Cutaneous Alternariosis in Immunosuppressed Patients Treated with Photodynamic Therapy and Oral Antifungals, a Synergistic Strategy

Cutaneous alternariosis is a rare condition, more frequently presented in immunocompromised patients, which usually requires long courses of systemic antifungals that may interact with other medications. The presented series shows three cases of cutaneous alternariosis in immunocompromised patients and organ transplant recipients that were successfully treated with photodynamic therapy and oral antifungals, allowing a reduction in the systemic treatment duration and therefore decreasing the risk of side effects and drug interactions.


Introduction
Alternaria is a dematiaceous fungus, with widespread presence in the natural environment, that causes phaeohyphomycosis, a relatively infrequent human infection.This melanized mold can lead to a diverse range of clinical conditions, varying from localized manifestations in specific organs to more widespread systemic infections [1].Alternaria infects mainly immunocompromised hosts; although there have been occasional instances of infections in individuals with healthy immune systems [2,3], these cases seldom progress to invasive diseases [4].
The skin and the subcutaneous tissues are the most frequently infected sites by this fungus, although other types of infections such as oculomycosis, sinus infections, onychomycosis, and invasive diseases have been described [1].
The treatment of cutaneous alternariosis includes, when feasible, the reduction of immunosuppression, in addition to long courses of antifungals, which often interact with the immunosuppressants taken by organ transplant recipients (OTRs).Excision of the lesions can also be an option in certain cases, often combined with systemic treatment [5].
Recently, some authors have shown the use of antimicrobial photodynamic therapy (PDT) as an effective treatment of phaeohyphomycosis [6,7], and also chromoblastomycosis [4,8].PDT is based on the use of photosensitizing molecules that end up generating Pharmaceuticals 2024, 17, 245 2 of 8 reactive oxygen species (ROS) that destroy the target cells when being irradiated with light of a suitable wavelength and a proper dose [9].Some reports have shown that PDT is effective in the treatment of cutaneous infections due to pathogens such as multi-resistant mycobacteria, in combination with systemic treatments [10].Moreover, it also offers the possibility of a reduction in the duration of systemic treatment of fungal infections such as onychomycosis [11].In relation with cutaneous alternariosis, Liu et al. [4] reported a case of an infection due to Alternaria alternata treated with oral itraconazole followed by PDT with complete healing.Moreover, the combination of systemic antifungal and PDT has also been described in other cutaneous fungal infections with promising results [6][7][8].
We present three additional cases of cutaneous alternariosis in immunocompromised patients, two of them OTRs, treated with systemic antifungals and PDT (Table 1), and a review of the literature about the role of PDT in the treatment of phaeohyphomycosis and chromoblastomycosis.In our series, the combination of both treatment modalities allowed shortening the antifungal treatment duration, diminishing the possibility of interactions with other drugs, especially in the ORTs, and side effects. 1 Thirty minutes of incubation followed by two hours of exposure to sunlight, once every 10 days. 2 Incubation time starting at 1 hour and progressively increasing to 3 hours, and subsequent illumination with Aktilite ® CL 128 increasing fluence from 37 to 74 J/cm 2 , once a week. 3Three hours of incubation followed by illumination with Aktilite ® CL 128 (fluence 37 J/cm 2 ), twice weekly. 4Thirty minutes of incubation and illumination with Aktilite ® CL 128 (fluence 74 J/cm 2 ) once a week, and exposure to daylight half an hour every day until fading of the blue color.

