Simple, low cost assay for detection of HIV-1 antiretroviral resistance in resource-limited settings

Antiretroviral therapy has become a reality in resource limited settings thanks to entities such as PEFAR and the Global Fund However contrary to the therapeutic choice flexibility in developed countries all patients go on reverse transcriptase inhibitor RTI based first line regimens with more expensive protease and integrase inhibitor based regimens being reserved as second line therapy for those who fail initial treatment RTI efficacy is compromised to a large degree by resistance Because of cost however resistance testing is not performed and a high percentage of individuals fail treatment because of pre existing resistance This has undermined the efficacy of treatment rollout and has created an urgent clinical need The solution is implementation of a simple inexpensive assay for detection of resistance to first line RTI based regimens We have exploited a novel polymerase with extraordinary requirement for base pairing at the end of the primer target template to create an allele specific AS PCR assay that uses standard PCR and simply scores for resistance by the presence of amplification products This format brings the cost of resistance testing down to one tenth of the cost of current gold standard genotyping assays and would make it feasible for the first time to initiate wide spread resistance testing in resource limited settings The proposed research will have specific aims Aim Design and optimize the AS PCR assay to detect the six mutations that at clinically relevant frequencies confer resistance to first line antiretroviral therapy commonly used in resource limited settings and assess the performance of the assay after modifying the primers by adding detection markers Aim Determine the limits of multiplexing to minimize the number of reactions and reduce assay cost Aim Assess the discriminatory capacity of the assay on validated HIV sample panels with multiple clades and determine level of concordance with patient samples previously characterized by commercial genotyping assays Completion of these aims will lead to a phase II submission focused on optimization of the assay for utilization in resource limited settings Drug resistance to antiretroviral therapy for HIV infection is a serious clinical problem without cost effective solutions in resource limited settings where standard genotypic resistance assays are unaffordable We have exploited a Taq polymerase that absolutely requires a terminal base match that with appropriate primers matched to resistance associated polymorphisms forms the basis for a low cost allele specific PCR resistance assay The presence of resistance will be scored by the presence of DNA amplification products that can be detected simply and cost effectively thereby making widespread resistance testing a possibility in resource limited settings