Korean J Lab Med 2010;30:147-52 DOI 10.3343/kjlm.2010.30.2.147 Original Article Clinical Microbiology Evaluation of the Efficacies of Rapid Antigen Test, Multiplex PCR, and Real-time PCR for the Detection of a Novel Influenza A (H1N1) Virus Yusun Hwang, M.D., Kyounghee Kim, M.T., and Miae Lee, M.D. Department of Laboratory Medicine, School of Medicine, Ewha Womans University, Seoul, Korea Background : In April 2009, a novel influenza A (H1N1) virus was detected in the US, and at the time of conducting this study, H1N1 infection had reached pandemic proportions. In Korea, rapid antigen tests and PCR assays have been developed to detect the H1N1 virus. We evaluated the efficacies of rapid antigen test, multiplex PCR, and real-time PCR for detecting the H1N1 virus. Methods : From August to September 2009, we tested 734 samples obtained from nasopharyngeal swab or nasal swab using rapid antigen test (SD Influenza Antigen, Standard Diagnostics, Inc., Korea) and multiplex PCR (Seeplex FluA ACE Subtyping, Seegene, Korea). We also tested 224 samples using the AdvanSure real-time PCR (LG Life Sciences, Korea) to compare the results obtained using real-time PCR with those obtained using multiplex PCR. Furthermore, 99 samples were tested using the AdvanSure real-time PCR and the AccuPower real-time PCR (Bioneer, Korea). Results : In comparison with the results of multiplex PCR, the sensitivity and specificity of the rapid antigen test were 48.0% and 99.8%, respectively. The concordance rate for multiplex PCR and the AdvanSure real-time PCR was 99.6% (k=0.991, P=0.000), and that for the AdvanSure real-time PCR and the AccuPower real-time PCR was 97.0% (k=0.936, P=0.000). Conclusions : The rapid antigen test is significantly less sensitive than PCR assay; therefore, it is not useful for H1N1 detection; however multiplex PCR, the AdvanSure real-time PCR, and the Accu- Power real-time PCR can be useful for H1N1 detection. (Korean J Lab Med 2010;30:147-52) Key Words : Novel influenza A (H1N1) virus, Rapid antigen test, Multiplex PCR, Real-time PCR 서 인플루엔자 A (H1N1) 바이러스는 1918년부터 1957년사이에유행하였고 1977년에재출현하여현재까지계절성독감으로유행하고있다. 2009년 4월미국에출현한돼지에서유래한신종인플루엔자 A (H1N1) (swine-origin influenza A [H1N1] virus) 는지속적으로사람간에전파되어 10만명이상의확진환자가발생하였고, 세계보건기구 (WHO) 는인플루엔자대유행경고를 6단계 (2개이상의 WHO 지역의최소 1개국이상에서지속적인 Received : November 19, 2009 Manuscript No : KJLM09-130 Revision received : February 12, 2010 Accepted : February 18, 2010 Corresponding author : Miae Lee, M.D. Department of Laboratory Medicine, Mokdong Hospital, School of Medicine, Ewha Womans University, 911-1 Mok-dong, Yangcheon-gu, Seoul 158-710, Korea Tel : +82-2-2650-5222, Fax : +82-2-2650-5222 E-mail : miae@ewha.ac.kr * 본연구는일부 LG 생명과학의연구비지원에의하여이루어진것임. 