Plasma Human Herpesvirus-6 DNA Research Articles

Once-Daily Foscarnet Is Effective for Human Herpesvirus 6 Reactivation after Hematopoietic Stem Cell Transplantation

Human herpesvirus 6 (HHV-6) reactivation is common after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is associated with higher mortality and increased transplantation-related complications. We hypothesized that preemptive treatment with a short course of foscarnet at a lower cutpoint of plasma HHV-6 viral load would be effective in treating early HHV-6 reactivation, preventing complications and precluding hospitalization of these patients. We reviewed outcomes of adult patients (age ≥18 years) who received preemptive treatment with once-daily foscarnet 60 to 90 mg/kg for 7 days for HHV-6 reactivation after allo-HSCT at our institution between May 2020 and November 2022. Plasma HHV-6 viral load was monitored by quantitative PCR twice monthly in the first 100 days post-transplantation and twice weekly after reactivation until resolution. Eleven patients with a median age of 46 years (range, 23 to 73 years) were included in the analysis. HSCT was performed with a haploidentical donor in 10 patients and with an HLA-matched related donor in 1 patient. The most common diagnosis was acute leukemia (9 patients). Myeloablative and reduced-intensity conditioning regimens were used in 4 and 7 patients, respectively. Ten of the 11 patients received post-transplantation cyclophosphamide-based graft-versus-host disease prophylaxis. The median follow-up was 440 days (range, 174 to 831 days), and the median time to HHV-6 reactivation was 22 days post-transplantation (range, 15 to 89 days). The median viral load at first reactivation was 3100 copies/mL (range, 210 to 118,000 copies/mL), and the median peak viral load was 11,300 copies/mL (range, 600 to 983,000 copies/mL). All patients received a short course of foscarnet at either 90 mg/kg/day (n = 7) or 60 mg/kg/day (n = 4). In all patients, plasma HHV-6 DNA was undetectable at completion of 1 week of treatment. No HHV-6 encephalitis or pneumonitis occurred. All patients achieved neutrophil and platelet engraftment after a median of 16 days (range, 8 to 22 days) and 26 days (range, 14 to 168 days), respectively, with no secondary graft failure. No complications related to foscarnet administration were noted. One patient with very high HHV-6 viremia had recurrent reactivation and received a second course of foscarnet as an outpatient. A short course of once-daily foscarnet is effective in treating early HHV-6 reactivation post-transplantation and may reduce the incidence of HHV-6-related and treatment-related complications and preclude hospitalization in these patients.

Kinetics and clinical significance of human herpesvirus 6 DNA shedding in saliva after allogeneic hematopoietic stem cell transplantation.

Little is known about the kinetics and clinical significance of saliva human herpesvirus-6 (HHV-6) DNA after hematopoietic stem cell transplantation (HSCT). In this observational study, we quantified HHV-6 DNA in serially collected plasma and saliva from allogeneic HSCT recipients. Associations between the status of salivary HHV-6 DNA and the development of HHV-6 encephalitis, depression, and oral mucosal graft-versus-host disease (GVHD) were retrospectively analyzed. A total of 787 plasma and 434 saliva samples were collected from 56 patients. The cumulative incidence of HHV-6 DNA in plasma and saliva at 60days after transplantation was 51.8% and 83.9%, respectively. The peak level of salivary HHV-6 DNA was significantly higher in patients who displayed plasma HHV-6 DNA than in those who did not (median, 51,584copies/mL vs 587copies/mL; P<.0001). Salivary HHV-6 DNA levels increased after positive plasma HHV-6 DNA was detected and remained high during observation period. Despite the frequent occurrence of positive salivary HHV-6 DNA, no patient developed depression. Positivity of salivary HHV-6 DNA was not significantly associated with the development of HHV-6 encephalitis (P=1.00, Fisher's exact test) or oral mucosal GVHD (P=.71, Grey's test). No significant relationship between salivary HHV-6 DNA and these diseases was found even when comparing higher HHV-6 DNA loads in saliva. Salivary HHV-6 DNA levels increased after HHV-6 DNA was detected in the blood. However, no epidemiological evidence was shown to support a role of salivary HHV-6 in the development of HHV-6 encephalitis, depression, and oral mucosal GVHD.

