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기관지 검체에서의 마이코박테리아 검출 및 동정을 위한 BacT/ALERT 3D System과 PCR-RFLP 병용

Evaluation of Combined Use of BacT/ALERT 3D Liquid Culture System and PCR-RFLP for Detection and Identification of Mycobacteria from Bronchial Specimens

이장호|이남용|기창석|정혜선
대한임상미생물학회 (Korean Society of Clinical Microbiology)|2009.03|Annals of Clinical Microbiology|제12권 제1호|pp. 37-42

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참고문헌

키워드

CultureMycobacteriaLiquid mediaPCRRFLPBronchial specimen

초록

배경: 본 연구에서는 기관지 검체에서 액체 배지 배양 자동화 장비 BacT/ALERT 3D liquid culture system (bioMérieux, USA)과 중합효소연쇄반응-제한절편길이다형성(PCR-restriction fragment length polymorphism, PCR-RFLP)을 이용하여 마 이코박테리아의 검출 및 동정을 시행하고, 기존의 고체 배지 배양과 비교하였다. 방법: 2006년 4월부터 2007년 9월까지, 총 3,037건의 기관지 검체(기관지 세척액 2,309예, 기관지폐포 세척액 728예)를 대상으로, BacT/ALERT MP 액체 배지(bioMérieux, USA)와 고체 배지(3% Ogawa; Shinyang, Korea)에 동시에 접종하여, 두 방법 간의 검출률과 검출 시간의 차이를 비교하였다. BacT/ALERT MP 액체 배지에서 양성이면, Myco-ID (M&D Inc., Korea)를 이용하여 PCR-RFLP 방법으로 균종을 동정하였다. 결과: 총 3,037개의 검체 중 518검체(17.1%)에서 항산균이 배양되었으며, Mycobacterium tuberculosis (MTB) 215건, nontuberculous mycobacteria (NTM) 303건이었다. 액체 배지에서는 492건(16.2%; 195 MTB, 297 NTM), 고체 배지에서는 416건 (13.7%; 187 MTB, 229 NTM)이 검출되었다(P＜0.001). 액체 배지에서만 검출된 경우는 102건(28 MTB, 74 NTM)이었고, 고체 배지에서만 검출된 경우는 26건(20 MTB, 6 NTM)이었다. 마이코박테리아를 검출하는 데 소요된 평균 시간은 액체 배지가 고체 배지보다 빨랐다(18.1 vs 29.3일; P＜0.001). 액체 배지에 접종 후 균을 동정하기까지 소요된 총 평균 시간은 21.8일이었다. 양성 액체 배지로 직접 PCR-RFLP를 시행한 결과, MTB (39.1%), M. avium (26.3%), M. abscessus (19.0%), M. intracellulare (12.6%) 등이 동정되었다. 결론: 액체배지 배양과 PCR-RFLP의 병용은 검출률을 높이고 검출 시간을 단축시킬 수 있었으며, 검출률을 최대화하기 위해 고체 배지와의 병용이 권고된다.

Abstract

Background: We evaluated BacT/ALERT 3D liquid culture system (bioMérieux, USA) and PCR-restriction fragment length polymorphism (RFLP) for recovery and direct identification of mycobacteria, and the results were compared with a conventional culture system using an egg-based solid medium. Methods: A total of 3,037 bronchial specimens (2,309 bronchial washing fluids and 728 bronchoalveolar lavages) were collected. Decontaminated specimens were inoculated to both BacT/ALERT MP liquid media and Ogawa solid media (3%, Shinyang, Korea). Recovery rate and detection time were compared between the two systems. Liquid media from positive cultures were centrifuged and the pellets were tested for direct identification of mycobacteria by PCR-RFLP using Myco-ID (M&D Inc., Korea). Results: A total of 518 isolates, including 215 M. tuberculosis (MTB) and 303 non-tuberculosis mycobacteria (NTM), were recovered. The liquid media detected 492 isolates (16.2%), including 195 MTB and 297 NTM), whereas the solid media detected 416 isolates (13.7%), including 187 MTB and 229 NTM (P＜0.001); 102 isolates (28 MTB and 74 NTM) were recovered only by the liquid media, while 26 (20 MTB and 6 NTM) isolates were recovered only by the solid media. The mean time to detection was 18.1 days by the liquid media and 29.3 days by the solid media (P＜0.001). The overall time to species identification from inoculation was 21.8 days. Direct PCR-RFLP from the liquid media identified 39.1% of MTB, 6.3% of M. avium, 19.05 of M. abscessus, and 12.6% of M. intracellulare respectively. Conclusion: Combined use of a liquid culture system and PCR-RFLP improved the recovery rate and shortened the detection time. However, solid media is still necessary to maximize the diagnostic efficiency.

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