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1 ORIGINAL ARTICLE Korean J Clin Lab Sci. 2017;49(3): pissn eissn Korean J Clin Lab Sci. Vol. 49, No. 3, September An Evaluation of the Rapid Antimicrobial Susceptibility Test by VITEK MS and VITEK 2 Systems in Blood Culture Kang-Gyun Park 1,2, Young-Bin Yu 2, Keundol Yook 3, Sang-Ha Kim 4, Sunghyun Kim 5, Young Kwon Kim 2 1 Department of Laboratory Medicine, Seoul St. Mary s Hospital, The Catholic University of Korea, Seoul, Korea 2 Department of Biomedical Laboratory Science, College of Medical Sciences, Konyang University, Daejeon, Korea 3 Department of Clinical Laboratory Science, Daejeon Health College, Daejeon, Korea 4 Department of Laboratory Medicine, Konyang University Hospital, Daejeon, Korea 5 Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan, Korea 혈액배양에서 VITEK MS 와 VITEK 2 System 을이용한신속항생제감수성시험의유용성평가 박강균 1,2, 유영빈 2, 육근돌 3, 김상하 4, 김성현 5, 김영권 2 1 가톨릭대학교서울성모병원진단검사의학과, 2 건양대학교의과학대학임상병리학과, 3 대전보건대학교임상병리과, 4 건양대학교병원진단검사의학과, 5 부산가톨릭대학교보건과학대학임상병리학과 The results of rapid antimicrobial susceptibility test (AST) in blood cultures were obtained by inoculating the bacteria directly into the VITEK MS and the VITEK 2 systems without subculturing in the blood culture positive medium. The obtained results were compared with the results using a standard method to evaluate their reliability and accuracy. The direct AST results in blood culture positive specimens were 97.9% (1,936/1,978), consistent with the standard AST results. Gram-positive bacteria showed a concordance rate of 97.2% (1,051/1,081), a very major error rate of 0.5% (5/1,081), a major error rate of 0.1% (1/1,081), and a minor error rate of 2.2% (24/1,081). Staphylococcus epidermidis was the main cause of discordance, and gentamicin (N=9) and fusidic acid (N=8) showed high errors. The overall concordance rate and minor error among the Gram-negative bacteria were 98.6% (885/897) and 1.4% (12/897), respectively. Escherichia coli and Pseudomonas aeruginosa were the major causative bacteria of Gram-negative bacteria. Among them, amoxicillin/clavulanic acid (N=3) showed high error. Direct AST met the CLSI criteria and shortened the reporting time by 24 hours; however, we found that there was a need to perform an addition test via disk diffusion for antimicrobials with very large errors. These results suggest that the method of direct AST