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ORIGINAL ARTICLE pissn 1225-7737/eISSN 2234-8042 YUJM 2012;29(2):83-8 http://medlib.yu.ac.kr/yujm 항산균배양에서 BACTEC MGIT 960 System 의유용성및 MGIT 양성배지에서결핵균진단을위한 TB Ag MPT64 면역발색법의유용성 이승훈 1, 이민정 1, 이정미 1, 임수진 1, 이승준 1, 김유은 1, 조유지 1,3, 정이영 1,3, 김호철 1,3, 이종덕 1,3, 김선주 2,3, 황영실 1,3 경상대학교의학전문대학원 1 내과학교실, 2 진단검사의학교실, 3 건강과학연구원 Usefulness of the BACTEC MGIT 960 System for Mycobacterial Culture and TB Ag MPT64 Immunochromatographic Assay to Identify Mycobacterium tuberculosis Seung Hun Lee 1, Min Jeong Lee 1, Jeong-Mi Lee 1, Su Jin Yim 1, Seung Jun Lee 1, You Eun Kim 1, Yu Ji Cho 1,3, Yi Yeong Jeong 1,3, Ho Cheol Kim 1,3, Jong Deog Lee 1,3, Sun Joo Kim 2,3, Young Sil Hwang 1,3 Departments of 1 Internal Medicine, 2 Laboratory Medicine, 3 Gyeongsang Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju, Korea Background: This study was conducted to evaluate the usefulness of the BACTEC MGIT (Mycobacterium Growth Indicator Tube) 960 system for mycobacteria culture and immunochromatographic assay to identify Mycobacterium tuberculosis (MTB) in positive MGIT culture. Methods: Mycobacteria-culture-positive cases were retrospectively analyzed from December 2010 to July 2011. The detection rates and the recovery times of the mycobacteria between the Ogawa media and the MGIT were compared. An immunochromatographic assay (ICA) (SD BIO-LINE) was also performed in the positive MGIT culture for identification, and the results were compared with those of the Ogawa media in the Korea National Tuberculosis Association. Results: Among the 261 patients (M:F, 168:93; mean age, 61.6±17.16 yrs), 450 specimens (sputa, 365; bronchial washing, 61; and pleural effusion, 24) were found positive with mycobacteria. Mycobacteria were grown both on the MGIT and Ogawa media in 310 cases (68.9%); only on the MGIT in 115 cases (22.6%); and only on the Ogawa media in 25 cases (5.5%) (p<0.05).the recovery time was 28.2±8.9 days in the Ogawa media and 11.1±5.8 days in the MGIT (p<0.05). Among the 127 cases from the positive MGIT culture, all 92 cases that were confirmed as MTB cases bythe Korea National Tuberculosis Association were identified as MTB by ICA, with 100% sensitivity. Conclusion: MGIT increases the detection rate and shortens the recovery time of mycobacteria in clinical respiratory specimens, and the TB Ag MPT64 kit using ICA is useful in