Automated quantification of Epstein-Barr virus in whole blood for post-transplant lymphoproliferative disorders monitoring.

BACKGROUND: Standardized and sensitive assays for Epstein Barr Virus (EBV) are needed to define universal cutoff for treatment initiation in allogeneic hematopoietic stem cells transplant recipients. In a context of accreditation and the availability of EBV international standard, we evaluated the Abbott RealTime EBV (RT) assay for EBV quantification in whole blood. METHODS: The RT assay was compared on 282 prospective clinical samples with the Artus EBV PCR Kit V1 assay (V1) and we analyzed the kinetics of EBV load in 11 patients receiving rituximab treatment. RESULTS: The estimated limit of detection was 88 IU/mL. The assay was linear (r2 = 0.9974) in the range of all samples tested (100 to 1,000,000 IU/mL). Intra-assay coefficients of variation (CV) ranged between 0.35 and 1.35%, and inter-assay CV between 3.40 and 4.5%. On samples above the limit of quantification, the two assays were strongly correlated. EBV RT values were on average 0.30 log10 IU/mL lower than those measured with the V1 assay. In patients treated with rituximab, the RT assay remained positive in 5 patients at the time it dropped below undetectable levels with the V1 assay. CONCLUSIONS: In conclusion, the RT assay is a reliable assay for EBV load in whole blood. Its sensitivity will enable to estimate the kinetics of EBV load and the impact of treatments to control EBV reactivations.

Multimodal techniques failed to detect cytomegalovirus in human glioblastoma samples.

The role of the human cytomegalovirus (HCMV) in gliomagenesis is largely debated. Contradictory data exist regarding the sensitivity and specificity of HCMV detection techniques, including immunohistochemistry (IHC), in situ hybridization (ISH), and RNA and DNA sequencing. The aim of this study is to detect HCMV in glioblastoma (GBM) tumor samples using IHC, ISH, and real-time PCR (qPCR), as well as to correlate the findings with serological status and HCMV DNA load in blood. Forty-seven patients with histopathological diagnosis of GBM and HCMV serological status were retrospectively reviewed. HCMV DNA quantification in whole blood was performed in 31 patients. The detection of HCMV in tumor samples was performed using IHC in 42 cases, ISH in 10 cases, and qPCR in 29 cases. All but two patients were taking high steroid doses at the time of biological testing. HCMV seroprevalence was 68%. Active infection with HCMV DNA detected in blood was diagnosed in 6 out of 21 (28%) seropositive patients. HCMV was not detected in GBM samples using IHC or ISH, while qPCR was positive in one case (also positive for blood HCMV DNA). These data do not support a crucial role of HCMV in GBM tumorigenesis. HCMV might be reactivated in GBM patients, due to steroid treatment.

Short article: Hepatitis B virus status in children born to HIV/HBV coinfected women in a French hospital: a cross-sectional study.

OBJECTIVES: We assessed hepatitis B virus (HBV) status in children born to HIV/HBV coinfected women with large access to antiretroviral therapy. METHODS: All HIV/HBV coinfected pregnant women from 01 January 2000 to 01 January 2012 were included in the retrospective study (NCT02044068). Antiretroviral therapy during pregnancy and injection of HBV immunoglobulin/vaccine to newborns was recorded. We assessed HBV status of children aged at least 2 years. RESULTS: Twenty-one women (35 children) were studied. Twenty-six children (74%) had HBsAb: 22 had received immunoglobulin and 24 had received a complete vaccine (with immunoglobulin in 21 cases); their mothers had been administered lamivudine or tenofovir/emtricitabine during eight and nine pregnancies, respectively. Eight children (23%) were negative for HBsAg, HBsAb, and HBcAb: four (11.5%) had received immunoglobulin and a complete vaccine; in two children, it was not known whether they had received an immunoglobulin injection; in one child, the vaccine was incomplete; and in the last one, it was not known whether he had received immunoglobulin/vaccine. Their mothers had been administered lamivudine or tenofovir/emtricitabine during five and two pregnancies, respectively. No infant has chronic HBV infection (HBsAg) after prenatal mothers' antiretroviral therapy combined with a complete postnatal HBV protection. One child had HBcAb and HBsAb: it was not known whether she had received an immunoglobulin injection; the vaccine was incomplete. The mother had been administered lamivudine during the last trimester of pregnancy. CONCLUSION: Antiretroviral therapy in HBV/HIV coinfected women following current national HBV guidelines may prevent mother-to-child-transmission of HBV. Negativity of surrogate markers of vaccine-induced protection is frequent; large studies on long-term protection are needed.

