The Susceptibility of Dermatophytes to Photodynamic Treatment with Special Focus on Trichophyton rubrum

Authors


Corresponding author email: threes.smijs@ou.nl (Threes G. M. Smijs)

Abstract

Owing to the accessibility of skin to light, many applications of photodynamic treatment (PDT) have been developed within dermatology. The recent increase of dermatological antimicrobial PDT investigations is related to the growing problem of bacterial and fungal resistance to antibiotics. This review focuses on the susceptibility of dermatophytic fungi, in particular Trichophyton rubrum, to PDT and shows its potential usefulness in treatment of clinical dermatophytoses. There are no data indicating significant differences in PDT susceptibility between various dermatophytes and it is unlikely that treatment problems of especially T. rubrum with current antimycotics would occur in case of PDT. Red light 5-aminolevulinic acid-mediated PDT is after repeated sessions successful in in vivo treatment of onychomycosis (fungal nail infection) caused by various dermatophytes. Regarding skin dermatophytoses, UVA-1 PDT with cationic porphyrins appears to be safe and efficient. Most effective toward T. rubrum ex vivo is 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) when combined with UVA-1 radiation or red light; this creates the possibility of efficiently treating nail infections and remaining spores in hair follicles. If the promising in vitro and ex vivo results could be transferred to clinical practice, then PDT has a good prospect to become a worthy alternative to established antifungal drugs.

Introduction

The concept of photodynamic treatment (PDT) refers to a treatment that requires light-activated agents, named photosensitizers, in combination with light of a proper wavelength and, depending on the reaction type, the presence of molecular oxygen (1,2). Owing to the accessibility of skin by light, scientists have intensively investigated the use of PDT for skin cancer and also for other skin conditions. As a result, many oncologic and nononcologic PDT applications have been developed within the field of dermatology (3–6) as recently reviewed by Babilas et al. (7). The nonmalignant skin diseases that have been (experimentally) treated with PDT, include psoriasis, lichen ruben planus, lichen sclerosus et atrophicus, scleroderma, alopecia areata, human papillomavirus infections and Darier’s disease (8–12).

During the last decade, an increase can be seen in research and application opportunities of antimicrobial PDT. This development has become an important treatment option for fungal diseases and various fungal species have already shown to be susceptible to PDT (13,14). Within the dermatologic area, it concerns mainly PDT of bacterial skin infections and fungal skin diseases (skin mycoses) caused by dermatophytes or yeast (15–22). The importance of this particular development within PDT is underlined by the worldwide increasing problem of microbial resistance to commonly used antibiotics (23,24). Many human pathogens are already resistant to current antimicrobial drugs. Fifty percent of the gram-positive Staphylococcus aureus bacteria, frequently causing bacterial skin infections, have become resistant to penicillin since the late 1950s (25). Moreover, the methicillin-resistant S. aureus is a difficult problem since 1980 (26). Fungal resistance to currently used antifungal drugs has also been described (27–29) particularly in case of the dermatophyte Trichophyton rubrum (30,31). This therapeutic failure can increase the prevalence of this disease and contributes to an ever negative image associated with fungal infections in humans. Moreover, the incidence of dermatophytic infections has increased because of a growth of the number of patients with a compromised immune system (32) or the patients suffering from diabetes mellitus (33,34). In particular, the fungal nail infection, onychomycosis, mainly caused by dermatophytes is approximately three times more prevalent among people with diabetes mellitus than in those without this disease.

The problem of microbial drug resistance, in general, has been recognized as a threat to public health. At the same time, it is a challenge for the development of new therapies like those based on PDT principles (35–37). These studies, however, mainly target bacteria. In this review, we focus on superficial skin mycoses caused by dermatophytes and the susceptibility of these microorganisms to PDT. Special attention is paid to the frequently occurring dermatophyte T. rubrum. This dermatophyte is also most frequently isolated from onychomycosis (38).

To address the contents of this review properly, it is important to understand subjects like dermatophytes, antifungal drugs and antimicrobial PDT. The reviewed articles comprise PDT applications to treat dermatophytes under in vitro, ex vivo and in vivo conditions. The in vivo studies regard both animal and case reports. We have summarized important conclusions and recommendations that are based on the reviewed studies.