론 지역사회에서의유행이있는단계 ) 로발표하였다 [1]. 신종인플루엔자 A (H1N1) 에의한감염의정도는중등도이지만, 첫환자발생이후 6주만에전세계로전파되었고인플루엔자유행계절인겨울로접어든남반구국가들에서환자가급증하는등감염력이매우크기때문에, 인플루엔자의증상을보이거나감염의위험에노출되었던환자는신속히진단하여격리하는것이감염의확산을막는방법으로생각된다 [2, 3]. 인플루엔자 A를검출하기위한검사로신속항원검사 (rapid antigen test) 들이개발되어임상에서사용되고있고, 미국질병통제센터 (Centers for Disease Control, CDC) 에서신종인플루엔자 A (H1N1) 검출을위한중합효소연쇄반응검사방법을발표한후검사실에서중합효소연쇄반응검사를이용해확진할수있게되었다 [4, 5]. 저자들은국내에서인플루엔자진단을위한신속항원검사와함께신종인플루엔자 A (H1N1) 를진단할수있는다중중합효소연쇄반응검사와실시간중합효소연쇄반응검사가개발되어이에대한평가를시행하고자하였다. 147
148 Yusun Hwang, Kyounghee Kim, and Miae Lee 대상및방법 1. 대상 2009년 8월부터 9월까지본원에서신종인플루엔자 A (H1N1) 감염이의심되어코인두면봉 (swab) 이나비강면봉으로채취된검체를대상으로하였다. 인플루엔자증상을보이는환자에서코인두면봉검체는 Flocked swab (Copan, Copan Diagnostics, Corona, CA, USA) 을이용하였고비강검체는멸균된면봉으로채취하였으며, 바이러스수송용배지로 Hank s buffered salt solution (HBSS) 을사용하였다. 734검체를대상으로신속항원검사와다중중합효소연쇄반응검사를시행하였고, 다중중합효소연쇄반응검사를기준으로하여신속항원검사의민감도와특이도를산출하였다. 다중중합효소연쇄반응검사가의뢰된검체중에서 224검체는다중중합효소연쇄반응검사와 AdvanSure 실시간중합효소연쇄반응검사 (LG Life Sciences, Seoul, Korea) 를동시에시행하여두검사간의일치도를평가하였다. 224검체중 99 검체는다중중합효소연쇄반응과 AdvanSure 실시간중합효소연쇄반응검사이외에 AccuPower 실시간중합효소연쇄반응검사 (Bioneer, Daejeon, Korea) 도시행하여두가지실시간중합효소연쇄반응검사사이의일치도를평가하였다. 2. 방법 1) 신속항원검사신속항원검사는 SD Influenza Antigen 키트 (Standard Diagnostics, Inc., Yongin, Korea) 를이용하여제조사의지침에따라검사를시행하였다. 이키트는대조물질, 인플루엔자 A에대한항체및인플루엔자 B에대한항체를스트립에흡착시킨후검체와반응시키는면역크로마토그래피원리를이용하는검사였다. 2) RNA 분리수송배지에운송된검체를 Viral Gene-spin Viral DNA/ RNA Extraction 키트 (intron Biotechnology, Inc., Seongnam, Korea) 를이용하여제조사의지침에따라 RNA를분리하였다. 3) 다중중합효소연쇄반응검사다중중합효소연쇄반응검사는 Seeplex FluA ACE Subtyping 키트 (Seegene, Seoul, Korea) 를이용하여제조사의지침에따라검사를시행한후전기영동에서밴드를판독하였다. 이키트는내부대조, influenza A virus의기질단백질 M유전자, Human influenza A virus subtype H3, Human influenza A virus subtype H1, Avian influenza A virus subtype H5 및 Swine influenza A virus subtype H1의특이적혈구응집소유전자에대한시발체가포함되어인플루엔자의아형에대한감별진단이가능하였다. 4) AdvanSure 실시간중합효소연쇄반응검사 AdvanSure Influenza A/Influenza A H1N1 키트 (LG Life Sciences) 를이용하여제조사의지침에따라실시간중합효소연쇄반응검사를시행하였다. 이키트는 CDC에서제안한검사방법 [4] 에기초하여인플루엔자 A의기질단백질에대한 M 유전자, 신종인플루엔자 A의특이적혈구응집소유전자및내부대조로 Rnase P 시발체를이용하여신종인플루엔자 A (H1N1) 를검출하였다. 5) AccuPower 실시간중합효소연쇄반응검사 AccuPower New Influenza A (H1N1) Real-Time RT- PCR 키트 (Bioneer) 를이용하여제조사의지침에따라검사를시행하였다. 이키트는 CDC에서제안한검사방법 [4] 에기초하여신종인플루엔자 A (H1N1) 에특이적인혈구응집소유전자, 인플루엔자 A에대해특이기질단백질 M 유전자그리고내부대조를이용하여신종인플루엔자 A (H1N1) 를검출하였다. 6) 염기서열분석각중합효소연쇄반응검사방법간결과가일치하지않은검체는클로닝후염기서열을분석하였으며, National Center for Biotechnology Information (NCBI) 에서 Basic Local Alignment Search Tool (BLAST) 을실시하여신종인플루엔자 A (H1N1) 를확인하였다 [6]. 7) 통계분석통계분석은 SPSS (version 17.0, SPSS Inc., Chicago, IL, USA) 프로그램을이용하여시행하였다. 중합효소연쇄반응검사법사이의일치율평가는 kappa 통계량을이용하였고통계적유의성은 P<0.05로하였다.
