RESUMEN
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.
Asunto(s)
Sangre/virología , Infecciones por Virus de Epstein-Barr/diagnóstico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Herpesvirus Humano 4/aislamiento & purificación , Trastornos Linfoproliferativos/virología , Carga Viral/métodos , Automatización de Laboratorios , ADN Viral/sangre , Infecciones por Virus de Epstein-Barr/sangre , Humanos , Límite de Detección , Trastornos Linfoproliferativos/prevención & control , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
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.
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Anticuerpos Antivirales/sangre , Neoplasias Encefálicas/virología , Infecciones por Citomegalovirus/virología , Citomegalovirus/inmunología , ADN Viral/sangre , Glioblastoma/virología , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/cirugía , Citomegalovirus/efectos de los fármacos , Citomegalovirus/genética , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/mortalidad , Infecciones por Citomegalovirus/cirugía , ADN Viral/genética , Femenino , Glioblastoma/inmunología , Glioblastoma/mortalidad , Glioblastoma/cirugía , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Estudios Retrospectivos , Estudios Seroepidemiológicos , Esteroides/administración & dosificación , Esteroides/efectos adversos , Análisis de Supervivencia , Activación Viral/efectos de los fármacosRESUMEN
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.
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Hepatitis B/epidemiología , Hepatitis B/transmisión , Transmisión Vertical de Enfermedad Infecciosa/estadística & datos numéricos , África del Sur del Sahara/etnología , Análisis de Varianza , Anticuerpos Antivirales/sangre , Asia/etnología , Secuencia de Bases , Análisis por Conglomerados , ADN Viral/sangre , Femenino , Hepatitis B/genética , Vacunas contra Hepatitis B/administración & dosificación , Humanos , Recién Nacido , Masculino , Datos de Secuencia Molecular , Paris/epidemiología , Filogenia , Embarazo , Estudios Retrospectivos , Medición de Riesgo , Análisis de Secuencia de ADN , Vacunación/estadística & datos numéricos , Carga ViralRESUMEN
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.
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Antivirales/metabolismo , Antivirales/farmacología , Artemisininas/metabolismo , Artemisininas/farmacología , Citomegalovirus/efectos de los fármacos , Artesunato , Biotransformación , Medios de Cultivo/química , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad MicrobianaRESUMEN
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.
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Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Neoplasias Hematológicas/complicaciones , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Células Asesinas Naturales/inmunología , Adolescente , Adulto , Enfermedad Crónica , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/virología , Femenino , Citometría de Flujo , Estudios de Seguimiento , Enfermedad Injerto contra Huésped/etiología , Neoplasias Hematológicas/mortalidad , Neoplasias Hematológicas/terapia , Humanos , Subgrupos Linfocitarios/inmunología , Masculino , Estadificación de Neoplasias , Pronóstico , Estudios Prospectivos , Tasa de Supervivencia , Acondicionamiento Pretrasplante , Trasplante Homólogo , Activación Viral/inmunología , Adulto JovenRESUMEN
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.
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Sangre/virología , Citomegalovirus/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Carga Viral/métodos , Automatización de Laboratorios/métodos , Citomegalovirus/genética , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Manejo de Especímenes/métodos , Carga Viral/normasRESUMEN
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.
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Infecciones por VIH/complicaciones , Virus de la Hepatitis E/inmunología , Virus de la Hepatitis E/aislamiento & purificación , Hepatitis E/epidemiología , Hepatitis E/patología , Transaminasas/sangre , Adulto , Afinidad de Anticuerpos , Femenino , Anticuerpos Antihepatitis/sangre , Hepatitis E/virología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Pakistán/epidemiología , Prevalencia , ARN Viral/sangre , ARN Viral/genética , Estudios Retrospectivos , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/virología , Citomegalovirus/efectos de los fármacos , Farmacorresistencia Viral/genética , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Órganos/efectos adversos , Adulto , Antivirales/uso terapéutico , Quimioprevención , Niño , Preescolar , Estudios de Cohortes , Citomegalovirus/genética , Infecciones por Citomegalovirus/prevención & control , ADN Polimerasa Dirigida por ADN/genética , Francia , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación , Fenotipo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Estudios Prospectivos , Proteínas Virales/genéticaRESUMEN
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.
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Bencimidazoles/farmacología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/metabolismo , Ribonucleósidos/farmacología , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Bencimidazoles/química , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/crecimiento & desarrollo , ADN Viral/metabolismo , Dimerización , Fibroblastos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Ribonucleósidos/química , Análisis de Secuencia de ADN , Proteínas Estructurales Virales/genética , Ensamble de Virus/efectos de los fármacosRESUMEN
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.
