In vitro effect of photodynamic therapy with different lights and combined or uncombined with chlorhexidine on Candida spp.

Candidiasis is very common and complicated to treat in some cases due to increased resistance to antifungals. Antimicrobial photodynamic therapy (aPDT) is a promising alternative treatment. It is based on the principle that light of a specific wavelength activates a photosensitizer molecule resulting in the generation of reactive oxygen species that are able to kill pathogens. The aim here is the in vitro photoinactivation of three strains of Candida spp., Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, using aPDT with different sources of irradiation and the photosensitizer methylene blue (MB), alone or in combination with chlorhexidine (CHX). Irradiation was carried out at a fluence of 18 J/cm2 with a light-emitting diode (LED) lamp emitting in red (625 nm) or a white metal halide lamp (WMH) that emits at broad-spectrum white light (420–700 nm). After the photodynamic treatment, the antimicrobial effect is evaluated by counting colony forming units (CFU). MB-aPDT produces a 6 log10 reduction in the number of CFU/100 μL of Candida spp., and the combination with CHX enhances the effect of photoinactivation (effect achieved with lower concentration of MB). Both lamps have similar efficiencies, but the WMH lamp is slightly more efficient. This work opens the doors to a possible clinical application of the combination for resistant or persistent forms of Candida infections

Photodynamic Therapy Combined with Antibiotics or Antifungals against Microorganisms That Cause Skin and Soft Tissue Infections: A Planktonic and Biofilm Approach to Overcome Resistances

The present review covers combination approaches of antimicrobial photodynamic therapy (aPDT) plus antibiotics or antifungals to attack bacteria and fungi in vitro (both planktonic and biofilm forms) focused on those microorganisms that cause infections in skin and soft tissues. The combination can prevent failure in the fight against these microorganisms: antimicrobial drugs can increase the susceptibility of microorganisms to aPDT and prevent the possibility of regrowth of those that were not inactivated during the irradiation; meanwhile, aPDT is effective regardless of the resistance pattern of the strain and their use does not contribute to the selection of antimicrobial resistance. Additive or synergistic antimicrobial effects in vitro are evaluated and the best combinations are presented. The use of combined treatment of aPDT with antimicrobials could help overcome the difficulty of fighting high level of resistance microorganisms and, as it is a multi-target approach, it could make the selection of resistant microorganisms more difficult

Association between preoperative levels of 25-hydroxyvitamin D and hospital-acquired infections after hepatobiliary surgery: A prospective study in a third-level hospital

Introduction Evidence implicates vitamin D deficiency in poorer outcomes and increased susceptibility to hospital-acquired infections (HAIs). This study examined the association between serum vitamin D levels and HAIs in a population of hepatobiliary surgery patients. Methods Participants in this prospective analytical observational study were patients who underwent hepatobiliary surgery in a tertiary hospital in Aragon, Spain, between February 2018 and March 2019. Vitamin D concentrations were measured at admission and all nosocomial infections during hospitalization and after discharge were recorded. Results The mean 25-hydroxyvitamin D concentration of the study population (n = 301) was 38.56 nmol/L, which corresponds to vitamin D deficiency. Higher vitamin D concentrations were associated with a decreased likelihood of developing a HAI in general (p = 0.014), and in particularly surgical site infection (p = 0.026). The risk of HAI decreased by 34% with each 26.2-nmol/L increase in serum vitamin D levels. Conclusions Vitamin D levels may constitute a modifiable risk factor for postoperative nosocomial infections in hepatobiliary surgery patients

Infección del sitio quirúrgico en cirugía hepatobiliopancreática y su relación con la concentración sérica de vitamina D

