- Email: [email protected]
CMV infection is usually associated with concurrent HHV-6 and HHV-7 antigenemia in liver transplant patients
Journal of Clinical Virology 25 (2002) S57 /S61 www.elsevier.com/locate/jcv
CMV infection is usually associated with concurrent HHV-6 and HHV-7 antigenemia in liver transplant patients I. Lautenschlager a,b,*, M. Lappalainen a, K. Linnavuori a, J. Suni a, K. Ho¨ckerstedt b a b
Department of Virology, Helsinki University Hospital and University of Helsinki, Haartmaninkatu 3, FIN-00290 Helsinki, Finland Department of Surgery, Transplantation and Liver Surgery Unit, Helsinki University Hospital and University of Helsinki, Kasarmikatu 11-13, FIN-00130 Helsinki, Finland
Abstract Human herpesvirus 6 and 7 (HHV-6, HHV-7) have been recently reported in liver transplant patients. HHV-6 may cause fever, neurological disorders and hepatitis. The clinical significance of HHV-7 is less clear. HHV-6 and -7 are closely related to cytomegalovirus (CMV), and interactions between the viruses have also been suggested. In this study, we investigated the post transplant HHV-6 and -7 antigenemia was in relation to symptomatic CMV disease after liver transplantation. Consecutive 34 adult liver allograft recipients transplanted during 1999 /2000 were included in the study. CMV infections were diagnosed by the frequent monitoring of pp65-antigenemia and by viral cultures. HHV-6 and -7 were demonstrated, by using immunoperoxidase staining and monoclonal antibodies against the virus specific antigens, in the mononuclear cells from the same blood specimens which were obtained for CMV pp65 monitoring. Altogether 322 blood specimens were analyzed. CMV disease was diagnosed in 12 (35%) patients during the first 3 months (first pp65 positive specimen mean 25 days, range 8 /61 days) after transplantation. Concurrent HHV-6 antigenemia was detected in 10/12 (mean 14 days, range 6 /22 days) and HHV-7 antigenemia in 9/12 patients (mean 25 days, range 10 /89 days) after transplantation. HHV-6 usually appeared slightly before CMV. All CMV infections were successfully treated with ganciclovir and the CMV /antigenemia subsided. HHV-6 and -7 antigenemia also responded to the antiviral treatment, but more slowly than CMV. In conclusion, CMV infection was usually associated with HHV6 and -7 antigenemia in liver transplant patients. The results support the suggestion that CMV, HHV-6 and -7 may have interactions. The clinical symptoms of CMV infection, may also be linked with HHV-6 or -7. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytomegalovirus; Human herpesvirus 6; Human herpesvirus 7; Liver transplantation
1. Introduction
* Corresponding author. Tel.: /358-9-47188484; fax: /3589-47188348 E-mail address: [email protected] (I. Lautenschlager).
Human herpesvirus 6 and 7 (HHV-6 and -7) belong to the b-herpesvirus family, together with cytomegalovirus (CMV). HHV-6 and -7 are known to cause febrile infectious syndromes of early childhood, known as exanthema subitum
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and roseola (Yamanishi et al., 1988; Tanaka et al., 1994). Both viruses, are common with a high seroprevalence in the adult population. HHV-6 and -7 are lymphotropic viruses but HHV-6 in particular, which uses the CD46 molecule as its receptor, may also infect other cell types, such as monocytes, epithelial and endothelial cells (Lusso, 1996; Santoro et al., 1999). HHV-7 uses the CD4 molecule as its receptor and is more strictly lymphotropic (Black and Pellet, 1999). Like other herpesviruses, HHV-6 and -7 can establish a latent infection for the lifetime of the host and can reactivate during immunosuppression. Although, it is known that CMV has a major impact on the outcome of transplant patients, the roles of other two members of the b-herpesvirus family have been acknowledged only during the last few years. HHV-6 infection has been recently described in liver transplant patients (Herbein et al., 1996; Singh et al., 1997; Lautenschlager et al., 1998). HHV-6 may cause fever and other clinical symptoms, such as neurological disorders and hepatitis (Lusso, 1996; Singh, 2000). We have previously demonstrated that HHV-6 may also infect the liver allograft and cause graft dysfunction (Lautenschlager et al., 1998). Some association between graft rejection and HHV-6 infection have also been recorded (Lautenschlager et al., 1998; Griffiths et al., 1999). HHV-7 has been found, together with HHV-6 and CMV, in transplant patients (Griffiths et al., 1999; Dockrell and Paya, 2001; Emery, 2001), but its clinical significance is less clear. HHV-6 and -7 are closely related to CMV with large genomic overlapping, and some interactions between CMV and HHV-6, as well as HHV-7, have also been suggested (Osman et al., 1996; Tong et al., 2000; Mendez et al., 2001). In several series of patients we, and others also have recorded an overlapping activation of the b-herpesviruses after liver transplantation (Dockrell et al., 1997; Humar et al., 2000; Lautenschlager et al., 2000a). In this study, we have investigated the post transplant HHV-6 and -7 antigenemia in relation to symptomatic CMV infections in adult liver transplant patients.
