Fluconazole-resistant Candida parapsilosis: fast detection of the Y132F ERG11p substitution, and a proposed microsatellite genotyping scheme.

OBJECTIVES: We propose fast and accurate molecular detection of the Y132F ERG11p substitution directly on pure-cultured C. parapsilosis isolates. We also assessed a discriminative genotyping scheme to track circulating genotypes. METHODS: A total of 223 C. parapsilosis isolates (one patient each) from 20 hospitals, located in Spain and Italy were selected. Isolates were fluconazole-resistant (n=94; harbouring the Y132F ERG11p substitution [n=85], the G458S substitution [n=6], the R398I substitution [n=2], or the wild-type ERG11 gene sequence) or fluconazole-susceptible (n=129). Two targeted-A395T-mutation PCR formats (conventional and real-time) were engineered and optimized on fluconazole-susceptible and fluconazole-resistant pure-cultured isolates, thus skipping DNA extraction. Two genotyping schemes were compared: Scheme 1 (CP1, CP4a, CP6, and B markers), and Scheme 2 (6A, 6B, 6C, CP1, CP4a, and CP6 markers). RESULTS: The screening performed using both PCR formats showed 100% specificity (fluconazole-susceptible isolates; n=129/129) and sensitivity (Y132F isolates; n=85/85) values, however, results were available in 3 and 1.5 hours with the conventional and real-time PCR formats, respectively. Overall, Scheme 1 showed higher genetic diversity than Scheme 2, as shown by the number of alleles detected (n=98; mean 23, range 13-38), the significantly higher observed and expected heterozygosity, and the probability of identity index (2.5x10-6). Scheme 2 markers did not provide further genotypic discrimination of Y132F fluconazole-resistant genotypes. CONCLUSION: Both proposed PCR formats allow to speed up the accurate detection of substitution Y132F ERG11p in C. parapsilosis isolates with 100% specificity and sensitivity. In addition, we recommend CP1, CP4a, CP6, and B microsatellite markers for genotyping fluconazole-resistant isolates.

Endogenous CO2 Overpressure Effect on Higher Alcohols Metabolism during Sparkling Wine Production.

Higher alcohols produced by yeast during the fermentation of sparkling wine must have the greatest impact on the smell and taste of wine. At present, the metabolic response to methanol and higher alcohols formation of Saccharomyces cerevisiae under endogenous CO2 overpressure has not been fully elucidated. In this work, a proteomics and metabolomics approach using a OFFGEL fractionator and the LTQ Orbitrap for the protein identification, followed by a metabolomic study for the detection and quantification of both higher alcohols (GC-FID and SBSE-TD-GC-MS) and amino acids (HPLC), was carried out to investigate the proteomic and metabolomic changes of S. cerevisiae in relation to higher alcohols formation under a CO2 overpressure condition in a closed bottle. The control condition was without CO2 overpressure in an open bottle. Methanol and six higher alcohols were detected in both conditions, and we have been able to relate to a total of 22 proteins: 15 proteins in the CO2 overpressure condition and 22 proteins in the control condition. As for the precursors of higher alcohols, 18 amino acids were identified in both conditions. The metabolic and proteomic profiles obtained in both conditions were different, so CO2 overpressure could be affecting the metabolism of higher alcohols. Furthermore, it was not possible to establish direct correlations in the condition under CO2 overpressure; however, in the condition without pressure it was possible to establish relationships. The data presented here can be considered as a platform that serves as a basis for the S. cerevisiae metabolome-proteome with the aim of understanding the behavior of yeast under conditions of second fermentation in the production of sparkling wines.

The Role of S. cerevisiae Sub1/PC4 in Transcription Elongation Depends on the C-Terminal Region and Is Independent of the ssDNA Binding Domain.

