증례 Lab Med Online Vol. 2, No. 2: 111-115, April 2012 임상미생물학 증식편에서 16S rrna 직접염기서열분석으로진단한 Haemophilus parainfluenzae 심내막염 Haemophilus parainfluenzae Infective Endocarditis Diagnosed by Direct 16S rrna Sequencing of Vegetation 오성희 1 조민철 1 김재욱 1 안동희 1 정문희 1 김미나 1 최상호 2 Sung-Hee Oh, M.D. 1, Min-Chul Cho, M.D. 1, Jae-Wook Kim, M.D. 1, Dongheui An, M.D. 1, Mun-Hui Jeong, M.D. 1, Mi-Na Kim, M.D. 1, Sang-Ho Choi, M.D. 2 울산의대서울아산병원진단검사의학과 1, 감염내과 2 Departments of Laboratory Medicine 1 and Internal Medicine 2, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea The HACEK group of microorganisms is responsible for approximately 3-6% of endocarditis cases and is a major cause of culture-negative endocarditis. Here, we report a case of Haemophilus parainfluenzae infective endocarditis that was diagnosed by direct PCR sequencing of 16S rrna from resected vegetation. A healthy 26-yr-old man was admitted to the emergency room (ER) on March 27, 2011 because of intermittent high fever. The patient was prescribed cefpodoxime for 5 days at the ER. Six and 11 sets of blood cultures were performed at the ER and in a general ward, respectively, using BACTEC Plus Aerobic/F (Becton-Dickinson, USA) and Lytic Anaerobic/F Plus (BD) together. Echocardiography revealed a large vegetation at the posterior mitral valve leaflet. After performing mitral valvoplasty on hospital day (HD) 11, the vegetation tissue was cultured in thioglycolate broth, blood agar, Brucella agar, and MacConkey agar for 7 days, but no organism was grown. Direct PCR sequencing of 16S rrna of the tissue revealed the presence of H. parainfluenzae. In the 17 sets of blood cultures, bacterial growth was detected in only 2 aerobic bottles of 5 sets taken at HD 9 after 10-day and 14-day incubation. The organism was identified as H. parainfluenzae by using the VITEK NHI card (biomerieux, France). Direct PCR sequencing of vegetation could be useful in diagnosing bacterial pathogens in infective endocarditis patients, especially in culture-negative cases. Key Words: Endocarditis, PCR, Tissue, 16S rrna, RNA sequence analysis 서론 감염성심내막염을확진하는데혈액배양에서원인균을검출하 는것은매우중요한기준이다 [1]. 하지만혈액배양이음성인감염 성심내막염은 2.5-30% 에달한다 [2]. 