Dermatophytes

General features

Dermatophytes are traditionally divided in three anamorphic genera, Trichophyton, Microsporum and Epidermophyton. The term dermatophyte is restricted to organisms that can cause an infection. Therefore, in medical mycology, the three genera are classified as the anamorphic class Hyphomycetes of the Deuteromycota (Fungi imperfecti) (39,40). Depending on their environment, dermatophytes produce different types of conidia (40). Epidermophyton spp. produce abundant macroconidia (cigar shaped conidia) of 20–60 μm in length and no microconidia (conida resembling in shape a liquid droplet). Only the type specie, Epidermophyton fluccossum, is pathogenic. Microsporum spp. produce both macroconidia of 6–160 μm in length and the smaller microconidia. Trichophyton spp. produce macroconidia of 8–80 μm and microconidia of approximately 3–5 μm in length. The latter are more abundant. In less optimal environmental conditions, chlamydospores are produced, whereas the cylindrical shaped, thick-walled, arthroconidia (6–12 × 3–6 um) are produced in infected skin (41).

Dermatophytosis

The term dermatophytosis refers to an infection by dermatophytic fungi of keratinized tissues, such as the cutaneous stratum corneum (SC), hair and nails. A persisting dermatophytosis is mostly caused by T. rubrum. However, other dermatophytes also cause chronic infections in persons with a compromised immune system (42). Local warm and moist conditions favor the development of dermatophytosis. To differentiate the various localizations of dermatophytosis, it is common to use the word tinea followed by the term of body site that is infected (43). Dermatophytes seldom penetrate the skin into the deeper epidermal or dermal parts of the skin and the infections are therefore mainly superficially localized.

The distribution of dermatophytes isolated from skin infections varies around the world and depends as described by Svejgaard (44) on three factors: (a) a poor living standard, (b) densely populated urban areas and (c) the increasing migration of people from infected areas which leads to reintroduction of the infection.

Trichophyton rubrum was first isolated from humans in 1910 by Bang (45–47) and since the middle of the last century is listed as the most common dermatophyte. It is followed by T. mentagrophytes—typically in central and north Europe associated with the incidence of tinea pedis and corporis (48) and tinea unquium (onychomycosis) (49). During the last century, a gradual replacement of other dermatophytes by T. rubrum has also been observed (48). In children, especially in Mediterranean countries, tinea capitis caused by Microsporum canis is most prevalent (50).

Infection, survival in human skin and host defences

Appearance of a dermatophytic infection is closely related to capability of the dermatophyte to overcome the host’s defence mechanisms. In the infection process, the fungal wall plays an essential role that probably correlates with virulence and is also the frequent target of many antifungal drugs (28,51,52). The outermost layer of the wall constitutes of β-glucan, composed of α-glucopyranose units with predominantly β-1,3 and β-1,6 linkages. The second layer contains galactomannans, complex glycoproteins consisting of α-mannopyranose, mannofuranose, galactofuranose attached to a peptide backbone. In T. rubrum galactomannan I, the galactofuranose units are missing and galactomannan II contains α-1,2 en α-1,6-linked mannopyranose en mannofuranose units (see Fig. 1). The third layer is known as chitin, a β-1,4-linked polymer of N-acetylglucosamine, giving the fungal wall its rigidity. The innermost layer of the wall is the cell membrane containing lipids, proteins and little carbohydrates. It resembles the cell membrane in eukaryotic cells, however, cholesterol is in the cell membrane of fungi replaced by ergosterol (53–55). As a result of the layered structure, the total wall thickness is approximately 100–300 nm, but it is thinner at the growing hyphal tips (56). In addition, the dermatophyte hyphal walls contain relatively high concentrations of (negatively charged) phosphoproteins (51).

Figure 1.

 Diagrammatic representation of the wall structure of Trichophyton rubrum, including the chemical structure of the main units present within the layers that are characteristic for the fungal wall. (A) Outermost layer of β-1,3-glucans and β-1,6-glucans. (B) Galactomannan, showing α-1,2 en α-1,6-linked mannopyranose en mannofuranose units. (C) Chitin microfibers, embedded within protein. (D) The cell membrane, containing ergosterol.

