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"10.29235/1814-6023-2019-16-4-454-467vestim-592Research ArticleКЛИНИЧЕСКАЯ И ЭКСПЕРИМЕНТАЛЬНАЯ МЕДИЦИНАCLINICAL AND EXPERIMENTAL MEDICINEСвязь механизмов генотипической резистентности Streptococcus pneumoniae к антибиотикам с фенотипической резистентностью и серотипамиAccociation between molecular mechanisms of antimicrobial resistance, pnenotypes and serotypes in Streptococcus pneumoniaeДавыдовА. В.DavydovA. V.

Давыдов Александр Владимирович – ассистент. 

пр. Дзержинского, 83, 220116, г. Минск

Alexander V. Davydov – Assistant. 

83, Dzerzhinski Ave., 220116, Minsk

alexander.davydovv@gmail.comТитовЛ. П.TitovL. P.

Титов Леонид Петрович – член-корреспондент, доктор медицинских наук, профессор, заведующий лабораторией. 

ул. Филимонова, 23, 220114, г. Минск

Leonid P. Titov – Corresponding Member, D. Sc. (Med.), Professor, Head of the Laboratory. 

23, Filimonov Str., 220114, Minsk

leonidtitov@tut.byХархальА. Н.KharkhalA. N.

Хархаль Анна Николаевна – младший научный сотрудник. 

ул. Филимонова, 23, 220114, г. Минск

Anna N. Kharkhal – Junior researcher. 

23, Filimonov Str., 220114, Minsk

anna-madlen69@yandex.byБарауляВ. Г.BaraulyaV. G.

Барауля Валентина Геннадьевна – заведующий отделением.

ул. П. Бровки, 13/1, 220013, г. Минск

Valentina G. Baraulya – Head of the Department.

13/1, Brovka Str., 220013, Minsk

aleftina90@mail.ruГусаковаЮ. В.GusakovaY. V.

Гусакова Юлия Владимировна – врач-бактериолог. 

ул. П. Бровки, 13/1, 220013, г. Минск

Yuliya V. Gusakova – Bacteriologist.

13/1, Brovka Str., 220013, Minsk

Белорусский государственный медицинский университетBelarusian State Medical UniversityРеспубликанский научно-практический центр эпидемиологии и микробиологииRepublican Scientific and Practical Center for Epidemiology and MicrobiologyМинский городской центр гигиены и эпидемиологииMinsk City Center for Hygiene and Epidemiology201905122019164454467Copyright © Давыдов А.В., Титов Л.П., Хархаль А.Н., Барауля В.Г., Гусакова Ю.В., 20192019Давыдов А.В., Титов Л.П., Хархаль А.Н., Барауля В.Г., Гусакова Ю.В.Davydov A.V., Titov L.P., Kharkhal A.N., Baraulya V.G., Gusakova Y.V.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/592

Изучение резистома пневмококка и получение данных о молекулярно-генетических механизмах резистентности и их распространении имеют важное значение при проведении сравнительных исследований и осуществлении эпидемиологического надзора за резистентностью.

Цель исследования – изучение генотипической резистентности к антибиотикам штаммов пневмококка, выделенных от пациентов с различными формами пневмококковой инфекции и бактерионосителей, а также ее взаимосвязи с фенотипической резистентностью к антибиотикам и клинико-эпидемиологическими характеристиками штаммов (серотип, форма вызываемой инфекции).

Материалом исследования являлись 546 штаммов пневмококка и 5 образцов биологического материала, выделенных/полученных от пациентов разного возраста (5 дней – 81 год) с различными формами пневмококковой инфекции (менингит и другие инвазивные формы – 28 пациентов; пневмония – 27; острый синусит – 18; острый средний отит – 118; конъюнктивит – 26) и бактерионосителей (331 пациент).

Генотипическая резистентность пневмококка к антибиотикам исследовалась посредством мультиплексной ПЦР с детекцией генов mefA, ermB и мутаций в генах пенициллин-связывающих белков: pbp1a (574T→N, 575S→T, 576Q→ G и 577F→Y); pbp2b (431T→K, 432Q→L) и pbp2x (338T→A).