Effects of Prophylactic Foscarnet on Human Herpesvirus-6 Reactivation and Encephalitis in Cord Blood Transplant Recipients: A Prospective Multicenter Trial with an Historical Control Group

Cord blood transplantation (CBT) is a distinct risk factor for human herpesvirus-6 (HHV-6) reactivation and HHV-6 encephalitis. In a prospective multicenter trial we investigated the effects of prophylactic foscarnet (90 mg/kg i.v. infusion from days 7 to 27 after CBT) on the occurrence of HHV-6 reactivation, HHV-6 encephalitis, and acute graft-versus-host disease (aGVHD) in CBT recipients. Between 2014 and 2016, 57 patients were included in a foscarnet-prophylaxis group. Outcomes were compared with an historical control group who received CBT between 2010 and 2014 (standard-treatment group, n = 63). The cumulative incidence of high-level HHV-6 reactivation, defined as plasma HHV-6 DNA ≥ 104 copies/mL, at 60 days after CBT was significantly lower in the foscarnet-prophylaxis group than in the standard-treatment group (18.3% versus 57.3%, P < .001). Multivariate analysis revealed that myeloablative preconditioning and standard treatment were significant risk factors for high-level HHV-6 reactivation. The cumulative incidence of HHV-6 encephalitis at 60 days after CBT was not different between the groups (foscarnet-prophylaxis group, 12.4%; standard-treatment group, 4.9%; P = .14). The cumulative incidences of grades II to IV and grades III to IV aGVHD at 60 days after CBT were not different between the groups (grades II to IV aGVHD: foscarnet-prophylaxis group, 42.0%; standard-treatment group, 40.5%; P = .96; grades III to IV aGVHD: foscarnet-prophylaxis group, 14.5%; standard-treatment group, 14.5%; P = 1.00). In the setting of this study foscarnet significantly suppressed systemic HHV-6 reactivation in CBT recipients but failed to prevent the development of HHV-6 encephalitis. Suppression of HHV-6 reactivation by foscarnet did not show any effects against the incidence of aGVHD.

Human Herpesvirus 6 (HHV-6) Reactivation and HHV-6 Encephalitis After Allogeneic Hematopoietic Cell Transplantation: A Multicenter, Prospective Study

The epidemiology of human herpesvirus 6 (HHV-6) encephalitis after allogeneic hematopoietic cell transplantation (HCT) and its relationship with HHV-6 reactivation have not been sufficiently characterized. This prospective, multicenter study of 230 allogeneic HCT recipients investigated the epidemiology of HHV-6 reactivation and HHV-6 encephalitis. Plasma HHV-6 DNA load was prospectively evaluated twice weekly until 70 days after HCT. Cumulative incidence of positive HHV-6 DNA and high-level HHV-6 reactivation (plasma HHV-6 DNA ≥10(4) copies/mL) at day 70 after HCT was 72.2% and 37.0%, respectively. Multivariate analysis identified myeloablative conditioning (hazard ratio [HR], 1.9; P = .004), umbilical cord blood transplantation (UCBT) (HR, 2.0; P = .003), and male sex (HR, 1.6; P = .04) as risk factors for displaying high-level HHV-6 reactivation. HHV-6 encephalitis occurred in 7 patients, and cumulative incidence at day 70 was 3.0%. None of the144 patients without high-level HHV-6 reactivation and 7 of 86 patients (8.1%) with high-level HHV-6 reactivation developed HHV-6 encephalitis (P = .0009). Prevalence of HHV-6 encephalitis was significantly higher among patients receiving UCBT than in patients with other sources (cumulative incidence at day 70, 7.9% vs 1.2%, P = .008). In each of 7 patients with HHV-6 encephalitis, central nervous system (CNS) symptoms developed concomitant with peak plasma HHV-6 DNA (range, 21 656-433 639 copies/mL). High levels of plasma HHV-6 DNA are associated with higher risk of HHV-6 encephalitis. UCBT is a significant risk factor for HHV-6 encephalitis. HHV-6 encephalitis should be considered if CNS dysfunction develops concomitant to high-level plasma HHV-6 DNA after allogeneic HCT.