Untreated highly viraemic pregnant women from Asia or sub-Saharan Africa often transmit hepatitis B virus despite serovaccination to newborns.

BACKGROUND & AIMS: Mother-to-child (MTC) hepatitis B virus (HBV) transmission has been mainly studied in Asia. The geographical origins of women and HBV genotypes differ in Europe. The aims were to determine the rate and risk factors of MTC HBV transmission from women with high HBV DNA loads in a maternity hospital in Paris, France. METHODS: Retrospective study of HIV-negative, HBs Ag-positive pregnant women with HBV DNA loads above 5 Log10 I.U/ml who were not given lamivudine or tenofovirDF during pregnancy between 2004 and 2011. RESULTS: Among 11 417 pregnant women, 437 (4%) showed a positive HBs Ag. Among these women, 52 had HBV DNA loads above 5 Log10 I.U/ml: 41, 10 and 1 born in Asia, sub-Saharan Africa and Europe respectively. Among the 52 women, 40 were eligible for the analysis: no antiviral therapy during pregnancy; children over 9 months old. Twenty-eight (70%) women were assessed, corresponding to 41 childbirths. Eleven children (27%) had positive HBs Ag, 14 (34%) had positive HBc and HBs Ab, 16 (39%) had positive HBs Ab only. The risk of having positive HBs Ag, according to maternal HBV DNA loads, was 14% for HBV DNA loads less or equal to 8 Log10 I.U/ml, 42% for HBV DNA loads over 8 Log10 I.U/ml, P = 0.04, but not related to the women's origin, HBV genotype. CONCLUSIONS: This study confirms that serovaccination does not fully protect newborns from MTC HBV transmission, when maternal HBV DNA loads exceed 5 Log10 I.U/ml, regardless of the women's origin or HBV genotype.

Hepatitis B virus (HBV) status of children born to HIV/HBV co-infected women in a French hospital: a cross-sectional study.

INTRODUCTION: Human Immunodeficiency Virus (HIV) Mother-To-Child-Transmission (MTCT) and prevention by combined antiretroviral therapy (cART) have been extensively studied. Hepatitis B Virus (HBV) MTCT from HIV/HBV co-infected women and prevention by antiretroviral therapy with dual activity have been poorly studied. The aim of the study was to assess HBV MTCT from HIV/HBV co-infected women in a developed country with a large access to cART. MATERIALS AND METHODS: HIV/HBV co-infected pregnant women attending the Obstetrics Department from 1st January 2000 to 1st January 2012 could be included in the study (NCT02044068). Antiretroviral therapy during pregnancy, injection of immunoglobulin and/or vaccine to newborns was retrospectively recorded. We assessed HBV status of children at least as old as two years. RESULTS: Forty nine (9.2%) from 530 HIV-infected women followed in the hospital were HIV/HBV co-infected. 34 (69.4%) had given birth to 57 children in the hospital. 13 of these women (22 children) were lost-to-follow-up, 21 women (35 children) could be studied. Twenty six children (74.3%) had HBs Ab at a protective level, 22 of them had received immunoglobulin at birth; 24 had received a complete vaccine schedule during the first six months of life (with immunoglobulin in 21 cases). The women had been given lamivudine or tenofovir/emtricitabine during eight and nine pregnancies respectively. Eight children (22.8%) were tested negative for HBs Ag, HBs Ab and HBc Ab: 4 (11.4%) had received immunoglobulin and a complete vaccine schedule; in two children, immunoglobulin was uncertain; in one child, the vaccine schedule was incomplete; in the last one, data about immunoglobulin and the vaccine schedule were lacking. The women had been given lamivudine or tenofovir/emtricitabine during five and two pregnancies respectively. One child had HBc Ab and HBs Ab, immunoglobulin was uncertain and the vaccine schedule was incomplete. The woman had been given lamivudine during the last trimester. CONCLUSIONS: Three quarters of the children were protected. HBs Ab were negative in more than a tenth of the children who had received immunoglobulin and a complete vaccine schedule, questioning on long-term protection and underlining the need of control.

