Post-antifungal effect of the combination of anidulafungin with amphotericin B and fluconazole against fluconazole-susceptible and -resistant Candida albicans

Background and Purpose: Invasive candidiasis is a life-threatening condition that kills a large number of immunocompromised patients each year worldwide. We used post-antifungal effect studies to analyze the activities of anidulafungin (AFG), as a clinically crucial antifungal drug, amphotericin B (AMB), and fluconazole (alone and in combinations) against FLC-susceptible and -resistant Candida albicans (C. albicans) isolates obtained from the cancer patients. Materials and Methods: We tested the phenomenon of post antifungal effects of FLC, AMB, AFG, and combinations of FLC+AFG, AFG+AMB, and FLC+AMB against 17 C. albicans isolates obtained from the oral cavity of cancer patients. Isolates that had not been exposed to antifungals, served as a control group. Colony counts were performed at 0, 2, 4, 6, and 24 h after a brief (1 h) exposure to antifungal. Results: The FLC had no detectable post-antifungal effect independent of antifungal concentration and resembled drug-free FLC (control). Significant variations in the post-antifungal effect were observed when all AMB and AFG were compared to FLC. The combination of AFG and AMB with FLC resulted in effective activity compared to FLC alone. Combination regimens were rated as indifferent in general. Interestingly, low dosages of the AFG displayed increasing fungistatic action as it approached a fungistatic endpoint against C. albicans isolates (n=17). Conclusion: Our findings suggested that brief exposure to AFG, in combination with FLC and AMB, at low concentrations of the medicines utilized, could be effective in the evaluation and optimization of new dosage regimens to manage candidiasis. However, future studies will determine the clinical utility of our findings.


Introduction
andida infection (candidiasis) is a lifethreatening condition that causes significant morbidity and mortality worldwide. The prevalence of candidiasis has risen considerably in recent years, owing primarily to an increase in the number of immunocompromised people [1]. Despite a global trend in candidiasis epidemiology toward an increasing prevalence of non-albicans Candida species, Candida albicans remains the most commonly reported pathogenic yeast [2,3]. Fluconazole (FLC) is the preferred antifungal for treating candidiasis owing to low toxicity, wide tissue distribution, and high solubility. However, candidiasis therapy is problematic due to frequent relapses and treatment failures [4]. One of the most critical factors contributing to the progressive development of azoleresistant fungi appears to be the widespread use of FLC for prophylaxis or pre-emptive treatment [5,6]. Therefore, the combination of antifungal regimens should be narrowed to avoid further emergence of resistance and treatment failure, based on in vitro activity and post-antifungal effect (PAFE) profiles. The therapeutic significance of in vitro PAFE, in conjunction with a drug's minimum inhibitory concentration (MIC) data, is linked to evaluating novel dosage regimens for new antifungal medications or combinations of agents in vivo during clinical use [7].
To explain the effect of azole-echinocandin and/or polyene combinations, we conducted PAFE studies to evaluate and compare the activities of anidulafungin (AFG) as a clinically important antifungal drug, amphotericin B (AMB), and FLC alone and in combination, against FLC-susceptible and -resistant C. albicans isolates derived from the cancer patients.

Fungal strains
This study was conducted on eight FLC-resistant clinical strains of C. albicans and nine FLC-susceptible strains. The isolates were taken from the oral cavity of patients with hematological malignancies and oncological disorders at the cancer center of Mazandaran University Hospital, Sari, Iran. Matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify all clinical isolates earlier [8]. These isolates had been stored at -70 °C at the reference culture collection of invasive fungi research center (IFRC, Sari, Iran) in cryo-tubes (Mast Diagnostics, Bootle, Merseyside, UK). The clinical strains were subcultured twice onto Sabouraud dextrose agar (SDA) before usage.
This study was approved by the Ethics Committee of Mazandaran University of Medical Sciences, Sari, Iran (Nr. 1298).

Determination of minimum inhibitory concentration
After 24 h of incubation at 35 °C, all Candida isolates were tested for antifungal susceptibility, according to the CLSI guidelines M27-A3 and M27-S4 documents, as validated recently by Pfaller et al. [9][10][11][12]. The MIC endpoint was set at 100% inhibition for AMB and greater than 50% inhibition for the other antifungal drugs. All of the tests were done twice in each round.

