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