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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 (r 2  = 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 log 10 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.

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.

Background: We describe a homosexual man who strongly controlled HIV-1 for ten years despite lack of protective genetic background., Methods: HIV-1 DNA was measured in blood and other tissues. Cell susceptibility was evaluated with various strains. HIV-1-specific (CD4 and CD8 activation markers and immune check points) and NK cells responses were assessed; KIRs haplotypes and HLA alleles were determined., Findings: Two HIV-1 RNA copies/mL of plasma were detected in 2009, using an ultra-sensitive assay. HIV-DNA was detected at 1.1 and 2 copies/10 6 PBMCs in 2009 and 2015 respectively, at 1.2 copies/10 6 cells in rectal cells in 2011. WBs showed weak reactivity with antibodies to gp160, p55 and p25 from 2007 to 2014, remaining incomplete in 2017. CD4 T cells were susceptible to various strains including HIV KON , a primary isolate of his own CRF02_AG variant. CD8 T cells showed a strong poly-functional response against HIV-Gag, producing mainly IFN-γ; a robust capacity of antibody-dependant cell cytotoxicity (ADCC) was observed in NK cells. Case patient was group B KIR haplotype. Neutralizing antibodies were not detected. CD4 and CD8 blood T cells showed normal proportions without increased activation markers. Phylogenetic analyses identified the same CRF02_AG variant in his partner. The patient and his partner were heterozygous for the CCR5ΔD32 deletion and shared HLA-B*07, C*07 non-protective alleles., Interpretation: This thorough description of the natural history of an individual controlling HIV-1 in various compartments for ten years despite lack of protective alleles, and of his partner, may have implications for strategies to cure HIV-1 infection., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Human cytomegalovirus (HCMV) resistance to antiviral drugs is a major drawback of repeated or long-duration treatment in immunocompromised patients. Resistance testing is usually performed by genotypic assays. For accurate interpretation of these assays, the role of new mutations in HCMV resistance has to be assessed. Two previously unknown UL54 single point mutations (D515Y and V787A) were characterized for phenotypic drug-resistance by marker transfer analysis using bacterial artificial chromosome (BAC) mutagenesis. Increases in 50% inhibitory concentrations of ganciclovir and foscarnet were found for both mutated recombinant strains showing that mutations D515Y and V787A induce resistance to both antivirals. Importantly, none of those impacted the viral growth kinetics. For a better understanding of their molecular resistance mechanisms, a 3D homology model was used to localize the mutated amino-acids in functional domains of UL54 and predict their impact on UL54 function and resistance. However, 3D homology model analysis has limits and phenotypic characterization using BAC-HCMV is still essential to measure the role of unknown mutations., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pre-emptive antiviral treatment efficiently prevents occurrence of cytomegalovirus (CMV) disease in allogeneic stem cell transplant recipients. However, successive treatment courses can be necessary. The current study was aimed at determining factors that could influence the response to antiviral treatment, in particular the donor CMV serostatus. A total of 147 consecutive CMV-seropositive recipients (R+) were included and prospectively monitored for 6 months after transplantation. Reactivation of CMV occurred in 111 patients, 61 of 78 with a CMV-positive donor (D+) and in 50 of 69 with a CMV-negative donor (D-) (p 0.45). Baseline viral loads and initial viral doubling times did not differ between D+/R+ and D-/R+. Fifteen D+/R+ and four D-/R+ had self-resolving CMV infections. A total of 92 patients received antiviral treatment and 81 (88%) had a significant decrease in CMV load under therapy. Repeated CMV episodes were observed in 67% of those and were significantly more frequent in D-/R+ than in D+/R+ (p <0001). Half-life of CMV under treatment was significantly longer in D-/R+ than in D+/R+. Treatment failure observed in eight recipients was associated with low leucocyte count at reactivation onset, and was significantly more frequent in D-/R+ (six patients) than in D+/R+ (two patients) (p <0.0001). CMV strains resistant to antivirals were found in two D-/R+. Donor CMV serostatus influenced neither CMV reactivation occurrence nor the kinetics of CMV DNA load in the early phase of CMV replication but had a significant impact on response to antiviral therapy. Virological drug-resistance remained rare., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

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.

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., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (Copyright© Ferrata Storti Foundation.)

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.

