Polar Origanum vulgare (Lamiaceae) extracts with antifungal potential against Sporothrix brasiliensis.

Oregano (Origanum vulgare) has anti-Sporothrix spp. activity, including against strains that are resistant to antifungal drugs. As the studies are limited to the essential oil, the aim of this study was to evaluate the chemical, antioxidant and cytotoxic properties of polar oregano extracts and their anti-Sporothrix brasiliensis activity. Aerial plant parts were used in the preparation of 10 min (INF10) and 60 min (INF60) infusions, a decoction (DEC) and a hydroalcoholic extract (HAE). Six phenolic acids and four flavonoids were identified and quantified through liquid-chromatography (LC-MS). Extracts in increasing order of total phenolic and flavonoid contents were HAE 40 mg/ml for the other extracts. This is the first report of oregano extracts showing antifungal activity against S. brasiliensis. Its use in the treatment of sporotrichosis may be considered upon toxicity and safe-use conditions are tested.

Plants from Lamiaceae family as source of antifungal molecules in humane and veterinary medicine.

This work aimed to review the main plants of Lamiaceae family with activity against pathogenic fungi of medical and veterinary interest. Published studies in the main international databases between January 2002 and June 2016 showed that 55 botanical species belonging to 27 genus presented antifungal activity in different forms of extractions, mainly essential oils. Pathogenic fungi of Aspergillus spp., Candida spp., Malassezia spp., Cryptococcus spp., Sporothrix spp., Microsporum spp., Trichophyton spp. and Epidermophyton spp. genus were in vitro sensitive to several plants of Lamiaceae family. Chemical molecules isolated were described as promising use as antifungals in mycoses, highlighting estragole, 1,8-cineole, terpineol-4, γ-terpinene, among others. However, it should be alert to need of universal standardization in the laboratories tests with natural products.

In Vitro Susceptibility of Sporothrix brasiliensis to Essential Oils of Lamiaceae Family.

This study evaluated the chemical, cytotoxic and anti-Sporothrix brasiliensis properties of commercial essential oils of rosemary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.) and marjoram (Origanum majorana L.). Chemical composition of the oils was identified through gas chromatography with flame ionization detector, and cytotoxicity was performed through MTT assay in VERO cell line. Anti-S. brasiliensis activity was performed according to the CLSI M38-A2 guidelines using isolates obtained from cats and dogs. The major compounds found were carvacrol in the oregano oil (73.9 %) and 1,8-cineole in rosemary and marjoram oils (49.4 and 20.9 %, respectively). All S. brasiliensis isolates were susceptible to the plant oils, including itraconazole-resistant ones. Marjoram and rosemary oils showed MIC90 of 0.56 and 1.12 mg ml-1, and MFC90 of 4.5 and 9 mg ml-1, respectively. For oregano oil, a strong antifungal activity was observed with MIC90 and MFC90 values ≤0.07 mg ml-1. The weakest cytotoxicity was observed for rosemary oil. Further studies should be undertaken to evaluate the safety and efficacy of these essential oils in sporotrichosis.

The use of (1-3) ß-glucan along with itraconazole against canine refractory sporotrichosis.

Sporotrichosis, caused by the Sporothrix schenckii fungal complex, is a zoonotic mycosis distributed worldwide. Itraconazole is the treatment of choice for domestic animals although some fungal isolates have shown resistance to this drug. The objective of this study was to report, for the first time, the use of (1-3) ß-glucan along with itraconazole in the treatment of a canine with sporotrichosis caused by Sporothrix brasiliensis. The animal had ulcerated and crusted lesions, especially on the nasal planum. Clinical samples were collected for a complete blood count, cytological analysis of the lesion, and fungal culture. Based on the results of the laboratory examination, and after the fungal culture, antibiotic therapy and treatment with itraconazole were initiated. Two additional fungal cultures were performed, which were positive. After 7 months of the animal treatment with itraconazole, the S. brasiliensis culture was still positive, so that the itraconazole was associated with (1-3) ß-glucan. After four weekly applications of glucan, the complete elimination of the fungus was observed based on the fungal culture negative results. The results show, therefore, that (1-3) ß-glucan with itraconazole promoted the case resolution, and it may be considered a promising alternative for the treatment of sporotrichosis in cases of resistance to conventional therapy.

