Multi-Pathogen Wastewater Surveillance enables Real-Time Targeted Public Health Interventions During the 2025 African Nations Championship Football Tournament

Technical Summary: Multi-Pathogen Wastewater Surveillance During the 2025 African Nations Championship

Problem Statement
Mass gatherings (MGs) create conditions conducive to the rapid spread of infectious diseases due to high population density and movement. While Wastewater-Based Surveillance (WBS) has been established as a critical tool in high-income settings, its operational feasibility and public health utility as a multi-pathogen surveillance mechanism in low- and middle-income countries (LMICs) during such events remain under-documented. Clinical surveillance often misses asymptomatic cases and faces logistical barriers, whereas WBS offers a non-invasive, cost-effective method to capture population-level pathogen shedding. This study addresses the gap in evidence regarding the implementation of multi-pathogen WBS in an LMIC context during a major sporting event.

Methodology
The study was conducted at the Mandela National Stadium in Namboole, Kampala, Uganda, during the African Nations Championship (CHAN) football tournament in August 2025.

• Study Design: A cross-sectional surveillance design targeting eight match days (August 4, 8, 11, 15, 18, 23, 26, and 29) to coincide with peak stadium occupancy.
• Sampling Strategy: 38 manholes, serving as tributary collection points for individual toilet facilities across the stadium, were sampled. Moore swabs (8-ply pads) were deployed at each manhole for 24-hour periods (7:00 a.m. to 7:00 a.m. the following day) to capture representative wastewater.
• Sample Processing: Collected swabs were transported to the Central Emergency Response and Surveillance Laboratory (CERSL). Pathogens were concentrated using Nanotrap® microbiome virus particles, followed by nucleic acid extraction using Qiagen kits.
• Detection: Extracted nucleic acids were analyzed via quantitative PCR (qPCR) using singleplex and multiplex real-time PCR kits. The panel targeted priority notifiable and epidemic-prone pathogens under Uganda's Integrated Disease Surveillance and Response (IDSR) system, including:
  • Enteric: Shigella spp., Salmonella spp., Rotavirus A/B/C, Enterovirus, Enteric Adenovirus, Vibrio cholerae.
  • Respiratory: SARS-CoV-2, Influenza A/B, Respiratory Syncytial Virus (RSV).
  • Emerging: Mpox (Monkeypox) virus.
• Data Analysis: Descriptive statistics were used to calculate positivity rates, co-detection patterns, and Cq value distributions. No formal inferential statistics were performed.

Key Results

• Overall Positivity: Of 304 samples collected, 259 (85.2%) tested positive for at least one pathogen.
• Pathogen Prevalence:
  • Enteric Pathogens: Predominated the detection profile. Shigella spp. was the most frequent (53.6%), followed by Rotavirus A (35.9%) and Enterovirus (32.2%). Rotavirus C and Enteric Adenovirus were also detected.
  • Mpox: Detected in 28.6% of samples (87/304) across multiple days, indicating circulation within the population despite low clinical reporting.
  • Respiratory Pathogens: Detected at lower frequencies: SARS-CoV-2 (11.8%) and Influenza B (8.2%). Influenza A, RSV, Rotavirus B, and Vibrio cholerae were not detected.
• Co-detection: Multi-pathogen presence was common; 69.1% of positive samples contained two or more pathogens.
• Temporal Variation: Pathogen diversity fluctuated daily, with up to eight distinct pathogens detected on a single day (August 23), coinciding with peak attendance.
• Cq Values: Enteric pathogens (e.g., Shigella, Rotavirus A) generally exhibited lower median Cq values (indicating higher nucleic acid concentrations) compared to respiratory pathogens like SARS-CoV-2 and Influenza B, which showed higher median Cq values.

Public Health Interventions
The surveillance findings directly triggered real-time public health actions during the tournament:

1. Risk Communication: Intensified messaging on handwashing and respiratory hygiene; media briefings were issued following Mpox detection.
2. Sanitation Reinforcement: Increased frequency of toilet cleaning, deployment of mobile handwashing stations, and targeted disinfection of high-touch surfaces using chlorine-based solutions.
3. Clinical Linkage: Strengthened syndromic surveillance with on-site triage and point-of-care testing for symptomatic individuals.
4. Environmental Assessment: Field investigations into food handling and wastewater infrastructure integrity.

Significance and Claims
The paper asserts that this study demonstrates the operational feasibility and public health utility of integrating multi-pathogen WBS into mass gathering preparedness frameworks within LMICs. Key claims include:

• Actionable Intelligence: WBS can provide near-real-time, population-level insights that inform targeted interventions (sanitation, hygiene, risk communication) without requiring individual consent or clinical testing.
• Scalability in Resource-Limited Settings: The study proves that complex, multi-pathogen surveillance can be successfully executed in open-city, resource-constrained environments using established laboratory protocols.
• Early Warning Capacity: The detection of Mpox and diverse enteric pathogens highlights WBS's potential to serve as an early warning system for emerging and re-emerging threats in crowded settings.
• Framework for Future Events: The authors propose a structured pathway for national integration of WBS, including pre-event baselining, real-time monitoring with alert thresholds, and post-event surveillance to strengthen epidemic intelligence.

The authors maintain a modest tone regarding limitations, noting the lack of pre-event baseline data, the absence of quantitative genome copy measurements, and the inability to directly link wastewater signals to specific clinical outcomes. However, they conclude that the successful implementation validates WBS as a scalable tool for enhancing data-driven decision-making during future mass gatherings and infectious disease threats.