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.

Urgent need for a non-discriminatory and non-stigmatizing nomenclature for monkeypox virus.

We propose a novel, non-discriminatory classification of monkeypox virus diversity. Together with the World Health Organization, we named three clades (I, IIa and IIb) in order of detection. Within IIb, the cause of the current global outbreak, we identified multiple lineages (A.1, A.2, A.1.1 and B.1) to support real-time genomic surveillance.

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.

Distribution of Chlamydia trachomatis ompA-genotypes over three decades in Portugal.

OBJECTIVES: Chlamydia trachomatis is classified into 15 major genotypes, A to L3, based on the diversity of ompA gene. Here, we evaluated and characterised the distribution and diversity of ompA-genotypes over 32 years (1990-2021) in Portugal. METHODS: The collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824 C. trachomatis-positive samples that were successfully ompA-genotyped between 1990 and 2021. An in-depth analysis of ompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed. RESULTS: ompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGV ompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples. ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV cases per year) and the non-negligible detection of ompA-genotype B in urogenital and anorectal specimens. CONCLUSIONS: This study provides a comprehensive landscape of C. trachomatis molecular surveillance in Portugal, highlighting the continued relevance of ompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding of C. trachomatis epidemiology, diversity and pathogenicity.

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.

Epidemiology and genetic diversity of invasive Neisseria meningitidis strains circulating in Portugal from 2003 to 2020.

Invasive meningococcal disease (IMD) continues to be a public health problem due to its epidemic potential, affecting mostly children. We aimed to present a detailed description of the epidemiology of IMD in Portugal, including insights into the genetic diversity of Neisseria meningitidis strains. Epidemiological analysis included data from the Portuguese National Reference Laboratory of Neisseria meningitidis during 2003 to 2020. Since 2012, N. meningitidis isolates have also been assessed for their susceptibility to antibiotics and were characterized by whole genome sequencing. During 2003-2020, 1392 confirmed cases of IMD were analyzed. A decrease in the annual incidence rate was observed, ranging from 1.99 (2003) to 0.39 (2020), with an average case fatality rate of 7.1%. Serogroup B was the most frequent (69.7%), followed by serogroups C (9.7%), Y (5.7%), and W (2.6%). Genomic characterization of 329 isolates identified 20 clonal complexes (cc), with the most prevalent belonging to serogroup B cc41/44 (26.3%) and cc213 (16.3%). Isolates belonging to cc11 were predominantly from serogroups W (77.3%) and C (76.5%), whereas cc23 was dominant from serogroup Y (65.7%). Over the past 4 years (2017-2020), we observed an increasing trend of cases assigned to cc213, cc32, and cc11. Regarding antimicrobial susceptibility, all isolates were susceptible to ceftriaxone and 61.8% were penicillin-nonsusceptible, whereas 1.4% and 1.0% were resistant to ciprofloxacin and rifampicin. This is the first detailed study on the epidemiology and genomics of invasive N. meningitidis infections in Portugal, providing relevant data to public health policy makers for a more effective control of this 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.

Genome-Scale Characterization of Mycobacterium abscessus Complex Isolates from Portugal.

The Mycobacterium abscessus complex (MABC) is an emerging, difficult to treat, multidrug-resistant nontuberculous mycobacteria responsible for a wide spectrum of infections and associated with an increasing number of cases worldwide. Dominant circulating clones (DCCs) of MABC have been genetically identified as groups of strains associated with higher prevalence, higher levels of antimicrobial resistance, and worse clinical outcomes. To date, little is known about the genomic characteristics of MABC species circulating in Portugal. Here, we examined the genetic diversity and antimicrobial resistance profiles of 30 MABC strains isolated between 2014 and 2022 in Portugal. The genetic diversity of circulating MABC strains was assessed through a gene-by-gene approach (wgMLST), allowing their subspecies differentiation and the classification of isolates into DCCs. Antimicrobial resistance profiles were defined using phenotypic, molecular, and genomic approaches. The majority of isolates were resistant to at least two antimicrobials, although a poor correlation between phenotype and genotype data was observed. Portuguese genomes were highly diverse, and data suggest the existence of MABC lineages with potential international circulation or cross-border transmission. This study highlights the genetic diversity and antimicrobial resistance profile of circulating MABC isolates in Portugal while representing the first step towards the implementation of a genomic-based surveillance system for MABC at the Portuguese NIH.

Differential Gene Expression of Malaria Parasite in Response to Red Blood Cell-Specific Glycolytic Intermediate 2,3-Diphosphoglycerate (2,3-DPG).

