Sloanea chocoana and S. pittieriana (Elaeocarpaceae): Chemical and Biological Studies of Ethanolic Extracts and Skincare Properties.

The Colombian Chocó is known for its rich biodiversity and to harbor plant species that are under-explored, including the genus Sloanea. This study aimed to analyze the chemical composition of derivatized ethanolic extracts from S. chocoana and S. pittieriana using BSTFA and TMCS through GC-MS, and to assess cell viability of immortalized human non-tumorigenic keratinocytes (HaCaT) and periodontal ligament fibroblast cells using crude extracts through MTS assay. Antioxidant and photoprotective properties were determined using DPPH assay and spectrophotometry. Antifungal activity of extracts against Candida species was developed following the CLSI standard M27, 4th ed. The sun protective factor (SPF) and UVA/UVB ratio values were calculated using the Mansur equation and the Boots star rating system. The critical wavelength (λc) was determined by calculating the integrated optical density curve's area. The transmission of erythema and pigmentation was calculated through equations that use constants to calculate the flux of erythema and pigmentation. The GC-MS analysis identified 37 compounds for S. chocoana and 38 for S. pittieriana, including alkaloids, triterpenoids, and polyphenolics, among others. Both extracts exhibited proliferative effects on periodontal ligament fibroblasts, did not affect the viability of HaCaT cells, and showed excellent antioxidant activities (46.1% and 43.7%). Relevant antifungal activity was observed with S. pittieriana extract against Candida albicans (GM-MIC: 4 µg/mL), followed by C. auris and C. glabrata (GM-MIC: 32 µg/mL), while S. chocoana extract was active against C. albicans and C. glabrata (GM-MIC: 16 and 32 µg/mL, respectively). High SPF values (31.0 and 30.0), λc (393.98 and 337.81 nm), UVA/UVB ratio (1.5 and 1.2), and low percentage of transmission of erythema and pigmentation were determined for S. chocoana and S. pittieriana, respectively. Results showed that species of Sloanea constitute a promising alternative as ingredients for developing skincare products, and exhaustive studies are required for their sustainable uses.

Lippia origanoides Essential Oil or Thymol in Combination with Fluconazole Produces Damage to Cells and Reverses the Azole-Resistant Phenotype of a Candida tropicalis Strain.

Candida tropicalis is one of the most pathogenic species within the genus. Increased antifungal resistance has been reported, which is in part due to the organism's ability to form biofilms. In natural products derived from plants, such as essential oils (EOs) or their major components, there is significant potential to develop new antifungals or to both enhance the efficacy and reduce the toxicity of conventional antifungals. This study aimed to evaluate the effect of combining an EO of Lippia origanoides or thymol with fluconazole on an azole-resistant C. tropicalis strain. Synergism was observed in the combination of fluconazole with the EO and with thymol, and minimal inhibitory concentrations for fluconazole decreased at least 32-fold. As a consequence of the synergistic interactions, mitochondrial membrane potential was reduced, and mitochondrial superoxide production increased. Alteration in nuclear morphology, cell surface, and ultrastructure was also observed. In conclusion, the synergistic interaction between L. origanoides EO or thymol with fluconazole reverted the azole-resistant C. tropicalis phenotype. These findings suggest that L. origanoides EO or thymol alone, or in combination with fluconazole, have the potential for development as antifungal therapies for this yeast, including resistant strains.

Preparation, Physicochemical Characterization, and Stability Study of Lippia origanoides Essential Oil-Based Nanoemulsion as a Topical Delivery System.

INTRODUCTION: Fungal diseases are a priority in research, development, and health care, according to the WHO, mainly due to Candida spp. Essential oils (EOs) of the genus Lippia have demonstrated broad antimicrobial biological activity. Previous studies identified the anti-Candida potential of a thymol/p-cymene chemotype EO from Lippia origanoides H.B.K coded "0018". Nanoemulsions favor the biological activity of EOs and overcome limitations such as low solubility, instability against oxidizing agents, pH, light, and low permeability. To develop, characterize, and adjust a prototype of an O/W nanoemulsion containing the "0018" EO from Lippia origanoides for its evaluation in an In vitro permeability study. METHOD: Nanoemulsions were obtained using a high energy high shear method. Their particle size distribution, Z potential, viscosity, pH, encapsulation efficiency (EE), thermodynamic stability and the Turbiscan Stability Index (TSI) were evaluated. The nanoemulsion prototype was adjusted to improve performance characteristics and microbiological efficacy. Thymol was used as an analyte in the EO quantification using UHPLC-DAD. RESULTS: An O/W nanoemulsion with hydrodynamic diameter <200 nm and polydispersity index <0.3, EE >95%, with TSI < 1.5, anti-Candida albicans efficiency >95% was obtained; permeable with a flow of 6.0264 µg/cm2/h and permeability coefficient of 1.3170x10-3 cm/h. CONCLUSION: A pharmaceutical formulation prototype is obtained that maintains the physical and physicochemical characteristics over time. Permeability is verified in an in-vitro model. It is proposed to evaluate its antifungal activity in preclinical or clinical studies as a contribution to the treatment of topical fungal diseases caused by Candida spp., through the use of biological resources and Colombian biodiversity.

