Conectar
by Anne-Fleur Griffon, Loeïza Rault, Clément Tanvet, Etienne Simon-Lorière, Myrielle Dupont-Rouzeyrol, Catherine Inizan
BackgroundComparing the in vitro fitness of dengue virus (DENV) isolates is a pivotal approach to assess the contribution of DENV strains’ replicative fitness to epidemiological contexts, including serotype replacements. Competition assays are the gold standard to compare the in vitro replicative fitness of viral strains. Implementing competition assays between DENV serotypes requires an experimental setup and an appropriate read-out to quantify the viral progeny of strains belonging to different serotypes.
MethodsIn the current study, we optimized an existing serotyping qRT-PCR by adapting primer/probe design and multiplexing the serotype-specific qRT-PCR reactions, allowing to accurately detect and quantify all four DENV serotypes. We next developed an in vitro competition assay to compare the replicative fitness of two DENV serotypes in the human hepatic cell line HuH7.
FindingsThe qRT-PCR was specific, and had a limit of detection below 7.52, 1.19, 3.48 and 1.36 genome copies/µL, an efficiency of 1.993, 1.975, 1.902, 1.898 and a linearity (R²) of 0.99975, 0.99975, 0.99850, 0.99965 for DENV-1, −2, −3 and −4, respectively. Challenge of this multiplex serotype-specific qRT-PCR on mixes of viral supernatants containing known concentrations of strains from two serotypes evidenced an accurate quantification of the amount of genome copies of each serotype. Quantification of the viral progeny of each serotype in the inoculum and the supernatant of competition assays using the serotype-specific multiplex qRT-PCR unveiled an enrichment of the supernatant in DENV-1 genome copies, uncovering the enhanced replicative fitness of this DENV-1 isolate.
ConclusionsThis optimized qRT-PCR combined with a relevant cellular model allowed to accurately quantify the viral progeny of two DENV strains belonging to two different serotypes in a competition assay, allowing to determine which strain had a replicative advantage. This reliable experimental setup is adaptable to the comparative study of the replicative fitness of any DENV serotypes.
Stroke is a leading cause of death and disability in the Caribbean, yet there is limited published information on the availability and utilisation of diagnostic imaging and treatment methods. Inequities in healthcare infrastructure, access to neuroimaging and acute treatment options may contribute to poorer outcomes following stroke, particularly in the low-resource settings that characterise most of the Caribbean region. The objective of this review is to map the literature on access to diagnostic and therapeutic modalities for adult stroke care in the Caribbean to identify potential limitations in acute treatment and examine how restricted access may impact outcomes. The resulting data can help inform strategies for improving access to stroke care in resource-limited communities.
We will apply a three-step strategy based on the Joanna Briggs Institute methodological framework: first, a limited search to identify relevant articles; second, selection of key search terms; third, implementation into a comprehensive search strategy. The query will range from 1 January 1995 to 1 June 2025 (date of final search). Search results will be extracted and screened by two independent reviewers, and findings will be presented in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews guidelines. We will consider studies focusing on ischaemic and haemorrhagic stroke in the Caribbean, emphasising access to diagnostic imaging, stroke centres, prehospital management and emergent treatment. Studies examining acute stroke management capacity within the region will be considered. Studies will be excluded if they: focus exclusively on primary stroke prevention, postacute care, longitudinal care pathways for stroke victims or paediatric populations; are unrelated to stroke diagnosis or treatment or are conducted outside the Caribbean.
This protocol aims to perform secondary analysis of previously published literature; therefore, ethical approval is not required. The results of this review will be disseminated through academic conferences and peer-reviewed publication.
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