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Browsing by Author "Duarte, Silvia"

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  • Development of an amplicon-based sequencing approach in response to the global emergence of mpox
    Publication . Chen, Nicholas F.G.; Chaguza, Chrispin; Gagne, Luc; Doucette, Matthew; Smole, Sandra; Buzby, Erika; Hall, Joshua; Ash, Stephanie; Harrington, Rachel; Cofsky, Seana; Clancy, Selina; Kapsak, Curtis J.; Sevinsky, Joel; Libuit, Kevin; Park, Daniel J.; Hemarajata, Peera; Garrigues, Jacob M.; Green, Nicole M.; Sierra-Patev, Sean; Carpenter-Azevedo, Kristin; Huard, Richard C.; Pearson, Claire; Incekara, Kutluhan; Nishimura, Christina; Huang, Jian Ping; Gagnon, Emily; Reever, Ethan; Razeq, Jafar; Muyombwe, Anthony; Borges, Vítor; Ferreira, Rita; Sobral, Daniel; Duarte, Silvia; Santos, Daniela; Vieira, Luís; Gomes, João Paulo; Aquino, Carly; Savino, Isabella M.; Felton, Karinda; Bajwa, Moneeb; Hayward, Nyjil; Miller, Holly; Naumann, Allison; Allman, Ria; Greer, Neel; Fall, Amary; Mostafa, Heba H.; McHugh, Martin P.; Maloney, Daniel M.; Dewar, Rebecca; Kenicer, Juliet; Parker, Abby; Mathers, Katharine; Wild, Jonathan; Cotton, Seb; Templeton, Kate E.; Churchwell, George; Lee, Philip A.; Pedrosa, Maria; McGruder, Brenna; Schmedes, Sarah; Plumb, Matthew R.; Wang, Xiong; Barcellos, Regina Bones; Godinho, Fernanda M.S.; Salvato, Richard Steiner; Ceniseros, Aimee; Breban, Mallery I.; Grubaugh, Nathan D.; Gallagher, Glen R.; Vogels, Chantal B.F.
    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.
    2023-06-13Journal article Open access Show more
  • A large outbreak of Legionnaires’ Disease in an industrial town in Portugal
    Publication . George, Francisco; Shivaji, Tara; Pinto, Catia Sousa; Serra, Luis Antonio Oliveira; Valente, João; Albuquerque, Maria João; Vicêncio, Paula Cristina Olivença; San-Bento, Ana; Diegues, Paulo; Nogueira, Paulo Jorge; Marques, Teresa; Rebelo, Helena; Costa, Filipa; Rodrigues, Raquel; Nunes, Alexandra; Borges, Vitor; Gomes, João Paulo; Sampaio, Daniel; Barreiro, Paula; Duarte, Silvia; Carpinteiro, Dina; Mendonça, Joana; Silva, Catarina; Vieira, Luís; Simões, Maria Joao; Gonçalves, Paulo; Nunes, Baltazar; Dias, Carlos; Machado, Jorge; Almeida, Fernando; Goncalves, Elsa A; Carvalho, Lucilia; Viterbo, Pedro; Jardim, Dilia; Lacasta, Nuno; Boavida, Filomena; Perez, Ana; Santana, Isabel; Matias, Paula; Banza, Nuno; Rabacal, Carlos
    Background: We describe the investigation and control of an outbreak of Legionnaires’ disease in Portugal in October, November and December 2014. Methods: Confirmed cases were individuals with pneumonia, laboratory evidence of Legionella pneumophila serogroup 1 and exposure, by residence, occupational or leisure to the affected municipalities. 49 possible sources were reduced to four potential sources, all industries with wet cooling system, following risk assessment. We geo-referenced cases’ residences and the location of cooling towers defining four study areas 10km buffer centered on each cooling tower system. We compared the number of cases with expected numbers, calculated from the outbreak's attack rates applied to 2011 census population. Using Stones’ Test, we tested observed to expected ratios for decline in risk, with distance up to 10km four directions. Isolates of Legionella pneumophila were compared using molecular methods. Results: We identified 403 cases, 377 of which were confirmed, 14 patients died. Patients became ill between 14 October and 2 December. A NE wind and thermal inversion were recorded during the estimated period of exposure. Disease risk was highest in people living south west from all of the industries identified and decreased with distance (p<0.001). 71 clinical isolates demonstrated an identical SBT profile to an isolate from a cooling tower. Whole genome sequencing identified an unusual L. pneumophila subsp. fraseri serogroup 1 as the outbreak causative strain, and confirmed isolates’ relatedness. Conclusions: Industrial wet cooling systems, bacteria with enhanced survival characteristics and a combination of climatic conditions contributed to the second largest outbreak of Legionnaires’ disease recorded internationally.
    2016-11-16Journal article Open access Show more
  • Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus
    Publication . Isidro, Joana; Borges, Vítor; Pinto, Miguel; Sobral, Daniel; Santos, João Dourado; Nunes, Alexandra; Mixão, Verónica; Ferreira, Rita; Santos, Daniela; Duarte, Silvia; Vieira, Luís; Borrego, Maria José; Núncio, Sofia; de Carvalho, Isabel Lopes; Pelerito, Ana; Cordeiro, Rita; Gomes, João Paulo
    The largest monkeypox virus (MPXV) outbreak described so far in non-endemic countries was identified in May 2022 (refs. 1-6). In this study, shotgun metagenomics allowed the rapid reconstruction and phylogenomic characterization of the first MPXV outbreak genome sequences, showing that this MPXV belongs to clade 3 and that the outbreak most likely has a single origin. Although 2022 MPXV (lineage B.1) clustered with 2018-2019 cases linked to an endemic country, it segregates in a divergent phylogenetic branch, likely reflecting continuous accelerated evolution. An in-depth mutational analysis suggests the action of host APOBEC3 in viral evolution as well as signs of potential MPXV human adaptation in ongoing microevolution. Our findings also indicate that genome sequencing may provide resolution to track the spread and transmission of this presumably slow-evolving double-stranded DNA virus.
