Human Herpesvirus 6 DNA in Plasma after Allogeneic Stem Cell Transplantation: Incidence and Clinical Significance

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1 MAJOR ARTICLE Human Herpesvirus 6 DNA in Plasma after Allogeneic Stem Cell Transplantation: Incidence and Clinical Significance Masao Ogata, 1 Hiroshi Kikuchi, 2 Takako Satou, 1 Rie Kawano, 1 Junji Ikewaki, 1 Kazuhiro Kohno, 1 Kenji Kashima, 3 Eiichi Ohtsuka, 4 and Jun-ichi Kadota 1 1 Division of Pathogenesis and Disease Control, Department of Infectious Diseases, 2 Blood Transfusion Center, and 3 Department of Pathology, Oita University Faculty of Medicine, and 4 Department of Hematology, Oita Prefectural Hospital, Oita, Japan Background. Human herpesvirus 6 (HHV-6) is increasingly recognized as an opportunistic and potentially life-threatening pathogen in recipients of allogeneic stem cell transplants (SCTs). Methods. To clarify the incidence and clinical relevance of active HHV-6 infection, serial titers of plasma HHV- 6 DNA were determined for 50 allogeneic SCT recipients, using real-time polymerase chain reaction. Results. HHV-6 DNA was detected in plasma from 24 patients (48%). HHV-6 DNA was most frequently apparent days after transplantation. An increased risk of a positive result for HHV-6 DNA was associated with transplantation from an allelic-mismatch donor ( P p.02) and administration of steroids ( P p.04). Steroid use was associated with high HHV-6 DNA loads ( P p.02). High HHV-6 DNA loads were correlated with delayed platelet engraftment ( P p.04). Among patients who had positive results for HHV-6 DNA, the HHV-6 DNA load was higher in plasma from those who developed limbic encephalitis ( n p 4) ( P!.0001). Conclusions. Active HHV-6 infection is not rare in SCT recipients. SCT from allelic-mismatch donors is associated with increased risk of active HHV-6 infection. Steroid therapy is associated with not only increased incidence of infection but also accelerated viral replication. Development of limbic encephalitis is associated with high HHV-6 DNA load. Human herpesvirus 6 (HHV-6) was first isolated in 1986 from patients with lymphoproliferative disorders [1]. This virus is now recognized as a ubiquitous infectious agent that infects most healthy children by 2 years of age [2, 3] and commonly causes exanthem subitum [4]. Like other herpesviruses, HHV-6 is thought to remain latent after primary infection, reactivating under conditions of immunosuppression. Although HHV- 6 preferentially infects CD4 + T lymphocytes, the potential range of cellular hosts is wide [5, 6]. Infection of primary fetal astrocytes has been confirmed in cell Received 25 April 2005; accepted 21 July 2005; electronically published 30 November Potential conflicts of interest: none reported. Financial support: Oita University Faculty of Medicine; Japanese Ministry of Education, Culture, Sports, Science, and Technology (grant-in-aid for scientific research ). Reprints or correspondence: Dr. Masao Ogata, Div. of Pathogenesis and Disease Control, Dept. of Infectious Diseases, Oita University Faculty of Medicine, Hasamamachi, Oita , Japan (mogata@med.oita-u.ac.jp). The Journal of Infectious Diseases 2006; 193: by the Infectious Diseases Society of America. All rights reserved /2006/ $15.00 culture [7], and HHV-6 may represent the most neuroinvasive member of the herpesvirus family [8]. HHV-6 has increasingly been recognized as an opportunistic and potentially life-threatening pathogen in those who have received liver transplants and allogeneic stem cell transplants (SCTs). The virus is associated with graft dysfunction [9, 10] and encephalitis [11] in liver transplant recipients and with rash [12 15], interstitial pneumonitis [16, 17], bone marrow suppression [18, 19], and encephalitis [20 32] in recipients of SCTs. An increasing number of clinical reports suggest that HHV-6 represents an important emerging pathogen responsible for central nervous system (CNS) complications accompanying liver transplantation and SCT. Several investigations of the incidence and clinical significance of HHV-6 infection in recipients of allogeneic SCT have been reported [15, 33 43]. These studies have used various methods to detect the virus, including virus isolation [15], qualitative polymerase chain reaction (PCR) [33 38] or quantitative PCR [39 42] for leukocytes, and antigenemia assay [37, 43]. However, findings about the incidence of active HHV-6 in- 68 JID 2006:193 (1 January) Ogata et al.

