- Email: [email protected]
Incidence of Reactive Antibodies Against Epstein-Barr in a Group of Renal Transplant Patients
Incidence of Reactive Antibodies Against Epstein-Barr in a Group of Renal Transplant Patients B. Geramizadeh, M. Aghdai, N. Azarpira, A. Behzade Behabahani, T. Heidari, M. Banihashemi, A.R. Raisjalali, J. Roozbeh, and A. Behzadi ABSTRACT Epstein-Barr virus (EBV) infection which is common among immunocompromised patients, may lead to life threatening lymphoproliferative diseases. In this study we examined the incidence and serologic status of EBV infection in 116 renal transplant patients including 84 males and 32 females as well as 72 normal volunteers. The time interval between transplantation and sampling was 1 month to 10 years. Twenty-two patients had a history of rejection. All cases were first transplants except for 3 second transplants. Four patients and no normals showed a positive PCR by a qualitative method. VCA IgM was positive in 11/116 patients (0.09%) and 3 of 72 (0.04%) normal volunteers. 99% (115/116) and 98% (65/72) of patients and normal controls were positive for VCA IgG. EA IgG was positive in 36/116 (31%) and 13/72(18%) of patients and normals, respectively. EBNA IgG was positive in 113/116 (97%) and 100% of patients versus normal controls, respectively. In all except one case with a positive VCA IgM there was a history of infectious mononucleosis-like syndrome. According to our previous data in more than 1000 renal transplant patients during more than 10 years, only one case of PTLD has been diagnosed (0.1%) which is lower than that reported. The high incidence of EBV seropositivity may contribute to this low incidence. The rate of EBV seropositivity in renal transplant patients was greater than in the normal population (P ⫽ .05). No association was observed between PCR and seropositivity and rejection or the type of treatment. After this study we began routine PCR and antibody testing in all renal transplant patients both pre- and posttransplant to determine the exact rate of reactivation versus primary infection which we plan to evaluate after 2 to 3 years. In conclusion we believe that the best easiest method to detect EBV infection in immunocompromised patients is VCA IgM ELISA; a qualitative PCR alone is not sufficient for this evaluation.
E
PSTEIN-BARR virus (EBV) infection, which is common in immunocompromised patients, can lead to life threatening lymphoproliferative diseases.1 These illnesses can be diagnosed early using PCR as well as antibodies against VCA, EBNA, and EA antigen reflecting seropositivity. We sought to report the incidence of these findings in our renal transplant patients.
informed consent. In addition 72 normal volunteers were evaluated by PCR and ELISA. DNA was extracted from 100 L buffy coat by the boiling method. For EBV detection the PCR used primers of 536 base pairs (RS42, KM29) with negative and positive controls. The threshold of assay detection was approximately 100 DNA copies /mL. PCR was done in a thermocycler with initial denaturation at 94°C for 2 minutes, then it was followed by 60.2°C (30S) annealing and 72°C
PATIENTS AND METHODS
From the Transplant Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran. Address reprint requests to B. Geramizadeh, Nemazee Hospital, Shiraz University of Medical Sciences, P.O. Box 719351119, Shiraz, Iran. E-mail: [email protected]
Blood samples were obtained from 116 renal transplant patients (84 male and 32 female) for PCR and ELISA antibody detection. All patients had undergone a single treatment protocol, after giving © 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.08.068
Transplantation Proceedings, 37, 3051–3052 (2005)
3051
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GERAMIZADEH, AGHDAI, AZARPIRA ET AL
Table 1. Incidence and Percentage of Positive Antibodies and Positive PCR in the Patients and Controls
EBV/PCR VCA IgM VCA IgG EA IgG EBNA IgG
Patients
Controls
4 (0.03%) 11 (0.09%) 115 (99%) 36 (31%) 113 (97%)
0 3 (0.04%) 65 (98%) 14 (12%) 72 (100%)
(1 minute) extension for 40 cycles with a final extension at 72°C for 5 minutes. PCR products run on 2% agarose gels containing ethidium bromide (30 min/120 mA) were visualized on an UV transilluminator. Viral capsid (VCA) IgM and IgG, early antigen (EA) IgG and EBNA IgG were also detected by ELISA methods in all renal transplant and normal sera. The treatment protocol was relatively the same in all the patients, as previously described.2,3 Age, sex, episodes of rejection, posttransplant interval, WBC count and hemoglobin level were all obtained from the clinical charts.
