A Test for the Rapid Detection of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus

Abstract

The cefazolin inoculum effect (CzIE) has been associated with therapeutic failures and mortality in invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections. A diagnostic test to detect the CzIE is not currently available. We developed a rapid (∼3 h) CzIE colorimetric test to detect staphylococcal-β-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin America and the United States. The cefazolin MIC determination at a high inoculum (10⁷ CFU/ml) was used as a reference standard (cutoff ≥16 μg/ml). All isolates underwent genome sequencing. A total of 257 (37.3%) of MSSA isolates exhibited the CzIE by the reference standard method. The overall sensitivity and specificity of the colorimetric test was 82.5% and 88.9%, respectively. Sensitivity in MSSA isolates harboring type A BlaZ (the most efficient enzyme against cefazolin) was 92.7% with a specificity of 87.8%. The performance of the test was lower against type B and C enzymes (sensitivities of 53.3% and 72.3%, respectively). When the reference value was set to ≥32 μg/ml, the sensitivity for isolates carrying type A enzymes was 98.2%. Specificity was 100% for MSSA lacking blaZ The overall negative predictive value ranged from 81.4% to 95.6% in Latin American countries using published prevalence rates of the CzIE. MSSA isolates from the United States were genetically diverse, with no distinguishing genomic differences from Latin American MSSA, distributed among 18 sequence types. A novel test can readily identify most MSSA isolates exhibiting the CzIE, particularly those carrying type A BlaZ. In contrast to the MIC determination using high inoculum, the rapid test is inexpensive, feasible, and easy to perform. After minor validation steps, it could be incorporated into the routine clinical laboratory workflow.

Keywords: BlaZ; CzIE rapid test; cefazolin inoculum effect; high inoculum.

FIG 1

Methodology of the CzIE rapid test. (A) Methodology of the rapid test (as described in the Materials and Methods section). (B) S. aureus quality controls. Tubes from left to right correspond to MSSA TX0117 (BlaZ-positive exhibiting the CzIE), ATCC 29213 (BlaZ-positive lacking the CzIE), and ATCC 25923 (BlaZ-negative lacking the CzIE). Change of color to red indicates a positive result (presence of CzIE), whereas a yellow color is suggestive of a negative result.

FIG 2

Receiver operational characteristic (ROC) curve and area under the ROC curve (AUC) to evaluate the diagnostic performance of the CzIE rapid test. (A) ROC curve for the complete set of MSSA isolates (n = 689). (B) ROC curves for BlaZ type A isolates (n = 186). The dotted lines represent random chance (AUC = 0.5).