EU surveys insights: analytical tools, future directions, and the essential requirement for reference materials in wastewater monitoring of SARS-CoV-2, antimicrobial resistance and beyond.

BACKGROUND: Wastewater surveillance (WWS) acts as a vigilant sentinel system for communities, analysing sewage to protect public health by detecting outbreaks and monitoring trends in pathogens and contaminants. To achieve a thorough comprehension of present and upcoming practices and to identify challenges and opportunities for standardisation and improvement in WWS methodologies, two EU surveys were conducted targeting over 750 WWS laboratories across Europe and other regions. The first survey explored a diverse range of activities currently undertaken or planned by laboratories. The second survey specifically targeted methods and quality controls utilised for SARS-CoV-2 surveillance. RESULTS: The findings of the two surveys provide a comprehensive insight into the procedures and methodologies applied in WWS. In Europe, WWS primarily focuses on SARS-CoV-2 with 99% of the survey participants dedicated to this virus. However, the responses highlighted a lack of standardisation in the methodologies employed for monitoring SARS-CoV-2. The surveillance of other pathogens, including antimicrobial resistance, is currently fragmented and conducted by only a limited number of laboratories. Notably, these activities are anticipated to expand in the future. Survey replies emphasise the collective recognition of the need to enhance the accuracy of results in WWS practices, reflecting a shared commitment to advancing precision and effectiveness in WWS methodologies. CONCLUSIONS: These surveys identified a lack of standardised common procedures in WWS practices and the need for quality standards and reference materials to enhance the accuracy and reliability of WWS methods in the future. In addition, it is important to broaden surveillance efforts beyond SARS-CoV-2 to include other emerging pathogens and antimicrobial resistance to ensure a comprehensive approach to protecting public health.

In Silico Analysis of the Effects of Omicron Spike Amino Acid Changes on the Interactions with Human Proteins.

The SARS-CoV-2 variant Omicron is characterized, among others, by more than 30 amino acid changes occurring on the spike glycoprotein with respect to the original SARS-CoV-2 spike protein. We report a comprehensive analysis of the effects of the Omicron spike amino acid changes in the interaction with human antibodies, obtained by modeling them into selected publicly available resolved 3D structures of spike-antibody complexes and investigating the effects of these mutations at structural level. We predict that the interactions of Omicron spike with human antibodies can be either negatively or positively affected by amino acid changes, with a predicted total loss of interactions only in a few complexes. Moreover, our analysis applied also to the spike-ACE2 interaction predicts that these amino acid changes may increase Omicron transmissibility. Our approach can be used to better understand SARS-CoV-2 transmissibility, detectability, and epidemiology and represents a model to be adopted also in the case of other variants.

SARS-CoV-2: Reinfection after 18 Months of a Previous Case with Multiple Negative Nasopharyngeal Swab Tests and Positive Fecal Molecular Test.

This short communication describes the reinfection after nearly 18 months of the same patient who was previously infected with coronavirus disease 2019 (COVID-19) and who showed multiple negative real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) results by nasal swabs for severe acute respiratory syndrome coronavirus (SARS-CoV-2) but positive results on a fecal sample. We previously noted how, in the presence of symptoms suggestive of pneumonia, visible on a chest computed tomography (CT) scan and confirmed by fecal molecular testing, it was possible to draw the diagnosis of SARS-CoV-2 infection. One year later, the same patient was again affected by SARS-CoV-2. This time, the first antigenic nasal swab showed readily positive results. However, the patient's clinical course appeared to be more attenuated, showing no signs of pulmonary involvement in the radiographic examinations performed. This case shows a novelty in the pulmonary radiological evaluation of new SARS-CoV-2 infection.

SARS-CoV-2 Detection in Fecal Sample from a Patient with Typical Findings of COVID-19 Pneumonia on CT but Negative to Multiple SARS-CoV-2 RT-PCR Tests on Oropharyngeal and Nasopharyngeal Swab Samples.