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.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Maribavir (MBV), a UL97 inhibitor, shows good oral bioavailability, low host cell toxicity, and theoretical benefits to inhibit cross-resistant viruses. We herein examined clinical and virological outcomes of 12 patients, including 3 bone marrow recipients and 9 organ recipients infected with resistant cytomegalovirus (CMV) and treated with MBV during 2011-2012. All received at least 800-mg daily doses. They had developed clinical (12/12) and/or virological (11/12) resistance to CMV infection. Based on a decrease of viral load in blood >1.5 log copies/mL half of them responded to MBV treatment. The individual changes varied from a rapid decrease in viral load (n = 4) to no response (n = 3) with some late response slowly decreasing viremia (n = 3). In 2 cases MBV was used as secondary prophylaxis. No clear parameter emerged as a clinical surrogate for nonresponse to MBV. These results contrast with the lack of efficacy in phase III trials of MBV prophylaxis among stem cell recipients, which were possibly due to low doses or inadequate timing of drug initiation in the study. Additional clinical and surrogate laboratory markers are needed to determine antiviral responses to guide MBV use. Dosage ranging studies might benefit future MBV use., (Copyright © 2013. Published by Elsevier Inc.)

Unlabelled: Monitoring cytomegalovirus circulating viral load is an important parameter of the follow-up in immunocompromised patients. It can be measured either by DNAemia or by pp65 antigenemia. The French national reference center for cytomegaloviruses organized an investigation of practice in 37 teacher hospital virology laboratories to assess the situation in France in 2010., Methods: A questionnaire was sent to collect following information: method used in routine for monitoring of circulating viral load of CMV, assay used, sample matrix and extraction method., Results: Thirty-six over thirty-seven laboratories filled the questionnaire. Among these, 67% used the quantitative PCR in routine, 11% antigenemia and 22% antigenemia or quantitative PCR; 87% of the laboratories use whole blood for quantitative PCR, whereas 10% and 3% use plasma and leukocytes respectively. Among the laboratories using DNAemia, 100% used real-time PCR assays, 91% use an automated extraction and 9% a manual extraction., Conclusion: Thus in France, measurement of DNAemia by real-time PCR is a tool, which gradually replaces the antigenemia for the monitoring of cytomegalovirus infection among immunocompromised patients. The very great diversity of the methods used justifies the installation of a national quality control on total blood, matrix used by 87% of the laboratories., (Copyright © 2011. Published by Elsevier SAS.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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.

Unlabelled: Occult hepatitis B is defined as the presence of hepatitis B virus (HBV) DNA in the absence of detectable HBs antigen. The prevalence of occult hepatitis B among patients HIV-infected is uncertain, varying between 0% and 89%, and the clinical consequences of the coinfection are poorly known. The aim of this study was to evaluate the frequency of occult hepatitis B among HIV-infected patients and determine risk factors., Methods: This retrospective study was conducted with plasma samples from 31HIV-infected patients untreated for HBV infection and for whom at least one sample was available. All patients were found to be carriers of isolated anti-HBc antibodies between 2000 and 2008, and HBV DNA was quantified in 51 samples (one to three per patient) by real-time PCR using the Qiagen HBV PCR kit., Results: HBV DNA was found in samples from seven patients (22%). Occult hepatitis B seemed to be more frequent among patients coinfected with HCV (p=0.047). The number of CD4 cells was significantly less in samples containing detectable HBV DNA than in those with no detectable HBV DNA., Conclusion: The prevalence of occult hepatitis B seemed high, and HBV DNA titers were weak (< 20UI/mL), among patients infected with HIV and carrying isolated anti-HBc antibodies. These results would support screening HIV-infected patients for the presence of HBV DNA if confirmed with a larger patient population., (Copyright 2009 Elsevier Masson SAS. All rights reserved.)

