대한내과학회지 : 제 82 권제 3 호 2012 http://dx.doi.org/10.3904/kjm.2012.82.3.263 특집 (Special Review) - 결핵 결핵의새로운진단법 서울대학교의과대학내과학교실, 분당서울대학교병원내과 임효정 이재호 New Diagnostic Methods for Tuberculosis Hyo-Jeong Lim and Jae Ho Lee Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and Lung Institute of Medical Research Center, Seoul National University College of Medicine, Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea Tuberculosis (TB) is one of the most important public issues for its global burden of mortality and morbidity. Rapid diagnosis and treatment of active TB are crucial in effective TB control because of lacking truly effective preventive strategy. In recent decades, drug-resistant TB (DR-TB) has emerged as an expanding threat, prompt and reliable laboratory detection of of DR-TB are urgently needed to limit the spread of them. Recently, nonmolecular and molecular assays has been developed for early detection of active TB with or without detection of drug resistance. Broth-based culture systems are capable of producing positive results in 3 weeks or less and many automated system can be widely used for drug susceptibility test. Nucleic acid amplication (NAA) assays have high specificity and positive predictive value, high sensitivity in smear positive specimens. Line probe assays shows high sensitivity for detecting resistance to rifampin and variables sensitivity for detecting resistance to isoniazid. XpertMTB/RIF assay, an automated rapid PCR device, showed high sensitivity and specificity for TB and rifampin resistance. Although new diagnostic methods can decrease turn-around time with considerable diagnostic accuracy and convenience, they are not a replacement for culture and conventional drug susceptibility test for diagnostic limitation in special situations. (Korean J Med 2012;82:263-268) Keywords: Nucleic acid amplication; Line probe assays; Xpert MTB/RIF 서론결핵은전세계적으로단일감염질환으로는가장높은유병률과사망률을보이는질환으로과거는물론이고현재까지도여전히공공보건의가장중요한문제가되고있다. 아직까지결핵에대한효과적인예방책이없기때문에결핵관리를위해가장중요한것은활동성결핵환자를신속히진단하고적절한치료를시행하여결핵균전파에의한새로운환자의발생을막는것이다. 문제는결핵의증상이비특이적이며흉부방사선검사만으로는진단하기어려운경우 Correspondence to Jae Ho Lee, M.D. Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and Lung Institute of Medical Research Center, Seoul National University College of Medicine, Department of Internal Medicine, Seoul National University Bundang Hospital, 82 Gumi-ro 173beon-gil, Bundang-gu, Seongnam 463-707, Korea Tel: +82-31-787-2120, Fax: +82-31-787-2204, E-mail: jhlee7@snubh.org - 263 -
