Nervous necrosis virus capsid protein and Protein A dynamically modulate the fish cGAS-mediated IFN signal pathway to facilitate viral evasion.

Nervous necrosis virus (NNV), an aquatic RNA virus belonging to Betanodavirus, infects a variety of marine and freshwater fishes, leading to massive mortality of cultured larvae and juveniles and substantial economic losses. The enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is widely recognized as a central component in the innate immune response to cytosolic DNA derived from different pathogens. However, little is known about the response of cGAS to aquatic RNA viruses. This study found that Epinephelus coioides cGAS (EccGAS) overexpression inhibited NNV replication, whereas EccGAS silencing promoted NNV replication. The anti-NNV activity of EccGAS was involved in interferon (IFN) signaling activation including tumor necrosis factor receptor-associated factor family member-associated NF-kappa-B activator-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor 3 (IRF3) nuclear translocation, and the subsequent induction of IFNc and ISGs. Interestingly, NNV employed its capsid protein (CP) or Protein A (ProA) to negatively or positively modulate EccGAS-mediated IFN signaling by simultaneously targeting EccGAS. CP interacted with EccGAS via the arm-P, S-P, and SD structural domains and promoted its polyubiquitination with K48 and K63 linkages in an EcUBE3C (the ubiquitin ligase)-dependent manner, ultimately leading to EccGAS degradation. Conversely, ProA bound to EccGAS and inhibited its ubiquitination and degradation. In regulating EccGAS protein content, CP's inhibitory action was more pronounced than ProA's protective effect, allowing successful NNV replication. These novel findings suggest that NNV CP and ProA dynamically modulate the EccGAS-mediated IFN signaling pathway to facilitate the immune escape of NNV. Our findings shed light on a novel mechanism of virus-host interaction and provide a theoretical basis for the prevention and control of NNV.IMPORTANCEAs a well-known DNA sensor, cGAS is a pivotal component in innate anti-viral immunity to anti-DNA viruses. Although there is growing evidence regarding the function of cGAS in the resistance to RNA viruses, the mechanisms by which cGAS participates in RNA virus-induced immune responses in fish and how aquatic viruses evade cGAS-mediated immune surveillance remain elusive. Here, we investigated the detailed mechanism by which EccGAS positively regulates the anti-NNV response. Furthermore, NNV CP and ProA interacted with EccGAS, regulating its protein levels through ubiquitin-proteasome pathways, to dynamically modulate the EccGAS-mediated IFN signaling pathway and facilitate viral evasion. Notably, NNV CP was identified to promote the ubiquitination of EccGAS via ubiquitin ligase EcUBE3C. These findings unveil a novel strategy for aquatic RNA viruses to evade cGAS-mediated innate immunity, enhancing our understanding of virus-host interactions.

A case study application of AQbD to the re-development and validation of an affinity chromatography analytical procedure for mAb titer quantitation.

Protein A (ProA) high-performance liquid chromatography (HPLC) is a common analytical procedure for measuring monoclonal antibody (mAb) titers due to its high specificity and efficiency. Accurate and reliable results of this procedure are imperative, as the quantitation of the total mAb present for in-process samples directly impacts downstream purification steps related to the removal of process-related impurities. This study aimed to improve a platform ProA HPLC analytical procedure which was previously developed using traditional approaches and was not always reliable. By retrospectively applying Analytical Quality by Design (AQbD) principles and statistical assessments of performance, a bias in the calibration standard due to protein-adsorption to common sample vial materials was identified. The inclusion of Tween® 20 into the mobile phase used as sample diluent was optimized to ensure procedure performance and improve analytical range. The resulting procedure robustness was evaluated using Design of Experiment (DoE) approaches and performance was verified against Analytical Target Profile (ATP) criteria as recommended by regulatory agencies. The resulting linearity displayed R2 values of 1.00 with intercept biases of 1.2 % (analyst 1) and 0.8 % (analyst 2), accuracy across all levels was reported at 99.2 % recovery, and intermediate precision was reported as 3.0 % RSD. Application of this new platform procedure has since reduced development timelines for new mAb products by 50 % and allowed for accurate titer determination to support >5 early phase product-specific process decisions without requiring extensive analytical procedure development. This work demonstrates the utility and relative ease of adopting AQbD concepts, even for established procedures, and supporting them with a lifecycle approach to managing procedure performance.

