Epstein-barr virus enfeksiyonlarının tanısında PCR sonuçlarının değerlendirilmesi
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Date
2012-10
Authors
Geçgel, Sanem Karadağ
Journal Title
Journal ISSN
Volume Title
Publisher
Ankara Microbioloji Derneği
Abstract
Epstein-Barr virus (EBV), öncelikle enfeksiyöz mononükleoz ve ayrıca çeşitli lenfomalar ve posttransplant lenfoproliferatif hastalığın (PTLD) etiyolojisinden sorumlu, latent enfeksiyon oluşturan bir herpes virustur. EBV enfeksiyonlarının laboratuvar tanısı; atipik lenfositlerin, heterofil antikorların, virusun kapsid antijeni (VCA), nükleer antijeni (EBNA) ve erken antijeni (EA)'ne karşı oluşan özgül antikorların ve viral DNA'nın saptanmasına dayanır. Ülkemizde yetişkin popülasyondaki seropozitiflik oranının çok yüksek (%80-95) olması nedeniyle, transplant alıcıları veya onkoloji hastaları gibi özellikle immünsüpresif hasta gruplarında EBV reaktivasyonunun belirlenmesinde rutin serolojik testler yetersiz kalabilir. Bu gibi durumlarda VCA IgG avidite testi ve moleküler yöntemlerden yararlanılmaktadır. Bu çalışmada, renal transplant ve çocuk onkoloji hastalarında EBV enfeksiyonlarının tanı ve takibinde serolojik yöntemlerin yanı sıra gerçek zamanlı polimeraz zincir reaksiyonu (Rt-PCR) ile viral DNA düzeylerinin belirlenmesi amaçlanmıştır. Çalışmaya, 62 erişkin renal transplant alıcısı, 37 çocuk onkoloji hastası ile EBV seropozitif immün kompetan sağlıklı 50 birey (28'i çocuk, 22'si erişkin) dahil edilmiştir. Transplant alıcılarından transplantasyon öncesi bir kez, transplantasyon sonrası üç kez (birinci hafta, birinci ay ve üçüncü ay) olmak üzere toplam dört kez; çocuk onkoloji hastalarından tedavi öncesi bir kez, immünsüpresif tedaviye başlandıktan sonra üç kez (birinci ay, üçüncü ay ve altıncı ay) olmak üzere toplam dört kez; kontrol grubundan ise bir kez kan örneği alınmıştır. Serum örneklerinde EBV serolojik profilleri Paul-Bunnel ve immünoblot testleri ile; VCA IgG avidite testi ELISA ile ve EBV-DNA düzeyi Rt-PCR yöntemiyle araştırılmıştır. Renal transplant grubundaki hastaların 3 (%4.8)'ünde EBV-DNA pozitif bulunmuştur. Bu hastalarda CD4/CD8 oranları transplantasyon öncesi değerlerine göre transplantasyon sonrası birinci hafta ve üçüncü ayda anlamlı olarak düşük bulunmakla birlikte PTLD ve organ reddi gelişmemiştir. Çocuk onkoloji grubundaki hastaların 3 (%8.1)'ünde EBV-DNA pozitif bulunmuş; bu durum Hodgkin lenfoma tanısıyla takip edilen iki çocukta hastalık, diğerinde ise reaktivasyon ile ilişkilendirilmiştir. Kontrol grubundaki olguların ise 10 (%20)'unda EBV-DNA pozitifliği tespit edilmiştir. İmmünoblot sonuçları akut enfeksiyon serolojisi ile uyumlu olan erişkin kontrol olgularında EBV-DNA pozitifliği, Paul-Bunnel pozitifliği ve düşük avidite sonuçları arasındaki uyum istatistiksel olarak anlamlıdır. Çocuk kontrol grubunda ise bu serolojik profil ile sadece düşük avidite arasındaki uyum anlamlı kabul edilmiştir. Sonuç olarak bu çalışmada erişkin renal transplant hastalarında transplantasyonu takip eden üç aylık dönemde EBV ile ilişkili PTLD ve akut rejeksiyon riskinin bulunmadığı; EBV ile ilişkili malignitesi bulunan çocuklarda tedavi öncesi Rt-PCR ile EBV-DNA araştırılmasının tanı, takip ve prognozu değerlendirmede yararlı olabileceği; immünsüpresif hastalarda EBV reaktivasyonunu saptamak için serolojik sonuçların avidite ve PCR testleri ile desteklenmesi gerektiği düşünülmüştür.
