ГЕНЕТИЧЕСКИЕ ВАРИАНТЫ ВИРУСА ГЕПАТИТА В У ПАЦИЕНТОВ С ХРОНИЧЕСКИМ ГЕПАТИТОМ В

Introduction. Biological properties of virus, such as susceptibility to antivirals, the clinical course of chronic hepatitis B (CHB), the probability of developing cirrhosis and hepatocellular carcinoma are determined by genotype and mutations in the genome of the hepatitis B virus (HBV).The aim of the study was evaluating of HBV genetic variants in patients with CHB from St. Petersburg hospitals.Material and methods. A total of 1414 CHB patients with positive polymerase chain reaction HBV genome in blood and/or liver tissue were observed. Genotype was determined in 298 patients, sequencing of the polymerase gene fragment was performed in 80 patients.Results. Viral DNA was detected in 323 (55.8%) patients with CHB. Genotype D was determined in 238 (80,1%), genotype A – in 49 (16,5%), C genotype – in 2 (0,7%) and mixed A+D – in 8 (2,7%) patients. Substitutions in YMDD-motif of the polymerase protein (M204I/V) as well as other primary and secondary resistance mutations to nucleotide analogues (lamivudine, telbivudine, entecavir) were found in four patients. Mutations in the reverse transcriptase (rt) region of polymerase gene were shown to affect the structure of surface protein. The substitution rtA181T in three patients resulted in formation of stop codon (sW172*) and premature termination of surface protein synthesis. The absence of HBeAg and the degree of fibrosis increase in 7 patients may be the result of mutations identified in core gene (G1896A, A1762T, G1764A).Conclusion. Study of the geographical distribution of HBV genotypes and identification of amino acid substitutions leading to decrease in serum markers concentration and emergence of resistance antivirals mutations is of great practical importance for predicting severity of the disease and effectiveness of antiviral therapy.Введение. Биологические свойства вируса, такие как чувствительность к противовирусным препаратам, клиническое течение хронического гепатита В (ХГВ), вероятность развития цирроза и гепатоцеллюлярной карциномы определяются генотипом и мутациями в геноме вируса гепатита В (ВГВ).Цель. Изучение генетических вариантов ВГВ у пациентов с ХГВ, находящихся на лечении в клиниках Санкт- Петербурга.Материалы и методы. Обследовано 1414 пациентов с ХГВ на наличие ВГВ в крови и/или ткани печени методом полимеразной цепной реакции. Генотип определен у 298 пациентов, секвенирование фрагмента гена полимеразы – у 80 пациентов.Результаты. Вирусная ДНК выявлена у 323 (55,8%) больных ХГВ. Генотип D обнаружен у 238 (80,1%), генотип А – у 49 (16,5%), генотип С– у 2 (0,7%) и микст D+А – у 8 (2,7%) пациентов. У 4 пациентов были выявлены замены в YMDD-мотиве полимеразы (M204I/V), а также найдены другие первичные и вторичные мутации устойчивости к аналогам нуклеотидов (ламивудину, телбивудину, энтекавиру). Показано, что мутации в области обратной транскиптазы (rt) полимеразного гена влияют и на поверхностный белок. У трех пациентов замена rtA181T привела к образованию стоп-кодона в S-гене и преждевременной терминации синтеза поверхностного белка (sW172*). Отсутствие HBeAg и увеличение степени фиброза у 7 пациентов может быть следствием выявленных мутаций в CORE гене (G1896A, A1762T, G1764A).Заключение. Исследование географического распределения генотипов вируса гепатита В и выявление аминокислотных замен, приводящих к снижению концентрации серологических маркеров и появлению мутаций устойчивости к противовирусным препаратам, имеет важное практическое значение для прогнозирования тяжести течения заболевания и эффективности противовирусной терапии

Эпидемиологические и клинические характеристики острых респираторных инфекций в Санкт-Петербурге в эпидемические сезоны 2017–2018 гг. и 2018–2019 гг.

