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1 DOI: /trd ISSN: (Print)/ (Online) Tuberc Respir Dis 2010;69: CopyrightC2010. The Korean Academy of Tuberculosis and Respiratory Diseases. All rights reserved. 결핵균과비결핵성항산균검출에 Real-time 의유용성 Original Article 1 경상대학교의학전문대학원내과학교실, 2 경상대학교건강과학연구원, 3 경상대학교의학전문대학원진단검사의학교실윤은영 1, 조수희 1, 고세일 1, 백종하 1, 김유은 1, 마정은 1, 이기동 1, 조유지 1, 정이영 1,2, 김호철 1,2, 이종덕 1,2, 김선주 2,3, 황영실 1,2 Usefulness of Real-time to Detect Mycobacterium tuberculosis and Nontuberculous Mycobacteria Eun Young Yun, M.D. 1, Su Hee Cho, M.D. 1, Se Il Go, M.D. 1, Jong Ha Baek, M.D. 1, You Eun Kim, M.D. 1, Jeong Eun Ma, M.D. 1, Gi Dong Lee, M.D. 1, Yu Ji Cho, M.D. 1, Yi Yeong Jeong, M.D. 1,2, Ho Cheol Kim, M.D. 1,2, Jong Deok Lee, M.D. 1,2, Sun-Joo Kim, M.D. 2,3, Young Sil Hwang, M.D. 1,2 1 Department of Internal Medicine, Gyeongsang National University School of Medicine, 2 Gyeongsang Institute of Health Sciences, Gyeongsang National University, 3 Department of Laboratory Medicine, Gyeongsang National University School of Medicine, Jinju, Korea Background: The purpose of this study was to evaluate recently developed real-time polymerase chain reaction () assay kit to detect Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) in respiratory specimens. Methods: We assessed the positive rate of the real-time assay to detect MTB and NTM in 87 culture-positive specimens (37 sputum, 50 bronchial washing), which were performed real-time by using Real-Q TM MTB&NTM Kit from January 2009 to June 2009, at Gyeongsang University Hospital. To compare the efficacy with the TB- assay, we evaluated 63 culture-positive specimens (19 sputum, 44 bronchial washing) for MTB or NTM, which were performed TB- by using ABSOLUTE TM MTB II Kit from March 2008 to August Results: Among 87 specimens tested using real-time, MTB and NTM were cultured in 58 and 29, respectively. The positive rate of real-time assay to detect MTB was 71% (22/31) and 92.6% (25/27) in AFB stain-negative and stain-positive specimens. For NTM, the positive rate of real-time was 11.1% (2/18) and 72.7% (8/11) in AFB stain-negative and stain-positive specimens. Among 63 specimens performed using TB-, MTB and NTM were cultured in 46 and 17, respectively. The positive rate of TB- was 61.7% (21/34) and 100% (12/12) in AFB stain-negative and stain-positive specimens. TB- was negative in all NTM-cultured 17 specimens. Conclusion: TB/NTM real-time assay is