Innovative strategies to control malaria are urgently needed. Exploring the interplay between Plasmodium sp. parasites and host red blood cells (RBCs) offers opportunities for novel antimalarial interventions. Pyruvate kinase deficiency (PKD), characterized by heightened 2,3-diphosphoglycerate (2,3-DPG) concentration, has been associated with protection against malaria. Elevated levels of 2,3-DPG, a specific mammalian metabolite, may hinder glycolysis, prompting us to hypothesize its potential contribution to PKD-mediated protection. We investigated the impact of the extracellular supplementation of 2,3-DPG on the Plasmodium falciparum intraerythrocytic developmental cycle in vitro. The results showed an inhibition of parasite growth, resulting from significantly fewer progeny from 2,3-DPG-treated parasites. We analyzed differential gene expression and the transcriptomic profile of P. falciparum trophozoites, from in vitro cultures subjected or not subjected to the action of 2,3-DPG, using Nanopore Sequencing Technology. The presence of 2,3-DPG in the culture medium was associated with the significant differential expression of 71 genes, mostly associated with the GO terms nucleic acid binding, transcription or monoatomic anion channel. Further, several genes related to cell cycle control were downregulated in treated parasites. These findings suggest that the presence of this RBC-specific glycolytic metabolite impacts the expression of genes transcribed during the parasite trophozoite stage and the number of merozoites released from individual schizonts, which supports the potential role of 2,3-DPG in the mechanism of protection against malaria by PKD.

Genomic surveillance of Neisseria meningitidis serogroup W in Portugal from 2003 to 2019.

In recent years, a change in the epidemiology of meningococcal disease caused by Neisseria meningitidis serogroup W (MenW) has been observed worldwide, with the emergence of new sublineages associated with a higher rate of fatal cases. The present study intends to describe the epidemiology of invasive meningococcal disease (IMD) due to MenW in Portugal between 2003 and 2019, and to genetically characterize population structure. Despite MenW has a low incidence in Portugal, having almost disappeared from 2008 to 2015, since 2016, the number of MenW cases has been steadily increasing at a rate of ~ twofold per year, with more than 80% of the characterized isolates belonging to clonal complex 11 (cc11). Core-genome phylogeny of 25 Portuguese (PT) MenW isolates showed a strain clustering mainly either with the Original UK or the UK 2013 sublineages. Our study also reported for the first time the presence of distinct prophages with a notable overrepresentation of an ~ 32-35-kb PS_1-like prophage found in MenW cc11 genomes. The presence of the PS_1-like prophage in almost all 4723 cc11 genomes selected from Neisseria PubMLST database regardless of the capsular group they belong to suggests an ancestral acquisition of this mobile element prior to capsular switching events. Overall, by mimicking the scenario observed worldwide, this study reinforces the importance of a close monitoring of MenW disease, especially from cc11, in order to promptly adapt the vaccination plan for IMD control in Portugal. Moreover, future studies are needed to understand the putative contribution of prophages to fitness and virulence of PT MenW strains.

Global gene expression analysis of Streptococcus agalactiae at exponential growth phase.

A comparative overview of the global gene expression levels of S. agalactiae reference strain NEM316 at the exponential growth phase was done through RNA-sequencing. The expression levels of 47 genes potentially linked to virulence evidenced that: i) the major nuclease, GBS_RS03720/gbs0661, presented higher mean expression values than the remainder of DNase genes; ii) the genetic pilus island PI-2a genes presented higher mean expression values than PI-1 coding genes; and, iii) three virulence-associated genes ranked among the top-100 most expressed genes (GBS_RS07760, GBS_RS09445 and GBS_RS03485). Among this top-100, genes encoding proteins involved in "Translation, ribosomal structure and biogenesis" represented 46%. Curiously, genes with no assigned function were grouped in the category of highly expressed genes. As very little is known about the molecular mechanisms behind the release of DNases, preliminary assays were developed to understand whether direct DNA exposure would affect gene expression at the exponential growth phase. No differentially expressed genes were detected, indicating that follow-up studies are needed to disclose the complex molecular pathways (and stimuli) triggering the release of DNases. In general, our insights on the global expression levels of NEM316 at exponential growth phase with and without DNA exposure should open novel research lines to decipher S. agalactiae puzzling adaptation and virulence mechanisms, such as DNase production.

Characterization of the Human Papillomavirus 16 Oncogenes in K14HPV16 Mice: Sublineage A1 Drives Multi-Organ Carcinogenesis.

