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• The QIAstat-Dx platform provides rapid results, may be performed at or near the point of care and can be used for syndromic testing. • Differential diagnoses of viral vesicular infections include chicken pox and disseminated herpes zoster caused by Varicella-zoster virus, herpes infections caused by herpes simplex virus 1, 2 and human herpesvirus 6; and Hand, foot and mouth disease caused by enterovirus. • Results of the QIAstat viral vesicular panel were highly sensitive and specific and demonstrated 99.6% concordance with laboratory developed tests. • The QIAstat-Dx viral vesicular panel, can play a significant role in rapid diagnosis of mpox and viral vesicular diseases, empowering fast and informed clinical decisions for patient centric treatment and public health response. The recent mpox outbreak has highlighted the need to rapidly diagnose the causative agents of viral vesicular disease to inform treatment and control measures. Common causes of vesicular disease include Monkeypox virus (MPXV), clades I and II, Herpes simplex viruses Type 1 and Type 2 (HSV-1, HSV-2), human herpes virus 6 (HHV-6), Varicella-zoster virus (VZV) and Enteroviruses (EVs). Here, we assessed a syndromic viral vesicular panel for rapid and simultaneous detection of these 7 targets in a single cartridge. The aim of this study was to evaluate the QIAStat-Dx ® viral vesicular (VV) panel and compare with laboratory developed tests (LDTs). Limit of detection, inter-run variability, cross-reactivity and specificity were assessed. Positive and negative percent agreement, and correlation between assays was determined using 124 clinical samples from multiple anatomical sites. The overall concordance between the QIAstat and LDTs was 96%. Positive percent agreement was 82% for HHV-6, 89% for HSV-1 and 100% for MPXV, HSV-2, EV and VZV. Negative percent agreement was 100% for all targets assessed. There was no cross-reactivity with Vaccinia, Orf, Molluscum contagiosum viruses, and a pooled respiratory panel. The QIAstat VV multi-target syndromic panel combine ease of use, rapid turnaround, good sensitivity and specificity for enhanced diagnosis, clinical care and public health responses. [ABSTRACT FROM AUTHOR]

Mild disease due to human herpesvirus-6 (HHV-6) has been reported in healthy children. Severe disease due to this virus can occur in immunocompromised patients but is rarely reported in previously healthy adults. We report the case of a previously healthy woman who presented with a skin rash, mild upper respiratory symptoms, and abdominal pain and succumbed to fulminant hepatic failure attributed to infection with HHV-6B. HHV-6 may be more commonly associated with fulminant hepatitis in immunocompetent patients than previously thought and should be considered in the differential diagnosis of patients presenting with skin rash, upper respiratory symptoms, and unexplained hepatitis. [ABSTRACT FROM AUTHOR]

Background The triggering agent of multiple sclerosis is still unknown and many viruses, including human herpesvirus-6 (HHV-6), are under suspicion. In earlier study we found patients who had HHV-6 reactive OCBs in their CSF. We wanted to investigate whether HHV-6 has an active role in diseases with demyelination. Objective To analyze the HHV-6-reactive cases in detail and investigate the possible independent role of HHV-6 in the development of central nervous system involvements with demyelination. Study design We studied serum and CSF samples that were collected over a period of one year, from all patients who had oligoclonal bands (OCB) in cerebrospinal fluid (CSF) and were examined in the Department of Neurology, University Central Hospital of Helsinki, Finland. Clinical evaluation was accomplished blinded of HHV-6 analysis and follow-up time was two years. All patients underwent MRI of the head and clinically indicated CSF analysis. Results The 17 patients with HHV-6-reactive OCBs were significantly younger and had significantly more IgG-OCBs in comparison to patients without HHV-6-reactive OCBs. Initial diagnoses in patients with HHV-6-reactive OCBs remained the same during the follow-up time. Conclusion Patients with HHV-6-positive OCBs appear to form a separable group. In progressive neurological diseases HHV-6 may have a role in long-term infection with demyelination. [ABSTRACT FROM AUTHOR]

Background and objectives Since HHV-6 reactivation after transplant has been reported to increase the risk of CMV infection, we tested this hypothesis in the HLA-haploidentical hematopoietic stem cell transplantation setting. Study design From February 2011 to October 2015, 75 patients received hematopoietic stem cell transplantation using a T-cell replete graft from a HLA-haploidentical donor at our Institution. Results Interestingly, 87% of HHV-6 reactivations were followed by a CMV reactivation, at a median of 15 days between the two viruses. Incidence of CMV reactivation was 14.5-fold higher in those patients with prior HHV-6 reactivation vs. those without it (p-value < 0.001). Conclusion The present results suggest that HHV-6 can be considered as a predicting indicator of cellular immunosuppression preceding the onset of CMV infection. [ABSTRACT FROM AUTHOR]

