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1 Korean J Clin Microbiol Vol. 14, No. 1, March, 2011 DOI: /KJCM Evaluation of Automated Blood Culture System for Body Fluids Culture Other Than Blood Tae Yeal Choi 1, Jung Oak Kang 1, Hyun Joo Pai 2 Departments of 1 Laboratory Medicine, 2 Internal Medicine, Hanyang University Medical School, Seoul, Korea Background: We investigated whether culture using an automated blood culture system enhances the recovery of bacteria and fungi from body fluids other than blood when compared to conventional solid media culture methods. Methods: A total of 734 specimens [ascites (n=457), bile (n=5), CAPD (n=28), CSF (n=32), joint fluids (n= 165), pericardial fluid (n=17), and pleural fluid (n=30)] were included in the study. Half of the volume of each specimen was inoculated directly into automated blood culture bottles (biomeriux, Marcy-I Etoile, France). The remaining volume was inoculated onto conventional solid media (sheep blood agar, chocolate agar, and phenylethyl alcohol agar) after centrifuging at 3,000 rpm for 10 min. Results: Clinically significant microorganisms were isolated from 62 specimens (8.5%) by automated blood culture and 61 specimens (8.3%) by the conventional solid media culture (kappa index: 0.81, 95% confidence interval: ). Contamination was observed in 11 (1.8%) of the automated blood culture specimens and 3 (0.4%) of the solid media culture specimens. The mean turnaround times of the automated blood cultures and the conventional solid media cultures were 3.7 and 2.8 days, respectively (P< ). Conclusion: Compared with conventional culture methods, no improvement in the recovery of clinically significant microorganisms was noted with the use of the automated blood culture system for the culture of body fluids other than blood. (Korean J Clin Microbiol 2011;14:13-17) Key Words: Culture of body fluids, Automated blood culture system, Solid media culture methods 서 체액 ( 복막액, 담즙, 복막투석액, 뇌척수액, 관절활액, 심낭액, 늑막액, 등 ) 에세균이나진균이감염되면임상적으로위중한상태에빠지므로원인균을신속정확하게배양하는것은매우중요한일이다 [1-3]. 혈액을제외한체액의배양은일반적으로고체배지를사용하여배양하여왔으나, 근자에혈액배양을위하여자동혈액배양법이사용된이후로혈액을제외한체액배양에도액체배지를이용한자동혈액배양법을사용하려는경향이있다 [2-5]. 그러나체액배양에자동혈액배양법을사용하는것이고체배지를이용한배양보다우수하리라는생각은아직논쟁의대상이되고있다 [6,7]. 체액배양에서자동혈액배양법의사용목적은고체배지에서배양하기어려운세균을진단할뿐만아니라신속히균의성장유무를알기위해서이다 [2-6]. 그러나현재고체배지에조성성분의발달로오히려액체배지 Received 4 May, 2010, Revised 28 June, 2010 Accepted 20 July, 2010 Correspondence: Tae Yeal Choi, Department of Laboratory Medicine, Hanyng University Hospital, 17 Haengdang-dong, Seongdong-gu, Seoul , Korea. (Tel) , (Fax) , ( ) tychoi@hanyang.ac.kr 론 보다균배양이뒤지지않는것으로보고되고있다 [6,7]. 뿐만아니라자동혈액배양법은균동정을위하여다시고체배지에계대배양하여야하는불편과균동정의지연을초래하고, 오염균의증가로인한업무량증가및추가로사용되는고가의자동혈액배양병사용등경제적인손실이있다 [7]. 이에연구자들은혈액을제외한체액배양에서동일검체로자동혈액배양법과고체배지법을동시에실시하여자동혈액배양법을평가하였다. 대상및방법 1. 대상검체 2007년 6월부터 2010년 2월까지복수 (457), 담즙액 (5), 복막투석액 (28), 뇌척수액 (32), 관절활액 (165), 심낭액 (17), 늑막액 (30) 등총 734검체를자동혈액배양병을이용한호기성및혐기성배양을실시하였으며, 또한기존에사용하던고체배지를이용하여호기성및혐기성배양을동시에실시하였다. 2. 검사방법채취검체의절반은호기성 BacT/ALERT R SA 및혐기성 13

