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 <i>Mycobacterium tuberculosis</i> complex (MTBC) isolates in São Paulo, Brazil, using the MTBDR<i>sl</i> v. 2.0 line-probe assay. MTBC isolates RIF- and/or INH-resistant by GenoType MTBDR<i>plus</i> or phenotypic testing (2019-2021) were subsequently tested by MTBDR<i>sl</i> 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 MTBDR<i>sl</i>; 582 (93.4%) showed no additional resistance, while 41 (6.6%) carried mutations. FQ resistance was detected in 38 isolates (92.7%), mostly in <i>gyrA</i> (<i>n</i> = 35). Three isolates with <i>gyrB</i> mutations were wild-type by sequencing. Two MDR isolates harbored the <i>rrs</i> a1401g mutation, and one also harbored <i>gyrA</i> D94G. Sequencing confirmed resistance in 38 of 41 isolates. Most MDR strains with second-line mutations (<i>n</i> = 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.