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QuantiFERON®-TB Gold PlusTest code(s) Test Codes 36970 (1 tube, non-incubated), 36971 (4 tubes, draw-site incubated)
Question 1. What is QuantiFERON®-TB Gold Plus?
The QuantiFERON-TB Gold Plus (QFT-Plus) assay is an in vitro diagnostic test that utilizes a peptide cocktail simulating ESAT-6 and CFP-10 proteins to stimulate cells in heparinized whole blood. Detection of interferon-γ (IFN-γ) by enzyme-linked immunosorbent assay (ELISA) is used to identify in vitro responses to these peptide antigens that are associated with Mycobacterium tuberculosis infection. QFT-Plus is an indirect test for M. tuberculosis infection (including disease) and is intended for use in conjunction with risk assessment, radiography, and other medical and diagnostic evaluations. This test does not differentiate between latent and active TB.1
CDC recommendations state that2,3
- IGRAs may be used in place of (but not in addition to) a TST in all situations in which CDC recommends TST as an aid in diagnosing adult M tuberculosis infections.
- “An IGRA is preferred for testing persons who have received Bacillus Calmette-Guérin (BCG) as a vaccine or for cancer therapy.”
- “An IGRA or a TST may be used without preference to test recent contacts of persons known or suspected to have active tuberculosis with special considerations for follow-up testing. IGRAs offer the possibility of detecting M tuberculosis infection with greater specificity than with a TST. If IGRAs are to be used in contact investigations, negative results obtained prior to 8 weeks after the end of exposure typically should be confirmed by repeat testing 8 to 10 weeks after the end of exposure.”
Up-to-date product-specific warnings and other information can be found at www.QuantiFERON.com.
Question 2. How does QuantiFERON-TB Gold Plus work?
The QuantiFERON-TB Gold Plus assay is a blood test for M tuberculosis infection that is based on measurement of a cell-mediated immune (CMI) response. A peptide cocktail simulating the mycobacterial proteins ESAT-6 and CFP-10 stimulate the patient’s T-cells in vitro to release IFN-γ, which is then measured using enzyme-linked immunosorbent assay (ELISA) technology. The test detects infections produced by the M tuberculosis complex, which includes M tuberculosis, as well as certain non-tuberculous mycobacteria, including M kansasii, M szulgai, and M marinum. BCG strains and the majority of other non-tuberculous mycobacteria do not harbor ESAT-6 and CFP-10 proteins; thus, patients either vaccinated with BCG or infected with most environmental mycobacteria should test negative. Results should always be interpreted in conjunction with other clinical and laboratory findings.
Question 3. What are the advantages of QuantiFERON-TB Gold Plus over TST?
- Results are not subject to reader bias
- Accuracy is not affected by prior BCG vaccination
- Requires only 1 patient visit (2-4 required for TST)
- Reduced staff time and avoidance of unnecessary follow-up, additional treatment, and treatment due to false positive TST results2
Question 4. How do you interpret the QuantiFERON-TB Gold Plus report?
An example of a QuantiFERON-TB Gold Plus report is shown below:
Nil or Negative Control Tube (gray cap)
The nil value comes from the negative control tube, which contains no additives; it is used to determine if the patient has a preexisting immune response (e.g., heterophile antibody effects or non-specific IFN-γ production), which could cause a false-positive reading on the test.
For a test to be valid, the nil tube must have a value of ≤8.0 IU/mL. In the example cited above, the nil value (0.07 IU/mL) is well within the recommended range of ≤8.0 IU/mL. The nil tube value is subtracted from the values of the mitogen, TB1, and TB2 tubes for the final result of the respective tubes.
Mitogen or Positive Control Tube (purple cap)
The mitogen, or positive control, tube contains a mitogen (phytohemagglutinin-P), which is a non-specific stimulator of T-cells. It is used to assure that the patient’s T-cells are capable of producing IFN-γ and also serves as a control for correct blood handling and incubation. The mitogen tube is also used to detect false-negative readings.
For a test to be valid, the mitogen tube must have an IFN-γ value at least 0.5 IU/mL higher than the value of the nil tube. This indicates that the test is functioning properly.
In the example cited above, the mitogen tube value minus the nil tube value is well above the minimum breakpoint of 0.5 IU/mL.
TB1 Antigen (green cap)
The inside of this tube is coated with M tuberculosis-specific antigens designed to elicit CMI responses from CD4+ T-helper lymphocytes.
TB2 Antigen (yellow cap)
The inside of this tube is coated with the M tuberculosis-specific antigens designed to elicit CMI responses from CD4+ T-helper lymphocytes and also contains an additional set of peptides targeted to the induction of CMI responses from CD8+ cytotoxic T-lymphocytes.
Results from each of the 4 tubes are evaluated in accordance with the algorithm below, providing a final result:
Calculation of Positive, Negative, or Indeterminate
The amount of IFN-γ produced by each tube (nil, mitogen, TB1 and TB2 antigen) is measured using a standard ELISA format. QuantiFERON-TB Gold Plus results are based on the amount of IFN-γ that is released in response to the antigens. The result “Negative” or “Positive” is calculated from these values using an FDA approved algorithm run on QuantiFERON software. “Indeterminate” results are generated when either of the control tubes (nil or mitogen) does not produce the intended values.
