단신 ISSN 2093-9272 일산병원학술지 2017;16(2):237-241 혈류감염진단을위한신속한검사법비교분석 국민건강보험일산병원진단검사의학과 김영아, 이상선, 손영준 Rapid Diagnostic Tests for Bloodstream Infection Young Ah Kim, Sang Sun Lee, Young Jun Son Department of Laboratory Medicine, National Health Insurance Service Ilsan Hospital, Goyang Korea Bloodstream infection (BSI) is a severe clinical state with high mortality and rapid identification of bloodstream pathogens and report of antimicrobial susceptibility play an essential role for the timely and effective treatment of patients with sepsis. Newly developed and introduced rapid tests for bloodstream infection are helpful to diagnose etiologic agents of bloodstream infection or to treat patient effectively. At the same time, it is also important to provide information that will help determine the antibiotic prescription in real time in order to improve clinical utility. Key Words: Sepsis, Matrix assisted laser desorption ionization time-of flight mass spectrometry, Molecular method, Antimicrobial stewardship 서론 패혈증 (sepsis) 은병원균이나독소가혈관속으로들어가순환하면서전신에심한중독증상이나, 급성염증을일으키는상태로최근변경된정의에서는패혈증을감염으로인한생명을위협하는장기부전으로정의하고있다. 1 발열이나호중구증가증등의감염을의심하는전신염증반응 (Systemic Inflammatory Response Syndrome, SIRS) 이있는경우감염을의심할수있지만, 좀더정확한진단을위해서는검사실에서원인균을확인하는것이필요하다. 미국질병관리본부의정의에따르면관련임상증상이있고, 임상적인진단을목적으로시행한혈액배양이나, 비배양검사에서 Staphylococcus aureus, Enterococcu spp. Escherichia coli, Pseudomona spp. Klebsiella spp. Candida spp. 등의흔한패혈증병원균이한번이상확인된경우검사실확인혈류감염 (laboratory confirmed bloodstream infection) 을진단할수있다. 2 책임저자 : 김영아 10444 경기도고양시일산동구일산로 100 국민건강보험일산병원진단검사의학과전화 : (031)900-0908 팩스 : (031)900-0912 E-mail : yakim@nhimc.or,kr 최근비배양검사도많이소개되고있지만, 대부분의검사실에서혈액배양은혈류감염진단에서가장중요한검사이다. 하지만혈액배양에서병원균의분리는검체채취법, 배양량, 배양의뢰횟수및배양법등에영향을많이받고, 특히혈액내에 1-10개정도의매우낮은농도로존재하는 3 병원균을검출하기위해서는충분한증식과정을거쳐야하므로최종결과를얻기까지많은시간이소요되는등의제한점이있다. 최근에는세균표면단백질을분석하여균종을동정하는질량분석기 Matrix Assisted Laser Desorption Ionization Time- Of-Flight Mass Spectrometry (MALDI-TOF MS) 를이용하여병원균동정을시행하는임상미생물검사실이늘어나고있는데, 배양에기반한전통적인생화학적방법에비해패혈증의원인균을평균 28시간정도단축하여동정할수있다. 4 또한지난 10년간눈부신기술적발전에힘입어 PCR, films array 및차세대염기분석 (next generation sequencing) 등분자유전학적방법을이용하여신속하게패혈증을진단하는검사법도꾸준히개발되고있다. 5 신속하게패혈증의원인균을확인하고, 감수성시험을통해치료효과가있는항생제를알게되면적절한항균제치료를빨리시행할수있어환자의예후가향상되고의료비용을절감할수있는반면, 원인균의동정및감수성시험결과가 Volume 16 Number 2 December 2017 237
