vspВопросы современной педиатрииCurrent Pediatrics1682-55271682-5535Издательство «ПедиатрЪ»10.15690/vsp.v12i6.871vsp-253Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESМОЛЕКУЛЯРНОЕ ТИПИРОВАНИЕ STREPTOCOCCUS PNEUMONIAE МЕТОДОМ МУЛЬТИПЛЕКСНОЙ ПОЛИМЕРАЗНОЙ ЦЕПНОЙ РЕАКЦИИ С УЧЕТОМ РАСПРОСТРАНЕННОСТИ СЕРОТИПОВ В РОССИЙСКОЙ ФЕДЕРАЦИИMOLECULAR TYPING OF STREPTOCOCCUS PNEUMONIAE BY THE MULTIPLEX POLYMERASE CHAIN REACTION ASSAY IN ACCORDANCE TO THE PREVALENCE OF SEROTYPES IN THE RUSSIAN FEDERATIONАлябьеваН. М.Alyab'evaN. M.

Alyabyeva Natalya Mikhaylovna, Junior Researcher at the Laboratory of Experimental Immunology and Virology

alyabieva@nczd.ruБлиноваТ. А.BlinovaT. A.alyabieva@nczd.ruПономаренкоО. А.PonomarenkoO. A.alyabieva@nczd.ruЛазареваА. В.LazarevaA. V.alyabieva@nczd.ruКатосоваЛ. К.KatosovaL. K.alyabieva@nczd.ruМаянскийН. А.MayanskiiN. A.alyabieva@nczd.ruНаучный центр здоровья детей РАМН, МоскваРоссияScientific Centre of Children Health of RAMS, MoscowRussian FederationНаучный центр здоровья детей РАМН, Москва; Первый Московский государственный медицинский университет им. И.М. СеченоваРоссияScientific Centre of Children Health of RAMS, Moscow; I.M. Sechenov First Moscow State Medical UniversityRussian Federation201307122013126Вопросы современной педиатрии3034Copyright © Алябьева Н.М., Блинова Т.А., Пономаренко О.А., Лазарева А.В., Катосова Л.К., Маянский Н.А., 20132013Алябьева Н.М., Блинова Т.А., Пономаренко О.А., Лазарева А.В., Катосова Л.К., Маянский Н.А.Alyab'eva N.M., Blinova T.A., Ponomarenko O.A., Lazareva A.V., Katosova L.K., Mayanskii N.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vsp.spr-journal.ru/jour/article/view/253

Цель исследования: сравнить классический серологический метод и метод молекулярного типирования S. pneumoniae с помощью мультиплексной полимеразной цепной реакции (М-ПЦР), модифицированной в соответствии с данными о циркулирующих в Российской Федерации серотипах. Материалы и методы: всего протестировано 420 изолятов пневмококка, преимущественно из нестерильных локусов. После микробиологической идентификации пневмококки серотипировали с помощью специфических антисывороток производства Statens Serum Institut (Дания) в реакциях латекс-агглютинации и/или набухания капсулы. Параллельно проводили серию М-ПЦР, максимально состоявшую из 7 последовательных реакций. Результаты: серологическим методом серотип определили у всех 420 штаммов S. pneumoniae; всего было идентифицировано 34 различных серотипа. При помощи М-ПЦР удалось типировать 95% (399/420) исследованных штаммов, причем 90% было типировано в первых трех М-ПЦР. Все ПЦР-нетипируемые изоляты (n = 21) обладали серотипами, которые не входили в состав М-ПЦР. Результаты серологического и молекулярного типирования совпали у 99,2% (396/399) изолятов; 3 штамма показали противоречивые результаты: серологическим методом у них определили серотип 19А, а методом ПЦР — 19F. Выводы: предложенная модификация М-ПЦР позволяет правильно определить серотип более чем у 90% циркулирующих в Российской Федерации штаммов пневмококка, включая все серотипы, входящие в состав полисахаридных конъюгированных пневмококковых вакцин.

