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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., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

This study evaluated combinations of amphotericin B with anidulafungin, caspofungin, and micafungin against 30 clinical isolates of Cryptococcus neoformans following the CLSI M27-A3 and the checkerboard microdilution method. The combination amphotericin B + micafungin showed 60% of synergistic effect against C. neoformans, while most of the other interactions were indifferent., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We tested 25 isolates of Pythium insidiosum to investigate their susceptibility to antibacterial drugs that act through inhibition of protein synthesis or other mechanisms of action. We observed that tetracycline, erythromycin, linezolid, nitrofurantoin, Synercid (quinupristin and dalfopristin), chloramphenicol, clindamycin, cetrimide, and crystal violet had inhibitory activity against P. insidiosum. Those in vitro results suggest that antibacterials that inhibit protein synthesis should be the primary antimicrobials investigated for the treatment of pythiosis in animals and humans., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Cryptococcus species are an encapsulated fungal pathogen that cause cryptococcal meningitis. There are limited therapeutic options for this infection. The management includes the use of different antifungals such as amphotericin B, flucytosine, or fluconazole, either alone or in combination. However, numerous therapeutic failures, as well as the limited effectiveness of such therapeutics, have been described. Diphenyl diselenide is a chemically synthesised molecule with was found to have antimicrobial activity. In this study, we evaluated the antifungal activities of fluconazole, amphotericin B and flucytosine, in combination with diphenyl diselenide against 30 clinical isolates of Cryptococcus spp. using CLSI M27-A3 method and the checkerboard microdilution technique. Our results show that the combination of flucytosine and diphenyl diselenide displayed 100% of synergism. However, when we analysed (PhSe) 2 plus AMB or FLZ we observed around 70% of indifference. Our results suggest that the combination of diphenyl diselenide with other antifungal agents deserves attention as a new option for the development of alternative therapies for cryptococcosis., (© 2019 Blackwell Verlag GmbH.)

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 MIC 90 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 (MIC 90 320μg/mL), thimol (MIC 90 320μg/mL) and cinnamaldehyde (MIC 90 160μg/mL) demonstrated antifungal activity against the samples tested., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The yeast Malassezia pachydermatis is a common commensal and occasional opportunistic pathogen of theskin microbiota of animals and humans. In this study, the susceptibility of M. pachydermatis isolates to fluconazole (FLC), itraconazole (ITZ), ketoconazole (KTZ), clotrimazole (CLZ), and miconazole (MCZ) alone and in combination with terbinafine (TRB), nystatin (NYS), and caspofungin (CSP) was evaluated in vitro based on the M27-A3 technique and the checkerboard microdilution method using Sabouraud dextrose broth with 1% tween 80 (SDB). Based on the mean FICI values, the main synergies observed were combinations of ITZ+CSP and CLZ+CSP (55.17%). The most significant combinations deserve in vivo evaluations because might provide effective alternative treatments against M. pachydermatis due to their synergistic interactions., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Rhodotorula species are emerging as opportunistic pathogens, causing catheter-associated fungemia in patients with compromised immunity. R. mucilaginosa is considered the most common species involved in human infections. Correct identification and susceptibility testing of Rhodotorula isolates recovered from the blood stream or central nervous system are essential to determine the best management of this unusual infection. The antifungal susceptibility tests showed that Rhodotorula was susceptible to low concentrations of amphotericin B (AMB) but was less susceptible to voriconazole. Combinations of AMB plus several non-antifungal medications were evaluated against 35 susceptible (Rm AMB-S) and resistant (Rm AMB-R) clinical Rhodotorula isolates using the broth microdilution checkerboard technique. We showed that in vitro exposure to increasing concentrations of AMB changed the susceptibility profile to these strains, which were named the Rm AMB-R group. The most synergistic interactions were AMB + simvastatin, followed by AMB + amlodipine and AMB + warfarin. Synergism and antagonism were observed in both groups for the combination AMB + cyclosporine A. AMB combined with a fluoroquinolone (AMB + levofloxacin) also demonstrated antagonism for the Rm AMB-S strains, but a high percentage of synergistic interactions was observed for the Rm AMB-R group. A combination drug approach can provide a different strategy to treat infections caused by AMB-resistant R. mucilaginosa.

