Development of a rapid diagnostic test for the cefazolin inoculum effect in methicillin-susceptible Staphylococcus aureus: Cefinase disk direct (CDD) test

Cefazolin is one of the first-line treatment for methicillin-susceptible Staphylococcus aureus (MSSA) infections worldwide. However, the cefazolin inoculum effect (CInE) can compromise its therapeutic efficacy, and the development of a rapid, practical diagnostic method to identify CInE-positive MSSA isolates is urgently required. To develop the novel Cefinase Disk Direct (CDD) test and evaluate its diagnostic performance. A total of 370 clinical MSSA isolates obtained from blood cultures across three Japanese hospitals were evaluated. CInE was determined by comparing MICs under standard and high-inoculum conditions. As a standardized alternative to the nitrocefin powder utilized in previous methods, we employed Cefinase disks for visual colorimetric detection. To validate diagnostic accuracy, we compared results obtained using direct inoculation (loopful) versus optical density (OD)-adjusted bacterial suspensions. Of all, 110 (29.7%) harbored the blaZ gene and 4 (1.1%) exhibited CInE; all CInE-positive strains were identified as blaZ type A. The CDD test demonstrated sensitivity of 100%, specificity of 94.4% (OD-adjusted) to 99.7% (loopful), positive predictive value of 80.0%, and negative predictive value of 100%. The spectrophotometric analysis resulted in sensitivity of 100%, specificity of 83.3%, and positive predictive value of 80.0%. The CDD test shortens the turnaround time to just 1.5 h. The proposed CDD test demonstrated satisfactory diagnostic performance for the detection of blaZ type A-harboring MSSA isolates. Further optimization is warranted to ensure that the assay becomes a rapid, cost-effective, and easily implementable solution for hospital laboratories worldwide.