Establishment of Pseudovirus-Based Reference Materials and Nationwide External Quality Assessment for Arboviruses during the 2025 Chikungunya Outbreak

BACKGROUND: Emerging arboviruses such as chikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV) pose growing public health challenges, demanding reliable diagnostic systems to support outbreak responses. During the July 2025 CHIKV outbreak in China, the absence of domestic nucleic acid reference materials (RMs) and external quality assessment (EQA) programs hindered diagnostic standardization. METHODS: We developed pseudovirus-based RNA RMs for CHIKV, DENV, and ZIKV and certified their concentrations using digital PCR across 9 laboratories for each virus, ensuring stability and commutability with WHO international standards and clinical samples. Leveraging these RMs, China conducted its first nationwide arbovirus nucleic acid EQA program. RESULTS: All three RMs were officially approved by China's State Administration for Market Regulation, with codes GBW(E)091403, GBW(E)091405, and GBW(E)091409 for CHIKV, DENV, and ZIKV, respectively. The EQA program involved 937 laboratories across 31 provinces and employed 51 amplification kits. Over 97.5% of laboratories achieved perfect scores and >99% were qualified, reflecting strong overall testing capacity. Crucially, the EQA revealed systematic gaps: institutional differences in testing scope and performance were evident, pooled testing in blood stations missed low CHIKV levels, and reagent-dependent differences in Cycle threshold values and amplification efficiency (78.9% to 146.4%) affected low-concentration CHIKV and DENV-2 detection. CONCLUSIONS: These findings underscore the power of large-scale EQA to expose hidden inter-laboratory and reagent inconsistencies. By integrating standardized RMs with nationwide EQA, this work provides a robust framework for diagnostic harmonization, drives quality improvement, and strengthens preparedness for future arboviral threats.