Journal of Soonchunhyang Medical Science 15(2) p.13~18 December 말라리아의신속진단 : 임상양상및혈액도말검사와항원검사의비교 1 김병철, 2 김태형, 1 박노진 순천향대학교의과대학서울병원 1 진단검사의학과,

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1 Journal of Soonchunhyang Medical Science 15(2) p.13~18 December 말라리아의신속진단 : 임상양상및혈액도말검사와항원검사의비교 1 김병철, 2 김태형, 1 박노진 순천향대학교의과대학서울병원 1 진단검사의학과, 2 내과 Rapid Diagnosis of Malaria: Clinical Manifestations and Comparison of Blood Smear Findings and Malarial Antigen Test Results 1 Byung Chul Kim, 2 Tae Hyung Kim, 1 Rojin Park Departments of 1 Laboratory Medicine and 2 Internal Medicine, College of Medicine, Soonchunhyang University, Seoul, Korea Abstract Background : Malarial infestation intermediated by Anopheles mosquito is one of the most prevalent parasitic diseases over the world and many people are infected by malarial plasmodium each year. The rapid diagnosis is needed to properly treat the malaria because plasmodium falciparum has a high mortality rate. Currently, we usually use a microscopic examination of blood smears for diagnosis of malaria and it has a high sensitivity and specificity. However, the result might depend on physician in change and this method needs much time for diagnosis of malaria. Methods : We evaluated 206 patients who were suspicious of malaria. We perforemed a microscopic examination of blood smears and antigen detection test. We investigated clinical symtoms such as fever pattern, thrombocytopenia, anemia and diagnosised as malaria who was positive in blood smears. After this we tried to follow up the patients. Results : As a result of a microscopic examination of blood smears and antigen detection test for 206 patients who were suspicious of malaria, 15 was diagnosed with malaria infestation and all of them showed a positive response to malarial antigen test. There were six patients who showed a positive reaction to antigen test, but negative results in blood smears. Malarial patients showed anemia, leukopenia or thrombocytopenia and revealed the symtom of splenomegaly, headache and abdominal pain. Conclusions : Malarial antigen test has a good relationship with a microscopic examination of blood smears and a high sensitivity and specificity. Because the result could be false positive or false negative, it might be helpful to perform antibody test together. Key words : Malaria, Microscopic examination of blood smears, Antigen detection test 1) 책임저자 : 박노진주소 : , 서울시용산구대사관길 22, 순천향대학교서울병원진단검사의학과 Tel : / Fax : 접수일 : 2009년 10월 27 일 / 게재확정일 : 2009년 12월 4

2 14 Journal of Soonchunhyang Medical Science Vol.15 No.2 December 2009 서론 말라리아는얼룩날개모기(Anopheles mosquito) 에 매개되는전세계적으로중요한기생충질환의하나로서 해마다많은인구가말라리아원충에감염된. 1) 우리나 라에서이미없어졌다고생각되었던삼일열말라리아 는 1993 년재출현이후국민건강을위협하고있다. 또한최근에는유행지역으로부터의해외여행객이증가 함에따라, 열대열말라리아의발생도증가하는추세 이다. 2,3) 경우사망률이 열대열말라리아는주요장기부전이동반될 20% 에이르는응급질환이다. 4) 말라리 아를적절히치료하기위해서는신속하고정확한진단 이필요하다. 5) Wright/Giemsa 통상말라리아의진단에는혈액도말을 염색한후현미경으로관찰하는말초 혈액도말법이표준으로사용되고있다. 이방법은말 초혈액도말슬라이드에서약 300개이상의영역을 고배율(1,000 배) 에서검사하여판단하여야하므로많 은시간과노력이필요하고발열시에 3회이상의검 사를추천하고있다. 또한검사자의숙련도에따라서 진단에소요되는시간이달라진다. 6) 최근에는말라리 아진단을위해효소면역법 (enzyme linked immuno- sorbent assay, ELISA), fluorescent antibody test, IFTA) 간접면역형광항체법(indirect 등의혈청학적방 법과중합효소연쇄반응을이용한여러가지방법들이 소개되었지만신속한진단을위해서는부적합하다. 7,8) 이연구에서는불명열환자를대상으로최근국내에서 개발된말라리아항원진단키트(SD, Yongin, Korea) 를 사용한항원및말초혈액도말검사를시행하고임상양 상을같이비교하여그유용성을평가하고자하였다. 1. 대상 대상및방법 2006년 9월부터 2008년 3월까지불명열을주소로 본원에내원한환자 206명을대상으로말초혈액도말 검사를 3 회, 말라리아항원검사를 1 회실시하였다. 2. 방법 1) 말초혈액도말검사및원충수계산 후층도말검사를통해말라리아를선별하고박층도말 검사로종류를감별하였다. 혈액도말슬라이드를 Wright- Giemsa 염색을하여고배율 ( 1000) 에서 300개이 상의영역을관찰하고말라리아원충의존재유무를확 인하였다. 단위용적당원충수( 수/L) 는 ( 도말원충수/ 혈 액도말상백혈구수) ( 백혈구수( 수/L)) 의식에의해 계산하였다. 9) 2) 말라리아항원검사 열대열말라리아특이항체및열대열및삼일열말 라리아에공통적인항체를이용하여열대열및삼일열 말라리아를감별진단할수있는에스디바이오라인 말라리아항원진단키트( 에스디, 용인, 한국) 를사용 하였다(Fig. 1). 검체로사용한전혈을접합체웰내에 서반응용액으로용혈시켜말라리아 pldh항원이 1차 적으로접합체웰에접합되어있는열대열및삼일열 말라리아의 pldh 항원에공통적인토끼항체와반응 하게하였다. 여기에검사용스트립을반응시키고면 역크로마토그래피원리에의해검액이나이트로셀룰로 스막을따라이동하게하였다. 이막의검사선1의위 치에는열대열말라리아의 pldh 항원에특이적인마 우스항체가, 검사선2의위치에는열대열및삼일열 말라리아의 pldh 항원에특이적인마우스항체가흡 착되어있다. 삼일열말라리아양성인경우에는검사 선2 에만, 열대열말라리아양성인경우에는검사선1과 2에항체-항원-항체복합체를형성하여직접샌드위치 (direct-sandwich) 된다. 3) 말라리아진단 원리에의해보라색으로발색하게 불명열로내원한환자 206 명을대상으로발열양상, 혈 소판감소증, 빈혈등의임상양상을조사하고, 말라리아 항원검사결과와상관없이말초혈액도말검사결과가 양성이면말라리아로진단하였다. 말라리아진단, 치 료후추적검사를실시하였다.