The study of human papillomavirus (HPV)-induced carcinogenesis uses multiple in vivo mouse models, one of which relies on the cytokeratin 14 gene promoter to drive the expression of all HPV early oncogenes. This study aimed to determine the HPV16 variant and sublineage present in the K14HPV16 mouse model. This information can be considered of great importance to further enhance this K14HPV16 model as an essential research tool and optimize its use for basic and translational studies. Our study evaluated HPV DNA from 17 samples isolated from 4 animals, both wild-type (n = 2) and HPV16-transgenic mice (n = 2). Total DNA was extracted from tissues and the detection of HPV16 was performed using a qPCR multiplex. HPV16-positive samples were subsequently whole-genome sequenced by next-generation sequencing techniques. The phylogenetic positioning clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). A comparative genome analysis of K14HPV16 samples revealed three mutations to the human papillomaviruses type 16 sublineage A1 representative strain. Knowledge of the HPV 16 variant is fundamental, and these findings will allow the rational use of this animal model to explore the role of the A1 sublineage in HPV-driven cancer.

Transcontinental Dissemination of the L2b/D-Da Recombinant Chlamydia trachomatis Lymphogranuloma venereum (LGV) Strain: Need of Broad Multi-Country Molecular Surveillance.

Previously, we identified a Chlamydia trachomatis lymphogranuloma venereum (LGV) recombinant strain possessing a non-LGV ompA genotype. Here, culture-independent genome sequencing confirms its circulation in Europe, Middle East, and North America, and unveils emergence of antibiotic resistance. Broad surveillance is needed.

Characteristics of SARS-CoV-2 variants of concern B.1.1.7, B.1.351 or P.1: data from seven EU/EEA countries, weeks 38/2020 to 10/2021.

We compared 19,207 cases of SARS-CoV-2 variant B.1.1.7/S gene target failure (SGTF), 436 B.1.351 and 352 P.1 to non-variant cases reported by seven European countries. COVID-19 cases with these variants had significantly higher adjusted odds ratios for hospitalisation (B.1.1.7/SGTF: 1.7, 95% confidence interval (CI): 1.0-2.9; B.1.351: 3.6, 95% CI: 2.1-6.2; P.1: 2.6, 95% CI: 1.4-4.8) and B.1.1.7/SGTF and P.1 cases also for intensive care admission (B.1.1.7/SGTF: 2.3, 95% CI: 1.4-3.5; P.1: 2.2, 95% CI: 1.7-2.8).

Cephalosporin-Resistant Neisseria gonorrhoeae Isolated in Portugal, 2019.

We report a multidrug-resistant Neisseria gonorrhoeae exhibiting resistance to ceftriaxone and cefixime, isolated in Portugal in 2019. Whole-genome sequencing was performed for typing and identification of genetic determinants of antimicrobial resistance. Because of its antimicrobial susceptibility profile, awareness should be raised for the circulation of this strain.

Lung abscess due to Neisseria meningitidis serogroup X-unexpected virulence of a commensal resulting from putative serogroup B capsular switching.

To report the first case of a lung abscess caused by Neisseria meningitidis (Nm) and to genetically characterize the rare underlying capsule switching event. The strain (PT NmX) was subjected to whole genome sequencing, and a comparative gene-by-gene analysis was performed based on 1605 N. meningitidis core loci that constitute the MLST core-genome scheme (cgMLST) V1.0. All ~ 9,600 genomes available on Neisseria PubMLST (until 30th November 2019) from all serogroups were used to better identify the genome make-up of the PT NmX strain. This strain was found to be highly divergent from other NmX reported worldwide and to belong to a new sequence type (ST-14273), with the finetype X: P1.19,15-1:F5-2. Moreover, it revealed a closer genetic proximity to strains from serogroup B than to other serogroups, suggesting a genome backbone associated with serogroup B, while it presents a capsule synthesis region derived from a NmX strain. We describe a new hybrid NmB/X isolate from a noninvasive meningococcal infection, causing lung abscess. Despite capsular switching events involving serogroup X are rare, it may lead to the emergence of pathogenic potential. Studies should continue to better understand the molecular basis underlying Neisseria strains' ability to spread to body compartments other than the tissues for which their tropism is already known.

Fifteen years of a nationwide culture collection of Neisseria gonorrhoeae antimicrobial resistance in Portugal.

