Studies on the indirect fluorescent antibody test for the serodiagnosis of paragonimiasis

의학과/박사[한글] 근래에 기생충병의 면역진단법에 있어서는 간접형광 항체법의 응용이 특히 주목을 끌어 오고 있다. 본 연구에 있어서는 폐디스토마 탈지성충입자 도말스라이드를 항원으로 사용 한 간접형광항체법을 폐흡충증(폐디스토마증)에 대한 면역진단법으로서 고안하였다. 이방 법의 이용도를 4군의 사람혈청 즉 25명의 진단이 확정된 폐흡충증환자 헐청, 80명의 비폐 디스토마성 기생충 감염자혈청, 18명의 비기생충성 폐질환자혈청 및 20명의 건강대조군혈 청을 사용하여 평가하였다. 그외에 8명의 폐흡충중 환자혈청으로서 완전치유후의 형광항 체반응성의 변동에 대하여 실험하였다. 또한 31명의 건조혈액여지도말 표본을 작성하여 혈청표본과의 항체가를 비교하였다. 아울러 폐디스토마와 간디스토마항원으로서 혈청흡수 실험을 실시하여 항원의 특이성을 조사하였다. 본실험성적은 다음과 같다. 즉 폐흡충증에 대한 간접형광항체법에 있어서는 진단이 확 증된 폐디스토마환자혈청은 모두가 항체가 1:16 이상으로서 양성반응을 나타냈으며 최고 항체가는 1:512로서 항체가곡선의 정점은 1: 32였다. 한편 기타기생충감염자혈청의 음성 율은 87.5%(70예)였고 의양성율은 항체가 1:4에서 7.5%(6예), 1 :8에서 3,75%(3예), 1:16 에서 1.25%(1예)였다. 의양성 혈청의 대부분은 간디스토마환자혈청이었다. 비기생충성 폐 질환자와 건강대조군의 혈청은 모두 음성 반응을 나타냈다. 폐디스토마와 간디스토마와의 교차반응에 있어서는 폐디스토마환자혈청 25예중 12예에 있어서 간디스토마항원에 대하여 각각 항체가 1:8에서 4예, 1:4에서 8예의 양성반응을 나 타냈으나 동속항원과의 반응은 3∼7배 높았다. 또한 간디스토마환자혈청 33예중 폐디스토 마 항원에 대하여 4예 즉 항체가 1:16에서 1예, 1:8에서 1예, 1:4에서 2예의 양성반응을 나타냈으나 동속항원에 대한 항체가는 2∼6배 높았다. 혈청흡수실험결과, 동속항원에 대한 반응은 모두 완전히 흡수되어 음전되었다. 또한 모 든 교차반응과 폐디스토마 완치후 혈청중 항체가 1:8인 혈청은 동속항원과 이속항원에 의 하여 모두 완전히 흡수되었다. 이 결과는 항체가 1:16 이상은 양성이고 1:8 이하는 음성 또는 비특이성반응임을 시사하는 것이다. 전기 판정기준에 의하여 폐디스토마에 대한 간접형광항체반응을 평가한 결과 폐흡충증 에 대한 진단은 완전히 적중되었고 의양성반응은 폐디스토마외 타 기생충성감염자 혈청중 에서 1.2%뿐이었다. 형광항체가와 폐디스토마충란 배출수와의 상관관계는 유의성이 없었 다. 또한 폐흡충증은 6개월∼7년전에 bithionol로서 치료한 혈청의 형광항체가와 폐디스 토마 충란배출수와의 상관관계는 유의성이 없었다. 폐흡충증은 6개월∼7년전에 bithionol로서 치료한 혈청의 형광항체법 검사 성적에 있는 완전치유된자의 혈청은 모두 항체가 1:8 이하로서 음성이었는데 반하여 비치유자혈청은 항체가가 1:16 ∼ 1:128로서 양성반응을 나타냈다. 한편 피내반응 구진크기는 완전치유자 와 비치유자간에 각별한 차이가 없었다. 손끝에서 채집한 건조혈액 여지도말표본의 추출액과 혈청표본과의 형광항체가에 있어서 의 정량적 비교결과는 양표본이 거의 유사한 항체가를 나타냈다. 결론적으로 이상 제시된 본 연구 결과는 폐디스토마의 탈지성충입자도말스라이드를 사 용한 형광항체법이 폐흡충증에 대한 임상적 및 역학적 연구에 있어서 충분히 사용될 수 있음을 시사하였다. 또한 본 방법은 감응도, 특이성 및 재현성에 있어 타진단법예 비하여 대단히 우수함을 입증하였다. 아울러 본 진단법은 패흡충증 치유판정 기준으로도 이용될 수 있음을 확증하였다고도 믿는 바이다. [영문] Immunodiagnostic methods, especially the intradermal and complement fixation teats popularized by Sadun et al.(1958) and Walton and Chyu (1959) have been widely used for the diagnosis of paragonimiasis in laboratory and field studios. However, those tests have net proved as satis-factory tools because the intradermal reactivity keeps positive lasting more than 10-20 years even after the elimination of the Parasite (Yokogawa et al. 1962), and dross reaction occur in other related parasitic infections (Sadun and Buck, 1960). The procedure of complement fixation test requires considerably greater technical competence than that needed to perform many of the other immunodiagnostic tests. The other serologic methods are gel-diffusion precipitin test and immunoelectrophores (Yogore,1965: Lee et al., 1973), but these methods have been utilized for special aims in laboratory. Therefore, a more simple, economical and reliable serologic methods have been required in endemic areas of paragonimiasis. During recent years, a considerable attention has been given to the potentiality of indirect fluorescent antibody test for the immunodiagnosis of parasitic diseases. The method of immunofluorescence on slide with particles of adult worms hart teen introduced for schistosomiasis mansoni by Camargo et al. (1965) and Hoshino et al.(1970), and for clonorchiasis by Im (1974) and Cho and Soh (1974). They reported that thin new method was very sensitive, easy to perform and economical in relation to antigen consumption. A few worms were sufficient for a larger number of tests in compare with other means. In the present study, the indirect fluorescent antibody (IFA) teat was developed for the immunodiagnosis of paragonimiasis by using delipidized adult Paragonimus westermani (P. westermani)particles, fixed on microscope slide, as antigen. The usefulness of this technique was assessed with 4 batches of human sera: 25 sera from proven paragonimiasis, 80 sera from parasitic infections other than P. westermani, 18 sera from non-parasitic lung diseases and 20 sera from healthy individuals. Additionally, 8 sera from persons of paragonimiasis were used to examine the changes of IFA reactivity after the complete elimination of P. westermani. 