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Sophie Alain

Publications and source records attributed to Sophie Alain.

At least 19 recordsLinked to original sources
PubMed2007

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.

Antiviral Agents↗
PubMed2006

Expertise of laboratories in viral load quantification, genotyping, and precore mutant determination for hepatitis B virus in a multicenter study.

A national evaluation study was performed in 14 specialized laboratories with the objective of assessing their capacities to provide (i) hepatitis B virus (HBV) viral loads (VL), (ii) HBV genotypes, and(iii) identification of precore/core mutants. The panel consisted of 12 HBV DNA-positive samples with VLs from 2.8 to 9.1 log(10) copies/ml, different HBV genotypes (A to F), and 3 mutant and 9 wild-type samples at nucleotide 1896. The coefficients of variation of the mean VLs ranged from 2.4% to 10.4% with the Cobas HBV Monitor assay, from 1.8% to 5.5% with the Cobas TaqMan 48, from 1.5 to 26.2% with RealArt HBV PCR, and from 0 to 7% with branched DNA (bDNA). The Cobas Monitor assay underestimated the VLs of genotype F samples, with differences ranging from 1.4 to 2.4 log(10) copies/ml. The accuracies of genotype determinations ranged from 33% to 100%, and those of precore mutant determinations ranged from 25 to 100%. This study showed some drawbacks of two widely used assays: (i) Cobas Monitor has a narrow dynamic range and underestimates genotype F sample VLs and (ii) bDNA shows poor sensitivity and may fail to identify patients with low VLs. With higher performance in terms of analytical sensitivity combined with a larger dynamic range and an ability to quantify the main genotypes equally, real-time PCR methods appear more appropriate for accurate monitoring of HBV DNA quantification. Furthermore, the clinical implications of HBV genotyping and the determination of precore/core mutants need to be clearly stated to justify the standardization of these methods.

France↗
PubMed2006

Unique NS5b hepatitis C virus gene sequence consensus database is essential for standardization of genotype determinations in multicenter epidemiological studies.

A multicenter study of NS5b hepatitis C virus (HCV) genotype determination involving 12 laboratories demonstrates that any laboratory with expertise in sequencing techniques would be able to provide a reliable HCV genotype for clinical and epidemiological purposes as long as they are provided a consensus reference sequence database.

Consensus Sequence↗
PubMed2006

A novel mutation in the UL54 gene of human cytomegalovirus isolates that confers resistance to foscarnet.

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.

Antiviral Agents↗
PubMed2005

[Anti-papillomavirus vaccines and prevention of cervical cancer: progress and prospects].

Human papillomaviruses (HPV) cause the development of various cutaneous and mucosal lesions. Some genotypes play a role in the genesis of cervical cancer, which is the second most common cancer in women. HPV types 16 and 18 account for 60 to 72% of all HPV-associated cervical cancers, while types 6 and 11 cause genital warts. Despite the various escape strategies viruses use to fight the natural immune system, more than 90% of the infections clear spontaneously. It should therefore be possible to prepare prophylactic vaccines. The HPV major capsid protein L1 self-assembles into virus-like particles (VLP). Immunization after parenteral vaccination with it provided very good protection against experimental infection in different animal models. The first clinical trials revealed the satisfactory tolerance and excellent immunogenicity of these vaccines. Two vaccine approaches were selected: one based on protection against cervical cancer from a bivalent VLP L1 vaccine containing the two genotypes most frequently involved in cervical cancer (type 16 and 18) and the other, protecting against warts as well as cervical cancer, with a quadrivalent HPV VLP L1 vaccine containing genotypes 6, 11, 16 and 18. Initial results with these vaccines show an efficacy of more than 90% against infection and 100% against the onset of dysplastic lesions. Despite these hopeful results, a vaccined strategy sould still be defined. Meanwhile, the cytology screening program should be carried on until the beginning of the vaccination.

Clinical Trials as Topic↗
PubMed2005

Phenotypic characterisation of cytomegalovirus DNA polymerase: a method to study cytomegalovirus isolates resistant 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.

Cytomegalovirus↗
PubMed2005

Early selection of a new UL97 mutant with a severe defect of ganciclovir phosphorylation after valaciclovir prophylaxis and short-term ganciclovir therapy in a renal transplant recipient.

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.

Acyclovir↗
PubMed2005

Comparison of hepatitis C virus NS5b and 5' noncoding gene sequencing methods in a multicenter study.

A national evaluation study was performed in 11 specialized laboratories with the objective of assessing their capacities to genotype hepatitis C virus (HCV) and define the applicability of a given genotyping method. The panel consisted of 14 samples positive for HCV RNA of different genotypes (including 3 samples with two different artificially mixed genotypes) and 1 HCV-negative sample. Seventeen sets of data were gathered from the 11 participating laboratories. The sensitivities ranged from 64.3 to 100% and from 42.7 to 85.7% for the methods that used sequencing of the NS5b region and the 5' noncoding (5' NC) region, respectively. When the data for the artificially mixed samples were excluded, NS5b genotyping gave correct results for 80% of the samples, 1.7% of the samples were misclassified, and 18.3% of the samples had false-negative results. By 5' NC-region genotyping methods, 58.3% of the results were correct, 29.7% were incomplete, 8.3% were misclassifications, 1.2% were false positive, and 2.4% were false negative. Only two procedures based on NS5b sequencing correctly identified one of the three samples with mixtures of genotypes; the other methods identified the genotype corresponding to the strain with the highest viral load in the sample. Our results suggest that HCV 5' NC-region genotyping methods give sufficient information for clinical purposes, in which the determination of the subtype is not essential, and that NS5b genotyping methods are more reliable for subtype determination, which is required in epidemiological studies.

