Oomicidal activity of polypyrrole nanoparticles against Pythium insidiosum.

This study evaluated in-vitro action of a new molecule, the polypyrrole nanoparticles (Ppy-NP), against Pythium insidiosum isolates using M38-A2/CLSI; the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Additionally, changes in the hyphae wall of P. insidiosum CBS 575.85 treated with Ppy-NP were evaluated by scanning electron microscopy (SEM). The MIC100 and MOC for all isolates ranged from 8 to 32 µg mL-1, and the MIC90 and MIC50 were 16 µg mL-1. The SEM showed structural damage to the hyphae of P. insidisoum treated with Ppy-NP, as hyphae surfaces with less turgidity were found, thereby showing scaling and ruptures compared to the control (untreated hyphae). Our findings highlighted the anti-P. insidiosum properties of Ppy-NP proved to be a promising candidate for research using pythiosis experimental models.

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.

New insights on evolutionary aspects of Pythium insidiosum and other peronosporaleans.

BACKGROUND: The evolution of pathogenic mechanisms is a major challenge, which requires a thorough comprehension of the phylogenetic relationships of pathogens. Peronosporaleans encompasses a heterogeneous group of oomycetes that includes some animal/human pathogens, like Pythium insidiosum. OBJECTIVE: We analysed here the phylogenetic positioning and other evolutionary aspects related to this species and other peronosporaleans, using a multi-locus approach with one mitochondrial and three nuclear genes. METHODOLOGY: Phylogenetic patterns of 55 oomycetes were inferred by maximum likelihood and Bayesian analysis, and a relaxed molecular clock method was applied to infer the divergence time of some peronosporaleans branches. RESULTS: Pythium insidiosum was monophyletic with a major and polytomous clade of American isolates; however, Pythium spp. was found to be paraphyletic with Phytopythium sp. and Phytophthora spp. In general, peronosporaleans subdivided into four lineages, one of which evidenced a close relationship of P insidiosum, P aphanidermatum and P arrhenomanes. This lineage diverged about 63 million years ago (Mya), whereas P insidiosum diversified at approximately 24 Mya. The divergence of American and Thai isolates seems to have occurred at approximately 17 Mya, with further American diversification at 2.4 Mya. CONCLUSION: Overall, this study clarifies the phylogenetic relationships of P insidiosum regarding other peronosporaleans in a multi-locus perspective, despite previous claims that phylogenomic analyses are needed to accurately infer the patterns and processes related to the evolution of different lineages in this group. Additionally, this is the first time that a molecular clock was applied to study the evolution of P insidiosum.

Intradermal injection of Pythium insidiosum protein antigens for improved diagnosis and treatment of pythiosis in an experimental model.

The oomycetous pathogen Pythium insidiosum is the causative agent of pythiosis, a life-threatening disease that affects animals and humans. This infectious disease is difficult to treat, and early and accurate diagnosis is critical for effective treatment. In this sense, this study aimed to evaluate the intradermal (ID) injection of P. insidiosum protein antigens (PiPA) for the diagnosis and treatment of pythiosis using an experimental model. For diagnostic purposes, PiPA were injected by the ID route in the following groups of rabbits: (a) control; (b) previously immunized with PiPA injected by the subcutaneous (SC) route; and (c) infected with P. insidiosum zoospores. For treatment purposes, rabbits with pythiosis were also treated with PiPA by the ID or SC routes. Mean induration sizes were different at 24 h and 72 h readings when compared to the control group. Sensitivity of the protocol was 100% at 24 h and 80% at 72 h, with 100% specificity in both readings. PiPA treatment using ID or SC routes did not result in significant differences in lesion sizes and cure rates; however, serum levels of interferon-gamma were higher in SC route. This study demonstrates the applicability of PiPA ID for diagnosis and treatment of pythiosis in an experimental model.

Post-weaning piglets fed with different levels of fungal mycotoxins and spray-dried porcine plasma have improved weight gain, feed intake and reduced diarrhea incidence.