В ходе исследования 551 изолята S. pneumoniae установлено, что у 60 % из них имеется как минимум один механизм резистентности к макролидам/линкозамидам, а распространенность гетерорезистентности (mefA + ermB) составила 22 %. Среди исследованных штаммов как минимум одну модификацию pbp имели 65 %, две модификации – 26, все три исследованные модификации pbp – 24 %. Доминирующим механизмом резистентности к макролидам является метилирование 23S РНК (ген ermB), который был обнаружен у 43 % генетически резистентных изолятов. Среди генетически резистентных к пенициллину изолятов пневмококка чаще всего встречались комбинации модификаций генов pbp 1a + 2x + 2b или pbp 1a + 2x (по 39–40 %) и отсутствовал генотип резистентности, обусловленный модификацией только pbp2b.

Studies on pneumococcal resistome and molecular antimicrobial resistance mechanisms are relevant and may be used in large-scale epidemiological researches and surveillance of antimicrobial resistance.

A study of antimicrobial molecular resistance in the pneumococcal strains, that were isolated from the patients having the different forms of the pneumococcal infection or carriage, and association of it with phenotypes, clinical and epidemiological features of the strains (serotype, form of the infection).

We studied 546 pneumococcal strains and 5 specimens, that were isolated/obtained from the patients of various age (5 days – 81 years) having the different forms of the pneumococcal infection (meningitis and other invasive forms – 28, pneumonia – 27, acute rhinosinusitis – 18, acute otitis media – 118, conjunctivitis – 26) or carriage (331).

We used multiplex PCR to detect the following molecular pneumococcal antimicrobial resistance determinants – genes mefA, ermB and mutations in the penicillin-binding proteins: pbp1a (574T→N, 575S→T, 576Q→G and 577F→Y); pbp2b (431T→K, 432Q→L) and pbp2x (338T→A).

Among studied strains 60 % of 551 possess at least one resistance mechanism to macrolides/lincosamides, while 22 % were heteroresistant (mefA + ermB). About 65 % of the strains carry at least one pbp modification, 26 % – two modifications and 24 % – three pbp modifications. 23S RNA methylase (ermB gene) were discovered as a dominating mechanism and was detected in 43 % of genetically resistant strains. Pbp 1a + 2x + 2b and pbp 1a + 2x were more frequent modifications among penicillin genetically resistant pneumococci, while pbp2b genotype was not detected.

