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1 Korean J Clin Microbiol Vol. 15, No. 2, June, Vaginal Candida and Microorganisms Related to Sexual Transmitted Diseases in Women with Symptoms of Vaginitis Kye Hyun Kim 1, Mi-Kyung Lee 2 1 Department of Obstetrics and Gynecology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, 2 Department Laboratory Medicine, Chung-Ang University College of Medicine, Seoul, Korea Background: The female genital tract is equipped to deal with a variety of foreign substances including a wide array of microorganisms. It is important to consider Candida-bacterial interactions in balance between healthy colonization versus vaginitis. The objectives of this study were to evaluate the association between microorganism distribution and vaginitis, and to investigate the possibility of an interaction between vaginal Candida and other microorganisms in female genital tract. Methods: A total of 516 vaginal secretions were collected between October 2008 and June 2010 from patients with suspected vaginitis. Identification of Candida species and detection of 6 fastidious microorganisms (Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, and Ureaplasma urealyticum) were performed using a VITEK 2 system (biomérieux, Inc., Hazelwood, MO, USA) and multiplex PCR (Seegene, Biotechnology, Inc., Seoul, Korea), respectively. Results: M. genitalium, U. urealyticum, and C. trachomatis were more often detected in association with vaginal candidiasis. A statistically significant association between Candida and M. genitalium was observed (P<0.05). N. gonorrhoeae was detected less often in women with vaginal candidiasis. Conclusion: The results of this study suggest the possibility that vaginal Candida may associate with some microorganisms in patients with vaginitis. Further studies will be required to define the Candida-bacterial interactions and its mechanisms. (Korean J Clin Microbiol 2012;15:49-53) Key Words: Bacteria, Candida, Microbial interactions, Trichomonas vaginalis, Vaginitis 서 여성의생식관은정자, 태아, 병원체를포함하는다양한이물질과접촉하게되며, 이들병원체에는바이러스, 세균, 진균, 기생충등이포함된다. 이러한다양한침입자들로부터생식관을보호하기위하여면역체계와숙주상피세포, 생식관에존재하는세균들과의복잡한상호작용이일어나게되며, 특히여성의하부생식관에는면역반응에영향을줄수있는공생균 (commensal bacteria) 이집락을형성하고있다 [1,2]. 그러므로이러한점막부위에존재하는세균들을확인하는것은면역체계가어떻게미생물무리에의하여영향을받는지를이해하는데결정적인단계일수있다. 생식관의미생물무리는임상적진단기준에근거하여크게 2종류로분류하고있는데, 즉건강인에서분리되는미생물무리와세균질증 (bacterial vaginosis) 과연관되는세균으로나눌수있다. 건강인에서분리되는미생물 Received 4 October, 2011, Revised 29 November, 2011 Accepted 30 November, 2011 Correspondence: Mi-Kyung Lee, Department of Laboratory Medicine, Chung-Ang University College of Medicine, 102 Heukseok-ro, Dongjak-gu, Seoul , Korea. (Tel) , (Fax) , ( ) cpworld@cau.ac.kr 론 무리의대부분이 Lactobacillus 종인반면, 세균질증에서는사람에따라매우다양하지만대부분의경우여러종류의혐기성세균, Gardnerella vaginalis, Mycoplasma hominis와같이배양이까다로운세균등이집락을형성하는경우가많은것으로알려져있다 [1,3-6]. 또한세균이외에도 Candida 종과 Trichomonas vaginalis 등도여성의생식관에존재하며감염성질염을일으킬수있다 [6]. 여성의생식관처럼많은종류의병원체가동시에존재할수있는부위에서의집락형성과감염과의기전을연구하는과정에서세균과진균과의상호작용, 즉질내정상상재균과칸디다성질염사이의관련성에관한연구가많이시도되었지만, 아직까지명확하게알려져있지않다. 주로 Lactobacillus 종에관한연구가가장많으며, Lactobacillus 종은비뇨생식상피세포에결합하여병원성진균의부착을차단하거나대사산물에의해질내의낮은 ph를유지함으로비침습성형태의 C. albicans 성장에적합한환경을제공함으로발병기전에관여하는것으로생각되고있다 [7-9]. 또한칸디다와같은효모가감염이아닌집락형성상태에서임상적인감염으로발전하는과정에다른상재균과의상호작용이관여할것으로보고된바있다 49