2. Patients and methods Thirty-four consecutive adult liver allograft recipients who were transplanted during 1999 / 2000 were included in the study. As basic immunosuppression, the patients received triple drug therapy of various combinations of steroids (methylpredinisolone 2 mg/kg/d at day one rapidly tapering to 0.25 mg/kg/d), azathioprine (2 /1 mg/ kg/d) and cyclosporin or tacrolimus (according to trough level), and high doses of methylprednisolone (3 mg/kg/d for 5 days) were used as antirejection treatment. Standard biopsy histology was performed to diagnose allograft rejection. Most patients (30/34, 88%) were CMV-seropositive. No routine antiviral prophylaxis was given, besides ganciclovir prophylaxis (10 mg/kg/d for 5 /10 days i.v.) when acute rejection occurred. To monitor the graft function, the following serial liver function tests were performed: serum alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase and bilirubin. CMV infections were diagnosed by the frequent monitoring of pp65-antigenemia (The et al., 1990) and by viral cultures. Peripheral blood samples for detection of CMV /antigenemia were obtained weekly during the patients’ hospital stay, and thereafter once in 1 /2 weeks up to 3 months after transplantation and in any case of suspected viral infection. EDTA-blood specimens of 10 ml were divided in two proportions and 5 ml was used for the CMV pp65 antigenemia test. CMV pp65 positive peripheral blood leukocytes were demonstrated in the cytocentrifuge preparations by immunoperoxidase staining and by a monoclonal antibody (Biotest Ag, Dreieich, Germany). The standard pp65 antigenemia test was used (The et al., 1995). The other 5 ml of each blood specimen was used to monitor HHV-6 and -7 antigenemia. Altogether 322 blood specimens were obtained. HHV-6 infections were demonstrated by the HHV-6 antigenemia test, which detects the virus specific antigens in blood mononuclear cells. The technical details of the immunohistochemical method have been described previously (Lautenschlager et al., 1998, 2000b). First, the peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque density gradient centrifugation and
I. Lautenschlager et al. / Journal of Clinical Virology 25 (2002) S57 /S61
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cytocentrifuged onto microscope slides. The presence of viral antigens in the cytopreparations was demonstrated by indirect immunoperoxidase staining and by monoclonal antibodies (MAB8533 and MAB8535, Chemicon Inc. Temecula, CA) against an early HHV-6 specific antigen and an HHV-6 variant B virion protein, as described previously (Lautenschlager et al., 2000b). The specificity of the antibodies has been described by Chemicon. Normal mouse-IgG was used as a negative control for non-specific binding. A peroxidase-conjugated rabbit anti-mouse (Dako, Copenhagen, Denmark) and a peroxidase-conjugated goat anti-rabbit antibody (Zymed, San Francisco, CA) were used as second and third antibodies. The reaction was revealed by 3-amino-9-ethyl carbazole solution containing hydrogen peroxide and Mayer’s haemalum was used for counterstaining. Concomitantly, HHV-7 specific antigens were demonstrated in the cytocentrifuge preparations of PBMC by using immunoperoxidase staining with two monoclonal antibodies (Biodesign International, Saco, ME) to detect the early and late (gp110/160) antigens of HHV-7, respectively. No crossreactivity between the antibodies or non-specific staining were recorded. HHV-6 serology was performed in parallel. The HHV-6 antibody assay was performed by means of an indirect immunofluorescence test using HHV-6 infected HSB-2 cells (a continuous immature T-lymphoblastoid cell line) as antigens, as described in detail previously (Linnavuori et al., 1992).