Abstract: Background: Human herpesvirus 6 (HHV-6) causes exanthema subitum and is associated with symptomatic reactivations in immunocompromised patients, particularly after hematopoietic stem cell transplantation. The detection of viral mRNA can help to make the difference between latent, chromosomally integrated and true replicating virus. It can also be a useful tool to investigate viral multiplication in different cell types. Objectives: To develop molecular tools for the detection and quantification HHV-6 transcripts that can be used in a clinical setting. Study-design: Two one-step reverse-transcriptase quantitative real-time PCR (RT-qPCR) were developed for the quantification of the immediate early U90 and the late U100 mRNAs. Viral mRNA loads were compared to viral DNA loads during infection in vitro and in blood samples collected from stem cell transplanted patients. Results: Analytical performances of the two quantitative real-time PCR were good. In vitro, kinetics of both transcripts was well correlated with DNA levels. Sixty blood samples from patients with active HHV-6 infection were analyzed. Overall agreement of qualitative results for HHV-6 DNA, U90 RNA and U100 RNA was good. HHV-6 DNA loads were significantly higher than mRNA loads. In clinical samples, the amounts of U100 and U90 mRNAs were low and their detection was mainly associated to viral DNA loads upper than 1000copies/ml of blood. Conclusion: The new assays are sensitive and reliable methods for the monitoring of viral transcription in vitro and in vivo. As their detection is associated to high DNA loads in vivo, they can be helpful tools for the diagnosis of active infection. [Copyright &y& Elsevier]

Abstract: Background: Human herpesvirus-6 (HHV-6) frequently reactivates in immunocompromized individuals. Most commonly HHV-6 DNA is detected without organ localized disease. This HHV-6 DNAemia usually occurs in patients who also have CMV reactivations. The question if reactivation of one virus causes reactivation of the other, or whether both viruses reactivate independently, cannot be answered in populations with high seroprevalence for both viruses. Our study is the first in which 35 patients have been included who were CMV seronegative prior to transplantation. Objective: We investigated the occurrence of HHV-6 reactivations in relation to the CMV-serostatus of renal transplantation donor and recipient. Study design: 9 consecutive patients receiving a renal transplantation were included. All available stored whole blood samples were tested for HHV-6 DNA by quantitative PCR. Details including CMV serostatus of donor and recipient were recorded. Results: CMV-seropositive recipients have a 68% chance of developing HHV-6 DNAemia if the kidney came from a CMV-seropositive donor, compared to 26% if the kidney came from a CMV-seronegative donor. CMV-seronegative recipients, who are bound to undergo primary CMV infections following transplantation with a renal graft from a CMV-seropositive donor, have 88% chance of developing HHV-6 DNAemia. If they receive a graft from a CMV-seronegative donor the chance of developing HHV-6 DNAemia is 22%. Conclusion: Receiving a renal transplant from a CMV-seropositive donor increases the chance of developing HHV-6 DNAemia. HHV-6 DNAemia is a sign of impending primary CMV infections following sero-discordant renal transplantations. [Copyright &y& Elsevier]

Abstract: Background: Human herpesvirus 6 (HHV-6) and human cytomegalovirus (HCMV) are major opportunistic pathogens in solid organ transplant (SOT) recipients. The use of antivirals for the treatment of HCMV disease can result in the development of drug resistance mutations in HCMV and also potentially in HHV-6. Objectives: The emergence of HHV-6 drug resistance mutations was evaluated in SOT recipients at the onset of HCMV disease and following treatment with ganciclovir (GCV) or valganciclovir (VGCV). Study design: Detection of HHV-6 was performed by real-time PCR from whole blood samples serially obtained from SOT recipients treated for HCMV disease with an induction dose of intravenous GCV or oral VGCV for 21 days followed by VGCV maintenance for 28 days in both arms. Baseline and last positive HHV-6 samples were tested for mutations in the genes encoding the protein kinase (U69) and the DNA polymerase (U38). Results: The rate of HHV-6 viraemia among SOT patients with HCMV disease at baseline was 3.2% (5/155). All isolates belonged to the HHV-6B species. Mutations L213I and Y479H were detected at baseline and at later times in the U69 kinase. Mutation L213I was previously reported as polymorphism whereas the role of mutation Y479H in drug resistance is unknown. Mutations D854E and E855Q found in the DNA polymerase were known as natural variants. Conclusions: The incidence of HHV-6 viraemia in SOT recipients with established HCMV disease before initiation of antiviral therapy was low. Treatment with GCV or VGCV did not induce the emergence of HHV-6 drug resistance mutations. [Copyright &y& Elsevier]