31 dried blood smears on filter paper were tested for comparison of IFA titers with serum samples. Absorption experiment was also performed using P. westermani and Clonorchis sinensis (C. sinensis) antigens to test specificity of the antigens. In the IFA teat for paragonimiasis all serum samples from proven paragonimiasis cases showed positive reaction at a dilution of 1:16 or greater, and up to 1:512 with a distinct mode at 1:32. On the other hand 87.5% (70) of 80 sera from parasitic infections other than paragonimiasia showed negative reaction and only 7.5% (6) was false positive at a dilution of 1:4, falling to 3.75% (3) at 1:8 and 1.25% (1) at 1:16. Those false positive sera were mostly from patients with clonorchiasis. Sera from patients with non-parasitic lung diseases and from healthy controls gave all negative reaction. In the occurence of cross-reactions between P. westermani and C. sinens, 12 of 25 sera from proven paragonimiasis patient gave IFA teat positive reaction against C. sinensis antigen : at titers of 1:8 in 4 and 1:4 in 8, although homologous antigen gave always 3-7 fold higher titer. Among 33 sera from clonorchiasis, Paragonimus antigen gave positive reactions in 4 cases : at titers of 1:16 in 1, 1:8 in 1 and 1:4 in 2, but 2-6 fold higher positive titers were shown with homologous antigen. In absorption experiment homologous reactivity baa completely removed. All the cases of cross reaction and a titer of 1:8 in serum from cured paragonimiasia case were absorbed by both homologous and heterologous antigen. The results indicate that IFA titers of 1:16 or more are regarded as positive, and a titer of 1:8 or less as absence of the infection or non-specific reaction. When IFA reactions for P. westermani were evaluated according to the above criterion, all serum samples from patients with proven paragonimiasis were correctly diagnosed, and false positive was only 1.2% among sera from individuals with parasitic infections other than paragonimiasis. In the comparison between IFA titers and number of eggs in sputum showed not significant level of correlation. In the results of IFA test in sera from paragonimlasis who were treated with bithionol during the past 6 months-7 years the cured cases showed all negative reaction(titer: 1:8 or less), whereas all the uncured cases revealed positive reactions with titers ranging from 1:16 to 1:128. However, there was no significant difference in wheal size by intradermal test between the cured and uncured cases. In the quantitative comparison of IFA titers between serum samples and dried blood specimens from paragonimiasis cases, the eluate, which was collected from finger-pricks and dried on filter paper. yielded almost the same results as was in the serum. The value of Spearman's rank correlation (rs) resulted in rs of + 0.638, a highly significant level of correlation. As a conclusion, the data in the present study suggest that the fluorescein-labelled antibody technique using delipidized adult worm particles which fixed on microscope slide as antigen can be utilized satisfactorily for the diagnosis of paragonimiasis in clinical and experimental studies. The procedure possesses a high degree of sensitivity, specificity and reproducibility as compared to the other tests. It was also confirmed that the IFA test can be used for the criteria of cure after the treatment for paragonimiasis.restrictio