Reverse transcriptase polymerase chain reaction (RT-PCR) negative results in the upper respiratory tract represent a major concern for the clinical management of coronavirus disease 2019 (COVID-19) patients. Herein, we report the case of a 43-years-old man with a strong clinical suspicion of COVID-19, who resulted in being negative to multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RT-PCR tests performed on different oropharyngeal and nasopharyngeal swabs, despite serology having confirmed the presence of SARS-CoV-2 IgM. The patient underwent a chest computed tomography (CT) that showed typical imaging findings of COVID-19 pneumonia. The presence of viral SARS-CoV-2 was confirmed only by performing a SARS-CoV-2 RT-PCR test on stool. Performing of SARS-CoV-2 RT-PCR test on fecal samples can be a rapid and useful approach to confirm COVID-19 diagnosis in cases where there is an apparent discrepancy between COVID-19 clinical symptoms coupled with chest CT and SARS-CoV-2 RT-PCR tests' results on samples from the upper respiratory tract.

DUGMO: tool for the detection of unknown genetically modified organisms with high-throughput sequencing data for pure bacterial samples.

BACKGROUND: The European Community has adopted very restrictive policies regarding the dissemination and use of genetically modified organisms (GMOs). In fact, a maximum threshold of 0.9% of contaminating GMOs is tolerated for a "GMO-free" label. In recent years, imports of undescribed GMOs have been detected. Their sequences are not described and therefore not detectable by conventional approaches, such as PCR. RESULTS: We developed DUGMO, a bioinformatics pipeline for the detection of genetically modified (GM) bacteria, including unknown GM bacteria, based on Illumina paired-end sequencing data. The method is currently focused on the detection of GM bacteria with - possibly partial - transgenes in pure bacterial samples. In the preliminary steps, coding sequences (CDSs) are aligned through two successive BLASTN against the host pangenome with relevant tuned parameters to discriminate CDSs belonging to the wild type genome (wgCDS) from potential GM coding sequences (pgmCDSs). Then, Bray-Curtis distances are calculated between the wgCDS and each pgmCDS, based on the difference of genomic vocabulary. Finally, two machine learning methods, namely the Random Forest and Generalized Linear Model, are carried out to target true GM CDS(s), based on six variables including Bray-Curtis distances and GC content. Tests carried out on a GM Bacillus subtilis showed 25 positive CDSs corresponding to the chloramphenicol resistance gene and CDSs of the inserted plasmids. On a wild type B. subtilis, no false positive sequences were detected. CONCLUSION: DUGMO detects exogenous CDS, truncated, fused or highly mutated wild CDSs in high-throughput sequencing data, and was shown to be efficient at detecting GM sequences, but it might also be employed for the identification of recent horizontal gene transfers.

Novel nuclear barcode regions for the identification of flatfish species.

The development of an efficient seafood traceability framework is crucial for the management of sustainable fisheries and the monitoring of potential substitution fraud across the food chain. Recent studies have shown the potential of DNA barcoding methods in this framework, with most of the efforts focusing on using mitochondrial targets such as the cytochrome oxidase 1 and cytochrome b genes. In this article, we show the identification of novel targets in the nuclear genome, and their associated primers, to be used for the efficient identification of flatfishes of the Pleuronectidae family. In addition, different in silico methods are described to generate a dataset of barcode reference sequences from the ever-growing wealth of publicly available sequence information, replacing, where possible, labour-intensive laboratory work. The short amplicon lengths render the analysis of these new barcode target regions ideally suited to next-generation sequencing techniques, allowing characterisation of multiple fish species in mixed and processed samples. Their location in the nucleus also improves currently used methods by allowing the identification of hybrid individuals.

JRC GMO-Matrix: a web application to support Genetically Modified Organisms detection strategies.

BACKGROUND: The polymerase chain reaction (PCR) is the current state of the art technique for DNA-based detection of Genetically Modified Organisms (GMOs). A typical control strategy starts by analyzing a sample for the presence of target sequences (GM-elements) known to be present in many GMOs. Positive findings from this "screening" are then confirmed with GM (event) specific test methods. A reliable knowledge of which GMOs are detected by combinations of GM-detection methods is thus crucial to minimize the verification efforts. DESCRIPTION: In this article, we describe a novel platform that links the information of two unique databases built and maintained by the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) at the Joint Research Centre (JRC) of the European Commission, one containing the sequence information of known GM-events and the other validated PCR-based detection and identification methods. The new platform compiles in silico determinations of the detection of a wide range of GMOs by the available detection methods using existing scripts that simulate PCR amplification and, when present, probe binding. The correctness of the information has been verified by comparing the in silico conclusions to experimental results for a subset of forty-nine GM events and six methods. CONCLUSIONS: The JRC GMO-Matrix is unique for its reliance on DNA sequence data and its flexibility in integrating novel GMOs and new detection methods. Users can mine the database using a set of web interfaces that thus provide a valuable support to GMO control laboratories in planning and evaluating their GMO screening strategies. The platform is accessible at http://gmo-crl.jrc.ec.europa.eu/jrcgmomatrix/ .

