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Association of HHV-6 and HHV-7 reactivation with the development of chronic allograft nephropathy
Journal of Clinical Virology 46 (2009) 29–32
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Association of HHV-6 and HHV-7 reactivation with the development of chronic allograft nephropathy S. Chapenko a,∗ , I. Folkmane a,b , I. Ziedina b , M. Chistyakovs a , R. Rozentals b , A. Krumina a , M. Murovska a a b
August Kirchenstein Institute of Microbiology and Virology, Riga Stradins University, Ratsupites St. 5, LV-1067 Riga, Latvia The Transplantology Laboratory, Riga Stradins University, Riga, Latvia
a r t i c l e
i n f o
Keywords: Renal transplantation Chronic allograft nephropathy Human herpesvirus-6 Human herpesvirus-7 Virus activation Acute rejection
a b s t r a c t Background: The long-term effect of HHV-6 and HHV-7 infections on chronic allograft nephropathy (CAN) development after renal transplantation is uncertain. Objectives: To determine HHV-6 and HHV-7 reactivation during the post-transplantation period and to evaluate its effect on CAN development in renal transplant patients. Study design: Eighty-one renal allograft recipients (28 with CAN, 53 with normal transplant function) were studied to determine the frequency of HHV-6 and HHV-7 reactivation during 36.4 ± 7.8 months after renal transplantation using nested PCR. HHV-6 variants were identified using restriction endonuclease analysis. Patients were monitored for the development of CAN. Results: The frequency of HHV-6 and/or HHV-7 plasma DNA was significantly higher in CAN patients (25/28, 89.3%) compared to control patients (15/50, 30.0%, p = 0.0001). CAN patients also had an increased incidence of dual active infections (20/25, 80% and 2/15, 13.3%, p = 0.007, respectively). In all 34 HHV-6 positive cases, the HHV-6B variant was identified. The presence of HHV-7 DNA in plasma preceded the presence of HHV-6 DNA. Early development of CAN and graft loss was detected only in patients with simultaneous HHV-6 and HHV-7 plasma DNA. Conclusions: Reactivation of HHV-6 and HHV-7 in renal graft recipients is a risk factor for CAN development. The presence of concurrent HHV-6 and HHV-7 DNA in the plasma is an unfavorable prognostic factor. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Chronic allograft nephropathy (CAN) is the leading cause of progressive renal failure and the most important cause of posttransplantation graft loss in renal transplant recipients.1 CMV disease and acute rejection (AR) episodes are suggested as risk factors for the development of CAN.2–5 Human herpesvirus-6 and -7 (HHV-6 and -7) belong to the -herpesviruses family, together with cytomegalovirus (CMV).6 Both viruses are ubiquitous with a high seroprevalence in adults. After primary infection, these viruses establish a latent or persistent infection that remains for the lifetime of the host. The viruses are lymphotropic, but HHV-6 in particular, which uses the CD46 molecule as its receptor, may also infect other cell types, such as monocytes, and epithelial and endothelial cells.7 HHV-6 isolates are classified into two variants (termed HHV-6A
Abbreviations: RT, renal transplantation; CAN, chronic allograft nephropathy; AR, acute rejection; ATG, antithymocyte globulin. ∗ Corresponding author. Tel.: +371 67427920. E-mail address: [email protected] (S. Chapenko). 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.05.018
and HHV-6B) on the basis of distinct genetic, antigenic and biological characteristics. The specific pathogenicity of each variant remains poorly understood.8,9 Most infections in renal transplant recipients are due to HHV-6B. HHV-7 uses the CD4 molecule as its receptor and is more strictly lymphotropic.10 The stimulus for reactivation of beta-herpesviruses in renal transplant recipients is an immunosuppressive regimen. In addition, these viruses possess immunomodulating properties, including the ability to alter the expression of immune activation molecules, modulate expression of several cytokines and chemokines and induce apoptosis in lymphocytes, which may contribute to immunosuppression. Productive infection of CD4 T cells results in cytopathic effects and cell destruction.11 The ubiquitous nature of HHV-6 and HHV-7 creates conditions for the development of concurrent infection and interaction between these viruses. The kinetics of the activation of these viruses during the post-transplantation period suggest that HHV-7 may act as a co-factor for HHV6 and CMV reactivation, while both HHV-6 and HHV-7 may act as co-factors in the pathogenesis of CMV disease and acute rejection.12–16 The aim of this study was to determine HHV-6 and HHV-7 activation during the post-transplantation period and to evaluate the
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S. Chapenko et al. / Journal of Clinical Virology 46 (2009) 29–32
Table 1 Demographic data of patients with CAN (group I) and control group patients (group II). Parameter
Group I (n = 28)
Group II (n = 53)
Age (years) Sex (F/M)
43.4 ± 10.7 18/10
42.0 ± 11.3 32/21
Primary renal disease (n) Glomerular Diabetes mellitus Tubulointerstitial Polycystic kidney disease Other
13 4 5 3 3
32 3 9 5 4
Donor age (years) Total HLA mismatch Cold ischemia time Acute rejection episodes CMV diseases
44.3 ± 11.2 4.2 ± 1.3 17.8 ± 3.4 14/28 5/28
43.2 ± 12.0 4.0 ± 1.7 16.4 ± 2.7 11/53 4/53
CAN: chronic allograft nephropathy.
possible effects of the activation of these viruses on CAN development in renal transplant patients.