Different stages in epidermal invasion have been excellently described by Hay (57). Briefly, the pathogenetic process starts with adherence of conidia, mostly the arthroconidia, to the skin surface. This usually takes approximately 2 h (58). Following the attachment to keratinized structures, the germination is taking place and subsequently hyphae proliferation and spreading of the mycelium into and through holes of the epidermal surface. Dermatophytes can use various proteins as foodstuff but as soon as keratin is offered as sole Carbon-source enzyme extrusions change from general proteinases, capable of hydrolyzing various proteins, to keratinases specialized in hydrolyzing keratin only (59). The pH optimum of proteinases and of some keratinases produced during initial stage is at acidic pH (60) corresponding to the pH of human skin surface which is approximately 5.5. In vitro studies with T. rubrum have, however, shown that the pH of the cultivation medium changes as a function of nutrients used to reach values of pH 8–9 where most of the keratinases have their optimal activity (61). Both proteolytic and keratinolytic activity appear to be important virulent factors for dermatophytes. However, for manifestation of an infection, various host defence strategies have to be defeated. As described by Wagner and Sohnle (43), the physicochemical skin characteristics provide a number of nonimmunological coetaneous defences. Skin surface exposures to UV radiation as well as low moist conditions contribute to this. Furthermore, the SC is continually renewed thereby removing infecting fungal organisms. In inflammatory host responses, chemotactic activity of neutrophils and macrophages are key factors. Present humoral immune responses to dermatophytosis are complex and fall beyond the scope of this review but have been superbly summarized by both Grappel et al. (53) and Wagner and Sohnle (43). It is, however, interesting to mention why especially T. rubrum can cause a chronic infection. The most important reason regards the differences (compared with other Trichophyton species) in cell-wall components, such as galactomannans. Trichophyton rubrum galactomannans may have immunosuppressive effects, inhibiting normal immune reactions (like lymphoproliferation). It was also demonstrated in vitro that T. rubrum arthroconidia were more resistant to antifungal drugs than the hyphae (see also next).

Antifungal therapy

Treatment of dermatophytoses involves the use of an antifungal drug in either a topical or oral application form or a combination of both (28,62–65). The latter is often applied in case of persistent onychomycosis (66,67). Oral antifungal drugs may give rise to side effects, such as hepatotoxicity and interaction with other drugs. The antimycotics can be divided into three chemical classes, the polyenes (amphotericin B and nystatin), the azoles and imidazoles (with main representatives itraconazole and miconazole) and the allylamines and thiocarbamates (such as terbinafine and tolnaftate) (68,69). In addition, griseofulvin (dimethoxycoumaran derivative) and cyclopiroxolamine (oxaboroles/hydroxamic acid) are occasionally used (69,70). Many of these chemicals inhibit the synthesis of the cell membrane component—ergosterol (allylamines, thiocarbamates and azoles) or they bind and disrupt the cell membranes (polyenes), interfere with the microtubule organization during mitosis (dimethoxycoumaran derivative) or act as iron chelator (oxaboroles/hydroxamic acid) (28). These kinds of inhibitions are mainly effective in growing, metabolic active microorganisms. That is why the effect of many current antimycotics on conidia is insufficient, which leads to relatively frequent recurrences and the necessity of lengthy treatment (71). The need of long-lasting treatment may become the cause of patient’s incompliance.

A new treatment strategy with a mechanism that includes fungal conidia kill is therefore desired. PDT can certainly be a good candidate since, as mentioned before, the dermatophytoses are infections of the superficial skin.

Photodynamic processes in antimicrobial PDT

The term “photodynamic” refers to photosensitization reactions that require molecular oxygen and occur in different biological systems (see Fig. 2) (72,73). A type I reaction results in the production of photosensitizer (S˙ and S+˙) and substrate (A˙ and A+˙) radical ions. Depending on the environment and molecular structure, the produced radicals may, in turn, interact with other neighboring molecules in redox reactions leading to cytotoxic oxidation products including superoxide (O2˙) and consequently hydroxyl (OH˙) radicals that react with various types of biomolecules (74–76). A type II reaction involves an energy transfer between 3S* and ground-state oxygen (3O2) producing a singlet (excited) state of oxygen (1O2) (1). Singlet oxygen is a powerful, short-lived (ca 100–250 ns in in vivo cell systems), electrophilic particle that reacts rapidly with electron-rich molecules present in a variety of biological molecules and cell structures (5,77).

Figure 2.

 Photosensitization processes that may occur upon light absorption by a photosensitizer molecule (S). Following the absorption of light, the photosensitizer is electronically promoted from its ground state (S) to an excited singlet state (1S*). By intersystem crossing, this short-lived 1S* state is transformed to the triplet excited state (3S*) which can participate in a type I (with substrate molecule A) or a type II reaction (with the ground state of molecular oxygen).

In microbial systems, in general, both type I and II pathways can lead to cell death as a result of oxidative damage caused by generated reactive oxygen species (ROS). The pathway that will dominate is determined by general circumstances, such as sensitizer concentration and cellular environmental conditions (low oxygen concentrations favor the type I reaction) and in particular, by the physicochemical characteristics of the photosensitizer and the chemical properties and morphology of the microbial target structures (36,78–81). The physiochemical properties of the photosensitizer determine its binding affinity to the cell wall of microorganisms. This outer surface is, in most cases, negatively charged, and that is why the positively charged photosensitizers are commonly more effective than those having a negative or no charge (36,82,83). Following a binding of the photosensitizer to the microbial wall, it may either be translocated to the inner cell membrane or remain outside to induce dark- and/or light-stimulated wall permeability alterations (81). Especially, porphyrin photosensitizers have too large molecules to cross the microbial wall of most micro-organisms. The cationic porphyrins that have been extensively studied as photosensitizer in antimicrobial PDT initiate their photodynamic action on micro-organisms mainly from outside. These kinds of sensitizers are furthermore easily quenched by oxygen and favor thus a type II photodynamic action.