YA Kim, et al. Rapid Diagnostic Tests for Bloodstream Infection 늦어지면광범위항생제에노출하는기간이늘어나내성균이분리될위험이높아진다. 6 따라서패혈증환자에서신속한배양결과보고가매우중요하다. 저자들은신속한패혈증진단을위하여새로개발진단검사법을전혈에서직접원인균을검출하는방법과양성혈액배양병에서의검출하는방법으로나누어알아보고, 항균제처방관리프로그램 (antimicrobial stewardship) 을병행하여신속패혈증검사법을효과적으로이용하는방법을소개하고자한다. 전혈을이용한혈류감염의신속진단을위한분자유전학적방법 혈액을배양하지않고패혈증의심환자의전혈에서직접패혈증의원인균을직접검출하는방법은통상적인배양법에비해매우빠르고민감하게배양이어려운미생물을동정하고, 내성및병독성유전자도함께검출할수있는것은장점이나, 고비용, 항균제내성이나자동화문제로혈액배양을대체하기힘는점, 오염, 표준화및임상적유용서에대한증거가아직까지충분하지못한점등은단점이다. 5 PCR 원리를이용하여혈액검체에서직접주요패혈증의원인균을검출하도록고안된상품화된검사법들로는 Sepsi- Test (Molzym, Bremen, Germany), SeptiFast (Roche Molecular System, Basel, Switzerland), MagicPlex (Seegene, Seoul, Korea), VYOO (SIRS-Lab, Jena, Germany) 등이있다. 5 SeptiFast (Roche) 는 1.5 ml의전혈을사용하여 3.5-5시간내에 19종의주요원인균과 meca 내성유전자를검출하여 methicillin 내성 S. aureus를확인할수있으며, 국내에서개발된 MagicPlex (Seegene) 의경우 1mL 전혈을이용하여 3-5시간내에 25종의주요원인균을종수준으로동정할뿐만아니라, 내성유전자 meca, vana 및 vanb 내성유전자의검출도가능하여 methicillin 내성 S. aureus 과 vancomycin 내성 Enterococcus spp. 를확인할수있다. 5 패혈증진단을위한혈액배양에기반한분자유전학적신속검사법 양성혈액배양병에서분자유전학적검사법을이용하여직접혈류감염원인균을동정하는것은앞서설명한전혈에서직접검출하는방법과마찬가지로여러가지제한점이있다. 즉, 혈액내에는헤모글로빈같은 PCR 억제제가존재하고, 인체에서유래한핵산의양이미생물에비해매우많으며, 핵산에오염에의한위양성가능성높고, 죽은세균으로인한핵산이존재한다는점이다. 5,7 최근의추출과증폭기술의발 전으로혈액에서직접원인균을검출할수있는여러가지분자유전학적검사법들이상품화되어있다. 형광제자리부합법 (fluorescence in situ hybridization, FISH) 을이용한방법으로는 PNA FISH (AdvanDx, Woburn, MA, USA) 와 Quick FISH (AdvanDx) 가 1-3시간내에총 13종의원인균, AccuProbe (Gen-Probe, San Diego, CA, USA) 가 1시간이내에 S. aureus, Enterococcus spp. 및주요 Streptococcus spp. 를검출한다. 7 마이크로어레이법 (microarray) 인 Verigene (Nanosphere, Northbrook, IL, USA) 는 2.5시간내에총 21종의원인균과 meca, vana, vanb, bla KPC, bla NDM, bla CTX-M, bla VIM, bla IMP, bla OXA12 를동시에확인하며, Prove-it Sepsis (Mobidiag, Esbo, Finland) 은 3.5시간내에총 73종의원인균과 meca 내성유전자도찾는다. 7 multiplex PCR 을이용한 FilmArray (Idaho Technology, Salt Lake City, UT, USA) 가 1시간내에총 24 종의원인균과내성유전자 meca, vana, vanb, bla KPC 를 realtime PCR 법인 Xpert MRSA/SA BC (Cepheid, Sunnyvale, CA, USA) 가 1시간내에 S. aureus 와 meca 내성유전자를, multiplex PCR인 StaphSR assay (BD GeneOhm, San Diego, CA, USA) 가 1-2시간내에 S. aureus와 meca 내성유전자를검출할수있다. 7 양성혈액배양병의침사를이용한혈류감염의신속질량분석기검사법 양성혈액배양병의배지를고체배지에하루배양하여순배양집락을얻은후질량분석기로동정하는것은이제많은검사실에서통상적으로사용하고있지만, 좀더신속한결과를얻기위해서양성혈액배양병에서침사를만들어질량분석기로동정및항균제감수성을시험할수있다. 국내의연구결과는아직많지않지만, 최근한연구에서융해 (lysis) 나여과 (filter) 로얻은침사를이용하여동정하면기존의생화학적방법과 81.8% 의일치하였다고한다. 8 이연구에서그람음성균의일치도가그람양성균보다높은것으로보고하였는데, 저자들은 streptococci 와 coagulase 음성 staphylococci에서동정되지않은경우가있었기때문으로설명하였다. 8 국외보고된주요연구결과를양성혈액병에서미생물을직접전처리질량분석기를이용하는방법과자동화된생화학적방법을이용하여미생물을동정법과의비교하여 Table 1 에정리하였다. 8-16 질량분석기의검사특성을고려하여혈액에서두종이상의복합미생물이분리된것은양성혈액배양병을이용하여평가한것은제외하였다. 그람양성세균의경우종 (species) 수준의일치도가 65.9% 에서 97.9% 로다양 238 Korean Journal of National Health Insurance Service Ilsan Hospital
