Pediatric Infection and Vaccine

The Korean Society of Pediatric Infectious Diseases (대한소아감염학회)
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Real-time Reverse Transcription Polymerase Chain Reaction Using Total RNA Extracted from Nasopharyngeal Aspirates for Detection of Pneumococcal Carriage in Children

소아에서 폐렴구균 집락률 측정을 위해 비인두 흡인 물의 총 RNA를 이용한 실시간 중합효소 연쇄반응법

  • Kim, Young Kwang (Department of Pediatrics, Chung-Ang University Hospital, Chung-Ang University College of Medicine) ;
  • Lee, Kyoung Hoon (Department of Pediatrics, Chung-Ang University Hospital, Chung-Ang University College of Medicine) ;
  • Yun, Ki Wook (Department of Pediatrics, Seoul National University Children's Hospital, Seoul National University College of Medicine) ;
  • Lee, Mi Kyung (Department of Laboratory Medicine, Chung-Ang University Hospital, Chung-Ang University College of Medicine) ;
  • Lim, In Seok (Department of Pediatrics, Chung-Ang University Hospital, Chung-Ang University College of Medicine)
  • 김영광 (중앙대학교병원 소아청소년과) ;
  • 이경훈 (중앙대학교병원 소아청소년과) ;
  • 윤기욱 (서울대학교 어린이병원 소아청소년과) ;
  • 이미경 (중앙대학교병원 진단검사의학과) ;
  • 임인석 (중앙대학교병원 소아청소년과)
  • Received : 2016.06.11
  • Accepted : 2016.09.21
  • Published : 2016.12.25
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Abstract

Purpose: Monitoring pneumococcal carriage rates is important. We developed and evaluated the accuracy of a real-time reverse transcription polymerase chain reaction (RT-PCR) protocol for the detection of Streptococcus pneumoniae. Methods: In October 2014, 157 nasopharyngeal aspirates were collected from patients aged <18 years admitted to Chung-Ang University Hospital. We developed and evaluated a real-time PCR method for detecting S. pneumoniae by comparing culture findings with the results of the real-time PCR using genomic DNA (gDNA). Of 157 samples, 20 specimens were analyzed in order to compare the results of cultures, real-time PCR, and real-time RT-PCR. Results: The concordance rate between culture findings and the results of real-time PCR was 0.922 (P<0.01, Fisher exact test). The 133 culture-negative samples were confirmed to be negative for S. pneumoniae using real-time PCR. Of the remaining 24 culture-positive samples, 21 were identified as S. pneumonia -positive using real-time PCR. The results of real-time RT-PCR and real-time PCR from 20 specimens were consistent with culture findings for all S. pneumoniae -positive samples except one. Culture and real-time RT-PCR required 26.5 and 4.5 hours to perform, respectively. Conclusions: This study established a real-time RT-PCR method for the detection of pneumococcal carriage in the nasopharynx. Real-time RT-PCR is an accurate, convenient, and time-saving method; therefore, it may be useful for collecting epidemiologic data regarding pneumococcal carriage in children.

