There is growing interest in the molecular surveillance of the Respiratory Syncytial Virus and the monitorization of emerging mutations that could impair the efficacy of antiviral prophylaxis and treatments. A simple, scalable protocol for viral nucleic acid enrichment could improve the surveillance of RSV. We developed a protocol for RSV-A and B amplification based on the Illumina CovidSeq workflow using an RSV primer panel. A total of 135 viral genomes were sequenced from nasopharyngeal samples through the optimization steps of this panel, while an additional 15 samples were used to test the final version. Full coverage of the G gene and over 95% of the coverage of the F gene, the target of the available RSV antivirals or monoclonal antibodies, were obtained. The F:K68N mutation, associated with decreased nirsevimab activity, was detected in our facility. Additionally, phylogenetic analysis showed several sublineages in the 2022–2023 influenza season in Europe. Our protocol allows for a simple and scalable simultaneous amplification of the RSV-A and B whole genome, increasing the yield of RSV sequencing and reducing costs. Its application would allow the world to be ready for the detection of arising mutations in relation to the widespread use of nirsevimab for RSV prevention.

To control the SARS-CoV-2 pandemic, healthcare systems have focused on ramping up their capacity for epidemiological surveillance through viral whole genome sequencing. In this paper, we tested the performance of two protocols of SARS-CoV-2 nucleic acid enrichment, an amplicon enrichment using different versions of the ARTIC primer panel and a hybrid-capture method using KAPA RNA Hypercap. We focused on the challenge of the Omicron variant sequencing, the advantages of automated library preparation and the influence of the bioinformatic analysis in the final consensus sequence. All 94 samples were sequenced using Illumina iSeq 100 and analysed with two bioinformatic pipelines: a custom-made pipeline and an Illumina-owned pipeline. We were unsuccessful in sequencing six samples using the capture enrichment due to low reads. On the other hand, amplicon dropout and mispriming caused the loss of mutation G21987A and the erroneous addition of mutation T15521A respectively using amplicon enrichment. Overall, we found high sequence agreement regardless of method of enrichment, bioinformatic pipeline or the use of automation for library preparation in eight different SARS-CoV-2 variants. Automation and the use of a simple app for bioinformatic analysis can simplify the genotyping process, making it available for more diagnostic facilities and increasing global vigilance.

Respiratory Syncytial Virus (RSV) is a leading cause of respiratory infections in infants and older adults. In Spain, surveillance is supported by the SiVIRA system and the RELECOV genomic sequencing network. The 2023–2024 season marked the first nationwide administration of nirsevimab, a monoclonal antibody for preventing severe RSV in infants. This study analyzes the genomic evolution of RSV during this period, focusing on potential escape mutations in the F protein. RSV-A showed high genetic diversity with eleven circulating lineages, while RSV-B was dominated by lineage B.D.E.1. Phylogenetic analysis revealed three distinct B.D.4.1.1 groups, one sharing mutations with B.D.E.1 in the nirsevimab binding site. Despite these changes, neutralization assays confirmed that nirsevimab and other monoclonal antibodies remained effective. No significant antigenic drift compromising immunoprophylaxis was observed. These findings support the continued efficacy of nirsevimab and highlight the importance of genomic surveillance for tracking RSV evolution and informing future immunization strategies.