Background: Candida parapsilosis may acquire resistance to echinocandins, a fact that prompts the search for new therapeutic options., Aims: The present study aimed to evaluate the in vitro activity of antifungal agents, alone and in combination, against four groups of C. parapsilosis strains: (1) echinocandin-susceptible (ES) clinical isolates (MIC ≤ 2μg/ml), (2) anidulafungin-resistant strains (MIC ≥ 8μg/ml), (3) caspofungin-resistant strains (MIC ≥ 8μg/ml), and (4) micafungin-resistant strains (MIC ≥ 8μg/ml)., Methods: Antifungal interactions were evaluated by a checkerboard micro-dilution method. The determination of the MIC to each drug for every isolate according to the Clinical and Laboratory Standards Institute documents M27 (2017) and M60 (2017) was also done., Results: The echinocandins-resistant (ER) strains showed higher MICs to the tested antifungals than the ES strains, except for amphotericin B, for which the ER groups remained susceptible., Conclusions: Most combinations showed indifferent interactions. The use of monotherapy still seems to be the best option. As resistance to echinocandins is an emergent phenomenon, further studies are required to provide clearer information on the susceptibility differences between strains to these antifungal agents., (Copyright © 2019 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.)

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.

The aim of this study was to evaluate the susceptibility of 20 clinical isolates of Fusarium spp. to classic antifungals [amphotericin B (AmB), itraconazole (ITR), voriconazole (VRC) and caspofungin (CAS)] and to non-antifungal agents [amiodarone (AMD), doxycycline (DOX) and moxifloxacin (MFX)] by the broth microdilution method. Combinations between these antifungal and non-antifungal agents were also evaluated to determine the fractional inhibitory concentration indices using the chequerboard technique. Synergistic interactions were observed for the following combinations (% synergism): AMD + VRC, 80%; MFX + AmB, 75%; AMD + AmB, 65%; DOX + VRC, 60%; MFX + VRC, 55%; DOX + AmB, 50%; and AMD + CAS, 30%. Synergism was not observed for associations with ITR. Antagonism was not seen in any combination. These findings suggest that the combinations of AMD, DOX or MFX with AmB or VRC to have potential for future in vivo investigations., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

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.

Context: Syzygium cumini (L.) Skeels (Myrtaceae) is a medicinal plant widely used in folk medicine for the treatment of diabetes mellitus (DM). However, studies on the use of this plant and of nanoparticle formulations against DM-related fungal infections are scarce., Objective: To evaluate the effect of the treatments with aqueous seed extract of S. cumini (ASc) and ASc-loaded polymeric nanoparticles (NPASc) on biochemical parameters in Candida albicans-infected diabetic rats., Materials and Methods: Male Wistar rats were divided into eight groups: Control, DM, C. albicans, C. albicans + ASc, C. albicans + NPASc, DM + C. albicans, DM + C. albicans + ASc and DM + C. albicans + NPASc. Rats were daily treated with ASc or NPASc (100 mg/kg) for 21 days. Biochemical parameters in serum and urine, advanced oxidation protein product (AOPP) and TBARS levels in the serum, kidney, liver and pancreas and N-acetyl-β-d-glucosaminidase (NAG) activities in kidney and urine were evaluated., Results: Biochemical and oxidative stress parameters increased in rats with DM and/or candidiasis. NPASc was more effective than ASc in decreasing glucose (56%), cholesterol (33%) and creatinine (51%) levels; serum (16%) and pancreatic (46%) AOPP and renal (48%) TBARS levels when compared with DM + C. albicans group. In C. albicans group, both treatments decreased NAG activity but did not decrease creatinine levels., Conclusions: These data suggest that the use of nanotechnology is able to improve plant extract properties such as antioxidant activity that may be useful in diabetes-related complications.

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.

Introduction:: This study evaluated the susceptibilities of oral candidiasis-derived Candida albicans, fluconazole-resistant (FR) Candida dubliniensis, and fluconazole-susceptible (FS) C. dubliniensis to synthetic antiseptics [chlorhexidine gluconate (CHX), cetylpyridinium chloride (CPC), and triclosan (TRC)] and natural compounds (carvacrol, eugenol and thymol)., Methods:: Susceptibility tests were performed based on the M27-A3 reference method. The fluconazole-resistant C. dubliniensis strains were obtained after prolonged in vitro exposure to increasing fluconazole concentrations. The geometric mean values for minimum inhibitory concentrations and minimum fungicidal concentrations were compared among the groups., Results:: Fluconazole-susceptible C. dubliniensis was more sensitive to CPC and TRC than FR C. dubliniensis and C. albicans were. However, eugenol and thymol were more active against FR C. dubliniensis. The fungicidal activities of CHX and TRC were similar for the three groups, and FR C. dubliniensis and C. albicans had similar sensitivities to CPC., Conclusions:: The resistance of C. dubliniensis to fluconazole affects its sensitivity the synthetic antiseptics and natural compounds that were tested.