Background: Factors associated with advanced liver disease have been incompletely explored in HIV/HBV coinfected patients., Objectives: To describe liver-related morbidity, mortality, and related risk factors, in HIV/HBV coinfected patients., Study Design: We followed-up 107 consecutive HIV/HBV coinfected patients. Clinical, biological and virological data were collected every 3 months. Liver-related mortality and a composite score were used to define advanced liver disease., Results: The patients were mainly sub-Saharan Africans (61%) or Europeans (33%). Forty-four percent of patients had liver biopsy, 78% of patients received lamivudine. Advanced liver disease (ALD) was diagnosed in 19/107 patients during follow-up (mean 4.8 years): 10 extensive fibrosis, 5 cirrhosis, 3 hepatocellular carcinoma resulting from cirrhosis, and 1 fulminant hepatitis following lamivudine withdrawal. Eleven patients died, 4 from HBV-related liver disease. In univariate analysis, male gender, mean HIV and HBV viral loads, and raised AST/ALT transaminases were associated with increased risk of ALD. The strongest associations, in a multivariate model, were mean AST transaminase and cumulated time receiving lamivudine, with a favourable effect. 39% of patients with increased mean AST presented with ALD, versus 7% when normal mean AST (Relative Risk 5.5)., Conclusions: During HIV/HBV coinfection, transaminase levels are strongly associated with ALD. Normal mean AST has a high negative predictive value, contrary to previously reported data in HIV/HCV patients., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

Six cases of cytomegalovirus retinitis (CMVR) after allogeneic hematopoietic stem cell transplantation were diagnosed between 2002 and 2005 in our center, whereas only one case was diagnosed between 1985 and 2001. Cumulative incidence reaches 2.2%, whereas this complication has been rarely described after hematopoietic stem cell transplantation. We aimed to describe clinical and biologic features of CMVR and search for risk factors associated with CMVR. CMVR was diagnosed on specific funduscopic examination in all patients either on visual symptoms (n=3) or on systematic ophthalmologic examination (n=3). CMVR occurred in the context of unrelated transplantation in patients with profound immune defect and multiple episodes of cytomegalovirus (CMV) reactivation. The combination of a CMV-seropositive recipient and CMV-seronegative donor was the leading risk factor.

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.

We report on a case of herpes simplex virus (HSV) type 1 colitis in a 69-year-old patient with common variable immunodeficiency syndrome. A treatment with polyvalent immunoglobulins was discontinued in April 2001. In March 2004 she developed chronic diarrhoea related to rectosigmoidal and caecal ulcerations. In November 2004, HSV was recovered in tissue culture from colonic biopsies. Valaciclovir was then started, leading the patient to clinical remission at day 4, and continued for a 6-week course (without any secondary antiviral prophylaxis). Colonic biopsies were negative for HSV by tissue culture and PCR within 3 weeks of antiviral treatment. Intravenous polyvalent immunoglobulin infusions were readministered within the third week of antiviral treatment. She has declared no clinical event since this period. Three months after the antiviral treatment was achieved, a rectosigmoidoscopy showed an ad-integrum macroscopic and histological mucosal healing whereas PCR was negative for HSV in the colonic tissue. As a large proportion of patients with common variable immunodeficiency syndrome present not only as a humoral immunodeficiency but also as a defect in the cellular immunity compartment (with T-cell deficits), HSV, as well as cytomegalovirus, should be investigated in patients with common variable immunodeficiency syndrome presenting colitis.

The human cytomegalovirus (HCMV) DNA polymerase (pUL54), a member of DNA polymerase family B, is a DNA-dependant polymerase, which possesses both polymerase and 3'-5'exonuclease enzymatic functions. pUL54 is the specific target of the anti-HCMV drugs currently used for the treatment of severe HCMV infections, including ganciclovir and its prodrug (valganciclovir), cidofovir, foscarnet. pUL54 is related to the DNA polymerases of other herpes viruses through a series of eight conserved domains. The DNA polymerase alterations involved in resistance to antiviral drugs are mostly located in these conserved domains. Some of those affect the enzymatic functions (polymerase and/or exonuclease) of the protein and/or the fitness of the mutated viruses. Phenotypic and genotypic assays are used to study the susceptibility to antiviral drugs of HCMV strains. The phenotype of mutated polymerase is a new tool to characterize mutations suspected to confer resistance to foscarnet. Significant advances have been made in the understanding of the molecular mechanisms of HCMVdrug resistance. However, cristallographic data are needed to design new antiviral agents.