2. Methods Eighty-one adult first-time renal allograft recipients, who were transplanted during 2002–2003 in the Transplantation Centre of the Stradins University Hospital of Riga, were enrolled in this study. All patients had received a cadaveric renal transplant. The patients were divided into two groups according to the graft function: with CAN (28/81) and with normal transplant function (53/81, control group). Demographic data of the patients is presented in Table 1. Initially, all patients had received an intravenous injection of methylprednisolone (up to 500 mg on the day of the transplantation), which was tapered gradually within the first four postoperative days. Maintenance therapy consisted of oral cyclosporine A, oral steroids (prednisolone), and mycophenolate mofetil in a fixed dosage of 0.75–1 g. Twentysix out of 28 patients with CAN and 50 out of 53 patients had received induction with monoclonal IL2R antibodies (basiliximab or daclizumab). AR episodes were diagnosed on the basis of a rise in serum creatinine (>25% from baseline), clinical evidence, and graft biopsy. Histological examination and classification of a core biopsy was done according to the Banff-97 scheme. AR episodes were treated with methylprednisolone. Steroid-resistant AR episodes (two recipients from the control group and three recipients with CAN) were subsequently treated with antithymocyte globulin (ATG) (4 mg/kg/day for 10–14 days). All patients with high (donor [D]+/recipient [R]−) and moderate (D+/R+, D−/R+) risk for CMV disease, as well as patients with ATG therapy, had received ganciclovir (GCV) 5 mg/kg/day orally for 3 months or intravenously for 2 weeks. Intravenous GCV has been used to treat the patients with CMV disease (5 mg/kg twice per day for 2 weeks). GCV dose was adjusted to renal function and to viral (CMV) infection activity. CAN was characterized by progressive loss of renal function, commonly associated with proteinuria and hypertension. Characteristic histopathologic features included interstitial fibrosis, tubular atrophy, transplant arteriopathy and chronic transplant glomerulopathy. EDTA blood samples were collected from the patients before transplantation and at 7–14 days, 3, 6, 9, 12 weeks and 6, 9, 12 18, 24, 30, 36 and 42 months after transplantation. Activation of HHV-6 and HHV-7, AR, and CAN development were monitored.
2.1. Nested polymerase chain reaction (nPCR) Nested polymerase chain reaction (nPCR) was used for the detection of viral sequences in DNA isolated from peripheral blood leukocytes (PBL) and plasma (markers of latent/persistent and active infection, respectively). Total DNA was isolated from 0.5 ml of fresh whole blood by phenol–chloroform extraction. For DNA purification from 200 l of cell free blood plasma, QIAamp DNA Blood Kit (Qiagen) was used. The plasma samples were treated with deoxyribonuclease I before DNA purification. To assure the quality of the PBL DNA, as well as exclude contamination of plasma DNA by cellular DNA, a globin PCR was performed. PCR amplification for the viruses was carried out in the presence of 1 g of PBL DNA and 10 l of plasma DNA (corresponding to 100 l of plasma). The detection of HHV-6, HHV-7 and CMV DNA was performed according to Secchiero et al.,17 Berneman et al.,6 and Studahl et al.,18 respectively. Positive (virus genomic DNA) and negative (DNA without virus-specific sequences) and water controls were included in each experiment. 2.2. Restriction endonuclease analysis Restriction endonuclease analysis was carried out using enzyme HindIII17 . This enzyme cuts HHV-6B 163 kbp amplimer into two fragments: 66 kbp and 97 kbp and does not cut HHV-6A amplimer. This difference shows the distinction between HHV-6A and HHV-6B virus variants. 2.3. Statistical method Statistical difference in the prevalence of latent/persistent and active CMV, HHV-6 and HHV-7 infection between tested groups was assessed by Fisher’s exact test. 3. Results 3.1. Clinical data Fifty-three out of 81 (65.4%) renal allograft recipients had good graft function, but in 28 (34.5%) recipients, CAN were diagnosed for a mean follow-up period of 36.4 ± 7.8 months after renal transplantation (RT). No significant difference in demographic data between the patients’ groups was detected (Table 1). 3.2. Prevalence of beta-herpesvirus DNA in PBLs As expected, beta-herpesvirus genomic sequences were found in all PBL DNA samples from CAN patients (28 out of 28), and in 50 out of 53 (94.3%) samples from control group patients without CAN before RT (Table 2). A significantly higher rate of dual (HHV-6, HHV-7) and triple (CMV, HHV-6, HHV-7) infections was found in CAN patients in comparison with control group patients (p = 0.002 and 0.0003, respectively). The rate of dual CMV and HHV-7 infection was significantly higher in the control group patients compared to the patients with CAN (p = 0.042). Single CMV and HHV-6 infections, as well as dual CMV and HHV-6 infections, were found only in the control group patients and not in the CAN group. In all 34 PBL DNA samples from the patients with HHV-6 infection, HHV-6B was the variant identified. No beta-herpesvirus genomic sequences were detected in plasma DNA samples from the patients before RT. 3.3. Prevalence of active beta-herpesvirus infection The frequency of plasma viremia was significantly higher in CAN patients (25/28, 89.3%) in comparison with the control group patients (15/50, 30.0%, p = 0.0001) after RT (Table 2). The frequency of simultaneous activation of HHV-6 and HHV-7 was significantly
S. Chapenko et al. / Journal of Clinical Virology 46 (2009) 29–32
31
Table 2 Prevalence of beta-herpesviruses DNA in patients with CAN and control group patients before and after RT by nPCR in PBMCs and in plasma. Viral infection
PBMCs before RT
Plasma after RT
CAN (n = 28)
Control group (n = 53)
Single CMV Single HHV-6 Single HHV-7 Dual CMV + HHV-6 Dual CMV + HHV-7 Dual HHV-6 + HHV-7 Triple CMV + HHV-6 + HHV-7 CMV+ HHV-6+ HHV-7+ Negative for all Positive for 1 or more
0/28 0/28 3/28 0/28 2/28 11/28 12/28
3/53 1/53 21/53 2/53 15/53 4/53 4/53
0/28 28/28
3/53 50/53
p-Value
0.009 0.042 0.002 0.0003
CAN (n = 25)
Control group (n = 15)
0/25 0/25 0/25 0/25 0/25 15/25 5/25 0 3 2 3/28 25/28
0/15 1/15 5/15 0/15 7/15 2/15 0/15
38/53 15/50
p-value
0.007
0.0001
RT: renal transplantation; CAN: chronic allograft nephropathy.
