The in vitro activity of caspofungin and micafungin was determined with and without farnesol in RPMI-1640 against Candida parapsilosis biofilms. Drug interactions were examined using the XTT colorimetric assay-based broth microdilution checkerboard method. Drug-drug interactions were assessed utilizing a fractional inhibitory concentration index (

Summary Background Candidaemia is a common life‐threatening disease among hospitalised patients, but the effect of the Candida biofilm‐forming ability on the clinical outcome remains controversial. Objective The aim was to determine the impact of biofilms, specifically focusing on biofilm mass and metabolic activity, on the mortality in candidaemia. Patients/Methods The clinical data of patients (n = 127) treated at the University of Debrecen, Clinical Centre, between January 2013 and December 2018, were investigated retrospectively. Biofilm formation was assessed using the crystal violet and XTT assays, measuring the biofilm mass and metabolic activity, respectively. Isolates were classified as low, intermediate and high biofilm producers both regarding biofilm mass and metabolic activity. The susceptibility of one‐day‐old biofilms to fluconazole, amphotericin B, anidulafungin, caspofungin and micafungin was evaluated and compared to planktonic susceptibility. Results Intermediate/high biofilm mass was associated with significantly higher mortality (61%). All Candida tropicalis, Candida parapsilosis and Candida glabrata isolates originating from fatal infections were intermediate/high biofilm producers, whereas this ratio was 85% for Candida albicans. Solid malignancy was associated with intermediate/high biofilm producers (P = .043). The mortality was significantly higher in infections caused by Candida strains producing biofilms with intermediate/high metabolic activity (62% vs. 33%, P = .010). The ratio of concomitant bacteraemia was higher for isolates forming biofilms with low metabolic activity (53% vs 28%, P = .015). Conclusions This study provides evidence that the Candida biofilms especially with intermediate/high metabolic activity are related to higher mortality in candidaemia.

Antifungal lock therapy has received significant interest in the last few years because the frequently usage of intravascular devices is associated with an increasing number of catheter‐related bloodstream infections caused by Candida species. Antifungal combinations with synergistic interaction can be a good choice for antifungal lock therapy; therefore, interactions were examined between two echinocandins (caspofungin and micafungin) and the chitin synthesis inhibitor nikkomycin Z against Candida albicans and C. parapsilosis biofilms. Susceptibility was evaluated using the XTT‐based checkerboard microdilution method, while the nature of interactions was assessed by calculating fractional inhibitory concentration indices and using the Bliss independence model. Mathematic‐based evaluations were supplemented with fluorescent LIVE/DEAD viability assay. The results obtained by statistical interaction analyses correlated well with the viability assay. The tested echinocandins with nikkomycin Z caused an extended cell death and the structure of the biofilm was sparse compared to the control, especially for C. albicans. The findings support the simultaneous usage of nikkomycin Z and caspofungin or micafungin in alternative therapies such as the antifungal lock therapy. Significance and Impact of the Study Antifungal lock therapy can be a potential therapeutic approach to eradicate the intraluminal Candida biofilms; however, there is no approved lock strategy against fungal species so far. The results of this study provide valuable evidence that nikkomycin Z acts synergistically in combination with caspofungin or micafungin against biofilms. In addition, this synergy was more pronounced for micafungin combined with nikkomycin Z. Therefore, nikkomycin Z can be considered as a potential agent in antifungal lock therapy especially with micafungin against C. albicans or C. parapsilosis biofilms.

Farnesol is a quorum-sensing molecule that inhibits biofilm formation in Candida albicans. Previous in vitro data suggest that, in combination with certain antifungals, farnesol may have an adjuvant anti-biofilm agent. However, the in vivo efficacy of farnesol is very questionable. Therefore, the in vitro and in vivo activity of fluconazole combined with farnesol was evaluated against C. albicans biofilms using fractional inhibitory concentration index (FICI) determination, time-kill experiments and a murine vulvovaginitis model. The median biofilm MICs of fluconazole-sensitive C. albicans isolates ranged between 4 -> 512 mg/L and 150-300 μM for fluconazole and farnesol, respectively. These values were 512 -> 512 mg/L and > 300 μM for fluconazole-resistant clinical isolates. Farnesol decreased the median MICs of fluconazole by 2-64-fold for biofilms. Based on FICI, synergistic interaction was observed only in the case of the sessile SC5314 reference strain (FICIs: 0.16-0.27). In time-kill studies, only the 512 mg/L fluconazole and 512 mg/L fluconazole + 75 μM farnesol reduced biofilm mass significantly at each time point in the case of all isolates. The combination reduced the metabolic activity of biofilms for all isolates in a concentration- and time-dependent manner. Our findings revealed that farnesol alone was not protective in a murine vulvovaginitis model. Farnesol was not beneficial in combination with fluconazole for fluconazole-susceptible isolates, but partially increased fluconazole activity against one fluconazole-resistant isolate, but not the other one.