배양음성심내막염은혈액배 Corresponding author: Mi-Na Kim, M.D. Department of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, 388-1 Pungnap-2dong, Songpa-gu, Seoul 138-736, Korea Tel: +82-2-3010-4511, Fax: +82-2-478-0884, E-mail: mnkim@amc.seoul.kr Received: September 5, 2011 Revision received: September 28, 2011 Accepted: September 28, 2011 This article is available from http://www.labmedonline.org 2012, Laboratory Medicine Online This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 양전항생제치료나감염의원인균이 HACEK (Haemophilus parainfluenzae, H. aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae) 군과같이배양하기까다로운균종일경우등이주원인이된다 [3]. 최근배양음성심내막염의경우증식편에서직접진균이나세균을광범위하게증폭한후염기서열을분석하는것이원인균을진단하는데이용되고있다 [3]. 이증례는혈액배양에서원인균검출이지연되어심장판막증식편으로부터 16S rrna 직접염기서열분석한후 H. parainfluenzae 에의한심내막염을진단할수있었기에보고하고자한다. 증례 2011년 3월 23일평소건강하였던 26세의남자가내원 1일전부터발생한간헐적인발열과오한, 두통을주소로응급실에내원하였다. 내원당시시행한혈액검사에서백혈구 11.5 10 3 /µl ( 호중구 eissn 2093-6338 www.labmedonline.org 111
87.7%), C-반응단백 15.78 mg/dl로증가하였고, 총빌리루빈 4.5 mg/dl, 직접빌리루빈 0.6 mg/dl였으며소변검사에서빌리루빈 1+, 잠혈반응 1+ 였다. 일시적인세균또는바이러스감염이원인으로추정되어경구용 3세대세팔로스포린계열인 cefpodoxime 100 mg을 5일간처방받은후퇴원하였다. 이후계속되는발열및오한으로이틀후응급실에다시내원하였으며, 당시이학적검사에서체온 38.7, 혈압 121/73 mmhg, 호흡수 20회 / 분, 맥박 103회 / 분이었고, 청진시심잡음은들리지않았다. 혈액검사상백혈구 6.1 10 3 /µl ( 호중구 86.7%), 혈소판 102 10 3 /µl, 적혈구침강속도 62 mm/hr, C-반응단백 9.63 mg/dl, 총빌리루빈 2.4 mg/dl, 직접빌리루빈 0.4 mg/dl이었다. 뇌척수액검사에서백혈구수는 2/µL로정상이었다. 흉부단순방사선촬영및뇌컴퓨터단층촬영에서다른특이소견은발견되지않았다. 환자는발열의원인을찾기위하여 3월 27일입원하였고, 3월 23일부터 27일까지 5일동안투여하던 cefpodoxime 을입원 2일째인 3월 28일부터중단하였다. 응급실에서시행한 3월 24일에 1쌍, 25일에 2쌍, 26일에 3쌍의혈액배양과, 입원 2일및 7일째시행한각각 3쌍의혈액배양은배양 5 일까지모두음성이었다. 혈액배양은 BACTEC Plus Aerobic/F (Becton-Dickinson, Sparks, MD, USA) 배지와 Lytic Anaerobic/F Plus 배지 (BD) 에동시접종하였고, BACTEC 9240 (BD) 에장착하여배양하였다. 입원 9일째시행한경흉부심초음파에서승모판후방판막에 11 15 mm 크기의증식물및컬러도플러에서승모판역류가관찰되었다. 다음날경식도심초음파로승모판후방판막에서 11 18 mm 크기의과운동성인증식물을확진하였다 (Fig. 1). 입원 9일째혈액배양 5쌍을시행한후, 느리게자라는까다로운그람음성균에의한감염성심내막염을의심하여 ceftriaxone 2,000 mg/day 정맥내주입치료를시작하였다. 입원 11일째증식물을제거하고판막성형술을시행하였다. 증식편은혈액한천배지, Mac- Conkey 한천배지, 브루셀라혈액한천배지, thioglycolate 액체배지에배양하였으나배양 7일째까지균은자라지않았다. 배양후남은조직에서 QIAamp DNA Mini (QIAGEN, Hilden, Germany) 를이용하여 DNA를추출하였고, 16S rrna의 30-1,370 번째염기부위를중합효소연쇄반응으로증폭한후직접염기서열분석을시행하였다. 1,327 bp 크기의염기서열을 GenBank에서 BLAST를통해검색한결과, H. parainfluenzae (GenBank Accession No. EU083530.1) 와 1,324/1,327 (99.8%) 의일치도를보였다. 차순위일치도를보인 H. paraphrophilus 와 1,300/1,327 (97.9%) 의상동성을보여 H. parainfluenzae 로동정할수있었다. 입원 9일째시행한혈액배양중호기성 2병에서배양 10일, 14일째그람음성간균이자랐고, VITEK NHI card (biomerieux, Marcy l Etoile, France) 로 H. parainfluenzae 를동정하였다 (bionumber 245700, 92%). 이균은 β-lactamase는음성이었고, Haemophilus Test Medium 배지를이용한디스크확산법에서 ampicillin, amoxicillin/clavulanate, azithromycin, cefuroxime, ceftriaxone, trimethoprim/sulfamethoxazole, meropenem 에감수성을보였다. 