RESULTS
During the 2 year study period, we evaluated 116 patients (32 female and 84 male). The time interval between transplant and sampling was between 1 month and 10 years. Twenty-two patients had a history of rejection. One hundred thirteen patients had their first transplant, three others, their second transplant. Except for one HBS antigen positive subject, all patients were HBS and HCV antigen negative. PCR was positive in 4 patients (0.03%) and none of the controls. VCA IgM was positive in 11/116 (0.09%) patients and 3/72 normals (0.04%). VCA IgG was positive in 115/116 (99%) patients and 65/72 normal individuals (98%). EBNA was positive in 113/116 (97%) and 72/72 (100%) of patients and normal individuals, respectively (Table 1). EA IgG was positive in 36/116 (31%) and 14/72 (12%) of patients and normal individuals, respectively. 8/22 (36%) patients, with a history of rejection showed either EA IgG or VCA IgM. In four patients both EA IgG and VCA IgM were positive. None of the PCR positive patients had any clinical symptoms, but 10/11 patients who had VCA IgM reported a history of nonspecific flu-like syndromes. There was no correlation between the posttransplant interval and seropositivity. The patients and controls were statistically equivalent with respect to sex, age and all other data except for the presence of EA IgG and VCA IgG which were significantly higher among the patients (P ⫽ .05). DISCUSSION
This is the first study to show the EBV status of renal transplant patients in Iran. It may be important to know the
EBV status of post renal transplant patients, because failure to control EBV viral replication can lead to posttransplant lymphoproliferative disease (PTLD), which has a mortality of 50% and leads to graft loss in 63% of cases.4 In our series about 1/3 of patients showed VCA IgM or EA IgG or both. Most patients with a positive VCA IgM had a history of infectious mononucleosis-like syndrome and 4 asymptomatic patients were PCR positive. According to our previous data in more than 1000 renal transplant patients who have had transplants for more than 10 years, only one case of PTLD has been discovered until now (0.1%). This is a small frequency than most previous reports.5,6 The high incidence of EBV seropositivity may contribute to the low incidence of PTLD7; for example, solid organ transplant recipients at one major center showed a 20-fold higher incidence of disease in seronegative recipients.1 EBV seropositivity in renal transplant patients was greater than in the normal population (P ⫽ .05), a finding which has also been previously reported.7 No association was found between PCR and seropositivity with rejection or its treatment. We suggest routine testing of EBV VCA IgM and EA IgG in all renal transplant patients both pre and post transplant at regular intervals. After this study we began performing routine PCR and antibody in all renal transplant patients both pre and post transplant to assess the rate of reactivation versus primary infection; this study is planned for 2 to 3 years. Also we are trying to determine the relationship between viral load and the low incidence of PTLD in our renal transplant patients.
REFERENCES 1. Kopf S, Tomshoff B: Surveillance of EBV infection as a risk factor for posttransplant lymphoproliferative disorder in pediatric renal transplant recipients. Pediatr Nephrol 19:365, 2004 2. Hurault de Ligny B: Viral infections in renal transplantation. Presse Med 30 (24 pt 2):16, 2001 3. Rickinson AB, Kieff E: Epstein-Barr Virus. In the fields of virology. Vol 2, 4th ed. Philadelphia: Lippincott Williams and Wilkins Company; 2001, p 2575 4. Collins MH, Monton KT, Leachey AM, et al: Posttransplant lymphoproliferative disease in children. Pediatr Transplant 5:2, 2001 5. Zaia JA: Infections in organ transplant recipients. In Clinical Virology. Churchill Livingston, on CD ROM 6. Nalesnik MA, Jaffe R, Starlz TE, et al: The pathology of posttransplant lymphoproliferative disease occurring in the setting of cyclosporine A. Prednisone Immunosuppression. Am J Pathol 133:173, 1998 7. Shroff R, Trompoter R, Cubitt D, et al: EBV monitoring in pediatric renal transplant recipients. Pediatric Nephrol 17:770, 2002
E
PSTEIN-BARR virus (EBV) infection, which is common in immunocompromised patients, can lead to life threatening lymphoproliferative diseases.1 These illnesses can be diagnosed early using PCR as well as antibodies against VCA, EBNA, and EA antigen reflecting seropositivity. We sought to report the incidence of these findings in our renal transplant patients.
informed consent. In addition 72 normal volunteers were evaluated by PCR and ELISA. DNA was extracted from 100 L buffy coat by the boiling method. For EBV detection the PCR used primers of 536 base pairs (RS42, KM29) with negative and positive controls. The threshold of assay detection was approximately 100 DNA copies /mL. PCR was done in a thermocycler with initial denaturation at 94°C for 2 minutes, then it was followed by 60.2°C (30S) annealing and 72°C
PATIENTS AND METHODS
From the Transplant Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran. Address reprint requests to B. Geramizadeh, Nemazee Hospital, Shiraz University of Medical Sciences, P.O. Box 719351119, Shiraz, Iran. E-mail: [email protected]
Blood samples were obtained from 116 renal transplant patients (84 male and 32 female) for PCR and ELISA antibody detection. All patients had undergone a single treatment protocol, after giving © 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.08.068
Transplantation Proceedings, 37, 3051–3052 (2005)
3051
3052
GERAMIZADEH, AGHDAI, AZARPIRA ET AL
Table 1. Incidence and Percentage of Positive Antibodies and Positive PCR in the Patients and Controls
EBV/PCR VCA IgM VCA IgG EA IgG EBNA IgG
Patients
Controls
4 (0.03%) 11 (0.09%) 115 (99%) 36 (31%) 113 (97%)
0 3 (0.04%) 65 (98%) 14 (12%) 72 (100%)
(1 minute) extension for 40 cycles with a final extension at 72°C for 5 minutes. PCR products run on 2% agarose gels containing ethidium bromide (30 min/120 mA) were visualized on an UV transilluminator. Viral capsid (VCA) IgM and IgG, early antigen (EA) IgG and EBNA IgG were also detected by ELISA methods in all renal transplant and normal sera. The treatment protocol was relatively the same in all the patients, as previously described.2,3 Age, sex, episodes of rejection, posttransplant interval, WBC count and hemoglobin level were all obtained from the clinical charts.
