Fluoroquinolone and Second-Line Injectable Resistance Among Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis Clinical Isolates: A Molecular Study from a High-Burden Setting

Drug-resistant tuberculosis (DR-TB) threatens global TB control. We investigated the prevalence and molecular characteristics of second-line drug resistance among rifampicin (RIF)- and/or isoniazid (INH)-resistant Mycobacterium tuberculosis complex (MTBC) isolates in São Paulo, Brazil, using the MTBDRsl v. 2.0 line-probe assay. MTBC isolates RIF- and/or INH-resistant by GenoType MTBDRplus or phenotypic testing (2019–2021) were subsequently tested by MTBDRsl for fluoroquinolone (FQ) and injectable drugs (capreomycin, amikacin, kanamycin) resistance. Isolates with inferred mutations underwent Sanger sequencing. Of 13,557 isolates, 728 (5.4%) were RIF- and/or INH-resistant (297 INH-R, 235 RIF-R, 196 MDR). Among them, 623 (85.6%) were tested by MTBDRsl; 582 (93.4%) showed no additional resistance, while 41 (6.6%) carried mutations. FQ resistance was detected in 38 isolates (92.7%), mostly in gyrA (n = 35). Three isolates with gyrB mutations were wild-type by sequencing. Two MDR isolates harbored the rrs a1401g mutation, and one also harbored gyrA D94G. Sequencing confirmed resistance in 38 of 41 isolates. Most MDR strains with second-line mutations (n = 32/33; 97%) were pre-XDR. Affected patients were predominantly male (68.4%), with pulmonary TB (92.1%), and unfavorable outcomes (39.5%). Second-line resistance prevalence was low overall, but FQ resistance was high among MDR isolates. Findings support integrating molecular and sequencing-based tools for accurate detection and management of DR-TB.