Korean J Clin Microbiol Vol. 10, No. 1, April, 2007 Evaluation of Strep A for Rapid Antigen Testing in Elementary Schoolchildren Eun-Ha Koh, Sunjoo Kim Department of Laboratory Medicine, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Korea Background: Rapid antigen tests (RAT) of group A streptococci (GAS) are easy to perform and can save two days of bacterial culture time. Performance of Strep A was analyzed in comparison with throat culture. Methods: Three consecutive throat swabs were taken from 308 healthy elementary schoolchildren. The first two swabs were tested for Strep A and Dipstick Strep A rapid antigen tests, and the third one was inoculated onto blood agar plate to grow GAS. Results: Sensitivity, specificity, positive predictive value and negative predictive value of Strep A were 79.3%, 88.9%, 72.2%, and 92.2% respectively. Those of Strep A were 58.5%, 93.8%, 77.4%, and 86.2% respectively. Conclusion: Strep A showed a significantly higher sensitivity and a slightly lower specificity compared to Strep A. SD Bioline Strep A RAT should be useful for the rapid diagnosis of bacterial pharyngitis and the optimum use of antibiotics. (Korean J Clin Microbiol 2007; 10:54-58) Key Words: Streptococcus pyogenes, Group A streptococcus, Rapid antigen test, Throat culture, Pharyngitis 서 상기도감염은매우흔한질환이며대부분은바이러스가원인이지만약 30% 에서는세균에의해발생한다 [1-3]. 세균성인두염의가장흔한원인은 A군연쇄구균이다 [4,5]. 저자들이연구한바에의하면세균성인두염환자의약 50% 에서 A군연쇄구균이분리되었다 [6]. A군연쇄구균인두염의정확한진단을위해서는인두배양이표준검사법 (gold standard) 이지만배양기등세균배양시설이필요하고, 세균동정을위해 1~2일이필요하므로진단결과를알기위해서는환자가다시병원을방문해야하는단점이있다. 세균배양시설이필요하지않은신속항원검사는환자가첫번째의사를방문했을때 A군연쇄구균인두염진단이가능해져즉시항생제치료여부를결정할수있다 [7,8]. 미국소아과학회 (American Academy of Pediatrics) 에서는세균성인두염진단시신속항원검사를먼저시행하여음성인경우에는세균배양을시행하도록권장하고있다 [9]. 진찰소 Received 17 January, 2007, Accepted 15 February, 2007 Correspondence: Sunjoo Kim, Department of Laboratory Medicine, Gyeongsang National University Hospital, 90, Chilam-dong, Jinju 660-702, Korea. (Tel) 82-55-750-8239, (Fax) 82-55-762-2696, (E-mail) sjkim8239@hanmail.net 론 견만으로상기도감염을진단하는경우부정확할수있으며, 경험많은의사라고하더라도증상이나징후만으로정확히진단할수있는가능성은 80% 미만이라는보고가있다 [3-5]. 대부분이바이러스성인두염인상기도감염에서검사를시행하지않고항생제를처방하는것은불필요한치료와의료비용상승, 그리고항생제오남용의문제로이어진다. 우리나라에서상기도감염의항생제남용은심각한수준이며, 이로인해항생제내성률이매우높다 [10]. 항생제처방비용과약제부작용, 내성문제의심각성을고려한다면, 정확한검사과정을거쳐 A군연쇄구균인두염이확정된후에항생제를투여하는것이바람직하다. 우리나라에서는개인의원에서세균배양이나신속항원검사를일상적으로시행하고있지않지만, 구미선진국에서는약 20년전부터검사실이나소아과의원등에서자주이용하고있다 [11]. 신속항원검사는라텍스응집반응을관찰하거나효소면역법혹은면역크로마토그래피법으로발색정도를확인하는것이다. 신속항원검사의평가는검사방법이나시약, 연구자에따라매우다양하게보고되고있으며, 예민도는 60~ 90%, 특이도는 80~95% 정도로알려져있다 [1,7,12-14]. 저자들은최근국내에서개발된세균성인두염신속항원검사 SD Bioline Strep A의제품평가를위해초등학생을대상으로본연구를시행하였다. 54
