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Candida auris is an emerging fungal pathogen responsible for hospital outbreaks of invasive candidiasis associated with high mortality. The treatment of these mycoses is a clinical challenge due to the high resistance levels of this species to current antifungal drugs, and alternative therapeutic strategies are needed. In this study, we evaluated the in vitro and in vivo activities of combinations of citral with anidulafungin, amphotericin B or fluconazole against 19 C. auris isolates. The antifungal effect of citral was in most cases similar to the effect of the antifungal drugs in monotherapy. The best combination results were obtained with anidulafungin, with synergistic and additive interactions against 7 and 11 of the 19 isolates, respectively. The combination of 0.06 μg/mL anidulafungin and 64 μg/mL citral showed the best results, with a survival rate of 63.2% in Caenorhabditis elegans infected with C. auris UPV 17-279. The combination of fluconazole with citral reduced the MIC of fluconazole from > 64 to 1–4 μg/mL against 12 isolates, and a combination of 2 μg/mL fluconazole and 64 μg/mL citral was also effective in reducing mortality in C. elegans. Amphotericin B combined with citral, although effective in vitro, did not improve the activity of each compound in vivo.

The pathogenic yeastCandida kruseiis more distantly related toCandida albicansthan clinically relevant CTG-cladeCandidaspecies. Its cell wall, a dynamic organelle that is the first point of interaction between pathogen and host, is relatively understudied, and its wall proteome remains unidentified to date. Here, we present an integrated study of the cell wall inC. krusei. Our comparative genomic studies and experimental data indicate that the general structure of the cell wall inC. kruseiis similar toSaccharomyces cerevisiaeandC. albicansand is comprised of β-1,3-glucan, β-1,6-glucan, chitin, and mannoproteins. However, some pronounced differences withC. albicanswalls were observed, for instance, higher mannan and protein levels and altered protein mannosylation patterns. Further, despite absence of proteins with high sequence similarity toCandidaadhesins, protein structure modeling identified eleven proteins related to flocculins/adhesins inS. cerevisiaeorC. albicans. To obtain a proteomic comparison of biofilm and planktonic cells,C. kruseicells were grown to exponential phase and in static 24-h cultures. Interestingly, the 24-h static cultures ofC. kruseiyielded formation of floating biofilm (flor) rather than adherence to polystyrene at the bottom. The proteomic analysis of both conditions identified a total of 32 cell wall proteins. In line with a possible role in flor formation, increased abundance of flocculins, in particular Flo110, was observed in the floating biofilm compared to exponential cells. This study is the first to provide a detailed description of the cell wall inC. kruseiincluding its cell wall proteome, and paves the way for further investigations on the importance of flor formation and flocculins in the pathogenesis ofC. krusei.AUTHOR SUMMARYThe yeastCandida kruseiis among the five most prevalent causal agents of candidiasis but its mechanisms underlying pathogenicity have been scarcely studied. This is also true for its cell wall structure, an essential organelle that governs primary host-pathogen interactions and host immune responses. Solid knowledge about cell wall synthesis and dynamics is crucial for the development of novel antifungal strategies against this pathogenic yeast. Here, through a combination of comparative genomics, protein structure modeling, and biochemical and proteomic analysis of purified walls, we present a detailed study of the cell wall composition inC. kruseiand identify important architectural differences compared toC. albicanscell walls. Cell walls ofC. kruseicontain higher mannan and protein levels with altered mannan branching patterns, governed by expansions and reductions in gene families encoding mannosyltransferases. We also show that, in contrast to otherCandidaspecies, static cultures produce floating biofilms. Comparative wall proteomic studies of these biofilms show increased abundance of flocculins and hydrolytic enzymes, protein classes implicated in biofilm formation and primary host-pathogen interactions leading to tissue colonization. In conclusion, our study uncovers important keys towards a better molecular understanding of the virulence mechanisms of the important pathogenC. krusei.

Mycoses are accountable for millions of infections yearly worldwide. Invasive candidiasis is the most usual, presenting a high morbidity and mortality.