Case 1
A 70-year-old male with a personal history of Chronic Obstructive Pulmonary Disease (COPD), requiring frequent courses of oral steroids (30-40 mg of prednisone for 10 to 14 days every month), diabetes mellitus type 2 with poor control, and larynx squamous cell carcinoma.The patient developed verrucous plaques on the left forearm for four months before consulting (Figure 1a,b).Moreover, he mentioned gardening often and outdoor farming activities.The histopathological analysis showed light acanthosis, with mild hyperkeratosis and foci of parakeratosis; at the dermal layer, there was granulomatous perifollicular inflammation with multinuclear, surrounding broken follicles (Figure 1c,d).Grocott (Figure 1e) and PAS staining revealed structures compatible with hyphae, and Ziehl-Nielsen was negative.Tissue culture showed a grey-olive green color and woolly texture colonies (Figure 1g).Microscopic study showed septate, brown hyphae compatible with those of Alternaria spp.(Figure 1f).Alternaria infectoriae was identified by PCR sequencing.The patient received one month of itraconazole 100 mg twice daily, and fivefour months Pharmaceuticals 2024, 17, 245 3 of 8 of 50 mg twice daily achieving partial improvement, followed by two sessions of daylight photodynamic therapy (DL-PDT) with 5-aminolevulinic acid (5-ALA, Ameluz ® ) consistent of 30 min of incubation followed by 2 hours of exposure to sunlight, once every 10 days.Complete healing was achieved after de DL-PDT, with no remission relapse after two months.The patient died shortly after due to COPD worsening.
The histopathological analysis showed light acanthosis, with mild hyperkeratosis and foci of parakeratosis; at the dermal layer, there was granulomatous perifollicular inflammation with multinuclear, surrounding broken follicles (Figure 1c,d).Grocott (Figure 1e) and PAS staining revealed structures compatible with hyphae, and Ziehl-Nielsen was negative.Tissue culture showed a grey-olive green color and woolly texture colonies (Figure 1g).Microscopic study showed septate, brown hyphae compatible with those of Alternaria spp.(Figure 1f).Alternaria infectoriae was identified by PCR sequencing.The patient received one month of itraconazole 100 mg twice daily, and fivefour months of 50 mg twice daily achieving partial improvement, followed by two sessions of daylight photodynamic therapy (DL-PDT) with 5-aminolevulinic acid (5-ALA, Ameluz ® ) consistent of 30 min of incubation followed by 2 hours of exposure to sunlight, once every 10 days.Complete healing was achieved after de DL-PDT, with no remission relapse after two months.The patient died shortly after due to COPD worsening.

(d) (H-E 20×
) Some yeast forms can also be identified.Grocott (e) (20×) and PAS staining revealed structures compatible with hyphae, and Ziehl-Nielsen was negative.(g) Macroscopic appearance of the culture showed a grey-olive green color and woolly texture colonies.(f) (40×) The septate, brown hyphae seen microscopically were compatible with Alternaria spp.Alternaria infectoriae was identified by PCR sequencing.Partial improvement was seen five months after Itraconazole (100 mg/12 h for 1 month and Itraconazole 50 mg/12 h for 4 months), which was followed by two sessions of 5-aminolevulinic acid (5-ALA) daylight photodynamic therapy, achieving resolution of the lesions (no photography available).The patient died shortly after due to COPD worsening.

Case
A 62-year-old male, treated with tacrolimus (0.1 mg/kg/day) and 10 mg/day of prednisone due to a kidney transplant, presented with a tumoral lesion on the lateral side of his right ankle two months ago (Figure 2a).Histopathology analysis showed hyphae compatible with phaeohyphomycotic infection, and culture confirmed Alternaria spp.Simultaneous treatment with voriconazole 400 mg/day for three months and PDT with methyl-aminolevulinate (MAL-PDT), Metvix ® , was started with 1 hour incubation, progressively increasing the time to 3 hours; and subsequent illumination with Aktilite ® increasing fluences from 37 to 74 J/cm 2 .MAL-PDT was performed once a week, with a total of 12 sessions, achieving complete resolution after six months, with no relapse after twelve months follow-up (Figure 2b).
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 4 of 9 dermal layer, there was granulomatous perifollicular inflammation with multinuclear, surrounding broken follicles.(d) (H-E 20×) Some yeast forms can also be identified.Grocott (e) (20×) and PAS staining revealed structures compatible with hyphae, and Ziehl-Nielsen was negative.(g) Macroscopic appearance of the culture showed a grey-olive green color and woolly texture colonies.(f) (40×) The septate, brown hyphae seen microscopically were compatible with Alternaria spp.Alternaria infectoriae was identified by PCR sequencing.Partial improvement was seen five months after Itraconazole (100 mg/12 h for 1 month and Itraconazole 50 mg/12 h for 4 months), which was followed by two sessions of 5-aminolevulinic acid (5-ALA) daylight photodynamic therapy, achieving resolution of the lesions (no photography available).The patient died shortly after due to COPD worsening.