identifying MTB in a positive MGIT culture. Key Words: MGIT, Mycobacterium tuberculosis, Immunochromatographic assay, TB Ag MPT64 Received: August 28, 2012, Accepted: October 19, 2012 교신저자 : 김호철, 660-302, 경상남도진주시칠암동 90 경상대학교의학전문대학원내과학교실 Tel: (055) 750-8684, Fax: (055) 750-8618 E-mail: hochkim@gnu.ac.kr 서론 결핵은전세계적인보건학적문제이며, 매년 9백만명이상의새로운환자가발생하고, 1백만명이상이사망하는심 YUJM VOLUME 29, NUMBER 2, DECEMBER 2012 83

이승훈등 각한감염성질환이다. 1 국내에서는 2009년한해동안결핵신환발병률이인구 10만명당 97명으로다른선진국에비해매우높은편이고, 20대젊은층과 70대이상의고령군에서높은신환발병률을보이는양극형태를보이고있다. 2 결핵은전염성질환이기때문에신속하고정확한진단및치료가필요하며, 진단을위해서는배양을통한균동정이필수적이다. 액체배지를이용한배양은고식적인고체배지보다상대적으로높은검출률을보이고신속하게결과를얻을수있는장점이있다. 3-5 이에미국질병관리본부와세계보건기구는액체배지를이용한항산균배양을권고하고있고, 6 국내에서도많은병원에서도입하여사용중이며, 이에대한연구결과가발표되었다. 7-11 Mycobacterium Growth Indicator Tube (MGIT) 960 System (Becton Dickinson, USA) 은액체배지에서항산균이자라면서산소가소모되어분리된형광물질을센서가감지하여결핵균을포함한항산균의배양여부를자동으로확인하는시스템으로가장흔히사용되고있는항산균액체배지시스템이다. 3,12 MGIT 시스템은배양여부를신속하게확인할수있지만, 결핵균과비결핵항산균의감별을위해서는추가적인검사가필요하게된다. 결핵균과비결핵항산균의감별검사는적절한치료를위해매우중요하며, MPT64 는결핵균과 Mycobacterium bovis 등일부아균주의배양검체나조직에서감지될수있는것으로알려져있고, 결핵균과비결핵항산균의감별진단시유용하게사용될수있다. 13-16 최근 Mouse monoclonal anti-mpt64 antibody를이용한면역발색법 (TB Ag MPT64; SD, Yong-In, Korea) 이개발되어 MGIT 양성배지에서결핵균과비결핵항산균을빠르고간단하게감별할것으로기대할수있다. 이에본연구는호흡기검체의항산균배양에서 MGIT 960 system 의유용성을평가하고면역발색법을이용한 TB Ag MPT64 키트가결핵균과비결핵항산균의감별에유용한지를평가하기위한것이다. 자를대상으로후향적으로조사하였다. 2. 항산균도말및배양검사검체를동량의 4% NaOH 과섞고실온에서 15분간두었다가인산완충용액 (0.067 M, ph 6.8) 2-3배를첨가하여다시섞어주었다. 검체는 4, 4,000 rpm에서 20분간원심분리한후침전물 (<1 ml) 을회수하여검사를시행하였다. 항산균도말은 Auramine 형광염색법을이용하여미국질병예방통제국의기준에따라판독하였다. 6 형광염색에서양성을보인경우 Ziehl-Neelsen 염색을시행하여다시확인하였다. 배양검사는 0.5 ml의검체를 3% Ogawa 배지에접종하고대기조성배양기에서 37 에서최대 8주까지배양하였으며, 매주 2회양성여부를육안으로확인한다음집락이관찰되면동정을시행하였다. MGIT 배지는제조사의지침에따라사용당일 PANTA (Becton Dickinson, Sparks, MD USA) 를보충하여검체 0.5 ml 를접종하였으며, MGIT 960 system 에서 6주간배양하였다. 장비에서양성신호를보이면 Ziehl- Neelsen과 auramine-rhodamine 염색으로항산균양성여부를확인하였다. 3. 항산균동정 Ogawa 배지와 MGIT 에서양성이확인되면항산균염색을실시하여항산균인것을확인하였다. Ogawa 배지에서결핵균과비결핵항산균의감별은육안적으로집락을확인한다음 TB Ag MPT64 키트를이용한면역발색법을이용하여동정하였다. MGIT에서양성인경우에도 TB Ag MPT64 키트를이용한면역발색법으로동정을하고, Ogawa 배지에서동정한결과와비교하였다. MGIT 양성이고항산균도말양성인검체는 Ogawa 배지에계대배양하여동정과약제감수성검사에이용하였다. 재료및방법 1. 대상환자 2010년 12월부터 2011년 7월까지경상대학교병원에내원하여결핵이의심되어호흡기검체 ( 객담, 기관지세척액, 늑막액 ) 로항산균배양검사를시행한환자중에서고체배지 (Ogawa) 또는액체배지 (MGIT 960) 에서항산균이배양된환 4. TB Ag MPT64 키트를이용한면역발색법 (Immunochromatographic assay) MGIT 에서양성인경우검체에서군락이보이는것을확인하고, 소량의집락을채취한후 TB Ag MPT64 키트에넣고약 15분후키트를확인하여빨간색선이두개가보일경우는결핵균으로, 빨간선이한개가보일경우 ( 대조선만보이는경우 ) 는비결핵항산균으로판단하였다. 대조선이보이지않으면재검사를시행하였다 (Fig. 1). TB Ag MPT64 키트를 84 YUJM VOLUME 29, NUMBER 2, DECEMBER 2012