Introducción La relación entre las infecciones nosocomiales en pacientes quirúrgicos y la vitamina D ha sido estudiada por algunos autores. Sin embargo, hasta la fecha no existe ningún estudio realizado sobre pacientes de cirugía hepatobiliar. El objetivo de nuestro trabajo es estudiar la infección del sitio quirúrgico (ISQ) en la unidad de cirugía hepatobiliar, y valorar su relación con la concentración sérica de vitamina D. Métodos Se llevó a cabo un estudio analítico observacional de pacientes sucesivos intervenidos en la unidad de cirugía hepatobiliar de nuestro centro durante un año. Se incluyeron las intervenciones relativas a enfermedad biliar, pancreática y hepática. Se determinaron los niveles de vitamina D al ingreso, así como las ISQ de tipo superficial, profunda y órgano-cavitaria diagnosticadas durante el estudio. El seguimiento del paciente se realizó durante al menos un mes tras la cirugía, dependiendo de la enfermedad. La estadística se realizó mediante el programa estadístico R v.3.1.3. Resultados La muestra quedó constituida por 321 pacientes, de los cuales el 25, 8% presentó ISQ a expensas fundamentalmente de las infecciones órgano-cavitarias que presentaron una incidencia del 24, 3%. Concentraciones séricas superiores a 33, 5 nmol/l demostraron reducir en un 50% el riesgo de ISQ. Conclusiones Las concentraciones elevadas de vitamina D en sangre demostraron ser un factor protector frente a las ISQ (OR: 0, 99). Nuestros resultados sugieren una relación directa entre la concentración sérica de vitamina D y la ISQ, justificando la realización de nuevos estudios prospectivos. Introduction: While several studies have examined the correlation between vitamin D concentrations and post-surgical nosocomial infections, this relationship has yet to be characterized in hepatobiliary surgery patients. We investigated the relationship between serum vitamin D concentration and the incidence of surgical site infection (SSI) in patients in our hepatobiliary surgery unit. Methods: Participants in this observational study were 321 successive patients who underwent the following types of interventions in the hepatobiliary surgery unit of our center over a 1-year period: cholecystectomy, pancreaticoduodenectomy, total pancreatectomy, segmentectomy, hepatectomy, hepaticojejunostomy and exploratory laparotomy. Serum vitamin D levels were measured upon admission and patients were followed up for 1 month. Mean group values were compared using a Student's T-test or Chi-squared test. Statistical analyses were performed using the Student's T-test, the Chi-squared test, or logistic regression models. Results: Serum concentrations >33.5 nmol/l reduced the risk of SSI by 50%. Out of the 321 patients analyzed, 25.8% developed SSI, mainly due to organ-cavity infections (incidence, 24.3%). Serum concentrations of over 33.5 nmol/l reduced the risk of SSI by 50%. Conclusions: High serum levels of vitamin D are a protective factor against SSI (OR, 0.99). Our results suggest a direct relationship between serum vitamin D concentrations and SSI, underscoring the need for prospective studies to assess the potential benefits of vitamin D in SSI prevention

Idoneidad del uso del MALDI-TOF MS para la identificación de Staphylococcus aureus y miembros del grupo de Staphylococcus intermedius (S.I.G.)

A pesar de que el género Staphylococcus es comúnmente aislado en humanos y animales, su identificación sigue siendo problemática. Este estudio evalúa la idoneidad del MALDI-TOF MS (Biotyper 3) para su identificación, comparando los resultados obtenidos mediante diferentes métodos: fenotípicos, MALDITOF MS (añadiendo o no ácido fórmico) y moleculares. Una colección de 37 cepas fue identificada por técnicas convencionales como S. aureus (n= 7), S. intermedius (n=1) y S. pseudintermedius (n=29). Los aislamientos provenían de perros, tanto sanos como enfermos (n=27), y humanos (n=10), a partir de diferentes muestras biológicas. Todas ellas fueron también identificadas por biología molecular y los cultivos puros fueron procesados en el MALDI-TOF MS. La información fue analizada con DAG_Stat. La sensibilidad, especificidad, eficiencia y el índice kappa se estimaron para cada especie bacteriana con un nivel de confianza del 95%, tomando la biología molecular como gold standard. Se detectaron Estafilococos de 27 perros y de sus dueños. Solamente una cepa de S. intermedius fue aislada, así que los parámetros relacionados con ella tienen que ser considerados con cautela. Todos los S. aureus fueron identificados correctamente. Usando MALDI-TOF MS con ácido fórmico, hubo una concordancia casi perfecta entre pruebas en la identificación de S. aureus y S. pseudintermedius. Cuando no se añadía ácido fórmico, la concordancia fue perfecta para S. aureus, mientras que para S. pseudintermedius fue buena. Este trabajo demuestra la validez, la utilidad y la fiabilidad del MALDI-TOF MS para la identificación de aislamientos bacterianos pertenecientes a Staphylococcus spp

