Human Herpesvirus-6 Load Research Articles

HHV-6 Infection and Chemokine RANTES Signaling Pathway Disturbance in Patients with Autoimmune Thyroiditis.

The aim of this study was to investigate the role of human herpesvirus-6 (HHV-6) in autoimmune thyroiditis (AIT) development. We examined the possible involvement of HHV-6 gene expression encoding immunomodulating proteins U12 and U51 in AIT development and their role in the modulation of chemokine signaling. One hundred patients with autoimmune thyroiditis following thyroidectomy were enrolled in this study. Nested polymerase chain reaction (nPCR) was used to detect the HHV-6 sequence in DNA samples. Reverse transcription PCR (RT-PCR) with three different HHV-6 gene targets (U79/80, U51 and U12) was to detect active infection markers. HHV-6 load was identified using a commercial real-time PCR kit. Immunohistochemistry was performed to investigate the expression of the HHV-6 antigen and RANTES (Regulated upon Activation, Normal T Cell Expressed and Secreted) in thyroid gland tissue. Different commercial immunosorbent assay kits were used for the detection of RANTES, IFNγ, IL-6, and TNFα levels in the AIT patient group and controls. We detected 98% presence of the HHV-6 genomic sequence in AIT patients’ thyroid gland tissues. Markers of active HHV-6 infection (HHV-6 U79/80, U12 and/or U51 mRNA) were predominant in AIT patients’ thyroid tissue samples in comparison with the control group (56% vs. 6%). Evidence from immunofluorescence microscopy showed that HHV-6 can persist in thyrocytes and can interact with RANTES. Visual confirmation of the intense immunofluorescence signal of RANTES detected in thyroid tissues could indicate high expression of this chemokine in the thyroid gland. On the other hand, immunosorbent assays showed very low RANTES levels in AIT patients’ peripheral plasma. These results indicate that RANTES level in AIT patients could be influenced by HHV-6 activation, which in turn may aid AIT development.

Influence of corticosteroid therapy on viral reactivation in drug‐induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms

Drug‐induced hypersensitivity syndrome (DIHS)/drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe adverse drug reaction characteristically associated with sequential reactivation of herpesviruses, such as human herpesvirus 6 (HHV‐6), Epstein‐Barr virus (EBV), and cytomegalovirus (CMV). Since systemic corticosteroids are thought to result in viral reactivation due to their immunosuppressive effects, we clarified the influence of systemic corticosteroid therapy on viral reactivation in DIHS/DRESS. Viral DNA in peripheral whole blood and serum sIL‐2R level were measured during the disease course in twenty DIHS/DRESS patients. Six of seven patients treated without corticosteroids experienced HHV‐6 viremia associated with elevated serum sIL‐2R levels. In contrast, high‐dose corticosteroids started within 1 week after onset tended to inhibit the occurrence of HHV‐6 reactivation with remarkable suppression of serum sIL‐2R level. Low‐dose corticosteroids or late‐start high‐dose corticosteroids did not suppress occurrence of HHV‐6 viremia and the increase of sIL‐2R levels. HHV‐6 load in the blood was clearly correlated with the serum sIL‐2R level. On the other hand, increased CMV load were found in patients treated with corticosteroids regardless of the start time. The frequency of detection of EBV DNA in peripheral blood was similarly observed in all groups. In conclusion, high‐dose corticosteroids started within 1 week tended to suppress HHV‐6 reactivation through suppression of T cell activation. However, CMV proliferation was promoted by corticosteroids regardless of the start time. These observations suggested that careful consideration should be given to the dose and timing of administration of systemic corticosteroids in the treatment of DIHS/DRESS.

