OBJECTIVE: To assess outcomes of keratoplasty performed in patients diagnosed with keratitis caused by Pythium insidiosum (PI).
METHODS: Retrospective review.
METHODS: Preoperative, intra operative and post operative data of patients diagnosed with PI keratitis and who underwent keratoplasty for their condition from January 2020 to December 2021 were collected from the central patient database of a tertiary eye care hospital in India. The data were analyzed for anatomic success, elimination of infection, graft survival, incidence of repeat keratoplasty, final visual acuity and varied complications.
RESULTS: In total, 16 eyes underwent penetrating keratoplasty for PI keratitis during the study period. Mean time to keratoplasty from onset of symptoms was 31.3 days and mean graft size was 10.4 mm. Nine out of the 16 cases had recurrence of infection following surgery, seven of which required a repeat keratoplasty for elimination of infection. Mean graft size for repeat keratoplasty performed in recurrent cases was 11.7 mm. Globe was successfully salvaged in 14 out of 16 patients (87.5 %). Three grafts remained clear at 6-month follow up while 11 grafts failed. Mean improvement in uncorrected visual acuity from 2.32 to 2.04 logMAR was observed at last follow up. Endo-exudates, graft infiltration, graft dehiscence, secondary glaucoma and retinal detachment were the various complications noted after keratoplasty.
CONCLUSIONS: PI keratitis is a tenacious and potentially blinding condition. Keratoplasty remains the choice of treatment in this condition, however recurrence of disease and graft failure are common. Large sized grafts, meticulous per-operative removal of infection, adjuvant cryotherapy, and intraoperative and post operative use of antibiotics can help in improving outcome of keratoplasty in these patients.

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BACKGROUND: To report the nursing experience of a case of corneal contact lens wearer receiving the 2nd keratoplasty due to corneal ulcer and perforation caused by Pythium insidiosum infection.
METHODS: A 30-year-old female patient had blurred vision after deep anterior lamellar keratoplasty for a right corneal ulcer. At the 5th week, the right eye appeared the symptoms, such as redness and pain. The anterior segment photography was performed on the eye, and the result showed that the epithelium was missing in the right eye lesion area, and a large number of longitudinal and transversal streaks were visible from the epithelium to the stroma, with fungus filaments to be discharged. Upon macro-genome sequencing of the corneal secretion, a P. insidiosum infection was observed. Then, the patient underwent the keratoplasty, and 3 weeks later, the corneal implant showed a tendency to dissolve, the sutures were partially loosened, and the eye was almost blind. Subsequently, the patient was admitted to our hospital and subject to the 2nd penetrating keratoplasty of the right eye (allograft). After surgery, linezolid and azithromycin injections were given through intravenous drip and local drip of the eye for anti-inflammation, and tacrolimus eye drops for antirejection.
RESULTS: Postoperatively, the patient showed signs of recovery with slight corneal edema and visible pupil, leading to discharge with improved vision. The corneal implant was normal 1 week after surgery and the vision of the right eye was hand move/before eye at the 6th month of follow-up. Continuous care and removal of sutures 3 months post-surgery contributed to a successful outcome, with the patient achieving hand motion vision 6 months after the procedure.
CONCLUSIONS: Corneal ulcer caused by P. insidiosum infection not only needs timely and effective keratoplasty intervention, but also requires perfect nursing measures.

A clinical case of an adult horse with invasive, ulcerative, proliferative, pyogranulomatous disease of the skin (tumor) in the shoulder region is presented. The mass had a granulomatous and crater-shaped appearance, with serosanguinous discharge and the presence of fistulas with caseous material. The tumor was removed by surgery and sent to the laboratory for diagnosis. Histopathology was performed using Grocott-Gomori methenamine silver stain. The presence of necrotic material, fibrosis, infiltrated cells, and brown-colored hyphae, characteristic of members of the genus Pythium, were observed. To identify the infecting species, conventional PCRs for the amplification of the ITS-1 was carried out. Histopathological and PCR tests confirmed infection by a Pythium insidiosum strain closely associated with previous records from the US and Central America. Our report represents the first molecularly confirmed case of equine pythiosis in Mexico.

Pythiosis is caused due to a filamentous eukaryotic micro-organism called Pythium insidiosum and the disease occurs commonly in horses and cattle. Subcutaneous pythiosis infection in humans is rare with no clear clinical guidelines for treatment. We present a case of a man in his 20s with non-resolving ulcers noted over lower extremity after exposure to swamp water draining animal remains. The patient received several courses of oral antibiotics with no improvement in symptoms before getting admitted to our institution. A diagnosis of subcutaneous pythiosis was made after deep wound culture following debridement detected P. insidiosum by use of PCR. Due to the rare incidence of such infection in humans and no clear guidelines available for treatment, the case was discussed with infectious disease specialists outside our institution and with veterinary physicians. An emergent approval for use of immunotherapy in conjunction with surgical debridement and antimicrobials was obtained from Food and Drug administration. The patient underwent successful treatment of infection and skin graft following treatment.