Penicillin susceptibility among invasive MSSA infections: a multicentre study in 16 Spanish hospitals

Objectives: To determine the prevalence of penicillin susceptibility among MSSA causing bloodstream infections (BSIs) in 16 Spanish hospitals and to characterize the penicillin-susceptible MSSA (MSSA-PENS) isolates. Methods: A total of 1011 Staphylococcus aureus isolates were collected from blood cultures in 16 Spanish hospitals during 2018–19 (6–12 months) and their susceptibility to 18 antimicrobials was determined. The MSSA-PENS isolates were selected and examined by PCR to determine the presence of the blaZ gene, other resistance genes and the genes lukF/lukS-PV, eta, etb and tst. The immune evasion cluster (IEC) type was also analysed. All the MSSA-PENS isolates were submitted to S. aureus protein A (spa) typing and the clonal complexes (CCs) were assigned according to their spa type. Results: The prevalence of MSSA was 74.6% (754/1011) and 14.9% (151/1011) were MSSA-PENS-blaZnegative. MSSA-PENS-blaZnegative isolates (n = 151) were ascribed to 88 spa types and 11 CCs. The most frequent CCs were CC5 (35/151) and CC398 (25/151), with t002-CC5 and t571-CC398 being the most common lineages. Pan-susceptibility was identified in 117 of the 151 MSSA-PENS-blaZnegative isolates (77.5%). In the remaining isolates, erythromycin and clindamycin resistance was the most frequent resistance found, although tobramycin, ciprofloxacin, fusidic acid, mupirocin and/or tetracycline resistance was also detected. Thirty-eight MSSA-PENS-blaZnegative isolates were IEC negative and four isolates were Panton–Valentine leucocidin (‘PVL’) positive. Conclusions: A high penicillin susceptibility rate was detected among MSSA, opening therapeutic opportunities for BSIs. The emergence of new successful MSSA-PENS clones could be responsible for these data. The detection among MSSA-PENS-blaZnegative isolates of the clonal lineage CC398 or the absence of an IEC raises questions about their possible animal origin, requiring further analysis

Prevalence and genetic characteristics of Staphylococcus aureus CC398 isolates from invasive infections in spanish hospitals, focusing on the livestock-independent CC398-MSSA clade

Background: Livestock-associated (LA)-CC398-MRSA is closely related to pigs, being unfrequently detected in human invasive infections. CC398-MSSA is emerging in human invasive infections in some countries, but genetic and epidemiological characteristics are still scarcely reported. Objectives: To determine the prevalence of Staphylococcus aureus (SA) CC398, both MRSA and MSSA, among blood cultures SA isolates recovered in Spanish hospitals located in regions with different pig-farming densities (PD) and characterize the recovered isolates. Methods: One thousand twenty-two SA isolates (761 MSSA, 261 MRSA) recovered from blood cultures during 6–12 months in 17 Spanish hospitals (2018–2019) were studied. CC398 lineage identification, detection of spa-types, and antibiotic resistance, virulence and human immune evasion cluster (IEC) genes were analyzed by PCR/sequencing. Results: Forty-four CC398-MSSA isolates (4.3% of SA; 5.8% of MSSA) and 10 CC398-MRSA isolates (1% of SA; 3.8% of MRSA) were detected. Eleven spa-types were found among the CC398-MSSA isolates with t571 and t1451 the most frequent spa-types detected (75%). Most of CC398-MSSA isolates were Immune-Evasion-Cluster (IEC)-positive (88.6%), tetracycline-susceptible (95.5%) and erythromycin/clindamycin–inducible-resistant/erm(T)-positive (75%). No statistical significance was detected when the CC398-MSSA/MSSA rate was correlated to PD (pigs/km2) (p = 0.108). On the contrary, CC398-MRSA isolates were all IEC-negative, predominately spa-t011 (70%), and the CC398-MRSA/MRSA rate was significantly associated to PD (p < 0.005). Conclusion: CC398-MSSA is an emerging clade in invasive infections in Spanish hospitals. CC398-MRSA (mostly t011) and CC398-MSSA (mostly t571 and t1451) show important differences, possibly suggesting divergent steps in host-adaptation evolutionary processes. While CC398-MRSA is livestock-associated (lacking IEC-system), CC398-MSSA seems to be mostly livestock-independent, carrying human-adaptation markers.

