Rapid identification and subsequent contextualization of an outbreak of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit using nanopore sequencing.

Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) are well described in the neonatal intensive care unit (NICU) setting. Genomics has revolutionized the investigation of such outbreaks; however, to date, this has largely been completed retrospectively and has typically relied on short-read platforms. In 2022, our laboratory established a prospective genomic surveillance system using Oxford Nanopore Technologies sequencing for rapid outbreak detection. Herein, using this system, we describe the detection and control of an outbreak of sequence-type (ST)97 MRSA in our NICU. The outbreak was identified 13 days after the first MRSA-positive culture and at a point where there were only two known cases. Ward screening rapidly defined the extent of the outbreak, with six other infants found to be colonized. There was minimal transmission once the outbreak had been detected and appropriate infection control measures had been instituted; only two further ST97 cases were detected, along with three unrelated non-ST97 MRSA cases. To contextualize the outbreak, core-genome single-nucleotide variants were identified for phylogenetic analysis after de novo assembly of nanopore data. Comparisons with global (n=45) and national surveillance (n=35) ST97 genomes revealed the stepwise evolution of methicillin resistance within this ST97 subset. A distinct cluster comprising nine of the ten ST97-IVa genomes from the NICU was identified, with strains from 2020 to 2022 national surveillance serving as outgroups to this cluster. One ST97-IVa genome presumed to be part of the outbreak formed an outgroup and was retrospectively excluded. A second phylogeny was created using Illumina sequencing, which considerably reduced the branch lengths of the NICU isolates on the phylogenetic tree. However, the overall tree topology and conclusions were unchanged, with the exception of the NICU outbreak cluster, where differences in branch lengths were observed. This analysis demonstrated the ability of a nanopore-only prospective genomic surveillance system to rapidly identify and contextualize an outbreak of MRSA in a NICU.

Gut colonization by extended-spectrum ß-lactamase-producing Escherichia coli in dairy herd in Brazil: successful dissemination of a One Health clone.

The overuse of antimicrobials in livestock has contributed to the emergence and selection of clinically relevant multidrug-resistant bacteria. In Brazil, there is no conclusive information on the occurrence of Escherichia coli producing extended-spectrum ß-lactamase (ESßL) in cattle breeding, which is an important sector of agribusiness in this country. Herein, we investigated the presence of ESßL-positive E. coli strains in dairy cattle from a commercial farm with routine practice of therapeutic cephalosporins. Ninety-five rectal swab samples were collected from healthy dairy calves and cows under treatment with ceftiofur. Samples were screened for the presence of ESßL producers, and positive isolates were identified by MALDI-TOF, with subsequent screening for genes encoding ESßL variants by PCR and sequencing. The presence of ESßL (CTX-M-15)-producing E. coli was confirmed in calves, and lactating and dry cows. Most ESßL strains with genetic homologies ≥ 90% were grouped into two major PFGE clusters, confirming the suscessful expansion of clonally related lineages in animals from different lactating cycles, on the same property. Four representatives CTX-M-15-positive E. coli strains had their genomes sequenced, belonging to the clonal complex (CC) 23 and sequence type (ST) 90. A phylogeographical landscape of ST90 was performed revealing a global One Health linkage. Our results highlight the intestinal microbiota of dairy cattle as a hotspot for the spread of critical priority ESßL-producing E. coli and demonstrate that ST90 is an international clone genomically adapted to human and animal hosts, which deserve additional investigation to determine its zoonotic potential and impact in food chain.

Easing genomic surveillance: A comprehensive performance evaluation of long-read assemblers across multi-strain mixture data of HIV-1 and Other pathogenic viruses for constructing a user-friendly bioinformatic pipeline.