The antifungal activity of tacrolimus in combination with antifungal agents against different fungal species has been previously reported. Here we report the in vitro interactions between tacrolimus and amphotericin B, fluconazole, itraconazole, and caspofungin against 30 clinical isolates of both fluconazole-susceptible and fluconazole-resistant Trichosporon asahii. For these analyses, we used the broth microdilution method based on the M27-A3 technique and checkerboard microdilution method. Tacrolimus showed no activity against T. asahii strains (minimal inhibitory concentrations, MICs>64.0μgmL -1 ). However, a larger synergistic interaction was observed by the combinations tacrolimus+amphotericin B (96.67%) and tacrolimus+caspofungin (73.33%) against fluconazole-susceptible isolates. Combinations with azole antifungal agents resulted in low rates of synergism for this group (fluconazole+tacrolimus=40% and itraconazole+tacrolimus=10%). Antagonistic interactions were not observed. For the fluconazole-resistant T. asahii group, all tested combinations showed indifferent interactions. The synergism showed against fluconazole-susceptible T. asahii isolates suggests that the potential antifungal activity of tacrolimus deserves in vivo experimental investigation, notably, the combination of tacrolimus with amphotericin B or caspofungin., (Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.)

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., (© 2016 The Society for Applied Microbiology.)

The naturally high minimum inhibitory concentration exhibited by echinocandins against Candida parapsilosis has been known since the first introduction of these antifungal agents. Despite this awareness, clinical failures have not been reported; consequently, the resistance of C. parapsilosis to echinocandins remains unexplored. We exposed 30 isolates of C. parapsilosis to echinocandins (caspofungin, micafungin, and anidulafungin) in vitro and studied the effects of this exposure. After 60 exposures, 80, 67, and 60 % of the isolates changed from susceptible to non-susceptible to caspofungin, micafungin, and anidulafungin, respectively. In addition, four strains exhibited cross-resistance to all three echinocandins. Based on the M27-A3 (CLSI, 2008) and M27-S4 (CLSI, 2012) techniques, the susceptibility of the resistant strains to other antifungal agents was assayed. All of the tested echinocandin-resistant strains were susceptible to amphotericin B, and the resistance rate to fluconazole, voriconazole, and flucytosine was 73.3, 43.3, and 20 %, respectively. The exposure of C. parapsilosis to the three echinocandins generated cross-resistant strains and an unexpected in vitro resistance to azoles and flucytosine.

Cryptococcus neoformans is encapsulated yeast that causes cryptococcosis. The cryptococcal meningitis may cause neuropsychiatric symptoms. Here, we evaluated the in vitro activity of amphotericin B (AMB), chlorpromazine (CLOR), and sertraline (SERT) alone or in combination against clinical isolates of C. neoformans considering the capsular induction in vitro. Susceptibility tests were carried out using the broth microdilution method in accordance with the CLSI document M27-A3. The combination [CLOR + AMB] exhibited synergism for 50 and 67 % of strains before capsular induction (group I) and after capsular induction (group II), respectively. The combination [SERT + AMB] showed 60 % of synergism against the both groups. Antagonism was not observed. Our results show the therapeutic potential of chlorpromazine and sertraline in combination with amphotericin B against neurocryptococcosis.

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., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The presence of melanin in the fungal cell is a major virulence factor of the genus Sporothrix since it protects the fungal cells against the defense systems., Aims: The present study aimed to investigate the interference of melanin in the susceptibility of Sporothrix brasiliensis and Sporothrix schenckii sensu stricto to amphotericin B and itraconazole, drugs recommended as therapy for disseminated and subcutaneous sporotrichosis, respectively., Methods: Yeast cells were cultivated in minimal medium with or without l-DOPA in order to induce the production of melanin. Microdilution and killing assay methods were used to determine the antifungal activity against yeast cells with different amounts of melanin., Results: The killing assay showed that melanization protected isolates within the S. schenckii complex from amphotericin B, particularly in the lower concentrations tested., Conclusions: Studies combining amphotericin B and inhibitors of melanin are required in order to avoid this effect., (Copyright © 2014 Asociación Española de Micología. Published by Elsevier Espana. All rights reserved.)

