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The rapid diagnosis of adenoviral ocular infections affords the opportunity to limit the transmission of virus within the community and avoid expensive, unnecessary, and ineffective therapy. This study evaluated the results of a 5-year experience with the Adenoclone test (Cambridge Biotech, Worcester, MA), an enzyme immunoassay, applied directly to conjunctival swabs obtained from infected eyes. The sensitivity of this test was determined on 372 consecutive adenovirus culture-positive ocular specimens. A subset of 106 specimens was evaluated, including a retrospective chart review to determine the relationship between the Adenoclone result and the time to viral cytopathic effect (CPE) in A549 cell culture, ocular titers (90% tissue culture infectious dose; TCID90), serotype, and associated clinical parameters. Overall, the sensitivity for Adenoclone was 38% (142 of 372), which improved to 65% (129 of 199) for samples positive in culture during the first week. A positive Adenoclone test result was associated with a shorter time to CPE in cell culture (p=0.0001). The mean ocular titers (log TCID90) associated with a positive test result were found to be at a significantly higher dilution than a negative result (-1.70 ± 0.93 vs. -0.88 ± 1.00, p<0.0001). A positive Adenoclone outcome was independent of the serotype but directly associated with a recent visit to an ophthalmologist's office, follicular conjunctivitis, and conjunctival chemosis. For the rapid diagnosis of adenoviral ocular infections, the Adenoclone test remains useful, but a more sensitive test based on nonradioactive amplification is eagerly anticipated.