External quality assurance of molecular serotype detection of dengue virus RNA among virus research and diagnostic laboratories in India

Background External Quality Assessment Schemes (EQAS) are essential for ensuring the accuracy, reliability, and comparability of molecular diagnostic testing across laboratory networks. To strengthen dengue molecular surveillance and laboratory quality systems in India, a Pan-Dengue Molecular Serotyping EQAS was developed and implemented among Virus Research and Diagnostic Laboratories (VRDLs) in India. Methods A nationwide EQAS was conducted in three rounds between December 2024 and February 2026. Proficiency testing panels consisting of five coded lyophilized serum samples (500 µL each) containing dengue-positive and dengue-negative specimens were prepared, characterized, validated, and distributed to participating laboratories. Homogeneity and stability assessments were performed prior to distribution. Participating laboratories tested the panels using their routine molecular diagnostic workflows and reported dengue detection and serotype identification results through the National Institute of Epidemiology (NIE) PT Panel Portal. Laboratory performance was evaluated based on concordance with assigned reference results. Results A total of 34, 40, and 56 laboratories enrolled in Round 1, Round 1 Phase-II, and Round 2, respectively. The corresponding response rates were 91.2%, 95.0%, and 94.6%. Complete concordance was achieved by all reporting laboratories in Round 1 (31/31) and Round 1 Phase-II (38/38). In Round 2, 50/53 laboratories (94.3%) achieved 100% concordance, while two and one laboratory achieved 80% and 60% concordance, respectively. The overall pass rate across all rounds was 97.5%. Conclusion The Pan-Dengue Molecular Serotyping EQAS highlighted a high level of proficiency among participating laboratories and effectively strengthened molecular diagnostic quality assurance within the national laboratory network. Regular implementation of such programmes will support reliable dengue surveillance, continuous quality improvement, and enhanced preparedness for dengue outbreaks in India.