higher in the CAN patients compared to the control group patients (p = 0.007). No significant difference in the frequency of CMV activation between patients with CAN and control group patients was revealed (5/14 and 7/24, respectively). All 25 CAN patients with viremia also had concurrent HHV-6 and HHV-7 or triple detection of herpesvirus DNA in the PBLs. Simultaneous activation of HHV6 and HHV-7 was found in 15 of them, and triple infections—in 5 patients. Single infections of CMV, HHV-6, and HHV-7 were 0, 3 and 2 patients, respectively. In the control group, only 25% (2/8) of the patients with concurrent HHV-6 and HHV-7 and triple infections in the PBLs had DNA in the plasma (Table 2). Simultaneous presence of CMV and HHV-7 DNA was more commonly found in the plasma of controls than in patients with CAN (Table 2). These patients had dual CMV and HHV-7 infections in PBLs. Only HHV-6 variant B was detected in the 23 positive plasma samples. The mean number of days before detecting HHV-7, HHV-6 and CMV reactivation was 12 days (range 7–23 days), 38 days (range 14–103 days), and 63 days (range 42–112 days), respectively. CMV disease was diagnosed in 5 patients with CAN and simultaneous CMV, HHV-6 and HHV-7 active infection and in 4 control group patients with simultaneous CMV and HHV-7 active infection. All patients had received intravenous GCV. 3.4. Acute rejection and HHV-6 and HHV-7 infection AR episodes were diagnosed in 25 out of 81 (30.9%) recipients. The frequency of AR episodes was significantly higher in CAN patients (14/28, 50.0%) in comparison with control group patients (11/53, 20.8%; p = 0.002). The mean time until AR episodes was 127 ± 53 days in patients with active viral infections and 113 ± 42 days in patients with latent infections. 3.5. CAN, acute rejection and activation of viral infections The mean time until the onset of clinical manifestations of CAN was 242 ± 63 days. This period had tendency to shorten in recipients with AR episodes and simultaneous activation of both HHV-6 and HHV-7 (163 ± 41 days) compared to recipients with AR episodes and activation of HHV-6 only (171 ± 52) or HHV-7 only (185 ± 31 days). Further statistical analysis was not performed because the case size was too small. Out of 28 recipients with CAN, 9 grafts were rejected. All 9 recipients had AR episodes and simultaneous HHV-6 and HHV-7 activation. 4. Discussion Chronic allograft nephropathy is an important cause of posttransplantation graft loss in the recipients of renal allografts. A large number of new effective immunosuppressive drugs are available
and antiviral therapy effectively prevents acute rejection and CMV disease.19–22 However, standards of the dose for prophylaxis and the optimal duration of antiviral prophylaxis or preemptive therapy to prevent the infection have not been established.23 In our experience, a low dose of GCV regimen (5 mg/kg/day for 2 weeks or orally for 3 months) has been used according to recommendations by the EBPG Expert Group on Renal Transplantation.24 Antiviral therapy also can inhibit reactivation of other herpesviruses, and maximize short-term graft survival in kidney transplant recipients. Ganciclovir is shown to be more effective in inhibiting CMV than HHV-6 or HHV-7.25–27 HHV-6 is reported as significant factor in CAN development2 ; however, the importance of HHV-7 infection in the pathogenesis of CAN is still unknown. Our data shows that latent/persistent HHV-6 and HHV-7 infection is widely prevalent in renal transplant recipients (92.6%) before renal transplantation, thus confirming the ubiquity of these viruses. We found a significantly higher prevalence of dual (HHV-6 and HHV-7) DNA by nPCR in PBL in CAN patients. Only the HHV-6B variant was detected in PBL and plasma DNA samples from the patients with HHV-6 infection, indicating that either the HHV-6A variant occurs infrequently in these patients or it may be limited to sites other than the peripheral blood. The frequency of simultaneous activation of HHV-6 and HHV7 was significantly higher in CAN patients, suggesting a possible involvement for HHV-6 and HHV-7 in the development of the diseases. In the CAN and control groups, patients with dual HHV-6 and HHV-7 and triple CMV, HHV-6 and HHV-7 infection had activation of HHV-7 before HHV-6 and CMV activation, confirming our previous reports.13,14 The development of CMV disease in the patients with CMV and HHV-7 and CMV, HHV-6 and HHV-7 infection suggests that HHV-6 and HHV-7 may be co-factors of this disease in consequence of a synergistic effect. The association of HHV-6 and HHV-7 infection with an increased risk for development of CMV disease in the renal transplant patients is reported previously.28–30 We did not observe CAN in patients with single CMV reactivation. We did not detect a significant difference in the frequency of CMV activation and CMV disease between patients with CAN and the control group patients. This finding may be due to the antiviral treatment. We did not detect an association between activation of these viral infections and AR detected that agree with the data of Ono et al.31 However, the data showing a significantly higher AR rate in CAN patients suggests that AR is an independent risk factor for CAN. The tendency to shorten the period of CAN development and loss of grafts in renal transplant patients with simultaneous activation of HHV-6 and HHV-7 suggest that both viruses may be involved in the CAN development via their immunomodulatory ability.
