The key role of Spain in the traffic of West Nile virus lineage 1 strains between Europe and Africa.

BACKGROUND: West Nile Virus (WNV) is a zoonotic arbovirus worldwide spread. Seasonal WNV outbreaks occur in the Mediterranean basin since the late 1990's with ever-increasing incidence. In Southern Spain WNV is endemic, as disease foci - caused by WNV lineage 1 (WNV-L1) strains - occur every year. On the contrary, WNV-L2 is the dominant lineage in Europe, so most European WNV sequences available belong to this lineage, WNV-L1 sequences being still scarce. METHODS: To fill this gap, this study reports the genetic characterisation of 27 newly described WNV-L1 strains, involved in outbreaks affecting wild birds and horses during the last decade in South-Western Spain. RESULTS: All strains except one belong to the Western Mediterranean-1 sub-cluster (WMed-1), related phylogenetically to Italian, French, Portuguese, Moroccan and, remarkably, Senegalese strains. This sub-cluster persisted, spread and evolved into three distinguishable WMed-1 phylogenetic groups that co-circulated, notably, in the same province (Cádiz). They displayed different behaviours: from long-term persistence and rapid spread to neighbouring regions within Spain, to long-distance spread to different countries, including transcontinental spread to Africa. Among the different introductions of WNV in Spain revealed in this study, some of them succeeded to get established, some extinguished from the territory shortly afterwards. Furthermore, Spain's southernmost province, Cádiz, constitutes a hotspot for virus incursion. CONCLUSION: Southern Spain seems a likely scenario for emergence of exotic pathogens of African origin. Therefore, circulation of diverse WNV-L1 variants in Spain prompts for an extensive surveillance under a One Health approach.

Effectiveness of COVID-19 vaccines administered in the 2023 autumnal campaigns in Europe: Results from the VEBIS primary care test-negative design study, September 2023-January 2024.

In autumn 2023, European vaccination campaigns predominantly administered XBB.1.5 vaccine. In a European multicentre study, we estimated 2023 COVID-19 vaccine effectiveness (VE) against laboratory-confirmed symptomatic infection at primary care level between September 2023 and January 2024. Using a test-negative case-control design, we estimated VE in the target group for COVID-19 vaccination overall and by time since vaccination. We included 1057 cases and 4397 controls. Vaccine effectiveness was 40 % (95 % CI: 26-53 %) overall, 48 % (95 % CI: 31-61 %) among those vaccinated < 6 weeks of onset and 29 % (95 % CI: 3-49 %) at 6-14 weeks. Our results suggest that COVID-19 vaccines administered to target groups during the autumn 2023 campaigns showed clinically significant effectiveness against laboratory-confirmed, medically attended symptomatic SARS-CoV-2 infection in the 3 months following vaccination. A longer study period will allow for further variant-specific COVID-19 VE estimates, better understanding decline in VE and informing booster administration policies.

Listeria monocytogenes from Food Products and Food Associated Environments: Antimicrobial Resistance, Genetic Clustering and Biofilm Insights.

Listeria monocytogenes, a foodborne pathogen, exhibits high adaptability to adverse environmental conditions and is common in the food industry, especially in ready-to-eat foods. L. monocytogenes strains pose food safety challenges due to their ability to form biofilms, increased resistance to disinfectants, and long-term persistence in the environment. The aim of this study was to evaluate the presence and genetic diversity of L. monocytogenes in food and related environmental products collected from 2014 to 2022 and assess antibiotic susceptibility and biofilm formation abilities. L. monocytogenes was identified in 13 out of the 227 (6%) of samples, 7 from food products (meat preparation, cheeses, and raw milk) and 6 from food-processing environments (slaughterhouse-floor and catering establishments). All isolates exhibited high biofilm-forming capacity and antibiotic susceptibility testing showed resistance to several classes of antibiotics, especially trimethoprim-sulfamethoxazole and erythromycin. Genotyping and core-genome clustering identified eight sequence types and a cluster of three very closely related ST3 isolates (all from food), suggesting a common contamination source. Whole-genome sequencing (WGS) analysis revealed resistance genes conferring resistance to fosfomycin (fosX), lincosamides (lin), fluoroquinolones (norB), and tetracycline (tetM). In addition, the qacJ gene was also detected, conferring resistance to disinfecting agents and antiseptics. Virulence gene profiling revealed the presence of 92 associated genes associated with pathogenicity, adherence, and persistence. These findings underscore the presence of L. monocytogenes strains in food products and food-associated environments, demonstrating a high virulence of these strains associated with resistance genes to antibiotics, but also to disinfectants and antiseptics. Moreover, they emphasize the need for continuous surveillance, effective risk assessment, and rigorous control measures to minimize the public health risks associated to severe infections, particularly listeriosis outbreaks. A better understanding of the complex dynamics of pathogens in food products and their associated environments can help improve overall food safety and develop more effective strategies to prevent severe health consequences and economic losses.