Abstract: Background: Human herpesvirus 6 (HHV-6) is an important cause of fulminant or acute viral myocarditis. However, insufficiency of standard antiviral treatment against HHV-6 is an emerging problem. Objectives: To describe the case of child with HHV-6 myocarditis who was treated by unloading with a left ventricular assist device and Artesunate. Study design: Case report supported by histological and viral diagnoses via a combination of histology/immunohistochemistry and polymerase chain reaction techniques performed on cardiac tissues before and after treatment. Results: Following therapeutic intervention, the clinical status and heart function improved. Endomyocardial biopsies revealed decreased levels of HHV-6B DNA in the myocardium and the disappearance of histological findings in support of lymphocytic myocarditis. Left ventricular assist device could be explanted. No adverse effects of Artesunate were noted. Conclusions: In addition to existing heart failure treatments, Artesunate can be considered as an effective candidate for clinical use in cases of HHV-6B associated myocarditis. [Copyright &y& Elsevier]

Abstract: Background: Cord Blood (CB) are increasingly used as an alternative stem cells source in adults for allogeneic Stem Cell Transplantation (allo-SCT). The risk of human herpesvirus (HHV-6) reactivation is significantly higher after CB transplant vs unrelated peripheral blood stem cells (PBSC) allo-SCT. Higher HHV-6 cell receptor CD46 expression on progenitor cells in CB may explain this difference. Objectives: To prospectively compare the HHV-6 cell receptor CD46 expression on various cell subsets of three freshly harvested blood sources on one hand and of three graft sources on the other hand. Study design: 52 samples were used for the purpose of this study. They were issued from peripheral blood (PB, n =10), G-CSF mobilised PB (GCSF-PB, n =10), cord blood (CB, n =10), unmanipulated bone marrow (uBM, n =5), leukapheresis product (LP, n =10) and thawed CB graft (n =7). CD46 expression was assessed by FACS analysis on total lymphocytes, monocytes, NK cells, T and B cells subsets, plasmacytoid (pDCs) dendritic cells and stem cells. Results: As all cell subsets were found CD46 positive, CD46 mean fluorescence intensity (MFI) was then considered for comparison between the three blood sources and the three graft sources. The most impressive result observed was that HHV-6 cell receptor CD46 expression was significantly reduced in almost all cell components of thawed CB graft compared to other graft sources. Conclusions: This original study shows strong differences in term of quantitative CD46 expression between several blood and grafts samples. Our results suggest that other factors than the qualitative CD46 expression play a role in the higher HHV-6 reactivation observed after CB transplant in adults. [Copyright &y& Elsevier]

Abstract: Background: Diagnosis of human herpesvirus-6A (HHV-6A), -6B (HHV-6B) or -7 (HHV-7) infections is often based on the measure of viral load in blood. Objectives: The aim of this study was to define usual values of HHV-6A, HHV-6B and HHV-7 loads in blood fractions (whole blood [WB], mononuclear cells [PBMCs], polymorphonuclear leukocytes [PMNLs]) of blood donors. Study design: HHV-6A, HHV-6B and -7 DNAs were quantitated using real-time PCR assays in WB, PBMCs and PMNLs separated on Ficoll or dextran gradients, respectively, for 200 blood donors. Viral loads were expressed as the number of viral genomic copies per million cells (Cop/M) for all fractions, and also per milliliter for WB. Results: HHV-6B DNA was rarely detected in WB (8%), PBMCs (16.5%), and PMNLs (10.5%), HHV-6A was never detected, whereas HHV-7 DNA was often present in WB (51.5%), PBMCs (62%) and PMNLs (51.5%). Median loads were low with 81 Cop/M in WB, 62 Cop/M in PBMCs and 34.5 Cop/M in PMNLs for HHV-6B, and 129 Cop/M in WB, 225 Cop/M in PBMCs and 62 Cop/M in PMNLs for HHV-7. Viral load expression per million cells and per mL were equivalent. One subject had chromosomally integrated HHV-6 with high viral loads ranging from 2.23×106 to 3.21×106 Cop/M in all compartments and plasma. Conclusions: These results allow to propose viral load in WB as a sensitive and suitable marker, with values for healthy subjects at approximately 100 Cop/M for both viruses. The prevalence of chromosomally integrated HHV-6 was 0.5%. [Copyright &y& Elsevier]

