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1 Biomedical Science Letters 2013, 19(4): 330~337 eissn : Original Article The Rapid Drug Susceptibility Testing of Mycobacterium tuberculosis by GenoType MTBDRplus in Contaminated Specimen Reun Heo 1 and Yoon-Sik Kim 2, 1 Department of Laboratory Medicine, Korean National Tuberculosis Association, Gwangju , Korea 2 Department of Biomedical Laboratory Science, Dongkang College, Gwangju , Korea There are several methods currently being used to diagnose tuberculosis in patients, such as smear, PCR, tuberculosis culture and X-ray. For a proper medical treatment, antimicrobial susceptibility test and rapid drug susceptibility testing have been operated. Tuberculosis bacilli usually need 3~8 weeks of culture period because of delay in RNA synthesis and require 15~22 hours for generation. After a germ raises in culture, we initiated antimicrobial susceptibility test for a proper treatment. It has some difficulties to give a proper prescription for a tuberculosis patient because antimicrobial susceptibility test requires 4 weeks. To supplement this, we are practicing drug susceptibility testing which allow us to know the sensibility of RMP and INH after 2 or 3 days. But this is only possible when more than 2 positive germ. Therefore, we should practice rapid drug susceptibility testing with culture test. But if media is contaminated by other germs except Mycobacterium tuberculosis, it's hard to interpret result about culture test and to practice antimicrobial susceptibility test and rapid drug susceptibility testing. Because we have to practice again smear, culture test after extracting specimen from the patient, time is consumed and proper patient treatment is postponed. To address these problems and quick patient treatment, rapid drug susceptibility testing is practiced by using GenoType MTDRplus method. As a result of this method we detected sensibility 10 and 7 cases and resistance 0 and 3 cases using RIM and INH respectively with other 1 case toward medicals out of the total 11 test. In conclusion rapid drug susceptibility testing can be used from the contaminated specimen after elimination of contaminated source from culture and proved that it can be practiced for rapid examination of a tuberculosis patient. Key Words: Mycobacterium tuberculosis, GenoType MTBDRplus, Antimicrobial susceptibility test 서 결핵균군중사람에결핵을일으키는원인균을 1882년 Robert Koch에의해서분리동정되었으며, 1896년 Lehmann와 Neumann에의해 Mycobacterium tuberculosis라명명되었고현재는유전자염기서열까지완전하게해독되었다 (Cole et al., 1998: Arnvig and Young, 2009). 