혈액배양검사는접종후 10 일째양성신호가감지되고동정및감수성검사결과보고까지 12 일이소요된반면, 조직편을직접염기서열분석하는방법으로 4일만에원인균을동정할수있었다. 수술을받은후 1일째, 자극에대한반응이느려지고질문을반복적으로해야대답하는등환자의의식수준이떨어져서뇌자기공명영상검사를시행하였고양쪽전두엽에서다병소의급성피질및피질하경색이관찰되었다 (Fig. Fig. 1. Transthoracic echocardiography showed a huge, hypermobile vegetation (demarcated by 4 crosses) on a posterior leaflet of the mitral valve at hospital day 9. Fig. 2. Multifocal acute cortical and subcortical infarcts in both cerebral hemispheres on brain MRI at hospital day 12. 112 www.labmedonline.org
2). 환자는 4주간의 ceftriaxone 2,000 mg/day 로정맥내주입치료를받은다음회복되어퇴원하였다. 고찰 이증례는 17쌍의혈액배양중 2쌍의호기성배양에서만 H. parainfluenzae 가분리되었고, 항생제투여중단 8일째시행한혈액배양에서 10일만에첫양성신호가감지되었다. 동정및항균제감수성결과보고까지총 12일이소요되었다. HACEK군은구강상재균에속하며 [4] 전체심내막염의원인중 3-6% 를차지한다 [5]. Haemophilus 종으로는 H. parainfluenzae 와 H. aphrophilus 가원인균의대부분을차지한다 [6]. Haemophilus 종은 X 또는 V 인자와같은특수한성장인자를필요로하는특징이있어배양이까다롭고성장까지시간이오래걸린다 [4]. H. parainfluenzae 는 V 인자가필요하다 [4]. BACTEC 9240과같은자동혈액배양기는영양요구도가높고느리게자라는균종도빠르고민감하게검출할수있어서 [7], 5일안에임상적으로중요한병원균들은대부분검출된다 [8]. 과거 HACEK군등에의한심내막염을진단하기위해 2주까지배양을연장하도록권장하기도하였지만 [9], 현재는배양시간을연장하여도배양양성률을높이는효과가없는것으로보고되고있다 [10-12]. Clinical and Laboratory Standards Institute (CLSI) 에서는심내막염진단에도표준적인 5일간의혈액배양을권장하며, 5일까지혈액배양이음성일때초콜릿한천배지에계대배양하는것을권장한다 [8]. 국내에서보고된 Haemophilus 종에의한심내막염증례들은자동화혈액배양기에서 1-5일만에균성장이검출되거나, 5-8일사이에동정결과가보고되었다 [13-15]. 따라서이증례에서오랫동안배양이지연된것은원인균이 Haemophilus 종인것만으로는설명할수없다. 이증례는첫혈액배양을시행할때이미 3세대세팔로스포린을투여중이었다. 입원 2일째항생제복용을중단하고, 입원 9일째시행한혈액배양에서배양 10일만에양성이었다. 채혈전항생제투여로배양양성률이 100% 에서 64% 까지감소하는등 [16], 채혈전항생제투여는배양음성심내막염의중요한원인이된다 [3]. 세균의세포벽합성을차단하는 β-lactam 계열의항생제들은 항생제후효과 가오래지속될수있다 [17]. 본증례에서동정된 H. parainfluenzae 는 β-lactamase 음성으로모든 β-lactam제제에감수성이었기때문에혈액배양전투여한 cefpodoxime 때문에초기혈액배양이실패한것으로추정된다. 따라서감염성심내막염이의심되는환자에서선행하는항생제투여로혈액배양이음성일때단기간치료받은환자는 3일, 장기간치료받은경우 6-7일까지항생제를중단한다음혈액배양을시도하는것이권장된다 [18]. 증례에서증식편의 16S rrna 직접염기서열분석으로 4일만에 원인균을동정할수있었다. 이는동일한날짜에실시한혈액배양에비해 8일정도더빨리결과를얻었다. Actinobacillus, H. parainfluenzae 등 HACEK군에의한혈액배양음성심내막염을증식편에서직접염기서열분석을시행하여진단한보고가있다 [19, 20]. 국내에서는증식편에서털곰팡이균사체를관찰하고 internal transcribed spacer (ITS) 1부위의염기서열을분석하여 Aspergillus fumigatus 에의한심내막염을진단한예가있다 [14]. 심내막염으로확진된환자에서증식편의세균과진균에대한직접염기서열분석검사의민감도, 특이도는 96.0%, 95.3% 에달했지만조직배양은각각 24.3%, 56.4% 로진단수행능이떨어진다 [21]. 염기서열분석대신 SeptiFast (Roche Diagnostics, Penzberg, Germany) 를혈액이나조직에적용한경우에도, 혈액배양보다민감도가훨씬높았다 [22]. 수술전항생제를투여받았던환자에서, 조직증균배양민감도가 10% 인반면, 조직의직접염기서열분석방법의민감도는 76% 로민감도차이가더욱커진다 [23]. 따라서혈액배양음성이지만초음파검사에서판막의증식물이확인된경우, 증식편에서직접 16S rrna 염기서열분석을하는것을심내막염진단기준의하나로포함할것을제안하고있다 [18]. 