Enhancement of the antidermatophytic activity of silver nanoparticles by Q-switched Nd:YAG laser and monoclonal antibody conjugation.

The objective of this research was to investigate the effect of silver nanoparticles (AgNPs), free or conjugated with monoclonal antibody and mediated by Q-switched Nd:YAG laser on five dermatophytes. The laser was applied for 45 s at 532 nm and 0.8 J/cm2. The application of AgNPs combined with laser caused an increase in fungal susceptibility compared to application of AgNPs alone. The MIC50 and MIC100 recorded 3 and 9 µg/ml in the case of E. floccosum (the most susceptible species), 10 and 19 µg/ml for T. rubrum (the most tolerant species), respectively. A decrease in keratinase activity reaching 76.1, 67.1, and 62.4% was attained in the case of M. gypseum, T. rubrum, and T. mentagrophyte, respectively, on application of 10 µg/ml AgNPs combined with Nd:YAG laser. Under the same conditions of application, a steady increase in leaked materials coupled with reduction in ergosterol synthesis was reached. The structural alterations occurred to the fungus were more observed on the application of AgNPs in combination with laser where the conidia and hyphae lost their cellular integrity, become flaccid, permanently destructed, and completely killed. The monoclonal antibody conjugated AgNPs did not result in significant variation in in vitro experiments compared with that produced by nonconjugated nanoparticles. However, the conjugates achieved significantly more curing of M. canis-inoculated guinea pigs compared with nonconjugated nanoparticles.

A randomised comparative study of 1064 nm Neodymium-doped yttrium aluminium garnet (Nd:YAG) laser and topical antifungal treatment of onychomycosis.

Conventional treatments for onychomycosis include oral and topical antifungal agents. Although Nd:YAG laser has been reported to be effective, controlled studies combined or compared with conventional treatments are needed. To evaluate the efficacy of 1064 nm Nd:YAG laser for onychomycosis and compare outcomes with those of topical antifungal treatment and combination therapy. Patients were randomly divided into three groups: Laser (L); laser with topical antifungal therapy (L + T); and topical antifungal treatment (T). Laser treatment consisted of three or four sessions at 4 week intervals. Outcomes were assessed clinically and mycologically. In 217 nails of 56 patients, 76% in the L group and 71.8% in the L + T group experienced clinical responses, and 15.2% and 22.5%, respectively, were cured at 24 weeks. The clinical and cure rates of both groups were significantly greater than those of the T group. Six patients of the L group developed new infections, as did one patient in the L + T group and two patients in the T group. 1064 nm Nd:YAG laser shows clinically good responses in onychomycosis. Addition of topical antifungal agent did not appear to improve efficacy, but may have protected against new infection.

Inactivation of human pathogenic dermatophytes by non-thermal plasma.

Non-thermal plasma (NTP) was tested as an in vitro deactivation method on four human pathogenic dermatophytes belonging to all ecological groups including anthropophilic Trichophyton rubrum and Trichophyton interdigitale, zoophilic Arthroderma benhamiae, and geophilic Microsporum gypseum. The identification of all strains was confirmed by sequencing of ITS rDNA region (internal transcribed spacer region of ribosomal DNA). Dermatophyte spores were suspended in water or inoculated on agar plates and exposed to NTP generated by a positive or negative corona discharge, or cometary discharge. After 15 min of exposure to NTP a significant decrease in the number of surviving spores in water suspensions was observed in all species. Complete spore inactivation and thus decontamination was observed in anthropophilic species after 25 min of exposure. Similarly, a significant decrease in the number of surviving spores was observed after 10-15 min of exposure to NTP on the surface of agar plates with full inhibition after 25 min in all tested species except of M. gypseum. Although the sensitivity of dermatophytes to the action of NTP appears to be lower than that of bacteria and yeast, our results suggest that NTP has the potential to be used as an alternative treatment strategy for dermatophytosis and could be useful for surface decontamination in clinical practice.

The effect of microwave irradiation on the vitality of various dermatophytes.

