Background:
Onychomycosis and tinea pedis (athlete’s foot) are infections of the nails and skin caused by pathogenic fungi collectively known as dermatophytes. These infections are difficult to treat, and patients often relapse; it is thought that a patient’s footwear becomes infected with these fungal organisms and, thus, is an important reservoir for reinfection. Therefore, it is important to find an effective means for killing the dermatophytes that may have colonized the inner surface of the shoes of patients with superficial fungal infections. In this study, we developed an in vitro model for culturing dermatophytes in footwear and used this model to evaluate the effectiveness of a commercial ultraviolet shoe sanitizer in eradicating the fungal elements residing in shoes.
Methods:
Leather and athletic shoes (24 pairs) were inoculated with either Trichophyton rubrum or Trichophyton mentagrophytes (107 colony-forming units/mL) strains and were placed at 35°C for 4 to 5 days. Next, we compared the ability of swabbing versus scraping to collect microorganisms from infected shoes. Following the optimized method, shoes were infected and were irradiated with one to three cycles of radiation. The inner surfaces of the shoes were swabbed or scraped, and the specimens were cultured for dermatophyte colony-forming units.
Results:
Leather and canvas shoes were infected to the same extent. Moreover, scraping with a scalpel was overall more effective than was swabbing with a cotton-tipped applicator in recovering viable fungal elements. Irradiation of shoes with one, two, or three cycles resulted in reduction of fungal colonization to the same extent.
Conclusions:
The developed infected shoe model is useful for assessing the effectiveness of ultraviolet shoe sanitizers. Also, ultraviolet treatment of shoes with a commercial ultraviolet C sanitizing device was effective in reducing the fungal burden in shoes. These findings have implications regarding breaking foot infection cycles. (J Am Podiatr Med Assoc 102(4): 309–313, 2012)
The prevalence of superficial fungal infections, most often caused by dermatophytes belonging to one of three genera, Trichophyton, Microsporum, and Epidermophyton, has been estimated to be as high as 25% in the worldwide population.[1] Dermatophytes are so named because they can metabolize the keratin in skin, nails, and hair, and most infections involve onychomycosis (nail infection) and tinea pedis (athlete’s foot).[1] Tinea pedis is often found in persons with onychomycosis and may be a precursor to the development of nail infection.[2] Dermatophyte propagules are transmitted from person to person or by contact with contaminated surfaces, such as shower stalls and dressing rooms at communal gyms.[3,4]
Footwear has been shown to be an important reservoir for harboring organisms that cause infections.[5–7] The presence of fungi in the shoes has been postulated as a major reason for reinfection. Thus, eliminating the infecting fungal spores from the shoes will likely break the cycle of reinfection. For the treatment of footwear, Tanaka et al[8] found that rinsing athletic shoes with either boiling or cold water was effective in removing dermatophytes but that neither method was effective in boots. Other suggested methods to disinfect footwear of fungal elements have included treatment with chemical disinfectants, such as chlorine, phenol, sodium dodecyl sulfate, and quaternary ammonium salts.[9] These methods are not shoe friendly, and, clearly, a less toxic and less destructive method for disinfecting shoes is needed.
Commercial ultraviolet shoe sanitizers have been developed as a means to irradiate the inner surfaces of any type of shoe. One such device uses an ultraviolet C lamp with a peak frequency of 253.7 nm, which has been used as a germicide for decades because of its ability to destroy microorganisms.[10] Ultraviolet C light has been shown to reduce bacterial and fungal surface contamination, and it is an integral component of the biosafety cabinets used by commercial and research laboratories to sterilize work spaces heavily contaminated with microorganisms, including dermatophytes.[11,12] However, to demonstrate the efficacy of the ultraviolet shoe sanitizer device in reducing the number of organisms residing in the shoe, a model for infecting shoes with dermatophytes and recovering the fungal conidia in shoes needed to be optimized. A previous model for recovery of dermatophytes from shoes[13] using adhesive tape for spore collection had the disadvantage of providing qualitative rather than quantitative results. In this study, we developed a model for infecting shoes and recovering dermatophytes, and we used this model to determine the efficacy of ultraviolet C irradiation to decrease fungal load.
