No Remdesivir Resistance Observed in the Phase 3 Severe and Moderate COVID-19 SIMPLE Trials.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC50 fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

Kinetics of cellular and humoral immunogenicity and effectiveness of SARS-CoV-2 booster vaccination in hematologic neoplasms.

Data on the effect of booster SARS-CoV-2 vaccination are mainly focused on humoral immunogenicity, while the kinetics of vaccine-induced cellular response and its correlation with effectiveness in hematologic patients are less explored. Our aim was to evaluate the longitudinal cellular and humoral immunogenicity induced by two and three doses of the mRNA-1273 SARS-CoV-2 vaccine in 270 patients with hematologic malignancies, and its relationship with the severity of breakthrough SARS-CoV-2 infection. Results indicate that at 23 weeks after the second dose, the seroconversion rate declined from 68.5% to 59.3%, with a reduction in median anti-S titers from 1577 to 456 BAU/mL, mainly in patients over 65 years of age or chronic lymphocytic leukemia (CLL) patients undergoing active therapy. Cellular immunogenicity, however, remained positive in 84.4% of cases. A third vaccine dose seroconverted 42.7% (41/96) and triggered cellular response in 36.7% (11/30) of previously negative patients. Notably, only 7.2% (15/209) of patients failed to develop both humoral and cellular response. Active therapy, anti-CD20 antibodies, lymphopenia, hypogammaglobulinemia, and low CD19+ cell count were associated with poor humoral response, while active disease, GvHD immunosuppressive therapy, lymphopenia, and low CD3+ , CD4+ , CD56+ cell count determined an impaired cellular response. After 13.8 months of follow-up, the incidence of SARS-CoV-2 infection was 24.8% (67/270), including 6 (9%) severe/critical cases associated with a weaker cellular (median interferon gamma (IFN-γ) 0.19 vs. 0.35 IU/mL) and humoral response (median anti-S titer <4.81 vs. 788 BAU/mL) than asymptomatic/mild cases. In conclusion, SARS-CoV-2 booster vaccination improves humoral response and COVID-19 severity is associated with impaired vaccine-induced immunogenicity.

Emergence of Delta and Omicron variants carrying resistance-associated mutations in immunocompromised patients undergoing sotrovimab treatment with long-term viral excretion.

OBJECTIVES: To monitor the early emergence of genetic mutations related to reduced susceptibility to monoclonal anti-body (mAb)-based treatment in immunocompromised patients with long-term viral excretion using whole-genome sequencing at a tertiary university hospital in Barcelona, Spain. METHODS: Serial severe acute respiratory syndrome coronavirus 2-positive samples (mid-December 2021-mid-March 2022) from eight immunosuppressed, fully vaccinated patients (for solid-organ transplantation or haematologic malignancies) with long-term viral excretion despite undergoing mAb therapy (sotrovimab) for coronavirus disease 2019 were selected. Whole-genome sequencing was performed following the ARTIC, version 4.1, protocol on the MiSeq platform. Mutations in the coding sequence of the spike protein with a frequency of ≥5% were studied. RESULTS: A total of 37 samples from the studied cases were analysed. All the cases, except one, were confirmed to have the Omicron variant BA.1; one had Delta (AY.100). Thirty-four different mutations were detected within the receptor-binding domain of the spike protein in 62.5% of patients, eight of which were not lineage related and located in the sotrovimab target epitope (P337L, E340D, E340R, E340K, E340V, E340Q, R346T and K356T). Except for P337L, all changes showed a significant increase in frequency or fixation after the administration of sotrovimab. Some of them have been associated with either reduced susceptibility to mAb therapy, such as those at position 340, or the acquisition of a new glycosylation site (346 and 356 positions). CONCLUSIONS: This study highlights the importance of monitoring for early in vivo selection of mutations associated with reduced susceptibility to mAb therapy, especially in immunocompromised patients receiving anti-viral drugs, whose immune response is not able to control viral replication, resulting in long-term viral shedding, and those receiving selective evolution pressure. Virologic surveillance of genetically resistant viruses to available anti-viral therapies is considered a priority for both patients and the community.

Commercialized kits to assess T-cell responses against SARS-CoV-2 S peptides. A pilot study in health care workers.

