A Novel Transdermal Application for Clearing Skin Colonization by Candida auris

Candida auris has demonstrated the ability to colonize the skin of hospitalized patients, possibly contributing to nosocomial spread. The objective of this study was to determine whether two novel transdermal agents could clear skin colonization established by C. auris.

ABSTRACT

Candida auris has demonstrated the ability to colonize the skin of hospitalized patients, possibly contributing to nosocomial spread. The objective of this study was to determine whether two novel transdermal agents could clear skin colonization established by C. auris. A murine skin colonization model was first optimized and then used to test fungal burden reduction following treatment with 1% terbinafine or 1% clotrimazole in a proprietary Advanced Penetration Technology formulation (APT). Both treatments significantly reduced fungal burden compared to that in control groups. These novel agents show promise as a topical means of preventing skin colonization by C. auris.

INTRODUCTION

The importance of Candida auris was initially underestimated due to the inability of laboratories to correctly distinguish this species from other Candida species. However, increased use of matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) for definitive identification has demonstrated the emergent impact of C. auris in hospital settings worldwide. Published reports have shown C. auris to be involved in bloodstream infections, with some contributing to fatal outcomes despite antifungal therapy (1). A report from a major hospital outbreak in London in 2015 to 2016 concluded that C. auris demonstrates high survival rates on environmental surfaces and the ability to rapidly colonize the skin of patients. This report also demonstrated that although the majority of the patients had only skin C. auris colonization, a significant percentage 44% (22/50) required the use of systemic antifungal treatment due to clinical manifestations of infection, including candidemia in eight patients (2). Skin colonization in patients is hypothesized to contribute to outbreaks in hospital settings from continual recontamination of environmental surfaces (3). Subsequent evidence demonstrating the importance of C. auris skin colonization as a risk for development of infection and increased transmission has led to current guidelines recommending that infection control measures should be the same for both infection and colonization with C. auris (4). The ability to clear C. auris isolates from the skin, not only from colonized patients but also from those who may have established skin wounds, is extremely important for the disruption of hospital transmission. Thus, finding means of skin decolonization and wound treatment are of vital importance in preventing C. auris nosocomial infections.

Recently, we used a guinea pig wound model of C. auris to show that oral dosing with 10 mg/kg ibrexafungerp (IBX) reduced the severity of lesions and significantly reduced the C. auris fungal burden in infected wounds compared with that in untreated controls (5). However, since this model was an infection model, we needed to develop a C. auris skin colonization model in order to determine whether an agent would be able to decolonize the skin. Initial experiments determined that a C. auris inoculum of 10⁸ blastospores/ml applied at 4 time points induced skin colonization of the ears and backs of mice without causing infection. The C. auris inoculum was considered to have resulted in skin colonization if the inoculum resulted in a sustained tissue fungal burden of the back and ears, while demonstrating no clinical manifestations of active C. auris infection. We then used this new model to evaluate the efficacy of two topical antifungal formulations. The antifungal formulations used for this study are nonprescription strength 1% terbinafine hydrochloride formulated with Advanced Penetration Technology (APT) and a nonprescription strength 1% clotrimazole formulated with APT.

The APT is a patent-pending, proprietary transdermal dual carrier formulation. This formulation provides improved dermal penetration and efficacy of topical active pharmaceutical ingredients (APIs). Additionally, the APT imparts both mechanical and biochemical effects on the microbe/fungal cell walls, providing a highly effective method of destruction of microbes. APT is intellectual property owned by Patient Focused Tele-Health, LLC, a Rockwall, Texas-based company. The company’s focus is improving over-the-counter (OTC) topical formulations, allowing consumers better therapeutic outcomes with nonprescription medications. The antifungal effect on skin colonized by C. auris of 1% terbinafine in APT (TAPT) and 1% clotrimazole in APT (CAPT) was determined by CFU per gram of skin tissue sampled.

RESULTS

Model development.

Figure 1 displays the average log CFU per gram values and standard deviations (SDs) for the ear and back skin samples taken at different time points following the 4th skin inoculation with C. auris. Normal (intact) skin appearance was noted throughout the 14-day observation period, indicating that skin infection did not develop. Based on these data, we determined that skin colonization was achieved after the fourth inoculation with C. auris (10⁸) and continued through day 14. Therefore, for the efficacy study, application of test compounds was initiated 24 h after the final inoculation (day 7).

