes) is identified. Identification will occur to the greatest extent possible, which is usually to the species level but can be to the subspecies level for some organisms (e.g. Mycobacterium abscessus subsp. abscessus) .

Collect

Specimen	Preferred Volume	Unacceptable Conditions	Collection Container	Total Stability Time-including transport
Sputum (expectorated or induced)	5-10 mL	<3 mL	Sterile, leak-proof container.	7 days refrigerated; 72 hours ambient.
Lower respiratory tract (e.g. bronchoalveolar lavage, bronchial washes etc.)	5-10 mL	<3 mL	Collect washing ans aspirates in a sputum trap. Place brushes in a sterile, leak-proof container with up to 5 mL sterile non- bacteriostatic saline.
Body Fluids (e.g. pericardial, synovial, pleural etc.)	2-4 mL	<2 mL	Sterile, leak-proof container.
Cerebrospinal Fluid (CSF)	2-4 mL	<2 mL refrigerated/frozen CSF.	Sterile, leak-proof container.	72 hours ambient. This source should not be refrigerated.
Fresh Tissue	1-2 g	<1 g or tissue submitted on a dry swab or fixed in formalin or other preservatives.	Sterile, leak-proof container with tissue submerged in sterile non-bacteriostatic saline.
Gastric lavage or wash	10 mL	<10 mL or a specimen that has not been neutralized within 1 hour of collection.	Sterile, leak-proof container. Neutralize specimen within 4 hours of collection with 100 mg of sodium carbonate.
Urine	5-10 mL	<3 mL, pooled urine specimen or a urine specimen that has been mixed with preservatives.	Sterile, leak-proof container.	7 days refrigerated.

Unacceptable Conditions

Sample collected in wax containers are not acceptable. Wax containers may produce false-positive smear results.
Swabs are not recommended for the isolation of mycobacteria, since they provide limited material and the hydrophobicity of the mycobacterial cell envelop often compromises a transfer from the swabs onto solid media or into broth media.
Swabs, stool, and blood cannot be used in the TB NAAT assay.
Specimen received in the lab 14 days post-collection.

Notes

The Centers for Disease Control and Prevention (CDC) recommends that NAAT should be performed on at least one respiratory specimen from each patient with signs and symptoms of pulmonary TB for whom a diagnosis of TB is being considered but has not yet been established, and for whom the test result would alter case management or TB control activities.

Smear is not performed for urine, stool, or blood specimens.
TB NAAT is not performed on swabs, stool, or blood specimens.

Performed

Typically all days.

Methodology

The specimen is screened for M. tuberculosis complex with a nucleic acid amplification test (TB NAAT). A smear for AFB is performed and the specimen is cultured on solid and liquid media (see AFB7 for more details). Mycobacteria or partially acid fast organisms are identified to the full extent possible, such as to the species or subspecies level (AFB4).

Reported

TB NAAT is reported within 1-3 days.
AFB Smear reported within 24 hours. Note that AFB smears are not performed for urine, stool, or blood.
The final report for the AFB culture is issued at 6 weeks. Solid media cultures are read at 1, 3, and 6 weeks. Liquid media cultures are continuously monitored. Positive cultures are reported at the time of positivity and negative cultures are reported at 1, 3, and 6 weeks. In some cases, media may be incubated for up to 8 weeks and if positive, a supplemental report will be issued.
Identification is reported within 3 days to 2 weeks, depending on the method(s) used.

Department

Infectious Disease Laboratory, Mycobacteriology

Synonyms

NAAT with AFB3, NAAT with AFB7 and AFB4, TB rule out, AFB2C, NAAT, culture, smear, identification, nucleic acid amplification testing, reverse hybridization testing, NTM

Related Tests

NAAT is a direct TB test component of AFB1; AFB3 is similar to AFB1, except it is primarily for NTM, since the TB NAAT test is not performed; AFB4 is the identification component of AFB1.

License Comments

TB NAAT: FDA approved for sputum, validated for some other sources.
Smear and culture (AFB7): Reference method, no special licensing required.
Identification (AFB4): waiver/single use permit is required for Identification of AFB for NYS isolates

Reference Interval

Negative for acid fast bacilli.

Interpretation

Test	Interpretations
AFB Smear	Fluorochrome stain read at 400X No AFB found Doubtful: Repeat specimen recommended. 1 to 2 AFB seen/ 70 fields observed. 1+ = 2-18 AFB/ 50 fields observed. 2+ = 4-36 AFB/ 10 fields observed. 3+= 4-36 AFB/ field observed. 4+= >36 AFB/ field observed.
NAAT	M. tuberculosis complex may be Detected, Not Detected, or Indeterminate. Rifampin resistance may be Detected, Not Detected or Indeterminate. In cases where the line probe assay is used instead of real-time PCR, rifampin resistance is Present, Not Present, or Indeterminate. Isoniazid may also be reported.
AFB Culture	Negative cultures are reported as negative growth. Positive cultures are reported as organisms recovered with a colony count where possible (CFU/mL).
AFB Identification	Organisms are identified to the species or subspecies level, where relevant.

CPT Code

This is a test algorithm that includes multiple tests. When it is ordered, TB NAAT, smear and culture are typically performed. If an organism is recovered, a reflex identification test will be ordered. The identification method used will depend on the organism.

Test	Method	CPT code	Billing Code	When is this applied?
NAAT or AFB2C	PCR	87556	AFB2C	On sources where real-time PCR can be used. See NAAT for more details.
NAAT or MTB4	PCR Line Probe	87798 x 2	MTB4S	On sources where the line probe can be used. See NAAT for more details.
AFB Smear	Fluorochrome Stain	87206	CXAFB1	All samples
AFB Culture	Specimen processing	87015	CXAFB2	All samples
AFB Culture and presumptive ID	Culture	87116	CXAFB3	All samples
AFB Identification	PCR/MALDI-TOF Mass spectrometry	87798 x 9	MASID (1st organism), MASI2 (2nd organism), MASI3 (3rd organism)	If appropriate organisms are recovered
AFB Identification	Line probe assay	87798 x 7	NTMID (1st organism), NTMD2 (2nd organism), NTMD3 (3rd organism)	If appropriate organisms are recovered
MTB Identification	Line probe assay	87556	MTB7T	If the appropriate organism is recovered
rpoB Identification	Sequencing	87153	RPOB	If the appropriate organism is recovered
16S Identification	Sequencing	87153	16S	If the appropriate organism is recovered