Effectiveness of photo-ozone therapy against equine Pythium insidiosum.

Cutaneous pythiosis is a life-threatening infectious disease. Low-level laser therapy (LLLT) and ozone (O3) have been used individually in the treatment of infected wounds. The goals of the study were a) to characterize the antimicrobial action of the photo-ozone therapy (LLLT-O3) against equine Pythium insidiosum, and b) to assess the cytotoxic potential of the LLLT-O3 in keratinocytes. Specimens of pathogen were isolated from 10 horses. After culturing, 120 hyphae plugs were distributed among four groups (n=30 hyphae plugs/group): LLLT (laser irradiation for 160 sec;), O3 (exposition to O3 for 15 min;), LLLT-O3 (LLLT and O3 treatments in sequence) and control (untreated plugs). The hyphae growth was measured during the first 14 days post-treatment. Where there was an absence of hyphae growth, the plug was recultured for an additional 7 days. The cytotoxic potential of the treatments against HaCaT keratinocytes was assessed by colorimetric assays. The LLLT-O3 and O3 treatments inactivated, respectively, 92.3% (28/30) and 30% (9/30) of the samples. No growth was detected after 7 days reculture of inactivated hyphae plugs on new media. Hyphae growth was visualized in 100% of the control and LLLT hyphae plugs. The viability of HaCaT cells was not affected by the isolated treatments (LLLT and O3), while the LLLT-O3 showed slight cytotoxic effect (20%) when compared to the control group (P<0.05). Photo-ozone therapy inactivated equine P. insidiosum hyphae with minimal cytotoxicity in skin cells in vitro.

Potential anti-Pythium insidiosum therapeutics identified through screening of agricultural fungicides.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Clinical manifestations of pythiosis include an eye, blood vessel, skin, or gastrointestinal tract infection. Pythiosis has been increasingly reported worldwide, with an overall mortality rate of 28%. Radical surgery is required to save patients' lives due to the limited efficacy of antimicrobial drugs. Effective medical treatments are urgently needed for pythiosis. This study aims to find anti-P. insidiosum agents by screening 17 agricultural fungicides that inhibit plant-pathogenic oomycetes and validating their efficacy and safety. Cyazofamid outperformed other fungicides as it can potently inhibit genetically diverse P. insidiosum isolates while exhibiting minimal cellular toxicities. The calculated therapeutic scores determined that the concentration of cyazofamid causing significant cellular toxicities was eight times greater than the concentration of the drug effectively inhibiting P. insidiosum. Furthermore, other studies showed that cyazofamid exhibits low-to-moderate toxicities in animals. The mechanism of cyazofamid action is likely the inhibition of cytochrome b, an essential component in ATP synthesis. Molecular docking and dynamic analyses depicted a stable binding of cyazofamid to the Qi site of the P. insidiosum's cytochrome b orthologous protein. In conclusion, our search for an effective anti-P. insidiosum drug indicated that cyazofamid is a promising candidate for treating pythiosis. With its high efficacy and low toxicity, cyazofamid is a potential chemical for treating pythiosis, reducing the need for radical surgeries, and improving recovery rates. Our findings could pave the way for the development of new and effective treatments for pythiosis.IMPORTANCEPythiosis is a severe infection caused by Pythium insidiosum. The disease is prevalent in tropical/subtropical regions. This infectious condition is challenging to treat with antifungal drugs and often requires surgical removal of the infected tissue. Pythiosis can be fatal if not treated promptly. There is a need for a new treatment that effectively inhibits P. insidiosum. This study screened 17 agricultural fungicides that target plant-pathogenic oomycetes and found that cyazofamid was the most potent in inhibiting P. insidiosum. Cyazofamid showed low toxicity to mammalian cells and high affinity to the P. insidiosum's cytochrome b, which is involved in energy production. Cyazofamid could be a promising candidate for the treatment of pythiosis, as it could reduce the need for surgery and improve the survival rate of patients. This study provides valuable insights into the biology and drug susceptibility of P. insidiosum and opens new avenues for developing effective therapies for pythiosis.

Pythium insidiosum: In vitro oomicidal evaluation of telithromycin and interactions with azithromycin and amorolfine hydrochloride.

