In vitro activity of antifungals in combination with essential oils against the oomycete Pythium insidiosum.

AIMS: The aim of this study was to investigate the in vitro susceptibility of Pythium insidiosum to combinations of the antifungal drugs terbinafine or itraconazole with Melaleuca alternifolia, Mentha piperita and Origanum vulgare essential oils (EOs). METHODS AND RESULTS: In vitro combinations of antifungal drugs with EOs were evaluated by checkerboard microdilution method against 20 Brazilian isolates of P. insidiosum. The tests were performed according to protocol M38-A2, and the interpretation of each combination result was based on the values of the fractional inhibitory concentration index. The combinations of itraconazole with EOs presented prominent synergistic effects against P. insidiosum isolates, and no antagonism was observed with these combinations. However, the combinations of terbinafine with EOs resulted in indifferent or antagonistic effects. CONCLUSIONS: The combination of plant-derived bioactive compounds with antifungal drugs may be an alternative therapy for the control of infections caused by P. insidiosum. Studies of new therapeutic protocols involving these proposed combinations are needed. SIGNIFICANCE AND IMPACT OF THE STUDY: The antimicrobial combinations using EOs with terbinafine or itraconazole can be an attractive therapeutic option for controlling P. insidiosum infections.

Different risk factors for candidemia occur for Candida species belonging to the C. parapsilosis complex.

Previous studies have demonstrated reduced virulence in the species that comprise the Candida parapsilosis complex. We investigated a cohort of 93 patients with candidemia caused by this complex. Most infections were caused by C. parapsilosis (80.6%), followed by C. orthopsilosis (18.3%) and C. metapsilosis (1.1%). Renal failure (P < 0.001) and chronic liver diseases (P = 0.019) were more frequently encountered with infections caused by the C. orthopsilosis group, suggesting an association with patients who had a greater state of immune suppression in comparison with infections caused by C. parapsilosis sensu stricto.

Efficacy of a Brazilian calcium montmorillonite against toxic effects of dietary aflatoxins on broilers reared to market weight.

1. The protective effect of a natural Brazilian calcium montmorillonite (CaMont) against aflatoxins was studied in broiler chickens. 2. A total of 1056-d-old Cobb male broilers were housed in experimental pens (22 chickens per pen) for 42 d. Three levels of CaMont (0, 2.5 and 5 g/kg) and two levels of aflatoxins (0 and 3 mg/kg) were assayed. Each treatment had 8 replicate pens of 22 broiler chickens each. 3. Of all the chickens tested in the experiment, the ones treated with aflatoxins were the most adversely affected. CaMont treatment at concentrations of 2.5 and 5 g/kg improved body weight of chickens at 42 d of age by 13.3% and 22.7%, increased daily feed intake by 9.7% and 24.7%, and improved the productive efficiency index of chickens by 53% and 66.5%, respectively. 4. Dietary CaMont positively affected parameters such as weight of liver, heart and gizzard; however, serum potassium concentration decreased by 15.3% compared with that of chickens given only the aflatoxin-contaminated diet. 5. CaMont did not cause adverse effects in chickens that did not receive aflatoxins. 6. CaMont at pH 8.5 partially reduced the toxic effects of aflatoxins in broilers when included at levels of 2.5 and 5 g/kg in the diet.

Iron chelation therapy as a treatment for Pythium insidiosum in an animal model.

