Modeling and Laboratory Experiments to Assess Detection of Plasmodium falciparum in Wastewater: Insights for Wastewater-Based Surveillance of Malaria.

Malaria remains a major global health challenge, with eradication efforts constrained by clinical surveillance limitations. Using our previously developed modeling framework, we quantified Plasmodium falciparum wastewater detection probabilities and the conditions necessary for feasible malaria wastewater-based epidemiology (WBE). Modeling predicts malaria WBE to be a challenge in nonendemic countries: given an estimated P. falciparum shedding of 5.31 log10 genome copies (GC)/day per infection, case rates would need to increase >1000-fold to achieve a 50% detection probability, even using eight PCR replicates at an ideally low process limit of detection (PLOD) of 3.0 log10 GC/L. In contrast, ∼84% of endemic African countries could achieve this detection probability under identical conditions. To put these predictions into perspective, we conducted P. falciparum spike-in experiments to evaluate how wastewater persistence and freeze-thawing affect modeled outcomes and whether the predicted PLOD is attainable through recovery testing. First-order wastewater decay behavior was observed (0.035-0.11 h-1 at 4-35 °C), resulting in minimal (<0.1 log10 GC/L) impact on modeling results. However, freeze-thawing caused >70% DNA loss, and recovery using the HA membrane filtration approach was <2% in most cases. Nonetheless, PLODs below the ideal value are achievable. Collectively, the dual modeling-experimental results help guide future malaria WBE applications.