MGIT and ICA to Detect M. Tuberculosis Fig. 1. Identification of the M. tuberculosis complex by the TB Ag MPT64 immunochromatic assay (ICA) kit. (A) positive two band indicating M. tuberculosis, (B) negative one band indicating nontuberculous mycobacterium. Table 1. Recovery rate of MGIT and Ogawa media No. (%) of positive cultures for MTB NTM All mycobacteria MGIT & Ogawa 226 (74.59) 84 (54.14) 310 (68.89) MGIT only 69 (22.77) 46 (31.29) 115 (25.56) Ogawa only 8 (2.64) 17 (11.57) 25 (5.55) Total 303 (100) 147 (100) 450 (100) MGIT: Mycobacteria Growth Indicator Tube, MTB: Mycobacterium tuberculosis, NTM:nontuberculous mycobacteria. 이용한면역발색법의유용성을검정하기위해 MGIT 양성배지중에서대한결핵협회에동정과약제감수성검사를의뢰하여최종적으로결핵균또는비결핵항산균으로감별된결과를확인하여 TB Ag MPT64 키트를이용한면역발색법의결과와동일한지확인하였다. 5. TB/NTM real-time PCR법 Real-QTM MTB & NTM 키트 (Biosewoom Inc., Seoul, Korea) 를이용하여제조사의지침에따라시행하였다. 전처리한검체 1mL를원심분리하여상층액을제거한다음인산완충용액과멸균된증류수를순서대로첨가하여각각원심분리를한다음침전물을얻었다. DNA 추출은침전물에추출완충용액을섞고, 56 에서 15분간반응시키고간헐적으로혼합한뒤, 100 에서 8분간가열한다음원심분리하여상층액 2.5 L를얻었다. PCR 은튜브에 2X PCR mixture를 12.5 L 넣고, 결핵균과비결핵항산균, Internal control (IC) primer/ probe 혼합액을넣은다음추출한검체 DNA 2.5 L를첨가하고, Roter-Gene 3,000/6,000 (QIAGEN GmbH, Hilden, Germany) 을이용하여 50 2분, 95 10분의변성단계후 95 15초, 67 45초동안 40주기로시행하였다. 결과는각각의채널에서파장을확인하여 CT값을구하고, 35 미만인경우에양성으로판독하였다. 6. 통계처리값은평균과표준편차로표시하였다. 각각배지의배양양성률의차이는 chi-square test 또는 Fisher s exact test를이용하였고, 배양시간의차이는 unpaired t-test를이용하였다. p값이 0.05 이하인경우의미있는것으로판단하였고, SPSS 18.0 (SPSS, Chicago, IL, US) 통계프로그램을이용하였다. 결과 1. 대상환자및배양결과총 2,154명의환자에서 3,698회항산균배양을실시하였으며, 261명의환자 ( 남 : 여 =168:93, 평균나이 61.6±17.16 세 ), 450 검체 ( 객담 365, 기관지세척액 61, 늑막액 24) 가항산균배양양성을보였다. 450검체중결핵균이 303 (67.3%) 검체에서동정되었고, 비결핵항산균이 147 (32.7%) 검체에서동정되었다. 2. 배양양성률 450개의검체에서 MGIT 와 Ogawa 배지에서동시에배양된경우는 310예 (68.9%) 였으며, MGIT배지또는 Ogawa 배지에서만배양된경우는각각 115예 (25.5%), 25예 (5.5%) 로 MGIT 배지에만배양된경우가의미있게많았다 (p<0.05). MGIT 에서배양양성률은 Ogawa 배지에비해결핵균및비결핵항산균모두에서높았다 (Table 1). 항산균도말검사에서음성을보인 270 검체에서 MGIT 배지와 Ogawa 배지배양양성을보인경우는각각 90.7%, 66.2% 로 MGIT 배지에서배양양성률이의미있게높았으며 (p<0.05), PCR과도말검사모두음성인 89예의검체에서도 MGIT 배지와 Ogawa 배지배양양성률은각각 95.5, 71.9% 로 MGIT 배지가배양양성률이의미있게높았다 (p<0.05, Table 2). 3. 배양양성검출시간 Ogawa 배지와 MGIT 배지에서항산균배양양성검출시간은평균 28.2±8.9일, 11.1±5.8일이었으며, 결핵균과비결핵성항산균모두에서의미있게 MGIT 배지에서짧았다 (p<0.05, Table 3). YUJM VOLUME 29, NUMBER 2, DECEMBER 2012 85