Eun-Ha Koh, Sunjoo Kim : Evaluation of Rapid Antigen Test 55 대상및방법 2006년 6월에서 9월사이에진주지역의초등학교세곳을방문하여인두염이없는건강한초등학생 308명을대상으로하였다. 연령은 9세에서 13세까지다양하였고, 각교실에서희망자를대상으로검체를 3회채취하였다. 먼저 아 소리를내게하여설압자로편도를잘보이게한후소독된면봉으로편도표면을문질러검체를채취하였다. 인후도말하여채취한면봉검체에대하여처음 2개는 Strep A와 Quidel Quick Vue Dipstick Strep A 신속항원검사를사용설명서대로시행하였다. 5명씩번갈아서신속항원검사순서를바꾸었다. 이들신속항원검사는 lateral flow type의간편한면역발색법이다. 발색반응결과는 2명이판정하였으며, 발색이강하거나약한경우양성으로, 발색이희미하거나없는경우에는음성으로판단하였다. 나머지 1개의면봉은면양혈액한천배지에접종한후 2시간이내에검사실로옮겨백금이로획선을그어균을확산시켰다. 35 o C에서하룻밤배양하여베타용혈성집락을확인하였고, 베타용혈성연쇄구균으로의심되는것은 bacitracin 디스크 (0.04 U) 감수성검사와라텍스응집법 (Seroiden Streptokit, Eiken, Tokyo, Japan) 으로 A, B, C, 그리고 G군을동정하였다. 세균동정은신속항원검사결과를모르는상태에서진행하였다. 세균배양결과를기준으로각신속항원검사의예민도, 특이 도, 양성및음성예측률을구하였다 (χ 2 -test). B, C, 그리고 G군베타용혈성연쇄구균분리자에서신속항원검사가양성으로나오는지살펴보았다. A군연쇄구균집락수가 10개미만이면 1+, 10~50개사이이면 2+, 50개이상이면 3+ 로표시하여, 집락수에따라신속항원검사결과가달라지는지살펴보았다. 본연구는경상대학교병원임상시험위원회의승인을얻었다. 결과총 308명의초등학생을대상으로시행한두가지 A군연쇄구균신속항원검사시약의평가에서 Strep A는예민도 79.3%, 특이도 88.9%, 양성예측률 72.2%, 음성예측률 92.2% 의효율성을보였다. 한편 Dipstick Strep A는예민도 58.5%, 특이도 93.8%, 양성예측률 77.4%, 음성예측률 86.2% 의결과를보였다 (Table 1, 2). B, C, G군 30균주에서위양성을보인경우는 Strep A가 5균주 (16.7%), Dipstick Strep A가 3균주 (10.0%) 있었다 (Table 3). 집락수에따른양성률을살펴보면집락정도가 1+ 일때 SD Bioline Strep A가 89.7% 로매우높은양성률을보였고, Quidel Quick Vue Dipstick Strep A는집락수가많아질수록양성률도증가하는양상을보였다 (Table 4). Table 1. Comparison of two rapid antigen tests with throat culture in schoolchildren Throat culture Strep A Rapid antigen test Dipstick Strep A Positive Negative Positive Negative Total GAS+ 65 17 48 34 82 GAS- 25 201 14 212 226 Abbreviation: GAS, group A streptococci. Table 3. Positive rates of Strep A and Dipstick Strep A rapid antigen tests in non-a beta-hemolytic streptococci Group No. Dipstick B 5 0 (0) 1 (20.0) C 16 4 (25.0) 1 (6.3) G 9 1 (11.1) 1 (11.1) Total 30 5 (16.7) 3 (10.0) Table 2. Performance of two rapid antigen tests in comparison with throat culture Dipstick P value* Sensitivity 79.3 58.5 0.007 Specificity 88.9 93.8 0.094 Positive predictive value 72.2 77.4 0.572 Negative predictive value 92.2 86.2 0.055 *χ 2 -test. Table 4. Positive rates of two rapid antigen tests according to colony forming units of group A streptococci CFU* No. Dipstick 1+ 29 26 (89.7) 14 (48.3) 2+ 40 29 (72.5) 26 (65.0) 3+ 11 10 (90.9) 8 (72.7) Total 80 65 (81.3) 48 (60.0) *1+, <10 colonies; 2+, 11~50 colonies; 3+, >51 colonies.