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Acknowledgements This work was supported in part by the National Research Program in Medicine “Multi-Disciplinary Research Consortium on Major Pathologies Threatening the Life Expectancy and Quality of Life of the Latvian Population” project no. 11 and grant “Role of blood-borne viruses infection in the development of posttransplant complications.” References 1. Pascual M, Theruvath T, Kawai T, Tolkoff-Rubin N, Cosimi AB. Strategies to improve long-term outcomes after renal transplantation. N Engl J Med 2002;346:580–90. 2. Tong CY, Barkan A, Peiris JC, Muir P, Herrington CS. The association of viral infection and chronic allograft nephropathy with graft dysfunction after renal transplantation. Transplantation 2002;74:576–8. 3. Sagedal S, Hartmann A, Nordal KP, Osnes K, Leivestad T, Foss A, et al. Impact of early cytomegalovirus infection and disease on long-term recipient and kidney graft survival. Kidney Int 2004;66:329–37. 4. Nett PC, Heisey DM, Fernandez LA, Sollinger HW, Pirsch JD. Association of cytomegalovirus disease and acute rejection with graft loss in kidney transplantation. Transplantation 2004;78:1036–41. 5. Boratynska M, Banasik M, Watorek E, Patrzatek D, Szyber P, Klinger M. Influence of cytomegalovirus disease on early and late renal graft function. Transplant Proc 2006;38:147–50. 6. Berneman ZN, Ablashi DV, Li G, Eger-Fletcher M, Reitz MS, Hung C-L, et al. Human herpesvirus 7 is a T-lymphotropic virus and is related to, but significantly different from human herpesvirus 6 and human cytomegalovirus. Proc Natl Acad Sci USA 1992;89:10552–6. 7. Santoro F, Kennedy PE, Locatelli G, Malnati MS, Berger EA, Lusso P. CD46 is a cellular receptor for human herpesvirus 6. Cell 1999;99:817–27. 8. Ablashi DV, Blahandran N, Josephs SF, Hung CL, Krueger GRF, Kramarsky B, et al. Genomic polymorphism, growth properties, and immunologic variations in human herpesvirus-6 isolates. Virology 1991;184:545–52. 9. Boutelleau D, Duros C, Bonnafous P, Caiola D, Karras A, Castro ND, et al. Identification of human herpesvirus 6 variants A and B by primer-specific realtime PCR may help to revisit their respective role in pathology. J Clin Virol 2006;35:257–63. 10. Black JB, Pellet PE. Human herpesvirus 7. Rev Med Virol 1999;9:245–62. 11. Lusso P. HHV-6 and the immunosystem: mechanisms of immunomodulation and viral escape. J Clin Virol 2006;37(Suppl. 1):4–10. 12. Osman HKE, Peiris JSM, Taylor CE, Warwicker P, Jarret RF, Madeley CR. Cytomegalovirus disease in renal allograft recipients: is human herpesvirus-7 a cofactor for disease progression? J Med Virol 1996;48:295–301. 13. Chapenko S, Folkmane I, Tomsone V, Amerika D, Rozentals R, Murovska M. Coinfection of two -herpesviruses (CMV and HHV-7) as an increased risk factor for “CMV disease” in patients undergoing renal transplantation. Clin Transplant 2000;14:486–92. 14. Chapenko S, Folkmane I, Tomsone V, Kozireva S, Bicans J, Amerika D, et al. Infection of -herpesviruses (CMV, HHV-6, HHV-7): role in postrenal transplantation complication. Transplant Proc 2001;33:2463–4. 15. Dockrell DH, Paya CV. Human herpesvirus-6 and -7 in transplantation. Rev Med Virol 2001;11:23–36.
16. Boutelliau D, Fernandez C, Andre E, Imbert-Marcille BM, Milpied N, Agut H, et al. Human herpesvirus (HHV)-6 and HHV-7: two closely related viruses with different infection profiles in stem cell transplantation recipients. J Infect Dis 2003;187:179–86. 17. Secchiero P, Carrigan DR, Asano Y, Benedetti L, Crowley RW, Komaroff AL, et al. Detection of human herpesvirus 6 in plasma of children with primary infection and immunosuppressed patients by polymerase chain reaction. J Infect Dis 1995;171:273–80. 18. Studahl M, Bergstrom T, Ekeland-Sjoberg K, Ricksten A. Detection of cytomegalovirus DNA in cerebrospinal fluid in immunocompetent patients as a sign of active infection. J Med Virol 1995;46, 274-80.19. 19. Paya C, Humar A, Dominguez E, Washburn K, Blumberg E, Alexander B, et al. Efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cytomegalovirus disease in solid organ transplant recipients. Am J Transplant 2004;4:611–20. 20. Varga M, Remport A, Czebe K, Peter A, Toronyi E, Fehervari I, et al. Cytomegalovirus infection after solid-organ transplantation, its risk factors, direct and indirect effects and prevention strategies. Orv Hetil 2008;149: 551–8. 21. Khoury JA, Storch GA, Bohl DL, Schuessler RM, Torrence SM, Lockwood M, et al. Prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients. Am J Transplant 2006;6:2134–43. 22. Hodson EM, Craig JC, Strippoli GF, Webster AC. Antiviral medications for preventing cytomegalovirus disease in solid organ transplant recipients. Cochrane Database Syst Rev 2008;16:CD003774. 23. Fishman JA, Emery V, Freeman R, Pascual M, Rostaing L, Schlitt HJ, et al. Cytomegalovirus in transplantation—challenging the status quo. Clin Transplant 2007;21:149–58. 24. The EBPG Expert Group on Renal Transplantation. European best practice guidelines for renal transplantation (Part I). Nephrol Dial Transplant 2000;15(Suppl. 7):71–4. 25. Brennan DC, Storch GA, Singer GG, Lee L, Rueda J, Schnitzler MA. The prevalence of human herpesvirus-7 in renal transplant recipients is unaffected by oral or intravenous ganciclovir. J Infect Dis 2000;181:1557–61. 26. Galarraga MC, Gomez E, de Ona M, Rodriguez A, Laures A, Boga JA, et al. Influence of ganciclovir prophylaxis on cytomegalovirus, human herpesvirus 6, and human herpesvirus 7 viremia in renal transplant recipients. Transplant Proc 2005;37:2124–6. 27. Humar A, Asberg A, Kumar D, Hartmann A, Moussa G, Jardine A, et al. An assessment of herpesvirus co-infection in patients with CMV disease: correlation with clinical and virologic outcomes. Am J Transplant 2009;9:374-381. 28. Kidd IM, Clark DA, Sabin CA, Andrew D, Hassan-Walker AF, Sweny P, et al. Prospective study of human betaherpesviruses after renal transplantation: association of human herpesvirus 7 and cytomegalovirus co-infection with cytomegalovirus disease and increased rejection. Transplantation 2000;15:2400–4. 29. Clark DA. Human herpesvirus 6 and human herpesvirus 7: emerging pathogens in transplant patients. Int J Hematol 2002;76(Suppl. 2):246–52. 30. Rasonable RR, Brown RA, Humar A, Covington E, Alecock E, Paya CV. Herpesvirus infections in solid organ transplant patients at high risk of primary cytomegalovirus disease. J Infect Dis 2005;15:1331–9. 31. OnoY, Ito Y, Kaneko K, Shibata-Watanabe Y, Tainaka T, Sumida W, et al. Simultaneous monitoring by real-time polymerase chain reaction of Epstein–Barr virus, human cytomegalovirus, and human herpesvirus-6 in juvenile and adult liver transplant recipients. Transplant Proc 2008;40:3578–82.