결핵균은 * Received: December 10, 2013 / Revised: December 23, 2013 Accepted: December 26, 2013 Corresponding author: Yoon Sik Kim. Department of Biomedical Laboratory Science, Dongkang College, Gwangju , Korea. Tel: , Fax: kys2982@naver.com C The Korean Society for Biomedical Laboratory Sciences. All rights reserved. 론 그람양성으로항산성 (acid fast) 염색성을나타낸다. 인공배지상에서발육한결핵균을보면분열증식한균들이서로엉켜붙어균덩어리가마치뱀처럼보인다 (Toman, 1979). 형태적으로도 20 nm 두께의세포벽에는 60% 이상이미콜산과같은독특한지방산이주축이되는지방화합물을함유하고있어염료가잘침투하지않아석탄산과같은매염제를첨가해주어야염료가지방에용해되어균세포내로확산되어염색이된다. 균세포내로침투한염료는산성알콜과같은강한탈색제로처리해도탈색되지않고남게되기때문에항산성염색성이라한다 (Barksdale and Kim, 1977). 굵기는 0.2~0.5 μm, 길이가 2~5 μm 정도되는막대균으로운동성도없고내생또는외생포자도생산하지않는다 (Grange, 1992). 결핵의진단방법으로는도말검사, 임상가검물과파라핀

2 포매조직을이용한 PCR 검사, 균배양검사, X-ray 촬영등으로폐결핵의진단과치료경과를관찰하고있으며적절한치료를위해서는약제감수성검사등을통해환자에서검출된균의약제내성여부를판단하여적절한처방으로환자를치료하는데사용된다 (Kim et al., 2000; Imperiale et al., 2012). 도말검사는비용이저렴하고검사결과를신속하게얻을수있다. 그러나광학현미경으로 1,000배율 300시야정도를검경하였을때 3~9개의균이있어야양성으로보고를낼수있는데최소한객담 1 ml 에 1~3만개이상의균이있어야현미경관찰이가능하기때문에민감도는배양검사보다떨어지는단점이있다 (Groothuis and Yates, 1991). 배양검사는균을검출할수있는민감도가도말검사에비해훨씬높아객담 1 ml에발육가능한균이 10개만있어도검출된다. 그리고균을분리배양해야정확한균종을동정할수있고각종항결핵제에대한감수성검사도실시할수있기때문에매우중요한검사방법이다. 그러나결핵균의세대증식시간이 15~22시간이라는특성상검사결과를얻기까지 3~8주정도의시간이걸리고검사조작과정이복잡하고어려워숙련된검사자에의해서만수행되고있으며많은기자재를필요로하는단점이있다 (Jin et al., 1989). 이러한문제점을보완한방법으로 DNA 중합효소연쇄반응검사법인 PCR 검사법이쓰이고있다 (Mullis and Faloona, 1987). PCR 검사법은결핵균처럼분리배양에오랜시간이걸리는병원균의검출에매우유용하다 (Saiki et al., 1988; Saiki et al., 1992). 약제감수성검사는환자치료에적절한약제를처방하는데매우유용하며특히초기치료에실패한환자에대해서는감수성검사결과가유용한재치료처방선정에도움이된다. 일반적인방법으로는절대농도법, 내성비례법, 내성비율법등이있으나대부분의검사실이절대농도법과내성비율법을이용한다 (Padungchan et al., 1986). 그러나약제감수성검사는 4주정도의시간이걸린다는단점이있다. 신속감수성검사법은약제감수성검사의검사결과동정까지시간이오래걸린다는단점을보완하고적절한처방을위해매우유용하게사용되어지고있으며최근 2가지이상의약제에내성이있는다제내성결핵이증가하여문제화되면서좀더빨리약제감수성검사결과를얻고자하는필요성대두되었다 (Jeon et al., 2008). 결핵의 1차치료약제중대표적인리팜피신 (rifampicin, RMP) 과아이나 (isoniazid, INH) 에대한내성은다제내성의보조지표로이용되고있기때문에현재이두약제에대한내성여부를 2~3일안에신속하게확 인할수있는검사법이결핵환자의치료에효과적으로사용되어지고있다 (Vijdea et al., 2008; Lei et al., 2010). 그러나소량의균으로는검사가불가능하기때문에배양검사를실시한후신속감수성검사를실시하게된다. 그러나배양중에배지가결핵균이외의다른세균에의해오염이된경우는배양검사에대한결과판독과약제감수성검사및신속감수성검사를실시할수없게된다. 그러면처음부터환자로검체를다시채취하여도말, 배양검사를재실시하게되므로인해시간이많이소요되어그만큼적절한환자치료가지연되게된다. 본연구에서는배양시발생되는오염에대한문제점을보완하여다제내성결핵환자의확산방지와신속한환자치료를위해도말검사양성검체중에서배양기간중에오염이된검체에서도신속감수성검사가가능한지를알아보기위한실험을실시하였다. 