Background: The development of new large-scale saliva pooling detection strategies can significantly enhance testing capacity and frequency for asymptomatic individuals, which is crucial for containing SARS-CoV-2. Objective: This study aims to implement and scale-up a SARS-CoV-2 screening method using pooled saliva samples to control the virus in critical areas and assess its effectiveness in detecting asymptomatic infections. Methods: Between August 2020 and February 2022, our laboratory received a total of 928,357 samples. Participants collected at least 1 mL of saliva using a self-sampling kit and registered their samples via a smartphone app. All samples were directly processed using AutoMate 2550 for preanalytical steps and then transferred to Microlab STAR, managed with the HAMILTON Pooling software for pooling. The standard pool preset size was 20 samples but was adjusted to 5 when the prevalence exceeded 2% in any group. Real-time polymerase chain reaction (RT-PCR) was conducted using the Allplex SARS-CoV-2 Assay until July 2021, followed by the Allplex SARS-CoV-2 FluA/FluB/RSV assay for the remainder of the study period. Results: Of the 928,357 samples received, 887,926 (95.64%) were fully processed into 56,126 pools. Of these pools, 4863 tested positive, detecting 5720 asymptomatic infections. This allowed for a comprehensive analysis of pooling’s impact on RT-PCR sensitivity and false-negative rate (FNR), including data on positive samples per pool (PPP). We defined Ctref as the minimum cycle threshold (Ct) of each data set from a sample or pool and compared these Ctref results from pooled samples with those of the individual tests (ΔCtP). We then examined their deviation from the expected offset due to dilution [ΔΔCtP = ΔCtP – log2]. In this work, the ΔCtP and ΔΔCtP were 2.23 versus 3.33 and –0.89 versus 0.23, respectively, comparing global results with results for pools with 1 positive sample per pool. Therefore, depending on the number of genes used in the test and the size of the pool, we can evaluate the FNR and effective sensitivity (1 – FNR) of the test configuration. In our scenario, with a maximum of 20 samples per pool and 3 target genes, statistical observations indicated an effective sensitivity exceeding 99%. From an economic perspective, the focus is on pooling efficiency, measured by the effective number of persons that can be tested with 1 test, referred to as persons per test (PPT). In this study, the global PPT was 8.66, reflecting savings of over 20 million euros (US $22 million) based on our reagent prices. Conclusions: Our results demonstrate that, as expected, pooling reduces the sensitivity of RT-PCR. However, with the appropriate pool size and the use of multiple target genes, effective sensitivity can remain above 99%. Saliva pooling may be a valuable tool for screening and surveillance in asymptomatic individuals and can aid in controlling SARS-CoV-2 transmission. Further studies are needed to assess the effectiveness of these strategies for SARS-CoV-2 and their application to other microorganisms or biomarkers detected by PCR.

Objective: To investigate the urethral carriage rate of Haemophilus parainfluenzae isolated in men who have sex with men and to describe the clinical characteristics of carriers. We performed an analysis of the resistance determinants, virulence factors and the genetic phylogeny of the isolates. Methods: Clinical data were obtained from 234 patients attending the infectious diseases unit at the Álvaro Cunqueiro Hospital (Vigo, Spain) between 2021 and 2023. H. parainfluenzae isolated in urethral samples was subjected to antimicrobial susceptibility testing and whole-genome sequencing. Results: The urethral carriage rate was 25%. None of the risk factors studied (received antimicrobial therapy in the previous month, HIV status, Pre-Exposure Prophylaxis (PrEP) use and age) were related to a higher carriage rate. Neither multidrug-resistant (MDR), the presence of a capsule, nor virulence factors were useful to distinguish asymptomatic carriage from acute urethritis (urethral discharge, dysuria, irritation). Nearly half the isolates (48%) met the criteria for MDR. Furthermore, the bla _CTXM-15 gene was found in two isolates that were highly resistant to ceftriaxone (CRO). The other 22 CRO-resistant strains had low minimum inhibitory concentrations and showed alterations in the transpeptidase domain of penicillin-binding protein 3. Moreover, resistance to CRO was high (37%). Most of our urethral strains grouped into one cluster, and this difference was statistically significant when the phylogenetic analysis was performed with samples from different anatomical origins. Conclusions: The increase in H. parainfluenzae strains isolated in the urethra belonging to only one cluster is worrying. Moreover, the isolation of strains increasingly resistant to different groups of antimicrobials highlights the need for close monitoring of the evolution of resistance and the transmission of these resistant strains in the future. Larger studies should be planned to establish the epidemiological relevance of these difficult-to-treat MDR bacteria.