Kistik ekinokokkozisli hastaların tanı ve takibinde yeni multiepitop rekombinant peptit antijeninin kist sıvısı antijeni ile karşılaştırılması, immun yanıtın kistin evreleri ve parazitin genotipiyle ilişkilendirilmesi
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Tarih
2020
Yazarlar
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Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Kistik Ekinokokkozis (KE) Echinococcus granulosus’un metasestod evresinin neden olduğu ihmal edilen zoonotik bir enfeksiyondur. KE her yaş grubundan insanı etkileyen önemli bir halk sağlığı problemidir ve Türkiye’nin de içinde bulunduğu pek çok ülkede endemik olarak görülmektedir. KE tanısında öncelikli olarak klinik bulgular ve görüntüleme yöntemleri kullanılmaktadır. Hastalığın erken dönem tanısında, klinik ve epidemiyolojik çalışmalarda görüntüleme yöntemlerinin ve serolojik testlerin birlikte kullanılmasının standart bir yaklaşım olacağı bildirilmiştir. Serolojik testlerde temel antijenik kaynak olarak en çok kist sıvısı (KS) antijenleri kullanılmaktadır. KS antijenlerinin standardizasyonu oldukça zordur. Bu nedenle farklı laboratuvarlarda farklı sensitivite ve spesifite değeri vermektedir. KE’nin cerrahi ve medikal tedavisinden sonra %2-25 oranında nüks ihtimalinin bulunduğu bildirilmiştir. KS antijenlerine karşı oluşan antikorların dolaşımda uzun süre kalması hasta takibi açısından da dezavantaj oluşturmaktadır.
Son yıllarda yapılan çalışmalarda rekombinant antijenlerde umut verici sonuçlar elde edildiği bildirilmiştir. Kist sıvısının son immunoproteom analizine göre, immunodominant epitoplarının bir kist evresinden (Dünya Sağlık Örgütü (DSÖ) sınıflandırmasına göre) diğerine değiştiği ve birden fazla tanımlanmış immunodominant epitop içeren antijen kullanımına ihtiyaç olduğu bildirilmiştir.
Bu amaçla çalışmamızda öncelikle AgB1, AgB2 ve Ag5’e ait immunodominant epitoplar belirlenmiş ve bu epitopları içeren gen bölgelerinin sıralı klonlaması ile multiepitop rekombinant peptit antijeni (recDipol) elde edilmiştir. RecDipol ve KS’nın tanı performansı, cerrahi veya Percutaneous- Aspiration- Injection- Respiration (PAIR) işlemi uygulanan 149 KE hastası ve 70 kontrol grubu (49 diğer paraziter enfeksiyonu bulunan hasta ve 21 sağlıklı birey) IgG-ELISA yöntemi kullanılarak karşılaştırılmıştır. Ayrıca 28 takip hastasının değişen sürelerde (6- 36 ay) Ultrasonografi ve serolojik yöntemlerle takibi yapılmıştır. Genotipik çeşitlilik ve immun yanıt arasındaki ilişkiyi değerlendirmek amacıyla genotip analizi yapılan 20 hastaya ait serum örnekleri KS ve recDipol antijenleri kullanılarak IgG-ELISA yöntemi ile değerlendirilmiştir.
KS ve recDipol’un sensitivitesi sırayla %82,55 ve %78,52 iken spesifitesi sırayla %91,43 ve %95,71 olarak değerlendirilmiştir. KS antijenin sensitivitesi DSÖ sınıflamasına göre aktif (CE1 ve CE2) ve geçiş (CE3A ve CE3B) evresinde recDipol’den yüksek iken aktif olmayan (CE4 ve CE5) kistlerde recDipol oldukça yüksek seropozitivite göstermişir. RecDipol’un sensitivitesinin kist evreleriyle ilişkisi istatistiksel olarak da anlamlı bulunmuştur (P= 0. 041).