in blood culture positive medium may be very useful in efficiently treating patients. Key words: VITEK MS system, VITEK 2 system, Blood culture, Rapid antimicrobial susceptibility test, Bloodstream infection Corresponding author: Young Kwon Kim Department Biomedical Laboratory Science, College of Medical Sciences, Konyang University, 158 Gwanjeodong-ro, Seo-gu, Daejeon 35365, Korea Tel: Fax: ykkim3245@konyang.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2017 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: August 23, 2017 Revised: August 28, 2017 Accepted: August 28, 2017 서론 패혈증은혈류에미생물이감염되어심각한전신반응을유 발하고사망률이높은질환이며, 환자에게경제적부담또한증가시키게된다 [1]. 최근국내에서이루어진다기관연구에서도중증패혈증에의한원내사망률은 34.3%, 중환자실내사망률

2 280 Kang-Gyun Park, et al. Rapid AST in Blood Culture 은 29.1% 로발표되었다 [2]. 자동화된혈액배양시스템을사용하여혈액배양양성미생물을고체배지에계대배양하여집락이형성될때가지일반적으로최소 12시간에서최대 48시간이소요된다 [3,4]. 고체배지에서증식된집락을채취하여자동화동정장비에서 24시간검사하면세균동정과항생제감수성시험 (antimicrobial susceptibility test, AST) 결과가보고되지만최종보고까지 48시간에서 72시간이소요된다. 혈류감염을일으키는미생물의신속한동정및 AST는임상에서항생제를선택하는데있어서중요한역할을한다 [5]. 혈류감염의원인세균을동정하고 AST 시간의단축은경험적인광범위항균제를투여하는대신효율적이고적절한항생제를투여할수있고, 이는환자의임상증상의회복을향상시키는데매우중요하다 [6]. 최근 matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI- TOF MS) 가다양한미생물의동정을위해임상미생물학검사실에도입되었는데, 이장비는몇분안에세균과효모를동정할수있지만, 검사를수행하기위해서는 시간배양된독립된집락이필요하다 [7]. 또한, 세균동정과 AST에소요되는시간을줄이기위해일부연구자들은계대배양을추가로수행하지않고, 혈액배양양성배지에서 MALDI-TOF MS 시스템으로직접미생물을동정하려는시도들이이어지고있다 [3,5-14]. 하지만, 혈액배양양성시료에서직접세균을동정하고 AST를수행하기위해서는시료의준비단계가필요하다. 혈액배양배지에는사람의혈액및배양액에서파생된단백질과같은잔해물들이포함되어있기때문에 MALDI-TOF MS 시스템으로세균동정시미생물의스펙트럼을간섭할수있다. 따라서, 혈구용해액인염화암모늄, 에탄올, 포름산, 아세토니트릴, 트리플루오로아세트산등이추가적으로사용되고있다 [5,6,11,13,14]. 또한, 용해여과법 [3,10], 겔분리법 [7], 시판용검사키트 [8] 를시료준비단계에사용하기도한다. 그러나, 이러한시료준비단계중일부는과정이복잡하고, 시간이많이소요되며, 가격이비싸기때문에경제적이지못하다 [13]. 또한아세토니트릴 (acetonitrile) 의경우, 아세토니트릴의강한냄새때문에시료를준비하고시약을조작하는과정에서흄후드를사용해야하는번거로움이있다. 일부연구에서혈액배양양성검체의직접 AST 수행을시도한바있다 [3,6,9]. 그결과, 직접법으로 MALDI TOF MS와 VITEK 2 시스템을병행할경우, 표준방법보다최대 24시간빠른결과를보고할수있다는연구결과를얻을수있었고, 이를통해혈액배양배지로부터병원균의직접동정뿐만아니라 AST도 유효성이있음을보고하였다 [3]. 하지만, 이러한방법들은에탄올, 포름산, 아세토니트릴 [6], 노동집약적용해여과법 [3] 을사용하거나그람음성막대균만을대상으로평가하였다 [9]. 따라서본연구에서는이러한제한점들을보완하기위해질량분석기와 AST 자동화장비를이용해혈액배양양성배지에서계대배양없이세균동정과 AST를동시에시행하였고, 이러한방법이기존의표준방법과비교했을때, 보다신속하고간단한검사를통해신뢰성있는결과를도출해낼수있는지신속 AST 의신뢰도과정확도를평가해보고자하였다. 재료및방법서울소재 1,300병상규모의상급의료기관에입원한환자의혈액배양검체중양성으로확인된 254개의검체를대상으로연구를수행하였다. 혈액배양양성시료를계대배양없이기존의표준방법과신속 AST 방법을병행하여수행하여그결과를비교하였다. 1. 추출용해액의제조추출용해액제조를위해 9.26% 염화암모늄 (ammonium chloride) 용액 (0.1 ml) 과 2% 사포닌 (saponin) 용액을혼합하였다. 