Аннотация
Для оценки значимости влияния мутаций лекарственной устойчивости ВГС к препаратам прямого противовирусного действия (ПППД) среди пациентов с хроническим гепатитом С (ХГС) разработан метод, позволяющий определить наличие
мутаций резистентности в генах NS5A, NS5B, NS3 методом ПЦР с последующим секвенированием. При разработке метода
использованы образцы плазмы крови 193 пациентов с диагнозом ХГС, проживающих на территории Санкт-Петербурга и
Ленинградской области. Для определения клинически значимых нуклеотидных замен в регионах NS3, NS5A, NS5B ВГС использована разработанная методика на основе двухэтапной ПЦР, а именно: продукт амплификации кодируемых регионами
белков NS3 область 3312- 4341 нт, NS5A область 6129- 6949 нт, NS5B область 8007- 8634 нт, получали методом «гнездовой» (nested) ПЦР с использованием на каждом этапе пары праймеров в зависимости от определенного ранее генотипа ВГС.
Для определения наличия мутаций устойчивости использовано программное обеспечение «Geno2pheno HCV resistance”. На
основании анализа данных выявлено, что необходимая вирусная нагрузка для проведения секвенирующей реакции и получения
нуклеотидной последовательности искомых фрагментов генома составляет 1,5х103
МЕ/мл и более. Разработанный способ
позволяет идентифицировать наличие мутаций лекарственной резистентности вируса к противовирусным препаратам
при наиболее распространенных в РФ субгенотипах ВГС (1, 2, 3) и может быть использован для определения стратегии лечения пациентов с ХГС и назначении повторного лечения при неуспехе первой линии терапии. Метод использован для оценки
распространённости мутаций устойчивости ВГС среди некоторых уязвимых групп населения: пациенты с моноинфекцией
ВГС и пациенты с коинфекцией ВИЧ/ВГС.
Annotation
To assess the significance of the impact of HCV drug resistance mutations on DAAs among patients with chronic hepatitis C (CHC),
a method was developed to determine the presence of clinically significant nucleotide substitutions in the NS5A, NS5B, NS3 genes
using PCR followed by sequencing. When developing the method, blood plasma samples were used from 193 patients diagnosed with
CHC living in St. Petersburg and the Leningrad region. To determine clinically significant nucleotide substitutions in the NS3, NS5A,
NS5B regions of HCV, we used a developed method based on two-step PCR, namely: amplification product of proteins encoded by
the regions, namely NS3 region 3312-4341 nt, NS5A region 6129-6949 nt, NS5B region 8007-8634 nt, was obtained by nested PCR
using a pair of primers at each stage depending on from the previously determined HCV genotype. To determine the presence of
significant nucleotide substitutions, the “Geno2pheno HCV resistance” software was used. Based on data analysis, it was revealed
that the required viral load to carry out a sequencing reaction and obtain the nucleotide sequence of the desired genome fragments for
determining MDR is 1.5×103
IU/ml or more. The developed method allows us to identify the presence of mutations of drug resistance of
the virus to antiviral drugs in the most common HCV subgenotypes in the Russian Federation (1, 2 and 3) and can be used to determine
the treatment strategy for patients with CHC and prescribe re-treatment if first-line therapy fails. This method was used to estimate the
prevalence of HCV resistance mutations among certain vulnerable populations: patients with HCV monoinfection and patients with
HIV/HCV coinfection.
Key words: chronic viral hepatitis C; HCV; drug resistance mutations; identification method, diagnostics; DAAs
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