Introduction

Invasive fungal infections pose an increasingly serious threat to the health of humans throughout the globe. Candida auris is an emerging pathogen that has been linked to severe invasive infections1 and high rates of patient mortality, having been detected in multiple nations since its initial identification in a female patient in Japan in 2009.2,3 Strikingly, most of these clinical C. auris isolates exhibit resistance to one or more of the antifungal drugs typically employed for the treatment of infections caused by Candida spp.4 Given these multidrug resistance characteristics, rapid spread throughout the globe, and potential to kill infected patients, there is a clear need for the design of novel therapeutic regimens that can more reliably combat C. auris and related fungal pathogens.

A variety of compounds have been explored in this therapeutic context, including both novel antifungal agents and drugs purported to synergistically enhance the efficacy of other antifungal drugs.5 The anthelmintic drug pyrvinium pamoate (PP) initially received US Food and Drug Administration (FDA) approval in 1955 for the treatment of pediatric pinworm infections. With the emergence of novel antiparasitic agents, the use of PP as an anthelmintic in the United States progressively diminished during the 1970s and 1980s. Despite the declining use of PP as an anthelmintic over the years, interest in PP has experienced a resurgence since the early 2000s due to its potential for targeting disease-causing organisms across various levels, including viruses, bacteria, fungi, and multicellular organisms.6–8 Notably, PP reportedly suppresses the growth of fluconazole (FLU)-resistant C. albicans and can synergize with FLU treatment to more effectively kill these pathogenic fungi.9 Whether PP similarly exerts single-agent or combination efficacy against C. auris, however, has yet to be tested. Accordingly, this study was designed to explore the antifungal activity of PP as a treatment for C. auris and other common Candida spp. both in vitro and in vivo.

Materials and Methods Fungal Isolates

For this study, 21 clinical Candida spp. isolates were used, including C. albicans (n=5), C. parapsilosis (n=2), C. tropicalis (n=2), C. glabrata (n=2), and C. auris (n=10). C. auris strains used in this study were obtained through the CDC and FDA Antibiotic Resistance Isolate Bank. These strains were subjected to morphological assessment and also underwent ITS and D1/D2 region sequencing to validate their strain designations. C. parapsilosis ATCC22019 was also incorporated into this study for quality control.

Antifungal Drug Selection

FLU, itraconazole (ITC), voriconazole (VOR), posaconazole (POS), and PP were purchased from Selleck Chemicals (TX, USA) and prepared as in M27-A4.10 All drugs were prepared at a stock concentration of 1600 µg/mL with DMSO.

Inoculum Preparation

Following growth on potato dextrose agar (PDA) for 2 days, yeast cells were harvested and suspended in sterile dH2O, followed by adjustment with a hemocytometer to a final concentration of 1–5×106 CFU/mL.

In vitro Antifungal Efficacy Testing

A microdilution checkerboard approach was performed as per the CLSI M27-A4 reference method to test the antifungal efficacy of PP either alone or in combination with azoles. Briefly, each of these drugs was subject to serial two-fold dilution using RPMI-1640 to a final concentration at twice the target concentration level, with respective working concentration ranges for PP and azoles of 0.25–32 µg/mL and 0.031–16 µg/mL. An inoculum concentration of 1–5×103 CFU/mL was established by diluting yeast cells with RPMI-1640 and then adding these fungi to 96-well plates followed by a 48h incubation at 35°C. Minimum inhibitory concentration (MIC) values were determined by identifying the lowest drug concentration capable of suppressing 50% of fungal growth relative to control treatment. Interactions among drugs were determined with the fractional inhibitory concentration index (FICI): FICI = (Ac/Aa) + (Bc/Ba), where Ac and Bc, respectively, denote the MIC values for the two antifungal drugs in combination, and Aa and Ba, respectively, denote the MIC values for those two antifungal agents when applied as single-agent treatments.11 Synergy, indifference, and antagonism were, respectively, defined by FICI values ≤ 0.5, 0.5< FICI ≤ 4.0, and > 4.0. All testing was repeated three times with triplicate analyses.