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern.

BACKGROUND: Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome. RESULTS: PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y's phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism. CONCLUSIONS: In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants.

Multiple lineages of SARS-CoV-2 have been identified featuring distinct sets of genetic changes that confer to the virus higher transmissibility and ability to evade existing immunity. The continuous evolution of SARS-CoV-2 may pose challenges for current treatment options and diagnostic tools. In this study, we have first evaluated the performance of the 14 WHO-recommended real-time reverse transcription (RT)-PCR assays currently in use for the detection of SARS-CoV-2 and found that only one assay has reduced performance against Omicron. We then developed a new duplex real-time RT-PCR assay based on the amplification of two ultra-conserved elements present within the SARS-CoV-2 genome. The new duplex assay successfully detects all of the tested SARS-CoV-2 variants of concern (including Omicron sub-lineages BA.4 and BA.5) from both clinical and wastewater samples with high sensitivity and specificity. The assay also functions as a one-step droplet digital RT-PCR assay. This new assay, in addition to clinical testing, could be adopted in surveillance programs for the routine monitoring of SARS-CoV-2's presence in a population in wastewater samples. Positive results with our assay in conjunction with negative results from an Omicron-specific assay may provide timely indication of the emergence of a novel SARS-CoV-2 variant in a certain community and thereby aid public health interventions.

Study of antibiotic resistance in freshwater ecosystems with low anthropogenic impact.

This study aimed to investigate the bacterial diversity and the background level of antibiotic resistance in two freshwater ecosystems with low anthropogenic impact in order to evaluate the presence of natural antimicrobial resistance in these areas and its potential to spread downstream. Water samples from a pre-Alpine and an Apennine river (Variola and Tiber, respectively) were collected in three different sampling campaigns and bacterial diversity was assessed by 16S sequencing, while the presence of bacteria resistant to five antibiotics was screened using a culturable approach. Overall bacterial load was higher in the Tiber River compared with the Variola River. Furthermore, the study revealed the presence of resistant bacteria, especially the Tiber River showed, for each sampling, the presence of resistance to all antibiotics tested, while for the Variola River, the detected resistance was variable, comprising two or more antibiotics. Screening of two resistance genes on a total of one hundred eighteen bacterial isolates from the two rivers showed that blaTEM, conferring resistance to ß-lactam antibiotics, was dominant and present in ~58 % of isolates compared to only ~9 % for mefA/E conferring resistance to macrolides. Moreover, ß-lactam resistance was detected in various isolates showing also resistance to additional antibiotics such as macrolides, aminoglycosides and tetracyclines. These observations would suggest the presence of co-resistant bacteria even in non-anthropogenic environments and this resistance may spread from the environment to humans and/or animals.

Mechanisms Leading to Gut Dysbiosis in COVID-19: Current Evidence and Uncertainties Based on Adverse Outcome Pathways.

Alteration in gut microbiota has been associated with COVID-19. However, the underlying mechanisms remain poorly understood. Here, we outlined three potential interconnected mechanistic pathways leading to gut dysbiosis as an adverse outcome following SARS-CoV-2 presence in the gastrointestinal tract. Evidence from the literature and current uncertainties are reported for each step of the different pathways. One pathway investigates evidence that intestinal infection by SARS-CoV-2 inducing intestinal inflammation alters the gut microbiota. Another pathway links the binding of viral S protein to angiotensin-converting enzyme 2 (ACE2) to the dysregulation of this receptor, essential in intestinal homeostasis-notably for amino acid metabolism-leading to gut dysbiosis. Additionally, SARS-CoV-2 could induce gut dysbiosis by infecting intestinal bacteria. Assessing current evidence within the Adverse Outcome Pathway framework justifies confidence in the proposed mechanisms to support disease management and permits the identification of inconsistencies and knowledge gaps to orient further research.

A qualitative RT-PCR assay for the specific identification of the SARS-CoV-2 B.1.1.529 (Omicron) Variant of Concern.

