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

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

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.

[Standards, Options and Recommendations 2005 for a good practice in enteral nutrition in oncology (summary report.)]. / Recommandations pour la pratique clinique: Standards, Options et Recommandations 2005. Bonnes pratiques pour la prise en charge diététique en cancérologie: la nutrition entérale (rapport abrégé). Validation du rapport original: avril 2005.

CONTEXT: The "Standards, Options and Recommendations" (SOR) project, which started in 1993, is a collaboration between the French Federation of Cancer Centers (FNCLCC), the 20 French Regional Cancer Centers, and specialists from French public universities, general hospitals and private clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and the outcome of cancer patients. OBJECTIVE: To develop good practice guidelines for a good practice of enteral nutrition in oncology, in collaboration with three French learned societies involved in this area. METHOD: The methodology is based on a literature review and critical appraisal by a multidisciplinary group of experts who define the CPGs according to the definitions of the Standards, Options and Recommendations project. Once the guideline has been defined, the document is submitted for review by independent reviewers. RESULTS: The good practices suggested in the document approach seven topics: indications and counter-indications, conditions of the installation of the enteral nutrition, monitoring, prevention of complications, education of the patient, specificities of enteral nutrition in children and at home. In the setting of enteral nutrition, feeding tubes, type of insertion, enteral nutrition products, material and techniques of administration are described as well as the criteria permitting their selection.

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.

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.

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.