Consenso latinoamericano sobre la importancia de un sistema de gestión de datos microbiológicos para apoyar el programa de optimización de uso de antimicrobianos (PROA): Recomendaciones basadas en la evidencia. Parte 1 / Latin American consensus on the importance of a microbiological data management system to support antimicrobial use optimization program (PROA): Evidence-based recommendations. Part 1

La resistencia antimicrobiana es una amenaza para los logros de la medicina moderna y una de las medidas más efectivas para contrarrestarla son los programas de optimización del uso de antimicrobianos (PROA), en el cual el laboratorio de microbiología es uno de los principales componentes. La aplicación efectiva de tecnología de la información en los procesos es fundamental, pero existe poca información en Latinoamérica sobre el desarrollo y la articulación de las herramientas tecnológicas para apoyar los PROA. Este consenso hace recomendaciones sobre la gestión de los datos microbiológicos para la toma de decisiones. En la Parte I, se presentan las recomendaciones en cuanto al uso de un sistema informatizado de gestión de datos microbiológicos en la práctica clínica, los requerimientos de datos y de reporte en el laboratorio de microbiología, y los contenidos del sistema de gestión de calidad avanzado en el laboratorio. En la Parte II, se discuten los requerimientos de información para la gestión de PROA en estadios intermedios, iniciales y avanzados por el laboratorio y la farmacia; así como la integración del equipo de PROA con el Comité de Prevención y Control de Infecciones y la información para la gestión de PROA a nivel gerencial.

Antimicrobial resistance is a threat to the achievements of modern medicine and one of the most effective measures to counteract it is antimicrobial use optimization programs (AMS), in which the microbiology laboratory is one of the main components. The effective application of information technology in the processes is fundamental, but there is little information in Latin America on the development and articulation of technological tools to support AMSs. This consensus makes recommendations on the management of microbiological data for decision making. In Part I, recommendations on the use of a computerized microbiological data management system in clinical practice, data and reporting requirements in the microbiology laboratory, as well as the contents of the advanced quality management system in the laboratory are presented. In Part II, the information requirements for AMS management in intermediate, initial, and advanced stages by the laboratory and pharmacy are discussed; as well as the integration of the AMS team with the Infection Prevention and Control Committee and the information for AMS management at the management level.

El farmacéutico clínico en el abordaje del paciente con sospecha de alergia a beta-lactámicos: una revisión sistemática / The role of clinical pharmacists in patients with suspected allergy to B-lactams: A systematic review

Objetivo analizar el papel desempeñado por el farmacéutico clínico y su impacto en el ámbito de los programas de optimización de antimicrobianos ante la sospecha de alergia a antibióticos beta-lactámicos.Método se realizaron 2 búsquedas bibliográficas independientes. Se encontraron un total de 35 artículos incluyéndose 12. Se analizaron los artículos incluidos y se recogieron variables de eficacia, seguridad y aplicabilidad de herramientas de evaluación a pacientes con sospecha de alergia a beta-lactámicos. Además, se analizó la variación en el consumo y en el perfil de prescripción de antibióticos alternativos. Resultados los estudios seleccionados analizaron cuestionarios, desetiquetado, test intradérmicos y pruebas de provocación oral realizados por farmacéuticos. Se hallaron diferencias significativas en la variable principal de eficacia en 4 estudios incluidos a favor de la intervención farmacéutica. En un estudio cuasi experimental, la utilización de cefazolina aumentó tras la intervención farmacéutica (65 vs. 28%; p < 0,01). En otro estudio cuasi experimental, la dosis diaria definida media de aztreonam y la media de días de terapéutica por 1.000 pacientes/día disminuyeron (21,23 vs. 9,05; p < 0,01) y (8,79–4,24; p = 0,016), pre y postintervención, respectivamente, aumentando las desescaladas antibióticas (p ≤ 0,01). En otro estudio, disminuyó la prescripción de antibióticos de uso restringido (42,5 vs. 17,9%; p < 0,01) y en otro, la utilización de antibióticos profilácticos prequirúrgicos alternativos a cefazolina (81,9 vs. 55,9%; p < 0,01). En otro estudio, el tiempo medio por entrevista fue de 5,2 minutos por paciente. No se reportaron eventos adversos en ningún estudio. Conclusiones la intervención del farmacéutico en la evaluación del paciente con sospecha de alergia a beta-lactámicos resulta eficaz, segura y aplicable a la práctica clínica (AU)

Objective To analyze the role played by the clinical pharmacist and its impact in antibiotic stewardship facing suspected allergy to beta-lactam antibiotics. Method We performed two different independent bibliographic searches. A total of 35 articles were found, and the final number included in the study was 12. We analysed the articles and collected variables of efficacy, safety and applicability of evaluation tools applied to patients with suspected allergy to beta-lactams. Also, the variation in the consumption and prescription profile of alternative antibiotics was analyzed. Results The selected studies analysed questionnaires, allergy delabeling, intradermal tests and oral challenge tests performed by pharmacists. Significant differences in the efficacy endpoint were found in 4 studies in favour of pharmaceutical intervention. In the study of Kwiatkowski et al, cefazolin use increased in surgical patients after pharmacist intervention (65 vs. 28%; p < 0.01). In a quasi-experimental study, the mean defined daily dose of aztreonam and the mean days of therapy per 1000 patients/day decreased (21.23 vs 9.05, p <0.01) and (8.79–4.24, p = 0.016), pre and post-intervention, respectively, increasing antibiotic de-escalations (p ≤ 0.01). In another quasi-experimental study, the prescription of restricted-use antibiotics decreased (42.5% vs. 17.9%, p < 0.01) and the use of pre-surgical prophylactic antibiotics alternative to cefazolin (81.9% vs 55.9%, p<0.01) in another study. Other study showed that the mean time per interview was 5.2 minutes per patient. No adverse events were reported in any study. Conclusion The pharmacist intervention in the evaluation of the patient with suspected allergy to beta-lactams is effective, safe and feasible to implement on daily clinical practice (AU)