Takibi yapılan 28 hastanın 1 (%3,5)’i KS ile 6 (%21,4)‘ sı recDipol ile tüm takip boyunca seronegatif olarak değerlendirilmiştir. Ayrıca 5 (%17,85) hastada KS antijeni ile 6 (%21,42) hasta da recDipol ile takip sonunda seronegatifleşme görüldü. Nüks ihtimali bulunan bir hastanın Serolojik Indeks (SI) değerinde her iki antijenle de artma görülmüştür.
Genotip analizi yapılan 20 hastanın (16 G1, 1 G2, 2 G3 ve 1 G1/3) kanları IgG-ELISA yöntemi ile analiz edilmiştir. G1 genotipi dışındaki hastaların hepsi her iki antijene karşı seropozitif iken, G1 genotipindeki hastaların %87,5’i KS ile %75’i recDipol ile seropozitif olarak değerlendirilmiştir.
Sonuç olarak; recDipol antijeni ile özellikle aktif olmayan (CE4 ve CE5) kistlerin tanısında ve spesifitesinde umut verici sonuçlar elde edilmiştir. RecDipol’un spesifik epitoplar içeren gen bölgelerinden oluşmasına rağmen KE hastalarının başlangıç kanlarında KS’na oranla daha fazla negatif sonuçlar elde edilmesi, recDipol’un tanı ve hasta takibindeki performansının sınırlılığı olarak değerlendirilmiştir.
Cystic Echinococcosis (CE) is a neglected zoonotic infection caused by the metasestod stage of Echinococcus granulosus. CE is an important public health problem that is affecting people in all age groups and appears to be endemic in many countries including Turkey. Clinical findings and imaging methods are primarily used in the diagnosis, however, synchronous use of imaging methods and serological tests in early diagnosis of the disease, clinical and epidemiological reserach will be a standard approach. Hydatid fluid (HF) is used as the main antigenic resource for serological diagnosis. Standardization of HF antigens is considerably difficult. This causing different sensitivity and specificity results in different laboratories. The recurrence rate of CE is 2-25% after surgical and medical treatment. Besides, long-term persistence of antibodies in circulation against HF is another problem in follow-up. Recently, promising results have been reported about recombinant antigens. According to immunoproteom analysis, immunodominant epitopes change from one cyst stage (according to World Health Organization (WHO) classification) to another and antigens are required that including multiple defined immunodominant epitopes. For this purpose, multiepitope recombinant peptide antigen (recDipol) was obtained by sequential cloning of immunodominant epitopes belong to AgB1, AgB2 and Ag5. Diagnosis performance of recDipol and HF was compared with 149 CE patients who had surgery or Percutaneous- Aspiration- Injection- Respiration (PAIR) procedure, and 70 control groups (49 patients with other parasitic infections and 21 healthy individuals) using the IgG-ELISA method. Besides, 28 patients were followed up with Ultrasonography and serological methods for varying periods (6-36 months). Sera samples of 20 patients with genotype analysis were analyzed in the termf of relationship with genotypic diversity and immune response using IgG-ELISA method. The sensitivity of HF and recDipol were evaluated as 82,55% and 78,52%, while specificity was 91,43% ve 95,71%, respectively. While the sensitivity of HF antigen is higher than recDipol in the active (CE1 and CE2) and transition (CE3A and CE3B) stage, recDipol showed very high seropositivity in the inactive (CE4 and CE5) cysts. The relationship with the sensitivity of recDipol and cyst stage was statistically meaningful (P = 0. 041). Out of 28 follow up patients, 1 (3,5%) with HF and 6 (21,4%) with recDipol were evaluated seronegative during the whole follow-up. In addition, seronegativeization was observed in 5 (17,85%) patients with HF antigen and in 6 (21,42%) patients with recDipol at the end of follow-up. Serological Indeks (SI) value of increased one patient who has the possibility of recurrence at the end of the follow-up period with both antigens. 20 patients with genotype analysis (16 G1, 1 G2, 2 G3 and 1 G1/3) were analyzed using IgG-ELISA. All patients except the G1 genotype were seropositive against both antigens, While 87,5% of patients in the G1 genotype were seropositive with HF and 75% with recDipol. As a result, promising results were obtained with recDipol antigen especially in the diagnosis of inactive (CE4 and CE5) cysts and specificity. Although recDipol consists of gene regions containing specific epitopes, negative results in the initial blood of CE patients were evaluated as the limited performance of recDipol in diagnosis and follow-up patients.
