Quantiferon and T spot for Mycobacterium tuberculosis Detection

Tuberculosis (TB) remains one of the most prevalent infectious diseases worldwide, with an estimated 9 million active infections annually, and 2 billion latent infections. Attributable mortality is approximately 2 million deaths globally per year. Overall, both active and latent TB infections have declined in the United States since 1998.

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.

TB blood assays are based on the principle of interferon-gamma being critical to regulation of cell-mediated immune response to MTB infection, hence their designation as interferon gamma release assays (IGRAs). Currently available FDA-approved IGRA assays for MTB include Quantiferon and T-spot TB. Both assays measure the interferon-gamma response to specific MTB proteins, including early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10).  Because these assays quantitate a biologic response, testing of a fresh blood specimen with viable white blood cells is crucial to obtaining accurate results.

Oxford Immunotec sells the T-SPOT.TB test, which received pre-market approval from the FDA in July 2008. A tube of blood is drawn and centrifuged. Mononuclear cells are added to a microtiter plate that contains solid phase antibodies to γ-interferon. Peptide antigens identical to ESAT-6, a protein virulence factor of M. tuberculosis, are introduced. T-cells that have been sensitized to M. tuberculosis secrete γ-interferon, which binds to the solid phase antibodies. Substrate is added and the amount of gamma interferon is quantified.

QuantiFERON-TB Gold (QFT-Gold), uses a cocktail of peptides including ESAT-6, CFP-10, and TB7.7(p4) to stimulate pre-sensitized T-cells to release γ-interferon, which is then measured via ELISA. QuantiFERON-TB Gold Plus (QFT-Plus) only uses ESAT-6 and CFP-10 as stimulatory peptides.

The CDC has published guidelines for use of blood assays for the diagnosis of latent and active TB (MMWR 2010;59, No. RR-5).

  • In general, an IGRA may be used in all situations for which TST is indicated. An IGRA is the test of choice in two instances; for people who have received BCG, either as a vaccine or as chemotherapy and under circumstances where the tested person is unlikely to return to have the TST read.
  • Either TST or IGRA may be used to test contacts of people with active TB infection, and in screening for occupational exposures.
  • Routine testing with both TST and an IGRA is not generally recommended. Exceptions include suspected active TB in immunocompromised patients, or indeterminate results from either test.
  • TST testing is preferred for children aged <5 years.

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 exclude infection in suspect cases.

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 as gray-zone instead of positive.

Specimen requirement is one green-top sodium heparin tube of blood, which must be transported at ambient temperature as soon as possible, since testing must begin within 30 hours of specimen collection.  

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