Chapter 4.5
Photodynamic therapy for pythiosis
Background – Pythiosis is a life-threatening disease caused by Pythium insidiosum. Photodynamic therapy (PDT) is an alternative treatment to surgery that uses the interaction of a photosensitizer, light and molecular oxygen to cause cell death.
Objectives – To evaluate the effect of PDT on the in vitro growth of P. insidiosum and in an in vivo model of pythiosis.
Methods – For in vitro studies, two photosensitizers were evaluated: a haematoporphyrin derivative (Photogem®) and a chlorine (Photodithazine®). Amphotericin B was also evaluated, and the control group was treated with sterile saline solution. All experiments (PDT, porphyrin, chlorine and light alone, amphotericin B and saline solution) were performed as five replicates. For in vivo studies, six rabbits were inoculated with 20,000 zoospores of P. insidiosum, and an area of 1 cm3 was treated using the same sensitizers. The PDT irradiation was performed using a laser emitting at 660 nm and a fluence of 200 J/cm2. Rabbits were clinically evaluated daily and histopathological analysis was performed 72 h after PDT.
Results – For in vitro assays, inhibition rates for PDT ranged from 60 to 100% and showed better results in comparison to amphotericin B. For the in vivo assays, after PDT, histological analysis of lesions showed a lack of infection up to 1 cm in depth.
Conclusions and clinical importance – In vitro and in vivo studies showed that PDT was effective in the inactivation of P. insidiosum and may represent a new approach to treating pythiosis.
Introduction
Pythium insidiosum is a fungus-like organism from the Stramenopila Kingdom, Phylum Oomycota.1 Several factors differentiate it from true fungi. Firstly, there is an absence of ergosterol in the cell membrane, which is the main target of antifungal drugs and explains why antifungal drugs are not very effective.2 Secondly, oomycetes do not have chitin in their cell wall; their cell structure includes cellulose, which is difficult for drugs to penetrate.3 Finally, the life cycle begins with parasitism of aquatic grasses by hyphae followed by the development of a sporangium, which matures and releases infective zoospores that encyst in plant or animal tissues.4
Pythiosis is a life-threatening disease that occurs mainly in tropical and subtropical areas of the world. Infections have been described in humans, some domestic and wild animals and recently in an aquatic bird.5–12 The most commonly affected animal is the horse, for which no breed, age or sex predilection has been documented. Lesions are tumour like, with serosanguinous exudate, ulceration, pruritus and odour. The disease is endemic in the Brazilian Pantanal, which potentially may have the highest incidence in the world.11 The literature describes more than 90 cases, but the true number is undoubtedly higher because this is not a notifiable disease. Dogs are the second most commonly affected species, presenting with both skin and gastrointestinal lesions. Over 100 cases of canine pythiosis (both forms) have been described in the USA alone.13,14
Surgery (aggressive surgical excision and amputation) is the most common treatment. Unfortunately, complete surgical excision is not possible due to the location of many lesions (e.g. distal limb of a horse), resulting in a high rate of local recurrence. Because of this, surgical excision is often coupled with concurrent antifungal drugs and immunotherapy.15–17
Photodynamic therapy (PDT) is a type of treatment where a dye reacts with a specific wavelength of light, resulting in the production of molecular oxygen in the target environment. The dye, termed the photosensitizer, absorbs light and starts photochemical reactions that transfer energy to molecular oxygen that in turn forms reactive oxygen species, which are highly toxic to cells, promoting their death.18,19,
This treatment has been widely used to treat cancer, multidrug-resistant strains of bacteria and other infectious diseases.18 Photodynamic therapy has been used to treat chromoblastomycosis20 and onychomycosis.21 Treatment of caries, denture disinfection and biofilm formation with PDT have been investigated in various studies.22–27 Hamblin et al.19 used PDT to treat experimentally infected wounds and reported success without damage to the surrounding healthy tissue. Photosensitizers are selective, because they accumulate in microbial cells and not in healthy host tissues.22,25
Pythiosis is a challenging disease to treat. There is little response to conventional or available antimicrobial therapies. Experimental therapies often appear promising in vitro, but when tested in vivo the results are less promising with relapses occurring when treatment is discontinued. This is an important equine disease and newer treatments are needed.13–16 The goal of this study was to evaluate the use of PDT on the in vitro growth of P. insidiosum and in an in vivo model of pythiosis to determine whether this is a potentially viable treatment option.