Objective: to analyze the epidemiological and clinical features of acute respiratory infections occurring during the St. Petersburg 2017–2018 and 2018–2019 epidemic seasons.Materials and methods: the study included 457 patients, treated in St. Petersburg clinics from 2017–2019, displaying symptoms of acute respiratory infection (ARI), including evaluation of their clinical histories. Pathogen types were determined by polymerase chain reaction (PCR). Data analysis was carried out using mathematical statistics methods using the Statistica 10 software package (StatSoft Inc.).Results: in this study, we examined the epidemiological and clinical features of acute respiratory infections in St. Petersburg occurring during two epidemic seasons, 2017–2018 and 2018–2019. The 2017–2018 season was characterized by a prevalence of infections caused by influenza B viruses and influenza A subtype H3N2 viruses. In the 2018–2019 season, there was a greater number of acute respiratory viral infections (ARVIs) and infections caused by influenza A subtype H1N1pdm; influenza B virus was detected only in isolated cases. In the 2017–2018 sore throats and muscle aches were a characteristic symptom of influenza A H1N1pdm infections, of bacterial infections – only sore throats. It was shown that throat pain and vasodilation of the scleral and soft palate vessels were significantly more frequent in the 2017–2018 season, compared to the 2018–2019 season. Cough and redness of the posterior pharyngeal wall were hallmark signs of ARVIs in the 2018–2019 season.Conclusion: according to the data, each epidemic season is characterized not only by its own type-specific acute respiratory infection frequencies, but also by different clinical manifestation frequencies. For global monitoring, treatment effectiveness evaluation, and refined study of acute respiratory infection clinical features, it is advisable to use approaches which incorporate accurate, specific, and rapid molecular biological methods capable of identifying a broad range of pathogens. Цель: сравнительный анализ эпидемиологических данных и клинических характеристик течения острых респираторных инфекций у больных в Санкт-Петербурге в эпидемические сезоны 2017–2018 гг. и 2018–2019 гг.Материалы и методы: в исследование включено 457 пациентов, находившихся на лечении в клиниках Санкт-Петербурга в 2017–2019 гг., с симптомами острых респираторных инфекций, проведен анализ эпидемиологических данных и клинических характеристик течения заболевания. Методом полимеразной цепной реакции определен вид патогена. Статистический анализ проведен с использованием методов математической статистики при помощи пакета Statistica 10, StatSoft Inc.Результаты. В сезон 2017–2018 гг. было характерно превалирование инфекций, вызванных вирусами гриппа В и А (H3N2). В сезоне 2018–2019 гг. было отмечено большее число случаев острых респираторных вирусных инфекций, в том числе гриппа, вызванного вирусом гриппа А (H1N1pdm); вирус гриппа В регистрировали в единичных случаях. Характерным симптомом в сезоне 2017–2018 гг. для гриппа А (H1N1pdm) были мышечные боли и боли в горле, для бактериальных инфекций – также боли в горле. Боли в горле и инъекции сосудов склер и мягкого неба достоверно чаще были отмечены в сезоне 2017–2018 гг. по сравнению с сезоном 2018–2019 гг. Кашель и гиперемия задней стенки глотки были характерными симптомами для ОРВИ в сезоне 2018–2019 гг.Заключение: рассмотренные два эпидемических сезона характеризовались не только различной частотой острых респираторных инфекций, но и различной частотой их клинических проявлений. Для глобального мониторинга, оценки эффективности лечения и более детального изучения клинических особенностей острых респираторных инфекций целесообразно использовать точные, специфичные и быстрые молекулярно-биологические методы с большим количеством определяемых патогенов.