intravenous ganciclovir, and the CMV antigenemia subsided within 2 weeks. Concurrent HHV-6 antigenemia was detected in 10/12 patients with CMV infection (mean 14 days, range 6 /22 days) post transplantation. The positive staining of PBMCs was recorded in lymphocytes but some monocytes also demonstrated HHV-6 antigens. The diagnosis of HHV-6 infection was confirmed by serology. HHV-6 antigenemia usually appeared slightly before CMV antigenemia. Symptoms, such as fever or graft dysfunction, could have been partly due to HHV-6 infection, too. In four cases, HHV-6 preceding CMV was more likely to have been the cause of graft dysfunction. In two additional patients, HHV-6 antigenemia was recorded without CMV infections and these also had graft dysfunction. HHV-7 antigenemia was recorded in 9/12 patients concomitantly with CMV disease (mean 25 days, range 10/89 days) after transplantation. The positive staining of PBMC was recorded in lymphocytes only. HHV-7 antigenemia usually appeared together with CMV antigenemia and could not be linked with any clinical symptoms other than CMV. In all three patients with primary CMV infection, both HHV-6 and -7 antigenemia appeared before CMV. HHV-6 and -7 antigenemia also responded to ganciclovir given to treat CMV infection, but the effect was less clear and slower than that of CMV. HHV-6 antigenemia usually lasted for several weeks, though HHV-7 antigenemia was shorter. However, the clinical signs of infection during the CMV episode could have been partly due to either HHV-6 or -7 or both.
3. Results
4. Discussion
CMV disease was diagnosed in 12 (35%) out of 34 patients during the first months (first pp65 positive specimen, mean 26 days, range 8/61 days) after transplantation. Most CMV infections (9/12, 75%) were reactivations/reinfections and only three were primary infections. The symptoms consisted of fever and graft dysfunction. Graft dysfunction was recorded in 10/12 patients. All CMV infections were successfully treated with
These results demonstrate that CMV infection is usually associated with HHV-6 and -7 antigenemia in liver transplant patients. In most cases HHV-6 activation was recorded prior to CMV disease but HHV-7 antigenemia usually appeared more or less concurrently with CMV. In all primary CMV infections, both HHV-6 and -7 preceded CMV. It has been reported by others that HHV-6 seroconversion is a risk factor predisposing the patient to
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severe CMV infection (Dockrell et al., 1997). Similar findings concern also the third b-herpesvirus, HHV-7, which is also suggested to be a cofactor for CMV disease progression (Osman et al., 1996). Interaction between the two b-herpesviruses was previously recorded in a case of primary CMV infection and HHV-6 reactivation (DesJardin et al., 1998). However, HHV-6 infection has also been found to be associated with CMV reactivations (Humar et al., 2000). These observations are in agreement with our results concerning both primary CMV infections and CMV reactivations. On the other hand, all herpesviruses tend to reactivate in immunosuppressed patients, and primary infections are known to be severe. The concurrent activation of CMV, HHV-6 and -7 may thus be just a consequence of the immunological condition of the transplant patients. Thus the three b-herpesvirus infections may appear concomitantly by chance and not necessarily due to their interactions. The concurrent appearance of the three b-herpesviruses in solid organ transplant patients has recently been reported by others (Griffiths et al., 1999; Dockrell and Paya, 2001; Mendez et al., 2001). The symptoms associated with CMV disease, such as fever and liver dysfunction or even hepatitis may also be linked with the other bherpesviruses, at least with HHV-6. However, in this patient population, only in four patients, the symptoms seemed to be associated with HHV-6 preceding CMV. We and others have reported the pathogenicity of HHV-6 in liver transplantation. It may infect the graft, and cause liver dysfunction, and possibly trigger acute and chronic rejection (Lautenschlager et al., 1998). HHV-6 may also infect epithelial and endothelial cells, which facilitates its involvement in development of transplant vasculopathy, also known as chronic rejection. Whether the involvement of HHV-6 in the rejection processes is similar to that of CMV (Lautenschlager, 1998), remains still unclear. HHV-7 may also be responsible for the clinical symptoms but its ability to act as a co-factor for the other bherpesviruses might be more important. Thus, the indirect effects of all three b-herpesviruses are possible and further studies are required to establish it.
