Early Detection of Emergent Extensively Drug-Resistant Tuberculosis by Flow Cytometry-Based Phenotyping and Whole-Genome Sequencing

Abstract

ABSTRACTA 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-frequencyMycobacterium tuberculosisdrug resistance. We assessed a reporter mycobacteriophage (Φ2GFP10)in vitroto detect drug-resistant subpopulations and predictM. tuberculosisbactericidal activity in this pilot study. Subsequently, we prospectively studied 20 TB patients with serial Φ2GFP10, Xpert MTB/RIF, andM. tuberculosisculture through end of treatment. WGS was performed, and single nucleotide polymorphisms (SNPs) were examined to detect mixed infection in selectedM. tuberculosisisolates. ResistantM. tuberculosisisolates were detected at 1:100,000, and changes in cytometry-gated events were predictive ofin vitroM. tuberculosisbactericidal activity using the Φ2GFP10 assay. Emergent drug resistance was detected in one patient by Φ2GFP10 at 3 weeks but not by conventional testing (M. tuberculosisculture 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. Φ2GFP10 can detect low-frequency drug-resistantM. tuberculosisand with WGS characterize emergentM. tuberculosisresistance. In areas of high TB transmission and drug resistance, rapid screening for heteroresistance should be considered.