Novel Quantitative Cost-effective Assay for the Diagnosis and Monitoring of HIV viral loads

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PROJECT SUMMARY Since the start of the AIDS epidemic, according to the United Nations Joint Programme on HIV/AIDS (UNAIDS), a total of 75.7 million people have been infected with HIV, and of which 32.7 million have died due to AIDS- related illnesses. In 2016, UNAIDS defined several targets for HIV testing and treatment to be achieved by 2020 to end the AIDS epidemic by 2030. Such milestones include the reduction of the yearly infection rate to 500,000 new infections per year, the reduction of yearly AIDS-related deaths to 5000,000 deaths per year, and the 90- 90-90 goal, requiring that 90% of HIV-infected individuals be aware of their status, of which 90% are receiving treatment, and of which 90% have undetectable viral loads. We have now reached the goal year, and, based on data from 2019, we are not projected to meet any of these milestones. Although the targets were reached in a handful of countries, most low- and middle-income countries continue to fall behind the global goal in part due to decreases in funding and income disparities. To achieve these goals, extensive HIV testing is required, not only to diagnose new cases but in order to monitor the viral loads of those living with HIV. Therefore, there is an urgent need for a specific virus detection platform that can be used in low-resources settings. Here, we propose to develop a new technology for the specific, sensitive, and rapid detection of HIV-1 and HIV-2, using a universal recognition point-of-care testing platform. This unique platform integrates electrochemical detection of specific viral genome sequences using four-way junction (4WJ) probes following a modify isothermal amplification of the viral RNA fragments using Nucleic Acid Sequence Based Amplification (NASBA). The complete analysis (RNA amplification and detection) is expected to be executed in less than one hour, which is comparable with a regular visit to the doctor’s office. In the proposed research we will: 1) Optimized the modified-tag isothermal amplification using NASBA; 3) Design an electrochemical biosensor by selecting appropriate fragments of the amplicons to be interrogated by the 4WJ probes; 4) Optimize and characterize the biosensor for virus direct detection of amplicons; 5) Study variable parameters for single- and multiplex biosensor analysis; 6) Apply the developed biosensors for virus detection of commercially available reference material; and 7) Validate our methodology using a reference method (RT-qPCR). The proposed technology promises to be a user-friendly point-of-care testing at limited-resource settings for timely diagnosis of HIV infection and monitoring to help stopping the proliferation of AIDS infection by 2030.