In conclusion, HHV-6 and -7 antigenemia were usually associated with CMV disease in liver transplant patients. The results support the suggestion that CMV, HHV-6 and -7 may have interactions. The clinical signs of CMV infection, such as fever and graft dysfunction, may also be linked with HHV-6 or -7. Further studies are needed to investigate the direct and indirect post transplantation effects of HHV-6 and -7.
Acknowledgements This work was supported by grants from the Sigrid Juselius Foundation (to KH and IL) and Helsinki University Hospital Research Funds (EVO) (to IL). The authors wish to thank Raisa Loginov, Marjatta Palovaara and Sinikka SuurNuuja for technical assistance and Stephen Venn for correcting the English text.
References Black JB, Pellet PE. Human herpesvirus 7. Rev Med Virol 1999;9:245 /62. DesJardin JA, Gibbon L, Cho E, Supran SE, Falagas ME, Werner BG, et al. Human herpesvirus 6 reactivation is associated with cytomegalovirus infection and syndromes in kidney transplant recipients at risk for primary cytomegalovirus infection. J Infect Dis 1998;178:1783 /6. Dockrell DH, Prada J, Jones MF, Patel R, Badley AD, Harmsen WS, et al. Seroconversion to human herpesvirus 6 following liver transplantation is a marker of cytomegalovirus disease. J Infect Dis 1997;176:1135 /40. Dockrell DH, Paya CV. Human herpesvirus-6 and -7 in transplantation. Rev Med Virol 2001;11:23 /36. Emery VC. Human herpesviruses 6 and 7 in solid organ transplant recipients. Clin Infect Dis 2001;32:1357 /60. Griffiths PD, Ait-Khaled M, Bearcroft CP, Clark DA, Quagila A, Davies SE, et al. Human herpesviruses 6 and 7 as potential pathogens after liver transplant: prospective comparison with the effect of cytomegalovirus. J Med Virol 1999;59:496 /501. Herbein G, Strasswimmer J, Altieri M, Woehl-Jaegle ML, Obert G. Longitudinal study of human herpesvirus 6 infection in organ transplant recipients. Clin Infect Dis 1996;22:171 /3. Humar A, Malkan G, Moussa G, Greig P, Levy G, Mazzulli T. Human herpesvirus-6 is associated with cytomegalovirus reactivation in liver transplant recipients. J Infect Dis 2000;181:1450 /3.
