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Healthcare workers (HCWs) are at increased risk of exposure to tuberculosis (TB). Traditionally, screening for latent TB infection (LTBI) is done using the tuberculin skin test (TST). Interferon-gamma release assays (IGRAs) are now increasingly being used for diagnosis of LTBI, but their role in HCW screening is unclear. A systematic review was conducted of all IGRA studies in HCWs to summarise their performance in cross-sectional and serial testing settings. By searching four electronic databases and other sources, all available studies using any one of the commercial IGRA assays in HCWs were retrieved and screened. 50 unique studies were identified which met the inclusion criteria including five from high TB incidence settings. Among 24 cross-sectional studies in low TB incidence settings, the pooled prevalence of positive IGRA using either test was significantly lower than for a positive TST. However, in high-incidence settings (n=2) there were no consistent differences in the prevalence of positive tests. IGRAs showed good correlation with occupational risk factors for TB exposure in low-incidence settings. Only 10 studies assessed use of IGRA for serial testing and all showed large variation in the rates of conversions and reversions, with no data suggesting that IGRAs are better at identifying the incidence of new TB infection than the TST. The use of IGRAs instead of TST for one-time screening may result in a lower prevalence of positive tests and fewer HCWs who require LTBI treatment, particularly in low TB incidence settings. However, the use of IGRAs for serial testing is complicated by lack of data on optimum cut-offs for serial testing and unclear interpretation and prognosis of conversions and reversions. Further longitudinal research will be required to inform guidelines on serial testing using IGRAs.