56 Korean J Clin Microbiol 2007;10(1):54-58 고찰인두배양은세균성인두염을진단하기위한표준검사법이지만, 검체채취나세균배양의경험과기술에따라결과에많은차이가있다. 특히검체채취과정은가장중요한데, 환자가입을크게벌리고 아 소리를내어편도가잘보이게한후양쪽편도혹은편도와 (tonsillar fossa) 부위를세게문질러야한다. 후인두 (posterior pharynx) 부위는균이적으므로굳이채취하지않아도된다 [15]. 목젖이나혀, 침, 구강점막등에서채취하면위음성을초래할수있으므로주의해야한다. 만약균수가적어서백금이에의해제대로확산되지못하고상재균과섞여있는경우순수분리하는것이매우어려우며, 이때는정확한집락수를파악하는것도어렵다. 배양기에사용하는가스는공기, CO 2 및질소 ( 혐기성 ) 를이용할수있는데, 상재균이잘자라는 CO 2 보다는공기가나은것으로보고되고있다 [16,17]. 저자들은공기를이용한배양기를사용하였는데, 비용이가장저렴하고단순한배양기를사용할수있으므로추천할만하다. 혐기성상태에서베타용혈성연쇄구균의분리율을높일수있다는보고가있지만 [11], 추가비용이나검사의편리성을고려하면일상적으로적용하기는어려울것으로생각한다. 상재균을억제하기위해서항균제가들어있는선택배지를사용할수도있는데, 집락이늦게자라는단점이있지만세균분리경험이적은사람들은사용하기편할것으로생각한다 [13,18,19]. 이러한배양조건이외에도, 집락판독이나순수분리기술, 동정방법등에의해서도배양양성률은영향을많이받는다 [20]. Rosenstein 등 [20] 은동일한검체를세균배양숙련도에따라서양성률을비교한결과, 유의한차이가나는것으로보고하였다. 면봉을한개사용하는것보다두개사용할경우, 인두배양양성률이올라간다는보고도있었다 [3,18,21]. 또한배지하나에서 A군과 B, C, G군이섞여있는경우선택한집락에따라서양성률에영향을미칠수있다 [22]. 심지어서로다른혈청형의 A군이 2가지이상섞여자랄수도있다 [23]. 이와같이인두배양이많은제한점과문제점을가지고있지만현재까지는세균성인두염진단의표준검사법으로사용되고있으며, 신속항원검사키트의효율성을비교하는기준이된다. 신속항원검사는세균배양에필요한시간을단축할수있으므로독성이강한균에의해감염이의심되거나화농성혹은삼출성인두염이있을때는신속한세균학적진단이가능해져적합한항생제치료를할수있다 [4,12]. A군연쇄구균인두염은비교적흔할뿐만아니라검체채취가용이하므로배양시설이나배지의멸균, 검사경험의부족등으로세균배양이힘든여건에서는신속항원검사법이추천된다 [1]. 신속항원검사는약 10~15분이면발색반응결과를쉽게판정할수있고, 복잡한검사장비가없어도되므로외래에서손쉽게시행할수있다. 본연구에서는건강한초등학생을대상으로 A군연쇄구군신속항원검사 Strep A와이미상품화된미국 Quidel 사의 Quick Vue Dipstick Strep A를비교하였다. 두가지모두원리는면역발색법이고, 약 5~10분만에신속하고간편하게결과판정이가능하였다. 대조밴드에서양성반응을보이고, 검사밴드에서동시에양성반응을보이는경우양성으로판정하였다. 본연구에서는밴드가약하더라도일단보이면양성으로판정하고, 매우희미한경우에는음성으로판정하였다. 희미하게발색반응을보이는경우판독하기어려워경험이필요하며, 판독자의주관이작용할수있는단점이있다 [13,14,24,25]. 만약 A군연쇄구균의역학적특징이나항생제내성률을조사하고자한다면신속항원검사만으로는알수가없다. 매회사용하는면봉마다채취되는균수가달라질수있으므로면봉두개를동시에사용하여검체를채취하는것을고려해볼수있다 [3,18,21]. 균수가양성률에큰영향을미치므로만약면봉두개로채취하여한가지의신속항원검사에사용한다면신속항원검사양성률을높일수있다 [21,25]. 신속항원검사는초등학생을대상으로평가하는것보다는인두염환자를대상으로평가하는것이바람직하다고생각한다. 한편초등학생에서 A군연쇄구균보균율이 15% 정도로높고 [26], 빠른시간에제품의품질을평가할수있는장점이있다. 보균자도인두염환자에비해집락수가충분하고 [26], 검체채취및배양, 신속항원검사를숙련된한두사람이시행함으로써검사자간오차를줄일수있는장점이있다. 인두배양이나신속항원검사로인두염환자와보균자를감별하는것은불가능하며, 이는임상적판단에의해서만가능하다 [1,2,4]. 외국문헌에비해서제품의효율성은약간낮게측정되었지만저자들이과거에평가했던라텍스응집법이나효소면역법에비해서는크게향상되었다 [27,28]. 외국문헌은대개인두염환자를대상으로오랜기간에걸쳐서시행한것이고, 본연구에서는하루에수십명씩연속해서검사한것이기때문에효율성에있어서차이를보일수있다. 앞으로인두염환자에서본제품의평가가다양하게이루어져야할것이다. Strep A는 Strep A에비해예민도가통계적으로유의하게높았고 (P<0.05), 특이도, 양성예측률및음성예측률은통계적유의성이없었다 (Table 2). 검체채취과정이나밴드를판독하는주관성, 베타용혈성연쇄구균이상재균과섞여있어균분리에실패하는경우등이결과에영향을미쳤다고볼수있다. B, C 그리고 G군에서신속항원검사양성으로나온경우위양성으로판정할수있다 (Table 3). 그러나베타용혈성연쇄구균이전혀분리되지않은경우에도위양성인경우가있어서, 반드시이들다른군과교차반응을하였다고볼수는없다 [25,29]. 실제로분리된균주를면봉에묻혀다시신속항원검사를시행한결과모두음성을보였다. 앞으로위양성으로나온이유에대해서추가연구가필요하다.
