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

Max R. O’Donnell, Michelle H. Larsen, Tyler S. Brown, Paras Jain, Vanisha Munsamy, Allison Wolf, Lorenzo Uccellini, Farina Karim, Tulio de Oliveira, Barun Mathema, William R. Jacobs, Alexander Pym

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


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.

Original languageEnglish (US)
Article numbere01834-18
JournalAntimicrobial agents and chemotherapy
Issue number4
StatePublished - Apr 2019


  • Extensively drug-resistant tuberculosis
  • Heteroresistance
  • Phage
  • South Africa
  • Tuberculosis
  • Whole-genome sequencing

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases


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