This study evaluated the synergistic interactions between amphotericin B (AMB) and azithromycin (AZM), daptomycin (DAP), linezolid (LNZ), minocycline (MINO), fluconazole (FLZ), flucytosine (5FC), linezolid (LZD), or tigecycline (TIG) against clinical isolates of Cryptococcus neoformans var. grubii before and after capsule induction. High synergism (>75%) was observed for the combinations, AMB+5FC, AMB+TIG, AMB+AZM, AMB+LZD and AMB+MINO but only in the strains after capsule induction. The results show that the presence of the capsule may lower the minimum inhibitory concentrations (MICs) of antifungal agents, but antimicrobial activity can be improved by combining antifungal and antibacterial agents., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The present study investigated the in vitro and the in vivo interactions among azithromycin, clarithromycin, minocycline, and tigecycline against Pythium insidiosum. In vitro antimicrobial activities were determined by the broth microdilution method in accordance with CLSI document M38-A2, and the antibiotic interactions were assayed using the checkerboard MIC format. In vivo efficacy was determined using a rabbit infection model. The geometric mean MICs of azithromycin, clarithromycin, minocycline, and tigecycline against P. insidiosum were, respectively, 1.91, 1.38, 0.91, and 0.79 μg/ml. By checkerboard testing, all combinations resulted in in vitro synergistic interactions (>60%). Antagonism was not observed. The in vivo studies showed that azithromycin (20 mg/kg/day twice daily) alone or in combination with minocycline (10 mg/kg/day twice daily) significantly decreased the fungal burden. This study demonstrates that azithromycin possesses potent curative efficacy against subcutaneous pythiosis in the rabbit model., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Sporothrix schenckii was reclassified as a complex encompassing six cryptic species, which calls for the reassessment of clinical and epidemiological data of these new species. We evaluated the susceptibility of Sporothrix albicans(n = 1) , S. brasiliensis(n = 6) , S. globosa(n = 1), S. mexicana(n = 1) and S. schenckii(n = 36) to terbinafine (TRB) alone and in combination with itraconazole (ITZ), ketoconazole (KTZ), and voriconazole (VRZ) by a checkerboard microdilution method and determined the enzymatic profile of these species with the API-ZYM kit. Most interactions were additive (27.5%, 32.5% and 5%) or indifferent (70%, 50% and 52.5%) for TRB+KTZ, TRB+ITZ and TRB+VRZ, respectively. Antagonisms were observed in 42.5% of isolates for the TRB+VRZ combination. Based on enzymatic profiling, the Sporothrix schenckii strains were categorized into 14 biotypes. Leucine arylamidase (LA) activity was observed only for S. albicans and S. mexicana. The species S. globosa and S. Mexicana were the only species without β-glucosidase (GS) activity. Our results may contribute to a better understanding of virulence and resistance among species of the genus Sporothrix in further studies.

In this study, we assessed the generation of reactive oxygen species (ROS) induced by subinhibitory concentration of fluconazole in susceptible and resistant Candida glabrata strains at stationary growth phase and measured their oxidative responses parameters: glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione-S-transferase (GST), consumption of hydrogen peroxide, and total glutathione, as well as oxidative damage in lipids, proteins, and DNA. Data showed that fluconazole increased generation of ROS and GPx and SOD enzymatic activity in treated cells; however, these enzymatic activities did not differ between resistant and susceptible strains. Susceptible strains exhibited higher GST activity than resistant, and when susceptible cells were treated with fluconazole, GST activity decreased. Fluconazole treatment cause oxidative damage only in DNA. There are a possible participation of ROS, as organic peroxides and O2(•-), in antifungal mechanism of fluconazole, which results in higher GPx and SOD enzymatic activities and oxidative DNA damage in C. glabrata., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We describe the in vitro activities of the combinations of carvacrol and thymol with antibiotics (azithromycin, clarithromycin, minocycline and tigecycline) and antifungal agents (amphotericin B, caspofungin, itraconazole and terbinafine) against 23 isolates of the oomycete Pythium insidiosum. The assays were based on the M38-A2 technique and checkerboard microdilution. Based on the mean FICI values, the main synergies observed were combinations of carvacrol+itraconazole and thymol+itraconazole (96%), thymol+clarithromycin (92%), carvacrol+clarithromycin (88%), thymol+minocycline (84%), carvacrol+minocycline (80%), carvacrol+azithromycin (76%), thymol+azithromycin (68%), carvacrol+tigecycline (64%) and thymol+tigecycline (60%). In conclusion, we found that combinations of carvacrol or thymol with these antimicrobial agents might provide effective alternative treatments for cutaneous pythiosis due to their synergistic interactions. Future in vivo experiments are needed to elucidate the safety and therapeutic potential of these combinations., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

In vitro interaction between tacrolimus (FK506) and four azoles (fluconazole, ketoconazole, itraconazole and voriconazole) against thirty clinical isolates of both fluconazole susceptible and -resistant Candida glabrata were evaluated by the checkerboard microdilution method. Synergistic, indifferent or antagonism interactions were found for combinations of the antifungal agents and FK506. A larger synergistic effect was observed for the combinations of FK506 with itraconazole and voriconazole (43%), followed by that of the combination with ketoconazole (37%), against fluconazole-susceptible isolates. For fluconazole-resistant C. glabrata , a higher synergistic effect was obtained from FK506 combined with ketoconazole (77%), itraconazole (73%), voriconazole (63%) and fluconazole (60%). The synergisms that we observed in vitro , notably against fluconazole-resistant C. glabrata isolates, are promising and warrant further analysis of their applications in experimental in vivo studies.