ASSOCIATION OF HUMAN HERPES VIRUS 6A INFECTION IN ENDOMETRIAL EPITHELIAL CELLS WITH MISCARRIAGE

Background: The microbial infections are major cause in abortion, of which viruses appear to be the most frequently involved pathogens. Human Herpes Virus-6 (HHV-6) has been implicated in cases of poor pregnancy outcome. Objective: Detection and quantification of HHV-6 viral load along with titer of IgM and IgG in endometrial epithelial cells of women with unexplained miscarriage (cases group) compared to the decidual endometrial tissue in women with full term conception (the control group). Methods: A total of 90 samples, 45 were taken by curettage from miscarriage women at first and second trimesters of pregnancy and 45 were taken from women with full term pregnancy during caesarian operation. HHV-6 DNA was extracted from uterus lining tissue samples, and then it was detected and measured by real time qPCR; in addition, 3 ml of blood were collected from each subject for detection of HHV-6 IgM and IgG antibodies. Results: HHV-6 DNA was detected in uterus lining tissue of 20% of abortion and 22.22% of controls (P value=0.79). The median viral load of HHV-6 per 106 of uterine cells in abortion group was 15, while for control group was 13 (P=0.54). HHV-6 load increased with increased number of previous abortions, and it increased with presence of recurrent abortion history. It showed significant differences in the median of HHV-6 DNA load between recurrent abortion and non-recurrent abortion groups (P value =0.033). Positive anti-HHV-6 IgM serum antibodies in HHV-6 DNA detected and no-detected subjects were 0%, and 9.85% respectively; the positive anti-HHV-6 IgG serum antibodies in detected HHV-6 DNA were 100%, while 83% in HHV-6 DNA non-detected subjects. The combination of positive/negative results of the anti-HHV-6 IgG and IgM serum antibodies in HHV-6 DNA detected versus non-detected group showed 100% of HHV-6 DNA detected were positive for IgG and negative for IgM, versus 73% in non-detected group. Conclusion: HHV-6 might associated with unexplained miscarriage. HHV-6 infection might increase with increased number of previous abortions, and increased with presence of recurrent abortion history. Keywords: Human Herpes Virus 6A, Miscarriage Citation: Dosh HS, Abdulamir AS, Abdul-Razzaq SH. Association of Human Herpes Virus 6A infection in endometrial epithelial cells with miscarriage. Iraqi JMS. 2019; 17(1): 57-65. doi: 10.22578/IJMS.17.1.9

Cytokine and cellular responses to human herpesvirus-6B in patients with B-acute lymphoblastic leukemia.

Primary infection with human herpesvirus-6 (HHV-6), is followed by its lifelong persistence in the host. Most T-cell responses to HHV-6 have been characterized using peripheral blood from healthy adults; however, the role of HHV-6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV-6 infection in patients with B-acute lymphoblastic leukemia (B-ALL) was analyzed. HHV-6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti- and pro-inflammatory cytokines (IL-4, IL-1, IL-6, IL-8, IL-12p70, IL-17a, TNF-α and IFN-γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV-6 virus loads in blood. Characterization of T-cell responses to HHV-6 showed low specific T-cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN-γ-producing T cells were detected in 0.03%-0.23% and in 0%-0.2% of CD4+T cells, respectively. Strong production of IL-6 was detected in medium supernatants of challenged T-cells whatever the HHV-6 status of the patients (973.51 ± 210.06 versus 825.70 ± 210.81 pg/mL). However, concentrations of TNF-α and IFN-γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV-6 in blood was identified, suggesting that HHV-6 is not strongly associated with development of B-ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV-6B specific immune responses may be below the detection threshold of the assays used.

