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We assessed the value of wastewater sequencing for monitoring influenza circulation in Colorado, USA, focusing on the emergence of influenza A(H3N2) subclade K. We detected subclade K in wastewater 31 days before clinical detection. Our results demonstrated that wastewater sequencing can provide real-time situational awareness for public health officials.
Seasonal influenza A and B viruses cause significant illness and death annually ( 1 , 2 ). During the early 2025–26 influenza season, an antigenetically drifted influenza A(H3N2) strain, known as subclade K, emerged with reduced antibody binding to contemporaneous vaccine strains ( 3 , 4 ). The emergence of subclade K was correlated with early seasonal influenza activity observed in several countries ( 5 ). Colorado was among 3 states in the United States to observe early influenza activity based on outpatient respiratory illness reported to ILINet ( 6 ) . By the week ending November 22, 2025, Colorado reported high activity.
The Colorado Department of Public Health and Environment supplemented conventional surveillance, including hospitalization rates, clinical specimen sequencing, and wastewater detection using digital PCR (dPCR), with wastewater sequencing for genetic surveillance. We sequenced the influenza A virus hemagglutinin (HA) gene from wastewater and assessed its added value for monitoring influenza trends during the respiratory illness season.
Figure 1 . Comparison of hospitalizations, clinical specimens sequenced, and wastewater detection in study of early-season detection of influenza A(H3N2) subclade K in wastewater, Colorado, USA, 2025–2026. A) Influenza hospitalizations of influenza A...
Colorado requires the reporting of influenza-associated hospitalizations. We retrieved data for influenza-associated hospitalizations with admission dates of September 1, 2025–February 14, 2026, from the statewide surveillance system. During that period, Colorado recorded 4,426 influenza A–associated hospitalizations and 110 influenza B–associated hospitalizations. Cases began increasing the week ending November 25, 2025, and peaked the week ending December 27, 2025 ( Figure 1 , panel A).
The state laboratory received up to 5 influenza A–positive clinical specimens weekly from hospitals of both inpatient and outpatient cases participating in the statewide influenza sentinel surveillance system. We subtyped specimens and sequenced those meeting sequencing criteria ( Appendix ); we sequenced 812 specimens during September 1, 2025–February 14, 2026. Of those specimens, 720 (89%) were influenza A(H3N2). Of those samples, we characterized 704 (98%) as influenza A(H3N2) subclade K (hereafter, subclade K) ( Figure 1 , panel B). The first sample characterized as subclade K was collected on October 12, 2025. Colorado’s right-size target is 50 samples per week to confidently provide situational awareness; we reached that threshold the week ending December 6, 2025.
Established in 2020, the Colorado Wastewater Surveillance Program monitors wastewater across the state for respiratory viruses and emerging pathogens…