This study evaluated the repositioning of the ketolide antibacterial telithromycin (TLT) against the oomycete Pythium insidiosum and verified the combination of TLT and the antimicrobials azithromycin (AZM) and amorolfine hydrochloride (AMR), which have known anti-P. insidiosum activity. Susceptibility tests of P. insidiosum isolates (n = 20) against the drugs were carried out according to CLSI protocol M38-A2, and their combinations were evaluated using the checkerboard microdilution method. The minimum inhibitory concentrations were 0.5-4 µg/mL for TLT, 2-32 µg/mL for AZM, and 16-64 µg/mL for AMR. For the TLT+AZM combination, 52.75 % of interactions were indifferent, 43.44 % were antagonistic, and 9.70 % were synergistic. As for interactions of the TLT+AMR combination, 60.43 % were indifferent, 39.12 % were antagonistic, and 10.44 % synergistic interactions. This study is the first to evaluate the repositioning of the antibacterial TLT against mammalian pathogenic oomycetes, and our results show that its isolated action is superior to its combinations with either AZM or AMR. Therefore, we recommend including TLT in future research to evaluate therapeutic approaches in different clinical forms of human and animal pythiosis.

Pythium insidiosum: insights into biofilm formation and antibiofilm activity of antifungal drugs.

In this study, we investigate the ability of Pythium insidiosum to form biofilms across various substrates and the antibiofilm efficacy of 8-hydroxyquinoline derivatives (8-HQs). Biofilms of P. insidiosum were cultured on polystyrene plates, contact lenses, and horsehair. We provide the first evidence of P. insidiosum's biofilm-forming capability, thus considerably expanding our understanding of its transmission and pathogenesis. Our results demonstrate that 8-HQs effectively inhibit biofilm formation and eradicate pre-existing biofilms, underscoring their potential as a novel treatment strategy for pythiosis, a disease currently lacking a gold-standard treatment. This finding has particular relevance for ocular pythiosis associated with contact lens usage and potential infection sources in animals. Our results contribute to the scientific knowledge base and directly impact innovative therapeutic interventions' development.

Anti-Pythium insidiosum activity of bioactive compounds from medicinal plants.

Pythiosis is a serious disease caused by the aquatic oomycete Pythium insidiosum that mainly affects mammals. Unlike fungal and bacterial resistance induced by the indiscriminate use of drugs, P. insidiosum has low susceptibility to antifungal drugs. In this sense, essential oils and their major components emerge as a promising treatment line for this disease. Given the above, this study sought to verify P. insidiosum (n = 34) susceptibility to the bioactive compounds eugenol, α-terpineol, menthol, and carvacrol and correlate them with the respective essential oils of Eugenia caryophyllata, Melaleuca alternifolia, Mentha piperita, and Origanum vulgare. The essential oils and bioactive compounds were purchased commercially and tested according to the Clinical and Laboratory Standards Institute protocol M38-A2. Our findings showed that eugenol, α-terpineol, and carvacrol had superior anti-P. insidiosum action than their respective essential oils, suggesting that they may be responsible for inhibitory activity against P. insidiosum. Notably, the major compound with the best anti-P. insidiosum activity was α-terpineol; nonetheless, menthol showed less activity than its essential oil. The results imply that essential oils and their major compounds may be important allies in treating pythiosis, expanding the perspectives of developing new drugs with anti-P. insidiosum activity.

Case Report: Poor Outcome Despite Aggressive Management in Pythium insidiosum Endophthalmitis.

Pythium insidiosum is a parasitic oomycete that can cause keratitis and closely resembles fungus, both clinically and morphologically. It requires a trained microbiologist for its differentiation from fungal filaments and has poor response to antifungal therapy. We present a case of primary isolation of the organism from the vitreous cavity in a case of endophthalmitis. The infection spread quickly and involved all the ocular tissues. The eye had poor visual outcome despite a sequence of rapidly completed retinal and corneal surgeries along with initiation of anti-Pythium therapy.

Successful management of colonic pythiosis in two dogs in Thailand using antifungal therapy.