2 14 Korean J Clin Microbiol 2011;14(1):13-17 BacT/ALERT R SN (biomeriux, Marcy-I Etoile, France) 혈액배양병에각각 5 ml씩분주후자동혈액배양기기 BacT/Alert 3D (biomeriux, Durham, USA) 에장착하여 5일간배양하였다. 나머지검체는 3,000 rpm으로원심분리하여침사를혈액한천배지, 쵸코렛배지및 phenylethyl agar에접종하고 2일간배양하였다. 3. 결과판정각검체에서원인균및오염균의판정은연속검사에서의동일균검출, 체액내세포수, 백분율및임상증상등을참조하였다. 배양결과보고시간은병원내전산결과를이용하였다. 4. 통계처리두방법간의비교는 Kappa index (Kappa index/agreement: <0.00/Less than chance, /Slight, /Fair, /Moderate, /Substantial, /Almost perfect) 를사용하였다. 결과전체 734검체중임상적으로의의가있는균의발견은자동혈액배양법 / 고체배지법에서각기 62 (8.5%)/61 (8.3%) 이었으며, 검체별로는복수 12 (2.6%)/11 (2.4%), 담즙 4 (80%)/4 (80%), 복막투석액 6 (21.4%)/6 (21.4%), 뇌척수액 3 (9%)/3 (9%), 관절활액 29 (17.6%)/30 (18.2%), 심낭액 0 (0.0%)/0 (0.0%), 늑막액 8 (26.7%)/7 (23.3%) 균주가분리되었다. 임상적으로의의있는세균종류는양쪽모두 Staphylococcus aureus, Escherichia coli, Candida spp., Klebsella spp., Streptococcus agalactiae, Stenotrophomonas spp., Streptococcus Group G, Enterococcus spp. 등이었다. 오염균발견은전체적으로자동혈액배양법과고체배지법에서각기 11 (1.4%) 과 3 (0.3%) 로자동혈액배양법에서오염이많이보고되었다. 오염균이배양된주된검체는복막투석액, 관절활액, 뇌척수액이었으며, 오염균의종류는 Staphylococcus hominis, Staphylococcus epidermidis, Staphylococcus cohnii, Staphylococcus capitis 등이었다 (Table 1). 자동혈액배양법과고체배지법에서배양결과의불일치는전체적으로는 24/734 (3.3%) 였으며, 각검체별로는복수 6/457 (1.3%), 담즙 0/5 (0.0%), 복막투석액 6/ 28 (21.4%), 뇌척수액 4/32 (12.5%), 관절활액 6/165 (3.6%), 심낭액 1/17 (5.9%), 늑막액 1/30 (3.3%) 였다 (Table 2). 두방법간에 Kappa index는검체에따라 로다소차이는있었으나전체검체의 Kappa index는 0.81 (95% confidence index: ) 로두방법간에균배양검출률에있어서차이가없었다 (Table 3). 배양결과보고시간은균이자라지않은경우는자동혈액배양법이 5일, 고체배지법은 2일이면 no growth 로결과가보고 Table 1. Number of isolates by automatic blood culture system (BC) and solid media culture methods (SM) Test Ascite Bile CAPD CSF JF PcF PF Total Clinically significant isolates S. aureus 1/0 0/1 1/0 18/19 2/2 22/22 Streptococcus spp.* 0/1 6/7 6/8 Enterococcus spp. 0/1 1/1 1/1 2/3 S. pneumoniae 1/1 1/1 Enterobacteriaceae 9/7 3/3 3/2 1/1 1/1 1/0 18/14 P. aeruginosa 2/3 1/0 3/3 6/5 Fungus 1/1 2/2 1/1 4/4 Nocardia spp. 1/1 1/1 M. abscessus 1/1 1/1 Bacteroides spp. 1/1 1/1 Subtotal 12/11 4/4 6/6 3/3 29/30 8/7 62/61 Contaminants CNS 1/0 3/1 2/0 3/1 1/0 10/2 Gram+bacilli 1/1 1/1 Subtotal 2/1 3/1 2/0 3/1 1/0 11/3 No growth 443/445 1/1 19/21 27/29 133/134 16/17 22/23 661/670 Total 457/457 5/5 28/28 32/32 165/165 17/17 30/30 734/734 *S. pyogenes, S. agalactiae, Streptococcus β-hemolysis Group G; E. faecalis, E. faecium; E. coli, K. pneumonia, E. cloacae, P. mirabilis, S. marcescens, Aeromonas spp.; Candida spp., C. neoformans; S. epidermidis, S. capitis, S. cohnii, S. hominis. Abbreviations: CAPD, continuous ambulatory peritoneal dialysis; CSF, cerebrospinal fluid; JF, joint fluid; PcF, pericardial fluid; PF, pleural fluid; CNS, coagulase-negative staphylococci.