재료및방법검체 2% Ogawa 배지에배양한검체총 18,241건중결핵균양성 396 (2.17%) 건, 음성 17,485 (95.85%) 건, 오염 173 (0.95%) 건, 비결핵항산균 (nontuberculous mycobacteria, NTM) 187 (1.02%) 건으로나타났다. 실험에이용된검체는배양시오염이발생된 173건검체중도말검사양성이나온 13건에대해재채담의뢰해재채담한결과도말검사음성 1건, 양성 11건, 미의뢰 1건등을보였다. 재채담한도말검사상양성으로나온검체의오염검체 11건을연구검체로이용하였다. 도말검사와배양도말검사는형광염색시약인 Auramine-Rhodamine으로염색하였고, 탈색액으로는 1% HCL-alcohol을사용하였으며, 대조염색액으로 0.2% 과망간산칼륨 (KMnO 4 ) 수용액을사용하였다. 배양용으로멸균된담통에받은객담에 4% NaOH를동량을가한다음 2% Ogawa 배지 2본에 0.1 ml씩접종하여발육유무를관찰하였다. 오염원제거 2% Ogawa 배지에서오염원이발견된즉시 4% NaOH 와 2.9% 구연산나트륨을동량으로첨가하여소화시켜균질화한용액 100 ml당 0.5 g N-Acetyl-L-Cysteine (NALC)- NaOH 분말을첨가하였다. 모아진검체를 50 ml 원심분리

3 Table 1. rpob gene of mutation and wild type Failing wild type probe(s) Codons analyzed probe rpob WT1 505~509 F505L T508A S509T rpob WT2 510~513 L511P * rpob WT2/WT3 510~517 Q513L * Q513P del rpob WT3/WT4 513~519 rpob MUT1 D516V D516Y del515 rpob WT4/WT5 516~522 del518 * N518l rpob WT5/WT6 518~525 S522L S522Q rpob WT7 526~529 rpob MUT2A H526Y rpob MUT2B H526D H526R H526P * H526Q * H526N H526L H526S H526C rpob WT8 530~533 rpob MUT3 S531L S531P S531Q * S531W L533P Fig. 1. Multiplex-PCR. M: 100 bp ladder (Seegene Cat. No. M0100), 1: Internal control, 2~5: Mycobacterium tuberculosis, 6: Mycobacteria, 7: Mycobacterium tuberculosis Failing wild type probe(s) Table 2. katg gene of mutation and wild type Codon analyzed 상층액을 PCR 반응에사용하였다. probe katg WT 315 katg MUT1 S315T1 Failing wild type probe(s) katg MUT2 Table 3. inha gene of mutation and wild type Codon analyzed Mmutation probe S315T2 inha WT1-15 inha MUT1 C15T -16 inha MUT2 A16G inha WT2-8 inha MUT3A T8C inha MUT3B T8A 신속감수성검사 시험관에분주한다음검체와동일한양으로 NALC 용액을첨가하여혼합하였다. 1, 000 g의속도로 15분동안원심분리하여검체를농축한다음조심스럽게상청액을제거하고최종양이 1~3 ml이되기까지멸균된피펫을사용하여 PBS (ph 6.8) 로침전물을재부유하여이용하였다. DNA 추출채취한검체를멸균된증류수 300 μl에재부유한다음부유한박테리아를 vortex로잘혼합하여 100 에서 20분간가열하여비활성화시킨다음 60 에서 15분동안초음파처리하였다. 10,000 g로 5분동안원심 분리하여 신속감수성검사에는 GenoType MTBDRplus kit (Hain Lifescience GmbH, Hehren, Germany) 를이용하여분석하였다. GenoType MTBDRplus법은 DNA STRIP 기술을기초로하여검체 1개당 PNM, 10x PCR Buffer, 25 mm MgCl 2, 1 U Host StarTaq, 멸균된증류수를혼합한 PCR 마스터혼합액 45.2 μl에 DNA 추출액 5 μl과혼합하여다중중합효소연쇄반응 (Multiplex-PCR) 을실시한후인형결핵균과비인형결핵균으로판정하였다 (Fig. 1). 인형결핵균으로판정된다중중합효소연쇄반응증폭산물은 rpob, katg, inha의야생형과돌연변이형에특이한탐침자가접합된스트립에역교잡시켜리팜피신과아이나내성을동시에진단하였다 (Table 1, 2, 3). 인형결핵균으로판정

Fig. 2. GenoType MTBDRplus an analysis table. Representative patterns obtained by the GenoType MTBDRplus test for isolates with dual mutations in the rpob gene based on sequencing.