пневмококкпневмококковая инфекцияStreptococcus pneumoniaeмолекулярно-генетические механизмы резистентности к антибиотикамmefAermBpbppneumococcuspneumococcal infectionStreptococcus pneumoniaemolecular resistance mechanisms to antimicrobialsmefAermBpbpReferencesБелошицкий, Г. В. Пневмококковые менингиты в Российской Федерации / Г. В. Белошицкий, И. С. Королева, Н. И. Кошкина // Эпидемиология и вакцинопрофилактика. – 2009. – № 2. – С. 21–26.Beloshitskii G. V., Koroleva I. S., Koshkina N. I. Pneumococcal meningitis in Russian Federation. Epidemiologiya i vaktsinoprofilaktika [Epidemiology and vaccine prevention], 2009, no. 2, pp. 21–26 (in Russian).Biological and epidemiological features of antibiotic-resistant Streptococcus pneumoniae in preand post-conjugate vaccine eras: a United States perspective / L. Kim [et al.] // Clin. Microbiol. Rev. – 2016. – Vol. 29, N 3. – P. 525–552. https://doi.org/10.1128/cmr.00058-15Kim L., McGee L., Tomczyk S., Beall B. Biological and epidemiological features of antibiotic-resistant Streptococcus pneumoniae in preand post-conjugate vaccine eras: a United States perspective. Clinical Microbiology Reviews, 2016, vol. 29, no. 3, pp. 525–552. https://doi.org/10.1128/cmr.00058-15Molecular mechanisms and epidemiology of resistance in Streptococcus pneumoniae in the Middle East region / G. El Moujaber [et al.] // J. Med. Microbiol. – 2017. – Vol. 66, N 7. – P. 847–858. https://doi.org/10.1099/jmm.0.000503El Moujaber G., Osman M., Rafei R., Dabboussi F., Hamze M. Molecular mechanisms and epidemiology of resistance in Streptococcus pneumoniae in the Middle East region. Journal of Medical Microbiology, 2017, vol. 66, no. 7, pp. 847–858. https://doi.org/10.1099/jmm.0.000503Molecular epidemiology of macrolide-resistant Streptococcus pneumoniae isolates in Europe / R. R. Reinert [et al.] // J. Clin. Microbiol. – 2005. – Vol. 43, N 3. – P. 1294–1300. https://doi.org/10.1128/jcm.43.3.1294-1300.2005Reinert R. R., Ringelstein A., van der Linden M., Cil M. Y., Al-Lahham A., Schmitz F.-J. Molecular epidemiology of macrolide-resistant Streptococcus pneumoniae isolates in Europe. Journal of Clinical Microbiology, 2005, vol. 43, no. 3, pp. 1294–1300. https://doi.org/10.1128/jcm.43.3.1294-1300.2005Central Asian and Eastern European surveillance of antimicrobial resistance. Annual report 2016 / World Health Organization. – Copenhagen : WHO, 2016. – 133 p.Central Asian and Eastern European surveillance of antimicrobial resistance. Annual report 2016. Copenhagen, World Health Organization, 2016. 133 p.Substitutions in PBP2b from β-lactam-resistant Streptococcus pneumoniae have different effects on enzymatic activity and drug reactivity / P. Calvez [et al.] // J. Biol. Chem. – 2017. – Vol. 292, N 7. – P. 2854–2865. https://doi.org/10.1074/jbc.m116.764696Calvez P., Breukink E., Roper D. I., Dib M., Contreras-Martel C., Zapun A. Substitutions in PBP2b from β-lactamresistant Streptococcus pneumoniae have different effects on enzymatic activity and drug reactivity. Journal of Biological Chemistry, 2017, vol. 292, no. 7, pp. 2854–2865. https://doi.org/10.1074/jbc.m116.764696Reinert, R. R. The antimicrobial resistance profile of Streptococcus pneumoniae / R. R. Reinert // Clin. Microbiol. Infect. – 2009. – Vol. 15, suppl. 3. – P. 7–11. https://doi.org/10.1111/j.1469-0691.2009.02724.xReinert R. R. The antimicrobial resistance profile of Streptococcus pneumoniae. Clinical Microbiology and Infection, 2009, vol. 15, suppl. 