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1 체외 Interferon-gamma 검사를이용한결핵감염의진단 종설 1 건국대학교의료원충주병원호흡기내과, 2 울산대학교의과대학서울아산병원호흡기내과 이정연 1, 심태선 2 Diagnosis of Mycobacterium tuberculosis Infection using Ex-vivo interferon-gamma Assay Jung Yeon Lee, M.D. 1, Tae Sun Shim, M.D. 2 1 Division of Pulmonary Medicine, Department of Internal Medicine, Konkuk University Medical Center, Chungju Hospital, 2 Division of Pulmonary & Critical Care Medicine, University of Ulsan College of Medicine, Asan Medical Center, Until recently, the tuberculin skin test (TST) has been the only tool available for diagnosing a latent TB infection. However, the development of new diagnostic tools, using the Mycobacterium tuberculosis (MTB)-specific early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) antigens, should improve the control of tuberculosis (TB) by allowing a more accurate identification of a latent TB infection (LTBI). Antigen-specific interferon-gamma (IFNγ) assays have greater specificity in BCG-vaccinated individuals, and as less biased by nontuberculous mycobacterial infections. Many comparative studies have suggested that those assays have a higher specificity than the TST, and the sensitivity of these assays are expected to remarkably improved if more MTB-specific antigens can become available. Nevertheless, the major obstacle to the widespread use of these tests is the limited financial resources. Similar to other diagnostic tests, the predictive value of IFN-γ assays depends on the prevalence of a MTB infection in the population being tested. Therefore, prospective studies will be meeded to establish the applicability of these new assays at multiple geographic locations among patients of different ethnicities, and to determine if the IFN-γ responses can indicate those with a high risk of progressing to active TB. (Tuberc Respir Dis 2006; 60: ) Key words: Interferon-gamma assay, Tuberculin test, Tuberculosis, Latent tuberculosis infection. 1. 서론 전세계인구의 3분의 1이결핵균에감염되어있으며, 결핵이발병하는환자의 90% 이상이이미감염되어있는사람에서발병한다는자료는활동성결핵뿐만이아니라잠복결핵환자의올바른진단및치료도결핵의조절및근절에중요한역할을한다는것을보여준다. 그러나잠복결핵치료의중요한문제점중하나는어떻게잠복결핵환자를올바르게진단하는 가이다. 전통적으로투베르쿨린검사 (tuberculin skin test, 이하 TST) 가잠복결핵의유일한진단법이었으 Address for correspondence: Tae Sun Shim, M.D. Address: Division of Pulmonary & Critical Care Medicine, University of Ulsan College of Medicine, Asan Medical Center, Pungnap-Dong, Songpa-Ku, Seoul, , South Korea Phone: Fax: shimts@amc.seoul.kr 나 TST는비씨지접종또는비결핵항산균감염과결핵감염을명확히구분하지못하여특이도가떨어지고, 특히잠복결핵의치료의적응증이되는면역억제환자에서는무반응 (anergy) 으로인하여민감도가떨어져유용성에제한점이있다. 최근말초혈액을이용한체외인터페론감마검사 (ex-vivo IFN-γ assay, 이하 IFN-γ 검사 ) 가기존의 TST보다잠복결핵의진단에더우수하다는보고가있어왔고, 일부검사는이미상업화되어시판중이다. 이검사들은결핵균- 특이항원을이용하므로실제결핵균감염과비씨지접종에의한 TST 위양성을구별할수있을것으로기대되므로국내처럼대다수의젊은사람이비씨지접종을받은환경에서는이검사가특히유용할가능성이있다. 이에결핵면역기전에서의 IFN-γ 의역할을잠시언급하고, 후반부에는체외 IFN-γ 검사를이용한잠복결핵의진단에관한문헌들을고찰해보고자한다. 497