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Association of HHV-6 and HHV-7 reactivation with the development of chronic allograft nephropathy S. Chapenko a,∗ , I. Folkmane a,b , I. Ziedina b , M. Chistyakovs a , R. Rozentals b , A. Krumina a , M. Murovska a a b
August Kirchenstein Institute of Microbiology and Virology, Riga Stradins University, Ratsupites St. 5, LV-1067 Riga, Latvia The Transplantology Laboratory, Riga Stradins University, Riga, Latvia
a r t i c l e
i n f o
Keywords: Renal transplantation Chronic allograft nephropathy Human herpesvirus-6 Human herpesvirus-7 Virus activation Acute rejection
a b s t r a c t Background: The long-term effect of HHV-6 and HHV-7 infections on chronic allograft nephropathy (CAN) development after renal transplantation is uncertain. Objectives: To determine HHV-6 and HHV-7 reactivation during the post-transplantation period and to evaluate its effect on CAN development in renal transplant patients. Study design: Eighty-one renal allograft recipients (28 with CAN, 53 with normal transplant function) were studied to determine the frequency of HHV-6 and HHV-7 reactivation during 36.4 ± 7.8 months after renal transplantation using nested PCR. HHV-6 variants were identified using restriction endonuclease analysis. Patients were monitored for the development of CAN. Results: The frequency of HHV-6 and/or HHV-7 plasma DNA was significantly higher in CAN patients (25/28, 89.3%) compared to control patients (15/50, 30.0%, p = 0.0001). CAN patients also had an increased incidence of dual active infections (20/25, 80% and 2/15, 13.3%, p = 0.007, respectively). In all 34 HHV-6 positive cases, the HHV-6B variant was identified. The presence of HHV-7 DNA in plasma preceded the presence of HHV-6 DNA. Early development of CAN and graft loss was detected only in patients with simultaneous HHV-6 and HHV-7 plasma DNA. Conclusions: Reactivation of HHV-6 and HHV-7 in renal graft recipients is a risk factor for CAN development. The presence of concurrent HHV-6 and HHV-7 DNA in the plasma is an unfavorable prognostic factor. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Chronic allograft nephropathy (CAN) is the leading cause of progressive renal failure and the most important cause of posttransplantation graft loss in renal transplant recipients.1 CMV disease and acute rejection (AR) episodes are suggested as risk factors for the development of CAN.2–5 Human herpesvirus-6 and -7 (HHV-6 and -7) belong to the -herpesviruses family, together with cytomegalovirus (CMV).6 Both viruses are ubiquitous with a high seroprevalence in adults. After primary infection, these viruses establish a latent or persistent infection that remains for the lifetime of the host. The viruses are lymphotropic, but HHV-6 in particular, which uses the CD46 molecule as its receptor, may also infect other cell types, such as monocytes, and epithelial and endothelial cells.7 HHV-6 isolates are classified into two variants (termed HHV-6A
Abbreviations: RT, renal transplantation; CAN, chronic allograft nephropathy; AR, acute rejection; ATG, antithymocyte globulin. ∗ Corresponding author. Tel.: +371 67427920. E-mail address: [email protected] (S. Chapenko). 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.05.018
and HHV-6B) on the basis of distinct genetic, antigenic and biological characteristics. The specific pathogenicity of each variant remains poorly understood.8,9 Most infections in renal transplant recipients are due to HHV-6B. HHV-7 uses the CD4 molecule as its receptor and is more strictly lymphotropic.10 The stimulus for reactivation of beta-herpesviruses in renal transplant recipients is an immunosuppressive regimen. In addition, these viruses possess immunomodulating properties, including the ability to alter the expression of immune activation molecules, modulate expression of several cytokines and chemokines and induce apoptosis in lymphocytes, which may contribute to immunosuppression. Productive infection of CD4 T cells results in cytopathic effects and cell destruction.11 The ubiquitous nature of HHV-6 and HHV-7 creates conditions for the development of concurrent infection and interaction between these viruses. The kinetics of the activation of these viruses during the post-transplantation period suggest that HHV-7 may act as a co-factor for HHV6 and CMV reactivation, while both HHV-6 and HHV-7 may act as co-factors in the pathogenesis of CMV disease and acute rejection.12–16 The aim of this study was to determine HHV-6 and HHV-7 activation during the post-transplantation period and to evaluate the
30
S. Chapenko et al. / Journal of Clinical Virology 46 (2009) 29–32
Table 1 Demographic data of patients with CAN (group I) and control group patients (group II). Parameter
Group I (n = 28)
Group II (n = 53)
Age (years) Sex (F/M)
43.4 ± 10.7 18/10
42.0 ± 11.3 32/21
Primary renal disease (n) Glomerular Diabetes mellitus Tubulointerstitial Polycystic kidney disease Other
13 4 5 3 3
32 3 9 5 4
Donor age (years) Total HLA mismatch Cold ischemia time Acute rejection episodes CMV diseases
44.3 ± 11.2 4.2 ± 1.3 17.8 ± 3.4 14/28 5/28
43.2 ± 12.0 4.0 ± 1.7 16.4 ± 2.7 11/53 4/53
CAN: chronic allograft nephropathy.