Prophylactic Foscarnet and Suppression of Pre-Engraftment Immune Reaction Are Essential to Overcome the Development of HHV-6 Encephalitis From the Experience of 496 Adult Cord Blood Transplants in Toranomon Hospital

Abstract 461 Background:Human herpes virus 6 (HHV-6) encephalitis is a growing concern after cord blood transplantation (CBT), which leads to permanent neurological sequelae that do not allow patients to get back into society. The pathogenic mechanism remains unknown and appropriate preventative strategies have not been established. Patients and methods:To evaluate incidences, risk factors and outcomes of HHV-6 encephalitis after CBT, we reviewed the medial records of 496 adult patients who underwent CBT for the first time at the Toranomon Hospital between 2002 and 2011, and who survived more than 7 days. Over the entire period, routine prophylaxis with acyclovir against reactivation of herpesviruses and pre-emptive therapy with ganciclovir or foscarnet (FCV) against cytomegalovirus disease were performed. From January 2006, prophylactic administration of FCV with monitoring of serum HHV-6 viral load against HHV-6 disease was introduced, the indication of which was decided at a physician's discretion. Plasma HHV-6 DNA copy numbers were measured using real-time PCR method, and the detection limit was 200 copies/mL. Immune reaction prior to neutrophil engraftment characterized by unexplained fever in the absence of documented infection with skin eruption, peripheral edema and body-weight gain was defined as pre-engraftment immune reaction (PIR), which was categorized as mild and severe, according to the degree of concomitant organ dysfunctions. HHV-6 encephalitis was defined as the neurological symptoms with positive PCR results for HHV-6 in cerebrospinal fluid (CSF) and the absence of other identified etiologies of encephalitis. Results:Forty-two patients developed HHV-6 encephalitis with a cumulative incidence of 8.5%. The incidence was significantly higher in the period from 2006 to 2011 compared to the period from 2002 to 2005 in spite of the introduction of prophylactic FCV after 2006 (11.1% vs. 4.3%, P=0.01), probably owing to more vigorous investigation. Among the 308 patients who received CBT after 2006, engraftment was achieved in 239 at a median of 20 (10–66) days after CBT, and PIR occurred in 133 patients at a median of 10 (4–24) days, the severity of which was classified into mild in 114 and severe in 19. 132 patients developed grade II-IV acute graft-versus-host disease (GVHD) at a median of 31 (9–91) days, and 182 received high-dose corticosteroids at >= 0.5mg/kg with a median starting point of 19.5 (0–1363) days. Serum HHV-6 PCR measurement was performed in 280 patients, 122 of who showed positive test results at a median of 20 (8–193) days. Prophylactic FCV was given in 234 patients from a median of 11 (0–35) days, with a median duration of 23 (2–79) days. HHV-6 encephalitis occurred in 34 of the 308 patients at a median of 22 (11–49) days, with a cumulative incidence of 11.2%. The median peak level of HHV-6 DNA was 20,000 (200–400,000) copies/mL in CSF, in contrast with 200 (0–89,300) copies/mL in plasma. PIR was identified as an independent significant risk factor for HHV-6 encephalitis (HR 3.13(1.52–6.44), P=0.002), which occurred more frequent in patients with severe PIR compared to those with mild one (HR 2.82(1.11–7.18), P=0.029). Another significant risk factor was high-level of HHV-6 DNA in plasma at >=10,000 copies/mL (HR 3.29(1.17–9.30), P=0.024), which was, however, observed in only 7 of the 34 patients with encephalitis. Neither grade II-IV acute GVHD nor the use of high-dose steroids influenced an incidence of the encephalitis (HR 2.2(0.90–5.37), P=0.083, and HR 0.98(0.47–2.03), P=0.95). Prophylactic FCV at >=60mg/kg showed a significant benefit in preventing HHV-6 encephalitis (HR 0.27(0.09–0.77), P=0.015), which substantially reduced the incidence to 1.6% if PIR was completely suppressed, whereas the incidence reached 23.4% in the presence of PIR if the FCV was not given (P<0.001). Although no patient directly died of HHV-6 encephalitis, the encephalitis tended to affect non-relapse mortality after CBT (HR 1.75(0.99–3.07), P=0.053). Conclusions:PIR seems to bring at least similar or more risk for HHV-6 encephalitis compared to high-level HHV-6 viral load. Prophylaxis with FCV at >=60mg/kg combined with the optimal suppression of PIR might provide a further reduction of HHV-6 encephalitis after CBT. Disclosures:No relevant conflicts of interest to declare.