useful to differentiate MTB and NTM in AFB stain-positive respiratory specimens and it is as effective in detecting MTB with TB-. Key Words: Mycobacterium tuberculosis; Mycobacteria, Atypical; Polymerase Chain Reaction, Real-Time 서 결핵은전세계적보건문제이며한국에서의유병률은지속적으로감소하고있지만아직도서구선진국에비해 Address for correspondence: Ho Cheol Kim, M.D. Department of Internal Medicine, Gyeongsang National University School of Medicine, 92, Chilam-dong, Jinju , Korea Phone: , Fax: hochkim@gnu.ac.kr Received: Jun. 11, 2010 Accepted: Aug. 5, 2010 론 서높은상황이다 1,2. 폐결핵은타인에게감염되는것을줄이고조직의파괴성병변이나후유증의발생을예방하기위해조기진단이중요하지만확진을위해서는미생물학적균의동정이필요하다. 항산균도말염색검사는간단한방법으로신속하게결과를확인할수있지만민감도가낮고결핵균과비결핵성항산균 (nontuberculous mycobacteria, NTM) 과의감별이안되며, 결핵균배양검사는검체에존재하는결핵균이아주적어도검출되는장점이있고특이도도높지만결과를확인하는데시간이오래걸리는단점이있다

2 Tuberculosis and Respiratory Diseases Vol. 69. No. 4, Oct 도말및배양검사의단점을보완한결핵균중합효소연쇄반응법 (TB-) 은검체내에소수의균이존재하더라도진단이가능하고신속한결과를얻을수있어조기진단에널리이용되고있으며, 민감도와특이도가높아결핵의진단에널리사용하고있다 4,5. 하지만위양성의위험이있고, 최근빈도가증가되고있는 NTM 을직접검출하지못하는단점이있다 6. 항산균도말양성의호흡기검체에서 NTM을조기에확인하는것은임상적으로중요한데그이유는폐결핵으로오인하여치료하는경우가있고, 폐결핵과치료약제도다르며최근그빈도가점점증가하는추세이기때문이다. 미국의경우항산균도말양성인호흡기검체에서 NTM 의빈도는약 25 30% 정도이고 7, 국내에서도 10% 이상차지하는것으로보고되고있으며과거에비해그빈도는점점증가하는추세이다 8,9. 현재임상에서는항산균도말염색결과가양성이고 TB- 이음성인경우에 NTM으로추정하고있다 실시간중합효소연쇄반응법 (real-time ) 은특정유전자의증폭산물에결합된형광물질의방출을실시간으로분석하여측정하는방법으로정량화할수있고중합효소연쇄반응법이후전기영동과같은추가적인과정이필요하지않기때문에오염의위험성을낮출수있다. 또한기존중합효소연쇄반응법에비해검사시간을단축할수있으며특이도를높일수있는다양한장점을가지고있다 13. 결핵균과 NTM 을동시에진단할수있는결핵균 / 비결핵성항산균실시간중합효소연쇄반응법 (TB/NTM real-time ) 이최근에개발되고상용화되면서호흡기검체에서결핵균의검출률을높이고조기에결핵균과 NTM 을감별할수있을것으로기대하고있으며그유용성에대한보고가국내에서도발표되고있다 본연구는객담및기관지세척액과같은호흡기검체에서결핵균과 NTM 검출에 TB/NTM real-time 의유용성을알아보기위한것이다. 대상및방법 1. 대상환자 2009년 1월부터 6월까지객담및기관지세척액에서 TB/NTM real-time 을시행하고배양에서결핵균또는 NTM이동정된 80명 ( 남자 54명, 여자 26명 ; 평균나이 61.7세 ) 을대상으로총 87예의검체 ( 객담 37예, 기관지세척액 50예 ) 를후향적으로분석하였다. 호흡기검체에서항산균도말검사는음성이면서 TB/NTM real-time 이 양성인경우와도말검사는양성이면서 TB/NTM real-time 이음성인경우, 항산균도말검사와 TB/NTM realtime 이모두음성또는양성인경우를조사하였다. 기존의 TB- 법과비교하기위해 2008 년 3월부터 8월까지 TB- 을시행하고객담및기관지세척액에서결핵균또는 NTM 이배양된 59명 ( 남자 39명, 여자 20명 ; 평균나이 61.3 세 ) 환자의 63예검체 ( 객담 19예, 기관지세척액 44예 ) 를대상으로후향적으로같은방법으로분석하였다. 2. 검체의처리와항산균도말및배양검사 검체와동량의 4% NaOH 를섞고실온에서 15분간두었다가인산완충용액 (0.067 M, ph 6.8) 2 3배를첨가하여다시섞어주었다. 검체를 4 o C에서 4,000 rpm으로 20분간원심분리한후침전물을회수하여검사를시행하였다. 항산균도말은 Ziehl-Neelsen 염색법을이용하여미국질병예방통제국의기준에따라판독하였고 3, 배양은 3% Ogawa 한천배지에접종하고 37 o C에서최대 8주까지배양하였다. 배양후집락이보이면 SD bioline TB Ag MPT64 Rapid (Standard Diagnostic Inc., Seoul, Korea) 를이용한면역크로마토그래피방법으로결핵균과 NTM을일차적으로구별하였다. 