Urine collection, storage, and pre-treatment. First morning mid-stream urine samples were collected in plastic containers and kept in a 4–8°C box during transport to the laboratory, which occurred within 24 hours to ensure timely processing. Urine samples were centrifuged at 1,500 rpm at 4°C for 15 min and the clarified supernatants were aliquoted into screw top vials and stored at -20°C until used in the urine OV-ES assay with urine pre-treated by TCA, freezing, heating, or alkaline as discussed below.