RESULTS
During the 2 year study period, we evaluated 116 patients (32 female and 84 male). The time interval between transplant and sampling was between 1 month and 10 years. Twenty-two patients had a history of rejection. One hundred thirteen patients had their first transplant, three others, their second transplant. Except for one HBS antigen positive subject, all patients were HBS and HCV antigen negative. PCR was positive in 4 patients (0.03%) and none of the controls. VCA IgM was positive in 11/116 (0.09%) patients and 3/72 normals (0.04%). VCA IgG was positive in 115/116 (99%) patients and 65/72 normal individuals (98%). EBNA was positive in 113/116 (97%) and 72/72 (100%) of patients and normal individuals, respectively (Table 1). EA IgG was positive in 36/116 (31%) and 14/72 (12%) of patients and normal individuals, respectively. 8/22 (36%) patients, with a history of rejection showed either EA IgG or VCA IgM. In four patients both EA IgG and VCA IgM were positive. None of the PCR positive patients had any clinical symptoms, but 10/11 patients who had VCA IgM reported a history of nonspecific flu-like syndromes. There was no correlation between the posttransplant interval and seropositivity. The patients and controls were statistically equivalent with respect to sex, age and all other data except for the presence of EA IgG and VCA IgG which were significantly higher among the patients (P ⫽ .05). DISCUSSION
This is the first study to show the EBV status of renal transplant patients in Iran. It may be important to know the
EBV status of post renal transplant patients, because failure to control EBV viral replication can lead to posttransplant lymphoproliferative disease (PTLD), which has a mortality of 50% and leads to graft loss in 63% of cases.4 In our series about 1/3 of patients showed VCA IgM or EA IgG or both. Most patients with a positive VCA IgM had a history of infectious mononucleosis-like syndrome and 4 asymptomatic patients were PCR positive. According to our previous data in more than 1000 renal transplant patients who have had transplants for more than 10 years, only one case of PTLD has been discovered until now (0.1%). This is a small frequency than most previous reports.5,6 The high incidence of EBV seropositivity may contribute to the low incidence of PTLD7; for example, solid organ transplant recipients at one major center showed a 20-fold higher incidence of disease in seronegative recipients.1 EBV seropositivity in renal transplant patients was greater than in the normal population (P ⫽ .05), a finding which has also been previously reported.7 No association was found between PCR and seropositivity with rejection or its treatment. We suggest routine testing of EBV VCA IgM and EA IgG in all renal transplant patients both pre and post transplant at regular intervals. After this study we began performing routine PCR and antibody in all renal transplant patients both pre and post transplant to assess the rate of reactivation versus primary infection; this study is planned for 2 to 3 years. Also we are trying to determine the relationship between viral load and the low incidence of PTLD in our renal transplant patients.
REFERENCES 1. Kopf S, Tomshoff B: Surveillance of EBV infection as a risk factor for posttransplant lymphoproliferative disorder in pediatric renal transplant recipients. Pediatr Nephrol 19:365, 2004 2. Hurault de Ligny B: Viral infections in renal transplantation. Presse Med 30 (24 pt 2):16, 2001 3. Rickinson AB, Kieff E: Epstein-Barr Virus. In the fields of virology. Vol 2, 4th ed. Philadelphia: Lippincott Williams and Wilkins Company; 2001, p 2575 4. Collins MH, Monton KT, Leachey AM, et al: Posttransplant lymphoproliferative disease in children. Pediatr Transplant 5:2, 2001 5. Zaia JA: Infections in organ transplant recipients. In Clinical Virology. Churchill Livingston, on CD ROM 6. Nalesnik MA, Jaffe R, Starlz TE, et al: The pathology of posttransplant lymphoproliferative disease occurring in the setting of cyclosporine A. Prednisone Immunosuppression. Am J Pathol 133:173, 1998 7. Shroff R, Trompoter R, Cubitt D, et al: EBV monitoring in pediatric renal transplant recipients. Pediatric Nephrol 17:770, 2002