Multiple Tests for Novel Influenza A (H1N1) 149 결과 1. 신속항원검사와다중중합효소연쇄반응검사의비교 734검체중에서신속항원검사의양성과음성은각각 50검체, 684검체였으며, 다중중합효소연쇄반응검사의양성과음성은각각 102검체, 632검체였다. 다중중합효소연쇄반응을기준으로하여산출한신종인플루엔자 A (H1N1) 에대한신속항원검사의민감도는 48.0% (49/102), 특이도는 99.8% (631/632) 였다. 다중중합효소연쇄반응검사에서신종인플루엔자 A (H1N1) 가검출되지는않았으나계절인플루엔자 A H3형 6검체와 H1형 1 검체가검출되었고, 그중계절인플루엔자 A H3형 1검체만신속항원검사에서검출되고나머지 6검체는신속항원검사에서음성으로나타났다 (Table 1). 2. 다중중합효소연쇄반응검사와 AdvanSure 실시간중합효소연쇄반응검사의비교 224검체중 105검체는다중중합효소연쇄반응검사와 Advan- Sure 실시간중합효소연쇄반응검사모두에서양성이었고 118 검체는모두에서음성으로두검사사이에 99.6% (κ=0.991, P=0.000) 의일치를보였다. 불일치를보인 1검체를염기서열분석한결과신종인플루엔자 A (H1N1) 로확인되었다 (Table 2). 서모두양성인검체가 59, 음성인검체가 37로나타나서 97.0% (κ=0.936, P=0.000) 의일치를보였으며, 3검체는불일치를보였다. 불일치를보인 3검체중 1검체는 AdvanSure 실시간중합효소연쇄반응검사만양성이었고 2검체는 AccuPower 실시간중합효소연쇄반응검사만양성이었는데, 염기서열분석을실시한결과모두신종인플루엔자 A (H1N1) 로확인되었다 (Table 3). 고찰인플루엔자를진단할수있는신속항원검사는간단하고빠르게결과를얻을수있으나신종인플루엔자 A (H1N1) 에대한민감도가 10-69% 로다양하게보고되고있다 [7-9]. 시약에따른민감도를보면 BinaxNOW Influenza A&B (Binax, Inc., Scarborough, ME, USA) 는 38.3-40%, Becton Dickinson Directigen EZ Flu A+B (Becton, Dickinson and Company, Sparks, MD, USA) 는 46.7-49%, Quidel QuickVue Influenza A+B (Quidel Corporation, San Diego, CA, USA) 는 53.3-69% 였다 [7, 8]. Ginocchio 등 [9] 에의하면바이러스배양검사와비교한신속항원검사의민감도는 BinaxNOW Influenza A&B가 9.6%, 3M Rapid Detection Flu A+B (3M Medical Diagnostics, St. Paul, MN, USA) 가 40.0% 였다. 본연구에서다중중합효소연쇄반응검사에대한신속항원검사의민감도는 48% 로 Directigen EZ Flu A+B와비슷하였다. 3. AdvanSure 실시간중합효소연쇄반응검사와 AccuPower 실시간중합효소연쇄반응검사의비교 다중중합효소연쇄반응검사와 AdvanSure 실시간중합효소연쇄반응검사를비교하였던 224검체중 99검체를두가지실시간중합효소연쇄반응검사사이에비교해본결과두검사에 Table 1. Comparison of the efficacies of the rapid antigen test and multiplex PCR for detecting the novel influenza A (H1N1) virus Multiplex PCR + - Total Rapid antigen test + 49 1* 50-53 631 684 Total 102 632 734 *One case showing a positive result in rapid antigen test was negative for novel influenza A (H1N1), but positive for seasonal influenza A (H3) in multiplex PCR; including 5 cases of seasonal influenza A (H3) and 1 case of seasonal influenza A (H1) detected using multiplex PCR. Table 2. Comparison of the efficacies of multiplex PCR and AdvanSure real-time PCR for detecting the novel influenza A (H1N1) virus Multiplex PCR + - *Novel influenza A (H1N1) was confirmed by sequencing. Total AdvanSure real-time PCR + 105 1* 106-0 118 118 Total 105 119 224 Table 3. Comparison of the efficacies of AdvanSure real-time PCR and AccuPower real-time PCR for detecting the novel influenza A (H1N1) virus AdvanSure real-time PCR Total + - AccuPower real-time PCR + 59 2* 61-1* 37 38 Total 60 39 99 *Sequencing revealed 3 cases of novel influenza A (H1N1).
