1 The proposed project will demonstrate proof-of-concept that a single-use, disposable nucleic acid amplification 2 device suitable for home self-testing can automatically and rapidly amplify and detect HIV-1 genomic RNA from 3 a finger-stick blood sample. The device will extract nucleic acid from plasma or whole blood, amplify HIV-1 4 RNA, and detect amplified product, providing sample to answer in < 30 minutes. The device will be simple and 5 easy to use, and therefore suitable as a home self-test. This proposed development is based on Click 6 Diagnostics? single-use, disposable PCR diagnostic device that will enter an NIH-funded clinical trial in 2018 for 7 the rapid detection of three sexually transmitted infections, N. gonorrhoeae, C. trachomatis, and T. vaginalis, 8 using self-collected vaginal swabs. The R61 phase of the project will develop and port a multiplex RT-PCR 9 HIV-1 assay onto Click Diagnostics? platform, and will qualitatively assess the presence of HIV-1 Group O and 10 Group M (subtype A-H) RNA at concentrations as low as 1000 copies/ml. As plasma represents the gold- 11 standard sample matrix for accurately detecting HIV RNA, we partnered with two companies to evaluate their 12 non-centrifugation based methods to separate plasma from finger-stick blood; these include a 13 superhydrophobic surface/membrane filtration system and a size-selection membrane filter system. Finally, we 14 will evaluate the possibility of direct amplification of HIV-1 RNA from whole finger-stick blood. This method will 15 not require plasma separation, and will selectively amplify only HIV-1 RNA (but not pro-viral DNA) from an 16 EDTA anticoagulated blood sample. Based on performance, one of these methods will be chosen for use with 17 the Click diagnostic test, and for potential future physical integration into the Click device. The R33 phase of 18 the project will focus on evaluation and optimization of HIV diagnostic using archived de-identified plasma 19 samples from HIV-1 infected patients representing a range of viral loads and HIV-1 Group O and Group M 20 subtypes. Click Diagnostics has partnered with the Department of Veterans Affairs, Palo Alto AIDS Research 21 Center to assist with device evaluation and testing. The testing will demonstrate that the integrated device can 22 detect HIV-1 RNA from patient de-identified plasma samples in the HIV-1 Early Infection AccuSet Performance 23 Panel spiked into EDTA anticoagulated whole blood. Limit of Detection (LoD) and inclusivity/exclusivity results 24 will be compared with, and are expected to be comparable to, commercially available comparator instrument- 25 based PCR assays. At the conclusion of proposed project, the device will be ready for: 1) physical integration 26 of plasma separation with the Click device; 2) a pre-submission meeting with the FDA prior to conducting 27 formal pre-analytical and pre-clinical studies; 3) a user study to determine the feasibility of the device for home 28 self-testing; and 4) a manufacturing readiness assessment preceding volume production of sufficient devices 29 for a pivotal clinical trial. The fully integrated Click HIV diagnostic will bring accurate and easy to use HIV RNA 30 self-testing devices into the home, thus markedly increasing access, while decreasing costs.
authorities increasingly recognize the value of HIV self-testing both by individuals at risk for de novo HIV infection and to monitor for treatment failure in those receiving HIV antiretroviral therapy. The proposed project will demonstrate proof of concept that a portable, disposable, fully integrated, and cost- effective nucleic acid amplification device can automatically and rapidly amplify and detect HIV-1 genomic RNA from a finger-stick blood sample. Click Diagnostics? HIV diagnostic will maintain the accuracy of traditional instrument-based diagnostics, while providing a device that an untrained individual can easily use and operate, thus markedly increasing access to HIV diagnostics by bringing HIV RNA self-testing into the home.