Association of active human herpesvirus-6 (HHV-6) infection with autoimmune thyroid gland diseases

ObjectivesViral infections frequently have been cited as important environmental factors implicated in the onset of autoimmune thyroiditis (AIT). The aim of this study was to determine the involvement of HHV-6 infection in the development of autoimmune thyroiditis. MethodsThis study included 45 patients (42 female and 3 male; median age 47.00 IQR 38.50–57.00) with histologically, laboratory, and clinically confirmed autoimmune thyroiditis, as well as 30 autopsied subjects (26 female and 4 male; median age 58.50, IQR 51.50–67.00) without thyroid pathologies and 30 healthy blood donors (25 female and 5 male; median age 33.50, IQR 27.75–44.25) as controls. Results were obtained by applying molecular virology and immunohistochemistry techniques. ResultsThe presence of persistent HHV-6 infection in AIT patients was significantly higher (p 0.0058) than in the control group (44/45 (98%) vs. 23/30 (77%), respectively). Also, a significantly higher frequency of HHV-6 activation marker (U79/80 mRNA) was found in patients’ thyroid gland tissue samples with AIT in comparison with the control group (18/44 (41%) vs. 1/17 (6%), respectively; p 0.0118). The median HHV-6 load was found to be higher in patients with active viral infection than in patients without it (2147, IQR 971–4188 vs. 551, IQR 145–1589 copies/1×106 cells; p 0.003). The presence of HHV-6 antigen expression was demonstrated in intrafollicular cellular clusters and immunohistochemistry indicated thyrocytes in the follicle wall. ConclusionsThese findings provide evidence of strong HHV-6 infection association with AIT development.

Identification of Viruses in Cases of Pediatric Acute Encephalitis and Encephalopathy Using Next-Generation Sequencing.

Acute encephalitis/encephalopathy is a severe neurological syndrome that is occasionally associated with viral infection. Comprehensive virus detection assays are desirable because viral pathogens have not been identified in many cases. We evaluated the utility of next-generation sequencing (NGS) for detecting viruses in clinical samples of encephalitis/encephalopathy patients. We first determined the sensitivity and quantitative performance of NGS by comparing the NGS-determined number of sequences of human herpesvirus-6 (HHV-6) in clinical serum samples with the HHV-6 load measured using real-time PCR. HHV-6 was measured as it occasionally causes neurologic disorders in children. The sensitivity of NGS for detection of HHV-6 sequences was equivalent to that of real-time PCR, and the number of HHV-6 reads was significantly correlated with HHV-6 load. Next, we investigated the ability of NGS to detect viral sequences in 18 pediatric patients with acute encephalitis/encephalopathy of unknown etiology. A large number of Coxsackievirus A9 and mumps viral sequences were detected in the cerebrospinal fluid of 2 and 1 patients, respectively. In addition, Torque teno virus and Pepper mild mottle viral sequences were detected in the sera of one patient each. These data indicate that NGS is useful for detection of causative viruses in patients with pediatric encephalitis/encephalopathy.

Detection frequency of human herpesviruses-6A, -6B, and -7 genomic sequences in central nervous system DNA samples from post-mortem individuals with unspecified encephalopathy.

In this autopsy-based study, human herpesvirus-6 (HHV-6) and -7 (HHV-7) genomic sequence frequency, HHV-6 variants, HHV-6 load and the expression of HHV-6 antigens in brain samples from the individuals, with and without unspecified encephalopathy (controls), using nested and real-time polymerase chain reactions, restriction endonuclease, and immunohistochemical analysis were examined. GraphPad Prism 6.0 Mann-Whitney nonparametric and chi-square test and Fisher's exact test were used for statistical analysis. The encephalopathy diagnoses were shown by magnetic resonance imaging made during their lifetime and macro- and microscopically studied autopsy tissue materials. Widespread HHV-6 and/or HHV-7 positivity was detected in the brain tissue of various individuals with encephalopathy, as well as in controls (51/57, 89.4% and 35/51, 68.6%, respectively; p = 0.009). Significantly higher detection frequency of single HHV-6 and concurrent HHV-6 + HHV-7 DNA was found in pia mater meninges, frontal lobe, temporal lobe, and olfactory tract DNAs in individuals with encephalopathy compared to the control group. HHV-6 load and higher frequency of the viral load >10 copies/10(6) cells significantly differed in samples from individuals with and without encephalopathy. The expression of HHV-6 antigens was revealed in different neural cell types with strong predominance in the encephalopathy group. In all HHV-6-positive autopsy samples of individuals with and without encephalopathy, HHV-6B was revealed. Significantly higher detection frequency of beta-herpesvirus DNA, more often detected HHV-6 load >10 copies/10(6) cells, as well as the expression of HHV-6 antigens in different brain tissue samples from individuals with encephalopathy in comparison with control group indicate on potential involvement of these viruses in encephalopathy development.