2 fection appear to be contradictory, and the role that HHV-6 plays in posttransplantation complications remains in question. Although isolation of virus offers a reliable method for detecting active viral infection [15], the process is both timeconsuming and labor-intensive. HHV-6 replication is indirectly reflected by the kinetics of virus load on the basis of quantitative PCR analysis. However, detection of the viral genome by PCR in clinical samples containing peripheral blood mononuclear cells (PBMCs) may reflect both latent and active viral infection, because the HHV-6 genome persists in PBMCs throughout life after primary infection [6]. Virus load, as determined by realtime PCR in cell-free body fluids such as plasma, is thus considered a good indicator of active HHV-6 infection [44, 45]. We hypothesized that quantification of HHV-6 DNA in plasma, using a real-time PCR assay, may be useful for identifying active HHV-6 infection in allogeneic SCT recipients. This assay might allow accurate elucidation of the clinical course and risk factors of HHV-6 activation and associations between posttransplantation complications and HHV-6 infection. PATIENTS, MATERIALS, AND METHODS Table 1. Characteristic Characteristics of stem cell transplant recipients. Value Age, median (range), years 40.5 (12 59) Sex, male 30 (60) Underlying disease Acute myeloid leukemia 14 (28) Acute lymphoblastic leukemia 5 (10) Acute mixed lineage leukemia 1 (2) Chronic myelogenous leukemia 8 (16) Myelodysplastic syndrome 5 (10) Adult T cell leukemia 9 (18) Non-Hodgkin lymphoma 6 (12) Multiple myeloma 1 (2) Severe aplastic anemia 1 (2) Disease stage at transplantation a Early 22 (44) Nonearly 28 (56) Pretransplantation conditioning Myeloablative 46 (92) TBI (12 Gy)/CY (120 mg/kg) 33 TBI (12 Gy)/CY (120 mg/kg)/arac (8 g/m 2 ) 4 TBI (12 Gy)/CY (120 mg/kg)/etoposide (40 mg/kg) 4 TBI (12 Gy)/CY (120 mg/kg)/bu (8 mg/kg) 1 BU (16 mg/kg)/cy (120 mg/kg) 2 AraC (8 g/m 2 )/CY (120 mg/kg)/etoposide (40 mg/kg) 1 TLI (7.5 Gy)/CY (200 mg/kg) 1 Nonmyeloablative 4 (8) FU (150 mg/kg)/bu (8 mg/kg)/atg (5 mg/kg) 2 FU (150 mg/kg)/bu (8 mg/kg)/tbi (2 Gy) 1 FU (150 mg/kg)/melpharan (100 mg/kg) 1 Transplant type Related bone marrow 11 (22) Related peripheral blood 8 (16) Unrelated bone marrow 31 (62) Matching of HLA Allele match 33 (66) Allele mismatch 17 (34) Antigen match 44 (88) Antigen mismatch 6 (12) GVHD prophylaxis Cyclosporine/methotrexate 46 (92) Tacrolimus/methotrexate 3 (6) Cyclosporine only 1 (2) Acyclovir prophylaxis 50 (100) NOTE. Data are no. (%), unless otherwise indicated. AraC, cytosine arabinoside; ATG, antithymocyte globulin; BU, busulfan; CY, cyclophosphamide; FU, fludarabine; GVHD, graft-vs.-host disease; TBI, total body irradiation; TLI, total lymphoid irradiation. a Early stage was defined as acute leukemia during the first or second remission and chronic myelogenous leukemia during the first chronic phase, and myelodysplastic syndrome was classified as refractory anemia or refractory anemia with ringed sideroblasts; all others were considered to be in the nonearly stage. Patients. This study involved consecutive patients who received an unmanipulated hematopoietic SCT from either a sibling or an unrelated donor at Oita Medical University between January 1995 and December Recipients of cord-blood SCTs, patients who died of any cause within 21 days of SCT, and patients who received a second transplant were excluded, resulting in a final total of 50 patients. Patient characteristics are summarized in table 1. Treatment regimens. Pretransplantation conditioning is shown in table 1. To prevent acute graft-versus-host disease (GVHD), 46 patients received