INTRODUCTION. Biology of Blood and Marrow Transplantation 13: (2007) 2007 American Society for Blood and Marrow Transplantation

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1 Biology of Blood and Marrow Transplantation 13: (2007) 2007 American Society for Blood and Marrow Transplantation /07/ $32.00/0 doi: /j.bbmt Risk Factors for Developing Human Herpesvirus 6 (HHV-6) Reactivation after Allogeneic Hematopoietic Stem Cell Transplantation and Its Association with Central Nervous System Disorders Akiko Yamane, 1 Takehiko Mori, 1 Shigeaki Suzuki, 2 Ai Mihara, 1 Rie Yamazaki, 1 Yoshinobu Aisa, 1 Tomonori Nakazato, 1 Takayuki Shimizu, 1 Yasuo Ikeda, 1 Shinichiro Okamoto 1 1 Division of Hematology and 2 Division of Neurology, Department of Medicine, Keio University School of Medicine, Tokyo, Japan Correspondence and reprint requests: Takehiko Mori, MD, PhD, Division of Hematology, Department of Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo , Japan ( tmori@sc.itc.keio.ac.jp). Received June 13, 2006; accepted September 8, 2006 ABSTRACT We prospectively evaluated the incidence of human herpesvirus 6 (HHV-6) DNAemia after allogeneic hematopoietic stem cell transplantation (HSCT) using quantitative plasma real-time polymerase chain reaction. Of 46 recipients of bone marrow or peripheral blood stem cell transplantation (BMT/PBSCT) from related (n 11) or unrelated donors (n 22), and cord blood transplantation (CBT) from unrelated donors (n 13), 22 (47.8%) developed HHV-6 DNAemia. HHV-6 DNA levels ranged from 200 to 200,000 copies/ml of plasma, and HHV-6 DNAemia was observed significantly more frequently after CBT than after BMT/PBSCT (92.3% vs 30.3%; P <.001). Multivariate analyses identified CBT (vs BMT/PBSCT), HLA mismatches between recipient and donor, and low anti HHV-6 IgG titer before transplantation as the only risk factors for developing HHV-6 DNAemia. Three patients developed central nervous system (CNS) disorders with detectable HHV-6 DNA in the cerebrospinal fluid; all of these patients simultaneously developed HHV-6 DNAemia. These results suggest that HHV-6 DNAemia is frequently observed after allogeneic HSCT, especially in patients with the aforementioned risk factors. Thus, together with the assessment of risk factors, monitoring of HHV-6 DNAemia could be a useful asset in diagnosing HHV-6 associated CNS disorders American Society for Blood and Marrow Transplantation KEY WORDS Human herpesvirus 6 Hematopoietic stem cell transplantation Cord blood transplantation Polymerase chain reaction Central nervous system disorders INTRODUCTION Human herpesvirus 6 (HHV-6), a member of the Betaherpesvirinae subfamily, has biological properties similar to those of cytomegalovirus (CMV) and HHV-7 [1,2]. After primary infection during early childhood, HHV-6 remains latent but may reactivate in immunocompromised patients. Some 80%-90% of adults have serum anti-human HHV-6 antibody or detectable HHV-6 in peripheral blood mononuclear cells (PBMCs) or saliva [3-6]. The incidence of HHV-6 reactivation has been reported to be 28%- 78% after hematopoietic stem cell transplantation (HSCT) [6-16]. Several retrospective studies have demonstrated that the incidence of HHV-6 reactivation is higher after allogeneic cord blood stem cell transplantation (CBT) than after allogeneic bone marrow transplantation (BMT) and/or peripheral blood stem cell transplantation (PBSCT) [17,18]. HHV-6 reactivation has also been associated with skin rash, fever, myelosuppression, and pneumonitis, and may cause graft-versus-host disease (GVHD) [9-14,17-20]. 100