OBJECTIVES: The aim of this study was to develop a RT-PCR assay for the specific detection of the SARS-CoV-2 Omicron Variant of Concern (VOC) as a rapid alternative to sequencing. METHODS: A RT-PCR was designed in silico and then validated using characterised clinical samples containing Omicron (both BA.1 and BA.2 lineages) and the Omicron synthetic RNA genome. As negative controls, SARS-CoV-2 positive clinical samples collected in May 2020, and synthetic RNA genomes of the isolate Wuhan Hu-1 and of the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Iota (B.1.526), Epsilon (B.1.429) and Delta (B.1.617.2) SARS-CoV-2 VOC were used. RESULTS: Experiments performed using as templates the synthetic RNA genomes demonstrate the high specificity of the PCR-method for the SARS-CoV-2 Omicron. Despite the synthetic RNAs were used at high copy numbers, specific signal was mainly detected with the Omicron synthetic genome. Only a non-specific late signal was detected using the Alpha variant genome, but these results were considered negligible as Alpha VOC has been replaced by the Delta and it is not circulating anymore in the world. Using our method, we confirmed the presence of Omicron on clinical samples containing this variant but not of other SARS-CoV-2 lineages. The method is highly sensitive and can detect up to 1 cp of the Omicron virus per µl. CONCLUSIONS: The method presented here, in combination with other methods in use for detection of SARS-CoV-2, can be used for an early identification of Omicron.

Gut as an Alternative Entry Route for SARS-CoV-2: Current Evidence and Uncertainties of Productive Enteric Infection in COVID-19.

The gut has been proposed as a potential alternative entry route for SARS-CoV-2. This was mainly based on the high levels of SARS-CoV-2 receptor expressed in the gastrointestinal (GI) tract, the observations of GI disorders (such as diarrhea) in some COVID-19 patients and the detection of SARS-CoV-2 RNA in feces. However, the underlying mechanisms remain poorly understood. It has been proposed that SARS-CoV-2 can productively infect enterocytes, damaging the intestinal barrier and contributing to inflammatory response, which might lead to GI manifestations, including diarrhea. Here, we report a methodological approach to assess the evidence supporting the sequence of events driving SARS-CoV-2 enteric infection up to gut adverse outcomes. Exploring evidence permits to highlight knowledge gaps and current inconsistencies in the literature and to guide further research. Based on the current insights on SARS-CoV-2 intestinal infection and transmission, we then discuss the potential implication on clinical practice, including on long COVID. A better understanding of the GI implication in COVID-19 is still needed to improve disease management and could help identify innovative therapies or preventive actions targeting the GI tract.

Effects of spike protein and toxin-like peptides found in COVID-19 patients on human 3D neuronal/glial model undergoing differentiation: Possible implications for SARS-CoV-2 impact on brain development.

The possible neurodevelopmental consequences of SARS-CoV-2 infection are presently unknown. In utero exposure to SARS-CoV-2 has been hypothesized to affect the developing brain, possibly disrupting neurodevelopment of children. Spike protein interactors, such as ACE2, have been found expressed in the fetal brain, and could play a role in potential SARS-CoV-2 fetal brain pathogenesis. Apart from the possible direct involvement of SARS-CoV-2 or its specific viral components in the occurrence of neurological and neurodevelopmental manifestations, we recently reported the presence of toxin-like peptides in plasma, urine and fecal samples specifically from COVID-19 patients. In this study, we investigated the possible neurotoxic effects elicited upon 72-hour exposure to human relevant levels of recombinant spike protein, toxin-like peptides found in COVID-19 patients, as well as a combination of both in 3D human iPSC-derived neural stem cells differentiated for either 2 weeks (short-term) or 8 weeks (long-term, 2 weeks in suspension + 6 weeks on MEA) towards neurons/glia. Whole transcriptome and qPCR analysis revealed that spike protein and toxin-like peptides at non-cytotoxic concentrations differentially perturb the expression of SPHK1, ELN, GASK1B, HEY1, UTS2, ACE2 and some neuronal-, glia- and NSC-related genes critical during brain development. Additionally, exposure to spike protein caused a decrease of spontaneous electrical activity after two days in long-term differentiated cultures. The perturbations of these neurodevelopmental endpoints are discussed in the context of recent knowledge about the key events described in Adverse Outcome Pathways relevant to COVID-19, gathered in the context of the CIAO project (https://www.ciao-covid.net/).