We have determined the in vitro activity of several antibacterial and antifungal drugs against Pythium insidiosum using broth microdilution (BMD), disk diffusion, and Etest methods. The largest zones of inhibition (disk diffusion) and the lowest BMD and Etest MICs were observed for azithromycin, clarithromycin, linezolid, mupirocin, doxycycline, minocycline, and tigecycline. The in vitro activities observed suggest that antibacterials, which act by inhibiting protein synthesis, are promising candidate therapies for the treatment of pythiosis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The high mortality rates associated with candidemia episodes and the emergence of resistance to antifungal agents necessitate the monitoring of the susceptibility of fungal isolates to antifungal treatments. The new, recently approved, species-specific clinical breakpoints (SS-CBPs)(M27-S4) for evaluating susceptibility require careful interpretation and comparison with the former proposals made using the M27-A3 breakpoints, both from CLSI. This study evaluated the susceptibility of the different species of Candida that were isolated from candidemias based on these two clinical breakpoints. Four hundred and twenty-two isolates were identified and, among them, C. parapsilosis comprised 46.68%, followed by C. albicans (35.78%), C. tropicalis (9.71%), C. glabrata (3.55%), C. lusitaniae (1.65%), C. guilliermondii (1.65%) and C. krusei (0.94%). In accordance with the M27-A3 criteria, 33 (7.81%) non-susceptible isolates were identified, of which 16 (3.79%) were resistant to antifungal agents. According to SS-CBPs, 80 (18.95%) isolates were non-susceptible, and 10 (2.36%) of these were drug resistant. When the total number of non-susceptible isolates was considered, the new SS-CBPs detected 2.4 times the number of isolates that were detected using the M27-A3 interpretative criteria. In conclusion, the detection of an elevated number of non-susceptible species has highlighted the relevance of evaluating susceptibility tests using new, species-specific clinical breakpoints (SS-CBPs), which could impact the profile of non-susceptible Candida spp. to antifungal agents that require continuous susceptibility monitoring.

The objective of this work was to propose coconut oil-core nanocapsules prepared from Eudragit(®) RS100, a cationic polymer, and to evaluate their potential for vaginal delivery of clotrimazole in candidiasis. Nanocapsule suspensions loaded with clotrimazole at 1.0 and 3.0mg/mL were prepared by interfacial deposition of Eudragit(®) RS100. The physicochemical characterization showed average diameter lower than 200 nm, low polydispersity index, positive zeta potential (+10.94 to +14.57 mV), acid pH values (5.4-5.7) and encapsulation efficiencies close to 100%. After 60 days of storage at room temperature and protected from light, the nanocapsules were reasonably stable. Photodegradation studies showed that nanoencapsulation improved clotrimazole stability against UV radiation. The in vitro drug release at pH 4.5 was characterized by a prolonged release with no burst effect. The nanocapsules were more active than free clotrimazole against Candida albicans and Candida glabrata strains susceptible and resistant to fluconazole. Hence, clotrimazole-loaded coconut oil-core nanocapsules represent promising alternatives to the treatment of vulvovaginal candidiasis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Introduction: Candida dubliniensis, a new species of Candida that has been recovered from several sites in healthy people, has been associated with recurrent episodes of oral candidiasis in AIDS and HIV-positive patients. This species is closely related to C. albicans. The enzymatic activity of C. dubliniensis in response to oxidative stress is of interest for the development of drugs to combat C. dubliniensis., Methods: Fluconazole- and amphotericin B-resistant strains were generated as described by Fekete-Forgács et al. (2000). Superoxide dismutase (SOD) and catalase assays were performed as described by McCord and Fridovich (1969) and Aebi (1984), respectively., Results: We demonstrated that superoxide dismutase (SOD) and catalase activities were significantly higher (p<0.05) in the fluconazole- and amphotericin B-resistant strains of C. dubliniensis and C. albicans than in the sensitive strains. The catalase and SOD activities were also significantly (p<0.01) higher in the sensitive and resistant C. albicans strains than in the respective C. dubliniensis strains., Conclusions: These data suggest that C. albicans is better protected from oxidative stress than C. dubliniensis and that fluconazole, like amphotericin B, can induce oxidative stress in Candida; oxidative stress induces an adaptive response that results in a coordinated increase in catalase and SOD activities.