Safety of pre‐engraftment prophylactic foscarnet administration after allogeneic stem cell transplantation

Human herpesvirus-6 (HHV-6) is a major cause of limbic encephalitis with a dismal prognosis after allogeneic hematopoietic stem cell transplantation (SCT). Because our previous trial of preemptive therapy with foscarnet sodium (phosphonoformic acid; PFA) failed to prevent HHV-6 encephalitis, we conducted a prospective study to examine the safety of prophylactic PFA administration and elucidate the changes in the plasma HHV-6 DNA levels in the early post-SCT period. Plasma HHV-6 DNA was measured thrice weekly from day 6. PFA, 90 mg/kg/day, was administered from days 7 to 21 after bone marrow or peripheral blood SCT and to day 25 after umbilical cord blood transplantation. Of the 10 patients enrolled, 2 dropped out of the study, 1 because of early death, and 1 with a low glomerular filtration rate. Grade 3 or greater adverse events occurred in 9 of the 10 prophylactic PFA patients and in 7 of the 10 control patients who had clinical backgrounds similar to the study subjects and underwent SCT during the same period. Neurological disorders developed in none of the study subjects but in 4 of the 10 control patients, including 2 with HHV-6 encephalitis. HHV-6 reactivation occurred in 3 of the 10 study subjects. The prophylactic PFA regimen was thus safe and it may reduce the risk of limbic encephalitis, but is not considered to be potent enough to prevent HHV-6 reactivation.

Preemptive therapy of human herpesvirus-6 encephalitis with foscarnet sodium for high-risk patients after hematopoietic SCT

Human herpesvirus-6 (HHV-6) is a major cause of limbic encephalitis with a dismal prognosis after allogeneic hematopoietic SCT (HSCT). A prospective, multicenter study was conducted to assess the safety and efficacy of preemptive therapy with foscarnet sodium (PFA) for the prevention of HHV-6 encephalitis. Plasma HHV-6 DNA was measured thrice weekly from day 7 until day 36 after umbilical cord blood transplantation (UCBT) or HSCT from HLA-haploidentical relatives. PFA, 90 mg/kg/day, was started when HHV-6 DNA exceeded 5 × 10(2) copies/mL. Mild and transient adverse events were associated with PFA in 7 of 8 patients. Twelve of 15 UCBT recipients became positive for HHV-6 DNAemia, defined by greater than 1 × 10(2) copies/mL of HHV-6 DNA in plasma. The virus exceeded 5 × 10(2) copies/mL in seven patients, whereas none of the five HLA-haploidentical HSCT recipients became positive. One patient developed mild limbic encephalitis just after initial PFA administration. Preemptive PFA therapy is safe, but as HHV-6 DNAemia can abruptly develop before neutrophil engraftment in UCBT recipients, prophylactic PFA administration from day 7 or earlier after UCBT may be needed.

Translational Challenges Of Human Herpesvirus 6 Chromosomal Integration

Translational Challenges Of Human Herpesvirus 6 Chromosomal Integration

Diagnostic assays for active infection with human herpesvirus 6 (HHV-6)

Background Human herpesvirus 6 (HHV-6) causes ubiquitous infection in early childhood with lifelong latency or persistence. Reactivation of HHV-6 has been associated with multiple diseases including encephalitis. Chromosomal integration of HHV-6 also occurs. Previous studies have suggested that the detection of HHV-6 DNA in plasma is an accurate marker of active viral replication. Objective We sought to determine whether PCR assays on plasma could correctly differentiate between primary HHV-6 infection, chromosomal integration of HHV-6 and latent HHV-6 infection. Study design We performed qualitative PCR, real-time quantitative PCR (RQ-PCR), and reverse-transcriptase PCR (RT-PCR) assays on samples of peripheral and cord blood mononuclear cells, as well as plasma, from groups of subjects with well defined HHV-6 infection, including subjects with chromosomally integrated HHV-6. Results and conclusions The detection of HHV-6 DNA in plasma was 92% sensitive compared to viral isolation for the identification of primary infection with HHV-6. All plasma samples from infants with chromosomally integrated HHV-6 had HHV-6 DNA detectable in plasma while only 5.6% were positive by RT-PCR. The specificity of plasma PCR for active replication of HHV-6 was 84% compared to viral culture while the specificity of RT-PCR was 98%. Our results demonstrate that qualitative or quantitative PCR of plasma is insufficient to distinguish between active viral replication and chromosomal integration with HHV-6. We found a higher specificity of RT-PCR performed on PBMC samples compared to PCR or RQ-PCR performed on plasma when evaluating samples for active HHV-6 replication.

Assessment of the Impact of Human Herpesvirus 6 (HHV-6) Replication On Active Citomegalovirus (CMV) Infection After Allogeneic Stem Cell Transplantation (Allo-SCT).