일반적으로심내막염에의한사망률이 16-19% 로높기때문에 [5], 원인균을신속하게동정하고적절한치료하는것은환자의예후에결정적인영향을준다 [24]. 심내막염환자에서수술적치료를하는경우는 40-60% 정도로알려져있다 [24]. 조직을획득할수있는경우세균이나진균을대상으로광범위중합효소연쇄반응을시행하고직접염기서열을분석하는것은신속하고민감하게원인균을검출하는데매우유용할것이다. 이증례는건강한성인의자연판막에서발생한심내막염으로, 심잡음이없었기때문에초기에심내막염을의심하지못하여진단이지연되었다. 심내막염의위험인자로구강또는치아병변, 선행하는치과시술, 류마티스열이나선천성심기형, 심장수술력과같은심장손상이나, 심장내인공장치물등이보고되고있다 [25]. 이증례에서이러한위험요인은밝혀지지않아진단이더어려웠다. Haemophilus 종에의한심내막염은분명한위험인자가없는경우가가장흔하다 [6]. 국내에서보고된 H. parainfluezae 에의한심내막염 5예가보고되었지만이중에서치과시술력이있었던 1 예, 비위생적인인슐린피하주사력 1예에서만위험인자를추정할수있었다 [13, 15, 26, 27]. H. parainfluenzae 심내막염의 60% 에서색전증이동반된다 [6]. 통상적으로아급성심내막염에서 25% 정도인것과비교했을때이는훨씬높은비율이며, H. parainfluenzae 증식물이잘부서지는특성때문으로추정된다 [6]. 이때가장흔하게침범되는장기는뇌, 사지, 비장, 폐이다 [6]. 이증례도입원시부터두통이있었고자기공명영상에서다발성뇌경색을보여 H. parainfluenzae 에의한심내막염의특성에맞는소견이었다. 이전에국내보고된 H. parainfluenzae 에의한심내막염증례보고에 www.labmedonline.org 113
서도급성뇌경색이합병되었다 [26]. 색전증과같은합병증의발생은사망률을높인다 [27]. H. parainfluenze 심내막염은위험인자가없는정상인에서발생할수있고, 색전증을잘동반하기때문에신속한원인균규명과치료가필요하다. 저자는증식편조직 16S RNA 직접염기서열분석으로 H. parainfluenzae 를동정하여심내막염원인균을진단한국내첫증례로보고하는바이다. 증식편을얻을수있을때, 조직에서직접광범위중합효소반응및직접염기서열분석을시행하는것이심내막염확진에유용할것으로생각한다. 요약 HACEK 군은전체심내막염중 3-6% 를차지하며, 배양음성심내막염의주요원인이다. 혈액배양이지연된 H. parainfluenzae 심내막염에서증식편을직접염기서열분석방법으로진단한증례를보고한다. 건강했던 26세남자가 2011년 3월 27일간헐적고열을주소로응급실에내원하여 cefpodoxime을 5일간처방받았다. 응급실과입원후병실에서각각 6쌍과 11쌍의혈액배양검사를실시하였고혈액은 BACTEC Plus Aerobic/F (Becton-Dickinson, USA) 와 Lytic Anaerobic/F Plus (BD, USA) 에세트로접종하였다. 심초음파검사에서승모판후방에큰증식물이관찰되어심내막염으로진단되었다. 입원 11일째증식물제거술및승모판성형술을시행한후증식편을 thioglycolate 액체배지, 혈액한천배지, 브루셀라혈액한천배지, MacConkey 배지에 7일간배양하였으나균은자라지않았다. 증식편에서 16S rrna를직접염기서열분석검사하여 H. parainfluenzae 를동정하였다. 입원 9일째시행한 5쌍의배양중 2쌍의호기성병에서혈액배양 10일, 14일만에균이배양되었다. VITEK NHI card (biomerieux, France) 를이용하여 H. parainfluenzae 로동정하였다. 감염성심내막염, 특히배양음성심내막염환자에서증식편을직접염기서열분석하여원인균을동정하는것은매우유용한진단법이될것이다. 참고문헌 1. Li JS, Sexton DJ, Mick N, Nettles R, Fowler VG Jr, Ryan T, et al. Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis. Clin Infect Dis 2000;30:633-8. 2. Lamas CC and Eykyn SJ. Blood culture negative endocarditis: analysis of 63 cases presenting over 25 years. Heart 2003;89:258-62. 3. Mandell GL, Bennett JE, et al. eds. Principles and practice of infectious diseases. 7th ed. Philadelphia, PA: Churchill Livingstone, 2010:1067-112. 4. Versalovic J, Carroll KC, et al. eds. Manual of clinical microbiology. 10th ed. Washington, DC: American Society for Microbiology, 2011: 588-98. 