Data regarding the susceptibility of Conidiobolus lamprauges is limited and there is no consensus about the optimal treatment for infections caused by Conidiobolus spp. In this context, the objective of this study was to evaluate the in vitro susceptibility of six C. lamprauges strains isolated from sheep conidiobolomycosis to amphotericin B, ketoconazole, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, micafungin, flucytosine, and terbinafine using the CLSI M38-A2 microdilution technique. Terbinafine was the most active (MIC range <0.06-0.5 μg/mL). Resistance or reduced susceptibility was observed for amphotericin B and azole and echinocandin antifungals. Additional studies are necessary to determine the therapeutic potential of terbinafine as monotherapy or in combination therapy with other antifungals., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Candidiasis is one of the most important among recurrent invasive yeast infections in patients, thus antifungal treatment becomes a challenge., Aims: The aim of this study was to evaluate the in vitro activity of clinical Candida albicans isolates from blood cultures to fluconazole, amphotericin B and anidulafungin, in a hospital from Rio Grande do Sul, Brazil., Methods: The susceptibility of 153 isolates to the 3 drugs mentioned was tested according to Clinical and Laboratory Standars Institute. Minimal inhibitory and fungicidal concentrations (MIC, MFC, respectively) of each drug were determined, as well as the epidemiological cutoff value (ECV)., Results: All of the isolates were susceptible to anidulafungin, MIC and MFC ≤ 1 μg/ml; however, when compared with ECV, 3% of the isolates exhibited higher values against fluconazole, 96% were susceptible, 3% susceptible dose-dependent, and 1% resistant. Also, it was observed that 21% of the isolates exhibited higher values than ECV. One isolate was resistant to amphotericin B; the other ones, susceptible, based on the MFC; furthermore, 1.5% of the isolates exhibited higher values., Conclusions: C. albicans isolates exhibited more susceptibility to anidulafungin, and 90% of them (MIC90) exhibited the lowest values against amphotericin B. Based on ECV and Pfaller classification, isolates could be resistant to fluconazole, demonstrating the importance of the combination of these parameters., (Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

Pythium insidiosum is an oomycete, a fungal like microorganism, which infects mammals, causing pythiosis in animals and humans, especially in tropical and subtropical regions around the world. The treatment for this infection is very difficult, and therapeutic options commonly comprise surgery, immunotherapy and antimicrobial drugs. The present report describes the clinical healing of a dog with gastrointestinal pythiosis by treatment with a combination of antifungals and immunotherapy, as well as reviews the cases reported in the literature that used some type of therapy for canine pythiosis. A 2.5-year-old male beagle initially showed sporadic vomiting episodes, and this symptom became more frequent 5 months after the onset of clinical signs. Celiotomy procedure found thickness of the stomach wall extending to the pylorus and duodenum. A biopsy was performed, and the diagnosis of pythiosis was made by mycological, histopathological analyses and molecular identification. Therapy was based on an association of terbinafine plus itraconazole during 12 months and immunotherapy for 2.5 months. The healing of the dog reported here allows us to propose the use of immunotherapy associated with antifungal therapy to treat canine gastrointestinal pythiosis. However, additional studies should be performed on a larger number of patients to establish a standard treatment protocol for canine pythiosis.

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.

Background: Infections caused by Fusarium are difficult to treat because these fungi show in vitro and in vivo resistance to practically all the antifungal agents available, which explains the high mortality rates. An attempt to overcome fungal resistance is the combination of antifungal agents, especially those with different mechanisms of action., Aims: Evaluate the in vitro interactions of combinations of voriconazole or itraconazole with other antifungal agents against 32 isolates of Fusarium spp.: Fusarium chlamydosporum, Fusarium oxysporum, Fusarium proliferatum and Fusarium solani., Methods: Drug interactions were assessed by a checkerboard microdilution method that also included the determination of the MIC of each drug alone according to CLSI (Clinical and Laboratory Standards Institute) document M38-A2, 2008., Results: The best combinations were voriconazole+terbinafine which showed synergism against 84% of Fusarium strains. Other synergistic combinations were voriconazole+itraconazole (50%), voriconazole+fluconazole (50%), voriconazole+miconazole (38%), voriconazole+flucytosine (22%) and voriconazole+ketoconazole (25%). The synergisms observed with itraconazole combinations were itraconazole+terbinafine (25%) and itraconazole+flucytosine (9.37%). The antagonisms observed were: voriconazole+fluconazole (3%) and itraconazole+flucytosine (12.5%)., Conclusions: The synergism showed by voriconazole+terbinafine was remarkable. To better elucidate the potential usefulness of our findings, new in vivo and in vitro studies deserve be performed., (Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