Favorable impact of natural killer cell reconstitution on chronic graft-versus-host disease and cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation.

Natural killer cells are the first lymphocyte subset to reconstitute, and play a major role in early immunity after allogeneic hematopoietic stem cell transplantation. Cells expressing the activating receptor NKG2C seem crucial in the resolution of cytomegalovirus episodes, even in the absence of T cells. We prospectively investigated natural killer-cell reconstitution in a cohort of 439 adult recipients who underwent non-T-cell-depleted allogeneic hematopoietic stem cell transplantation between 2005 and 2012. Freshly collected blood samples were analyzed 3, 6, 12 and 24 months after transplantation. Data were studied with respect to conditioning regimen, source of stem cells, underlying disease, occurrence of graft-versus-host disease, and profiles of cytomegalovirus reactivation. In multivariate analysis we found that the absolute numbers of CD56(bright) natural killer cells at month 3 were significantly higher after myeloablative conditioning than after reduced intensity conditioning. Acute graft-versus-host disease impaired reconstitution of total and CD56(dim) natural killer cells at month 3. In contrast, high natural killer cell count at month 3 was associated with a lower incidence of chronic graft-versus-host disease, independently of a previous episode of acute graft-versus-host disease and stem cell source. NKG2C(+)CD56(dim) and total natural killer cell counts at month 3 were lower in patients with reactivation of cytomegalovirus between month 0 and month 3, but expanded greatly afterwards. These cells were also less numerous in patients who experienced later cytomegalovirus reactivation between month 3 and month 6. Our results advocate a direct role of NKG2C-expressing natural killer cells in the early control of cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation.

Stability and antiviral activity against human cytomegalovirus of artemisinin derivatives.

OBJECTIVES: Artesunate, a derivative of dihydroartemisinin, itself a product of artemisinin, inhibits the replication of cytomegalovirus in vitro. In vivo, artesunate undergoes rapid conversion into the active metabolite dihydroartemisinin. The in vitro stability of the compounds and the antiviral activity of dihydroartemisinin are of great concern for the interpretation of in vitro testing. The aim of the study was to measure artesunate conversion into dihydroartemisinin in culture medium and to evaluate the stability and antiviral activity of artemisinin derivatives, according to culture conditions. METHODS: Conversion of artesunate into dihydroartemisinin was measured in culture medium with or without fetal calf serum, in the presence or absence of fibroblast monolayers, at different times. The stability of artemisinin derivatives was determined in serum-enriched medium. Concentrations of each compound inhibiting viral DNA synthesis by 50% were determined in fibroblasts cultured in serum-free or serum-enriched medium, after addition of compound as a single dose or fractional doses. RESULTS: Conversion of artesunate into dihydroartemisinin in serum-free or serum-enriched medium was non-equimolar. The half-lives of artesunate, dihydroartemisinin and artemisinin were 10.3 ± 0.9, 5.2 ± 0.5 and 11.2 ± 1.2 h, respectively. Activity of dihydroartemisinin and artesunate was markedly reduced in serum-starved cells. Unexpectedly, dihydroartemisinin displayed a lower activity than artesunate. Addition of both compounds as fractional doses increased their activity. Artemisinin had no anticytomegaloviral activity. CONCLUSIONS: Artemisinin derivatives were shown to be unstable in vitro and their addition as fractional doses could partly compensate for this instability. Importantly, the cellular physiological condition was a determinant of their antiviral activity.

Dynamics of cytomegalovirus populations harbouring mutations in genes UL54 and UL97 in a haematopoietic stem cell transplant recipient.