Apart from exogenously applied photosensitizers, the endogenous photosensitizer, protoporphyrin IX, produced from its precursor 5-aminolevulinic acid (ALA) in the heme biosynthesis pathway is also important in the case of antimicrobial PDT application (84–87).

PDT of dermatophytes

Despite the theoretical advantages that PDT can offer as treatment strategy for superficial skin infections, there has been only a limited number of studies dealing with the PDT of dermatophytoses. Most of them consist of in vitro and in vivo experiments. Trichophyton rubrum and Trichophyton mentagrophytus have been the most frequent targets. As the most important causative agent of onychomycosis, T. rubrum is a grateful and inspiring subject for this kind of research.

First, we will discuss the in vitro PDT studies and then we will deal with several ex vivo studies. Finally, we will pay attention to reports on various forms of clinical dermatophytoses treated with PDT. In general, we have aimed at the mechanisms that can lead to a fungicidal effect (a complete fungal kill) after one treatment.

In vitro studies

The in vitro studies on PDT of dermatophytes are summarized in Table 1 (15,20,88–93). This table shows that such studies have been reported since the end of 20th century. A striking difference between older and more recent studies is the much higher light dose (approximately 10-fold) used in the last 10 years. Before that time, however, UVA and once a blue light source were used. Apparently, various dermatophyte strains are more susceptible to UVA PDT compared with visible light when cultivated in vitro. For instance, you can compare the study of Propst and Lubin (15) who used the wavelength of 445 nm which is just outside the UVA range with the studies of Horio (90) and Romagnoli et al. (88) where UV radiation from, respectively, 300 to 420 and 320 to 400 nm was utilized. Although Propst and Lubin applied a comparable light dose, for a fungicidal effect, they had to use a higher photosensitizer concentration of 3 mm, nevertheless, the fungicidal effect was obtained only in case of proflavine (see Table 1). Except for methylene blue, all wavelengths used in this study matched the photosensitizer’s absorption properties. Interesting is the reduced fungal growth caused by a 300–420 nm UVA radiation source as reported by Horio (90).

Table 1. In vitro studies on PDT of dermatophytes, emphasizing a fungicidal result.
DermatophytePhotosensitizerLightPDT effectReferences
λ (nm)Dose (J cm−2)Fluency rate (mW cm−2)Source
  1. *5-Aminolevulinic acid; †not reported; ‡deuteroporphyrin monomethyl ester (DP mme), 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B), deuteroporphyin (DP), hematoporphyrin (HP), Photofrin, zinc phthalocyanine (ZnPc), phthalocyanine tetrasulfonate (PcS4), aluminium (III) phthalocyanine chloride tetrasulfonate (AlPcS4); §quinolino-[4,5,6,7-efg]-7-demethyl-8-deethylmesoporphyrin dimethylester; ¶demethylchlortetracycline; **tetrachlorosalicylanilide; ††tribromosalicylanilide.