목적: 폐렴구균은 주요 비인두 상재균으로, 주위 조직을 침범하여 침습성 감염을 일으킬 수 있어 보균율에 대한 감시가 중요하다. 본 연구에서는 임상에서 비인두 흡인물로부터 추출하고 남은 RNA를 이용하여 폐렴구균을 확인할 수 있는 실시간 중합효소 연쇄반응(real-time reverse transcription polymerase chain reaction [RT-PCR])법을 구축하고, 보균율 측정에 있어서의 정확성과 이점을 확인하고자 하였다. 방법: 2014년 9월부터 10월까지 중앙대학교병원에 입원하여 호흡기 바이러스 RT-PCR 검사를 시행받은 18세 이하의 소아들로부터 비인두 흡인물을 채취하였다. 먼저 배양법과 genomic DNA (gDNA)를 이용한 real-time PCR을 시행하여 폐렴구균 검출률의 정확성을 확인하였다. 이 중 처음 20개의 검체를 이용하여, 고전적인 배양법과 gDNA를 이용한 real-time PCR, 그리고 RNA를 이용한 real-time RT-PCR법을 시행하고 이를 비교 분석하였다. 결과: 총 157개의 검체에서 시행한 real-time PCR 검사는 기존의 배양검사와 일치율이 0.922 (P<0.01, Fisher exact test)로 매우 높았다. 배양검사에서 음성인 133개의 검체는 real-time PCR에서도 모두 음성을 보였다. 24개의 배양 양성 검체 중 21개의 검체는 real-time PCR에서도 양성이었지만, 나머지 검체는 음성 결과를 보였다. 20개의 검체에서 시행한 real-time RT-PCR 검사는 1개 검체를 제외하고 배양법 및 real-time PCR과 결과가 일치하였다. 한편, 배양법을 시행하고 결과를 확인하기까지는 총 26.5시간, real-time RT-PCR 검사에는 총 4.5시간이 소요되었다. 결론: 본 연구는 비인두 집락균 확인을 위한 real-time RT-PCR법의 확립과, 폐렴구균 보균율 측정에 있어서의 real-time RT-PCR 검사의 정확성 및 편의성을 보여주었다. Real-time RT-PCR 검사법은 주요 세균들의 보균율 연구에 있어서 시간과 노력을 줄일 수 있는 좋은 방법이며, 폐렴구균의 역학자료 수집에 큰 도움이 될 것으로 기대한다.