2 50 Korean J Clin Microbiol 2012;15(2):49-53 [10]. 이에여성의생식관에존재하는진균과다른미생물간의상호작용가능성을연구하기위하여, 질염증상을가진여성의질내칸디다와성매개병 (sexually transmitted disease, STD) 과관련되는것으로알려져있는 T. vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, M. hominis, Mycoplasma genitalium 및 Ureaplasma urealyticum을검출하여칸디다배양유무에따른균종분포간관련성을분석하였다. 대상및방법 2008년 10월부터 2010년 6월까지질염증상을주소로중앙대학교용산병원산부인과를방문하여질분비물배양이의뢰된총 516건의검체를대상으로하였으며, 중앙대학교용산병원임상시험심사위원회의승인을받았다. 환자의질분비물은 Stuart Agar Gel Transport Swabs (COPAN Italia, Brescia, Italy) 으로채취하여검사실로수송후, 일상적미생물배양과분자유전학적검출을시행하였다. 혈액한천배지에서효모양진균이자랐을경우 Sabouraud dextrose agar (SDA) 에계대배양하여 VITEK 2 ID-YST system (biomérieux, Inc., Hazelwood, MO, USA) 을이용하여제조사의지침에따라칸디다균종동정을시행하였다. 배양이까다로운 T. vaginalis, C. trachomatis, N. gonorrhoeae, M. hominis, M. genitalium 및 U. urealyticum 검출을위하여 QIAamp DNA Mini kit (QIAgen Korea, Seoul, Korea) 로 DNA를추출하였고, Seeplex STD6 ACE Detection kit (Seegene, Biotechnology, Inc., Seoul, Korea) 을사용하여 GeneAmp PCR system 9700 (Applied biosystems, Inc., Foster City, CA, USA) 에서제조사의지시에따라다중 PCR을시행하였다. PCR 산물은 2% 아가로오스겔에서전기영동한후 Image system (ChemiDoc XRS system, Bio-Rad Laboratories, Hercules, CA, USA) 으로분절의유무와크기를확인하였다. 통계분석은 Statcalc (Epi Info version 3.5.1, Centers for Disease Control and Prevention, GA, USA) 을사용하여 chi-square 검증을하였으며, P<0.05를통계적으로유의한것으로판정하였다. 분석값이 5 이하인경우는 Fisher s exact test 의 P값을사용하였다. 결과총 516건의질분비물에서배양된칸디다종은 86균주 (16.7%) 였으며, 이중 C. albicans가 74균주 (14.3%) 로칸디다종의대부분을차지하였고 (74/86균주, 86.0%), 그다음으로 C. glabrata, C. tropicalis 순으로분리되었다. 다중 PCR에의한 T. vaginalis, C. trachomatis, N. gonorrhoeae, M. hominis, M. genitalium 및 U. urealyticum의검출결과는 M. hominis가 99검체 (19.2%) 로가장많이검출되었고, U. urealyticum 87검체 (16.9%), C. trachomatis 41검체 (7.9%), N. gonorrhoeae 13검체 (2.5%), M. genitalium 12검체 (2.3%), 그리고 T. vaginalis 7검체 (1.4%) 의순으로검출되었다. 여성생식관에서칸디다종과상기 6종류의미생물간상호작용가능성을평가하기위하여칸디다종배양유무에따른 T. vaginalis, C. trachomatis, N. gonorrhoeae, M. hominis, M. genitalium 그리고 U. urealyticum의검출빈도를분석하였다. 칸디다종이동시에배양되지않은경우에비하여칸디다종이동시에배양되었을경우검출빈도가높은균종은 M. genitalium, U. urealyticum, C. trachomatis이었고, 이중 M. genitalium만이통계적으로유의하게많이검출되었으며 (P<0.05) 이때동시에배양된칸디다종은모두 C. albicans였다. 이와는반대로 N. gonorrhoeae는칸디다종이배양되지않는경우보다칸디다종이동시에배양되는경우에서검출빈도가낮았다. T. vaginalis와 M. hominis는칸디다종의동시배양유무에따른검출빈도의차이를보이지않았다 (Table 1). Table 1. Microorganisms related to sexual transmitted diseases in women who were or were not vaginal carriers of Candida species Accompanying microorganism C. albicans Candida spp. (total) Growth (n=74) No growth (n=442) P value Growth (n=86) No growth (n=430) P value TV CT NG MH MG UU Total 1 (1.4%) 7 (9.5%) 1 (1.4%) 15 (20.3%) 5 (6.8%) 16 (21.6%) 45 (60.8%) 6 (1.4%) 34 (7.7%) 2 (2.7%) 84 (19.0%) 7 (1.6%) 71 (16.1%) 214 (48.4%) (1.2%) 9 (10.5%) 1 (1.2%) 17 (19.8%) 5 (5.8%) 16 (18.6%) 49 (57.0%) 6 (1.4%) 32 (7.4%) 12 (2.8%) 82 (19.1%) 7 (1.6%) 71 (16.5%) 210 (48.8%) Abbreviations: CT, Chlamydia trachomatis; MG, Mycoplasma genitalium; MH, Mycoplasma hominis; NG, Neisseria gonorrhoeae; TV, Trichomonas vaginalis; UU, Ureaplasma urealyticum.