Material and methods
In vitro assays
Pythium isolation and culture.
An isolate of P. insidiosum was obtained from a horse diagnosed with pythiosis at São Paulo, School of Veterinary Medicine and Animal Science at Universidade Estadual Paulista, UNESP/Botucatu, Brazil. Cultures were maintained on Sabouraud dextrose agar (SDA; Difco, Sparks, MD, USA), incubated at 37°C aerobically and recultured weekly. For experimental purposes, standardized fragments 5 mm in diameter were obtained from the borders of the culture and subcultured onto SDA. Five replicates were performed for each assay (saline solution, chlorine alone and porphyrin alone, amphotericin B, light alone and PDT) and plates were incubated at 37°C for 30 days.
Light source.
The light source was a light-emitting diode (LED)-based system capable of emitting light at 530 nm for haematoporhyrin trials and at 660 nm for chlorine trials, developed by the Technological Support Laboratory of the University of São Paulo – LAT/USP. The intensity was 65 mW/cm2.
Photosensitizers.
Two photosensitizers were used in this study at three different concentrations as follows: porphyrin (Photogem®; Photogem LLC Co., Moscow, Russia) at concentrations of 10, 15 and 25 mg/mL; and chlorine (Photodithazine®; VETA-GRAND Company, Moscow, Russia) at concentrations of 0.7, 1.0 and 1.3 mg/mL.
Treatment groups.
The control group consisted of standardized fragments treated with sterile saline solution cultured on SDA and incubated at 37°C.
For the amphotericin B group, 10 μL of amphotericin B (Eurofarma, Sao Paulo, Brazil) at a concentration of 100 μg/mL was added to standardized fragments of P. insidiosum cultured on SDA and incubated at 37°C. Amphotericin B was used in order to compare a common antifungal drug used in pythiosis treatment with photodynamic therapy.
The effect of dyes alone was evaluated at maximum concentrations of 25 mg/mL for porphyrin and 1.3 mg/mL for chlorine. Ten microlitres of each sensitizer concentration was added to standardized fragments of the Pythium cultured on SDA and incubated at 37°C. No washing was done after incubation.
In the light only group, fragments were exposed to light with a fluence of 30 J/cm2 and irradiated for 461 s at 530 and 660 nm.
For photodynamic treatment, the standardized fragments of the Pythium culture were placed in Petri dishes with SDA and treated with either haematoporphyrin (10, 15, and 25 mg/mL) or chlorine (0.7, 1.0 and 1.3 mg/mL) and incubated in the dark for 20 min at 37°C. This was followed by the light irradiation. For porphyrin and chlorine, a wavelength of 530 and 660 nm was used, respectively. Irradiation lasted for 461 s, and immediately afterwards the plates were incubated at 37°C.
Analysis.
Petri dishes with treated fragments of P. insidiosum were viewed 48 h after each treatment. The growth area was measured using the software ImageJ® (image processing and analysis in JAVA; http://rsbweb.nih.gov/ij/), and the inhibition rate was calculated as follows:
where T represents the growth area of fragments from the treatment group and C the growth area of fragments from the control group. Cultures were incubated at 37°C for 30 days after PDT, to look for a possible growth recovery. After 30 days, the fragments treated with PDT as described above and control fragments were submitted for analysis by scanning electron microscopy.
Statistical analyses were performed using Kruskal-Wallis and Dunn’s tests to compare the treatment groups. A P-value of <0.05 was considered significant.
In vivo assays
This study was approved by the Ethical Committee of the Universidade Estadual Paulista ‘Júlio de Mesquita Filho’, UNESP/Botucatu, Brazil.