3 Tae Yeal Choi, et al. : Automated Blood Culture System for Body Fluids 15 Table 2. Discrepancy of results in automated blood culture system and solid media culture methods Solid media Liquid media Ascite Bile CAPD CSF JF PcF PF Total Abbreviations: CAPD, continuous ambulatory peritoneal dialysis; CSF, cerebrospinal fluid; JF, joint fluid; PcF, pericardial fluid; PF, pleural fluid; +, growth;, no growth. Table 3. Agreement (Kappa index)* of automated blood culture system and solid media culture methods Specimens (n) Kappa index.95 confidence interval Ascite (457) Bile (5) CAPD (28) CSF (32) Joint (165) Pericardial (17) NC NC Pleural (30) Total (734) *Kappa index/agreement: <0.00/Less than chance, / Slight, /Fair, /Moderate, /Substantial, /Almost perfect. Abbreviation: NC, no calculation. 되었다. 균이배양된경우는자동혈액배양법은평균 3.7 (SD 1.3일 ) 일, 고체배지법은 2.8 (SD 1.0일 ) 일로자동혈액배양법이 0.9일 (SD 1.1일 ) 늦게보고되었다 (P=0.003). 경제적으로는직접소모품만계산하더라도자동혈액배양법에서는호기성및혐기성액체배양병이각기 1개씩추가로소모되었다. 고 체액배양에서액체배지를이용한자동혈액배양법의사용목적은첫째, 검체내에항균제가있을경우액체배지에서희석및흡착되며, 둘째, 검체내에균수가적은경우양성률을높이기위하여많은양의검체를접종할수있기때문이다 [6]. 특히복막투석액과척수액검체는검체내균수가적어액체배지에많은양의검체를접종하면 Propionibacterium spp. 와 coagulase-negative staphylococci (CNS) 가더많이배양되었다 [7]. Cetin 등 [5] 은 BACTEC blood culture system (Becton Dickinson Diagnostic Instrument System, Sparks, Md) 을이용하여뇌척수액이나관절활액을배양한결과기존의고체배지를이용한방법보다 Brucella spp., Streptococcus spp., Aeromonas hydrophila 를더많이발견하였다. 그러나이들의논문에서는균배양결 찰 과의통계학적인차이를제시하지는않았고, 배양된균에대한오염균여부가명확히구분되어있지않았다. Gibb [6] 은복막투석액과뇌척수액검체는자동혈액배양법에서많은양의검체를접종할수가있어고체배지배양법보다우수하다하였으나, 일반적으로액체배지에검체접종시액체배지와검체와의비율이 5 10:1 이적합하므로접종량이그이상이될때는오히려균배양성적이안좋다 [8]. Azap 등 [3] 은검체양이많은경우는 50 ml tube 등을사용하여 3,500 rpm에서 10분원심분리후침사를여러장의고체배지에접종하면보다많은양의검체를접종할수있으며, 검체내항생제도제거할수있어고체배지사용을강조하고있다. Gibb [6] 은 Neisseria meningitidis 및 Haemophilus influenzae 등배양이어려운세균도고체배지구성성분의발달로고체배지사용이액체배지사용보다우수하다고하였다. 본연구결과에서는전체적으로임상적으로의의있는균의배양결과는자동혈액배양방법과고체배지배양법에서차이가없었다. 그러나배양결과불일치율이복막투석액에서 21.4%, 뇌척수액 12.5% 로높았으며, 담즙액에서는 0%, 복수는 1.3% 로낮았다. 이는복막투석액및뇌척수액내균의농도가낮고담즙및복수액내균농도가높기때문인것으로생각된다 [6]. 연구자도상대적으로균농도가낮은복막투석액같이검체양이많은경우는많은양의검체모두를원침하여침사를고체배지에접종하는것이더타당하리라생각한다. 배양된원인균의분포도 S. aureus, E. coli, Candia spp., K. pneumoniae, S. agalactiae 등으로배양방법에따른배양된균종의차이가없었다. 관절활액에서 Nocardia spp. 와 Mycobacterium abscessus가액체배지에서 2일만에배양되었는데임상검체의그람염색및 AFB 염색에서쉽게발견되었을뿐만아니라혈액한천배지및쵸코렛배지에서도동일균이 2일만에배양되었다. 이것으로보아현재국내에서사용되고있는고체배지의성능이자동혈액배양법의액체배지보다못하지않았다. 본연구에서혐기성세균의분리가저조한것은최근혈액이나체액에서혐기성세균의감염이전체적으로감소하는추세이고, Clostridium spp., Propionibacterium acne 등은배양이되더라도오염균일경우가많으므로, 복수및수술후감염등의혐기성균의감염이의