Per the package insert, “The magnitude of the measured IFN-γ level cannot be correlated to stage or degree of infection, level of immune responsiveness, or likelihood for progression to active disease. A positive TB response in persons who are negative to mitogen is rare, but has been seen in patients with TB disease. This indicates the IFN-γ response to TB antigens is greater than that to mitogen, which is possible as the level of Mitogen does not maximally stimulate IFN-γ production by lymphocytes.”4
Question 5. What are conversion and reversion?
Conversion: With respect to TB antigens, conversion is the point at which IFN-γ becomes detectable above the established threshold or cutoff from a previous negative or unknown result.
Reversion: The opposite of conversion—a result that goes from above the established cutoff to below the cutoff (positive to a negative). While there are no data derived using the QuantiFERON-TB Gold Plus test, there are numerous publications on conversion/reversion based on the QuantiFERON-TB Gold test.
In one study of the QuantiFERON-TB Gold assay, among individuals with quantitative IFN-γ values just above or just below the cut-off threshold, reversions and conversions were common.5 Per the package insert, it is prudent to confirm a positive QuantiFERON-TB Gold Plustest by the same or alternate method before starting therapy if M tuberculosis complex infection is not suspected.
Question 6. What causes an indeterminate response?
The 2 main causes of indeterminate results are1:
- Lack of incubating the samples properly (such as not incubating the sample at all or incubating longer than recommended)
- Insufficient or overly aggressive mixing of blood collection tubes
Other less common causes of indeterminate results are1:
- Presence of heterophile antibodies in the patient sample
- Compromised immune status of the individual being tested
- Intrinsic IFN-γ secretion
- Recent patient illness
- Recent vaccinations
- Lymphocytes responding indiscriminately (recent patient bouts with poison ivy, rheumatoid arthritis, etc)
- Lack of response to phytohemagglutinin (rare, <1 in 1,000 patients)
- Storage of filled blood collection tubes outside the recommended temperature range
- Compromised mitogen transport tubes
- Insufficient lymphocytes
- Inability of the patient’s lymphocytes to generate IFN-γ
- Immune-modulating therapies, especially in patients who are also receiving high-dose steroids
If an indeterminate result is obtained, the physician may choose to redraw a specimen or perform other procedures as appropriate.1
Question 7. Can QuantiFERON-TB Gold Plus testing be used to monitor TB therapy?
No. QuantiFERON-TB Gold Plus testing cannot be used to monitor TB therapy.6
Question 8. Does prior TST affect a QuantiFERON-TB Gold Plus result?
Yes, a prior TST can boost IGRA results. However, boosting does not occur in uninfected individuals, because they do not have an established immune response to TB. Furthermore, the TST, despite its in vivo application, does not cause sensitization or establish a cell-mediated response. Therefore, it cannot cause subsequent boosting among persons without prior TB infection. The implications of a boosted response are unknown. However, a boosted response is thought to reflect the induction of a weak memory response from multiple points of stimulation. Boosting is a common phenomenon when a TST is repeated. Each TST can boost subsequent TST responses, due to remote TB infection, as well as infection with non-tuberculous mycobacteria or vaccination with BCG.1,7,8
Question 9. Can IGRAs be given to persons receiving vaccinations?
The effect of live virus vaccination on IGRAs has not been studied. However, it is advisable that the IGRA be drawn on the same day as vaccination or 4 to 6 weeks after the administration of a live-virus vaccine.
Question 10. Can an IGRA be used for children?
Yes. An IGRA can be used for children 2 years of age or older. It is preferred for children who have had the BCG vaccine or are unlikely to return for a TST to be read. For children younger than 2 years of age, the TST is the preferred test to detect TB infection.9
- FAQs for health professionals: QuantiFERON®-TB Gold Plus. QuantiFERON Website. www.quantiferon.com/wp-content/uploads/2017/10/PROM-11178-001_1107769_BRO-QFT-TB-Gold-Plus-FAQ-HCPs-0717-US.pdf. Published July 2017. Accessed June 2018.
- 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:e1-e33.
- Mazurek GH, Jereb J, Vernon A, et al. Updated guidelines for using interferon gamma release assays to detect Mycobacterium tuberculosisinfection—United States, 2010. MMWR Recomm Rep. 2010;59:1-25.
- QFT-Plus Package Inserts. QuantiFERON Website.https://www.quantiferon.com/us/products/quantiferon-tb-gold-plus-us/package-inserts. Accessed June 2018.
- Dorman SE, Belknap R, Graviss EA, et al. Interferon-grelease assays and tuberculin skin testing for diagnosis of latent tuberculosis infection in healthcare workers in the United States. Am J Respir Crit Care Med. 2014;189:77-87.
- Pollock NR, Kashino SS, Napolitano DR, et al. Evaluation of the effect of treatment of latent tuberculosis infection on QuantiFERON-TB Gold assay results. Infect Control Hosp Epidemiol. 2009;30:392-395.
- King TC, Upfal M, Gottlieb A, et al. T-SPOT.TB interferon-γ release assay performance in healthcare worker screening at nineteen US hospitals. Am J Resp Crit Care Med. 2015;192:367-373.
- van Zyl-Smit RN, Zwerling A, Dheda K, et al. Within-subject variability of interferon-g assay results for tuberculosis and boosting effect of tuberculin skin testing: a systematic review. PLoS One. 2009;4(12):e8517. doi:10.1371/journal.pone.0008517
- American Academy of Pediatrics. Tuberculosis. In: Kimberlin DW, Brady MT, Jackson MA, et al, eds. Red Book: 2018 Report of the Committee on Infectious Diseases. 31st ed. Itasca, IL: American Academy of Pediatrics; 2018: 829-853.