Quantitation of human herpesvirus-6 (HHV-6) DNA in a cord blood transplant recipient with chromosomal integration of HHV-6.

Chromosomal integration of the human herpesvirus-6 (HHV-6) genome (CIHHV-6) is an important consideration if HHV-6 DNA is detected during the course of transplantation. A 4-year-old girl with refractory anemia with excess blasts type-2 was diagnosed with CIHHV-6 before a cord blood transplantation. HHV-6 DNA was serially quantitated by polymerase chain reaction assay in the transplant period. The possibility of HHV-6 reactivation in a transplant recipient with CIHHV-6 was suspected in our case.

Transmission of chromosomally integrated human herpesvirsus 6 (HHV-6) variant A from a parent to children leading to misdiagnosis of active HHV-6 infection.

Only a handful of cases of chromosomally integrated human herpesvirus 6 (CI-HHV-6) have been reported, suggesting that this phenomenon is rare. We here present a familial case of HHV-6 variant A (HHV-6A) transmission through a generation, which was identified in the setting of allogeneic hematopoietic stem cell transplantation (HSCT). A 31-year-old man with myelodysplastic syndrome underwent allogeneic HSCT from a human leukocyte antigen-identical sibling, and was found to be continuously yielding high copy numbers of HHV-6A DNA in plasma evaluated by real-time polymerase chain reaction (PCR). Antiviral therapy with ganciclovir or foscarnet failed to decrease the copy numbers. HHV-6A DNA was detected in the patient's buccal mucosa and hair follicles, and was also detected in the plasma, whole blood, and buccal mucosa of the patient's father and 2 siblings, but not in his mother. The sequences of HHV-6A DNA isolated from all family members were identical. Since monitoring of HHV-6 by PCR has been widely introduced to the field of HSCT, transplant physicians should be aware of such an alternative form of HHV-6 transmission, particularly when HHV-6A is detected.

Inverse relationship between human herpesvirus-6 and -7 detection after allogeneic and autologous stem cell transplantation.

Human herpesvirus-6 (HHV-6) and -7 were analyzed in 25 and 18 patients with allogeneic (allo) and autologous (auto) stem cell transplantation (SCT), respectively, by weekly examination of viral DNA in peripheral mononuclear cells using semiquantitative PCR and serologic tests up to 12 weeks after SCT. HHV-6 DNA was detected in 29.6% and 27.9% of samples after allo- and auto-SCT, respectively. The proportions of HHV-6-DNA-positive samples increased in week 3 and 4 after allo-SCT, and in week 1 to 3 after auto-SCT. The frequency of HHV-7 DNA detection, however, was higher after auto-SCT (24.7%) than allo-SCT (12.8%) (P 10(2) copies of HHV-6 DNA (/10(5) cells) on two consecutive occasions were allo-SCT recipients and three showed clinical episodes. Conversely, three of five patients with continuous reactivation of HHV-7 were auto-SCT recipients. Thus, the frequencies of HHV-6 and -7 DNA detection showed an inverse relationship comparing allo- and auto-SCT, suggesting a different mechanism may regulate HHV-6 and -7 reactivation.

Allogeneic peripheral stem cell transplantation using positively selected CD34+ cells from HLA-mismatched donors.

We examined five children who underwent allogeneic peripheral stem cell transplantation (PSCT) using positively selected CD34+ cells from three or two loci-mismatched donors. CD34+ cells mobilized from peripheral blood were separated by immunomagnetic beads. CD34+ cells at 2.2-6.2 x 10(6)/kg were transplanted into three patients with refractory leukemia, a patient with relapsed medulloblastoma and a patient with Fanconi's anemia following a conditioning regimen which included irradiation, alkylating agents and antithymocyte globulin treatment. The number of infused CD3+ cells included in grafts was 2.3-22.7 x 10(4)/kg. Four patients achieved engraftment and hematopoietic reconstitution (> 5 x 10(8)/l of neutrophils on day 10 or 11). Graft rejection was observed in the patient with Fanconi's anemia, but a rapid engraftment was obtained after second PSCT. Although no prophylactic agents other than ATG (included in the conditioning regimen) were used, greater than grade I acute GVHD was not observed, but limited chronic GVHD was observed in two patients. The two patients with leukemia relapsed on days 103 and 210, respectively, and the patient with medulloblastoma died of disease on day 159. The patient with Fanconi's anemia died of fungal infection. CMV and HHV-6 diseases developed in four and two patients, respectively. Thus, although SCT using positively selected peripheral CD34+ cells may be an alternative approach for overcoming graft rejection and GVHD from HLA- mismatched donors, persistent immune deficiency attributing to extremely low numbers of T cells in grafts can potentially lead to reactivation of herpes viruses.