In vivo Antifungal Efficacy Testing

To assess the antifungal efficacy of PP and/or azoles when used to treat C. auris infections in vivo, G. mellonella larvae were prepared as in a prior report.8 Briefly, sixth instar larvae (300–350 mg, 2–3 cm, Chengdu Pets and Insects Company, Sichuan, China) were raised in the dark. C. auris AR385 was cultured for 2 days on PDA at 37°C, after which yeast cells were harvested, resuspended in dH2O at 1×108 CFU/mL, and each larva was injected with 10 μL of this C. auris suspension, with saline instead being injected into control larvae. In total, seven treatment groups were established (PP, ITC, VOR, POS, PP+ITC, PP+VOR, and PP+POS). There are twenty larvae in each group. Therapeutic (1ug per agent) or control solutions were performed via the last left leg with a Hamilton syringe (25 gauge, 50 μL) after cleaning the area using alcohol. Rates of larval survival were assessed every 24 h for 120 h post-infection. Analyses were repeated three times with triplicate analyses.

Statistical Analysis

GraphPad Prism 9.0 was used for all statistical testing and figure preparation. G. mellonella survival was assessed using Kaplan–Meier curves and log-rank (Mantel-Cox) tests. A P-values below 0.05 were considered statistically significant.

Results Analysis of the in vitro Single-Agent and Combination Antifungal Activity of PP

When employed as a single-agent treatment, the antifungal activity of PP was relatively poor, with MICs from 2 μg/mL to >32 μg/mL (Table 1) for the tested Candida spp. However, synergistic activity was detected when combining PP and azoles for the treatment of several of these Candida isolates (Tablea 1 and 2). The combination of PP + ITC exhibited synergistic activity against one C. albicans strain (20%) and four C. auris strains (40%), while PP + VOR synergistically suppressed the growth of three C. albicans strains (60%), one C. tropicalis strain (50%), and five C. auris strains (50%). More obviously, the combination of PP + POS synergistically inhibited four C. albicans strains (80%), one C. tropicalis strain (50%), one C. glabrata strain (50%), and nine C. auris strains (90%). PP also exhibited synergistic activity when combined with FLU and used to treat four C. albicans isolates (80%) and one C. tropicalis isolate (50%). PP and the tested azoles failed to exhibit any synergistic activity against C. parapsilosis in all experiments.

Table 1 Minimum Inhibitory Concentration (MIC) Values Corresponding to the Combined Use of Pyrvinium Pamoate and Azoles Against Candida Spp.

Table 2 Observed in Vitro Drug Interactions

Analysis of the in vivo Single-Agent and Combination Antifungal Activity of PP

G. mellonella larval survival was next analyzed for groups treated with PP (10%), ITC (30%), VOR (20%), POS (26.67%), PP+ITC (46.67%), PP+VOR (40%), and PP+POS (48.33%) (Figure 1). Single-agent PP treatment failed to prolong the survival of C. auris AR385-infected larvae. However, larval survival was significantly enhanced when combining PP with POS or VOR, exceeding the survival benefits afforded by POS or VOR alone (P< 0.05). The combination of PP and ITC also exhibited a trend towards increased survival rates relative to ITC treatment alone, although the difference was not significant.

Figure 1 C. auris AR385-infected larval survival was monitored for different treatment groups (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05).

Note: The curves were consisted of untouched growth control group, sterile saline group, yeast cells only group, PP treated group, ITC treated group, POS treated group, VRC treated group, PP with ITC treated group, PP with POS treated group, and PP with VRC treated group.

Discussion

Many cases of C. auris infection have been documented since 2009 when it was first identified in the external ear canal of a Japanese patient who subsequently experienced the infiltration of C. auris into her bloodstream.3C. auris infections have since been reported in over 20 countries,12 typically presenting in the form of nosocomial infections of a wide range of sites including the blood, skin, urine, bile, nares, and wounded tissues.13,14 Antifungal drug treatment is often insufficient to effectively treat C. auris, underscoring the need for novel interventional approaches such as combination therapies that can expand the overall spectrum of available antifungal drugs by simultaneously enhancing therapeutic efficacy and reducing the severity of treatment-related adverse effects.