150 Yusun Hwang, Kyounghee Kim, and Miae Lee 대부분의신속항원검사는인플루엔자 A에양성인경우신종인플루엔자 A (H1N1) 와계절인플루엔자를구분할수없다. 본연구에서도신속항원검사에서양성이었으나다중중합효소연쇄반응검사에의해인플루엔자 A H3형으로진단된 1검체가있었다. 본격적인계절인플루엔자의유행시에신종인플루엔자 A는계절인플루엔자 A와항바이러스내성양상이다르므로이를쉽게감별할수있는시약이필요하다고생각된다. 인플루엔자감염을확진하기위한방법은바이러스배양검사와중합효소연쇄반응검사가있지만, 바이러스배양은결과를얻기까지통상적으로일주일이상의시간이소요되기때문에분자진단방법이빠르고예민하고특이도가높아서바이러스배양을대체할수있으며, 특히인플루엔자대유행시는빠르고예민하고자동화장비를이용할경우대량의검사를수행할수있는방법이라고하였다 [10]. 2009년신종인플루엔자 A (H1N1) 대유행시에 CDC에서신종인플루엔자 A (H1N1) 에대해적혈구응집소유전자, 인플루엔자 A에대해기질단백질 M 유전자, Rnase P 내부대조를시발체로이용한통상적과실시간중합효소연소반응검사방법을제시하여각검사실에서신종인플루엔자 A (H1N1) 의진단에이용할수있게되었다 [4]. 이 CDC 방법을기초로하여전세계적으로자가제조또는상품화된통상적중합효소연쇄반응법, 다중중합효소연쇄반응법, 실시간중합효소연쇄반응법및 nucleic acid sequence-based amplification (NASBA) 등의분자진단법이개발되어신종인플루엔자 A (H1N1) 를확진하고있다 [5, 11-16]. 이들연구에따르면분자진단법은빠르고, 예민하고, 바이러스배양이나염기분석으로확인된계절인플루엔자 A와 B, 인플루엔자 A 아형및다른호흡기바이러스등에대해교차반응을보이지않았다고하였다. 실시간중합효소연쇄반응의검출한계는 2 10-2~-3 50% tissue culture infective doses (TCID 50 ) 정도라고하였고, 4개다중중합효소연쇄반응검사와실시간중합효소연쇄반응검사를동시에시행하여 2개이상에서양성인경우를참고방법으로하여실시간중합효소연쇄반응의민감도와특이도를 90.9% 와 100% 라고하였다 [5, 12, 13]. 실시간 NASBA는기존의바이러스배양과 CDC TaqMan 검사및상품화된실시간중합효소연소반응검사에서 2개이상양성인경우를참고방법으로하여민감도와특이도가각각 100% 라고하였다 [11]. 국내는상품화된중합효소연쇄반응검사로 Seegene의 Seeplex FluA ACE Subtyping 키트, LG Life Sciences의 Advan- Sure Influenza A/Influenza A H1N1 키트, Bioneer의 Accu- Power new Influenza A (H1N1) Real-Time PCR 키트등이개발되어신종인플루엔자 A (H1N1) 의확진에이용하고있는 데이에대한임상적평가가없었다. 본연구에서사용한다중중합효소연쇄반응검사는신종인플루엔자 A (H1N1) 이외의계절성인플루엔자 A (H3, H1) 와조류인플루엔자 (H5) 에대한시발체를사용해양성인경우인플루엔자 A의아형을알수있다. 그러나통상적중합효소연쇄반응검사는역전사과정에수기법이필요하고검사소요시간이길며, 전기영동이필요하여밴드가약한경우는판독이어려울수있고, 한번에많은검사를시행하기가어렵다. 본연구에사용된두가지실시간중합효소연쇄반응검사는신종인플루엔자 A (H1N1) 를검출할수있으나인플루엔자 A 의아형을구분할수는없다. 하지만역전사및실시간중합효소연쇄반응검사가동시에진행되어검사과정이간단하고오염이적으며, 검사소요시간이짧다. 또한 threshold cycle 값을측정하여바이러스양을반정량할수있으며한번에많은검사를시행할수있다. 본연구에서다중중합효소연쇄반응검사와 AdvanSure 실시간중합효소연쇄반응검사를동시에검사한 224 검체에서두검사간에일치도가 99.6% (223/224, κ=0.991, P=0.000) 로거의완벽히일치하였고, 두가지실시간중합효소연쇄반응검사를비교한 99검체도 97.0% (96/99, κ=0.936, P=0.000) 의거의완벽한일치를보였다. 불일치결과를보인검체는 RNA 농도가매우낮아서클로닝후염기서열분석을시행하였는데, 모두신종인플루엔자 A (H1N1) 로확인되었다. 국내에서개발된세가지중합효소연쇄반응검사는검사법간에일치율이매우높으므로, 검사실의검사건수등을고려하여세가지중합효소연쇄반응법중에선택하여이용한다면신종인플루엔자 A (H1N1) 의진단에유용할것으로생각된다. 