Mycotoxins are responsible for economic losses in the swine production industry, especially during post-weaning, when piglets are physiologically immature. Spray-dried porcine plasma (SDPP), added to pig diets, may help reduce losses due to mycotoxins. This work investigates the effects of SDPP in post-weaning piglets fed with diets containing natural contaminants or with more contaminants (co-contamination by mycotoxins). Fifty-six castrated weaned piglets were used in a randomized 2 (0 and 6% of SDPP) x 2 (natural contamination or co-contamination with mycotoxin) factorial design, with seven experimental units of two piglets each. The natural contaminants were 0.95 µg/kg aflatoxins +450 µg/kg fumonisins. The co-contaminated diet contained 300 µg/kg aflatoxins +8000 µg/kg fumonisins. Animals were fed 15 days with experimental diets. Feed intake, weight gain, feed efficiency, diarrhea incidence, and economic feasibility of SDPP treatement were evaluated in three periods of five days each. There was no interaction (P < 0.05) between mycotoxins levels and SDPP. Feed intake, weight gain and feed efficiency were higher (P < 0.05) in diets supplemented with SDPP. Animals fed with SDPP showed lower (P < 0.05) diarrhea incidence in the 1-10 day and 1-15 day periods. The experimental dose of mycotoxins reduced (P < 0.05) weight gain at 11-15 days. SDPP proved to be economical feasible over the total experimental period (1-15 days). Spray-dried plasma improved weight gain, feed intake and reduced diarrhea incidence in piglets post-weaning, but did not correlate with various levels of mycotoxins.

In vitro interactions of azoles and echinocandins against clinical strains of Aspergillus flavus.

Combinations of an azole (itraconazole, voriconazole, or posaconazole) with an echinocandin (caspofungin, micafungin, or anidulafungin) were tested against 20 clinical isolates of Aspergillus flavus according to EUCAST guidelines. The interactions were determined using two endpoints-minimal effective concentration (MEC) and minimal inhibitory concentration (MIC)-via calculation of the fractional inhibitory concentration (FIC) index. A higher prevalence of synergistic interactions was observed for MIC, whereas indifference was the most frequent outcome according to MEC among the 20 strains. Combined treatment of A. flavus with these two important classes of antifungals should be explored further in in vivo studies.

Comparison Between Etest and Broth Microdilution Methods for Testing Itraconazole-Resistant Aspergillus fumigatus Susceptibility to Antifungal Combinations.

The checkerboard broth microdilution assay (BMD) is the most frequently used method for the in vitro evaluation of drug combinations. However, its use to evaluate the effect of antifungal drugs on filamentous fungi is sometimes associated with endpoint-reading difficulties, and different degrees of interaction are assigned to the same drug combination. We evaluated combinations of the azoles, itraconazole, posaconazole, and voriconazole, with the echinocandins, anidulafungin, caspofungin, and micafungin, against 15 itraconazole-resistant Aspergillus fumigatus clinical strains via the checkerboard BMD and Etest assay. Readings after 24 and 48 h, considering the two reading endpoints, the minimum inhibitory concentration (MIC) and minimum effective concentration (MEC), were performed for both methods. Our results showed that the correlation coefficients between the BMD and Etest methods were quite diverse to the drug combinations tested. The highest correlation coefficients of the Etest with the BMD assays (MEC and MIC reading) were the Etest-MIC reading at 24 h and the Etest-MEC reading at 48 h. Improvements in experimental conditions may increase the correlation between the two methods and ensure that Etest assay can be safely used in the evaluation of antifungal combinations against Aspergillus species.

In vitro susceptibility of the oomycete Pythium insidiosum to metallic compounds containing cadmium, lead, copper, manganese or zinc.

Pythium insidiosum is an aquatic oomycete that causes pythiosis, an important and severe disease of difficult treatment that affects humans, domestic and wild animals. This infection is often described in horses in Brazil and humans in Thailand. In clinical practice, we have observed many cases that do not respond to available therapies, indicating the need to explore alternative therapeutic approaches. In this sense, studies using metal compounds in conjunction with available antimicrobial agents have been demonstrated greater antimicrobial activity. Thus, in this research, we tested in vitro activities of metallic compounds containing cadmium, lead, copper, manganese, or zinc against 23 isolates of P. insidiosum. The assays were performed by broth microdilution based on CLSI M38-A2 document. The minimum inhibitory and fungicidal concentrations were established for all isolates. Copper acetate and cadmium acetate showed the highest inhibitory effects, with minimal inhibitory concentration ranging from 4-64 µg/ml and 16-256 µg/ml, respectively. The mean geometric for minimal fungicidal concentrations were, respectively, 26 µg/ml and 111.43 µg/ml for copper acetate and cadmium acetate. These results suggest that copper and cadmium can inhibit P. insidiosum growth, highlighting the greater inhibitory activity of copper acetate. In addition, our results propose that copper and/or cadmium compounds can be used in upcoming researches to formulate effective new complexed drugs against P. insidiosum in in vitro and in vivo experimental models.