NEW METHOD FOR DETERMINING HEPATITIS B VIRUS RESISTANCE MUTATIONS M204I/V TO NUCLEOS(T)IDE ANALOGUES IN PATIENTS WITH CHRONIC HEPATITIS B

Аnalogues of nucleos(t)ides (AN) such as lamivudine (LAM), telbivudine (TBV), adefovir (ADP), entecavir (ENT) are widely used for the treatment of chronic hepatitis B (CHB). However, the prolonged treatment using these drugs often leads to the development of drug resistance. The most common substitutions in the reverse transcriptase are methionine for valine (rtM204V), or methionine for isoleucine (rtM204I) at position 204. Early AN-resistant mutations detection is of great importance to determine the treatment strategy of patients with CHB. Currently there are many highly sensitive methods for detection of drug resistance mutations, such as next-generation sequencing, reverse hybridizationbased line probe assay (LiPA), mass spectrometry. However, these methods require expensive equipment and reagents, and they are not widely used in clinical laboratories. The aim of this study was to develop a simple and accurate real-time PCR method for detection of rtM204I/V mutation. This method showed high specificity and sensitivity (1000 copies/ml), it is less laborious and does not require additional equipment, fast and cost effective compared to other methods. HBV mutations of resistance to AN were determined in 5 groups of patients with CHB. Patients of the first group received monotherapy with pegylated interferon (n = 12), the second group — lamivudine (n = 10), the third group — telbivudine (n = 7), the fourth group — entecavir (n = 15). The fifth group consisted of patients who did not receive antiviral therapy (n = 3). The frequency of mutations in HBV polymerase YMDD-motif was determined among 47 patients with CHB: it was 10% for lamivudine treated patients, 20% — for entecavir, 28% — for telbivudine. YIDD/YVDD motifs were identified in two patients and YMDD/YIDD — in one patient. Real-time PCR method for the detection of AN-resistant rtM204I/V mutations in HBV polymerase can be used in routine diagnostics for primary screening of patients not responding to AN treatment. The application of this method can reduce the number of samples for in-depth study of primary and compensatory mutations of resistance to AN by sequencing method. The developed method versus Sanger-sequencing is fast, economical, and provides the detection of minor variants of HBV populations

Genetic variants of hepatitis B virus in patients with chronic hepatitis B

Introduction. Biological properties of virus, such as susceptibility to antivirals, the clinical course of chronic hepatitis B (CHB), the probability of developing cirrhosis and hepatocellular carcinoma are determined by genotype and mutations in the genome of the hepatitis B virus (HBV).The aim of the study was evaluating of HBV genetic variants in patients with CHB from St. Petersburg hospitals.Material and methods. A total of 1414 CHB patients with positive polymerase chain reaction HBV genome in blood and/or liver tissue were observed. Genotype was determined in 298 patients, sequencing of the polymerase gene fragment was performed in 80 patients.Results. Viral DNA was detected in 323 (55.8%) patients with CHB. Genotype D was determined in 238 (80,1%), genotype A – in 49 (16,5%), C genotype – in 2 (0,7%) and mixed A+D – in 8 (2,7%) patients. Substitutions in YMDD-motif of the polymerase protein (M204I/V) as well as other primary and secondary resistance mutations to nucleotide analogues (lamivudine, telbivudine, entecavir) were found in four patients. Mutations in the reverse transcriptase (rt) region of polymerase gene were shown to affect the structure of surface protein. The substitution rtA181T in three patients resulted in formation of stop codon (sW172*) and premature termination of surface protein synthesis. The absence of HBeAg and the degree of fibrosis increase in 7 patients may be the result of mutations identified in core gene (G1896A, A1762T, G1764A).Conclusion. Study of the geographical distribution of HBV genotypes and identification of amino acid substitutions leading to decrease in serum markers concentration and emergence of resistance antivirals mutations is of great practical importance for predicting severity of the disease and effectiveness of antiviral therapy

Epidemiological and clinical features of acute respiratory infections occurring in St. Petersburg during the 2017–2018 and 2018–2019 epidemic seasons