Gastrointestinal pythiosis is a severe, progressive and often a fatal disease, which is caused by the aquatic pathogen Pythium insidiosum. Treatment is challenging due to the disease's resistance to antifungal drugs. Surgical resection is frequently attempted in cases of pythiosis; however, it can be technically challenging. This report presents two dogs with decreased appetite, abdominal pain, progressive haematochezia, tenesmus and significant weight loss. With the medical histories of both being young canines, living in areas with access to natural water resources and with the main chronic gastrointestinal symptoms having not responded to symptomatic treatment, pythiosis was taken into consideration. Abdominal ultrasound revealed severe, diffuse thickening and loss of normal layering of the colonic wall. These findings led to a differential diagnosis between intestinal neoplasia and fungal disease. Full-thickness biopsies were later performed, and immunohistochemistry staining was suggested for colonic pythiosis. Medical treatment for pythiosis was successful with a combination of oral terbinafine and prednisolone. However, therapy with itraconazole in case 1 did not improve the clinical signs, and in case 2, itraconazole was used after all clinical signs have improved for clinical control. Since then, there has been no recurrence of clinical signs until the time of preparing this report (19 months for case 1, 11 months for case 2 since the cessation of treatment). The treatment was successful based on clinical signs and ultrasonographic data, and the disease remission was not confirmed by advance imaging, monitoring of pythiosis enzyme-linked immunosorbent essay concentration or repeat sampling.

Severe skin and soft tissue pythiosis acquired in a hot spring in the southwestern United States, a case report and review of North American cases.

BACKGROUND: Human pythiosis, caused primarily by the aquatic oomycete, Pythium insidiosum, is an emerging but uncommon infection in North America. The infection is frequently life-threatening and is often initially unrecognized due to its rarity and similar presentation to certain fungal infections. METHODS: We report a case of skin and soft tissue pythiosis in a patient without significant underlying comorbidities acquired in a New Mexico hot spring and review its successful treatment. We also review all reported pythiosis cases in North America. RESULTS: Eleven confirmed cases of human pythiosis acquired in North America were identified. The majority of cases occurred in children (64%), ten of eleven cases were acquired in the southern U.S., Mexico, Central America or the Caribbean and four of the eleven individuals succumbed to the infection. CONCLUSIONS: With recognition and aggressive surgical and medical treatment good clinical outcomes can be achieved when treating human pythiosis.

Melaleuca alternifolia formulations in the treatment of experimental pythiosis.

Essential oils (EO) are aromatic compounds from the plant secondary metabolism. Melaleuca alternifolia EO is well known for its medicinal properties and promising use as an antimicrobial agent. Pythiosis is a difficult-to-treat and emerging disease caused by the oomycete Pythium insidiosum. This study evaluated a nanoemulsion formulation of M. alternifolia (NEMA) in topical and intralesional application to treat experimental pythiosis. Dermal toxicity tests were performed on M. alternifolia EO in Wistar rats. Pythiosis was reproduced in rabbits (n = 9) that were divided into groups: group 1 (control), cutaneous lesions with daily topical application of a non-ionizable gel-based formulation and intralesional application of sterile distilled water every 48 h; group 2 (topical formulation), lesions treated daily with topical application of a non-ionizable gel-based formulation containing 5 mg/ml of NEMA; and group 3 (intralesional formulation), lesions treated with NEMA at 5 mg/ml in aqueous solution applied intralesionally/48 h. The animals were treated for 45 days, and the subcutaneous lesion areas were measured every 5 days. M. alternifolia EO showed no dermal toxicity. The lesion areas treated with intralesional NEMA reduced at the end of treatment, differing from groups 1 and 2 (P < 0.05). In the topically treated group, the lesion areas did not differ from the control group, although the number of hyphae significantly reduced (P < 0.05). Under the experimental conditions of this study, the NEMA formulations presented a favorable safety profile. However, further studies are required to evaluate if this safety applies to higher concentrations of NEMA and to validate its use in clinical pythiosis.

Successful management of pediatric pythium insidiosum keratitis with cyanoacrylate glue, linezolid, and azithromycin: Rare case report.