Background The pathogen Candida auris is rapidly gaining clinical importance because of its resistance to antifungal treatments and its persistence in hospital environments. Early and accurate diagnosis of C. auris infections is crucial, however, the fungus has often been misidentified by commercial systems. Objectives To develop conventional and real-time PCR methods for accurate and rapid identification of C. auris and its discrimination from closely related species by exploiting the uniqueness of certain glycosylphosphatidylinositol-modified protein-encoding genes. Methods Species-specific primers for two unique putative GPI protein-encoding genes per species were designed for C. auris, C. haemulonii, C. pseudohaemulonii, C. duobushaemulonii, C. lusitaniae, and C. albicans. Primers were blind tested for their specificity and efficiency in conventional and real-time multiplex PCR set-up. Results All primers combinations showed excellent species specificity. In multiplex mode, correct identification was aided by different sized amplicons for each species. Efficiency of the C. auris primers was validated using a panel of 155 C. auris isolates, including all known genetically diverse clades. In real-time multiplex PCR, different melting points of the amplicons allowed the distinction of C. auris from four related species. C. auris limit of detection was 5 CFU/reaction with a threshold value of 32. The method was also able to detect C. auris in spiked blood and serum. Conclusions PCR identification based on unique GPI protein-encoding genes allows for accurate and rapid species identification of C. auris and related species without need for expensive equipment when applied in conventional PCR set-up.

Background Candida parapsilosis is the second or third most frequently isolated Candida species related to nosocomial infections, even overtaking Candida albicans in some hospitals. C. parapsilosis constitutes a complex of closely related species: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis. Accurate detection of these species is of importance, as the incidence of C. orthopsilosis has been reported to surpass that of Candida krusei. Objective To evaluate the diagnostic utility of two PCR-RFLP methods targeting the SADH and FKS1 genes and to determine the prevalence of cryptic species in 96 bloodstream isolates of C. parapsilosis from 93 patients. Methods Restriction patterns of the SADH and FKS1 genes were analysed, and sequencing of the D1/D2 regions of the ribosomal RNA was used to evaluate the reliability of both PCR-RFLP methods. Results In our study, 77 C. parapsilosis sensu stricto, 13 C. orthopsilosis and five C. metapsilosis were identified by sequencing. Both PCR-RFLP methods demonstrated strong agreement with D1/D2 sequencing in the identification of C. parapsilosis and C. orthopsilosis, while both methods were unable to identify the C. metapsilosis isolates. Moreover, unexpected restriction patterns were observed for two isolates on SADH PCR-RFLP and for four isolates on FKS1 PCR-RFLP. Mixed bloodstream infections of C. parapsilosis sensu stricto and C. orthopsilosis were detected for three patients, for which differential growth characteristics were observed. Conclusion The molecular method chosen for identification could have an impact on determination of the real prevalence of C. metapsilosis in candidaemia, and mixed fungaemias can remain undetected.

[EN] Mycoses are accountable for millions of infections yearly worldwide. Invasive candidiasis is the most usual, presenting a high morbidity and mortality. Candida albicans remains the prevalent etiologic agent, but the incidence of other species such as Candida parapsilosis, Candida glabrata and Candida auris keeps increasing. These pathogens frequently show a reduced susceptibility to commonly used antifungal drugs, including polyenes, triazoles and echinocandins, and the incidence of emerging multi-drug-resistant strains of these species continues to increase. Therefore, the need to search for new molecules that target these pathogenic species in a different manner is now more urgent than ever. Nature is an almost endless source of interesting new molecules that could meet this need. Among these molecules, antimicrobial peptides, present in different sources in nature, possess some advantages over conventional antifungal agents, even with their own drawbacks, and are considered as a promising pharmacological option against a wide range of microbial infections. In this review, we describe 20 antimicrobial peptides from different origins that possess an activity against Candida. This research was funded by the Spanish Ministry of Science and Innovation (PID2020-117983RB-I00) and by the Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza (IT1607-22). A.P.-R. was funded by a Ph.D. grant from the University of the Basque Country (PIF17/167).