2 JY Lee et al. : Diagnosis of Mycobacterium tuberculosis infection using ex-vivo interferon-gamma assay 2. 결핵에대한세포면역에서 IFN-γ 의역할결핵균에대한면역기전은주로세포매개성면역, 특히 Th1 면역이중요한역할을하는것으로알려져있다. Th1 면역기전을간단히살펴보면가지세포 (dendritic cell) 나대식세포와같은항원제시세포 (antigen-presenting cell) 가결핵균을탐식한후 IL- 12를분비하여 T 림프구를자극한다. 결핵균항원에감작된 T 림프구는 IFN-γ 를분비하고분비된 IFN-γ 는결핵균을포식하고있는대식세포를자극한다. 자극된대식세포는반응성산소기 (reactive oxygen species) 또는반응성질소기 (reactive nitrogen intermediate) 등의생성을통하여결핵균을사멸시키게된다. 한편결핵균을탐식한대식세포및 T 림프구는 TNF-α를분비하여 T 림프구를자극하고또한동시에대식세포를자극한다. 이러한방어기전에서 IFNγ가가장중추적인역할을한다. 결핵에서 IFN-γ 의역할은 IFN-γ knock-out 마우스연구를통하여잘알려져있고 1, 사람에서도 IFN-γ 또는 IL-12 경로의장애가있는사람에서중증마이코박테리아증이발병한보고들이있어서 IFN-γ 가결핵에대한방어기전에서가장중요한역할을하는사이토카인임은잘알려져있는사실이다 활동성결핵의진단및치료에서 IFN-γ 의역할결핵의진단에서 IFN-γ 와관련하여가장많은연구가되어있는것은결핵성흉수에서흉수 IFN-γ 의측정이다. 결핵성흉수의경우균이음성인경우가많아다른보조진단방법들에많이의존하게된다. 림프구-우세성흉수에서흉수 adenosine deaminase (ADA) 의측정이결핵성과악성흉수의감별에유용하게사용되고있는데흉수 IFN-γ 농도측정은 ADA 와비교하여비슷하거나높은진단율을보여주고있다 3-5. 그외에도결핵성심낭염, 결핵성복막염등의진단에도 IFN-γ 의측정이유용한것으로보고되고 있으며 6,7 기관지폐포세척액혹은객담내의 IFN-γ 측정을통해폐결핵의중증도및치료반응을평가할 수있는지표로도사용될수있는것으로보고되고있다 8,9. 치료면에서는다제내성결핵에서 IFN-γ 를항결핵치료의보조제로사용한여러보고가있었으나아직그효과는증명되지않았다 국내에서도 3편의연구보고가있었으나극히제한된환자에서만효과를보여주어면역기전의장애가확인되지않은일반적인중증, 진행성다제내성결핵환자에서보편적으로효과가있을지는미지수이다. 한편분무용 IFN-γ-b1 (Actimmune R ) 을생산한 InterMune 이라는제약회사에서 2000년에다제내성결핵환자를대상으로다기관 3상연구를시작하였으나성공적인결과를얻지못한상태로연구를종료하였다. 4. 체외 IFN-γ 검사를이용한결핵감염의진단 1) 투베르쿨린검사 TST와 IFN-γ 검사는결핵균항원에대한세포면역반응을평가한다는면에서는비슷하지만면역반응의다른관점을보는검사이다 14. 이는 IFN-γ 유전자 knock-out 마우스에서도 purified protein derivatives ( 이하 PPD) 에의한지연과민반응은발생하나 IFN-γ 는생성되지않는다는점에서알수있다. TST에사용되는 PPD는결핵균배양상청액의침전물로약 200개이상의항원을포함하는데이중일부는여러비결핵항산균에도포함되어있기때문에결핵균에비특이적이다. 또한검사를위하여 PPD를반복주사하는것자체가 booster 반응을야기하여위양성결과를초래함으로써특이도가낮아지는원인이된다 15. 한편활동성결핵환자에서는약 75-90%, 미만성결핵에서는약 50% 이하의민감도를보인다고보고되고있음에도불구하고 15 TST를기반으로하여잠복결핵을치료한결과활동성결핵의발병을약 60% 정도줄였다는보고가있었으며 16 비용이적게들고, 특별한검사장비없이쉽게시행할수있는장점등으로인해현재까지도 TST를잠복결핵의진단에이용하고있는것이현실이다. 498

3 Tuberculosis and Respiratory Diseases Vol. 60. No. 5, Mar ) 체외 IFN-γ 검사 IFN-γ 검사는말초혈액을채취하여결핵균항원으로자극한후결핵균항원을인지할수있는 T 림프구에의한 IFN-γ 생성능을확인하는검사이다. 즉, IFN-γ 가생성되면체내에결핵균항원을인지할수있는 T 림프구가있으므로결핵균에감염되었음을의미한다. 처음에개발된 QuantiFERON R -TB (Cellestis limited, Carnegie Victoria, Australia; 이하 QFT) 검사는결핵균에비특이적인 PPD를항원으로사용하였지만비씨지접종에의한위양성을구분하기에더용이하다는결과가보고되면서 2001년에미국식품의약품안전청 (Food & Drug Administration; FDA) 으로부터잠복결핵의진단목적으로허가를받았다. 이후 PPD 대신에결핵균특이항원인 ESAT-6 (early secreted antigenic target 6) 와 CFP-10 (culture filtrate protein 10) 을사용한 2세대검사인 QuantiFERON R -TB Gold (Cellestis limited, Carnegie Victoria, Australia; 이하 QFT-G) 가개발되어 2005년미국 FDA로부터결핵감염의진단목적으로승인을받았다. 또한, 미국 CDC (Centers for Disease Control) 에서는결핵감염의진단에 TST를대체하여사용할수있다고기술하고있다 QFT-G의또다른형태인 3 세대검사 QuantiFERON R -TB Gold In Tube ( 이하 ; QFT-IT) 는 ESAT-6와 CFP-10 항원외에도 TB7.7(p4) 펩타이드항원이추가된합성펩타이드를이용한검사로써소량의검체 ( 약 2ml) 로도진단이가능하고활동성결핵환자에서 QFT-G에비해특이항원에대한 IFN-γ 의반응이강하게나타나는것으로알려져있어민감도가높을것으로기대되고있으며, 소아환자및특이항원에대한반응정도가약한면역억제환자에서좀더유용할가능성이있다 20. 