Abstract: Background: Human Herpesvirus 6 (HHV-6) is an important cause of fulminant or acute viral myocarditis in immunocompromised or immunocompetent patients. However the physiopathological mechanisms of HHV-6 related acute myocarditis and the involvement of subsequent HHV-6 reactivation phases in the development of chronic cardiomyopathies remain to be assessed. Objectives: To describe a case of fatal HHV-6 chronic myocarditis in an immunocompetent adult. Study design: Case report and detailed histological and viral diagnoses by combination of histology/immunohistochemistry and polymerase chain reaction techniques on cardiac tissues. Results: Histopathological analysis of ventricular tissues showed large interstitial and scarring fibrotic areas with a moderate mononuclear cell infiltrate compatible with histological aspect of chronic myocarditis. Detection of both HHV-6 by real-time PCR and viral glycoproteins in mononuclear and endothelial cells by immunohistochemistry evidenced an ongoing cardiac HHV-6 replication with viral late protein synthesis activity. Conclusions: This case report indicates that HHV-6 can establish a chronic active myocarditis leading to heart failure in immunocompetent subjects. [Copyright &y& Elsevier]

Abstract: Background: Clinical manifestations of human herpesvirus-6 (HHV-6) have not been clearly defined, and the role of HHV-6 in human disease remains to be fully elucidated. Objective: To determine the frequency of HHV-6 infections at Rennes Teaching Hospital and to describe all possible symptoms of such infections. Study design: We systematically analyzed in a retrospective study all the samples between May 2003 and December 2004 from patients with HHV-6 by polymerase chain reaction (PCR). Clinical records of patients with positive HHV-6 PCR were recorded. Diagnosis of HHV-6 infection was accepted if all other possible diagnoses had been eliminated. Results: Over the study period, 1591 PCRs were performed from various tissues, including blood, cerebrospinal fluid, ascitis and tissue biopsies. Forty-three samples from 25 patients tested positive (3%). We describe three groups of clinical manifestations of HHV-6 infection. The first group consisted of neurological complications (32% of patients), including convulsions, encephalitis and chronic psychiatric disorders in immunocompetent patients. The second group consisted of clinical problems relating to gastrointestinal tract, which was found in 9 of our patients (36%). All of these patients were immunocompromised. Four of them presented colitis, and one of them died one month after liver transplantation because of this colitis. The last group of clinical symptoms was associated with maternal–fetal infection leading to abortion following HHV-6 seroconversion during pregnancy. Conclusion: Three clinical types of HHV-6 infections are described: neurological manifestations including encephalitis in non-immunocompromised patients, digestive problems in immunosuppressed patients and severe maternal–fetal infection. [Copyright &y& Elsevier]

Abstract: Background: Human herpesvirus-6 (HHV-6) has been associated with a diverse spectrum of central nervous system (CNS) diseases and reported glial tropism. Objective: To determine if HHV-6 is present in a series of pediatric brain tumors. Study design: Pediatric gliomas from 88 untreated patients represented in a tissue microarray (TMA) were screened for HHV-6 by nested polymerase chain reaction (PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) and compared to non-glial tumors (N =22) and control brain (N =32). Results were correlated with tumor grade and overall survival. Results: HHV-6 U57 was detected by nested PCR in 68/120 (57%) tumors and 7/32 (22%) age-matched non-tumor brain (P =0.001). HHV-6 U31 was positive in 73/120 (61%) tumors and 11/32 (34%) controls (P =0.019). Seventy-two percent (43/60) of tumors were HHV-6 Variant A. HHV-6 U57 was confirmed by ISH in 83/150 (54%) tumors and 10/32 (31%) controls (P =0.021), revealing a non-lymphocytic origin of HHV-6. HHV-6A/B gp116/64/54 late antigen was detected by IHC in 50/124 (40%) tumors and 6/32 (18%) controls (P =0.013). Interestingly, 58% of low grade gliomas (N =67) were IHC positive compared to 19% of high grade gliomas (N =21, P =0.002) and 25% of non-gliomas (N =36, P =0.001). HHV-6A/B gp116/64/54 antigen co-localized with glial fibrillary acidic protein, confirming the astrocytic origin of antigen. Overall, there was no primary association between HHV-6A/B gp116/64/54 antigen detection and survival (P =0.861). Conclusions: We provide the first reported series of HHV-6 detection in pediatric brain tumors. The predominance of HHV-6 in glial tumors warrants further investigation into potential neurooncologic disease mechanisms. [Copyright &y& Elsevier]

Abstract: Background: The long-term effect of HHV-6 and HHV-7 infections on chronic allograft nephropathy (CAN) development after renal transplantation is uncertain. Objectives: To determine HHV-6 and HHV-7 reactivation during the post-transplantation period and to evaluate its effect on CAN development in renal transplant patients. Study design: Eighty-one renal allograft recipients (28 with CAN, 53 with normal transplant function) were studied to determine the frequency of HHV-6 and HHV-7 reactivation during 36.4±7.8 months after renal transplantation using nested PCR. HHV-6 variants were identified using restriction endonuclease analysis. Patients were monitored for the development of CAN. Results: The frequency of HHV-6 and/or HHV-7 plasma DNA was significantly higher in CAN patients (25/28, 89.3%) compared to control patients (15/50, 30.0%, p =0.0001). CAN patients also had an increased incidence of dual active infections (20/25, 80% and 2/15, 13.3%, p =0.007, respectively). In all 34 HHV-6 positive cases, the HHV-6B variant was identified. The presence of HHV-7 DNA in plasma preceded the presence of HHV-6 DNA. Early development of CAN and graft loss was detected only in patients with simultaneous HHV-6 and HHV-7 plasma DNA. Conclusions: Reactivation of HHV-6 and HHV-7 in renal graft recipients is a risk factor for CAN development. The presence of concurrent HHV-6 and HHV-7 DNA in the plasma is an unfavorable prognostic factor. [Copyright &y& Elsevier]