FIG 1.

Tissue fungal burden (method development) for right ear, left ear, and back skin samples inoculated with C. auris.

Clinical observations.

Throughout the treatment period, the skin of the ears and backs of mice demonstrated a normal appearance and showed no signs of infection or inflammation. No adverse effects from treatment were observed in any of the colonized mice (Fig. 2). Figure 3a and b show histopathology of mouse skin stained with periodic acid-Schiff (PAS). As can be seen, in this colonization model, C. auris failed to penetrate the skin layers (Fig. 3a). Also, it appears C. auris yeast cells were dislodged during processing (Fig. 3b).

FIG 2.

Clinical appearance of the skin of treated mice inoculated with C. auris and treated with 1% terbinafine in APT or 1% clotrimazole in APT.

FIG 3.

Histopathology of PAS-stained mouse skin colonized with C. auris (magnification, ×200). C. auris yeast cells are indicated by arrows.

Evaluation of the efficacy of 1% TAPT and 1% CAPT: tissue fungal burden.

Table 1 shows the fungal burden of the three colonization sites, expressed as the average log CFU gram (± SD). For the right ear, mice in the untreated control and vehicle control groups showed the highest tissue fungal burdens (average ± SD of 7.79 ± 0.1 and 7.58 ± 0.1 log CFU/g, respectively), while the 1% TAPT- and 1% CAPT-treated groups showed tissue fungal burdens of 4.59 ± 0.1 and 4.43 ± 0.1 log CFU/g, respectively. This reduction in CFU in the treated groups after 7 days of treatment was significant compared to values in the vehicle and untreated control groups (P values of <0.0001).

TABLE 1.

Tissue fungal burden with P values compared to the untreated control

Group	Animal no.	Right ear			Left ear			Back
		Log CFU/g		Log CFU/g		Log CFU/g
		Each animal	Avg ± SD	P value	Each animal	Avg ± SD	P value	Each animal	Avg ± SD	P value
Untreated	1	7.96	7.79 ± 0.1		7.84	7.72 ± 0.1		7.74	7.33 ± 0.2
	2	7.79			7.66			7.34
	3	7.69			7.76			7.11
	4	7.77			7.65			7.27
	5	7.77			7.71			7.18
Vehicle	1	7.50	7.58 ± 0.1	0.0353	7.61	7.76 ± 0.1	0.9422	7.77	7.66 ± 0.2	0.1003
	2	7.59			7.80			7.41
	3	7.59			7.68			7.54
	4	7.72			7.82			8.02
	5	7.51			7.89			7.57
1% TAPT	1	4.69	4.59 ± 0.1	<0.0001	4.49	4.40 ± 0.1	<0.0001	4.00	4.29 ± 0.2	<0.0001
	2	4.66			4.33			4.50
	3	4.49			4.48			4.51
	4	4.71			4.37			4.14
	5	4.39			4.35			4.29
1% CAPT	1	4.31	4.43 ± 0.1	<0.0001	4.47	4.36 ± 0.1	<0.0001	4.51	4.51 ± 0.1	<0.0001
	2	4.51			4.14			4.61
	3	4.40			4.33			4.39
	4	4.36			4.49			4.63
	5	4.58			4.37			4.43

For the left ear, the mice in the untreated and vehicle groups again showed the highest tissue fungal burden (average ± SD of 7.72 ± 0.1 and 7.76 ± 0.1 log CFU/g, respectively), while the 1% TAPT- and 1% CAPT-treated groups demonstrated averages (± SDs) of 4.40 ± 0.1 and 4.36 ± 0.1 log CFU/g, respectively. Compared to values in the vehicle and untreated control groups, the CFU reduction in treated groups was also significant (P values of < 0.0001).