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Adenosina Trifosfatasas/química , Citomegalovirus/enzimología , Empaquetamiento del ADN , ADN Viral/metabolismo , Endodesoxirribonucleasas/química , Proteínas Virales/química , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Antivirales/química , Antivirales/farmacología , Dominio Catalítico , Secuencia Conservada , Citomegalovirus/efectos de los fármacos , Citomegalovirus/genética , Citomegalovirus/crecimiento & desarrollo , Empaquetamiento del ADN/efectos de los fármacos , ADN Viral/efectos de los fármacos , Diseño de Fármacos , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Polimorfismo Genético , Conformación Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacos , Dedos de ZincRESUMEN
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.
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Antivirales/uso terapéutico , Retinitis por Citomegalovirus/epidemiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Adulto , Preescolar , Farmacorresistencia Viral , Humanos , Incidencia , Estudios RetrospectivosRESUMEN
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.
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Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Infecciones por VIH/virología , Virus de la Hepatitis B/fisiología , Hepatitis B/virología , Lamivudine/farmacología , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/virología , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Farmacorresistencia Viral , VIH/efectos de los fármacos , VIH/genética , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Hepatitis B/complicaciones , Hepatitis B/tratamiento farmacológico , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Humanos , Linfoma no Hodgkin/inmunología , Masculino , Activación Viral/efectos de los fármacosRESUMEN
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.
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Antivirales/farmacología , Colorimetría/métodos , Citomegalovirus/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , Foscarnet/farmacología , Proteínas Virales/metabolismo , Dominio Catalítico/genética , Citomegalovirus/enzimología , Citomegalovirus/genética , ADN Polimerasa Dirigida por ADN/genética , Digoxigenina , Farmacorresistencia Viral , Concentración 50 Inhibidora , Mutación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Proteínas Virales/genéticaRESUMEN
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.
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Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/genética , Farmacorresistencia Viral/genética , Foscarnet/farmacología , Mutación , Proteínas Virales/genética , Citomegalovirus/genética , Citomegalovirus/crecimiento & desarrollo , Infecciones por Citomegalovirus/virología , Fibroblastos/virología , Humanos , Pruebas de Sensibilidad Microbiana , Recombinación GenéticaRESUMEN
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.
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Colitis/virología , Inmunodeficiencia Variable Común/complicaciones , Herpes Simple , Herpesvirus Humano 1 , Anciano , Anticuerpos Antivirales/sangre , Antivirales/uso terapéutico , Colitis/inmunología , Colon/inmunología , Colon/virología , Inmunodeficiencia Variable Común/inmunología , Femenino , Herpes Simple/tratamiento farmacológico , Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangreRESUMEN
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.
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Coinfección , Infecciones por VIH/virología , Hepatitis B/transmisión , Hepatitis B/virología , Transmisión Vertical de Enfermedad Infecciosa , Complicaciones Infecciosas del Embarazo/virología , Adolescente , Fármacos Anti-VIH/uso terapéutico , Antirretrovirales/uso terapéutico , Niño , Preescolar , Estudios Transversales , Femenino , Francia , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , Hepatitis B/diagnóstico , Hepatitis B/tratamiento farmacológico , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/sangre , Vacunas contra Hepatitis B/uso terapéutico , Humanos , Inmunoglobulinas/uso terapéutico , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Embarazo , Complicaciones Infecciosas del Embarazo/diagnóstico , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Estudios Retrospectivos , Factores de Tiempo , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Citomegalovirus/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Farmacorresistencia Viral/genética , Foscarnet/farmacología , Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , ADN Polimerasa Dirigida por ADN/genética , Variación GenéticaRESUMEN
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.
Asunto(s)
Infecciones por Citomegalovirus/virología , Citomegalovirus/genética , Citomegalovirus/aislamiento & purificación , Citosina/análogos & derivados , Evolución Molecular , Linfoma de Células T/complicaciones , Epidemiología Molecular , Organofosfonatos , Anciano , Antivirales/farmacología , Antivirales/uso terapéutico , Cidofovir , Infecciones por Citomegalovirus/tratamiento farmacológico , Citosina/farmacología , Citosina/uso terapéutico , ADN Viral/análisis , ADN Viral/química , ADN Viral/aislamiento & purificación , Farmacorresistencia Viral/genética , Femenino , Foscarnet/farmacología , Foscarnet/uso terapéutico , Ganciclovir/farmacología , Ganciclovir/uso terapéutico , Humanos , Mutación Missense , Compuestos Organofosforados/farmacología , Compuestos Organofosforados/uso terapéutico , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADNRESUMEN
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.