Current global efforts to confront the HIV/AIDS epidemic focus on the prevention of new infections and the care for those who are living with HIV. Highly active antiretroviral therapy (HAART) which has dramatically improved the outlook of HIV/AIDS patients is expensive although it is gradually being made available to developing countries through charitable organizations and pharmaceutical companies. For it to be effective, however, HAART has to be monitored to avoid the risk of rapid development and transmission of resistant strains. Monitoring HIV viral load, or the concentration of viral RNA in the plasma, is part of the standard care of HIV-infected individuals. Two critical decisions in HIV/AIDS patient care are primarily based on HIV viral load measurements: when to initiate drug therapy and when to switch or adjust drug therapy regimen. Similarly, HBV and HCV viral loads have also been used to monitor disease progression and treatment efficacy. Improvements of nucleic acid detection technologies such as the introduction of real-time PCR have considerably simplified viral load testing. However, preparation of nucleic acids from serum or plasma samples remains a difficult and expensive process that, in part, hinders the adoption of these technologies in developing countries. We have now developed a virus capture reagent - magnetic particles coated with a chemical polymer, which seems to be highly effective for isolating and concentrating HIV-1, HBV and HCV from even highly diluted human serum samples, e.g., 20 RNA copies or 25 IU/mL. Isolated viruses could be lysed in a PCR compatible buffer and directly used for detection using PCR or other technologies. The proposed studies are aimed to critically evaluate this reagent and to develop a protocol for simplified sample preparation that is less expensive, more effective and suitable for resource-poor regions. If successfully developed, the entire sample preparation will take less than 30 minutes with 5-10 minute labor using only a magnet. With this sample preparation method, it should be possible to perform a real-time PCR viral load test with less than 15 minutes labor and without using an expensive automated sample preparation system. The proposed studies will evaluate the feasibility of using polymer coated magnetic particles to isolate and concentrate viruses (HIV-1, HBV and HCV) from human serum or plasma samples as a simplified sample preparation method that is less expensive, more effective and suitable for resource-poor regions. Clinical and contrived samples (standards) will be used to establish sensitivity and consistency of the reagent in detecting the viral load. ? ? ?