2 HHV-6 Reactivation after Allogeneic HSCT 101 Central nervous system (CNS) disorders, such as encephalitis, have also been linked to HHV-6, although the causative relationship and incidence have not yet been fully clarified [8,12,21-24]. To elucidate the optimal management of HHV-6 reactivation and HHV-6 diseases after allogeneic HSCT, we prospectively monitored HHV-6 DNA in plasma (HHV-6 DNAemia) using quantitative real-time polymerase chain reaction (PCR). Through this prospective study, we identified several risk factors for developing HHV-6 DNAemia, including pretransplantation anti HHV-6 IgG titers, which, to the best of our knowledge, has not been reported previously. We believe that identifying HHV-6 DNAemia may be useful in detecting HHV-6 associated CNS disorders. PATIENTS AND METHODS Patients and Transplantation Procedures A total of 46 patients undergoing allogeneic HSCT for hematologic malignancies at Keio University Hospital between July 2003 and May 2005 were evaluable for analysis. Patient characteristics are given in Table 1. Eleven patients received BMT or PBSCT from a HLA-identical sibling (n 9) or HLA-mismatched related donor (n 2) (rbmt/pbsct), 22 patients received BMT from an unrelated donor (ubmt), and 13 patients received cord blood transplantation (CBT) from an unrelated donor. The grafts were not manipulated ex vivo. The patients were conditioned with either a myeloablative regimen, comprising 12 Gy of total body irradiation (TBI) or busulfan plus cyclophosphamide (Bu-Cy), or a reduced-intensity regimen, comprising fludarabine plus melphalan or fludarabine plus cyclophosphamide. For GVHD prophylaxis, all but 1 recipients of ubmt and CBT received tacrolimus with or without methotrexate, and 9 of 11 recipients of rbmt/pbsct received cyclosporine A with or without methotrexate. HLA disparity differed significantly among the 3 groups, including a 1- or 2-locus mismatch in 11 of the 13 CBT recipients (Table 1). Intravenous acyclovir, 750 mg/day, was administered from 3 days before transplantation until posttransplantation day 14 for herpes simplex virus prophylaxis. For CMV prophylaxis, patients were monitored by CMV antigenemia assay or real-time PCR after neutrophil engraftment, and ganciclovir or foscarnet were started preemptively when positive results of CMV antigenemia and/or real-time PCR were obtained, as reported previously [25,26]. Table 1. Patient Characteristics Total (n 46) rbmt/pbsct (n 11) ubmt (n 22) CBT (n 13) P Value Age, years.82 Median (range) 47 (20-63) 48 (33-59) 43.5 (31-61) 50 (20-63) Sex.59 Male/female 21/25 4/7 12/10 5/8 Underlying disease.29 Acute leukemia Myelodysplastic syndrome Myeloproliferative disease Non-Hodgkin s lymphoma Multiple myeloma Conditioning regimen.64 Myeloablative Reduced-intensity HLA disparity <.0001 Complete match locus mismatch locus mismatch GVHD prophylaxis <.0001 Cyclosporin A methotrexate Tacrolimus methotrexate HHV-6 IgG titer*.44 < > Pretreatment CMV serologic status.59 Recipient and/ or donor Recipient /donor CMV reactivation.62 Positive Negative The CMV serologic status of the CBT donor was determined to be negative. *HHV-6 IgG titer was examined in 38 patients.

