A Validated National Framework for Multi-Pathogen Wastewater-Based Epidemiological Surveillance in Kenya: From Pathogen Prioritisation to Real- Time Public Health Intelligence

Abstract Sub-Saharan Africa carries a disproportionate burden of global infectious diseases. However, its surveillance architecture is still reactive, clinic-centered, and blind to community-level transmission from asymptomatic and undiagnosed carriers. Wastewater-based epidemiology (WBE) addresses this blind spot by measuring the aggregate pathogen signal shed from the contributing population. Here, we describe a validated national protocol for multipathogen WBE in Kenya, delivered as an end-to-end, quality-assured framework. In the protocol, we integrate five components: (i) a structured seven-criterion pathogen prioritisation matrix, applied to a 64-pathogen candidate list drawn from Kenya's Integrated Disease Surveillance and Response (IDSR), the WHO R&D Blueprint, and the Africa CDC priority-pathogen list, and refined through a country-specific feasibility gate that yields the operational surveillance panel and the five antimicrobial-resistance (AMR) marker sets described below; (ii) a 57-site national environmental sampling network spanning Nairobi, Mombasa, Kisumu, Garissa, and Isiolo counties, integrating ILRI's established environmental sites and the WHO Global Polio Eradication Initiative (GPEI) environmental surveillance network; (iii) standardised methods for passive Moore-swab and grab collection, viral concentration on Nanotrap Magnetic Virus Particles (hydrogel-based affinity capture on paramagnetic beads), nucleic-acid extraction, multi-pathogen screening by TaqMan Array Card (TAC), absolute quantification by digital PCR (dPCR), and broad-spectrum characterisation through metagenomic sequencing; (iv) bacterial culture with whole-genome sequencing for AMR profiling and viral whole-genome sequencing for variant tracking; and (v) a real-time geospatial analytics platform (Geo App) built on ArcGIS Online for visualisation, thresholded alerting, and bilingual (English/Kiswahili) situation reporting. The full cycle from collection to an actionable public health report is executable within 7 calendar days, a turnaround consistent with public health response requirements. The framework is designed for adaptation across low- and middle-income settings and is aligned with emerging global WBE reporting standards.