Early detection of emergent extensively drug-resistant tuberculosis by flow cytometry-based phenotyping and whole-genome sequencing

A critical gap in tuberculosis (TB) treatment is detection of emergent drug resistance. We hypothesized that advanced phenotyping with whole-genome sequencing (WGS) will detect low-frequency Mycobacterium tuberculosis drug resistance. We assessed a reporter mycobacteriophage (²GFP10) in vitro to detect drug-resistant subpopulations and predict M. tuberculosis bactericidal activity in this pilot study. Subsequently, we prospectively studied 20 TB patients with serial ²GFP10, Xpert MTB/RIF, and M. tuberculosis culture through end of treatment. WGS was performed, and single nucleotide polymorphisms (SNPs) were examined to detect mixed infection in selected M. tuberculosis isolates. Resistant M. tuberculosis isolates were detected at 1:100,000, and changes in cytometry-gated events were predictive of in vitro M. tuberculosis bactericidal activity using the ²GFP10 assay. Emergent drug resistance was detected in one patient by ²GFP10 at 3 weeks but not by conventional testing (M. tuberculosis culture and GeneXpert). WGS revealed a phylogeo-graphically distinct extensively drug-resistant tuberculosis (XDR-TB) genome, identical to an XDR-TB isolate from the patient’s spouse. Variant lineage-specific SNPs were present early, suggesting mixed infection as the etiology of emergent resistance with temporal trends providing evidence for selection during treatment. ²GFP10 can detect low-frequency drug-resistant M. tuberculosis and with WGS characterize emergent M. tuberculosis resistance. In areas of high TB transmission and drug resistance, rapid screening for heteroresistance should be considered.