3 102 A. Yamane et al. Serologic Examination for HHV-6 A fluorescent antibody method was used to detect anti HHV-6 antibody in serum. Briefly, sera were first diluted 10 times, then serially diluted twofold in phosphate-buffered saline, after which they were reacted with HHV-6 antigen fixed on a slide. HHV-6 antigen was prepared from NP40-solubilized lymphocytes infected with the virus. After incubation for 1 hour at 37 C, the slides were washed and then reacted with fluorescein isothiocyanate conjugated rabbit anti-human IgG antibody (Dako, Glostrup, Denmark). Fluorescence was detected by fluorescent microscopy, and the endpoint titer was determined as the reciprocal of the maximum dilution at which fluorescence was detected. Titers 10 were considered positive. Of the 38 patients examined, all but 1 were seropositive for HHV-6. Sample Collection and Real-Time PCR for HHV-6 Plasma samples were obtained weekly from posttransplantation week 1 to week 8 or until HHV-6 DNA became undetectable by real-time PCR in patients who had exhibited positive PCR results. Three patients failed to provide a blood sample during posttransplantation week 1 or 2. Cerebrospinal fluid (CSF) samples were also obtained when patients with positive results for HHV-6 DNA in plasma developed any CNS symptoms. The results of plasma real-time PCR were provided to each physician, and CNS symptoms were monitored daily as part of routine care. Realtime PCR was performed as reported previously [16,25-27]. Briefly, DNA extracted from plasma using a QIAamp Blood Mini Kit (Qiagen, Valencia, CA) was subjected to PCR. Sequences of PCR primers and a probe for HHV-6 variant A (HHV-6A) were selected from the U89 immediate-early protein region of HHV-6, and those for variant B (HHV-6B) were selected from the U90 IE-A transactivator region. The sequences of forward primers were 5=-GTACAG- CCTCAGTGACAGATCTG-3= for both HHV-6A and -6B; the reverse primers were 5=-AGGAACCA- TCTTGTTCTGTCCCTT-3= for HHV-6A and 5=- GGTCATACCAGGAAGCGTTTCG-3= for HHV- 6B. The Taqman probe selected between the primers was dual-labeled with FAM (6-carboxyfluorescein) at the 5= end as a reporter dye and with TAMRA (6-carboxy-tetramethylrhodamine) at the 3= end as a quenching dye, with a sequence of 5=-CAGCCCC- GATAAAAGGTCACAGACAAAAGA-3=. The PCR reaction was performed using TaqMan Universal PCR Master Mix (PE Biosystems, Foster City, CA), and nuclease degradation of the probe was detected as an increase in fluorescence intensity by an ABI PRISM 7700 (PE Biosystems). The quantification of HHV-6 was carried out with a serially diluted standard ranging from 10 to copies, and the gene copy numbers were calculated by Sequence Detection System version software (PE Biosystems). The sensitivity of this assay was 200 copies/ml of plasma. Statistical Analyses Differences between the groups were assessed by Fisher s exact test, the Kruskal-Wallis test, and the Mann-Whitney U test as appropriate. The cumulative incidence of HHV-6 DNAemia was calculated according to the Kaplan-Meier method, and comparisons were made using the log-rank test. Multivariate analysis was performed using the Cox proportional hazards model with stepwise regression, using the criteria to remove factors with P values.10. P values.05 were considered statistically significant. RESULTS Detection of HHV-6 DNA in Plasma HHV-6 DNA in plasma became positive (HHV-6 DNAemia) in at least one sample in 22 (47.8%) of 46 patients (Figure 1A). The detected HHV-6 strains Figure 1. (A) Cumulative incidence of the detection of HHV-6 DNA in plasma (HHV-DNAemia) after allogeneic HSCT. (B) Cumulative incidence of HHV-6 DNAemia according to the sources of stem cells: CBT, BMT, or PBSCT. HHV-6 DNAemia was more frequently observed after CBT than after BMT/PB- SCT (P.001).