Background: Determining the appropriate computational requirements and software performance is essential for efficient genomic surveillance. The lack of standardized benchmarking complicates software selection, especially with limited resources. Methods: We developed a containerized benchmarking pipeline to evaluate seven long-read assemblers-Canu, GoldRush, MetaFlye, Strainline, HaploDMF, iGDA, and RVHaplo-for viral haplotype reconstruction, using both simulated and experimental Oxford Nanopore sequencing data of HIV-1 and other viruses. Benchmarking was conducted on three computational systems to assess each assembler's performance, utilizing QUAST and BLASTN for quality assessment. Results: Our findings show that assembler choice significantly impacts assembly time, with CPU and memory usage having minimal effect. Assembler selection also influences the size of the contigs, with a minimum read length of 2,000 nucleotides required for quality assembly. A 4,000-nucleotide read length improves quality further. Canu was efficient among de novo assemblers but not suitable for multi-strain mixtures, while GoldRush produced only consensus assemblies. Strainline and MetaFlye were suitable for metagenomic sequencing data, with Strainline requiring high memory and MetaFlye operable on low-specification machines. Among reference-based assemblers, iGDA had high error rates, RVHaplo showed the best runtime and accuracy but became ineffective with similar sequences, and HaploDMF, utilizing machine learning, had fewer errors with a slightly longer runtime. Conclusions: The HIV-64148 pipeline, containerized using Docker, facilitates easy deployment and offers flexibility to select from a range of assemblers to match computational systems or study requirements. This tool aids in genome assembly and provides valuable information on HIV-1 sequences, enhancing viral evolution monitoring and understanding.

Avian Influenza outbreaks: Human infection risks for beach users - One health concern and environmental surveillance implications.

Despite its popularity for water activities, such as swimming, surfing, fishing, and rafting, inland and coastal bathing areas occasionally experience outbreaks of highly pathogenic avian influenza virus (HPAI), including A(H5N1) clade 2.3.4.4b. Asymptomatic infections and symptomatic outbreaks often impact many aquatic birds, which increase chances of spill-over events to mammals and pose concerns for public health. This review examined the existing literature to assess avian influenza virus (AIV) transmission risks to beachgoers and the general population. A comprehensive understanding of factors governing such crossing of the AIV host range is currently lacking. There is limited knowledge on key factors affecting risk, such as species-specific interactions with host cells (including binding, entry, and replication via viral proteins hemagglutinin, neuraminidase, nucleoprotein, and polymerase basic protein 2), overcoming host restrictions, and innate immune response. AIV efficiently transmits between birds and to some extent between marine scavenger mammals in aquatic environments via consumption of infected birds. However, the current literature lacks evidence of zoonotic AIV transmission via contact with the aquatic environment or consumption of contaminated water. The zoonotic transmission risk of the circulating A(H5N1) clade 2.3.4.4b virus to the general population and beachgoers is currently low. Nevertheless, it is recommended to avoid direct contact with sick or dead birds and to refrain from bathing in locations where mass bird mortalities are reported. Increasing reports of AIVs spilling over to non-human mammals have raised valid concerns about possible virus mutations that lead to crossing the species barrier and subsequent risk of human infections and outbreaks.

Reemergence of Clade IIb-Associated Mpox, Germany, July-December 2023.

In July 2023, clade IIb-associated mpox reemerged in Germany at low levels, mainly affecting men who have sex with men. We report a representative case and phylogeny of available genome sequences. Our findings underscore the need for standardized surveillance and indication-based vaccination to limit transmission and help prevent endemicity.

Displacement of Hospital-Acquired, Methicillin-Resistant Staphylococcus aureus Clones by Heterogeneous Community Strains in Kenya over a 13-Year Period.