cyclosporine administered intravenously beginning on day 1 (1.5 mg/kg, twice daily), with a target trough level of ng/ml, combined with methotrexate (10 mg/m 2 on day +1 and 7 mg/m 2 on days +3 and +6); 3 patients received methotrexate (as above) combined with tacrolimus as a continuous intravenous infusion, starting on day 1 (0.03 mg/kg/day), with a target level of ng/ml. Another patient received cyclosporine alone as continuous infusion (3 mg/kg/day). Each patient was isolated in a room with laminar air flow. Prophylaxis for bacteria, fungal, and Pneumocystis carinii infection comprised levofloxacin, fluconazole, and sulfamethoxazole-trimethoprim. All patients were administrated oral acyclovir at 1000 mg/day from day 7 to day +35 to prevent infection with herpes simplex virus. Irradiated leukocyte-depleted blood products were used for blood-component substitution throughout the pre- and posttransplantation course. Diagnosis and grading of acute GVHD was based on the clinical criteria described by Thomas et al. [46], with histological confirmation as required. Both GVHD of greater than grade I and GVHD accompanied by pyrexia were treated using steroids, usually starting with 2 mg/kg intravenous methylprednisolone for 2 days, with subsequent dose tapering. To diagnose reactivation of cytomegalovirus (CMV), testing for CMV antigenemia, nested PCR, and/or real-time PCR were performed once a week after engraftment, as described elsewhere [47]. The decision to use preemptive therapy with ganciclovir for prevention of CMV disease was based on positive results from these Plasma HHV-6 DNA Load after Allogeneic SCT JID 2006:193 (1 January) 69

3 tests. Neutrophil engraftment was defined as an absolute neutrophil count of neutrophils/l, while platelet en- 9 graftment was considered present with a self-sustained platelet count of platelets/l. Specimen collection. After written, informed consent was obtained from each patient, EDTA-treated peripheral blood was collected. Blood-sample collection was started at a median of 3 days after transplantation (range, 0 10 days) and lasted until a median of 54.5 days (range, days). The median number of blood samples per patient was 10 (range, 5 11). Collection intervals between samples were as follows: 3 days ( n p 2), 4 days ( n p 1), 6 days ( n p 1), 7 days ( n p 386), 8 days ( n p 1), 13 days ( n p 2), or 14 days ( n p 4). A total of 447 samples obtained from 50 patients were examined using real-time PCR. Oligonucleotide primers and TaqMan probe. Oligonucleotide primers and a TaqMan probe were selected in accordance with the methods described by Locatelli et al. [48]. The forward primer, TAQ6A (5 -CGCTAGGTTGAGGATGATCGA- 3 ), and the reverse primer, TAQ6E (5 -CAAAGCCAAATTAT- CCAGAGCG-3 ), which amplify a 133-bp fragment, were designed in the highly conserved U67 open-reading frame of HHV-6. A TaqMan probe (5 -[FAM]CACCAGACGTCACACC- CGAAGGAAT[TAMURA]-3 ) had a complementary sequence 28 bp downstream from the internal region of the forward primer. These primers and the TaqMan probe were purchased from Applied Biosystems. Preparation of HHV-6 standard DNA template. Viral DNA was extracted from the plasma (200 ml) from an HHV- 6 infected patient, using a QIAamp DNA Blood Mini kit (Qiagen), in accordance with the manufacturer s instructions. HHV- 6 DNA derived from the patient was amplified using TAQ6E and TAQ6A (see above). The PCR mixture contained 10 Ex Taq buffer, dntp mixture (each at 2.5 mmol/l), 500 nmol/l each primer (TAQ6E and TAQ6A), 2.5 U of Ex Taq (Takara), and 5% (vol/vol) DNA template. The cycling profile comprised a first denaturation step of 2 min at 95 C; a second step of 26 cycles