Unveiling a Listeria monocytogenes Outbreak in a Rabbit Farm: Clinical Manifestation, Antimicrobial Resistance, Genomic Insights and Environmental Investigation.

Listeria monocytogenes poses a threat to both human and animal health. This work describes an L. monocytogenes outbreak in a Portuguese rabbit farm, detailing the isolates' clinical manifestations, necropsy findings, and phenotypic and genomic profiles. Clinical signs, exclusively observed in does, included lethargy and reproductive signs. Post-mortem examination of does revealed splenomegaly, hepatomegaly with a reticular pattern, pulmonary congestion, and haemorrhagic lesions in the uterus, with thickening of the uterine wall and purulent greyish exudates. Positive L. monocytogenes samples were identified in fattening and maternity units across different samples, encompassing does and environmental samples. Core-genome Multi Locus Sequence Typing (cgMLST) analysis confirmed the outbreak, with the 16 sequenced isolates (lineage II, CC31, and ST325) clustering within a ≤2 allelic difference (AD) threshold. Antimicrobial susceptibility testing for five antibiotics revealed that 15 out of 19 outbreak isolates were resistant to sulfamethoxazole-trimethoprim (SXT). Concordantly, all SXT-resistant sequenced isolates were found to exclusively harbour a plasmid containing a trimethoprim-resistance gene (dfrD), along with loci linked to resistance to lincosamides (lnuG), macrolides (mphB), and polyether ionophores (NarAB operon). All sequenced outbreak isolates carried the antibiotic resistance-related genes tetM, fosX, lin, norB, lmrB, sul, and mprF. The outbreak cluster comprises isolates from does and the environment, which underscores the ubiquitous presence of L. monocytogenes and emphasizes the importance of biosecurity measures. Despite limited data on listeriosis in rabbit farming, this outbreak reveals its significant impact on animal welfare and production.

Recurrence, Microevolution, and Spatiotemporal Dynamics of Legionella pneumophila Sequence Type 1905, Portugal, 2014-2022.

We investigated molecular evolution and spatiotemporal dynamics of atypical Legionella pneumophila serogroup 1 sequence type 1905 and determined its long-term persistence and linkage to human disease in dispersed locations, far beyond the large 2014 outbreak epicenter in Portugal. Our finding highlights the need for public health interventions to prevent further disease spread.

Raw milk cheeses from Beira Baixa, Portugal-A contributive study for the microbiological hygiene and safety assessment.