Thrombotic microangiopathy associated with reactivation of human herpesvirus-6 following high-dose chemotherapy with autologous bone marrow transplantation in young children.

Thrombotic microangiopathy (TMA) is a serious complication of BMT. Several factors are important in the etiology of TMA, such as cyclosporin A, GVHD, irradiation, intensive conditioning chemotherapy and infection, which cause damage to vascular endothelial cells leading to activation of these cells. We describe two young children with TMA following high-dose chemotherapy with autologous BMT. Development of TMA was accompanied by reactivation of HHV-6, which was identified by both an increase in the copy number of HHV-6 DNA in the peripheral blood and a significant increase in antibody titers to HHV-6. Thus, it was suggested that reactivation of HHV-6 together with high-dose chemotherapy played an important role in the pathogenesis of TMA in these patients. Since HHV-6 is known to infect vascular endothelial cells, and CMV which is virologically closely related to HHV-6, has been reported to be a pathogen that causes TMA, infection with HHV-6 of vascular endothelial cells may induce TMA via damage and activation of these cells.

Foscarnet therapy for ganciclovir-resistant cytomegalovirus retinitis after stem cell transplantation: effective monitoring of CMV infection by quantitative analysis of CMV mRNA.

We report three pediatric patients with ganciclovir-resistant cytomegalovirus (CMV) retinitis who were successfully treated with foscarnet. The patients were recipients of hematopoietic stem cell transplantation (SCT) from HLA-mismatched donors. Because these patients had developed or experienced progressive CMV retinitis during ganciclovir therapy, they received foscarnet therapy at 60 mg/kg every 8 h. Their retinitis resolved promptly after initiating foscarnet therapy, suggesting foscarnet's effectiveness in treating ganciclovir-resistant CMV infection. The amount of CMV mRNA was quantitatively measured using an NASBA technique, which amplified the beta2.7 transcripts specific for CMV replication. This technique was useful for monitoring disease activity in a more rapid and sensitive manner than the PCR assay for CMV DNA.

Ganciclovir is effective for prophylaxis and treatment of human herpesvirus-6 in allogeneic stem cell transplantation.

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

Remission of progressive multifocal leukoencephalopathy following highly active antiretroviral therapy in a patient with HIV infection.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease resulting from lytic infection of oligodendrocytes by the papovavirus JC (JCV). PML has also been recognized as an AIDS-defining illness. The incidence of PML has increased since 1987 and it occurs in up to 4% of patients with AIDS. To date, there is no treatment available for PML and it usually results in death within 3-6 months of diagnosis. However, there are some reports of remission of PML after antiretroviral therapy. We report a 12-year-old child with hemophilia B and developing AIDS with the onset of PML. With highly active antiretroviral therapy, PML subsided with an increase of CD4 count from 10 to 300/microl in spite of about 1.0 X 10(4) human immunodeficiency virus (HIV)-1-RNA copies. He has survived more than 1 year without specific therapy against JCV. Highly active antiretroviral therapy appears to have improved his prognosis in HIV-associated PML.

Cross-reactivity of anti-HIV-1-p17-derivative peptide (P30-52) antibody to Env V3 peptide.

Strong antibody responses are often seen in human immunodeficiency virus type 1 (HIV-1) carriers, but it is not known whether these antibodies are effective in the inhibition of disease progression. In this study, we examined antigenic epitopes for anti-HIV-1 p17 antibody (p17 Ab) in an HIV-1 carrier's serum, and found that the residues of amino acid numbers 1 to 12 (P1-12), 12 to 29 (P12-29) and 30 to 52 (P30-52) of p17 were highly recognized in the serum. Our examination of purified antibodies from the patient using the p17-derivative-peptide-immunoaffinity columns showed that the reactivity of anti-p30-52 Ab (p30-52Ab) was high for p30-52 and the naive protein, p17. In addition, this P30-52Ab cross-reacted with the third variable region of the envelope glycoprotein (Env V3). To confirm this cross-reactivity, we immunized mice with P30-52, and established a monoclonal antibody (MAb), 8H10. We found that 8H10 was also reactive to Env V3. It is unclear whether this cross-reactivity of P30-52 Ab can function as the inhibitor of HIV-1, but these results will be of help in clarifying the interaction of Env protein with HIV-1 gag polyprotein and the relationship of the decline of the p17 antibody titer with the disease progression in HIV-1 carriers.

