Introduction

Fungal infections in general and Candida spp infections, in particular, are a major cause of nosocomial invasive disease and a severe concern to human health globally [1-3]. These infections are typically associated with predisposing immune-deficient status, prolonged hospitalization, broad-spectrum antibiotics, and prophylactic use of antifungal agents such as fluconazole [4,5]. Candidemia is the most common form of invasive candidiasis. However, Candida's invasiveness does not necessarily represent its detection in the heart, kidney, and other internal organs because of its absence in the blood [6,7]. Hence, CDC estimates the actual burden of invasive candidiasis might be twice as high as the estimate for candidemia [8].

Although C. albicans is the principal agent of nosocomial infection, non-albicans Candida sp infections have been associated with higher mortality and drug resistance in the last decades [4,6,7]. Of these non-albicans species, C. auris is a rapidly growing global public health challenge. It is the most serious, emerging, and a multidrug-resistant fungal pathogen that is rapidly spreading worldwide and causing mortality in 30-60% of the infected patients [9-17]. The earliest Candida isolate, identified in 2011 as C. auris, was taken from a bloodstream sample of a patient from Korea in 1996 [18]. C. auris, as such, was first reported in 2009 from ear cultures (and hence "auris") of one patient in Japan [19] and 15 patients from University Hospitals South Korea [20]. In a decade since then, the infection has spread to all six continents [10,14-17,21,22]. Epidemiological data have shown that India and Pakistan have the highest cases of C. auris infections, primarily reported in overcrowded and tertiary care trauma hospitals [10,17,23]. Phylogenetic analysis of the globally isolated C. auris and related fungal strains has revealed that the climate change/global warming-related swamps, the widespread use of azoles, and the birds' migration-mediated transmission may have contributed to the simultaneous emergence of C. auris in three different continents [17,24].

High mortality caused by invasive and systemic mycosis in general, the emergence of multidrug-resistant invasive C. auris infections, and limitations of early detection and correct identification of C. auris together have left the health system with limited therapeutic options. Hence, the C. auris infection is the most challenging unmet medical need [11,16,21,25,26]. This situation is precipitously felt in the ongoing Covid-19 infection pandemic [27,28]. Typical C. auris isolates display reduced susceptibility to azoles, polyenes, echinocandins, and fluconazole [3,11,14,16,29-32]. Several studies have also shown that C. auris isolates also display increased resistance/reduced susceptibility patterns to the second generation and improved azoles antifungals, such as voriconazole, posaconazole, itraconazole, and isavuconazole [30,31,33]. Some of the biggest challenges in antifungal/anti- C. auris infections therapy regimens are i) a limited antifungal spectrum, ii) inadequate potency, iii) significant toxicity, and iv) the rapid onset of preexisting antifungal drug resistance even before it is marketed [3,30,34]. In many instances, the use of these antifungal agents results in poor treatment outcomes in the absence of apparent alternatives [12,35]. Amphotericin-B, a polyene macrolide antibiotic, was discovered in the 1950s with its potent broad-spectrum antifungal [36] and antileishmanial activities [37]. Because of the absence of its genetically confirmed resistance in 60 years of its clinical use, Amphotericin-B has been the mainstay for treating the highly fatal group of fungal infections [36].

