TABLE 1.
Validation of the breakpoints for dBLIM result interpretation and evaluation of different variables of the dBLIM protocola
| Evaluation | Variable | Diam range (mm), mean, for the |
Evidence | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| np-ESBL/ACBL/CARB group with: |
ESBL group with: |
Carbapenemase group with: |
|||||||||
| MEM at 10 μg | CTX at 5 μg | CAZ at 10 μg | MEM at 10 μg | CTX at 5 μg | CAZ at 10 μg | MEM at 10 μg | CTX at 5 μg | CAZ at 10 μg | |||
| Validation of interpretative breakpoints | 17–20, 18.3 | 14–16, 15.2 | 15–19, 18.1 | 6–12, 7.3 | 6–12, 7.2 | 6–10, 6.5 | Breakpoints and interpretative criteriab allow one to correctly differentiate np-ESBL/ACBL/CARB, ESBL, and carbapenemase groups. This validation phase shows 100% sensitivity and specificity for both ESBL and carbapenemase detection. | ||||
| Validation of DBLIM steps and parameters | |||||||||||
| Vol of BC broth (ml) | 5 | 17–20, 18.5 | 14–16, 15.3 | 18–20, 18.6 | 6–12, 7.1 | 6–12, 7.3 | 6–12, 6.6 | No difference in diagnostic sensitivity and specificity. However, a 10-ml volume may ensure a higher sensitivity. | |||
| 10 | 18–20, 18.6 | 14–16, 15.2 | 17–19, 18.5 | 6–10, 6.9 | 6–10, 7 | 6–10, 6.4 | |||||
| Centrifugation step(s): time and speed of rotation | 1st: 900 × g for 15 min; 2nd and 3rd: 5,000 × g for 15 min | 17–19, 18.3 | 14–16, 15 | 18–20, 18.6 | 6–12, 7 | 6–12, 7.1 | 6–9,6.3 | No difference in diagnostic sensitivity and specificity. The second option allows a reduction of step preparation time. | |||
| 1st: 900 × g for 10 min; 2nd and 3rd: 6,000 × g for 10 min | 18–20, 18.5 | 14–15, 14.8 | 18–19, 18.4 | 6–11, 6.9 | 6–11, 7 | 6–10, 6.4 | |||||
| Washing step | No | 16–18, 17.4 | 6–15, 11.7 | 15–19, 17.3 | 6, 6 | 6–12 6.6 | 6, 6 | Three false-positive ESBLs among the np-ESBL/ACBL/CARB group. The absence of a washing step is associated with smaller inhibition zone diameters and a reduction of specificity for ESBL detection. | |||
| Yes | 17–20, 18.6 | 14–16, 15.1 | 18–20, 18.7 | 6–12, 7.1 | 6–12, 7.1 | 6–8, 6.2 | |||||
| Extraction buffer | H2O | 18–19, 18.4 | 15–16, 15.6 | 18–19, 18.6 | 6–11, 7.2 | 6–18, 12.2 | 6–16, 10.6 | No difference in diagnostic sensitivity and specificity for ESBL activity detection. Metallo-β-lactamase activity is not detected using H2O or Tris HCl. Levels of MEM hydrolysis on cation-adjusted Mueller-Hinton broth and Tris HCl-Zn were similar for all carbapenemase classes. | |||
| Cation-adjusted Mueller-Hinton broth | 18–20, 18.6 | 14–16, 14.9 | 17–20, 18.3 | 6–11, 7.2 | 6–12, 7.2 | 6–15, 10.2 | |||||
| Tris HCl, 0.5 M | 17–20, 18.5 | 14–15, 14.7 | 17–20, 18.3 | 6–10, 7.1 | 6–17, 11.3 | 6–14, 9.7 | |||||
| Tris HCl, 0.5 M, + ZnSO4, 0.1 M (990 + 10 μl) | 18–19, 18.3 | 14–16, 14.8 | 18–19, 18.1 | 6–9, 6.5 | 6–12, 7 | 6, 6 | |||||