3 Kye Hyun Kim and Mi-Kyung Lee : Vaginal Candida and Microorganisms with Vaginitis 51 고찰출생직후신생아의질은무균상태이며, 출생후수일이지난후에어머니로부터이동해온 lactobacilli에의해집락이형성되게된다. 대부분의경우일생을통하여정상상재균으로서 lactobacilli가우세하게유지되며, 또한주기적으로호르몬자극의영향을받아균무리분포가변화하게된다 [3]. 지금까지질내미생물무리의구성에관한대부분의자료들은배양에기초한방법으로시도된정성적이고반정량적인기술적연구들이었다 [6,7,10]. 그러나배양에기초한연구들은배양이까다로운미생물을분리하거나검출하는데실패할가능성이있으며, 배양으로분리된일부균종은동정에필요한표현형적방법이나적절한배지가없어동정을할수없는단점을가지고있었다. 최근분자유전학적기법들이개발되고도입되어, 생식관에존재하는비정상적인집락화뿐아니라정상질균무리의구성에대한새로운정보를제공하게되어배양에근거한방법들로부터얻은기존의자료들에추가적인정보가가능하게되었다 [11]. 배양과는독립적으로시도되는분자유전학적방법들은배양에기초한방법들의제한점을극복하여다양한정보를줄수있지만, DNA를증폭하여얻게되는대부분의결과들이가장우세한미생물을검출하는경향이있으며 1% 미만으로존재하는미생물은검출하지못할수도있다 [12]. 그러므로균무리구성에관한연구를위하여배양과분자유전학적방법들을상호보완적으로사용하는것이가장바람직할것으로생각된다. 본연구에서는질염증상을보이는여성질분비물의 16.7% 에서칸디다가배양되어동정되었으며, 분리된칸디다종의 86% 는 C. albicans였다. 이는질염증상을가진여성의 17-39% 에서칸디다가분리되었고 [5,6,13], 이중 C. albicans가가장많다는이전보고 [7,13,14] 와유사한결과였다. 또한다중 PCR로확인해본 6종류의배양이까다로운질내미생물중, 생식기 mycoplasmas 중에서세균질증과가장밀접하게관련되는것으로알려져있는 M. hominis가가장많이검출되었으며 [3,4], 이는이전분리빈도 ( %) 와유사하였다 [4,15,16]. 그러나이전연구들에서세균질증과의관련성은명확하지않으나질분비물에서의분리빈도가 % 로 M. hominis보다높은것으로보고된 U. urealyticum [4,16] 은 M. hominis보다낮게검출되었다. 그밖에 T. vaginalis, C. trachomatis, N. gonorrhoeae 의검출빈도도각각 1.4%, 7.9%, 2.5% 로이전보고들과차이를보였다 [6,16-18]. 본연구에서배양이까다로운질내미생물들의검출빈도가기존보고와다른것은대상환자군, 지역, 검체채취방법및검출방법의차이등으로생각된다. 여성의생식관에존재하는진균과다른미생물간의상호작용가능성을연구하기위하여생식관에서가장많이분리되는진균인칸디다배양유무에따른 6종류의질내미생물검출빈 도를분석한결과, 칸디다가존재할때 C. trachomatis, M. genitalium, U. urealyticum은더많이검출된반면, N. gonorrhoeae 는더적게검출되었다. 칸디다유무에따라통계적으로유의한차이의검출빈도를보인균종은 M. genitalium였으며, 이는 C. albicans가존재할때더욱의미가있었다. 1999년 Hermann 등은 1,400병상의 3차의료기관에서 1986년부터 1988년까지 3 년동안약 67,700건의각종임상검체에서분리된효모양진균과세균을분석하여, 진균단독또는진균과세균이함께분리된그룹 (F-group) 과세균만분리되거나무균인그룹 (N-group) 으로나누어비교하였다 [10]. 모든유형의검체에서 Enterobacter종이 F-group에서일정하게많이분리되어 Enterobacter 종이칸디다와관련된혼합감염에관련이될것으로추측하였으며, 소변과생식기검체에서는임상적으로중요한세균중 Enterobacter종, Escherichia coli, Pseudomonas aeruginosa, Streptococcus faecalis가 F-group에서의미있게많이분리되었다. N. gonorrhoeae는본연구의결과와유사하게 N-group에서많이분리되었으며, 통계적으로유의한차이를보였다. 그러나아직까지칸디다유무에따른균무리의분포차이가미생물간의상승또는억제작용인지, 우연히특정환자들에서발생하는특수한상황인지, 집락화와감염사이의중요한기전중의하나인지등에관하여평가할수는없었다. 또한호흡기감염에서의 P. aeruginosa와 C. albicans의상호작용가능성을이해하기위한연구에서, P. aeruginosa와 C. albicans를함께배양하였을때 P. aeruginosa가 C. albicans의균사에조밀한균막 (dense biofilm) 을형성하여결국 C. albicans 를사멸시키는것으로보고된바있다 [19]. 이때 P. aeruginosa 는 C. albicans가효모양모양으로존재할때에는 C. albicans에부착하지도못하고사멸시키지도못하는것으로확인되었다. 그러므로본연구가비록일부균종의분포차이만을분석하였고균종간분포차이에관한근거나관련유전학적연구를시행하지못한제한점을가지고있지만, 칸디다와다른미생물간의상호작용연구는흥미로운주제이며, 특히 C. albicans가진균감염에서가장문제가되는균종중의하나이기때문에진균과세균의상호작용에관한연구의대부분은 C. albicans와세균간의상호작용에관한내용이될것으로생각된다. 또한이들간의상호작용에서진균의독력을연구하는것은첫째, 많은경우에서 C. albicans가주로자신에게있던상재균으로부터감염이된다는사실, 둘째, 단일균종에의한감염과진균과세균의혼합감염은명백히다른성상을가진다는것, 셋째, 칸디다와세균사이의관계를연구하고이해하는것은칸디다의생태를이해하고나아가칸디다감염을조절하는새로운전략을개발하는데도움이될수있을것이기때문에중요하다고여겨지고있다 [20]. 결론적으로진균과다른미생물간의상호작용가능성을연구하기위하여시도한본연구를통하여질염증상을가진여

4 52 Korean J Clin Microbiol 2012;15(2):49-53 성의질내칸디다와일부균종의관련가능성을추측할수있었으며, 향후칸디다와미생물간의상호작용확인과기전을밝히기위한추가적인다양한연구가필요하다고사료되었다. 감사의글 이논문은 2009년도정부 ( 교육과학기술부 ) 의재원으로한국연구재단의지원을받아수행된기초연구사업임 ( ). 참고문헌 1. Mirmonsef P, Gilbert D, Zariffard MR, Hamaker BR, Kaur A, Landay AL, et al. The effects of commensal bacteria on innate immune responses in the female genital tract. Am J Reprod Immunol 2011;65: Wira CR, Fahey JV, Sentman CL, Pioli PA, Shen L. Innate and adaptive immunity in female genital tract: cellular responses and interactions. Immunol Rev 2005;206: Forsum U, Holst E, Larsson PG, Vasquez A, Jakobsson T, Mattsby-Baltzer I. Bacterial vaginosis--a microbiological and immunological enigma. APMIS 2005;113: Keane FE, Thomas BJ, Gilroy CB, Renton A, Taylor-Robinson D. The association of Chlamydia trachomatis and Mycoplasma genitalium with non-gonococcal urethritis: observations on heterosexual men and their female partners. Int J STD AIDS 2000;11: Tibaldi C, Cappello N, Latino MA, Masuelli G, Marini S, Benedetto C. Vaginal and endocervical microorganisms in symptomatic and asymptomatic non-pregnant females: risk factors and rates of occurrence. Clin Microbiol Infect 2009;15: Karaca M, Bayram A, Kocoglu ME, Gocmen A, Eksi F. Comparison of clinical diagnosis and microbiological test results in vaginal infections. Clin Exp Obstet Gynecol 2005;32: Sobel JD and Chaim W. Vaginal microbiology of women with acute recurrent vulvovaginal candidiasis. J Clin Microbiol 1996;34: Boris S, Suárez JE, Vázquez F, Barbés C. Adherence of human vaginal lactobacilli to vaginal epithelial cells and interaction with uropathogens. Infect Immun 1998;66: Boris S and Barbés C. Role played by lactobacilli in controlling the population of vaginal pathogens. Microbes Infect 2000;2: Hermann C, Hermann J, Munzel U, Rüchel R. Bacterial flora accompanying Candida yeasts in clinical specimens. Mycoses 1999;42: Lamont RF, Sobel JD, Akins RA, Hassan SS, Chaiworapongsa T, Kusanovic JP, et al. The vaginal microbiome: new information about genital tract flora