Amphotericin-B, despite having the merit of fungicidal potency [36], poses dose-limiting nephrotoxicity leading to renal failure [38-40]. Two distinctly different Amphotericin-B liposomal formulations have been developed to overcome its inherent nephrotoxicity. These liposomal formulations are ideal antifungal agents for empirical, prophylactic, and case-targeted systemic and invasive mycosis [41-43]. Evolving Amphotericin-B parenteral formulations from a deoxycholate suspension to lipid complex [44] and then to liposomal preparations have led to incremental lowering of dose-limiting toxicity and consequent improved clinical outcomes [41,45-47]. However, the therapeutic usage of liposomal formulations of Amphotericin-B in C. auris has not been, hitherto, examined. The liposomal Amphotericin-B preparations, proven for higher antifungal efficacy [48], are typically not included for antifungal susceptibility testing for Candida spp [49-53], including C. auris [26,32,54,55]. As such, significant controversy and difficulty in judging the breakpoint of Amphotericin-B for Candida spp exist, and specific breakpoints have not been proposed due to interlaboratory variations [49-53]. The applicability of Amphotericin-B therapy, in general, whether it would pose dose-dependent nephrotoxicity, is judged based on the antifungal susceptibility pattern of Candida spp to Amphotericin-B-deoxycholate [56]. Although the most accurate way to determine antifungal agents, including Amphotericin-B susceptibility, is the CLSI recommended broth dilution method [57], routine laboratories heavily rely on additional methods, such as E-test and Vitek-2 systems, to report identification and/or AFST for yeast. A recent report on comparative findings on AFST of 102 C. auris strains against Amphotericin-B using the CLSI-Broth microdilution, Vitek-2, and E-test methods showed inconsistent MIC results [26]. In this study, Vitek-2 and E-test revealed high MICs and low MICs, respectively, for the same C. auris strains. Thus, in general, C. auris antifungal susceptibility patterns are interpreted using breakpoints established for other Candida species [17]. The cutoff points for susceptibility for C. auris against Amphotericin-B, is carried out using Amphotericin-B deoxycholate suspension [32]. Based on such inconsistent results, MDR C. auris infections are presumed to be recalcitrant to Amphotericin-B, including the improved yet untested liposomal Amphotericin-B in the AFST. This practice is followed even though the resistance to Amphotericin-B in Candida spp is rare [36,58] and the mechanism of this resistance is presently not fully understood for C. auris [59]. To that end, another study has, however, shown that the CLSI-based MIC of a novel liposomal Amphotericin-B in Saline (FUNGISOME®) is 2-16 folds lower than Amphotericin-B deoxycholate for several species of yeast and molds, which did not include C. auris [60]. These studies underscore the fact that the AFST for Amphotericin-B against yeast and molds in general and C. auris, in particular, should be revisited.

Presently, there is no information on whether C. auris strains generally show differential susceptibility patterns to these improved preparations of liposomal Amphotericin-B compared to variant forms of available Amphotericin-B formulations and other antifungal agents. This existing knowledge gap prompted us to investigate and compare the susceptibility patterns of 10 C. auris and one C. glabrata against seven antifungal drugs, including three each of different Amphotericin-B formulations (Amphotericin-B-deoxycholate, Liposomal Amphotericin-B: AmBisome®, and Liposomal Amphotericin-B in Saline: FUNGISOME®), and echinocandins (Caspofungin, Micafungin, and Anidulafungin), and one azole, Posaconazole. The present study showed that the liposomal preparation of Amphotericin-B in Saline, FUNGISOME®, displayed uniform higher efficacy against all MDR C. auris strains.

Materials and Methods

Isolates and growth conditions

A total of 10 multidrug-resistant C. auris strains CDC-0381-0390), and one C. glabrata (CDC-0317) were obtained from the antibiotic-resistant strain collection of CDC. All strains were grown and maintained on Sabouraud's dextrose agar or broth.

Antifungal agents

Three formulations of Amphotericin-B, Micafungin, Caspofungin, Anidulafungin, Posaconazole were obtained as indicated. Amfocare™ (Colloidal dispersion of Amphotericin-B in deoxycholate, Bangalore Pharmaceutical), Liposomal AmBisome® (Mylan), Liposomal in Saline (FUNGISOME®, Lifecare Innovations), Caspofungin (Casfung™, Glenmark), Micafungin (Micona™, Glenmark), Anidulafungin (Andulfa™, Gufic /Intas), and Posaconazole (Posatral™, Mylan). All antifungal agents other than those obtained from Sigma were of pharmaceutical grades and were reconstituted in saline, PBS, or distilled water per the respective manufacturers' instructions. FUNGISOME® preparation was sonicated for 45 min using a temperature-controlled water sonicator bath per the manufacturer instructions before use.

Antifungal susceptibility test (AFST)

AFST was carried out using the Clinical and Laboratory Standard Institute broth microdilution method (CLSI-BMD)-M270 A3-guidelines [57]. Briefly, RPMI-1640 medium with glutamine without bicarbonate containing 0.165M MOPS pH 7.0 (Sigma) was used for 2-fold serial dilution (32 µg/ml-to 0.0156 µg/ml) and also to dilute the stock solutions of antifungal agents in microtitre plates. Overnight cultures of Candida species grown on Sabouraud's dextrose agar were suspended in RPMI broth and adjusted to O.D._620nm of 0.1 (equivalent to McFarland Standard 0.5). 100 µl of each culture was added to the individual wells containing 100 µl of serially diluted antifungal agents. Antifungal agent- and Candida species-free controls were included as blank background readings. Similarly, wells containing Candida species without any antifungal agents were treated as no (0%) inhibition. Microtiter plates with tests and controls samples were incubated at 35 °C for 24 h. All experiments were carried out in three individual biological replicates. Growth inhibition of individual Candida species against different antifungal agents was observed visually as well as spectrophotometrically (PolarStar Galaxy, BMG, O.D._620nm). Average background readings obtained from 6-12 wells/plate were subtracted from the test results for further calculations. Minimum inhibition concentration (MIC) for all Amphotericin-B variants was defined as the lowest concentration at which 100% inhibition was achieved (both visually and by culture confirmation). The MIC endpoints were defined as the lowest concentration that caused 50% growth inhibition relative to growth control (No inhibition) for the rest of the antifungal agents. Percent Inhibition of Candida species by individual antifungal drugs at different concentrations was calculated using formula = 100 X (1-O.D. of test sample/average O.D. of control wells receiving respective Candida sp.). Inhibition curves for each strain for all antifungals were plotted using GraphPad prism 6, and 50 % and 90% inhibition values were determined. Fungicidal activity of echinocandins (Micafungin and Anidulafungin) was determined as described above for Amphotericin-B based on optical density as well as culture negativity.