Aim: to compare two methods of S. pneumoniae molecular typing: classic serological method and the multiplex polymerase chain reaction (M-PCR) assay modified in accordance to the date on the serotypes circulating in Russian Federation. Patients and methods: 420 pneumococcal isolates mainly from non-sterile loci were analyzed. After microbiological identification pneumococci were serologically typed with the means of specific antiserum produced by Staten Serum Institute (Denmark) in latex agglutination test and/or capsular swelling method. At the same time we performed series of the M-PCR, which consisted of 7 consecutive reactions at the most. Results: serotype was identified by the means of serological method in all 420 strains of S. pneumoniae; in total we determined 24 different serotypes. By the means of the M-PCR we succeeded in identification of 95% (399/420) examined strains, and 90% of them were typed during the first 3 reactions. All isolates failed to be typed by M-PCR (n =21) have serotypes not included into the composition of the M-PCR. The results of serological and molecular typing were identical in 99,2% (396/399) of the isolates; 3 strains showed contradictory results: serotype 19A was found on serological assay and serotype 19F — on PCR assay. Conclusions: the introduced modification of M-PCR allows correct identification of pneumococcal serotype more than in 90% of strains circulating in the Russian Federation, including all serotypes of pneumococcal polysaccharide conjugate vaccine.

Streptococcus pneumoniaeсеротипмультиплексная ПЦРStreptococcus pneumoniaeserotypemultiplex PCRReferencesReinert R. R., Paradiso P., Fritzel B. Advances in pneumococcal vaccines: the 13-valent pneumococcal conjugate vaccine received market authorization in Europe. Exp. Rev. Vac. 2010; 9: 229–236.. Reinert R. R., Paradiso P., Fritzel B. Advances in pneumococcal vaccines: the 13-valent pneumococcal conjugate vaccine received market authorization in Europe. Exp. Rev. Vac. 2010; 9: 229–236.Katosova L. K., Spichak T. V., Kim S. S., Yatsyshina S. B., Zubkova I. V., Praded M. N. Voprosy diagnostiki v pediatrii [Pediatric Diagnostics]. 2009; 2: 27–31.L.K. Katosova, T.V. Spichak, S.S. Kim, S.B. Yatsyishina, I.V. Zubkova, M.N. Praded. Issues of Pediatric Diagnosis. 2009; 2: 27-31.Sidorenko S. V., Lobzin Yu. V., Kharit S. M., Korolyova I. S., Tatochenko V. K. Pnevmokokkovaya infektsiya i sovremennye vozmozhnosti eye profilaktiki — epidemiologicheskiy obzor situatsii v mire i v Rossii [Pneumococcal infection and advanced features for its prevention: An epidemiological overview of the situation in the world and in Russia]. Voprosy sovremennoy pediatrii — Current Pediatrics. 2010; 9 (1): 62–69.S.V. Sidorenko, Y.V. Lobzin, S.M. Kharit, I.S. Koroleva, V.K. Tatochenko. Pneumococcal infection and the modern means of preventing thereof – epidemiological review of the situation in Russia and around the world. Issues of Modern Pediatrics. 