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Quantiferon and T spot for Mycobacterium tuberculosis Detection

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.

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 (TST) 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 M. 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). Currently available FDA-approved IGRA assays for MTB include QuantiFERON TB Gold (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.

The T-SPOT TB test received pre-market approval from the FDA in July 2008. In this test, a tube of blood is drawn and centrifuged. Mononuclear cells are added to a microtiter plate that contains solid phase antibodies to gamma interferon. Peptide antigens identical to ESAT-6, a protein virulence factor of M. tuberculosis, are then introduced. T-cells, which have been sensitized to M. tuberculosis, secrete gamma interferon, which binds to the solid phase antibodies. Substrate is added and the amount of gamma interferon is quantified using a T.SPOT using an enzyme-linked immunospot (ELISPOT) method.

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). This assay was designed to stimulate both CD4 and CD8 positive T cells and have increased sensitivity for both active disease and latent infection. Qiagen plans to discontinue QFT-Gold by July 2018.

Both QFTGold and QFT Plus assays include identical mitogen and nil tubes. The mitogen tube contains phytohemaglutanin, which serves as a positive control for T-cell activity, while the nil tube measures the background level of circulating interferon gamma in the patient. The notable difference between the two versions is the mixture of peptides used to stimulate T cells.

Tube 1 of the QFT-Gold assay uses Mycobacterium tuberculosis long peptides of ESAT-6, CFP-10 and TB7.7 proteins 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.The QFT-Plus assay eliminated the TB7.7 antigen.

Cutoff values, and result interpretation are the same for QFT-Plus and QFT-Gold assays with one exception.  If either one or both of the 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, Quantiferon results are usually >10 in patients with active TB. Results between 1 and 10 should be considered significant and receive some kind of follow-up based on their medical history and clinical findings. Intermediate results are most often seen in employee health 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 usually repeat as negative and are most likely false positive.  Some laboratories report these values as gray-zone instead of positive.

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

  1. 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.

IGRAs are much better in detecting latent tuberculosis than TST in persons who have received BCG (bacillus Calmette-Guérin) vaccination because a positive TST is not meaningful. Most countries outside the U.S. administer BCG vaccination routinely. Persons who are not likely to return to have a TST read include undocumented immigrants, homeless persons, prisoners released before their TST is read.

If even one of these conditions is not met, the tuberculin skin test, or TST, could be used. TST is much less expensive than IGRA. At this time, TST is also preferred in children under age five.

Testing is warranted in those 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. There is an active program to test some who go through the formal process to immigrate to the U.S., including refugees. In this program IGRA is used because most come from countries where BCG vaccination is administered.

Many health care institutions require staff to be screened for latent M. tuberculosis infection, even though many employees are in a low-risk group. . Typically the tuberculin skin test is performed annually in this population. Studies have shown that in this very low-risk population in the U.S., serial testing could lead to false-positive results. Conversion to a positive test occurs most commonly with IGRA.

Neither IGRA nor TST can distinguish active from latent tuberculosis. CDC recommends that persons with a positive TST 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.

Specimen requirement for the QFT-Plus test is whole blood collected directly into four separate tubes, as is the protocol for the QFT-Gold assay, 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.


  1. 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
  2. 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
  3. Lewinsohn DM, et al. Clin Infect Dis. 2017;64[2]:e1–e33
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