title: Evaluation of fluoromycobacteriophages for detecting drug resistance in Mycobacterium tuberculosis.
abstract: We tested a new method for detecting drug-resistant strains of Mycobacterium tuberculosis that uses a TM4 mycobacteriophage phAE87::hsp60-EGFP (EGFP-phage) engineered to contain the gene encoding enhanced green fluorescent protein (EGFP). After promising results in preliminary studies, the EGFP-phage was used to detect isoniazid (INH), rifampin (RIF), and streptomycin (STR) resistance in 155 strains of M. tuberculosis, and the results were compared to the resazurin microplate technique, with the proportion method serving as the reference standard. The resazurin technique yielded sensitivities of 94% for INH and RIF and 98% for STR and specificities of 97% for INH, 95% for RIF, and 98% for STR. The sensitivity of EGFP-phage was 94% for all three antibiotics, with specificities of 90% for INH, 93% for RIF, and 95% for STR. The EGFP-phage results were available in 2 days for RIF and STR and in 3 days for INH, with an estimated cost of ∼2$ to test the three antibiotics. Using a more stringent criterion for resistance improved the specificity of the EGFP-phage for INH and RIF without affecting the sensitivity. In preliminary studies, the EGFP-phage could also effectively detect resistance to the fluoroquinolones. The EGFP-phage method has the potential to be a valuable rapid and economic screen for detecting drug-resistant tuberculosis if the procedure can be simplified, if it can be adapted to clinical material, and if its sensitivity can be improved.
authors.affiliation: Laboratorio de Genética Molecular, CMBC, IVIC, Km 11, Carr Panamericana, Caracas 1020A, Venezuela.
authors.forename: Liliana
authors.initials: L
authors.lastname: Rondón
authors.forename: Mariana
authors.initials: M
authors.lastname: Piuri
authors.forename: William R
authors.initials: WR
authors.lastname: Jacobs
authors.forename: Jacobus
authors.initials: J
authors.lastname: de Waard
authors.forename: Graham F
authors.initials: GF
authors.lastname: Hatfull
authors.forename: Howard E
authors.initials: HE
authors.lastname: Takiff
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D005470:Fluorometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D050296:Microbial Viability / Q000187:drug effects*; D009160:Mycobacteriophages / Q000235:genetics / Q000254:growth & development*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000302:isolation & purification / Q000821:virology*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins