ORIGINAL ARTICLE Comparison of the Anyplex II STI-7 and Seeplex STD6 ACE Detection Kits for the Detection of Sexually Transmitted Infections Se Jin Mo

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1 ORIGINAL ARTICLE Comparison of the Anyplex II STI-7 and Seeplex STD6 ACE Detection Kits for the Detection of Sexually Transmitted Infections Se Jin Moon, Jung-Eun Choi, and Kwang-Il Park Department of Laboratory Medicine, U2Bio Laboratory, Seoul, Korea Corresponding author: Se Jin Moon Department of Laboratory Medicine, U2Bio Laboratory, 275 Hwarang-ro, Seongbuk-gu, Seoul , Korea Tel Fax Background: Sexually transmitted infections (STI) encompass a variety of clinical syndromes caused by many pathogens that are transmitted through sexual activity. Multiplex PCR is frequently used to detect STI. In this study, two multiplex real-time PCR-based assays were used to detect STI in clinical specimens, and the concordance of the results obtained by each method was evaluated. Methods: A total of 626 specimens were tested using the Anyplex II STI-7 (Seegene, Korea) and Seeplex STD6 ACE Detection kits (Seegene). Results: Among the 626 individuals tested, 227 (44.2%) tested positive for STI by using Anyplex II STI-7. The prevalence rates of the various infectious microorganisms detected were as follows: Chlamydia trachomatis (C. trachomatis), 19.2% (120/626); Neisseria gonorrhoeae (N. gonorrhoeae), 5.6% (35/626); Trichomonas vaginalis (T. vaginalis), 0.2% (1/626); Mycoplasma genitalium (M. genitalium), 8.1% (51/626); Mycoplasma hominis (M. hominis), 2.9% (18/626); Ureaplasma urealyticum (U. urealyticum), 17.6% (110/626); and Ureaplasma parvum, 3.7% (23/626). The concordance rates for the STI-7 and STD6 assays in detecting the various types of microorganism were as follows: C. trachomatis, (99.5%); N. gonorrhoeae, (99.7%); T. vaginalis, (100%); M. genitalium, (100%); M. hominis, (100%); and U. urealyticum (99.2%). Conclusions: A high degree of concordance was observed between the results obtained using the Anyplex II STI-7 kits and those obtained using the Seeplex STD6 ACE Detection kits. ( J Lab Med Qual Assur 2013;35:87-92) Key Words: Sexually transmitted diseases, Multiplex polymerase chain reaction, Sexually transmitted infections-7 pissn: X eissn: Received May 23, 2013, Revision received July 24, 2013, Accepted August 21, 2013 서론 성매개감염 (sexually transmitted infections) 은성접촉을통해서전파되는감염증으로다양한원인미생물들로인해일어난다 [1,2]. 성매개감염은감염되어도무증상을보이는경우가많으며, 치료하지않을경우만성적으로진행하여합병증을일으키며, 타인에게전파시킬수도있다 [3,4]. 하지만성매개감염의원인미생물들은배양에적합한조건이서로상이하고검출에많은시간을요구되는경우가많아, 빠른진단과치료에어려움을주는경우가많다. 그로인해다중핵산증폭검사가진단에유용하게사용되고있다 [2,5-7]. 이연구는성매개감염원인균 인 Chlamydia trachomatis (C. trachomatis), Neisseria gonorrhoeae (N. gonorrhoeae), Trichomonas vaginalis (T. vaginalis), Mycoplasma genitalium (M. genitalium), Mycoplasma hominis (M. hominis), Ureaplasma urealyticum (U. urealyticum), Ureaplasma parvum (U. parvum) 을한번에진단할수있도록개발된 multiplex real-time polymerase chain reaction (PCR) 인 Anyplex II STI-7 Detection Kit (STI-7; Seegene, Seoul, Korea) 를기존 conventional PCR을이용한 Seeplex STD6 ACE Detection kit (STD6, Seegene) 와비교하여성매개감염진단에서의유용성을평가하고자하였다. STD6는성매개감염의주요원인미생물인 C. Copyright 2013 The Korean Association of Quality Assurance for Clinical Laboratory 87

2 trachomatis, N. gonorrhoeae, T. vaginalis, M. genitalium, M. hominis, U. urealyticum 에특이적인시동체를사용하여각원인미생물을동시에증폭하여결과를확인할수있는분자진단검사이며 [8], STI-7은실시간중합효소연쇄반응법을이용하며, tagging oligonucleotide cleavage and extension (TOCE) 기술이적용되었다 [9-11]. STI-7은 STD6와동일목적유전자를이용하며추가적으로 U. parvum 을확인할수있다. 대상및방법 1. 대상 2013년 2월 1일부터 4월 31일까지 1차비뇨기과의원 103개, 2차병원 3개, 산부인과의원 7개에서성매개성감염검사가의뢰된검체중에서중복되지않는 626예를대상으로하였다. 남자는 561명, 여자는 65명이었다. 2. 방법환자의검체는소변일경우 50 ml tube에채취하였고, swab 의경우 Digene cervical sampler (Qiagen, Hilden, Germany) 로채취하여수송후, Seeprep12 NA common kit (Biotrin International Ltd., Dublin, Ireland) 와 Chelex 100 (Bio-Rad, Hercules, CA, USA) 를사용하여제조사의지시에따라 DNA를추출하였으며, 동일한방법으로추출된 DNA로각각의키트로 PCR을진행하였다. PCR은 GeneAmp PCR system 2720 (Applied Biosystems Inc., Foster City, CA, USA) 으로, Seeplex STD6 ACE Detection (Seegene, Seoul, Korea) 을사용하여 94 o C에서 15분, 94 o C에서 30초, 63 o C에서 90초, 72 o C 에서 90초로 40번반복후 72 o C에서 10분간반응하였다. PCR 산물은 2% agarose gel 전기영동을통해확인하였다. Real time PCR은 Anyplex II STI-7 Detection (STI-7, Seegene) 을사용하여 CFX 96 real-time thermocycler (Bio-Rad) 를이용하였으며, 반응은 95 o C에서 15분, 50 cycles at 95 o C에서 30초, 60 o C에서 60초, 72 o C에서 30초간을 50회반복하는조건으로수행되었으며, 30, 40, 50번째반복후결과를측정한후제조사의지침에따라판독하였다. 각원인미생물별로사용된유전자부위는 C. trachomatis 는 pctt1, M. genitalium 은 gyra, M. hominis 는 gap, N. gonorrhoeae 는 pora pseudogene, T. vaginalis 는 tub1, U. urealyticum 은 ureg-d였으며, STI-7도같은유전자를사용하였으며추가된 U. parvum의유전자는 UreA였다. PCR 반응을확인하기위한내부대조물질 Table 1. Distribution of microorganisms in the clinical samples that tested positive for sexually transmitted infections Pathogen Prevalence No. of cases (n=626) Male (n=561) Female (n=65) C. trachomatis (20.0) 8 (12.3) N. gonorrhoeae (6.2) 0 (0) Trichomonas vaginalis (0) 1 (1.5) M. genitalium (7.5) 9 (13.8) M. hominis (2.0) 7 (10.8) U. urealyticum (16.8) 16 (24.6) U. parvum (2.3) 10 (15.4) Not detected One microorganism Two microorganisms* Three microorganisms Abbreviations: N. gonorrhoeae, Neisseria gonorrhoeae; C. trachomatis, Chlamydia trachomatis; M. genitalium, Mycoplasma genitalium; U. parvum, Ureaplasma parvum; U. urealyticum, Ureaplasma urealyticum; M. hominis, Mycoplasma hominis. *N. gonorrhoeae+c. trachomatis (n=11); C. trachomatis+m. genitalium (n=4); U. parvum+c. trachomatis (n=15); U. urealyticum+m. hominis (n=6); U. urealyticum+m. genitalium (n=8); other infections (n=22 ). Three microorganisms (n=6); four microorganisms (n=1). 88 J Lab Med Qual Assur 2013;35:

3 은 Arabidopsis 의 Cesa3 유전자였다. STD6와 STI-7은유전자부위는동일하지만검사특성에따라시동체를다르게선정하였다. 3. 염기서열분석 STD-7과 STD6에서서로다른결과를보인 9예와 STI-7에서만검출되는 U. parvum 양성검체 23예중 4예를대상으로염기서열을분석하였다. 염기서열분석은 PCR 산물을해당원인미생물의유전자에특이적인시동체를이용하여 BigDye Terminator v3.1 Cycle Sequencing Kits (Life technologies, Carlsbad, CA, USA) 로염기서열확인을하기위한증폭과정을거친후, 반응이끝나면제조사에서권장하는방법으로반응에참여하지않은 dntp와반응물을제거한후, ABI 3730xl DNA (Life technologies) 를이용하여염기서열결과를얻은후 BLAST ( ncbi.nlm.nih.gov/blast) 에서검색하여해당원인미생물유전자의염기서열과일치하는지를확인하였다. 결과 검사를시행한총 626예의검사에서양성결과를보인것은 277예 (44.2%) 였다. 원인미생물별로각각 C. trachomatis 120예 (19.2%), N. gonorrhoeae 35예 (5.6%), T. vaginalis 1예 (0.2%), M. genitalium 51예 (8.1%), M. hominis 18예 (2.9%), U. urealyticum 110예 (17.6%), U. parvum 23예 (3.7%) 의결과를보였다 (Table 1). 단일미생물양성은 204예 (32.6%) 였으며 2가지미생물양성은 66예 (10.5%), 3종이상미생물양성은 7예 (1.1%) 로그중에서 C. trachomatis 와 N. gonorrhoeae 중복감염은 11예였으며, 모두남자환자에서검출되었다. C. trachomatis 와 M. genitalium 중복감염은 4예였다 (Table 1). STI-7과 STD6 검사결과의일치율은 C. trachomatis 99.5% (Tables 2, 3), N. gonorrheae 99.7% (Table 2), T. vaginalis 100%, M. genitalium 100%, M. hominis 100%, U. urealyticum 99.2% (Table 3) 였다. 두검사결과간차이를보인 C. trachomatis 3예, N. gonorrhoeae 2예, U. urealyticum 4예였으며, C. trachomatis 3예는 STI-7 에서는단독으로양성결과를보였고, N. gonorrhoeae 2 예는 STI-7에서 C. trachomatis와중복감염을보였으나 STD6에서는 C. trachomatis만관찰되었다. U. urealyticum 4예는 STI-7에서모두단독양성결과를보였다. 일치하지않은 9예는염기서열분석을시행한결과모두 STI-7 결과와일치하였다. 또한 STI-7에서만검출가능한 U. parvum 는양성결과를보인 23예중에서 4예를무작위로선택하여염기서열분석을진행하였고모두 U. parvum 으로확인되었다. 고찰 성매개감염은치료하지않는경우불임이나골반내염증등의합병증을일으킬수있고, 다른사람에게감염을전파시킬수있으므로정확한진단과그에따른적절한치료가필요하다 [1,2,12]. 또한감염되어도증상이없는경우가많으며복합감염을보이는경우가많기때문에 [13], 한번의검사로여러균종의감염을확인할수있는다중 PCR이진단에도움을주고있다. 또한국내성병진료지침에서는원인미생물에따라적절한항생제를선택하여치료할것을권장하고있다 [1]. 이처럼적절한항생제를선택하기위해서는감염의원인이되는원인미생물을정확하게규명하는것이필요하며, 다중 PCR은진단및치료후추적관찰에도도움을줄수있을것으로생각된다. 이번연구에서는 C. trachomatis, N. gonorrhoeae, T. vaginalis, M. genitalium, M. hominis, U. urealyticum, U. parvum 의양성률은각각 19.2%, 5.6%, 0.2%, 8.1%, 2.9%, 17.6%, 3.7% 를보였으며 (Table 1), 이는건강검진환자를대상으로실시되었던두국내연구에서 [13,14] 보고된 C. trachomatis 5.4%, 5,6%, N. gonorrhoeae 4.3%, 0.4%, M. genitalium 0.3%, U. urealyticum 21.2%, 22,1%, M. hominis 11.1%, 11.6%, T. vaginalis 0.9%, 1,1% 와는다르게 C. trachomatis 와 N. gonorrhoeae 가높은양성률을보였다. 두국내연구는모두검사방법으로 STD6를이용하여실험하였으며, 결과는 N. gonorrhoeae 를제외하고모두유사한양성률을보였다. 이는이번연구대상은성매개감염이의심되어 1, 2차병원을방문한환자군이었고다른연구들은 3차병원을방문한증상이없는건강검진환자였다는점에서차이를보였다고생각된다. 세연구결과모두 T. vaginalis 감염양성률은 0.1% 로유사하게나타났다. 양성결과중 2가지이상의균종이나타난 73예중 C. trachomatis 와 N. gonorrheae 중복감염은 11예였고이는전체 N. gonorrheae 양성환자의 31.4% 에해당한다. C. trachomatis 와 M. genitalium 중복감염은 5예에서관찰되었다. 성매개감염진료지침에서는임균과클라미디아동 J Lab Med Qual Assur 2013;35:

4 Table 2. Comparison of results obtained using the Anyplex II STI-7 and Seeplex STD6 kits for the detection of C. trachomatis, N. gonorrhoeae, and T. vaginalis N of samples (n=626) Seeplex STD6 ACE Detection Anyplex II STI-7 Detection C. trachomatis N. gonorrhoeae T. vaginalis Pos Neg Total Abbreviations: C. trachomatis, Chlamydia trachomatis; N. gonorrhoeae, Neisseria gonorrhoeae; T. vaginalis, Trichomonas vaginalis; Pos., positive; Neg., negative. Table 3. Comparison of results obtained using the Anyplex II STI-7 and Seeplex STD6 kits for the detection of M. genitalium, M. hominis, and U. urealyticum N of samples (n=626) Seeplex STD6 ACE Detection Anyplex II STI-7 Detection M. genitalium M. hominis U. urealyticum Pos Neg Total Abbreviations: M. genitalium, Mycoplasma genitalium; M. hominis, Mycoplasma hominis; U. urealyticum, Ureaplasma urealyticum; Pos., positive; Neg., negative. 90 J Lab Med Qual Assur 2013;35:

5 시감염은 30-50% 로흔하다고보고되고있다. 기존국내및국외연구결과에서성매개감염진단에서우수한민감도와특이도를보였다고보고된 [13-17] STD 6종과의비교실험결과 STI-7종검사는 C. trachomatis, N. gonorrhoeae, T. vaginalis, M. genitalium, M. hominis, U. urealyticum 는각각각각 99.5%, 99.7%, 100%, 100%, 100%, 99.2% 의일치율을보였다 (Tables 2, 3). 두검사결과간차이를보인 C. trachomatis 3예, N. gonorrhoeae 2예, U. urealyticum 4예를대상으로염기서열분석을시행한결과 STI-7 검사결과와일치하였다. 두검사결과간차이를보인 C. trachomatis 3예의경우 STI-7에서는단독양성결과를보였지만 STD6에서는음성결과를보여위음성으로생각되었다. 또한 N. gonorrhoeae 2예는 STI-7에서 C. trachomatis 와중복감염을보였으나 STD6에서는 C. trachomatis 만관찰되었으므로임상적치료의방향에영향을줄수있을것으로생각된다. 임상검체를대상으로한비교평가에서양성검출률이향상된것으로보아 STD6 제품보다 STI-7이민감도가향상된것으로보이지만이연구만으로는판단하기힘들며, 이에대해서는더많은연구가필요할것으로생각된다. 단이번연구대상환자중여성에서는 N. gonorrhoeae 가모두음성을보였으므로여성에대한 N. gonorrhoeae 의진단적가치에대해서는추가적인연구가필요할것으로보인다. 다중중합효소연쇄반응인 STD6와달리 STI-7에적용된 TOCE 기술은표적유전자에특이적인서열을가지고결합되어있던 Pitcher가유전자증폭과정에서 Taq polymerase 에의해절단되고, 절단된 Pitcher는 Catcher 와결합후 Catcher 에서발산되는형광을융해온도분석법을통하여검출하는다중실시간중합효소연쇄반응이다. 두검사는 STD6와비교하였을때 STI-7검사는실시간중합효소연쇄반응으로진행되므로기존의 PCR방법과비교하면 PCR 후전기영동을통한결과의확인과정이필요없어검사시간을단축할수있으며더편리하였다. 또한 PCR 산물을개봉하지않고도결과를분석할수있으므로 PCR 산물로인한오염으로부터검사결과의오류를줄이는데도움을줄수있을것으로생각된다. 또한 STI-7종검사는기존 STD 6종의검출항목에서 U. parvum 이추가되었다. Ureaplasma 종은성매개질환과관계가있다고알려져있으나상재균으로존재하는경우가있어서단순히 PCR 진단결과만으로치료를시작하지않고임상증상을고려하여치료를선택한다 [1,2,18]. U. parvum 은요로감염인경우남성불임과관계가있다는보고는있 지만 [19], U. urealyticum 과는달리성매개감염치료지침이정해져있지않고 [1,2], STI-7종검사는 U. parvum 과 U. urealyticum 의구분이가능하기때문에각원인균에대해임상적인증상을추적관찰하는연구가진행된다면보다자세한임상적의미를구분할수있을것으로생각된다. 결론적으로 STI-7는기존에평가된 STD6과비교하여높은일치율을보였으므로성매개감염균의진단및치료에도움을줄수있을것으로생각된다. REFERENCES 1. Korea Centers for Disease Control and Prevention. Sexually transmitted infections Korean guidelines jsp? m enuid s = H O M E M NU0004-M NU0085- MNU0088&fid=51&q_type=title&q_value=%EC%84% B1%EB%A7%A4%EA%B0%9C&cid=9935&pageNum=1 (Accessed May 23, 2013). 2. Workowski KA, Berman S; Centers for Disease Control and Prevention (CDC). Sexually transmitted diseases treatment guidelines, MMWR Recomm Rep 2010;59: Choi JH, Jeung IC, Pak YG, Park DC. Prevalence and risk factors of chlamydia trachomatis and Neisseria gonorrhea among Korean women. Korean J Obstet Gynecol 2007;50: Lee IS. Historical changes and the present situation of sexually transmitted diseases. J Korean Med Assoc 2008; 51: Naber KG, Schaeffer AJ, Heyns CF, Matsumoto T, Shoskes DA, Bjerklund Johansen TE. Urogenital infections. Arnhem: European Association of Urology, Holmes KK, Sparling PF, Mardh PA. Sexually transmitted diseases. 4th ed. New York: McGraw-Hill Medical, Lee SR, Chung JM, Kim YG. Rapid one step detection of pathogenic bacteria in urine with sexually transmitted disease (STD) and prostatitis patient by multiplex PCR assay (mpcr). J Microbiol 2007;45: Horii T, Ohtsuka H, Osaki M, Ohkuni H. Use of a dual priming oligonucleotide system to detect multiple sexually transmitted pathogens in clinical specimens. Lett Appl Microbiol 2009;49: Mackay IM, Arden KE, Nitsche A. Real-time PCR in J Lab Med Qual Assur 2013;35:

6 virology. Nucleic Acids Res 2002;30: Chun JY. High multiplex molecular diagnostics: shifting the diagnostics paradigm. Seegene Bull 2012;1: Lee DH. TOCE: innovative technology for high multiplex real-time PCR. Seegene Bull 2012;1: Hillis SD, Wasserheit JN. Screening for chlamydia: a key to the prevention of pelvic inflammatory disease. N Engl J Med 1996;334: Kim SJ, Lee DS, Lee SJ. The prevalence and clinical significance of urethritis and cervicitis in asymptomatic people by use of multiplex polymerase