Development of the Infected Shoe Model
Materials
Isolates
One clinical strain each of Trichophyton rubrum and Trichophyton mentagrophytes, taken from the culture collection at the Center for Medical Mycology (Case Western Reserve University, Cleveland, Ohio), was used in this study. Isolates were removed from frozen stock (−80°C) and were subcultured to potato dextrose agar or cereal agar (T rubrum strain) and were incubated at 35°C until good conidiation was achieved.
Substrate
Newly purchased pairs each of leather and athletic shoes were used in this study.
Methods
Infection of Shoe Material
Dermatophyte strains were inoculated into leather and athletic shoes to assess their ability to grow on different substrate surfaces. Initially, inoculum was prepared by harvesting conidia to sterile saline and adjusting to a concentration of 103 colony-forming units (CFUs)/mL using a hemacytometer. Inoculum was then applied by sterile cotton-tipped applicator to the inner surfaces of one trial pair of athletic shoes and was allowed to dry.
Recovery of Dermatophyte Conidia From Infected Shoes
The insoles of the infected shoes were sampled by two different collection methods: scraping with a scalpel blade or swabbing with a sterile cotton-tipped applicator. Subsequently, the tops of the shoes were removed from the soles and were sampled in the same manner. Swabs and scalpel blades were used to inoculate the surface of potato dextrose agar (Difco Laboratories Inc, Detroit, Michigan) plates, which were then struck for isolation. After 4 to 5 days of incubation at 30°C, colonies were counted and CFUs were recorded for each culturing method. This method resulted in the recovery of few colonies by either swabbing or scraping with a scalpel, and, thus, the inoculum size was increased to 107 conidia/mL for the remainder of the experiments with canvas and leather shoes.
Figure 1.
Comparison of the ability of shoe material to support dermatophyte growth. Error bars represent SD. CFU indicates colony-forming unit.
Results
Test Material
Figure 1 compares the difference in fungal growth of untreated control shoes between canvas and leather substrates. For T mentagrophytes, the mean ± SD log CFUs for canvas and leather were 2.79 ± 0.42 and 3.04 ± 0.40, respectively. There was no significant difference between canvas and leather (P = .305). For T rubrum, the mean ± SD log CFUs for canvas and leather were 1.14 ± 1.06 and 1.85 ± 1.09, respectively. There was no significant difference between canvas and leather (P = .281).
Culturing Method
Table 1 and Figure 2 compare the difference in fungal growth between scraping the infected surfaces with a scalpel versus swabbing the infected surfaces with a cotton-tipped applicator. In the growth controls of T mentagrophytes, scraping the test material with a scalpel blade resulted in a total colony count nearly 2.5 times greater than that obtained by swabbing with a sterile cotton-tipped applicator. The mean ± SD log CFUs for scraping and swabbing were 3.06 ± 0.47 and 2.77 ± 0.33, respectively. There was no significant difference between the scraping and swabbing methods (P = .255).
For T rubrum growth controls, scraping the test material with a scalpel blade resulted in a total colony count nearly eight times greater than that obtained by swabbing with a sterile cotton-tipped applicator. The mean ± SD log CFUs for scraping and swabbing were 2.21 ± 0.60 and 0.78 ± 1.02, respectively. The scraping method yielded significantly greater CFUs than the swabbing method (P = .015).
The previous studies showed that the use of an inoculum containing 107 conidia/mL subsequently harvested by scraping with a scalpel blade were conditions for a successful model of dermatophyte infection of canvas and leather shoes.
Determination of the Irradiation Schedule
Once the parameters for shoe infection and recovery of fungal elements were determined, we proceeded to use the method to optimize the irradiation treatment schedule for infected shoes.
Methods
Two pairs each of leather and canvas shoes were infected with T mentagrophytes or T rubrum and were subsequently used to determine optimum irradiation schedules. One shoe of each pair was treated by exposure to the sanitizer (SteriShoe; Shoe Care Innovations Inc, Redwood City, California) for one, two, or three cycles of irradiation (a cycle is 45 min). The other shoe of each pair acted as a growth control and was not exposed to the ultraviolet C radiation. The device was placed firmly against the toe of the shoe and was locked into place. The shoe with the inserted device was then enclosed in a black cloth bag and left undisturbed for the entire irradiation cycle(s).