Human Herpesvirus-6 and Polyomaviruses DNAemia in Children and Young Adult Patients after Kidney Transplantation

Aims: Human herpesvirus-6 (HHV-6) and polyomaviruses (HPyV) establish latency and reactivate in immunocompromised hosts. The prevalence of their infections was investigated in pediatric and young adult kidney transplant recipients. Materials & methods: Blood was obtained from 83 patients. Quantitative real-time polymerase chain reactions were conducted to quantify HHV-6 and HPyV loads; nested PCR and reverse transcriptase-PCR assays were conducted to differentiate HHV-6A from 6B and to evaluate the presence of transcripts. Results: HHV-6 and HPyV DNAemia were detected in 19.3 and 18.1% patients, respectively. No association between HHV-6 and HPyV DNA presence, age of patients and time from transplant was observed. Conclusion: HHV-6 infection in immunosuppression setting was as common as those of HPyVs that are commonly recognized as opportunistic agents.

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation.

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

Quantitative analysis of human herpesvirus-6 and human cytomegalovirus in blood and saliva from patients with acute leukemia.

Human herpesvirus-6 (HHV-6) and human cytomegalovirus (HCMV) DNAs were quantified by real-time PCR assays in blood and saliva obtained from 50 patients with acute leukemia at the time of diagnosis (50 of each matrix), aplasia (65 of each matrix), remission (55 of each matrix), and relapse (20 of each matrix) to evaluate which biological matrix was more suitable to identify a viral reactivation, search for a possible link between HHV-6 and HCMV reactivations, and evaluate the relations between viral loads and count of different leukocyte types in blood. The median HHV-6 loads were 136; 219; 226, and 75 copies/million cells in blood at diagnosis, aplasia, remission and relapse, respectively. The HCMV loads were 193 and 317 copies/million cells in blood at diagnosis and remission. In the saliva samples, the HHV-6 loads were 22,165; 15,238; 30,214, and 17,454 copies/million cells at diagnosis, aplasia, remission, and relapse, respectively. The HCMV loads were 8,991; 1,461; 2,980, and 4,283 copies/million cells at diagnosis, aplasia, remission, and relapse, respectively. The HHV-6 load in the blood was correlated to the counts of polymorphonuclear leukocytes (R(2) = 0.5; P < 0.0001) and lymphocytes (R(2) = 0.4; P = 0.001) and was not correlated to the monocyte counts (R(2) = 0.07; P = 0.7). Saliva appears to be a more sensitive biological matrix than whole blood in the detection of HHV-6 or HCMV reactivations. The HHV-6 and HCMV reactivations were linked only in saliva.

The dynamics of herpesvirus reactivations during and after severe drug eruptions: their relation to the clinical phenotype and therapeutic outcome.

Drug-induced hypersensitivity syndrome/drug rash with eosinophilia and systemic symptoms (DIHS/DRESS) and Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) represent contrasting poles of severe drug eruptions, and sequential reactivations of several herpesviruses have exclusively been demonstrated in the former. No previous studies, however, were extended beyond the acute stage. We sought to investigate whether herpesvirus reactivations could also be observed in SJS/TEN and beyond the acute stage of both diseases. Patients with SJS (n = 16), SJS/TEN overlap (n = 2), TEN (n = 10), and DIHS/DRESS (n = 34) were enrolled. We performed a retrospective analysis of Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and cytomegalovirus (CMV) DNA loads sequentially determined by real-time polymerase chain reaction during a 2-year period after onset. Persistently increased EBV loads were detected in SJS during the acute stage and long after resolution, but not in others. In contrast, high HHV-6 loads were exclusively detected in DIHS/DRESS during the acute stage. The dynamics of herpesvirus reactivation varied in DIHS/DRESS according to the use of systemic corticosteroids: While EBV loads were higher in patients not receiving systemic corticosteroids, CMV and HHV-6 loads were higher in those receiving them. Distinct patterns of herpesvirus reactivation according to the pathological phenotype and to the use of systemic corticosteroids were observed during the acute stage and follow-up period, which may contribute, at least in part, to the difference in the clinical manifestations and long-term outcomes. Systemic corticosteroids during the acute stage may improve the outcomes in DIHS/DRESS.