Screening of alkaloids and withanolides isolated from Solanaceae plants for antifungal properties against non-wild type Sporothrix brasiliensis.

Antifungal resistance has often been found in animal sporotrichosis in Southern Brazil. The biological potential of compounds from plants of the Solanaceae family against infectious diseases is known, however, it is still unknown against Sporothrix brasiliensis. This study evaluated the anti-Sporothrix brasiliensis activity, synergism, cytotoxicity, and action mechanism of steroidal lactones (withanolides) and alkaloids isolated from these plants. Pure compounds of withanolide D (WNOD), physalin F (PHYF), withanicandin (WNIC), nicandin B (NICB), solasonine (SSON), and solamargine (SMAR) were tested against 12 Sporothrix brasiliensis isolated from cats (n = 11) and dogs (n = 2) through M38-A2 CLSI. For the compounds with the best activity, a checkerboard assay for synergism, sorbitol protection, and ergosterol effect for action mechanism; and MTT test for cytotoxicity were performed. The withanolides WNOD, PHYF, WNIC, and NICB were not antifungal, but SSON (MIC 0.125-1 mg/mL) and SMAR (MIC 0.5-1 mg/mL) were both fungistatic and fungicidal (MFC 0.5-1 mg/mL for both) against wild-type (WT) and non-WT isolates. The activity of SSON and SMAR was indifferent when combined with itraconazole. In the mechanism of action, cell wall and plasma membrane by complexation with ergosterol seemed to be two target structures of SSON and SMAR. SSON was selected for cytotoxicity, whose cell viability in MDBK cells ranged from 28.85 % to 101.75 %, and was higher than 87.49 % at concentrations ≤0.0015 mg/ml. Only the steroidal alkaloids SSON and SMAR were active against non-WT isolates, being promising antifungal candidates for the treatment of feline and canine sporotrichosis with low susceptibility to itraconazole.

A comparative study between integrative practices and preappointement gabapentin on serum cortisol in cats.

Clinical appointment generates stress in feline patients, influencing the frequency of veterinary care with the species. The purpose of this study was to assess serum cortisol in cats submitted to oral gabapentin and integrative practices during clinical care. Twenty cats were evaluated in three clinical appointments, one week apart. All cats were submitted to treatments: placebo (PL), gabapentin (GA), and integrative practices (IP) (music therapy, pheromone therapy, and chromotherapy). GA and PL were administered by the owners 90 min before transportation to the veterinary teaching hospital, and IP were applied 30 min before clinical care. Cats were physically examined at all timepoints, and blood samples were collected for cortisol measurement. Serum cortisol levels ranged from 0.49 µg/dL to 17.99 µg/dL. Mean cortisol concentrations when cats received PL (7.6 µg/dL) were higher than when cats received GA (4.9 µg/dL) and IP (4.1 µg/dL). There was a statistical difference in cortisol levels when cats receiving PL and GA were compared (p = 0.03) and between PL and IP (p = 0.005). The study showed that feline serum cortisol levels were lower when cats received the treatments to IP (integrative practices) and GA (gabapentin), demonstrating that these are applicable methods for reducing stress of feline patients in clinical evaluation.

An evaluation of techniques to diagnose Dioctophyme renale in dogs.