[Human herpesvirus-6 and -7 infection and bone marrow transplantation (BMT)].

In our all patients, the antibodies to HHV-6 and -7 were positive before BMT. HHV -6 and -7 DNA were sometimes detected after BMT, and HHV-6 infection after BMT caused fever, interstitial peumonitis, diarrhea, and myelosuppression. However, HHV -7 didn't induce any clinical symptoms. For the diagnosis of the HHV-6 or -7 infection, we used the virus isolation, semiquantitative PCR, and 4-hold elevation of the antibodies to HHV-6 or -7. Ganciclovir, foscarnet, high dose gamma-globulin and high dose acyclovir were useful for the treatment of HHV-6 infection after BMT. HHV-6 is an important agent for the fever of unknown origin, interstitial peumonitis, diarrhea, and myelosuppression after BMT.

Chronic hepatitis in an infant, in association with human herpesvirus-6 infection.

A 20-month-old boy was investigated for persistent liver dysfunction. Liver histologic findings showed chronic hepatitis. The presence of human herpesvirus-6 DNA in liver tissue was demonstrated both by in situ hybridization and by polymerase chain reaction. Human herpesvirus-6 may be a causative agent of chronic hepatitis in this case.

Human herpesvirus 7 infection associated with central nervous system manifestations.

The clinical features of infection with human herpesvirus 7 (HHV-7) are not well described. Exanthem subitum is the only illness that is confirmed to be caused by HHV-7. We report two children who had exanthem subitum associated with central nervous system manifestations. Two strains of HHV-7 were isolated sequentially from peripheral blood mononuclear cells and saliva of the some child who had exanthem subitum complicated with acute hemiplegia in childhood. Two strains were confirmed to be HHV-7 by means of monoclonal antibodies to human herpesvirus 6 (HHV-6) and HHV-7, polymerase chain reaction, and DNA analysis. During the convalescent period, the antibody titer to HHV-7 rose from less than 1:10 to 1:320, whereas the antibody titer to HHV-6 remained less than 1:10. Another child with exanthem subitum complicated by acute hemiplegia had serologic evidence of primary HHV-7 infection. These two cases demonstrate a new relationship between HHV-7 and central nervous system symptoms.

Frequent detection of the human herpesvirus 6-specific genomes in the livers of children with various liver diseases.

This study was performed to investigate the frequency of human herpesvirus 6 (HHV-6) infection of the liver in children with a variety of liver diseases and to evaluate the role of HHV-6 infection in pediatric patients with prolonged non-B non-C hepatitis. Detection of the HHV-6 genomes in liver, in peripheral blood mononuclear cells (PBMC), and in plasma was performed by PCR or by in situ hybridization. Liver biopsy materials from 48 patients, in whom HHV-6 infection was serologically confirmed, were available for PCR analysis. Sequences of the HHV-6B genome were detectable in the livers of 36 of 48 patients (75%). The presence of the genome was not associated with serum transaminase activities. The genome was detectable in PBMC of 22 of 31 (71%) patients tested. In these 31 patients HHV-6 was detected in both the livers and PBMC of 20, was detected in PBMC but not in the livers of 2, was detected in the livers but not in PBMC of 3, and was detected in neither of samples of 6. In situ hybridization of the livers of six patients showed the presence of the HHV-6B genome in the nuclei of hepatocytes. The anti-HHV-6 immunoglobulin M antibody was detectable in 2 of 9 of the non-B non-C hepatitis patients, whereas none of the 22 patients with etiology-defined liver diseases tested positive. Cell-free viral DNA was not detectable in either group of patients. Our results showed that HHV-6B is frequently present in the livers of children with a variety of liver diseases but do not support the assumption that HHV-6B infection of the liver is associated with prolonged non-B non-C hepatitis.