Lesion induction.
Experimental infections were induced by subcutaneous injection of 1 mL (20,000 zoospores/mL) of P. insidiosum into the dorsal thoracic region of six New Zealand rabbits. This resulted in a large lesion of approximately 10-30 cm2 in size and 4-5 cm in depth. During this time rabbits were examined daily to ensure that the experimental infection was not resulting in a loss of body condition or causing signs of illness other than skin lesions. Investigators were prepared to administer pain medication or euthanize any rabbit if the infection caused suffering. For the study, a randomly selected area of 1 cm2 within the main lesion was used for irradiation. As the mean light penetration for 660 nm is approximately 1 cm, it was estimated that the volume of tissue treated was 1 cm3.
One of the rabbits was randomly selected as an infected, untreated control animal. Skin biopsy specimens from treated animals and the control animal were collected 72 h after treatment.
Light source.
A homogeneous optical fibre coupled to laser equipment emitting at 660 nm was used for chlorine assays. The intensity was set at 150 mW/cm2 with a fluence of a 200 J/cm2 obtained by 1333 s of exposure. For porphyrin assays, a LED device was used, emitting at 630 nm, with an intensity of 150mW/cm2 and fluence of 200 J/cm2, as for chlorine.
Photosensitizer.
Chlorine at 1.0 mg/kg and porphyrin at 1.5 mg/kg was administered by intravenous injection into the auricular vein. Intravenous administration was chosen in order to obtain adequate distribution of photosensitizer in the lesion, given that topical application of the photosensitizer may not penetrate the lesion.
Photodynamic therapy treatment.
Rabbits were sedated with a combination of ketamine hydrochloride (Dopalen®; Vetbrands, Paulínia/SP, Brazil) and xylazine hydrochloride (Rompum®; Bayer, São Paulo/SP, Brazil), at a dose of 5mg/kg of each agent, and anaesthetized with 1.7% isoflurane in oxygen using a mask. Four hours after administration of the photosensitizer, rabbits were anaesthetized and the skin lesion was irradiated. Dye photobleaching was monitored through fluorescence spectroscopy as previously described.28 (Successful photobleaching means that the photosensitizer is completely degraded and no fluorescence is observed after PDT.)
Analysis.
Three days (72 h) after treatment, the animals were euthanized and tissue biopsy specimens were collected, fixed in 10% neutral buffered formalin and routinely processed. Tissues were stained with haematoxylin and eosin or Gomori-Grocott’s methenamine silver stain.
Results
In vitro assays
As noted in the Material and methods, the inhibition rate was calculated 48 h post-PDT and plates were incubated for 30 days post-treatment to look for evidence of regrowth.
Figure 1 shows the inhibition rates for the three concentrations of porphyrin (10, 15 and 25 mg/mL) with light and dye controls and amphotericin B. For porphyrin, PDT resulted in inhibition rates of more than 60% for all three concentrations; however, the greatest inhibition was found using 10 mg/mL of porphyrin, because all fragments were inactivated. In the 15 and 25 mg/mL groups, one of five fragments showed recovery growth in 7 days. Light alone and dye control (25 mg/mL) showed discrete inhibition rates in the first 48 h, but regrowth was noted in 7 days. A porphyrin PDT group (10 mg/mL) showed statistically significant inhibition rates compared with the untreated controls, light and dye only and Amphotericin B. The 10 mg/mL group showed statistically significant more inhibition compared with the amphotericin B group. No statistically significant difference was found between untreated controls, the dye only, light and amphotericin B groups.
Figure 1. In vitro analyses of inhibition rates of hyphal growth with porphyrin at 48 h after each treatment. The group treated with the lower concentration of porphyrin showed a higher inhibition rate when compared with the two other concentrations evaluated. The photodynamic therapy (PDT) treatment also showed best results when compared with the amphotericin B group. *Groups for which P < 0.05 the inhibition rate showed statistical significance when compared with the untreated control group; and + groups that showed statistical significance when compared with the Amphotericin B group (P < 0.05).
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