We determined antibiotic susceptibility and employed Oxford Nanopore whole-genome sequencing to explore strain diversity, resistance, and virulence gene carriage among methicillin-resistant Staphylococcus aureus (MRSA) strains from different infection sites and timepoints in a tertiary Kenyan hospital. Ninety-six nonduplicate clinical isolates recovered between 2010 and 2023, identified and tested for antibiotic susceptibility on the VITEK ID/AST platform, were sequenced. Molecular typing, antibiotic resistance, and virulence determinant screening were performed using the relevant bioinformatics tools. The strains, alongside those from previous studies, were stratified into two periods covering 2010-2017 and 2018-2023 and comparisons were made. Mirroring phenotypic profiles, aac(6')-aph(2″) [aminoglycosides]; gyrA (S84L) and grlA (S80Y) [fluoroquinolones]; dfrG [anti-folates]; and tet(K) [tetracycline] resistance determinants dominated the collection. While the proportion of ST239/241-t037-SCCmec III among MRSA reduced from 37.7% to 0% over the investigated period, ST4803-t1476-SCCmec IV and ST152-t355-SCCmec IV were pre-eminent. The prevalence of Panton-Valentine leucocidin (PVL) and arginine catabolic mobile element (ACME) genes was 38% (33/87) and 6.8% (6/87), respectively. We observed the displacement of HA-MRSA ST239/241-t037-SCCmec III with the emergence of ST152-t355-SCCmec IV and a greater clonal heterogeneity. The occurrence of PVL+/ACME+ CA-MRSA in recent years warrants further investigations into their role in the CA-MRSA virulence landscape, in a setting of high PVL prevalence.

Detection and Characterisation of SARS-CoV-2 in Eastern Province of Zambia: A Retrospective Genomic Surveillance Study.

Mutations have driven the evolution and development of new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with potential implications for increased transmissibility, disease severity and vaccine escape among others. Genome sequencing is a technique that allows scientists to read the genetic code of an organism and has become a powerful tool for studying emerging infectious diseases. Here, we conducted a cross-sectional study in selected districts of the Eastern Province of Zambia, from November 2021 to February 2022. We analyzed SARS-CoV-2 samples (n = 76) using high-throughput sequencing. A total of 4097 mutations were identified in 69 SARS-CoV-2 genomes with 47% (1925/4097) of the mutations occurring in the spike protein. We identified 83 unique amino acid mutations in the spike protein of the seven Omicron sublineages (BA.1, BA.1.1, BA.1.14, BA.1.18, BA.1.21, BA.2, BA.2.23 and XT). Of these, 43.4% (36/83) were present in the receptor binding domain, while 14.5% (12/83) were in the receptor binding motif. While we identified a potential recombinant XT strain, the highly transmissible BA.2 sublineage was more predominant (40.8%). We observed the substitution of other variants with the Omicron strain in the Eastern Province. This work shows the importance of pandemic preparedness and the need to monitor disease in the general population.

Accurate Recapitulation of Chikungunya Virus Complete Coding Sequence Phylogeny Using Variable Genome Regions for Genomic Surveillance.

Chikungunya virus (CHIKV) is transmitted by mosquito bites and causes chikungunya fever (CHIKF). CHIKV has a single-stranded RNA genome and belongs to a single serotype with three genotypes. The Asian lineage has recently emerged in the Western Hemisphere, likely due to travel-associated introduction. Genetic variation accumulates in the CHIKV genome as the virus replicates, creating new lineages. Whole genome sequencing is ideal for studying virus evolution and spread but is expensive and complex. This study investigated whether specific, highly variable regions of the CHIKV genome could recapitulate the phylogeny obtained with a complete coding sequence (CDS). Our results revealed that concatenated highly variable regions accurately reconstructed CHIKV phylogeny, exhibiting statistically indistinguishable branch lengths and tree confidence compared to CDS. In addition, these regions adequately inferred the evolutionary relationships among CHIKV isolates from the American outbreak with similar results to the CDS. This finding suggests that highly variable regions can effectively capture the evolutionary relationships among CHIKV isolates, offering a simpler approach for future studies. This approach could be particularly valuable for large-scale surveillance efforts.

Genomic surveillance of SARS-CoV-2 evolution by a centralised pipeline and weekly focused sequencing, Austria, January 2021 to March 2023.