2. 표준방법을이용한항생제감수성시험혈액을채취하여혈액배양용배지인산소성배양배지 (Plus aerobic/f), 무산소성배양배지 (Lytic/10 Anaerobic/F), 소아용배양배지 (Peds Plus/F) (Becton Dickinson, Franklin Lakes, NJ, USA) 에접종하여혈액배양자동화시스템인 BACTEC FX (Becton Dickinson) 에서배양하였다. 혈액배양양성시시료는 sheep blood agar (Asan BAP I; Asan Pharmaceutical, Seoul, Korea) 와 MacConkey agar (Asan Mac II; Asan Pharmaceutical) 에각각 100 L씩접종하여계대배양하였다. Sheep blood agar는 37 C, 5% CO 2, 암실환경에서 24시간배양하였고, MacConkey agar는 37 C, 암실환경에서 24시간배양하였다. 무산소성배양은 sheep blood agar에 100 L를접종하여 37 C 무산소성환경에서 48시간배양하였다. 배양후고형배지에형성된세균집락들은 AST용 VITEK 2 AST-N225 (biomerieux) 카드 Enterococci, Staphylococci, Streptococci, Enterobacteriaceae, Non-fermentative Gram-negative bacteria (NFB) 에접종하여 AST를시행하였다. 최소발육억제농도 (MIC) 결과값은 CLSI 지침에따라감수성 (susceptible), 중

3 Korean J Clin Lab Sci. Vol. 49, No. 3, September 등도도내성 (intermediate) 또는내성 (resistant) 의임상범주로분류하였다 [15]. 3. 혈액배양에서직접법을이용한항생제감수성시험 VITEK 2 시스템에의해총 120개의임상분리균주 ( 그람양성알균 69주, 그람음성막대균 51주 ) 는동정에따른 AST 카드패널로분석을수행하였다. 무산소성배지의경우, 혈액배양양성배지에서 10 ml를채취하여 15 ml 용량의시험관에넣고 4,500 g에서 3분간원심분리하고, 상층액을버리고남아있는침사에 0.45% NaCl로 2 회세척하고, 마지막으로 0.45% NaCl 10 ml에현탁시켜나일론메쉬 (66 m) 로세포파편을제거하였다. 필터처리한현탁액은 4,500 g에서 3 분간원심분리하고침사는 0.45% NaCl에재부유하였다. 재부유한현탁액은 CLSI 지침에따라 McFarland (McF) 0.5농도로맞추어 VITEK 2 시스템을사용하여 AST를실시하였다. 스펙트럼분석을위해 VITEK MS IVD system을사용하였고, 검사결과는제조자가제공한라이브러리 v2.0과비교하여분석하였다. VITEK MS에서표적슬라이드의결과값이 90% 이상의신뢰수준을보였을경우에만유효한결과로사용하였다. 4. 통계분석직접법과표준방법의 AST 수행결과비교는 CLSI에서사용 하고있는용어인매우중대한오류 (very major error: false susceptibility), 대오류 (major error: false resistance), 소오류 (minor error: susceptible/resistance versus intermediate susceptibility) 로평가하였다. 혈액배양배지에따른평가는산소성 / 무산소성병의성능을통계적으로비교하기위해 Chi-square test 또는 Fisher's exact test를사용하였다. 결과 1. 혈액배양에서직접법과표준방법에의한항생제감수성시험결과비교직접법을이용한 AST 검사의결과와표준방법을이용한 AST 결과를비교하였을때, 97.9% (1,936/1,978) 의일치율을보였다. 매우중대한오류 (very major error), 중대한오류 (major error), 사소한오류의비율 (minor error) 은각각 0.25% (5/1,978), 0.05% (1/1,978), 1.8% (36/1,978) 를나타냈다 (Table 1). 2. 직접법과표준방법에의한항생제감수성시험간불일치결과분석혈액배양에서직접법과표준방법에의한 AST 결과를분석하였을때, 그람양성세균에서는 97.2% (1,051/1,081) 의일치율을보였고, 매우중대한오차율은 0.5% (5/1081), 중대한오차 Table 1. Comparison of results between direct and standard antimicrobial susceptibility test in blood culture Microorganisms Antimicrobial susceptibility test No. of test Agreement Minor error Major error Very major error Gram-positives 1,081 (100%) 1,051 (97.2%) 24 (2.2%) 1 (0.1%) 5 (0.5%) Staphylococcus aureus Staphylococcus epidermidis Staphylococcus haemolyticus Staphylococcus capitis Staphylococcus hominis Staphylococcus saprophyticus Enterococcus faecium Enterococcus faecalis Streptococcus pneumoniae Gram-negatives 897 (100%) 885 (98.6%) 12 (1.4%) 0 (0%) 0 (0%) Escherichia coli Klebsiella pneumoniae Enterobacter cloacae Enterobacter aerogenes Klebsiella oxytoca Serratia marcescens Providencia stuartii Pseudomonas aeruginosa Acinetobacter baumannii Total 1,978 (100%) 1,936 (97.9%) 36 (1.8%) 1 (0.05%) 5 (0.25%)

4 282 Kang-Gyun Park, et al. Rapid AST in Blood Culture Table 2. List of cases that have discordant results in between direct and standard antimicrobial susceptibility test Microorganisms Antimicrobials (No. of cases) Minor error Major error Very major error Staphylococcus aureus Erythromycin (1) - Gentamicin (2) Staphylococcus epidermidis Fusidic acid (7), Gentamicin (4), - Trimethoprim/Sulfamethoxazole (2) Ciprofloxacin (2), Teicoplanin (2) Staphylococcus haemolyticus Ciprofloxacin (1), Erythromycin (1) - - Staphylococcus capitis Gentamicin (1), Fusidic acid (1) - Gentamicin (1) Staphylococcus hominis Gentamicin (1) - - Enterococcus faecium Ampicillin/ Sulbactam (1) - - Enterococcus faecalis Erythromycin (2) Erythromycin (1) - Escherichia coli Amoxicillin/Clavulanic acid (2), - - Piperacillin/Tazobactam (1), Cefepime (1) Klebsiella pneumoniae Amoxicillin/Clavulanic acid (1), Ertapenem (1) - - Serratia marcescens Tigecycline (1) - - Providencia stuartii Piperacillin/Tazobactam (1), Imipenem (1) - - Pseudomonas aeruginosa Amikacin (1), Cefepime (1), Aztreonam (1) - - Total 율은 0.1% (1/1,081), 사소한오차율은 2.2% (24/1,081) 의결과를나타냈다. 두방법간불일치를야기한주요원인균은 Staphylococcus epidermidis였고, 그중 gentamicin (N=9) 과 fusidic acid (N=8) 에서높은오류를나타냈다. 그람음성세균중두방법에의한 AST 결과의전체적인일치율은 98.6% (885/897) 였고, 사소한오류는 1.4% (12/897) 였다. 그람음성세균중두방법간불일치를야기한주요원인균은 Escherichia coli와 Pseudomonas aeruginosa였으며, 그중 amoxicillin/clavulanic acid (N=3) 에서높은오류를나타냄을확인하였다 (Table 2). 고찰패혈증은중환자실내에서높은유병률뿐만아니라, 중환자실내사망의주요원인으로알려져있다 [16]. 미국에서중증패혈증은인구 10만명당 300례정도발생하였으며, 연령의증가에따라그빈도가증가하여 85세이상의노인에서는어린이에비하여약 100배이상발생한다고보고된바있다 [17]. 최근국내에서이루어진다기관연구에서도중증패혈증에의한원내사망률은 34.3%, 중환자실내사망률은 29.1% 로발표하였다 [2]. 최근몇년동안유명인들이패혈증으로인한급성심부전이나다발성장기부전등으로진행돼사망에이르게되면서패혈증에대한사회적관심이높아졌지만, 뇌졸중이나심근경색증보다사망률이높음에도여전히인식부족으로인해폐렴등감염병이패혈증으로악화하는경우가많았다. 이와같이패혈증은다양한감염증이동반될수있고, 여러종류의미생물종이원인이되기때문에그원인체를정확히규명해내기위해서는혈 액배양이필수적이다. 