2010; 9 (1): 62-69.McIntosh E. D., Reinert R. R. Global prevailing and emerging pediatric pneumococcal serotypes. Exp. Rev Vac. 2011; 10 (1): 109–129. doi: 10.1586/erv.10.145.McIntosh E. D., Reinert R. R. Global prevailing and emerging pediatric pneumococcal serotypes. Exp. Rev Vac. 2011; 10(1): 109–129. doi: 10.1586/erv.10.145.Reinert R. R., Taishi B. Novye dannye po effektivnosti 13-valentnoy pnevmokokkovoy konyugirovannoy vaktsiny v otnoshenii invazivnykh pnevmokokkovykh infektsiy, pnevmoniy, ostrogo srednego otita i nazofaringealnogo nositelstva [New data on the effectiveness of 13-valent pneumococcal conjugate vaccine against invasive pneumococcal disease, pneumonia, acute otitis media and nasopharyngeal carriage]. Pediatricheskaya Farmakologiya — Pediatric Pharmacology. 2012; 3: 12–18.R.R. Raynert, B. Tayshi. New data on efficacy of the 13-valent pneumococcal conjugate vaccine against invasive pneumococcal infections, pneumonias, acute otitis media and nasopharyngeal carriage. Pediatric Pharmacology. 2012; 3: 12-18.Rodgers G. L., Arguedas A., Cohen R., Dagan R. Global serotype distribution among S. pneumoniae isolates causing otitis media in children: potential implications for pneumococcal conjugate vaccines. Vaccine. 2009; 27: 3802–3810.Rodgers G. L., Arguedas A., Cohen R., Dagan R. Global serotype distribution among S. pneumoniae isolates causing otitis media in children: potential implications for pneumococcal conjugate vaccines. Vaccine. 2009; 27: 3802–3810.Rodgers G. L., Arguedas A., Cohen R., Dagan R. Global serotype distribution among S. pneumoniae isolates causing otitis media in children: potential implications for pneumococcal conjugate vaccines. Vaccine. 2009; 27: 3802–3810.Rodgers G. L., Arguedas A., Cohen R., Dagan R. Global serotype distribution among S. pneumoniae isolates causing otitis media in children: potential implications for pneumococcal conjugate vaccines. Vaccine. 2009; 27: 3802–3810.Mayanskiy А. N. Streptokokki: mikrobiologiya i patologiya [Streptococci: Microbiology and pathology]. Voprosy diagnostiki v pediatrii — Pediatric Diagnostics. 2010; 1: 9–19.A.N. Mayanskiy. Streptococcus: microbiology and pathology. Issues of Pediatric Diagnosis. 2010; 1: 9-19.Katosova L. K., Tatochenko V. K., Arova A. A., Keshikbaeva A. A., Baturo A. P., Kuznetsova T. A., Levin A. B. Serotipy Streptococcus pneumoniae u detey, bolnykh ostroy pnevmoniyey i plevritom [Serotypes of Streptococcus pneumoniae in children with acute pneumonia and pleuritis]. Zhurnal mikrobiologii, epidemiologii i immunobiologii — Journal of Epidemiology, Microbiology and Immunobiology. 1990; 5: 23–28.L.K. Katosova, V.K. Tatochenko, A.A. Arova, A.A. Keshikbaeva, A.P. Baturo, T.A. Kuznetsova, A.B. Levin. Streptococcus pneumoniae serotypes in children with acute pneumonia or pleuritic. Journal of Microbiology, Epidemiology and Immunobiology. 1990; 5: 23-28.Mayanskiy А. N., Alyabyeva N. M., Katosova L. K., Grechukha T. A., Pinelis V. G., Namazova-Baranova L. S. Opredeleniye kapsulnykh serotipov pnevmokokka metodom multipleksnoy PTsR [Determination of capsular pneumococcal serotypes using the method of multiplex PCR]. Voprosy diagnostiki v pediatrii — Pediatric Diagnostics. 2010; 2 (6): 6–10.N.A. Mayanskiy, N.M. Alyab’eva, L.K. Katosova, T.A. Grechukha, V.G. Pinelis, L.S. Namazova-Baranova. Identification of capsule pneumococcal serotypes using multiplex PCR assay. Issues of Pediatric Diagnosis. 