Due to specific bacterial microbiota, raw milk cheeses have appreciated sensory properties. However, they may pose a threat to consumer safety due to potential pathogens presence. This study evaluated the microbiological contamination of 98 raw milk cheeses from Beira Baixa, Portugal. Presence and enumeration of Coagulase Positive Staphylococci (CPS), Listeria monocytogenes, Salmonella spp., pathogenic Escherichia coli, and indicator microorganisms (non-pathogenic E. coli and Listeria spp.) was attained. E. coli antimicrobial resistance (AMR) was also evaluated. PCR and/or Whole genome sequencing (WGS) was used to characterize E. coli, Salmonella spp. and L. monocytogenes isolates. Sixteen cheeses (16.3%) were classified as Satisfactory, 59 (60.2%) as Borderline and 23 (23.5%) as Unsatisfactory/Potential Injurious to Health. L. monocytogenes, CPS > 104 cfu g-1, Extraintestinal pathogenic E. coli (ExPEC) and Salmonella spp. were detected in 4.1%, 6.1%, 3.1% and 1.0% of the samples, respectively. Listeria innocua (4.1%) and E. coli > 104 cfu g-1 (16.3%) were also detected. AMR E. coli was detected in 23/98 (23.5%) of the cheese samples, of which two were multidrug resistant. WGS identified genotypes already associated to human disease and Listeria spp. cluster analysis indicated that cheese contamination might be related with noncompliance with Good Hygiene Practices during cheese production.

INSaFLU-TELEVIR: an open web-based bioinformatics suite for viral metagenomic detection and routine genomic surveillance.

BACKGROUND: Implementation of clinical metagenomics and pathogen genomic surveillance can be particularly challenging due to the lack of bioinformatics tools and/or expertise. In order to face this challenge, we have previously developed INSaFLU, a free web-based bioinformatics platform for virus next-generation sequencing data analysis. Here, we considerably expanded its genomic surveillance component and developed a new module (TELEVIR) for metagenomic virus identification. RESULTS: The routine genomic surveillance component was strengthened with new workflows and functionalities, including (i) a reference-based genome assembly pipeline for Oxford Nanopore technologies (ONT) data; (ii) automated SARS-CoV-2 lineage classification; (iii) Nextclade analysis; (iv) Nextstrain phylogeographic and temporal analysis (SARS-CoV-2, human and avian influenza, monkeypox, respiratory syncytial virus (RSV A/B), as well as a "generic" build for other viruses); and (v) algn2pheno for screening mutations of interest. Both INSaFLU pipelines for reference-based consensus generation (Illumina and ONT) were benchmarked against commonly used command line bioinformatics workflows for SARS-CoV-2, and an INSaFLU snakemake version was released. In parallel, a new module (TELEVIR) for virus detection was developed, after extensive benchmarking of state-of-the-art metagenomics software and following up-to-date recommendations and practices in the field. TELEVIR allows running complex workflows, covering several combinations of steps (e.g., with/without viral enrichment or host depletion), classification software (e.g., Kaiju, Kraken2, Centrifuge, FastViromeExplorer), and databases (RefSeq viral genome, Virosaurus, etc.), while culminating in user- and diagnosis-oriented reports. Finally, to potentiate real-time virus detection during ONT runs, we developed findONTime, a tool aimed at reducing costs and the time between sample reception and diagnosis. CONCLUSIONS: The accessibility, versatility, and functionality of INSaFLU-TELEVIR are expected to supply public and animal health laboratories and researchers with a user-oriented and pan-viral bioinformatics framework that promotes a strengthened and timely viral metagenomic detection and routine genomics surveillance. INSaFLU-TELEVIR is compatible with Illumina, Ion Torrent, and ONT data and is freely available at https://insaflu.insa.pt/ (online tool) and https://github.com/INSaFLU (code).

ReporType: A Flexible Bioinformatics Tool for Targeted Loci Screening and Typing of Infectious Agents.