We characterised by pyrosequencing, the dynamics of cytomegalovirus populations harbouring mutations A594V in gene UL97 and A834P and Q578H in gene UL54 in a haematopoietic stem cell transplant recipient. Unexpected re-emergence of A594V and decrease of A834P under CMX001 were shown to depend on both the selection pressure exerted by the antiviral treatments and the immune response.

Fully automated quantification of cytomegalovirus (CMV) in whole blood with the new sensitive Abbott RealTime CMV assay in the era of the CMV international standard.

Fully standardized reproducible and sensitive quantification assays for cytomegalovirus (CMV) are needed to better define thresholds for antiviral therapy initiation and interruption. We evaluated the newly released Abbott RealTime CMV assay for CMV quantification in whole blood (WB) that includes automated extraction and amplification (m2000 RealTime system). Sensitivity, accuracy, linearity, and intra- and interassay variability were validated in a WB matrix using Quality Control for Molecular Diagnostics (QCMD) panels and the WHO international standard (IS). The intra- and interassay coefficients of variation were 1.37% and 2.09% at 5 log10 copies/ml and 2.41% and 3.80% at 3 log10 copies/ml, respectively. According to expected values for the QCMD and Abbott RealTime CMV methods, the lower limits of quantification were 104 and <50 copies/ml, respectively. The conversion factor between international units and copies (2.18), determined from serial dilutions of the WHO IS in WB, was significantly different from the factor provided by the manufacturer (1.56) (P = 0.001). Results from 302 clinical samples were compared with those from the Qiagen artus CMV assay on the same m2000 RealTime system. The two assays provided highly concordant results (concordance correlation coefficient, 0.92), but the Abbott RealTime CMV assay detected and quantified, respectively, 20.6% and 47.8% more samples than the Qiagen/artus CMV assay. The sensitivity and reproducibility of the results, along with the automation, fulfilled the quality requirements for implementation of the Abbott RealTime CMV assay in clinical settings. Our results highlight the need for careful validation of conversion factors provided by the manufacturers for the WHO IS in WB to allow future comparison of results obtained with different assays.

Eight flavonoids and their potential as inhibitors of human cytomegalovirus replication.

The drugs currently available for treatment of severe human cytomegalovirus (HCMV) infections suffer from many drawbacks, particularly toxicity, and potential teratogenicity contraindicating their use in target populations such as pregnant women. The emergence of drug-resistant strains is still a problem for disease management, particularly in immunosuppressed populations where antivirals are used for extended periods of time. The flavonoid family of drugs contains promising candidates as they have low toxicity and inhibit different targets to currently available antivirals. We report here that, unlike their chalcon homologs, four flavonoids (baicalein, quercetin, quercetagetin and naringenin) inhibit various stages of HCMV replication, the most active anti-HCMV compound being baicalein and the less active and less selective being quercetagetin. These drugs could provide potential inhibitors of virus replication alone or in combination, without increased toxicity.

Hepatitis E virus infection in HIV-infected patients with elevated serum transaminases levels.

Increases in aminotransferases levels are frequently encountered in HIV-positive patients and often remain unexplained. The role in this setting and natural history of hepatitis E in HIV-infected patients are unknown. The aim of the study was to assess HEV infection in HIV-infected patients attending a Parisian hospital, with a current or previous cryptogenic hepatitis.191 plasma samples collected from 108 HIV-infected patients with elevated aminotransferases levels were retrospectively tested for the presence of hepatitis E virus (HEV) infection markers: anti-HEV IgM antibodies, anti-HEV IgG antibodies, anti-HEV IgG avidity index and plasma HEV RNA.One acute infection, documented by positive tests for anti-HEV IgM antibody, low anti-HEV IgG avidity index and plasma HEV RNA (genotype 3e), and three past infections were diagnosed, without any observed case of persistent infection. The acute hepatitis was benign and resolved spontaneously within two weeks. This infection was probably contracted locally. Acute HEV hepatitis can occur in HIV-infected patients but rarely explains cryptogenic hepatitis, at least in an urban HIV population, regardless geographic origin and CD4 counts.