이외에유럽에서는 ESAT-6와 CFP- 10 항원을사용하는 T SPOT-TB (Oxford Immunotec, Oxford, UK; 이하 T SPOT) 검사가개발되어잠복결핵진단의목적으로승인을받았다. 위검사방법들의주된장점은결핵균-특이항원을사용하므로비씨지접종이나비결핵항산균감염에의한위양성을줄여특이도를높일수있다는점이다. 민감도에대한보고는 다양하나기존의 TST에비하여민감도도더높을것으로기대되고있다 ( 특히면역억제환자에서도민감도가높은것으로보고되고있다 ) 21. 지금까지잠복결핵을대상으로한대부분의연구에서는 TST 결과와의일치도가약 60-80% 로보고되고있다 이검사들의다른장점으로는판독자에의한판독오류의가능성이적고, 환자가한번만병원을방문하여채혈하면되며, 반복된 PPD 주사로인한 booster 효과가없다는점등이다. 그렇지만아직은 TST에비하여고가의비용이필요하고, 다양한역학상황에서도동일하게적용될수있을지가미지수이고, 이검사에의하여결핵균감염으로판명된사람이실제로향후결핵이발병할확률이높은지등은추후임상경과를지켜보아야할것이다. 또한 TST와마찬가지로결핵감염의위험도 (risk-stratified) 에따라판정기준치 (cutoff value) 도차등화해야한다는주장도제기되고있다 19. (1) QuantiFERON R -TB Gold 검사전혈 (whole blood) 을이용한 IFN-γ 검사법은 1990 년 Wood 등에의하여소결핵의진단을위하여처음으로개발되었으며 25, 이후인간에서도유사한검사법이개발되어 QuantiFERON R -TB (QFT) 라는이름으로상품화되었다 26. 이검사는전혈을이용하며자극항원으로 PPD를사용하고, 시간배양한후상청액에분비된 IFN-γ 를 ELISA (enzyme-linked immusorbent assay) 법으로측정하는검사이다. Mazurek 등은이검사를이용하여 1,226명의결핵감염의위험인자가있거나, 활동성결핵이있거나의심되는환자를대상으로한연구에서 TST와의일치율이 83.1% 임을보였고, 특히비씨지접종군에서 TST 양성 /QFT 음성일가능성이 7배나높음을보여서이검사가비씨지접종에의한결핵감염진단의위양성을줄일수있는것으로보고하였다 14. 그러나결핵환자에서는 TST가 QFT에비해높은양성률 ( 민감도 ) 을보였기때문에미국 CDC는 QFT 검사를잠복결핵의진단목적으로만사용을제한하였고, 활동성결핵이의심되는경우에는과거와같이 TST를시행할것을권고하였다 27. 반면에 Fietta 등은활동성결핵환자에 499

4 JY Lee et al. : Diagnosis of Mycobacterium tuberculosis infection using ex-vivo interferon-gamma assay Figure 1. Dot plot of individual responses to CFP-10* and ESAT-6 for 118 culture-positive patients with TB (a) and 213 subjects with a low risk for TB exposure (b) using QFT-G kit. The dashed line represents the cutoff of 0.35 IU/ml for IFN-γ. The specificity of the test for the low-risk group was 98.1% and the sensitivity for patients with M. tuberculosis infection was 89.0%17. Adopted from reference 17. *early secretory antigenic target 6, culture filtrate protein 10, tuberculosis, QuantiFERON-TB Gold 서 QFT 검사가 TST보다민감도가높음을보고하였는데그이유로고령, 간경화등의기저질환을가진환자들이많이포함되었기때문으로분석하였고, 따라서 QFT 검사가면역기능이저하된환자에서는결핵감염을진단하는데 TST보다더유용할가능성을제시하였다 28. 그렇지만이연구에서결핵감염의고위험군중비씨지접종한군에서 QFT 검사가 TST보다높은양성률을보여비씨지접종의영향을배제하지못했을가능성이제시되었다 28. 이후 Johnson 등은 TST 음성인학생들을대상으로비씨지접종전과비씨지접종 5개월후에 TST, QFT 검사및결핵균-특이항원을이용한체외 IFN-γ 검사를시행하였는데양성전환율이각각 13%, 20%, 0% 로보고되어 29, QFT 검사는비씨지접종의영향을받을뿐만아니라결핵감염의진단에있어서 TST 보다우수하지못함을보고하여결핵균에특이적인항원을이용한검사법의개발이필요하게되었다. QuantiFERON R -TB Gold (QFT-G) 는 QFT 검사의단점을보완하기위하여 BCG나대부분의다른마 이코박테리아에는존재하지않고 (M. kansasii, M. szulgai, M. flavescens, M. marinum 는예외 ) 결핵균에만존재하는 RD1 (region of difference 1) 유전자분절부위에서생성되는 ESAT-6 와 CFP-10 항원을사용하였다 21, 명의비씨지를접종받은결핵감염의위험도가낮은일본인을대상으로한 Mori 등의연구결과 98.1% 의높은특이도를보여주었으며, 118 명의배양양성결핵환자를대상으로한 QFT-G 검사의민감도는 TST와비교하여유의하게높은것으로 (89% vs. 65.8%, 각각, Fig. 1) 보고되었다 17. 