BackgroundThe COVID-19 pandemic was largely driven by genetic mutations of SARS-CoV-2, leading in some instances to enhanced infectiousness of the virus or its capacity to evade the host immune system. To closely monitor SARS-CoV-2 evolution and resulting variants at genomic-level, an innovative pipeline termed SARSeq was developed in Austria.AimWe discuss technical aspects of the SARSeq pipeline, describe its performance and present noteworthy results it enabled during the pandemic in Austria.MethodsThe SARSeq pipeline was set up as a collaboration between private and public clinical diagnostic laboratories, a public health agency, and an academic institution. Representative SARS-CoV-2 positive specimens from each of the nine Austrian provinces were obtained from SARS-CoV-2 testing laboratories and processed centrally in an academic setting for S-gene sequencing and analysis.ResultsSARS-CoV-2 sequences from up to 2,880 cases weekly resulted in 222,784 characterised case samples in January 2021-March 2023. Consequently, Austria delivered the fourth densest genomic surveillance worldwide in a very resource-efficient manner. While most SARS-CoV-2 variants during the study showed comparable kinetic behaviour in all of Austria, some, like Beta, had a more focused spread. This highlighted multifaceted aspects of local population-level acquired immunity. The nationwide surveillance system enabled reliable nowcasting. Measured early growth kinetics of variants were predictive of later incidence peaks.ConclusionWith low automation, labour, and cost requirements, SARSeq is adaptable to monitor other pathogens and advantageous even for resource-limited countries. This multiplexed genomic surveillance system has potential as a rapid response tool for future emerging threats.

Genomic surveillance during the first two years of the COVID-19 pandemic - country experience and lessons learned from Türkiye.

Background: Türkiye confirmed its first case of SARS-CoV-2 on March 11, 2020, coinciding with the declaration of the global COVID-19 pandemic. Subsequently, Türkiye swiftly increased testing capacity and implemented genomic sequencing in 2020. This paper describes Türkiye's journey of establishing genomic surveillance as a middle-income country with limited prior sequencing capacity and analyses sequencing data from the first two years of the pandemic. We highlight the achievements and challenges experienced and distill globally relevant lessons. Methods: We tracked the evolution of the COVID-19 pandemic in Türkiye from December 2020 to February 2022 through a timeline and analysed epidemiological, vaccination, and testing data. To investigate the phylodynamic and phylogeographic aspects of SARS-CoV-2, we used Nextstrain to analyze 31,629 high-quality genomes sampled from seven regions nationwide. Results: Türkiye's epidemiological curve, mirroring global trends, featured four distinct waves, each coinciding with the emergence and spread of variants of concern (VOCs). Utilizing locally manufactured kits to expand testing capacity and introducing variant-specific quantitative reverse transcription polymerase chain reaction (RT-qPCR) tests developed in partnership with a private company was a strategic advantage in Türkiye, given the scarcity and fragmented global supply chain early in the pandemic. Türkiye contributed more than 86,000 genomic sequences to global databases by February 2022, ensuring that Turkish data was reflected globally. The synergy of variant-specific RT-qPCR kits and genomic sequencing enabled cost-effective monitoring of VOCs. However, data analysis was constrained by a weak sequencing sampling strategy and fragmented data management systems, limiting the application of sequencing data to guide the public health response. Phylodynamic analysis indicated that Türkiye's geographical position as an international travel hub influenced both national and global transmission of each VOC despite travel restrictions. Conclusion: This paper provides valuable insights into the testing and genomic surveillance systems adopted by Türkiye during the COVID-19 pandemic, proposing important lessons for countries developing national systems. The findings underscore the need for robust testing and sampling strategies, streamlined sample referral, and integrated data management with metadata linkage and data quality crucial for impactful epidemiological analysis. We recommend developing national genomic surveillance strategies to guide sustainable and integrated expansion of capacities built for COVID-19 and to optimize the effective utilization of sequencing data for public health action.

Mathematical modeling of SARS-CoV-2 variant substitutions in European countries: transmission dynamics and epidemiological insights.