Epstein-Barr virus (EBV), a herpesvirus leading to latent infections, is principally responsible for infectious mononucleosis, and also plays role in the etiology of various lymphomas and post-transplantation lymphoproliferative disease (PTLD). Laboratory diagnosis of EBV infections depends on the detection of atypical lymphocytes, heterophile antibodies, specific antibodies against viral capsid (VCA), nuclear (EBNA) and early (EA) antigens, and of the viral DNA. Since the seropositivity rate in adult population is very high (80-95%) in our country, routine serologic tests may be insufficient to characterize EBV reactivation in immunosuppressive subjects, such as transplant recipients or oncology patients. In those cases VCA IgG avidity test and molecular methods are more useful. This study was conducted to determine the role of viral DNA levels detected by real-time polymerase chain reaction (Rt-PCR) and serological tests for the diagnosis and follow up of EBV infections in renal transplant recipients and pediatric oncology patients. A total of 62 adult renal transplant recipients, 37 children with oncological diseases, and 50 EBV-seropositive immunocompetent healthy subjects (28 children, 22 adults) as controls, were included in the study. Four blood samples, once before transplantation and three times thereafter (at first week, first and third months) were collected from transplant recipients; in pediatric oncology patients blood samples were collected four times, once before immunosuppressive treatment and three times thereafter (at first, third and sixth months), while the control group had a single blood sample collected. Serological profiles for EBV were searched by Paul-Bunnel and immunoblotting tests; VCA IgG avidity by ELISA and viral load by Rt-PCR. EBV-DNA was found positive in 3 (4.8%) of the renal transplant patients. While in these patients the CD4/CD8 ratio was significantly lowered in the first week and third month post-transplant, no PTLD or organ rejection developed. EBV-DNA was positive in 3 (8.1%) of the pediatric oncology patients. This positivity was attributed to Hodgkin's disease in two of these cases and to reactivation in the third case. EBV-DNA positivity was present in 10 (20%) of the control subjects. In the adult controls whose immunoblot results were compatible with the serologic pattern of an acute infection, the correlation among positive EBV-DNA, positive Paul-Bunnel and low IgG avidity results was statistically significant. As for children in the control group, this serologic profile was significantly correlated with low IgG avidity only. The data obtained from this study indicated that no risk of EBV-related PTLD or acute rejection was found in the first three months in the adult renal transplant patients. In children with EBV-related malignancy the search for EBV-DNA by RtPCR before therapy may be useful in the diagnosis, follow-up and prognostic evaluation. Serologic results should be supported by IgG avidity and PCR in order to ascertain the presence of EBV reactivation in immunosuppressive patients.
Epstein-Barr virus (EBV), a herpesvirus leading to latent infections, is principally responsible for infectious mononucleosis, and also plays role in the etiology of various lymphomas and post-transplantation lymphoproliferative disease (PTLD). Laboratory diagnosis of EBV infections depends on the detection of atypical lymphocytes, heterophile antibodies, specific antibodies against viral capsid (VCA), nuclear (EBNA) and early (EA) antigens, and of the viral DNA. Since the seropositivity rate in adult population is very high (80-95%) in our country, routine serologic tests may be insufficient to characterize EBV reactivation in immunosuppressive subjects, such as transplant recipients or oncology patients. In those cases VCA IgG avidity test and molecular methods are more useful. This study was conducted to determine the role of viral DNA levels detected by real-time polymerase chain reaction (Rt-PCR) and serological tests for the diagnosis and follow up of EBV infections in renal transplant recipients and pediatric oncology patients. A total of 62 adult renal transplant recipients, 37 children with oncological diseases, and 50 EBV-seropositive immunocompetent healthy subjects (28 children, 22 adults) as controls, were included in the study. Four blood samples, once before transplantation and three times thereafter (at first week, first and third months) were collected from transplant recipients; in pediatric oncology patients blood samples were collected four times, once before immunosuppressive treatment and three times thereafter (at first, third and sixth months), while the control group had a single blood sample collected. Serological profiles for EBV were searched by Paul-Bunnel and immunoblotting tests; VCA IgG avidity by ELISA and viral load by Rt-PCR. EBV-DNA was found positive in 3 (4.8%) of the renal transplant patients. While in these patients the CD4/CD8 ratio was significantly lowered in the first week and third month post-transplant, no PTLD or organ rejection developed. EBV-DNA was positive in 3 (8.1%) of the pediatric oncology patients. This positivity was attributed to Hodgkin's disease in two of these cases and to reactivation in the third case. EBV-DNA positivity was present in 10 (20%) of the control subjects. In the adult controls whose immunoblot results were compatible with the serologic pattern of an acute infection, the correlation among positive EBV-DNA, positive Paul-Bunnel and low IgG avidity results was statistically significant. As for children in the control group, this serologic profile was significantly correlated with low IgG avidity only. The data obtained from this study indicated that no risk of EBV-related PTLD or acute rejection was found in the first three months in the adult renal transplant patients. In children with EBV-related malignancy the search for EBV-DNA by RtPCR before therapy may be useful in the diagnosis, follow-up and prognostic evaluation. Serologic results should be supported by IgG avidity and PCR in order to ascertain the presence of EBV reactivation in immunosuppressive patients.
Description
Keywords
Microbiology, Epstein-barr virus, Immunosuppression, Paul-bunnel, Immunoblotting, VCA, IgG avidity, Real-time, PCR, Ebv, Load, Immunofluorescence, Transplantation, Assay, Dna, Immünoblot, Epstein-Barr virus, Immünsüpresyon, Paul-bunnel, Immünoblot, VCA, IgG avidite, Gerçek zamanlı PCR
Citation
Geçgel, S. K. vd. (2012). "Epstein-barr virus enfeksiyonlarının tanısında PCR sonuçlarının değerlendirilmesi". Mikrobiyoloji Bülteni, 46(4), 594-606.