Saccharomyces cerevisiae Sub1 (ScSub1) has been defined as a transcriptional stimulatory protein due to its homology to the ssDNA binding domain (ssDBD) of human PC4 (hPC4). Recently, PC4/Sub1 orthologues have been elucidated in eukaryotes, prokaryotes, and bacteriophages with functions related to DNA metabolism. Additionally, ScSub1 contains a unique carboxyl-terminal region (CT) of unknown function up to date. Specifically, it has been shown that Sub1 is required for transcription activation, as well as other processes, throughout the transcription cycle. Despite the progress that has been made in understanding the mechanism underlying Sub1's functions, some questions remain unanswered. As a case in point: whether Sub1's roles in initiation and elongation are differentially predicated on distinct regions of the protein or how Sub1's functions are regulated. Here, we uncover some residues that are key for DNA-ScSub1 interaction in vivo, localized in the ssDBD, and required for Sub1 recruitment to promoters. Furthermore, using an array of genetic and molecular techniques, we demonstrate that the CT region is required for transcription elongation by RNA polymerase II (RNAPII). Altogether, our data indicate that Sub1 plays a dual role during transcription-in initiation through the ssDBD and in elongation through the CT region.

Educational Programs for the Promotion of Health at School: A Systematic Review.

CONTEXT: Health promotion programs generate healthy changes in the educational community. However, not all of them meet the expected objectives due to multiple factors that affect their development, such as the teachers overload work, the lack of specific training, the lack of time to carry out health promotion activities, the lack of flexibility of the programs, and their non-inclusion in the training programs of the centers. OBJECTIVE: To know the scope of the strategies and programs that promote healthy habits among students in compulsory educational stages. DATA SOURCES: a systematic review of articles in English, using the Web of Science (WOS), Medline, and PsycINFO databases.

Disseminated lomentosporiosis in a heart transplant recipient: Case report and review of the literature.

BACKGROUND: Lomentospora prolificans (formerly S prolificans) is a saprophyte fungi that causes opportunistic infections in solid organ transplant (SOT) recipients. Resulting disseminated infections are difficult to treat and have a high mortality. Indications for antifungal prophylaxis after heart transplantation (HT) include CMV disease, reoperation, renal replacement therapy, extracorporeal membrane oxygenation (ECMO), and high environmental exposure to Aspergillus spores. However, the risk of breakthrough infections, such as Lomentosporiosis, remains a cause of concern. METHODS: We report the clinical findings, microbiology, treatment and outcome of a disseminated Lomentosporiosis in a heart transplant recipient with ECMO and antifungal prophylaxis. RESULTS: A 25-year-old male with complex grown-up congenital heart disease (GUCHD) was admitted for HT. He presented severe post-surgical complications including acute kidney injury and right heart and respiratory failure requiring venoarterial-ECMO, continuous renal replacement therapy (CCRT) and later on (+14) a ventricular assist device (VAD). Ganciclovir, cotrimoxazole, and antifungal prophylaxis with anidulafungin at standard doses had been started on day + 3 post HT. The patient presented seizures (+4), pancytopenia with mild neutropenia (days + 6 to + 11), influenza B (+7), and bacteremic Pseudomonas aeruginosa ventilator associated pneumonia (VAP) (+10). On days + 14 to + 16 Lomentospora prolificans was recovered from blood cultures, broncho aspirate, catheter tip, and skin biopsy. Despite treatment with L-AMB, voriconazole and terbinafine the patients died on day 17 after HT. Necropsy revealed disseminated infection with fungal invasion in central nervous system, heart, lung, cutaneous, and subcutaneous tissue. Broth microdilution tests demonstrated resistance to all antifungals. CONCLUSIONS: Lomentosporiosis is a rare complication that may emerge as a breakthrough invasive fungal infection in heart transplant recipients on ECMO despite antifungal prophylaxis.

Utility of 1,3 ß-d-Glucan Assay for Guidance in Antifungal Stewardship Programs for Oncologic Patients and Solid Organ Transplant Recipients.

The implementation of 1,3 ß-d-glucan (BDG) has been proposed as a diagnostic tool in antifungal stewardship programs (ASPs). We aimed to analyze the influence of serum BDG in an ASP for oncologic patients and solid organ transplant (SOT) recipients. We conducted a pre-post study. In the initial period (PRE), the ASP was based on bedside advice, and this was complemented with BDG in the post-period (POST). Performance parameters of the BDG assay were determined. Antifungal (AF) use adequacy was evaluated using a point score. Clinical outcomes and AF costs were also compared before and after the intervention. Overall, 85 patients were included in the PRE-period and 112 in the POST-period. Probable or proven fungal infections were similar in both groups (54.1% vs. 57.1%; p = 0.67). The determination of BDG contributed to improved management in 75 of 112 patients (66.9%). The AF adequacy score improved in the POST-period (mean 7.75 vs. 9.29; p < 0.001). Median days of empiric AF treatment was reduced in the POST-period (9 vs. 5 days, p = 0.04). All-cause mortality (44.7% vs. 34.8%; p = 0.16) was similar in both periods. The cost of AF treatments was reduced in the POST-period with a difference of 779.6 €/patient. Our data suggest that the use of BDG was a cost-effective strategy that contributed to safely improving the results of an ASP for SOT and oncologic patients.