- The Korean Journal of Medicine: Vol. 82, No. 3, 2012 - 가많고, 검체를얻을수없는경우도드물지않아서진단이늦어지는경우가많다는점이다. 결핵이의심되는경우에는흉부 X-선검사를시행하고객담도말및배양검사를시행하고, 배양검사에서결핵균이동정되었을때확진한다. 항산균도말검사는전염력이높은환자를신속하게선별할수있다는장점이있지만, 민감도는배양과비교시 25-80% 정도로낮은편이다 [1-3]. 또한국내에서도비결핵항산균의분리빈도가증가하는추세로, 객담도말검사결과만으로는활동성결핵으로진단하기불충분하다 [4,5]. 결핵확진검사인배양검사에서는결핵균이자라기까지 2-6주정도소요되기때문에항산균도말검사에서음성이나온경우에는임상적으로진단을해서치료시작여부를결정해야한다. 최근에항산균도말검사보다높은민감도를보이면서기존의배양검사보다신속하게결핵을진단할수있으며, 약제내성여부를함께평가할수있는다양한검사법이개발되어진단적유용성에대한평가가이루어지고있다. 최근발표된여러연구결과들을바탕으로결핵의새로운진단법에대해서알아보고자한다 (Fig. 1). 본론 Acid Fast bacilli smear microscopy 항산균염색으로는 carbolfuchsin을이용한염색과형광염색이있다. Carbolfuchsin을이용한검사는우리가흔하게알고있는 Ziehl-Neelsen (ZN) 염색과 kinyoun 염색이있으며광학현미경을사용하여 1,000배의배율로관찰한다. ZN 염색후광학현미경에서관찰하는것의한계를극복하기위해형광물질염색이시도되었는데, 형광염색은 auramine O나 rhodamine을이용한염색법으로형광현미경을이용해 250 배혹은 450배에서관찰한다. 염색에필요한시간이짧고 ZN 염색에비해 10% 정도민감도가높으며, 보다저배율에서관찰가능하며, 검사자의업무량이적은장점이있어서선진국에서특히검사량이많은기관에서사용을권고하였다. 그러나진단을위해서형광현미경과고가의광원, 어두운방등이필요하기때문에실제로설비가잘갖춰지지않은많은국가들에서는널리시행되지못하였다 [6]. 최근에형광현미경의장점은지니면서광원으로더저렴하고, 전기 LINE PROBE ASSAY XDR Figure 1. Algorithm for use of selected noncommercial culture and drug susceptibility testing methods from WHO Policy Framework 2010. - 264 -
- Hyo-Jeong Lim, et al. New diagnostic methods for tuberculosis - 소모가적은발광다이오드현미경이개발되어결핵진단에사용되고있다. 발광다이오드현미경은형광물질을염색에이용하지만, 광원으로는 LED (light-emitting diode) 기술을이용하는검사법으로기존의광학현미경또는형광현미경에비해높은민감도를보이면서동등한특이도를보임이알려졌다 [7-9]. 이를바탕으로 2010년부터 WHO에서는형광현미경대신발광다이오드현미경을사용할것을권고하고있지만, 아직 ZN 검사를대체하지는못한다 [10]. 아직국내의결핵진료지침에서는이러한새로운현미경사용에대한언급이없는실정이다 [11]. 항산균배양검사항산균배양검사는결핵을확진할수있는검사이다. 배양검사에사용되는배지의종류는크게고체배지와액체배지로나눌수있는데, 고체배지는계란성분의배지인 Löwenstein-Jensen 배지가표준배지이며, 한천고체배지인 Middlebrook 7H10/11이있다. 고체배지는오래전부터사용되어온방법으로안정적이지만, 배양여부를확인하는데 3-8주의시간이소요되는한계가있었다. 최근액체배지의개발로배양양성보고에소모되는시간을줄일수있게되었는데, 기존액체배지 (Middlebrook 7H12) 뿐만아니라다양한자동화된액체배지 (BACTEC460, MGIT, Septi-Check, Myco-ESP culture System II, BacT/ALERT MB Susceptibility Kit) 들이개발되었다. 이들액체배지의경우, 도말양성인경우 2주, 도말음성인검체의경우약 3주면배양양성이보고된다 [12,13]. 액체배지는비싸고, 세균이나비결핵항산균에의한교차감염의비율이높은단점이있긴하지만, 빠른결과보고와높은양성률때문에국내여러기관에서도널리도입되고있으며, 2011년결핵진료지침에도고체배지와액체배지에각각접종할것을권고하고있다 [11]. 다양한종류의자동화된액제배지는결핵의진단뿐아니라약제내성결핵의신속한진단에도이용되고있다. Mycobacteria growth indicator tube (MGIT) MGIT system 은변형된액체배지인 Middlebrook 7H9 broth가들어있는유리튜브로서바닥에형광물질을묻은실리콘이있다. 