with denaturation for 20 s at 95 C, annealing for 30 s at 58 C, and extension for 30 s at 72 C; and a final extension step of 10 min at 72 C. The amplified 133-bp fragment was then cloned into pgem-t easy vector (Promega). Cloned plasmids were sequenced by ABI PRISM 377 (Applied Biosystems). Detected sequences were compared with published sequences for complete HHV-6 variant B strain HST genome (NCBI database accession number, AB ) and variant A strain U1102 genome (NCBI database accession number, X ) and were used to standardize the quantification of real-time PCR. Real-time PCR. Plasma samples for quantification of HHV- 6 DNA were separated from EDTA-treated whole blood by centrifugation (1750 g for 10 min) and filtration using a filter with 0.22-mm pores. Real-time PCR was performed according to the RESULTS methods described by Locatelli et al. [48]. DNA fragments were amplified with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) in a 50-mL reaction mixture containing 10 ml of DNA samples, 25 ml of TaqMan Universal PCR master mix (Applied Biosystems), 300 nmol/l each primer (TAQ6E and TAQ6A), and 200 nmol/l TaqMan probe. Thermal cycling conditions were as follows: 50 C for 2 min, 95 C for 10 min, and 40 cycles of 95 C for 15 s and 58 C for 1 min. The detection limit of HHV-6 DNA was 50 copies/ml of plasma. Statistical analyses. Statistical tests were performed using Statview software for the Macintosh (version 5; Abacus Concepts). Univariate analyses of variables associated with positive results for HHV-6 DNA were performed by x 2 analysis, Fisher s exact test, and Student s t test. The time to first positive PCR result (in days), according to donor types, was estimated by cumulative incidence, and differences between groups were analyzed using log-rank tests. P!.05 was considered to be significant in all analyses. Incidence of positive results for HHV-6 DNA in plasma. Stable engraftment occurred in all patients. During the period after transplantation (median, 54.5 days; range, days), 24 (48%) of the 50 recipients had positive results for HHV-6 DNA in plasma (figure 1A). The median time to onset of positive HHV-6 DNA results was 18 days (range, 0 48 days) after transplantation. In addition, the onset of positive HHV-6 DNA results was concentrated at 0 9 days after neutrophil engraftment (figure 1B). HHV-6 DNA was most frequently apparent at days after transplantation (figure 2A and 2B). Of the 24 patients who had positive results, 15 (62.5%) demonstrated HHV- 6 DNA in only 1 sample, 6 (25%) in 2 samples, 2 (8.3%) in 3 samples, and 1 (4.2%) in 5 samples. The median maximum plasma HHV-6 DNA load among patients with positive results was copies/ml of plasma (range, ,465.0 copies/ml of plasma). Risk factors for positive HHV-6 DNA results. Univariate analysis revealed that transplantation from an allelic-mismatch donor and administration of steroids after SCT were associated with increased risk of positive results for HHV-6 DNA (P p.02 and P p.04, respectively, x 2 test) (table 2). Patients who received an SCT from an unrelated donor were more likely to display HHV-6 positive results, but no significant difference was identified ( P p.07, x 2 test). Figure 1C and 1D show differences in cumulative incidence of positive results according to donor type. Transplantation from an allelic-mismatch donor was again identified as a significant risk factor for a positive result for HHV-6 DNA ( P p.01, log-rank test). Influence of steroid treatment on HHV-6 DNA in plasma. Among the 24 patients who had positive results for HHV-6 DNA in plasma, 7 (29.2%) did not receive steroids, 5 (20.8%) 70 JID 2006:193 (1 January) Ogata et al.