Results

In the present study, seven different antifungal agents against 11 different Candida species, 10 of which belonged to multidrug-resistant C. auris, and one C. glabrata were subjected to AFST analysis. MIC₅₀ and MIC₉₀ of all antifungals except Amphotericin-B formulations are shown in Table 1 and three Amphotericin-B formulations in Table 2 based on inhibitory concentration 50 (IC₅₀) and IC₉₀ of individual strain. As shown in Table 1, all C. auris strains except CDC-0381 C. auris (IC₉₀: 0.016 µg/ml) and CDC-0390 C. auris (IC₉₀: 0.14 µg/ml) were found to be uniformly resistant to Caspofungin showing MIC > 32 µg/ml. Most strains except two strains of C. auris CDC-0383 (IC₉₀: > 32 µg/ml) and CDC-0384 (IC₉₀- > 32 µg/ml) were found to be susceptible to another echinocandin antifungal antibiotic, Micafungin (MIC₅₀-0.014-0.016 µg/ml, MIC₉₀-0.55 µg/ml). Similar to Micafungin, all Candida strains (C. auris and C. glabrata) except strains CDC-0383 and CDC-0384 (IC₉₀- > 32 µg/ml) showed high susceptibility to Anidulafungin (MIC₅₀-0.014-0.016, MIC₉₀-0.24 µg/ml). C. glabrata was found to be resistant to Posaconazole (no inhibition at the highest concentration 12.5 µg/ml). MIC of Posaconazole was based on > 50% growth inhibition although the inhibition of none of the strains reached beyond 79% (growth inhibition range 53-79%). The inhibitory concentration of Posaconazole for various strains ranged between 0.09-0.78 µg/ml. (MIC₅₀-0.09-0.19 µg/ml; MIC₉₀-0.78 µg/ml).

Since echinocandins are fungicidal for Candida species, the concentration at which no observed growth of C. auris noted was taken as the fungicidal concentration. For Caspofungin, the fungicidal concentration could not be determined as all strains except one strain were resistant (> 32 µg/ml). However, the fungicidal concentration of Micafungin (0.0156-0.125 µg/ml) and Anidulafungin (0.0156-0.75 µg/ml) was evaluated based on 100% growth inhibition and culture negativity. In this evaluation, two highly resistant strains (CDC0383 and CDC 0384) were not included.

Inhibitory concentrations (I.C.) for all Amphotericin-B preparations were measured at 100% growth inhibition (Table 2). Amphotericin-B deoxycholate preparation (Amfocare™) showed varied efficacy for all strains except CDC-0386 and CDC-0387 C. auris strains (20%, IC > 32 µg/ml) in the range of 0.25-32 µg/ml (MIC₅₀-0.25-0.5 µg/ml, MIC₉₀-32.0 µg/ml). Compared to Amfocare™, AmBisome™ showed 8-16 fold lower efficacy to inhibit five of 10 C. auris strains (MIC₅₀ 2.0-16.0 µg/ml, MIC₉₀ 32 µg/ml) and one CDC-0317 C. glabrata strain (IC 1.0 µg/ml). Five remaining strains (50%) of C. auris (CDC386-CDC390) showed no inhibition of growth at the highest concentration of AmBisome® (32 µg/ml). Compared to these two Amphotericin-B preparations, FUNGISOME® showed uniform higher efficacy by 8-32 folds. All C. auris strains, including those strains (CDC0383 and CDC0384), which showed uniform resistance patterns to all antifungal agents included in the present study, were completely inhibited at much lower concentrations (MIC₅₀ 0.125-0.25 µg/ml. MIC₉₀-1.3 µg/ml).