형광물질은산소에민감하여처음에는배지내의풍부한산소에의해발광이억제되어있다가객담을접종했을때결핵균이자라나산소를소모할경우형광물질이발광하여 UV 램프하에서오렌지색으로보이도록고안되었으며, 최근에는전자동화된시스템인 BACTEC MGIT 960 이개발되었다. 약제내성여부를진단하는데에도사용할수있는데, 이는배양된결핵균을약제가들어있는배지에접종한후색깔의변화를확인한다. 최근메타분석에의하면항결핵약제일차약제에대해서 BACTEC MGIT 960 system의정확도와예측도가모두우수하였다 [14-16]. 내성검사를얻을때까지걸리는시간은짧게는 5-8.2일정도이다. Microscopic observation drug susceptibility (MODS) assay MODS는결핵균이고체배지보다액체배지에서빠르게자라며, 특징적으로형성하는 cord 모양을액체배지에서조기에현미경적으로관찰가능한점을결핵진단및약제내성결핵의진단에이용하는검사법이다 [17]. Middlebrook 7H9 액체배지, 성장보충제, 오염균의과성장을막기위한각종항균제가채워진 24 well plate 에검체를넣고, 오염되지않도록봉한뒤매일 inverted light microscopy를이용하여결핵균의특징적인모양인 cord가형성이되었는지확인하여결핵균을조기에진단한다. 균배양까지소요되는시간도약 7일정도에불과하며, 24개의 well 중일부에다양한농도의항결핵약제를넣은뒤검체를접종하여, isoniazid 와 rifampin에대한내성검사를빠르게시행할수있다는장점이있다. 가격이비싸지않고, 간편하다는장점이있으나매일직접관찰해야하고숙련된인력이필요하며, biosafety Level 3 이상의실험실이갖춰져야하기때문에이미결핵진단에필요한요건들을갖춘비교적큰검사실이적당하며, 이때문에아직방법적으로표준화되지못하였다. 균배양민감도는 87.4-97.8% 이고 [18] isoniazid 내성에대한민감도는 90.3%, 특이도는 98.6%, rifampin 내성에대한민감도는 98%, 특이도는 99.4% 였다 [17,19]. Nitrate reductase assay (NRA) 고체배지를사용한검사법으로결핵균이 nitrate를 nitrite 로환원시키는것에바탕을둔검사로, LJ 배지에 nitrate를넣은뒤, 결핵균이자랄경우 Griess 반응에의해배지의색이붉은빛으로변하는것으로진단한다. 약제내성을평가하는데에도사용될수있는데항균제처리한뒤결핵균을접종한뒤, 접종한지 10일째되는날 nirate 를환원시키는능 - 265 -
- 대한내과학회지 : 제 82 권제 3 호통권제 619 호 2012 - 력을측정한다. 내성균주는 nitrate를 nitrite로환원시켜배지의색이 pink-red color로변하며, 감수성있는균이자란배지는무색으로남는다. NRA 는 rifampin 내성에대한민감도와특이도가각각 99%, 100%, isoniazid 에대한민감도와특이도는 94%, 100% 일정도로매우우수하다 [20,21]. 검사가쉽고저렴하며숙달된인력이나고급장비가필요없는등맹유용하며장점이많으나아직까지는배양된결핵균의 1차약제내성검사에만이용되고있으며연구가더필요하다. 결핵균핵산증폭검사 (nucleic acid amplification, NAA testing) 결핵균핵산증폭검사는결핵균에만특이하게존재하는 DNA나 RNA를중합효소연쇄반응 (polymerase chain reaction, PCR) 을이용하여측정하는것으로 24-48시간내에결과를알수있다. 일반적으로매우높은특이도를가지고있으나상대적으로낮은민감도를가지고있는데항산균도말검사와비교하였을때비결핵항산균이흔한환경에서도말양성검체에서는높은양성예측률 (> 95%) 을보이나, 도말음성배양양성인검체의경우 50-80% 에서양성률을보인다. Amplified mycobacterium tuberculosis direct (MTD) test (Gen- Probe, San diego, CA) 와 Amplicor M. tuberculosis test (Roche Diagnostics, Basel, Switzerland) 등이임상적으로결핵이의심되는환자의도말양성호흡기계검체에서사용이 FDA에서공인되었으며, 이외에도 In- house PCR method 등다양한형태의 NAA assay 가결핵진단에이용되고있다. CDC 는결핵이의심되지만확진되지않은환자에서호흡기계검체에서 NAA를시행할것을추천하고있으며 [22], 개정된국내결핵지침에서도결핵균핵산증폭검사를 1) 폐결핵이의심되나도말검사가음성일때혹은 2) 항산균도말검사가양성이지만비결핵항산균의가능성이있을때실시할것을추천하였고, 3) 폐외결핵인경우에도도움이된다고기술하였다. 최근에는이러한분자학적기술의발달로진단과약제감수성검사를동시에시행하게되었다. Line probe assay (LPA) Reverse hybridization 검사로서결핵균또는검체에서직접 DNA를추출하여이를 PCR로증폭하여 oligonucleotide probe 를이용하여 labeling을하여 strip에나타나는색깔의변화를확인하는검사법으로결핵의진단과약제내성여부를확인 하는데사용한다 [18]. 