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Background Second-line drug resistance (SLD) among tuberculosis (TB) patients is a serious emerging challenge towards global control of the disease. We characterized SLD-resistance conferring-mutations among TB patients with rifampicin and/or isoniazid (RIF and/or INH) drug-resistance tested at the Uganda National TB Reference Laboratory (NTRL) between June 2017 and December 2019. Methods This was a descriptive cross-sectional secondary data analysis of 20,508 M . tuberculosis  isolates of new and previously treated patients’ resistant to RIF and/or INH. DNA strips with valid results to characterise the SLD resistance using the commercial Line Probe Assay Genotype  MTBDRsl  Version 2.0 Assay (Hain Life Science, Nehren, Germany) were reviewed. Data were analysed with STATAv15 using cross-tabulation for frequency and proportions of known resistance-conferring mutations to injectable agents (IA) and fluoroquinolones (FQ). Results Among the eligible participants, 12,993/20,508 (63.4%) were male and median (IQR) age 32 (24–43). A total of 576/20,508 (2.8%) of the  M. tuberculosis  isolates from participants had resistance to RIF and/or INH. These included; 102/576 (17.7%) single drug-resistant and 474/576 (82.3%) multidrug-resistant (MDR) strains. Only 102 patients had test results for FQ of whom 70/102 (68.6%) and 01/102 (0.98%) had resistance-conferring mutations in the  gyrA  locus and  gyrB  locus respectively. Among patients with FQ resistance,  gyrAD94G  42.6% (30.0–55.9) and  gyrA A90V  41.1% (28.6–54.3) mutations were most observed. Only one mutation, E540D was detected in the  gyrB  locus. A total of 26 patients had resistance-conferring mutations to IA in whom, 20/26 77.0% (56.4–91.0) had A1401G mutation in the  rrs  gene locus. Conclusions Our study reveals a high proportion of mutations known to confer high-level fluoroquinolone drug-resistance among patients with rifampicin and/or isoniazid drug resistance. Utilizing routinely generated laboratory data from existing molecular diagnostic methods may aid real-time surveillance of emerging tuberculosis drug-resistance in resource-limited settings.

Molecular characterisation of second-line drug resistance among drug resistant tuberculosis patients tested in Uganda: a two and a half-year’s review