Human herpesvirus‐6 encephalopathy after hematopoietic stem cell transplantation and class I human leukocyte antigen

Human herpesvirus-6 (HHV-6) encephalopathy is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although reactivation of HHV-6 is often observed after allo-HSCT, encephalopathy only affects a few patients with HHV-6 reactivation. Human leukocyte antigen (HLA) class I is expressed by most somatic cells, and a relationship between some class I alleles and neurological diseases has been reported. The HHV-6 load at two, three, and four weeks after allo-HSCT was examined. HHV-6 encephalopathy was diagnosed from symptoms, results of cerebrospinal fluid examination, and magnetic resonance imaging findings. The relation between HHV-6 reactivation or encephalopathy and the HLA class I status of the recipients was investigated. In 130 patients, 147 allo-HSCT transplantation procedures were carried out. HHV-6 reactivation and encephalopathy occurred in 56 and nine procedures, respectively. HLA mismatch (p = 0.008) and unrelated donor (p = 0.001) were associated with HHV-6 reactivation, but not with HHV-6 encephalopathy. HHV-6 encephalopathy was more frequent in patients with HLA-B*40:06 (p = 0.027). In addition, HLA-A*26:01 and HLA-B*40:06 were found to be associated with each other (p = 0.089), while HLA-B*40:06 and HLA-C*08:01 showed a significant association (p < 0.001). The HLA class I alleles of recipients may be associated with the occurrence of HHV-6 encephalopathy after allo-HSCT.

Successful ganciclovir therapy in a patient with human herpesvirus-6 encephalitis after unrelated cord blood transplantation: usefulness of longitudinal measurements of viral load in cerebrospinal fluid

There have been no reports of human herpesvirus-6 (HHV-6) encephalitis treatment based on both HHV-6 DNA load and the antiviral agent's concentration in the cerebrospinal fluid (CSF). A 20-year-old male with a hematological malignancy developed HHV-6 encephalitis 15 days after unrelated cord blood transplantation (UCBT). He had fever, chest pain, memory impairment, and insomnia. His CSF showed no increased cell counts, but the amount of HHV-6 DNA was elevated to 2.0 × 10(6) copies/ìgDNA. Magnetic resonance imaging (MRI) of the head revealed abnormal high-intensity signals in the left limbic system on T2-weighted and diffusion-weighted images. Intravenous administration of ganciclovir (GCV) was initiated at 5 mg/kg every 12 h on day 18, and was continued until day 137. The amount of HHV-6 DNA in the plasma became undetectable on day 25. The HHV-6 load in the CSF decreased to 1.5 × 10(3) copies/ìgDNA on day 32, and reached undetectable levels on day 53. The mean concentration of GCV 1 h after an infusion of 5 mg/kg was 4.12 mg/mL in plasma and 0.7 mg/mL in CSF. The chest pain and insomnia disappeared on days 35 and 47, respectively. Memory defects recovered up to day 85. Serial quantification of HHV-6 DNA in CSF may be useful for successful treatment with GCV in post-transplant HHV-6 encephalitis.