Candida albicans is the major etiological agent of invasive candidiasis but the increasing prevalence of emerging species of Candida, such as Candida glabrata and phylogenetically closely related species, Candida nivariensis and Candida bracarensis, requires special attention. Differences in virulence among these species and their therapeutic responses using in vivo non-mammalian models are scarcely analysed. The aim of this study was analyse the survival of G. mellonella and host-pathogen interactions during infection by C. glabrata, C. nivariensis and C. bracarensis. Moreover, therapeutic responses to echinocandins were also assessed in the G. mellonella model of candidiasis. These three species produced lethal infection in G. mellonella; C. glabrata was the most virulent species and C. bracarensis the less. Haemocytes of G. mellonella phagocytised C. bracarensis cells more effectively than those of the other two species. Treatment with caspofungin and micafungin was most effective to protect larvae during C. glabrata and C. nivariensis infections while anidulafungin was during C. bracarensis infection. The model of candidiasis in G. mellonella is simple and appropriate to assess the virulence and therapeutic response of these emerging Candida species. Moreover, it successfully allows for detecting differences in the immune system of the host depending on the virulence of pathogens. This research was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) [SAF2017-86188-P and PID2020-117983RB-I00] and from the Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza [GIC15/78 IT-990-16]. Ainara Hernando-Ortiz was funded by Ph.D. grants from the University of the Basque Country (PIF 16/39).

El objetivo de este trabajo es dar a conocer el origen y la trayectoria del equipo docente estructurado IKAsasun (edIKDit-18-2, equipo docente para la Enseñanza Multidisciplinar Biosanitaria). El equipo está compuesto por personal docente investigador adscrito a los Campus de Leioa (Facultad de Medicina y Enfermería) y de Vitoria-Gasteiz (Facultad de Farmacia) de la Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU). La docencia que se imparte comprende a distintas disciplinas de Grados mayoritariamente relacionados con las Ciencias de la Salud (Enfermería, Farmacia, Fisioterapia, Medicina, Nutrición Humana y Dietética, Odontología, Ciencia y Tecnología de los Alimentos y Ciencias Ambientales). Desde el inicio de la andadura como equipo docente en el año 2015, el grupo ha participado en dos Proyectos de Innovación Educativa (PIE) subvencionados por la UPV/EHU (PIE nº 40 y nº 96). Estos proyectos están dirigidos a ayudar al alumnado a desarrollar, de una manera eficaz, las competencias específicas y transversales propias de cada Grado y a conseguir que se involucren en su propio aprendizaje. En ambos proyectos se ha incidido en la importancia de trabajar en equipos multidisciplinares, apostando por la utilización de metodologías de aprendizaje activo. En el primero de ellos (PIE nº 40; 2015-2017), se planteó la participación activa del alumnado en la resolución de casos clínicos complejos que requieren un abordaje multidisciplinar para garantizar la asistencia integral al paciente. En el segundo proyecto (PIE nº 96; 2018-2020), se llevó a cabo un abordaje multidisciplinar de temas relacionados con la salud biomédica y científica desde distintos Grados, y posterior divulgación enfocada a diferentes sectores de la sociedad. Los resultados obtenidos tras el desarrollo de ambos proyectos han sido muy alentadores, y alumnado, profesorado y personas externas colaboradoras han valorado muy positivamente la experiencia, que ha quedado reflejada en varias publicaciones y comunicaciones a congresos. The aim of this work is to provide an insight into the origin and background of the IKAsasun structured teaching team (edIKDit-18-2, teaching team for Multidisciplinary Biosanitary Education). Team is formed by an interdisciplinary group of teachers and researchers that belongs to the Campuses of Leioa (Faculty of Medicine and Nursing) and Vitoria-Gasteiz (Faculty of Pharmacy) of the University of the Basque Country (UPV/EHU). The teaching activity of the group covers different disciplines of the Degrees mostly related to Health Sciences (Nursing, Pharmacy, Physiotherapy, Medicine, Human Nutrition and Dietetics, Dentistry, Environmental Sciences and Food Sciences and Technology). Since the beginning of the journey as a teaching team in 2015, the group has participated in two educational innovation projects (PIE) funded by the UPV/EHU (PIE no. 40 and 96) aimed at helping students to effectively develop the specific and transversal competences of each degree, and to get them involved in their own learning. Theseprojects have emphasized the importance of working in multidisciplinary teams, promoting the implementation of active learning methodologies. In the first project (2015-2017), we proposed the resolution of complex clinical cases that require a multidisciplinary approach to ensure integral patient care. In the second one (2018-2020), we proposed a multidisciplinary approach to issues related to biomedical and scientific health from different Degrees, and their subsequent dissemination focusing on different sectors of society. The results obtained after the development of both projects have been very encouraging and both students, teaching staff and external collaborators have valued the experience very positively, which has been reflected in several publications and communications to conferences.