In-line buffer exchange in the coupling of Protein A chromatography with weak cation exchange chromatography for the determination of charge variants of immunoglobulin G derived from chinese hamster ovary cell cultures.

Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension (1D) and WCX in the second dimension (2D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development.

The role of clinical pharmacists in patients with suspected allergy to ß-lactams: A systematic review. / El farmacéutico clínico en el abordaje del paciente con sospecha de alergia a beta-lactámicos: una revisión sistemática.

OBJECTIVE: To analyze the role played by the clinical pharmacist and its impact in antibiotic stewardship facing suspected allergy to beta-lactam antibiotics. METHOD: We performed two different independent bibliographic searches. A total of 35 articles were found, and the final number included in the study was 12. We analysed the articles and collected variables of efficacy, safety and applicability of evaluation tools applied to patients with suspected allergy to beta-lactams. Also, the variation in the consumption and prescription profile of alternative antibiotics was analyzed. RESULTS: The selected studies analysed questionnaires, allergy delabeling, intradermal tests and oral challenge tests performed by pharmacists. Significant differences in the efficacy endpoint were found in 4 studies in favour of pharmaceutical intervention. In the study of Kwiatkowski et al, cefazolin use increased in surgical patients after pharmacist intervention (65 vs. 28%; p < 0.01). In a quasi-experimental study, the mean defined daily dose of aztreonam and the mean days of therapy per 1000 patients/day decreased (21.23 vs 9.05, p <0.01) and (8.79-4.24, p = 0.016), pre and post-intervention, respectively, increasing antibiotic de-escalations (p ≤ 0.01). In another quasi-experimental study, the prescription of restricted-use antibiotics decreased (42.5% vs. 17.9%, p < 0.01) and the use of pre-surgical prophylactic antibiotics alternative to cefazolin (81.9% vs 55.9%, p<0.01) in another study. Other study showed that the mean time per interview was 5.2 minutes per patient. No adverse events were reported in any study. CONCLUSION: The pharmacist intervention in the evaluation of the patient with suspected allergy to beta-lactams is effective, safe and feasible to implement on daily clinical practice. The standardization of protocols to clarify the history of allergies and development of evaluation tools represent simple screenings to perform delabelling or refer to the Immunoallergology service, improving penicilins use and reducing the need for second line antibiotics. More studies are needed to standardize the desensitization tests made by pharmacists. However, despite these results, the involvement and leadership of the pharmacist in this area is limited and constitutes a future challenge for the profession.

[Translated article] The role of clinical pharmacists in patients with suspected allergy to ß-lactams: A systematic review.

OBJECTIVE: To analyze the role played by the clinical pharmacist and its impact in antibiotic stewardship facing suspected allergy to beta-lactam antibiotics. METHOD: We performed 2 different independent bibliographic searches. A total of 35 articles were found, and the final number included in the study was 12. We analyzed the articles and collected variables of efficacy, safety, and applicability of evaluation tools applied to patients with suspected allergy to beta-lactams. Also, the variation in the consumption and prescription profile of alternative antibiotics was analyzed. RESULTS: The selected studies analyzed questionnaires, allergy delabeling, intradermal tests, and oral challenge tests performed by pharmacists. Significant differences in the efficacy endpoint were found in 4 studies in favor of pharmaceutical intervention. In the study of Kwiatkowski et al., cefazolin use increased in surgical patients after pharmacist intervention (65% vs 28%; P < .01). In a quasi-experimental study, the mean defined daily dose of aztreonam and the mean days of therapy per 1000 patients/day decreased (21.23 vs 9.05, P <.01) and (8.79-4.24, P = .016), pre- and post-intervention, respectively, increasing antibiotic de-escalations (P = < .01). In another quasi-experimental study, the prescription of restricted use antibiotics decreased (42.5% vs 17.9%, P < .01)and the use of pre-surgical prophylactic antibiotics alternative to cefazolin (81.9% vs 55.9%, P < .01)in another study. Other study showed that the mean time per interview was 5.2 min per patient. No adverse events were reported in any study. CONCLUSION: The pharmacist intervention in the evaluation of the patient with suspected allergy to beta-lactams is effective, safe, and feasible to implement on daily clinical practice. The standardization of protocols to clarify the history of allergies and development of evaluation tools represent simple screenings to perform delabeling or refer to the Immunoallergology service, improving penicilins use and reducing the need for second-line antibiotics. More studies are needed to standardize the desensitization tests made by pharmacists. However, despite these results, the involvement and leadership of the pharmacist in this area is limited and constitutes a future challenge for the profession.