Exploring the Potential of Extracts from Sloanea medusula and S. calva: Formulating Two Skincare Gels with Antioxidant, Sun Protective Factor, and Anti-Candida albicans Activities.

Sloanea is a plant genus, native to tropical regions, used in medicinal practices for its anti-inflammatory properties. This study aimed to determine the antioxidant activity, sun protective factor (SPF), and antifungal of extracts obtained from two species of Sloanea and to develop extract-based gels with antioxidants, photoprotective, and anti-Candida albicans effects. Ethanolic extracts from S. medusula and S. calva collected in Chocó, Colombia, were used for antioxidant activity and SPF determination using the DPPH assay and the Mansur equation, respectively. Extracts were characterized using HPLC-MS and used to prepare the gels. The viscosity of the extract-based gels was evaluated using an MCR92 rheometer. In addition, the anti-Candida activity of extracts against five yeasts and anti-C. albicans of gels were evaluated following the Clinical and Laboratory Standards Institute M27, 4th Edition. High DPPH radical scavenging activity (42.4% and 44.7%) and a high SPF value (32.5 and 35.4) were obtained for the extracts of S. medusula and S. calva, respectively. Similarly, extract-based gels showed significant DPPH radical scavenging activity of 54.5% and 53.0% and maximum SPF values of 60 and 57. Extract from S. medusula showed an important antifungal activity against C. albicans (minimal inhibitory concentration (MIC) of 2 µg/mL). In contrast, S. calva extract was active against C. krusei, C. albicans (MIC of 2 µg/mL) and C. tropicalis (MIC of 4 µg/mL). Sloanea medusula gel (0.15%) exhibited an important C. albicans growth inhibition (98%), while with S. calva gel (0.3%) growth inhibition was slightly lower (76%). Polyphenolic and triterpenoid compounds were tentatively identified for S. medusula and S. calva, respectively. Both extracts can be considered promising sources for developing photoprotective gels to treat skin infections caused by C. albicans.

Antifungal susceptibility profile of Trichosporon inkin: About three cases of White Piedra.

Trichosporon spp. usually cause systemic or superficial infections. Three cases of White Piedra produced by Trichosporon inkin are described. The in vitro antifungal activity to fluconazole, amphotericin B, ketoconazole and caspofungin against the three clinical isolates were evaluated. Sensitivity to fluconazole and ketoconazole was evidenced. However, the treatment of this mycosis is still a challenge.

Lippia origanoides derivatives in vitro evaluation on polymicrobial biofilms: Streptococcus mutans, Lactobacillus rhamnosus and Candida albicans.

OBJECTIVE: This work evaluated the Lippia origanoides derivatives in vitro effect on polymicrobial biofilms of Streptococcus mutans, Lactobacillus rhamnosus and Candida albicans. Additionally, the cytotoxic effect of the oils on human skin keratinocytes (HaCaT) and fibroblasts of the periodontal ligament (FLP) cell lines was evaluated. DESIGN: The minimum inhibitory concentration, the inhibitory activity on monomicrobial (S. mutans) and polymicrobial biofilm (S. mutans, L. rhamnosus and C. albicans) of L. origanoides four essential oils and terpenes (thymol and carvacrol) were evaluated. The cytotoxic effect of each one of the compounds was measured, and all the tests were compared against chlorhexidine. RESULTS: All the evaluated compounds reached an inhibition percentage of S. mutans monomicrobial biofilms formation of 100 % at 600 µg/mL (p < 0.0001). The highest concentration (2 MIC) eradicated 100 % of S. mutans-preformed biofilms after 5 min L. origanoides carvacrol + thymol and thymol chemotypes showed marked reductions in topography, the number of microbial cells and extracellular matrix on polymicrobial biofilm. The cytotoxic effect of the compounds was very similar to chlorhexidine. CONCLUSIONS: L. origanoides essential oils have an inhibitory effect on mono and polymicrobial biofilms. The oils present a similar cytotoxic effect to chlorhexidine on HaCaT and FLP cell lines. However, including these compounds in formulations for clinical use is an exciting proposal yet to be investigated.