3 박노진 : 말라리아의임상양상및혈액도말검사와항원검사 15 Table1. Laboratory findings of patients positive to malarial Ag test Laboratory data Range P. vivax (n=13) P. falciparum (n=2) Microscopy (-)/Ag (+) (n=6) Patients (%) Patients Patients Hgb (g/dl) <12 (F),13 (M) 10 (76.9%) 3(50%) < (46.2%) 1(50%) WBC (/ul) (53.8%) 1(50%) 5(83.3%) > (0%) 1(16.7%) <100,000 6 (46.2%) 1(50%) 1(16.7%) Platelet (/ul) ,000 3 (23.0%) 1(50%) 1(16.7%) >140,000 4 (30.8%) 4(66.7%) Total bilirubin < (76.9%) 1(50%) 4(66.7%) (mg/dl) (23.1%) 1(50%) 2(33.3%) ALT (IU/L) <40 9 (69.2%) 2(100%) 4(66.7%) 40 4 (30.8%) 2(33.3%) Abbreviation: P, plasmodium; Ag, antigen test; F, female; M, male. Table 2. Symptoms and signs of malarial patients Clinical symptoms and signs P. vivax (n=13) P. falciparum (n=2) Microscopy (-) /Ag (+) (n=6) N (%) N (%) N (%) Fever 13 (100%) 2(100%) 6(100%) Splenomegaly 4 (30.8%) 2(100%) 0(0%) Headache 8 (61.6%) 2(100%) 1(16.7%) Abdominal pain 4 (30.8%) 1(50%) 3(50%) Abbreviation: P, plasmodium; Ag, antigen test; N, number. 결과 1) 말라리아환자의검사실소견 13 명의삼일열말라리아환자에서빈혈(M, <13g/dL; F, <12g/dl) 10 명(76.9%), 백혈구감소증(<4000/L) 6 명(46.2%), 혈소판감소증(<140,000/L) 9 명(69.2%), 빌 리루빈혈증(>1.2g/dL) 3 명(23.1%), ALT 증가(>40IU/L) 4 명(30.8%) 으로나타났다. 열대열말라리아환자 2명 모두혈소판감소증을보였다. 그중 1명은백혈구감소 증, 나머지 1 명은빌리루빈혈증을보였다. 임상적으로 말라리아가의심되었지만혈액도말에서원충을발견할 수없었던 6명의환자에서빈혈 3 명, 혈소판감소증 2 명, 빌리루빈혈증 2 명이보였다(Table 1). 2) 말라리아환자의임상양상 환자들은모두발열을보였다. 13명의삼일열말라리 아환자중 4 명 (30.8%) 이비장종대, 8 명(61.6%) 이 두통, 4 명(30.8%) 이복통을보였다. 2명의열대열말 라리아환자모두비장종대와두통을호소하였다. 그

