HIV viral load measurement in low-resource settings is an area of persistent need. A combination of two technologies holds great promise for improving access to HIV viral load measurement among underserved patient populations. The first is branched DNA (bDNA), a gold-standard nucleic acid chemistry which labels each strand of viral RNA with as many as 10,000 signaling molecules to enable detection of as few as 75 copies of viral RNA in a 1 mL plasma sample with low risk of test failure from sample contamination. The second is SCA microfluidic technology for high-performance on-cartridge sample/reagent processing with a static interface between cartridge and instrument. The starting point for this Phase II project is the successful Phase I work to shorten the bDNA assay's duration while simultaneously adapting the assay to an enclosed-cartridge format. The newly developed cartridge- format bDNA assay, the EO-NAT HIV Assay, includes re-engineered capture probes and signaling chemistry while retaining the original assay's multi-tiered probe configuration and high-specificity synthetic bases. Running in enclosed cartridges with cartridge-integrated SCA micropumps, the primary hybridization step in the bDNA assay has been speeded up eight-fold: whereas the primary hybridization step takes 16 hours running on conventional reference laboratory instruments, in the EO-NAT HIV cartridge-format assay this step requires only 120 minutes. Total test turnaround time has been reduced to less than four hours, a duration which facilitates treatment of HIV/AIDS patients with limited access to care, for whom achieving favorable clinical outcomes often depends on carrying out viral load testing and developing a management plan in the same visit. The Phase II project work incorporates two additional technological elements into the EO-NAT HIV assay. Detection of hybridized bDNA complexes is by a specialized form of electrochemiluminescence with sub- nanomolar concentrations of signaling molecules and multiplexing. Sample preparation, including lysing and preconcentration, is by cartridge-integrated photopolymerized monoliths. During the second year of Phase II, cartridges integrating all four technological elements-cartridge-optimized bDNA chemistry, SCA microfluidic technology, mECL detection, and extraction monoliths-are produced. These cartridges and prototype EO- NAT instruments are tested on clinical specimens to validate the clinical sensitivity and specificity of the system. In the final portion of Phase II, pilot ISO 13485-compliant manufacturing is ramped up in preparation for filings with regulatory agencies. The central hypothesis of this project is that the EO-NAT HIV Assay running on EO-NAT instruments detects as few as 50 copies of HIV RNA in 20 microliter samples with 98% sensitivity (n=100) and exhibits specificity greater than 90% in testing of negative samples (n=100).
Plasma viral load, determined by complex blood tests, is an indication of how sick an HIV/AIDS patient is and how well he or she is responding to treatment. Regular viral load measurement is important in caring for HIV/AIDS patients, but these tests are currently not available for everyone. This project develops a new system which makes viral load measurement less expensive and more readily available to doctors and patients who live far from sophisticated medical facilities.