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Hui Xia,1 Yuanyuan Song,1 Yang Zheng,1 Shengfen Wang,1 Bing Zhao,1 Wencong He,1 Dongxin Liu,2 Xichao Ou,1 Yang Zhou,1 Yanlin Zhao1 1National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China; 2Institute of Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, People’s Republic of ChinaCorrespondence: Yanlin Zhao, National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Road, Changping District, Beijing, People’s Republic of China, Tel +86 10-58900517, Email zhaoyl@chinacdc.cnObjective: To compare the ability of detection of borderline rifampicin resistance in Mycobacterium tuberculosis between molecular assay and phenotypic drug susceptibility tests.Methods: Fifty-seven isolates with His445Leu, Asp435Tyr, Leu452Pro, Leu430Pro, His445Asn, Ile491Phe, and His445Ser mutations in rpoB gene identified by whole-genome sequencing conferring borderline rifampicin resistance were included. Molecular-based Xpert MTB/RIF, phenotypic Löwenstein–Jensen (L-J) medium-based drug susceptibility test (DST) with a critical concentration of 40.0μg/mL and minimal inhibitory concentration (MIC) assay were performed to detect borderline rifampicin resistance.Results: When using Xpert MTB/RIF, 48/57 (84.2%) isolates exhibited resistance to rifampicin. 25/57 (43.9%) and 33/57 (57.9%) isolates showed rifampicin resistance by L-J medium-based DST with 4 and 6 weeks of incubation, respectively. 30/57 (52.6%) and 40/57 (70.2%) strains were resistant to rifampicin by MIC method at cutoff values of 1.0 and 0.5μg/mL, respectively. The detection rate of rifampicin resistance of Xpert MTB/RIF was significantly higher than that of phenotypic methods (p < 0.001). Of the 57 isolates with borderline rpoB mutations, 5 (8.8%) had MICs of 0.25 or 0.12μg/mL, 22 (38.6%) had MICs of 0.5μg/mL or 1.0μg/mL, and 30 (52.6%) other isolates showed MICs ≥ 2.0μg/mL.Conclusion: Molecular-based Xpert MTB/RIF showed superior ability to detect borderline rifampicin resistance over phenotypic DST methods. Extending the incubation time of L-J DST or lowering the cutoff value of the MIC method can improve borderline rifampicin resistance detection.Keywords: Mycobacterium tuberculosis, rifampicin, borderline resistance, phenotypic drug susceptibility test