Parámetros de detección precoz de infección fúngica por aspergillus en el paciente hematológico

PO-267 Introducción: La infección fúngica invasiva (IFI) por Aspergillus es una de las principales micosis invasivas en el paciente hematológico y una importante causa de morbimortalidad. Es más frecuente en pacientes con leucemia aguda mieloide, trasplante de progenitores hematopoyéticos alogénico, enfermedad injerto contra receptor activa o neutropenia prolongada. Supone un reto diagnóstico, siendo preciso el empleo de múltiples técnicas microbiológicas y radiológicas. Avances recientes en el diagnóstico, en la profilaxos y en el tratamiento antifúngico precoz, han reducido considerablemente la mortalidad asociada a esta infección. Objetivos: Determinar si los marcadores diagnósticos empleados en la práctica clínica habitual permiten un diagnóstico precoz de IFI, si la determinación de bismetilgliotoxina (bmGT) en suero permite un diagnóstico precoz en pacientes con antígeno de galactomanano (AGA) en suero negativo y la exactitud diagnóstica de estos marcadores. ..

Spread of a SARS-CoV-2 variant through Europe in the summer of 2020.

Following its emergence in late 2019, the spread of SARS-CoV-21,2 has been tracked by phylogenetic analysis of viral genome sequences in unprecedented detail3–5. Although the virus spread globally in early 2020 before borders closed, intercontinental travel has since been greatly reduced. However, travel within Europe resumed in the summer of 2020. Here we report on a SARS-CoV-2 variant, 20E (EU1), that was identified in Spain in early summer 2020 and subsequently spread across Europe. We find no evidence that this variant has increased transmissibility, but instead demonstrate how rising incidence in Spain, resumption of travel, and lack of effective screening and containment may explain the variant’s success. Despite travel restrictions, we estimate that 20E (EU1) was introduced hundreds of times to European countries by summertime travellers, which is likely to have undermined local efforts to minimize infection with SARS-CoV-2. Our results illustrate how a variant can rapidly become dominant even in the absence of a substantial transmission advantage in favourable epidemiological settings. Genomic surveillance is critical for understanding how travel can affect transmission of SARS-CoV-2, and thus for informing future containment strategies as travel resumes. © 2021, The Author(s), under exclusive licence to Springer Nature Limited

The first wave of the COVID-19 epidemic in Spain was associated with early introductions and fast spread of a dominating genetic variant