5' Untranslated Regions↗
PubMed2004

Serological pattern 'anti-hepatitis B core alone' in HIV or hepatitis C virus-infected patients is not fully explained by hepatitis B surface antigen mutants.

We determined the frequency of hepatitis B virus (HBV) occult infection and studied S gene mutations in HIV or hepatitis C virus-co-infected patients with persistent pattern 'anti-hepatitis B core alone'. HBV DNA was particularly frequent in HIV-positive patients. Sequencing of the S gene showed that only two HIV-infected patients harboured mutants in the major hydrophilic loop; the absence of hepatitis B surface antigen detection could not be explained by diagnostic escape mutants.

AIDS-Related Opportunistic Infections↗
PubMed2004

Screening of pregnant women for hepatitis B markers in a French Provincial University Hospital (Limoges) during 15 years.

During the 15 years from January 1984 to December 1998 the Limoges University Hospital screened 22,859 pregnant patients for hepatitis B surface antigen (HBs Ag) and identified 149 positives. The overall prevalence (0.65%) was intermediate between prevalences observed among women of French origin (0.29%), French West Indies islands (5.68%) and of foreign origin particulary South East Asian origin (7.14%) and Sub Saharan African origin (6.52%). Hepatitis B Virus (HBV) replication markers was detected with relative low frequence (HBe Ag: 14.4%; HBV-DNA: 13.7-20%) among HBs Ag positive mothers. Markers of delta hepatitis virus was found among 10.5% of the HBs Ag carrier pregnant women. During the 15 years study period variations of the global prevalence were not statistically significant. Universal prenatal screening and infant immunisation could greatly contribute to the control of HBV infection if the polemic about the hepatitis B vaccination recently propagated in France will not have a negative effect on the acceptance and national programme of vaccination.

Africa South of the Sahara↗
PubMed2004

Comparison of sequential cytomegalovirus isolates in a patient with lymphoma and failing antiviral therapy.

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.

Aged↗
PubMed2004

Natural polymorphism of cytomegalovirus DNA polymerase lies in two nonconserved regions located between domains delta-C and II and between domains III and I.

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.

Amino Acid Sequence↗
PubMed2004

Expertise of French laboratories in detection, genotyping, and quantification of hepatitis C virus RNA in serum.

Before initiating new large-scale therapeutic trials for hepatitis C virus (HCV)-infected patients, the French Health Authorities for HCV research decided to organize an evaluation of the expertise of laboratories that could be engaged to undertake molecular biology assays in such trials; 21 experienced laboratories participated in this national evaluation of laboratory expertise, which was performed in two successive rounds. The first round evaluated the laboratories for their abilities to detect HCV RNA in serum, determine genotypes, and quantify HCV RNA loads. The results observed by qualitative assays for HCV RNA detection were 100% sensitivity and 100% specificity for all laboratories. The genotyping results were 100% concordant for 9 laboratories and greater than 90% for 10 laboratories. By contrast, large coefficients of variation were observed for quantitative determination of HCV RNA loads, leading to a second round with standardized quantitative assays only. The dispersion of the results was larger by the AMPLICOR HCV Monitor assay than by the branched-DNA assay (mean coefficients of variation, 57.4 and 16.9%, respectively). In the majority of cases, discrepancies between the results of the two tests were found for samples with high viral loads. These results indicate the usefulness of validating, by controlling for expertise, both the reliabilities of laboratories involved in multicenter work and the standardized assays chosen for use in the evaluation of the biological impacts of new therapies.

Analysis of Variance↗
PubMed2003

Herpes simplex virus DNA in corneal transplants: prospective study of 38 recipients.

Herpes simplex virus (HSV) infection of the eye can induce epithelial and stromal keratitis and may also lead to postoperative endothelial failure in keratoplasty. Clinical symptoms and/or virus culture of corneal scrapings most frequently provide the basis for diagnosis of ocular HSV infection, and although HSV DNA has been shown to be present in the cornea, its role in success or failure of corneal grafts remains unclear. In this study, a PCR assay was used to detect HSV DNA in corneal buttons of 38 corneal graft recipients and in donor scleral remnants, retaining one-half of each sample for subsequent viral isolation. Recipients were followed up clinically for a period of 6 months after keratoplasty. All recipients but three were found to be HSV seropositive. Eight recipient corneal buttons contained detectable HSV DNA (7 HSV-1, 1 HSV-2, the latter case confirmed by viral culture). Two donor corneas were found positive for HSV-1 DNA, with negative cultures, and endothelial graft failure occurred in one of the matching recipients after 4 months. One recipient with no history of herpes contracted herpetic keratitis 4 months after keratoplasty, even though the corneal button and donor scleral remnants contained no detectable HSV DNA. The study confirms previous observations of HSV DNA in the corneal tissue of HSV seropositive patients apparently unrelated to any clinical manifestation of herpes infection. However, as demonstrated by culture, HSV remains infectious and may therefore induce donor-to-host infection in corneal recipients.

Adult↗