Head-to-head comparison of two interferon-gamma tests for tuberculosis infection

Tuberculosis remains one of the most formidable infectious disease challenges of our time, and the World Health Organization estimates that a quarter of the global population carries Mycobacterium tuberculosis, often in a dormant form that can reactivate without warning. Detecting this hidden reservoir of infection has become a central pillar of global TB control, particularly as immunocompromising conditions, biological therapies, and migration patterns drive rising incidence even in developed nations. Now, a large real-world study published in Pharmacia has put the two most widely used interferon-gamma release assays, QuantiFERON-TB Gold Plus and T-SPOT.TB, under the microscope in a head-to-head comparison spanning more than 7,000 patient samples, offering clinicians fresh guidance on which test to trust and when.

Both assays rest on the same biological foundation: the cell-mediated immune response that T lymphocytes mount against specific Mycobacterium tuberculosis antigens. When sensitized T cells encounter peptides from the TB-specific proteins ESAT-6 and CFP-10, they release interferon-gamma, a signaling molecule whose presence reveals antigen-specific immune memory. QuantiFERON-TB Gold Plus, manufactured by Qiagen, incubates whole venous blood overnight with these antigens and then measures the accumulated IFNγ using an enzyme-linked immunosorbent assay, or ELISA. T-SPOT.TB, from Oxford Immunotec, takes a different route: it first isolates peripheral blood mononuclear cells from the blood, standardizes their number, and then counts the individual IFNγ-secreting T cells with an enzyme-linked immunospot assay, known as ELISPOT. QuantiFERON additionally includes peptides recognized by TB-specific CD8+ T lymphocytes, broadening the immune cell populations it stimulates beyond the CD4+ compartment.

The study, conducted at the Ramus laboratory between January 1 and December 31, 2024, processed a total of 7,392 patient samples: 6,887 with QuantiFERON-TB Gold Plus and 505 with T-SPOT.TB. The outcome distributions differed markedly between the two platforms. QuantiFERON yielded negative results in an expected 92.3 percent of samples and positive results in only 5.3 percent, reflecting the low TB prevalence in Bulgaria, a high-income country with established tuberculosis control programs. T-SPOT.TB, by contrast, produced 59.8 percent negative and 24.6 percent positive results, a substantially higher positivity rate that hints at its greater analytical sensitivity in populations with low T-cell counts, such as immunosuppressed patients.

Perhaps the most striking differences emerged in the gray zones of testing. Borderline and indeterminate results, the categories that most frustrate clinicians, hovered around 2 percent for QuantiFERON but reached 5.7 percent and 9.5 percent, respectively, for T-SPOT.TB. The indeterminate rate for T-SPOT.TB dropped to 5.3 percent when 21 patients on biological therapy were followed up and their results subsequently converted to negative. QuantiFERON recorded indeterminate results in only 0.4 percent of cases overall, rising to 1.4 percent among patients receiving biological therapy, while 0.5 percent of its results were classified as borderline and 0.1 percent produced no valid outcome. T-SPOT.TB, meanwhile, classified 5.7 percent of results as borderline and failed to generate a valid result in 0.4 percent of tests.

To probe the clinical meaning of these differences, the researchers analyzed 94 patients who underwent both tests, either simultaneously or within defined intervals of up to one year, along with 12 patients who were tested multiple times with a single method. The dual-tested cohort had a mean age of 52 years, ranging from 21 to 85, and comprised 44 females and 50 males. Concordance between the two assays appeared in 51 individuals, or 54.3 percent: 31 had concordant positive results, 17 had concordant negative results, and 3 showed indeterminate results in both tests. Discrepancies surfaced in the remaining 43 patients, or 45.7 percent, a proportion that demands careful clinical interpretation.

The analysis of these discordant cases revealed a reassuring pattern. In 61 percent of the discrepancies, or 26 of 43 patients, results shifted from indeterminate or borderline to negative, or the reverse, within one to six months, a degree of variability the authors deem acceptable under existing guidelines. Among the 17 individuals, or 39 percent, whose results changed from positive to negative or indeterminate, further investigation showed that all had initiated biological therapy. This finding carries significant weight: immunosuppressive treatments can suppress the T-cell response that both assays measure, converting a previously detectable antigen-specific reaction into an undetectable one. Among the 12 patients repeatedly tested with T-SPOT.TB over intervals of four to eight months, three showed consistent positive results, two showed consistent negative results, and seven experienced changes, all of whom were on biological therapy.

Statistical analysis quantified the agreement between the two platforms. Cohen’s kappa, a measure of inter-rater reliability, came in at 0.312, indicating significant but fair agreement, far from the perfect concordance of 1. However, when indeterminate and borderline results were omitted from the calculation, the agreement became substantial, with a kappa of 0.697, much closer to perfect. Under this refined analysis, 85.7 percent of samples were concordant while 14.3 percent were discordant. Sensitivity and specificity also diverged depending on which test served as the reference: T-SPOT.TB, compared to QuantiFERON, showed a sensitivity of 83.78 percent and a specificity of 89.47 percent, while QuantiFERON, compared to T-SPOT.TB, demonstrated a sensitivity of 93.94 percent and a specificity of 73.91 percent.

