Optimizing diagnosis of Mycobacterium tuberculosis latent infection: ELISpot as a technique of choice for samples with high negative control values in QuantiFERON assay

A quarter of the world's population is considered to be affected by latent tuberculosis infection (LTBI) by the World Health Organization, which recommends that individuals with LTBI receive anti-tuberculosis treatment, aiming to avoid progression to active tuberculosis and transmission. Moreover, patients undergoing immunosuppressive therapy, such as anti-TNFα antibodies, constitute a growing high-risk group for which LTBI screening prior to treatment initiation is strongly advised. Improving LTBI diagnosis and patient management relies on the accuracy of broadly deployed laboratory tests, such as interferon-gamma release assays (IGRAs). The QuantiFERON-TB test is one of the most widely used IGRAs worldwide. In some cases, results may be defined as indeterminate when the negative control (Nil) exceeds 8 IU/mL, delaying or preventing appropriate patient care. This scenario was observed in 57 (0.8%) patients out of the 6,600 tested in Grenoble University Hospital from November 2022 to November 2025. Subsequent re-evaluation with either QuantiFERON-TB and/or the ELISpot-based T-SPOT.TB assays was possible for 34 patients. Among the 15 samples tested again with QuantiFERON-TB, 12 finally resulted in being positive, while three remained indeterminate. By contrast, all 19 patients tested with T-SPOT.TB returned unequivocally positive results. We suggest that repeating IGRA testing is essential when QuantiFERON-TB results are indeterminate due to elevated Nil values, with the T-SPOT.TB assay being a choice solution. Our results highlight an impactful strategy for strengthening LTBI diagnosis, enabling timely chemoprophylaxis, preventing progression to active disease, and contributing to global tuberculosis control.IMPORTANCEInterferon-gamma release assays (IGRAs), including QuantiFERON-TB and ELISpot-based T-SPOT.TB, are essential laboratory tools for latent tuberculosis infection (LTBI) diagnosis. However, indeterminate QuantiFERON results with elevated Nil values (>8 IU/mL) are a challenge to timely clinical management. Our findings demonstrate that systematic retesting with IGRAs is essential, with most cases yielding positive results upon repeat testing. T-SPOT.TB appears to be the most reliable alternative for resolving these indeterminate outcomes. This approach enhances LTBI detection, supports prompt chemoprophylaxis, and contributes to preventing progression to active tuberculosis.