전염성결핵환자접촉자연구에서도 TST와의높은일치율을보여주었고, 비씨지접종을한군과하지않은군에서비슷한양성률을보여비씨지접종에영향받지않음을간접적으로보여주었다 18. 이연구에서는또한 QFT-G와 QFT 검사를비교하여전자는비씨지접종에영향을받지않는반면에후자는비씨지접종의영향을받음을보여주었다 18. 국내인을대상으로한연구에서도결핵감염의위험도가증가함에따라검사양성일확률이 TST의경우 1.52배증가하는데비하여 QFT-G는 5.31배증가하는것으로보고되어 BCG 접종률이높은국내에서도 QFT-G가결핵감염의진단에유용할가능성을보여주었다 31. 그러나 Ferrara 등은 QFT-G가면역억제환자를대상으로한경우에는양성, 음성의판정이불가능한 indeterminate result 가많이관찰됨을보고하여이검사의단점으로지적되었다 32. Indeterminate result 는양성대조군항원 (phytohemaglutinin) 에도충분히자극되지않았거나음성대조군에기저 IFN-γ 분비가많은경우를의미한다 17. Brock 등도 HIV양성환자들을대상으로잠복결핵의선별검사에 QFT-IT검사를시행하였는데중증의면역억제 (CD4 세포 < 100/uL) 인경우그렇지않은경우에비해 indeterminate result 가유의하게높음 (24% vs. 2.8%, p<0.05) 을보고하여 QFT-IT 역시면역억제정도에따라검사의유용성에제한이있을가능성이있음을제시하였다 33. 결핵의유병률이높은남아프리카지역에서 358명의건강한성인을대상으로 QFT, QFT-G, QFT-IT 검사를비교한연구결과양성률은각각 215 (60%), 137 (38%), 201 (56%) 명이었으며각검사간일치도 500

5 Tuberculosis and Respiratory Diseases Vol. 60. No. 5, Mar 또한낮게 (kappa = ) 나타나같은 ELISA방법을사용하더라도각세대간검사특징및유병률에따라결과에큰차이가있음을제시하였으며, 특히 QFT-G, QFT-IT의일치도가낮게나타나이에대해서는추가적인연구가필요할것으로생각된다 34. (2) T SPOT-TB 검사 (Fig. 2) QFT-G는분비된 IFN-γ 의농도를 ELISA 방법으로측정하는데비하여 T SPOT-TB 검사 (T SPOT) 는 ELISPOT (enzyme-linked immunospot analysis) 기법을이용하며, 항원의자극에의하여 IFN-γ 가분비되면바닥에고정되어있는 IFN-γ 항체와결합하게되며 T 림프구주위로분비되어 IFN-γ 항체와결합하므로염색후에는한개의점으로보이게되는데이를 spot-forming cell count (SFC) 이라고한다 30. 많은보고들은상업화된 T SPOT을이용한것이아니나같은항원을사용하였고, 같은 ELISPOT 법을이용하였으므로 T SPOT 검사를사용한것에준하여기술한다. 대부분의 ELISPOT을이용한연구는재조합항원 보다는 15mer의펩타이드의조합 (peptide pool) 을항원으로사용하였다 21,30,35. 보고에따라펩타이드나재조합항원사이에차이가없기도하였고 30,36, 펩타이드는항원특이 T 림프구를검출한반면에재조합항원은 T 림프구검출에실패한결과를보고하기도하였다 37. 아마도이는재조합 ESAT-6 항원이외부에서주어질경우 MHC class I 항원제시경로를통하여항원이제시되지못하기때문인것으로생각되었다 21,38. Lalvani 등은 ESAT-6 펩타이드만을항원으로사용하여 ELISPOT 법으로 92% (43/47) 의특이도및배양양성결핵환자에서 96% (45/47) 의민감도를보여주었다 35. ESAT-6 및 ESAT-6/CFP-10을이용한 ELISPOT 연구에서도전염성결핵환자와의접촉의강도및노출기간과 ELISPOT 검사간에강한상관관계를보여주었다 39,40. 이후결핵유병률이높은건강인도인 (BCG 접종여부모름 ) 과유병률이낮은건강영국인 (82.5% 가비씨지접종 ) 을대상으로 ESAT- 6/CFP-10을이용한 ELISPOT 검사연구에서양성률이각각 80% (80/100), 0% (0/40) 로역학상황과잘일 A. B. Figure 2. Representative positive (A) and negative (B) results of T SPOT-TB analysis. The positive result (A) shows many spots in the ESAT-6* or CFP-10 stimulated wells, whereas the negative result (B) shows less than 5 spots in the ESAT-6 or CFP-10 stimulated wells. *early secretory antigenic target 6, culture filtrate protein

6 JY Lee et al. : Diagnosis of Mycobacterium tuberculosis infection using ex-vivo interferon-gamma assay 치하였으며 30, TST와 PPD를이용한 ELISPOT 검사결과는양군에서차이가없어서 ESAT-6/CFP -10 을이용한 ELISPOT 검사는비씨지접종의영향을배제하고실제잠복결핵을진단할수있는유용한방법으로평가되었다 30. 반면에결핵의유병률이높은잠비아인을대상으로한연구에서는낮은민감도가문제점으로제기되었는데이는기준치 (cutoff value) 의설정이기존의연구와차이가있었기때문일가능성이있어서향후다양한역학상황에서의연구가필요할것으로생각된다 22. 