Keywords

References

  1. Dahlblom V, Soderstrom M. Bacterial interactions in the nasopharynx: effects of host factors in children attending daycare centers. J Infect Public Health 2012;5:133-9. https://doi.org/10.1016/j.jiph.2011.11.007
  2. Gray BM, Converse GM 3rd, Dillon HC Jr. Epidemiologic studies of Streptococcus pneumoniae in infants: acquisition, carriage, and infection during the first 24 months of life. J Infect Dis 1980;142:923-33. https://doi.org/10.1093/infdis/142.6.923
  3. Syrjanen RK, Kilpi TM, Kaijalainen TH, Herva EE, Takala AK. Nasopharyngeal carriage of Streptococcus pneumoniae in Finnish children younger than 2 years old. J Infect Dis 2001;184:451-9. https://doi.org/10.1086/322048
  4. Levy PY, Ollivier M, Drancourt M, Raoult D, Argenson JN. Relation between nasal carriage of Staphylococcus aureus and surgical site infection in orthopedic surgery: the role of nasal contamination. A systematic literature review and meta-analysis. Orthop Traumatol Surg Res 2013;99:645-51. https://doi.org/10.1016/j.otsr.2013.03.030
  5. Kim SM, Hur JK, Lee KY, Shin YK, Park SE, Ma SH, et al. Epidemiological study of pneumococcal nasal carriage and serotypes among Korean children. Korean J Pediatr 2004;47:611-6.
  6. Paule SM, Pasquariello AC, Hacek DM, Fisher AG, Thomson RB Jr, Kaul KL, et al. Direct detection of Staphylococcus aureus from adult and neonate nasal swab specimens using real-time polymerase chain reaction. J Mol Diagn 2004;6:191-6. https://doi.org/10.1016/S1525-1578(10)60509-0
  7. Chan YR, Morris A. Molecular diagnostic methods in pneumonia. Curr Opin Infect Dis 2007;20:157-64. https://doi.org/10.1097/QCO.0b013e32808255f1
  8. Wood JB, Peters TR. Streptococcus pneumoniae (Pneumococcus). In: Kliegman R, Behrman RE, Nelson WE, editors. Nelson textbook of pediatrics. 20th ed. Philadelphia: Elsevier Saunders, 2016:1322-7.
  9. Feigin RD, Cherry JD. Pneumococcal infections. In: Feigin RD, Cherry JD, Demmler-Harrison GJ, Kaplan SL, Steinbach WJ, Hotez PJ, editors. Feigin & Cherry's textbook of pediatric infectious diseases. 7th ed. Philadelphia: Saunders, 2013:1198-246.
  10. Shin JH, Han HY, Kim SY. Detection of nasopharyngeal carriages in children by multiplex reverse transcriptasepolymerase chain reaction. Korean J Pediatr 2009;52:1358-63. https://doi.org/10.3345/kjp.2009.52.12.1358
  11. Jung CL, Lee MA, Chung WS. Clinical evaluation of the multiplex PCR assay for the detection of bacterial pathogens in respiratory specimens from patients with pneumonia. Korean J Clin Microbiol 2010;13:40-6. https://doi.org/10.5145/KJCM.2010.13.1.40
  12. Al-Marzooq F, Imad MA, How SH, Kuan YC. Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia. Trop Biomed 2011;28:545-56.
  13. Caliendo AM. Multiplex PCR and emerging technologies for the detection of respiratory pathogens. Clin Infect Dis 2011;52 Suppl 4:S326-30. https://doi.org/10.1093/cid/cir047
  14. Brugger SD, Hathaway LJ, Muhlemann K. Detection of Streptococcus pneumoniae strain cocolonization in the nasopharynx. J Clin Microbiol 2009;47:1750-6. https://doi.org/10.1128/JCM.01877-08
  15. Carvalho Mda G, Tondella ML, McCaustland K, Weidlich L, McGee L, Mayer LW, et al. Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA. J Clin Microbiol 2007;45:2460-6. https://doi.org/10.1128/JCM.02498-06
  16. Simoes AS, Tavares DA, Rolo D, Ardanuy C, Goossens H, Henriques-Normark B, et al. lytA-based identification methods can misidentify Streptococcus pneumoniae. Diagn Microbiol Infect Dis 2016;85:141-8. https://doi.org/10.1016/j.diagmicrobio.2016.03.018
  17. Park HK, Lee SJ, Yoon JW, Shin JW, Shin HS, Kook JK, et al. Identification of the cpsA gene as a specific marker for the discrimination of Streptococcus pneumoniae from viridans group streptococci. J Med Microbiol 2010;59(Pt 10):1146-52. https://doi.org/10.1099/jmm.0.017798-0
  18. Keith ER, Podmore RG, Anderson TP, Murdoch DR. Characteristics of Streptococcus pseudopneumoniae isolated from purulent sputum samples. J Clin Microbiol 2006;44:923-7. https://doi.org/10.1128/JCM.44.3.923-927.2006
  19. Kurola P, Erkkila L, Kaijalainen T, Palmu AA, Hausdorff WP, Poolman J, et al. Presence of capsular locus genes in immunochemically identified encapsulated and unencapsulated Streptococcus pneumoniae sputum isolates obtained from elderly patients with acute lower respiratory tract infection. J Med Microbiol 2010;59(Pt 10):1140-5. https://doi.org/10.1099/jmm.0.016956-0
  20. Stensballe LG, Trautner S, Kofoed PE, Nante E, Hedegaard K, Jensen IP, et al. Comparison of nasopharyngeal aspirate and nasal swab specimens for detection of respiratory syncytial virus in different settings in a developing country. Trop Med Int Health 2002;7:317-21. https://doi.org/10.1046/j.1365-3156.2002.00867.x
  21. Ohrmalm L, Wong M, Rotzen-Ostlund M, Norbeck O, Broliden K, Tolfvenstam T. Flocked nasal swab versus nasopharyngeal aspirate for detection of respiratory tract viruses in immunocompromised adults: a matched comparative study. BMC Infect Dis 2010;10:340. https://doi.org/10.1186/1471-2334-10-340
  22. Satzke C, Turner P, Virolainen-Julkunen A, Adrian PV, Antonio M, Hare KM, et al. Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae: updated recommendations from the World Health Organization Pneumococcal Carriage Working Group. Vaccine 2013;32:165-79. https://doi.org/10.1016/j.vaccine.2013.08.062