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Introduction <p>Wastewater-based surveillance (WBS) is a vital epidemiological tool for monitoring community-level pathogen transmission. Viral concentrations in wastewater are commonly normalized using fecal indicators such as Pepper Mild Mottle Virus (PMMoV) RNA; however, in tropical urban settings, PMMoV RNA signal may be influenced by seasonal rainfall, stormwater infiltration, and sanitation infrastructure, potentially compromising its reliability. This study aimed to characterize the seasonal and site-specific variability of PMMoV RNA in Kampala, Uganda, and to evaluate its suitability as a fecal indicator under tropical urban conditions.</p> Methods <p>Moore swab-based influent wastewater samples were collected weekly between March 2023 and May 2024 at four wastewater treatment facilities in the Kampala Metropolitan Area. PMMoV RNA was quantified using RT-qPCR and expressed as log₁₀ genomic copies per 100 mL (GC/100 mL). Between-facility differences were assessed using the Kruskal-Wallis H test with Dunn’s post-hoc correction. The association between PMMoV RNA and monthly average rainfall was quantified using Pearson’s correlation. A linear mixed model (LMM) with rainfall and site as fixed effects and a random intercept for site was fitted to partition the variance into rainfall-driven and structural sources.</p> Results <p>PMMoV RNA was detected in 239 of 244 samples (97.95%) across all four facilities. Concentrations exhibited significant spatial and temporal variability (Kruskal-Wallis H = 9.83, <i>p</i> = 0.020), driven by sewershed structure, including infrastructure type, hydraulic configuration, and inflow susceptibility rather than population size alone (per capita analysis: H = 84.29, <i>p</i> &lt; 0.0001). Naalya wastewater stabilization pond showed the highest and most stable concentrations (mean = 6.09 log₁₀ GC/100 mL; IQR = 1.03), while Bugolobi fecal sludge treatment plant showed the lowest central tendency (mean = 5.27) and greatest variability (IQR = 2.60), reflecting intermittent sludge inputs. A strong, significant negative correlation was identified between PMMoV RNA and monthly average rainfall (Pearson <i>r</i> = − 0.623, <i>p</i> = 0.013). The LMM confirmed rainfall as an independent, site-invariant predictor (β = −0.0077 log₁₀ GC/100 mL per mm, <i>p</i> &lt; 0.001), with a 100&#xa0;mm increase in rainfall corresponding to an approximately 6-fold reduction in PMMoV RNA concentration.</p> Conclusion <p>PMMoV RNA concentrations in Kampala’s wastewater are systematically and substantially modulated by rainfall and catchment infrastructure, with seasonal dilution sufficient to bias the interpretation of normalized pathogen signals if left unadjusted. These findings challenge the assumption of proportional dilution underpinning ratio-based normalization and highlight the need for seweshed characterization as a foundation for site selection and interpretation, alongside rainfall-adjusted interpretation frameworks, site-specific validation, and complementary fecal indicators in tropical urban WBS programs.</p>

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Seasonal and spatial variability of PMMoV in tropical urban wastewater-based surveillance: implications for accurate SARS-CoV-2 monitoring in Kampala, Uganda

  • 期刊论文

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  • 发表日期：

    2026年5月7日

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  • Andrew Nsawotebba,
  • Valeria Nakintu,
  • Innocent Morunyanga,
  • Jonathan Kabazzi,
  • Jordan Magala,
  • Samuel Jefferson Mutyaba,
  • Noah C. Hull,
  • Victor Vusi Mabasa,
  • Denis Smith Akejo,
  • Fatim Cham,
  • Susan Nabadda

摘要

Introduction

Wastewater-based surveillance (WBS) is a vital epidemiological tool for monitoring community-level pathogen transmission. Viral concentrations in wastewater are commonly normalized using fecal indicators such as Pepper Mild Mottle Virus (PMMoV) RNA; however, in tropical urban settings, PMMoV RNA signal may be influenced by seasonal rainfall, stormwater infiltration, and sanitation infrastructure, potentially compromising its reliability. This study aimed to characterize the seasonal and site-specific variability of PMMoV RNA in Kampala, Uganda, and to evaluate its suitability as a fecal indicator under tropical urban conditions.

Methods

Moore swab-based influent wastewater samples were collected weekly between March 2023 and May 2024 at four wastewater treatment facilities in the Kampala Metropolitan Area. PMMoV RNA was quantified using RT-qPCR and expressed as log₁₀ genomic copies per 100 mL (GC/100 mL). Between-facility differences were assessed using the Kruskal-Wallis H test with Dunn’s post-hoc correction. The association between PMMoV RNA and monthly average rainfall was quantified using Pearson’s correlation. A linear mixed model (LMM) with rainfall and site as fixed effects and a random intercept for site was fitted to partition the variance into rainfall-driven and structural sources.

Results

PMMoV RNA was detected in 239 of 244 samples (97.95%) across all four facilities. Concentrations exhibited significant spatial and temporal variability (Kruskal-Wallis H = 9.83, p = 0.020), driven by sewershed structure, including infrastructure type, hydraulic configuration, and inflow susceptibility rather than population size alone (per capita analysis: H = 84.29, p < 0.0001). Naalya wastewater stabilization pond showed the highest and most stable concentrations (mean = 6.09 log₁₀ GC/100 mL; IQR = 1.03), while Bugolobi fecal sludge treatment plant showed the lowest central tendency (mean = 5.27) and greatest variability (IQR = 2.60), reflecting intermittent sludge inputs. A strong, significant negative correlation was identified between PMMoV RNA and monthly average rainfall (Pearson r = − 0.623, p = 0.013). The LMM confirmed rainfall as an independent, site-invariant predictor (β = −0.0077 log₁₀ GC/100 mL per mm, p < 0.001), with a 100 mm increase in rainfall corresponding to an approximately 6-fold reduction in PMMoV RNA concentration.

Conclusion

PMMoV RNA concentrations in Kampala’s wastewater are systematically and substantially modulated by rainfall and catchment infrastructure, with seasonal dilution sufficient to bias the interpretation of normalized pathogen signals if left unadjusted. These findings challenge the assumption of proportional dilution underpinning ratio-based normalization and highlight the need for seweshed characterization as a foundation for site selection and interpretation, alongside rainfall-adjusted interpretation frameworks, site-specific validation, and complementary fecal indicators in tropical urban WBS programs.