Pediatric Infection and Vaccine

The Korean Society of Pediatric Infectious Diseases (대한소아감염학회)
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Molecular Diagnosis of Streptococcus pneumoniae in Middle Ear Fluids from Children with Otitis Media with Effusion

삼출성 중이염 소아의 중이액에서 폐구균의 분자적 진단

  • Byun, Sung Wan (Department of Otolaryngology-Head and Neck Surgery, Medical Research Institute, Ewha Womans University School of Medicine) ;
  • Kim, Han Wool (Department of Pediatrics, Medical Research Institute, Ewha Womans University School of Medicine) ;
  • Yoon, Seo Hee (Department of Pediatrics, Medical Research Institute, Ewha Womans University School of Medicine) ;
  • Park, In Ho (Center for Vaccine Evaluation and Study, Medical Research Institute, Ewha Womans University School of Medicine) ;
  • Kim, Kyung-Hyo (Department of Pediatrics, Medical Research Institute, Ewha Womans University School of Medicine)
  • 변성완 (이화여자대학교 의학전문대학원 이비인후과학교실) ;
  • 김한울 (이화여자대학교 의학전문대학원 소아과학교실) ;
  • 윤서희 (이화여자대학교 의학전문대학원 소아과학교실) ;
  • 박인호 (이화여자대학교 의학전문대학원 의과학연구소 백신효능연구센터) ;
  • 김경효 (이화여자대학교 의학전문대학원 소아과학교실)
  • Received : 2015.03.19
  • Accepted : 2015.07.01
  • Published : 2015.08.25
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Abstract

Purpose: The long-term administration of antibiotics interferes with bacterial culture in the middle ear fluids (MEFs) of young children with otitis media with effusion (OME). The purpose of this study is to determine whether molecular diagnostics can be used for rapid and direct detection of the bacterial pathogen in culture-negative MEFs. Methods: The specificity and sensitivity of both polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to the lytA gene of Streptococcus pneumoniae were comparatively tested and then applied for pneumococcal detection in the clinical MEFs. Results: The detection limit of the PCR assay was approximately $10^4$ colony forming units (CFU), whereas that of LAMP was less than 10 CFU for the detection of S. pneumoniae. Both PCR and LAMP did not amplify nucleic acid at over $10^6$ CFU of H. influenzae or M. catarrhalis, both of which were irrelevant bacterial species. Of 22 culture-negative MEFs from children with OME, LAMP positivity was found in twelve MEFs (54.5%, 12/22), only three of which were PCR-positive (25%, 3/12). Our results showed that the ability of LAMP to detect pneumococcal DNA is over four times higher than that of PCR (P<0.01). Conclusions: As a high-resolution tool able to detect nucleic acid levels equivalent to <10 CFU of S. pneumoniae in MEFs without any cross-reaction with other pathogens, lytA -specific LAMP may be applied for diagnosing pneumococcus infection in OME as well as evaluating the impact of a pneumococcal conjugate vaccine against OME.

목적: 장기간의 항생제 치료는 중이염 어린이 환자의 중이액으로부터 원인균이 배양되는 것을 방해한다. 본 연구는 배양 음성 중이액으로부터 분자적 진단에 의한 신속한 균 검출 가능성 여부를 확인하고자 하였다. 방법: 폐구균 lytA 유전자를 표적으로 하는 PCR과 LAMP로 민감도와 특이도를 비교 결정하고, 임상중이액에서의 폐구균 검출에 적용하였다. 결과: PCR 기법에 의한 폐구균 검출 최소한계는 약 $10^4$ 집락형성단위(CFU)이고, LAMP의 검출 최소한계는 10 CFU에서 결정되었다. 한편 두 가지 검사법 모두 Haemophilus influenzae 와 Moraxella catarrhalis 에 대해 $10^6$ CFU 이상에서도 DNA를 증폭하지 않았다. 22개의 배양음성 중이액 중에서 12개 검체가 LAMP-양성(54.5%, 12/22)으로 확인되었고, 이들 12개 LAMP-양성 검체 중, 3개의 검체만이 PCR-양성으로 확인되었다(25%, 3/12). 본 연구의 결과는 LAMP 기법의 폐구균 검출 해상력이 PCR 기법에 비교하여 4배 이상 높음을 보여준다(P<0.01). 결론: lytA -특이 LAMP 기법은, 중이액 내의 타 병원균과는 교차반응 없이 10 CFU 폐구균의 DNA를 검출할 수 있는 고해상 기술로서, 중이액 폐구균 검출 및 폐구균 백신의 보급에 따른 백신 효과 평가에 적용이 기대된다.

Keywords

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