Programa de optimización del uso de antimicrobianos en un Servicio de Medicina Intensiva: análisis retrospectivo observacional de los resultados 15 meses después de su implementación / Antimicrobial stewardship program in an Intensive Care Unit: A retrospective observational analysis of the results 15 months after its implementation

Objetivo. Determinar el grado de aceptación de un Pro grama de Optimización del Uso de Antimicrobianos (PROA) en un Servicio de Medicina Intensiva (SMI), y evaluar su efecto sobre el consumo de antibióticos, indicadores de calidad y re sultados clínicos. Pacientes y métodos. Descripción retrospectiva de las intervenciones propuestas por un PROA. Comparación de uso de antimicrobianos, indicadores de calidad y seguridad frente a un periodo sin PROA. Se realizó en un SMI polivalente de un Hospital Universitario mediano (600 camas). Se estudió a pacientes ingresados por cualquier causa en el SMI durante el periodo PROA en los que se hubiera obtenido una muestra di rigida al diagnóstico de una potencial infección, o se hubieran iniciado antimicrobianos. Se elaboraron recomendaciones no impositivas para mejorar la prescripción antimicrobiana (es tructura audit and feedback) y se procedió a su registro du rante periodo PROA (15 meses, octubre 2018–diciembre 2019). Comparación de indicadores en un periodo con PROA (abril– junio 2019) y sin PROA (abril–junio 2018). Resultados. Se emitieron 241 recomendaciones sobre 117 pacientes, el 67% de ellas de tipo desescalada terapéutica. La aceptación de las recomendaciones fue elevada (96.3%). En el periodo PROA se redujo el número medio de antibióticos por paciente (3.3±4.1 vs 2.4±1.7, p=0.04) y los días de tratamiento (155 DOT/100 PD vs 94 DOT/100 PD, p <0.01) (AU)

Objective. We aim to evaluate the adherence rate to an Antimicrobial Stewardship Program (ASP) in an Intensive Care Unit (ICU), and to assess its effect on the use of antibiotics, quality indicators and clinical outcomes. Patients and methods. Retrospective description of the interventions proposed by the ASP. We compared antimi crobial use, quality and safety indicators in an ASP versus a non-ASP period. The study was performed in a polyvalent ICU of a medium-size University Hospital (600 beds). We studied patients admitted to the ICU for any cause during the ASP pe riod, provided that a microbiological sample aiming to diag nose a potential infection has been drawn, or antibiotics have been started. We elaborated and registered of non-mandatory recommendations to improve antimicrobial prescription (audit and feedback structure) and its registry during the ASP peri od (15 months, October 2018-December 2019). We compared indicators in a period with ASP (April-June 2019) and without ASP (April-June 2018). Results. We issued 241 recommendations on 117 pa tients, 67% of them classified as de-escalation type. The rate of adherence to the recommendations was high (96.3%). In the ASP period, the mean number of antibiotics per patient (3.3±4.1 vs 2.4±1.7, p=0.04) and the days of treatment (155 DOT/100 PD vs 94 DOT/100 PD, p <0.01) (AU)

Suspensión de antibióticos a través de un programa de optimización de tratamiento antibiótico en pacientes atendidos en Urgencias con baja sospecha de infección / Antibiotic discontinuation through an antibiotic treatment optimization program in emergency department patients with low suspicion of infection

Introducción: Una de las estrategias más importantes de los PROA en los Servicios de Urgencias (SU) es el diagnóstico adecuado de infección para evitar la prescripción inadecuada. Nuestro objetivo es evaluar a los pacientes que reciben antibiótico a pesar de no tener datos objetivos de infección. Métodos: Realizamos un estudio transversal de los pacientes ingresados en el SU del Hospital Universitario Fundación Alcorcón durante 2 meses (mayo del 2019 y marzo del 2021) en los que se recomendó suspender el antibiótico a través del PROA. Se analizaron las características clínicas y epidemiológicas, y el seguimiento a 30 días para valorar los reingresos y la mortalidad. Resultados: Se analizaron 145 pacientes. Se recomendó suspender el antibiótico en 25. El 44% de ellos tenían diagnóstico de infección urinaria. La recomendación de suspensión se aceptó en el 88%. Ningún paciente falleció y uno reingresó. Conclusiones: Un porcentaje importante de pacientes tenían prescrito antibiótico a pesar de no tener criterios de infección, siendo la evolución clínica tras la de prescripción favorable.(AU)