4 HHV-6 Reactivation after Allogeneic HSCT 103 Figure 2. HHV-6 DNA levels in plasma after allogeneic HSCT. Approximately 80% of positive samples and samples with high HHV-6 DNA copies ( 10,000 copies/ml) were distributed between weeks 2 and 4 after transplantation. were exclusively HHV-6B; HHV-6A was not detected in any patients. The median time of the first detection of HHV-6 DNAemia was posttransplantation week 3 (range, week 2-week 5). Of the 22 patients with HHV-6 DNAemia, 11 (50%) had 1 positive sample and the other 11 had 2 or more positive samples. HHV-6 DNA was more frequently detected in recipients of CBT (92.3%) than in recipients of rbmt/ PBSCT or ubmt (30.3%, P.001; Figure 1B). Only 1 of the 11 rbmt/pbsct recipients developed HHV-6 DNAemia. Among the 22 patients with HHV-6 DNAemia, 4 had a second HHV-6 DNAemia at posttransplantation week 7 or 8, defined as a reactivation after clearance of the first reactivation. Plasma Levels of HHV-6 DNA after Transplantation In patients with HHV-6 DNAemia, the number of HHV-6 DNA copies ranged from 200 to 200,000/mL of plasma. The relationship between the time after transplantation and the HHV-6 DNA levels in plasma is shown in Figure 2. Twenty-nine of 36 (80.6%) HHV-6 DNA positive samples and 4 of 5 (80%) samples with high HHV-6 DNA copies ( 10,000 copies/ ml) were distributed at posttransplantation weeks 2-4. The median duration of HHV-6 DNAemia was 1.5 weeks (range, 1-3 weeks). The median maximum number of HHV-6 DNA copies in each patient did not differ significantly between CBT recipients (750 copies/ml; range, ,000 copies/ml) and rbmt/pbsct or ubmt recipients (850 copies/ml; range, ,000 copies/ml; P.67). Two patients exhibited HHV-6 DNAemia at posttransplantation week 8; subsequent monitoring confirmed clearance in both cases. Risk Factors for Developing HHV-6 DNAemia The stem cell source significantly affected the incidence of HHV-6 DNAemia, as shown in Figure 1B. In addition, univariate analysis identified HLA mismatch between patient and donor, tacrolimus for GHVD propylaxis (vs cyclosporine A), lower anti HHG-6 IgG titer before transplantation ( 40 vs 40), and CMV reactivation as risk factors for developing HHV-6 DNAemia (Table 2). Other factors, including age, sex, underlying diseases, conditioning regimen, and pretransplantation CMV serologic status, had no significant effect on the incidence of HHV-6 DNAemia (data not shown). Among these factors, cord blood as a stem cell source, HLA mismatch, and low anti HHV-6 IgG titer before transplantation remained as significant risk factors as identified by multivariate analysis (Table 2). Association between CNS Disorders and HHV-6 DNA in Plasma and CSF Among our 46 patients, 4 (patients 30, 38, 39, and 45) manifested CNS symptoms and underwent CSF Table 2. Factors Affecting HHV-6 DNAemia after Allogeneic Hematopoietic Stem Cell Transplantation Factors Univariate P Value Multivariate Hazard Rate 95% CI P Value Stem cell source CBT versus BMT/PBSCT/uBMT < Acute GVHD Grade 0-I versus II-IV.269 HLA disparity Complete match versus 1-2 locus mismatch GVHD prophylaxis Tacrolimus versus cyclosporine A.010 NS HHV-6 IgG titer* < 40 versus > CMV infection Positive versus negative.033 NS The results of univariate analyses using the log-rank test and multivariate analyses using the Cox proportional hazards model with stepwise regression method of factors affecting the cumulative incidence of HHV-6 DNAemia are shown. CI indicates confidence interval; NS, not significant. *HHV-6 IgG titer was examined in 38 patients.

