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Objective: To investigate the effect of pregnancy and Human immunodeficiency virus (HIV) infection on detection performances of tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube (QFTGIT) for the diagnosis of latent tuberculosis infection (LTBI) among women living in high TB and HIV endemic setting. Method: A cross-sectional study was conducted among women with and without pregnancy and HIV infection. Three-hundred twenty women were enrolled in this study and were diagnosed using TST and QFTGIT for the detection of LTBI. Results: Overall prevalence of LTBI among the enrolled women was 55.6%, 46.3% and 51.1% as determined by TST, QFTGIT and concordant TST/QFTGIT results, respectively. Our study revealed that pregnancy or HIV infection reduced the rate of detection of LTBI by TST and QFTGIT tests, with the utmost effect observed in HIV-positive pregnant women. Additionally, we observed that the concordance between TST and QFTGIT among women increased with the presence of pregnancy and/or HIV infection. A history of contact with TB patients was significantly associated with positivity of TST and QFTGIT. Conclusion: This study demonstrated that both pregnancy and HIV infection profoundly affected the detection performance of TST and QFTGIT, which may be associated with immunosuppression of anti-mycobacterial immunity in women with pregnancy and/or HIV infection.