Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021

Acute flaccid myelitis; Enterovirus D68; SurveillanceMielitis flàcida aguda; Enterovirus D68; VigilànciaMielitis flácida aguda; Enterovirus D68; VigilanciaWe report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe

Flexible multiplex PCR to detect SARS-CoV-2, coronavirus OC43 and influenza A virus in nasopharyngeal swab samples

Introduction Quantitative reverse transcription PCR (RT-qPCR) is the leading tool to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given that it will almost certainly continue to coexist with other respiratory viruses in the coming years, our study aimed to design a multiplex PCR system not affected by supplier outages and with reduced cost compared to the existing commercially available kits. Methods and results In this study, combinations of four primers/probe sets were used to construct a flexible RT-qPCR assay which is capable of discriminating between SARS-CoV-2 and the seasonal human coronavirus HCoV-OC43, or even influenza A virus. Additionally, the human RPP30 gene was used as an internal control. To demonstrate the robustness of the assay, it was applied to a collection of 150 clinical samples. The results showed 100% sensitivity and specificity compared to the automatized system used at the hospital and were better when indeterminate samples were analysed. Conclusions This study provides an efficient method for the simultaneous detection of SARS-CoV-2, HCoV-OC43 and influenza A virus, and its efficacy has been tested on clinical samples showing outstanding results. Significance and impact of the study The multiplex RT-qPCR design offers an accessible and economical alternative to commercial detection kits for hospitals and laboratories with limited economic resources or facing situations of supply shortage.This research was funded by Basque Government, grants numbers 2020333042 and IT1362‐19. LM‐S have received a predoctoral Grant from Basque Government

Epidemiological and Clinical Complexity of Amoxicillin-Clavulanate- Resistant Escherichia coli

Two hundred twelve patients with colonization/infection due to amoxicillin-clavulanate (AMC)-resistant Escherichia coli were studied. OXA-1- and inhibitor-resistant TEM (IRT)-producing strains were associated with urinary tract infections, while OXA-1 producers and chromosomal AmpC hyperproducers were associated with bacteremic infections. AMC resistance in E. coli is a complex phenomenon with heterogeneous clinical implications

Spanish Multicenter Study of the Epidemiology and Mechanisms of Amoxicillin-Clavulanate Resistance in Escherichia coli

We conducted a prospective multicenter study in Spain to characterize the mechanisms of resistance to amoxicillin-clavu-lanate (AMC) in Escherichia coli. Up to 44 AMC-resistant E. coli isolates (MIC>32/16 g/ml) were collected at each of theseven participant hospitals. Resistance mechanisms were characterized by PCR and sequencing. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and by multilocus sequence typing. Overall AMC resistance was 9.3%. The resistance mechanisms detected in the 257 AMC-resistant isolates were OXA-1 production (26.1%), hyperpro-duction of penicillinase (22.6%), production of plasmidic AmpC (19.5%), hyperproduction of chromosomic AmpC(18.3%), and production of inhibitor-resistant TEM (IRT) (17.5%). The IRTs identified were TEM-40 (33.3%), TEM-30(28.9%), TEM-33 (11.1%), TEM-32 (4.4%), TEM-34 (4.4%), TEM-35 (2.2%), TEM-54 (2.2%), TEM-76 (2.2%), TEM-79(2.2%), and the new TEM-185 (8.8%). By PFGE, a high degree of genetic diversity was observed although two well-defined clusters were detected in the OXA-1-producing isolates: the C1 cluster consisting of 19 phylogroup A/sequence type 88[ST88] isolates and the C2 cluster consisting of 19 phylogroup B2/ST131 isolates (16 of them producing CTX-M-15). Each of the clusters was detected in six different hospitals. In total, 21.8% of the isolates were serotype O25b/phylogroup B2 (O25b/B2). AMC resistance in E. coli is widespread in Spain at the hospital and community levels. A high prevalence of OXA-1 was found. Although resistant isolates were genetically diverse, clonality was linked to OXA-1-producing isolates of the STs 88 and 131. Dissemination of IRTs was frequent, and the epidemic O25b/B2/ST131 clone carried many different mechanisms of AMC resistance