Candida albicans is the major etiological agent of invasive candidiasis but the increasing prevalence of emerging species of Candida, such as Candida glabrata and phylogenetically closely related species, Candida nivariensis and Candida bracarensis, requires special attention. Differences in virulence among these species and their therapeutic responses using in vivo non-mammalian models are scarcely analysed. The aim of this study was analyse the survival of G. mellonella and host-pathogen interactions during infection by C. glabrata, C. nivariensis and C. bracarensis. Moreover, therapeutic responses to echinocandins were also assessed in the G. mellonella model of candidiasis. These three species produced lethal infection in G. mellonella; C. glabrata was the most virulent species and C. bracarensis the less. Haemocytes of G. mellonella phagocytised C. bracarensis cells more effectively than those of the other two species. Treatment with caspofungin and micafungin was most effective to protect larvae during C. glabrata and C. nivariensis infections while anidulafungin was during C. bracarensis infection. The model of candidiasis in G. mellonella is simple and appropriate to assess the virulence and therapeutic response of these emerging Candida species. Moreover, it successfully allows for detecting differences in the immune system of the host depending on the virulence of pathogens.

The aims of this study were to characterize the antifungal activity of amphotericin B against Candida auris in a static in vitro system and to evaluate different dosing schedules and MIC scenarios by means of semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) modelling and simulation. A two-compartment model consisting of a drug-susceptible and a drug-resistant subpopulation successfully characterized the time-kill data and a modified Emax sigmoidal model best described the effect of the drug. The model incorporated growth rate constants for both subpopulations, a death rate constant and a transfer constant between both compartments. Additionally, the model included a parameter to account for the delay in growth in the absence or presence of the drug. Amphotericin B displayed a concentration-dependent fungicidal activity. The developed PK/PD model was able to characterize properly the antifungal activity of amphotericin B against C. auris. Finally, simulation analysis revealed that none of the simulated standard dosing scenarios of 0.6, 1 and 1.5 mg/kg/day over a week treatment showed successful activity against C. auris infection. Simulations also pointed out that an MIC of 1 mg/L would be linked to treatment failure for C. auris invasive infections and therefore, the resistance rate to amphotericin B may be higher than previously reported. This research was funded by Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza, GIC15/78 IT-990-16 and by FIS, Spain, PI17/01538. U.C. was funded by a Ph.D. grant from the University of the Basque Country, PIF 17/266.

Candida auris is an emerging multidrug-resistant fungal pathogen responsible for nosocomial outbreaks of invasive candidiasis. Although several studies on the pathogenicity of this species have been reported, the knowledge on C. auris virulence is still limited. This study aims to analyze the pathogenicity of C. auris, using one aggregating isolate and eleven non-aggregating isolates from different clinical origins (blood, urine and oropharyngeal specimens) in two alternative host models of candidiasis: Caenorhabditis elegans and Galleria mellonella. Furthermore, possible associations between virulence, aggregation, biofilm-forming capacity, and clinical origin were assessed. The aggregating phenotype isolate was less virulent in both in vivo invertebrate infection models than non-aggregating isolates but showed higher capacity to form biofilms. Blood isolates were significantly more virulent than those isolated from urine and respiratory specimens in the G. mellonella model of candidiasis. We conclude that both models of candidiasis present pros and cons but prove useful to evaluate the virulence of C. auris in vivo. Both models also evidence the heterogeneity in virulence that this species can develop, which may be influenced by the aggregative phenotype and clinical origin.