Activity of Combined Antifungal Agents Against Multidrug-Resistant Candida glabrata Strains.

In this study, we evaluated the in vitro activity of echinocandins, azoles, and amphotericin B alone and in combination against echinocandin/azole-sensitive and echinocandin/azole-resistant Candida glabrata isolates. Susceptibility tests were performed using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute document M27-A3. The checkerboard method was used to evaluate the fractional inhibitory concentration index of the interactions. Cross-resistance was observed among echinocandins; 15% of the isolates resistant to caspofungin were also resistant to anidulafungin and micafungin. Synergistic activity was observed in 70% of resistant C. glabrata when anidulafungin was combined with voriconazole or posaconazole. Higher (85%) synergism was found in the combination of caspofungin and voriconazole. The combinations of caspofungin with fluconazole, posaconazole and amphotericin B, micafungin with fluconazole, posaconazole and voriconazole, and anidulafungin with amphotericin B showed indifferent activities for the majority of the isolates. Anidulafungin combined with fluconazole showed the same percentage of synergism and indifference (45%). Antagonism was detected in 50% of isolates when micafungin was combined with amphotericin B. Combinations of echinocandins and antifungal azoles have great potential for in vivo assays which are required to evaluate the efficacy of these combinations against multidrug-resistant C. glabrata strains.

Antifungal activities of diphenyl diselenide and ebselen alone and in combination with antifungal agents against Fusarium spp.

Herein, we describe the in vitro activity of a combination of the organoselenium compounds diphenyl diselenide and ebselen alone and in combination with amphotericin B, caspofungin, itraconazole, and voriconazole against 25 clinical isolates of Fusarium spp. For this analysis, we used the broth microdilution method based on the M38-A2 technique and checkerboard microdilution method. Diphenyl diselenide (MIC range = 4-32 µg/ml) and ebselen (MIC range = 2-8 µg/ml) showed in vitro activity against the isolates tested. The most effective combinations were (synergism rates): ebselen + amphotericin B (88%), ebselen + voriconazole (80%), diphenyl diselenide + amphotericin B (72%), and diphenyl diselenide + voriconazole (64%). Combination with caspofungin resulted in low rates of synergism: ebselen + caspofungin, 36%, and diphenyl diselenide + caspofungin, 28%; combination with itraconazole demonstrated indifferent interactions. Antagonistic effects were not observed for any of the combinations tested. Our findings suggest that the antifungal potential of diphenyl diselenide and ebselen deserves further investigation in in vivo experimental models, especially in combination with amphotericin B and voriconazole.

In Vitro Activity of Melaleuca alternifolia (Tea Tree) in Its Free Oil and Nanoemulsion Formulations Against Pythium insidiosum.

Pythium insidiosum is an important aquatic oomycete which can cause pythiosis in both animals and humans. This microorganism shows low susceptibility to antifungal drugs available. This study analyzed the in vitro antimicrobial activity of Melaleuca alternifolia in its free oil (FO) and nanoemulsion (NE) formulations against Brazilian P. insidiosum isolates. The antimicrobial activity evaluation was performed by the broth microdilution method according to CSLI M38-A2 document adapted to phytopharmaceuticals. Twenty-six P. insidiosum isolates were evaluated, and the minimum inhibitory concentration was determined at 100 % growth inhibition. Melaleuca alternifolia essential oil or FO was obtained commercially. The NE containing 1 % M. alternifolia essential oil was prepared by the spontaneous emulsification method. All P. insidiosum isolates evaluated showed minimum inhibitory concentrations (MIC) ranging from 531.5 to 2125 µg/mL for the FO formulation; MIC50 and MIC90 showed values between 1062.5 and 2125 µg/mL, respectively. When the NE formulation was evaluated, MIC values ranged from 132.7 to 2125 µg/mL and both MIC50 and MIC90 corresponded to 1062.5 µg/mL. FO and NE formulations of M. alternifolia showed antimicrobial activity against P. insidiosum. This study demonstrated that M. alternifolia oil can be an additional therapy in pythiosis treatment; however, further studies are needed to evaluate the applicability of the plant essential oils in the treatment of clinical pythiosis.

In Vitro and In Vivo Efficacy of Amphotericin B Combined with Posaconazole against Experimental Disseminated Sporotrichosis.

We evaluated the combination of posaconazole with amphotericin B in vitro and in a murine model of systemic infections caused by Sporothrix brasiliensis and Sporothrix schenckii sensu stricto. In vitro data demonstrated a synergistic effect, and although posaconazole alone was effective against sporotrichosis, efficacy in terms of survival and burden reduction was increased with the combination. This combination might be an option against disseminated sporotrichosis, especially when itraconazole or amphotericin B at optimal doses are contraindicated.