Trichophyton interdigitaleALA* (0–100 mm)635100100Paterson Lamp (Photohterapeutics Ltd.)Fungistatic, 79% kill at mostDonnely et al. (89)
Trichophyton rubrum (liquid cultured)ALA (0–100 mm)NR† (broad band white)10–12836.8Quartz-halogen Zeiss KL 2500 LCDFungistatic, reduced number of colonies or reduced fungal colony diameterKamp et al. (20)
Trichophyton rubrum (liquid cultured)Various porphyrins‡ (0–90 μg mL−1)NR (broad band white)10830“MASSIVE” (no. 74900/21), 13 max.500W-230 V-R7s, IP 44Fungicidal (20 μg mL−1) with Sylsens B, DP, DP mme and HPSmijs et al. (92)
Trichophyton rubrum (microconidia and hyphae suspensions)Sylsens B‡, DP mme‡, QDD§ (0–50 μm)580–87010830“MASSIVE” (no. 74900/21), 13 max.500W-230 V-R7s, IP 44 with cutt-off filter at 600 nmFungicidal for microconidia: Sylsens B at 1 μm, DP mme at a concentration >5 μm Fungicidal for hyphae: Sylsens B at 10 μm, DP mme at 40 μmSmijs et al. (93)
Trichophyton rubrum
Trichophyton verrucosum
Trichophyton mentagrophytes
Trichophyton violaceum
Microsporum canis
Microsporum gypseum
Epidermophyton floccosum
(spore solutions)
Hematoporphyrin derivative, Methylene blue, Toluidine blue (10−3 to 10−7m)NR (visible light)72–14440Oriel solar simulatorFungicidal (10−3 m) with hematoporphyrin and methylene blue to M. canis, T. mentagrophytes and T.verrucosumOuf et al. (91)
Epidermophyton floccosum Trichophyton mentagrophytes
Microsporum cookie
Trichophyton tonsurans
Microsporum gypseum
MIcrosporum canis
5-(4-hydroxy-1-butinyl)-2,2′-bithienyl (0–50 μg mL−1)320–4002.70.5UVA black light, Sylvania F20, T 12-BLB (peak at 350 nm)Fungicidal to E. floccosum (50 μg mL−1)Romagnoli et al. (88)
Trichophyton mentagrophytesChlorpromazine, perphenazine, Promethazine, anthracene
DMCT¶, sulphaniamide, trimethylpsoralen, Tolbutamide, TCSA**, hydrochlorothaiazide, chlorothiazide, trichlomethiazide, methyclothiazide, TBS†† (0.1–1% solutions)
300–42010.80.45, 3UVA black light, Toshiba FL 20S, BLBFungicidal with 0.002% chlorpromazine, perphenazine, TBS, with 0.0002% TCSA, and with 0.02% prometazine, anthracene, trimethylpsoralen and sulfanilamide solution
UVA alone reduced fungal growth
Horio (90)
Microsporum gypseum
Trichophyton mentagrophytes
Methylene blue, neutral red, proflavine hemisulfate (3 mm)455∼1.1∼1.8NRFungicidal for proflavine (3 mm)Propst and Lubin (15)

Despite the promising results obtained with long-wavelength UV radiation, these kinds of studies were not continued probably because of the fear of possible carcinogenic effects of this radiation. However, the risk of skin cancerogenicity can be minimized by exclusion of UVB (290–320 nm) and UVA-2 (320–340 nm) radiations (see also paragraph 5) (94). In particular, the fungicidal results obtained by Horio might be partly due to a UVB-effect. However, in one of our ex vivo studies, we also observed a fungicidal effect of UVA-1 (340–400 nm) radiation toward T. rubrum (95).

Most recently, our own research (93) showed interesting differences in PDT susceptibility for different T. rubrum growth phases. The fungal spores in suspension appeared to be far more susceptible to PDT than mature fungal colonies in a liquid culture. They could easily be killed by photosensitizers which bind to the outer wall of fungal elements. Of note is also the study of Ouf et al. (91) in which T. rubrum could not be completely killed by visible light PDT when methylene blue, hematoporphyrin derivative or toluidine blue was used as photosensitizer. This study, however, reveals some more interesting information. It is the only study that we are aware of that focused on seven different dermatophyte strains and included the determination of the production of metabolic enzymes (keratinase, phosphatases, amylase and lipase) before and after PDTs. Although the highest (fungicidal) PDT efficacy of all three photosensitizers (3 mm) was found when M. canis, T. mentagrophytes and T. verrucossum were treated, the corresponding data were not supported by statistical analysis. Statistically grounded differences between the data given for the natural enzyme keratinase production in the different strains and a correlation of these data to differences in PDT effectiveness are, unfortunately, both missing. Keratinase enzyme production decreased as a result of all PDT combinations in all strains. The highest reduction occurred in the strains with the highest susceptibility to PDT. There are no in vitro studies that report a fungicidal effect of the endogenous protoporphyrin IX production resulting from ALA pretreatment. Few investigations have reported on a photosenitizer’s dark toxicity potential. In one of our papers, we mentioned that next to photodynamic action, the PDT results could in several cases at least partly be ascribed to an effect of the photosensitizer itself in the absence of light. The fungicidal 10−3 m hematoporphyrin derivative concentration that was used in the study of Ouf et al. was, unfortunately, not supported by data that could exclude such a dark effect.

Ex vivo studies

Table 2 lists the ex vivo studies conducted by our group (95–98). To resemble the clinical situation we utilized in these experiments, T. rubrum grown on the SC of isolated human skin. In this ex vivo model, we were able to investigate the PDT susceptibility of various fungal growth stages to (red) light PDT. These studies showed the importance of fungal adherence to a keratinized structure, like the SC, in fungal virulence. When compared with in vitro, we noticed that the susceptibility of mature mycelium decreased while conidia displayed an equally high susceptibility to PDT. Adherence of the dermatophyte to a keratinized structure is an important stage in the pathogenesis of dermatophytosis whereby proteolytic enzyme activity is changed to a specific keratolytic activity enabling the dermatophyte to use keratin as sole carbon source (99).