최근에는 Shams등이 413명의건강접촉자를대상으로전향적인연구에서접촉계수 (contact score) 의증가에따른검사양성률이 TST보다 ELISPOT 검사에서유의하게높음을보여주었으며, 특히미국에서출생한경우나비씨지접종을하지않은경우이러한차이가더욱유의하게나타남을보여주어검사결과의해석에있어서각지역의비씨지접종률이나결핵의유병률또한중요하게작용함을알수있었다 41. Richeldi 등은단기간 ( 평균 6시간 ) 다제내성결핵환자에노출된 92명의건강인을대상으로 TST, PPD, 혹은 RD1 펩타이드를이용한 ELISPOT 검사를시행하여그결과를비교하였는데 TST와달리 ELISPOT 검사는환자에의노출정도와높은상관성을보여주어 RD1 ELISPOT 검사가단기간의접촉에의한결핵감염을진단하는데있어서도유용함을보여주었다 42. 접촉자 92명중 1명은면역억제제를사용중인환자로활동성결핵이증명되었는데이환자에서 TST는음성이었으나 RD1 ELISPOT 검사는양성으로결핵의진단을가능하게하였다. 면역억제환자, 특히 HIV (human immunodeficiency virus) 감염환자에서도 CD4 T 림프구감소에따라면역반응이떨어질것이라는예상과는달리 ESAT-6 항원에대한반응이좋은것으로보고되었고 30, Chapman 등도 HIV 양성이 ESAT-6와 CFP-10 펩타이드를이용한 ELISPOT 검사의민감도를떨어뜨리지만그정도가 PPD를이용한 ELISPOT 검사나 TST보다훨씬적음을보고하였다 21. 따라서, TST는 HIV 양성환자또는면역억제제를사용하는환자처럼잠복결핵의진단및치료가중요한환자에서민감 도가낮은단점이있으나 15,43 T SPOT 검사는이런단점을해결해줄수있을것으로기대되고있다. 한연구에의하면 T SPOT는면역억제환자에서도 indeterminate result 결과가많지않은것 (<5%) 으로보고되었으며 44, 최근혈액암환자를대상으로한접촉자연구에서도 T SPOT-TB 검사가 TST에비해양성률이높았고, indeterminate result 역시낮게 (4.3%) 나타나면역억제상태에서도 T SPOT 검사가잠복결핵의진단에유용하게사용될수있음을제시하였다 45. (3) QuantiFERON-TB Gold 와 T SPOT-TB 검사법의비교양검사법의차이는 Table 1과같다. T SPOT은말초혈액에서단핵구를따로분리하고세포수를측정하여야하므로전혈을사용하는 QFT-G에비하여시간과비용이더많이드는단점이있지만반면에혈청에이미분비되어있던 IFN-γ 의영향을배제할수있는장점이있다. 또한배양후의실험과정은오히려 T SPOT이더간단하다고할수있다. QFT-G 는 ELISA법을이용하고 ELISA 판독기는실험실에서흔히사용되고있으므로설치에큰부담이없을수있으나 T SPOT은 SFC의수를측정하기위하여고가의 ELISPOT 판독장비를필요로한다. 그러나고가의 ELISPOT 판독기대신현미경이나확대경을사용할수도있다. 또한 QFT-G는 96 well plate가 8 well strip들로나누어져있어서소수의검체를검 Table 1. Comparison between QuantiFERON-TB Gold and T SPOT-TB tests. QuantiFERON-TB Gold T SPOT-TB Material Mononuclear cells Whole blood (1 ml) (well) (2.5x105) ESAT-6*/CFP-10 ESAT-6/CFP-10 Stimulants peptides peptides Methods ELISA ELISPOT Incubation Less than 24 hours Less than 24 hours time Equipments ELISA reader ELISPOT reader *early secretory antigenic target 6, culture filtrate protein 10, tuberculosis, enzyme-linked immunosorbent assay, enzyme-linked immunospot assay 502

7 Tuberculosis and Respiratory Diseases Vol. 60. No. 5, Mar 사할때편리한장점이있지만매검사마다표준농도를구하기위하여 8-well이추가로필요하므로소수의검체를검사할때는많은 well이소모되는단점이있다. 반면에 T SPOT-TB 는표준농도를구하기위한 well이필요없고각검체당 4개의 well만사용하면된다. 한개의 plate를 1주내에여러번반복하여배양하더라도결과에는영향을미치지않는것으로보고되어있으므로소수의검체씩여러번반복하여사용하는것도가능하다. 최근 T SPOT도 96 well이 strip으로나누어져여러번나누어검사하는데편리하도록제조되고있다. ELISA와 ELISPOT 검사법을비교한자료들을살펴보면, Scholvinck 등 46 은 ESAT-6와 CFP-10 항원을이용한결과를비교하였는데 ELISPOT 법은 T SPOT에서사용하는펩타이드대신항원단백질을사용하였으며, ELISA법은 QFT-G 와달리 72시간배양하는방법을사용하여양검사법의정확한비교라고하기는어렵다. 이연구에서활동성결핵환자 13 명에서 ELISPOT 법은 100%, ELISA 법은 92.3% 의민감도를보였고, 잠복결핵환자에서는각각 87%, 91% 의민감도를보여양검사결과는높은일치도를보였다. Goletti 등도활동성결핵환자를대상으로한연구에서 ELISPOT 및선택된 RD1 펩타이드를사용한 ELISA 법과 QFT-G를비교하였고각검사의민감도는 74%, 81%, 89%, 각검사의특이도는 93%, 90% 68% 를나타내어선택된 RD1 펩타이드를사용한 ELISA법이상업화된 kit를이용한 QFT-G에비해민감도에는큰차이가없으면서특이도가높음을보여주었다 47. 그러나, QFT-G의경우잠복결핵을포함한결핵감염의진단에사용할수있도록 cutoff 값을설정한것에비해 Goletti 등의연구에서는선택된 RD1 펩타이드를사용한 ELISA법에서는활동성결핵을진단하기에가장적절한 cutoff값을이용하여민감도및특이도를구하였으므로 cutoff의차이에의하여 QFT- G의특이도가낮게나왔을가능성을언급하고있다 47. 