possible effects of the activation of these viruses on CAN development in renal transplant patients.
2. Methods Eighty-one adult first-time renal allograft recipients, who were transplanted during 2002–2003 in the Transplantation Centre of the Stradins University Hospital of Riga, were enrolled in this study. All patients had received a cadaveric renal transplant. The patients were divided into two groups according to the graft function: with CAN (28/81) and with normal transplant function (53/81, control group). Demographic data of the patients is presented in Table 1. Initially, all patients had received an intravenous injection of methylprednisolone (up to 500 mg on the day of the transplantation), which was tapered gradually within the first four postoperative days. Maintenance therapy consisted of oral cyclosporine A, oral steroids (prednisolone), and mycophenolate mofetil in a fixed dosage of 0.75–1 g. Twentysix out of 28 patients with CAN and 50 out of 53 patients had received induction with monoclonal IL2R antibodies (basiliximab or daclizumab). AR episodes were diagnosed on the basis of a rise in serum creatinine (>25% from baseline), clinical evidence, and graft biopsy. Histological examination and classification of a core biopsy was done according to the Banff-97 scheme. AR episodes were treated with methylprednisolone. Steroid-resistant AR episodes (two recipients from the control group and three recipients with CAN) were subsequently treated with antithymocyte globulin (ATG) (4 mg/kg/day for 10–14 days). All patients with high (donor [D]+/recipient [R]−) and moderate (D+/R+, D−/R+) risk for CMV disease, as well as patients with ATG therapy, had received ganciclovir (GCV) 5 mg/kg/day orally for 3 months or intravenously for 2 weeks. Intravenous GCV has been used to treat the patients with CMV disease (5 mg/kg twice per day for 2 weeks). GCV dose was adjusted to renal function and to viral (CMV) infection activity. CAN was characterized by progressive loss of renal function, commonly associated with proteinuria and hypertension. Characteristic histopathologic features included interstitial fibrosis, tubular atrophy, transplant arteriopathy and chronic transplant glomerulopathy. EDTA blood samples were collected from the patients before transplantation and at 7–14 days, 3, 6, 9, 12 weeks and 6, 9, 12 18, 24, 30, 36 and 42 months after transplantation. Activation of HHV-6 and HHV-7, AR, and CAN development were monitored.
2.1. Nested polymerase chain reaction (nPCR) Nested polymerase chain reaction (nPCR) was used for the detection of viral sequences in DNA isolated from peripheral blood leukocytes (PBL) and plasma (markers of latent/persistent and active infection, respectively). Total DNA was isolated from 0.5 ml of fresh whole blood by phenol–chloroform extraction. For DNA purification from 200 l of cell free blood plasma, QIAamp DNA Blood Kit (Qiagen) was used. The plasma samples were treated with deoxyribonuclease I before DNA purification. To assure the quality of the PBL DNA, as well as exclude contamination of plasma DNA by cellular DNA, a globin PCR was performed. PCR amplification for the viruses was carried out in the presence of 1 g of PBL DNA and 10 l of plasma DNA (corresponding to 100 l of plasma). The detection of HHV-6, HHV-7 and CMV DNA was performed according to Secchiero et al.,17 Berneman et al.,6 and Studahl et al.,18 respectively. Positive (virus genomic DNA) and negative (DNA without virus-specific sequences) and water controls were included in each experiment. 2.2. Restriction endonuclease analysis Restriction endonuclease analysis was carried out using enzyme HindIII17 . This enzyme cuts HHV-6B 163 kbp amplimer into two fragments: 66 kbp and 97 kbp and does not cut HHV-6A amplimer. This difference shows the distinction between HHV-6A and HHV-6B virus variants. 2.3. Statistical method Statistical difference in the prevalence of latent/persistent and active CMV, HHV-6 and HHV-7 infection between tested groups was assessed by Fisher’s exact test. 3. Results 3.1. Clinical data Fifty-three out of 81 (65.4%) renal allograft recipients had good graft function, but in 28 (34.5%) recipients, CAN were diagnosed for a mean follow-up period of 36.4 ± 7.8 months after renal transplantation (RT). No significant difference in demographic data between the patients’ groups was detected (Table 1). 3.2. Prevalence of beta-herpesvirus DNA in PBLs As expected, beta-herpesvirus genomic sequences were found in all PBL DNA samples from CAN patients (28 out of 28), and in 50 out of 53 (94.3%) samples from control group patients without CAN before RT (Table 2). A significantly higher rate of dual (HHV-6, HHV-7) and triple (CMV, HHV-6, HHV-7) infections was found in CAN patients in comparison with control group patients (p = 0.002 and 0.0003, respectively). The rate of dual CMV and HHV-7 infection was significantly higher in the control group patients compared to the patients with CAN (p = 0.042). Single CMV and HHV-6 infections, as well as dual CMV and HHV-6 infections, were found only in the control group patients and not in the CAN group. In all 34 PBL DNA samples from the patients with HHV-6 infection, HHV-6B was the variant identified. No beta-herpesvirus genomic sequences were detected in plasma DNA samples from the patients before RT. 3.3. Prevalence of active beta-herpesvirus infection The frequency of plasma viremia was significantly higher in CAN patients (25/28, 89.3%) in comparison with the control group patients (15/50, 30.0%, p = 0.0001) after RT (Table 2). The frequency of simultaneous activation of HHV-6 and HHV-7 was significantly