Case 2
A 62-year-old male, treated with tacrolimus (0.1 mg/kg/day) and 10 mg/day of prednisone due to a kidney transplant, presented with a tumoral lesion on the lateral side of his right ankle two months ago (Figure 2a).Histopathology analysis showed hyphae compatible with phaeohyphomycotic infection, and culture confirmed Alternaria spp.Simultaneous treatment with voriconazole 400 mg/day for three months and PDT with methyl-aminolevulinate (MAL-PDT), Metvix ® , was started with 1 hour incubation, progressively increasing the time to 3 hours; and subsequent illumination with Aktilite ® increasing fluences from 37 to 74 J/cm 2 .MAL-PDT was performed once a week, with a total of 12 sessions, achieving complete resolution after six months, with no relapse after twelve months follow-up (Figure 2b).

Case 3
An 81-year-old male, recipient of a kidney transplant, under treatment with tacrolimus (0.2 mg/kg/day) and 5 mg/day of prednisone, was referred to the dermatology clinic for four ulcerated lesions surrounded by grey-blue macules on his right leg for the past month (Figure 3a).A skin biopsy showed findings consistent with deep fungal infection and Alternaria spp.grew in the tissue culture.It was decided to treat the patient with MAL-PDT, Metvix ® , with three hours of incubation followed by illumination with Aktilite ® 37 J/cm 2 (twice weekly).After 11 sessions, he showed partial improvement, but the culture was still positive; therefore, the photosensitizer was switched to methylene blue 1% solution (MB-PDT) with 30 minutes of incubation under occlusion and illuminated with

Case 3
An 81-year-old male, recipient of a kidney transplant, under treatment with tacrolimus (0.2 mg/kg/day) and 5 mg/day of prednisone, was referred to the dermatology clinic for four ulcerated lesions surrounded by grey-blue macules on his right leg for the past month (Figure 3a).A skin biopsy showed findings consistent with deep fungal infection and Alternaria spp.grew in the tissue culture.It was decided to treat the patient with MAL-PDT, Metvix ® , with three hours of incubation followed by illumination with Aktilite ® 37 J/cm 2 (twice weekly).After 11 sessions, he showed partial improvement, but the culture was still positive; therefore, the photosensitizer was switched to methylene blue 1% solution (MB-PDT) with 30 minutes of incubation under occlusion and illuminated with Aktilite ® (fluence 74 J/cm 2 ) once a week, and exposure to daylight for half an hour every day until the disappearance of the blue color.After 10 sessions of MB-PDT, since the biggest lesion persisted, voriconazole 400mg per day was added to MB-PDT (Figure 3b).After 1.5 months, the lesion was clinical and microbiologically cured, and voriconazole also had to be discontinued due to acute renal failure.No recurrence had been presented after three years of follow-up (Figure 3c).
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 5 of 9 Aktilite ® (fluence 74 J/cm 2 ) once a week, and exposure to daylight for half an hour every day until the disappearance the blue color.After 10 sessions of MB-PDT, since the biggest lesion persisted, voriconazole 400mg per day was added to MB-PDT (Figure 3b).After 1.5 months, the lesion was clinical and microbiologically cured, and voriconazole also had to be discontinued due to acute renal failure.No recurrence had been presented after three years of follow-up (Figure 3c).