패혈증이나균혈증환자는매우위험한상태이므로혈액배양을통한신속한세균동정결과는환자의치료에매우중요하며생존확률과도밀접하게연관되어있다. 최근에는신속하고정확하게병원체를동정하기위한자동화시스템이도입되고있다. 이에본연구에서는혈액배양에서신속한세균동정과 AST 결과를얻기위해혈액배양양성배지에서계대배양없이세균을 VITEK MS와 Vitek 2 시스템에직접접종하였으며, 도출된결과를표준방법과비교하여그신뢰도와정확도를평가하였다. 본연구에서 AST의정확도는그람양성세균과그람음성세균모두에서우수하게나타났으며, AST의일치율은다른연구들에비해우수하거나유사한수준으로나타났다 [3,6,9]. Machen 등이보고한연구에의하면그람음성막대균중심각한오류의원인이되는주요세균은 Proteus spp. 로 ampicillin, cefazolin, ceftazidime, ceftriaxone 등의다양한항생제에서나타난다고보고하였지만, 본연구에서는 Proteus spp. 가포함되지않았다. 그람양성세균은 97.2% (1,051/1,081) 의일치율을보였으며, 매우중대한오차율은 0.5% (5/1081), 중대한오차율은 0.1% (1/1,081), 사소한오차율은 2.2% (24/1,081) 의결과를나타냈다. 불일치의주요원인균은 S. epidermidis였으며, 그중 gentamicin (N=9) 과 fusidic acid (N=8) 에서높은오류를나타냈다. 그람음성세균중전체적인일치율은 98.6% (885/897) 였고, 사소한오류는 1.4% (12/897) 였다. 그람음성세균의불일치주요원인균은 E. coli와 P. aeruginosa였으며, 그중 amoxicillin/clavulanic acid (N=3) 에서높은오류를나타냈다. 그람양성세균에서는 Staphylococcus spp. 에서매우

5 Korean J Clin Lab Sci. Vol. 49, No. 3, September 중대한오류가발견되었으며, 관련항생제는 gentamicin 과 trimethoprim/sulfamethoxazole 이었다. 이결과는 Machen [14] 등이보고한내용과일치하였다. 향후추가적인연구에서는본연구에서주된오류의원인이되었던 streptococci와 coagulase negative staphylococci (CoNS) 에의한중대한오류의원인분석과이를개선하기위한노력이필요할것으로사료된다. 본연구에서평가한직접법에의한 AST는 CLSI 기준을충족하였고, 결과보고시간을 24시간단축할수있었지만, 매우큰오류가있는항균제에대해서는디스크확산법으로추가적인검사를시행할필요성이있다는것을알수있었다. 이러한연구결과들을토대로혈액배양액에서직접 AST를실시하는방법은 90% 이상의높은정확도를가지고결과보고시간을크게줄일수있기때문에환자의신속하고정확한치료에매우유용할것으로사료된다. 요약본연구에서는혈액배양에서신속한세균동정과항생제감수성시험 (antibiotic susceptibility test, AST) 결과를얻기위해혈액배양양성배지에서계대배양없이세균을 VITEK MS와 VITEK 2 시스템에직접접종하였으며, 도출된결과를표준방법과비교하여, 그신뢰도와정확도를평가하였다. 혈액배양양성시료에서직접결과는표준방법 AST 결과와비교하였을때, 97.9% (1,936/1,978) 의전체적인일치율을보였다. 그람양성세균은 97.2% (1,051/1,081) 의일치율을나타냈으며, 매우중대한오차율은 0.5% (5/1081), 중대한오차율은 0.1% (1/1,081), 사소한오차율은 2.2% (24/1,081) 의결과를나타냈다. 두방법간불일치의주요원인균은 Staphylococcus epidermidis이었고, 그중 gentamicin (N=9) 과 fusidic acid (N=8) 에서높은오류를나타냈다. 그람음성세균중전체적인일치율은 98.6% (885/897) 였고, 사소한오류는 1.4% (12/897) 였다. 그람음성세균의불일치주요원인균은 Escherichia coli와 Pseudomonas aeruginosa였으며, 그중 amoxicillin/clavulanic acid (N=3) 에서높은오류를나타냈다. 직접법에의한 AST 방법은 CLSI 기준을충족하였고, 결과보고시간을 24시간단축할수있었지만, 매우큰오류가있는항생제에대해서는디스크확산법으로추가적인검사를시행한후보고해야한다는것을알수있었다. 