OBJECTIVES: Iron plays an important role in the pathogenesis of Pythium insidiosum. Human pythiosis frequently occurs in iron-overloaded thalassaemic patients and experimentally infected animals develop iron deficiency anaemia. Therefore, we sought to determine the in vitro and in vivo activities of the iron chelator deferasirox against P. insidiosum. METHODS: In vitro, the MIC and minimum fungicidal concentration (MFC) values of deferasirox for 17 strains of P. insidiosum were determined in accordance with CLSI document M38-A2. In vivo studies were carried out in 20 inoculated rabbits divided into four groups: placebo, immunotherapy obtained from vortexed P. insidiosum cultures (14 day intervals), deferasirox (15 mg/kg/day) and a combination of immunotherapy and deferasirox. Five non-infected animals were used as controls. RESULTS: The MIC and MFC values of deferasirox for P. insidiosum ranged from 12.5 to 50 mg/L and from 50 to 100 mg/L, respectively. Treatment with deferasirox alone ameliorated anaemia and normalized the serum iron levels and hepatic iron concentration in the animals. However, the mean lesion size, although decreased, did not differ significantly from that in the placebo group. The results of immunotherapy plus iron chelation therapy were worse than those of immunotherapy alone. Moreover, the disease spread to the lung tissue in 5 out of 10 deferasirox-treated animals. CONCLUSIONS: Despite its limited in vitro and in vivo activity, deferasirox improved iron deficiency anaemia in P. insidiosum-infected rabbits. Further studies are needed to investigate the immunomodulatory properties observed in this study and the benefits and drawbacks of using iron-chelating drugs as an adjuvant therapy in pythiosis.

Antifungal activity of nanocapsule suspensions containing tea tree oil on the growth of Trichophyton rubrum.

The aim of this study was to evaluate, for the first time, the antifungal efficacy of nanocapsules and nanoemulsions containing Melaleuca alternifolia essential oil (tea tree oil) in an onychomycosis model. The antifungal activity of nanostructured formulations was evaluated against Trichophyton rubrum in two different in vitro models of dermatophyte nail infection. First, nail powder was infected with T. rubrum in a 96-well plate and then treated with the formulations. After 7 and 14 days, cell viability was verified. The plate counts for the samples were 2.37, 1.45 and 1.0 log CFU mL(-1) (emulsion, nanoemulsion containing tea tree oil and nanocapsules containing tea tree oil, respectively). A second model employed nails fragments which were infected with the microorganism and treated with the formulations. The diameter of fungal colony was measured. The areas obtained were 2.88 ± 2.08 mm(2), 14.59 ± 2.01 mm(2), 40.98 ± 2.76 mm(2) and 38.72 ± 1.22 mm(2) for the nanocapsules containing tea tree oil, nanoemulsion containing tea tree oil, emulsion and untreated nail, respectively. Nail infection models demonstrated the ability of the formulations to reduce T. rubrum growth, with the inclusion of oil in nanocapsules being most efficient.

In vitro interactions between amphotericin B and other antifungal agents and rifampin against Fusarium spp.

Fusarium species are common hyaline soil saprophytes and plant pathogens that are opportunistic fungal pathogens of immunocompromised patients. The treatment for fusariosis remains uncertain with an unfavourable prognosis; new possibilities for treatment, such as various synergistic drug interactions, must be uncovered. In this study, we evaluated the in vitro interactions of amphotericin B with caspofungin, ketoconazole, 5-flucytosine, itraconazole, miconazole, rifampin, fluconazole, terbinafine and voriconazole against isolates of Fusarium spp. using the chequerboard method with interactions evaluated by fractional inhibitory concentration indices. The highest percentages of synergistic interactions were observed for the combinations of amphotericin B and caspofungin (68.7%), amphotericin B and rifampin (68.7%), amphotericin B plus 5-flucytosine (59.3%) and amphotericin B with voriconazole (37.5%). The pattern of susceptibility to antifungal agents among Fusarium species and their consequence on the effects of drug combinations are also discussed.

Efficacy of miltefosine therapy against subcutaneous experimental pythiosis in rabbits.

We evaluated the in vitro activity of miltefosine against 29 Pythium spp. and the in vivo therapeutic response of 2mg/kg/day of miltefosine given orally to rabbit with pythiosis induced experimentally. The MICs (in µg/mL) of miltefosine was medium-dependent and ranged from 0.5 to 2 and 32-64 on RPMI 1640 and Mueller Hinton broth, respectively. The treatment with miltefosine demonstrated significantly lower subcutaneous lesion areas compared to the control group but was not sufficient for the complete remission of the lesions. This study indicates that miltefosine has limited efficacy against pythiosis and furthers in vitro and in vivo studies are necessary to determine the possible potential of this drug in the treatment of pythiosis.