Abstract: Background: Viral load in whole blood is the main virological marker for assessing HHV-6 infection and is used as an indication to begin antiviral therapy. Results are usually expressed as the number of genomic equivalent copies (gec) per mL of blood, although HHV-6 DNA in blood is mainly localized in lymphocytes and polymorphonuclear leukocytes. Objectives: Since leukocyte counts vary in immunocompromised patients, especially in stem cell transplant recipients, the aim of this study was to compare HHV-6 load expressed as gec per mL with load expressed as gec per million cells (mc), using quantitative real-time PCR for HHV-6 and cell DNA. Study design: 194 blood samples from 101 patients were analyzed. Leukocyte count was obtained for 142 samples. Results: The two modes of expression were incompletely correlated (p <0.0001; R 2 =0.732). To understand this relative discrepancy, samples were classified according to hematological criteria (normal leukocyte count, leukopenia, agranulocytosis, lymphopenia). The expression modes were correlated in all cases except for agranulocytosis (p =0.21; R 2 =0.087). Moreover, the median of ratio between gec per mc and gec per mL ranged from 0.5 when leukocyte count was normal, to 8.2 in cases of agranulocytosis. HHV-6 load follow-up suggested that in agranulocytosis expressing results as gec per mc tended to provide a more representative result. Conclusions: The different expression of HHV-6 load in whole blood, as either gec per mL or gec per mc resulted in different estimations of infection in the case of agranulocytosis. In this situation, the latter mode of expression is preferred. [Copyright &y& Elsevier]

Abstract: Background: Human herpesvirus-6 (HHV-6) has been associated with a wide spectrum of diseases. (r)-9-[4-Hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) is an acyclic guanosine analogue that is structurally similar to acyclovir and is in clinical development for treatment of herpesvirus infections. H2G has been found to have activity against HSV type 1, HSV type 2, and HHV-6 in lymphoblast cell lines. A new anti-viral duplex drug, 3′-azido-3′-deoxythymidylyl-(5′→2-O)-3-O-octadecyl-sn-glycerol (AZT–lipid–PFA), linking zidovudine (AZT) and foscarnet (PFA) via a lipophilic octadecylglycerol residue (lipid) also exhibits anti-viral activities against HIV, HSV type 1 and HCMV. Objective: To assess the efficacy of H2G and AZT–lipid–PFA conjugate against HHV-6. Study design: Drug-associated toxicity and proliferative response were evaluated. We conducted in vitro experiments to determine the efficacy of H2G and an AZT–lipid–PFA conjugate in interfering with expression HHV-6 viral transcript in primary human peripheral blood mononuclear cells (PBMC). Results: Both H2G and AZT–lipid–PFA were effective at inhibiting expression of HHV-6 gene transcript at comparable concentrations. Additionally, while AZT–lipid–PFA treatment was toxic to cells at concentrations above 5μM, H2G treatment was associated with minimal cytotoxicity. Conclusion: These data suggest the potential application of these anti-viral compounds in controlling HHV-6 infection. [Copyright &y& Elsevier]

Abstract: Background: Viral resistance to antiviral drugs can cause serious complications in immunosuppressed patients. We isolated from an allogeneic stem cell transplant (SCT) recipient an antiviral-resistant human herpesvirus 6 (HHV-6) strain with mutations that caused amino acid substitutions. Objective: To study the impact of mutations in the U38 and U69 genes of the ganciclovir (GCV)-resistant HHV-6 strain associated with the death of the SCT recipient. Study design: Viruses were obtained from blood taken during symptomatic disease. Mutations in the genes for U69 protein kinase and U38 DNA polymerase were analyzed and the effects of the U69 mutations on GCV resistance were assayed using a recombinant baculovirus system. Results: Increasing HHV-6 antigenemia occurred after 2–3 months of preemptive GCV therapy, followed by symptomatic HHV-6 disease that ended in fatal fungus-related septic shock. The HHV-6 strain isolated from the patient was 100-fold more resistant to GCV than was a wild-type strain. New mutations were found in HHV-6 genes U38 (P462S and A565V) and U69 (L202I and L213I). The mutation of U38 P462S corresponds to a mutation in the UL54 gene (P522S) of a GCV-resistant HCMV. The U69 mutations did not alter GCV sensitivity in baculovirus GCV-resistant assay system. Conclusions: Drug-resistant mutations arising during preemptive therapy may complicate post-transplant HHV-6 disease in SCT recipients. The increased copy number during GCV treatment of this new GCV-resistant HHV-6 strain correlated with mutations in the U69 and U38 genes. Since the kinase mutation did not alter sensitivity to GCV when tested in the in vitro system, it is likely that the substitutions in the polymerase related to GCV resistance. [Copyright &y& Elsevier]