Comparative Study of the Proteins Involved in the Fermentation-Derived Compounds in Two Strains of Saccharomyces cerevisiae during Sparkling Wine Second Fermentation.

Sparkling wine is a distinctive wine. Saccharomyces cerevisiae flor yeasts is innovative and ideal for the sparkling wine industry due to the yeasts' resistance to high ethanol concentrations, surface adhesion properties that ease wine clarification, and the ability to provide a characteristic volatilome and odorant profile. The objective of this work is to study the proteins in a flor yeast and a conventional yeast that are responsible for the production of the volatile compounds released during sparkling wine elaboration. The proteins were identified using the OFFGEL fractionator and LTQ Orbitrap. We identified 50 and 43 proteins in the flor yeast and the conventional yeast, respectively. Proteomic profiles did not show remarkable differences between strains except for Adh1p, Fba1p, Tdh1p, Tdh2p, Tdh3p, and Pgk1p, which showed higher concentrations in the flor yeast versus the conventional yeast. The higher concentration of these proteins could explain the fuller body in less alcoholic wines obtained when using flor yeasts. The data presented here can be thought of as a proteomic map for either flor or conventional yeasts which can be useful to understand how these strains metabolize the sugars and release pleasant volatiles under sparkling wine elaboration conditions.

Biological Processes Highlighted in Saccharomyces cerevisiae during the Sparkling Wines Elaboration.

Sparkling wines elaboration has been studied by several research groups, but this is the first report on analysis of biological processes according to the Gene Ontology terms (GO terms) and related to proteins expressed by yeast cells during the second fermentation of sparkling wines. This work provides a comprehensive study of the most relevant biological processes in Saccharomyces cerevisiae P29, a sparkling wine strain, during the second fermentation under two conditions (without and with endogenous CO2 overpressure) in the middle and the end of second fermentation. Consequently, a proteomic analysis with the OFFGEL fractionator and protein identification with LTQ Orbitrap XL coupled to HPLC were performed. The classification of biological processes was carried out using the tools provided by the Saccharomyces Genome Database. Results indicate that a greater number of biological processes were identified under condition without CO2 overpressure and in the middle of the fermentation versus the end of the second fermentation. The biological processes highlighted under condition without CO2 overpressure in the middle of the fermentation were involved in the carbohydrate and lipid metabolic processes and catabolic and biosynthetic processes. However, under CO2 overpressure, specific protein expression in response to stress, transport, translation, and chromosome organization and specific processes were not found. At the end of fermentation, there were higher specific processes under condition without CO2 overpressure; most were related to cell division, growth, biosynthetic process, and gene transcription resulting in increased cell viability in this condition. Under CO2 overpressure condition, the most representative processes were related to translation as tRNA metabolic process, chromosome organization, mRNA processing, ribosome biogenesis, and ribonucleoprotein complex assembly, probably in response to the stress caused by the hard fermentation conditions. Therefore, a broader knowledge of the adaptation of the yeast, and its behavior under typical conditions to produce sparkling wine, might improve and favor the wine industry and the selection of yeast for obtaining a high-quality wine.

Autophagic Proteome in Two Saccharomyces cerevisiae Strains During Second Fermentation for Sparkling Wine Elaboration.

A correlation between autophagy and autolysis has been proposed in order to acceleratethe acquisition of wine organoleptic properties during sparkling wine elaboration. In this context, aproteomic analysis was carried out in two industrial Saccharomyces cerevisiae strains (P29,conventional sparkling wine strain and G1, implicated in sherry wine elaboration) with the aim ofstudying the autophagy-related proteome and comparing the effect of CO2 overpressure duringsparkling wine elaboration. In general, a detrimental effect of pressure and second fermentationdevelopment on autophagy-related proteome was observed in both strains, although it was morepronounced in flor yeast strain G1. Proteins mainly involved in autophagy regulation andautophagosome formation in flor yeast G1, and those required for vesicle nucleation and expansionin P29 strain, highlighted in sealed bottle. Proteins Sec2 and Sec18 were detected 3-fold underpressure conditions in P29 and G1 strains, respectively. Moreover, 'fingerprinting' obtained frommultivariate data analysis established differences in autophagy-related proteome between strainsand conditions. Further research is needed to achieve more solid conclusions and design strategiesto promote autophagy for an accelerated autolysis, thus reducing cost and time production, as wellas acquisition of good organoleptic properties.