S. Chapenko et al. / Journal of Clinical Virology 46 (2009) 29–32
31
Table 2 Prevalence of beta-herpesviruses DNA in patients with CAN and control group patients before and after RT by nPCR in PBMCs and in plasma. Viral infection
PBMCs before RT
Plasma after RT
CAN (n = 28)
Control group (n = 53)
Single CMV Single HHV-6 Single HHV-7 Dual CMV + HHV-6 Dual CMV + HHV-7 Dual HHV-6 + HHV-7 Triple CMV + HHV-6 + HHV-7 CMV+ HHV-6+ HHV-7+ Negative for all Positive for 1 or more
0/28 0/28 3/28 0/28 2/28 11/28 12/28
3/53 1/53 21/53 2/53 15/53 4/53 4/53
0/28 28/28
3/53 50/53
p-Value
0.009 0.042 0.002 0.0003
CAN (n = 25)
Control group (n = 15)
0/25 0/25 0/25 0/25 0/25 15/25 5/25 0 3 2 3/28 25/28
0/15 1/15 5/15 0/15 7/15 2/15 0/15
38/53 15/50
p-value
0.007
0.0001
RT: renal transplantation; CAN: chronic allograft nephropathy.
higher in the CAN patients compared to the control group patients (p = 0.007). No significant difference in the frequency of CMV activation between patients with CAN and control group patients was revealed (5/14 and 7/24, respectively). All 25 CAN patients with viremia also had concurrent HHV-6 and HHV-7 or triple detection of herpesvirus DNA in the PBLs. Simultaneous activation of HHV6 and HHV-7 was found in 15 of them, and triple infections—in 5 patients. Single infections of CMV, HHV-6, and HHV-7 were 0, 3 and 2 patients, respectively. In the control group, only 25% (2/8) of the patients with concurrent HHV-6 and HHV-7 and triple infections in the PBLs had DNA in the plasma (Table 2). Simultaneous presence of CMV and HHV-7 DNA was more commonly found in the plasma of controls than in patients with CAN (Table 2). These patients had dual CMV and HHV-7 infections in PBLs. Only HHV-6 variant B was detected in the 23 positive plasma samples. The mean number of days before detecting HHV-7, HHV-6 and CMV reactivation was 12 days (range 7–23 days), 38 days (range 14–103 days), and 63 days (range 42–112 days), respectively. CMV disease was diagnosed in 5 patients with CAN and simultaneous CMV, HHV-6 and HHV-7 active infection and in 4 control group patients with simultaneous CMV and HHV-7 active infection. All patients had received intravenous GCV. 3.4. Acute rejection and HHV-6 and HHV-7 infection AR episodes were diagnosed in 25 out of 81 (30.9%) recipients. The frequency of AR episodes was significantly higher in CAN patients (14/28, 50.0%) in comparison with control group patients (11/53, 20.8%; p = 0.002). The mean time until AR episodes was 127 ± 53 days in patients with active viral infections and 113 ± 42 days in patients with latent infections. 3.5. CAN, acute rejection and activation of viral infections The mean time until the onset of clinical manifestations of CAN was 242 ± 63 days. This period had tendency to shorten in recipients with AR episodes and simultaneous activation of both HHV-6 and HHV-7 (163 ± 41 days) compared to recipients with AR episodes and activation of HHV-6 only (171 ± 52) or HHV-7 only (185 ± 31 days). Further statistical analysis was not performed because the case size was too small. Out of 28 recipients with CAN, 9 grafts were rejected. All 9 recipients had AR episodes and simultaneous HHV-6 and HHV-7 activation. 4. Discussion Chronic allograft nephropathy is an important cause of posttransplantation graft loss in the recipients of renal allografts. A large number of new effective immunosuppressive drugs are available
and antiviral therapy effectively prevents acute rejection and CMV disease.19–22 However, standards of the dose for prophylaxis and the optimal duration of antiviral prophylaxis or preemptive therapy to prevent the infection have not been established.23 In our experience, a low dose of GCV regimen (5 mg/kg/day for 2 weeks or orally for 3 months) has been used according to recommendations by the EBPG Expert Group on Renal Transplantation.24 Antiviral therapy also can inhibit reactivation of other herpesviruses, and maximize short-term graft survival in kidney transplant recipients. Ganciclovir is shown to be more effective in inhibiting CMV than HHV-6 or HHV-7.25–27 HHV-6 is reported as significant factor in CAN development2 ; however, the importance of HHV-7 infection in the pathogenesis of CAN is still unknown. Our data shows that latent/persistent HHV-6 and HHV-7 infection is widely prevalent in renal transplant recipients (92.6%) before renal transplantation, thus confirming the ubiquity of these viruses. We found a significantly higher prevalence of dual (HHV-6 and HHV-7) DNA by