3, pp. 7–11. https://doi.org/10.1111/j.1469-0691.2009.02724.xВнебольничная пневмония у взрослых: практические рекомендации по диагностике, лечению и профилактике / А. Г. Чучалин [и др.] // Инфекц. болезни: новости, мнения, обучение. – 2013. – № 2 (3). – С. 91–123.Chuchalin A. G., Sinopal’nikov A. I., Kozlov R. S., Tyurin I. E., Rachina S. A. Community-acquired pneumonia in adults: practical recommendations on diagnosis, therapy and prophylaxis. Infektsionnye Infektsionnye bolezni: novosti, mneniya, obuchenie [Infectious diseases: news, opinions, training], 2013, no. 2 (3), pp. 91–123 (in Russian).Antimicrobial susceptibility and analysis of macrolide resistance genes in Streptococcus pneumoniae isolated in Hamadan / M. N. Mosleh [et al.] // Iran. J. Basic Med. Sci. – 2014. – Vol. 17, N 8. – P. 595–599.Mosleh M. N., Gharibi M., Alikhani M. Y., Saidijam M., Vakhshiteh F. Antimicrobial susceptibility and analysis of macrolide resistance genes in Streptococcus pneumoniae isolated in Hamadan. Iranian Journal of Basic Medical Sciences, 2014, vol. 17, no. 8, pp. 595–599.Quadriplex real-time polymerase chain reaction (lytA, mef, erm, pbp2bwt) for pneumococcal detection and assessment of antibiotic susceptibility / V. Srinivasan [et al.] // Diagn. Microbiol. Infect. Dis. – 2011. – Vol. 71, N 4. – P. 453–456. https://doi.org/10.1016/j.diagmicrobio.2011.08.017Srinivasan V., du Plessis M., Beall B. W., McGee L. Quadriplex real-time polymerase chain reaction (lytA, mef, erm, pbp2bwt) for pneumococcal detection and assessment of antibiotic susceptibility. Diagnostic Microbiology and Infectious Disease, 2011, vol. 71, no. 4, pp. 453–456. https://doi.org/10.1016/j.diagmicrobio.2011.08.017Rapid identification of penicillin and macrolide resistance genes and simultaneous quantification of Streptococcus pneumoniae in purulent sputum samples by use of a novel real-time multiplex PCR assay / K. Y. Fukushima [et al.] // J. Clin. Microbiol. – 2008. – Vol. 46, N 7. – P. 2384–2388. https://doi.org/10.1128/jcm.00051-08Fukushima K. Y., Yanagihara K., Hirakata Y., Sugahara K., Morinaga Y., Kohno S., Kamihira S. Rapid identification of penicillin and macrolide resistance genes and simultaneous quantification of Streptococcus pneumoniae in purulent sputum samples by use of a novel real-time multiplex PCR assay. Journal of Clinical Microbiology, 2008, vol. 46, no. 7, pp. 2384– 2388. https://doi.org/10.1128/jcm.00051-08World Health Organization. Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae : WHO manual / World Health Organization, Centers for Disease Control and Prevention. – Pub. 2. – WHO/IVB.11.09, 2011. – 311 p.World Health Organization. Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae : WHO manual. World Health Organization, Centers for Disease Control and Prevention. Pub. 2. WHO/IVB.11.09, 2011. 311 p.Серотиповая характеристика штаммов Streptococcus pneumoniae, выделенных у детей с острыми средними отитами и синуситами / А. В. Давыдов [и др.] // Здравоохранение. – 2016. – № 3. – С. 12–20.Davydov A. V., Titov L. P., Klyuiko N. L., Gurinovich V. V. Serotype characteristic of Streptococcus pneumoniae strains isolated from children with acute otitis media and sinusitis. Zdravookhranenie [Healthcare], 2016, no. 3, pp. 12–20 (in Russian).Давыдов, А. В. Использование мультиплексной ПЦР для серогенотипирования штаммов Streptococcus pneumoniae в рамках осуществления мониторинга пневмококковой инфекции в Беларуси / А. В. Давыдов, Л. П. Титов, А. Н. Хархаль // Молекулярная диагностика 2017 : сб. тр. IX Всерос. науч.-практ. конф. с междунар. участием (Москва, 18–20 апр. 2017 г.) : в 2 т. / Федер. служба по надзору в сфере защиты прав потребителей и благополучия человека [и др.] ; под ред. В. И. Покровского. – М., 2017. – Т. 2. – С. 336–338.Davydov A. V., Titov L. P., Kharkhal’ A. N. The usage of multiplex PCR for serogenotyping the strains of Streptococcus pneumoniae as a part of the monitoring for pneumococcal infection in Belarus. Molekulyarnaya diagnostika 2017: sbornik trudov IX Vserossiiskoi nauchno-prakticheskoi konferentsii s mezhdunarodnym uchastiem (Moskva, 18–20 aprelya 2017 goda). Tom 2 [Molecular diagnostics 2017: proceedings of the IX All-Russian scientific and practical conference with international