The skin from the backs of mice in the untreated and vehicle-control groups also showed the highest tissue fungal burden (average ±SD of 7.12 ± 0.1 and 7.16 ± 0.1 log CFU/g, respectively), while the 1% TAPT- and 1% CAPT-treated groups demonstrated averages (± SDs) of 3.80 ± 0.1 and 3.76 ± 0.1 log CFU/g, respectively. As with the ear samples, the back-skin samples from the treated groups showed a significant reduction in tissue fungal burden compared to those in the vehicle and untreated control groups (P values of <0.0001).

No statistically significant difference in efficacy between the two antifungal agents (P value of >0.05) was noted in any of the tissue samples.

DISCUSSION

Candida auris is emerging as a frequent cause of nosocomial infections, and skin colonization of both health care workers and patients is suspected to be a major contributing factor. In the study by Schelenz and colleagues (2), no health care workers had C. auris colonization of the hands, but eight patients with candidemia did demonstrate skin colonization. Other studies have shown that hands play an important role in the transmission of Candida species either by direct contact with infected patients or by contact with contaminated surfaces (6). The ability to clear C. auris from the skin, not only from colonized patients but also from those who may have established skin wounds, is extremely important to the disruption of hospital transmission.

The choice of antifungal therapy is also a critical factor for C. auris patients, since many of these patients will have serious underlying health issues. For example, in an effort to characterize the epidemiology of this newly identified pathogen, Lockhart et al. studied isolates from 54 patients in Japan, Venezuela, South Africa, India, and Pakistan; 61% of these patients had developed bloodstream infections, although 41% were receiving systemic antifungal treatment (7). This publication also indicated that C. auris strains infecting these patients were resistant to conventional antifungals, in particular, fluconazole and amphotericin B, with certain strains having a pan-resistant profile (8). In this cohort of patients, the mortality rate was 59%. Therefore, use of an effective drug is vital to restoring the health of patients infected with C. auris as well as for the prevention of further transmission.

Relying on surface disinfectants to eliminate the reservoir of C. auris in the hospital environment is also not a foolproof strategy. CDC guidelines for managing patients with C. auris infections include maintaining a minimum 3-ft spatial separation between patients, changing all personal protective equipment (PPE) by personnel between patients, and cleaning and disinfecting environmental surfaces and shared equipment on a more frequents basis (9). In that regard, studies by Cadnum et al. (10) showed that not all disinfecting agents are equally effective at killing C. auris on surfaces. In fact, the quaternary ammonium compounds tested were significantly less effective against Candida species than against methicillin-resistant Staphylococcus aureus (MRSA) (P ≤ 0.02), with no significant differences between C. auris and other Candida species (10).

Alternatively, transdermal delivery of an effective antifungal against C. auris would be an easier, much less costly approach for widespread use. TAPT and CAPT are formulations of 1% terbinafine and clotrimazole, respectively, in a proprietary Advanced Penetration Technology formulation (APT). APT is a transdermal carrier with dual mechanical and biochemical actions that work together to inhibit microbial growth, including that of bacterial and fungal biofilms.

Our data show that both 1% terbinafine in APT and 1% clotrimazole in APT demonstrated significant antifungal activity against C. auris in this newly developed murine model of skin colonization. Importantly, the MIC value for terbinafine and clotrimazole against the C. auris strain used was 2 μg/ml. Since the proposed topical formulations are 1% terbinafine and 1% clotrimazole (i.e., 10 mg/ml) the concentration applied to the skin is well above the MIC values. In this regard, drug levels that are up to 10× the MIC values are considered effective. Furthermore, the 3-log reduction in colony counts from all three sites (right and left ears and back) following treatment mirrors that of previously published in vivo data describing successful decolonization of the skin by Staphylococcus aureus (11–13). This reduction in skin fungal burden may be a significant factor in reducing the transmission of C. auris from potential “super spreaders” among health care workers in a hospital setting, even though complete eradication may not be attained. The results suggest that APT formulations may provide a safe and easy method to decolonize the skin of patients as well as health care workers.

MATERIALS AND METHODS

Animal care and use statement.

All procedures in the protocol were in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare. Mice were used upon review and approval of an addendum to our existing protocol by the Case Western Reserve University (CWRU) Institutional Animal Care and Use Committee (IACUC).

Procurement and housing of animals.

