Syndromic approach for rapid detection and differentiation of human pathogenic alphaviruses

Background: Knowledge of epidemiology, pathogenesis, and public health burden is scarce for many arthropod-borne viruses (arboviruses). Insufficient knowledge is partly attributable to the lack of exhaustive laboratory diagnostics due to resource limitations. Among arboviruses, arthritogenic and encephalitogenic alphaviruses are globally widespread, can cause severe disease, and can co-occur regionally. Objectives: We developed and validated a multiplexed real-time reverse transcription-PCR assay for the detection of all alphaviruses commonly causing human disease except Barmah Forest virus. Study design: The assay combines five antigenic complex-specific assays and one Chikungunya virus (CHIKV)-specific assay in a single parallelized reaction. Results: Comparisons with previously published PCR-based protocols for broad alphavirus detection using 20 different human-pathogenic alphaviruses revealed a significantly higher sensitivity of the new multiplexed assay (Fisher’s exact test, p < 0.0001). Detection limits with the new assay ranged from 0.83 cps/μl of extracted O’nyong-nyong virus to 33.05 cps/μl of extracted Western equine encephalitis virus. Antigenic complexes could be clearly differentiated by reactivity, Ct values (t-test, p < 0.0025) and signal intensities (t-test, p < 0.0001), even when testing high alphavirus concentrations potentially capable of causing false-positive PCR results. Testing of high-titred cell culture supernatants of eight important non-alphaviral arboviruses, of 4308 serum samples collected from febrile patients in Benin and Peru, of seven CHIKV-positive diagnostic samples from Brazil, and of non-targeted alphaviruses confirmed excellent diagnostic performance by the new assay, including improved detection of CHIKV, Mayaro and Venezuelan equine encephalitis virus in clinical specimens. Conclusions: Short turn-around time, applicability in resource-limited settings, antigenic complex determination, and higher sensitivity compared to previously available tests make the new assay a useful tool for alphavirus surveillance and routine patient diagnostics.