In response to the pressing need for continuous monitoring of emergence and circulation of pathogens through genomics, it is imperative to keep developing bioinformatics tools that can help in their rapid characterization and classification. Here, we introduce ReporType, a versatile bioinformatics pipeline designed for targeted loci screening and typing of infectious agents. Developed using the snakemake workflow manager, ReporType integrates multiple software for read quality control and de novo assembly, and then applies ABRicate for locus screening, culminating in the production of easily interpretable reports for the identification of pathogen genotypes and/or screening of specific genomic loci. The pipeline accommodates a range of input formats, from Illumina or Oxford Nanopore Technology (ONT) reads (FASTQ) to Sanger sequencing files (AB1), or FASTA files, making it flexible for application in multiple pathogens and with different purposes. ReporType is released with pre-prepared databases for some viruses and bacteria, yet it remains easily configurable to handle custom databases. ReporType performance and functionality were validated through proof-of-concept exercises, encompassing diverse pathogenic species, including viruses such as measles, Newcastle disease virus (NDV), Dengue virus (DENV), influenza, hepatitis C virus (HCV) and Human T-Cell Lymphotropic virus type 1 (HTLV-1), as well as bacteria like Chlamydia trachomatis and Legionella pneumophila. In summary, ReporType emerges as a simple, dynamic and pan-pathogen tool, poised to evolve in tandem with the ever-changing needs of the fields of pathogen genomics, infectious disease epidemiology, and one health bioinformatics. ReporType is freely available at GitHub.

APOBEC3 deaminase editing in mpox virus as evidence for sustained human transmission since at least 2016.

Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa. However, in May 2022, human cases of mpox were detected spreading internationally beyond countries with known endemic reservoirs. When the first cases from 2022 were sequenced, they shared 42 nucleotide differences from the closest mpox virus (MPXV) previously sampled. Nearly all these mutations are characteristic of the action of APOBEC3 deaminases, host enzymes with antiviral function. Assuming APOBEC3 editing is characteristic of human MPXV infection, we developed a dual-process phylogenetic molecular clock that-inferring a rate of ~6 APOBEC3 mutations per year-estimates that MPXV has been circulating in humans since 2016. These observations of sustained MPXV transmission present a fundamental shift to the perceived paradigm of MPXV epidemiology as a zoonosis and highlight the need for revising public health messaging around MPXV as well as outbreak management and control.

Viral genetic clustering and transmission dynamics of the 2022 mpox outbreak in Portugal.

Pathogen genome sequencing during epidemics enhances our ability to identify and understand suspected clusters and investigate their relationships. Here, we combine genomic and epidemiological data of the 2022 mpox outbreak to better understand early viral spread, diversification and transmission dynamics. By sequencing 52% of the confirmed cases in Portugal, we identified the mpox virus sublineages with the highest impact on case numbers and fitted them into a global context, finding evidence that several international sublineages probably emerged or spread early in Portugal. We estimated a 62% infection reporting rate and that 1.3% of the population of men who have sex with men in Portugal were infected. We infer the critical role played by sexual networks and superspreader gatherings, such as sauna attendance, in the dissemination of mpox virus. Overall, our findings highlight genomic epidemiology as a tool for the real-time monitoring and control of mpox epidemics, and can guide future vaccine policy in a highly susceptible population.

ReporTree: a surveillance-oriented tool to strengthen the linkage between pathogen genetic clusters and epidemiological data.

BACKGROUND: Genomics-informed pathogen surveillance strengthens public health decision-making, playing an important role in infectious diseases' prevention and control. A pivotal outcome of genomics surveillance is the identification of pathogen genetic clusters and their characterization in terms of geotemporal spread or linkage to clinical and demographic data. This task often consists of the visual exploration of (large) phylogenetic trees and associated metadata, being time-consuming and difficult to reproduce. RESULTS: We developed ReporTree, a flexible bioinformatics pipeline that allows diving into the complexity of pathogen diversity to rapidly identify genetic clusters at any (or all) distance threshold(s) or cluster stability regions and to generate surveillance-oriented reports based on the available metadata, such as timespan, geography, or vaccination/clinical status. ReporTree is able to maintain cluster nomenclature in subsequent analyses and to generate a nomenclature code combining cluster information at different hierarchical levels, thus facilitating the active surveillance of clusters of interest. By handling several input formats and clustering methods, ReporTree is applicable to multiple pathogens, constituting a flexible resource that can be smoothly deployed in routine surveillance bioinformatics workflows with negligible computational and time costs. This is demonstrated through a comprehensive benchmarking of (i) the cg/wgMLST workflow with large datasets of four foodborne bacterial pathogens and (ii) the alignment-based SNP workflow with a large dataset of Mycobacterium tuberculosis. To further validate this tool, we reproduced a previous large-scale study on Neisseria gonorrhoeae, demonstrating how ReporTree is able to rapidly identify the main species genogroups and characterize them with key surveillance metadata, such as antibiotic resistance data. By providing examples for SARS-CoV-2 and the foodborne bacterial pathogen Listeria monocytogenes, we show how this tool is currently a useful asset in genomics-informed routine surveillance and outbreak detection of a wide variety of species. CONCLUSIONS: In summary, ReporTree is a pan-pathogen tool for automated and reproducible identification and characterization of genetic clusters that contributes to a sustainable and efficient public health genomics-informed pathogen surveillance. ReporTree is implemented in python 3.8 and is freely available at https://github.com/insapathogenomics/ReporTree .

