Anidulafungin Susceptibility Testing of Candida glabrata Isolates from Blood Cultures by the MALDI Biotyper Antibiotic (Antifungal) Susceptibility Test Rapid Assay

Abstract

Echinocandins are the recommended first-line antifungals for treatment of invasive candidiasis. The increasing number of Candida glabrata strains resistant against echinocandins is an emerging health care concern. The rapid detection of resistant C. glabrata isolates is an urgent requirement for clinical laboratories. In this study, we developed the MALDI Biotyper antibiotic (antifungal) susceptibility test rapid assay (MBT ASTRA) for the rapid detection of anidulafungin-resistant C. glabrata isolates directly from positive blood cultures. Of 100 C. glabrata strains, MBT ASTRA classified 69 as susceptible and 29 as resistant. Microdilution assays performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, used as a standard reference, identified 65 susceptible, 9 intermediate, and 26 resistant isolates. Sequencing of hot spot 1 and hot spot 2 regions of the FKS1 and FKS2 genes classified 86 susceptible and 14 resistant isolates. The MBT ASTRA had sensitivity and specificity of 80% and 95%, respectively, compared to the microdilution method. Positive and negative agreement of MBT ASTRA was calculated at 100% and 80%, respectively, compared with the molecular sequencing approach. Together, these results revealed a high accuracy of MBT ASTRA compared to microdilution according to the CLSI and PCR analysis, resulting in a categorical agreement of 90% and 83%, respectively. The validity of MBT ASTRA was 98%. Importantly, MBT ASTRA provided antifungal susceptibility testing (AFST) within 6 h that was both accurate and reliable compared to the other two approaches, which require at least 24 h or are costly. Therefore, this method has the potential to facilitate clinical AFST rapidly at low sample costs for clinical labs already equipped with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Keywords: C. glabrata; MBT ASTRA; anidulafungin; positive blood samples; rapid antifungal susceptibility testing.

FIG 1

Venn diagram of 35 C. glabrata isolates found resistant by PCR, the CLSI method, and the MBT ASTRA for AFST. The number of resistant strains detected by each method is shown. In total, 14 isolates resistant against anidulafungin were detected by all three methods. There were no isolates detected as resistant only by PCR. Fourteen resistant isolates were detected by CLSI microdilution and the MBT ASTRA but not by PCR. The CLSI method detected 7 resistant isolates (6 intermediate and 1 resistant) that were found to be susceptible by PCR and MBT ASTRA. Three isolates were detected as susceptible by PCR and the CLSI method but not by MBT ASTRA. However, 2 out of these 3 isolates could not pass the growth control cutoff and were not considered for data analysis.

FIG 2

Example of RG and MIC distribution of 36 C. glabrata isolates against anidulafungin derived from positive blood cultures. RG values (y axis) of 36 susceptible and resistant isolates were plotted against the MIC values (x axis). The horizontal dashed line indicates the suggested cutoff for MBT ASTRA-derived RG values. The vertical dashed line indicates the MIC breakpoint defined by CLSI. Fourteen susceptible isolates (green circles) and 14 resistant isolates (red squares) were separated similarly by the MBT ASTRA and CLSI method. Eight isolates with discrepant results between these two methods are shown by triangles. One isolate was detected as resistant by the MBT ASTRA (light blue triangle) that was susceptible by the CLSI method. In contrast, CLSI microdilution detected 6 intermediate and 1 resistant C. glabrata isolate (dark blue triangles) that all were susceptible by the MBT ASTRA.