Antiviral activity of ganciclovir and artesunate towards human cytomegalovirus in astrocytoma cells.

Antimalarial drug artesunate inhibits cytomegalovirus (HCMV) replication in human fibroblasts. Astrocytes, the major cell type of the brain, support cytomegalovirus (HCMV) replication. The aim of the study was to assess the antiviral activity of artesunate in astrocytoma cell line U373MG in comparison with ganciclovir. The antiviral concentration inhibiting by 50% (IC(50)) the synthesis of viral DNA was measured by real-time PCR in parallel in U373MG and MRC-5 cells. Reference HCMV strains susceptible and resistant to ganciclovir, and clinical isolates were tested. Ganciclovir and artesunate had similar activity in U373MG cells and MRC-5 fibroblasts. The artesunate IC(50)s in U373MG cells (1.5-2.25 µM) were at least 36-fold lower than the 50% cytotoxicity concentrations. Then, the anti-HCMV activity of artesunate was demonstrated in a cancer cell line.

Drug-resistant cytomegalovirus in transplant recipients: a French cohort study.

OBJECTIVES: Cytomegalovirus (CMV) drug resistance is a therapeutic challenge in the transplant setting. No longitudinal cohort studies of CMV resistance in a real-life setting have been published in the valganciclovir era. We report findings for a French multicentre prospective cohort of 346 patients enrolled at initial diagnosis of CMV infection (clinical trial registered at clinicaltrials.gov: NCT01008540). PATIENTS AND METHODS: Patients were monitored for detection of CMV infection for ≥2 years. Real-time detection of resistance by UL97 and UL54 gene sequencing and antiviral phenotyping was performed if viral replication persisted for >21 days of appropriate antiviral treatment. Plasma ganciclovir assays were performed when resistance was suspected. RESULTS: Resistance was suspected in 37 (10.7%) patients; 18/37 (5.2% of the cohort) had virological resistance, associated with poorer outcome. Most cases involved single UL97 mutations, but four cases of multidrug resistance were due to UL54 mutations. In solid organ transplant recipients, resistance occurred mainly during primary CMV infection (odds ratio 8.78), but also in two CMV-seropositive kidney recipients. Neither CMV prophylaxis nor antilymphocyte antibody administration was associated with virological resistance. CONCLUSIONS: These data show the feasibility of surveying resistance. Virological resistance was frequent in patients failing antiviral therapy. More than 1/5 resistant isolates harboured UL54 mutations alone or combined with UL97 mutations, which conferred a high level of resistance and sometimes were responsible for cross-resistance, leading to therapeutic failure.

Rapid determination of antiviral drug susceptibility of human cytomegalovirus by real-time PCR.

A quantitative real-time PCR-based assay was developed for determination of cytomegalovirus (HCMV) susceptibility to antiviral drugs. After HCMV isolate-growth for 4 days, antiviral drug susceptibility was determined by measuring the reduction of intracellular HCMV DNA in the presence of increasing concentrations of either ganciclovir, or foscarnet or cidofovir. The 50% inhibitory concentration (IC(50)) was the drug concentration that reduced the number of HCMV genome copies by 50%. The IC(50) values were measured for seven HCMV reference strains sensitive or resistant to one or more antiviral drugs. The antiviral susceptibility of 21 HCMV isolates was then tested and the results were consistent with prior determination of their phenotype and/or genotype by plaque reduction assay and sequencing. The real-time PCR susceptibility assay reported here was found to be highly reproducible, simpler to perform than the plaque reduction assay, and amenable to use in the routine diagnostic virology laboratory.

Putative functional domains of human cytomegalovirus pUL56 involved in dimerization and benzimidazole D-ribonucleoside activity.