Objective: to analyze the epidemiological and clinical features of acute respiratory infections occurring during the St. Petersburg 2017–2018 and 2018–2019 epidemic seasons.Materials and methods: the study included 457 patients, treated in St. Petersburg clinics from 2017–2019, displaying symptoms of acute respiratory infection (ARI), including evaluation of their clinical histories. Pathogen types were determined by polymerase chain reaction (PCR). Data analysis was carried out using mathematical statistics methods using the Statistica 10 software package (StatSoft Inc.).Results: in this study, we examined the epidemiological and clinical features of acute respiratory infections in St. Petersburg occurring during two epidemic seasons, 2017–2018 and 2018–2019. The 2017–2018 season was characterized by a prevalence of infections caused by influenza B viruses and influenza A subtype H3N2 viruses. In the 2018–2019 season, there was a greater number of acute respiratory viral infections (ARVIs) and infections caused by influenza A subtype H1N1pdm; influenza B virus was detected only in isolated cases. In the 2017–2018 sore throats and muscle aches were a characteristic symptom of influenza A H1N1pdm infections, of bacterial infections – only sore throats. It was shown that throat pain and vasodilation of the scleral and soft palate vessels were significantly more frequent in the 2017–2018 season, compared to the 2018–2019 season. Cough and redness of the posterior pharyngeal wall were hallmark signs of ARVIs in the 2018–2019 season.Conclusion: according to the data, each epidemic season is characterized not only by its own type-specific acute respiratory infection frequencies, but also by different clinical manifestation frequencies. For global monitoring, treatment effectiveness evaluation, and refined study of acute respiratory infection clinical features, it is advisable to use approaches which incorporate accurate, specific, and rapid molecular biological methods capable of identifying a broad range of pathogens

VIRUS-SPECIFIC HUMORAL IMMUNE RESPONSE ISOTYPIC STRUCTURE IN ADULT PATIENTS HOSPITALIZED WITH INFLUENZA A

The aim of this investigation was a comparative analysis of isotypic structure of specific antiviral systemic humoral immune response in hospitalized patients with influenza caused by virus A(H3N2) or A(H1N1), including the A(H1N1)pdm09. Paired acute and convalescent phase sera from 109 adult patients aged 18 to 67 years with laboratoryconfirmed influenza A were analyzed by ELISA. Purified surface glycoproteins of influenza A viruses of different subtypes containing the hemagglutinin and neuraminidase were used as antigen for sensitization of plates in ELISA.The absence of type-specific conserved internal proteins in antigenic material allowed to carry out a subtype-specific differentiation of antibodies against influenza viruses in ELISA. Regardless of the subtype of influenza A viruses caused the disease, the most pronounced response was observed by subtype-specific IgG1 (70–90% of seroconversions). It has been shown for the first time that low activity of virus-induced IgG2 (6–9% of seroconversions) is a peculiarity of the immune response both to primary or recurrent infections with A(H1N1)pdm09. In patients repeatedly suffered by «seasonal» influenza A(H1N1) in 2007/2008 or influenza A(H3N2) in 2012–2014 IgG2 seroconversion’s rates were 40–59% (р < 0,05). Reaction virusspecific IgG3 was also weaker in patients with influenza A(H1N1)pdm09 (29–44% of seroconversions) than in subjects with influenza A(H1N1) or A(H3N2) (65% and 56% of seroconversions, respectively). Geometric mean titers of virus neutralizing antibodies identified during recovery phase in patients with primary and secondary influenza A(H1N1)pdm09 (1/28 and 1/103, respectively) were significantly lower than in patients recovered from influenza A(H1N1) or A(H3N2) (GMT were 1/594 and 1/378, respectively). It was shown that the surface glycoproteins of influenza A viruses may be an allergens. Virus-specific IgE seroconversion rates were comparable in all groups reaching 25–45%. The high activity of virus-induced serum IgA was detected in patients with influenza A(H3N2) or A(H1N1)pdm09 (60–79% of seroconversions). Thus, study of virus-specific activity of various immunoglobulin isotypes provides important information about the formation of adaptive antiviral immune response to influenza A viruses, and also estimate the contribution of its protective and immunopathogenic components to pathogenesis of the disease