결론으로 SD Influenza Antigen키트를이용한신속항원검사는신종인플루엔자 A (H1N1) 와계절성인플루엔자 A를감별할수없고, 중합효소연쇄반응검사에비해민감도가매우낮아음성인경우는중합효소연쇄반응검사등의확인이필요하여신종인플루엔자 A (H1N1) 진단의선별검사로의의가적다. 신종인플루엔자 A (H1N1) 에대한다중중합효소연쇄반응검사와 AdvanSure와 AccuPower 실시간중합효소연쇄반응검사는일치율이 97.0-99.6% 로높아신종인플루엔자 A (H1N1) 진단에유용할것으로생각된다. 요약배경 : 2009년 4월신종인플루엔자 A (H1N1) 바이러스가미국에서발견되었고대유행상태에도달하였다. 국내에서신
Multiple Tests for Novel Influenza A (H1N1) 151 종인플루엔자 A (H1N1) 를진단하기위한신속항원검사와중합효소연쇄반응검사들이개발되었다. 저자들은국내에서개발된신속항원검사, 다중중합효소연쇄반응검사와실시간중합효소연쇄반응검사를이용하여신종인플루엔자 A (H1N1) 의검출능력을평가하고자하였다. 방법 : 2009년 8월부터 9월까지코인두면봉이나비강면봉으로채취한 734검체를대상으로하여신속항원검사 (SD Influenza Antigen, Standard Diagnostics, Inc., Korea) 와다중중합효소연쇄반응검사 (Seeplex FluA ACE Subtyping, Seegene, Korea) 를시행하였다. 저자들은 224검체를대상으로 AdvanSure 실시간중합효소연쇄반응검사 (LG Life Sciences, Korea) 를시행하여다중중합효소연쇄반응검사결과와비교하였다. 실시간중합효소연쇄반응검사간의비교를위해 99검체를대상으로 AdvanSure 실시간중합효소연쇄반응검사와 AccuPower 실시간중합효소연쇄반응검사 (Bioneer, Korea) 를시행하였다. 결과 : 다중중합효소연쇄반응검사와비교했을때신속항원검사의민감도는 48.0% 였고특이도는 99.8% 였다. 다중중합효소연쇄반응검사와 AdvanSure 실시간중합효소연쇄반응검사사이의일치도는 99.6% (κ=0.991, P=0.000) 였고, 두가지실시간중합효소연쇄반응검사간의일치도는 97.0% (κ=0.936, P= 0.000) 였다. 결론 : 신속항원검사는중합효소연쇄반응검사에비해민감도가상당히낮아신종인플루엔자 A (H1N1) 의검출에유용하지않다고생각된다. 반면다중중합효소연쇄반응검사, Advan- Sure 실시간중합효소연쇄반응검사및 AccuPower 실시간중합효소연쇄반응검사는신종인플루엔자 A (H1N1) 의검출에유용할것이다. 참고문헌 1. WHO. World now at the start of 2009 influenza pandemic. http:// www.who.int/mediacentre/news/statements/2009/h1n1_pandemic_phase6_20090611/en/ (Updated on Jun 2009). 2. Peiris JS, Poon LL, Guan Y. Emergence of a novel swine-origin influenza A virus (S-OIV) H1N1 virus in humans. J Clin Virol 2009; 45:169-73. 3. WHO. Assessing the severity of an influenza pandemic. http:// www.who.int/csr/disease/swineflu/assess/disease_swineflu_ass ess_20090511/en/index.html (Updated on May 2009). 4. WHO. WHO information for laboratory diagnosis of pandemic (H1N1) 2009 virus in humans-revised. http://www.who.int/csr/ resources/publications/swineflu/who_diagnostic_recommendationsh1n1_20090521.pdf (Updated on Nov 2009). 5. Pabbaraju K, Wong S, Wong AA, Appleyard GD, Chui L, Pang XL, et al. Design and validation of real-time reverse transcription-pcr assays for detection of pandemic (H1N1) 2009 virus. J Clin Microbiol 2009;47:3454-60. 