Together, these results showed that if the conventional AFST also includes available liposomal Amphotericin-B formulations along with the traditional Amphotericin-B colloidal form, the interpretation as "resistant" susceptibility pattern of the same strain to Amphotericin-B would likely differ to "susceptible". The corrected AFST, in turn, would provide appropriate therapeutic recommendation.

Discussion

This is the first study showing a comparative susceptibility pattern of multidrug-resistant C. auris against two different liposomal formulations of Amphotericin-B (AmBisome® and FUNGISOME®), Amphotericin-B-deoxycholate (Amfocare™), echinocandins, and an azole antifungal agents. Several new azoles group of antifungals, such as Voriconzole, Posaconazole, Itraconzole, Fluconazole and Isavuconazole are now available [4,30,61]. Most fungal species, including Candida, Aspergillus, Fusarium, and Mucor are resistant to Fluconazole [29,62]. Presuming so, fluconazole was not included for investigation in the present study. Since the echinocandins, such as Caspofungin, Anidualfungin, and Micafungin have shown efficacy or success rate of 50-75% [63,64] and Posaconzole ~92% [43,65,66,79,80], they were included them to compare their AFST with conventional Amphotericin-B deoxycholate and two available open-label liposomal formulations of Amphotericin-B.

Compared to the fungistatic antifungal drugs, azoles, and echinocandins, the Amphotericin-B is fungicidal and accorded distinction as a "Gold Standard" [67]. However, its high tendency to cause dose-dependent nephrotoxicity and infusion-related complications dampen its use [38,39,68]. Amphotericin-B-sensitive and -resistant C. auris strains have been recently reported in Colombia, and India and were found to be regionally restricted [26,59]. A recent multicenter study of AFST patterns of C. auris strains has shown that ~8% (27 of 350 strains) displayed resistance to Amphotericin-B [54]. A comparison of EUCAST (European Committee for Antimicrobial Susceptibility Testing) and CLSI reference-based microdilution MICs of antifungals for several C. auris strains has revealed tentative cutoff values for Amphotericin-B susceptibility (CLSI-MIC₅₀-0.5 µg/ml, MIC₉₀-2.0 µg/ml, EUCAST- MIC₅₀/MIC₉₀-1.0 µg/ml). In all these studies, AFST patterns for Amphotericin-B were measured using the conventional Amphotericin-B- deoxycholate. The mechanism of resistance of Candida species, including C. auris, to azoles has been attributed to point mutations occurring in the ergosterol biosynthesis genes (ERG11, ERG2) and to echinocandins in FKS1 gene [54,69,70]. Similarly, along with mutations in ergosterol biosynthesis genes [71,72], a strong association of four nonsynonymous (missense/nonsense) mutations in the protein-coding region responsible for transcription factor and membrane transport have been attributed to Amphotericin-B resistance [59]. However, this observed association does not represent the functional Amphotericin-B resistance. Several reported Amphotericin-B AFST assays are carried out with a generic form of Amphotericin-B employing the E-test or Vitek AST-YS07 methods, however, these methods do not provide a uniform measurement of AFS pattern for Amphotericin-B [26]. Hence, the present comparative study is based on the CLSI BDM method.

Resistance to Amphotericin-B is rare, and thus, the development of resistance to Amphotericin-B, unlike other antifungals, has not been a significant factor in the treatment of patients [36]. To minimize inherent nephrotoxicity of Amphotericin-B in conventional colloidal formulation, liposomal Amphotericin-B (AmBisome®) and further a novel liposomal Amphotericin-B have been developed [41,44,46-48] (Table 3).

Liposomal Amphotericin-B (AmBisome®) is composed of hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylglycerol (DSPG), and cholesterol [41,44]. The lipid composition, suspension medium, shape size, stability pharmacokinetics, and toxicity of this preparation substantially differ from the newer liposomal formulation, FUNGISOME® [45-48]. FUNGISOME® has been proven to be safe and with high efficacy against systemic fungal infection, including candidiasis, mucrormycosis, cryptococcosis, aspergillosis, and visceral leishmaniasis [38,73-77]. AmBisome® still has higher toxicity, and hence in many previous studies, voriconazole and Posaconazole were preferred over AmBisome® for the treatment of fungal infections [42,78-80]. In the unique formulation of FUNGISOME®, Amphotericin-B is encapsulated in 20-200 nm size predominantly unilamellar liposomes composed of phosphatidylcholine and cholesterol and stabilized in normal saline suspension. FUNGISOME® is sonicated before the infusion to maximize the number of smaller liposomes [48]. Previous studies have shown that the MIC of FUNGISOME® is 2-16 fold lower than the reference drug Amphotericin-B-deoxycholate against various yeasts, including Candida and Cryptococcus neoformans, dimorphic fungi, filamentous fungi, Zygomycetes, dematiaceous fungi, and dermatophytes [60]. The latter study included MIC studies of Candida spp viz. albicans, tropicalis, guillermondii, haemulonii, krusei, parapsilosis, dubliniensis, and glabrata showed susceptibility to FUNGISOME® [60]. This study, however, did not include AmBisome® in the comparative AFST analysis. In another multicenter study, the MIC of AmBisome® against yeasts and filamentous fungi has been reported to be 4-5 times higher than reference drug Amphotericin-B-deoxycholate [81]. However, these two susceptibility studies lack comparative analysis using C. auris. In this regard, the present study makes a significant contribution to C. auris AFST analysis and its potential application for targeted therapy.