Differential Analysis of Proteins Involved in Ester Metabolism in two Saccharomyces cerevisiae Strains during the Second Fermentation in Sparkling Wine Elaboration.

The aromatic metabolites derived from yeast metabolism determine the characteristics of aroma and taste in wines, so they are considered of great industrial interest. Volatile esters represent the most important group and therefore, their presence is extremely important for the flavor profile of the wine. In this work, we use and compare two Saccharomyces cerevisiae yeast strains: P29, typical of sparkling wines resulting of second fermentation in a closed bottle; G1, a flor yeast responsible for the biological aging of Sherry wines. We aimed to analyze and compare the effect of endogenous CO2 overpressure on esters metabolism with the proteins related in these yeast strains, to understand the yeast fermentation process in sparkling wines. For this purpose, protein identification was carried out using the OFFGEL fractionator and the LTQ Orbitrap, following the detection and quantification of esters with gas chromatograph coupled to flame ionization detector (GC-FID) and stir-bar sorptive extraction, followed by thermal desorption and gas chromatography-mass spectrometry (SBSE-TD-GC-MS). Six acetate esters, fourteen ethyl esters, and five proteins involved in esters metabolism were identified. Moreover, significant correlations were established between esters and proteins. Both strains showed similar behavior. According to these results, the use of this flor yeast may be proposed for the sparkling wine production and enhance the diversity and the typicity of sparkling wine yeasts.

Empatía médica en residentes y tutores de medicina familiar y comunitaria. La visión del profesional y del paciente / Clinical empathy among family and community medicine residents and tutors. The view of physicians and patients

OBJETIVO: Determinar el grado de empatía de residentes y tutores de medicina de familia. Saber si existe relación entre la empatía autopercibida por los médicos y la valorada por sus pacientes. DISEÑO: Estudio observacional transversal mediante encuesta. Emplazamiento: Unidad Docente de Atención Primaria, Madrid. PARTICIPANTES: Se envió una encuesta por correo electrónico a todos los tutores y residentes de familia de la Unidad Docente. Respondieron 50 residentes (39,4%) y 41 tutores (45%). Un total de 428 pacientes fueron captados de forma oportunista en un Centro de Salud. Se entrevistó a sus médicos. Mediciones principales: La empatía se midió mediante la Escala de Empatía Médica de Jefferson y la Escala de Percepciones de los Pacientes sobre la Empatía Médica de Jefferson. RESULTADOS: Los tutores puntuaron 2,53 puntos más alto en empatía cognitiva que los residentes (p = 0,04). Las puntuaciones de la empatía emocional de los tutores son menores en aquellos de mayor edad (r = -0,32; p < 0,05). Los residentes españoles (el 82%) y sin experiencia laboral previa puntuaron más alto en empatía global (p = 0,02). Los residentes de último año obtuvieron calificaciones significativamente más bajas en empatía que el resto de sus compañeros. Se observó correlación positiva (r = 0,72; p = 0,01) entre la autopercepción de la empatía del profesional y la referida por sus pacientes. CONCLUSIONES: Los residentes con experiencia profesional previa, los de último año de residencia y los de origen latinoamericano presentan puntuaciones más bajas de empatía. Existe fuerte relación entre la empatía autopercibida por los médicos y la visión que sus pacientes tienen sobre la misma