Abstract 2225Poster Board II-202 Introduction:In spite of significant advances in the diagnosis and treatment of CMV infection post-allogeneic SCT it continues to be associated with significant morbidity and mortality. The human herpesvirus 6 (HHV-6) reactivation has been associated with different complications, including delayed neutrophil and platelet engraftment, interstitial pneumonia, skin rash, severe graft versus host disease (GvHD), and central nervous system disorders. HHV-6 is considered an immunomodulatory and immunosuppressive agent, and thereby, it may the increase the risk of active CMV infection and disease in the transplantation setting Aims:To determine if the HHV-6 facilitates the CMV reactivation or influences the kinetic of CMV replication and its specific immune replication during the first 100 days after allogeneic hematopoietic SCT. Patients and Methods:Between Nov-05 and Feb-08, sixty-eight patients were monitored for plasma HHV-6 and CMV DNAemia by PCR once/week until day +100 after Allo-SCT. Characteristics of patients: median age: 45 yo (18-70); sex (M/F): 37/31. Diagnosis: AML/MDS(n=27), ALL(n=10), lymphoproliferative syndrome(n=17), MM (n=4), CML (n=3), Others(n=7). CMV seropositive (R/D)(%): 88/60. Cell source: PBSC: 53(78%);CB:13(19%);BM:2(3%). Conditioning treatment: RIC: 40 pts, myeloablative: 28 pts. pp65 antigenemia in PMN leucocytes (Light Diagnostics, Chemicon Intern). Positive if ≥1cel+/200.000. Plasma DNA-CMV: PCR-RT Abbott or Amplicor CMV Monitor Roche. Plasma DNA-HHV6: Q-PCR Alert Amplimix, Nanogen Adv. Diag., detection limit: 10 copies/mL plasma. Enumeration of pp65 and IE-1 IFNγ CD4+ and CD8+ T cells was performed by intracellular cytokine staining. (BD Fastimmune, BD-Beckton Dickinson). Pre-emptive antiviral treatment was initiated if pp65 Ag +. Results:HHV-6 DNAemia occurred in 27 out of 68 pts (40%), after a median time of 20 days post-SCT with a peak value of 345 copies (26-13,661) reached on the fourth week in the 52% of patients, with a median duration of 10 days (3-35). Twenty-three out of the 27 patients were HHV-6 seropositive before transplantation. Clearance of most episodes (21 out of 27) occurred in the absence of (val)ganciclovir treatment, which was initiated in the course of the remaining 6 episodes because of the development of antigenemia-positive active CMV infection. HHV-6 DNAemia was significantly associated with subsequent CMV DNAemia in univariate (P=.01), but not in multivariate analysis (P=.056) that included transplant from URD, HLA non identical donor, myeloablative conditioning, CBT, use of prednisone as GVHD prophylaxis and plasma HHV-6 DNA detection. HHV-6 DNAemia was not predictive of development of CMV DNAemia. Timing and kinetics of active CMV infection were comparable in patients either with or without a preceding episode of HHV-6 DNAemia. Occurrence of HHV-6 DNAemia had no impact on the level of CMV-specific T-cell immunity reconstitution early after transplant. The receipt of a graft from an unrelated donor was the principal variable associated with HHV-6 and CMV coactivation. Conclusion:The results of this study suggest that a basal immunosuppressive state, specially the receipt of a graft from an unrelated donor, leads to HHV-6 and CMV coactivation, and argue against an effect of active HHV-6 infection on CMV replication either by a direct interaction or indirectly by suppressing CMV-specific T-cell responses. Disclosures:No relevant conflicts of interest to declare.

Preemptive Therapy of HHV-6 Encephalitis with Foscarnet Sodium for High Risk Patients After Hematopoietic Stem Cell Transplantation.