김영아외. 혈류감염신속진단 하였는데, 이는 streptococci의낮은일치도가주로영향을주는것으로생각되었다. 그람음성세균의경우종수준의일치 도가 74.7% 에서 100% 를보였는데, 장내세균 (Enterobacteriaceae) 에서의일치도는매우좋은것으로판단되었다. Table 1. Comparison of identification between MALDI-TOF with the direct sample preparation method and the standard biochemical method in mono-microbial blood cultures No (%) of correct identifications at species level Preparation MALDI Standard Ref Gram-positive, 108/146 (73.9) Gram-negative, 100/108 (92.6) Total 208/254 (81.8) Lysis-filtration Vitek-MS Vitek-2 8 Enterobacteriaceae, 232/255 (90.9) Non-fermentor 20/25 (80.0) Other Gram-negative organisms 9/12 (75) Vitek-MS: Gram-positive, 51/75 (68) Gram-negative, 95/106(89.6) Total 146/188(80.7) Biotyper: Gram-postive, 54/75 (72) Gram-negative, 94/106(88.7) Total 148/188(81.8) Gram-positive, 46/47 (97.9) Gram-negative, 45/49 (91.8) Yeast 3/4 (75) Total 94/100 (94) Staphylococci, 51/58 (87.9) Enterococci, 16/16 (100) Streptococci, 6/14 (42.9) Enterobacteriaceae, 32/35 (91.4) Non-Enterobacteriaceae, 5/11 (45.5) Yeast 3/5 (60) Total, 117/146 (80.1) Gram-positive, 53/74 (71.6) Gram-negative, 41/46 (90) Other 0/2 (0) Total 95/122 (77.9) Gram-positive, 16/17 (94.1) Gram-negative, 21/21 (100) Anaerobe, 0/7 (0) Yeast, 0/3 (0) Total 85/109 (78) Gram-positive, 44/56 (79) Gram-negative, 44/45 (97) Total 88/101 (87.1) Gram-positive, 29/44 (65.9) Gram-negative, 65/87 (74.7) Yeast, 3/9 (33.3) Total 97/140 (69.3) Spin-lysis with formic acid extraction Sepsityper Biotyper Phoenix 9 Vitek-MS Biotyper Vitek-2 Phoenix Lysis-filtration Vitek-MS Vitek-2 Microscan Sepsityper Biotyper Vitek-2, 16SrRNA Ammonium chloride lysis 10 11 12 Biotyper Vitek-2 13 Tween 80 lysis Biotyper Phoenix 14 Centrifugationwashing Biotyper Phoenix 15 Lysis-filtration Vitek-MS Vitek-2 16 MALDI, Matrix assisted laser desorption ionization time-of-flight mass spectrometry; N, test number; Ref, Reference; Sepsityper (Bruker Daltonik, Bremen, Germany); MALDI-TOF Vitek-MS system (biomérieux, Marcy l'etoile, France); Biotyper MALDI-TOF MS (Bruker Daltonik, Bremen, Germany); Vitek-2 (biomérieux); Phoenix ((BD Diagnostic Systems, Sparks, MD, USA); Microscan (Siemens, West Sacramento, CA, USA); 16SrRNA, 16SrRNA gene sequencing Volume 16 Number 2 December 2017 239
YA Kim, et al. Rapid Diagnostic Tests for Bloodstream Infection Table 2. Comparison of antimicrobial susceptibility results between the direct sample preparation and the standard method in mono-microbial blood cultures N Agreement MIE MAE VMAE Preparation AST Ref 1,978 1,936 (97.9%) 36 (1.8%) 1 (0.05%) 5 (0.25%) Lysis-filtration Vitek-2 8 1,012 946 (93.5%) 36 (3.6%) 17 (1.7%) 13 (1.3%) Lysis-filtration Vitek-2 11 1,084 1,072 (98.9%) 8 (0.8%) 4 (0.4%) 0 (0%) Centrifugation-washing Phoenix E-test 15 891 860 (96.5%) 22 (2.5%) 5 (0.55%) 4 (0.45%) Lysis-filtration Vitek-2 16 N, number; MIE, minor error; MAE, major error; VMAE, very major error; AST, antimicrobial sensitivity test; Ref, Reference 양성혈액배양병의침사를이용한신속항균제감수성시험 혈액배양결과가나오기전에선제적으로시행한경험적항균제처방 (conservative antibiotic treatment) 을최종항균제처방 (definitive antibiotic treatment) 로변경하는데는최종항균제감수성시험결과가필수적이다. 