BACKGROUND: It is crucial to assess the levels of protection generated by natural infection or SARS-CoV-2 vaccines, mainly in individuals professionally exposed and in vulnerable groups. Measuring T-cell responses may complement antibody tests currently in use as correlates of protection. Our aim was to assess the feasibility of a validated assay of T-cell responses. METHODS: Twenty health-care-workers (HCW) were included. Antibody test to SARS-CoV-2 N and S-proteins in parallel with a commercially available whole-blood-interferon-gamma-release-assay (IGRA) to S-peptides and two detection methods, CLIA and ELISA were determined. RESULTS: IGRA test detected T-cell responses in naturally exposed and vaccinated HCW already after first vaccination dose. The correlation by the two detection methods was very high (R>0.8) and sensitivity and specificity ranged between 100 and 86% and 100-73% respectively. Even though there was a very high concordance between specific antibody levels and the IGRA assay in the ability to detect immune response to SARS-CoV-2, there was a relatively low quantitative correlation. In the small group primed by natural infection, one vaccine dose was sufficient to reach immune response plateau. IGRA was positive in one, with Ig(S) antibody negative vaccinated immunosuppressed HCW illustrating another advantage of the IGRA-test. CONCLUSION: Whole-blood-IGRA-tests amenable to automation and constitutes a promising additional tool for measuring the state of the immune response to SARS-CoV-2; they are applicable to large number of samples and may become a valuable correlate of protection to COVID-19, particularly for vulnerable groups at risk of being re-exposed to infection, as are health-care-workers.

Host-dependent editing of SARS-CoV-2 in COVID-19 patients.

A common trait among RNA viruses is their high capability to acquire genetic variability due to viral and host mechanisms. Next-generation sequencing (NGS) analysis enables the deep study of the viral quasispecies in samples from infected individuals. In this study, the viral quasispecies complexity and single nucleotide polymorphisms of the SARS-CoV-2 spike gene of coronavirus disease 2019 (COVID-19) patients with mild or severe disease were investigated using next-generation sequencing (Illumina platform). SARS-CoV-2 spike variability was higher in patients with long-lasting infection. Most substitutions found were present at frequencies lower than 1%, and had an A → G or T → C pattern, consistent with variants caused by adenosine deaminase acting on RNA-1 (ADAR1). ADAR1 affected a small fraction of replicating genomes, but produced multiple, mainly non-synonymous mutations. ADAR1 editing during replication rather than the RNA-dependent RNA polymerase (nsp12) was the predominant mechanism generating SARS-CoV-2 genetic variability. However, the mutations produced are not fixed in the infected human population, suggesting that ADAR1 may have an antiviral role, whereas nsp12-induced mutations occurring in patients with high viremia and persistent infection are the main source of new SARS-CoV-2 variants.

Characterisation of the sewage virome: comparison of NGS tools and occurrence of significant pathogens.

NGS techniques are excellent tools to monitor and identify viral pathogens circulating among the population with some limitations that need to be overcome, especially in complex matrices. Sewage contains a high amount of other microorganisms that could interfere when trying to sequence viruses for which random PCR amplifications are needed before NGS. The selection of appropriate NGS tools is important for reliable identification of viral diversity among the population. We have compared different NGS methodologies (Untargeted Viral Metagenomics, Target Enrichment Sequencing and Amplicon Deep Sequencing) for the detection and characterisation of viruses in urban sewage, focusing on three important human pathogens: papillomaviruses, adenoviruses and enteroviruses. A full picture of excreted viruses was obtained by applying Untargeted Viral Metagenomics, which detected members of four different vertebrate viral families in addition to bacteriophages, plant viruses and viruses infecting other hosts. Target Enrichment Sequencing, using specific vertebrate viral probes, allowed the detection of up to eight families containing human viruses, with high variety of types within the families and with a high genome coverage. By applying Amplicon Deep Sequencing, the diversity of enteroviruses, adenoviruses and papillomaviruses observed was higher than when applying the other two strategies and this technique allowed the subtyping of an enterovirus A71 C1 strain related to a brainstem encephalitis outbreak occurring at the same time in the sampling area. From the data obtained, we concluded that the different strategies studied provided different levels of analysis: TES is the best strategy to obtain a broad picture of human viruses present in complex samples such as sewage. Other NGS strategies are useful for studying the virome of complex samples when also targeting viruses infecting plants, bacteria, invertebrates or fungi (Untargeted Viral Metagenomics) or when observing the variety within a sole viral family is the objective of the study (Amplicon Deep Sequencing).

