Chompoonek Yurayart

Pythium insidiosum, the causative agent of pythiosis, remains an important pathogen in tropical and subtropical regions, where delayed diagnosis is associated with severe disease and poor clinical outcomes. We developed a loop-mediated isothermal amplification coupled with lateral flow dipstick (LAMP-LFD) assay for the rapid detection of P. insidiosum. The assay employs dual-labeled LAMP amplicons that are directly detected by a lateral flow dipstick, enabling simple visual interpretation without agarose gel electrophoresis. Analytical performance was evaluated using genomic DNA from 102 clinically relevant isolates, comprising 51 P. insidiosum isolates and 51 non-target organisms. The LAMP-LFD assay correctly detected all P. insidiosum isolates, achieving an inclusivity of 100.0% (95% CI: 93.0–100.0%), and correctly excluded 48 of 51 non-target organisms, yielding an exclusivity of 94.1% (95% CI: 83.8–98.8%). The assay demonstrated a detection limit of 10 fg of genomic DNA per reaction, representing at least 1000-fold greater analytical sensitivity than the reference multiplex PCR (m-PCR) assay. Clinical validation using 47 blinded animal tissue specimens showed a sensitivity of 90.3%, specificity of 87.5%, accuracy of 89.4%, and good agreement with the reference standard (culture and ITS sequence analysis) (κ = 0.76). In comparison, the m-PCR assay achieved a sensitivity of only 41.9%, indicating substantially lower clinical detection performance. In conclusion, direct LFD-based detection preserves the analytical performance of the established P. insidiosum LAMP assay while providing a clear and easily interpretable visual endpoint. The LAMP-LFD assay represents a rapid, sensitive, and practical tool for screening pythiosis, offering improved diagnostic performance and more reliable result interpretation.

Pythiosis is an emerging infectious disease caused by the oomycete Pythium insidiosum, affecting humans and animals primarily in subtropical and tropical regions. The pathogen is commonly found in swampy environments, and exposure can lead to diverse clinical manifestations. In humans, ocular and vascular infections predominate, whereas in animals, cutaneous/subcutaneous or gastrointestinal disease is more common. Medical therapy is frequently ineffective, and many patients require extensive surgical intervention. Advanced cases may progress to fatal outcomes. Therefore, early and accurate detection is critical for improving clinical management. This study evaluated a colorimetric loop-mediated isothermal amplification (c-LAMP) assay compared with an established multiplex PCR (m-PCR) assay for the detection of P. insidiosum in clinical specimens from animals with and without pythiosis. When tested on 47 frozen tissue samples, c-LAMP demonstrated superior diagnostic performance, with markedly greater sensitivity (83.9% vs. 41.9%), higher accuracy (78.7% vs. 61.7%), and a shorter turnaround time (65 vs. 180 min). However, c-LAMP yielded five false-positive results, likely due to nonspecific amplification or contamination. Improved sample-handling practices increased the specificity from 68.8% to 93.8%. In contrast, m-PCR showed perfect specificity (100.0%) but substantially lower sensitivity, resulting in a high false-negative rate. In conclusion, these preliminary findings suggest that c-LAMP is a promising rapid screening tool for suspected pythiosis, particularly in resource-limited settings. Nevertheless, confirmatory testing remains necessary for positive or equivocal c-LAMP results.