Post-antifungal effect (PAFE)
The post-antifungal effect investigations were carried out as previously stated [13]. Briefly, the PAFEs were determined for each strain alone or in the combination with three drugs (1×MIC, 4×MIC, 16×MIC of FLC alone; 0.25×MIC, 1×MIC, and 4×MIC of AMB alone; 0.125×MIC, 1×MIC, and 4×MIC of AFG alone; 1×MIC-FLC+1×MIC-AFG; 4×MIC-FLC+4×MIC-AFG; 1×MIC-FLC+1×MIC-AmB; 4×MIC-FLC+4×MIC-AMB; 1×MIC-AFG+1×MIC-AMB; 4×MIC-AFG+4×MIC-AMB. A hemocytometer slide was used to modify the turbidity concentration of yeast cell suspensions in sterile distilled water (1×10 6 CFU/mL). Afterwards, 1 mL of the yeast suspension was added to 9 mL of RPMI 1640 medium with and without the drug (control). Antifungal agents were removed by three cycles of repeated centrifugation (2000 rpm, 10 min) and washing with sterile PBS after a brief exposure to concentrations of antifungal agents (1 h at 35 °C). The supernatant was decanted entirely after the final centrifugation, and the fungal pellets were suspended in 9 mL warm RPMI. The solutions were incubated with gentle agitation at 35 °C. At the predesigned time points (0, 2, 4, 6, and 24 h), a 100 μL suspension from each solution was serially diluted and a 30 μL aliquot was covered onto an SDA plate for CFU counting. After a 48-hour incubation period at 35 °C, colonies were counted [13]. For each isolate, PAFE studies were carried out twice. PAFE refers to the time it takes for antifungal drug-treated cells to recover from the drug's inhibitory effect, as measured by an increase in CFU/ml of culture. The fungicidal activity was defined as a ≥3 log10 (99.9%) reduction in CFU/ml from the starting inoculum size, and the fungistatic activity was defined as a <99.9% reduction in CFU/mL from the starting inoculum size [14]. Synergy was defined as a ≥2 log10 increase in the killing activity of the combination. In contrast, antagonism was defined as a ≥2 log10 decrease in killing activity of combinations compared to the most active medication alone at the same concentration.
The interaction would be classified as indifferent if the variation was less than 100-fold [15]. Plots showing averaged colony counts (log10 CFU/ml) over time were created and compared to a drug-free control (control).

Statistical analysis
The SPSS software (version 16.0) was used to analyze the data. T-test was used to examine the changes in PAFE following the exposure to various amounts of the three antifungals and their combinations.

Post-antifungal effect
Figures 1-4 represent the post-antifungal effects of the three antifungal medications and their combination on 17 FLC-susceptible and -resistant C. albicans isolates after 1 h of exposure to and removal of the medicines. Regardless of antifungal concentration, the FLC did not show any significant PAFE (with curves similar to drug-free for FLCsusceptible and -resistant C. albicans isolates) (P≥0.001). All AFG concentrations and AFG+FLC regimens showed fungistatic efficacy (P<0.001) against the FLC-resistant C. albicans isolates (n=8) (Figure 1, Figure 2A). Furthermore, there was some fungistatic activity at doses of one and four times the MICs of AMB alone (significantly at 6 h) (Figure 1).
At all doses, the PAFEs of AMB with FLC combinations generated neither fungicidal nor fungistatic activity (P≥0.001) (Fig 1, Fig 2C). At all concentrations, AFG appeared to be slightly more effective than AMB alone, as well as in combination with AFG at one and four times the concentration of AMB (Fig 2B). The AMB-combined regimens did not improve the rate or degree of activity supplied by AFG; therefore, they were classed as indifferent ( Figure 2B).
Furthermore, we discovered that AFG with FLC combination was the most effective drug at 1 × MIC (significant within the first 6 h) (P<0.001) (Figure 1).   Figure 4). AFG at 4×MIC and in combination with FLC at 1×MIC both produced similar results and were the most effective concentrations (P<0.001) (Figure 3). For FLC-susceptible C. albicans isolates, the addition of AMB at one and four times the concentrations increased FLC activity ( Figure 4C).
When the medications were examined separately and in combination, FLC-susceptible and -resistant C. albicans isolates showed substantially identical patterns. When compared to FLC-susceptible C. albicans isolates, the combination of AFG with FLC and AFG alone appeared to be marginally more active against FLC-resistant C. albicans isolates. For all isolates, AMB and AFG did not show significant dosedependent PAFE. When comparing FLC alone and control with all concentrations of AMB and AFG, substantial variations in PAFE were identified. AFG reached a fungistatic endpoint at all doses against C. albicans isolates (n=17), although interestingly low concentrations displayed higher fungistatic activity. The combination of AFG and FLC demonstrated effective action when compared to FLC alone.

