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%.
Highlights
Цель исследования: разработка новой тест-системы для ускоренного фенотипического определения лекарственной чувствительности клинических изолятов микобактерий туберкулеза (МБТ) к противотуберкулезным препаратам (ПТП) первого и второго рядов на основе применения литических микобактериофагов
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
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
Summary
Цель исследования: разработка новой тест-системы для ускоренного фенотипического определения лекарственной чувствительности клинических изолятов МБТ к противотуберкулезным препаратам (ПТП) первого и второго рядов на основе применения литических микобактериофагов. Культуры МБТ (108 клинических изолятов после первичного культивирования в системе Bactec MGIT) инкубировали 48 ч в присутствии ПТП, затем 48 ч после добавления литического микобактериофага D29. Одновременно лекарственная чувствительность/устойчивость всех клинических изолятов МБТ после рекультивирования анализировалась в системе Bactec MGIT, который был принят в качестве эталонного метода. Определение на основе применения литического микобактериофага лекарственной чувствительности/устойчивости 108 изолятов МБТ к четырем препаратам первого ряда и 90 изолятов к шести препаратам второго ряда ПТП продемонстрировало высокий уровень совпадения с результатами в системе Bactec MGIT. 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
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