In Vitro Activity of Essential Oils Distilled from Colombian Plants against Candidaauris and Other Candida Species with Different Antifungal Susceptibility Profiles.

Multi-drug resistant species such as Candida auris are a global health threat. This scenario has highlighted the need to search for antifungal alternatives. Essential oils (EOs), or some of their major compounds, could be a source of new antifungal molecules. The aim of this study was to evaluate the in vitro activity of EOs and some terpenes against C. auris and other Candida spp. The eleven EOs evaluated were obtained by hydro-distillation from different Colombian plants and the terpenes were purchased. EO chemical compositions were obtained by gas chromatography/mass spectrometry (GC/MS). Antifungal activity was evaluated following the CLSI standard M27, 4th Edition. Cytotoxicity was tested on the HaCaT cell line and fungal growth kinetics were tested by time-kill assays. Candida spp. showed different susceptibility to antifungals and the activity of EOs and terpenes was strain-dependent. The Lippia origanoides (thymol + p-cymene) chemotype EO, thymol, carvacrol, and limonene were the most active, mainly against drug-resistant strains. The most active EOs and terpenes were also slightly cytotoxic on the HaCaT cells. The findings of this study suggest that some EOs and commercial terpenes can be a source for the development of new anti-Candida products and aid the identification of new antifungal targets or action mechanisms.

Antifungal Activity of Amphiphilic Perylene Bisimides.

Perylene-based compounds, either naturally occurring or synthetic, have shown interesting biological activities. In this study, we report on the broad-spectrum antifungal properties of two lead amphiphilic perylene bisimides, compounds 4 and 5, which were synthesized from perylene-3,4,9,10-tetracarboxylic dianhydride by condensation with spermine and an ammonium salt formation. The antifungal activity was evaluated using a collection of fungal strains and clinical isolates from patients with onychomycosis or sporotrichosis. Both molecules displayed an interesting antifungal profile with MIC values in the range of 2-25 µM, being as active as several reference drugs, even more potent in some particular strains. The ammonium trifluoroacetate salt 5 showed the highest activity with a MIC value of 2.1 µM for all tested Candida spp., two Cryptococcus spp., two Fusarium spp., and one Neoscytalidium spp. strain. Therefore, these amphiphilic molecules with the perylene moiety and cationic ammonium side chains represent important structural features for the development of novel antifungals.

Differential Diagnosis of Fungal Pneumonias vs. Tuberculosis in AIDS Patients by Using Two New Molecular Methods.

Opportunistic fungal pneumonias (OFP) are the main cause of death in AIDS patients worldwide. Diagnosis of these infections is often late as tuberculosis (TB) is frequently the first suspicion. In addition, diagnostic tools have limitations and are unavailable in disadvantaged regions. To perform the differential diagnosis of the main fungi causing OFP in AIDS patients (Histoplasma capsulatum, Cryptococcus neoformans/C. gattii and Pneumocystis jirovecii) vs. the Mycobacterium tuberculosis complex (MTBC), two new assays were developed: (i) a multiplex real-time PCR (MRT-PCR) and (ii) a simple and cost-effective method based on real-time PCR and the analysis of melting curves after amplification (MC-PCR). Both of the techniques were optimized and standardized "in vitro", showing a suitable reproducibility (CV ranged between 1.84 and 3.81% and 1.41 and 4.83%, respectively), a 100% specificity and detection limits between 20 and 2 fg of genomic DNA per 20 µL of reaction. A validation study was performed by retrospectively using 42 clinical samples from 37 patients with proven fungal infection or TB, and 33 controls. The overall sensitivity for the MRT-PCR assay and the MC-PCR assay was 88% and 90.4%, respectively. Both techniques were fast, sensitive and reproducible, allowing for the detection of these pathogens and the performance of a differential diagnosis.

Onychomycosis caused by the environmental mold Neoscytalidium dimidiatum in Colombia, and in vitro antifungal susceptibility evaluation.