3. TB/NTM real-time 방법 Real-QTM MTB&NTM Kit (Biosewoom Inc., Seoul, Korea) 를이용하여제조사의지침에따라시행하였다. 전처리한검체 1 ml을원심분리하여상층액을제거한다음인산완충용액과멸균된증류수를순서대로첨가하여각각원심분리를한다음침전물을얻었다. DNA 추출은침전물에 extraction buffer 를섞고, 56 o C에서약 15분간보관하고간헐적으로혼합한뒤, 100 o C에서 8분간가열한다음원심분리하여상층액 2.5 μl 를얻었다. 은튜브에 2 mixture를 12.5 μl 을넣고, MTB와 NTM, Internal control (IC) primer/probe 혼합액을넣은다음추출한검체 DNA 2.5 μl 를첨가하고, Roter-Gene TM 3000/6000 (QIAGEN GmbH, Hilden, Germany) 을이용하여 50 o C 2분, 95 o C 10분의변성단계후 95 o C 15초, 67 o C 45초동안 40주기로시행하였다. 결과는각각의채널에서파장을확인하여 CT 값을구하고 35 미만인경우에양성으로판독하였다. MTB와 NTM을구별하는방법은 MTB signal은나오지않고 mycobacterai genus-specific signal만나오는경우에 NTM으로추정하였다. 251

3 EY Yun et al: Real-time to detect MTB and NTM 4. TB- 방법 ABSOLUTETM MTB II Kit (Biosewoom Inc.) 를이용하여제조사의지침에따라시행하였다. 전처리와 DNA 추출은 real-time 과동일한방법으로실시하였다. 은튜브에 1차 혼합물를 15 μl, 1차 효소를 0.5 μl 넣고추출한검체 DNA 4.5 μl 를첨가하였다. Roter-Gene TM 3000/6000 을이용하여 95 o C 5분의변성단계후 94 o C 30초, 68 o C 30초, 72 o C 30초의 주기로 35회한후 72 o C 5분동안시행하였다. 1차 이끝난후튜브에 2차 혼합물을 18 μl, 1 차 효소 0.5 μl 와 1차산물 1.5 μl 를첨가한다음 Roter-Gene TM 3000/6000을이용하여 95 o C 5분의변성단계후 94 o C 30 초, 65 o C 30초, 72 o C 30초의 주기로 25회한후 72 o C 5분동안시행하였다. 반응후반응물 7 μl 를 2% 아가로스젤에전기영동하여 산물의크기를확인하였다. 230 bp와 473 bp가나오거나혹은 230 bp 밴드만나올경우결핵균으로판정하였다. 5. 통계처리 SPSS version 12.0 (SPSS Inc., Chicago, IL, USA) 프로그램을이용하였다. 검사간의민감도비교는교차분석을통하여 chi-sqaure test를시행하였으며, p값이 0.05 이하인경우의미있는것으로판단하였다. Table 1. Detection rate of MTB and NTM in TB/NTM real-time Culture result positive for MTB (%) NTM (%) negative (%) Total M. tuberculosis 58 AFB stain (-) 22 (71) - 9 (29) 31 AFB stain (+) 25 (92.6) - 2 (7.4) 27 NTM 29 AFB stain (-) - 2 (11.1) 16 (88.9) 18 AFB stain (+) - 8 (72.7) 3 (27.3) 11 MTB: Mycobacterium tuberculosis; NTM: nontuberculous mycobacterium; AFB: acid fast bacilli; : polymerase chain reaction. 결과 TB/NTM real-time 을시행한검체에서결핵균과 NTM이배양된경우는각각 58예, 29예이었다. 결핵균이배양된 58예중에서 47예 (81%) 가 TB real-time 양성이었고, NTM이배양된 29예중에서 NTM real-time 이양성인경우는 10예 (34.4%) 이었다. 도말음성이고결핵균이배양된 31예에서 TB real-time 양성은 22예 (71%), 음성은 9예 (29%) 이었으며, 도말양성 27예중에서 TB real-time 양성은 25예 (92.6%), 음성은 2예 (7.4%) 이었다 (Table 1). NTM이배양된 29예중에서도말음성은 18예이었고이중 NTM real-time 양성은 2예 (11.1%), 음성은 16예 (88.9%) 이었고, 도말양성 11예중에서 NTM Table 2. Results of cases of cultured NTM No. Specimen NTM real-time AFB stain Culture 1 Sputum (+) Negative M. intracellulare type 1 2 Sputum (-) 1+ M. intracellulare type 1 3 Bronchial washing (-) Negative M. chelonae 4 Bronchial washing (+) 1+ M. intracellulare type 1 5 Bronchial