A simple and sensitive 'in-house' method for determining genotypic drug resistance in HIV-1

Antiretroviral combination therapy is a major advance in the treatment of HIV infection, but development of antiretroviral resistance is still an important cause of treatment failure. Therefore, resistance testing was recommended recently for follow-up of HIV-1 infected individuals. The aim of this study was to develop a new genotypic resistance assay because simple and affordable assays with sufficient sensitivity for different genetic subtypes and low copy number samples are still lacking. Different methods and primers for RNA extraction from plasma, cDNA synthesis, nested PCR and sequencing on an ABI310 automated sequencer were evaluated and optimised. The PCR was designed to amplify a fragment covering the protease and the first half of the reverse transcriptase (RT), which harbour most known resistance mutations to licensed antiretroviral drugs. Resistance mutations were identified using resistance analysis tools available over the Internet. The optimised assay had a sensitivity of approximately 200 RNA copies per ml. The method was evaluated on plasma samples from treated patients infected with different subtypes of HIV-1 and appeared to have similar sensitivity for all subtypes. Samples that had failed previously in routine testing were analysed successfully with the new assay. The new assay is more sensitive and robust than the current routine method.