Introduction: One of the most important strategies of PROA in the Emergency Department (ED) is the accurate diagnosis of infection to avoid inappropriate prescription. Our objective is to evaluate patients who receive antibiotics despite not having objective data of infection. Methods: We carried out a cross-sectional study of patients admitted to the ED of the Hospital Universitario Fundación Alcorcón in which it was recommended to suspend the antibiotic through the PROA. Clinical and epidemiological characteristics and 30-day follow-up were analyzed to assess readmissions and mortality. Results: 145 patients were analyzed. It was recommended to suspend the antibiotic in 25. 44% of them had a diagnosis of urinary infection. The suspension recommendation was accepted by 88%. No patient died and one was readmitted. Conclusions: An important percentage of patients are prescribed antibiotics despite not having infection criteria, the clinical evolution after suspension of antibiotics was favorable.(AU)

Impacto del tratamiento antimicrobiano domiciliario endovenoso (TADE) como parte de un programa de optimización del uso de antimicrobianos (PROA) específico para pediatría / Impact of an outpatient parenteral antimicrobial treatment (OPAT) as part of a paediatric-specific PROA program

Introducción: El tratamiento antimicrobiano domiciliario endovenoso (TADE) es una alternativa a la hospitalización para pacientes pediátricos bien seleccionados. Presentamos un programa TADE basado en un modelo de autoadministración e integrado en el programa de optimización del uso de antimicrobianos (PROA) de un hospital pediátrico de tercer nivel. Material y métodos: Estudio descriptivo, retrospectivo y unicéntrico que incluye todos los pacientes menores de 20años tratados con TADE prescrito en un hospital pediátrico entre 2019 y 2020. Se analizaron los datos sobre los días de ingreso ahorrados y una estimación económica del coste oportunidad que supone el programa TADE para el hospital. Resultados: Cincuenta y siete pacientes realizaron un total de 106 episodios de tratamiento. En el 74,5% hubo una evolución clínica favorable. El principal motivo de interrupción prematura fue una mala evolución de la infección (37,1%). Se liberaron 2,62 camas diarias, suponiendo un beneficio económico de 1.069.963€. Conclusión: Un programa TADE integrado en el PROA pediátrico de nuestro hospital y basado en un modelo de autoadministración se ha mostrado un programa seguro y efectivo y aporta importantes ventajas a nivel económico.(AU)

Introduction: Outpatient parenteral antimicrobial treatment (OPAT) is an alternative to in-patient care in carefully selected patients. This study presents a self-administration OPAT program integrated within the pediatric antibiotic stewardship program (ASP) in a pediatric tertiary care center. Material and methods: Descriptive, retrospective and unicentric study. Data from all patients under 20years of age who were prescribed treatment by a pediatric unit during 2019 and 2020 were included. Data regarding number of saved beds and estimating the opportunity cost of the OPAT program for the hospital were analyzed. Results: Fifty-seven patients received 106 episodes of treatment. Favorable clinical outcome occurred in 74.5% of the episodes. The main cause of premature interruption was unfavorable clinical outcome of the infection (37.1%). A total of 2.62 beds/day were saved, resulting in an economic benefit of 1,069,963€. Conclusion: A self-administration OPAT program integrated within the pediatric ASP has proven to be safe and effective and provides economic benefits.(AU)

Antimicrobial stewardship in hospitals: Expert recommendation guidance document for activities in specific populations, syndromes and other aspects (PROA-2) from SEIMC, SEFH, SEMPSPGS, SEMICYUC and SEIP / Programas de optimización del uso de antimicrobianos en hospitales: guía de recomendaciones de expertos para actividades en poblaciones específicas, síndromes y otros aspectos (PROA-2) de la SEIMC, SEFH, SEMPSPGS y SEIP

In 2012, The Spanish Societies of Infectious Diseases and Clinical Microbiology (SEIMC), Hospital Pharmacy (SEFH), and Preventive Medicine, Public Health and Healthcare Management (SEMPSGS) lead a consensus document including recommendations for the implementation of antimicrobial stewardship (AMS) programs (AMSP; PROA in Spanish) in acute care hospitals in Spain. While these recommendations were critical for the development of these programs in many centres, there is a need for guidance in the development of AMS activities for specific patient populations, syndromes or other specific aspects which were not included in the previous document or have developed significantly since then. The objective of this expert recommendation guidance document is to review the available information about these activities in these patient populations or circumstances, and to provide guidance recommendations about them. With this objective the SEIMC, SEFH, SEMPSPGS, the Spanish Society of Intensive Care Medicine (SEMICYUC) and the Spanish Pediatric Infectious Disease Society (SEIP) selected a panel of experts who chose the different aspects to include in the document. Because of the lack of high-level evidence in the implementation of the activities, the panel opted to perform a narrative review of the literature for the different topics for which recommendations were agreed by consensus. The document was open to public consultation for the members of these societies for their comments and suggestions, which were reviewed and considered by the panel.(AU)

