Evaluation of a Bacteriophage-Based Assay (Phage Amplified Biologically Assay) as a Rapid Screen for Resistance to Isoniazid, Ethambutol, Streptomycin, Pyrazinamide, and Ciprofloxacin among Clinical Isolates of Mycobacterium tuberculosis

Abstract

Rapid molecular assays for the detection of mutations associated with rifampin resistance in Mycobacterium tuberculosis are commercially available. However, they are complex and expensive and have predictive values of 90 to 95%. Molecular assays for other drugs are less predictive of resistance. Ideally, assays based on phenotypic markers should be used for susceptibility testing, but these can take weeks to complete. We previously described a rapid phenotypic assay, the phage amplified biologically (PhaB) assay, for the rapid determination of rifampin and isoniazid susceptibility in clinical isolates of M. tuberculosis. In this study, we extended the assay to the study of ethambutol, pyrazinamide, streptomycin, and ciprofloxacin. After the optimization of antibiotic concentrations and incubation conditions, the assay was applied to each drug for a total of 157 isolates. The correlations between the results of the PhaB assay and the resistance ratio method were 94% for isoniazid, 96% for streptomycin, 100% for ciprofloxacin, 88% for ethambutol, and 87% for pyrazinamide. For ciprofloxacin, ethambutol, and pyrazinamide, significantly better correlations were found when a 90% reduction in plaque count was used as the cutoff. Turnaround times for the PhaB assay were 2 to 3 days, compared with 10 days for the resistance ratio method. We believe that this low-cost assay may have widespread applicability for the rapid screening of drug resistance in M. tuberculosis isolates, especially in developing countries.

Detection of viral aerosols by use of real-time quantitative PCR

“…Phage UX174, phage f2, and phage SM701 were culture collections in our laboratory. Phage D29 was prepared in M. smegmatis by the standard method and stored, as described by Eltringham et al (1999).…”

Performance of a rapid phage-based test, FASTPlaqueTB™, to diagnose pulmonary tuberculosis from sputum specimens in South Africa

“…[9][10][11][12][13] The FASTPlaqueTB™ test (Biotec Laboratories Ltd., Ipswich, UK) is based on novel phage amplification technology in which mycobacteriophage (phage specific for mycobacteria) are used as indicators of the presence of viable Mycobacterium tuberculosis in a clinical specimen. [14][15][16][17][18][19] This test utilises mycobacteriophage to reflect the presence of viable M. tuberculosis. After phage infection, a virucidal solution is added which destroys all phage that have not infected the tubercle bacilli.…”

Molecular Techniques in the Diagnosis of Mycobacterium tuberculosis and the Detection of Drug Resistance

“…One such RT-PCR assay, which measured a reduction in inducible heat-shock protein dnaK mRNA levels in susceptible isolates exposed to rifampicin, showed 96% (46/48) correlation in susceptible isolates and 97% (35/36) correlation in resistant isolates. 29 Alternative RNA-based approaches have included measuring levels of pre-16S rRNA stem sequences through hybidization to specific radiolabeled probes for rifampicin and ciprofloxacin 32 and quantitative analysis of 85B mRNA for rifampicin and isoniazid. 33…”