4 16 Korean J Clin Microbiol 2011;14(1):13-17 심되는경우를제외하고는호기성배양에치중하여검체를사용하는것이바람직하다고생각된다 [9]. Dunbar 등 [10] 은뇌척수액배양에서자동혈액배양법은 6.2%, 기존의고체배지사용은 3.8% 의오염률을나타내어자동혈액배양법에서오염률이높아불필요한검사의진행과검사비의증가를초래하는것으로보고하였다. Meredith 등 [7] 도뇌척수액의액체배양은 CNS, Propionibacterium spp. 의오염이많으므로반드시고체배지와함께배양하여야하여야한다고하였다. 본연구에서도복막투석액, 늑막액, 뇌척수액등에서오염이많았다. 전체적으로도오염이고체배지에서는 3건인데비하여자동혈액배양법에서는 11건으로자동혈액배양법에서오염이많았다. 고체배지에서오염률이낮은이유중에하나는오염확률이높은균이소량 (1 2 집락 ) 배양된경우는환자의임상증상및체액내세포수등을참조하여 no growth 로보고한경우도있지않았나생각된다. 오염균의종류는주로 S. epidermidis 등의 CNS로다른연구자들의보고와유사하였다 [2-5]. Cetin 등 [5] 은 BACTEC blood culture system을이용하여체액을배양한결과고체배지를이용한배양방법보다균배양시간이짧다고 ( 자동혈액배양법 : 9.5시간 / 고형배지사용 : 38.9시간 ) 보고하였으나, 이것은액체배양의경우자동혈액배양기기에서균검출시간이고, 혈액배양병에서균배양이확인되면균동정을위하여반드시고체배지에계대배양하여야하기때문에통상 18 24시간더소요된다. 연구자의결과에서도 no growth 의경우고체배지의경우는최소 2일이면보고를할수있으나액체배지의경우는 5일까지기다려야하였다. 균배양양성의경우도자동혈액배양법 3.7일로고형배지는 2.8일보다 0.9일늦게보고되었다 (P=0.003). 체액내에세균을증명하기위하여최근분자생물학적방법인중합효소연쇄반응을시도할수있으나오히려세균오염에의한위양성의결과가있을수있어기존의고체배지를사용한배양법을대치할수는없는것으로생각된다 [11,12]. 그러나관절활액에서임균 [13] 이나 Chlamydia trachomatis [14], Mycobacterium tuberculosis [15] 및 Borrelia burgdorferi [16] 등의특수균의발견은중합효소연쇄반응법이유용한것으로보고되고있다. 결론적으로혈액이외의체액배양에서자동혈액배양법을이용한액체배지사용이기존의고체배지사용보다균배양양성률이우수하지도않았으며, 세균오염의증가, 검사결과보고의지연및검사비용의증가가있었다. 그러므로혈액을제외한체액배양에자동혈액배양법의무분별한사용은자제하여야한다. 감사의글본연구결과의통계처리를하여주신한양대학교의과대학 예방의학교실최보율교수님께감사드립니다. 참고문헌 1. Margaretten ME, Kohlwes J, Moore D, Bent S. Does this adult patient have septic arthritis? JAMA 2007;297: Bourbeau P, Riley J, Heiter BJ, Master R, Young C, Pierson C. Use of the BacT/Alert blood culture system for culture of sterile body fluids other than blood. J Clin Microbiol 1998;36: Azap OK, Timurkaynak F, Sezer S, Cağir U, Yapar G, Arslan H, et al. Value of automatized blood culture systems in the diagnosis of continuous ambulatory peritoneal dialysis peritonitis. Transplant Proc 2006;38: Akcam FZ, Yayli G, Uskun E, Kaya O, Demir C. Evaluation of the Bactec microbial detection system for culturing miscellaneous sterile body fluids. Res Microbiol 2006;157: Cetin ES, Kaya S, Demirci M, Aridogan BC. Comparison of the BACTEC blood culture system versus conventional methods for culture of normally sterile body fluids. Adv Ther 2007;24: Gibb AP. Plates are better than broth for recovery of fastidious organisms from some specimen material. J Clin Microbiol 1999; 37: Meredith FT, Phillips HK, Reller LB. Clinical utility of broth cultures of cerebrospinal fluid from patients at risk for shunt infections. J Clin Microbiol 1997;35: Clinical and laboratory standards institute. Principles and procedures for