Eun-Ha Koh, Sunjoo Kim : Evaluation of Rapid Antigen Test 57 최근에베타용혈을보이지않고알파용혈을보이는 A군연쇄구균이보고되어 [30,31], 이경우신속항원검사를무조건위양성으로판정하기어려울수있음을시사하였다. 대개집락수가많을수록신속항원검사양성률도높아질것을기대할수있는데 [11,12,14,21,25,28], 특이하게집락수가 1+ 에서 Strep A는 89.7% 의매우높은양성률을보였다 (Table 4). 이는 Strep A가적은수의균도검출할수있을정도로감도가높다고볼수도있고, 혹은상재균속에베타용혈균이섞여있을때집락수를정확히측정하기어려운점도고려할수있다. 국내에서는개인의원에서세균배양시설은거의전무할뿐만아니라 2차및 3차병원에서도인두배양성적이만족할만하지않다. 이는배양시설의미비, 세균배양의경험부족, 세균동정에드는비용등미생물검사실요인과검체채취를제대로하지못해편도의세균을면봉에충분히묻히지못하는채취자요인, 그리고경험적으로인두염의원인을추정하여항생제치료를하는의사의요인으로나누어볼수있다. 인두염때문에 3차병원으로직접찾아오는경우는별로없고대부분개인의원을방문하게되는데항생제투여전 A군연쇄구균에의한인두염인지정확한진단이먼저이루어져야한다. 만약항생제를사용한후 3차병원을방문하는경우인두배양양성률은현저히낮아질것이다. 현재대부분인두염환자들이정확한세균배양및감수성결과없이항생제를처방받게되고이로인해내성률상승및다제내성균이출현하게된다. 이를예방하기위해서는의사와환자모두심각한항생제내성문제를인식하고, 적절한항생제사용지침을통해불필요한항생제사용을최소화하려는노력이필요하다. 인두염환자에서증상및징후와더불어인두배양혹은간편하고예민도가높은신속항원검사를이용해 A군연쇄구균을증명한다면정확한진단이가능하고항생제오남용을줄일수있을것이다. 감사의글 본연구는보건복지부보건의료기술진흥사업의지원 (A05-0191-A40102-05N1-00000A) 에의하여이루어졌으며, 저자는본제품의개발에참여했음. 참고문헌 1. DiMatteo LA, Lowenstein SR, Brimhal B, Reiquam W, Gonzales R. The relationship between the clinical features of pharyngitis and the sensitivity of a rapid antigen test: evidence of spectrum bias. Ann Emerg Med 2001;38:648-52. 2. Hall MC, Kieke B, Gonzales R, Belongia EA. Spectrum bias of a rapid antigen detection test for group A β-hemolytic streptococcal pharyngitis in a pediatric population. Pediatrics 2004;114: 182-6. 3. Gieseker KE, Roe MH, MacKenzie T, Todd JK. Evaluating the American Academy of Pediatrics diagnostic standard for Streptococcus pyogenes pharyngitis: backup culture versus repeat rapid antigen testing. Pediatrics 2003;111:e666-70. 4. Bisno AL, Gerber MA, Gwaltney JM Jr, Kaplan EL, Schwartz RH. Practice guidelines for the diagnosis and management of group A streptococcal pharyngitis. Infectious Diseases Society of America. Clin Infect Dis 2002;35:113-5. 5. Breese BB. A simple scorecard for the tentative diagnosis of streptococcal pharyngitis. Am J Dis Child 1997;131:514-7. 6. Jung HJ, Lee NY, Kwon OY, Maeng KY, Kim S. Epidemioloigcal characteristics of group A streptococci using emm genotyping from throat cultures in patients with acute pharyngitis in children. Korean J Pediatr Infect Dis 2003;10:178-85. 7. Johansson L and Mansson NO. Rapid test, throat culture and clinical assessment in the diagnosis of tonsillitis. Fam Pract 2003; 20:108-11. 8. DeNeef P. Comparison of tests for streptococcal pharyngitis. J Fam Pract 1986;3:551-5. 