There is an increasing need for novel drugs and new strategies for the therapy of invasive candidiasis. This study aimed to develop and characterize liposome-based nanoparticles of carvacrol, cinnamaldehyde, citral, and thymol with anti-Candida activities. Dioctadecyldimethylammonium bromide- and monoolein-based liposomes in a 1:2 molar ratio were prepared using a lipid-film hydration method. Liposomes were assembled with equal volumes of liposomal stock dispersion and stock solutions of carvacrol, cinnamaldehyde, citral, or thymol in dimethyl sulfoxide. Cytotoxicity was tested on RAW 264.7 macrophages. In vitro antifungal activity of liposomes with phytocompounds was evaluated according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology using clinical isolates of Candida albicans, Candida auris, Candida dubliniensis, and Candida tropicalis. Finally, the ability of macrophage cells to kill Candida isolates after addition of phytocompounds and their nanoparticles was determined. Nanoparticles with 64 mu g/ml of cinnamaldehyde, 256 mu g/ml of citral, and 128 mu g/ml of thymol had the best characteristics among the formulations tested. The highest encapsulation efficiencies were achieved with citral (78% to 83%) and carvacrol (66% to 71%) liposomes. Carvacrol and thymol in liposome-based nanoparticles were nontoxic regardless of the concentration. Moreover, carvacrol and thymol maintained their antifungal activity after encapsulation, and there was a significant reduction (similar to 41%) of yeast survival when macrophages were incubated with carvacrol or thymol liposomes. In conclusion, carvacrol and thymol liposomes possess high stability, low cytotoxicity, and antifungal activity that act synergistically with macrophages., This work was supported by the Consejeria de Educacion, Universidades e Investigacion (GIC15/78 IT-990-16) of Gobierno Vasco-Eusko Jaurlaritza, the Fundacion ONCE Oportunidad al Talento, and the Fondo Social Europeo (to C.M.-A.), and the Federation of European Microbiological Societies (FEMS) by Research and Training grant FEMS-GO-2017-020 (to K.M.-C.).

Candida auris is an emerging multidrug-resistant fungal pathogen responsible for nosocomial outbreaks of invasive candidiasis. Although several studies on the pathogenicity of this species have been reported, the knowledge on C. auris virulence is still limited. This study aims to analyze the pathogenicity of C. auris, using one aggregating isolate and eleven non-aggregating isolates from different clinical origins (blood, urine and oropharyngeal specimens) in two alternative host models of candidiasis: Caenorhabditis elegans and Galleria mellonella. Furthermore, possible associations between virulence, aggregation, biofilm-forming capacity, and clinical origin were assessed. The aggregating phenotype isolate was less virulent in both in vivo invertebrate infection models than non-aggregating isolates but showed higher capacity to form biofilms. Blood isolates were significantly more virulent than those isolated from urine and respiratory specimens in the G. mellonella model of candidiasis. We conclude that both models of candidiasis present pros and cons but prove useful to evaluate the virulence of C. auris in vivo. Both models also evidence the heterogeneity in virulence that this species can develop, which may be influenced by the aggregative phenotype and clinical origin. This work was supported by the Euskal Herriko Unibertsitatea [PIF 16/39]; Euskal Herriko Unibertsitatea [PIF17/167]; Eusko Jaurlaritza [GIC15/78 IT-990-16]; Ministerio de Economia y Competitividad [SAF2017-86188-P].

[EN]Oral candidiasis is frequently associated with Candida biofilms. Biofilms are microbial communities related to persistent, recalcitrant and difficult to-treat infections. Conventional treatments are not sufficient to overcome biofilm-associated candidiasis; thus, the search of new antifungal compounds is necessary. In the current study, we have evaluated the effect of three phytocompounds, carvacrol, cinnamaldehyde and thymol, against Candida planktonic and sessile cells. Reduction in biofilm biomass and metabolic activity was assessed during adhesion and mature biofilm phases. Candida albicans was the most biofilm-producing Candida species. All phytocompounds tested were fungicidal against Candida planktonic cells. Cinnamaldehyde was the most active in inhibiting biofilm adhesion, but carvacrol and thymol significantly reduced both mature biofilm biomass and metabolic activity. These results highlight the role of cinnamaldehyde, carvacrol and thymol as promising alternatives for the treatment of candidiasis due to their antibiofilm capacities, and stress the necessity to continue studies on their safety, toxicity and pharmacodynamics and pharmacokinetics. This work was supported by Gobierno Vasco-Eusko Jaurlaritza, Spain [grant number GIC15/78 IT-990-16, 2016] and Fundacion ONCE "Oportunidad al Talento", Spain and Fondo Social Europeo, Spain [CMA, 2018].