SeqCOVID-Spain consortium: Álvaro Chiner-Oms, Irving Cancino-Muñoz, Mariana G. López, Manuela Torres-Puente, Inmaculada Gómez-Navarro, Santiago Jiménez-Serrano, Jordi Pérez-Tur, Darío García de Viedma, Laura Pérez-Lago, Marta Herranz, Jon Sicilia, Pilar Catalán-Alonso, Julia Suárez González, Patricia Muñoz, Mireia Coscolla, Paula Ruiz-Rodríguez, Fernando González-Candelas, Iñaki Comas, Lidia Ruiz-Roldán, María Alma Bracho, Neris García-González, Llúcia Martínez Priego, Inmaculada Galán-Vendrell, Paula Ruiz-Hueso, Griselda De Marco, María Loreto Ferrús-Abad, Sandra Carbó-Ramírez, Giuseppe D’Auria, Galo Adrian Goig, Juan Alberola, Jose Miguel Nogueira, Juan José Camarena, David Navarro, Eliseo Albert, Ignacio Torres, Maitane Aranzamendi Zaldumbide, Óscar Martínez Expósito, Nerea Antona Urieta, María de Toro, María Pilar Bea-Escudero, Jose Antonio Boga, Cristian Castelló-Abietar, Susana Rojo-Alba, Marta Elena Álvarez-Argüelles, Santiago Melón, Elisa Martró, Antoni E. Bordoy, Anna Not, Adrián Antuori, Anabel Fernández-Navarro, Andrés Canut-Blasco, Silvia Hernáez Crespo, Maria Luz Cordón Rodríguez, Maria Concepción Lecaroz Agara, Carmen Gómez-González, Amaia Aguirre-Quiñonero, José Israel López-Mirones, Marina Fernández-Torres, Maria Rosario Almela-Ferrer, Ana Carvajal, Juan Miguel Fregeneda-Grandes, Héctor Argüello, Gustavo Cilla Eguiluz, Milagrosa Montes Ros, Luis Piñeiro Vázquez, Ane Sorarrain, José María Marimón, José J. Costa-Alcalde, Rocío Trastoy, Gema Barbeito Castiñeiras, Amparo Coira, María Luisa Pérez del Molino, Antonio Aguilera, Begoña Palop-Borrás, Inmaculada de Toro Peinado, Maria Concepción Mediavilla Gradolph, Mercedes Pérez-Ruiz, Mirian Fernández-Alonso, Jose Luis del Pozo, Oscar González-Recio, Mónica Gutiérrez-Rivas, Jovita Fernández-Pinero, Miguel Ángel Jiménez Clavero, Begoña Fuster Escrivá, Concepción Gimeno Cardona, María Dolores Ocete Mochón, Rafael Medina-Gonzalez, José Antonio Lepe, Verónica González Galán, Ángel Rodríguez-Villodres, Nieves Gonzalo Jiménez, Jordi Reina, Carla López-Causapé, Maria Dolores Gómez-Ruiz, Eva M. Gonzalez-Barbera, José Luis López-Hontangas, Vicente Martín, Antonio J. Molina, Tania Fernandez-Villa, Ana Milagro Beamonte, Nieves Felisa Martínez-Cameo, Yolanda Gracia-Grataloup, Rosario Moreno-Muñoz, Maria Dolores Tirado Balaguer, José María Navarro-Marí, Irene Pedrosa-Corral, Sara Sanbonmatsu-Gámez, Antonio Oliver, Mónica Parra Grande, Bárbara Gómez Alonso, Francisco José Arjona Zaragozí, Maria Carmen Pérez González, Francisco Javier Chamizo López, Ana Bordes-Benítez, Núria Rabella, Ferran Navarro, Elisenda Miró, Antonio Rezusta, Alexander Tristancho, Encarnación Simarro Córdoba, Julia Lozano-Serra, Lorena Robles Fonseca, Álex Soriano, Francisco Javier Roig Sena, Hermelinda Vanaclocha Luna, Isabel Sanmartín, Daniel García-Souto, Ana Pequeño-Valtierra, Jose M. C. Tubio, Javier Temes, Jorge Rodríguez-Castro, Martín Santamarina García, Manuel Rodríguez-Iglesias, Fátima Galán-Sanchez, Salud Rodríguez-Pallares, José Manuel Azcona-Gutiérrez, Miriam Blasco-Alberdi, Alfredo Mayor, Alberto L. García-Basteiro, Gemma Moncunill, Carlota Dobaño, Pau Cisteró, Oriol Mitjà, Camila González-Beiras, Martí Vall-Mayans, Marc Corbacho-Monné, Andrea Alemany, Cristina Muñoz-Cuevas, Guadalupe Rodríguez-Rodríguez, Rafael Benito, Sonia Algarate, Jessica Bueno, Andrea Vergara-Gómez, Miguel J. Martínez, Jordi Vila, Elisa Rubio, Aida Peiró-Mestres, Jessica Navero-Castillejos, David Posada, Diana Valverde, Nuria Estévez, Iria Fernández-Silva, Loretta de Chiara, Pilar