적절한항균제처방이패혈증환자의예후에매우중요한인자임은여러연구에서잘알려져있다. 17,18 따라서양성혈액배양병침사를이용한신속항균제감수성시험은결과보고시간을기존의검사법보다하루앞당길수있어임상적으로매우유용하리라생각된다. 하지만아직까지는정확성문제를간과할수없는데, 위내성 (false resistance) 으로보고하는 major error 발생시내성세균의확산을위해제한적으로사용해야하는고가의활성이높은항균제를남용할위험이있고, 위감수성 (false susceptible) 으로보고할수있는 very major error 발생시치료실패를야기할위험이있다. 기존의평가에따르면양성혈액배양병침사를이용한신속항균제감수성시험법은배양후순집락 (colony) 으로시험하는기존방법과일치율이 93.5% 에서 98.9% 로매우우수하였고, major error 0.05-1.7%, very major error 0-1.3% 수준을보여, 8,11,15,16 기존의방법을대체할수있을것으로판단되었다. European Clinical Antimicrobial Susceptibility Testing (EUCAST) 에서도한종류의세균만자란양성혈액배양병에서 agar diffusion 법으로항균제감수성시험을시행할때기존의 breakpoint를적용할수있는지검토하는데, 전체적인일치율은 95.7% 로매우좋았고, very major error는 1.3% 였다. 19 이연구에서는 Enterococcu spp. 의고농도 gentamicin 내성과 Staphylococcu spp. 의 rifampicin내성이오류가높았다고하며, major error와 minor error는모두 3.4% 이었다고보고하였다. 19 결론적으로패혈증의임상적중요성을생각해볼때신속하게원인균을검출하고효과적인항생제치료에대한정보 를제공하는혈류감염진단을위한신속한검사법을개발하고검사실에도입하는데노력을기울어야하겠다. 또한이렇게얻은결과가임상적인결정에실질적인도움이될수있도록실시간으로항생제처방을결정하는데도움이되는정보를동시에제공하는것도매우중요하다. 다만신속한혈류감염진단을위해새로개발된검사들은대개전통적인방법보다좀더신속하고정확하지만, 검사비용이고가인경우가많아한정된의료자원을비용효과적으로이용하기위해서는적용하기전에임상적인효용성을충분히분석할필요가있다. REFERENCES 1. Singer M, Deutschman CS, Seymour CW, et al. The third international consensus definitions for sepsis and septic shock (sepsis- 3). JAMA 2016;315:801-10. 2. Horan TC, Andrus M, Dudeck MA. CDC/NHSN surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting. Am J Infect Control 2008;36:309-32. 3. Wain J, Diep TS, Ho VA, et al. Quantitation of bacteria in blood of typhoid fever patients and relationship between counts and clinical features, transmissibility, and antibiotic resistance. J Clin Microbiol 1998;36:1683-7. 4. Huang AM, Newton D, Kunapuli A, et al. Impact of rapid organism identification via matrix-assisted laser desorption/ionization time-of-flight combined with antimicrobial stewardship team intervention in adult patients with bacteremia and candidemia. Clin Infect Dis 2013;57:1237-45. 5. Opota O, Jaton K, Greub G. Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood. Clinl Microbiol Infect 2015;21:323-31. 6. Buehler SS, Madison B, Snyder SR, et al. Effectiveness of practices to increase timeliness of providing targeted therapy for inpatients with bloodstream infections: a laboratory medicine best practices systematic review and meta-analysis. Clin Microbiol Rev 2016;29:59-103. 7. Opota O, Croxatto A, Prod'hom G, et al. Blood culture-based diagnosis of bacteraemia: state of the art. Clin Microbiol Infect 2015;21:313-22. 8. Jo SJ, Park KG, Han K, et al. Direct identification and antimi- 240 Korean Journal of National Health Insurance Service Ilsan Hospital