Malassezia pachydermatis is a yeast pathogen associated with recurrent skin and ear infections in dogs, often complicated by biofilm formation and reduced antifungal susceptibility. We aimed to evaluate the in vitro antifungal activity of essential oils and nanoemulsions of Zingiber cassumunar and Cymbopogon citratus compared with conventional antifungal agents against planktonic and biofilm forms of M. pachydermatis. Preliminary screening of six plant extracts was performed using 12 clinical isolates identified Z. cassumunar and C. citratus for nanoemulsion formulation. Antifungal susceptibility testing of conventional antifungal agents and nanoemulsions was subsequently conducted using 31 clinical isolates, and nanoemulsions were prepared by high-pressure homogenization. Both essential oils exhibited antifungal activity, and nanoemulsion formulations showed enhanced inhibitory effects compared with the crude oils. Biofilm-associated cells demonstrated reduced susceptibility, particularly to conventional antifungal agents. Terbinafine was the most potent agent against planktonic cells but showed reduced efficacy in biofilms. Nanoemulsions of Z. cassumunar and C. citratus exhibited improved activity against both forms. These findings suggest that nanoemulsification may enhance the in vitro antifungal performance of essential oils against M. pachydermatis biofilms. However, further studies, including mechanistic investigations and in vivo evaluations, are required to confirm their therapeutic potential and safety.

Healthy and asymptomatic cats may serve as reservoirs of fungal pathogens, facilitating transmission through direct contact or environmental contamination, and they may represent an underrecognized source of subclinical fungal infection in humans, particularly among cat owners and veterinarians. We evaluated the prevalence of fungal species in healthy cats and their owners, assessed potential cat–human transmission, identified feline lifestyle factors associated with Microsporum canis carriage, and evaluated antifungal susceptibility of the most prevalent isolated fungi. We collected 59 cat facial hair and 59 owner nail samples for fungal isolation and identification. Five fungal species were identified, M. canis, Aspergillus flavus, A. niger, A. fumigatus, and A. terreus, which were found in both cats and humans. Aspergillus spp. were the most frequently detected fungi in both groups. Significant associations between cats and owners were observed for M. canis (p = 0.010) and A. niger (p = 0.050). Long-haired cats showed a significantly higher prevalence of carrying M. canis (p = 0.024), while other lifestyle factors were not associated with fungal carriage. The antifungal susceptibility profiles of the tested fungi were broadly similar between feline and human isolates; however, resistance to itraconazole and amphotericin B was detected among Aspergillus spp. Healthy cats and their owners frequently share fungal species, especially M. canis, which suggests possible household zoonotic transmission. Long-haired cats are at higher risk of M. canis carriage.

Feline cryptococcosis is a common systemic mycosis that typically begins in the nasal cavity and can extend to the eyes, skin, and CNS. Objective monitoring during therapy remains limited. This study aimed to test whether serial serum cryptococcal antigen titers (CALAS) and a simple six-domain clinical score can track response, and to describe clinicopathologic trends during amphotericin B and fluconazole treatment. We retrospectively reviewed cases from 2014 to 2023 and analyzed a single-center cohort of 35 cats. Management used amphotericin B plus fluconazole for ~3–4 months, then fluconazole maintenance. Monthly assessments included CALAS (log₂), the clinical score (0–18), and routine hematology/biochemistry; mixed-effects models tested change over time. CALAS fell (mean log₂ 12.00 to 6.79), total leukocytes decreased (16.85 × 10⁹/L to 10.90 × 10⁹/L) with a similar neutrophil decline, alanine aminotransferase remained stable, and blood urea nitrogen/creatinine increased in some cats. The clinical score improved from 3.83 to 0.68. Seven cats developed azotemia during amphotericin B; the drug was stopped, renal values normalized in three, and four remained azotemic at the last follow-up. These findings support the use of CALAS and a simple score as practical monitoring tools and underscore the need for routine renal surveillance.