Dermatomycosis is one of the most common dermatological infectious diseases. In recent years, the incidence of tinea pedum, a fungal infection of the feet, was increasing due to changing lifestyles. The risk of tinea pedum infections is associated with the use of sport shoes as well as contact with public sports facilities. Transmission of dermatomycosis occurs almost exclusively through indirect contacts, meaning that contagious material initially contaminates the patients' environment and subsequently facilitates the spread of infection to others. A suitable disinfection procedure for 'fungal reservoirs' is very important in order to reduce the risk of reinfection of tinea pedum. This study investigates the effect of microwave radiation on various dermatophytes- (Trichophyton rubrum, T. rubrum var. nigricans, T. interdigitale and Microsporum canis infected cork and polyethylene sponge shoe insoles. The contaminated insoles were irradiated with various intensities and durations of microwaves. In each case, 10 colonies on cork and polyethylene sponge insoles were irradiated with the same intensity and duration, and subsequently compared with those of corresponding non-irradiated control groups. Results of three independent experiments were statistically verified using Chi-squared test for significance. We found significant differences between the various dermatophytes on polyethylene sponge insoles and also partly on cork insoles for the same irradiation intensity and duration. We were also able to show that a complete growth inhibition of all four dermatophytes occurs on both types of insoles after a 30 s exposure at 560 W, including a maximum temperature of 60 °C.

Heat profiles of laser-irradiated nails.

Onychomycosis is a worldwide problem with no tendency for self-healing, and existing systemic treatments achieve disease-free nails in only 35 to 76% of cases. Recently, treatment of nail fungus with a near-infrared laser has been introduced. It is assumed that fungal eradication is mediated by local heat. To investigate if laser treatment has the potential to eradicate fungal hyphae and arthrospores, laser heat application and propagation needs to be studied in detail. This study aimed to measure nail temperatures using real-time videothermography during laser irradiation. Treatment was performed using 808- and 980-nm linear scanning diode lasers developed for hair removal, enabling contact-free homogeneous irradiation of a human nail plate in one pass. Average and peak temperatures increased pass by pass, while the laser beam moved along the nail plates. The achieved mean peak temperatures (808 nm: 74.1 to 112.4°C, 980 nm: 45.8 to 53.5°C), as well as the elevation of average temperatures (808 nm: 29.5 to 38.2°C, 980 nm: 27.1 to 32.6°C) were associated with pain that was equivalent to that of hair removal procedures and was not significantly different for various wavelengths. The linear scanning laser devices provide the benefits of contact-free homogeneous heating of the human nail while ensuring adequate temperature rises.

Inactivation of pathogenic dermatophytes by ultraviolet irradiation in swimming pool thermal water.

The effect of ultraviolet (UV) radiation from low-pressure mercury lamp against some pathogenic dermatophytes species such as Epidermophyton floccosum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton tonsurans and Trichophyton violaceum suspended in thermal water was evaluated in laboratory-scale condition at various times. The main results showed that within 120 s of exposure, all species of dermatophytes are completely inactivated, which was evidenced by the absence of fungal regrowth, while after 60 s only T. tonsurans was recovered, with a reduction of 3.28 log. Shorter exposure times were not enough to completely inactivate all dermatophytes species. The samples treated with UV radiation for 120 s did not give evidence of fungal regrowth indicating that this disinfectant action is persistent over time. In conclusion, UV radiation can be proposed to reduce the risk of infection by dermatophytes eventually present in swimming pools that use thermal water.

Susceptibilities of the dermatophytes Trichophyton mentagrophytes and T. rubrum microconidia to photodynamic antimicrobial chemotherapy with novel phenothiazinium photosensitizers and red light.

