Tuberculosis (TB) is a potentially deadly infectious disease caused by the Mycobacterium tuberculosis (MTB) bacterium. TB is a leading cause of infectious disease morbidity and mortality worldwide and is often difficult to diagnose, given that individuals with MTB infection may develop symptoms and signs of disease (active TB) or may have no clinical evidence of TB (latent TB infection [LTBI]). Laboratory testing is used to screen for and diagnose TB; accurate diagnosis is extremely important to determine proper treatment and for appropriate disease reporting. Immunologic tests indicate the presence of MTB and are recommended for patients at high risk for LTBI. Diagnostic tests indicate MTB infection in patients with suspected active TB. Some tests (eg, culture and nucleic acid amplification [NAAT]) can confirm TB diagnosis and provide evidence for possible drug resistance and susceptibility.
Quick Answers for Clinicians
Latent tuberculosis infection (LTBI) is diagnosed using laboratory tests that identify a positive immune response to Mycobacterium tuberculosis (MTB). These tests are recommended for individuals at high risk for TB exposure. Active tuberculosis (TB) is evaluated using a combination of medical history, physical examination, radiographic imaging, and laboratory testing. Tests such as an acid-fast-bacilli (AFB) smear, culture, and nucleic acid amplification tests (NAATs) indicate the presence of MTB. Additional laboratory testing is required to identify drug resistance or susceptibility. A definitive diagnosis of TB requires positive culture results.
Nontuberculous mycobacteria (NTM) include over 160 different bacterial species. The clinical symptoms and acid-fast-bacilli (AFB) smear results for diseases caused by NTM and Mycobacterium tuberculosis (MTB) are often indistinguishable. As a result, patients infected with NTM often receive unnecessary TB treatment. Improper diagnosis and treatment complicate patient management and contribute to the development of drug resistance. As such, the accurate diagnosis of TB and NTM infection is extremely important for the implementation of appropriate treatment and disease reporting. Laboratory approaches such as culture and nucleic acid-based testing are essential for accurate diagnosis.
People living with HIV are much more likely to become sick with tuberculosis (TB). In fact, TB is one of the leading causes of death among people living with HIV worldwide. HIV infection is the strongest risk factor for progression of latent TB infection (LTBI) to TB disease. Because of their elevated risk, frequent TB screening is recommended for persons living with HIV. Additionally, persons diagnosed with LTBI or TB disease who do not know their HIV status should receive testing to better understand their risk and help inform proper medical management.
Diagnosis of extrapulmonary tuberculosis (TB) can be challenging. Most established TB tests have been optimized for sputum samples and have low sensitivity in different sample types. Additionally, nonsputum samples are often paucibacillary, which further reduces test sensitivity. Because of this, negative test results cannot confirm the absence of extrapulmonary Mycobacterium tuberculosis (MTB) infection. A combination of acid-fast-bacilli (AFB) staining, culture, tissue biopsy, and histologic, immunologic, and nucleic acid-based tests may be required to diagnose extrapulmonary TB.
Indications for Testing
Risk of Latent Infection
Individuals who are at increased risk for TB infection should undergo laboratory testing. Those with elevated risk include people who have spent time with someone who has TB disease, people from a country where TB is common, healthcare workers who care for high-risk patients, infants and adolescents who have been exposed to high-risk adults, or people who work in high-risk settings such as correctional facilities or long-term care facilities. Many institutions require LTBI testing before employment or enrollment.
Suspected Active Tuberculosis
Individuals with clinical suspicion for TB disease should receive diagnostic testing. Pulmonary TB should be suspected in persons who experience coughing for longer than 3 weeks, hemoptysis, or chest pain. Other symptoms of TB disease include unexplained weight loss, loss of appetite, night sweats, fever, or fatigue.
Tests for Latent Infection
LTBI tests investigate the presence of MTB bacterium in an individual by measuring immune response. Two main types of tests are used to determine the presence of LTBI: skin and blood tests. Screening tests cannot differentiate between LTBI and TB disease and should not be used in patients with active disease symptoms. Generally, screening tests should not be used for testing persons with a low risk of LTBI or TB disease. A tuberculin skin test (TST) or an interferon-γ release assay (IGRA) can be used interchangeably for persons with moderate to high risk of infection, although clinical evaluation may help determine that one test is better suited for a specific situation.
Mantoux Tuberculin Skin Test
A TST is performed by injecting tuberculin into the lower arm. The reaction should be evaluated at a second appointment 2-3 days after the initial injection. A positive result indicates the presence of MTB. A negative result indicates that TB infection is unlikely. The TST is the preferred test for children 5 years or younger who are at high risk for infection and disease progression. A TST is not recommended for persons who have been vaccinated for TB with bacille Calmette-Guérin (BCG) because of an increased risk of false-positive results. Persons who have had a severe reaction to a TST previously should instead undergo IGRA testing in future evaluations.
Interferon-γ Release Assay Blood Test
IGRAs measure the immune response to MTB. IGRA testing is preferred in individuals who may have difficulty returning for a follow-up appointment and in those who have received BCG as a vaccine or for cancer therapy. IGRA testing is relatively fast and does not require a follow-up appointment to determine results. However, IGRAs should not be used for children 5 years or younger.
Tests for Active Tuberculosis
A definitive diagnosis of active TB requires bacteriologic confirmation. For pulmonary TB, the preferred sample type is freshly expectorated sputum; a volume of 5-10 mL is preferred, but testing can be performed on volumes as low as 3 mL. If the patient is not spontaneously producing sputum, induced sputum is also acceptable. The recommended sample type varies for extrapulmonary TB. For a complete and accurate medical evaluation, samples should be tested with a combination of the tests described below. These tests are generally ordered concurrently.