Human herpesvirus 6 (HHV-6) is an ubiquitous virus that can reactivate in immunocompromised hosts, resulting in diverse clinical sequelae. We describe a case of fatal acute HHV-6 myocarditis in a patient who underwent allogeneic haemopoietic stem cell transplantation (HSCT). To our knowledge, this is the first reported case of biopsy proven HHV-6 myocarditis post-HSCT. [ABSTRACT FROM AUTHOR]

Abstract: Human herpesvirus type 6 (HHV6) is known to reactivate after hematopoetic stem cell transplantation (HSCT) and has been suggested to be associated with increased mortality and severe clinical manifestations, including graft versus host disease (GvHD). The exact etiological role of HHV6 reactivation in increased morbidity and mortality after HSCT remains unclear. This review will focus on the current available evidence of HHV6 reactivation after HSCT and its immuno-modulatory capacities, with particular emphasis on the severe complication GvHD. At present, no effective specific antiviral treatment for HHV6 reactivation has been identified. The currently available antiviral agents are outlined, as well as possible future strategies for the treatment of HHV6 reactivation. Non-toxic, specific treatment or prevention of HHV6 reactivation might improve the safety and efficacy of the HSCT procedure. [Copyright &y& Elsevier]

Abstract: Background: A more rapid and easier method is needed for monitoring human herpesvirus 6 (HHV-6) infections. The loop-mediated isothermal amplification method (LAMP) can detect viral DNA with high specificity, efficiency, and speed under isothermal conditions. LAMP requires only simple equipment that is available in hospital laboratories. Objectives: We evaluated LAMP as a means of detecting HHV-6 DNA directly from patients’ sera. Results: The sensitivity of the HHV-6 LAMP protocol without heat denaturation was 1000 copies/tube; with heat denaturation 10 copies/tube were detected. Three hundred serum samples from children with fever were analyzed. Using HHV-6 isolation as a definition of HHV-6 infection, the sensitivity, specificity, positive predictive value, and negative predictive value of the HHV-6 LAMP method without DNA extraction were 95.5%, 95.2%, 94.0%, and 96.4%, respectively. Conclusion: Direct detection of HHV-6 DNA in serum with a modified HHV-6 LAMP could be used for rapid diagnosis of exanthem subitum (ES). [Copyright &y& Elsevier]

Abstract: Background: Human herpesvirus 6 (HHV-6) variants A and B are distinct viruses that differ in their biological properties and association to disease. Diagnostic tools able to discriminate between these two variants and between active or latent HHV-6 infection are much needed. In our effort to develop variant-specific antibodies against HHV-6 immediate-early (IE) proteins, we had previously generated P6H8, a monoclonal antibody (mAb) reacting specifically with the immediate-early 2 (IE2) proteins from HHV-6A. Objectives: To characterize the P6H8 HHV-6 variant specific mAb and evaluate its potential as part of a variant-specific diagnostic tool for HHV-6A infection. Consequently, our objective was to map the epitope recognized by P6H8. Study design: In order to map P6H8 reactivity by Western blotting, we generated deletion mutants of IE2 protein as well as various GST-IE2 fusion proteins. HHV-6A infected cells were used to demonstrate P6H8 reactivity against native IE2. A synthetic peptide corresponding to the P6H8 epitope was used to confirm our results and block P6H8 reactivity. Results: We mapped the P6H8 epitope to amino acids 1078–1089 of HHV-6A IE2. A peptide (FTPFYYQSSRTR) recreating this epitope was effective in blocking the recognition of both native and recombinant IE2 by P6H8. Conclusions: Our work provides a precise characterization of the P6H8 mAb and its specificity toward the IE2 protein of HHV-6 variant A which could prove useful for the differential diagnostic of active infection by HHV-6. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Background and objectives: Human herpesvirus-6 (HHV-6) is the causative agent of exanthem subitum. Both HHV-6 variants, A and B, have been associated with central nervous system (CNS) diseases, suggesting a wide neuropathogenic potential. We describe a case of recurrent bilateral anterior optic neuritis with HHV-6 active infection associated with clinical relapses. Case report: A 23-year old woman presented with progressive visual impairment, bilateral papillitis and painful ocular movements. Nested polymerase chain reaction (PCR) for DNA viruses, HHV-6 variant specific real time quantitative PCR, serological analysis and retrotranscription PCR (RT-PCR) for HHV-6 mRNA transcripts were performed. Nested PCR in PBMC and CSF samples was negative for all viruses but positive for HHV-6 DNA, subtyped as HHV-6B. The disease had a relapsing/remitting course. During relapses PBMC samples remained positive for HHV-6 DNA, and HHV-6 active infection was confirmed by the presence of anti-HHV-6 IgM and of HHV-6 U27 mRNA transcript. High viremia levels and relapses were overlapping. After the last relapse, the patient was successfully treated with gancyclovir. Conclusions: The case reported here suggests a possible association of HHV-6 in bilateral optic neuritis. HHV-6 could be monitored when bilateral optic neuritis is identified, in order to establish an appropriate antiviral therapy. [Copyright &y& Elsevier]