Dioctophyme renale is a nematode with zoonotic potential that affects the kidneys of carnivorous, wild, and domestic mammals. In this study, we sought to evaluate the indirect ELISA method against routine methods used to diagnose dioctophimosis. Hence, 38 dogs parasitized by D. renale, as confirmed by surgery, were selected. The dogs were evaluated by abdominal ultrasound and urinalysis, and their sera were tested by indirect ELISA using D. renale adult secretion and excretion antigen (DES). Five dogs were followed up with serum collections on day 0 (day of surgery) and 30, 60, and 90 days after surgery to evaluate antibody kinetics. Abdominal ultrasound and indirect ELISA successfully diagnosed 37 dogs parasitized by D. renale, while urinalysis diagnosed 29 animals. The positive animals were parasitized with 1-7 parasites; 17 dogs were infected by male and female parasites, 15 only by female parasites, and six were parasitized only by male parasites. When assessing specificity and sensitivity, all techniques showed 100% specificity and 81.6%, 97.4%, and 97.4% sensitivity for urinalysis, ultrasound, and ELISA, respectively (p < 0.001). The five positive dogs that were followed up after surgery showed a progressive decrease in mean absorbances in indirect ELISA (0.644, 0.516, 0.511, and 0.440, respectively). This study demonstrated that the indirect ELISA using the DE antigen could diagnose dioctophimosis regardless of the number, sex, and location of the parasites, with the potential to be used in epidemiological research and implementing immunological and molecular studies, opening new lines of research on D. renale.

Dioctophyme renale é um nematódeo que possui potencial zoonótico e acomete o rim de mamíferos carnívoros, silvestres e domésticos. Neste estudo busca-se avaliar a técnica de ELISA indireto frente metodologias de rotina utilizadas no diagnóstico da dioctofimose. Para isto, 38 cães participaram do estudo, sendo todos parasitados por D. renale, confirmados por cirurgia. Esses cães foram avaliados por ultrassom abdominal, urinálise e seus soros testado por ELISA indireto utilizando antígeno de excreção e secreção (DES) de adultos de D. renale. Destes, cinco cães foram acompanhados com coletas de soro, no dia zero (dia da cirurgia) e após 30, 60 e 90 dias da cirurgia para avaliar a cinética dos anticorpos. O ultrassom abdominal e ELISA indireto apresentaram sucesso no diagnóstico de 37 cães parasitados por D. renale, enquanto que a urinálise diagnosticou 29 animais. Os animais positivos possuíam de um a sete parasitos, 17 cães apresentaram infecções por macho e fêmeas, 15 somente por fêmeas e seis foram parasitados apenas por machos. Na avaliação da especificidade e sensibilidade, todas as técnicas apresentaram 100% de especificidade e 81,6%, 97,4%, 97,4% de sensibilidade para urinálise, ultrassom e ELISA, respectivamente (p < 0,001). Os cinco cães positivos que foram acompanhados após a cirurgia apresentaram diminuição progressiva nas médias de absorbâncias no ELISA indireto (0,644, 0,516, 0,511 e 0,440, respectivamente). O estudo demonstrou que o ELISA indireto utilizando o antígeno DES poderia diagnosticar dioctofimose, independentemente do número, sexo e localização dos parasitos, com potencial para ser utilizada em estudos epidemiológicos e na implementação de estudos imunológicos e moleculares, abrindo novas linhas de pesquisa sobre Dioctophyme renale.

Nephrectomy in 52 dogs for the treatment of Dioctophyme renale infection - Knowing the enemy to win.

Dioctophyme renale (D. renale) is a nematode that parasitizes the kidney of mammals. Treatment is often surgical, with removal of the affected organ. This retrospective study aims to evaluate the epidemiological, clinical, and surgical aspects, the interval between diagnosis and treatment, the occurrence of pre- and intraoperative complications, and the postoperative survival time of dogs parasitized by D. renale undergoing therapeutic nephrectomy. Records of fifty-two dogs treated in a single hospital service were analyzed. We collected epidemiological data, laboratory results, diagnostic method, anesthetic protocol, surgical technique and time, type of antimicrobial prophylaxis, pre- and intraoperative complications, location and number of parasites, and postoperative survival time. Of the 52 dogs undergoing right nephrectomy by laparotomy, 61.5 % were female and 63.4 % were adults. Although the most common clinical sign was hematuria (25 %), 61.5 % of the patients were asymptomatic. Eosinophilia and increased serum urea were the only laboratory changes found. The interval between diagnosis and surgery was 27.4 ± 23 days and no patient showed changes suggestive of surgical emergency. The most common surgical approach was the right paracostal (61.5 %), and a continuous suture pattern was predominant. Intraoperative complications occurred in 9.6 % of the procedures, varying from mild to severe hemorrhage. Mean postoperative survival was 835.5 ± 428 days. Dioctophymosis was effectively controlled by nephrectomy of the affected kidney, allowing a mean survival of more than 830 days. No serious complications caused by intervals between diagnosis and treatment have been reported. This is the largest retrospective study evaluating dogs infected with D. renale that were surgically treated.