Background: Countries across Europe have faced similar evolutions of SARS-CoV-2 variants of concern, including the Alpha, Delta, and Omicron variants. Materials and methods: We used data from GISAID and applied a robust, automated mathematical substitution model to study the dynamics of COVID-19 variants in Europe over a period of more than 2 years, from late 2020 to early 2023. This model identifies variant substitution patterns and distinguishes between residual and dominant behavior. We used weekly sequencing data from 19 European countries to estimate the increase in transmissibility ( Δ ß ) between consecutive SARS-CoV-2 variants. In addition, we focused on large countries with separate regional outbreaks and complex scenarios of multiple competing variants. Results: Our model accurately reproduced the observed substitution patterns between the Alpha, Delta, and Omicron major variants. We estimated the daily variant prevalence and calculated Δ ß between variants, revealing that: ( i ) Δ ß increased progressively from the Alpha to the Omicron variant; ( i i ) Δ ß showed a high degree of variability within Omicron variants; ( i i i ) a higher Δ ß was associated with a later emergence of the variant within a country; ( i v ) a higher degree of immunization of the population against previous variants was associated with a higher Δ ß for the Delta variant; ( v ) larger countries exhibited smaller Δ ß , suggesting regionally diverse outbreaks within the same country; and finally ( v i ) the model reliably captures the dynamics of competing variants, even in complex scenarios. Conclusion: The use of mathematical models allows for precise and reliable estimation of daily cases of each variant. By quantifying Δ ß , we have tracked the spread of the different variants across Europe, highlighting a robust increase in transmissibility trend from Alpha to Omicron. Additionally, we have shown that the geographical characteristics of a country, as well as the timing of new variant entrances, can explain some of the observed differences in variant substitution dynamics across countries.

Estimation of SARS-CoV-2 fitness gains from genomic surveillance data without prior lineage classification.

The emergence of SARS-CoV-2 variants with increased fitness has had a strong impact on the epidemiology of COVID-19, with the higher effective reproduction number of the viral variants leading to new epidemic waves. Tracking such variants and their genetic signatures, using data collected through genomic surveillance, is therefore crucial for forecasting likely surges in incidence. Current methods of estimating fitness advantages of variants rely on tracking the changing proportion of a particular lineage over time, but describing successful lineages in a rapidly evolving viral population is a difficult task. We propose a method of estimating fitness gains directly from nucleotide information generated by genomic surveillance, without a priori assigning isolates to lineages from phylogenies, based solely on the abundance of single nucleotide polymorphisms (SNPs). The method is based on mapping changes in the genetic population structure over time. Changes in the abundance of SNPs associated with periods of increasing fitness allow for the unbiased discovery of new variants, thereby obviating a deliberate lineage assignment and phylogenetic inference. We conclude that the method provides a fast and reliable way to estimate fitness advantages of variants without the need for a priori assigning isolates to lineages.

Genomic surveillance of SARS-CoV-2 and emergence of XBB.1.16 variant in Rajasthan.

PURPOSE: Genomic surveillance of positive SARS-CoV-2 samples is important to monitor the genetic changes occurring in virus, this was enhanced after the WHO designation of XBB.1.16 as a variant under monitoring in March 2023. From 5th February till May 6, 2023 all positive SARS-CoV-2 samples were monitored for genetic changes. METHODS: A total of 1757 samples having Ct value <25 (for E and ORF gene) from different districts of Rajasthan were processed for Next Generation Sequencing (NGS). The FASTA files obtained on sequencing were used for lineage determination using Nextclade and phylogenetic tree construction. RESULTS AND CONCLUSIONS: Sequencing and lineage identification was done in 1624 samples. XBB.1.16 was the predominant lineage in 1413 (87.0%) cases while rest was other XBB (207, 12.74%) and other lineages (4, 0.2%). Of the 1413 XBB.1.16 cases, 57.47% were males and 42.53% were females. Majority (66.53%) belonged to 19-59 year age. 84.15% of XBB.1.16 cases were infected for the first time. Hospitalization was required in only 2.2% cases and death was reported in 5 (0.35%) patients. Most of the cases were symptomatic and the commonest symptoms were fever, cough and rhinorrhea. Co-morbidities were present in 414 (29.3%) cases. Enhanced genomic surveillance helped to rapidly identify the spread of XBB variant in Rajasthan. This in turn helped to take control measures to prevent spread of virus and estimate public health risks of the new variant relative to the previously circulating lineages. XBB variant was found to spread rapidly but produced milder disease.

Genomic Surveillance for SARS-CoV-2 Variants: Dominance of XBB Replacement - China, January-June 2023.