Unlike echinocandins and azoles, all Amphotericin-B formulations - (conventional, lipid, and liposomal) have a distinctly different mechanism of drug targeting and action, pharmacokinetics, nephrotoxicity, potency, and dose as reflected in MIC. This study clearly shows that different formulations of Amphotericin-B cannot be treated the same as a generic Amphotericin-B formulation. Thus, two liposomal Amphotericin-B preparations, FUNGISOME® and AmBisome®, individually and together are different from the reference, Amphotericin-deoxycholate. As shown in Table 1 and Table 2, all MDR C. auris strains are susceptible to FUNGISOME®. In fact, the present findings show that in comparison to AmBisome®, FUNGISOME® showed 8 to 32-fold reduced MIC (higher efficacy) against most C. auris strains. Additionally, in the present study, Posaconazole and Micafungin were found to be effective against most strains, except for two strains (CDC383-CDC384) of South Africa origin. These resistant strains were found to be susceptible to FUNGISOME®, indicating that FUNGISOME® can potentially offer substantial therapeutic benefits. Such resistant C. auris strains and other similar resistant fungal pathogens would be reported as “resistant” and not suited for Amphotericin-B, if the therapy is based on the conventional Amphotericin-B AFST.

It is intriguing that despite having known Amphotericin-B-associated dose-dependent nephrotoxicity, the liposomal formulation of FUNGISOME® and not AmBisome® is more effective In vitro as revealed in this study. One explanation is that the multilamellar converting to unilamellar (Table 3) structure of liposomes in the FUNGISOME preparation may allow slow release of Amphotericin-B sufficient to offer optimum fungicidal concentrations for a prolonged period providing its extended fungicidal action In vitro. Clinically, this property may remain more beneficial as its serum level below toxicity would avoid dose-dependent nephrotoxicity. Clinically, both AmBisome® and FUNGISOME® have independently shown high efficacy against fungal infection as compared to conventional Amphotericin-B deoxycholate [48,73,82] although there is no clinical study that has shown side by side comparative efficacy of these two liposomal Amphotericin-B for any fungal infection including Candida auris. Amphotericin-B also serves as immunomodulator enabling the host to defend against the fungal/parasitic infection [83,84]. KALSOME, a FUNGISOME comparable Amphotericin-B preparation, used for leishmaniasis seems also has immunomodulatory and potentially protective effects [85]. At present, it is unknown whether the increased efficacy of certain liposomal Amphotericin-B preparations is mediated in part by their liposomal formulation-specific beneficiary immunomodulatory activities.

In conclusion, although the number of C. auris strains used in the present study are limited, they, irrespective of their origins or clad type, showed a uniform susceptibility to FUNGISOME® as compared to the other two Amphotericin-B formulations, and the pattern of susceptibility was well within the sensitive breakpoint limits [32]. This study emphasizes that the perceived notion of non-usefulness of Amphotericin-B based on its potential cytotoxic nature and/or conventional AFST for the treatment of C. auris infection is not justifiable and should be revisited both at laboratory assay and clinical therapy levels. Merits of liposomal Amphotericin-B preparations and their potential therapeutic applications to treat fungal infection may be universally recognized based on the improved and accurate reporting of AFST stated in the present study. The proposed modified BD-AFST approach is more relevant in COVID-19 pandemic times when several fatal cases of Covid-19 patients were found to be infected with incurable fungal pathogens such as C. auris and other Candida spp., Aspergillus and Mucor [27,28,86].

Author Contributions

Conceptualization, methodology, validation of the data, writing, editing and project administration, V. P.

Funding

This research received no external funding. The author has no financial conflict.

Acknowledgments

The author acknowledges the technical assistance of Mr. David Ordough during the initial phase of this study.

Conflicts of Interest

The authors declare no conflict of interest.

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