Susceptibility profile of Candida rugosa (Diutina rugosa) against antifungals and compounds of essential oils.

Candida rugosa (recently reclassified Diutina rugosa) is an emerging pathogen affecting humans and animals. Candida resistance to existing drugs is an important factor to be monitored, as well as the need of researching alternatives to conventional antifungals. Here, we evaluated the in vitro effects of some antifungals and major components of essential oils by the broth microdilution method (CLSI M27-A3) against fifteen C. rugosa strains from animals isolated and molecular identificated. The results showed MIC90 of: 0.125µg/mL to ketoconazole and voriconazole, 0.25µg/mL to micafungin, 0.5µg/mL to anidulafungin, 1µg/mL to caspofungin, 2µg/mL to amphotericin B, itraconazole and flucytosin, 8µg/mL to fluconazole, 16µg/mL to nystatin and >128µg/mL to terbinafine. The compounds carvacrol (MIC90 320µg/mL), thimol (MIC90 320µg/mL) and cinnamaldehyde (MIC90 160µg/mL) demonstrated antifungal activity against the samples tested.

In vitro activity of carvacrol, cinnamaldehyde and thymol combined with antifungals against Malassezia pachydermatis.

Malassezia pachydermatis is an important opportunistic agent of dermatitis and otitis in dogs. M. pachydermatis is generally treated with topical therapies using combinations of antifungal, antimicrobial and anti-inflammatory agents. We investigated the in vitro activities of carvacrol (CRV), cinnamaldehyde (CIN) and thymol (THY) alone and in combination with antifungal agents (fluconazole, itraconazole, ketoconazole, clotrimazole, miconazole, terbinafine and nystatin) against M. pachydermatis. The assays were performed according to the Clinical and Laboratory Standards Institute (CLSI), using Sabouraud dextrose broth and checkerboard microdilution. The mean fractional inhibitory concentration index (FICI) showed primary synergies for the combinations carvacrol+nystatin, thymol+nystatin, and carvacrol+miconazole (80%). In conclusion, the results obtained indicate that the phytochemicals tested showed relevant in vitro anti-M. pachydermatis activity. Future in vivo experiments are needed to elucidate the safety and therapeutic potential of these combinations.

Comparison of antioxidant activities and total polyphenolic and methylxanthine contents between the unripe fruit and leaves of Ilex paraguariensis A. St. Hil.

Ilex paraguariensis is used in Brazil as a stimulating beverage called "mate". Leaves and immature fruit extracts of Ilex paraguariensis were evaluated for their radical scavenging capacity, total methylxanthine and polyphenol contents. Antimicrobial activity of two enriched saponin fractions obtained from the fruits were also evaluated. The radical scavenging activity of the fractioned extracts was determined spectrophotometrically using 1,1-diphenylpicrylhydrazyl free radical (DPPH). The IC50o of L-ascorbic acid, ethyl acetate and n-butanol fractions from the leaves and ethyl acetate fraction from the fruits were 6.48 microg/mL, 13.26 microg/mL, 27.22 microg/mL, and 285.78 microg/mL, respectively. Total methylxanthine content was 1.16 +/- 0.06 mg/g dry weight in the fruits and 8.78 +/- 0.01 mg/g in the leaves. Total polyphenol content varied from 86.82 +/- 3 x 10(-4) to 199.91 +/- 3 x 10(-3) mg/g in leaf fractions and from 54.25 +/- 1 x 10(-3) to 110.36 +/- 4 x 10(-4) mg/g in fruit fractions. Enriched saponin fractions from the fruits showed no antimicrobial activity. To our knowledge, this are the first data available on the antioxidant/antimicrobial activities and polyphenol/methylxanthine contents of Ilex paraguariensis fruits.