nPCR in PBL in CAN patients. Only the HHV-6B variant was detected in PBL and plasma DNA samples from the patients with HHV-6 infection, indicating that either the HHV-6A variant occurs infrequently in these patients or it may be limited to sites other than the peripheral blood. The frequency of simultaneous activation of HHV-6 and HHV7 was significantly higher in CAN patients, suggesting a possible involvement for HHV-6 and HHV-7 in the development of the diseases. In the CAN and control groups, patients with dual HHV-6 and HHV-7 and triple CMV, HHV-6 and HHV-7 infection had activation of HHV-7 before HHV-6 and CMV activation, confirming our previous reports.13,14 The development of CMV disease in the patients with CMV and HHV-7 and CMV, HHV-6 and HHV-7 infection suggests that HHV-6 and HHV-7 may be co-factors of this disease in consequence of a synergistic effect. The association of HHV-6 and HHV-7 infection with an increased risk for development of CMV disease in the renal transplant patients is reported previously.28–30 We did not observe CAN in patients with single CMV reactivation. We did not detect a significant difference in the frequency of CMV activation and CMV disease between patients with CAN and the control group patients. This finding may be due to the antiviral treatment. We did not detect an association between activation of these viral infections and AR detected that agree with the data of Ono et al.31 However, the data showing a significantly higher AR rate in CAN patients suggests that AR is an independent risk factor for CAN. The tendency to shorten the period of CAN development and loss of grafts in renal transplant patients with simultaneous activation of HHV-6 and HHV-7 suggest that both viruses may be involved in the CAN development via their immunomodulatory ability.
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Acknowledgements This work was supported in part by the National Research Program in Medicine “Multi-Disciplinary Research Consortium on Major Pathologies Threatening the Life Expectancy and Quality of Life of the Latvian Population” project no. 11 and grant “Role of blood-borne viruses infection in the development of posttransplant complications.” References 1. Pascual M, Theruvath T, Kawai T, Tolkoff-Rubin N, Cosimi AB. Strategies to improve long-term outcomes after renal transplantation. N Engl J Med 2002;346:580–90. 2. Tong CY, Barkan A, Peiris JC, Muir P, Herrington CS. The association of viral infection and chronic allograft nephropathy with graft dysfunction after renal transplantation. Transplantation 2002;74:576–8. 3. Sagedal S, Hartmann A, Nordal KP, Osnes K, Leivestad T, Foss A, et al. Impact of early cytomegalovirus infection and disease on long-term recipient and kidney graft survival. Kidney Int 2004;66:329–37. 4. Nett PC, Heisey DM, Fernandez LA, Sollinger HW, Pirsch JD. Association of cytomegalovirus disease and acute rejection with graft loss in kidney transplantation. Transplantation 2004;78:1036–41. 5. Boratynska M, Banasik M, Watorek E, Patrzatek D, Szyber P, Klinger M. Influence of cytomegalovirus disease on early and late renal graft function. Transplant Proc 2006;38:147–50. 6. Berneman ZN, Ablashi DV, Li G, Eger-Fletcher M, Reitz MS, Hung C-L, et al. Human herpesvirus 7 is a T-lymphotropic virus and is related to, but significantly different from human herpesvirus 6 and human cytomegalovirus. Proc Natl Acad Sci USA 1992;89:10552–6. 7. Santoro F, Kennedy PE, Locatelli G, Malnati MS, Berger EA, Lusso P. CD46 is a cellular receptor for human herpesvirus 6. Cell 1999;99:817–27. 8. Ablashi DV, Blahandran N, Josephs SF, Hung CL, Krueger GRF, Kramarsky B, et al. Genomic polymorphism, growth properties, and immunologic variations in human herpesvirus-6 isolates. Virology 1991;184:545–52. 9. Boutelleau D, Duros C, Bonnafous P, Caiola D, Karras A, Castro ND, et al. Identification of human herpesvirus 6 variants A and B by primer-specific realtime PCR may help to revisit their respective role in pathology. J Clin Virol 2006;35:257–63. 10. Black JB, Pellet PE. Human herpesvirus 7. Rev Med Virol 1999;9:245–62. 11. Lusso P. HHV-6 and the immunosystem: mechanisms of immunomodulation and viral escape. J Clin Virol 2006;37(Suppl. 1):4–10. 12. Osman HKE, Peiris JSM, Taylor CE, Warwicker P, Jarret RF, Madeley CR. Cytomegalovirus disease in renal allograft recipients: is human herpesvirus-7 a cofactor for disease progression? J Med Virol 1996;48:295–301. 13. Chapenko S, Folkmane I, Tomsone V, Amerika D, Rozentals R, Murovska M. Coinfection of two -herpesviruses (CMV and HHV-7) as an increased risk factor for “CMV disease” in patients undergoing renal transplantation. Clin Transplant 2000;14:486–92. 14. Chapenko S, Folkmane I, Tomsone V, Kozireva S, Bicans J, Amerika D, et al. Infection of -herpesviruses (CMV, HHV-6, HHV-7): role in postrenal transplantation complication. Transplant Proc 2001;33:2463–4. 15. Dockrell DH, Paya CV. Human herpesvirus-6 and -7 in transplantation. Rev Med Virol 2001;11:23–36.
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