4 16 Journal of Soonchunhyang Medical Science Vol.15 No.2 December 2009 Table 3. The results of malarial blood smear and antigen test Ag test * Blood smear Positive Negative Positive 15 6 Negative *Sensitivity 100%, Specificity 96.9% Abbreviation: Ag, antigen. Table 4. The comparison of number of parasites between Park s study* and this study according to parasite density This study Previous Park s study Number of No. of No. of parasites (/ul) Ag (+) Ag (+) patients patients Not detected < > Total *Park s study is referenced by 17) Not observed through the 300 oil immersion fields. Abbreviation: Ag, antigen test. 중 1 명은복통을보였다. 혈액도말검사에서원충을발 견할수없었던 6명의환자중 1 명은두통, 3명은복 통을보였다 (Table 2). 3) 말초혈액도말검사 불명열환자 206명의말초혈액도말검사에서 15명이 말라리아로진단되었다. 13 명이삼일열, 2명이열대열 말라리아이었다(Table 3). 내원당시실시한말초혈액 도말에서말라리아의원충수는다양하게나타났으며, <50/L 2 명, /L 2 명, /L 3 명, /L 2 명, /L 3 명, >2000/L 3명이었다 (Table 4). 4) 말라리아항원검사 말초혈액도말검사를통해말라리아로진단된 을포함하여 이들중 15명 21 명이말라리아항원검사에양성이었다. 6명은혈액도말에서말라리아원충을발견할 수없었다(Table 3). 항원검사의민감도는 100%, 특 이도는 96.9% 였다. 5) 치료후추적검사 약물치료후실시한혈액도말검사에서 3명은 7일간 약물치료후에도말라리아원충이계속관찰되었고 6 명은약물치료후3 일이내에, 6명은약물치료후 4-7 일이내에원충을발견할수없었고항원검사에도음

5 박노진 : 말라리아의임상양상및혈액도말검사와항원검사 17 성이었다. 도말검사에서음성이고항원검사에양성인 6 명의환자들은임상적으로말라리아가의심되어약물 치료를시행하였다. 약물치료 3일후실시한항원검사 에모두음성반응을보였다. 고찰 말라리아는해마다많은사람들이감염되고높은사 망률을보이는응급질환으로신속한진단이필요하다. 10) 현재말라리아진단에주로사용되고있는말초혈 액도말검사는민감도와특이도가높은검사이다. 11) 그러나판독하는의사에따라결과판정이달라질수 있고많은시간을필요로하기때문에신속한진단이 필요하지만인력이부족한곳에서이를보완해줄수 있는검사법이요구되고있다 ) 말라리아환자중 내원당시삼일열 12명이빈혈또는혈소판감소증을 보였다. 6 명은빈혈과혈소판감소증이동반되었고, 이 들중 3 명은비장종대를보였다. 말라리아로인해골 수기능이억제되고비장에서적혈구가파괴되었음을 시사한다. 15) 혈소판수의감소는말라리아원충이혈 소판을직접파괴하거나면역글로불린의작용으로비 장을통해파괴되기때문에일어난다고생각되고있으 며, 최근에 M-colony stimulating factor의감소와 P-selectin 과의관련성이제기되고있다. 16) 말라리아 환자들은임상적으로발열(100%), 비장종대(40%), 두 통(66.7%), 복통(33.3%) 등의양상을보였다. 혈액도 말검사에서양성인말라리아환자들은모두항원검사 에양성이었다. 동일한항원검사를사용한박등 17) 의 연구에따르면원충수 2000/L 이상인환자 1명과원 충수 50/L 이하인환자 1명이항원검사에음성을보 였는데, 이연구에서는원충수 1000/L 이상인환자와 원충수 50/L 이하인환자중항원검사에음성반응을 보인사람은없었다. 혈액도말검사를통해말라리아로 진단된 15명의환자중 12명은약물치료후실시한 혈액도말검사에서일주일이내에음성반응을보였다. 일주일간의치료후에도말라리아원충이지속적으로 관찰된 3명의환자는치료도중에퇴원하여더이상 추적관찰이되지않았다. 본연구에서혈액도말검사 에서말라리아음성이었던환자 6명이항원검사에양 성반응을보인점은이전의박등 17) 의연구에서는볼 수없었던결과이다. 혈액도말검사결과가정상이었지 만항원검사에양성이었던 6명의환자들중 2명은혈 소판감소증과발열양상으로미루어말라리아가강하게 의심되어, 3 일간하이드록시퀴노론을투여하였다. 이 후, 재시행한항원검사에서음성반응을보였으며, 혈 소판감소증도점차회복되었다. 나머지 4명의항원검 사양성반응은위음성으로추정하였다. pldh를이용 한말라리아항원검사는환자의혈액내에말라리아 pldh 항원과유사한구조의단백질이존재하거나, 다 른전염성질환, 또는오래된검체가위양성의원인이 될수있다. 18,19) 이번연구에서불명열환자 206명을 대상으로말초혈액도말검사와말라리아항원검사를 함께시행하여항원검사에양성인 21명의환자중 15 명이도말검사에서도양성인소견을보임으로써민감 도 100%, 특이도 96.9% 를나타내었다. 이러한말라리 아항원검사법은민감도와특이도가높지만항말라리 아약제를투여하여원충의농도가낮아지거나저밀도 말라리아혈증에서는말라리아항원의검출이어려워 진다는단점이있다. 20) 그렇지만말라리아항체는항 말라리아약제를투여한환자나저밀도말라리아혈증 환자에서도지속될수있기때문에, 신속하고정확한 말라리아의진단을위해서는환자의임상양상과빈혈, 혈소판감소증등의검사소견과함께말라리아항체검 사를함께시행하면많은도움이될것이다. 참고문헌 1.2World Health Organization. World Malaria Situation World Health Statist Quart ; 45: , Shin KS, Kim JS, Ryu SW, Suh IB, Lim CS : A case of Plasmodium vivaxinfection diagnosed after treatment of imported falciparum malaria. Korean J Lab Med A 1 21: Looareesuwan S, White NJ, Chittamas S, Bunnag D, Harinasuta T : High rate of

