DETECTING AND QUANTITATING UNCULTURABLE HUMAN PATHOGENS

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This proposal is to develop a rapid method for separating, identifying and counting small numbers of viral pathogens from tissues, blood, urine, water supplies, and natural waters, with emphasis on agents currently unculturable. In nature there are few particles of viral dimensions, which have both a narrow range of both sedimentation rates and isopycnic banding densities which are not viruses. Hence it is feasible to develop centrifugal systems to concentrate particles in the desired size range, and, using a new centrifuge tube under development, to concentrate and isopycnically band viral particles from several milliliters to a band having a volume of a cubic micron in a specially designed small-diameter density gradient. Using blue laser illumination the particles may be detected by light scattering or fluorescence using new fluorescent dyes which penetrate viruses and stain DNA or RNA. With these dyes, single virions have been detected. We believe the system is applicable to the search for previously undetected viruses, and to clinically detecting and quantitating viremias in HIV and hepatitis A, B, and C and other infections, and to the problem of routinely distinguishing viral from bacterial infections. In Phase I a prototype system will be evaluated using model non-pathogenic viruses. PROPOSED COMMERCIAL APPLICATION: For research purposes we estimate a world market of approximately 200 machines, and for clinical diagnosis a market of over 2,000 machines.