이승훈등 Table 2. Recovery of MGIT and Ogawa media for negative AFB stain and negative PCR specimens No. (%) of positive cultures for MGIT Ogawa Negative for AFB stain 245 (90.7) 179 (66.2) (n=270) Negative for AFB stain and PCR (n=98) 94 (95.9) 73 (74.5) AFB: acid fast bacilli, PCR: polymerase chain reaction, MGIT: Mycobacteria Growth Indicator Tube, MTB: Mycobacterium tuberculosis, NTM: nontuberculous mycobacteria. Table 3. Duration of time (days) to detect mycobacteria in MGIT and Ogawa media Days to detection (range) MTB (N=303) NTM (N=147) Total (N=450) Ogawa 26.9±8.1 (11-58) 31.5±10.2 (5-56) 28.2±8.9 (5-56) MGIT 11.7±5.5 (1-38) 9.6± 6.1 (4-51) 11.1±5.8 (1-38) MGIT: Mycobacteria Growth Indicator Tube, MTB: Mycobacterium tuberculosis, NTM: nontuberculous mycobacteria. 4. MGIT 양성배지에서 TB Ag MPT64 키트를이용한면역발색법의유용성 MGIT 배지에서양성을보이고대한결핵협회에서동정과약제감수성검사를시행한 127개의검체중결핵균으로판정된경우는 92예였으며, 검체모두에서 TB Ag MPT64 키트가결핵균양성으로나왔다. 또한비결핵항산균이동정된 35예에서는모두결핵균음성으로나왔다 (Table 4). 고 찰 본연구는항산균배양에액체배지인 MGIT 의유용성과 MGIT 배양양성일경우결핵균감별을위한면역발색법키트의유용성을평가한것으로항산균배양에 MGIT 가고체배지에비해신속하고높은배양양성률을보여기존연구와부합되는결과를보였으며, MGIT 양성배지에서면역발색법을이용한 TB Ag MPT64 키트가결핵균과비결핵항산균을감별하는데유용한것으로나타났다. 결핵균과비결핵항산균의배양양성률은 MGIT 가 Ogawa 배지에비해월등히높았고, 도말음성, PCR 음성인검체에서도배양양성률은상당한차이를보였다. 하지만고체배지에서만항산균이배양된경우도 450예의검체에서 25예정도를차지하므로항산균배양을위해서는액체배지와고체배지를병합해서 Table 4. TB Ag MPT64 ICA assay in MGIT positive media MGIT culture result MTB NTM + 92 0 ICA - 0 35 ICA: immunochromatographic assay, MGIT: Mycobacteria Growth Indicator Tube, MTB: Mycobacterium tuberculosis, NTM: nontuberculous mycobacteria. 사용하는것이가장적절한방법으로생각할수있겠다. 본연구에서 MGIT 항산균배양검출기간은평균 11.1일 (1-38일 ) 걸렸다. 이결과는기존에국내에발표된항산균의평균검출기간인 11.4-12.8일과비슷한수준이다. 7-8,17 항산균배양시간단축은임상적으로중요한의미를가진다. 고체배지의경우는배양및약제내성검사를확인하는데약 4달정도의시간이걸리는경우가많은데, 이경우약제내성결핵이의심되는환자는내성여부를확인하는데시간이많이걸리므로치료에어려움을겪는경우가있을수있다. 하지만액체배지는배양시간을단축할수있으므로치료결정을신속하게할수있는장점이있다. 액체배지를사용하는 MGIT 에서는배지내오염균이증식하기쉬운영양성분이풍부하여, 18 고체배지에비해오염률이높은것으로알려져있다. 12,17,19 본연구에서 MGIT 의오염률은 5.1% 로 Ogawa 배지의 3.5% 에비해높은것으로나타났지만, 외국에서보고된 6.4-17.1% 의 5,12,17,20-22 오염률에비해비교적낮은수치를보이고있다. 국내에서발표된 MGIT 의오염률은본연구와비슷한 5% 정도를보고 9,23 하기도하고, 10% 이상의오염률이보고되어있기도하다. 8,24 오염률을줄이는것이임상적으로는중요한데, MGIT에섞는항생제 PANTA (Polymyxin B, Amphotericin B, Nalidixic acid, Trimethoprim, Azlocillin) 의조합을 vancomycin, amphotericin B, nalidixic acid로교체하여검출률과검출시간의차이없이오염률을 10.7% 에서 5.4% 로줄일수있었다는보고가있다. 25 또한, 최근에는단순히 PANTA 의농도를 2배증가시킴으로써오염률을 2배이상감소시킬수있다고보고되고있다. 26 MGIT 는항산균이자라게되면배지내에있는산소가소모되면서분리되는형광물질을센서가감지하여항산균의배양여부를알수있는것으로양성이나온다고해도결핵균과비결핵항산균을감별할수는없다. 국내에서도예전에비해비결핵항산균의비율이점점높아지고있는상황에서항산균의배양을높이기위한액체배지의이용도중요하지 86 YUJM VOLUME 29, NUMBER 2, DECEMBER 2012