Candida auris is an emergent fungal pathogen that causes severe infectious outbreaks globally. The public health concern when dealing with this pathogen is mainly due to reduced susceptibility to current antifungal drugs. A valuable alternative to overcome this problem is to investigate the efficacy of combination therapy. The aim of this study was to determine the in vitro interactions of isavuconazole with echinocandins against C. auris. Interactions were determined using a checkerboard method, and absorbance data were analyzed with different approaches: the fractional inhibitory concentration index (FICI), Greco universal response surface approach, and Bliss interaction model. All models were in accordance and showed that combinations of isavuconazole with echinocandins resulted in an overall synergistic interaction. A wide range of concentrations within the therapeutic range were selected to perform time-kill curves. These confirmed that isavuconazole–echinocandin combinations were more effective than monotherapy regimens. Synergism and fungistatic activity were achieved with combinations that included isavuconazole in low concentrations (≥0.125 mg/L) and ≥1 mg/L of echinocandin. Time-kill curves revealed that once synergy was achieved, combinations of higher drug concentrations did not improve the antifungal activity. This work launches promising results regarding the combination of isavuconazole with echinocandins for the treatment of C. auris infections. This research was funded by Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza, GIC15/78 IT-990-16 and by FIS, Spain, PI17/01538. UC was funded by a Ph.D. grant from the University of the Basque Country UPV/EHU, PIF 17/266.

Although Candida albicans remains the major etiological agent of invasive candidiasis, Candida glabrata and other emerging species of Candida are increasingly isolated. This species is the second most prevalent cause of candidiasis in many regions of the world. However, clinical isolates of Candida nivariensis and Candida bracarensis can be misidentified and are underdiagnosed due to phenotypic traits shared with C. glabrata. Little is known about the two cryptic species. Therefore, pathogenesis studies are needed to understand their virulence traits and their susceptibility to antifungal drugs. The susceptibility of Caenorhabditis elegans to different Candida species makes this nematode an excellent model for assessing host-fungus interactions. We evaluated the usefulness of C. elegans as a nonconventional host model to analyze the virulence of C. glabrata, C. nivariensis, and C. bracarensis. The three species caused candidiasis, and the highest virulence of C. glabrata was confirmed. Furthermore, we determined the efficacy of current antifungal drugs against the infection caused by these species in the C. elegans model. Amphotericin B and azoles showed the highest activity against C. glabrata and C. bracarensis infections, while echinocandins were more active for treating those caused by C. nivariensis. C. elegans proved to be a useful model system for assessing the pathogenicity of these closely related species.

[EN] Background: Morphological, physical and chemical properties of both implants and prostheses can determine the biofilm formation on their surface and increase the risk of biological complications. The aim of this study was to evaluate the capacity of biofilm formation of Candida albicans on different materials used to manufacture abutments and prostheses. Material and Methods: Biofilm formation was analyzed on cp grade II titanium, cobalt-chromium alloy and zirconia, silicone, acrylic resin (polymethylmethacrylate) and nano-hybrid composite. Some samples were partially covered with lithium disilicate glass ceramic to study specifically the junction areas. C. albicans was incubated in a biofilm reactor at 37 °C with agitation. The biofilm formation was evaluated at 24 and 48 hours. In addition, the morphology of the biofilm was evaluated by scanning electron microscopy. Results: C. albicans developed biofilms on the surface of all materials tested. Cobalt-chromium alloy showed the lowest density of adhered biofilm, followed by zirconia and titanium. Silicone and resin showed up to 20 times higher density of biofilm. A higher biofilm formation was observed when junctions of materials presented micropores or imperfections. Conclusions: The biofilm formed in the three materials used in the manufacture of abutments and prostheses showed no major differences, being far less dense than in the resins. Two clinical recommendations can be made: to avoid the presence of resins in the subgingival area of implant prostheses and to design prostheses placing cobalt-chromium alloy/ceramic or titanium/ceramic junctions as far as possible from implants. ES, GQ, and EE have received research grant support from the Consejería de Educación, Universidades e Investigación del Gobierno Vasco-Eusko Jaurlaritza (GIC15 78 IT-990-16), the Ministerio de Economía y Competitividad del Gobierno de España, MINECO (SAF2017-86188-P) and the Universidad del País Vasco/Euskal Herriko Unibertsitatea, UPV/EHU (UFI 11/25). The authors report no conflicts of interest related to this study. The authors thank for technical and human support provided by Ramón Santamaría (Corus dental) and Analytical and High-Resolution Microscopy Service in Biomedicine of SGIker-UPV/EHU.