chain reaction. Korean J Urol 2011;52: Lee SJ, Park DC, Lee DS, Choe HS, Cho YH. Evaluation of Seeplex STD6 ACE Detection kit for the diagnosis of six bacterial sexually transmitted infections. J Infect Chemother 2012;18: Yu N, Lee MK. Clinical implications of multiplex PCR detection of fastidious microorganisms in vaginitis patients. Korean J Clin Microbiol 2011;14: Kim WJ, Lee K, Kim DL. PCR-based investigation of infection patterns in patients with pelvic inflammatory diseases in Jeju. Lab Med Online 2013;3: Samra Z, Rosenberg S, Madar-Shapiro L. Direct simultaneous detection of 6 sexually transmitted pathogens from clinical specimens by multiplex polymerase chain reaction and auto-capillary electrophoresis. Diagn Microbiol Infect Dis 2011;70: Abele-Horn M, Wolff C, Dressel P, Pfaff F, Zimmermann A. Association of Ureaplasma urealyticum biovars with clinical outcome for neonates, obstetric patients, and gynecological patients with pelvic inflammatory disease. J Clin Microbiol 1997;35: Gdoura R, Kchaou W, Chaari C, Znazen A, Keskes L, Rebai T, et al. Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis and Mycoplasma genitalium infections and semen quality of infertile men. BMC Infect Dis 2007;7:129. 성매개감염진단을위한 Anyplex II STI-7 키트와 Seeplex STD6 ACE 키트의비교문세진 최정은 박광일유투바이오진단의학연구소진단검사의학과 배경 : 성매개감염은성접촉을통해전파되는감염증으로많은원인미생물들이존재한다. 이연구는새롭게출시된 Anyplex II STI-7 Detection Kit (STI-7; Seegene, Korea) 의유용성을평가해보고자하였다. 방법 : 본검사실에의뢰된성매개감염이의심되는 626 예의검체를대상으로 STI-7 검사 (Seegene) 와 Seeplex STD6 ACE Detection 검사 (STD6; Seegene) 를각각시행하였다. 결과 : 검사를시행한총 626 예에서 STI-7 에서양성결과를보인것은 277 예 (44.2%) 였으며미생물별로 Chlamydia trachomatis 120 예 (19.2%), Neisseria gonorrheae (N. gonorrheae) 35 예 (5.6%), Trichomonas vaginalis (T. vaginalis) 1 예 (0.2%), Mycoplasma genitalium (M. genitalium) 51 예 (8.1%), Mycoplasma hominis (M. hominis) 18 예 (2.9%), Ureaplasma urealyticum (U. urealyticum) 110 예 (17.6%) 의양성결과를보였으며, STI-7 검사는 STD6 와비교하여 N. gonorrheae, T. vaginalis, M. genitalium, M. hominis, U. urealyticum 에서 99.5%, 99.7%, 100%, 100%, 100%, 99.2% 의일치율을보였다. 결론 : 이번연구에서 STI-7 검사는기존의 STD6 와높은일치율을보였다. (J Lab Med Qual Assur 2013;35:87-92) 교신저자 : 문세진우 ) 서울시성북구화랑로 275, 유투바이오진단의학연구소진단검사의학과 Tel: 02) Fax: 02) moon8112sj@naver.com 92 J Lab Med Qual Assur 2013;35:

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untitled Ann Clin Microbiol Vol. 20, No. 1, March, 2017 https://doi.org/10.5145/acm.2017.20.1.1 pissn 2288-0585 eissn 2288-6850 Comparative Evaluation of Multiplex Real-Time PCR Assays for Six Pathogens of Sexually