4 Fig. 2. GenoType MTBDRplus an analysis table. Representative patterns obtained by the GenoType MTBDRplus test for isolates with dual mutations in the rpob gene based on sequencing. The positions of the olignucleotides and the marker lines are given. The targeted genes and specificity are shown from left to right, as for the MTBDR assay, conjugate control (CC); amplification control (AC); M. tuberculosis complex-specific control (TUB). Table 4. Results from polymerase chain reaction Polymerase chain reaction Negative Positive Doubt (NTM) 11 2 PCR analysis were determined as described in materials and methods. 검체를 GenoType TBDRplus법을이용하여교잡과정을거쳐증폭산물을 rpob, katg, inha의야생형과돌연변이형에특이한탐침자가접합된스트립에역교잡시켜리팜피신과아이나에대한내성검사를실시한뒤 GenoType MTBDRplus 결과분석표및판정지를참조하여결과를분석하였다 (Fig. 2, 3). 결 과 오염된검체의 DNA 중합효소연쇄반응검사 (PCR) 도말검사양성중배양검사오염으로판정되어진검체를인형결핵균인지비결핵항산균인지를감별하기위해오염원을제거하고검체 1개당 PCR 마스터혼합액을조제한다음모든 PCR 시험관에 PCR 마스터혼합액 45 μl씩을분주하고추출된 DNA 5 μl씩을각각의 PCR 시험관에분주한후 PCR 반응을시켜실시한결과총 13 건중인경결핵균 11건, 음성 ( 비결핵항산균의심 ) 2건의 Table 5. Results of rapid drug susceptibility testing Rapid drug susceptibility test Sensibility Resistance Other RMP (ill defined) INH 7 3 결과를얻을수있었다 (Table 4). 신속감수성검사신속감수성검사는배양검사오염으로판정된검체를오염원을제거하고, PCR 검사를통해인형결핵균으로판정된 11검체를 GenoType MTBDRplus법을이용분석한결과는 CC, AC, TUB, Locus Control은모두양성일때나머지밴드로결과를판독하고 AC 음성일경우는 PCR 단계가잘못되었거나 PCR 저해제의영향을받았을가능성이있으므로재시험을수행하였다. rpob는모든 WT가양성이고 MUT가음성이면리팜피신감수성으로, WT가음성이고 MUT가양성과 WT이음성이고 MUT가음성이면리팜피신내성으로판정하였다. katg는모든 WT가양성이고 MUT가음성이면아이나감수성으로, WT가음성이고 MUT가양성과 WT가음성이고 MUT가음성이면아이나내성으로판정하였다. inha는모든 WT가양성이고 MUT가음성이면아이나감수성, WT가음성이고 MUT가양성과 WT가음성 MUT가음성이면아이나내

Fig. 3. Rapid drug susceptibility test a result table. RMP/INH: 1 S/S, 2 S/S, 3 nontuberculous mycobacteria, 4 S/S, 5 S/R, 6 S/S, 7 S/R, 8 S/S, 9 S/R, 10 S/S, 11 S/S, 12 Negative control.

5 Fig. 3. Rapid drug susceptibility test a result table. RMP/INH: 1 S/S, 2 S/S, 3 nontuberculous mycobacteria, 4 S/S, 5 S/R, 6 S/S, 7 S/R, 8 S/S, 9 S/R, 10 S/S, 11 S/S, 12 Negative control. sensibility (S); resistance (R). 성으로판정하였다 (Fig. 3). 이러한결과를기준으로검사한결과검체 11건중리팜피신내성은 0건, 감수성은 10건, 아이나내성은 3건, 감수성은 7건, 원인불명 1건의결과를얻었다 (Table 5). 신속감수성검사결과보고일까지첫번째채담검체와재채담검체와의소요일수비교검체중에도말검사결과양성으로배양검사오염으로판정된경우해당검체에대해재의뢰요구를하여재의뢰된검체를다시도말검사와배양검사그리고신속감수성검사를실시하는데걸리는소요일수와처음의뢰되었던검체가배양검사에서오염이되었다하더라도오염원을제거하고신속감수성검사를실시하여검사결과를보고하기까지의소요일수를비교분석한결과대부분의검체의오염확인은배양후평균 11.8일째발생하였으며, 재채담하여검체의결과를분석하기까지지체된 평균시간은 12.2일이더소요됨을알수있었다 (Table 6). 고찰대한결핵협회자료를보면우리나라는 1965년실태조사때는연간결핵감염위험률이 5.3% 이던것이체계적인결핵관리사업등으로유병률이꾸준히감소하여 1995년에는 0.5% 로감소되었다. 그러나보건소등록환자를대상으로한약제내성률은 1999년에는 11.3% 였으며 2004년에는 12.8% 로변화가있었고다제내성환자의비율도 1.6% 에서 2.7% 로유의하게증가하였다 (Bai et al., 2007). 우리나라의결핵은비록감소추세에있지만다른국가와비교할때아직심각한것은분명하다. 최근고령층의증가, 외국인근로자의지속적인유입, HIV 감염자의증가, 북한주민의유입과교류활성화등을고려하면향후에는감소추세의둔화내지일시적인증가추세로전