Discussion
Cutaneous alternariosis is caused by the dematiaceous fungus Alternaria, which is frequently isolated in opportunistic deep cutaneous mycoses [5].Alternaria is widely distributed and can be found in plants, water, soil water, or objects, among others [2].The most frequent transmission mechanism in cutaneous infections is direct inoculation during or after a traumatism, commonly causing phaeohyphomycosis.However, cases of chromoblastomycosis due to Alternaria, characterized by the histopathologic finding of sclerotic or muriform cells, have also been reported [4,12,13].The clinical presentation is usually non-specific and asymptomatic and may present as small papules or nodules, multinodular plaques, ulcers, cellulitis, or even blisters, usually in exposed areas of outdoor workers, as they are more prone to trauma [5,6].
Histopathological study often shows a dense dermal suppurative granulomatous infiltrate and fungal structures, which are more evident with different stains such as Grocott, Periodic acid-Schiff (PAS), or Masson-Fontana.These findings are enough to guide the management, which commonly involves empirical treatment with high doses of antifungals.Confirmation of the fungal species requires culture or direct sequencing [5].
Surgery can be an effective treatment option; however, it might not be enough or possible in some cases such as in large and multifocal cases that require a combination with systemic antifungals [5], or certain locations in which the surgery would be too aggressive, or in which healing would be difficult.
High doses of antifungals for several months are recommended to treat cutaneous alternariosis.Itraconazole 400 mg/day or voriconazole 400 mg/day are two of the most common options [5].Nevertheless, the use of these treatments in OTRs requires close monitoring due to their interaction with calcineurin inhibitors, as well as other adverse reactions such as hepatotoxicity.A combination of surgery plus different antifungals is

Discussion
Cutaneous alternariosis is caused by the dematiaceous fungus Alternaria, which is frequently isolated in opportunistic deep cutaneous mycoses [5].Alternaria is widely distributed and can be found in plants, water, soil water, or objects, among others [2].The most frequent transmission mechanism in cutaneous infections is direct inoculation during or after a traumatism, commonly causing phaeohyphomycosis.However, cases of chromoblastomycosis due to Alternaria, characterized by the histopathologic finding of sclerotic or muriform cells, have also been reported [4,12,13].The clinical presentation is usually non-specific and asymptomatic and may present as small papules or nodules, multinodular plaques, ulcers, cellulitis, or even blisters, usually in exposed areas of outdoor workers, as they are more prone to trauma [5,6].
Histopathological study often shows a dense dermal suppurative granulomatous infiltrate and fungal structures, which are more evident with different stains such as Grocott, Periodic acid-Schiff (PAS), or Masson-Fontana.These findings are enough to guide the management, which commonly involves empirical treatment with high doses of antifungals.Confirmation of the fungal species requires culture or direct sequencing [5].
Surgery can be an effective treatment option; however, it might not be enough or possible in some cases such as in large and multifocal cases that require a combination with systemic antifungals [5], or certain locations in which the surgery would be too aggressive, or in which healing would be difficult.
High doses of antifungals for several months are recommended to treat cutaneous alternariosis.Itraconazole 400 mg/day or voriconazole 400 mg/day are two of the most common options [5].Nevertheless, the use of these treatments in OTRs requires close monitoring due to their interaction with calcineurin inhibitors, as well as other adverse reactions such as hepatotoxicity.A combination of surgery plus different antifungals is commonly needed to achieve cure rates around 90% due to adverse events to the antifungal drugs or lack of response to them [5], which is emerging as a concern even in immunocompetent patients [2].PDT has been shown to be effective in different types of infections including bacterial infections [9], fungal infections [11,14], multi-resistant mycobacteria, or other pathogens [10,15].The antimicrobial efficacy of PDT has been extensively explored through numerous in vitro studies since the 1990s, and unlike traditional antibiotics, PDT's diverse cellular targets make resistance development less likely [16].
Recently, there have been reports of encouraging outcomes achieved through the utilization of PDT (using methylene blue as a photosensitizer rather than ALA) in the management of chromoblastomycosis [17,18].Moreover, several authors have also shown the utility of PDT with ALA as an adjuvant of antifungal therapy for the treatment of different phaeohyphomycosis and chromoblastomycosis (Table 2).
Yang and colleagues [19] documented a challenging case of chromoblastomycosis caused by F. monophora that showed improvement following treatment with ALA-PDT.Moreover, Liu et al. [4] described a 50-year-old man with a chromoblastomycosis because of Alternaria alternata, successfully treated with a short course of systemic itraconazole and subsequent 5-aminolevulinic acid-photodynamic therapy.
The presented case series shows similar results of PDT in phaeohyphomycosis due to Alternaria and highlights the challenges of diagnosing and managing opportunistic co-infection in an immunocompromised host.As previously described in fungal infections such as onychomycosis [11], PDT with MAL or 5-ALA potentiates the effects of the systemic antifungals, both sequentially or simultaneously, permitting a reduction in the duration of the systemic treatment, and therefore, diminishing the possibility of drug adverse events and interactions.In addition, the beneficial effects of PDT on host tissues, such as growth factor stimulation and immune response enhancement, potentially foster improved wound healing, particularly in common presentations of this condition such as chronic ulcers [20].