5 104 A. Yamane et al. examination. Patient 45 was diagnosed with bacterial meningitis and did not exhibit HHV-6 DNA in plasma or CSF at the onset, whereas HHV-6 DNA was detected in CSF in the other 3 patients, at levels ranging from 200 to 600 copies/ml. In these 3 patients, CSF examination demonstrated no elevation of leukocytes, along with normal levels of protein and glucose. Cultures of CSF for bacteria and fungus were negative. Magnetic resonance imaging of the brain and spinal cord revealed no abnormal findings, except for a T2-weighted high-intensity, 1-cm lesion in the left frontal cortex in patient 30. CNS disorders due to HHV-6 were diagnosed in these 3 patients; these patients characteristics and outcomes are summarized in Table 3. Their neurologic symptoms included severely impaired consciousness, headache, and dysesthesia. Although these patients were HHV-6 seropositive before transplantation, they had low antibody titers against HHV-6. Weekly monitoring detected HHV-6 DNAemia at the onset of CNS symptoms, with detection preceding the onset of CNS disorders in only 1 patient (patient 39). All patients were treated with intravenous ganciclovir. The symptoms of patients 38 and 39 improved after intravenous ganciclovir 10 mg/kg/day, ultimately achieving complete resolution, with HHV-6 DNA becoming undetectable in both plasma and CSF. Patient 30, in whom ganciclovir was not initiated immediately after the onset of CNS symptoms, died without experiencing improvement in these symptoms. Relationship between HHV-6 DNAemia and Antiviral Therapy for CMV Infection/Disease Of the 34 patients with CMV reactivation, 27 received ganciclovir or foscarnet as preemptive therapy for CMV infection (n 25) or therapy for CMV disease (n 2). In these patients, the median time of the diagnosis of CMV infection or disease was posttransplantation week 6 (range, week 1-week 9), significantly later than the detection of HHV-6 DNAemia (P.0001). Thirteen patients developed HHV-6 DNAemia before initiation of antiviral therapy, whereas only 2 patients developed HHV-6 DNAemia after initiation of antiviral therapy. The remaining 12 patients did not develop HHV-6 DNAemia during the study period. DISCUSSION HHV-6 reactivation is often observed after allogeneic HSCT; however, its clinical significance has yet to be fully elucidated. To clarify this issue, several investigators have attempted to evaluate the incidence of HHV-6 reactivation after allogeneic HSCT using various methods, including virus isolation, PCR, and antigenemia assays [6-16]. The reported incidence of HHV-6 reactivation after allogeneic HSCT varies widely, ranging from 28% to 78% [6-15]. This significant variation may be explained by the sensitivity of the tests used in each study. Another explanation could be the differences in the clinical samples subjected to analysis (PBMCs or cell-free serum/plasma), because PCR can detect latently infected HHV-6 DNA in PBMCs, possibly leading to false-positive results. Therefore, in the present study, we investigated HHV-6 DNAemia using cell-free plasma in allogeneic HSCT recipients so that we could exclude the detection of latent infection and further evaluate the longitudinal viral load. Twenty-two of 46 patients (47.8%) developed HHV-6 reactivation, with a significantly higher rate of reactivation in CBT recipients compared with BMT or PBSCT recipients. This finding is consistent with previous reports [17,18] and can be explained by the absence of primed HHV-6 specific T cells and the immunologic immaturity after CBT. In addition to the stem cell sources, multivariate analyses also identified HLA mismatches between patient and donor and low anti HHV-6 IgG titers before transplantation as significant factors associated with HHV-6 reactivation. Patient donor HLA mismatches have been reported to cause prolonged immune deficiency after allogeneic HSCT, which could contribute to HHV-6 reactivation [16,28]. The rate of HHV-6 DNAemia was significantly higher in patients with a lower anti HHV-6 IgG titer before transplantation compared with those Table 3. Clinical Characteristics and Outcomes of Patients with HHV-6-Associated CNS Disorders Patient Stem Cell Source HHV-6 IgG* CNS Symptoms HHV-6 Plasma** HHV-6 CSF** Treatment Outcome Complication 30 UBMT 20 Impaired consciousness (3 rd ) 40,000 (3rd) 200 (3rd) Ganciclovir Death Liver failure 38 CBT 40 Headache (3rd) 20,000 (3rd) 200 (3rd) Ganciclovir Improved Mild GVHD 39 CBT 20 Dysesthesia (3 rd ) 400 (2nd) 600 (3rd) Ganciclovir Improved Moderate GVHD CNS, central nervous system; HHV-6, human herpesvirus 6; CSF, cerebrospinal fluid; CBT, cord blood transplantation; ubmt, bone marrow transplantation from unrelated donor; GVHD, graft-versus-host disease. *Titers were determined before transplantation. **Copies/mL. The parentheses indicate the posttransplantation week of the onset of CNS disorders or first detection of HHV-6 DNA.