    2022-08Journal article Open access Show more
  • Viral genetic clustering and transmission dynamics of the 2022 mpox outbreak in Portugal
    Publication . Borges, Vítor; Duque, Mariana Perez; Martins, João Vieira; Vasconcelos, Paula; Ferreira, Rita; Sobral, Daniel; Pelerito, Ana; de Carvalho, Isabel Lopes; Núncio, Maria Sofia; Borrego, Maria José; Roemer, Cornelius; Neher, Richard A.; O’Driscoll, Megan; Rocha, Raquel; Lopo, Sílvia; Neves, Raquel; Palminha, Paula; Coelho, Luís; Nunes, Alexandra; Isidro, Joana; Pinto, Miguel; Santos, João Dourado; Mixão, Verónica; Santos, Daniela; Duarte, Silvia; Vieira, Luís; Martins, Fátima; Machado, Jorge; Veríssimo, Vítor Cabral; Grau, Berta; Peralta-Santos, André; Neves, José; Caldeira, Margarida; Pestana, Mafalda; Fernandes, Cândida; Caria, João; Pinto, Raquel; Póvoas, Diana; Maltez, Fernando; Sá, Ana Isabel; Salvador, Mafalda Brito; Teófilo, Eugénio; Rocha, Miguel; Moneti, Virginia; Duque, Luis Miguel; e Silva, Francisco Ferreira; Baptista, Teresa; Vasconcelos, Joana; Casanova, Sara; Mansinho, Kamal; Alves, João Vaz; Alves, João; Silva, António; Alpalhão, Miguel; Brazão, Cláudia; Sousa, Diogo; Filipe, Paulo; Pacheco, Patrícia; Peruzzu, Francesca; de Jesus, Rita Patrocínio; Ferreira, Luís; Mendez, Josefina; Jordão, Sofia; Duarte, Frederico; Gonçalves, Maria João; Pena, Eduarda; Silva, Claúdio Nunes; Guimarães, André Rodrigues; Tavares, Margarida; Freitas, Graça; Cordeiro, Rita; Gomes, João Paulo
    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.
    2023-09-11Journal article Open access Show more
  • Whole-genome-based characterization of invasive Haemophilus influenzae isolates from a pre- and post-vaccine era in Portugal
    Publication . Gonzalez Diaz, Aida; Pinto, Miguel; Bettencourt, Célia; Duarte, Silvia; Marti, Sara; Gomes, João Paulo; Bajanca-Lavado, Paula
    Introduction: Haemophilus influenzae (Hi) is responsible for severe invasive infections in both adults and children. Since the introduction in the year 2000 of the Hib vaccine, the incidence of disease has substantially declined, even though it doesn’t protect against non-typeable Hi (NTHi) isolates. Although not all NTHi are pathogenic, these are known to possess important virulence factors to promote colonization and host cells interactions, ultimately leading to disease. The application of WGS technology allows the uncovering of Hi population structure, including novel insights into its genomic features. Aims: This study aims to fully characterize, by WGS, Hi isolates from a pre- and post-vaccine era, from 1992 to 2015, in Portugal. Materials and Methods: Ninety invasive Hi isolates from the Portuguese NIH collection were selected for WGS. More than half were NTHi (63.3%) and 32.2% of the strains belong to a pre-vaccine era. Genomes were assembled and both sequence type (ST) and serotype were determined by PCR and confirmed in silico. A core-single nucleotide polymorphism-based phylogenetic tree was reconstructed to analyze overall genomic diversity between strains. Strains were further characterized by identifying the presence and genetic profile of genes related to antibiotic resistance and virulence factors, namely genes involved in adherence, host immune evasion, iron acquisition and lipooligosaccharides (LOS). Results: Preliminary results show high ST heterogeneity among NTHi, contrasting with the homogeneity of ST for Hib strains (all ST6, except one). Core-SNP-based analysis revealed that all strains were distinguishable by more than 140,000 single nucleotide variant sites, with a highest genetic diversity observed between NTHi (overall ~35,000 nucleotide differences). Interestingly, although all Hib segregated together, the ST282 Hib strain possessed a distinct genome profile, diverging by ~17,200 nucleotide differences from ST6, while these overall diverged between them by ~2,480. Differential presence of important virulence factors was observed among strains, namely for hia/hsf, hmw1/hmw2, hap and iga, with distinct genomic profiles observed between strains, requiring in-depth analysis. Curiously, 90% of NTHi had the lgtA LOS-coding gene which was absent in all Hib. Additionally, five genes coding for other LOS were found to be simultaneously present or absent among NTHi strains, most belonging to a post-vaccine era, indicating a potential cluster of circulating strains. Conclusions: Overall, we expect that the integrative analysis of all Hi isolates will strengthen the characterization of the genomic features in pre- and post-vaccine era, ultimately contributing to the understanding of the scenario of strains circulating in Portugal throughout more than 20 years.
    2017-09Conference object Restricted access Show more