Multicenter External Quality Assessment of Molecular Methods for Detection of Human Herpesvirus 6

The purpose of this study was to evaluate the performance of laboratories for the detection and quantification of human herpesvirus 6 (HHV-6) by an external quality assessment (EQA) evaluation. The HHV-6 EQA panel consisted of eight samples containing various concentrations of HHV-6 type A (strain GS) or type B (strain Z29), two samples containing other herpesviruses (i.e., human cytomegalovirus [HCMV] and Epstein-Barr virus [EBV]), and two HHV-6-negative samples. Panel samples were prepared in human plasma, heat inactivated, and lyophilized. Panel distribution, data management, and analysis were coordinated by Quality Control for Molecular Diagnostics (QCMD), Glasgow, United Kingdom. Fifty-one laboratories participated and submitted 57 data sets. Eleven (19.3%) data sets were generated using conventional in-house assays, 11 (19.3%) data sets using commercial real-time PCR assays, and 35 (61.4%) data sets using in-house real-time PCR assays. The presence of HHV-6 DNA at viral loads exceeding 6,000 copies/ml was detected by all participants, and over 80% of the participants still reported correct qualitative results for the sample containing just over 200 copies/ml. The false-positivity rate was 1.8% for both the negative samples and the samples containing HCMV or EBV DNA. The majority (23/33; 69.7%) of quantitative data sets were generated using in-house real-time PCR assays. The standard deviations of the geometric means of the samples ranged from 0.5 to 0.7 log(10). The results of this first international EQA demonstrate encouraging analytical sensitivity for the detection of HHV-6-DNA in human plasma, although we observed extensive interlaboratory variation of quantitative HHV-6 DNA results. Standardization needs to be improved to allow further elucidation of the clinical significance of HHV-6 loads.

Évaluation de la plate-forme de PCR en temps réel LightCycler ® 480 pour la mesure des charges virales CMV, EBV, HHV-6 et BKV dans le sang total

Objective The Roche LightCycler ® 480 (LC480) system was evaluated for quantitative molecular diagnosis of opportunistic viral infections caused by human cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and BK virus (BKV), in comparison with “in-house” real-time PCR assays. Patients and methods A total of 253 whole blood specimens obtained from transplant recipients were tested. Results Both the “in-house” and the LC480 methods were highly correlated (Spearman correlation coefficient Rho ≥ 0.85; p < 0.0001) with an excellent overall qualitative agreement (90.5 %) and no significant quantitative difference between both techniques for the four viruses tested. The accuracy of the LC480 protocols were confirmed further by the results obtained with the 44 samples from the Quality Control for Molecular Diagnosis (QCMD) 2008 proficiency panel. Conclusion The LC480 system constitutes a suitable and versatile real-time PCR platform in a routine laboratory setting for the diagnosis and monitoring of opportunistic viral infections in transplant recipients, by measuring HCMV, EBV, HHV-6, and BKV loads in whole blood samples.

Different expression of human herpesvirus-6 (HHV-6) load in whole blood may have a significant impact on the diagnosis of active infection

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.

Use of the Roche LightCycler ® 480 system in a routine laboratory setting for molecular diagnosis of opportunistic viral infections: Evaluation on whole blood specimens and proficiency panels

The Roche LightCycler ® 480 (LC480) system was evaluated for quantitative molecular diagnosis of opportunistic viral infections caused by human cytomegalovirus (HCMV), Epstein–Barr virus (EBV), human herpesvirus 6 (HHV-6) and BK virus (BKV), in comparison with “in-house” real-time PCR assays. A total of 253 whole blood specimens obtained from transplant recipients were tested. Both the “in-house” and the LC480 methods were highly correlated (Spearman correlation coefficient Rho ≥ 0.85; p < 0.0001) with an excellent overall qualitative agreement (90.5%) and no significant quantitative difference between both techniques for the four viruses tested. The accuracy of the LC480 protocols were confirmed further by the results obtained with the 44 samples from the Quality Control for Molecular Diagnosis (QCMD) 2008 proficiency panel. The LC480 system constitutes a suitable and versatile real-time PCR platform in a routine laboratory setting for the diagnosis and monitoring of opportunistic viral infections in transplant recipients, by measuring HCMV, EBV, HHV-6, and BKV loads in whole blood samples.

