Drug-Resistant M. tuberculosis Genome Sequencing

We selected 337 clinical isolates of M. tuberculosis with diverse drug susceptibility patterns. Strains were collected both retrospectively and prospectively from 2008 to 2013 from all 11 districts of KwaZulu-Natal (Table 1). Strains were chosen for study inclusion on the basis of a predetermined drug resistance pattern so that the dataset was heavily weighted toward drug-resistant strains rather than accurately reflecting the epidemiology of the region. Written informed consent was obtained from study participants prior to cohort enrollment. Biomedical Research Ethics Council (BREC) approval from the University of KwaZulu-Natal was granted for whole genome sequencing of clinical strains. On all study isolates, drug susceptibility testing (DST) was performed by the critical concentration method, using the WHO recommended concentrations [27 ]. The following drugs were assayed in all strains, with their respective critical concentration in parentheses (in μg/mL): rifampicin (1.0), isoniazid (0.2 and/or 1.0), streptomycin (2.0), kanamycin (6.0), and ofloxacin (2.0). Extended DST was performed for key isolates (Table 1) with the following drugs: capreomycin (10.0), ethambutol (7.5), and ethionamide (10.0). Pyrazinamide resistance testing was performed using PZA MGIT (100.0) or Nicotinamide (500.0). Subject data included age, gender, AFB smear, and HIV status, when available. Study participants were assigned GPS coordinates corresponding to their home provincial district or site of sputum collection.
We also selected for sequencing three historical strains previously collected in KwaZulu-Natal for resequencing [25 (link),24 (link)]: KZN4207 (drug susceptible, collected in Durban in 1995), KZN1435 (MDR, collected in Durban in 1994), and KZN605 (XDR, collected in Tugela Ferry in 2005).