Clinical and environmental surveillance of poliovirus type 2 outbreak using a novel specific real-time quantitative PCR assay, Israel, 2022-2023

Background An increase in the number of circulating vaccine derived poliovirus (VDPV) outbreaks is reported worldwide in recent years, necessitating improved surveillance and diagnostic tools. The current standard protocol for polio detection is lengthy, labor-intensive, and inefficient for direct wastewater testing. Methods Here we describe a multiplex poliovirus-2 (PV2) specific assay that directly detects PV2 RNA derived from Sabin-2 vaccine in wastewater and stool samples. The new PV2-specific assay was used for direct detection of PV2 in environmental and clinical samples. Results In-silico analysis suggested that the assay is specific for PV2 originated from Sabin-2 vaccine and identifies 98.3% of available sequences, including some with <95% similarity to the vaccine reference sequence. We show that the assay is specific for PV2 and does not identify 19 other enterovirus strains, including sabin-1 and Sabin-3 RNA. The new assay enabled fast detection of PV2 in suspected clinical samples, which was subsequently confirmed by isolation and sequencing. By analyzing 183 concentrated wastewater samples, we further show that the new assay specifically and directly identifies PV2 RNA, in the presence of PV1, PV3, and other enteroviruses RNA. Conclusions The assay provides quantitative results, allowing continuous monitoring of viral load dynamics. Comparison of the standard isolation protocol and the new assay showed excellent agreement in terms of specificity and sensitivity. This specific and rapid assay may therefore be a valuable tool for monitoring cVDPV2 outbreaks in real time, and promote the global efforts for eradication and containment poliovirus circulation