이러한연구결과들을토대로혈액배양시료에서직접 AST를실시하는방법은정확하고결과를보고하는데까지소요되는시간을크게감소시킬수있기때문에환자의정확하고효율적인치료에유용하게활용될수있을것으로사료된다. Acknowledgements: None Funding: None Conflict of interest: None REFERENCES 1.Watson RS, Carcillo JA, Linde-Zwirble WT, Clermont G, Lidicker J, Angus DC. The epidemiology of severe sepsis in children in the United Stated. Am J Respir Crit Care Med. 2003; 167(5): Kim JH, Hong SK, Kim KC, Lee MG, Lee KM, Jung SS, Choi HS, Lee JH, Jung KS, Lee SS, Cho JH, Koh SO, Park MS, Seo KW, Koh Y. Influence of full-time intensivist and the nurse-to-patient ratio on the implementation of severe sepsis bundles in Korean intensive care units. J Crit Care. 2012;27:414.e Machen A, Drake T, Wang YF. Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDI-TOF VITEK mass spectrometry and the VITEK2 system. PLoS One. 2014;9:e Schneiderhan W, Grundt A, Wörner S, Findeisen P, Neumaier M. Work flow analysis of around-the-clock processing of blood culture samples and integrated MALDI-TOF mass spectrometry analysis for the diagnosis of bloodstream infections. Clin Chem. 2013;59(11): Prod hom G, Bizzini A, Durussel C, Bille J, Greub G. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets. J Clin Microbiol. 2010;48(4): Romero-Gómez MP, Gómez-Gil R, Paño-Pardo JR, Mingorance J. Identification and susceptibility testing of microorganism by direct inoculation from positive blood culture bottles by combining MALDI-TOF and Vitek-2 Compact is rapid and effective. J Infect. 2012;65(6): Stevenson LG, Drake SK, Murray PR. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2009;48(2): Kok J, Thomas LC, Olma T, Chen SC, Iredell JR. Identification of bacteria in blood culture broths using matrix-assisted laser desorption-ionization Sepsityper TM and time of flight mass spectrometry. PLoS One. 2011;6:e Ling TK, Liu ZK, Cheng AF. Evaluation of the VITEK 2 system for rapid direct identification and susceptibility testing of gram-negative bacilli from positive blood cultures. J Clin Microbiol. 2003;41(10): Fothergill A, Kasinathan V, Hyman J, Walsh J, Drake T, Wang YF. Rapid identification of bacteria and yeasts from positive-blood-culture bottles by using a lysis-filtration method and matrix-assisted laser desorption ionization-time of flight mass spectrum analysis with the SARAMIS database. J Clin Microbiol. 2013;51(3): Lavergne RA, Chauvin P, Valentin A, Fillaux J, Roques-Malecaze C, Arnaud S, et al. An extraction method of positive blood cul-

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( )Kju225.hwp 비임균성비뇨생식기감염에서 의진단적효용성 Usefulness of the Mycofast Test (MYCOFAST Evolution 2) for the Diagnosis of Nongonococcal Genitourinary Infections Hang Ro Park, Yang Hyun Kim, Ho Jae Lee, Jea Sang Oh, Hyoung Jin