Clinical and mycological analysis of dog's oral cavity.

The oral microbiota of humans and animals is made up of a wide variety of yeasts and bacteria, but microbiota of dogs is not totally described. Although such identification is an important step to establish the etiopathogenesis and adequate therapy for the periodontal disease The aim of this study was to evaluate and correlate oral alterations with the presence of yeasts in oral cavity of female dogs. After clinical evaluation samples from healthy and from dogs with oral diseases were obtained from three different oral sites by swabs, curettes, millimeter periodontal probes and HA membrane tip in cellulose ester. Yeast identification was performed through macroscopic and microscopic colony features and biochemical tests. Dental calculus was the most prevalent occurrence in the oral cavity of 59 females. However, the isolation of yeasts was significantly higher (p < 0.05) in animals suffering from halitosis. Eleven yeast species were identified, namely: Malassezia pachydermatis, Rhodotorula spp., Candida albicans, C. catenulata, C. famata, C. guilliermondii, C. parapsilosis, C. intermedia, Trichosporon asahii, T. mucoides and Cryptococcus albidus. It could be concluded that the yeasts are part of the microbiota from the different sites of the oral cavity of the female canines studied without causing any significant alterations except halitosis.

Are γ-terpinene, 1,8-cineole, p-coumaric acid, and quercetin active against wild-type and non-wild-type Sporothrix brasiliensis to itraconazole?

The emergence of itraconazole (ITZ)-resistant Sporothrix brasiliensis in feline and canine cases in southern Brazil has hampered the clinical cure of animal sporotrichosis, encouraging the search for therapeutic alternatives. The promising use of plants extracts from Lamiaceae family is known; however, there are no studies with its major compounds, as γ-terpinene (γTER), 1,8-cineole (1,8CIN), p-coumaric acid (pCOU), and quercetin (QUER). For the first time, we evaluated the antifungal, synergistic, cytotoxic activities and action mechanism of these compounds against S. brasiliensis. For this, 28 S. brasiliensis from cats (n = 24) and dogs (n = 4) and standard strains of S. brasiliensis and S. schenckii (n = 4) were tested by M38-A2 (CLSI), revealing non-wild-type (WT) isolates to ITZ on 54.2% (13/24) and 75% (03/04) of feline and canine isolates, respectively. Of the compounds, γTER stood out against all isolates (MIC/MFC 0.75 to > 3 mg/ml; MIC50 3 mg/ml). However, 1,8CIN, pCOU, and QUER showed little or no activity (MIC50 > 3 mg/ml). Thus, γTER was selected for checkerboard assay, whose combination with ITZ showed synergistic (WT isolates) and indifferent (non-WT isolates) interaction. For action mechanism (sorbitol protection and ergosterol effect), γTER acted in membrane by complexing with fungal ergosterol and at the cell wall level, showing two possible pathways as antifungal target. Finally, cytotoxicity (MTT assay) showed that γTER was the safest compound on MDBK cells, even at a concentration of 3 mg/ml (90.16%). Our findings support that γTER is a potent antifungal candidate for the control of sporotrichosis, including against non-WT S. brasiliensis.

Are polar extracts and essential oil from Origanum vulgare Linn. (oregano) an alternative against itraconazole-resistant dermatophytes from veterinary cases?