PURPOSE: Pythium insidiosum causes a rare sight-threatening keratitis and is a devastating ocular pathology with a high morbidity. It is frequently mistaken as fungal keratitis. Here we highlight a rare case of pediatric Pythium insidiosum keratitis which was successfully managed using an antibiotic combination of linezolid and azithromycin with cyanoacrylate glue. CASE DESCRIPTION: A 9-year-old young male child presented to our clinic with defective vision, pain, redness in the right eye for 5 days post stick injury. In the right eye, Snellen's best-corrected visual acuity (BCVA) was 6/12 which deteriorated to hand movements within 5 days of treatment. Ocular examination revealed 6 × 5 mm dry-looking mid stromal corneal infiltrate with feathery margin involving the visual axis. The clinical picture was suggestive of fungal keratitis. Corneal scraping and smear examination with 10% KOH and Gram stain revealed long slender hyaline hyphae with sparse septations. Before the culture result, the patient was started on 5% Natamycin and 1% Itraconazole hourly, but still, the infiltrate progressed. Further, P. Insidiosum keratitis was considered as the differential, which was confirmed on blood agar culture. After receiving culture results, the patient was managed with 0.2% Linezolid and 1% Azithromycin hourly. Due to the rapid progression of infiltrate, corneal melt, and younger age, cyanoacrylate glue, and bandage contact lens were used. On the last follow-up, the BCVA recovered to 6/12. CONCLUSION AND IMPORTANCE: Prompt diagnosis, clinical awareness, and a specific treatment regime is needed for managing this devastating corneal entity. Cyanoacrylate glue due to its antibacterial properties can be a potential rescuer and can be considered for managing these cases.

Successful potassium iodide treatment for rhinofacial pythiosis in sheep.

BACKGROUND: Pythiosis in sheep is an important disease in Brazil, which could cause rhinitis, dermatitis and alimentary tract inflammation. It is caused by the aquatic oomycete, Pythium insidiosum. The rhinofacial pythiosis causes facial deformity and upper respiratory tract clinical signs associated with necroproliferative masses occupying the rostral nasal cavity and hard palate. Little is known regarding the therapy, prophylaxis and pathogenesis of this disease. METHODOLOGY: During the 6-year study, we examined 13 sheep presenting rhinofacial pythiosis. The diagnosis was performed through biopsy of the rhinofacial lesions followed by histopathology and immunohistochemistry using specific antibodies against P insidiosum, polymerase chain reaction and an indirect enzyme-linked immunosorbent assay. RESULTS: This study presents the clinical findings of a potassium iodide treatment of rhinofacial pythiosis in sheep. All sheep were treated with 10 ml of 10% potassium iodide solution, administered orally every day during 63-120 (mean 85) days. Among treated sheep, 84.6% demonstrated complete recovery. CONCLUSION: Potassium iodide therapy may treat rhinofacial pythiosis in sheep.

Vascular pythiosis caused by Pythium aphanidermatum: the first case report in Asia.

BACKGROUND: Pythium, soil-borne plant pathogens, are in the class Oomycetes. They are not true fungi, but are related to diatom and algae. There are two human pathogens including P. insidiosum and P. aphanidermatum. To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia. CASE PRESENTATION: A 47-year-old Thai woman, living in North Thailand, with ß thalassemia/hemoglobin E presented with acute recurrent arterial insufficiency of both legs. Emergent embolectomy with clot removal was performed. The pathology of the clot exhibited noncaseous granulomatous inflammation with many fungal hyphal elements. PCR identified P. aphanidermatum with 100% identity. Final diagnosis is vascular pythiosis. Unfortunately, the patient eventually expired after treatment with itraconazole, terbinafine, azithromycin, and doxycycline. CONCLUSIONS: To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia.

In vitro anti-Pythium insidiosum activity of amorolfine hydrochloride and azithromycin, alone and in combination.