Abstract 4655Umbilical cord blood from unrelated donors has been successfully used as an alternative hematopoietic stem cell source to treat hematologic malignancies in patients lacking HLA-matched donors. However, umbilical cord blood transplantation (UCBT) is associated with a higher risk of engraftment failure and more delayed immunological recovery than bone marrow transplantation and peripheral blood stem cell transplantation (PBSCT). Recently, human herpesvirus-6 (HHV-6) has been recognized as an important pathogen in allogeneic hematopoietic stem cell transplantation (HSCT). In particular, HHV-6 reactivation often causes limbic encephalitis with a dismal prognosis. We conducted a prospective, multicenter study to assess the safety and efficacy of preemptive therapy with foscarnet sodium (PFA) to prevent HHV-6 encephalitis after HSCT. Materials and methodsEligible patients were aged from 16 to 75 years with hematologic disorders refractory to conventional therapy and considered to require UCBT or HLA 1-haplotype mismatched HSCT (haplo HSCT) due to the unavailability of an HLA-identical relatives or a suitable unrelated donor. Informed consent was obtained from all subjects according to the Declaration of Helsinki, and this study protocol was approved by the institutional ethical committee. The amount of plasma HHV-6 DNA was measured 3 times per week between day 7 and day 36 after UCBT or PBSCT from HLA-haploidentical relative donors. PFA, 90 mg/kg/day, was given when the amount of HHV-6 DNA exceeded 5 ×102 copies/ml. ResultsOf 20 cases registered between September 2007 and January 2009, 12 of 15 UCBT recipients (80%) became positive for HHV-6 DNAemia, and 7 cases exceeded 5×102 copies/ml, while none of the 5 patients who received haplo HSCT became positive (UCBT vs. haplo HSCT; p<0.004). HHV-6 reactivation occurred earlier in patients who eventually required PFA due to an increase in the HHV-6 DNA copy number greater than 5×102 copies/ml than in patients who did not require PFA treatment (median date of developing HHV-6 DNAemia, day 17 vs. day 22 after HSCT, p<0.02). PFA was given to 7 patients whose HHV-6 DNA copy number exceeded 5×102 copies/ml on day 15 to day 20 (median, day 17) after UCBT. The amount of HHV-6 DNA in the plasma decreased the day after PFA administration in 4 of 7 patients, while the other 3 patients required 3-4 days until the copy number decreased. Two patients showed an increase in HHV-6 DNA copy number greater than 1×104 /ml prior to the initiation of PFA without accompanying symptoms suggestive of encephalitis. One patient developed limbic encephalitis with mild symptoms just after initial PFA administration, but the encephalitis resolved without any neurologic sequelae. Mild and transient adverse effects were associated with PFA in only 2 of 8 patients. ConclusionPFA administration guided by the HHV-6 copy number in the early posttransplant period is safe and may reduce the risk of severe limbic encephalitis. Disclosures:Nakao:Alexion: Research Funding.

Human herpesvirus-6 (HHV-6) DNA in plasma reflects the presence of infected blood cells rather than circulating viral particles

Background The presence of HHV-6 DNA in plasma or serum is considered a good marker of active infection. However, it is ignored whether this DNA corresponds to virus particles produced by lymphoid tissue infection or virus-free DNA released from infected circulating blood cells. Objectives To investigate whether HHV-6 DNA in whole plasma is nonencapsidated and its amount is correlated to cellular and human herpesvirus-7 (HHV-7) DNA loads in plasma subfractions as well as in corresponding peripheral blood mononuclear cells (PBMCs). Study design Whole plasma samples from immunocompromised patients were submitted to a DNase-resistance test. Plasma samples from a second group of patients were split up into three subfractions: P1 (pellet of clarification), P2 (pellet of ultracentrifugation), and S (supernatant of ultracentrifugation). HHV-6, HHV-7, and cellular DNA loads were determined in each fraction and PBMCs using specific real-time PCR. Results Among 14 whole plasma samples, the majority of HHV-6 DNA detected was unprotected against DNase, i.e. nonencapsidated. The study of 35 other plasma samples revealed that cellular DNA was present in all subfractions from all samples whereas HHV-6 DNA was detected in 13 P1, 12 P2, 10 S fractions, and HHV-7 DNA in only one P1 fraction. Accordingly, median HHV-6 DNA load was significantly higher in P1 than in P2 and S fractions. The detection of HHV-6 DNA in plasma subfractions was statistically associated with a higher HHV-6 viral load in PBMCs ( p ≤ 0.0003). Conclusions Taken together, these data tend to favour the hypothesis of a release of HHV-6 and cellular DNA into plasma following the lysis of infected PBMCs. HHV-6 DNA in plasma does not necessarily reflect the amount of virus produced by the active infection of distant lymphoid tissue and organs.