Acid-Fast Bacilli Smear
For all patients with suspected pulmonary TB, the CDC recommends performing an acid-fast-bacilli (AFB) smear on three different sputum specimens. A positive AFB smear can indicate TB disease, but does not confirm MTB infection. This is because some nontuberculous mycobacteria (NTM) are also acid fast. AFB smears are rapid first-line tests, and their results correlate with the likelihood of TB transmission, but accurate species identification requires positive culture or NAAT results.
Nucleic Acid Amplification Tests
The CDC recommends performing a NAAT on the initial respiratory specimen from patients with suspected pulmonary TB. NAATs are rapid and highly specific, although they are often less sensitive than culture. A NAAT, unlike an AFB smear, can distinguish MTB from NTM. A positive NAAT result with or without a positive AFB smear is considered sufficient evidence for TB diagnosis.
NAATs and other molecular tests (such as sequencing) can detect various drug-resistance genes. Molecular tests are recommended for rapid identification of drug resistance and susceptibility. Diagnostic test results and patient history should be taken into account when considering which NAAT is most appropriate.
Mycobacterial culture is a recommended test for the diagnosis of TB, and a positive culture is considered confirmatory. The CDC recommends that all specimens be cultured, regardless of AFB smear results. However, because of the relatively long turnaround time for culture (up to 6-8 weeks), a positive culture is not always necessary to begin or continue treatment. Once growth is detected, species identification can be performed if necessary.
Once MTB growth is confirmed, drug susceptibility testing should be performed. Culture-based drug susceptibility testing is considered the gold standard to identify drug-resistant TB. At a minimum, susceptibility for isoniazid, rifampin, pyrazinamide, and ethambutol should be evaluated. As in diagnosis, culture is the most sensitive and specific test to elucidate drug resistance and susceptibility, but it is limited by its relatively long turnaround time. Patients who do not respond adequately to treatment or who continue to have positive culture results after 3 months of therapy should have drug resistance and susceptibility testing repeated.
Tests for Extrapulmonary Tuberculosis
ARUP Laboratory Tests
IGRA test to aid in the detection of latent disease among persons at increased risk for TB
Positive predictive value is less in low-risk populations
May be used in persons who have received BCG vaccine
More information about the QuantiFERON-TB Gold Plus assays and sample types can be found here
Gold standard test for diagnosing the presence of mycobacteria organisms in any suspected tissue
Includes susceptibility testing
Identify mycobacterial isolates, including requests for speciation within the M. tuberculosis complex
Order for AFB culture of blood or bone marrow specimen
Comprehensive panel includes AFB culture and stain; positive smears reflex to polymerase chain reaction (PCR) amplification of M. tuberculosis complex species and rifampin resistance
Panel includes PCR testing to detect M. tuberculosis isolates and determine possible resistance to rifampin treatment
Qualitative Polymerase Chain Reaction
Order for M. tuberculosis complex speciation and susceptibility testing
If a significant organism is identified, then the appropriate susceptibility panel will be added
Susceptibility testing may not be performed on all isolates (varies by species)
Identification/Susceptibility. Methods may include biochemical, mass spectrometry, nucleic acid probe, polymerase chain reaction, or sequencing.
Order for susceptibility of clinically significant isolates of M. tuberculosis complex
Macrobroth Dilution/Microbroth Dilution
Order when phenotypic drug susceptibility testing is required for M. tuberculosis treatment
For genotypic (DNA sequencing) resistance testing, refer to TB drug resistance by sequencing test
Monitor respiratory specimens in previously diagnosed patients
This test should NOT be ordered without culture in previously undiagnosed patients
For the panel test that includes culture and stain, refer to Acid-Fast Bacillus (AFB) Culture and AFB Stain
Order to culture acid-fast bacteria and fungi from blood or bone marrow specimens
Lewinsohn DM, Leonard MK, LoBue PA, et al. Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children. Clin Infect Dis. 2017;64(2):111-115.PubMed
U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Tuberculosis: diagnosing latent TB infection & disease. [Last reviewed: Apr 2016; Accessed: Feb 2020]Online
Sarro YD, Kone B, Diarra B, et al. Simultaneous diagnosis of tuberculous and non-tuberculous mycobacterial diseases: Time for a better patient management. Clin Microbiol Infect Dis. 2018;3(3).PubMed
U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Tuberculosis: the connection between TB and HIV. [Last reviewed: Jun 2016; Accessed: Feb 2020]Online
U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Tuberculosis: TB and HIV coinfection. [Last reviewed: Mar 2016; Accessed: Feb 2020]Online
U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Tuberculosis (TB): who should be tested. [Last reviewed: Apr 2016; Accessed: Feb 2020]Online
U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Testing for TB Infection. [Last reviewed: Apr 2016; Accessed: Feb 2020]Online
U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Tuberculosis fact sheets: interferon-gamma release assays (IGRAs) – blood tests for TB infection. [Last reviewed: May 2016; Accessed: Feb 2020]Online
Lee JY. Diagnosis and treatment of extrapulmonary tuberculosis. Tuberc Respir Dis (Seoul). 2015;78(2):47–55.PubMed
Body BA, Beard MA, Slechta ES, et al. Evaluation of the Vitek MS v3.0 Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry System for Identification of Mycobacterium and Nocardia Species. J Clin Microbiol. 2018;56(6):e00237-18.
Lozier BK, Haven TR, Astill ME, et al. Detection of acetylcholine receptor modulating antibodies by flow cytometry. Am J Clin Pathol. 2015;143(2):186-192; quiz 305.