Foscarnet is currently licensed for the treatment of human cytomegalovirus (HCMV) infection. Mutations proven to confer resistance to foscarnet have mostly been mapped to regions II, III and VI of the HCMV UL54-encoded DNA polymerase. We previously showed that sequential foscarnet-resistant HCMV isolates recovered from a patient with lymphoma had change N495K in region delta-C of the DNA polymerase. To evaluate the impact of change N495K on HCMV sensitivity to foscarnet, a recombinant HCMV strain carrying the mutation was produced by homologous recombination. The recombinant virus showed a 3.4-fold increase in foscarnet resistance, and remained sensitive to ganciclovir and cidofovir. In addition, the recombinant strain showed a reduction of infectious virus yield compared with its parent strain. Change N495K should be added to the list of mutations conferring resistance to foscarnet and be taken into account in the genotypic diagnosis of antiviral resistance.

We described a colorimetric method to determine the biochemical phenotype of wild-type and mutated cytomegalovirus (HCMV) DNA polymerases by measuring the incorporation of digoxigenin-labelled nucleotides into the growing DNA chain. Mutations V715M and E756K, which are known to confer foscarnet-resistance, were used as controls. Mutation N495K and a combination of changes K415R and S291P, both observed in foscarnet-resistant isolates, were studied. The mutations were introduced by site-directed mutagenesis into wild-type gene UL54 cloned in an expression vector and then polymerases were synthesised by using a commercially available coupled transcription-translation system. The polymerase activity was measured with and without foscarnet. The activity of polymerases containing the V715M or E756K mutations was inhibited by foscarnet at concentrations 70- and 30-fold higher than that of wild-type polymerase, respectively. Change N495K and combination of K415R and S291P, induced a five- and ten-fold decrease in susceptibility to foscarnet, respectively. The results of this non-radioactive assay were consistent with those obtained with the conventional radioactive assay. Therefore, this novel phenotypic method could be useful for the characterisation of mutations that confer HCMV resistance to foscarnet.

A phenotypic method was developed to test mutations in the human cytomegalovirus (HCMV) DNA polymerase gene (UL54) suspected to confer resistance to foscarnet. This method was used to determine the biochemical phenotype of wild-type and mutated HCMV DNA polymerases that had been synthesised in vitro as follows. The UL54 genes were amplified from foscarnet-resistant and -sensitive isolates by PCR and the products were cloned into an expression vector under the control of a T7 promoter. Mutations were introduced by site-directed mutagenesis into wild-type gene UL54 and then polymerases were synthesised by using a commercially available coupled transcription/translation system. Polymerase activity was measured with and without foscarnet by detecting the incorporation of digoxigenin-labelled nucleotides into the growing DNA chain. The results of this non-radioactive assay were consistent with those obtained with the conventional radioactive assay. It was found that the activity of polymerases containing the V715M or E756K mutations was inhibited by foscarnet at concentrations 70- and 30-fold higher than that of wild-type polymerase, respectively. Change N495K and a combination of changes K415R and S291P, both observed in foscarnet-resistant isolates, induced a 5- and 10-fold decrease in susceptibility to foscarnet, respectively. This non-radioactive phenotypic assay could be useful for the characterisation of mutations that confer HCMV resistance to foscarnet.

We describe the emergence of a new ganciclovir resistance mutation in the UL97 gene of human cytomegalovirus, deletion of codon 601, after valaciclovir and short-term ganciclovir therapy following kidney transplantation. Its role in ganciclovir resistance was supported by decreased ganciclovir phosphorylation in a recombinant vaccinia virus system.

Background: The prevalence and clinical significance of cytomegalovirus infection is reportedly high in patients with refractory inflammatory bowel disease but is unknown in unselected patients with active disease., Methods: In patients admitted for active inflammatory bowel disease, we prospectively studied the presence and significance of cytomegalovirus infection using anti-cytomegalovirus antibodies, cytomegalovirus viraemia and antigenaemia and cytomegalovirus inclusions and cytomegalovirus immunochemistry staining in ileocolonic biopsies., Results: A total of 64 patients were included (ulcerative colitis, n = 23; Crohn's disease, n = 41), 18 of whom had been on high-dose oral steroids and 11 on immunosuppressants. Anti-cytomegalovirus IgG and IgM were positive in 42 (66%) and 3 (5%) patients respectively. Blood or urine cytomegalovirus replication markers were found in 4 (6%) patients, all of whom had ulcerative colitis. Three patients had cytomegalovirus viraemia and received anti-viral treatment with ganciclovir. Only one of these patients had cytomegalovirus antigenaemia and also associated biopsy-proven cytomegalovirus colitis, probably as a primary cytomegalovirus infection. This patient is the only one who benefitted from anti-viral therapy., Conclusions: Cytomegalovirus infection is infrequent in in-patients with active inflammatory bowel disease. Systematic search of cytomegalovirus replication markers should not be performed. Isolated viraemia without associated antigenaemia or direct demonstration of cytomegalovirus in ileocolonic biopsies does not warrant anti-viral therapy.