participation (Moscow, April 18–20, 2017). Vol. 2]. Moscow, 2017, pp. 336–338 (in Russian).Antimicrobial wild type distributions of microorganisms [Electronic resource] // EUCAST. – Mode of access : https://mic.eucast.org/Eucast2/SearchController/search.jsp?action=performSearch&BeginIndex=0&Micdif=mic&NumberIndex=50&Antib=-1&Specium=12. – Date of access : 02.12.2017.Antimicrobial wild type distributions of microorganisms. EUCAST. Available at: https://mic.eucast.org/Eucast2/SearchController/search.jsp?action=performSearch&BeginIndex=0&Micdif=mic&NumberIndex=50&Antib=1&Specium=12 (accessed 02.12.2017).Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standard. – 10th ed. CLSI document M07-A10. – Wayne, PA : Clinical and Laboratory Standards Institute, 2015. – 94 p.Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standard. 10th ed. CLSI document M07-A10. Wayne, PA, Clinical and Laboratory Standards Institute, 2015. 94 p.CLSI. Performance Standards for Antimicrobial Susceptibility Testing. CLSI supplement M100 / CLSI. – Pub. 27th. – Wayne, PA : Clinical and Laboratory Standards Institute, 2017. – 224 p.CLSI. Performance Standards for Antimicrobial Susceptibility Testing. CLSI supplement M100. CLSI. Pub. 27th. Wayne, PA, Clinical and Laboratory Standards Institute, 2017. 224 p.Predominance of 23S rRNA mutants among non-erm, non-mef macrolide-resistant clinical isolates of Streptococcus pneumoniae collected in the United States in 1999–2000 / T. A. Davies [et al.] // Antimicrob. Agents Chemother. – 2005. – Vol. 49, N 7. – P. 3031–3033. https://doi.org/10.1128/aac.49.7.3031-3033.2005Davies T. A., Bush K., Sahm D., Evangelista A. Predominance of 23S rRNA mutants among non-erm, non-mef macrolide-resistant clinical isolates of Streptococcus pneumoniae collected in the United States in 1999–2000. Antimicrobial Agents and Chemotherapy, 2005, vol. 49, no. 7, pp. 3031–3033. https://doi.org/10.1128/aac.49.7.3031-3033.2005Susceptibilities to telithromycin and six other agents and prevalence of macrolide resistance due to L4 ribosomal protein mutation among 992 Pneumococci from 10 central and Eastern European countries / K. Nagai [et al.] // Antimicrob. Agents Chemother. – 2002. – Vol. 46, N 2. – P. 371–377. https://doi.org/10.1128/aac.46.2.371-377.2002Nagai K., Appelbaum P. C., Davies T. A., Kelly L. M., Hoellman D. B., Andrasevic A. T. [et al.]. Susceptibilities to telithromycin and six other agents and prevalence of macrolide resistance due to L4 ribosomal protein mutation among 992 Pneumococci from 10 central and Eastern European countries. Antimicrobial Agents and Chemotherapy, 2002, vol. 46, no. 2, pp. 371–377. https://doi.org/10.1128/aac.46.2.371-377.2002Kargar, M. M. Multi-drug resistance and molecular pattern of erythromycin and penicillin resistance genes in Streptococcus pneumoniae / M. Kargar, M. Baghernejad, D. S. Ghorbani // Afr. J. Biotechnol. – 2012. – Vol. 11, N 4. – P. 968–973. https://doi.org/10.5897/ajb11.2783Kargar M., Baghernejad M., Ghorbani D. S. Multi-drug resistance and molecular pattern of erythromycin and penicillin resistance genes in Streptococcus pneumoniae. African Journal of Biotechnology, 2012, vol. 11, no. 4, pp. 968–973. https://doi.org/10.5897/ajb11.2783New aspects of the interplay between penicillin binding proteins, murm, and the two-component system CiaRH of penicillin-resistant Streptococcus pneumoniae serotype 19A isolates from Hungary / I. Schweizer [et al.] // Antimicrob. Agents Chemother. – 2017. – Vol. 61, N 7. – P. e00414–e00417. https://doi.org/10.1128/aac.00414-17Schweizer I., Blättner S., Maurer P., Peters K., Vollmer D., Vollmer W., Hakenbeck R., Denapaite D. New aspects of the interplay between penicillin binding proteins, murM, and the two-component system CiaRH of penicillin-resistant Streptococcus pneumoniae serotype 19A isolates from Hungary. Antimicrobial Agents and Chemotherapy, 2017, vol. 61, no. 7, pp. e00414–e00417. https://doi.org/10.1128/aac.00414-17Central Asian and Eastern European surveillance of antimicrobial resistance. Annual report 2017 / World Health Organization. – Copenhagen : WHO, 2018. – 135 p.Central Asian and Eastern European surveillance of antimicrobial resistance. Annual report 2017. Copenhagen, World Health Organization, 2018. 