4 Figure 1. Kaplan-Meier plot of the probability of having positive results for human herpesvirus 6 (HHV-6) DNA on testing of plasma. A, Overall cumulative incidence of positive results for HHV-6 DNA after stem cell transplantation (SCT). The cumulative rate by 70 days after transplantation was 48%. B, Overall cumulative incidence of positive results from the time of neutrophil engraftment. The day of engraftment is expressed as day 0. Of the 24 patients with positive results, 17 (70.8%) had positive results for HHV-6 DNA 0 9 days after neutrophil engraftment. C, Cumulative incidence of positive results for HHV-6 DNA in plasma for patients who received transplants from related donors (RD; n p 19) vs. unrelated donors (UD; n p 31). The cumulative incidences at day 70 were 31.6% and 58.1%, respectively ( P p.08, log-rank test). D, Cumulative incidence of positive results for HHV-6 DNA in plasma for patients who received transplants from allelic-match donors vs. allelic-mismatch donors. Cumulative incidences at day 70 were 36.4% and 70.6%, respectively ( P p.01, log-rank test). had positive results for HHV-6 DNA before beginning steroid treatment, and 12 (50.0%) began having positive results after receiving steroids. The kinetics of plasma HHV-6 DNA loads among patients in each group are shown in figure 2C 2E. In the 5 patients who developed positive results before receiving steroids, administration of steroids was started at a median of 42 days (range, days) after transplantation. Analysis of the 12 patients who first had positive results for HHV-6 DNA after receiving steroids revealed that steroid treatment was started a median of 13.5 days (range, days) after transplantation, and HHV-6 DNA was identified a median of 3 days (range, 0 19 days) after start of steroid therapy (figure 2E). Positive results for HHV-6 DNA were observed 10 days after start of steroid therapy in 11 (91.7%) of these 12 patients. All 12 patients had positive results for HHV-6 DNA during steroid administration. The mean ( SD) maximum plasma load of HHV-6 DNA was significantly higher for the 12 patients who had positive results for HHV-6 DNA during steroid treatment (25, ,532.2 copies/ml) than for the 12 patients who had positive results during the period in which steroids were not administered Plasma HHV-6 DNA Load after Allogeneic SCT JID 2006:193 (1 January) 71

5 Figure 2. A, Kinetics of human herpesvirus 6 (HHV-6) DNA loads in plasma among patients who had positive results for HHV-6 by polymerase chain reaction (PCR; n p 24). The shaded area indicates values below the threshold for detection (!50 copies/ml of plasma). B, Rate of positive results for HHV-6 by PCR in each posttransplantation period. Nos. above bars, no. of patients with positive results/no. tested. C, Kinetics of HHV-6 DNA loads in plasma among patients who did not receive steroids and who had positive results for HHV-6 DNA ( n p 7). D, Kinetics of HHV-6 DNA loads in plasma among patients who had positive results for HHV-6 DNA before beginning steroid treatment ( n p 5). The day of start of steroid treatment is day 0. Steroids were started a median of 42 days (range, days) after stem cell transplantation (SCT). E, Kinetics of HHV-6 DNA loads in plasma among patients who had positive results for HHV-6 DNA after beginning steroid treatment ( n p 12). The day of starting steroid treatment is day 0. Steroid treatment was started a median of 13.5 days (range, days) after SCT. Steroids were administrated for treatment of graft-vs.-host disease (above grade I or accompanied by pyrexia), usually beginning with intravenous methylprednisolone at 2 mg/kg for 2 days, with subsequent tapering. (including 7 patients who did not receive steroids and 5 patients who developed positive results before steroid treatment) ( copies/ml; P p.02, Student s t test). Association of HHV-6 DNA with rash. Rash was observed in 35 (70%) of the 50 SCT recipients and was clinically and histologically diagnosed as cutaneous GVHD in all patients. Positive results for HHV-6 were observed in 19 (54.3%) of the 35 SCT recipients who experienced rash (table 2). For 6 of these 19 patients, plasma HHV-6 DNA was detected in a sample at the time of the rash. Immunostaining for HHV-6 gp60/110 envelope glycoprotein was performed using skin specimens from 3 of the 6 patients who had HHV-6 DNA detected in plasma concomitant to the presence of rash. No expression of HHV-6 antigen was identified in skin specimens in any samples (data not shown). Association of HHV-6 DNA with engraftment. All patients received granulocyte colony-stimulating factor until neutrophil recovery. Positive results for HHV-6 DNA were not correlated with delayed neutrophil or platelet engraftment (table 2). Associations between high HHV-6 loads and engraftment were 72 JID 2006:193 (1 January) Ogata et al.