6 Table 6. Comparison of the time required both first sputum and second sputum (unit: day) Number Contamination day A Results PCR R.D.S B day Interim closing C Request day Total day D Delay day E No request No test No test A : smears + culture day : Interim closing + Request day B : Rapid Drug Susceptibility Testing : Total day - Interim closing C : Contamination day + R.D.S day 환할것으로보고있으며, 특히유의할점은다제내성환자의증가와 10~20대등을비롯한젊은층의결핵환자증가에관심을가지고지켜봐야할것으로본다. 결핵의주전염원은객담도말양성인폐결핵환자가기침, 재채기, 말, 노래등을할때결핵균이들어있는비말핵이배출되어이공기중에떠있다가다른사람의호흡기도로들어가서호흡세기관지나폐포에도달하면감염을일으킨다 (Collins et al., 1985; Grange, 1988). 도말검사는기법이간단하고저렴하며어디서나쉽게이용할수있고결과가빨라환자관리에편리하며, 도말검사로발견되는환자가그지역사회의결핵감염원이므로빨리찾아내어치료해야할뿐만아니라임상증상도심해치료를시급하게요하는환자들이다 (Blair et al., 1976; Toman, 1979). 배양검사는결핵균을동정할수있어서확실한진단이가능하고소수의균도검출할수있으며약제감수성검사를위해서는반드시필요하지만균의세대증식기간이길어결과보고까지시간이오래걸리는단점이있다 (Kim et al., 1989; Padungchan et al., 1986). 약제감수성검사는환자치료시에각항결핵제에대한감수성여부를검사하여적절한치료처방을위해아주유용한검사다. 그러나시간이오래걸린다는단점이있다 (Kent and Kubica, 1985). DNA 중합효소연쇄반응법은고온균으로부터얻은 DNA 중합효소를이용한 DNA를증폭합성법은민감도와특이도가매우높아각종병리가검물내병원 균검출에널리이용된다 (Saiki et al., 1988; Arrud et al., 1993). 신속감수성검사는다중중합효소연쇄반응후증폭산물을 rpob, katg, inha의야생형과돌연변이형에특이한탐침자가접합된스트립에역교잡시켜리팜피신과아이나내성을동시에진단하는것으로 2~3일만에내성여부를알수있기때문에신속하고적절한처방을하는데매우유용하게쓰인다 (Pietrzak et al., 1994; Vijdea et al., 2008). 본연구는요즘다제내성결핵균이증가하여사회적, 국가적으로민감한상황에당면하고있음을인식하여조기에내성균감염자를찾아내어신속하고적절하게치료할수있게하여더이상의내성균의전파를막고환자에게도빠른완치로행복하고건강한삶을영위할수있도록도움을주기위해서실시하게되었다. 현재까지도말검사결과양성인검체중에배양검사에서잡균에의한오염으로판독이불가능한경우에는다시재채담의뢰를하여검사를할수밖에없는문제점이있었다. 그만큼시간적, 경제적으로환자에게피해가가는것을방지하고환자의빠른완치를위해서오염된균주에서오염원을제거하여신속감수성검사를실시하게되었고처음의뢰되어서배양중에오염되었던검체를신속감수성검사를실시하여그결과를얻기까지의시간과오염된검체의대상자에게재채담을요구하여다시도말검사, 배양검사의과정을거치면서신속감수성검사를하기까지의소요

7 되는일수를서로비교 분석한결과재채담하여검체의결과를분석하기까지지체된평균시간은 12.2일이더소요됨을알수있었다. 배양중에오염된검체를분리하여도말검사결과를확인후양성인경우에는오염원을제거하여다중중합효소연쇄반응을실시하여인형결핵균인지를확인하였더니인형결핵균 11건, 비결핵항산균 1건, 검사불능검체 1건등으로확인되었다 (Table 4). 인형결핵균으로확인된검체의다중중합효소연쇄반응증폭산물을 rpob, katg, inha의야생형과돌연변이형에특이한탐침자가접합된스트립에역교잡시켜리팜피신과아이나내성여부를검사하였더니 Table 5와같이리팜피신에는 0건이내성이고 10건이감수성이였고, 아이나에서는 3건이내성이고 7건이감수성으로보였으며검사불능 1건의결과를얻었다. 첫번째의뢰된검사의도말검사의결과와재의뢰시도말검사결과를비교한결과총오염된 13건중도말검사결과일치가 11건, 불일치가 1건, 미의뢰 1건의결과를얻게되었다. 