5. Steckelberg JM, Melton LJ 3rd, Ilstrup DM, Rouse MS, Wilson WR. Influence of referral bias on the apparent clinical spectrum of infective endocarditis. Am J Med 1990;88:582-8. 6. Darras-Joly C, Lortholary O, Mainardi JL, Etienne J, Guillevin L, Acar J. Haemophilus endocarditis: report of 42 cases in adults and review. Haemophilus Endocarditis Study Group. Clin Infect Dis 1997;24:1087-94. 7. Rohner P, Pepey B, Auckenthaler R. Comparative evaluation of BACTEC aerobic Plus/F and Septi-Chek Release blood culture media. J Clin Microbiol 1996;34:126-9. 8. Clinical and Laboratory Standards Institute. Principles and procedures for blood cultures; Approved guideline. CLSI document M47-A. Wayne, PA: Clinical and Laboratory Standard Institute, 2007. 9. Washington JA 2nd. Blood cultures: principles and techniques. Mayo Clin Proc 1975;50:91-8. 10. Baron EJ, Scott JD, Tompkins LS. Prolonged incubation and extensive subculturing do not increase recovery of clinically significant microorganisms from standard automated blood cultures. Clin Infect Dis 2005; 41:1677-80. 11. Doern GV, Davaro R, George M, Campognone P. Lack of requirement for prolonged incubation of Septi-Chek blood culture bottles in patients with bacteremia due to fastidious bacteria. Diagn Microbiol Infect Dis 1996;24:141-3. 12. Forward KR. An evaluation of extended incubation time with blind subculture of blood cultures in patients with suspected endocarditis. Can J Infect Dis Med Microbiol 2006;17:186-8. 13. Huh JH, Bae SY, Kim JS, Lee KN, Lee CK. A case of Haemophilus parainfluenzae endocarditis. Korean J Clin Microbiol 2009;12:78-81. 14. Hong KW, Lee JA, Park HW, Kwon HL, Cho SJ, Kim JS, et al. A case of infective endocarditis caused by Aspergillus fumigatus in a liver transplant recipient. Korean J Med 2008;75:115-8. 15. Ryu KH, Choi HJ, Park SH, Park SH, Lee MA. Two cases of Haemophilus parainfluenzae endocarditis. Infect Chemother 2003;35:345-9. 16. Pazin GJ, Saul S, Thompson ME. Blood culture positivity: suppression by outpatient antibiotic therapy in patients with bacterial endocarditis. Arch Intern Med 1982;142:263-8. 17. Graig WA and Gudmundsson S. Postantibiotic effects. In: Lorian V. ed. Antibiotics in laboratory medicine. 4th ed. Baltimore, Maryland: Williams & Wikins, 1996:296-329. 18. Horstkotte D, Follath F, Gutschik E, Lengyel M, Oto A, Pavie A, et al. Guidelines on prevention, diagnosis and treatment of infective endo- 114 www.labmedonline.org