6 Table 2. Factors associated with positive results for human herpesvirus 6 (HHV-6) DNA in plasma after stem cell transplantation (SCT). Factor HHV-6 positive (n p 24) HHV-6 negative (n p 26) HHV-6 positive among those with factor, % Recipient age, median (range), years 42.5 (21 56) 38 (12 59).24 a Disease phase Early b Nonearly Pretransplantation conditioning Myeloablative c Nonmyeloablative TBI (12 Gy) based c Non TBI (12 Gy) based Donor Related b Unrelated Matching of HLA Antigen match c Antigen mismatch Allele match b Allele mismatch Steroid administration after SCT Yes b No Posttransplantation complication Limbic encephalitis Yes c No Skin rash Yes b No GVHD grade II IV Yes b No Cytomegalovirus reactivation Yes b No Engraftment Neutrophil, median (range), days 14 (11 27) 14.5 (11 25).99 a Platelet (by day 30) Yes b No 11 8 NOTE. Data are no. of patients, unless otherwise noted. GVHD, graft-vs.-host disease; TBI, total body irradiation. a Student s t test. b x 2 analysis. c Fisher s exact test. P also investigated. Among patients in whom HHV-6 loads exceeded the median value ( n p 12), neutrophil engraftment oc- curred at a median of 14.5 days (range, days), and platelet engraftment by day 30 was confirmed in 4 of these patients (33.3%). Comparison of these results with results for the other 38 patients (median neutrophil engraftment, day 14.5; platelet engraftment by day 30, 71.1%) revealed that high levels of HHV-6 DNA were not significantly associated with neutrophil engraftment ( P p.37, Student s t test) but were significantly associated with delayed platelet engraftment ( P p.04, Fisher s exact test). Encephalopathy. Encephalopathy developed in 8 patients. HHV-6 DNA was not detected in plasma from 4 patients during the course of encephalopathy, which was attributed, in these cases, to calcineurin inhibitor associated encephalopathy (n p 2), CNS leukemia ( n p 1), or idiopathic encephalopathy (n p 1). The remaining 4 patients had limbic encephalitis. Each of these 4 patients received a bone marrow transplant from an Plasma HHV-6 DNA Load after Allogeneic SCT JID 2006:193 (1 January) 73

7 Table 3. History of patients who displayed limbic encephalitis after stem cell transplantation. Variable Patient 1 Patient 2 Patient 3 Patient 4 Age, years (sex) 43 (M) 53 (M) 54 (F) 34 (M) Primary disorder Adult T cell leukemia Acute myeloid leukemia Acute myeloid leukemia Chronic myelogenous leukemia Diseases status at transplantation Refractory Refractory Second CR Chronic phase Matching of HLA Class II allelic mismatch Identical Class II allelic mismatch Class I allelic mismatch GVHD prophylaxis Cyclosporine/methotrexate Cyclosporine/methotrexate Cyclosporine/methotrexate Tacrolimus/methotrexate GVHD Grade II Grade I Grade I Grade IV Steroid therapy before onset Yes a Yes a Yes a Yes b Time to onset, days After transplantation After engraftment After start of steroid therapy PCR analysis at onset HHV-6 in CSF Positive NE NE NE HSV in CSF Negative NE NE Negative VZV in CSF Negative NE NE NE HSV in plasma NE Negative Negative NE VZV in plasma NE Negative Negative NE Clinical presentation EEG Short-term memory loss, loss of consciousness, seizure Diffuse lower frequencies of background activity (7 days after onset) Short-term memory loss, loss of consciousness, seizure, carbon dioxide narcosis Background activity was normal, but voltage was low (on day of onset) Short-term memory loss Lower frequencies of background activity in left frontoparietal and occipital lobes (5 days after onset) Short-term memory loss, mental confusion, hemiplegia, coma, seizure Evaluation impossible c (9 days after onset) Therapy Ganciclovir Ganciclovir Ganciclovir Ganciclovir, acyclovir Outcome Alive in CR for 24 months, but severe short-term memory difficulties remained CR for 38 months until death in an accident, but severe short-term memory difficulties remained Alive in CR for 20 months, but severe short-term memory difficulties remained Died 21 days after onset NOTE. All patients received total body irradiation (12 Gy) and cyclophosphamide (120 mg/kg); all patients received unrelated bone marrow transplants. CR, complete remission; CSF, cerebrospinal fluid; EEG, electroencephalogram; GVHD, graft-vs.-host disease; HHV, human herpesvirus; HSV, herpes simplex virus; NE, not evaluated; PCR, polymerase chain reaction; VZV, varicella-zoster virus. a Starting with 2 mg/kg methylprednisolone for 2 days, with subsequent dose tapering. b Symptoms accompanying GVHD did not improve with 2 mg/kg methylprednisolone, and methylprednisolone pulse therapy (20 mg/kg/day for 3 days and tapered) was initiated. c Due to contamination of electromyography.