Adherence to Human Colon Cells by Multidrug Resistant Enterobacterales Strains Isolated From Solid Organ Transplant Recipients With a Focus on Citrobacter freundii

Enterobacteria species are common causes of hospital-acquired infections, which are associated with high morbidity and mortality rates. Immunocompromised patients such as solid organ transplant (SOT) recipients are especially at risk because they are frequently exposed to antibiotics in the course of their treatments. In this work, we used a collection of 106 Escherichia coli, 78 Klebsiella pneumoniae, 25 Enterobacter spp., and 24 Citrobacter spp. multidrug resistant strains isolated from transplant patients (hepatic, renal or renal/pancreatic) in order to examine their ability to adhere in vitro to HT-29 human colon cells, and to determine if some adhesive characteristics are associated with prevalence and persistence of these strains. A total of 33 E. coli (31%), 21 K. pneumoniae (27%), 7 Enterobacter spp. (28%), and 5 Citrobacter spp. (21%), adhered to the colon epithelial cells. Two main adherence patterns were observed in the four species analyzed, diffuse adherence, and aggregative adherence. Under transmission electronic microscopy (TEM), most bacteria lacked visible fimbria on their surface, despite their strong adherence to epithelial cells. None of the strains studied was able to induce any cytotoxic effect on HT-29 cells although some of them strongly colonizing both cells and glass coverslips at high density. Some of the strains failed to adhere to the epithelial cells but adhered strongly to the cover-slide, which shows that microscopy studies are mandatory to elucidate the adherence of bacteria to epithelial cells in vitro, and that quantitative assays using colony forming unit (CFUs) counting need to be supplemented with pictures to determine definitively if a bacterial strain adheres or not to animal cells in vitro. We report here, for the first time, the aggregative adherence pattern of two multidrug resistant (MDR) Citrobacter freundii strains isolated from human patients; importantly, biofilm formation in Citrobacter is totally dependent on the temperature; strong biofilms were formed at room temperature (RT) but not at 37°C, which can play an important role in the colonization of hospital surfaces. In conclusion, our results show that there is a great variety of adhesion phenotypes in multidrug-resistant strains that colonize transplanted patients.This research was supported by Plan Nacional de I+D+i and Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias PI13/01191 to MF and PI16/01103 to JR-V), Subdirección General de Redes y Centros de Investigación Cooperativa, Spanish Ministry of Economy and Competitiveness, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0007, RD16/0016/0010, RD16/0016/0012, RD16/0016/0011, RD16/0016/0008, and RD16/0016/0002) co-financed by the European Development Regional Fund A way to achieve Europe ERDF, and Ministerio de Ciencia e Innovación, Acciones de dinamización ≪Redes de Investigación≫RED2018-102469-

Molecular characterization of multidrug resistant Enterobacterales strains isolated from liver and kidney transplant recipients in Spain

The objective of this study was to analyse the mechanisms of resistance to carbapenems and other extended-spectrum-?-lactams and to determine the genetic relatedness of multidrug-resistant Enterobacterales (MDR-E) causing colonization or infection in solid-organ transplantation (SOT) recipients. Prospective cohort study in kidney (n= 142), liver (n= 98) or kidney/pancreas (n= 7) transplant recipients between 2014 and 2018 in seven Spanish hospitals. We included 531 MDR-E isolates from rectal swabs obtained before transplantation and weekly for 4?6 weeks after the procedure and 10 MDR-E from clinical samples related to an infection. Overall, 46.2% Escherichia coli, 35.3% Klebsiella pneumoniae, 6.5% Enterobacter cloacae, 6.3% Citrobacter freundii and 5.7% other species were isolated. The number of patients with MDR-E colonization post-transplantation (176; 71.3%) was 2.5-fold the number of patients colonized pre-transplantation (71; 28.7%). Extended spectrum ?-lactamases (ESBLs) and carbapenemases were detected in 78.0% and 21.1% of MDR-E isolates respectively. In nine of the 247 (3.6%) transplant patients, the microorganism causing an infection was the same strain previously cultured from surveillance rectal swabs. In our study we have observed a low rate of MDR-E infection in colonized patients 4?6 weeks post-transplantation. E. coli producing blaCTX-M-G1 and K. pneumoniae harbouring blaOXA-48 alone or with blaCTX-M-G1 were the most prevalent MDR-E colonization strains in SOT recipients.Acknowledgements The authors thank Mª Jesús Lecea and Laura Álvarez for technical assistance. Tis research was supported by ‘Plan Nacional de I+D+i and Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias 13/01191), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, and the Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0007, RD16/0016/0010, RD16/0016/0012, RD16/0016/0011, RD16/0016/0008, RD16/0016/0002). Te study was co-fnanced by the European Development Regional Fund “A way to achieve Europe” and the Operative Program Intelligent Growth 2014‐2020