Photodynamic antimicrobial chemotherapy (PACT) is a promising alternative to conventional chemotherapy that can be used to treat localized mycosis. The development of PACT depends on identifying effective and selective PS for the different pathogenic species. The in vitro susceptibilities of Trichophyton mentagrophytes and Trichophyton rubrum microconidia to PACT with methylene blue (MB), toluidine blue O (TBO), new methylene blue N (NMBN), and the novel pentacyclic phenothiazinium photosensitizer S137 were investigated. The efficacy of each PS was determined based on its minimal inhibitory concentration (MIC). Additionally, we evaluated the effect of PACT with NMBN and S137 on the survival of the microconidia of both species. S137 showed the lowest MIC. MIC for S137 was 2.5 µM both for T. mentagrophytes and T. rubrum, when a light dose of 5 J cm(-2) was used. PACT with NMBN (10 µM and 20 J cm(-2)) resulted in a reduction of 4 logs in the survival of the T. rubrum and no survivor of T. mentagrophytes was observed. PACT with S137 at 1 µM and 20 J cm(-2) resulted in a reduction of approximately 3 logs in the survival of both species. When a S137 concentration of 10 µM was used, no survivor was observed for both species at all light doses (5, 10 and 20 J cm(-2)).

Ultraviolet C inactivation of dermatophytes: implications for treatment of onychomycosis.

BACKGROUND: Onychomycosis responds to systemic antifungals and sometimes to topical lacquers, but alternative treatments are desirable. Topical application of germicidal ultraviolet (UV) C radiation may be an acceptable and effective therapy for infected nails. OBJECTIVES: To test the ability of UVC to inactivate dermatophyte suspensions in vitro and to sterilize a novel ex vivo model of nail infection. METHODS: Trichophyton rubrum, T. mentagrophytes, Epidermophyton floccosum and Microsporum canis suspensions were irradiated with UVC (254 nm) at a radiant exposure of 120 mJ cm(-2) and surviving colony-forming units quantified. T. rubrum infecting porcine hoof slices and human toenail clippings was irradiated with UVC at radiant exposures of 36-864 J cm(-2). RESULTS: In vitro studies showed that 3-5 logs of cell inactivation in dermatophyte suspensions were produced with 120 mJ cm(-2) UVC irradiation. Depending on factors such as the thickness and infectious burden of the ex vivo cultures, the radiant exposure of UVC needed for complete sterilization was usually in the order of tens to hundreds of J cm(-2). Resistance of T. rubrum to UVC irradiation did not increase after five cycles of subtotal inactivation in vitro. CONCLUSIONS: UVC irradiation may be a less invasive treatment option for onychomycosis, when the appropriate consideration is given to safety.

The effects of laser irradiation on Trichophyton rubrum growth.

The effects of various laser wavelengths and fluences on the fungal isolate, Trichophyton rubrum, were examined in vitro. Standard-size isolates of T. rubrum were irradiated by using various laser systems. Colony areas were compared for growth inhibition on days 1, 3, and 6 after laser irradiation. Statistically significant growth inhibition of T. rubrum was detected in colonies treated with the 1,064-nm Q-switched Nd:YAG laser at 4 and 8 J/cm(2) and 532-nm Q-switched Nd:YAG laser at 8 J/cm(2). Q-switched Nd:YAG laser at 532- and 1,064-nm wavelengths produced significant inhibitory effect upon the fungal isolate T. rubrum in this in vitro study. However, more in vitro and in vivo studies are necessary to investigate if lasers would have a potential use in the treatment of fungal infections of skin and its adnexa.

Study of solar photosensitization processes on dermatophytic fungi.

The antifungal activity of solar simulator was evaluated in presence of haematoporphyrin derivative (HPD), methylene blue (MB) and toluidine blue O (TBO) as photosensitizers. Seven dermatophytes were used as test fungi. The solar simulator at fluence rate 400 W/m2 for 30 minutes induced marked inhibition for spore germination of the photosensitized fungi. The rate of inhibition varied according to the fungal species and concentration of the photosensitizer. There was an increase in percentage inhibition of spore germination as the concentration of HPD or MB increased. Complete inhibition for spore germination of Trichophyton. verrucosum, T. mentagrophytes, and Miccrosporum canis was induced when these species were pretreated with 10(-3) M of HPD or MB before irradiation. Epidermophyton floccosum, T. rubrum, M. gypseum and T. violaceum were less sensitive to irradiation when pretreated with HPD or MB. On contrary, the maximum reduction in percentage spore germination was induced at the lowest concentration (10(-7) M) of TBO. The tested dermatophytes were mostly capable of producing different enzymes (keratinase, phosphatases, amylase, lipase). The separate application of radiation or photosensitizer was ineffective or exerted slight inhibition on enzyme production. However, the activity of the enzymes was drastically inhibited when the fungi were irradiated after their treatment with photosensitizer. T. verrucosum and T. mentagrophytes were the most sensitive. In a trail to apply a control measure against dermatomycosis using solar simulator radiation, the results revealed that the radiation was successful in curing the MB-photosensitized guinea pigs, artificially infected with T. verrucosum, T. mentagrophytes or M. canis. The percentage of recovery reached 100% in some treatments.