Although the conventional therapy against dermatophytosis is based on antifungal drugs and environmental disinfection, the emergence of itraconazole(ITZ)-resistant dermatophytes has encouraged the search for active compounds, such as Origanum vulgare L. (oregano) essential oil (EO). However, little is known about its effect in polar extracts or the mechanism of action of these extracts and EO. We investigated the antifungal activity of four polar extracts and one EO from oregano against ITZ-susceptible and ITZ-resistant dermatophytes and their action mechanism. Polar extracts were prepared as infusions at 10 (INF10) and 60 (INF60) minutes, decoction (DEC) and hydroalcoholic extract (HAE); EO was purchased. All extracts and itraconazole were tested against Microsporum gypseum, M. canis, M. nanum, Trichophyton mentagrophytes and T. verrucosum isolated from cats, dogs and cattle (n = 28), and humans (n = 2) (M38-A2, CLSI). Among polar extract, DEC stood out as antifungal, followed by INF10 and INF60; HAE was little active. For EO, all isolates were susceptible, including ITZ-resistant dermatophytes. EO was selected for action mechanism assays, and acted in cell wall and plasmatic membrane by complexing with fungal ergosterol. By chromatographic analysis, 4-hydroxibenzoic acid was the most prevalent compound in all polar extracts, followed by syringic acid and caffeic acid; luteolin was present only in HAE. For EO, carvacrol was the major compound (73.9%); followed by γ-terpinene (3.6%) and thymol (3.0%). These findings showed that the extract type of oregano has influenced the antifungal action on dermatophytes, highlight EO and DEC, that are promising as antifungal agent, including against ITZ-resistant dermatophytes.

Inhibitory effect of sodium hypochlorite and chlorhexidine digluconate in clinical isolates of Sporothrix schenckii.

The susceptibility of Sporothrix schenckii isolates from clinical cases of canine, feline and human sporotrichosis, and from the environment, was evaluated with 4% sodium hypochlorite and 6.6% chlorhexidine digluconate using the broth microdilution, agar diffusion and direct exposure techniques. The minimal inhibitory concentration was smaller than 0.8% for chlorhexidine digluconate and between 8% and 4% for sodium hypochlorite. Inhibition zones were not found in agar diffusion for sodium hypochlorite, and zones averaging 1.9 mm were found for chlorhexidine digluconate. In the direct exposure test, sodium hypochlorite demonstrated best performance at 20 min of contact, as chlorhexidine digluconate presented little antimicrobial activity.

Bioisosteric modification on benzylidene-carbonyl compounds improved the drug-likeness and maintained the antifungal activity against Sporothrix brasiliensis.

Considering the emergence of antifungal resistance on Sporothrix brasiliensis, we aimed to assess new benzylidene-carbonyl compounds against feline-borne S. brasiliensis isolates. The compounds were designed as bioisosteres from previously reported benzylidene-ketones generating the p-coumaric (1), cinnamic (2), p-methoxycinnamic (3) and caffeic acid (4) analogues. The corresponding compounds were tested against feline isolates of S. brasiliensis with sensitivity (n = 4) and resistance (n = 5) to itraconazole (ITZ), following the M38-A2 protocol (CLSI, Reference method for broth dilution antifungal susceptibility testing of filamentous fungi M38-A2 Guideline, 2008). Eleven analogues showed activity against all fungal strains with minimum inhibitory concentrations (MIC) ≤1 mg/ml (1a-d, 2e, 3b, 3e, 4, 4a and 5e) and fungicidal concentrations (MFC) ≤1 mg/ml (1b, 1d, 3e and 4a), whereas 3 was the less active with both MIC and MFC values above 1 mg/ml. Compound 3e (4-methoxy-N-butylcinnamamide) was the most potent (MICrange 0.08-0.16 mg/ml; MFCrange 0.32-0.64 mg/ml) from the set, suggesting a different role of the substituents in ester and amide derivatives. The designed compounds proved to be important prototypes with improved drug-likeness to achieve compounds with higher activity against ITZ-resistant S. brasiliensis.

Ovicidal activity of the hydroalcoholic extract of Brazilian peppertree (Schinus terebinthifolia Raddi) against Ancylostoma spp. from naturally parasitized dogs.

This study aimed to evaluate the ovicidal activity of the hydroalcoholic extract of Schinus terebinthifolia (SCH; T1) against Ancylostoma spp. and its influence of storage time in the extract stored for 36 months (T36). Eggs of Ancylostoma spp. were obtained from naturally parasitized dogs, and used for the larval hatchability test, where the eggs were exposed to T1 and T36 extracts of SCH (15-0.625 mg/mL). In T1, all concentrations inhibited more than 80% of the eggs, being 100% at concentrations between 15 and 5 mg/mL (p > 0.05). At T36, all concentrations were active, even the ones between 2.5 and 0.625 mg/mL, with 100% inhibition (p < 0.05), revealing that the storage time maintained the ovicidal action. By LC-MS, T36 presented ethyl gallate, myricitrin, and gallic acid as major compounds. These findings support the promising use of SCH extract as an ovicide against Ancylostoma spp., even stored for 36 months of shelf life.