Each assay carries distinct methodological advantages that shape its clinical niche. T-SPOT.TB, as an ELISPOT-based assay, standardizes the number of peripheral blood mononuclear cells, which may provide greater sensitivity in immunocompromised patients or those with lymphopenia, since it reduces the impact of low cell counts on the result. QuantiFERON, as a whole-blood ELISA-based assay, is technically simpler, less labor-intensive, and more easily standardized, making it better suited for high-throughput laboratory settings. Its inclusion of antigen tubes designed to stimulate both CD4+ and CD8+ T-cell responses may offer advantages in certain clinical contexts. The choice between them, the authors suggest, should depend on patient characteristics, laboratory capacity, and clinical indication rather than on any universal superiority of one platform.

The study also spotlighted a compelling clinical case: a 55-year-old man with a persistent subfebrile condition and swelling in the area of one testicle, who consulted a urologist and underwent periodic antibiotic therapy with isoniazid without lasting effect. Roughly a decade earlier, he had tested positive by QuantiFERON. When retested twice within about a month, both QuantiFERON and T-SPOT.TB returned positive results, pointing to extrapulmonary tuberculosis after a pulmonary form of the disease was ruled out. Six months of specific anti-TB therapy produced a lasting effect on his clinical symptoms, and he has remained stable since, with no chills, fatigue, or fever. A specialist in extrapulmonary tuberculosis indicated that the test may remain positive for a prolonged period, illustrating how IGRA positivity can persist even after successful treatment.

The broader context reinforces the value of these findings. A health technology assessment by Ontario Health, encompassing 12 systematic reviews and more than 500 primary studies, demonstrated that IGRAs serve as effective rule-in tests for latent tuberculosis infection due to consistently high specificity, outperforming the tuberculin skin test in reducing false positives, particularly among immunocompromised populations, children, elderly individuals, and BCG-vaccinated persons. Economic analyses from five Canadian studies revealed that IGRA testing proved cost-effective or cost-saving for high-risk latent TB populations, with healthcare providers expressing strong preference for the single-visit requirement compared to the multiple visits demanded by skin testing. Yet the ability of IGRAs to yield indeterminate results in immunocompromised patients signals the need for further clinical investigation when skin tests may produce false negatives.

Both assays share inherent limitations. Neither can reliably differentiate between latent TB infection and active TB disease, and both depend on a robust host immune response, meaning false negatives may occur in immunocompromised individuals, young children, or patients with advanced disease. A recent systematic review and meta-analysis of IGRAs in people living with HIV, covering 6,525 patients from 45 studies, found that while both tests demonstrated acceptable specificity, their sensitivity was lower, particularly for latent TB infection, suggesting IGRAs may be more reliable for detecting active TB than for ruling out latent infection in immunocompromised populations. The variation in antigen-specific T-cell responses observed in the current cohort may also reflect differences in exposure history, BCG vaccination status, or concurrent infections.

From a clinical perspective, discrepancies between QuantiFERON and T-SPOT.TB results necessitate careful interpretation in conjunction with epidemiological risk factors, radiological findings, and clinical presentation. For borderline or discordant cases, repeating the IGRA or performing both tests sequentially may improve diagnostic confidence, particularly in high-risk groups. The retrospective nature of the study and the lack of detailed clinical data on individual disease activity, concomitant medications, and duration of biological therapy represent limitations that future prospective designs should address through detailed patient stratification and combined analysis of qualitative and quantitative results.

The bottom line for clinicians is clear: the results from both IFNγ-based tests are comparable, with acceptable variation over time, and either method can be relied upon in routine laboratory diagnostics. However, significant changes in results, whether a sharp conversion from positive to negative or the emergence of indeterminate readings, should always be interpreted in light of ongoing specific or biological therapy. As tuberculosis control efforts intensify worldwide and biological therapies continue to expand across medicine, this real-world evidence offers laboratories and physicians a practical roadmap for navigating the nuances of interferon-gamma testing in the patients who need it most.

Subject of Research: Comparative diagnostic performance of two interferon-gamma release assays for Mycobacterium tuberculosis infection

Article Title: Comparative results of two IGRA tests for determining the specific response against Mycobacterium tuberculosis infection

Article References: Stoilov, P., Mihova, A., Mihaylov, R., Zlateva, S., Dimcheva, P., Pentchev, E., Kalcheva, M., Pencheva, B., Naseva, E., & Velikova, T. (2026). Comparative results of two IGRA tests for determining the specific response against Mycobacterium tuberculosis infection. Pharmacia, 73, Article e167649. https://doi.org/10.3897/pharmacia.73.e167649

Image Credits: AI Generated

DOI: 10.3897/pharmacia.73.e167649

Keywords: tuberculosis, IGRA, QuantiFERON, T-SPOT.TB, interferon-gamma, diagnostics, latent TB infection, biological therapy, Comparative, results, tests, determining