In 2024-2025, Réunion Island experienced a large outbreak driven by East-Central-South African (ECSA)-2 lineage of chikungunya virus (CHIKV), leading to the implementation of a vaccination campaign using live-attenuated IXCHIQ (VLA1553). Currently, distinguishing vaccine-derived viremia from wild-type infection relies on genomic sequencing, which is resource-intensive and not routinely available in many diagnostic laboratories. To address this gap, we developed a duplex real-time RT-qPCR assay specifically designed to differentiate VLA1553 from wild-type ECSA strains circulating in La Réunion. The assay targets the non-structural protein 3 hypervariable region, including the 61-amino-acid deletion characteristic of the VLA1553 backbone. Analytical performance was assessed using a panel of CHIKV isolates representing different lineages and a VLA1553-like strain generated using an Infectious Subgenomic Amplicon system. Clinical performance was evaluated using samples from recently vaccinated individuals and patients infected with ECSA-2 strains, and compared with two RT-qPCR assays routinely used by the French National Reference Centers for arboviruses. The duplex assay proved to be specific, detecting wild-type ECSA strains exclusively in the FAM channel and the vaccine strain exclusively in the HEX channel, with no cross-reactivity. It demonstrated high sensitivity, with LOD95 values of 0.9 and 1.1 copies/µL for the vaccine-specific and wild-type-specific targets, respectively. All clinical samples were correctly classified, including one vaccinated individual simultaneously infected with a circulating wild-type strain, as confirmed by sequencing. This duplex assay provides a rapid and reliable method to distinguish vaccine-derived from naturally-acquired CHIKV viremia, supporting more accurate clinical and epidemiological investigations in settings where vaccination and viral circulation overlap.
Pezzi et al. (2026) studied this question.