Detection of Echinocandin-Resistant Candida glabrata in Blood Cultures Spiked with Different Percentages of FKS2 Mutants

Abstract

Infections caused by the coexistence ofCandida glabrataechinocandin-resistant and echinocandin-susceptible cells may be possible, and the detection ofFKSmutants when the proportions ofFKSmutants are underrepresented poses a problem. We assessed the role of EUCAST and methods directly performed on positive blood cultures—Etest (ETDIR) and anidulafungin-containing agar plate assays—for detecting resistance inC. glabrataisolates containing different amounts of echinocandin-susceptible and -resistantCandida glabrataisolates. We studied 10 pairs ofC. glabrataisolates involving parental echinocandin-susceptible isolates and isogenic echinocandin-resistantFKSmutant isolates. Three inocula per pair (1 × 103to 5 × 103, 1 × 102to 5 × 102, and 10 to 50 CFU/ml) spanning suspensions with different amounts of susceptible/resistant isolates (9/1, 5/5, and 1/9 proportions for each the three inocula) were prepared. The suspensions were spiked in Bactec bottles and incubated until they were positive, and the three methods were compared. The EUCAST method showed echinocandin resistance when the bottles were spiked with susceptible/resistant isolates at 5/5 and 1/9 proportions; the results for the suspensions with a 9/1 proportion of susceptible/resistant isolates were susceptible for three pairs. We observed with the ETDIRresistance to both echinocandins in all pairs (resistance to micafungin and anidulafungin; MICs, ≥0.064 mg/liter and ≥0.125 mg/liter, respectively) and a double ring of growth inhibition in two pairs. The anidulafungin-containing plates showed fungal growth in the 90 spiked blood cultures at 48 h. Testing of echinocandin susceptibility with the ETDIRdirectly on the positive blood culture bottles is a reliable and rapid method to detect echinocandin resistance inC. glabrata. On the other hand, resistance can be missed with the EUCAST method when resistant isolates are underrepresented.

Pan-Echinocandin-Resistant Candida glabrata Bloodstream Infection Complicating COVID-19: A Fatal Case Report

“…Thus, we documented that isolate 2 ("breakthrough" isolate), compared to isolate 1 ("parental" isolate), had increased MIC values of anidulafungin, caspofungin and micafungin, and all values were higher than the CLSI resistance breakpoints [22]. As specifically shown for C. glabrata and echinocandins [50], the automated blood culture systems currently used to detect bloodstream infections allow for the reliable recovery of isolate populations composed of echinocandin-resistant and echinocandin-susceptible cells. However, in cases with a low proportion of resistant cells, picking up single colonies to perform standard antifungal susceptibility testing may result in missed detections of echinocandin resistance [50].…”

“…As specifically shown for C. glabrata and echinocandins [ 50 ], the automated blood culture systems currently used to detect bloodstream infections allow for the reliable recovery of isolate populations composed of echinocandin-resistant and echinocandin-susceptible cells. However, in cases with a low proportion of resistant cells, picking up single colonies to perform standard antifungal susceptibility testing may result in missed detections of echinocandin resistance [ 50 ]. In our case, taking advantage of morphologically different C. glabrata colonies [ 51 ] from the patient’s blood culture that yielded isolate 2, we were able to detect echinocandin resistance by testing more than one colony.…”

In vitro activity of ibrexafungerp against Candida species isolated from blood cultures. Determination of wild-type populations using the EUCAST method

“…Species-specific ibrexafungerp wtUL were defined as two two-fold dilutions higher than the MIC 50 (MIC value at which 50% of the isolates were inhibited) [11], following the principles applied for ECOFFs calculation using the eye-ball method (MIC value at which 99% of the isolates were inhibited), and the ECOFFinder program (99% of the modelled population included, ECOFF 99% from now on) (12). FKS1 and FKS2 hotspots (HS) were sequenced for C. albicans, C. glabrata, C. tropicalis, and C. krusei isolates [13] showing echinocandin resistance, J o u r n a l P r e -p r o o f ibrexafungerp non-wild type phenotypes (MIC above wtUL), or isolates with ibrexafungerp MICs one two-fold dilution below wtULs. All isolates showing a non-wild type phenotype were retested.…”

Amphotericin B Induction with Voriconazole Consolidation as Salvage Therapy for FKS -Associated Echinocandin Resistance in Candida glabrata Septic Arthritis and Osteomyelitis

“…Until such methods are available, other innovative methods must suffice. For instance, emerging methods of subculturing of positive blood culture broth directly onto Etest strips or anidulafungin-impregnated agar can more rapidly identify resistance (16). Surrogate markers to determine response to therapy in clinical trials, as opposed to more traditional clinical or culture based methods, are critically needed.…”