En 2012, las Sociedades Españolas de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Farmacia Hospitalaria (SEFH) y Medicina Preventiva, Salud Pública y Gestión Sanitaria (SEMPSPGS) lideraron un documento de consenso que incluía recomendaciones para la implementación de Programas de optimización del uso de antimicrobianos (PROA) en hospitales de agudos en España. Si bien estas recomendaciones fueron críticas para el desarrollo de estos programas en muchos centros, actualmente es necesario establecer unas guías para la implementación de las actividades de los PROA en determinadas poblaciones de pacientes, síndromes clínicos y otros aspectos específicos que no se incluyeron en el documento previo o que desde entonces se han desarrollado significativamente. El objetivo de esta guía de recomendaciones de expertos es revisar la información disponible acerca de esas actividades en estas poblaciones o circunstancias de pacientes y proporcionar unas recomendaciones que sirvan de guía sobre ellas. Con este objetivo, la SEIMC, la SEFH y la SEMPSPGS, así como la Sociedad Española de Medicina Intensiva, Crítica y Unidades Coronarias (SEMICYUC) y la Sociedad Española de Infectología Pediátrica (SEIP), seleccionaron un panel de expertos que eligieron los diferentes aspectos a incluir en el documento. Debido a la ausencia de evidencia de alto nivel en la implementación de las diferentes actividades, el panel optó por realizar una revisión narrativa de la literatura de los diferentes aspectos, en los que las recomendaciones se acordaron por consenso. El documento se abrió para consulta pública a los miembros de estas sociedades para sus comentarios y sugerencias, que fueron revisadas y consideradas por el panel.(AU)

Legionella pneumophila and Free-Living Nematodes: Environmental Co-Occurrence and Trophic Link.

Free-living nematodes harbor and disseminate various soil-borne bacterial pathogens. Whether they function as vectors or environmental reservoirs for the aquatic L. pneumophila, the causative agent of Legionnaires' disease, is unknown. A survey screening of biofilms of natural (swimming lakes) and technical (cooling towers) water habitats in Germany revealed that nematodes can act as potential reservoirs, vectors or grazers of L. pneumophila in cooling towers. Consequently, the nematode species Plectus similis and L. pneumophila were isolated from the same cooling tower biofilm and taken into a monoxenic culture. Using pharyngeal pumping assays, potential feeding relationships between P. similis and different L. pneumophila strains and mutants were examined and compared with Plectus sp., a species isolated from a L. pneumophila-positive thermal source biofilm. The assays showed that bacterial suspensions and supernatants of the L. pneumophila cooling tower isolate KV02 decreased pumping rate and feeding activity in nematodes. However, assays investigating the hypothesized negative impact of Legionella's major secretory protein ProA on pumping rate revealed opposite effects on nematodes, which points to a species-specific response to ProA. To extend the food chain by a further trophic level, Acanthamoebae castellanii infected with L. pneumphila KV02 were offered to nematodes. The pumping rates of P. similis increased when fed with L. pneumophila-infected A. castellanii, while Plectus sp. pumping rates were similar when fed either infected or non-infected A. castellanii. This study revealed that cooling towers are the main water bodies where L. pneumophila and free-living nematodes coexist and is the first step in elucidating the trophic links between coexisting taxa from that habitat. Investigating the Legionella-nematode-amoebae interactions underlined the importance of amoebae as reservoirs and transmission vehicles of the pathogen for nematode predators.

Protein sociology of ProA, Mip and other secreted virulence factors at the Legionella pneumophila surface.

The pathogenicity of L. pneumophila, the causative agent of Legionnaires' disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted "protein sociology" of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

Antimicrobial stewardship in hospitals: Expert recommendation guidance document for activities in specific populations, syndromes and other aspects (PROA-2) from SEIMC, SEFH, SEMPSPGS, SEMICYUC and SEIP.

In 2012, The Spanish Societies of Infectious Diseases and Clinical Microbiology (SEIMC), Hospital Pharmacy (SEFH), and Preventive Medicine, Public Health and Healthcare Management (SEMPSGS) lead a consensus document including recommendations for the implementation of antimicrobial stewardship (AMS) programs (AMSP; PROA in Spanish) in acute care hospitals in Spain. While these recommendations were critical for the development of these programs in many centres, there is a need for guidance in the development of AMS activities for specific patient populations, syndromes or other specific aspects which were not included in the previous document or have developed significantly since then. The objective of this expert recommendation guidance document is to review the available information about these activities in these patient populations or circumstances, and to provide guidance recommendations about them. With this objective the SEIMC, SEFH, SEMPSPGS, the Spanish Society of Intensive Care Medicine (SEMICYUC) and the Spanish Pediatric Infectious Disease Society (SEIP) selected a panel of experts who chose the different aspects to include in the document. Because of the lack of high-level evidence in the implementation of the activities, the panel opted to perform a narrative review of the literature for the different topics for which recommendations were agreed by consensus. The document was open to public consultation for the members of these societies for their comments and suggestions, which were reviewed and considered by the panel.