Whether valaciclovir (VCV) prophylaxis could be responsible for ganciclovir (GCV)-resistance of Human cytomegalovirus (HCMV) in transplantation has never been documented. A multicentric retrospective pilot study was undertaken to detect GCV-resistance through mutations within the UL97 gene in renal transplant recipients who experienced active HCMV infection and received valacyclovir prophylaxis. Twenty-three patients who experienced HCMV antigenaemia or DNAemia during or at the end of prophylaxis were included. UL97 genotyping was carried out on peripheral blood samples, using a nested in-house PCR, which amplified the full-length UL97 gene. One patient has a resistance-related mutation (M460I); the major risk factor for emergence of resistance in this patient was the presence of early and persistent antigenaemia. GCV-resistance during VCV-prophylaxis was rare after renal transplantation. However, special attention must be paid to patients developing early active HCMV infection under prophylaxis., (Copyright 2004 Wiley-Liss, Inc.)

We described the natural polymorphism of cytomegalovirus DNA polymerase in 42 unrelated isolates susceptible to ganciclovir, foscarnet, and cidofovir. All variations, including an eight-amino-acid deletion, were located between domains delta-C and II and between domains III and I, suggesting that these specific residues are not involved in enzymatic functions.

Background: Long-term anti-cytomegalovirus (CMV) treatments in immunocompromised patients are hampered by resistance to antiviral drugs. Longitudinal changes in the resistance genotype may depend on changes in selective pressure and the complexity of CMV isolates., Objective: To evaluate longitudinal changes in the CMV resistance genotype and phenotype along with strain-specific variability in a patient with non-Hodgkin's lymphoma in whom successive anti-CMV treatments failed., Study Design: The resistance phenotype and genotype of seven CMV isolates collected from one patient during a 2-year follow-up period were retrospectively analysed. In parallel, we used glycoprotein B (gB) genotyping, and a- and UL10-13-sequence analysis to study CMV interstrain variability., Results: The patient was infected by at least three CMV strains plus variants of the parental strains. Resistance to ganciclovir, cidofovir and foscarnet was successively detected during the follow-up period. UL97 protein kinase changes responsible for resistance to ganciclovir were initially detected at residues 591 and 592, and then at position 594. Decreased sensitivity to foscarnet coincided with the appearance of amino acid substitution N495K in DNA polymerase, whereas cross-resistance to ganciclovir and cidofovir was due to the L501I substitution., Conclusions: The CMV isolates obtained from our patient were complex mixtures of strains. Changes in resistance genotypes depended on resistance selective pressure and were not linked to interstrain variation.

Background: The hypothetical responsibility of sperm donation in cytomegalovirus (CMV) transmission to recipients and precautions to prevent this transmission are widely discussed. The aim of this French CECOS Federation study was to evaluate both the reality and the importance of the CMV risk due to donor sperm and the relevance of measures used to screen it., Methods: We conducted a prospective multicentric study. CMV was detected by rapid and conventional cultures and by PCR in the frozen sperm of donors who met the normal criteria required of semen donors, irrespective of their CMV serological status., Results: 635 samples from 231 donors (39.4% IgG(+)) were obtained and tested by culture; 551 samples from 197 donors were also tested by PCR. From those samples, 0.78% were culture(+), 1.57% culture(+) and/or PCR(+); 3.3% of seropositive donors and 0.72% of initially seronegative donors were culture(+), but in the latter seroconversion occurred during the quarantine period; of the 197 PCR-tested donors, 3.5% (6.2/1.7) were PCR(+), 3.3% (5.3/1.45) culture(+) and/or PCR(+). PCR(+) samples can be culture(-) and vice versa. The most strongly positive sample corresponded to an initially seronegative donor., Conclusion: The best strategy to prevent potential CMV risk is to test donors for CMV IgG and IgM antibody at the outset and after a 6 month period of quarantine and to reject initially IgM seropositive donors or donors who seroconvert during the quarantine period.