AIM: To determine the degree of clinical empathy among family medicine residents and tutors. To gauge whether there is a relationship between physicians' self-perceived empathy levels and their patients' assessments. Study design: Observational, cross-sectional survey. LOCATION: Primary Care Teaching Unit. Madrid. PARTICIPANTS: A survey was sent by email to all the Teaching Unit's family medicine tutors and residents. Responses were received from 50 residents (39.4%) and 41 tutors (45%). In addition, 428 patients were opportunistically recruited at a healthcare centre and their doctors were also interviewed. Primary measurement instruments: Empathy was measured using the Jefferson Scale of Empathy and the Jefferson Scale of Patient Perceptions of Physician Empathy. RESULTS: The tutors scored 2.53 points higher for cognitive empathy than the residents (P = .04). Emotional empathy scores declined among older tutors (r = -0.32; P = .05). The Spanish students (82% of the total) without previous work experience scored higher for overall empathy (P = .02). Final-year residents recorded significantly worse empathy assessments than the other residents. A positive correlation (r = 0.72; P = .01) was observed between physicians' self-perceived empathy and their patients' perceptions. CONCLUSIONS: Residents with previous work experience, final-year residents and those of Latin American origin score lower for empathy. There is a strong relationship between physicians' self-perceived empathy and their patients' views of their empathy levels

Correction: Increasing incidence of mucormycosis in a large Spanish hospital from 2007 to 2015: Epidemiology and microbiological characterization of the isolates.

[This corrects the article DOI: 10.1371/journal.pone.0179136.].

A retrospective cohort of invasive fusariosis in the era of antimould prophylaxis.

Mould-active prophylaxis is affecting the epidemiology of invasive mycoses in the form of a shift toward less common entities such as fusariosis. We analyze the characteristics of invasive fusariosis and its association to antifungal prophylaxis in a retrospective cohort (2004-2017) from a tertiary hospital in Madrid, Spain. Epidemiological, clinical, microbiological, and antifungal consumption data were retrieved. Isolates were identified to molecular level, and antifungal susceptibility was tested. Eight cases of invasive fusariosis were diagnosed. Three periods were identified according to incidence: <2008 (three cases), 2008-2013 (zero cases), >2014 (five cases). All except one case involved breakthrough fusariosis. During the earliest period, the episodes occurred while the patient was taking itraconazole (two) or fluconazole (one); more recently, while on micafungin (three) or posaconazole (one). Early cases involved acute leukemia at induction/consolidation, recent cases relapsed/refractory disease (P = .029). Main risk factor for fusariosis (62.5%) was prolonged neutropenia (median 44 days). Galactomannan and beta-D-glucan were positive in 37.5% and 100% of cases, respectively. All isolates except F. proliferatum presented high minimal inhibitory concentrations (MICs) against the azoles and lower MIC to amphotericin B. Most patients received combined therapy. Mortality at 42 days was 62.5%. Resolution of neutropenia was associated with survival (P = .048). Invasive fusariosis occurs as breakthrough infection in patients with hematologic malignancy, prolonged neutropenia, and positive fungal biomarkers. Recent cases were diagnosed in a period of predominant micafungin use in patients who had more advanced disease and protracted neutropenia and for whom mortality was extremely high. Resolution of neutropenia was a favorable prognostic factor.

[Clinical empathy among family and community medicine residents and tutors. The view of physicians and patients]. / Empatía médica en residentes y tutores de medicina familiar y comunitaria. La visión del profesional y del paciente.

AIM: To determine the degree of clinical empathy among family medicine residents and tutors. To gauge whether there is a relationship between physicians' self-perceived empathy levels and their patients' assessments. STUDY DESIGN: Observational, cross-sectional survey. LOCATION: Primary Care Teaching Unit. Madrid. PARTICIPANTS: A survey was sent by email to all the Teaching Unit's family medicine tutors and residents. Responses were received from 50 residents (39.4%) and 41 tutors (45%). In addition, 428 patients were opportunistically recruited at a healthcare centre and their doctors were also interviewed. PRIMARY MEASUREMENT INSTRUMENTS: Empathy was measured using the Jefferson Scale of Empathy and the Jefferson Scale of Patient Perceptions of Physician Empathy. RESULTS: The tutors scored 2.53 points higher for cognitive empathy than the residents (P=.04). Emotional empathy scores declined among older tutors (r= -0.32; P=.05). The Spanish students (82% of the total) without previous work experience scored higher for overall empathy (P=.02). Final-year residents recorded significantly worse empathy assessments than the other residents. A positive correlation (r=0.72; P=.01) was observed between physicians' self-perceived empathy and their patients' perceptions. CONCLUSIONS: Residents with previous work experience, final-year residents and those of Latin American origin score lower for empathy. There is a strong relationship between physicians' self-perceived empathy and their patients' views of their empathy levels.