I. Lautenschlager et al. / Journal of Clinical Virology 25 (2002) S57 /S61 Lautenschlager I. Role of cytomegalovirus infection in the processes of organ allograft rejection: clinical studies and experimental models. In: Scholz M, Rabenau HF, Doerr HW, Cinatl J, editors. CMV-related immunopathology. Monographs in virology. Basel: Karger AG, 1998:142 /57. Lautenschlager I, Ho¨ckerstedt K, Linnavuori K, Taskinen E. Human herpesvirus-6 infection after liver transplantation. Clin Infect Dis 1998;26:702 /7. Lautenschlager I, Linnavuori K, Lappalainen M, Suni J, Ho¨ckerstedt K. HHV-6 reactivation is often associated with CMV infection in liver transplant patients. Transplant Int 2000a;13:S351 /3. Lautenschlager I, Linnavuori K, Taskinen E, Ho¨ckerstedt K. Human herpesvirus-6 antigenemia after liver transplantation. Transplantation 2000b;69:2561 /6. Linnavuori K, Peltola H, Hovi T. Serology versus clinical signs or symptoms and main laboratory findings in the diagnosis of exanthema subitum infantum. Pediatrics 1992;89:103 /6. Lusso P. Human herpesvirus 6 (HHV-6). Antivir Res 1996;31:1 /21. Mendez JC, Dockrell DH, Espy MJ, Smith TF, Wilson JA, Harmsen WS, et al. Human b-herpesvirus interactions in solid organ transplant recipients. J Infect Dis 2001;183:179 / 84. Osman HKE, Peiris JSM, Taylor CE, Warwicker P, Jarret RF, Madeley CR. Cytomegalovirus disease in renal allograft
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recipients: is human herpesvirus-7 a cofactor for disease progression? J Med Virol 1996;48:295 /301. 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. Singh N, Carrigan DR, Gayowski T, Marino IR. Human herpesvirus-6 infection in liver transplant recipients. Documentation of pathogenicity. Transplantation 1997;64:674 / 8. Singh N. Human herpesviruses-6, -7 and -8 in organ transplant recipients. Clin Microbiol Infect 2000;6:453 /9. Tanaka K, Kondo T, Torigoe S, Okada S, Mukai T, Yamanishi K. Human herpesvirus 7: another causal agent for roseola (exanthem subitum). J Pediatr 1994;125:1 /5. The TH, van der Bij W, van der Berg AP, van der Giessen M, Weits J, Sprenger HG, et al. Cytomegalovirus antigenemia. Rev Infect Dis 1990;12:S734 /44. The TH, van der Berg AP, Harmsen MC, van der Bij W, van Son WJ. The cytomegalovirus antigenemia assay: a plea for standardization. Scand J Infect Dis 1995;99(Suppl):25 /9. Tong CY, Bakran A, Williams H, Cheung CY, Peiris JS. Association of human herpesvirus 7 with cytomegalovirus disease in renal transplant recipients. Transplantation 2000;70:213 /6. Yamanishi K, Okuno T, Shiraki K, Takahashi M, Kondo T, Asanao Y, et al. Identification of human herpesvirus-6 as causal agent for eaxanthem subitum. Lancet 1988;I:1065 /7.
CMV infection is usually associated with concurrent HHV-6 and HHV-7 antigenemia in liver transplant patients I. Lautenschlager a,b,*, M. Lappalainen a, K. Linnavuori a, J. Suni a, K. Ho¨ckerstedt b a b
Department of Virology, Helsinki University Hospital and University of Helsinki, Haartmaninkatu 3, FIN-00290 Helsinki, Finland Department of Surgery, Transplantation and Liver Surgery Unit, Helsinki University Hospital and University of Helsinki, Kasarmikatu 11-13, FIN-00130 Helsinki, Finland
Abstract Human herpesvirus 6 and 7 (HHV-6, HHV-7) have been recently reported in liver transplant patients. HHV-6 may cause fever, neurological disorders and hepatitis. The clinical significance of HHV-7 is less clear. HHV-6 and -7 are closely related to cytomegalovirus (CMV), and interactions between the viruses have also been suggested. In this study, we investigated the post transplant HHV-6 and -7 antigenemia was in relation to symptomatic CMV disease after liver transplantation. Consecutive 34 adult liver allograft recipients transplanted during 1999 /2000 were included in the study. CMV infections were diagnosed by the frequent monitoring of pp65-antigenemia and by viral cultures. HHV-6 and -7 were demonstrated, by using immunoperoxidase staining and monoclonal antibodies against the virus specific antigens, in the mononuclear cells from the same blood specimens which were obtained for CMV pp65 monitoring. Altogether 322 blood specimens were analyzed. CMV disease was diagnosed in 12 (35%) patients during the first 3 months (first pp65 positive specimen mean 25 days, range 8 /61 days) after transplantation. Concurrent HHV-6 antigenemia was detected in 10/12 (mean 14 days, range 6 /22 days) and HHV-7 antigenemia in 9/12 patients (mean 25 days, range 10 /89 days) after transplantation. HHV-6 usually appeared slightly before CMV. All CMV infections were successfully treated with ganciclovir and the CMV /antigenemia subsided. HHV-6 and -7 antigenemia also responded to the antiviral treatment, but more slowly than CMV. In conclusion, CMV infection was usually associated with HHV6 and -7 antigenemia in liver transplant patients. The results support the suggestion that CMV, HHV-6 and -7 may have interactions. The clinical symptoms of CMV infection, may also be linked with HHV-6 or -7. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytomegalovirus; Human herpesvirus 6; Human herpesvirus 7; Liver transplantation
1. Introduction
* Corresponding author. Tel.: /358-9-47188484; fax: /3589-47188348 E-mail address: [email protected] (I. Lautenschlager).