8 Figure 3. Magnetic resonance imaging of the brain for the 4 patients who developed limbic encephalitis, showing axial images at the level of the basal ganglia. Arrows indicate signal hyperintensities in the region of the hippocampus. Shown are T2-weighted imaging (A) and diffusion imaging (B) for patient 1, on day +21 after stem cell transplantation (SCT), 6 days after onset of neurological symptoms. C, T2-weighted fluid attenuated inversion recovery imaging for patient 2 on day +24 after SCT, on the day of onset of neurological symptoms. D, T2-weighted imaging for patient 3 on day +40 after SCT, 20 days after onset of neurological symptoms. E, T2-weighted imaging for patient 4 on day +43 after SCT, 10 days after onset of neurological symptoms. unrelated donor, and 3 transplants were from allelic-mismatch donors (table 3). All 4 patients experienced impairment of short-term memory during days Symptoms in 3 of 4 cases appeared 10 days after neutrophil engraftment and 7 days after start of steroid therapy. Each patient was receiving calcineurin inhibitor (cyclosporine or tacrolimus) and steroids at the onset of symptoms. Two patients (patients 1 and 4) were receiving morphine, but no patient was receiving fentanyl at the time of onset of symptoms. Magnetic resonance imaging showed bilateral signal abnormalities in the region of the hippocampus in all 4 patients (figure 3). Figure 4 shows a schematic representation of the clinical courses of patients who developed limbic encephalitis. Development of neurological symptoms corresponded to the peak load of HHV-6 DNA in each patient. Three patients who continued to receive ganciclovir remained alive but retained severe short-term memory difficulties. Another patient (patient 4) who showed prolonged positive results for HHV-6 DNA in plasma (figure 4) died of encephalitis. Compared with patients who had positive results for HHV- 6 DNA but did not develop limbic encephalitis ( n p 20), those who developed limbic encephalitis tended to be more likely to have received steroid treatment by day 27 (100% vs. 40%), although this association was not statistically significant (P p.09, Fisher s exact test). Other features, including nonearly stage Plasma HHV-6 DNA Load after Allogeneic SCT JID 2006:193 (1 January) 75

9 Figure 4. Clinical courses of the 4 patients who developed limbic encephalitis. ACV, acyclovir; BMT, bone marrow transplantation; GCV, ganciclovir; TBI/CY, total body irradiation (12 Gy)/cyclophosphamide (120 mg/kg); WBC, white blood cell. of primary disorders, SCT from an unrelated donor, SCT from an allelic-mismatch donor, and patient age, were not associated with development of limbic encephalitis ( P 1.99, P p.53, P p.59 [Fisher s exact test], and P p.12 [Student s t test], respectively). The maximum plasma load of HHV-6 DNA was significantly higher in patients with limbic encephalitis (n p 4) than in patients who had positive results for HHV-6 but did not develop limbic encephalitis ( n p 20) ( P!.0001, Student s t test) (figure 5). DISCUSSION Active replication of HHV-6 is clearly not rare in SCT recipients. Steroid administration and SCT from an allelic-mismatch donor appear to be associated with increased risk of active HHV- 6 infection. Of importance, HHV-6 DNA may appear immediately after steroid therapy is begun, and virus loads are significantly increased, compared with those in patients who do not receive steroids. Steroid administration may not only increase the risk of HHV-6 infection but may also accelerate viral replication. Patients who received a transplant from an allelicmismatch donor tended to be more likely to develop GVHD of greater than grade I (allelic-mismatch donor, 41.1%; allelicmatch donor, 24.2%) and receive steroid treatment (allelicmismatch donor, 64.7%; allelic-match donor, 51.5%). In patients who received SCT from an allelic-mismatch donor, GVHD itself and intensive immunosuppressive treatment against GVHD may impair HHV-6 specific immune responses, and this may be associated with an increased risk of active HHV-6 infection following SCT. The onset and duration of active HHV-6 infection observed in the present study differed from those seen in previous studies using PCR analysis for leukocytes. Positive PCR results for HHV-6 in leukocytes were widely distributed from early to late periods after transplantation in those studies [33 37, 39 41]. In the present study, however, HHV-6 DNA was typically identified at days after transplantation. Because HHV-6 remains latent in PBMCs, PCR analysis for leukocytes may yield false-positive results [44]. Although most samples were col- 76 JID 2006:193 (1 January) Ogata et al.