Our findings support the development of new alternative therapeutic strategies against C. parapsilosis biofilms.

The activity of fluconazole, amphotericin B, caspofungin and micafungin was determined using XTT-based fungal damage assays against planktonic cells, early and mature biofilms of Candida kefyr. Median MICs of planktonic cells were 0.25 mg/l, 0.25 mg/l, 0.5 mg/l, and 0.06 mg/l for fluconazole, amphotericin B, caspofungin, and micafungin, respectively. Fluconazole showed at least 50% fungal damage at ≥4 mg/l (51.5% ± 6.63% to 78.38% ± 1.44%) and at ≥128 mg/l (57.88% ± 9.2% to 67.25% ± 9.59%), while amphotericin B produced an even higher anti-biofilm effect at ≥0.5 mg/l (64.63% ± 6.79% to 79.5% ± 5.9%) and at ≥0.12 mg/l (77.63% ± 8.43% to 92.75% ± 1.89%) against early and mature biofilms, respectively. In case of micafungin, 50% fungal damage was observed at ≥0.06 mg/l (66.88% ± 10.16% to 98.63% ± 1.24%) and ≥0.25 mg/l (74.13% ± 10.77% to 99.38% ± 0.38%) for early and mature biofilms, respectively. Caspofungin-exposed cells showed an unexpected susceptibility pattern, that is, planktonic cells showed significantly decreased susceptibility at concentrations ranging from 0.015 mg/l to 1 mg/l compared to biofilms (P < .05-.01). The damage in planktonic cells and biofilms was comparable at higher concentrations. For planktonic cells and biofilms, 50% fungal damage was observed first at 0.5 mg/l (59.75% ± 3.16%) and at 0.06 mg/l (70.25% ± 10.95%), respectively. This unexpected pattern was confirmed using scanning electron microscopy. The unusual susceptibility pattern observed at lower caspofungin concentrations may explain the poorer outcome of caspofungin-treated C. kefyr infections documented in certain patient populations. As this phenomenon was markedly less apparent in case of micafungin, these data suggest that micafungin may be a more reliable option than caspofungin for the treatment of C. kefyr infections.

Echinocandins are recommended as first-line agents against invasive fungal infections caused by Candida glabrata, which still carry a high mortality rate. Dose escalation of echinocandins has been suggested to improve the clinical outcome against C. glabrata. To address this possibility, we performed in vitro and in vivo experiments with caspofungin against four WT C. glabrata clinical isolates, a drug-susceptible ATCC 90030 reference strain and two echinocandinresistant strains with known FKS mutations. MIC values for the clinical isolates in RPMI 1640 were #0.03 mg l 21 but increased to 0.125-0.25 mg l 21 in RPMI 1640+50 % serum. In RPMI 1640+50 % serum, the replication of C. glabrata was weaker than in RPMI 1640.Caspofungin in RPMI 1640 at 1 and 4 mg l 21 showed a fungicidal effect within 7 h against three of the four clinical isolates but was only fungistatic at 16 and 32 mg l 21 (paradoxically decreased killing activity). In RPMI 1640+50 % serum, caspofungin at ¢1 mg l 21 was rapidly fungicidal (within 3.31 h) against three of the four isolates. In a profoundly neutropenic murine model, all caspofungin doses (1, 2, 3, 5 and 20 mg kg 21 daily) decreased the fungal tissue burdens significantly (P,0.05-0.001) without statistical differences between doses, but the mean fungal tissue burdens never fell below 10 5 cells (g tissue) 21 .The echinocandin-resistant strains were highly virulent in animal models and all doses were ineffective. These results confirm the clinical experience that caspofungin dose escalation does not improve efficacy.

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