Table 2. Ex vivo studies on PDT of dermatophytes, emphasizing a fungicidal result.
DermatophytePhotosensitizerLightPDT effectReferences
λ (nm)Dose J cm−2Fluency rate (mW cm−2)Source
  1. Presented studies have all used an ex vivo skin model with isolated human stratum corneum (SC) for dermatophytic growth. The model offers the possibility to apply photodynamic treatment (PDT) at different time points after spore inoculation and to investigate the susceptibility of various dermatophytic growth stages to PDT.

Trichophyton rubrum (on isolated human SC at 8 and 72 h after spore inoculation)Sylsens B (0–15 μm)350–4001846BioSun Med UVA-1 cold light unit (BioSun Silt- service)Fungicidal to the fungal conidia at the 8 h conidial growth stage (5 μm) and to the 72 h full mycelium growth stage (10 μm)Smijs et al. (95)
Trichophyton rubrum (on isolated human SC at 8, 17, 24, 48 and 72 h after spore inoculation)Sylsens B, DP mme580–87010830“MASSIVE” (no. 74900/21), 13 max.500W-230 V-R7s, IP 44 with cutt-off filter at 600 nmFungicidal with Sylsens B in all experiments with:
– 1 μm for the 8 h conidial growth stage
– 5 μm for the 17 h and 24 conidial growth stage
– 120 μm for the 48 h mycelium growth stage provided incubation is at pH 5.2
Fungicidal with DP mme in all experiments:
– 80 μm for the 8 h conidial growth stage
– 160 μm for the 17 h conidial growth stage provided incubation is at pH 7.4
Smijs et al. (96)
Smijs et al. (97)
Smijs et al. (98)

In the PDT results in Table 2 are the fungicidal effects obtained for the full mycelium growth stage (represented in the model by 72 h after spore inoculation) left out. Although a fungicidal effect could be obtained with 160 μm Sylsens B (and a 108 J cm−2 red light dose), it occurred only in 65% of the treatments. This percentage could be increased significantly to approximately 90% when a keratinase enzyme inhibitor was added to the incubation mixture prior to light application.

When we refer to the lower efficacy of current antimycotics toward fungal (arthroconidia) spores, the found high susceptibility of especially the T. rubrum (microconidia) spores toward PDT may be of importance.

In vivo studies

Investigations that have focused on PDT of dermatophytoses in either humans or in animal studies are given in Table 3 (15,18,91,100,100–104). Not surprisingly, many of them focused on the treatment of onychomycosis caused by T. rubrum. Several researches have used ALA PDT combined with a red light source and the results have been quite promising. Many cases described a 100% mycological and clinical absence of the signs of dermatophytosis after the treatment. In case of onychomycosis, ALA PDT effectiveness seems to depend on the presence of an urea nail pretreatment to increase the nail’s permeability. Despite the observed onychomycosis treatment success, it should be mentioned that the treatment still requires a long time because of the necessity of repeated PDT sessions. Even then, a 100% cure rate after follow up in all cases has been reported so far only in the study by Piraccini et al. (100) and Watanabe et al. (101). Watanabe et al. used the ALA-methyl ester but only two (onychomycosis) patients were included and the causative agent was not mentioned. The paper by Piraccini et al. (100) described only one patient with a nail infection caused by T. rubrum.