Ganglio centinela en cáncer de mama. Experiencia en nuestros primeros 100 casos / Sentinel Node in Breast Cancer. Our first 100 cases experience

Introducción Desde los trabajos presentados por Giuliano et al . en 1994 y sus posteriores validaciones, la biopsia del ganglio centinela se ha convertido en el standard de la evaluación axilar en estadios tempranos del cáncer de mama. Es de vital importancia respaldar esta técnica con altas tasas de identificación y de correlación anatomopatológica intraoperatoria/diferida. Objetivos 1) Determinar nuestra tasa de identificación usando solo azul patente. 2) Determinar la correlación entre el estudio intraoperatorio y diferido. 3) Determinar la tasa de recurrencia axilar. Material y método Se reclutaron 100 pacientes con cáncer de mama T1 T2 N0 o cdis extenso de alto grado diagnosticadas por punción con aguja fina (paf) o Core Biopsy. Se utilizó solo Azul Patente al 1%, inyección subareolar, masaje circular por 10 minutos, incisión axilar. Se reconoce como Ganglio Centinela al ganglio o a los ganglios teñidos de azul o con su linfático aferente con colorante. El estudio intraoperatorio se realizó por sección en fresco e impronta citológica. El diferido por coloración con Hematoxilina y Eosina. Las pacientes fueron seguidas por el cirujano actuante y/u oncología clínica. Resultados Se evaluaron 100 pacientes. Nuestra tasa de detección fue del 98%. Los falsos negativos intraoperatorios por impronta citológica fueron del 3%. Luego de un seguimiento promedio de 63,8 meses, no se detectaron recidivas axilares. El número total de Ganglios Centinela positivos fue del 26,5%. El promedio de Ganglios hallados fue de 1,2. Conclusiones La Biopsia de Ganglio Centinela usando Azul Patente como único método es una operación confiable, de bajo costo y al alcance de todo centro con interés de desarrollar dicha técnica.

Introduction Since Giuliano et al. publications in 1994 and forward validations, sentinel node biopsy (snp) has become the standard procedure for staging the axilla in early breast cancer. Therefore, it is of vital importance to back this technique up by a high rate of identification and high intraoperative/final pathology correlation. Objectives 1) To determine our identification rate by only using Patent Blue dye. 2) To determine the correlation between intraoperative and final pathology results. 3) To determine axillary recurrence. Materials and method A hundred patients have been recruited. Each was diagnosed with T1 T2 N0 Breast Cancer or extense high grade Ductal Carcinoma In situ (dcis) by fine needle aspiration (fna) and/or Core Biopsy. Patent Blue 1% was injected subareolary. In addition, circular massage was performed during 10 minutes and an axillary incision was made. The node or nodes dyed in blue or with coloured lymphatic afferent have been acknowledged as sentinel nodes. The node was analyzed intraoperatively by touch imprint citology. The final study was done with Hematoxilin Eosin coloration. Further follow up was in charge of the surgeon and/or clinical oncologist. Results A hundred patients were assesed. Our identification rate was 98%. The pathology intraoperative false negative rate was 3%. Afer an average of 63,8 months follow up, no axillary recurrence was detected. 26,5% of positive sentinel nodes was found. The average of sentinel nodes found was 1,2. Conclusions snb by using only patent blue dye is a low cost reliable technique, and available for every institution interested in its development.

Molecular epidemiology and molecular characterization of respiratory syncytial viruses at a tertiary care university hospital in Catalonia (Spain) during the 2013-2014 season.

BACKGROUND: Human respiratory syncytial virus (HRSV) is the main cause of lower respiratory tract infections among infants and young children. OBJECTIVES: The molecular epidemiology and characterization of HRSV strains detected at a Spanish tertiary hospital during the 2013-2014 season is reported. STUDY DESIGN: Phylogenetic analysis and molecular characterization of HRSV laboratory-confirmed respiratory samples were performed, based on coding sequences of G and F proteins. RESULTS: HRSV infection was laboratory-confirmed in respiratory samples from 320 patients of which 223 (70%) were less than 2 years of age and none undergoing Palivizumab treatment. HRSV was detected at varying levels throughout the season with a maximum rate in the week 52/2013, right before the beginning of the influenza epidemic. Whilst both HRSV groups were found co-circulating, HRSV-B group clearly predominated. The phylogenetic analyses from 139 HVR-2 sequences revealed that most characterized strains belonged to ON1 and BA9 genotypes. Three different phylogenetic subgroups could be distinguished within these genotypes. In addition, three strains (out of the 52 randomly selected) were carrying amino acid substitutions in the epitope A of the F protein, one of them previously related to Palivizumab resistance. CONCLUSIONS: The results of the present study highlight the importance of a continuous HRSV surveillance to monitor not only the introduction of new genotypes on circulation but also the emergence of viral variants with new genetic characteristics that can affect the antigenicity features and the susceptibility to the only current prophylaxis treatment and also for the future development of HRSV vaccines.