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Recurrent Campylobacter jejuni Infections with In Vivo Selection of Resistance to Macrolides and Carbapenems: Molecular Characterization of Resistance Determinants.

We present two independent cases of recurrent multidrug-resistant Campylobacter jejuni infection in immunocompromised hosts and the clinical challenges encountered due to the development of high-level carbapenem resistance. The mechanisms associated with this unusual resistance for Campylobacters were characterized. Initial macrolide and carbapenem-susceptible strains acquired resistance to erythromycin (MIC > 256mg/L), ertapenem (MIC > 32mg/L), and meropenem (MIC > 32mg/L) during treatment. Carbapenem-resistant isolates developed an in-frame insertion resulting in an extra Asp residue in the major outer membrane protein PorA, within the extracellular loop L3 that connects ß-strands 5 and 6 and forms a constriction zone involved in Ca2+ binding. The isolates presenting the highest MIC to ertapenem exhibited an extra nonsynonymous mutation (G167A|Gly56Asp) at PorA's extracellular loop L1. IMPORTANCE Carbapenem susceptibility patterns suggest drug impermeability, related to either insertion and/or single nucleotide polymorphism (SNP) within porA. Similar molecular events occurring in two independent cases support the association of these mechanisms with carbapenem resistance in Campylobacter spp.

Comparative complete scheme and booster effectiveness of COVID-19 vaccines in preventing SARS-CoV-2 infections with SARS-CoV-2 Omicron (BA.1) and Delta (B.1.617.2) variants: A case-case study based on electronic health records.

Background: Information on vaccine effectiveness in a context of novel variants of concern (VOC) emergence is of key importance to inform public health policies. This study aimed to estimate a measure of comparative vaccine effectiveness between Omicron (BA.1) and Delta (B.1.617.2 and sub-lineages) VOC according to vaccination exposure (primary or booster). Methods: We developed a case-case study using data on RT-PCR SARS-CoV-2-positive cases notified in Portugal during Weeks 49-51, 2021. To obtain measure of comparative vaccine effectiveness, we compared the odds of vaccination in Omicron cases versus Delta using logistic regression adjusted for age group, sex, region, week of diagnosis, and laboratory of origin. Results: Higher odds of vaccination were observed in cases infected by Omicron VOC compared with Delta VOC cases for both complete primary vaccination (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.8 to 2.4) and booster dose (OR = 5.2; 95% CI: 3.1 to 8.8), equivalent to reduction of vaccine effectiveness from 44.7% and 92.8%, observed against infection with Delta, to -6.0% (95% CI: 29.2% to 12.7%) and 62.7% (95% CI: 35.7% to 77.9%), observed against infection with Omicron, for complete primary vaccination and booster dose, respectively. Conclusion: Consistent reduction in vaccine-induced protection against infection with Omicron was observed. Complete primary vaccination may not be protective against SARS-CoV-2 infection in regions where Omicron variant is dominant.

Listeria monocytogenes, Escherichia coli and Coagulase Positive Staphylococci in Cured Raw Milk Cheese from Alentejo Region, Portugal.