Discussion
Although the Candida genus has over 150 species, C. albicans is the most prevalent cause of candidiasis isolated from clinical samples. Severe candidiasis has been much more common in recent years, owing to a large population of high-risk persons who utilized chemotherapeutic, immunosuppressive, and broadspectrum antifungal medications [1,16]. The longterm use of FLC, as the first-line therapy for prophylaxis and treatment in immunocompromised patients, has been linked to the development of drug resistance in Candida species [17]. Therefore, innovative therapeutic techniques, such as combination medications, may be a viable option for enhancing clinical outcomes, increasing efficacy, and lowering antifungal toxicity. In vitro evaluation of the efficiency of these combinations could help researchers find the most effective, powerful, and safe antifungal agents for treating severe infectious diseases.
When comparing all concentrations of AMB and AFG to the control group, we found significant changes in PAFE, whereas FLC did not produce any measurable PAFE. The low growth inhibitory action of FLC against C. albicans isolates in vitro is one rationale for the absence of significant PAFE after exposure to FLC, as established in many prior investigations [18][19][20]. As previously stated, echinocandins do not contain ergosterol; therefore, these antifungals should not cause antagonism in combination with azole drugs, such as FLC. At one and four times the MIC, the combination of AFG with FLC showed improved activity and resulted in fungistatic activity with no antagonistic interaction. However, the combination of AFG with AMB was not superior to AFG alone and was classed as indifferent. The concentrations employed in studies mentioning the fungicidal activity of AMB and AFG against C. albicans isolates varied significantly from the ones examined in the current study [21,22]. The PAFE phenomenon, on the other hand, is highly dependent on the fungus species, antifungal drug class, inoculum size, drug concentration, research methodology, and drug exposure time [23].
The PAFE phenomenon, on the other hand, is highly dependent on the fungus species, antifungal drug class, inoculum size, drug concentration, research methodology, and drug exposure time [21].
Although the PAFEs of AFG, FLC, and AMB against various Candida species have been evaluated in a few studies, to our knowledge, limited data is comparing the PAFEs of these antifungals and their combinations against fluconazole-susceptible andresistant Candida albicans isolated from the oral cavity of cancer patients [7,18,20,22]. AMB showed a prolonged PAFE of more than 12 h against C. albicans in the first study examining the PAFE of echinocandins (caspofungin) when evaluated at concentrations ranging from 0.125 to 4 times the MICs [13]. PAFEs of AFG, FLC, and AMB against clinical isolates of C. glabrata, C. guilliermondii, C. krusei, C. tropicalis, and C. parapsilosis were determined in another investigation. FLC displayed no measurable PAFE regardless of the concentration, and AFG revealed fungicidal activity against C. krusei, C. glabrata, and C. parapsilosis at four and 16 times the MICs, and AMB elicited a consistently high PAFE in C. tropicalis [20,22]. Ellepola [21] found that the mean duration of AMB-induced PAFE was lowest for Candida albicans and highest for Candida parapsilosis, with intermediate values for C. guilliermondii, C. glabrata, C. krusei, and C. tropicalis. AMB also had the longest PAFE against C. albicans, C. krusei, and C. glabrata, which were all dependent on antifungal drug concentrations and exposure periods [24]. In PAFE tests, Nguyen et al. [22] discovered that 1 h exposure of C. albicans, C. glabrata, C. parapsilosis, and C. krusei isolates to AFG, at four and 16 times the MICs, resulted in fungicidal levels for >12h, following the drug washout. Furthermore, Gil-Alonso et al. [25] previously demonstrated that micafungin produced extended PAFE (37.5 h) against all C. albicans strains at two times the MICs. It has been shown previously that FLC produced a significant decrease against C. albicans isolates; however, researchers have demonstrated that fluconazole showed no measurable PAFE, regardless of the tested concentration, which was consistent with the results obtained in the current study [13,[18][19][20]. Our findings suggested that AMB and AFG had a fungistatic activity, somewhat independent of concentration, whereas FLC did not elicit significant PAFE at any of the concentrations tested with all C. albicans isolates. AFG and AMB have been described as exhibiting fungistatic activity, independent of the PAFEs concentration. Although fungistatic, FLC did not produce any measurable PAFE. Antifungal combinations of AFG, FLC, and AMB have demonstrated encouraging efficacy against a variety of fungal isolates (Candida species, Cryptococcus neoformans, and Aspergillus species), with no evidence of antagonism [15,26]. Interestingly, in fluconazole-susceptible and -resistant C. albicans, the combination of AFG and FLC was superior to FLC alone (at all concentrations), whereas AFG alone was superior to the combination of AFG and FLC in all isolates. There were no notable changes when AFG and AMB were combined. In fact, the combination of AFG and AMB resulted in general apathy. In fluconazole-resistant Candida albicans, AFG and AMB alone outperformed the combination of AFG and AMB. Since the effectiveness of medicine on C. albicans isolates is detected at low concentrations, PAFEs derived from the combination of AFG and FLC may have clinical significance.

Conclusion
To our knowledge, this is the first investigation of PAFE created by the combination of these three medications on fluconazole-susceptible and -resistant Candida albicans isolated from oral cavities of cancer