6 HHV-6 Reactivation after Allogeneic HSCT 105 with higher titers. To the best of our knowledge, no report to date has shown the relationship between pretransplantation anti HHV-6 antibody titer and posttransplantation HHV-6 DNAemia. It is plausible that the neutralizing and suppressing activity of anti HHV-6 IgG exerts an effect against HHV-6 replication [29]. With respect to the pretransplantation serologic status of HHV-6, the present study included 1 patient who was HHV-6 seronegative before transplantation. This patient developed HHV-6 DNAemia between posttransplantation weeks 2 and 3; we speculated that transmission of HHV-6 through transplanted cord blood or transfused blood cells was the source of the HHV-6 DNAemia in this patient [3,30,31]. In the present study, patients received acyclovir until day 14 for the prophylaxis against herpes simplex virus and were allowed to receive ganciclovir or foscarnet for CMV infection/disease. There is a possibility that these agents affected the incidence and clinical course of HHV-6 DNAemia after HSCT. Acyclovir is less active against HHV-6, and ganciclovir and foscarnet are potential antiviral agents for HHV-6. Thus, these drugs could have played a role in suppressing HHV-6 reactivation. Although 27 of 46 patients (58.7%) received ganciclovir or foscarnet for CMV infection/disease, the time of detection of HHV-6 DNAemia (posttransplantation week 3) was significantly earlier than that of the onset of CMV infection/ disease (posttransplantation week 6). Thus, these agents may have had little effect on the incidence of HHV-6 DNAemia. However, they possibly may have altered the clinical course of HHV-6 infection by suppressing viral reactivation, which may have led us to underestimate the clinical significance of HHV-6 DNAemia after allogeneic HSCT. In the present study, 4 patients developed CNS symptoms during the study period. One patient was diagnosed with bacterial meningitis, but the other 3 patients were diagnosed with HHV-6 associated CNS disorders. Diagnosis was based on positive results for HHV-6 DNA in CSF and the absence of other identified causes of CNS disorders. Although a definition of CNS disorders associated with HHV-6 has yet to be established, CNS symptoms resolved completely after initiating anti HHV-6 therapy in 2 of our 3 patients, strongly suggesting the causative role of HHV-6 in CNS disorders in these patients. A possible reason for treatment failure in the 1 patient in whom CNS symptoms did not resolve was the delay in examining CSF and the resulting delay in initiation of antiviral therapy. The patient was in serious condition due to severe acute GVHD, and CSF examination could not be performed in a timely manner. Weekly monitoring was able to detect HHV-6 DNAemia at the onset of CNS disorders; however, it was not able to detect it before onset. Our experience suggests that prompt examination of CSF is essential for the early diagnosis and treatment of HHV-6 associated CNS disorders, and that weekly HHV-6 DNAemia monitoring could be a useful asset for diagnosing HHV-6 associated CNS disorders. The small number of patients with HHV-6 associated CNS disorders makes it difficult to draw any definite conclusions. However, HHV-6 DNAemia monitoring may have less predictive value, limiting the application of preemptive therapy using HHV-6 DNAemia for preventing HHV-6 associated CNS disorders. In the present study, we used real-time PCR to quantitatively evaluate serial changes in HHV-6 DNAemia early after allogeneic HSCT. In contrast to the significant difference in the incidence of HHV-6 DNAemia between CBT and BMT/ PBSCT recipients, HHV-6 DNA levels did not differ significantly between the 2 groups. The low incidence of HHV-6 associated CNS disorders (3 of 22 patients with HHV-6 DNAemia) makes it difficult to statistically evaluate the effect of viral load on the development of HHV-6 associated CNS disorders. However, HHV-6 associated CNS disorders developed in 2 of the 5 patients with high-level HHV-6 DNAemia ( 10,000 copies/ml) but in only 1 of 16 patients with low-level HHV-6 DNAemia ( 10,000 copies/ml). Therefore, highlevel HHV-6 DNAemia might be an important parameter for identifying patients at higher risk for developing HHV-6 associated CNS disorders. Further studies are needed to evaluate the relationship between viral load and the development of HHV-6 associated CNS disorders. We conclude that HHV-6 reactivation is frequently observed after allogeneic HSCT, especially in patients with some or all of the risk factors described earlier. 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