Neoscytalidium dimidiatum is a plant pathogen, but can also cause onychomycosis. We compared clinical and epidemiological data of cases of onychomycosis caused by N. dimidiatum and Trichophyton rubrum. We also evaluated the in vitro antifungal susceptibility of N. dimidiatum clinical isolates. It was not possible to establish any statistical differences between groups, except the place of residence and the number of affected nails. The results suggest that onychomycosis caused by N. dimidiatum is clinically similar to that caused by T. rubrum; besides, N. dimidiatum has been shown to have low sensitivity to itraconazole, but high to terbinafine. LAY SUMMARY: Cases of onychomycosis caused by Neoscytalidium dimidiatum were studied and compared to cases of onychomycosis caused by T. rubrum. The individuals affected were adults, and the clinical characteristics were not different between groups; accordingly, mycological diagnosis is mandatory.

Development and Validation of an In-House Library for Filamentous Fungi Identification by MALDI-TOF MS in a Clinical Laboratory in Medellin (Colombia).

Identification of filamentous fungi by conventional phenotypic methods are time-consuming, and a correct identification at the species level is prone to errors. Therefore, a more accurate and faster time-to-results, and cost-effective technique, is required, such as the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). In this study, we describe the development of an in-house spectra library for the identification of filamentous fungi frequently isolated from patients with infections. An in-house spectra library was constructed using 14 reference strains grown in solid medium. Clinical isolates were identified either by the in-house spectra library or the Biotyper commercial library from Bruker Daltonics. Fungal identification was carried following the Biotyper's established scores: ≤1.699: not reliably identified (NRI); 1.700-1.999: genus-level; ≥2.000: species-level. Clinical isolates were identified, with the in-house library, at species- and genus-level at 88.70% (55) and 3.22% (2), respectively. While 4.80% (3) was NRI and 3.22% (2) was discrepant concerning sequencing. On the contrary, identification up to species and genus-level with the commercial library was 44.44% (16) and 22.22% (8), respectively. NRI and the discrepancy was 30.55% (11) and 2.77% (1), respectively. For the reaming 26 isolates, 16 from Neoscytalidium dimidiatum and 10 from Sporothrix spp., respectively, the absence of spectrum and the specific spectra within the Sporothrix complex in the commercial library resulted in the inability to obtain an identification. In conclusion, the current results advocate the importance that each clinical microbiological laboratory needs to develop an ad hoc library associated with the MALDI-TOF MS fungal identification to overcome the limitations of the available commercial libraries.

ITS rDNA Gene Analysis Versus MALDI-TOF MS For Identification of Neoscytalidium dimidiatum Isolated from Onychomycosis and Dermatomycosis Cases in Medellin (Colombia).

Within the Neoscytalidium genus, N. dimidiatum, N. oculus, N. orchidacearum, and N. novaehollandiae have been recognized. Although these species are frequently found in soil, N. dimidiatum has been identified as an etiologic agent of onychomycosis or dermatomycosis, and N. oculus has been identified as an etiologic agent of an ocular lesion. All these species can be cultured in vitro, but their morphological identification by macroscopic and microscopic traits is difficult and imprecise due to their similarity. In this study, 34 isolates of Neoscytalidium spp. from 32 onychomycosis and two dermatomycosis cases in Medellin (Colombia) were identified at the species level using sequencing of the ITS1+5.8S+ITS2 nuclear rDNA region and MALDI-TOF mass spectrometry (MS). Neoscytalidium dimidiatum strain MUM 17.21 was used to construct the reference spectrum in the in-house library to identify the clinical isolates by MALDI-TOF MS. Additionally, N. dimidiatum PPC-216 and PLAB-055 strains were used to validate the in-house constructed reference spectra. Although four groups were observed in the dendrogram obtained from the proteins of each isolate profile, MALDI-TOF MS and sequencing results are in accordance, since all isolates were identified as N. dimidiatum.

Mechanisms of skin aging / Mecanismos de envejecimiento de la piel / Mecanismos de envelhecimento da pele

SUMMARY Skin aging is an inevitable biological phenomenon of human life that results from either the age-dependent decline of cell function (intrinsic aging) or from cumulative exposure to external harmful influences (extrinsic aging). Intrinsic and extrinsic factors act synergistically to induce skin changes that manifest clinically as burns, erythema, hyperpigmentation, telangiectasia, skin dryness or sagging, coarse wrinkles, skin texture changes or eventually as skin cancer. The molecular mechanisms of both types of skin aging are similar. This review focuses on intrinsic and extrinsic mechanisms of skin aging, and on current and new perspectives for prevention and treatment options extracted from natural products.