본연구자의경험에의하면활동성결핵환자를대상으로하여 ROC (receiver operator characteristic) curve 분석을통해계산된 cutoff를이용하여민감도 및특이도를구해보면기존의 cutoff 수치에비해 QFT-G와 T SPOT 검사간의일치도가향상되는것을관찰할수있었다 48. 이러한현상이물론검사법자체에의한차이일수도있으나한편으로는 TST 결과판독때와마찬가지로환자의면역상태나각지역의결핵의유병률등에따라 cutoff 값의조정이필요할가능성을제시하여준다. 그러나, 이를임상에적용하기위해서는다수의환자를대상으로한대규모전향적연구가필요할것이다. 최근 QFT-G 검사와 T SPOT 검사를직접비교한보고자료를살펴보면, 본저자의연구에서는 T SPOT 검사 (95.4%) 가 TST (66.7%) 나 QFT-G (70.1%) 에비해높은민감도를, QFT-G (91.6%) 가 TST (78.6%) 에비해높은특이도를나타내었고, 주로면역억제자에서 QFT-G 및 T SPOT 검사간의민감도차이가큰것을알수있었다 48. Ferrara 등은잠복결핵이나활동성결핵이의심되는환자를대상으로두검사를임상에서시행한자료를비교하였는데, T SPOT 검사가 QFT-G 에비해민감도가높고 indeterminate result 의비율은낮은것으로보고하였다 49. 그러나잠복결핵의확진방법이아직은없기때문에이러한결과가 T SPOT의민감도가높아서인지혹은특이도가낮아서인지추가확인이필요하다. (4) 치료후추적검사로 IFN-γ 검사의유용성 Stuart 등은활동성결핵및잠복결핵환자를대상으로각각 12개월간의병합치료및 isoniazid 예방치료를시행하였으나 QFT 검사결과에차이가없었고 50, 또다른 QFT 연구에서도 1개월간의항결핵치료는 QFT 결과에영향을미치지않았다 28. 그러나 ESAT-6 또는 ESAT-6/CFP-10을이용한 ELISPOT 연구에서는항결핵치료후 IFN-γ 를분비하는 T 림프구의수가감소함을보여주었다 30,36. Carrara 등도 ESAT-6 펩타이드를이용한 ELISPOT 연구에서항결핵치료 3개월시점에세균학적및임상적호전을보이는환자에서는 IFN-γ 를분비하는 T 림프구의수가감소하였고반응이없는환자에서는감소하지않아치료에대한반응을평가하는데유용할가능성을제시하였다 51. 반면에이연구에서도 PPD를이용 503

8 JY Lee et al. : Diagnosis of Mycobacterium tuberculosis infection using ex-vivo interferon-gamma assay 한 ELISPOT 검사는치료 3개월시점에일관되지않은반응을나타내었다 51. 최근불가리아인을대상으로한연구에서도비록대상환자수는적었으나 37.5% (3/8) 의환자에서치료후양성에서음성으로전환됨을보고하기도하였다 52. 한편, Aiken등이잠비아인을대상으로한연구에서는 T SPOT 검사를이용하여 6 개월간의항결핵치료를완료한환자 ( 총 82명 ) 의대부분은치료전에비해치료 12개월째추적검사에서는양성에서음성으로전환되거나 SFC가감소됨을보고하여 53 치료반응의평가에이용가능함을제시하여주었다 (10 SFC를 cutoff 로사용 ). QFT-G를이용하여치료의반응을평가한보고는아직없으나검사법을개발한 Rothel 박사에의하면 QFT-G도 ESAT-6와 CFP-10에대한 IFN-γ 반응이치료후에감소한다고하였다 ( 개인교신 ). 이는일반적으로알려진사실, 즉결핵치료초기에는결핵자체에의한면역기능억제로잠복결핵환자보다 IFN-γ 분비능이낮다가치료하면서면역기능이회복되어 IFN-γ 분비능이증가하는현상, 과모순되는결과를보여준다. 실제로일부연구에서는항결핵치료하면서 IFN-γ 분비가증가하는것을보여주었다 54. 현재상품화되어있는두가지의 IFN-γ 검사법을이용하였을때치료후 IFN-γ 분비능이감소할것이라는이론적인근거는두가지검사모두 16-24시간의배양기간을원칙으로하기때문이다. 이짧은기간동안에는결핵균의항원을인지할수있는효과세포 (effector cell) 가있어야만 IFN-γ 를분비할수있다고생각하고있다. 즉결핵이치료되면서대부분의결핵균이사멸함에따라효과세포는대부분기억세포 (memory cell) 로바뀌기때문이다. 기억세포가다시결핵균항원에노출되면효과세포로바뀌어 IFN-γ 를분비하게되기때문에 IFN-γ 를분비하기위해서는 16-24시간의배양시간보다는더오랜시간이필요하다. 효과세포의존재는최근항원에노출되어야만지속적으로유지될것이므로 55 ELISPOT 양성은체내에살아있는균이존재함을, 즉잠복결핵을의미하고과거에결핵균에노출되었던것만으로는양성반응을보이지못한다 30. 물론체내에살아있는균이없어도가지세포 (dendritic cell) 내에있는 ESAT-6 와 CFP-10 항원이지속적으로분비될가능성도고려할수있으나항결핵치료후항원특이 T 림프구의수가급격히감소하는것으로보아항원특이 T 림프구는체내에살아있는결핵균의존재에의해서유지될가능성이많다 30. 따라서 PPD와같이결핵균에비특이적인항원을사용하였거나 56 아니면 ESAT-6 같은결핵균-특이항원을사용하였더라도 24시간이상장기간배양하였으므로다양한결과를나타냈을가능성이있다 54,57. 반면 QFT-G나 T SPOT은 24시간이내로배양하므로기억세포들이 IFN-γ 를분비하기에는시간이부족하여치료후결핵균수가줄어들면서효과세포의수가감소하여 IFN-γ 반응이줄어드는것으로생각된다 58,59. 결핵균항원특이항체를생성하는 B 세포도치료에따라균배양음전보다도빠르게감소하는것으로보고되었다 60. 그러나이러한이론적인배경과보고에도불구하고본연구자의경험으로는결핵환자의치료시 QFT-G 는치료종료시농도가치료시작시와비교하여변화가없거나오히려증가하는양상을보이기도하였다. T SPOT은많은예에서 SFC가감소하는양상을보이기는하였으나완전히음성으로변하는경우는아주적었다 61. 향후이에대한추가연구가필요하다. (5) 활동성결핵의진단검사로서 IFN-γ 검사의유용성 Pathan 등은건강접촉자, 폐외결핵, 도말음성폐결핵및도말양성폐결핵군을비교하였을때항원의양은순서대로증가할것이나 ESAT-6 특이림프구의수는역상관관계가있음을보여주었다 36. 이에대한설명으로는이 T 림프구들이체내에서결핵균을억제하는역할을하기때문일것으로생각된다. 즉 T 림프구반응이강한사람 ( 건강접촉자 ) 은결핵균의성장을잘억제하여건강접촉자로유지될것이고, T 림프구의반응이약한사람은균수가증가하여결핵 ( 도말양성폐결핵 ) 이발병할것이다 36. 그러나아직까지활동성결핵과잠복결핵을구분하기위한 IFNγ 또는 SFC의기준치 (cutoff value) 를제시한연구는없었다. 504

