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Interferon-γ release assays for the diagnosis of infection with Mycobacterium tuberculosis have been increasingly used in recent years and are endorsed by national guidelines, but experience regarding their use in children is still limited.We retrospectively evaluated the routine use of the QuantiFERON-TB Gold In-Tube assay (QFT-IT) in a pediatric tertiary care center with a high prevalence of immunocompromising conditions. The relationship between age, immune status, and likelihood of an indeterminate test result was analyzed using logistic regression analysis and fractional polynomials.Two hundred thirty-seven tests from 237 children were included in the analysis. Fifty-nine children (25%) were immunocompromised by our definition. An indeterminate test result was obtained in 83 children (35%). The likelihood of an indeterminate test result was inversely correlated with age (P < 0.001) for children who were not known to be immunocompromised, and decreased by 13% per year of age. Impaired immunity (P < 0.001) was independently associated with a higher probability of an indeterminate QFT-IT. Among 161 children with a documented tuberculin skin test, 89% had a concordant QFT-IT (κ = 0.71). Twelve of 16 patients with culture-proven TB had a positive QFT-IT.These data suggest that QFT-IT may not provide a determinate test result in a substantial proportion of children in a tertiary care setting due to the combination of young age and primary and acquired immune deficiencies.