RESUMEN El envejecimiento cutáneo es un fenómeno biológico inevitable y puede ser el resultado de la disminución de la función celular como consecuencia del proceso normal de envejecimiento (envejecimiento intrínseco) o de la acumulación de los efectos dañinos debido a la exposición a factores nocivos externos (envejecimiento extrínseco). Los factores intrínsecos y extrínsecos actúan sinérgicamente para inducir cambios en la piel que se manifiestan clínicamente como quemaduras, eritema, hiperpigmentación, telangiectasias, sequedad de la piel o flacidez, arrugas profundas, cambios de textura o cáncer de piel. Los mecanismos moleculares de ambos tipos de envejecimiento de la piel son similares. Esta revisión se centra en una descripción general de los principales mecanismos intrínsecos y extrínsecos del envejecimiento de la piel así como en las medidas actuales de fotoprotección y en las nuevas perspectivas en cuanto a la prevención y al tratamiento basados en productos naturales.

RESUMO O envelhecimento cutâneo e um fenômeno biológico inevitável e pode ser o resultado da diminuição da função celular como consequência do processo normal do envelhecimento (envelhecimento intrínseco) ou do cúmulo dos efeitos daninhos devido à exposição com fatores nocivos externos (envelhecimento extrínseco). Os fatores intrínsecos e extrínsecos atuam sinergicamente para induzir mudanças na pele, que clinicamente manifestam-se como queimaduras, eritema, hiperpigmentação, telangiectasias, pele seca, rugas profundas, perda da tonalidade natural ou câncer de pele. Os mecanismos moleculares dos dois tipos de envelhecimento são similares. Esta revisão, centra-se na descrição geral dos principais mecanismos intrínsecos e extrínsecos do envelhecimento da pele, assim como nas medidas atuais de foto proteção e nas perspectivas referentes à prevenção e tratamento baseados no uso de produtos naturais.

Role of catalase overproduction in drug resistance and virulence in Candida albicans.

AIM: To investigate the role of Cat1 overproduction in Candida albicans. MATERIALS & METHODS: Strains overproducing the CAT1 gene were constructed. RESULTS: Cells overproducing CAT1 were found to be more resistant to some oxidants and mammalian phagocytic cells. They also showed reduced intracellular reactive oxygen species generated by amphotericin B or ciclopirox olamine. CAT1 overproduction did not change the minimum inhibitory concentration of fungal cells to fungistatic or fungicidal azoles nor to amphotericin B although increased twofold the minimum inhibitory concentration to caspofungin. The role of Cat1 overproduction in virulence and colonization was also analyzed in mouse models. CONCLUSION: The overproduction of Cat1 protects against oxidants, phagocytes and certain antifungals at subinhibitory concentration but does not increase virulence in a systemic infection mouse model.

Corrigendum: Impact of Resistance to Fluconazole on Virulence and Morphological Aspects of Cryptococcus neoformans and Cryptococcus gattii Isolates.

[This corrects the article on p. 153 in vol. 7, PMID: 26909069.].

Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host.

We have morphologically characterizedCandida tropicalisisolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of ß-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization ofGalleria mellonellalarvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.

Impact of Resistance to Fluconazole on Virulence and Morphological Aspects of Cryptococcus neoformans and Cryptococcus gattii Isolates.

Cryptococcus sp. are responsible for around 1 million cases of meningitis every year. Fluconazole (FLU) is commonly used in the treatment of cryptococcosis, mainly in immunocompromised patients and the resistance is usually reported after long periods of treatment. In this study, the morphological characterization and virulence profile of FLU-susceptible and FLU-resistant clinical and environmental isolates of C. neoformans and C. gattii were performed both in vitro and in vivo using the Galleria mellonella model. FLU-susceptible isolates from C. neoformans were significantly more virulent than the FLU-resistant isolates. FLU-susceptible C. gattii isolates showed a different virulence profile from C. neoformans isolates where only the environmental isolate, CL, was more virulent compared with the resistant isolates. Cell morphology and capsule size were analyzed and the FLU-resistant isolates did not change significantly compared with the most sensitive isolates. Growth at 37°C was also evaluated and in both species, the resistant isolates showed a reduced growth at this temperature, indicating that FLU resistance can affect their growth. Based on the results obtained is possible suggest that FLU resistance can influence the morphology of the isolates and consequently changed the virulence profiles. The most evident results were observed for C. neoformans showing that the adaptation of isolates to antifungal selective pressure influenced the loss of virulence.