Chemical, antioxidant and cytotoxic profile of hydroalcoholic extracts of plants from Southern Brazil and their activity against pathogenic fungi isolated from dogs and cats with sensitivity and resistance to conventional antifungals.

We aimed to evaluate the chemical, antioxidant, cytotoxic, and antifungal activities of hydroalcoholic extracts of native plants from Southern Brazil: Schinus terebinthifolia (SCH), Persicaria hydropiperoides (PER), Eugenia uniflora (EUG) and Equisetum hyemale (EQU). Ethyl gallate, quercetin, and quinic acid were prevalent compounds identified by LC-MS. For total phenolic/flavonoid contents and the antioxidant potential against ABTS/DPPH radicals, the ascending order was EQU < PER < EUG < SCH. All extracts were low cytotoxic and kept a high Vero cell viability (>75%) at concentrations up to 12.5 mg/mL (MTT assay). By M38-A2/M27-A3 (CLSI) against 68 clinical isolates of animals and strains of Malassezia pachydermatis, Sporothrix brasiliensis, Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes, all extracts (MIC/MFC ≤3.13-100 mg/mL) were active, except EUG. SCH inhibited and killed S. brasiliensis (MIC/MFC50/90 3.12-12.5 mg/mL) and dermatophytes (MIC/MFC 6.25-25 mg/mL) resistant to ketoconazole and itraconazole. These findings support the promising use of the selected plant extracts as antifungal agents.

Multiresistant bacteria isolated from domestic and wild animals with skin lesions were susceptible to native plants from Southern Brazil.

We evaluated the chemical composition, toxicity, and antibacterial activity of Schinus terebinthifolia (SCH), Eugenia uniflora (EUG), Persicaria hydropiperoides (PER), Equisetum hyemale (EQU), Solidago chilensis (SOL), and Baccharis trimera (BRA). These plants were tested (7.5-0.01 mg/mL) against Gram-positive (G+; n = 32) and Gram-negative (G-; n = 26) isolates from animals (M07-A9, CLSI). Antibiogram (disk diffusion), chromatographic analysis (UPLC), and toxicity assay (HET-CAM) were also performed. A high incidence of resistance was noted, in which 18.4% (07/38) of G+ (Staphylococcus intermedius/Enterococcus faecium) and 17.7% (06/34) of G- (Pseudomonas aeruginosa/Escherichia coli/Proteus mirabilis) were multidrug-resistant. All bacteria were sensitive (MIC50) to SCH (both 3.75 mg/mL), EUG (3.75 mg/mL and 7.5 mg/mL, respectively) and PER (both 7.5 mg/mL). SCH/EUG/PER highlighted as antibacterial, probably due to the major compounds (ethyl gallate, quinic acid, quercetin). These extracts showed normal embryonic development (SCH/EUG: 7.5-0.94 mg/mL). These findings highlighted the promising use of native plants for therapeutic purposes.

Jabuticaba [Plinia peruviana (Poir.) Govaerts]: a Brazilian fruit with a promising application against itraconazole-susceptible and -resistant Sporothrix brasiliensis.