Clotrimazole is a common choice for the treatment of vulvovaginal infections, but its low solubility and some side effects pose a challenge to its application. This work evaluated the feasibility to formulate clotrimazole-loaded cationic nanocapsules using Eudragit® RS100 and medium chain triglycerides as polymer and oily core, respectively, by the method of interfacial deposition of a preformed polymer. The physicochemical characteristics of nanocapsule formulations were evaluated at 0 day and 60 days after preparation. Particle size, zeta potential, polydispersity index, pH and drug content were stable during this period. In addition, nanocapsules were able to protect clotrimazole from photodegradation under UV radiation. By the dialysis bag diffusion technique, the nanosized formulations showed prolonged release of clotrimazole by anomalous transport and first order kinetics. A microbiological study was carried out by the microdilution method and showed that nanocapsules (mean size: 144 nm; zeta potential: +12 mV) maintained the antifungal activity of clotrimazole against Candida albicans and Candida glabrata strains susceptible and resistant to fluconazole., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The rise of Candida spp. resistant to classic triazole antifungal agents has led to a search for new therapeutic options. Here, we evaluated combinations of antifungals in a checkerboard assay against two groups of Candida glabrata strains: one containing fluconazole-susceptible clinical isolates (FS) and another containing fluconazole-resistant laboratory derivative (FR). The most synergistic combination observed was amphotericin B + flucytosine (synergistic for 61.77 % of FS strains and 76.47 % of FR strains). The most antagonistic combination observed was ketoconazole + flucytosine (FS 61.77 % and FR 55.88 %). Surprisingly, most combinations evidenced indifferent interactions, and the best synergism appeared when amphotericin B and flucytosine were combined against both groups of isolates.

The present study investigated the in vitro inhibitory activity of terbinafine, itraconazole, caspofungin, fluvastatin and ibuprofen against 15 isolates of Pythium insidiosum in double and triple combinations and determined in vivo correlations using rabbits with experimental pythiosis. The minimal inhibitory concentration (MIC) was determined in accordance with the Clinical and Laboratory Standards Institute M 38-A2 protocol (2008), and the in vitro interactions were evaluated using a checkerboard microdilution method. For the in vivo study, 20 rabbits inoculated with P. insidiosum zoospores were divided into four groups: group 1 was treated with terbinafine and itraconazole; group 2 was treated with terbinafine, itraconazole and fluvastatin; group 3 was treated with terbinafine and caspofungin; and group 4 was the control group. Combinations of terbinafine with caspofungin or ibuprofen were synergistic for 47% of the isolates, and antagonism was not observed in any of the double combinations. The triple combinations were mostly indifferent, but synergism and antagonism were also observed. In the in vivo study, the histological aspect of the lesions was similar among the groups, but group 2 showed the lowest amount of hyphae and differed significantly from the other groups., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The extensive use of azole antifungal agents has promoted the resistance of Candida spp to these drugs. Candida glabrata is a problematic yeast because it presents a high degree of primary or secondary resistance to fluconazole. In Brazil, C. glabrata has been less studied than other species. In this paper, we compared the activity of three major classes of antifungal agents (azoles, echinocandins and polyenes) against fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata strains. Cross-resistance between fluconazole and voriconazole was remarkable. Among the antifungal agents, the echinocandins were the most effective against FS and FR C. glabrata and micafungin showed the lowest minimal inhibitory concentrations.

In the present study we used two groups of Candida dubliniensis strains: one containing fluconazole-susceptible clinical isolates and another containing fluconazole-resistant laboratory derivative from the former to examine the changes on susceptibility accompanying the development of resistance to fluconazole. Our findings confirmed the ability of C. dubliniensis isolates to become resistant to fluconazole and indicated that this resistance was crossed with ketoconazole, itraconazole, ravuconazole and terbinafine. We also tested combinations of terbinafine, amphotericin B, itraconazole and voriconazole against both groups of isolates in a checkerboard assay. Surprisingly, most combinations evidenced indifferent interactions, and the best synergism appeared when terbinafine and itraconazole were combined against the fluconazole-resistant group.