Plasma HHV-6 viral load-guided preemptive therapy against HHV-6 encephalopathy after allogeneic stem cell transplantation: a prospective evaluation

Human herpesvirus 6 (HHV-6) causes life-threatening encephalopathy in recipients of allogeneic SCT, but no consensus has been reached regarding appropriate preventive methods. This study evaluated a plasma HHV-6 viral load-guided preemptive approach against HHV-6-associated encephalopathy. Plasma real-time PCR assay was performed once a week. Among 29 patients, 19 developed positive plasma HHV-6 DNA. Median maximum plasma HHV-6 DNA was 4593.5 copies/ml plasma (range, 150.0-127 891.0 copies/ml plasma). In one of eight events with low-level HHV-6 DNA (defined as <1000 copies/ml plasma) and four of seven events with mid-level HHV-6 DNA (1000-9999.5 copies/ml plasma), HHV-6 loads in plasma subsequently continued increasing. Ganciclovir was administered against six of nine patients with high-level HHV-6 DNA (> or =10,000 copies/ml plasma). High-level HHV-6 DNA resolved similarly in both groups with or without ganciclovir therapy. Among the nine patients with high-level HHV-6 DNA two developed encephalopathy. As encephalopathy developed before the detection of high-level HHV-6 DNA in plasma, these two patients had not received preemptive ganciclovir therapy. In conclusion, our preemptive approach against HHV-6-associated encephalopathy cannot prevent all cases of HHV-6 encephalopathy in SCT recipients due to the dynamic kinetics of plasma HHV-6 viral load.

Human herpesvirus 6‐associated retrobulbar optic neuritis in an HIV‐infected patient: response to anti‐herpesvirus therapy and long‐term outcome

Like other herpesviruses, human herpesvirus 6 (HHV-6) can reactivate in immunocompromised patients. A case is described of an HIV-1-infected patient who developed bilateral retrobulbar optic neuritis associated with HHV-6 infection. A 59-year-old woman, infected with HIV for 18 years, interrupted antiretroviral treatment because of therapeutic failure and severe metabolic complications. She presented subsequently with blurred vision and ophthalmological examination showed visual loss due to optic neuritis. Her CD4+ count was 285 cells/mm(3) and her plasma HIV-1 RNA level was 5.5 log(10) copies (cp)/ml. Magnetic resonance imaging of the brain was normal. HHV-6 loads were 3.2 log cp/ml in cerebrospinal fluid (CSF) and 6.3 log cp/10(6) peripheral blood mononuclear cells. Combined intravenous treatment was started with foscarnet and ganciclovir then changed to cidofovir and long-term valganciclovir. Her ocular condition improved gradually despite little decrease of the HHV-6 load in the CSF. Salvage antiretroviral treatment was then administered, with marked immunological and virological responses, contributing to further progressive ocular improvement. HHV-6-related optic neuritis has not been described previously in HIV-infected patients. Anti-HHV-6 treatment improved the patient's vision, but immune restoration seems to remain essential for long-term recovery.

158: Clinical outcome of human herpes-6 reactivation after haematopoietic stem cell transplantation in children