본연구에서지금까지는배양중에오염이발생되면그검체를판독불가로판정하고살균처리후폐기하고다시재채담검체를의뢰를요구하여처음부터검사를다시해야하는번거로움과시간적, 경제적인문제를야기시켰으나배양중오염이되더라도오염원을제거하고신속감수성검사를실시할수있음을확인하고, 이러한실험결과를통해인형결핵환자의보다빠른치료와적절한처방으로결핵환자를치료에큰도움이될수있기를기대한다. REFERENCES Arnvig KB, Young DB. Identification of small RNAs in Mycobacterium tuberculosis. Mol Microbiol : Arruda S, Bomfim G, Knights R, Huima-Byron T, Riley LW. Cloning of an Mycobacterium tuberculosis DNA fragment associated with entry and survival inside cells. Science : Bai GH, Park YK, Choi YW, Bai JI, Kim HJ, Chang CL, Lee JK, Kim SJ. Trend of anti-tuberculosis drug resistance in Korea, Int J Tuberc Lung Dis : Barksdale L, Kim KS. Mycobacterium. Bacteriol Rev : Blair EB, Brown GL, Tull AH. Computer files and analyses of laboratory data from tuberculosis patients. II. Analyses of six years' data on sputum specimens. Am Rev Respir Dis : Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature : Collins CH, Grange JM, Noble WC, Yates MD. Mycobacterium marinum infections in man. J Hyg (Lond) : Grange JM. Lupus and lepros. Int J Lepr Other Mycobact Dis : Grange JM. The mystery of the mycobacterial 'persistor'. Tuber Lung Dis : Groothuis DG, Yates MD (ED). Diagnostic and public health mycobacteriology, 2nd ed. Bureau of hygiene and tropical diseases. London Imperiale BR, Zumárraga MJ, Weltman G, Zudiker R, Cataldi AA, Morcillo NS. First evaluation in Argentina of the GenoType MTBDRplus assay for multidrug-resistant Mycobacterium tuberculosis detection from clinical isolates and specimens. Rev Argent Microbiol : Jeon DS, Shin DO, Kang HS, Sung NM, Kweon KS, Shin E, Kim KS, Lee MH, Park SK. Trend of Multidrug and Extensively Drug Resistant Tuberculosis in a Tuberculosis Referral Hospital, 2001~2005. Tuberc Respir Dis : Jin BW, Hong YP, Kim SJ. A contace study to evaluate the BCG vaccination tuberculosis programme in Seoul Tubercle : 241. Kent PT, Kubica GP. Public Health Mycobaceriology. a Guide for the Level III Laboratory. Atlanta, Georgia, USA: DHHS, CDC Kim EJ, Choi WS, Hwang SY. Detection of Mycobacterim tuberculosis by PCR from trace clinical specimens and paraffin-embedded tissue. Korean J Biomed Lab Sci : Kim SJ, Bai GH, Hwang HD. Tubercle Bacilli in the Sputum Specimens. Tuberc Respir Dis : Lei Z, Yuanxing Y, Lina D, Tingting W, Xingbo S, Xiaojun L, Binwu Y, Lanlan W. Application of Genotype MTBDRplus in rapid detection of the Mycobacterium tuberculosis complex

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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