Photodynamic inactivation of the dermatophyte Trichophyton rubrum.

Dermatophytes are fungi that can cause infections (known as tinea) of the skin, hair and nails because of their ability to use keratin. Superficial mycoses are probably the most prevalent of infectious diseases worldwide. One of the most distinct limitations of the current therapeutic options is the recurrence of the infection and duration of treatment. The present study shows that Trichophyton rubrum in suspension culture is susceptible to photodynamic treatment (PDT), a completely new application in this area. T. rubrum could be effectively killed with the use of the light-activated porphyrins deuteroporphyrin monomethylester (DP mme) and 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B). The photodynamic efficacy was compared with that of some other photosensitizers that are well known in the field of PDT: the porphyrins deuteroporphyrin and hematoporphyrin, the drug Photofrin and several phthalocyanines. It was demonstrated that with the use of broadband white light, the phthalocyanines and Photofrin displayed a fungistatic effect for about 1 week, whereas all the porphyrins caused photodynamic killing of the dermatophyte. Sylsens B was the most effective sensitizer and showed no dark toxicity; therefore, in an appropriate formulation, it could be a promising candidate for the treatment of various forms of tinea. For Sylsens B and DP mme, which displayed the best results, a concentration-dependent uptake by T. rubrum was established.

UV susceptibility and negative phototropism of dermatophytes.

High doses of ultraviolet radiation (UVR) have well-known inhibitory effects upon dermatophytes. In the present study, the effect of repetitive low doses of UVR on mycelial growth of dermatophytes was tested. Pellets of Trichophyton rubrum, T. mentagrophytes and Microsporum canis were placed between two thin layers of Sabouraud glucose agar. Obverse, reverse or both sides of these 'sandwich' agars were irradiated for 10 days twice daily with 0.13 or 0.17 J cm-2 UVB. To simulate microaerophilic conditions, one or both agar sides were covered by transparent airtight plastic lids. In addition, T. rubrum was also grown as usual on plates of Sabouraud glucose agar without any covering, and irradiated on its obverse side twice daily with UVA (13.5 J cm-2), UVB (0.17, 0.34 or 0.69 J cm-2) or infrared light, or once only with 3.8-15.1 J cm-2 UVB. As a result, thallus diameters of all strains were found to be reduced by repetitive UVB irradiation under both aerobic and microaerophilic growth conditions. T. rubrum was unaffected by infrared irradiation, responded with an increased pigmentation to UVA (13.5 J cm-2 twice daily) and was inhibited by a single dose of 15.1 J cm-2 UVB. Negative phototropism of dermatophytes is a new observation. It may be biologically relevant as a mechanism to evade harmful doses of UVR.

In vitro studies on the antifungal effect of PUVA.

The effect of PUVA therapy upon 12 dermatophyte strains, 3 of Candida albicans, and 4 of mould fungi was evaluated by the agar dilution method on Sabouraud agar. 8-MOP, 30 microgram/ml medium plus UVA, 4 Joule/cm2 proved to be fungicidal dose to all the strains studied, with the exception of three of the mould fungi. For dematophytes and Candida albicans the MIC was between 1 and 3 microgram, 8-MOP plus UVA, 4 J; and 3 microgram when the UVA dose was reduced to 1--2 J. For mould fungi it was higher. The MFC varied within the range 2 to 30 microgram, when the UVA dose was fixed at 4 J. It reached 16 J when the dose of 8-MOP was kept at 4 microgram. The wide range in the MFC of the dermatophytes could be due to resistant colonies. 8-MOP alone had a distinctly inhibitory effect upon dermatophytes, but not upon Candida albicans or mould fungi.