10 Figure 5. Comparison of maximum plasma human herpesvirus 6 (HHV- 6) DNA loads between stem cell transplant recipients who developed limbic encephalitis ( n p 4) and those with positive results for HHV-6 DNA in plasma but without limbic encephalitis ( n p 20) ( P!.0001, Stu- dent s t test). lected weekly, most patients had positive results for HHV-6 on only 1 or 2 occasions. These findings resemble those obtained using the virus isolation method to monitor active HHV-6 infection in SCT recipients [15]. Previous reports have suggested that HHV-6 is associated with rash [12 15], bone marrow suppression [18, 19], and encephalitis [20 32] after SCT. Although 35 of the 50 recipients in the present study developed rash, HHV-6 DNA was detectable at the time of rash in only 6 patients. Regarding neutrophil and platelet recovery, no differences were noted between recipients with and those without HHV-6 infection, but high levels of HHV-6 DNA were correlated with delayed platelet recovery. The most important, life-threatening complication induced by active HHV-6 infection after SCT is encephalitis. Previous case reports have shown that HHV-6 encephalitis displays various unique characteristics: short-term memory dysfunction [25, 28, 29], symptoms appearing close to the time of engraftment [21, 26, 28, 29, 31, 32], and signal hyperintensity in the limbic area on T2-weighted magnetic resonance imaging [24, 25, 28, 29, 31, 32]. In the present study, limbic encephalitis developed in 4 patients. Symptoms and clinical courses for these patients were compatible with the previously reported characteristics of HHV-6 encephalitis. HHV-6 DNA in cerebrospinal fluid was confirmed in only 1 patient the other 3 patients did not undergo that examination. However, times at which neurological symptoms developed were in accordance with the time at which the plasma load of HHV-6 DNA peaked in each patient. Encephalopathy in these patients was probably attributable to HHV-6 infection. The maximum plasma load of HHV-6 DNA was significantly higher in patients with limbic encephalitis than in patients without limbic encephalitis. Quantification of plasma levels of HHV-6 DNA may prove to be helpful in diagnosing HHV-6 encephalitis. A high virus load in plasma, reflecting high levels of viral replication, may suggest a risk of developing HHV- 6 encephalitis. Although more research is necessary, a number of virus copies that allows prediction of HHV-6 encephalitis may be copies/ml of plasma. A MEDLINE search identified 17 cases reported as HHV-6 encephalitis that developed 100 days after SCT [20, 21, 23, 24, 26, 28 32]. A review determined that encephalitis occurred a median of 24 days after transplantation (range, days), whereas 8 (72.7%) of 11 patients for whom donor information was available received transplants from an HLA-mismatch donor, and encephalitis occurred after steroid treatment in 9 (81.8%) of the 11 patients for whom information was available. The onset of encephalitis described in these reports corresponded with the period in which plasma HHV-6 DNA became detectable in the present study. Risk factors for active HHV-6 infection confirmed in the present study may correspond with risk factors for the development of HHV-6 encephalitis. HHV-6 infection is reportedly more frequent in cord-blood SCT recipients [42]. Patients who underwent cord-blood SCT were not added to the present study, because few such cases were handled by our institute. Engraftment occurred in 3 of the 5 cord-blood SCT recipients during 8 weeks after transplantation in our institute. The maximum plasma load of HHV- 6 DNA in these 3 recipients was high, compared with that in patients in the present study (10,860.5 copies/ml, 20,950.5 copies/ml, and 372,696.0 copies/ml; P p.004, Student s t test). Of interest, positive identification of plasma HHV-6 DNA was never made in the 2 cord-blood SCT recipients with graft failure. Conversely, HHV-6 appeared close to the time of engraftment in the SCT recipients examined in the present study. Engraftment may be essential for HHV-6 reactivation after SCT. In conclusion, HHV-6 DNA positive results were typically apparent days after transplantation. Active HHV-6 infection typically occurred close to the time of neutrophil engraftment but was transient in most cases. SCT from an allelicmismatch donor and steroid administration were associated with increased risk of active HHV-6 infection. Development of limbic encephalitis was associated with high HHV-6 load. Ste- Plasma HHV-6 DNA Load after Allogeneic SCT JID 2006:193 (1 January) 77

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