Mycobacteriophage-based test system for phenotypic drug sensitivity of clinical isolates of tuberculous mycobacteria

Abstract

The issue of rapid phenotypic drug sensitivity testing of clinical isolates of tuberculous mycobacteria remains relevant.The objective of the study is to develop a new test system for rapid phenotypic drug sensitivity testing of MTB clinical isolates based on lytic mycobacteriophages, capable of testing resistance to first and second line drugs.Subjects and methods. Cultures of MTB (108 clinical isolates after primary culturing in Bactec MGIT) were incubated for 48 hours with addition of anti-tuberculosis drugs, then for 48 hours more after adding lytic mycobateriophage D29. The subsequent multiplex reaction of the polymerase chain reaction in real time allowed performing quantitative analysis of MTB DNA and mycobacteriophage DNA. The drug sensitivity of the tested sample was assessed by ranges of the differences in fluorescence threshold levels corresponding to the amount of mycobacteriophages between the control and test sample. At the same time, the drug sensitivity/resistance of all MTB clinical isolates after repeated culture was tested by Bactec MGIT which was adopted as a reference method.Results. Lytic mycobacteriophage-based drug sensitivity testing of 108 MTB isolated to four first line drugs and 90 isolates to six second line anti-tuberculosis drugs demonstrated a high level of concordance with the results of the Bactec MGIT system. It provided 99.5% sensitivity and 100% specificity of the method for 3 first-line drugs; it was slightly lower for ethambutol (86 and 96.9%, respectively), while for second line drugs, its sensitivity made 94.83% and specificity - 98.85%.