using molecular based techniques. BJOG 2011;118: Donachie SP, Foster JS, Brown MV. Culture clash: challenging the dogma of microbial diversity. ISME J 2007;1: Anderson MR, Klink K, Cohrssen A. Evaluation of vaginal complaints. JAMA 2004;291: Ventolini G and Baggish MS. Recurrent fungal vulvovaginitis and its association with vaginal bacterial colonization. J Gynecol Sure 2001;19: Mårdh PA, Elshibly S, Kallings I, Hellberg D. Vaginal flora changes associated with Mycoplasma hominis. Am J Obstet Gynecol 1997;176: Lee HH, Ju KS, Lee KH, Won NH. Detection of Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum in the cervical swab and paraffin tissue with female genital tract infection. Korean J Obstet Gynecol 1999;42: Ryu JS, Chung HL, Min DY, Cho YH, Ro YS, Kim SR. Diagnosis of trichomoniasis by polymerase chain reaction. Yonsei Med J 1999;40: Franceschi S, Smith JS, van den Brule A, Herrero R, Arslan A, Anh PT, et al. Cervical infection with Chlamydia trachomatis and Neisseria gonorrhoeae in women from ten areas in four continents. A cross-sectional study. Sex Transm Dis 2007;34: Hogen DA and Kolter R. Pseudomonas-Candida interactions: An ecological role for virulence factors. Science 2002;296: Wargo MJ and Hogan DA. Fungal--bacterial interactions: a mixed bag of mingling microbes. Curr Opin Microbiol 2006;9:

5 Kye Hyun Kim and Mi-Kyung Lee : Vaginal Candida and Microorganisms with Vaginitis 53 = 국문초록 = 질염증상을가진여성에서질내칸디다와성병관련미생물 1 성균관대학교의과대학강북삼성병원산부인과학교실, 2 중앙대학교의과대학진단검사의학교실 김계현 1, 이미경 2 배경 : 여성의생식관은여러가지미생물을포함하는다양한이물질과접촉하게되며, 감염이아닌집락형성과질염사이의균형에서칸디다- 세균의상호작용을고려하는것이중요하다. 본연구의목적은질염과미생물분포간연관성을평가하고여성생식관에존재하는칸디다와다른미생물간의상호작용가능성을연구하는것이다. 방법 : 2008년 10월부터 2010년 6월까지외음질염이의심되는환자로부터채취한총 516건의질분비물을대상으로하였다. 칸디다종동정과배양이까다로운 6균종의미생물 (Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, 그리고 Ureaplasma urealyticum) 검출은각각 VITEK 2 system (biomérieux, Inc., Hazelwood, MO, USA) 과다중 PCR (Seegene, Biotechnology, Inc., Seoul, Korea) 을사용하여시행하였다. 결과 : M. genitalium, U. urealyticum, 그리고 C. trachomatis가질칸디다증과관련하여더많이검출되었고, M. genitalium은통계적으로유의하게많이검출되었다 (P<0.05). N. gonorrhoeae는질칸디다증이있는경우에더적게검출되었다. 결론 : 본연구는질염에서질내칸디다가일부균종과관련될가능성을시사한다. 그러므로향후칸디다와미생물간의상호작용확인과기전을밝히기위한추가적인다양한연구가필요하다고사료되었다. [ 대한임상미생물학회지 2012;15:49-53] 교신저자 : 이미경, , 서울시동작구흑석로 102 중앙대학교의과대학진단검사의학교실 Tel: , Fax: cpworld@cau.ac.kr

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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