Evaluation of real-time PCR assay for diagnosis of Tuberculosis = 결핵 진단을 위한 real-time PCR kit의 성능평가

국문 초록 (Abstract)

결핵 (TB)은 결핵균 (Mycobacterium tuberculosis, MTB)에 의해 주로 발생하고, 결핵균은 4개의 다른 마이코박테리아 종과 함께 결핵균 복합체 (Mycobacterium tuberculosis complex, MTBC)에 속한 병원성 박테리아 종입니다. MTBC에 포함되지 않은 마이코박테리아는 비결핵항산균 (nontuberculous mycobacteria, NTM)으로 알려져 있으며 폐렴을 비롯한 여러 폐 질환을 유발한다. 최근 결핵으로 의심되는 호흡기 환자의 검체에서 NTM의 발생이 증가하여 결핵의 진단 및 예방 접종에 있어 MTBC와 NTM의 정확한 구분과 개별 탐지가 중요하다. 결핵의 진단에 사용되고 있는 여러 가지 방법 중에서 실시간 PCR(Real-time PCR)은 신속성, 정확성 및 취급 용이성 때문에 MTBC 및 NTM 검출에 자주 이용된다. 최근 실시간 PCR을 이용하여 결핵균 및 비결핵항산균을 진단할 수 있는 새로운 키트가 출시되었고, 이 연구에서 우리는 객담, 기관지 세척 및 배양 검체를 대상으로 하여 새로운 키트에 대한 분석적 성능 및 임상적 성능을 평가하였다. 분석 성능을 평가하기 위해 표준 DNA 샘플을 사용하여 검출 한계 (LOD), 반응성 및 반복성 테스트를 수행하였고, 임상적 성능시험을 평가하기 위해 612 개의 샘플을 수집하여 국내 대형병원에서 임상 시험을 실시했다. LOD는 probit analysis (95 % positive)에 의해 MTBC는 0.584 copies / μL 와 NTM은 47.836 copies / μL로 확인되었다. 반응성 시험은 설정한 모든 박테리아 DNA를 검출하였고, 재현성 시험에서 0.36에서 1.59까지의 변동 계수(Coefficient Variation, CV)를 확인함으로써, 반복된 시험에서 일정하고 안정된 결과가 확인되었다. 임상 시험에서 민감도와 특이도는 MTBC와 NTM 각각 98.6 % -100 %와 98.8 % -100 %였다. 이 결과로 인해 결핵의 진단에 있어 평가 대상 키트의 유용함을 입증하였다.

다국어 초록 (Multilingual Abstract)

Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis (MTB), which is a pathogenic mycobacterial species grouped under Mycobacterium tuberculosis complex (MTBC) with four other pathogenic mycobacterial species. The mycobacteria not included in MTBC are known as nontuberculous mycobacteria (NTM), and cause several pulmonary diseases including pneumonia. Currently, NTM occurrences in TB-suspected respiratory specimens have increased, due to which, precise detection of MTBC and NTM is considered critical for the diagnosis and vaccination of TB. Among the various methods available, real-time PCR is frequently adopted for MTBC/NTM detection due to its rapidness, accuracy, and ease of handling. In this study, we evaluated a new real-time PCR kit for analytical and clinical performance on sputum, bronchial washing, and culture specimens. For assessing its analytical performance, limit of detection (LOD), reactivity, and repeatability test were performed using DNA samples. To evaluate clinical performance, 612 samples were collected and clinically tested at a tertiary hospital. LOD was confirmed as 0.584 copies/μL for MTBC and 47.836 copies/μL for NTM by probit analysis (95% positive). For the reactivity test, all intended strains were detected and, in the repeatability test, stable and steady results were confirmed with coefficient of variation ranging from 0.36 to 1.59. For the clinical test, sensitivity and specificity were 98.6%-100% and 98.8%-100% for MTBC and NTM, respectively. The results proved the usefulness of the kit in TB diagnosis. Furthermore, it could be adopted for the assessment of vaccine efficacy.

목차 (Table of Contents)

• Ⅰ. Abstract ················································································································5
• Ⅱ. Introduction ········································································································· 7
• Ⅲ. Materials and Methods ······················································································ 18
• Ⅳ. Results ················································································································ 23
• Ⅴ. Discussion ··········································································································· 32
• Ⅵ. References ········································································································· 36
• Ⅶ. Acknowledgement ····························································································· 44
• Ⅷ. 영문 논문제출서 ··································································································· 45
• Ⅸ. 영문 인준서 ········································································································· 46
• Ⅹ. Abstract in Korean ······························································································ 47