Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection

• DENV and CHIKV co-circulate and result in overlapping clinical presentations.
• A single-reaction pan-DENV–CHIKV rRT-PCR was designed.
• The pan-DENV–CHIKV rRT-PCR was evaluated using serum samples from Nicaragua.
• The pan-DENV–CHIKV rRT-PCR proved as sensitive as individual reference tests.
• This DENV–CHIKV multiplex test may reduce costs and improve molecular workflow.

BackgroundDengue virus (DENV) and chikungunya virus (CHIKV) now co-circulate throughout tropical regions of the world, with billions of people living at risk of infection. The differentiation of these infections is important for epidemiologic surveillance as well as clinical care, though widely-used molecular diagnostics for DENV and CHIKV require the performance of two to four separate PCR reactions for detection.ObjectivesIn the current study, we sought to develop and evaluate a single-reaction, multiplex real-time RT-PCR (rRT-PCR) for the detection and differentiation of DENV and CHIKV (the pan-DENV–CHIKV rRT-PCR).Study designFrom an alignment of all available CHIKV complete genome sequences in GenBank, a new CHIKV rRT-PCR was designed for use in multiplex with a previously described assay for pan-DENV detection. Analytical evaluation was performed in accordance with published recommendations, and the pan-DENV–CHIKV rRT-PCR was clinically compared to reference molecular diagnostics for DENV and CHIKV using 182 serum samples from suspected cases in Managua, Nicaragua.ResultsThe pan-DENV–CHIKV rRT-PCR had a dynamic range extending from 7.0 to 2.0 log10 copies/μL for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9–37.4 copies/μL. The pan-DENV–CHIKV rRT-PCR detected DENV in 81 patients compared to 75 using a reference, hemi-nested DENV RT-PCR, and it demonstrated perfect agreement with a reference CHIKV rRT-PCR (54 positive samples).ConclusionsThe single-reaction, multiplex format of the pan-DENV–CHIKV rRT-PCR, combined with sensitive detection of both viruses, has the potential to improve detection while decreasing testing costs and streamlining molecular workflow.