9. American Academy of Pediatrics, Committee on Infectious Diseases. Group A Streptococcal Infections. In: Pickering KL, ed. The Red Book. Elk Groove Village, IL: American Academy of Pediatrics, 2000:526-36. 10. Koh EH and Kim S. Analysis of phenotypic and genotypic distribution of erythromycin and clindamycin resistance of group A streptococci from acute pharyngitis. Korean J Lab Med 2004;24:126-30. 11. Gerber MA, Randolph MF, Chanatry J, Wright LL, DeMeo KK, Anderson LR. Antigen detection test for streptococcal pharyngitis: evaluation of sensitivity with respect to true infections. J Pediatr 1986;108:654-8. 12. Edmonson MB and Farwell KR. Relationship between the clinical likelihood of group A streptococcal pharyngitis and the sensitivity of a rapid antigen-detection test in a pediatric practice. Pediatrics 2005;115:280-5. 13. Wegner DL, Witte DL, Schrantz RD. Insensitivity of rapid antigen detection methods and single blood agar plate culture for diagnosing streptococcal pharyngitis. JAMA 1992;267:695-7. 14. Nerbrand C, Jasir A, Schalen C. Are current rapid detection tests for group A streptococci sensitive enough? Evaluation of 2 commercial kits. Scand J Infect Dis 2000;34:797-9. 15. Kim SJ. Optimal site of throat swab for the isolation of β-hemolytic streptococci. J Korean Med Sci 1993;8:453-7. 16. Kellogg JA. Suitability of throat culture procedures for detection of group A streptococci and as reference standards for evaluation of streptococcal antigen detection kits. J Clin Microbiol 1990;28:165-9. 17. Roddey OF, Clegg HW, Clardy LT, Martin ES, Swetenburg RL. Comparison of a latex agglutination methods for identification of group A streptococci in a pediatric office laboratory. J Pediatr 1986;108:347-51. 18. Gieseker K, MacKenzie T, Roe MH, Todd JK. Comparison of two rapid Streptococcus pyogenes diagnostic tests with a rigorous culture standard. Pediatr Infect Dis J 2002;21:922-7. 19. Kim SJ. Evaluation of streptocccus selective agar (ssa) for recovery of group A streptococci from throat cultures. J Clin Pathol Quality Control 1997;19:185-90. 20. Rosenstein BJ, Markowitz M, Gordis L. Accuracy of throat cultures processed in physician s offices. J Pediatr 1970;76:606-9.