Traditional cheeses are part of the Portuguese gastronomic identity, and raw milk of autochthonous species is a common primary ingredient. Here, we investigated the presence of Listeria monocytogenes, Coagulase Positive Staphylococci (CPS) and pathogenic Escherichia coli, as well as of indicator microorganisms (E. coli and other Listeria spp.) in 96 cured raw milk cheeses from the Alentejo region. Whole genome sequencing (WGS) of pathogenic E. coli and Listeria spp. as well as antimicrobial resistance (AMR) screening of E. coli isolates was also performed. L. monocytogenes, CPS > 104 cfu/g and Extraintestinal E. coli were detected in 15.6%, 16.9% and 10.1% of the samples, respectively. Moreover, L. monocytogenes > 102 cfu/g and Staphylococcal enterotoxins were detected in 4.2% and 2.2% of the samples, respectively. AMR was observed in 27.3% of the E. coli isolates, six of which were multidrug resistant. WGS analysis unveiled clusters of high closely related isolates for both L. monocytogenes and L. innocua (often correlating with the cheese producer). This study can indicate poor hygiene practices during milk collection/preservation or during cheese-making procedures and handling, and highlights the need of more effective prevention and control measures and of multi-sectoral WGS data integration, in order to prevent and detect foodborne bacterial outbreaks.

Comparative Effectiveness of COVID-19 Vaccines in Preventing Infections and Disease Progression from SARS-CoV-2 Omicron BA.5 and BA.2, Portugal.

We estimated comparative primary and booster vaccine effectiveness (VE) of SARS-CoV-2 Omicron BA.5 and BA.2 lineages against infection and disease progression. During April-June 2022, we implemented a case-case and cohort study and classified lineages using whole-genome sequencing or spike gene target failure. For the case-case study, we estimated the adjusted odds ratios (aORs) of vaccination using a logistic regression. For the cohort study, we estimated VE against disease progression using a penalized logistic regression. We observed no reduced VE for primary (aOR 1.07 [95% CI 0.93-1.23]) or booster (aOR 0.96 [95% CI 0.84-1.09]) vaccination against BA.5 infection. Among BA.5 case-patients, booster VE against progression to hospitalization was lower than that among BA.2 case-patients (VE 77% [95% CI 49%-90%] vs. VE 93% [95% CI 86%-97%]). Although booster vaccination is less effective against BA.5 than against BA.2, it offers substantial protection against progression from BA.5 infection to severe disease.

Molecular Capture of Mycobacterium tuberculosis Genomes Directly from Clinical Samples: A Potential Backup Approach for Epidemiological and Drug Susceptibility Inferences.

The application of whole genome sequencing of Mycobacterium tuberculosis directly on clinical samples has been investigated as a means to avoid the time-consuming need for culture isolation that can lead to a potential prolonged suboptimal antibiotic treatment. We aimed to provide a proof-of-concept regarding the application of the molecular capture of M. tuberculosis genomes directly from positive sputum samples as an approach for epidemiological and drug susceptibility predictions. Smear-positive sputum samples (n = 100) were subjected to the SureSelectXT HS Target Enrichment protocol (Agilent Technologies, Santa Clara, CA, USA) and whole-genome sequencing analysis. A higher number of reads on target were obtained for higher smear grades samples (i.e., 3+ followed by 2+). Moreover, 37 out of 100 samples showed ≥90% of the reference genome covered with at least 10-fold depth of coverage (27, 9, and 1 samples were 3+, 2+, and 1+, respectively). Regarding drug-resistance/susceptibility prediction, for 42 samples, ≥90% of the >9000 hits that are surveyed by TB-profiler were detected. Our results demonstrated that M. tuberculosis genome capture and sequencing directly from clinical samples constitute a potential valid backup approach for phylogenetic inferences and resistance prediction, essentially in settings when culture is not routinely performed or for samples that fail to grow.

Development of an amplicon-based sequencing approach in response to the global emergence of human monkeypox virus.

The 2022 multi-country monkeypox (mpox) outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and rapid pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR cycle threshold below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.