Neisseria gonorrhoeae antimicrobial resistance (AMR) and gonorrhea disease burden remain major public health concerns worldwide. To contribute to the supranational demands to monitor and manage the spread of antimicrobial-resistant N. gonorrhoeae, the Portuguese NIH promoted the creation of the National Laboratory Network for Neisseria gonorrhoeae Collection (PTGonoNet). The present study reports the N. gonorrhoeae major AMR trends observed from 2003 up to 2018. All isolates described in the present study constitute the opportunistic ongoing N. gonorrhoeae isolate collection supported by the National Reference Laboratory for Sexually Transmitted Infections of the Portuguese NIH, enrolling strains isolated in 35 different public and private laboratories. Minimum inhibitory concentrations were determined using E-tests for azithromycin, benzylpenicillin, cefixime, ceftriaxone, ciprofloxacin, gentamicin, spectinomycin and tetracycline. Molecular typing was determined using NG-MAST. AMR data of 2596 country-spread isolates show that 87.67% of all N. gonorrhoeae isolates presented decreased susceptibility to at least one antimicrobial. A continuous decreased susceptibility and resistance to penicillin, tetracycline and ciprofloxacin can be observed along the years. However, no decreased susceptibility to cephalosporins was observed until 2018, while for azithromycin, this was always low. The most common observed NG-MAST genogroups were G1407, G7445, G225, G2, and G1034. This study evidences the advantages of a nationwide collection of isolates and of centralized AMR testing to respond to supranational (EURO-GASP) requirements while providing unprecedented data on AMR in the context of 15 years of surveillance.

Towards a rapid sequencing-based molecular surveillance and mosaicism investigation of Toxoplasma gondii.

Advances in molecular epidemiology of Toxoplasma gondii are hampered by technical and cost-associated hurdles underlying the acquisition of genomic data from parasites. In order to implement an enhanced genotyping approach for molecular surveillance of T. gondii, we applied a multi-locus amplicon-based sequencing strategy to samples associated with human infection. This approach, targeting genome-dispersed polymorphic loci potentially involved in adaptation and virulence, genetically discriminated almost all 68 studied strains and revealed a scenario of marked genomic mosaicism. Two-thirds (n = 43) of all strains were classified as recombinant, although recombination seemed to be linked to the classical archetypal lineage. While 92% of the Sag2 archetype I strains revealed genetic mosaicism, only 45% of Sag2 archetype II strains were identified as recombinant. Contrarily to the virulence-associated archetype I, most type II strains (regardless of their recombination background) were non-virulent in mouse. Besides Sag2, some of the newly studied loci (namely the type I/I-like alleles of Sag1, B17, PK1, and Sag3 and type III/III-like alleles of TgM-A) constitute promising candidates to rapidly infer T. gondii mouse virulence. Our successful attempt to capture microsatellite length variation launches good perspectives for the straightforward transition from the laborious intensive historical method to more informative next-generation sequencing (NGS)/bioinformatics-based methodologies. Overall, while T. gondii whole-genome sequencing will be hardly feasible in most laboratories, this study shows that a discrete loci panel has the potential to improve the molecular epidemiology of T. gondii towards a better monitoring of circulating genotypes with clinical importance.

Dynamics of mercury in the plankton of a hydroelectric reservoir, Western Amazon.

The energy transfer in the aquatic food chain is an important way for mercury (Hg) to enter other trophic levels. The objective of this work was to evaluate the Hg concentrations in plankton upstream and downstream of the Samuel Hydroelectric Reservoir, Rondônia, Brazil. Phytoplankton and zooplankton samples were collected with 20-µm and 68-µm nylon nets. An aliquot was removed for taxonomic analysis and another for total mercury determination, performed by cold vapor atomic absorption spectroscopy. Water physical-chemical parameters were also measured. The Hg concentrations in total plankton (phytoplankton and zooplankton samples) obtained at the three sampling upstream stations showed the same behavior, with the highest values registered in June 2005 (232 µg kg-1, 118 µg kg-1, 128 µg kg-1). The lowest values at stations J1 and M1 were recorded in November 2005 (4 µg kg-1 and 22 µg kg-1, respectively), while the lowest values at stations M4 and M8 were recorded in October 2005 (22 µg kg-1 and 5 µg kg-1, respectively). The Hg results found in the plankton in this study corroborate the results of other recent studies in the same region. The statistical analyses revealed that Hg concentrations in plankton do not explain the distribution of these organisms at the four sampling stations of Samuel Reservoir. Graphical Abstract.

Imipenem Resistance in Clostridium difficile Ribotype 017, Portugal.

We describe imipenem-resistant and imipenem-susceptible clinical isolates of Clostridium difficile ribotype 017 in Portugal. All ribotype 017 isolates carried an extra penicillin-binding protein gene, pbp5, and the imipenem-resistant isolates had additional substitutions near the transpeptidase active sites of pbp1 and pbp3. These clones could disseminate and contribute to imipenem resistance.