Human herpesvirus 6 infection and transient acquired myelodysplasia in children

To demonstrate that primary human herpesvirus 6 (HHV-6) infection in childhood can cause hematopoietic dysplasia that mimics a myelodysplastic syndrome (MDS) in severe cases. Seven immunocompetent children, who presented at admission with concomitant cytopenias in blood and morphologic features of dysplasia in bone marrow, were evaluated. Diagnosis of acute HHV-6 infection was secondary made by detection of HHV-6 DNA in plasma, bone marrow, or cerebrospinal fluid and measurement of plasma antibody titers. Peripheral blood and bone marrow aspirate smears were examined at diagnosis and during follow-up. Morphologic recognition of myelodysplasia was made according to the recommendations of the Third MIC Cooperative Group. Anemia was the most frequent cytopenia (five of seven cases). Bi- or tri-lineage dysplasia was observed in the marrow samples. Granulocytic and erythroid cells were always affected with dysgranulopoiesis and dyserythropoiesis scores equal to or higher than 3. Myelodysplasia was not due to a clonal disorder and disappeared gradually within 1 or 2 months. Our results indicate that severe HHV-6 infection may induce reversible myelodysplastic changes. These findings contribute to elucidate the pathogenicity of HHV-6 and furthermore suggest that HHV-6 infection must also be considered as a cause of dysplasia in the differential diagnosis of MDS.

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

Human herpesvirus 6 (HHV-6) is increasingly recognized as an opportunistic and potentially life-threatening pathogen in recipients of allogeneic stem cell transplants (SCTs). 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. HHV-6 DNA was detected in plasma from 24 patients (48%). HHV-6 DNA was most frequently apparent approximately 14-27 days after transplantation. An increased risk of a positive result for HHV-6 DNA was associated with transplantation from an allelic-mismatch donor (P = .02) and administration of steroids (P = .04). Steroid use was associated with high HHV-6 DNA loads (P = .02). High HHV-6 DNA loads were correlated with delayed platelet engraftment (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 = 4) (P < .0001). 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.

Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification.

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.

Human herpesvirus 6 infection in febrile infants ninety days of age and younger.

The importance of human herpesvirus 6 (HHV-6) as a pathogen in febrile infants </=90 days of age is unknown. To determine whether febrile infants 90 days of age and younger evaluated for sepsis have evidence of HHV-6 DNA in plasma or cerebrospinal fluid (CSF). Febrile infants </=90 days of age were tested for HHV-6 DNA using a real time quantitative fluorescent probe polymerase chain reaction assay. Eighty samples from 47 infants were tested for HHV-6 DNA; 5 of 47 infants (10.6%) had HHV-6 DNA in plasma. In 2 of the 5 infants with HHV-6 DNA in plasma, HHV-6 DNA was also detected in the CSF. Both infants with evidence of HHV-6 DNA in plasma and CSF had HHV-6 Variant A infection. The quantity of HHV-6 DNA detected ranged from 70 to 169,000 DNA copies/ml. One infant with HHV-6 variant B infection had concomitant Escherichia coli bacteremia and urinary tract infection. Approximately 10% of febrile infants </=90 days of age evaluated for sepsis had evidence of HHV-6 infection. HHV-6 Variant A and B infections were seen in these young infants. HHV-6 DNA was found in infants with and without another explanation for fever. Quantification of viral DNA may be important in determining the relevance of HHV-6 DNA in clinical specimens.

A prospective case control study of the association of Gianotti-Crosti syndrome with human herpesvirus 6 and human herpesvirus 7 infections.

Gianotti-Crosti syndrome (GCS) is known to be associated with hepatitis B and Epstein-Barr virus (EBV) infections. Apart from a single case report based on serology alone, there are no published data on an association between GCS and human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) primary infections or reactivations. Our aim was to investigate the association between GCS and HHV-6 and HHV-7 infections. Ten patients diagnosed with GCS at a primary care practice over an 18-month period were recruited. Controls were age- and sex-matched patients with unrelated symptoms requiring venepuncture for other indications. Blood specimens were collected from patients and controls at presentation, and from patients 4 weeks later. Virologic evidence of HHV-6 and HHV-7 infection was sought in peripheral blood leukocytes and plasma using polymerase chain reaction (PCR) for viral DNA, reverse transcriptase polymerase chain reaction (RT-PCR) for HHV-6 U91 mRNA transcripts, and serology. Serology for EBV and hepatitis B virus was done. In contrast to the 10 controls, 2 patients (both infants) with clinically diagnosed GCS had evidence of active HHV-6 infection. This was demonstrated by detection of viral DNA in the absence of antibody in the acute plasma specimens and HHV-6 DNA viral loads of more than 5.3 log10 genome copies/5 microl in the whole blood specimens, a profile previously shown to be diagnostic of recent primary HHV-6 infection. None of the patients had evidence of recent EBV or hepatitis B infection. We conclude that primary HHV-6 infection may be associated with GCS in some infants.