BACKGROUND: Benzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89 (the large and small subunits of the HCMV terminase, respectively) their mechanism of action is not yet fully understood. We aimed here to better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives. METHODS: The HCMV pUL56 protein was studied by sequence analysis of the HCMV UL56 gene and herpesvirus counterparts combined with primary structure analysis of the corresponding amino acid sequences. RESULTS: The UL56 sequence analysis of 45 HCMV strains and counterparts among herpesviruses allowed the identification of 12 conserved regions. Moreover, comparison with the product of gene 49 (gp49) of bacteriophage T4 suggested that the pUL56 zinc finger is localized close to the dimerization site of pUL56, providing a spatial organization of the catalytic site that allows recognition and cleavage of DNA. CONCLUSIONS: This study provides a basis to investigate the mechanism of concatemeric DNA cleavage and a biochemical basis for DNA packaging inhibition by benzimidazole derivatives.

New functional domains of human cytomegalovirus pUL89 predicted by sequence analysis and three-dimensional modelling of the catalytic site DEXDc.

INTRODUCTION: Benzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89, the large and small subunits of the HCMV terminase respectively, their mechanism of action is not yet fully understood. METHODS AND RESULTS: To better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives, we studied the HCMV pUL89 protein by a genetic approach combined with primary structure analysis. The pUL89 sequence analysis of 25 HCMV strains and counterparts among herpesviruses allowed identification of 12 conserved regions. We also built a three-dimensional model of the pUL89 ATPasic catalytic site, including ATPase motor motifs 1, II and III, that may facilitate the development of future antiviral drugs active against HCMV. Finally, we identified several putative functional domains in pUL89, such as pUL89 zinc finger (pUL89-ZF), DNA cutting sites and portal binding sites, that are probably involved in CMV DNA cleavage and packaging.

Reactivation of lamivudine-resistant occult hepatitis B in an HIV-infected patient undergoing cytotoxic chemotherapy.

BACKGROUND: Reactivation of occult hepatitis B virus (HBV) infection is a well-known complication of cytotoxic chemotherapy. Lamivudine prophylaxis is recommended to reduce the incidence and severity of hepatitis in this context. CASE REPORT: An HIV-infected patient positive for HBs antigen became positive for HBc antibody alone under lamivudine given as part of antiretroviral therapy. He was treated with chemotherapy for non-Hodgkin's lymphoma while under lamivudine. Then, he developed HBV-related hepatitis that led to delay chemotherapy. He received adefovir that induced a dramatic decline in HBV DNA load and a normalisation of hepatic enzyme levels. However, the patient died of a relapse of lymphoma. Retrospective analysis of stored plasma samples showed evidence of lamivudine-resistant occult hepatitis before the onset of chemotherapy and reactivation of the HBV mutant. CONCLUSION: To our knowledge, this is the first report of occult hepatitis reactivation due to lamivudine-resistant mutant selected under lamivudine therapy in an HIV-infected patient. Our study underlines the need to carefully investigate lamivudine resistance in HIV-infected patients with occult infection under lamivudine therapy. Those patients should be monitored with the addition of anti-viral agents effective against the mutant strain.

Development and validation of a non-radioactive DNA polymerase assay for studying cytomegalovirus resistance to foscarnet.

Phenotypic characterisation of the human cytomegalovirus (HCMV) pUL54 DNA polymerase is a useful tool for testing for mutations in the UL54 gene thought to render HCMV resistant to foscarnet. In this study, an in-house non-isotopic method for assessing polymerase enzymatic activity in the presence and absence of foscarnet was developed and its utility for HCMV polymerase phenotyping evaluated. Polymerase activity was assessed by monitoring the incorporation of digoxigenin-labelled nucleotides into the growing DNA chain and foscarnet concentrations inhibiting enzymatic activity by 50% were determined. HCMV DNA polymerases were synthesised in vitro by expression of UL54 under the control of the T7 promoter. Mutations of interest were introduced into the wild-type UL54 gene by site-directed mutagenesis. Mutated polymerases and polymerases from HCMV reference strains were studied. The activity of polymerases containing mutations known to confer resistance to foscarnet (V715M, T700A and N495K) was inhibited by concentrations of foscarnet eight to 14 times higher than those required to inhibit wild-type polymerases. Our in-house non-radioactive phenotypic assay was sensitive and reproducible. It is also easy to perform and could provide a convenient method for characterising mutations conferring resistance to foscarnet in HCMV.