¹Department of Developmental Medicine (Pediatrics) D-5, Osaka University Graduate School of Medicine, Osaka, Japan

²Department of Microbiology C-1, Osaka University Graduate School of Medicine, Osaka, Japan

Correspondence to: Dr S Tokimasa, Department of Developmental Medicine (Pediatrics) D-5, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan

Human herpesvirus 6 (HHV-6) infection and disease are serious complications of allogeneic hematopoietic stem cell transplantation (allo-SCT). Ganciclovir (GCV) is effective against HHV-6 in vitro but the antiviral susceptibility of HHV-6 has not been well characterized in vivo. We retrospectively compared the HHV-6 reactivation rate in pediatric allo-SCT recipients with and without GCV prophylaxis. The HHV-6 reactivation rate at 3 weeks after allo-SCT in patients without prophylactic GCV administration was significantly higher than that in those receiving prophylactic GCV (11/28 vs 0/13, P < 0.01). Five of 36 patients without prophylactic GCV showed clinical manifestations including skin rash, interstitial pneumonitis, persistent thrombocytopenia, enterocolitis and thrombotic microangiopathy, respectively. HHV-6-associated symptoms were observed in one of the 13 patients receiving prophylactic GCV. This patient showed fever, diarrhea and graft rejection concomitantly with a sudden increase of HHV-6 DNA copy number. Patients who received GCV for treatment of HHV-6 infection showed an improvement in symptoms and/or decrease of HHV-6 copy number. Thus, GCV is effective for treating HHV-6 disease after allo-SCT in vivo.

Bone Marrow Transplantation (2002) 29, 595-598. DOI: 10.1038/sj/bmt/1703423

human herpes virus-6; ganciclovir; stem cell transplantation; prophylaxis; graft failure

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is frequently used for treatment of hematological malignancies, immunodeficiency diseases, solid tumors, and metabolic disorders. Viral infections are major causes of mortality and morbidity after allo-SCT. Herpesviruses, especially human cytomegalovirus (CMV) disease, remain the cause of some of the most serious complications including interstitial pneumonitis and graft failure. Human herpesvirus-6 (HHV-6) shares many biological characteristics with CMV, including viral protein products (~67% homology),¹ antigenic reactivity, and in vitro growth characteristics.² Several studies have suggested that HHV-6 is associated with fever, skin rash,³ graft-versus-host disease (GVHD),⁴ interstitial pneumonitis,⁵ encephalitis,^6,7 thrombotic microangiopathy (TMA),⁸ and suppression of engraftment,⁹ in bone marrow transplant recipients.

Ganciclovir (GCV) has been shown to be clinically effective for the prevention and treatment of CMV infection in a variety of immunocompromised hosts.¹⁰ There have been several studies suggesting the efficacy of GCV when given as prophylactic therapy or early treatment. Early GCV treatment has been shown to reduce CMV disease mortality in patients with laboratory evidence of on-going CMV replication.¹¹ GCV has also been given at engraftment to CMV-seropositive patients and patients receiving a CMV- seropositive graft. Both early treatment and prophylactic therapy are effective for preventing CMV disease.

GCV is also effective against HHV-6 in vitro¹² but the antiviral susceptibility of HHV-6 has not been well characterized in vivo. To prevent CMV disease, we have administered prophylactic GCV for patients undergoing allo-SCT from January 1994 to August 1997. We retrospectively analyzed the incidence and morbidity of HHV-6 infection in allo-SCT recipients with or without prophylactic GCV administration by examining viral DNA using semiquantitative PCR.

Materials and methods

Patients

Forty-nine consecutive patients undergoing allo-SCT between June 1989 and April 2000 were analyzed. Patient characteristics are shown in Table 1. A combination of cyclosporine A and methotrexate or methotrexate alone was administered for GVHD prophylaxis. Gammaglobulin 100-200 mg/kg i.v. was administered weekly until discharge. Oral acyclovir (ACV) (250 mg/m² twice daily) was administered for prophylaxis against herpes simplex and varicella zoster viruses.

Thirteen patients received prophylactic GCV (5 mg/kg i.v. twice daily for 7 days until SCT and 5 mg/kg once daily from the time of engraftment until day 120 post-SCT) (prophylactic therapy). Thirty-six patients did not receive prophylactic GCV but some of them were given early GCV treatment when asymptomatic HHV-6 infection was detected during regular monitoring (early GCV treatment). The two groups of patients with and without prophylactic GCV were well matched with respect to age, gender, donor, and graft sources (Table 1).

HHV-6 infection was defined by compatible clinical symptoms together with the detection of HHV-6 DNA at a high level (3+ or 4+; see later), when no other pathogens including CMV were detected. Patients with evidence of HHV-6 infection or with HHV-6 reactivation at a high level in peripheral blood mononuclear cells (PBMCs) on two consecutive occasions were treated with i.v. GCV 5 mg/kg every 12 h (dose adjusted if necessary for renal impairment) for 14 days followed by maintenance therapy with 5 mg/kg daily or 3 times a week for 4 weeks, depending on the level of immunosuppression and the absolute neutrophil counts. Informed consent was obtained from the patients and/or their parents.

Samples

EDTA or heparinized blood samples were collected at weekly intervals from 2 weeks before until up to 12 weeks after SCT or until death. PBMCs were obtained by centrifugation on Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) step gradient.