Human herpesvirus 6 infection after living related liver transplantation

The aim of the study was to investigate human herpesvirus-6 (HHV-6) infection after liver transplantation from living related donors, and to evaluate the reliability of the presence of HHV-6 DNA in plasma by the polymerase chain reaction (PCR) for monitoring active HHV-6 infection. EDTA peripheral blood was collected from 47 donor and recipient (16 males and 31 females, age 1–320 months) pairs at the time of transplantation and biweekly from these recipients after transplantation until 2 months after operation. Isolation of HHV-6 and serological assays were carried out to evaluate active HHV-6 infection in this study. The presence of the viral DNA in plasma was tested by nested PCR. Four clinical events, such as unexplained fever, thrombocytopenia, rejection, and central nervous system (CNS) involvement, were evaluated for clinical features of the virus infection. Risk factors for the virus activity after liver transplantation were also examined. HHV-6 activity was detected in 23 (49%) of the 47 recipients approximately 2–4 weeks after transplantation. All 9 isolates were HHV-6 variant B. The presence of the viral DNA in plasma correlated well with virus isolation and serology (P < 0.01). Only unexplained fever was associated statistically with HHV-6 activity after liver transplantation (P < 0.01). If the recipient was seronegative to HHV-6 before transplantation, the recipient was more likely to develop the active virus infection after liver transplantation (P = 0.11). HHV-6 activity occurred in one-half of the recipients approximately 2–4 weeks after liver transplantation, and there was a close association between HHV-6 activity and unexplained fever following transplantation. Detection of the viral DNA in plasma by PCR is useful for monitoring active HHV-6 infection in these patients. Seronegative recipients were more likely to have evidence of active HHV-6 infection after liver transplantation. J. Med. Virol. 62:52–59, 2000. © 2000 Wiley-Liss, Inc.

Human herpesvirus 6 infection after living related liver transplantation

The aim of the study was to investigate human herpesvirus-6 (HHV-6) infection after liver transplantation from living related donors, and to evaluate the reliability of the presence of HHV-6 DNA in plasma by the polymerase chain reaction (PCR) for monitoring active HHV-6 infection. EDTA peripheral blood was collected from 47 donor and recipient (16 males and 31 females, age 1-320 months) pairs at the time of transplantation and biweekly from these recipients after transplantation until 2 months after operation. Isolation of HHV-6 and serological assays were carried out to evaluate active HHV-6 infection in this study. The presence of the viral DNA in plasma was tested by nested PCR. Four clinical events, such as unexplained fever, thrombocytopenia, rejection, and central nervous system (CNS) involvement, were evaluated for clinical features of the virus infection. Risk factors for the virus activity after liver transplantation were also examined. HHV-6 activity was detected in 23 (49%) of the 47 recipients approximately 2-4 weeks after transplantation. All 9 isolates were HHV-6 variant B. The presence of the viral DNA in plasma correlated well with virus isolation and serology (P < 0.01). Only unexplained fever was associated statistically with HHV-6 activity after liver transplantation (P < 0. 01). If the recipient was seronegative to HHV-6 before transplantation, the recipient was more likely to develop the active virus infection after liver transplantation (P = 0.11). HHV-6 activity occurred in one-half of the recipients approximately 2-4 weeks after liver transplantation, and there was a close association between HHV-6 activity and unexplained fever following transplantation. Detection of the viral DNA in plasma by PCR is useful for monitoring active HHV-6 infection in these patients. Seronegative recipients were more likely to have evidence of active HHV-6 infection after liver transplantation.

Early diagnosis of primary human herpesvirus 6 infection in childhood: serology, polymerase chain reaction, and virus load.

Qualitative and quantitative polymerase chain reaction (PCR) for human herpesvirus 6 (HHV-6) DNA in whole blood and plasma was correlated with serology and clinical assessment in 143 children hospitalized for undifferentiated febrile illness to evaluate options for diagnosis of primary HHV-6 infection on the acute blood specimen. PCR and serology for HHV-7 were done in parallel to define serologic cross-reactions. Using HHV-6 seroconversion as the reference standard, detection of HHV-6 DNA in whole blood in the absence of antibody in the plasma was the most reliable evidence of primary HHV-6 infection. Detection of HHV-6 DNA in plasma and a high virus load in whole blood (>3.3 log10 copies/5 microL) had a sensitivity of 90% and 100%, respectively, in diagnosing primary HHV-6 infection. However, both were occasionally found in patients with other infections, possibly associated with HHV-6 reactivation. Maternal antibody may confound interpretation of serology in patients under 3 months of age.