Introduction: In the first half of 2023, a global shift was observed towards the predominance of XBB variants. China faced a significant epidemic between late 2022 and early 2023 due to Omicron subvariants BA.5.2 and BF.7. This study aims to depict the evolving variant distribution among provincial-level administrative divisions (PLADs) in China and explore the factors driving the predominance of XBB replacement. Methods: Sequences from local and imported coronavirus disease 2019 (COVID-19) cases recorded between January 1 and June 30, 2023, were included. The study analyzed the changing distribution of viral variants and assessed how the prior dominance of specific variants, XBB subvariants, and imported cases influenced the prevalence of the XBB replacement variant. Results: A total of 56,486 sequences were obtained from local cases, and 8,669 sequences were from imported cases. Starting in April, there was a shift in the prevalence of XBB from imported to local cases, with varying dominance among PLADs. In PLADs previously high in BF.7, the rise of XBB was delayed. A positive correlation was found between XBB proportions in imported cases from January to March and local cases in April. The distribution pattern of XBB subvariants differed between local and imported cases within the same PLAD. No significant differences were noted in the replacement rates of XBB subvariants. Conclusions: The timing of XBB dominance differed among various PLADs in China in the first half of 2023, correlating closely with the prevalence of XBB variants among imported cases.

DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus.

BACKGROUND: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. RESULTS: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/µL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. CONCLUSIONS: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

SARS-CoV-2 wastewater variant surveillance: pandemic response leveraging FDA's GenomeTrakr network.

Wastewater surveillance has emerged as a crucial public health tool for population-level pathogen surveillance. Supported by funding from the American Rescue Plan Act of 2021, the FDA's genomic epidemiology program, GenomeTrakr, was leveraged to sequence SARS-CoV-2 from wastewater sites across the United States. This initiative required the evaluation, optimization, development, and publication of new methods and analytical tools spanning sample collection through variant analyses. Version-controlled protocols for each step of the process were developed and published on protocols.io. A custom data analysis tool and a publicly accessible dashboard were built to facilitate real-time visualization of the collected data, focusing on the relative abundance of SARS-CoV-2 variants and sub-lineages across different samples and sites throughout the project. From September 2021 through June 2023, a total of 3,389 wastewater samples were collected, with 2,517 undergoing sequencing and submission to NCBI under the umbrella BioProject, PRJNA757291. Sequence data were released with explicit quality control (QC) tags on all sequence records, communicating our confidence in the quality of data. Variant analysis revealed wide circulation of Delta in the fall of 2021 and captured the sweep of Omicron and subsequent diversification of this lineage through the end of the sampling period. This project successfully achieved two important goals for the FDA's GenomeTrakr program: first, contributing timely genomic data for the SARS-CoV-2 pandemic response, and second, establishing both capacity and best practices for culture-independent, population-level environmental surveillance for other pathogens of interest to the FDA. IMPORTANCE: This paper serves two primary objectives. First, it summarizes the genomic and contextual data collected during a Covid-19 pandemic response project, which utilized the FDA's laboratory network, traditionally employed for sequencing foodborne pathogens, for sequencing SARS-CoV-2 from wastewater samples. Second, it outlines best practices for gathering and organizing population-level next generation sequencing (NGS) data collected for culture-free, surveillance of pathogens sourced from environmental samples.

Presence of phage-plasmids in multiple serovars of Salmonella enterica.

