Phenotypic discordance in rifampicin resistance detection among Mycobacterium tuberculosis isolates from China: insights from whole-genome sequencing and a structured literature review

Phenotypic drug susceptibility testing (pDST) for rifampicin (RIF) resistance in Mycobacterium tuberculosis (MTB) can yield discordant results between solid and liquid culture methods, complicating accurate detection of drug-resistant tuberculosis (DR-TB). We evaluated RIF pDST concordance in 265 clinical isolates (July 2016 through June 2023) tested on Lowenstein-Jensen (L-J) medium and the Mycobacteria Growth Indicator Tube (MGIT) 960 system. Discordant isolates underwent repeat testing and whole-genome sequencing (WGS). Genotypic data were interpreted with validated pipelines. A structured literature review synthesized published data on discordant RIF phenotypes and associated rpoB variants. Overall, 11.7% (31/265) of the isolates were discordant, with 17 L-J resistant/MGIT-susceptible, and 14 the reverse. Discrepancies persisted on retesting in 59% (16/27). WGS of 10 sequenced discordant isolates showed that all harbored rpoB mutations associated with rifampicin resistance, largely within the rifampicin resistance-determining region (RRDR), including Leu452Pro (5/10) and Asp435Tyr (1/10), plus compound patterns (Asp435Tyr + Asn437 Asp; Leu430Pro + Asn437 Asp; Met434Val + Asp435 Gly). All isolates belonged to the East Asian/Beijing lineage and shared a synonymous rpoB Ala1075Ala; one had a synonymous rpoA Tyr341Tyr. Putative compensatory rpoC mutations (Lys71Asn, Glu49Gly) were detected. The literature review corroborated frequent association of Leu452Pro, Asp435Tyr, Leu430Pro, and His445 variants with inter-method discordance and previously reported low-level RIF resistance near critical concentrations. RIF pDST discordance exceeding 10% reflects inherent methodological limits and the variable phenotypic expression of specific rpoB mutations, notably Leu452Pro, which may fall below MGIT detection thresholds. In high-suspicion or phenotypically discordant cases, WGS may improve resistance classification and help inform treatment decisions and transmission control. IMPORTANCE Accurate detection of RIF resistance underpins rapid initiation of effective TB therapy and containment of transmission. However, more than 1/10 MTB isolates in our cohort produced reproducible, method-dependent discordance between solid (L-J) and liquid (MGIT) pDST tests. Such discordance risks misclassifying genuinely resistant strains as susceptible, especially when low-level resistance mutations (notably rpoB Leu452Pro and certain Asp435 and Leu430 variants) sit near critical concentration thresholds used in automated liquid systems. Reliance on a single phenotypic platform may therefore delay appropriate multidrug-resistant TB regimens and foster ongoing spread. Among the sequenced discordant isolates, WGS identified resistance-associated rpoB mutations in all cases, with putative compensatory rpoC mutations detected in some isolates, supporting its potential value as an adjudication tool. Integrating rapid genotypic methods when phenotypic results conflict, or when clinical suspicion remains high, may improve regimen selection and laboratory quality assurance.