A request was made to the animal resource center (ARC) at CWRU to order the required number of female BALB/c mice (Charles River Laboratories, Wilmington, MA) with a body weight of ∼20 g. Animals were allowed to acclimate for a minimum of 5 days prior to use. Environmental controls for the animal room were set to maintain a temperature of 16 to 22°C, a relative humidity of 30% to 70%, and a 12/12-h light-dark cycle.

Development of the C. auris colonization model.

A clinical isolate of C. auris, MRL number 35368, was used as the skin colonizer, due to its elevated MICs to caspofungin, micafungin, and voriconazole (each 1 mg/liter). Broth microdilution susceptibility testing was performed for this strain against terbinafine and clotrimazole according to the Clinical and Laboratory Standards Institute (CLSI) M27-S4 guidelines (14). Against C. auris MRL number 35368, terbinafine and clotrimazole both demonstrated an MIC value of 2 μg/ml.

Sabouraud dextrose broth (SDB) was inoculated with fresh colonies of C. auris and incubated at 37°C overnight. C. auris cells were harvested by centrifugation and normal saline (0.85% NaCl) washes. A challenge inoculum of 10⁸ blastospores/ml was prepared using a hemacytometer. To check the inoculum count, 10-fold dilutions of the C. auris working conidial suspensions were plated onto Sabouraud dextrose agar (SDA) medium. The plates were incubated at 37°C for 2 days, and the colony counts determined.

An initial experiment was performed to determine if a C. auris inoculum of 10⁸ blastospores/ml applied at 4 time points could induce skin colonization of the ears and backs of mice without causing skin infection. The C. auris inoculum was considered to cause skin colonization if the inoculum resulted in a sustained tissue fungal burden of the back and ears, while demonstrating no clinical manifestations (i.e., redness of the skin or swelling) of active C. auris infection.

Two days prior to the first skin inoculation, the hair from the backs of the mice was clipped and depilated using Nair. On day 0, a 1.5-cm² stencil was used to mark the area of the back, and this area was then tape stripped 4 times using autoclave tape in order to disrupt the integrity of the skin and to encourage colonization. The tape was allowed to sit for approximately 5 s, and then removed; this was repeated 4 times. C. auris inoculum (10⁸ blastospores/ml) was applied to the pinna of the ears using a soaked cotton tipped applicator and to the back until the entire demarcated square was visibly wet (5 applications with the swab). All areas were similarly inoculated on days 2, 4, and 6 for a total of 4 inoculations.

Mice were observed following inoculation for up to 14 days. At the designated time periods, 5 mice were euthanized and assessed for tissue fungal burden in both ears and back.

Evaluation of the efficacy of 1% TAPT and 1% CAPT.

The developed C. auris colonization model was used to determine the efficacy of 1% TAPT and 1% CAPT in decolonization the skin of animals. Mice were randomized into the following groups (n = 5): (i) untreated control, (ii) vehicle plus 1% APT, (iii) 1% terbinafine in APT (TAPT), and (iv) 1% clotrimazole in APT (CAPT).

Beginning on day 7 (Table 2), test compounds were administered once a day for 7 days. Each ear was treated topically with 5 μl, while the back was treated topically with 40 μl. Animals were monitored daily for any signs of toxicity (i.e., lethargy, weight loss, and general failure to thrive) and skin appearance. Mice were euthanized 1 day after the final treatment application (day 14). The pinnae of the right and left ears and a 10-mm biopsy punch of skin from the back were collected aseptically and weighed. Tissues were homogenized and serially diluted in normal saline. The homogenates were cultured for 48 h on SDA plates to determine the CFU; tissue fungal burden was expressed as log CFU per gram of tissue. At the end of the study, all animals were euthanized and disposed to the Animal Resource Center for incineration.

TABLE 2.

Treatment schedule

Group	Experimental day:a
	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14
Treated animals	Inoc	Inoc	Inoc	Inoc	Tx	Tx	Tx	Tx	Tx	Tx	Tx	Euthanize

^aInoc, inoculation; Tx, treatment.

Statistical analysis.

Differences in average log CFU in skin samples from the ears and back were compared using a one-way analysis of variance (ANOVA) with a post hoc Bonferroni correction. A P value of <0.05 was considered statistically significant.