DNA analysis

Methods for detection of HHV-6 DNA and amplification of viral DNA extracted from peripheral blood mononuclear cells have been previously described.¹³ The number of copies of viral genome per 10⁵ PBSCs was estimated based on semiquantative PCR as described and expressed as follows: (1+), 1 to 10 copies; (2+), 10 to 10² copies; (3+), 10² to 10³ copies; and (4+), >10³ copies.

Statistical analysis

Fisher's exact test and the chi-square independent test for categorical data were used to compare the characteristics of the two groups of patients.

Results

Viral DNA

Before allo-SCT, high-level HHV-6 DNA was detected in none of four patients receiving 7 days prophylactic GCV and in three of 19 patients not receiving GCV. During the study period after allo-SCT, HHV-6 DNA at a high level (>10² copies per 10⁵ cells (3+ or 4+)) was detected in 5/13 patients (38%) on prophylactic GCV and 21/36 patients (58%) off it. The HHV-6 reactivation rate 3 weeks after allo-SCT in the patients not on prophylactic GCV was significantly higher than that in patients on prophylactic GCV (11/28 vs 0/13, P < 0.01). Kinetics of the reactivation rate of HHV-6 DNA is shown in Figure 1.

Clinical manifestations

Clinical manifestations related to HHV-6 infection observed during the course of allo-SCT are summarized in Table 2. In patient UPN 1020, who received prophylactic GCV, high-grade fever and diarrhea developed at the time of an increase in neutrophil count, which was accompanied by a sudden increase in HHV-6 DNA copy numbers in bone marrow cells as well as in PBMCs. The patient received therapeutic GCV, which resulted in a decrease in HHV-6 copy number, but the neutrophil count dramatically decreased and the graft was rejected. Another five patients not on prophylactic GCV developed symptoms concurrently with an increase in HHV-6 copy number. In addition to fever, these symptoms included generalized skin rash (UPN 1033) similar to that observed with exanthem subitum, thrombocytopenia (UPN 1015), interstitial pneumonitis (UPN 1018), enterocolitis (UPN 1053), and thrombotic microangiopathy (UPN 1071). The first three patients have been described in detail.¹⁴ In all patients, no other pathogens including CMV were detected. UPN 1033, 1053 and 1071 received treatment with GCV and showed improvement in symptoms with a decrease of HHV-6 copy number.

Discussion

In this study, we report clinical episodes associated with HHV-6 disease or reactivation in six patients undergoing allo-SCT. Four of them received treatment with GCV for their HHV-6 infection and experienced resolution of clinical symptoms and/or a decrease of HHV-6 copy number. This observation provides virologic evidence for the in vivo anti-HHV-6 activity of GCV and suggests potential clinical benefit from inhibition of HHV-6 replication. The efficacy of GCV against HHV-6 has been reported in patients with encephalitis⁷ and secondary graft failure.¹⁵ Our observation confirmed the efficacy of GCV against HHV-6.

The frequency of high-level HHV-6 DNA detection (3+ or 4+) was lower in patients who received prophylactic GCV following allo-SCT as compared to those on no prophylaxis. These findings suggest that prophylactic administration of GCV is effective in reducing the incidence of HHV-6 reactivation and infection, at least in the early post-transplantation period when HHV-6 reactivation and infection occur frequently.^16,17 This leads to a potential reduction in the morbidity associated with HHV-6 infection such as bone marrow failure, pneumonitis, and TMA. Although Wang and collegues² reported that high-dose ACV prophylaxis reduced HHV-6 DNA levels in SCT, the IC₅₀ of ACV for HHV-6 is 100 mol/l which is difficult to achieve in the clinical setting.¹⁸ However, the IC₅₀ of GCV for HHV-6 is 1.0-2.5 mol/l,¹² which is attainable in vivo for prophylaxis and treatment of HHV-6 infection.

Several studies have shown that GCV prophylaxis can reduce the frequency of CMV infections after allo-SCT but that survival was not improved.^10,19,20,21 This apparent lack of reduction in transplant-related mortality would appear to be related to the significantly higher incidence of invasive fungal and gram-negative infections, and late CMV disease in the prophylaxis groups.²² These studies did not specifically investigate HHV-6 infection and disease. However, the occurrence of HHV-6 reactivation and infection is generally earlier than CMV.^16,17 Therefore, it may be feasible to provide GCV prophylaxis against HHV-6 infection for a week before SCT and in the few weeks after engraftment in high-risk SCT such as T cell-depleted SCT and cord blood transplantation (Sashihara et al, manuscript in preparation).

In summary, prophylactic GCV reduced the HHV-6 reactivation rate after allo-SCT. GCV treatment for patients with HHV-6 disease relieved symptoms with a concomitant decrease in HHV-6 DNA copy number. This study thus suggests a clinical benefit of GCV against HHV-6 infection in allo-SCT.

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Figure 1 Kinetics of detection rate of HHV-6 DNA at a high level (>10² copies per 10⁵ cells (3+ or 4+)) before and after allo-SCT. Open and solid circles represent the percent of patients with high copy number of HHV-6 in the groups with and without GCV prophylaxis, respectively.

Table 1 Patient characteristics

Table 2 Characteristics of patients with HHV-6 infection