Fusarium spp is an opportunistic fungal pathogen responsible for causing invasive hyalohyphomycosis in immunocompromised patients. Due to its susceptibility pattern with a remarkable resistance to antifungal agents the treatment failures and mortality rates are high. To overcome this situation, combination therapy may be considered which must be subjected to in vitro tests. In vitro activities of amphotericin B, itraconazole, and voriconazole associated with azithromycin, ciprofloxacin, fluvastatin, ibuprofen, metronidazole, and also the combination of amphotericin B plus rifampin against 23 strains of Fusarium spp. through the checkerboard technique based on M38-A2 [Clinical and Laboratory Standards Institute (2008). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard, 2nd ed. (CLSI document M38-A2) (ISBN 1-56238-668-9). Wayne, PA: CLSI] were evaluated. The best synergistic interactions with amphotericin B were with ibuprofen (43.5%) (FICI [fractional inhibitory concentration index] range = 0.25-2). Combinations with voriconazole showed synergism, mainly with ciprofloxacin (30.4%) (FICI range = 0.25-3) and metronidazole (30.4%) (FICI range = 0.1-4); however, all the combinations with itraconazole were indifferent. In general, antagonistic interactions were not registered. Our results showed that in vitro synergisms obtained by some combinations studied deserve attention since they were better than those showed by the antimycotic., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We report on in vitro antifungal activity and the structure-activity relationship of diphenyl diselenide [(PhSe)(2) ] and its synthetic analogues, (p-Cl-C(6) H(4) Se)(2), (m-CF(3)-C(6) H(4)Se)(2) and (p-CH(3)O-C(6) H(4)Se)(2), against 116 strains of pathogenic fungi. (PhSe)(2) showed the highest inhibitory activity against Candida albicans (minimum inhibitory concentration of 4-32 μg ml(-1) ), Candida dubliniensis (2-16 μg ml(-1)), Aspergillus spp. (0.5-64 μg ml(-1)) and Fusarium spp. (2-16 μg ml(-1)). Its minimum fungicidal concentration (MFC) varied among C. albicans (4-64 μg ml(-1)), C. dubliniensis (2-32 μg ml(-1) ) and Fusarium spp. (4-64 μg ml(-1)). Antifungal activity was decreased by the introduction of functional groups to the (PhSe)(2) molecule: (PhSe)(2) >(p-CH(3)O-C(6)H(4) Se)(2) >(m-CF(3)-C(6)H(4)Se)(2) >(p-Cl-C(6) H(4)Se)(2)., (© 2011 Blackwell Verlag GmbH.)

Objectives: The first aim of this study was to evaluate the in vitro efficacies of fluconazole, ketoconazole, itraconazole and voriconazole on M. pachydermatis growth inhibition. This study also evaluated M. pachydermatis azole cross-resistance, comparing wild clinical isolates and the same isolates with in vitro-induced fluconazole resistance., Methods: Two techniques were used: (1) a broth microdilution method based on protocol M27-A3 from the Clinical and Laboratory Standards Institute to determine the minimum inhibitory concentration (MIC) and (2) the Fekete-Forgács method to induce fluconazole resistance in vitro. The isolates were divided into two groups: group 1 included fluconazole-susceptible clinical isolates (n=30) and group 2 contained the same isolates with in vitro-induced fluconazole resistance (n=30)., Results: The two groups exhibited differences in susceptibility (p<0.001). Group 1 isolates were susceptible to azoles: ketoconazole (MIC 0.01-1.0 μg/mL), itraconazole (MIC 0.01-1.0 μg/mL), voriconazole (MIC 0.01-4.0 μg/mL), and fluconazole (MIC 0.01-4.0 μg/mL). Group 2 isolates demonstrated a wider range of MICs to azoles: ITZ (MIC 0.06-64.0 μg/mL), KTZ (MIC 0.25-32.0 μg/mL), VRZ (MIC 2.0-128.0 μg/mL), and FLZ (MIC 64.0-128.0 μg/mL)., Conclusions: It was shown that FLZ-resistant M. pachydermatis isolates exhibit cross-resistance to other azoles, reinforcing the importance of susceptibility tests as a guide for the therapeutic prescription of antifungals in medical and veterinary mycology., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We studied 40 strains of the species complex formerly classified as the single species Sporothrix schenckii to identify new species within this complex and evaluate their antifungal susceptibility profiles. Based on phenotypic tests (ability to grow at 37°C, colony diameters, and pigmentation of the colonies, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), here we report the identification of S. albicans, S. brasiliensis, S. luriei, and S. schenckii; two isolates of these species were detected as itraconazole-resistant strains.

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., (© 2009 Blackwell Verlag GmbH.)

Pythium insidiosum is a zoosporic organism which causes pythiosis in humans and animals. This study aimed to report the paradoxical growth of Brazilian P. insidiosum strains when submitted to in vitro susceptibility tests with caspofungin. The growth at concentrations above the minimal inhibitory concentration (MIC) ranged from 16 to 128 μg/ml and it was observed in 50% of the isolates tested. This paradoxical growth in the presence of caspofungin has been observed with Candida and Aspergillus strains, however, the phenomenon involving oomycetes was described here for the first time., (Copyright © 2010. Published by Elsevier B.V.)