Objective To evaluate the importance of Candida glabrata, Candida parapsilosis and their close-related species, Candida bracarensis, Candida nivariensis, Candida metapsilosis and Candida orthopsilosis in patients with oral candidiasis and, to determine the in vitro activities of antifungal drugs currently used for the treatment. Methods One hundred fourteen isolates of C. glabrata and 97 of C. parapsilosis, previously identified by conventional mycological methods, were analysed by molecular techniques. In vitro antifungal susceptibility to fluconazole, itraconazole, miconazole, and nystatin was evaluated by CLSI M44-A2 disk diffusion test, and by CLSI M27-A3 microdilution for fluconazole. Results All C. glabrata isolates were identified as C. glabrata sensu stricto, 93 out of 97 C. parapsilosis isolates as C. parapsilosis sensu stricto, three as C. orthopsilosis and one as C. metapsilosis. Candida glabrata was mainly isolated in mixed cultures but C. parapsilosis complex was more frequent in pure culture. Candida metapsilosis and C. orthopsilosis were isolated as pure culture and both species were susceptible to all antifungal agents tested. Most C. glabrata isolates were susceptible to miconazole and nystatin, but resistant to fluconazole and itraconazole. Azole cross resistance was also observed. Candida parapsilosis isolates were susceptible to fluconazole although azole cross resistance to miconazole and itraconazole was observed. Conclusion This study highlights the importance of accurate identification and antifungal susceptibility testing of oral Candida isolates in order to have an in-depth understanding of the role of C. glabrata and C. parapsilosis in oral candidiasis.

The emerging multidrug-resistant pathogenic yeast Candida auris causes life-threatening invasive infections and shows a capacity for hospital transmission that is uncommon in other Candida species. Rapid and accurate diagnosis of C. curls infections is crucial; however, the fungus is frequently misidentified. Here, we present a rapid and easily applicable PCR assay for reliable identification of C. auris by designing primers from unique GPI protein-encoding genes. Specificity of the used primers for C. auris was verified with a panel of 19 different Candida species including the clinically most relevant and phylogenetically closely related species. Efficacy of the PCR approach was validated by correctly identifying 112 C. auris isolates from an outbreak in a Spanish hospital, 20% of which were not reliably identified by MALDI-TOF MS, and 27 genotypically diverse C. auris isolates originating from hospitals in various countries, in a test that included (blind) negative controls. By employing two GPI protein primer pairs in a single PCR, a double screening can be performed, which enhances the robustness of the PCR assay and avoids potential false negatives due to recent evolutionary events, as was observed for two isolates. Our PCR method, which is based on the uniqueness of selected GPI protein-encoding genes, is useful for easy, low-cost, and accurate identification of C. auris infections in a clinical setting.

