Clofazimine and Linezolid Resistance in Multidrug-Resistant Tuberculosis: Insights from a Single-Centre Study in Iran

Abstract

Background: Detection of drug resistance in multidrug-resistant tuberculosis (MDR-TB) is essential for effective treatment. This retrospective cross-sectional study compared a World Health Organization (WHO)-recommended proportional method with molecular techniques, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real-time polymerase chain reaction (RT-PCR), to assess clofazimine and linezolid resistance in MDR-TB isolates collected between 2024 and 2025 at a national referral center in Tehran, Iran. Objectives: By evaluating phenotypic and genotypic approaches, we aimed to describe resistance patterns and genetic diversity, as well as the value of molecular diagnostics in MDR-TB management. Methods: The study included consecutive clinical and laboratory-confirmed MDR-TB isolates from patients with confirmed first-line drug resistance based on symptoms, specialist examinations, and radiologic findings. Molecular identification used IS6110-based PCR and hsp65 spacer region PCR-RFLP. Drug susceptibility testing (DST) followed the WHO proportional method on Lowenstein-Jensen and Middlebrook media as the phenotypic reference standard. Primers targeted resistance genes rv0678 (clofazimine) and rrl and rplC (linezolid), analyzed by PCR-RFLP and RT-PCR; isolates with indeterminate or missing results were excluded. Results: Mutations were detected in the rv0678 gene (17.2%) and in the rrl or rplC genes (17.2%), associated with resistance to clofazimine and linezolid, respectively. The PCR-RFLP of rv0678 (720 bp) with NlaIII enzyme showed three distinct patterns: Eighty percent had 80/120/520 bp, 6.7% had 80/120/160/520 bp, and 10% had 80/120/190/520 bp. BsrI enzyme produced uniform 300/400 bp patterns. For rrl (860 bp), BsrI showed consistent 105/140/250/260 bp patterns; BbvI revealed 93.3% had a 60/190/290/320/510/530/780 bp pattern, 6.7% lacked the 190 bp fragment. HaeIII enzyme produced 100/770/780 bp in 93.3% and 100/780 bp in 6.7%. The rplC gene (400 bp) was highly conserved. BbvI enzyme produced a 75/100/290 bp pattern in 93.3% and a 70/100/290 bp pattern in 6.7%. Despite mutations, only one isolate showed phenotypic resistance to both clofazimine and linezolid, indicating complex resistance mechanisms; no significant genotype-phenotype correlation was observed. Conclusions: Many MDR-TB isolates carried mutations in genes linked to clofazimine and linezolid resistance, but these did not consistently translate into phenotypic resistance, suggesting complex mechanisms. The retrospective design and small single-centre sample limit generalizability. Future studies using whole genome sequencing (WGS) are recommended to clarify discrepancies and improve MDR-TB treatment strategies.