Pythium insidiosum infections have been widely studied in an attempt to develop an effective therapeutic protocol for the treatment of human and animal pythiosis. Several antifungal agents are still prescribed against this oomycete, although they present contradictory results. To evaluate the susceptibility profile and to verify the morphological alterations in P. insidiosum isolates treated with amorolfine hydrochloride and azithromycin, alone or in combination. Susceptibility tests for P. insidiosum isolates (n = 20) against amorolfine hydrochloride (AMR) and azithromycin (AZM) were performed according to Clinical and Laboratory Standards Institutes (CLSI) protocol M38-A2. Combinations of both drugs were evaluated using the checkerboard microdilution method. Additionally, transmission and scanning electron microscopy were performed in order to verify the morphological alterations in P. insidiosum isolates in response to these drugs. All P. insidiosum isolates had a minimum inhibitory concentration (MIC) ranging from 16 to 64 mg/l and 8 to 64 mg/l for amorolfine hydrochloride and azithromycin, respectively. Synergistic interactions between the drugs were not observed, with antagonism in 59.8% of isolates, and indifferent interactions in 36.2%. Electron microscopy showed changes in the surface of P. insidiosum hyphae, disorganization of intracellular organelles, and changes in the plasma membrane and cell wall of oomycetes treated with the drugs. This is the first study to demonstrate in vitro anti-P. insidiosum effect of amorolfine hydrochloride. These results indicate the therapeutic potential of this drug against cutaneous and subcutaneous forms of pythiosis, but further studies are necessary to confirm this potential.

Improved Outcome of Pythium Keratitis With a Combined Triple Drug Regimen of Linezolid and Azithromycin.

PURPOSE: To describe the clinical features, microbiological profile, and outcome of a series of cases of Pythium keratitis treated with topical and oral linezolid and topical azithromycin eye drops. METHODS: This was a retrospective interventional case series of microbiologically and/or histopathologically proven cases of Pythium keratitis seen between October 2016 and December 2019. All patients received a combination of topical linezolid and/or azithromycin eye drops with oral linezolid. Analysis of demographic data, predisposing risk factors, microbiological results, treatment regimen, visual acuity, surgical intervention, and final outcome was performed. A subgroup analysis of cases >6 mm in size was performed. Success was defined as complete resolution on medical management. Failure was defined as worsening of infection necessitating therapeutic penetrating keratoplasty or evisceration. RESULTS: Of 21 cases, 2 were lost to follow up, 1 was diagnosed on histopathology, and 1 received only topical linezolid. Characteristic microbiological features were noted on 10% potassium hydroxide calcofluor white wet mount in 20/21 (95.23%) and Gram stain in 18/21 (85.71%). On triple drug regimen, 14/17 cases (82.35%) resolved. Average time to resolution was 87.64 ± 44.44 days. More than 60% infiltrates (13/21) were large, and 66.66% infiltrates resolved in 109.3 ± 57.06 days. Of the 5 failures, 4 needed therapeutic keratoplasty and 1 needed evisceration. All grafts failed. CONCLUSIONS: The dual topical drug regimen with oral linezolid has good cure rates (over 80%) for Pythium keratitis over prolonged duration. It is recommended to persevere with medical therapy even in large infiltrates because more than two thirds resolved.

Linezolid shows high safety and efficacy in the treatment of Pythium insidiosum keratitis in a rabbit model.

Efficacy and safety of three antibiotics (Linezolid-LZ, 0.2%; Azithromycin-AZ, 1%; Tigecycline-TG, 1%) were determined in the treatment of Pythium insidiosum keratitis in rabbits. Infection of right eye of 38 rabbits was induced by standard intracorneal injection of P. insidiosum zoospores (left eye, intracorneal saline). Corneal infection developed in all right eyes. One hourly eye drops of one of the three antibiotics was instilled in both eyes (3 groups of 12 rabbits each) except in controls. Half of the rabbits in each group received intracorneal injection of the respective antibiotic after 4 days of starting eye drops. Clinical scoring of eyes was done over next 3 weeks. The reduction in scores post-treatment was significant for each drug (LZ: p < 0.025, AZ: p < 0.025, TG: p < 0.01). Scores with LZ (median change of 3) was significantly (p = 0.013) higher than TG (median change of 2) and comparable (p = 0.06) to AZ (median change of 3). Reduction in clinical scores in eyes receiving intracorneal antibiotics was not significantly different from the eyes that did not receive intracorneal antibiotics (p = 0.73). While no adverse effect of LZ was seen in the control corneas, 66-100% of rabbits showed reaction to AZ and TG. Histopathology showed severe inflammation in all infected corneas and intraocular extension in some of the rabbits with poor response. The success rate was noted to be 16.7%, 25% and 50% in AZ, TG and LZ respectively (p = 0.45). LZ demonstrated superior efficacy and safety and can be considered for trial in human disease.