MGIT and ICA to Detect M. Tuberculosis 만, 빠른감별이필요하게되는데단순한항산균염색은감별이되지않고 niacin 검사와같은전통적인생화학적검사는일정한결과가나오지않는경우가있다. 27 또한 PCR, 화학발광탐식자법 (Chemiluminescent DNA probes), 핵산증폭 (nucleic acid amplification), 고성능액체크로마토그래피 (high-performance liquid chromatography), 16S rrna 유전자서열분석등은유용한방법이지만, 사용이복잡하고비용이많이든다는단점이있다. 28,29 본연구에서면역발색법을이용한 TB Ag MPT64 키트는결핵균과비결핵항산균의감별에매우높은예민도와특이도를가진것으로나왔다. 또한검사결과를확인하는데 15 분정도의시간으로충분하며검사시특별한기술이필요하지않고비용이저렴하다는장점이있다. 본연구에서사용한 mouse monoclonal anti-mpt64 antibody에서 MPT-64 항원은 M. tuberculosis 와 M. bovis 의일부균만이배양액에서발견되는것으로알려져있어특이도가뛰어나임상적으로주요한항산균인결핵균을빠르고간단한방법으로동정할수있는방법으로여겨진다. 13,15,30 비결핵항산균중에서 MPT- 65 면역발색법에양성을보일수있는균은 Mycobacterium marinum 의일부균종또는 Mycobacterium flavescens 균종에양성을보일수있는것으로되어있지만, 결핵균과는육안적으로감별이쉽게되는것으로되어있다. 31 본연구는항산균배양에 MGIT 는고체배지에비해배양양성률이높고검출시간을단축할수있는유용한방법이며, MGIT 양성배지에서면역발색법을이용한 TB Ag MPT64 키트가결핵균과비결핵항산균의감별에유용하다는것을보여주고있다. 참고문헌 1. World Health Oragnization. Tuberculosis [Internet]. Geneva: World Health Organization; 2011 [cited 2011 Oct 7]. Available from: http://www.who.int/mediacentre/factsheets/fs104/en/ 2. Korea Center for Disease Control and Prevention. Annual report on the notified tuberculosis patients in Korea 2009. Seoul: Korea Centers for Disease Control and Prevention; 2010. p. 9-37. 3. Badak FZ, Kiska DL, Setterquist S, Hartley C, O Connell MA, Hopfer RL. Comparison of mycobacteria growth indicator tube with BACTEC 460 for detection and recovery of mycobacteria from clinical specimens. J Clin Microbiol 1996;34:2236-9. 4. Cornfield DB, Beavis KG, Greene JA, Bojak M, Bondi J. Mycobacterial growth and bacterial contamination in the mycobacteria growth indicator tube and BACTEC 460 culture systems. J Clin Microbiol 1997;35:2068-71. 5. Hanna BA, Ebrahimzadeh A, Elliott LB, Morgan MA, Novak SM, Rusch-Gerdes S, et al. Multicenter evaluation of the BACTEC MGIT 960 system for recovery of mycobacteria. J Clin Microbiol 1999;37:748-52. 6. Diagnostic standards and classification of tuberculosis in adults and children. Am J Respir Crit Care Med 2000;161:1376-95. 7. Bae E, Im JH, Kim SW, Yoon NS, Sung H, Kim MN, et al. Evaluation of combination of BACTEC mycobacteria growth indicator tube 960 system and Ogawa media for mycobacterial culture. Korean J Lab Med 2008;28:299-306. Korean. 