최초로사용된 LPA는 INNO-LiPA Rif TB (Innogenetics NV) 였으며, 2005년에발표된메타분석결과에따르면결핵의진단에민감도는 95% 이상, 특이도는 100% 였으며, 임상검체에서는민감도가 80-100% 까지다양하게보고된바있다 [23]. GenoType MTBDR (Hain Lifescience) 가두번째개발된 LPA였는데, rifampin 및 isoniazid 내성을진단하는데만족도가낮아, 더많은 rpob, inha mutation을찾아내는 GenoType MTBDR plus로보완되었으며, isoniazid 내성진단에더높은민감도를보임이증명되었다 [24,25]. 또한최근에는 fluoroquinolone, ethambutol, kanamycin, amikacin 등에대한내성을진단하는검사인 GenoType MTBDRsl 이개발되어 2차약제에대한내성진단에고무적인결과를보여주고있다 [26]. 현재까지여러연구결과를토대로할때도말음성검체에서는진단에제한이있고, rifampin 내성을확인하는데는유용하나 INH 내성에대해서는진단의변화폭이커서 LPA는 rifampin 내성에대한검사로추천된다. Xpert MTB/RIF (cepheid) 자동화된분자학적검사로플라스틱카트리지내에세포용해, 핵산추출및증폭, 증폭탐지에필요한모든반응용제가들어있어객담을전처치하여 MTB/RIF 카트리지에넣고이를 GeneXpert 기기에넣기만하면자동적으로 PCR을실행시켜결핵균확인과 rifampin 내성여부를 2시간내에알수있게된다 [27]. 도말양성환자에서는 98.2-100% 민감도를도말음성 / 배양양성환자에서는 71.7-72.5% 의민감도를보였다. 2번째객담으로 12.6%, 세번째객담으로는 5.1% 의민감도를증가시킬수있어 3개의객담검체를이용하는경우에는 90.2% 의민감도를나타내며, 특이도는 99.2% 에이른다. Rifampin 내성에대한민감도, 특이도는각각 97.6%, 100% 로높았으나가격이비싸고, isoniazid 내성을확인할수없다는단점이있다 [27]. 2011년 WHO는 MDR TB 관리를위해서적극적으로사용할것을권고하고있다. 인터페론감마분비능검사과거결핵균에감작된 T 림프구에결핵균특이항원을자극하여분비되는인터페론감마를측정하는검사로현재사용되고있는검사법은 Quantiferon-TB Gold In-Tube (Cellestis) 와 T-SPOT.TB (oxford immunotec) 가있다. Quantiferon-TB Gold In-Tube 방법은환자의말초혈액을채취해서결핵균 - 266 -
- 임효정외 1 인. 결핵의새로운진단법 - 항원 (ESAT-6, CFP-10, TB7.7) 이들어있는튜브, 음성대조튜브 (Nil), 양성대조튜브 (Mitogen) 에각각첨가하여 16-24 시간동안자극하여분비된인터페론감마를 ELISA를통하여측정한다. T-SPOT.TB 의경우에는채취한전혈에서말초단핵구를추출하여 ESAT-6 와 CFP-10 항원으로자극한후 ELISPOT 방법으로 spot의숫자를세어서진단한다. 결핵감염여부를진단하는것으로, 결핵감염률이 1% 미만인국가에서는임상적으로결핵이의심되어시행한경우양성일경우결핵가능성이높지만, 우리나라와같이결핵감염률이높은국가에서는활동성결핵의진단에대한유용성은떨어진다. 결 최근개발된여러분자학적또는비분자학적검사들을통해결핵을좀더신속하고간단하게진단하는것이가능해졌다. 기존의배양검사에걸리던시간을감소시킬수있을뿐만아니라최근결핵관리에서가장중요한문제인내성결핵의진단역시기존의약제감수성결과에비해빠르게진단할수있어결핵관리에긍정적인기여를할것으로기대된다. 그러나이러한새로운검사법은여전히결핵균배양검사를대체할수는없으며, 기존의약제감수성검사역시대체할수없다. 이들새로운검사들은도말음성결핵또는폐외결핵등특수한상황에서의결핵에서진단적유용성에차이가있으며, 여건과인력이갖추어진실험실이필요하며광범위내성결핵의진단을위해서는여전히현재의약제감수성결과가필요하기때문이다. 또한이러한검사기술의발달이곧효과적인결핵관리로이어지는것은아니기때문에결핵의신속하고정확한진단법에대한연구와함께결핵환자의관리등전반적인결핵관리사업에도함께노력을기울여야하겠다. 중심단어 : 결핵의새로운진단법 ; 핵산증폭검사 ; 엑스퍼트티비 론 REFERENCES 1. Aber VR, Allen BW, Mitchison DA, Ayuma P, Edwards EA, Keyes AB. Quality control in tuberculosis bacteriology: 1. Laboratory studies on isolated positive cultures and the efficiency of direct smear examination. Tubercle 1980;61: 123-133. 