Human herpesvirus 6 and 7 (HHV-6 and -7) belong to the b-herpesvirus family, together with cytomegalovirus (CMV). HHV-6 and -7 are known to cause febrile infectious syndromes of early childhood, known as exanthema subitum
1386-6532/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 2 ) 0 0 1 0 1 - 4
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and roseola (Yamanishi et al., 1988; Tanaka et al., 1994). Both viruses, are common with a high seroprevalence in the adult population. HHV-6 and -7 are lymphotropic viruses but HHV-6 in particular, which uses the CD46 molecule as its receptor, may also infect other cell types, such as monocytes, epithelial and endothelial cells (Lusso, 1996; Santoro et al., 1999). HHV-7 uses the CD4 molecule as its receptor and is more strictly lymphotropic (Black and Pellet, 1999). Like other herpesviruses, HHV-6 and -7 can establish a latent infection for the lifetime of the host and can reactivate during immunosuppression. Although, it is known that CMV has a major impact on the outcome of transplant patients, the roles of other two members of the b-herpesvirus family have been acknowledged only during the last few years. HHV-6 infection has been recently described in liver transplant patients (Herbein et al., 1996; Singh et al., 1997; Lautenschlager et al., 1998). HHV-6 may cause fever and other clinical symptoms, such as neurological disorders and hepatitis (Lusso, 1996; Singh, 2000). We have previously demonstrated that HHV-6 may also infect the liver allograft and cause graft dysfunction (Lautenschlager et al., 1998). Some association between graft rejection and HHV-6 infection have also been recorded (Lautenschlager et al., 1998; Griffiths et al., 1999). HHV-7 has been found, together with HHV-6 and CMV, in transplant patients (Griffiths et al., 1999; Dockrell and Paya, 2001; Emery, 2001), but its clinical significance is less clear. HHV-6 and -7 are closely related to CMV with large genomic overlapping, and some interactions between CMV and HHV-6, as well as HHV-7, have also been suggested (Osman et al., 1996; Tong et al., 2000; Mendez et al., 2001). In several series of patients we, and others also have recorded an overlapping activation of the b-herpesviruses after liver transplantation (Dockrell et al., 1997; Humar et al., 2000; Lautenschlager et al., 2000a). In this study, we have investigated the post transplant HHV-6 and -7 antigenemia in relation to symptomatic CMV infections in adult liver transplant patients.