Recent changes in the aetiology and epidemiology of invasive candidiasis have serious implications for current and future diagnosis, treatment and prognosis. The aim of the current review was to discuss the epidemiology of invasive candidiasis, the distribution of Candida species in different regions of the world, the medical concerns of the changing aetiology and the emergence of antifungal resistance. Overall burden of invasive candidiasis remains high, especially in vulnerable persons, such as the elderly, immunosuppressed or debilitated patients. Moreover, there is a progressive shift in the aetiology of invasive candidiasis from Candida albicans to other species of Candida, probably related to the increased use of azole drugs with a clear trend towards increased antifungal resistance. Finally, the emergence and rise of multiresistant species, such as Candida auris or Candida glabrata, is a major threat making necessary invasive candidiasis worldwide surveillances. These changes have serious implications for the diagnosis, treatment and prognosis of invasive candidiasis. Updated knowledge of the current local epidemiology of invasive candidiasis is critical for the clinical management.

Candida can be implicated in the pathology of chronic periodontitis.To analyze the oral Candida carriage in patients suffering from chronic periodontitis (CP) and its correlation with the severity of this condition.Microbiological samples were taken from 155 patients using the oral rinse (OR) technique and by using paper points in the periodontal pockets (GPP). These patients were divided into 3 groups: 89 patients without CP (control), 47 with moderate CP, and 19 with severe CP. Samples were cultured in a Candida chromogenic agar for Candida. Species were identified by microbiological and molecular methods.Candida was isolated in the OR of 45 (50.6%), 21 (44.7%), and 11 (57.9%) patients, respectively, and in the GPP of 32 (36%), 14 (29.2%), and 10 (42.6%) patients from the control, moderate CP and severe CP groups, respectively. Candida was isolated more frequently and in a greater burden in OR than in GPP (p0.01). Candida albicans was the most prevalent species. GPP of patients with CP had poor fungal biodiversity (p0.01).Colonization by Candida was present in the samples of patients without CP, and with both moderate and severe CP. Nonetheless, patients with severe CP had a higher rate of Candida colonization, especially by C. albicans.

Caries and chronic periodontitis are common oral diseases where a higher Candida colonization is reported. Antifungal agents could be adjuvant drugs for the therapy of both clinical conditions. The aim of the current study has been to evaluate the in vitro activities of conventional and new antifungal drugs against oral Candida isolates from patients suffering from caries and/or chronic periodontitis. In vitro activities of amphotericin B, fluconazole, itraconazole, miconazole, nystatin, posaconazole and voriconazole against 126 oral Candida isolates (75 Candida albicans, 18 Candida parapsilosis, 11 Candida dubliniensis, six Candida guilliermondii, five Candida lipolytica, five Candida glabrata, four Candida tropicalis and two Candida krusei) from 61 patients were tested by the CLSI M27-A3 method. Most antifungal drugs were highly active, and resistance was observed in less than 5% of tested isolates. Miconazole was the most active antifungal drug, being more than 98% of isolates susceptible. Fluconazole, itraconazole, and the new triazoles, posaconazole and voriconazole, were also very active. Miconazole, fluconazole and voriconazole have excellent in vitro activities against all Candida isolates and could represent suitable treatment for a hypothetically adjunctive therapy of caries and chronic periodontitis.

Healthcare-associated infections (HAIs) can be caused by microorganisms present in common practice instruments generating major health problems in the hospital environment. The aim of this work was to evaluate the disinfection capacity of a portable ultraviolet C equipment (UV Sanitizer Corvent&reg, UVSC-) developed to disinfect different objects. For this purpose, six pathogens causing HAIs: Acinetobacter baumannii, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans, were inoculated on slides and discs of different biomaterials (borosilicate, polycarbonate, polyurethane, silicone, Teflon and titanium) and exposed to ultraviolet C radiation. UVSC disinfection was compared with ethanol and chlorhexidine antimicrobial activities following the standards EN14561 and EN14562. Disinfection, established as a reduction of five logarithms from the initial inoculum, was achieved with the UVSC at 120 s of exposure time, with and without the presence of organic matter. The disinfectant effect was observed against S. aureus, P. aeruginosa, E. coli, B. subtilis and C. albicans (reduction &gt, 99.999%). Disinfection was also achieved with 70% ethanol and 2% chlorhexidine. As conclusion, UVSC was effective disinfecting the most contaminated surfaces assayed, being a promising alternative for disinfecting hospital materials and inanimate objects that cannot be immersed in liquid biocides, reducing the risk of pathogen transmission.