2. Siddiqi K, Lambert ML, Walley J. Clinical diagnosis of smear-negative pulmonary tuberculosis in low-income countries: the current evidence. Lancet Infect Dis 2003;3: 288-296. 3. 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 1999: this statement was endorsed by the Council of the Infectious Disease Society of America, September 1999. Am J Respir Crit Care Med 2000;161(4 Pt 1):1376-1395. 4. Kim S, Park EM, Kwon OJ, et al. Direct application of the PCR restriction analysis method for identifying NTM species in AFB smear-positive respiratory specimens. Int J Tuberc Lung Dis 2008;12:1344-1346. 5. Park YS, Lee CH, Lee SM, et al. Rapid increase of nontuberculous mycobacterial lung diseases at a tertiary referral hospital in South Korea. Int J Tuberc Lung Dis 2010; 14:1069-1071. 6. Steingart KR, Henry M, Ng V, et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis 2006;6:570-581. 7. Marais BJ, Brittle W, Painczyk K, et al. Use of lightemitting diode fluorescence microscopy to detect acid-fast bacilli in sputum. Clin Infect Dis 2008;47:203-207. 8. Minion J, Sohn H, Pai M. Light-emitting diode technologies for TB diagnosis: what is on the market? Expert Rev Med Devices 2009;6:341-345. 9. Anthony RM, Kolk AH, Kuijper S, Klatser PR. Light emitting diodes for auramine O fluorescence microscopic screening of Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2006;10:1060-1062. 10. Strategic and technical advisory group on tuberculosis (STAG-TB). Report of the Ninth meeting. World Health Organization (WHO). 2009. 11. Joint Committee for the Development of Korean Guidelines for Tuberculosis; Korea Centers of Disease Control and Prevention. Korean Guidelines for Tuberculosis. 2011. 12. Sharp SE, Lemes M, Sierra SG, Poniecka A, Poppiti RJ Jr. Löwenstein-Jensen media: no longer necessary for mycobacterial isolation. Am J Clin Pathol 2000;113:770-773. 13. Kanchana MV, Cheke D, Natyshak I, Connor B, Warner A, Martin T. Evaluation of the BACTEC MGIT 960 system for the recovery of mycobacteria. Diagn Microbiol Infect Dis 2000;37:31-36. 14. Sanders CA, Nieda RR, Desmond EP. Validation of the use of Middlebrook 7H10 agar, BACTEC MGIT 960, and BACTEC 460 12B media for testing the susceptibility of Mycobacterium tuberculosis to levofloxacin. J Clin Microbiol - 267 -
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