Table 3.   Summary of various forms of dermatophytoses treated with PDT. Both case and animal studies are included.
DermatophyteDisease (number of cases)PhotosensitizerLightCure rateCure rate after follow upReferences
λ (nm)Dose (J cm−2)Fluency rate (mW cm−2)Source
Trichophyton rubrumOnychomycosis (30)ALA (20%, MEDAC GmBh)
3 PDT sessions at a 14 days interval
570–6704040Waldmann PDT 1200100% absence of clinical signs after 12 months: (5/30)100% absence of clinical and mycological signs after 18 months: 11/30Sotiriou et al. (103)
Trichophyton rubrumTinea cruris (10)ALA (20%, MEDAC GmBh)
2 PDT sessions at a 14 days interval
570–6705050Waldmann PDT 1200100% (6/10)100% (4/10) after 8 weeksSotiriou et al. (104)
Trichophyton mentagrophytes var.interdigitales (6×)
Trichophyton rubrum (4×)
Tinea pedis (10)ALA cream
3 PDT sessions at a 14 days interval
570–6705050Waldmann PDT 1200100% absence mycological signs after 3 treatments: (6/10, 3×T. rubrum)3/10 after 8 weeks (2×T. rubrum)Sotiriou et al. (102)
Trichophyton rubrumOnychomycosis (1)ALA cream (160 mg g−1, PhotoCure)
3 PDT sessions at a 15 days interval
Nail pretreatment with 40% urea
63037NRAklilite CL 128, PhotoCureClinical and mycological cured after 12 monthsClinical and mycological cured after 24 monthsPiraccini et al. (100)
NROnychomycosis (2)ALA-methyl ester (20% solution in aqueous cream)
Nail pretreatment with 20% urea
630100 (6, 7 times)NRExcimer- dye laser, Hamamatsu Photonics KKNo dermatophyte detection (KOH), 2/2No clinical signs after 6 months: 1/2
No clinical signs after 3 months: 1/2
Watanabe et al. (101)
Trichophyton mentagrophytes (4×)
Trichophyton rubrum (2×)
Interdigital mycosis (6)ALA (20% solution in Eucerin cream)
1–4 treatments
570–70075100Osram 250 W and cut-off filter at 600 nmNo clinical signs after 1 treatment: 3/6, all T. metagrophytes
No clinical signs after 4 treatments: 1/6 (T. rubrum)
No clinical signs after 4 weeks: 1/6Calzavara-Pinton et al. (18)
Trichophyton verrucosum
Trichophyton mentagrophytes
Microsporum canis
Artificially infected guinea pigs (NR) and treated 3 weeks after inoculation for 3 days in successionMethylene blue (10−5 m)NR
visible light
36,72 or 144 daily40Oriel solar simulatorNo complete cure after 30, 45 days (all strains)Complete cure for only
T. verrucosum after 60 days
Ouf et al. (91)
Trichophyton mentagrophytesArtificially infected albino guinea pigs (54 infected sites) and treated 1 hour and 3 days after infectionProflavine hemisulfate (3 mm)455∼5∼1.3NRNo complete cureNo complete cure at 11 days after inoculationPropst and Lubin (15)

The animal studies performed by Ouf et al. (91) and Propst and Lubin (15) supported their in vitro findings (see Table 1) that were carried out parallel to these studies. Both research groups used guinea pigs artificially infected with various dermatophyte strains. Propst and Lubin, however, observed only slightly faster healing of the PDT treated lesions when compared with the untreated sites. In the study of Ouf et al. where methylene blue was used, only one of the tested dermatophytes, T. verrucosum appeared to be completely cured after 60 days of intense treatment. In both studies, however, most of the promising in vitro results could not be achieved in an in vivo situation.

General discussion and conclusion

In this review, we have concentrated on the susceptibility of various dermatophyte strains to PDT and the potential usefulness of such treatments as an alternative for the use of current antimycotics. We hold the opinion that the persistent character of the dermatophytosis with T. rubrum could be overcome when optimized PDT could be used. The papers we are referring to have not provided clear evidence for differences in susceptibility of various dermatophytes toward PDT. A lower susceptibility may be expected, for instance, for T. metagrophytes where the curly hyphae structures may lead to an incomplete photosensitizer binding to the targeted fungal wall. In addition, metabolic differences and variations in the keratinase enzyme production between various strains may contribute to these differences too. It is therefore unlikely that treatment problems of especially T. rubrum to current antimycotics will occur in the case of PDT.

When evaluating the affectivity of PDT on dermatophytes and the treatment efficacy of clinical dermatophytosis, we deal separately with those obtained for the nail and the skin infections. In vivo human studies focussing on skin infections with the use of an exogenously applied photosensitizer have not been reported so far, but the results carried out for ALA studies could be promising. The same successful ALA-mediated PDT results have been obtained for the treatment of onychomycosis whereby also the difficult T. rubrum infections can be effectively cured. The problem is, however, that several treatments are still required and the total cure in almost all reports is lacking. In the case of onychomycosis, low nail plate permeability of ALA may be the cause of insufficient therapeutic effects. Since the physicochemical skin and nail barrier characteristics differ widely, it is desired to address this subject in subsequent (ALA) onychomycosis PDT studies.

With regard to cutaneous applications, it is necessary to transfer the promising results with exogenously applied porphyrins to clinical studies. Successful candidates are relatively new compounds and the lack of preclinical toxicity studies may temporarily hinder this effort.

