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Objectives: To assess the performance of interferon-gamma release assays (IGRAs) in the differential diagnosis between Mycobacterium avium complex (MAC) and tuberculosis (TB) in children affected with subacute/chronic submandibular/cervical lymphadenitis., Study Design: Multicenter observational study comparing children with microbiologically confirmed MAC lymphadenitis from the European NontuberculouS MycoBacterial Lymphadenitis in childrEn study with children with TB lymphadenitis from the Spanish Network for the Study of Pediatric TB database., Results: Overall, 78 patients with MAC and 34 with TB lymphadenitis were included. Among MAC cases, 44 out of 74 (59.5%) had positive tuberculin skin test (TST) results at the 5-mm cut-off, compared with 32 out of 33 (97%) TB cases (P < .001); at the 10-mm cut-off TST results were positive in 23 out of 74 (31.1%) vs 26 out of 31 (83.9%), respectively (P < .001). IGRA results were positive in only 1 out of 32 (3.1%) patients with MAC who had undergone IGRA testing, compared with 21 out of 23 (91.3%) TB cases (P < .001). Agreement between TST and IGRA results was poor in MAC (23.3%; κ = 0.017), but good in TB cases (95.6%; κ = 0.646). IGRAs had a specificity of 96.9% (95% CI 84.3%-99.8%), positive predictive value of 95.4% (95% CI 78.2%-99.8%), and negative predictive value of 93.9% (95% CI 80.4%-98.9%) for TB lymphadenitis., Conclusions: In contrast to TST, IGRAs have high specificity, negative predictive value, and positive predictive value for TB lymphadenitis in children with subacute/chronic lymphadenopathy, and consequently can help to discriminate between TB and MAC disease. Therefore, IGRAs are useful tools in the diagnostic work-up of children with lymphadenopathy, particularly when culture and polymerase chain reaction results are negative., (Copyright © 2021 Elsevier Inc. All rights reserved.)