| Vol of extraction buffer (μl) | 500 | 18–20, 18.7 | 14–16, 15.2 | 18–20, 18.8 | 6–11, 7 | 6–12, 7.2 | 6–12, 6.6 | No difference in diagnostic sensitivity and specificity. A 1,000-μl volume can allow one to test more than 2 antibiotic disks. | |||
| 1,000 | 18–20, 18.6 | 14–16, 15.1 | 17–19, 18.5 | 6–10, 6.9 | 6–11, 7.1 | 6–10, 6.4 | |||||
| Inoculum of E. coli ATCC 25922(μl) on 9-cm-diam MHA disks | 200 at 1 McF suspension and streaking using a sterile swab | 18–20, 18.5 | 14–16, 15.4 | 18–20, 18.6 | 6–12, 7.2 | 6–12, 7.1 | 6–9, 6.4 | No difference in diagnostic sensitivity and specificity. The second option eases the reading of inhibition zone diameters. | |||
| 400 at a 0.5 McF suspension and streaking using a 10-μl inoculation loop | 18–19, 18.3 | 14–16, 15.2 | 18–20, 18.5 | 6–11, 7.1 | 6–10, 7 | 6–10, 6.4 | |||||
| Length of incubation (h) of bacterial suspensions with antibiotic disks | 2 | 18–20, 18.7 | 14–16, 15.2 | 18–20, 18.8 | 6–12, 7.1 | 6–11, 7.1 | 6–9, 6.3 | No difference in diagnostic sensitivity and specificity. Inhibition zones were small with 3 h of incubation; therefore, using the validated breakpoints, a 2-h incubation may ensure a higher specificity. | |||
| 3 | 16–17, 16.4 | 13–15, 13.9 | 15–17, 16.8 | 6, 6 | 6, 6 | 6, 6 | |||||
| Time (h) to reading of inhibition zones | 6 | 18–19, 18.5 | 14–16, 15.4 | 18–20, 18.7 | 6–11, 7 | 6–12, 7.2 | 6–10, 6.4 | No difference in diagnostic sensitivity and specificity and no significant difference in inhibition zone diameters. | |||
| 18 | 18–20, 18.8 | 15–16, 15.5 | 17–20, 18.8 | 6–12, 7.1 | 6–12, 7.2 | 6–11, 6.4 | |||||
| Evaluation of CAZ for β-lactamase activity detection | 18–19, 18.2 | 14–16, 15.4 | 14–16, 14.6 | 17–19, 18.5 | 6–11, 7 | 6–15 12.8 | 6–11, 7 | 6–9, 6.4 | 6–14, 9.8 | There is overlap of CAZ inhibition zone diameters between the ESBL and np-ESBL/ACBL/CARB groups (5 of 10 ESBL-producing isolates). CAZ is not effectively in vitro hydrolyzed by ESBL enzymes. Therefore, it is not an appropriate substrate for β-lactamase activity detection. | |
a
Bold and underlined values denote relevant differences between examined variables. Abbreviations: np-ESBL/ACBL/CARB, extended-spectrum-β-lactamase/AmpC-type-β-lactamase/carbapenemase-nonproducing; MEM, meropenem; CTX, cefotaxime; BC, blood culture; EB, Enterobacterales; ATU, area of technical uncertainty; McF, McFarland standard. Ten BCs were tested for each group and each method.
b
Breakpoints and interpretation criteria were as follows. For np-ESBL/ACBL/CARB EB, the MEM diameter was ≥15 mm and the CTX diameter was ≥14 mm; for ESBL-producing EB, the MEM diameter was ≥15 mm and the CTX diameter was ≤12; and for carbapenemase-producing EB, the MEM diameter was ≤12. For disks with ATUs, the MEM diameter was 13 to 14 and the CTX diameter was 13.