washing (+) Negative M. intracellulare type 1 6 Bronchial washing (-) Negative M. intracellulare type 1 7 Sputum (+) 4+ M. abscessus 8 Sputum (+) 1+ M. gordonae type 1 9 Bronchial washing (-) Negative M. intracellulare type 1 10 Sputum (-) Negative M. szulgai 11 Bronchial washing (+) 1+ M. avium 12 Bronchial washing (+) 1+ M. intracellulare type 1 13 Sputum (-) 3+ M. abscessus NTM: nontuberculous mycobacterium; AFB: acid fast bacilli; : polymerase chain reaction. 252

4 Tuberculosis and Respiratory Diseases Vol. 69. No. 4, Oct Table 3. Detection rate of MTB and NTM in TB- Culture result positive (%) negative (%) Total M. tuberculosis 46 AFB stain (-) 21 (61.7) 13 (38.3) 34 AFB stain (+) 12 (100) 0 (0) 12 NTM 17 AFB stain (-) 0 13 (100) 13 AFB stain (+) 0 4 (100) 4 MTB: Mycobacterium tuberculosis; NTM: nontuberculous mycobacterium; AFB: acid fast stain; : polymerase chain reaction. real-time 양성은 8예 (72.7%), 음성은 3예 (27.3%) 이었다 (Table 1). NTM이배양된 29예중에서균동정을의뢰하여확인한예는 Table 2와같다. TB- 검사를시행한검체에서결핵균이배양된경우는 46예이었고도말검사음성은 34예, 양성은 12예이었다. 도말음성인 34예중에서 TB- 양성은 21예 (61.7%), 음성은 13예 (38.3%) 이었고도말양성인 12예는 TB- 이모두양성이었다. NTM이배양된 17예는 TB- 이모두음성이었고, 항산균도말검사가양성으로나와 NTM으로간주한경우는 4예이었고 (Table 3) Mycobacterium abscessus가 2예, M. gordonae과 M. intracellulare가각각 1예이었다. 고 폐결핵과 NTM에의한폐질환은임상증상이나방사선소견으로감별하기쉽지않기때문에실제임상에서는항산균도말검사가양성으로나와폐결핵으로치료하다가배양에서 NTM이동정되어약제를변경하거나중단하는경우를흔히보게된다 18. 항산균배양양성의검체에서 NTM 의검출률은점점증가하는추세인데 8,9, 본연구에서도배양양성검체에서는 30% (46/150예) 이었고, 도말양성의경우에는 24.4% (12/49 예 ) 를차지하였다. 이런점으로볼때항산균도말이양성이나와도 NTM의가능성을항상염두에두어야하며적절한치료와방침을결정하기위해조기감별이중요하다고할수있다. 현재임상에서는항산균도말검사가양성이면서 TB- 이음성이면 NTM으로간주하고임상결정에유용한것으로보고되고있지만 10,11, 최근상용화된 TB/NTM real-time 검사는호흡기검체에서결핵균과 NTM을직접감별하고검출률 찰 도올릴수있을것으로기대된다. 본연구의결과로보면 TB/NTM real-time 검사는도말음성보다는도말양성검체에서결핵균과 NTM을감별하는데더유용할것으로생각된다. NTM이배양된검체에서도말양성의경우 real-time 양성률은 72.7% (8/11 예 ) 이었지만, 도말음성의경우는 11.1% (2/18예 ) 로도말양성검체에비해상대적으로매우낮은양성률을보였다. Chang 등 17 의보고에서도도말음성검체 8예중에서 1예에서만 NTM real-time 이양성으로나왔고, Kim 등 15 은 6예의도말음성검체에서 3예가 NTM real-time 양성으로보고하고있다. 본연구와국내의연구결과를보면도말음성검체에서 NTM에대한 real-time 의민감도는낮은것으로생각된다. TB/NTM real-time 검사는본연구에서결핵균을진단하는데도높은민감도를보였다. 도말양성검체에서 TB real-time 은 92.6% (25/27 예 ) 의양성률을보였고도말음성검체에서도 71% (22/31예 ) 의양성률을보였는데이것은 TB- 검사를시행한도말음성검체에서의양성률 61.7% (13/21 예 ) 보다높은것이다 (p<0.05). Kim 등 15 의보고에서도본연구와비슷한 70% 정도의양성률을보였다. 본연구의결과로보면결핵균검출에대한 real-time 민감도도기존의 TB- 만큼우수할것으로생각되지만두검사간의민감도를비교하기위해서는동일한검체를이용한검사가필요할것으로생각된다. 본연구에서사용한 ABSOLUTETM MTB II 키트는 nested 방법으로 1차 산물의특정한부위를 2차 로다시증폭하여전기영동에의해균의유무를확인하는방법으로기존의 법에비해오염의위험도는높지만민감도를향상시킨검사법이다. 