Strengthening National Wastewater Surveillance: Insights from a 162-Laboratory Proficiency Testing Program in China

Wastewater-based epidemiology (WBE) has rapidly emerged as an indispensable and scalable approach for monitoring SARS-CoV-2 circulation within communities worldwide. To systematically assess and further strengthen the reliability, robustness, and comparability of national surveillance data, a nationwide and large-scale interlaboratory proficiency testing (PT) initiative was organized in December 2023 by the Chinese Center for Disease Control and Prevention. A total of 162 laboratories, spanning four hierarchical administrative tiers (provincial, capital city, prefectural, and county levels), were systematically evaluated using standardized SARS-CoV-2 RNA reference materials. Among them, 93.8% of participating laboratories achieved satisfactory analytical performance, 3.7% yielded questionable outcomes, and 2.5% were classified as unsatisfactory. Substantial methodological heterogeneity, especially in virus concentration protocols, nucleic acid extraction reagents, and PCR assay selection, was found to significantly affect detection accuracy and sensitivity. Laboratories using optimized polyethylene glycol (PEG) or automated high-affinity precipitation (AHAP) methods consistently showed superior detection sensitivity and analytical reliability. In contrast, increasing sample volumes did not yield significant improvement in performance, possibly due to the concurrent enrichment of PCR inhibitors. This comprehensive study provides an in-depth and systematic evaluation of the current WBE analytical landscape in China, identifies critical technical challenges, and proposes actionable, evidence-based recommendations to standardize, harmonize, and improve the national SARS-CoV-2 wastewater surveillance infrastructure.