9 Tuberculosis and Respiratory Diseases Vol. 60. No. 5, Mar 이론적으로나현재까지의연구결과를토대로하였을때체외 IFN-γ 검사가활동성결핵과잠복결핵을구분하는데국내에서이용되기는어려울것으로보인다. 그러나두가지가능성을제시한다면다음과같다. 첫째, 결핵이의심되는임상상또는방사선학적소견이있는면역적격자에서체외 IFN-γ 검사가음성이라면결핵을배제하는데유용할가능성이있다. 이러한목적으로이용되기위해서는 IFN-γ 검사의민감도가높아야만한다. ESAT-6 만을이용한 Lalvani 등의 ELISPOT 연구에서는민감도 96% 30, QFT-G를이용한 Mori 등의연구에서는민감도 89% 17 를보여주었지만아직체외 IFN-γ 검사와 TST 중어느방법이결핵감염을진단하는데더민감도가높은지에는이견이있는상태이다. 향후결핵균에특이적인항원들이더발견되어이항원들을기존의 ESAT-6와 CFP-10에포함시키면민감도를더높일가능성이있다. 향후이에대한추가연구가필요할것이다. 둘째, 결핵감염의위험성이적은군에서는결핵감염의진단자체를결핵진단의보조도구로사용할수있다. 예로일반적으로어린이는결핵균에감염되었을가능성이낮기때문에 IFN-γ 검사로결핵감염이확인되면결핵이의심되는병이실제로결핵일가능성이높아지게된다. 또한결핵의유병률이낮은지역에서도마찬가지이다. Liebeschuetz 등은 HIV 와결핵감염률이높은아프리카에서결핵이의심되는어린이를대상으로 ELISPOT 검사법의활동성결핵진단의유용성을평가하였다. ELISPOT 검사의민감도는 83% 로 TST의민감도 63% 보다유의하게높았고, 이러한결과는특히어린이에서결핵진단이어른에서보다어렵기때문에더의미가크다고할수있겠다 62. Ravn 등은활동성결핵이의심되는환자를대상으로하였을때 QFT-G가항산균도말검사에비해활동성결핵을진단하는데더민감도가높고 (85% vs. 42%, 각각 ) 특히폐외결핵에서는민감도가각각 92%, 31% 로더높아, 항산균도말및배양검사와 QFT-G 검사를병합하면민감도를 96% 까지올릴수있다고하였으나, 이연구가결핵감염률이낮은덴마아크에서이루어진연구이기때문에가능했던것으 로보인다 63. 예로국내에서이검사가시행된다면성인폐렴환자를대상으로하더라도많은환자가이미결핵균에감염되어있으므로 IFN-γ 검사에서양성으로나올것으로예측된다. 결핵은감염의각단계에따라합성, 분비되는항원이달라지므로이를이용하여활동성결핵과잠복결핵혹은최근감염과진구성감염을구분하고자하는시도가진행되고있다. 그러나, 아직결핵의유병률이높은지역에서결핵이의심되는성인을대상으로결핵진단의목적으로 IFN-γ 검사를시행한결과는보고된바없다. 6) 향후전망체외 IFN-γ 검사를이용한접촉자연구에서처음에는 ESAT-6 39 만을항원으로사용하다가이후 ESAT-6 와 CFP-10을같이사용하였다 40. 이처럼항원을복합으로사용하면민감도를높일수있는데 23,64 이는개인마다 HLA-DR형이다르므로항원에대한반응이다르기때문인것으로알려져있어 23 향후결핵균에특이적인항원의추가적인발견은체외 IFNγ 법의민감도를더욱높여줄수있을것으로기대되고있다 65,66. 최근스위스에서발표된비용-효과연구에의하면접촉자추적검사에 TST를이용하는것에비해 T SPOT만사용하거나, TST와 T SPOT을병합한경우 (TST양성인경우 T SPOT으로확진 ) 가더비용- 효과적인절감을가져왔다고보고하였다. 이는정확한잠복결핵의진단으로인하여불필요한예방치료를줄임으로써효율의상승을가져온것으로분석되었다 67. 그러나각지역에따라잠복결핵치료의정책이다르므로이에따라비용-효과분석결과는다를것이다. 향후이에대한연구들이지속되리라생각된다. Doherty 등은말초혈액의단핵세포를 ESAT-6 로자극하여 5일간배양후상청액의 IFN-γ 를 ELISA로측정한결과비록대상군수는적었지만결핵환자접촉자중에서 IFN-γ 반응이높았던군에서 2년추적검사후결핵의발병률이높아서 ESAT-6 를이용한체외 IFN-γ 검사법이결핵감염뿐만아니라향후 505

10 JY Lee et al. : Diagnosis of Mycobacterium tuberculosis infection using ex-vivo interferon-gamma assay 발병할위험군을찾아내는방법으로도도움이될것으로보고하였다 68. 향후체외 IFN-γ 검사를시행한환자들을장기간추적하여이결과와환자의임상상의관계를밝히려는노력또한필요할것이다. 이외에상기에서언급한바와같이활동성결핵진단에의유용성, 항결핵치료반응의평가에서의유용성, 면역억제자에서의결핵감염진단에서의유용성등에대한연구가지속되리라생각된다. 5. 결론 최근개발되어상업화된체외 IFN-γ 검사들은이론적으로나지금까지의연구결과를토대로하여볼때결핵감염의진단에서 TST 보다유용할것으로생각된다. 주된장점은비씨지접종또는비결핵항산균감염에의한위양성을줄일수있다는점이지만 TST보다민감도가높을가능성도제시되고있고, 새로운결핵균-특이항원들이개발되어기존의항원들에추가된다면민감도는더욱높아질가능성도있다. 그러나세계각지역마다결핵의역학상황에차이가있으므로각상황에맞는적절한사용이필요할것으로생각되며, 아직은비용이제한점으로작용할가능성이많다. 이와관련된향후활동성결핵진단에서의유용성, 치료반응평가에있어서의유용성, 각지역의역학또는인종등에따른기준치 (cutoff value) 의설정의차이등추가적인분야들에대한연구가지속되어야할것으로생각한다. 