Browsing by Issue Date, starting with "2023-06-13"
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- Development of an amplicon-based sequencing approach in response to the global emergence of mpoxPublication . 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.
- Leukocyte Imbalances in Mucopolysaccharidoses PatientsPublication . Lopes, Nuno; Maia, Maria L.; Pereira, Cátia S.; Mondragão-Rodrigues, Inês; Martins, Esmeralda; Ribeiro, Rosa; Gaspar, Ana; Aguiar, Patrício; Garcia, Paula; Cardoso, Maria Teresa; Rodrigues, Esmeralda; Leão-Teles, Elisa; Giugliani, Roberto; Coutinho, Maria F.; Alves, Sandra; Macedo, M. FátimaMucopolysaccharidoses (MPSs) are rare inherited lysosomal storage diseases (LSDs) caused by deficient activity in one of the enzymes responsible for glycosaminoglycans lysosomal degradation. MPS II is caused by pathogenic mutations in the IDS gene, leading to deficient activity of the enzyme iduronate-2-sulfatase, which causes dermatan and heparan sulfate storage in the lysosomes. In MPS VI, there is dermatan sulfate lysosomal accumulation due to pathogenic mutations in the ARSB gene, leading to arylsulfatase B deficiency. Alterations in the immune system of MPS mouse models have already been described, but data concerning MPSs patients is still scarce. Herein, we study different leukocyte populations in MPS II and VI disease patients. MPS VI, but not MPS II patients, have a decrease percentage of natural killer (NK) cells and monocytes when compared with controls. No alterations were identified in the percentage of T, invariant NKT, and B cells in both groups of MPS disease patients. However, we discovered alterations in the naïve versus memory status of both helper and cytotoxic T cells in MPS VI disease patients compared to control group. Indeed, MPS VI disease patients have a higher frequency of naïve T cells and, consequently, lower memory T cell frequency than control subjects. Altogether, these results reveal MPS VI disease-specific alterations in some leukocyte populations, suggesting that the type of substrate accumulated and/or enzyme deficiency in the lysosome may have a particular effect on the normal cellular composition of the immune system.