Mobile wastewater and environmental surveillance in low-resource settings: validating an integrated laboratory workflow for pathogen detection

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Abstract Mobile laboratories (MLs) offer a flexible platform for on-site wastewater and environmental surveillance, particularly in regions with limited laboratory capacity. Effective ML deployment, however, requires optimized molecular workflows that match local infrastructure, equipment, and workforce constraints. Here, we present an integrated ML workflow that combines Oxford Nanopore Technologies (ONT) sequencing for shotgun metagenomics, multiplex metabarcoding for microbial community profiling, and Biomeme-based qPCR for targeted pathogen detection. We introduce and validate a multiplexed ONT small subunit rRNA amplicon sequencing approach that enables simultaneous profiling of bacteria, archaea, and microeukaryotes in complex environmental samples. Sample pretreatment and nucleic acid extraction was tested for combined chemical–mechanical lysis followed by magnetic bead purification. Workflow performance was assessed by analyzing a known mock community (ZymoBIOMICS) and wastewater samples spiked with inactivated monkeypox virus (MPXV), demonstrating accurate taxonomic representation and sensitive MPXV detection. The multiplex metabarcoding workflow enabled rapid, high-resolution community profiling and showed stronger concordance with qPCR-based pathogen detection than whole-genome and whole-transcriptome amplification workflows. MPXV tiled amplicon sequencing was successfully incorporated into the ML protocol, supporting near-real-time genomic characterization in field settings. Across diverse environmental matrices, a multiple displacement amplification (MDA)-based metagenomic workflow combined with ML nucleic acid extraction reliably reproduced the composition of the mock community. Overall, integrating ONT sequencing, MDA-based metagenomics, multiplex metabarcoding, and mobile qPCR provides an efficient surveillance platform within a One Health framework. While key components of the workflow were validated under controlled laboratory conditions, pilot testing on environmental samples from sub-Saharan Africa demonstrates its applicability to real-world matrices. Graphical abstract

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