58 Korean J Clin Microbiol 2007;10(1):54-58 21. Kurtz B, Kurtz M, Roe M, Todd J. Importance of inoculum size and sampling effect in rapid antigen detection for diagnosis of Streptococcus pyogenes pharyngitis. J Clin Microbiol 2000;38: 279-81. 22. Kim SJ and Maeng KY. Simultaneous isolation of group A and group B β-hemolytic streptococci from the throat culture of two children. J Clin Pathol Quality Control 1994;16:129-32. 23. Kim YJ and Kim SJ. Serial evaluation of serotypes of group A streptococci isolated from throat culture of normal school children. Korean J Clin Microbiol 1999;2:14-8. 24. Fox JW, Cohen DM, Marcon MJ, Cotton WH, Bonsu BK. Performance of rapid streptococcal antigen testing varies by personnel. J Clin Microbiol 2006;44:3918-22. 25. Lindbaek M, Hoiby EA, Lermark G, Steinsholt IM, Hjortdahl P. Which is the best method to trace group A streptococci in sore throat patients: culture or GAS antigen test? Scand J Prim Health Care 2004;22:233-8. 26. Kim SJ, Maeng KY, Lee HI, Cho YK, Yun HS. Bacteriological survey of beta-hemolytic streptococci from the throats of elementary school children in Chinju compared with the results of children in Kangwon, Chungnam and Seoul. J Korean Pediatr 1996; 39:238-45. 27. Cha SH and Kim SJ. Rapid detection test for Streptococcus pyo genes in normal carriers of group A beta-hemolytic streptococci. J Korean Pediatr 1994;37:812-5. 28. Kim SJ. Comparison of enzyme immunoassay and latex agglutination for direct detection of Streptococcus pyogenes from the throats. Korean J Infect Dis 1995;27:397-402. 29. Chapin KC, Blake P, Wilson CD. Performance characteristics and utilization of rapid antigen test, DNA probe, and culture for detection of group A streptococci in an acute care clinic. J Clin Microbiol 2002;11:4207-10. 30. Schroeder S and Procop GW. False positive strep A antigen test. Pediatr Infect Dis J 2000;19:1114-5. 31. Rubin LG and Mirkin GS. Apparent false positive detection of group A Streptococcus antigen resulting from pharyngeal infection with a nonhemolytic Streptococcus pyogenes. Pediatr Infect Dis J 2000;19:672-4. = 국문초록 = 초등학생에서세균성인두염신속항원검사 Strep A 의평가 경상대학교의과대학진단검사의학교실, 건강과학원고은하, 김선주 배경 : A군연쇄구균을검출할수있는신속항원검사는시행하기간편하고, 배양시간을절약하는장점이있다. 국내에서개발된신속항원검사 Strep A 시약을평가하고자본연구를시행하였다. 방법 : 건강한초등학생 308명으로부터 3회인후도말을시행하였다. 처음두개면봉은 Strep A와 Quidel Quick Vue Dipstick Strep A 신속항원검사를시행하였고, 세번째면봉은세균배양을하였다. 결과 : 세균배양결과를기준으로 Strep A의예민도, 특이도, 양성예측률및음성예측률은 79.3%, 88.9%, 72.2% 및 92.2% 였다. Dipstick Strep A의예민도, 특이도, 양성예측률및음성예측률은 58.5%, 93.8%, 77.4% 및 86.2% 였다. 결론 : Strep A는 Dipstick Strep A에비해예민도가유의하게높았으며, 특이도는약간낮았다. 신속항원검사 Strep A는세균성인두염을빠르게진단하고, 적합한항균제치료를하는데유용할것으로판단한다. [ 대한임상미생물학회지 2007;10:54-58] 교신저자 : 김선주, 660-702, 경남진주시칠암동 90 번지경상대학교병원진단검사의학과 Tel: 055-750-8239, Fax: 055-762-2696 E-mail: sjkim8239@hanmail.net