Factors Modulating COVID-19: A Mechanistic Understanding Based on the Adverse Outcome Pathway Framework.

Addressing factors modulating COVID-19 is crucial since abundant clinical evidence shows that outcomes are markedly heterogeneous between patients. This requires identifying the factors and understanding how they mechanistically influence COVID-19. Here, we describe how eleven selected factors (age, sex, genetic factors, lipid disorders, heart failure, gut dysbiosis, diet, vitamin D deficiency, air pollution and exposure to chemicals) influence COVID-19 by applying the Adverse Outcome Pathway (AOP), which is well-established in regulatory toxicology. This framework aims to model the sequence of events leading to an adverse health outcome. Several linear AOPs depicting pathways from the binding of the virus to ACE2 up to clinical outcomes observed in COVID-19 have been developed and integrated into a network offering a unique overview of the mechanisms underlying the disease. As SARS-CoV-2 infectibility and ACE2 activity are the major starting points and inflammatory response is central in the development of COVID-19, we evaluated how those eleven intrinsic and extrinsic factors modulate those processes impacting clinical outcomes. Applying this AOP-aligned approach enables the identification of current knowledge gaps orientating for further research and allows to propose biomarkers to identify of high-risk patients. This approach also facilitates expertise synergy from different disciplines to address public health issues.

Expanding the Galaxy's reference data.

Summary: Properly and effectively managing reference datasets is an important task for many bioinformatics analyses. Refgenie is a reference asset management system that allows users to easily organize, retrieve and share such datasets. Here, we describe the integration of refgenie into the Galaxy platform. Server administrators are able to configure Galaxy to make use of reference datasets made available on a refgenie instance. In addition, a Galaxy Data Manager tool has been developed to provide a graphical interface to refgenie's remote reference retrieval functionality. A large collection of reference datasets has also been made available using the CVMFS (CernVM File System) repository from GalaxyProject.org, with mirrors across the USA, Canada, Europe and Australia, enabling easy use outside of Galaxy. Availability and implementation: The ability of Galaxy to use refgenie assets was added to the core Galaxy framework in version 22.01, which is available from https://github.com/galaxyproject/galaxy under the Academic Free License version 3.0. The refgenie Data Manager tool can be installed via the Galaxy ToolShed, with source code managed at https://github.com/BlankenbergLab/galaxy-tools-blankenberg/tree/main/data_managers/data_manager_refgenie_pull and released using an MIT license. Access to existing data is also available through CVMFS, with instructions at https://galaxyproject.org/admin/reference-data-repo/. No new data were generated or analyzed in support of this research.

Toxin-like peptides in plasma, urine and faecal samples from COVID-19 patients.

Background: SARS-CoV-2 that causes COVID-19 disease and led to the pandemic currently affecting the world has been broadly investigated. Different studies have been performed to understand the infection mechanism, and the involved human genes, transcripts and proteins. In parallel, numerous clinical extra-pulmonary manifestations co-occurring with COVID-19 disease have been reported and evidence of their severity and persistence is increasing. Whether these manifestations are linked to other disorders co-occurring with SARS-CoV-2 infection, is under discussion. In this work, we report the identification of toxin-like peptides in COVID-19 patients by application of the Liquid Chromatography Surface-Activated Chemical Ionization - Cloud Ion Mobility Mass Spectrometry.   Methods: Plasma, urine and faecal samples from COVID-19 patients and control individuals were analysed to study peptidomic toxins' profiles. Protein precipitation preparation procedure was used for plasma, to remove high molecular weight proteins and efficiently solubilize the peptide fraction; in the case of faeces and urine, direct peptide solubilization was employed.   Results: Toxin-like peptides, almost identical to toxic components of venoms from animals, like conotoxins, phospholipases, phosphodiesterases, zinc metal proteinases, and bradykinins, were identified in samples from COVID-19 patients, but not in control samples.  Conclusions: The presence of toxin-like peptides could potentially be connected to SARS-CoV-2 infection. Their presence suggests a possible association between COVID-19 disease and the release in the body of (oligo-)peptides almost identical to toxic components of venoms from animals. Their involvement in a large set of heterogeneous extra-pulmonary COVID-19 clinical manifestations, like neurological ones, cannot be excluded. Although the presence of each individual symptom is not selective of the disease, their combination might be related to COVID-19 by the coexistence of the panel of the here detected toxin-like peptides. The presence of these peptides opens new scenarios on the aetiology of the COVID-19 clinical symptoms observed up to now, including neurological manifestations.