객담이나기관지세척액과같은호흡기검체에서도말음성인경우에결핵진단을위한 TB- 검사의민감도는연구자마다차이가있지만약 40 60% 정도로보고되고있으며 19-21, 국내의연구 22 도비슷한민감도를보고하고있고본연구에서는검체수가충분하지않지만약 60% 정도로비교적높은양성률을보였다. 일반적으로도말양성이지만 TB- 이음성인경우에 NTM 을의심할수있고임상적으로유용한것으로보고되고있는데 10-12, 본연구에서도도말양성이지만 TB- 이음성으로나온 4예에서모두 NTM이배양되었고, 도말음성이지만전기영동에서결핵균과는다른양상을보여 NTM으로보고된경우가 3예가있었다. 결과에는기술되지는않았지만 TB- 이양성으로나와결핵으로간주하 253

5 EY Yun et al: Real-time to detect MTB and NTM 였으나배양에서는 Mycobacterium spp. 로나온경우가 1예가있었으며이것은 에서 MTB와 NTM 밴드가동시에나오는경우에 MTB 로판정을내리게되므로배양에서는 NTM이배양될수있을것으로생각된다. 본연구에서는도말양성인검체에서 TB real-time 이음성인경우가 27예중에서 2예로위음성률이 7.4% 로나왔으며, Chang 등 17 의보고에서도 1예에서위음성을보고하였지만 Kim 등 15 의연구에서는 82예의도말양성검체모두가 real-time 이양성으로나온것으로보고하고있다. TB- 에서는위음성으로나온경우는본연구에서관찰되지않았는데결핵균에대한 real-time 과 TB- 의민감도와특이도는 Jung 등 16 의보고에의하면각각 86 93% 정도로비슷한수준을보이는것으로보고하고있다. 결론적으로 TB/NTM real-time 은항산균도말양성의호흡기검체에서결핵균과 NTM을감별하는데유용하며, 결핵균의검출에도기존 TB- 만큼우수한것으로판단된다. 참고문헌 1. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA 1999;282: Kim HJ. Current situation of tuberculosis and its control in Korea. J Korean Med Assoc 2006;49: Diagnostic Standards and Classification of Tuberculosis in Adults and Children. This official statement of the American Thoracic Society and the Centers for Disease Control and Prevention was adopted by the ATS Board of Directors, July This statement was endorsed by the Council of the Infectious Disease Society of America, September Am J Respir Crit Care Med 2000;161: Cousins DV, Wilton SD, Francis BR, Gow BL. Use of polymerase chain reaction for rapid diagnosis of tuberculosis. J Clin Microbiol 1992;30: Park SS, Kwak KR, Hwang JY, Yun SM, Ryue CC, Chang CH, et al. Clinical utility of amplified mycobacterium tuberculosis direct test in the diagnosis of pulmonary tuberculosis. Tuberc Respir Dis 1999;47: Balasingham SV, Davidsen T, Szpinda I, Frye SA, Tønjum T. Molecular diagnostics in tuberculosis: basis and implications for therapy. Mol Diagn Ther 2009;13: Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175: Lee JY, Choi HJ, Lee H, Joung EY, Huh JW, Oh YM, et al. Recovery rate and characteristics of nontuberculous mycobacterial isolates in a university hospital in Korea. Tuberc Respir Dis 2005;58: Koh WJ, Kwon OJ, Yu CM, Jeon KM, Suh GY, Chung MP, et al. Recovery rate of nontuberculous mycobacteria from acid-fast-bacilli smear-positive sputum specimens. Tuberc Respir Dis 2003;54: Lee JS, Ji HS, Hong SB, Oh YM, Lim CM, Lee SD, et al. Clinical utility of polymerase