2010; 2(6): 6-10.Techasaensiri C., Messina A. F., Katz K., Ahmad N., Huang R., McCracken G. H., Jr. Epidemiology and evolution of invasive pneumococcal disease caused by multidrug resistant serotypes of 19A in the 8 years after implementation of pneumococcal conjugate vaccine immu nization in Dallas, Texas. Pediatr. Infect. Dis. J. 2010; 29 (4): 294–300.Techasaensiri C., Messina A. F., Katz K., Ahmad N., Huang R., McCracken G. H., Jr. Epidemiology and evolution of invasive pneumococcal disease caused by multidrug resistant serotypes of 19A in the 8 years after implementation of pneumococcal conjugate vaccine immunization in Dallas, Texas. Pediatr. Infect. Dis. J. 2010; 29 (4): 294–300.Miller E., Andrews N. J., Waight P. A., Slack M. P., George R. C. Herd immunity and serotype replacement 4 years after sevenvalent pneumococcal conjugate vaccination in England and Wales: an observational cohort study. Lancet Infect. Dis. 2011; 11 (10): 760–768.Miller E., Andrews N. J., Waight P. A., Slack M. P., George R. C. Herd immunity and serotype replacement 4 years after sevenvalent pneumococcal conjugate vaccination in England and Wales: an observational cohort study. Lancet Infect. Dis. 2011; 11 (10): 760–768.Konradsen H. B. Validation of serotyping of Streptococcus pneumoniae in Europe. Vaccine. 2005; 23: 1368–1373.Konradsen H. B. Validation of serotyping of Streptococcus pneumoniae in Europe. Vaccine. 2005; 23: 1368–1373.Pai R., Gertz R. E., Beall B. Sequential multiplex PCR approach for determining capsular serotypes of Streptococcus pneumoniae isolates. J. Clin. Microbiol. 2006; 44: 124–131.Pai R., Gertz R. E., Beall B. Sequential multiplex PCR approach for determining capsular serotypes of Streptococcus pneumoniae isolates. J. Clin. Microbiol. 2006; 44: 124–131.Kozlov R. S., Chagaryan A. N., Kozlova L. V., Muravyov A. A. Serologicheskaya kharakteristika i chuvstvitelnost k antibiotikam pnevmokokkov, vydelennykh u detey v vozraste do 5 let v otdelnykh regionakh Rossyskoy Federatsii [Serological characterization and antibiotic sensitivity of pneumococci selected from children under 5 in certain regions of the Russian Federation]. Klinicheskaya mikrobiologiya i antimikrobnaya terapiya — Clinical Microbiology and Antimicrobial Therapy. 2011; 13 (2): 177–187.R.S. Kozlov, A.N. Chagaryan, L.V. Kozlova, A.A. Muravyov. Serological description and antibiotic sensitivity of the pneumococci identified in children under 5 years of age in different regions of the Russian Federation. Clinical microbiology and antimicrobial therapy. 2011; 13(2): 177-187.Mayanskiy А. N., Alyabyeva N. M., Ivanenko A. M., Ponoma renko O. A., Katosova L. K., Lazareva A. V., Kulichenko T. V., NamazovaBaranova L. S. Bakterialnaya etiologiya ostrogo srednego otita u detey do 5 let: rol Streptococcus pneumoniae [Bacterial etiology of acute otitis media in children under 5: The role of Streptococcus pneumoniae]. Voprosy diagnostiki v pediatrii — Pediatric Diagnostics. 2013; 5 (1): 5–13.N.A. Mayanskiy, N.M. Alyabyeva, A.M. Ivanenko, O.A. Ponomarenko, L.K. Katosova, A.V. Lazareva, T.V. Kulichenko, L.S. Namazova-Baranova. Bacterial etiology of acute otitis media in children under 5 years of age: role of Streptococcus pneumoniae. Issues of Pediatric Diagnosis. 2013; 5(1): 5-13.Boronina L. G., Samatova E. V. Epidemicheskie osobennosti Streptococcus pneumoniae, vydelennogo u detey, pri neinvazivnykh pnevmokokkovykh infektsiyakh i nosoglotochnom bakteroinositelstve [Epidemic features of Streptococcus pneumoniae, selected from children with non-invasive pneumococcal infections and nasopharyngeal bacteria carriage]. Voprosy diagnostiki v pediatrii — Pediatric Diagnostics. 