Mefenoxam, Itraconazole, and Terbinafine Combination Therapy for Management of Pythiosis in Dogs (Six Cases).

Pythium insidiosum is an oomycete that encysts in the skin or gastrointestinal tract, leading to pythiosis. Pythiosis is reported in tropical and subtropical climates, affecting dogs and rarely cats. Surgical resection is the treatment of choice; however, cases present late in the disease and lesions are often nonresectable. Medical management is typically unsuccessful, with uncommon exceptions; however, mefenoxam, an agricultural fungicide, has in vitro efficacy against P insidiosum. We describe the use of mefenoxam, itraconazole, and terbinafine (MIT) in five dogs with gastrointestinal pythiosis and one dog with cutaneous pythiosis. Two of the gastrointestinal cases had disease extending to surgical margins and received MIT: resolution of clinical signs and seronegativity occurred after 189-193 days. Another case underwent surgical resection and MIT. The dog improved but subsequently developed a rectal mass, which responded to addition of prednisone and immunotherapy. Two cases were treated with MIT alone, and response varied. Efficacy of MIT in cutaneous pythiosis could not be determined. MIT may result in improved survival and seronegativity in dogs with incompletely resected gastrointestinal pythiosis. Mefenoxam is EPA registered, and extralabel use under the Animal Medicinal Drug Use Clarification Act does not apply. Additional research is recommended before use.

Role of Topical Ethanol in the Treatment of Pythium insidiosum Keratitis-A Proof of Concept.

PURPOSE: To study the efficacy and safety profile of topical absolute ethanol in the treatment of Pythium insidiosum keratitis. METHOD: Microbiological, clinical, and histopathological assessments were performed to study the effects of absolute ethanol on P. insidiosum keratitis. In addition, infrared spectroscopy was performed to assess the corneal penetration of ethanol. RESULTS: Microbiological tests revealed that ethanol inhibited the growth of P. insidiosum at concentrations even as low as 20% as compared to Candida albicans and Aspergillus flavus, where minimal growth was noted. However, at 40%, 60%, 80%, and 99.9% of ethanol, complete inhibition of growth was noted for all organisms. Histopathology of the absolute ethanol-treated cadaveric cornea showed the compaction of collagen and no stromal necrosis. Infrared spectroscopy revealed secondary structural changes in collagen in the ethanol-treated cadaveric corneas as compared to controls. Clinically, 1 case with a recurrence of P. insidiosum after therapeutic penetrating keratoplasty resolved with the topical application of absolute ethanol, and the other case, where corneal scraping had grown Pythium within 24 hours, failed to grow the organism from the corneal button which was treated with absolute alcohol preoperatively. After therapeutic penetrating keratoplasty, there was no recurrence, and the graft epithelized well. CONCLUSIONS: Ethanol can be considered an option for treating P. insidiosum keratitis; however, the exact dose and strength of ethanol which will be most effective needs further work.

In vivo effect of minocycline alone and in combination with immunotherapy against pythium insidiosum.

This study examined the effect of minocycline alone and in combination with immunotherapy against pythiosis. Twenty rabbits, aged three months old and subcutaneously inoculated with Pythium insidiosum zoospores were divided into four groups (n = 5): treated with minocycline (10 mg/kg/day twice daily), treated with immunotherapy (34 mg subcutaneously every 14 days), treated with minocycline plus immunotherapy, and untreated (control group). The treatments were started 30 days after inoculation and continued for 70 days. The subcutaneous nodular injury areas in infected groups were measured every seven days after the beginning of treatment. Only the rabbits that developed lesions were selected for this study. When compared with the control group over 70 days, the minocycline and minocycline plus immunotherapy groups of rabbits with pythiosis showed significantly reduced injuries. The histopathology showed the presence of inflammation, macrophages and eosinophils. Grocott's staining revealed irregular hyphae-like structures that were ramified and occasionally septate. Our results suggest that minocycline has fungistatic activity and that the combination of minocycline and immunotherapy is more effective than the individual therapies tested.