Correlación de gasto cardiaco obtenido por termodilución y por monitor ultrasónico (USCOM) en pacientes con choque séptico / Correlation of cardiac output obtained by termodulution and by ultrasonic monitor in patients with septic shock (USCOM) / Correlação do débito cardíaco obtido pela termodulação e pelo monitor ultrasônico (USCOM) em pacientes com choque septico

Resumen: El catéter en la arteria pulmonar (CAP) es un dispositivo utilizado en unidades de cuidados intensivos (UCI) para medir las presiones en el corazón y los vasos sanguíneos pulmonares como parte del monitoreo hemodinámico, principalmente en pacientes de cirugía cardiaca. El dispositivo USCOM se trata de una técnica no invasiva que utiliza la tecnología Doppler para obtener las medidas de volumen sistólico y sus derivados. Se realiza la siguiente comparación de medición de GC entre estos dos dispositivos en pacientes con choque séptico. Se realizó un estudio tipo observacional, prospectivo, longitudinal y comparativo en pacientes con choque séptico entre 18 y 60 años de edad ingresados en la UTI en el periodo de mayo-junio del 2017. Ante la disminución del uso del catéter de la arteria pulmonar debido a la controversia de no mejorar la mortalidad en los pacientes de las unidades de terapia intensiva (UTI), la colocación de dicho catéter ha caído en desuso; sin embargo, el GC medido por el catéter de Swan-Ganz sigue siendo el «estándar de oro¼ para la medición en tiempo real del GC y las resistencias sistémicas y pulmonares. La medición del GC por CAP versus USCOM se correlaciona de tal forma que puede emplearse en la medición por USCOM en un paciente con choque séptico, al cual no se le pretenda invadir para determinar sus condiciones hemodinámicas.

Abstract: The pulmonary artery catheter (CAP) is a device used in intensive care units (ICUs) to measure pressures in the heart and pulmonary blood vessels as part of hemodynamic monitoring primarily in cardiac surgery patients. The USCOM device is a non-invasive technique that uses Doppler technology to obtain measurements of systolic volume and its derivatives. The following CO measurement comparison is performed between these two devices in patients with septic shock. An observational, prospective, longitudinal and comparative study was conducted in patients with septic shock aged between 18 and 60 years admitted to intensive care in the period May-June 2017. In view of the decrease in the use of the pulmonary artery catheter due to the controversy of not improving the mortality in the patients of the Intensive Care Units, the placement of this catheter has fallen into disuse; however, cardiac output measured by the Swan Ganz catheter remains the «gold standard¼ for real-time measurement of cardiac output and systemic and pulmonary resistance. The CO measurement by PAC versus USCOM correlates, in such a way, that USCOM measurement can be used in a patient with septic shock, who is not expected to invade to determine their hemodynamic conditions.

Resumo: O cateter de artéria pulmonar (CAP) é um dispositivo utilizado em unidades de terapia intensiva (UTI) para medir as pressões nos vasos sanguíneos cardíacos e pulmonares, como parte da monitorização hemodinâmica, principalmente em pacientes submetidos a cirurgia cardíaca. O dispositivo USCOM é uma técnica não invasiva que utiliza a tecnologia Doppler para obter medidas do volume sistólico e seus derivados. A seguinte comparação da medição do DC é feita entre esses dois dispositivos em pacientes com choque séptico. Foi realizado um estudo observacional, prospectivo, longitudinal e comparativo em pacientes com choque séptico com idade entre 18 e 60 anos internados na unidade de terapia intensiva no período de maio a junho de 2017. Dada a diminuição do uso do cateter de artéria pulmonar devido à controvérsia de não melhorar a mortalidade nos pacientes das Unidades de Terapia Intensiva, a colocação do referido cateter caiu em desuso; no entanto, o débito cardíaco medido pelo cateter de Swan Ganz continua sendo o «padrão ouro¼ para a medição em tempo real do débito cardíaco e resistências sistêmicas e pulmonares. A medida do DC por CAP vs USCOM está correlacionada, de tal forma que a medida por USCOM pode ser usada em um paciente com choque séptico, que não se destina a invadir para determinar suas condições hemodinâmicas.