Background and Aim: Skin antisepsis plays a crucial role in pre-operative skin preparation, with chlorhexidine gluconate and alcohol being historically the preferred choice. However, concerns have risen regarding the development of bacterial resistance to chlorhexidine. Polyhexamethylene biguanide (PHMB) combined with Tris-ethylenediaminetetraacetic acid (Tris-EDTA) has recently emerged as a skin and wound antiseptic. This study aimed to compare the antibacterial efficacy and local safety of 2% chlorhexidine gluconate with 70% alcohol (CG+Alc) and 0.3% PHMB with 6% Tris and 1.86% EDTA (PHMB+Tris-EDTA) for pre-operative skin preparation in dogs. Materials and Methods: Twenty-four adult dogs underwent aseptic preparation on both sides of their ventral abdomens, with one side receiving CG+Alc and the other side receiving PHMB+Tris-EDTA, assigned randomly. Skin swab samples were collected pre-antisepsis and at 3-, 10-, and 60-min post-antisepsis to quantify bacterial colony-forming units (CFUs). Local skin reactions (erythema and edema) were evaluated after hair clipping, pre-antisepsis, and at 3-, 10-, 30-, and 60-min post-antisepsis. Results: There was no significant difference in bacterial CFU counts between the two antiseptic groups pre-antiseptic. Both solutions significantly reduced CFU counts (p < 0.05) at all post-antisepsis sampling times compared with pre-antisepsis. However, dogs treated with PHMB+Tris-EDTA showed a significantly higher incidence of edema at 10 min (p = 0.02) and 30 min (p = 0.003) and a higher incidence of erythema at 10 min (p = 0.043) post-antisepsis compared with CG+Alc. No skin reactions were observed in either group at 60 min post-antisepsis. Conclusion: CG+Alc and PHMB+Tris-EDTA reduced bacterial counts in pre-operative skin preparation in dogs. However, acute transient skin reactions were observed more frequently following the application of PHMB+Tris-EDTA. Keywords: alcohol, antisepsis, chlorhexidine gluconate, dogs, polyhexamethylene biguanide, skin preparation, tris-ethylenediaminetetraacetic acid.

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.

Aflatoxin B1 (AFB1) is the most potent naturally occurring carcinogen for humans and animals produced by the common fungus Aspergillus flavus (A. flavus). Aflatoxin (AF) contamination in commodities is a global concern related to the safety of food and feed, and it also impacts the agricultural economy. In this study, we investigated the AFB1-inhibiting activity of a new benzaldehyde derivative, 2-[(2-methylpyridin-3-yl)oxy]benzaldehyde (MPOBA), on A. flavus. It was found that MPOBA inhibited the production of AFB1 by A. flavus, with an IC₅₀ value of 0.55 mM. Moreover, the inhibition of conidiation was also observed at the same concentration. The addition of MPOBA resulted in decreased transcript levels of the aflR gene, which encodes a key regulatory protein for the biosynthesis of AF, and also decreased transcript levels of the global regulator genes veA and laeA. These results suggested that MPOBA has an effect on the regulatory mechanism of the development and differentiation of conidia, leading to the inhibition of AFB1 production. In addition, the cytotoxicity study showed that MPOBA had a very low cytotoxic effect on the Madin-Darby canine kidney (MDCK) cell line. Therefore, MPOBA may be a potential compound for developing practically effective agents to control AF contamination.

Sporotrichosis, an invasive fungal infection caused by Sporothrix schenckii, has emerged in Southeast Asia, affecting cats and posing a potential zoonotic risk to humans. We evaluated 38 feline sporotrichosis cases in and around Bangkok, Thailand, from 2017 to 2021. The isolates were phenotypically and genotypically characterized. The cats infected with sporotrichosis were mainly young adults, males, and domestic short hairs with uncontrolled outdoor access, and they lived in Bangkok. All isolates showed low thermotolerance and converted to the yeast phase at 35 °C. Based on the internal transcribed spacer region of rDNA sequences, our strains belonged to S. schenckii sensu stricto and clustered with clinical clade D. Based on the concatenated tree of calmodulin and beta-tubulin genes, five groups of S. schenckii were generated, and the monophyletic clade, Group II, of Thai strains was recognized. In vitro antifungal susceptibility testing demonstrated that the MIC50 of our isolates to amphotericin B, itraconazole, and posaconazole were within the limit of the species-specific epidemiological cutoff values, suggesting that the organisms were the wild type. Addressing the outbreak of feline sporotrichosis in Thailand by providing guidelines for diagnosis and effective treatment may help control the spread of disease and reduce the risk of cat-transmitted sporotrichosis to humans.