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Method for the Detection of Minority Populations of Drug Resistant HIV

Background
Antiretroviral therapy (ART) reduces mortality and morbidity in HIV-infected individuals. With successful therapy HIV RNA becomes undetectable, but drug resistance may occur.  Specific HIV mutations are associated with resistance and these mutations can be detected through standard genotypic resistance tests, which have the ability to detect mutations only when they are present in approximately 20% of the virus population within an individual. Importantly, it is now known that the presence of certain resistance mutations, even at very low concentrations within a patient?s virus population (1% or more), can contribute to virological failure.  These drug resistant minor variants can reflect the early emergence of acquired resistance during therapy, and can also be transmitted to newly infected individuals. These minor variants are not detected by standard drug resistance assays and methods to detect minor variants that contribute to HIV virological failure are needed.  These assays would need to detect mutations causing resistance to each of the antiretroviral drug classes (NRTI, NNRTI, PI and INI) in all HIV subtypes, and must be inexpensive, since large numbers of patients would need to be screened.   
Project Goal
The goal of this solicitation is to develop an assay to detect minor populations of resistant variants in blood specimens from HIV-infected individuals with HIV RNA viral loads above 1000 copies/ml.  The test must detect resistant variants that comprise 1% or more of the virus population or quasispecies, and must detect mutations causing resistance to NNRTIs, NRTIs, PIs and INIs in all subtypes of HIV. Sensitivity for qualitative detection of the minor variant at 1% or more must be at least 95% and specificity at least 98%, but the method must also yield quantitative results, showing the percentage of each resistant variant in the overall quasispecies. Methods that detect a set of relevant point mutations and methods that collect full sequences are both acceptable. For methods that detect point mutations, a set of relevant mutations should be proposed in the application, but will be finalized in cooperation with DAIDS program staff.  The method must be appropriate for use in centralized clinical laboratories with a target turn-around-time of less than 1 week and an initial target cost of $100 or less.      
Phase I activities
?       Development of a method for the quantitative detection of minor populations of HIV resistance mutations comprising 1% or more of the viral quasispecies
a.      Must detect major mutations causing resistance to all drug classes (NNRTI, NRTI, PI and INI)
b.      Sensitivity must be at least 95%, specificity at least 98%
c.      Must be suitable for clinical laboratory use, with turn-around time of less than 1 week
d.      Cost must be less than $100 per test
?       Initial testing of the product with combinations of drug-susceptible and ?resistant laboratory strains of HIV spiked into HIV-negative blood
?       Additional testing on clinical isolates
Phase II activities
?       Validation testing with controls as well as clinical isolates, including assessment of sensitivity, specificity, precision, accuracy, and linearity
?       Production of the test under good manufacturing practices (GMP)
?       Development of a quality control program to ensure lot-to-lot consistency
 
Multi-site evaluation

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