Abstract: Background: Post-transplant lymphoproliferative disease (PTLD) causes significant morbidity and mortality in transplantation. The clinical significance of Epstein-Barr virus (EBV) in the development of PTLD is clear, but not all EBV-reactivations cause PTLD. Objectives: We retrospectively analyzed EBV-DNAemia in liver transplant patients by a quantitative TaqMan-based real-time plasma PCR. Study design: Altogether 1284 specimens, obtained from 105 patients for frequent monitoring of cytomegalovirus (CMV) and human herpesvirus-6 and -7 (HHV-6, HHV-7) during the post-transplant year, were retrospectively tested for EBV-DNA. Results: Altogether, 14/105 (13%) patients showed EBV-DNAemia, which usually occurred within 3 months after transplantation and subsided within a few weeks. EBV-DNAemia occurred concurrently with CMV in 10/14, with HHV-6 in 11/14, and with all three betaherpesviruses in 4/14 cases. The peak viral loads were relatively low (median 2100 EBV-DNAcopies/ml, range 568–6600), except in one patient who first had low-level EBV-DNA (562–3022copies/ml) in the early post-transplant period, but on day 175 after transplantation developed high-level DNAemia (9851–86,975copies/ml) which continued for 6 months and developed into PTLD at 6 months after transplantation. Conclusion: Low-level EBV-DNAemia is common after liver transplantation, often occurring together with betaherpesviruses, but seldom leads to high viral loads or PTLD. However, monitoring of EBV-DNA levels in the patients can be useful. [Copyright &y& Elsevier]

Abstract: Severe pulmonary hypertension (PH) mimicking idiopathic PH is an increasingly recognized complication of human immunodeficiency virus (HIV) infection. PH shares several histopathologic features with Kaposi''s sarcoma (KS), the most common malignancy in AIDS patients, and molecular evidence of the vasculotropic Kaposi''s sarcoma-associated herpesvirus or human herpesvirus 8 (HHV-8) has been found in the lung tissue of patients with the disease. Although the prevalence of HHV-8 infection is increased among HIV-infected patients, no clinical association between KS and PH has ever been reported. Herein, we described a 30-year-old HIV-infected female co-infected with HHV-8 who developed severe PH coincident with occult KS. The clinical presentation of KS was unusual and remained masqueraded for years as an indolent cervical lymphadenopathy, without the typical cutaneous lesions. This is the first ever-reported case of PH associated with KS. Although the co-occurrence of both diseases in this patient could have been just a coincidence, the observation may also indicate that a relationship between HHV-8 infection and HIV-associated PH exists. Coinfection with HHV-8 and occult lymphadenopatic KS should be considered in HIV-infected patients developing PH. [Copyright &y& Elsevier]