135 p.Serotypes and antibiotic resistance of non-invasive Streptococcus pneumoniae circulating in pediatric hospitals in Moscow, Russia / N. Mayanskiy [et al.] // Int. J. Infect. Dis. – 2014. – Vol. 20. – P. 58–62. https://doi.org/10.1016/j.ijid.2013.11.005Mayanskiy N., Alyabieva N., Ponomarenko O., Lazareva A., Katosova L., Ivanenko A., Kulichenko T., NamazovaBaranova L., Baranov A. Serotypes and antibiotic resistance of non-invasive Streptococcus pneumoniae circulating in pediatric hospitals in Moscow, Russia. International Journal of Infectious Diseases, 2014, vol. 20, pp. 58–62. https://doi.org/10.1016/j.ijid.2013.11.005Чувствительность к антибиотикам штаммов Streptococcus pneumoniae, выделенных от пациентов с менингитом в Беларуси / А. В. Давыдов [и др.] // Здравоохранение. – 2018. – № 1. – С. 22–32.Davydov A. V., Titov L. P., Kharkhal’ A. N., Klyuiko N. L., Baraulya V. G., Rogacheva T. A. Antibiotic susceptibility of Streptococcus pneumoniae strains isolated from patients with meningitis in Belarus. Zdravookhranenie [Healthcare], 2018, no. 1, pp. 22–32 (in Russian).Чувствительность к антибиотикам штаммов Streptococcus pneumoniae, выделенных от пациентов с пневмонией в Беларуси / А. В. Давыдов [и др.] // Мед. новости. – 2017. – № 12. – С. 74–82.Davydov A. V., Titov L. P., Klyuiko N. L., Levshina N. N., Kharkhal’ A. N., Madzharova O. A., Belanovskaya L. I. Antibiotic susceptibility of Streptococcus pneumoniae strains isolated from patients with pneumonia in Belarus]. Meditsinskie novosti [Medical news], 2017, no. 12, pp. 74–82 (in Russian).Чувствительность к антибиотикам и связь с серотипами штаммов Streptococcus pneumoniae у детей с острым средним отитом и острым синуситом в Беларуси / А. В. Давыдов [и др.] // Клин. микробиол. и антимикроб. химиотер. – 2018. – Т. 20, № 3. – С. 206–215.Davydov A. V. Titov L. P., Klyuiko N. L., Gurinovich V. V., Lazarev A. V. Antimicrobial susceptibility and association with serotypes of Streptococcus pneumoniae isolates in children with acute otitis media and acute sinusitis in Belarus. Klinicheskaya mikrobiologiya i antimikrobnaya khimioterapiya [Clinical microbiology and antimicrobial chemotherapy], 2018, vol. 20, no. 3 (in Russian).Phenotype, genotype, and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae strains, in Sousse, Tunisia / M. Marzouk [et al.] // Med. Mal. Infect. – 2014. – Vol. 44, N 10. – P. 478–482. https://doi.org/10.1016/j.medmal.2014.07.016Marzouk M., Ferjani A., Amamou S., Alibi S., Haj Ali M., Boukadida J. Phenotype, genotype, and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae strains, in Sousse, Tunisia. Médecine et Maladies Infectieuses, 2014, vol. 44, no. 10, pp. 478–482. https://doi.org/10.1016/j.medmal.2014.07.016Molecular epidemiology of penicillin-non-susceptible Streptococcus pneumoniae isolates from children with invasive pneumococcal disease in Germany / R. R. Reinert [et al.] // Clin. Microbiol. Infect. – 2007. – Vol. 13, N 4. – P. 363–368. https://doi.org/10.1111/j.1469-0691.2006.01676.xReinert R. R., van der Linden M., Seegmüller I., Al-Lahham A., Siedler A., Weißmann B., Toschke A. M., von Kries R. Molecular epidemiology of penicillin-non-susceptible Streptococcus pneumoniae isolates from children with invasive pneumococcal disease in Germany. Clinical Microbiology and Infection, 2007, vol. 13, no. 4, pp. 363–368. https://doi.org/10.1111/j.1469-0691.2006.01676.x

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