carditis executive summary; the task force on infective endocarditis of the European society of cardiology. Eur Heart J 2004;25:267-76. 19. Kelesidis T, Kelesidis I, Lewinski MA, Humphries R. Establishing diagnosis of Haemophilus parainfluenzae as etiology of culture-negative endocarditis using DNA sequence analysis on tissue specimen. Am J Med 2011;124:e9-e10. 20. Westling K and Vondracek M. Actinobacillus (Aggregatibacter) actinomycetemcomitans (HACEK) identified by PCR/16S rrna sequence analysis from the heart valve in a patient with blood culture negative endocarditis. Scand J Infect Dis 2008;40:981-3. 21. Marín M, Muñoz P, Sánchez M, del Rosal M, Alcalá L, Rodríguez-Créixems M, et al. Molecular diagnosis of infective endocarditis by real-time broad-range polymerase chain reaction (PCR) and sequencing directly from heart valve tissue. Medicine (Baltimore) 2007;86:195-202. 22. Fernández AL, Varela E, Martínez L, Martínez A, Sierra J, González- Juanatey JR, et al. Evaluation of a multiplex real-time PCR assay for detecting pathogens in cardiac valve tissue in patients with endocarditis. Rev Esp Cardiol 2010;63:1205-8. 23. Voldstedlund M, Nørum Pedersen L, Baandrup U, Klaaborg KE, Fuurst-ed K. Broad-range PCR and sequencing in routine diagnosis of infective endocarditis. APMIS 2008;116:190-8. 24. Murdoch DR, Corey GR, Hoen B, Miró JM, Fowler VG Jr, Bayer AS, et al. Clinical presentation, etiology, and outcome of infective endocarditis in the 21st century: the International Collaboration on Endocarditis- Prospective Cohort Study. Arch Intern Med 2009;169:463-73. 25. Winn WC, Allen SD, et al. eds. Koneman s color atlas and textbook of diagnostic microbiology. 6th ed. Baltimore, MD: Lippincott Williams & Wilkins, 1997:429-52. 26. Kang MH, Lim KR, Kim TS, Kim SH, Kim GH, Oh WS, et al. A case of early valve replacement for Haemophilus parainfluenzae endocarditis complicated with acute cerebral infarctions. Infect Chemother 2011;43:270-4. 27. Yang DJ, Ryu YS, Chung MY, Park EK, Park JY, Cho YB, et al. A case of Haemophilus parainfluenzae-related tricuspid valve endocarditis in an unhygenically insulin-injected patient. Korean J Med 2009;77(S): S1170-3. 28. Thuny F, Avierinos JF, Tribouilloy C, Giorgi R, Casalta JP, Milandre L, et al. Impact of cerebrovascular complications on mortality and neurologic outcome during infective endocarditis: a prospective multicentre study. Eur Heart J 2007;28:1155-61. www.labmedonline.org 115