Biofilm formation by multidrug resistant Enterobacteriaceae strains isolated from solid organ transplant recipients

Solid organ transplant (SOT) recipients are especially at risk of developing infections by multidrug resistant bacteria (MDR). In this study, the biofilm-forming capability of 209 MDR strains (Escherichia coli n = 106, Klebsiella pneumoniae n = 78, and Enterobacter spp. n = 25) isolated from rectal swabs in the first 48 hours before or after kidney (93 patients), liver (60 patients) or kidney/pancreas transplants (5 patients) were evaluated by using a microplate assay. Thirty-nine strains were isolated before transplant and 170 strains were isolated post-transplant. Overall, 16% of E. coli strains, 73% of K. pneumoniae strains and 4% Enterobacter strains showed moderate or strong biofilm production. Nine strains isolated from infection sites after transplantation were responsible of infections in the first month. Of these, 4 K. pneumoniae, 1 E. coli and 1 Enterobacter spp. strains isolated pre-transplant or post-transplant as colonizers caused infections in the post-transplant period. Our results suggest that in vitro biofilm formation could be an important factor for adhesion to intestine and colonization in MDR K. pneumoniae strains in SOT recipients, but this factor appears to be less important for MDR E. coli and Enterobacter spp.Acknowledgements: The authors thank Dr. Fidel Madrazo (Electron Microscopy Unit, Technology Support Services, IDIVAL) for helping with confocal microscopy. This research was supported by ‘Plan Nacional de I + D + i and Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias PI 13/01191 to MCF and PI 16/01103 to JRV), Subdirección General de Redes y Centros de Investigación Cooperativa, Spanish Ministry of Economy and Competitiveness, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015) and (REIPI RD16/0016) co-financed by the European Development Regional Fund “A way to achieve Europe” ERDF

Detection of cytomegalovirus drug resistance mutations in solid organ transplant recipients with suspected resistance

BACKGROUND: Current guidelines recommend that treatment of resistant cytomegalovirus (CMV) in solid organ transplant (SOT) recipients must be based on genotypic analysis. However, this recommendation is not systematically followed. OBJECTIVES: To assess the presence of mutations associated with CMV resistance in SOT recipients with suspected resistance, their associated risk factors and the clinical impact of resistance. STUDY DESIGN: Using Sanger sequencing we prospectively assessed the presence of resistance mutations in a nation-wide prospective study between September 2013-August 2015. RESULTS: Of 39 patients studied, 9 (23%) showed resistance mutations. All had one mutation in the UL 97 gene and two also had one mutation in the UL54 gene. Resistance mutations were more frequent in lung transplant recipients (44% p=0.0068) and in patients receiving prophylaxis >/=6 months (57% vs. 17%, p=0.0180). The mean time between transplantation and suspicion of resistance was longer in patients with mutations (239 vs. 100days, respectively, p=0.0046) as was the median treatment duration before suspicion (45 vs. 16days, p=0.0081). There were no significant differences according to the treatment strategies or the mean CMV load at the time of suspicion. Of note, resistance-associated mutations appeared in one patient during CMV prophylaxis and also in a seropositive organ recipient. Incomplete suppression of CMV was more frequent in patients with confirmed resistance. CONCLUSIONS: Our study confirms the need to assess CMV resistance mutations in any patient with criteria of suspected clinical resistance. Early confirmation of the presence of resistance mutations is essential to optimize the management of these patients

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe

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