6. National Center for Biotechnology Information (NCBI). Sequences from pandemic (H1N1) 2009 viruses. http://www.ncbi.nlm.nih. gov/genomes/flu/flu.html (Updated on Nov 2009). 7. Centers for Disease Control and Prevention (CDC). Evaluation of rapid influenza diagnostic tests for detection of novel influenza A (H1N1) Virus-United States, 2009. MMWR Morb Mortal Wkly Rep 2009;58:826-9. 8. Vasoo S, Stevens J, Singh K. Rapid antigen tests for diagnosis of pandemic (Swine) influenza A/H1N1. Clin Infect Dis 2009;49:1090-3. 9. Ginocchio CC, Zhang F, Manji R, Arora S, Bornfreund M, Falk L, et al. Evaluation of multiple test methods for the detection of the novel 2009 influenza A (H1N1) during the New York City outbreak. J Clin Virol 2009;45:191-5. 10. Petric M, Comanor L, Petti CA. Role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics. J Infect Dis 2006;194(S2):S98-110. 11. Ge Y, Cui L, Qi X, Shan J, Shan Y, Qi Y, et al. Detection of novel swine origin influenza A virus (H1N1) by real-time nucleic acid sequencebased amplification. J Virol Methods 2010;163:495-7. 12. LeBlanc JJ, Li Y, Bastien N, Forward KR, Davidson RJ, Hatchette TF. Switching gears for an influenza pandemic: validation of a duplex reverse transcriptase PCR assay for simultaneous detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus. J Clin Microbiol 2009;47:3805-13. 13. Poon LL, Chan KH, Smith GJ, Leung CS, Guan Y, Yuen KY, et al. Molecular detection of a novel human influenza (H1N1) of pandemic potential by conventional and real-time quantitative RT-PCR assays. Clin Chem 2009;55:1555-8. 14. Whiley DM, Bialasiewicz S, Bletchly C, Faux CE, Harrower B, Gould AR, et al. Detection of novel influenza A(H1N1) virus by real-time RT-PCR. J Clin Virol 2009;45:203-4. 15. He J, Bose ME, Beck ET, Fan J, Tiwari S, Metallo J, et al. Rapid multiplex reverse transcription-pcr typing of influenza A and B virus, and subtyping of influenza A virus into H1, 2, 3, 5, 7, 9, N1 (human),
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