Diagnóstico micológico: de los métodos convencionales a los moleculares / Mycological diagnosis: from conventional methods to molecular ones

Resumen: la incidencia de las infecciones fúngicas ha aumentado en las últimas décadas como consecuencia del incremento en el número de pacientes con factores predisponentes. Tanto el diagnóstico como el tratamiento de estas infecciones continúan siendo un reto para el personal de la salud. Aunque las especies de los géneros Candida, Aspergillus y Cryptococcus continúan siendo las más frecuentes, es notoria la emergencia de especies que no se habían reconocido anteriormente como patógenas para el hombre. Ante este panorama, el diagnóstico adecuado y oportuno de estas infecciones es de gran relevancia para la instauración de un tratamiento eficaz y, de esta manera, para impactar en la morbilidad y mortalidad que suelen causar algunas micosis, principalmente en los individuos inmunocomprometidos. Con esta revisión se pretende mostrar de manera general aspectos clínicos, epidemiológicos y, principalmente, relacionados con el diagnóstico de las infecciones fúngicas más frecuentes, partiendo de los métodos convencionales hasta las técnicas moleculares que actualmente se tratan de implementar en busca de un la prueba estándar de referencia que pueda superar la sensibilidad, la especificidad, la rapidez y el costo-efectividad de los métodos que se han utilizado hasta ahora en el diagnostico micológico.

Abstract: fungal infections incidence has risen in recent decades due to the increase of many predisposingfactors. The diagnosis and treatment of such infections continue to be a challenge for clinicians. Although some species of the genus Candida, Aspergillus and Cryptococcus remain as the most frequent, have emerged more fungal species not formerly recognized as pathogenic. Therefore, there is an increasing need for an appropriate and quick diagnosis of such infections in order to start an effective treatment and thus to impact on morbidity in the majority of patients and on mortality in the immunosuppressed. This review focuses in clinical and epidemiological features, but also in the diagnostic of most common fungal infections, based on conventional methods and new molecular techniques to search an standard test that be to more sensitive and specific, faster and cost-effective.

The production of reactive oxygen species is a universal action mechanism of Amphotericin B against pathogenic yeasts and contributes to the fungicidal effect of this drug.

Amphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species (Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Cryptococcus neoformans, and Cryptococcus gattii). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.

Comparison of the ATB® Fungus 2 with the AFST-EUCAST for in vitro susceptibility testing of Candida spp

The increase of diseases caused by Candida spp., and the treatment failures, has underscored the need for testing the susceptibilities to antifungal agents. The commercial panel ATB® Fungus 2 was compared with the reference testing method of the European Subcommittee on Antifungal Susceptibility Testing of the Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST) for the evaluation of the susceptibility of isolates of Candida spp. to three agents. The percentage of agreement was calculated based on the minimum inhibitory concentrations. There was a high correlation for AMB (100% қ = 1.0 Bhapkar coefficient p = 1.0); while it was lower with azoles (85%, қ = 0.41, p = Bhapkar coefficient 0.02 and 83.0%, қ = 0.15, Bhapkar coefficient p = 0.0006, respectively). The ATB® Fungus 2 and AFST-EUCAST are fully comparable methods for testing the susceptibility to AMB and to lesser extend comparable for ITR and FCA.

El aumento de infecciones por Candida spp. y de las fallas en los tratamientos, suscitan la necesidad de pruebas de susceptibilidad. Se comparó la marca comercial ATB® Fungus 2 con la técnica estándar del Subcomité para las Pruebas de Sensibilidad Antifúngica de la Unión Europea, de la Sociedad de Microbiología Clínica y Enfermedades Infecciosas (AFST-EUCAST) para evaluar la susceptibilidad de aislamientos de Candida spp. a tres antifúngicos. Con base en las concentraciones inhibitorias mínimas se calculó el porcentaje de acuerdo. La concordancia para anfotericina B (AMB) fue alta (100% қ = 1.0, Coeficiente de Bhapkarp = 1.0); para itraconazol (ITR) y fluconazol (FCA) fue inferior (85% қ = 0.41, Coeficiente de Bhapkarp =0.02 y 83.0 %, қ = 0.15, Coeficiente de Bhapkarp = 0.0006, respectivamente). Por lo tanto, ambas técnicas son comparables para la evaluación de la susceptibilidad a AMB; con los azoles el porcentaje de acuerdo es menor.