Hydroalcoholic extract from Jabuticaba peels was evaluated for the chemical profile, antioxidant potential, cytotoxicity, and anti-Sporothrix brasiliensis activities against both dimorphic phases. Higher phenolic content (14.91 ± 0.97) compared to flavonoid (2.05 ± 1.00) associated with the ellagic acid (1.93 ± 0.03; LC-MS), and a good scavenging ability against ABST and DPPH radicals was noted. On MDBK cells, elevated cell viability (>90%) was demonstrated between 0.39 and 0.097 mg/ml (MTT assay). Mycelial (CLSI M38-A2) and yeast (CLSI M27-A3) phases of 18 isolates of Sporothrix brasiliensis from cats (n = 8), dogs (n = 8) and humans (n = 2) were used. They were identified itraconazole-susceptible and itraconazole-resistant isolates in both phases, which were all inhibited (MIC of ≤1.56-6.25 mg/ml for both phases) and killed (MFC of ≤1.56-12.5 mg/ml for mycelial; ≤1.56-50 mg/ml for yeast) by Jabuticaba. For the first time, these findings highlighted the potential usefulness of hydroalcoholic extract from Jabuticaba peel as a promising antifungal against sporotrichosis.

Susceptibility and resistance of Sporothrix brasiliensis to branded and compounded itraconazole formulations.

Itraconazole is the first drug of choice for the treatment of sporotrichosis and it is available at different concentrations for veterinary patients. However, therapeutic failure has been reported, limiting clinical treatment. This study evaluated the in vitro efficacy of brand-name and compounded itraconazole formulations against Sporothrix brasiliensis and estimated the itraconazole content in each tested formulation. Oral capsules were acquired from two brand-name products for human (H-IND) and veterinary (V-IND) uses, and three from compounding pharmacies in Pelotas, RS, for human (H-COMP1/H-COMP2) and veterinary (V-COMP) uses. Capsule purity was analyzed by liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS). Antifungal activity was determined against 29 Sporothrix brasiliensis by the M38-A2 guideline of CLSI. H-IND/H-COMP1/H-COMP2 had high efficacy against S. brasiliensis (approximately 70% of total isolated susceptible), V-COMP showed moderate efficacy (51.7%), and V-IND was the least effective formulation (37.9%). Thirty-four percent of the total isolates were resistant to all formulations. Furthermore, itraconazole content did not match the concentration indicated by the manufacturers, ranging from 387.70 to 7.81 µg/mg (H-COMP2 > V-COMP > H-IND > H-COMP1 > V-IND). Therefore, it is possible that the formulations showed different in vitro efficacy due to the difference in their itraconazole contents. Given the emergence of antifungal resistance for all formulations, the choice product to be used must follow susceptibility testing. Stringent quality control measures are recommended for product manufactures to assure drug content uniformity.

Feline sporotrichosis: a case series of itraconazole-resistant Sporothrix brasiliensis infection.

The treatment of feline sporotrichosis is a challenge for veterinary clinicians since refractory cases may occur, due either to patient and/or to pharmacological management errors or due to the development of antifungal resistance. Thus, we aimed to describe the therapeutic history of feline cases infected by itraconazole-resistant Sporothrix brasiliensis in an endemic region of Southern Brazil. Medical records of cats attended at the Veterinary Clinic Hospital (Pelotas/RS, Brazil) between 2016 and 2017 were reviewed. Twelve cases of infection by S. brasiliensis with that showed high minimum inhibitory concentration (MIC) values (≥ 4 µg/mL) to itraconazole by M38-A2 of CLSI were selected. At the hospital consultation, disseminated (cats 1-l0, 12) and localized (cat 11) skin lesions remained in the cats, even after treatment with fluconazole, ketoconazole (02/12), and itraconazole (ITZ, 09/12) performed before this study. High doses (25-100 mg/kg/day) of ITZ for up to 4 months (03/12, cats 2, 6, 12) or over 12 months (05/12, cats 1, 5, 7, 8, 11) did not provide a clinical cure, except for the association of ITZ plus potassium iodide (01/12, cat 12) for 3 months, which proved useful in infections with itraconazole-resistant S. brasiliensis. However, the combined issues of abandonment of therapy by owners for financial reasons, difficulties surrounding therapy administration (03/12, cats 6, 11, 12), and the inappropriate choice of medication (01/12, cat 6), together reflect the reality of this endemic region, which greatly compromises clinical healing. This study highlighted the occurrence of refractory cases by itraconazole-resistant S. brasiliensis in cats from Southern Brazil, as well as the abandonment of treatment and therapeutic errors. We warn of the need for antifungal susceptibility tests to adapt therapeutic protocols in feline sporotrichosis.