This study revealed clear evidence of synergistic interactions between PP and the four tested azole drugs (ITC, VOR, POS, FLU) against many of the tested clinical Candida isolates. Consistently, the in vivo treatment of C. auris-infected larvae with both PP and azoles resulted in improved survival outcomes for all tested combinations other than PP + ITC relative to azole treatment alone. Given these results, combining PP with azoles represents a promising means of facilitating better C. auris-related survival outcomes. Additional studies will be essential to clarify the mechanistic basis for this result.

The azole resistance mechanisms associated with C. auris drug resistance mechanisms have yet to be established. One possibility is that these fungi may overexpress the ABC and MFS efflux pumps. Consistently, elevated levels of activity for ABC-type transporters have been documented in C. auris as compared to C. glabrata or C. haemulonii.4 Second, these fungi may harbor advantageous ERG11 point mutations, as supported by a study of 44 C. auris isolates that frequently identified FLU resistance-related point mutations within the ERG11 gene sequences in these isolates.15 Third, ERG11 overexpression may contribute to azole resistance, in line with data supporting higher levels of ERG11expression in FLU-treated C. auris as compared to controls.15 Recent data have also provided support to a model of adaptive aneuploidy in which C. albicans azole resistance has been tied to aneuploidy impacting the two left arms of chromosome 5.16 The stress associated with antifungal treatment has the potential to induce such aneuploidy, in turn enabling these fungi to better resist these antimicrobial agents.17 Genomic analyses have shown that many conserved orthologous antifungal resistance-related genes are present in both C. auris and C. albicans, and aneuploid C. auris isolates were recently documented to proliferate when exposed to azole stress. This suggests an important role for aneuploidy in shaping the antifungal resistance of C. auris.18

One prior study found that PP was able to effectively inhibit the ability of FLU-resistant C. albicans strain I(5L), which harbors two left chromosome 5 copies, to grow. PP was also able to enhance the antifungal effects of FLU when used to treat these fungi, suggesting that PP is a robust tool that can help overcome azole resistance resulting from aneuploidy.9,16 These results suggested that PP may also be able to more broadly enhance the antifungal potency of a range of agents, potentially via overcoming aneuploidy-associated changes in the characteristics of C. auris. Here, the synergistic antifungal impacts of PP and azoles were more robust when used to treat C. auris and C. albicans relative to other Candida spp., likely due to the higher odds of aneuploidy occurring in C. albicans and C. auris. Another potential mechanism that may underlie this synergistic activity may be the multifaceted metabolic effects of PP when used to treat C. auris, limiting iron availability to these pathogens while enhancing the nutritional status and functionality of immune cells. This induction of metabolic dysfunction can compromise the ability of these fungi to utilize macronutrients, thereby impairing their growth and viability.19

In summary, these findings indicate that PP may represent an effective treatment option for C. auris as it can overcome azole resistance. One limitation of this analysis, however, is that the number of clinical Candida spp. isolates used herein was relatively limited. Subsequent studies will necessitate the more comprehensive profiling of the combined effects of PP and azoles against a wider range of isolates with various genotypic and phenotypic characteristics to better understand the mechanistic basis for their synergistic activity and their potential for clinical application.

Conclusion

Combining PP with azoles represents a promising approach to effectively disrupting the growth of azole-resistant C. auris and other Candida spp. such that it may be a promising anti-Candida therapeutic option.

Ethical Approval

This study was approved by the ethics committee of Jingzhou Hospital Affiliated to Yangtze University [approval no. 2024-167-01]. We certify that the study was performed in accordance with the 1964 declaration of HELSINKI and later amendments.

Acknowledgments

We thank JT from the Department of Medical Mycology, Shanghai Skin Disease Hospital, Tongji University School of Medicine, Shanghai for kindly provided us with the isolates studied.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This work was Funded by Open Research Fund Program of the State Key Laboratory of Virology of China[Grant No.2023KF006].

Disclosure

The authors report no conflicts of interest in this work.

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