Cutaneous, respiratory and hepatic aspergillosis in Brazilian white Pekin mallards (Anas platyrhynchos).

Aspergillosis is one of the most frequent mycosis affecting avian species. Here is reported an outbreak of aspergillosis affecting 60-day-old white Pekin mallards (Anas platyrhynchos). About 10% of animals in a lot of 200 mallards from a commercial husbandry presented respiratory disorders and skin lesions at slaughter. Three out of 13 animals sent to diagnosis showed, simultaneously, airsacculitis, lung and liver presenting white nodules with variable diameters and elevated, yellowish brown, crusted, multifocal skin lesions located at the base of the feather follicles in the breast. Histopathological examination of lung and liver samples revealed nodules of different sizes with small areas of necrosis surrounded by intense granulomatous inflammation and the presence of fungal hyphae. The skin samples showed dermatitis surrounding a severe necrotizing folliculitis, associated with fungal hyphae. Mycological evaluation of tissues allowed the isolation of Aspergillus fumigatus from the skin samples and Aspergillus flavus from lungs and liver samples. The application of quicklime (CaO) in the litter as part of the disinfection procedures could have contributed to the development of skin lesion in the mallards, predisposing the fungal installation in the damaged site. The occurrence of cutaneous lesions associated with A. fumigatus is a rare manifestation of aspergillosis in birds, and this appears to be the first case reported in white Pekin mallards.

In vitro photodynamic inactivation of Sporothrix schenckii complex species.

Photodynamic therapy has been applied successfully against cutaneous and subcutaneous mycoses. We applied methylene blue as a photosensitizing agent and light emitting diode (InGaAlP) against Sporothrix schenckii complex species in an in vitro assay. The viability of the conidia was determined by counting colony-forming units. Methylene blue in conjunction with laser irradiation was able to inhibit the growth of all tested samples. The in vitro inhibition of Sporothrix spp. isolates by laser light deserves in vivo experimental and clinical studies since it may be a promising treatment for cutaneous and subcutaneous sporotrichosis.

In vitro reproduction of the life cycle of Pythium insidiosum from kunkers' equine and their role in the epidemiology of pythiosis.

Pythium insidiosum is an important pathogen of mammals' species, including humans. Equine is the main species affected by this oomycete. P. insidiosum requires an aquatic environment to develop its life cycle, and the susceptible hosts are contaminated when they contact the microorganism in swampy areas. The equine pythiosis is characterized by the formation of irregular masses within the cutaneous lesions, called kunkers, which easily detach from the lesion. From these structures, it is possible to isolate P. insidiosum in pure cultures. The present study aimed to reproduce in vitro the life cycle of P. insidiosum from kunkers of equine clinical lesions. Fifteen kunkers from different horses were tested. It was observed that the discharge of zoospores occurred after 24-48 h of incubation at 37 °C in, respectively, 40 and 47 % of the kunkers evaluated. Only two samples showed no development of the asexual cycle of P. insidiosum under the conditions tested. It was possible to demonstrate that kunkers are able to restart the asexual cycle of P. insidiosum. Based on our in vitro results, we highlight the importance of these structures in the epidemiology of the pythiosis, since kunkers can be a potential source of contamination of this oomycete for aquatic environments.

Equidae pythiosis in Brazil and the world: a systematic review of the last 63 years (1960-2023).

This systematic review compiles reports of clinical pythiosis in horses, mules and donkeys from 1960 to 2023 worldwide, focusing on Brazil. We searched databases and included 71 articles detailing clinical characteristics, geographic distribution, epidemiology, diagnostic methods, therapies, and outcomes. The results showed that publications on equine pythiosis have significantly increased since 2010. Brazil reported the highest incidence, comprising 55% of cases, predominantly in the southern, northeastern, and central-western regions during summer and autumn. Cutaneous pythiosis was the most prevalent form, generally presenting as single lesions in the appendicular region, and affected females more than males. Diagnosis typically involved histopathology, used alone or with other methods. Various treatments have been employed, with surgery, often combined with chemotherapy and immunotherapy, being the most common. Notably, 80.84% of treated animals recovered, highlighting the effectiveness of these therapies in enhancing survival rates. The limitations of the study included the lack of data in published case reports, which made it difficult to collect and calculate epidemiological data. Additionally, we recognize that pythiosis in Brazil is underreported, since this disease does not have mandatory notification and several cases are not registered and/or reported in the literature. Lastly, it is hypothesized that equid pythiosis may be more widespread than currently known, and its real occurrence in Brazil remains uncertain.