참고문헌 1.Cooper AM, Dalton DK, Stewart TA, Griffin JP, Russell DG, Orme IM. Disseminated tuberculosis in interferon gamma gene-disrupted mice. J Exp Med 1993;178: Newport MJ, Huxley CM, Huston S, Hawrylowicz CM, Oostra BA, Williamson R, et al. A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection. N Engl J Med 1996; 335: Wongtim S, Silachamroon U, Ruxrungtham K, Udompanich V, Limthongkul S, Charoenlap P, et al. Interferon gamma for diagnosing tuberculous pleural effusions. Thorax 1999;54: Ogawa K, Koga H, Yang B, Fukuda M, Ohno H, Yamamoto Y, et al. Differential diagnosis of tuberculous pleurisy by the measurement of cytokine concentration in pleural effusion. Kekkaku 1996;71: Barnes PF, Fong SJ, Brennan PJ, Twomey PE, Mazumder A, Modlin RL. Local production of tumor necrosis factor and IFN-gamma in tuberculous pleuritis. J Immunol 1990;145: Ribera E, Martinez Vasquez JM, Ocana I, Ruiz I, Jiminez JG, Encabo G, et al. Diagnostic value of ascites gamma interferon levels in tuberculous peritonitis: comparison with adenosine deaminase activity. Tubercle 1991;72: Burgess LJ, Reuter H, Carstens ME, Taljaard JJ, Doubell AF. The use of adenosine deaminase and interferon-gamma as diagnostic tools for tuberculous pericarditis. Chest 2002;122: Ribeiro-Rodrigues R, Resende Co T, Johnson JL, Ribeiro F, Palaci M, Sa RT, et al. Sputum cytokine levels in patients with pulmonary tuberculosis as early markers of mycobacterial clearance. Clin Diagn Lab Immunol 2002;9: Tsao TC, Huang CC, Chiou WK, Yang PY, Hsieh MJ, Tsao KC. Levels of interferon-gamma and interleukin-2 receptor-alpha for bronchoalveolar lavage fluid and serum were correlated with clinical grade and treatment of pulmonary tuberculosis. Int J Tuberc Lung Dis 2002;6: Koh WJ, Kwon OJ, Suh GY, Chung MP, Kim H, Lee NY, et al. Six-month therapy with aerosolized interferon-gamma for refractory multidrug-resistant pulmonary tuberculosis. J Korean Med Sci 2004;19: Suarez-Mendez R, Garcia-Garcia I, Fernandez-Olivera N, Valdes-Quintana M, Milanes-Virelles MT, Carbonell D, et al. Adjuvant interferon gamma in patients with drug-resistant pulmonary tuberculosis: a pilot study. BMC Infect Dis 2004;4: Condos R, Rom WN, Schluger NW. Treatment of multidrug-resistant pulmonary tuberculosis with interferon-gamma via aerosol. Lancet 1997;349: Kim EK, Shim TS, Lee JY, Oh YM, Lim CM, Lee SD, et al. The adjuvant effect of subcutaneous interferon-gamma in the treatment of refractory multidrug-resistant pulmonary tuberculosis. Tuberc Respir Dis 2004;57: Mazurek GH, LoBue PA, Daley CL, Bernardo J, Lardizabal AA, Bishai WR, et al. Comparison of a whole-blood interferon gamma assay with tuberculin 506

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