Increase of SARS-CoV-2 RNA load in faecal samples prompts for rethinking of SARS-CoV-2 biology and COVID-19 epidemiology.

Background Scientific evidence for the involvement of human microbiota in the development of COVID-19 disease has been reported recently. SARS-CoV-2 RNA presence in human faecal samples and SARS-CoV-2 activity in faeces from COVID-19 patients have been observed. Methods Starting from these observations, an experimental design was developed to cultivate in vitro faecal microbiota from infected individuals, to monitor the presence of SARS-CoV-2, and to collect data on the relationship between faecal bacteria and the virus. Results Our results indicate that SARS-CoV-2 replicates in vitro in bacterial growth medium, that the viral replication follows bacterial growth and it is influenced by the administration of specific antibiotics. SARS-CoV-2-related peptides have been detected in 30-day bacterial cultures and characterised. Discussion Our observations are compatible with a 'bacteriophage-like' behaviour of SARS-CoV-2, which, to our knowledge has not been observed or described before. These results are unexpected and hint towards a novel hypothesis on the biology of SARS-CoV-2 and on the COVID-19 epidemiology. The discovery of possible new modes of action of SARS-CoV-2 has far-reaching implications for the prevention and the treatment of the disease.

Variant Analysis of SARS-CoV-2 Genomes from Belgian Military Personnel Engaged in Overseas Missions and Operations.

More than a year after the first identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of the 2019 coronavirus disease (COVID-19) in China, the emergence and spread of genomic variants of this virus through travel raise concerns regarding the introduction of lineages in previously unaffected regions, requiring adequate containment strategies. Concomitantly, such introductions fuel worries about a possible increase in transmissibility and disease severity, as well as a possible decrease in vaccine efficacy. Military personnel are frequently deployed on missions around the world. As part of a COVID-19 risk mitigation strategy, Belgian Armed Forces that engaged in missions and operations abroad were screened (7683 RT-qPCR tests), pre- and post-mission, for the presence of SARS-CoV-2, including the identification of viral lineages. Nine distinct viral genotypes were identified in soldiers returning from operations in Niger, the Democratic Republic of the Congo, Afghanistan, and Mali. The SARS-CoV-2 variants belonged to major clades 19B, 20A, and 20B (Nextstrain nomenclature), and included "variant of interest" B.1.525, "variant under monitoring" A.27, as well as lineages B.1.214, B.1, B.1.1.254, and A (pangolin nomenclature), some of which are internationally monitored due to the specific mutations they harbor. Through contact tracing and phylogenetic analysis, we show that isolation and testing policies implemented by the Belgian military command appear to have been successful in containing the influx and transmission of these distinct SARS-CoV-2 variants into military and civilian populations.

A roadmap for the generation of benchmarking resources for antimicrobial resistance detection using next generation sequencing.

Next Generation Sequencing technologies significantly impact the field of Antimicrobial Resistance (AMR) detection and monitoring, with immediate uses in diagnosis and risk assessment. For this application and in general, considerable challenges remain in demonstrating sufficient trust to act upon the meaningful information produced from raw data, partly because of the reliance on bioinformatics pipelines, which can produce different results and therefore lead to different interpretations. With the constant evolution of the field, it is difficult to identify, harmonise and recommend specific methods for large-scale implementations over time. In this article, we propose to address this challenge through establishing a transparent, performance-based, evaluation approach to provide flexibility in the bioinformatics tools of choice, while demonstrating proficiency in meeting common performance standards. The approach is two-fold: first, a community-driven effort to establish and maintain "live" (dynamic) benchmarking platforms to provide relevant performance metrics, based on different use-cases, that would evolve together with the AMR field; second, agreed and defined datasets to allow the pipelines' implementation, validation, and quality-control over time. Following previous discussions on the main challenges linked to this approach, we provide concrete recommendations and future steps, related to different aspects of the design of benchmarks, such as the selection and the characteristics of the datasets (quality, choice of pathogens and resistances, etc.), the evaluation criteria of the pipelines, and the way these resources should be deployed in the community.