Background: Although human herpes virus 6 (HHV6) is known to reactivate during haematopoietic cell transplantation (HCT) and is suggested to be associated with severe clinical manifestations in adults, the clinical significance in children remains controversial. In this study we investigated the incidence of HHV6 reactivation and HCT-associated morbidity and mortality in children.Methods: Between 1/2004 and 4/2006 59 patients, median age 6.6y (0.1-18.1), underwent 68 allogeneic HCTs. By quantitative PCR HHV6, EBV, CMV and adenovirus (AdV)-plasma loads were measured once a week. Clinical features, engraftment, number of transfusions, HCT-associated mortality and morbidity (like GvHD) were monitored. Antiviral treatment for HHV-6 reactivation was only given from 4/2005 for those with clinical symptoms assumed to be associated with HHV6. HHV6 reactivations were grouped in group I (no HHV6), group II (loads <1000cp/mL) and group III (loads >1000cp/mL). CMV, EBV and AdV-reactivations were treated according to local guidelines. A risk factor analyses was performed using logistic regression.Results: 36 HLA-id and 23 HLA non-id grafts were used: 44 bone marrow/PBSCs and 15 cord blood grafts. Median follow up was 17 (5-35)mths. HHV6 reactivation occurred in 40/59 (67%) with 33/40 (82%) occurring within the first 30 d post-HCT. 23/59 (39%) had HHV6 loads above 1000cp/mL (group III). Groups did not differ regarding sex, age, donor source or HLA-disparity. In multivariate analysis HHV6 reactivation was associated with a lower survival in group III (OR 0,16; range 0,03-0,89; p=0.035) as well as with more multiple viral reactivations (p=0,049: OR 5,5; range 1,1-29) were seen in this group. In 11/13 multiple viral reactivation HHV6 was the first viral reactivation. There was a non-significant trend for more grade 2-4 acute-GvHD (p=0,058 OR 4,3; range 0,8-18) in group III. HHV6 didnot influence period of neutro-trombocytopenia.Conclusion: HHV6 reactivation is common after HCT in children and is associated with a higher rate of multiple viral reactivations, aGvHD and with a lower survival rate. Although the exact role of HHV6 reactivation in transplantation associated morbidity and mortality has to be elucidated, early detection and initiation of therapy might influence the outcome. Background: Although human herpes virus 6 (HHV6) is known to reactivate during haematopoietic cell transplantation (HCT) and is suggested to be associated with severe clinical manifestations in adults, the clinical significance in children remains controversial. In this study we investigated the incidence of HHV6 reactivation and HCT-associated morbidity and mortality in children. Methods: Between 1/2004 and 4/2006 59 patients, median age 6.6y (0.1-18.1), underwent 68 allogeneic HCTs. By quantitative PCR HHV6, EBV, CMV and adenovirus (AdV)-plasma loads were measured once a week. Clinical features, engraftment, number of transfusions, HCT-associated mortality and morbidity (like GvHD) were monitored. Antiviral treatment for HHV-6 reactivation was only given from 4/2005 for those with clinical symptoms assumed to be associated with HHV6. HHV6 reactivations were grouped in group I (no HHV6), group II (loads <1000cp/mL) and group III (loads >1000cp/mL). CMV, EBV and AdV-reactivations were treated according to local guidelines. A risk factor analyses was performed using logistic regression. Results: 36 HLA-id and 23 HLA non-id grafts were used: 44 bone marrow/PBSCs and 15 cord blood grafts. Median follow up was 17 (5-35)mths. HHV6 reactivation occurred in 40/59 (67%) with 33/40 (82%) occurring within the first 30 d post-HCT. 23/59 (39%) had HHV6 loads above 1000cp/mL (group III). Groups did not differ regarding sex, age, donor source or HLA-disparity. In multivariate analysis HHV6 reactivation was associated with a lower survival in group III (OR 0,16; range 0,03-0,89; p=0.035) as well as with more multiple viral reactivations (p=0,049: OR 5,5; range 1,1-29) were seen in this group. In 11/13 multiple viral reactivation HHV6 was the first viral reactivation. There was a non-significant trend for more grade 2-4 acute-GvHD (p=0,058 OR 4,3; range 0,8-18) in group III. HHV6 didnot influence period of neutro-trombocytopenia. Conclusion: HHV6 reactivation is common after HCT in children and is associated with a higher rate of multiple viral reactivations, aGvHD and with a lower survival rate. Although the exact role of HHV6 reactivation in transplantation associated morbidity and mortality has to be elucidated, early detection and initiation of therapy might influence the outcome.