First Enterovirus D68 (EV-D68) cases detected in hospitalised patients in a tertiary care university hospital in Spain, October 2014.

Several outbreaks of Enterovirus 68 (EV-D68) have recently been reported in the USA and Canada, causing substantial hospitalisation of children with severe respiratory disease. The acute flaccid paralysis detected in the USA and Canada among children with EV-D68 infection has raised concerns about the aetiological role of this EV serotype in severe neurological disease. The circulation of EV-D68 in the general European population seems to be low, but European Centre for Disease Prevention and Control (ECDC) recommends being vigilant to new cases, particularly in severely ill hospitalised patients. In October 2014, enteroviruses were detected in respiratory samples collected from five hospitalised patients, children and adults. Phylogenetic analysis of partial VP1 sequences confirmed that the detected enteroviruses belonged to the D68 serotype, which were also similar to strains reported in USA (2014). However, all five patients developed respiratory symptoms, but only one required ICU admission. None of the patients described had symptoms of neurological disease. Other considerations related to the detection methods used for the diagnosis of respiratory enteroviruses are also discussed. In conclusion, additional evidence has been provided that supports the role of EV-D68 in respiratory infections in hospitalised patients.

Clinical risk factors are more relevant than respiratory viruses in predicting bronchiolitis severity.

BACKGROUND: The role of respiratory viruses in the pathogenesis of bronchiolitis was re-evaluated with the use of molecular methods such as PCR for virus detection. Whether specific viruses or the classical clinical risk factors are more important in determining severe bronchiolitis is not well established. AIM: To analyze the specific viruses and clinical variables that can predict severe bronchiolitis at admission. METHODS: Nasopharyngeal aspirates were prospectively collected from 484 children <12 months admitted to the pediatrics ward or PICU at Universitary Hospital Sant Joan de Déu (Barcelona, Spain) for bronchiolitis from October 2007 to October 2008. Clinical and demographic data were collected. Sixteen respiratory viruses were studied using PCR. Severity was assessed with a bronchiolitis clinical score (BCS). RESULTS: Four hundred ten infants that tested positive for respiratory viruses were analyzed. Mixed viral infections did not increase the severity of the disease. Rhinovirus was associated with severe BCS in univariate analysis (P = 0.041), but in the multivariate logistic regression including viruses and clinical data only bronchopulmonary dysplasia (OR 7.2; 95% CI 1.2-43.3), congenital heart disease (OR 4.7; 95% CI 1.1-19.9), prematurity (OR 2.6; 95% CI 1.3-5.1), and fever (OR 1.8, 95% CI 1.1-3.1) showed statistical significance for predicting severe BCS. CONCLUSIONS: Classical clinical risk factors have more weight in predicting a severe BCS in infants with acute bronchiolitis than the involved viruses.

Influenza C virus surveillance during the first influenza A (H1N1) 2009 pandemic wave in Catalonia, Spain.

Although particular attention is paid to influenza A and B virus isolates during influenza surveillance, influenza C virus (FLUCV) coexisted during the first influenza A (H1N1) 2009 pandemic wave during the 2009-2010 season. From 27 April 2009 to 9 May 2010, 12 strains of FLUCV were detected in specimens collected from 1713 nonhospitalized patients with upper respiratory tract illness using a molecular method. Half of the patients with FLUCV infection were older than 14 years. The most frequent symptoms were cough and fever, similar to other viral respiratory infections. Phylogenetic analysis of the hemagglutinin-esterase gene revealed that the strains belonged to the C/Kanagawa/1/76-related and C/Sao Paulo/378/82-related lineages, demonstrating their co-circulation in Catalonia. In addition to regular virological surveillance that provides information about the incidence and the exact role of FLUCV in acute viral respiratory infections in the general population, the genetic lineage identification offers additional data for epidemiological purposes.