Abstract: Background: Human herpesvirus 6 (HHV-6) isolates are classified into two variants, termed HHV-6A and HHV-6B, on the basis of distinct genetic, antigenic and biological characteristics, but the specific pathogenicity of each variant remains poorly understood. Objectives: To design a rapid, sensitive and specific real-time variant-specific PCR (VS-PCR) method to differentiate both variants in biological specimens. Study design: The VS-PCR was adapted from a real-time PCR assay, based on TaqMan® technology, previously developed for the genome quantitation of both HHV-6 variants [Gautheret-Dejean A, Manichanh C, Thien-Ah-Koon F, Fillet AM, Mangeney N, Vidaud M, et al. Development of a real-time polymerase chain reaction assay for the diagnosis of human herpesvirus-6 infection and application to bone marrow transplant patients. J Virol Meth 2002;100:27–35], a consensual reverse primer (Taq2) being changed into two variant-specific primers named H6A and H6B. This method was applied to a large set of biological specimens obtained in different pathological contexts. Results: The sensitivity threshold was about 10copies/well for HHV-6A-specific PCR (PCR-A) and 1copy/well for HHV-6B-specific PCR (PCR-B). Both assays showed a linear dynamic range from 10 to 100,000 copies of HHV-6 DNA. Regarding the specificity and the capacity of discrimination of each assay, one variant could be detected and identified in the presence of more than 1000 times higher concentrations of the other variant in virus mixtures. The comparison of the results obtained with this VS-PCR with those previously obtained with a classic PCR method allowed us to validate our new technique on a wide panel of biological samples, including numerous patients with severe HHV-6-related symptoms. The high prevalence of HHV-6B was confirmed in healthy individuals and immunocompromised patients. HHV-6A was identified in distinct samples from several patients exhibiting neurological disorders. Conclusions: We developed a new VS-PCR assay, able to differentiate HHV-6A and HHV-6B in biological samples, even in the case of mixed infections. Our study confirms the wide prevalence of HHV-6B and highlights the potential greater neuropathogenic role of HHV-6A in immunocompromised patients and young infants. [Copyright &y& Elsevier]

Human herpesvirus 6 and 7 (HHV-6, HHV-7) have been recently reported in liver transplant patients. HHV-6 may cause fever, neurological disorders and hepatitis. The clinical significance of HHV-7 is less clear. HHV-6 and -7 are closely related to cytomegalovirus (CMV), and interactions between the viruses have also been suggested. In this study, we investigated the post transplant HHV-6 and -7 antigenemia was in relation to symptomatic CMV disease after liver transplantation. Consecutive 34 adult liver allograft recipients transplanted during 1999–2000 were included in the study. CMV infections were diagnosed by the frequent monitoring of pp65-antigenemia and by viral cultures. HHV-6 and -7 were demonstrated, by using immunoperoxidase staining and monoclonal antibodies against the virus specific antigens, in the mononuclear cells from the same blood specimens which were obtained for CMV pp65 monitoring. Altogether 322 blood specimens were analyzed. CMV disease was diagnosed in 12 (35%) patients during the first 3 months (first pp65 positive specimen mean 25 days, range 8–61 days) after transplantation. Concurrent HHV-6 antigenemia was detected in 10/12 (mean 14 days, range 6–22 days) and HHV-7 antigenemia in 9/12 patients (mean 25 days, range 10–89 days) after transplantation. HHV-6 usually appeared slightly before CMV. All CMV infections were successfully treated with ganciclovir and the CMV–antigenemia subsided. HHV-6 and -7 antigenemia also responded to the antiviral treatment, but more slowly than CMV. In conclusion, CMV infection was usually associated with HHV-6 and -7 antigenemia in liver transplant patients. The results support the suggestion that CMV, HHV-6 and -7 may have interactions. The clinical symptoms of CMV infection, may also be linked with HHV-6 or -7. [Copyright &y& Elsevier]

Background: Human herpes virus-6 (HHV-6) was first isolated in 1986. It has been shown to cause exanthema subitum and has been associated with various other diseases. HHV-6 infection is widespread, and more than 90% of the population have antibodies against HHV-6 at the age of 2 years. Once acquired, the virus remains latent in the body. This makes it difficult to draw any conclusions about a causal relationship between the demonstration of HHV-6 and a specific disease. Objectives: This work was to develop a μ-capture HHV-6 IgM enzyme linked immuno sorbent assay (ELISA) for use in routine diagnosis and for wide scale patient population analysis. Study design: A μ-capture HHV-6 IgM ELISA was established. A total of 682 sera consisting of 585 sera from Danish blood donors and 97 sera from patients with autoimmune antibodies were analysed in the HHV-6 IGM ELISA. One hundred and ninety-two sera had earlier been analysed for total HHV-6 antibody content in a competitive ELISA, 94 sera were analysed for cytomegalovirus (CMV) IgM and 57 sera for Epstein Barr virus (EBV) antibodies, using different ELISA assays. The results for 12 primary infections with HHV-6 are also reported. Results: A HHV-6 IgM optical density (OD)-ratio was calculated according to a constant positive control. An empirical cut off of 0.5 HHV-6 IgM OD-ratio was chosen (with regard to the 10 HHV-6 seroconverters), which resulted in a specificity of 97.5% of the HHV-6 IgM ELISA. Two of the three donor sera with HHV-6 IgM OD-ratios more than 1.05 had total HHV-6 antibody titers significantly above the group with IgM OD-ratios below 0.7 consisting with HHV-6 reactivation. There was no cross reactions to EBV or CMV IgM positive sera. Conclusion: The HHV-6 IgM ELISA seems valid to diagnose primary HHV-6 infection in particular in combination with the HHV-6 total antibody assay. [Copyright &y& Elsevier]