ABSTRACT
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.
Subject(s)
Cytomegalovirus/isolation & purification , DNA-Directed DNA Polymerase/isolation & purification , Drug Resistance, Viral/genetics , Foscarnet/pharmacology , Cytomegalovirus/genetics , Cytomegalovirus Infections/virology , DNA-Directed DNA Polymerase/genetics , Genetic VariationABSTRACT
Although second-line generic antiretroviral drugs are of great value in developing countries, there are concerns regarding their quality. We studied a generic Lopinavir/ritonavir (200/50 âmg; Arga-L, India) marketed in the Republic of Congo but not prequalified by WHO. Despite adequate quantitative and qualitative drug content, Arga-L had a bio-availablility of 10% compared with Kaletra. To avoid selection of drug-resistant HIV, rigorous pharmacological monitoring of generic drugs not prequalified by WHO must be a priority.
Subject(s)
Anti-Retroviral Agents/pharmacokinetics , Anti-Retroviral Agents/therapeutic use , Drugs, Generic/pharmacokinetics , Drugs, Generic/therapeutic use , HIV Infections/drug therapy , Congo , Developing Countries , Humans , Lopinavir/pharmacokinetics , Lopinavir/therapeutic use , Ritonavir/pharmacokinetics , Ritonavir/therapeutic use , Treatment OutcomeSubject(s)
Actinobacillus Infections/diagnosis , Actinobacillus Infections/microbiology , Aggregatibacter actinomycetemcomitans/isolation & purification , Endocarditis, Bacterial/diagnosis , Endocarditis, Bacterial/microbiology , Actinobacillus Infections/blood , Actinobacillus Infections/drug therapy , Adult , Anti-Infective Agents/therapeutic use , Ceftriaxone/therapeutic use , Cephalosporins/therapeutic use , Ciprofloxacin/therapeutic use , Drug Therapy, Combination/therapeutic use , Endocarditis, Bacterial/blood , Endocarditis, Bacterial/drug therapy , Female , HumansABSTRACT
Klebsiella pneumoniae KOL, a clinical strain resistant to various beta-lactams, was isolated from the stools of a patient from Greece. This strain harbored a new pI 9.1 plasmid-mediated AmpC beta-lactamase with unusually high levels of hydrolytic activity for cefoxitin and cefotetan that we named MOX-2. Sequencing of bla(MOX-2) revealed 93.2, 92.9, 92.7, and 73.1% identities with the deduced amino acid sequences of CMY-8, MOX-1, CMY-1, and the AmpC beta-lactamase of Aeromonas sobria, respectively.