2. Patients and methods Thirty-four consecutive adult liver allograft recipients who were transplanted during 1999 / 2000 were included in the study. As basic immunosuppression, the patients received triple drug therapy of various combinations of steroids (methylpredinisolone 2 mg/kg/d at day one rapidly tapering to 0.25 mg/kg/d), azathioprine (2 /1 mg/ kg/d) and cyclosporin or tacrolimus (according to trough level), and high doses of methylprednisolone (3 mg/kg/d for 5 days) were used as antirejection treatment. Standard biopsy histology was performed to diagnose allograft rejection. Most patients (30/34, 88%) were CMV-seropositive. No routine antiviral prophylaxis was given, besides ganciclovir prophylaxis (10 mg/kg/d for 5 /10 days i.v.) when acute rejection occurred. To monitor the graft function, the following serial liver function tests were performed: serum alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase and bilirubin. CMV infections were diagnosed by the frequent monitoring of pp65-antigenemia (The et al., 1990) and by viral cultures. Peripheral blood samples for detection of CMV /antigenemia were obtained weekly during the patients’ hospital stay, and thereafter once in 1 /2 weeks up to 3 months after transplantation and in any case of suspected viral infection. EDTA-blood specimens of 10 ml were divided in two proportions and 5 ml was used for the CMV pp65 antigenemia test. CMV pp65 positive peripheral blood leukocytes were demonstrated in the cytocentrifuge preparations by immunoperoxidase staining and by a monoclonal antibody (Biotest Ag, Dreieich, Germany). The standard pp65 antigenemia test was used (The et al., 1995). The other 5 ml of each blood specimen was used to monitor HHV-6 and -7 antigenemia. Altogether 322 blood specimens were obtained. HHV-6 infections were demonstrated by the HHV-6 antigenemia test, which detects the virus specific antigens in blood mononuclear cells. The technical details of the immunohistochemical method have been described previously (Lautenschlager et al., 1998, 2000b). First, the peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque density gradient centrifugation and
I. Lautenschlager et al. / Journal of Clinical Virology 25 (2002) S57 /S61
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cytocentrifuged onto microscope slides. The presence of viral antigens in the cytopreparations was demonstrated by indirect immunoperoxidase staining and by monoclonal antibodies (MAB8533 and MAB8535, Chemicon Inc. Temecula, CA) against an early HHV-6 specific antigen and an HHV-6 variant B virion protein, as described previously (Lautenschlager et al., 2000b). The specificity of the antibodies has been described by Chemicon. Normal mouse-IgG was used as a negative control for non-specific binding. A peroxidase-conjugated rabbit anti-mouse (Dako, Copenhagen, Denmark) and a peroxidase-conjugated goat anti-rabbit antibody (Zymed, San Francisco, CA) were used as second and third antibodies. The reaction was revealed by 3-amino-9-ethyl carbazole solution containing hydrogen peroxide and Mayer’s haemalum was used for counterstaining. Concomitantly, HHV-7 specific antigens were demonstrated in the cytocentrifuge preparations of PBMC by using immunoperoxidase staining with two monoclonal antibodies (Biodesign International, Saco, ME) to detect the early and late (gp110/160) antigens of HHV-7, respectively. No crossreactivity between the antibodies or non-specific staining were recorded. HHV-6 serology was performed in parallel. The HHV-6 antibody assay was performed by means of an indirect immunofluorescence test using HHV-6 infected HSB-2 cells (a continuous immature T-lymphoblastoid cell line) as antigens, as described in detail previously (Linnavuori et al., 1992).
intravenous ganciclovir, and the CMV antigenemia subsided within 2 weeks. Concurrent HHV-6 antigenemia was detected in 10/12 patients with CMV infection (mean 14 days, range 6 /22 days) post transplantation. The positive staining of PBMCs was recorded in lymphocytes but some monocytes also demonstrated HHV-6 antigens. The diagnosis of HHV-6 infection was confirmed by serology. HHV-6 antigenemia usually appeared slightly before CMV antigenemia. Symptoms, such as fever or graft dysfunction, could have been partly due to HHV-6 infection, too. In four cases, HHV-6 preceding CMV was more likely to have been the cause of graft dysfunction. In two additional patients, HHV-6 antigenemia was recorded without CMV infections and these also had graft dysfunction. HHV-7 antigenemia was recorded in 9/12 patients concomitantly with CMV disease (mean 25 days, range 10/89 days) after transplantation. The positive staining of PBMC was recorded in lymphocytes only. HHV-7 antigenemia usually appeared together with CMV antigenemia and could not be linked with any clinical symptoms other than CMV. In all three patients with primary CMV infection, both HHV-6 and -7 antigenemia appeared before CMV. HHV-6 and -7 antigenemia also responded to ganciclovir given to treat CMV infection, but the effect was less clear and slower than that of CMV. HHV-6 antigenemia usually lasted for several weeks, though HHV-7 antigenemia was shorter. However, the clinical signs of infection during the CMV episode could have been partly due to either HHV-6 or -7 or both.