Antibiotic discontinuation through an antibiotic treatment optimization program in emergency department patients with low suspicion of infection.

INTRODUCTION: One of the most important strategies of PROA in the Emergency Department (ED) is the accurate diagnosis of infection to avoid inappropriate prescription. Our objective is to evaluate patients who receive antibiotics despite not having objective data of infection. METHODS: We carried out a cross-sectional study of patients admitted to the ED of the Hospital Universitario Fundación Alcorcón in which it was recommended to suspend the antibiotic through the PROA. Clinical and epidemiological characteristics and 30-day follow-up were analyzed to assess readmissions and mortality. RESULTS: 145 patients were analyzed. It was recommended to suspend the antibiotic in 25. 44% of them had a diagnosis of urinary infection. The suspension recommendation was accepted by 88%. No patient died and one was readmitted. CONCLUSIONS: An important percentage of patients are prescribed antibiotics despite not having infection criteria, the clinical evolution after suspension of antibiotics was favorable.

Impact of an outpatient parenteral antimicrobial treatment (OPAT) as part of a paediatric-specific PROA program.

INTRODUCTION: Outpatient parenteral antimicrobial treatment (OPAT) is an alternative to in-patient care in carefully selected patients. This study presents a self-administration OPAT program integrated within the pediatric antibiotic stewardship program (ASP) in a pediatric tertiary care center. MATERIAL AND METHODS: Descriptive, retrospective and unicentric study. Data from all patients under 20 years of age who were prescribed treatment by a pediatric unit during 2019 and 2020 were included. Data regarding number of saved beds and estimating the opportunity cost of the OPAT program for the hospital were analyzed. RESULTS: Fifty-seven patients received 106 episodes of treatment. Favorable clinical outcome occurred in 74.5% of the episodes. The main cause of premature interruption was unfavorable clinical outcome of the infection (37.1%). A total of 2.62 beds/day were saved, resulting in an economic benefit of 1,069,963 €. CONCLUSION: A self-administration OPAT program integrated within the pediatric ASP has proven to be safe and effective and provides economic benefits.

Implantación e impacto de un programa de control antibiótico y colonización por bacterias multirresistentes en una unidad de trasplante hepático / Implementation and impact of an antibiotic control program and multidrug-resistant bacterial colonization in a liver transplant unit

Introducción: las infecciones por bacterias multirresistentes constituyen una importante causa de morbimortalidad pre coz en los pacientes trasplantados.Propósito: se presenta un estudio unicéntrico, retrospectivo, con objetivo de evaluar la implantación de un programa de optimización del uso de antibióticos y de control epidemio lógico (PROA) en la colonización e infección por bacterias multirresistentes tras el trasplante hepático (TH).Métodos: se analizaron la colonización por bacterias multi rresistentes y las infecciones en el primer año postrasplante hepático (post-TH) en un grupo de 76 pacientes trasplanta dos en dos etapas, anterior y posterior al PROA, entre los años 2016 y 2019. Se analizaron variables clínicas relacio nadas con infección, reingresos y supervivencia a un año.Resultados: se produjo una buena adherencia al PROA. Las infecciones en el primer año post-TH fueron la causa más fre cuente de reingreso. La incidencia de infecciones fue similar en ambos periodos, con una media de 1,25 y 1,5 episodios de infección bacteriana por paciente/año con 19 episodios infecciosos bacterianos, seis por bacterias multirresistentes y de resistencia extendida (MDR-XDR) en la etapa pre-PROA y 18 episodios infecciosos bacterianos, cinco por MDR-XDR. en la etapa posterior. Se objetivó un descenso del 37 % post TH de colonización rectal por MDR-XDR durante el año 2019.Conclusión: las políticas de vigilancia epidemiológica y optimización de antibióticos son necesarias como estrategia de control del incremento de colonización e infección por bac terias multirresistentes en unidades de trasplante hepático. Son necesarios estudios a largo plazo para evaluar mejor el impacto de estos programas (AU)

Indicadores basados en consumo en una Unidad de Cuidados Intensivos sin implantación de PROA / Indicators based on consumption in an intensive care unit without antimicrobial stewardship programs implementation