chain reaction for the differentiation of nontuberculous mycobacteria in patients with acid-fast bacilli smear-positive specimens. Tuberc Respir Dis 2005;58: Yu CM, Koh WJ, Ryu YJ, Jeon K, Choi JC, Kang EH, et al. Usefulness of test for M. tuberculosis for the differentiation of pulmonary tuberculosis and nontuberculous mycobacterial lung disease in patients with smear-positive sputum. Tuberc Respir Dis 2004;57: Kim CS, Son HD, Park MR, Seo JY, Cho DI, Rheu NS. Usefulness of study in AFB smear negative patients on admission. Tuberc Respir Dis 1997;44: Lee MK. Real-time polymerase chain reaction () in microbiology. Infect Chemother 2004;36: Lee SD, Lee HY, Kim HC, Kim SY. Mycobacterium tuberculosis and nontuberculous mycobacteria by assay. Korean J Clin Microbiol 2007;10: Kim YJ, Park MY, Kim SY, Cho SA, Hwang SH, Kim HH, et al. Evaluation of the performances of AdvanSure TB/NTM real time kit for detection of mycobacteria in respiratory specimens. Korean J Lab Med 2008;28: Jung CL, Kim MK, Seo DC, Lee MA. Clinical usefulness of real-time and amplicor MTB assays for diagnosis of tuberculosis. Korean J Clin Microbiol 2008; 11: Chang HE, Heo SR, Yoo KC, Song SH, Kim SH, Kim HB, et al. Detection of mycobacterium tuberculosis complex using real-time polymerase chain reaction. Korean J Lab Med 2008;28: Koh WJ, Kwon OJ. Diagnosis and treatment of nontuberculous mycobacterial lung disease. Korean J Med 254

6 Tuberculosis and Respiratory Diseases Vol. 69. No. 4, Oct ;74: Wobeser WL, Krajden M, Conly J, Simpson H, Yim B, D'costa M, et al. Evaluation of Roche Amplicor assay for Mycobacterium tuberculosis. J Clin Microbiol 1996;34: Bergmann JS, Woods GL. Clinical evaluation of the Roche AMPLICOR Mycobacterium tuberculosis test for detection of M. tuberculosis in respiratory specimens. J Clin Microbiol 1996;34: Mo EK, Kyung TY, Kim DG, Park MJ, Lee MG, Hyun IG, et al. The clinical utility of polymerase chain reaction in the bronchoalveolar lavage fluid for the detection of mycobacteria. Tuberc Respir Dis 1998;45: Baek SH, Lee JM, Kang MJ, Son JW, Lee SJ, Kim DG, et al. How reliable is sputum test in the diagnosis of pulmonary tuberculosis when sputum smear is negative? Tuberc Respir Dis 2001;50:

Lumbar spine

Lumbar spine CT 32 111 DOI : 10.3831/KPI.2010.13.2.111 Lumbar Spine CT 32 Received : 10. 05. 23 Revised : 10. 06. 04 Accepted : 10. 06. 11 Key Words: Disc herniation, CT scan, Clinical analysis The Clinical