8. Choi YM, Lee MH. Evaluation of the BACTEC MGIT 960 system for the recovery of mycobacteria. Korean J Clin Pathol 2000;20:56-61. Korean. 9. Yi JY, Kim JP, Shin JH, Suh SP, Ryang DW. Detection of Mycobacterium tuberculosis using BACTEC mycobacteria growth indicator tube (MGIT) 960 system: comparison with BACTEC 460 TB system and Ogawa media. Korean J Clin Pathol 2000;20:384-91. Korean. 10. Joung US, Jeong J, Lee SH, Kim SR. Comparison of mycobacterial culture by Mycobacterium growth indicator tube and Ogawa media. Korean J Clin Microbiol 2004;7:135-8. Korean. 11. Bang HI, Choi TY, Shin JW. Comparison of Ogawa media, BACTEC MGIT 960 System and TB/NTM real-time PCR for detecting Mycobacterium species. Tuberc Respir Dis 2011; 71:249-53. 12. Tortoli E, Cichero P, Piersimoni C, Simonetti MT, Gesu G, Nista D. Use of BACTEC MGIT 960 for recovery of mycobacteria from clinical specimens: multicenter study. J Clin Microbiol 1999;37:3578-82. 13. Abe C, Hirano K, Tomiyama T. Simple and rapid identification of the Mycobacterium tuberculosis complex by immunochromatographic assay using anti-mpb64 monoclonal antibodies. J Clin Microbiol 1999;37:3693-7. 14. Hasegawa N, Miura T, Ishii K, Yamaguchi K, Lindner TH, Merritt S, et al. New simple and rapid test for culture confirmation of Mycobacterium tuberculosis complex: a multicenter study. J Clin Microbiol 2002;40:908-12. 15. Nagai S, Wiker HG, Harboe M, Kinomoto M. Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis. Infect Immun 1991; 59:372-82. 16. Purohit MR, Mustafa T, Wiker HG, Mørkve O, Sviland L. Immunohistochemical diagnosis of abdominal and lymph node tuberculosis by detecting Mycobacterium tuberculosis complex specific antigen MPT64. Diagn Pathol 2007;2:36. 17. Lee JJ, Suo J, Lin CB, Wang JD, Lin TY, Tsai YC. Comparative evaluation of the BACTEC MGIT 960 system with solid medium for isolation of mycobacteria. Int J Tuberc Lung Dis 2003;7:569-74. 18. Stager CE, Libonati JP, Siddiqi SH, Davis JR, Hooper NM, Baker JF, et al. Role of solid media when used in conjunction with the BACTEC system for mycobacterial isolation and identification. J Clin Microbiol 1991;29:154-7. 19. Somoskövi A, Ködmön C, Lantos A, Bártfai Z, Tamási L, Füzy J, et al. Comparison of recoveries of Mycobacterium tuberculosis using the automated BACTECMGIT 960 system, the YUJM VOLUME 29, NUMBER 2, DECEMBER 2012 87

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