BACKGROUND: Pythiosis is a rare disease with high mortality, with over 94% of cases reported from Thailand and India. Prompt diagnosis and surgery improves patient outcomes. Therefore, continuing professional development (CPD) is essential for early recognition. However, a needs assessment related to a pythiosis CPD program has not been performed.
OBJECTIVE: We conducted a needs assessment to develop a pythiosis CPD program.
METHODS: We conducted a survey study with 267 King Chulalongkorn Memorial Hospital residents (141 internal medicine (IM) residents and 126 surgery residents). A 30-item survey consisting of a knowledge assessment, demographic section, and an attitudes portion was distributed both electronically and via paper. The data was analyzed with descriptive and inferential statistics.
RESULTS: Sixty-seven percent completed the survey (110/141 IM residents, 70/126 surgery residents). The mean score [95% confidence interval] on the knowledge assessment was 41.67% [39.64%-43.69%] across all objectives. The three domains with the highest scores were pythiosis risk factors (67.22% correct), microbiologic characteristics (50.83%), and radiographic interpretation (50.56%). The three domains with the lowest scores were laboratory investigation (15.00%), epidemiology (29.17%), and symptomatology (30.83%). Most participants noted that the program should be online with both synchronous and asynchronous sessions, with a preferred length of 60-90 minutes per session.
CONCLUSIONS: The pythiosis CPD program should emphasize education regarding symptomatology, laboratory investigation, and epidemiology, all of which are critical for the early detection of pythiosis to decrease mortality from this devastating disease. Most respondents felt this program was necessary and should be implemented in a virtual blended format.

Unlike most pathogenic oomycetes, Pythium insidiosum infects humans and animals instead of plants. P. insidiosum has three clinically relevant genotypes/clades that cause a severe disease called pythiosis. To develop strategies for infection control, it is necessary to understand the biology and pathogenesis of this pathogen. Investigating the evolutionary mechanisms behind the host-specific adaptation is vital, and comparative genomic analysis can help with this. To facilitate genomic analysis, an online bioinformatics tool called P. insidiosum (Pins) Gene Table v2.0 was developed. This tool includes genomic data from 37 genetically diverse P. insidiosum strains and four related species. The database contains 732,686 genes, grouped into 80,061 unique clusters and further divided into core and variable categories at genus, species, and genotype levels. A high-resolution phylogenomic relationship among P. insidiosum strains and other oomycetes was projected through hierarchical clustering and core gene analyses. 3156 P. insidiosum-specific genes were shared among all genotypes and may be responsible for causing disease in humans and animals. After comparing these species-specific genes to the MvirDB database, 112 had significant matches with 66 known virulence proteins, some of which might be involved in vascular occlusion, which is a pathological feature of pythiosis. The correlation of genotypes, geographic origins, and affected hosts of P. insidiosum suggests that clade-I strains are more specific to animals, while clade-II/III strains are more specific to humans. The clade-specific genes might link to host preference. In summary, Pins Gene Table v2.0 is a comprehensive genome database accessible to users with minimal bioinformatics experience for the analysis of P. insidiosum genomes.

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.

The mechanisms of Pythium insidiosum-antigen (PIA) immunotherapy activating a patient\'s immune system are unknown. We evaluated the interleukin-8 (IL-8) serum levels during P. insidiosum infection and after vaccination with PIA in vascular pythiosis cases. Furthermore, we studied the anti-P. insidiosum activity of neutrophils stimulated with various concentrations of PIA ex vivo in 3 strains of P. insidiosum isolated from vascular pythiosis patients. IL-8 serum levels were evaluated using the ELISA technique. We assessed the effect of PIA-stimulated neutrophils on the viability of zoospores using MTT assay, visualized neutrophil extracellular trap (NET) formation via microscopy, and measured the levels of double-stranded DNA (dsDNA) using PicoGreen dsDNA quantitation assay in 3 strains of P. insidiosum isolated from vascular pythiosis patients. Serum levels of IL-8 gradually lowered from the early to the end phases of vaccination with PIA among the surviving group of vascular pythiosis cases. Neutrophils stimulated with 0.01 µg/ml PIA reduced zoospore viability significantly compared to PIA-unstimulated neutrophils for strain 1 and strain 3 (p < .05). Neutrophils stimulated with 0.01, 0.1, 1, and 10 µg/ml PIA exhibited significantly lower zoospore viability than PIA-unstimulated neutrophils for strain 2 (p < .05). IL-8 can be used as a biomarker for monitoring vascular pythiosis cases treated with the PIA vaccine. Also, anti-P. insidiosum activity of PIA-stimulated neutrophils was probably due to the disruption of cellular activity in zoospores rather than the mechanisms based on the formation of NETs.

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.