김영아외. 혈류감염신속진단 crobial susceptibility testing of bacteria from positive blood culture bottles by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and the Vitek 2 system. Ann Lab Med. 2016;36:117-23. 9. Gray TJ, Thomas L, Olma T, et al. Rapid identification of Gramnegative organisms from blood culture bottles using a modified extraction method and MALDI-TOF mass spectrometry. Dig Microbiol Infect Dis 2013;77:110-2. 10. Chen JHK, Ho P, Kwan GSW, et al. Direct bacterial identification in positive blood cultures by use of two commercial matrix assisted laser desorption ionization-time of flight mass spectrometry systems. J Clin Microbiol 2013;51:1733-9. 11. 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 lysisfiltration method with MALDI-TOF VITEK mass spectrometry and the VITEK2 system. PlosOne 2014;9:e87870. 12. Martinez RM, Bauerle ER, Fang FC, et al. Evaluation of three rapid diagnostic methods for direct identification of microorganisms in positive blood cultures. J Clin Microbiol 2014;52: 2521-9. 13. Prod'hom G, Bizzini A, Durussel C, et al. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets. J Clin Microbiol 2010;48:1481-3. 14. Leli C, Cenci E, Cardaccia A, et al. Rapid identification of bacterial and fungal pathogens from positive blood cultures by MALDI-TOF MS. Int J Med Microbiol. 2013;303:205-9. 15. Maelegheer K, Nulens E. Same-day identification and antibiotic susceptibility testing on positive blood cultures: a simple and inexpensive procedure. Eur J Clin Microbiol Infect Dis. 2017;36:681-7. 16. Wattal C, Oberoi JK. Microbial identification and automated antibiotic susceptibility testing directly from positive blood cultures using MALDI-TOF MS and VITEK 2. Eur J Clin Microbiol Infect Dis 2016;35:75-82. 17. Perez KK, Olsen RJ, Musick WL, et al. Integrating rapid pathogen identification and antimicrobial stewardship significantly decreases hospital costs. Arch Pathol Lab Med 2013;137:1247-54. 18. Doern GV, Vautour R, Gaudet M, et al. Clinical impact of rapid in vitro susceptibility testing and bacterial identification. J Clin Microbiol 1994;32:1757-62. 19. Stokkou S, Geginat G, Schlüter D, et al. Direct disk diffusion test using European Clinical Antimicrobial Susceptibility Testing breakpoints provides reliable results compared with the standard method. Eur J Microbiol Immunol 2015;5:103-11. Volume 16 Number 2 December 2017 241