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 drug resistance. We assessed a reporter mycobacteriophage (ΦGFP10) to detect drug-resistant subpopulations and predict bactericidal activity in this pilot study. Subsequently, we prospectively studied 20 TB patients with serial ΦGFP10, Xpert MTB/RIF, and culture through end of treatment. WGS was performed, and single nucleotide polymorphisms (SNPs) were examined to detect mixed infection in selected isolates. Resistant isolates were detected at 1:100,000, and changes in cytometry-gated events were predictive of bactericidal activity using the ΦGFP10 assay. Emergent drug resistance was detected in one patient by ΦGFP10 at 3 weeks but not by conventional testing ( culture and GeneXpert). WGS revealed a phylogeographically 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. ΦGFP10 can detect low-frequency drug-resistant and with WGS characterize emergent resistance. In areas of high TB transmission and drug resistance, rapid screening for heteroresistance should be considered.