Cystic Echinococcosis (CE) is a neglected zoonotic infection caused by the metasestod stage of Echinococcus granulosus. CE is an important public health problem that is affecting people in all age groups and appears to be endemic in many countries including Turkey. Clinical findings and imaging methods are primarily used in the diagnosis, however, synchronous use of imaging methods and serological tests in early diagnosis of the disease, clinical and epidemiological reserach will be a standard approach. Hydatid fluid (HF) is used as the main antigenic resource for serological diagnosis. Standardization of HF antigens is considerably difficult. This causing different sensitivity and specificity results in different laboratories. The recurrence rate of CE is 2-25% after surgical and medical treatment. Besides, long-term persistence of antibodies in circulation against HF is another problem in follow-up. Recently, promising results have been reported about recombinant antigens. According to immunoproteom analysis, immunodominant epitopes change from one cyst stage (according to World Health Organization (WHO) classification) to another and antigens are required that including multiple defined immunodominant epitopes. For this purpose, multiepitope recombinant peptide antigen (recDipol) was obtained by sequential cloning of immunodominant epitopes belong to AgB1, AgB2 and Ag5. Diagnosis performance of recDipol and HF was compared with 149 CE patients who had surgery or Percutaneous- Aspiration- Injection- Respiration (PAIR) procedure, and 70 control groups (49 patients with other parasitic infections and 21 healthy individuals) using the IgG-ELISA method. Besides, 28 patients were followed up with Ultrasonography and serological methods for varying periods (6-36 months). Sera samples of 20 patients with genotype analysis were analyzed in the termf of relationship with genotypic diversity and immune response using IgG-ELISA method. The sensitivity of HF and recDipol were evaluated as 82,55% and 78,52%, while specificity was 91,43% ve 95,71%, respectively. While the sensitivity of HF antigen is higher than recDipol in the active (CE1 and CE2) and transition (CE3A and CE3B) stage, recDipol showed very high seropositivity in the inactive (CE4 and CE5) cysts. The relationship with the sensitivity of recDipol and cyst stage was statistically meaningful (P = 0. 041). Out of 28 follow up patients, 1 (3,5%) with HF and 6 (21,4%) with recDipol were evaluated seronegative during the whole follow-up. In addition, seronegativeization was observed in 5 (17,85%) patients with HF antigen and in 6 (21,42%) patients with recDipol at the end of follow-up. Serological Indeks (SI) value of increased one patient who has the possibility of recurrence at the end of the follow-up period with both antigens. 20 patients with genotype analysis (16 G1, 1 G2, 2 G3 and 1 G1/3) were analyzed using IgG-ELISA. All patients except the G1 genotype were seropositive against both antigens, While 87,5% of patients in the G1 genotype were seropositive with HF and 75% with recDipol. As a result, promising results were obtained with recDipol antigen especially in the diagnosis of inactive (CE4 and CE5) cysts and specificity. Although recDipol consists of gene regions containing specific epitopes, negative results in the initial blood of CE patients were evaluated as the limited performance of recDipol in diagnosis and follow-up patients.
Açıklama
Anahtar Kelimeler
Echinococcus Granulosus, Kistik Ekinokokkozis, Multiepitop Rekombinant Antijen, Serolojik Tanı, Hasta Takibi, Genotip, Cystic Echinococcosis, Multiepitope Recombinant Antigen, Serodiagnosis, Follow Up, Genotype