For the treatment of superficial skin infections, UVA light sources have been proven excellent source candidates. Particularly, when low doses of UVA-1 are used like in our recent ex vivo studies (95), the treatment can be considered safe. The light unit (340–550 nm) used in these studies has been routinely used for phototherapy of several skin diseases (105–107). The ability of UVA-1 radiation (340–400 nm) to cause skin erythema is approximately 103 to 104 lower than that of UVB (290–320 nm). The absorption by the skin of UVB leads to an inflammatory skin reaction. Whereas 6–12 J cm−2 can cause serious superficial skin burns, the same dose of UVA-1 does not generate any macroscopic or microscopic changes in the skin epidermis or underlying dermis (94). Furthermore, only an occasional sunburn cell was observed in human epidermis after repeated exposure to 35 J cm−2 (108). Furthermore, UVB mainly acts through direct absorption causing damage to DNA bases, while in contrast UVA-1 irradiation is not absorbed by DNA. The carcinogenic risk of UVA-1, mediated by the production of ROS, is minimized when low UVA-1 doses are applied for short-lasting treatments. This is exactly what should be aspired—a treatment strategy for dermatophytosis involving a single PDT. Regarding the effect of light only, as also observed in one of our own ex vivo studies, the research of Landsman et al. (109) and Dai et al. (110) are worth mentioning. Dai et al. reported the successful use of UVC radiation for onychomycosis treatment, while Landsman et al. mentioned the unique photolethal effects of 870–930 nm in the treatment of onychomycosis. The meso-substituted cationic porphyrin photosensitizer mainly used in our research possesses more interesting features when it comes to both skin and nail PDT, namely: (a) the ability of this compound to bind selectively to fungal elements including conida at physiologic pH of the skin, (b) the hydrophilic character preventing complete skin penetration, (c) the effectiveness also toward dermatophytes other than T. rubrum, such as Trichophyton mentagrophytes, M. canis and Trichophyton tonsurans (Smijs, unpubl. results) and (d) the ability to develop an effective treatment also with red light and thus creating a possibility to kill remaining spores in hair follicles and affect nail infections. Moreover, the use of the exogenous photosensitizer for skin infections instead of the endogeneous produced protoporphyrin IX minimizes the risk on phototoxicity to keratinocytes. The latter effect has been investigated in vitro by Zeina et al. (111). Exogenously applied hydrophilic photosensitizers that bind effectively to the target fungal elements will not reach the living part of the epidermis. One restriction regarding our ex vivo model is worth mentioning. A model is of course a simplification of reality to be used with a particular purpose. Shortage of this specific model concerns the inability to mimic a situation when large skin areas are affected by a dermatophytic infection and when all fungal walls should be foreseen by a high concentration of photosensitizer molecules. The minimal required number of the exogenously applied photosensitizer molecules per unit microbial surface area should be established. Furthermore, PDT studies should focus specifically on arthroconidia that are abundant in clinical situations but difficult to produce under in vitro conditions (112).

When taking this and other abovementioned recommendations into consideration, PDT can certainly offer a good prospect as alternative antifungal therapy thereby helping to solve the growing problem of antibiotic resistance.

Authors’ Biographies

Threes Smijs originally studied chemistry and environmental science. Her early research (Leiden University Medical Centre, LUMC, and Leiden University) focused on the metabolism of xenobiotics. She started her research on photodynamic treatment (PDT) in 2000 at the LUMC, primarily focusing on cancer and various synthetic porphyrin photosensitizers. She became fascinated by the specific growth of dermatophytic fungi on human skin and in 2003, started her own research group investigating the susceptibility of the dermatophyte Trichophyton rubrum to PDT. She joined the LUMC Department of Dermatology in 2004 working closely with Dr. Stan Pavel. At 2008, she received her Ph.D. (Leiden) and was assigned as assistant professor to both the University of Leiden and the Open Universiteit Nederland. Apart from educational tasks, she continues her research on PDT of dermatophytes with the specific aim to develop a single PDT of onychomycosis caused by, in particular, T. rubrum. In addition to this, she has recently started a chemical research project that focuses on silver nanoparticles, skin and risk assessment.

Stan Pavel finished his medical studies at Charles University in Prague (Czechoslovakia) in 1969, and was appointed at the Department of Medical Biochemistry. In 1981, he moved to the Netherlands. He received two PhDs: in biochemistry (1978, Charles University, Prague) and in dermatology (1988, University of Amsterdam). From 1991 till 2010, he was employed as a senior staff member of the Department of Dermatology at the University of Leiden. His research has always been focussed on two areas: photodermatology and biochemistry of skin pigmentation. He was one of the first dermatologists in the Netherlands involved in the research area of photodynamic therapy. Presently, he is appointed as Associate Professor of Dermatovenerology at the Medical Faculty of Charles University in Pilsen, Czech Republic. Dr. Pavel has published more than 240 full papers, of which more than 160 in peer-reviewed journals.

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