blood cultures; Approved guideline. CLSI document M47-A. CLSI, Wayne, PA: Ortiz E and Sande MA. Routine use of anaerobic blood cultures: are they still indicated? Am J Med 2000;108: Dunbar SA, Eason RA, Musher DM, Clarridge JE 3rd. Microscopic examination and broth culture of cerebrospinal fluid in diagnosis of meningitis. J Clin Microbiol 1998;36: Jalava J, Skurnik M, Toivanen A, Toivanen P, Eerola E. Bacterial PCR in the diagnosis of joint infection. Ann Rheum Dis 2001; 60: Clarke MT, Roberts CP, Lee PT, Gray J, Keene GS, Rushton N. Polymerase chain reaction can detect bacterial DNA in aseptically loose total hip arthroplasties. Clin Orthop Relat Res 2004;(427): Liebling MR, Arkfeld DG, Michelini GA, Nishio MJ, Eng BJ, Jin T, et al. Identification of Neisseria gonorrhoeae in synovial fluid using the polymerase chain reaction. Arthritis Rheum 1994;37: Taylor-Robinson D, Gilroy CB, Thomas BJ, Keat AC. Detection of Chlamydia trachomatis DNA in joints of reactive arthritis patients by polymerase chain reaction. Lancet 1992;340(8811): van der Heijden IM, Wilbrink B, Schouls LM, van Embden JD, Breedveld FC, Tak PP. Detection of mycobacteria in joint samples from patients with arthritis using a genus-specific polymerase chain reaction and sequence analysis. Rheumatology (Oxford) 1999; 38: Nocton JJ, Dressler F, Rutledge BJ, Rys PN, Persing DH, Steere AC. Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis. N Engl J Med 1994;330:

5 Tae Yeal Choi, et al. : Automated Blood Culture System for Body Fluids 17 = 국문초록 = 자동혈액배양법을이용한혈액이외체액배양의평가 한양대학교의과대학 1 진단검사의학교실, 2 내과학교실최태열 1, 강정옥 1, 배현주 2 배경 : 연구자들은혈액이외의체액배양에서자동혈액배양방법이세균과진균배양에있어서기존의고체배양법과비교하여우수한지알아보기위하여연구하였다. 방법 : 전체적으로 734검체 ( 복수 457, 담즙 5, 복막투석액 28, 뇌척수액 32, 관절활액 165, 심낭액 17, 늑막액 30) 가본연구에사용되었다. 검체의절반은직접자동혈액배양병 (biomeriux, Marcy-I Etoile, France) 에접종하였고, 나머지는 3,000 rpm 에서 10분원심분리후기존의고체배지에접종하였다. 결과 : 임상적으로의의가있는미생물의배양양성은자동혈액배양법에서 62 (8.5%), 고체배지법 61 (8.3%) 로방법상의차이가없었다 (kappa index: 0.81, 95% confidence index: ). 세균오염은자동혈액배양법 11 (1.8%), 고체배지법 3 (0.4%) 이었다. 결과보고까지의시간은자동혈액배양법과고체배지법이각각 3.7일, 2.8일이었다 (P=0.003). 결론 : 혈액을제외한체액배양에서자동혈액배양이기존의고형배지보다임상적으로의의있는균발견에향상이없었다. [ 대한임상미생물학회지 2011;14:13-17] 교신저자 : 최태열, , 서울시성동구행당동 17 한양대학교병원진단검사의학과 Tel: , Fax: tychoi@hanyang.ac.kr

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untitled Ann Clin Microbiol Vol. 18, No. 1, March, 2015 http://dx.doi.org/10.5145/acm.2015.18.1.1 pissn 2288-0585 eissn 2288-6850 Evaluation of the Usefulness of MacConkey Agar and Colistin-Nalidixic Acid Blood