Objetivo: Medir los indicadores del uso hospitalario de antibióticos basados en datos de consumo, comparando datos entre 2018 y 2019 en una Unidad de Medicina Intensiva de un hospital de tercer nivel sin implantación de Programas de Optimización de Antibióticos (PROA). Métodos: Asignar un valor a cada indicador evaluado en base a datos de consumo empleando los datos del programa de gestión del Servicio de Farmacia y las DDD porcada 100 estancias. En base a la diferencia de las medias obtenidas entre 2018 y 2019 se calculó la significación estadística mediante la t-Student de medidas pareadas. Resultados: Se evaluaron 13 indicadores, de los cuales sólo 2(15%) presentaron diferencias estadísticamente significativas, el consumo de fluorquinolonas y el ratio fluconazol/equinocandinas, mostrando una evolución positiva. Conclusiones: El empleo de estos indicadores deberían estandarizarse para la evaluación de las políticas antibióticas de los centros, lo que serviría para establecer comparaciones entre centros de similares características o bien la evolucióntemporal para un mismo centro y/o servicio. Esto permitiríadetectar puntos críticos y establecer acciones de mejora, entre ellas la creación de equipos PROA, especialmente en unidades de pacientes críticos. (AU)

Objective: The aim of our study is tocalculate the indicators of hospital useof antimicrobial agents based on consumption, comparing data from 2018with data from 2019 in an IntensiveCare Unit of a third level hospital without an stewardship program.Methods: Retrospective study in whichwe assigned a value to each indicatorbased on consumption using DDD per100 bed-stays. Data was obtainedusing the pharmacy management software. Statistical analysis was performed by t-Student test based on thedifference of means obtained in 2018and 2019 respectively.Results: 13 indicators were evaluated,only 2 of them (15%) showed an statistically significant difference betweenperiods, the consumption of fluoroquinolones and the fluconazole/ echinocandin ratio, both showing a positiveevolution.Conclusions: The use of these indicatorsshould be standardized in order to evaluate antibiotic policies, which will helpestablishing comparisons between centers of specific characteristics or studying the temporal evolution for thesame center and/or service. This willallow detecting critical points and establishing improvement actions, includingthe creation of stewardship programs,especially in critical care units. (AU)

Functional characterization of a gamma-glutamyl phosphate reductase ProA in proline biosynthesis and promoting expression of type three secretion system in Ralstonia solanacearum.

Ralstonia solanacearum RSc2741 has been predicted as a gamma-glutamyl phosphate reductase ProA catalyzing the second reaction of proline formation from glutamate. Here, we experimentally demonstrated that proA mutants were proline auxotrophs that failed to grow in a minimal medium, and supplementary proline, but not glutamate, fully restored the diminished growth, confirming that ProA is responsible for the biosynthesis of proline from glutamate in R. solanacearum. ProA was previously identified as one of the candidates regulating the expression of genes for type three secretion system (T3SS), one of the essential pathogenicity determinants of R. solanacearum. Supplementary proline significantly enhanced the T3SS expression both in vitro and in planta, indicating that proline is a novel inducer of the T3SS expression. Deletion of proA substantially impaired the T3SS expression both in vitro and in planta even under proline-supplemented conditions, indicating that ProA plays additional roles apart from proline biosynthesis in promoting the expression of the T3SS genes. It was further revealed that the involvement of ProA in the T3SS expression was mediated through the pathway of PrhG-HrpB. Both the proA mutants and the wild-type strain grew in the intercellular spaces of tobacco leaves, while their ability to invade and colonize tobacco xylem vessels was substantially impaired, which was about a 1-day delay for proA mutants to successfully invade xylem vessels and was about one order of magnitude less than the wild-type strain to proliferate to the maximum densities in xylem vessels. It thus resulted in substantially impaired virulence of proA mutants toward host tobacco plants. The impaired abilities of proA mutants to invade and colonize xylem vessels were not due to possible proline insufficiency in the rhizosphere soil or inside the plants. All taken together, these results extend novel insights into the understanding of the biological function of ProA and sophisticated regulation of the T3SS and pathogenicity in R. solanacearum.

Zinc Metalloprotease ProA from Legionella pneumophila Inhibits the Pro-Inflammatory Host Response by Degradation of Bacterial Flagellin.

The environmental bacterium Legionella pneumophila is an intracellular pathogen of various protozoan hosts and able to cause Legionnaires' disease, a severe pneumonia in humans. By encoding a wide selection of virulence factors, the infectious agent possesses several strategies to manipulate its host cells and evade immune detection. In the present study, we demonstrate that the L. pneumophila zinc metalloprotease ProA functions as a modulator of flagellin-mediated TLR5 stimulation and subsequent activation of the pro-inflammatory NF-κB pathway. We found ProA to be capable of directly degrading immunogenic FlaA monomers but not the polymeric form of bacterial flagella. These results indicate a role of the protease in antagonizing immune stimulation, which was further substantiated in HEK-BlueTM hTLR5 Detection assays. Addition of purified proteins, bacterial suspensions of L. pneumophila mutant strains as well as supernatants of human lung tissue explant infection to this reporter cell line demonstrated that ProA specifically decreases the TLR5 response via FlaA degradation. Conclusively, the zinc metalloprotease ProA serves as a powerful regulator of exogenous flagellin and presumably creates an important advantage for L. pneumophila proliferation in mammalian hosts by promoting immune evasion.