Seroepidemiological study of human herpesvirus-6 and -7 in children of different ages and detection of these two viruses in throat swabs by polymerase chain reaction.

The presence of human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) in throat swabs of 62 children of different age groups (group I, ages 0-5 month; group II, ages 6-11 months, group III, ages 12-23 months, group IV, age 2-8 years) and 28 adults was detected by polymerase chain reaction. The detection rate of HHV-6 DNA was the highest (87%) in children aged 1-year-old and decreased with age, whereas the detection rate of HHV-7 increased with age and reached a maximum in adults. HHV-6B was detected in almost all samples except for two children who secreted only HHV-6A. When the antibody prevalence was determined in the four groups of children, HHV-6 antibody was detected in 8/12 (66.7%), 10/12 (83.3%), 15/16 (93.8%), and 13/14 (92.9%), respectively. Antibody to HHV-7 in these groups was detected in 6/12 (50.0%), 4/12 (33.3%), 12/16 (75.0%), and 13/14 (92.9%), respectively. Detection of HHV-6 DNA in throat swabs of triplets who had the sequential onset of exanthem subitum was attempted by using samples sequentially collected from these children after the onset of the disease in the first patient. HHV-6 DNA with high copy numbers was detectable during the acute and convalescent phases of the disease in all patients, but no DNA was detected in samples collected before the onset of disease.

Cytomegalovirus hepatitis confirmed by in situ hybridization in 3 immunocompetent infants.

We present 3 cases of immunocompetent infants with CMV infection who showed prolonged liver dysfunction. In all cases the CMV genome was detectable in hepatocytes using the in situ hybridization method. Combination therapy with ganciclovir (GCV) and hyperimmune gammaglobulin (HGG) was instituted in 2 cases and successfully suppressed the replication of CMV, with sustained improvement in liver function. In 1 of these cases, signals for CMV DNA were undetectable in the liver 12 months after termination of combination therapy. These results help to confirm the etiology of CMV for persistent hepatitis in immunocompetent infants using the in situ hybridization method and also show the efficacy of combination therapy with a virostatic agent, GCV, and an immune-modulating agent, HGG.

Reactivation of human herpesvirus 6 by infection of human herpesvirus 7.

We have attempted to reactivate human herpesvirus 6 (HHV-6) by infection with HHV-7 using childhood exanthem subitum patients in vitro. Peripheral blood mononuclear cells (PBMCs) were collected from children who had a history of exanthem subitum(ES) by HHV-6 and were infected by human herpesvirus 7 (HHV-7) in vitro. The antigen positive rate to HHV-6 started to increase 7 days after the infection and reached a maximum by Day 15 using an immunofluorescence antibody test. The copy number of HHV-6 DNA also increased in the samples in 10 days after infection in vitro. No antigen or increase in DNA was detected in PBMCs, that were mock-infected or infected with supernatant of stock virus after ultracentrifugation, suggesting that an infection by HHV-7 is necessary to reactivate HHV-6. In the paired sera samples during the acute and the convalescent phases of ES, seven to ten bands, that were specific for HHV-6, were recognized in samples from the acute phase, and at least 5 dominant polypeptides were found more intensively after HHV-7 infection.

Detection of human herpesvirus 7 (HHV-7) DNA in breast milk by polymerase chain reaction and prevalence of HHV-7 antibody in breast-fed and bottle-fed children.

Twenty-nine breast milk mononuclear cell samples were analyzed for human herpesvirus 7 (HHV-7) DNA, human herpesvirus 6 (HHV-6) DNA, and human cytomegalovirus (HCMV) DNA by polymerase chain reaction (PCR). In addition, peripheral blood mononuclear cell samples from 13 puerperants were analyzed for HHV-7 DNA by PCR, and seropositivity of HHV-7 was also analyzed in breast-fed and bottle-fed children. HHV-7 DNA was detected in 3 of 29 breast milk samples. HCMV DNA was also detected in 3 of 29 breast milk samples, but HHV-6 DNA was not detected. HHV-7 DNA was detected in 11 of 13 samples of peripheral blood mononuclear cells. Though the seropositivity rate for HHV-7 in breast-fed children was slightly higher than that in bottle-fed children at 18 and 24 months old, the difference was not statistically significant. From these results, we speculate that breast-feeding may be one of the transmission routes of HHV-7, although this is not the main route.