Safety and Stability of Samples Stored on Filter Paper for Molecular Arbovirus Diagnosis

Abstract: Expanding urbanization, climate change, and population growth contribute to increased transmission and spread of arthropod-borne viruses (arboviruses), many of which cause severe disease in humans. Pathogenic arboviruses include dengue, Zika, tick-borne encephalitis, and sindbis viruses, which together threaten more than half the global population. Thus, there is a constant need for safe, specific, and sensitive molecular tests to identify early-stage infections for accurate diagnosis and molecular epidemiological data for disease prevention and control. The study tested the biosafety of using FTA™ cards when working with pathogenic arboviruses by conducting an infectivity assay using sindbis virus. Conditions for RNA extraction and storage of arboviruses on FTA were analyzed by measuring viral RNA (vRNA) stability using a SYBR-Green, Pan-Flavi RT-qPCR method composed of degenerate primers able to detect a variety of flaviviruses. Data from a Pan-Flavi RT-qPCR study comprising of 222 clinical blood and serum samples collected from a 2018 dengue virus outbreak in Hanoi (Vietnam) was analyzed to establish applicability of FTA for molecular epidemiology and diagnosis. Results showed that sindbis virus infectivity was inhibited by FTA-adsorption. FTA-adsorbed arboviruses were extracted with the highest yield using Trizol extraction and were preserved at storage at 4-20ºC for up to 30 days. The results showed that clinical blood samples acquired higher yields of vRNA for molecular testing than serum samples and that it may be possible to perform sequencing for genomic analysis. The study suggests that FTA cards may facilitate the storage and transportation of adsorbed arboviruses for downstream molecular epidemiological and diagnostic tests.