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Current methods for in situ diagnosis of catheter-related bloodstream infections require concurrent collection of central venous catheter (CVC) and peripheral vein (PV) blood cultures. Both the pain and inconvenience of PV cultures are undesirable.A prospective study was conducted (August 2002 to March 2004) to assess the accuracy of diagnosing catheter-related bloodstream infections based on the difference in time to detection of blood cultures drawn concurrently from 2 lumens of a multilumen CVC. This difference in time to detection between 2 lumens was compared with results of the standard criterion with paired CVC and PV blood cultures.Twenty-one infectious episodes were categorized as catheter-related bloodstream infections and 38 as non-catheter-related bloodstream infections. With a cutoff in difference in time to detection between 2 lumens of ≥180 minutes, the sensitivity of this test to diagnose a catheter-related bloodstream infection was 61% (95% confidence interval, 39–80%) and the specificity was 94% (95% confidence interval, 82–99%). In 4 of 7 episodes with false-negative results, the colony counts in cultures from both lumens were >400 colony-forming units/mL (maximal value reported), indicating the limitation of this method when both lumens of the catheter are colonized. With the pretest probability of catheter-related bloodstream infections ranging from 28% to 54%, the positive predictive value of a difference in time to detection between 2 lumens of ≥180 minutes for diagnosis of catheter-related bloodstream infections ranged from 81% to 93% and the negative predictive value ranged from 67% to 86%.Within the context of its limitations, this novel method provides an alternative for diagnosing catheter-related bloodstream infections among patients with a CVC, without PV cultures.