6 18 Journal of Soonchunhyang Medical Science Vol.15 No.2 December 2009 Plasmodium vivax relapse following treatment of falciparum malaria in Thailand. Lancet 2: , Shin ES, Woo JH, Kim YO, Bae IG, Ko YS, Jang JJ, et al.: Two cases of falciparum malaria with acute respiratory distress syndrome. Tuberculosis and Respiratory Diseases 45: 88-95, World Health Organization : WHO expert committee on malaria. World Health Organization, Technical Reports series 892: 1-25, Sun YK, Kim YA, Lim HS : Evaluation of point-of-care kit for diagnosis of malaria in Korea. Korean J Clin Pathol 21: 204-9, LiJ Wirtz RA, McConkey GA, Sattabongkot J, Waters AP, Rogers MJ : Plasmodium genusconserved primers for spieces identi- fication and quantitation. Exp Parasitol 81: , Kim YR, Choi HK, Bae JS, Kim JS, Kwon JA, Suh IB : Evaluation of nested multiplex PCR in the diagnosis of malaria infection. Korean J Lab Med 22: , Laserson KF, Petralanda I, Hamlin DM, Almera R, Fuentes M, Carrasquel A : Use of the polymerase chain reaction to directly detect malaria parasites in blood samples from the Venezuelan Amazon. Am J Trop Med Hyg 50: , Kwiatkowski D and Marsh K : Development of a malaria vaccine. Lancet 350: , Lim HS, Kim HS : Evaluation of diagnostic methods of re-emerging malaria in Korean patients. Yonsei Med J 42: 84-90, Payne D : Use and limitations of light microscopy for diagnosing malaria at primary health care level. Bulletin of the World Health Organization 66: 621-6, World Health Organization : Severe and complicated malaria. Trans R Soc Trop Med Hyg 84: 23-5, World Health Organization : New perspectives malaria diagnosis. World Health Organization. Geneva 10-39, Collins WE, Jeffery GM, Roberts JM : A retrospective examination of anemia during infection of humans with Plasmodium vivax. Am J Trop Med Hyg 68: 410-2, Yamaguchi S, Kubota T, Yamagishi T, Okamoto K, Izumi T, Takada M : Severe thrombocytopenia suggesting immunological mechanism in two cases of vivax malaria. Am J Hematol 56: 183-6, Park TS, Kim JH, Kang CI, Lee BH, Jeon BR, Lee SM : Diagnostic usefulness of SD malaria antigen and antibody kits for differential diagnosis of vivax malaria in pa- tients with fever of unknown origin. Korean J Lab Med 26: 241-5, Shin KS, Ma KR, Lim CS : Diagnosis of malaria using automatic hematology analyzer. J Lab Med & Quality Assurance 26: 171-6, Chung JY, Kim MY, Lim KJ, Sohn MJ, Kho WG : Evaluation of LG malaria anti-pvtmfor diagnosis of plasmodium vivax malaria in the republic of Korea. Korean J Clin Pathol 21: 67-71, Lee GW, Kim TH, Park RJ : P.Vivax malaria after successful treatment of P.falciparum malaria. J Soonchunhyang Med Col 10: , 2004.

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