3. Results
4. Discussion
CMV disease was diagnosed in 12 (35%) out of 34 patients during the first months (first pp65 positive specimen, mean 26 days, range 8/61 days) after transplantation. Most CMV infections (9/12, 75%) were reactivations/reinfections and only three were primary infections. The symptoms consisted of fever and graft dysfunction. Graft dysfunction was recorded in 10/12 patients. All CMV infections were successfully treated with
These results demonstrate that CMV infection is usually associated with HHV-6 and -7 antigenemia in liver transplant patients. In most cases HHV-6 activation was recorded prior to CMV disease but HHV-7 antigenemia usually appeared more or less concurrently with CMV. In all primary CMV infections, both HHV-6 and -7 preceded CMV. It has been reported by others that HHV-6 seroconversion is a risk factor predisposing the patient to
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severe CMV infection (Dockrell et al., 1997). Similar findings concern also the third b-herpesvirus, HHV-7, which is also suggested to be a cofactor for CMV disease progression (Osman et al., 1996). Interaction between the two b-herpesviruses was previously recorded in a case of primary CMV infection and HHV-6 reactivation (DesJardin et al., 1998). However, HHV-6 infection has also been found to be associated with CMV reactivations (Humar et al., 2000). These observations are in agreement with our results concerning both primary CMV infections and CMV reactivations. On the other hand, all herpesviruses tend to reactivate in immunosuppressed patients, and primary infections are known to be severe. The concurrent activation of CMV, HHV-6 and -7 may thus be just a consequence of the immunological condition of the transplant patients. Thus the three b-herpesvirus infections may appear concomitantly by chance and not necessarily due to their interactions. The concurrent appearance of the three b-herpesviruses in solid organ transplant patients has recently been reported by others (Griffiths et al., 1999; Dockrell and Paya, 2001; Mendez et al., 2001). The symptoms associated with CMV disease, such as fever and liver dysfunction or even hepatitis may also be linked with the other bherpesviruses, at least with HHV-6. However, in this patient population, only in four patients, the symptoms seemed to be associated with HHV-6 preceding CMV. We and others have reported the pathogenicity of HHV-6 in liver transplantation. It may infect the graft, and cause liver dysfunction, and possibly trigger acute and chronic rejection (Lautenschlager et al., 1998). HHV-6 may also infect epithelial and endothelial cells, which facilitates its involvement in development of transplant vasculopathy, also known as chronic rejection. Whether the involvement of HHV-6 in the rejection processes is similar to that of CMV (Lautenschlager, 1998), remains still unclear. HHV-7 may also be responsible for the clinical symptoms but its ability to act as a co-factor for the other bherpesviruses might be more important. Thus, the indirect effects of all three b-herpesviruses are possible and further studies are required to establish it.
In conclusion, HHV-6 and -7 antigenemia were usually associated with CMV disease in liver transplant patients. The results support the suggestion that CMV, HHV-6 and -7 may have interactions. The clinical signs of CMV infection, such as fever and graft dysfunction, may also be linked with HHV-6 or -7. Further studies are needed to investigate the direct and indirect post transplantation effects of HHV-6 and -7.
Acknowledgements This work was supported by grants from the Sigrid Juselius Foundation (to KH and IL) and Helsinki University Hospital Research Funds (EVO) (to IL). The authors wish to thank Raisa Loginov, Marjatta Palovaara and Sinikka SuurNuuja for technical assistance and Stephen Venn for correcting the English text.
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