Results
Efficacy of Ultraviolet C Shoe Sanitizer
Figure 3 shows the fungal burden for treated and growth control shoes. After irradiation, the percentages of fungal reduction for shoes treated with one, two, and three cycles compared with the growth controls were 83.9%, 77.6%, and 85.4%, respectively, when testing T mentagrophytes. As expected, the growth controls showed the highest mean ± SD fungal burden at 2.91 ± 0.41. The mean (SD) log CFUs for one, two, and three cycles were 2.04 ± 0.46, 2.12 ± 0.14, and 2.44 ± 0.19, respectively. Treatment with one and two cycles significantly reduced the fungal burden compared with the growth control (P = .003 and .008, respectively).
When testing T rubrum, percentage efficacies for shoes treated with one, two, and three cycles compared with growth controls were 88.8%, 75.6%, and 68.3%, respectively. Growth controls showed the highest mean ± SD fungal burden at 1.49 ± 1.09. The mean ± SD log CFUs for one, two, and three cycles were 0.55 ± 0.64, 1.29 ± 1.23, and 0.70 ± 0.82, respectively. There was no significant difference in CFUs between treated and growth control shoes (P > .05) due to the standard deviation, but there was a mean T rubrum CFU reduction in the treated shoes of 76.28%.
Table 1.
Total CFUs Obtained by Two Sampling Methods in Growth Control and Treated Shoes
Figure 2.
Comparison of dermatophyte recovery methods from growth control shoes. Error bars represent SD. CFU indicates colony-forming unit.
Discussion
Ultraviolet C irradiation has been used for many years in various settings to disinfect environmental surfaces, among them being hospital isolation rooms and laboratory biosafety cabinets.[11,14] More recently, the use of this technology has been investigated for the reduction of bacterial biofilms on indwelling catheters[15] and even for the decontamination of carpet surfaces.[16] To our knowledge, this is the first published method for establishing a dermatophyte-infected shoe model to study the effectiveness of ultraviolet C irradiation.
First, we identified the necessary parameters for a successful shoe model. The data show that we could recover T mentagrophytes and T rubrum from leather and canvas inoculated substrates and that there was no significant difference in the colony counts retrieved from the two types of shoes. However, we had more success retrieving T mentagrophytes than T rubrum from shoes, as indicated by the higher colony counts yielded by T mentagrophytes–inoculated shoes. This finding may be attributable to the propensity toward greater conidia production in T mentagrophytes strains, although this is unlikely. More likely is a possible differential in the growth rate between the two species, as visible colonies of T mentagrophytes from clinical specimens tend to be produced more quickly on culture media than do those of T rubrum. Furthermore, we demonstrated that scraping the interior surface of the shoe with a scalpel blade was significantly more effective than was swabbing for the retrieval of dermatophyte conidia. Based on the previous findings, we conclude that the following parameters are necessary for successful shoe inoculation and retrieval of fungi from infected shoes: use of an inoculum containing 107 conidia/mL and harvesting by scraping with a scalpel blade.
Figure 3.
Comparison of the efficacy of one, two, and three cycles of irradiation. CFU indicates colony-forming unit.
Once the parameters for a successful infected shoe model were identified, we used this model to evaluate the commercial ultraviolet C shoe sanitizer device. These data demonstrate that one, two, and three treatment cycles with the shoe sanitizer reduced the dermatophyte fungal load in shoes. This reduction was significant by treatment with one and two cycles of irradiation. Treatment with two and three cycles did not result in significant additional reduction of fungal growth of either T mentagrophytes or T rubrum compared with treatment with one cycle. Thus, this lack of reduction in fungal growth with additional exposure time indicates that one cycle should be satisfactory.
Conclusions
This study evaluated several parameters for establishing a dermatophyte-infected shoe model intended to evaluate an ultraviolet C shoe sanitizer, including type of shoe material, inoculum size, and methods for dermatophyte recovery. Overall, this infected shoe model has been demonstrated to be a successful method by which the efficacy of ultraviolet C irradiation in reducing dermatophyte contamination of footwear can be determined. Furthermore, by using this model, the SteriShoe ultraviolet shoe sanitizer was shown to be effective in reducing the fungal burden in shoes. These findings have implications regarding the ability to address the footwear environment as a means of breaking the foot infection cycle.
Financial Disclosure
This study was funded by Shoe Care Innovations, Inc, manufacturer of SteriShoe Ultraviolet shoe sanitizer.
Conflict of Interest
Due to a conflict of interest, JAPMA editor, Warren Joseph, DPM, was not involved in the review or decision-making process of this paper. Dr. Ghannoum has no conflicts to report.
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