2013; 5 (1): 22–26.L.G. Boronina, E.V. Samatova. Epidemic peculiarities of the Streptococci pneumoniae identified in children at non-invasive pneumococcal infections and nasopharyngeal carriage. Issues of Pediatric Diagnosis. 2013; 5(1): 22-26.Miernyk K., Debyle C., Harker-Jones M., Hummel K. B., Hennessy T. et al. Serotyping of Streptococcus pneumoniae isolates from nasopharyngeal samples: use of an algorithm combining microbiologic, serologic, and sequential multiplex PCR techniques. J. Clin. Microbiol. 2011; 49: 3209–3214. doi: 10.1128/ JCM.00610-11.Miernyk K., Debyle C., Harker-Jones M., Hummel K. B., Hennessy T. et al. Serotyping of Streptococcus pneumoniae isolates from nasopharyngeal samples: use of an algorithm combining microbiologic, serologic, and sequential multiplex PCR techniques. J. Clin. Microbiol. 2011; 49: 3209–3214. doi: 10.1128/ JCM.00610-11.Croucher N. J., Harris S. R., Fraser C., Quail M. A., Burton J., van der Linden M., McGee L., von Gottberg A., Song J. H., Ko K. S. et al. Rapid pneumococcal evolution in response to clinical interventions. Science. 2011; 331 (6016): 331–434. doi: 10.1126/science.1198545.Croucher N. J., Harris S. R., Fraser C., Quail M. A., Burton J., van der Linden M., McGee L., von Gottberg A., Song J. H., Ko K. S. et al. Rapid pneumococcal evolution in response to clinical interventions. Science. 2011; 331 (6016): 331–434. doi: 10.1126/science.1198545.Moore M. R., Gertz R. E., Jr, Woodbury R. L., BarkocyGallagher G. A., Schaffner W., Lexau C. et al. Population snapshot of emergent Streptococcus pneumoniae serotype 19A in the United States, 2005. J. Infect. Dis. 2008; 197: 1016–1027. doi: 10.1086/528996.Moore M. R., Gertz R. E., Jr, Woodbury R. L., Barkocy-Gallagher G. A., Schaffner W., Lexau C. et al. Population snapshot of emergent Streptococcus pneumoniae serotype 19A in the United States, 2005. J. Infect. Dis. 2008; 197: 1016–1027. doi: 10.1086/528996.Wyres K. L., Lambertsen L. M., Croucher N. J., McGee L., von Gottberg A., Linares J., Jacobs M. R., Kristinsson K. G., Beall B. W., Klugman K. P., Parkhill J., Hakenbeck R., Bentley S. D., Brueggemann A. B. Pneumococcal capsular switching: a historical perspective. J. Infect. Dis. 2013; 207 (3): 439–449.Wyres K. L., Lambertsen L. M., Croucher N. J., McGee L., von Gottberg A., Linares J., Jacobs M. R., Kristinsson K. G., Beall B. W., Klugman K. P., Parkhill J., Hakenbeck R., Bentley S. D., Brueggemann A. B. Pneumococcal capsular switching: a historical perspective. J. Infect. Dis. 2013; 207 (3): 439–449.Миронов К. О., Платонов А. Е., Козлов Р. С. Идентификация и серотипирование российских штаммов Streptococcus pneumoniae с применением методик, основанных на ПЦР. Клин. микробиол. и антимикробн. тер. 2011; 13 (4): 304–313.K.O. Mironov, A.E. Platonov, R.S. Kozlov. Identification and serotyping of Russian Streptococcus pneumoniae strains using the PCR-based techniques. Clinical microbiology and antimicrobial therapy. 2011; 13(4): 304-313.Pai R., Limor J., Beall B. Use of pyrosequencing to differentiate Streptococcus pneumoniae serotypes 6A and 6B. J. Clin. Microbiol. 2005; 43 (9): 4820–4822.Pai R., Limor J., Beall B. Use of pyrosequencing to differentiate Streptococcus pneumoniae serotypes 6A and 6B. J. Clin. Microbiol. 2005; 43(9): 4820–4822.

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