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Quantiferon and T spot for Mycobacterium tuberculosis Detection

The United States had 7,174 reported cases of tuberculosis in 2020. Fifty-two percent of the tuberculosis cases were in four states: New York (9%), Florida (6%), Texas (13%) and California (24%). These states have the most immigration from Mexico and the Pacific Islands.

There are two reasons to screen for tuberculosis

  1. To detect active disease and cure it to stop transmission
  2. To find latent disease and treat it to prevent progression to active disease

In the United States, the greater focus is to find individuals with latent infection. Approximately 72 percent of people who have latent TB were born in another country.

Three tests are available in the United States for diagnosis of TB and latent TB. One is the TB skin test and two are interferon-gamma release assays. The tuberculin skin test was developed in the early 1900s. This test involves intradermal injection of a purified protein derivative from an attenuated Mycobacterium tuberculosis strain, followed by visual assessment for the presence or absence of a delayed-type hypersensitivity reaction at the injection site 48 to 72 hours later. Tuberculin skin testing was a mainstay of latent and active TB infection prior to availability of blood assays for Mycobacterium tuberculosis (MTB) in 2001. Disadvantages of TST include the challenge of proper administration and interpretation, as well as false-positive results due to non-tuberculous mycobacteria infection and BCG administration.

Interferon-gamma release assays (IGRA) have become the preferred method for detecting latent disease. IGRA are in vitro T-cell–based assays that measure interferon gamma release by sensitized T cells in response to highly specific Mycobacteria tuberculosis antigens.

IGRA are based on the principle that individuals who are infected with TB have primed memory T cells, which, upon re-exposure to specific TB antigens, will be activated and stimulated to release interferon-gamma. Hence these assays are designated as interferon gamma release assays (IGRAs). FDA-approved IGRA assays for MTB include QuantiFERON TB Gold Plus (Qiagen) and T-spot TB (Oxford Immunotec) assays. Because these assays quantitate a biologic response, testing of a fresh blood specimen with viable white blood cells is crucial to obtaining accurate results.

T-spot TB test received pre-market approval from the FDA in July 2008. A single tube of blood is drawn and centrifuged. Mononuclear cells are separated using Ficoll separation and are added to microtiter plate wells that contain solid phase antibodies to gamma-interferon. TB antigens ESAT-6 and CFP-10 are added to separate wells. T-cells which have been sensitized to M. tuberculosis secrete gamma-interferon, which then binds to the solid phase antibodies. Substrate is added and the amount of gamma interferon is quantified using an enzyme-linked immunospot (ELISPOT) assay. T-Spot tubes are sent to Oxford’s mains laboratory in Memphis, TN.

The original QuantiFERON-TB Gold (QFT-Gold) assay used a cocktail of peptides to stimulate pre-sensitized CD4 T-cells to release gamma interferon. In June of 2017, Qiagen released a newer version, called QuantiFERON-TB Gold Plus, (QFT-Plus), which is their fourth-generation assay. This assay was designed to stimulate both CD4 and CD8 positive T cells and have increased sensitivity for both active disease and latent infection. Whole blood is collected into four tubes.

The mitogen tube (purple top) contains phytohemagglutinin, which serves as a positive control for T-cell activity, while the nil tube (gray top) measures the background level of circulating interferon gamma.

The Green and Yellow tubes contain TB antigens. Tube 1 contains long peptides of ESAT-6, CFP-10 that are recognized by MCH class II molecules for presentation to CD4-positive T cells. Tube 2 has both long and short peptides for ESAT- and CFP-10 for stimulation of MHC class II and class I molecules on CD4 and CD8 T cells, respectively.

If either one or both QFT-Plus TB antigen tubes are equal to or greater than 0.35 IU/mL and are at least 25% of the nil tube value, the patient is considered positive. In general, QFT-Plus results are usually >10 in patients with active TB. Results between 1 and 10 should be considered significant and be followed-up based on their medical history and clinical findings. Intermediate results are most often seen in employee health screening and patients that have migrated from a region of the world with a higher incidence of TB. Low-positive results between the cut-off of 0.35 and 0.99 and 85 percent of them repeat as negative and are most likely false-positive.  Some laboratories report these as gray zone instead of positive.

Neither IGRA nor tuberculin skin tests can distinguish active from latent tuberculosis. CDC recommends that persons with a positive skin test or IGRA be evaluated for the likelihood of TB infection. A diagnosis of latent TB requires that active TB be excluded by history & physical examination, chest X-ray, and cultures when indicated. Although both sensitivity and specificity of the IGRA tests is high, negative results are not sufficient by themselves to exclude infection in suspect cases.

Four conditions need to be met to justify primary use of an IGRA:

  1. A person age five or older who is likely to be infected with M. tuberculosis
  2. A low or intermediate risk of disease progression
  3. A decision that testing for latent infection is warranted
  4. A person who has received BCG vaccination or is unlikely to return to have his or her tuberculin skin test read.

If even one of these conditions is not met, the tuberculin skin test could be used. TST is also preferred in children under age five.

Most countries outside the U.S. administer Bacillus Calmette-Guérin (BCG) vaccination routinely. IGRAs are much better in detecting latent tuberculosis in vaccinated individuals than the tuberculin skin test because the vaccine produces positive skin test results but does not interfere with IGRA.

TB testing is warranted for individuals who have had a known exposure to a person infected with M. tuberculosis, those entering the U.S. from an area with a high prevalence of TB, HIV-infected patients, and those who are immunocompromised from other conditions, such as chronic renal failure or intravenous drug use. Prisoners could also qualify for testing.

CDC and the National Tuberculosis Controllers Association published guidelines for TB screening, testing and treatment of US health care workers on May 27, 2019. These guidelines stated that all health care workers should be tested at hire, up on transfer, at exposure and post-exposure. These guidelines eliminated mandatory annual screening.

Specimen requirement for QFT-Plus is whole blood collected directly into four separate tubes or into a single lithium heparin-tube, which can then be aliquoted into the four separate tubes in the laboratory. The specimen must be transported at ambient temperature as soon as possible since testing must begin within 30 hours of specimen collection.


Moon HW, Gaur RL, Tien SS, et al. 2017. Evaluation of QuantiFERON-TB Gold-Plus in healthcare workers in a low-incidence setting. J Clin Micro 55(6):1650-1657

Telisinghe L, Amofa-Sekyi M, Maluzi K, et al. 2017. The sensitivity of the QuantiFERON-TB Gold Plus assay in Zambian adults with active tuberculosis. Int J Tuberc Lung Dis 21(6):690-696

Lewinsohn DM, et al. Clin Infect Dis. 2017;64[2]:e1–e33

 Sosa LE, Njie GJ, Lobato MN, et al. Tuberculosis Screening, Testing, and Treatment of U.S. Health Care Personnel: Recommendations from the National Tuberculosis Controllers Association and CDC, 2019. MMWR Morb Mortal Wkly Rep 2019;68:439–443. DOI:

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