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.

E-ADA activity in lymphocytes of an experimental model of pythiosis treated with immunotherapy.

Pythiosis is a life-threatening disease caused by the oomycete Pythium insidiosum. Some authors have suggested the involvement of a Th2-like immune response in the infected host, which leads to extensive tissue damage. The switch from a Th2 to a Th1 response pattern is one hypothesis to explain the curative properties of immunotherapy. Taking into account the importance of immunotherapy for pythiosis treatment and the contribution of adenine nucleotides in the immunoregulation of the host, we evaluated the ecto-adenosine deaminase (E-ADA; EC 3·5.4·4) activity in lymphocytes from rabbits inoculated with P. insidiosum. Rabbits were inoculated with 1 milliliter of zoospores subcutaneously injected into the lateral thorax; after developing lesions, the rabbits received eight doses of immunotherapy. E-ADA activity was measured in lymphocytes and the adenine nucleotides and adenosine levels were quantitatively determined in serum. Rabbits with characteristic lesions of pythiosis showed a decreased E-ADA activity (82·36%), a decreased adenosine triphosphate concentration (54·04%) and a higher adenosine concentration (2·51 fold), when compared with controls, after 28 days of inoculation. However, after the immunotherapy, the rabbits showed an increase in the E-ADA activity when compared with control (78·62%), contributing for the change in the immune response. Our results reinforce the hypothesis that the change from a Th2 to a Th1 immune response with the participation of the purinergic system could be responsible for the curative properties of immunotherapy.

In vitro influence of temperature on the biological control activity of the fungus Duddingtonia flagrans against Haemonchus contortus in sheep.

Recently, research for alternative methods to combat gastrointestinal parasites has increased, and the biological control activity of the fungus Duddingtonia flagrans stands out. In this study, the possible influence of temperature on the nematophagous activity of D. flagrans, after gastrointestinal passage, against Haemonchus contortus in sheep was analysed. Four female sheep, between 2 and 3 years of age and weighing between 40 and 50 kg, were used. Two sheep were parasitised with H. contortus, while two other sheep were dewormed. Before the collection of faeces, one of the dewormed animals received a dosage of 1 × 10(6) chlamydospores of D. flagrans, lyophilised in gelatin capsules, for three consecutive days. The faeces were collected with collector bags, mixed, and then separated as samples with (fungus; 800 eggs per gram (EPG) of faeces) or without fungus (control; 900 EPG). Each sample (five replicates) was maintained in a biochemical oxygen demand incubator under different temperatures (5, 10, 15, 20, 25, 30, or 35 °C) for 21 days, followed by determination of the larval recovery. Compared to the control group, the best temperature for fungal action was 30 °C, while no larvae were recovered at 5 °C. At 10 °C, fungal action was detected, yet there was no significant difference in the percent larval reduction between all temperatures, demonstrating that larval presence seems to be the main factor affecting the nematophagous action of D. flagrans. Temperature does not appear to be a limiting factor in the biological control activity of D. flagrans against H. contortus, but larval presence, which was not observed at 5 °C, is mandatory. At low temperatures, which are typically suboptimal conditions for fungal and larval development, the lyophilised D. flagrans reduced the number of H. contortus larvae, which demonstrates the biological control potential and the potential use of D. flagrans in the subtropics.

Antifungal activities of diphenyl diselenide alone and in combination with fluconazole or amphotericin B against Candida glabrata.

Here, we evaluated combinations of diphenyl diselenide [(PhSe)2] with fluconazole and amphotericin B in a checkerboard assay against clinical Candida glabrata strains. Minimal inhibitory concentration (geometric mean) ranged from 0.25 to >64 (5.16 µg/mL) for (PhSe)2, 1 to 32 (5.04 µg/mL) for fluconazole and 0.06 to 0.5 (0.18 µg/mL) for amphotericin B. Synergistic (76.66 %) and indifferent (23.34 %) interactions were observed for (PhSe)2 + amphotericin B combination. (PhSe)2 + fluconazole combination demonstrated indifferent (50 %) and antagonistic (40 %) interactions, whereas synergistic interactions were observed in 10 % of the isolates. New experimental in vivo protocols are necessary and will promote a better understanding of the antimicrobial activity of (PhSe)2 against C. glabrata and its use as an adjuvant therapy with antifungal agents.