Evidence is accumulating in the literature that the horizontal spread of antimicrobial resistance (AMR) genes mediated by bacteriophages and bacteriophage-like plasmid (phage-plasmid) elements is much more common than previously envisioned. For instance, we recently identified and characterized a circular P1-like phage-plasmid harbouring a bla CTX-M-15 gene conferring extended-spectrum beta-lactamase (ESBL) resistance in Salmonella enterica serovar Typhi. As the prevalence and epidemiological relevance of such mechanisms has never been systematically assessed in Enterobacterales, in this study we carried out a follow-up retrospective analysis of UK Salmonella isolates previously sequenced as part of routine surveillance protocols between 2016 and 2021. Using a high-throughput bioinformatics pipeline we screened 47 784 isolates for the presence of the P1 lytic replication gene repL, identifying 226 positive isolates from 25 serovars and demonstrating that phage-plasmid elements are more frequent than previously thought. The affinity for phage-plasmids appears highly serovar-dependent, with several serovars being more likely hosts than others; most of the positive isolates (170/226) belonged to S. Typhimurium ST34 and ST19. The phage-plasmids ranged between 85.8 and 98.2 kb in size, with an average length of 92.1 kb; detailed analysis indicated a high amount of diversity in gene content and genomic architecture. In total, 132 phage-plasmids had the p0111 plasmid replication type, and 94 the IncY type; phylogenetic analysis indicated that both horizontal and vertical gene transmission mechanisms are likely to be involved in phage-plasmid propagation. Finally, phage-plasmids were present in isolates that were resistant and non-resistant to antimicrobials. In addition to providing a first comprehensive view of the presence of phage-plasmids in Salmonella, our work highlights the need for a better surveillance and understanding of phage-plasmids as AMR carriers, especially through their characterization with long-read sequencing.

Investing in health preparedness, response and resilience: a genomics costing tool focused on next generation sequencing.

The world has seen unprecedented gains in the global genomic surveillance capacities for pathogens with pandemic and epidemic potential within the last 4 years. To strengthen and sustain the gains made, WHO is working with countries and partners to implement the Global Genomic Surveillance Strategy for Pathogens with Pandemic and Epidemic Potential 2022-2032. A key technical product developed through these multi-agency collaborative efforts is a genomics costing tool (GCT), as sought by many countries. This tool was developed by five institutions - Association of Public Health Laboratories, FIND, The Global Fund to Fight AIDS, Tuberculosis and Malaria, UK Health Security Agency, and the World Health Organization. These institutions developed the GCT to support financial planning and budgeting for SARS-CoV-2 next-generation sequencing activities, including bioinformatic analysis. The tool costs infrastructure, consumables and reagents, human resources, facility and quality management. It is being used by countries to (1) obtain costs of routine sequencing and bioinformatics activities, (2) optimize available resources, and (3) build an investment case for the scale-up or establishment of sequencing and bioinformatics activities. The tool has been validated and is available in English and Russian at https://www.who.int/publications/i/item/9789240090866. This paper aims to highlight the rationale for developing the tool, describe the process of the collaborative effort in developing the tool, and describe the utility of the tool to countries.

Investigating the cause of a 2021 winter wave of COVID-19 in a border region in eastern Germany: a mixed-methods study, August to November 2021.

It is so far unclear how the COVID-19 winter waves started and what should be done to prevent possible future waves. In this study, we deciphered the dynamic course of a winter wave in 2021 in Saxony, a state in Eastern Germany neighbouring the Czech Republic and Poland. The study was carried out through the integration of multiple virus genomic epidemiology approaches to track transmission chains, identify emerging variants and investigate dynamic changes in transmission clusters. For identified local variants of interest, functional evaluations were performed. Multiple long-lasting community transmission clusters have been identified acting as driving force for the winter wave 2021. Analysis of the dynamic courses of two representative clusters indicated a similar transmission pattern. However, the transmission cluster caused by a locally occurring new Delta variant AY.36.1 showed a distinct transmission pattern, and functional analyses revealed a replication advantage of it. This study indicated that long-lasting community transmission clusters starting since early autumn caused by imported or locally occurring variants all contributed to the development of the 2021 winter wave. The information we achieved might help future pandemic prevention.

Influenza Virus Genomic Surveillance, Arizona, USA, 2023-2024.

Influenza viruses are constantly evolving and are therefore monitored worldwide in the hope to reduce the burden of disease by annual updates to vaccine recommendations. We conducted genomic sequencing of 110 influenza A and 30 influenza B viruses from specimens collected between October 2023 and February 2024 in Arizona, USA. We identified mutations in the hemagglutinin (HA) antigenic sites as well as the neuraminidase (NA) gene in our samples. We also found no unique HA and NA mutations in vaccinated yet influenza-infected individuals. Real-time genomic sequencing surveillance is important to ensure influenza vaccine effectiveness.