vestimИзвестия Национальной академии наук Беларуси. Серия медицинских наукProceedings of the National Academy of Sciences of Belarus, Medical series1814-60232524-2350The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1814-6023-2023-20-2-158-167vestim-913Research ArticleКЛИНИЧЕСКАЯ И ЭКСПЕРИМЕНТАЛЬНАЯ МЕДИЦИНАCLINICAL AND EXPERIMENTAL MEDICINEИспользование ПЦР с гидролизными зондами для выявления мутаций устойчивости к фторхинолонам у Mycobacterium tuberculosisPCR with hydrolysis probes for detection of fluoroquinolone resistance mutations in Mycobacterium tuberculosishttps://orcid.org/0000-0003-1846-6100СлизеньВ. В.SlizenV. V.

Слизень Вероника Вячеславовна – канд. мед. наук, доцент

Долгиновский тракт, 157, 220080, г. Минск

Veronika V. Slizen – Ph. D. (Med.), Associate Professor

157, Dolginovski Tract, 220080, Minsk

veronal@tut.byСурковаЛ. К.SurkovaL. K.

Суркова Лариса Константиновна – д-р мед. наук, профессор, заведующий отделом

Долгиновский тракт, 157, 220080, г. Минск

Larissa K. Surkova – D. Sc. (Med.), Professor, Head of the Department

157, Dolginovski Tract, 220080, Minsk

niipulm@tut.byhttps://orcid.org/0000-0001-5683-2322ГуревичГ. Л.GurevichG. L.

Гуревич Геннадий Львович – член-корреспондент, д-р мед. наук, профессор, директор

Долгиновский тракт, 157, 220080, г. Минск

Gennady L. Gurevich – Corresponding Member, D. Sc. (Med.), Professor, Director

157, Dolginovski Tract, 220080, Minsk

niipulm@tut.byРеспубликанский научно-практический центр пульмонологии и фтизиатрииRepublican Scientific and Practical Center for Pulmonology and Phthisiatry202305062023202158167Copyright © Слизень В.В., Суркова Л.К., Гуревич Г.Л., 20232023Слизень В.В., Суркова Л.К., Гуревич Г.Л.Slizen V.V., Surkova L.K., Gurevich G.L.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestimed.belnauka.by/jour/article/view/913

Ранняя диагностика резистентности к фторхинолонам дает возможность своевременно начать адекватную терапию и повышает шансы на благоприятный исход заболевания. Использование молекулярно-генетических методов исследования позволяет получить в течение 1–2 сут результаты определения устойчивости Mycobacterium tuberculosis к противотуберкулезным лекарственным средствам, в то время как при использовании классических методов результаты становятся известны почти через 1–2 мес. 

Цель работы – разработка метода детекции M. tuberculosis и точечных мутаций в 90, 91, 94-м кодонах gyrA гена, ассоциированных с устойчивостью к фторхинолонам. 

Исследовано 88 культур микобактерий: M. tuberculosis (n = 81), M. tuberculosis H37Rv, M. chelonae (n = 1), M. gordonae (n = 1), M. fortuitum (n = 1) и 3 изолята других нетуберкулезных микобактерий. Типы мутаций в гене gyrA изучены с помощью разработанного метода ПЦР в реальном времени, стандартного метода GenoType MTBDRsl (HAIN, Германия) и секвенирования по Сэнгеру. Анализ мутаций в gyrA гене 78 изолятов M. tuberculosis показал, что доминирующими были мутации p.Asp94Gly и p.Ala90Val, которые идентифицированы у 21 и 27 изолятов соответственно: в структуре мутаций на их долю приходится 64 %. У M. tuberculosis встречались также мутации p.ASP94ALA и p.ASP94TYR/HIS – у 6 (8,0 %) и 9 (12,0 %) изолятов соответственно. У одного штамма присутствовала мутация в 88-м кодоне и у одного штамма найдена двойная мутация (ALA90VAL и ASP94GLY). Результаты по выявлению мутаций разработанным методом ПЦР в реальном времени совпадают с результатами GenoType MTBDRsl и секвенирования. Разработанный метод ПЦР в реальном времени демонстрирует высокую частоту совпадений с данными фенотипического определения устойчивости к офлоксацину и результатами тестирования с помощью стандартного метода GenoType MTBDRsl и секвенирования. С помощью данного метода можно выявлять возбудителей туберкулеза, дискриминировать мутации p.ALA90VAL, p.SER91PRO, p.ASP94ALA, p.ASP94TYR/HIS, p.ASP94GLY, p.ASP94ASN, что позволяет диагностировать устойчивость к фторхинолонам. 

Early diagnostics of resistance to fluoroquinolones facilitates early start of adequate therapy and increases the chance of a favorable outcome of the tuberculosis. Application of genetic methods permits to obtain within 1–2 days the results of Mycobacterium tuberculosis resistance detection to anti-tuberculosis drugs, unlike the classical methods requiring up to 1−2 month.

The aim of the study is to develop a method for the M. tuberculosis identification and detection of point mutations in codons 90, 91, 94 of the gyrA gene associated with the resistance to fluoroquinolones.

There were 88 cultures of mycobacteria studied: M. tuberculosis (n = 81), M. tuberculosis H37Rv, M. chelonae (n = 1), M. gordonae (n = 1), M. fortuitum (n = 1), and other three isolates of non-tuberculosis mycobacteria isolated from patients in the Republican Scientific and Practical Center for Pulmonology and Phthisiatry. The types of mutations in the gyrA gene were studied by the standard GenoTypeMTBDRsl method (HAIN, Germany), Sanger sequencing, and by the developed real-time PCR method. Based on the analysis of mutations in the gyrA gene in 78 isolates of M. tuberculosis, the dominant mutations were found to be mutations Asp94Gly and Ala90Val, which were identified in 21 and 27 isolates correspondingly: they accounted for 64 % of all mutations. M. tuberculosis also harbored mutations p.ASP94ALA and p.ASP94TYR/HIS in 6 (8.0 %) and 9 (12.0 %) isolates, respectively. One strain harbored a mutation at triplet 88 and one strain had a double mutation (p.ALA90VAL and p.ASP94GLY). The developed real-time PCR method demonstrated a high frequency of coincidence of results with the phenotypic determination of resistance to ofloxacin and the results testing by the standard GenoTypeMTBDRsl method and sequencing.

The developed method is accomplished to identify M. tuberculosis, and discriminate mutations p.ALA90VAL, p.SER91PRO, p.ASP94ALA, p.ASP94TYR/HIS, p.ASP94GLY, p.ASP94ASN providing diagnostics of resistance to fluoroquinolones.

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The authors declare that there are no conflicts of interest present.