Gallego-García, Nair Varela, Ulises Gómez-Pinedo, Mónica Gozalo-Margüello, Maria Eliecer Cano García, José Manuel Méndez-Legaza, Jesus Rodríguez-Lozano, María Siller, Daniel Pablo-Marcos, Maria Montserrat Ruiz-García, Antonio Galiana, Judith Sánchez-Almendro, Maria Isabel Gascón Ros, Cristina Juana Torregrosa-Hetland, Eva María Pastor Boix, Paloma Cascales Ramos, Pedro Luis Garcinuño Enríquez, Salvador Raga Borja, Julia González Cantó, Olalla Martínez Macias, Adolfo de Salazar, Laura Viñuela González, Natalia Chueca, Federico García, Cristina Gómez-Camarasa, Amparo Farga Martí, Rocío Falcón, Victoria Domínguez-Márquez, Anna M. Planas, Israel Fernández-Cádenas, Maria Ángeles Marcos, Carmen Ezpeleta, Ana Navascués, Ana Miqueleiz Zapatero, Manuel Segovia, Antonio Moreno-Docón, Esther Viedma, Raúl Recio Martínez, Irene Muñoz-Gallego, Sara Gonzalez-Bodi, Maria Dolores Folgueira, Jesús Mingorance, Elias Dahdouh, Fernando Lázaro-Perona, María Rodríguez-Tejedor, María Pilar Romero-Gómez, Julio García-Rodríguez, Juan Carlos Galán, Mario Rodríguez-Dominguez, Laura Martínez-García, Melanie Abreu Di Berardino, Manuel Ponce-Alonso, Jose Maria González-Alba, Ivan Sanz-Muñoz, Diana Pérez San José, Maria Gil Fortuño, Juan B. Bellido-Blasco, Alberto Yagüe Muñoz, Noelia Hernández Pérez, Helena Buj Jordá, Óscar Pérez Olaso, Alejandro González Praetorius, Nora Mariela Martínez Ramírez, Aida Ramírez Marinero, Eduardo Padilla León, Alba Vilas Basil, Mireia Canal Aranda, Albert Bernet Sánchez, Alba Bellés Bellés, Eric López González, Iván Prats Sánchez, Mercè García-González, Miguel José Martínez-Lirola, Manuel Ángel Rodríguez Maresca, Maria Teresa Cabezas Fernández, María Eugenia Carrillo Gil, Maria Paz Ventero Martín, Carmen Molina Pardines, Nieves Orta Mira, María Navarro Cots, Inmaculada Vidal Catalá, Isabel García Nava, Soledad Illescas Fernández-Bermejo, José Martínez-Alarcón, Marta Torres-Narbona, Cristina Colmenarejo, Lidia García-Agudo, Jorge A. Pérez García, Martín Yago López, María Ángeles Goberna Bravo, Victoria Simón García, Gonzalo Llop Furquet, Agustín Iranzo Tatay, Sandra Moreno-Marro, Noelia Lozano Rodríguez, Amparo Broseta Tamarit, Juan José Badiola Díez, Amparo Martínez-Ramírez, Ana Dopazo, Sergio Callejas, Alberto Benguría, Begoña Aguado, Antonio Alcamí, Marta Bermejo Bermejo, Ricardo Ramos-Ruíz, Víctor Manuel Fernández Soria, Fernando Simón Soria & Mercedes Roig CardellsThe coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (Re < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.This work was mainly funded by the Instituto de Salud Carlos III project COV20/00140, with additional funding by Spanish National Research Council project CSIC-COV19-021, Ministerio de Ciencia project PID2019-104477RB-100, ERC StG 638553 and ERC CoG 101001038 to I.C., and BFU2017-89594R to F.G.C. M.C. is supported by Ramón y Cajal program from Ministerio de Ciencia and grants RTI2018-094399-A-I00 and Generalitat Valenciana (Regional Government) project SEJI/2019/011. We gratefully acknowledge Hospital Universitari Vall d’Hebron, Instituto de Salud Carlos III, IrsiCaixa AIDS Research Lab and all the international researchers and institutions that submitted sequenced SARS-CoV-2 genomes to the GISAID’s EpiCov Database (Supplementary Table 1), as an important part of our analyses has been made possible by the sharing of their work. We also thank Unidad de Bioinformática y Estadística, Centro de Investigación Príncipe Felipe, for allowing us to use the Computer Cluster to perform some of the bioinformatic analysis.Peer reviewe