Conclusions
PDT has been shown to be effective for the treatment of various types of infections including cutaneous fungal infections.The presented case series, as well as previous reports, supports its role as a simple and effective treatment for cutaneous alternariosis and other cutaneous fungal infections, which has a synergistic effect with oral antifungals.Combination of PDT with systemic antifungals allows an important reduction in the treatment duration and the healing time.By shortening the systemic antifungal treatments, the possible interactions with other drugs are also reduced, increasing the safety of the treatment.This is particularly important in certain populations, such as organ transplant recipients, who need immunosuppressive treatments that require monitoring when taking certain systemic antifungals and are more susceptible to these types of opportunistic infections.

Figure 1 . 1 .
Figure 1.Case 1. (a,b) Erythematous verrucous plaques on the left forearm.(c) (H-E 8×) Histopathological analysis showed light acanthosis, with mild hyperkeratosis and foci of parakeratosis; at the Figure 1.Case 1. (a,b) Erythematous verrucous plaques on the left forearm.(c) (H-E 8×) Histopathological analysis showed light acanthosis, with mild hyperkeratosis and foci of parakeratosis; at the dermal layer, there was granulomatous perifollicular inflammation with multinuclear, surrounding broken follicles.(d) (H-E 20×) Some yeast forms can also be identified.Grocott (e) (20×) and PAS staining revealed structures compatible with hyphae, and Ziehl-Nielsen was negative.(g) Macroscopic appearance of the culture showed a grey-olive green color and woolly texture colonies.(f) (40×) The

Figure 2 .
Figure 2. Case 2. (a) Erythematous-violaceus tumoral lesion on the lateral side of the right ankle.(b) Resolution six months after simultaneous treatment with voriconazole 400 mg/day for three months and photodynamic therapy with methyl-aminolevulinate (Metvix ® ).Resolution after six months and no recurrence after twelve months follow-up (c).

Figure 2 .
Figure 2. Case 2. (a) Erythematous-violaceus tumoral lesion on the lateral side of the right ankle.(b) Resolution six months after simultaneous treatment with voriconazole 400 mg/day for three months and photodynamic therapy with methyl-aminolevulinate (Metvix ® ).Resolution after six months and no recurrence after twelve months follow-up (c).

Figure 3 .
Figure 3. Case 3. (a) Ulcerated lesions on the right leg surrounded by grey-blue macules.(b) Partial resolution after 11 sessions of photodynamic therapy with methyl-aminolevulinate (Metvix ® ) (MAL-PDT) and 10 sessions of photodynamic therapy with methylene blue (MB-PDT).(c) Complete healing after 1.5 months of simultaneous treatment with voriconazole 400 mg/day and MB-PDT (6 sessions), leaving mild erythema and residual hyperpigmentation.

Figure 3 .
Figure 3. Case 3. (a) Ulcerated lesions on the right leg surrounded by grey-blue macules.(b) Partial resolution after 11 sessions of photodynamic therapy with methyl-aminolevulinate (Metvix ® ) (MAL-PDT) and 10 sessions of photodynamic therapy with methylene blue (MB-PDT).(c) Complete healing after 1.5 months of simultaneous treatment with voriconazole 400 mg/day and MB-PDT (6 sessions), leaving mild erythema and residual hyperpigmentation.