The genogroup II genotype 4 (GII.4) noroviruses are a major cause of viral gastroenteritis. Since the emergence of the Sydney_2012 variant, no novel norovirus GII.4 variants have been reported. The high diversity of noroviruses and periodic emergence of novel strains necessitates continuous global surveillance. The aim of this study was to assess the diversity of noroviruses in selected wastewater samples from Pretoria, South Africa (SA) using amplicon-based next-generation sequencing (NGS). Between June 2018 and August 2020, 200 raw sewage and final effluent samples were collected fortnightly from two wastewater treatment plants in Pretoria. Viruses were recovered using skimmed milk flocculation and glass wool adsorption-elution virus recovery methods and screened for noroviruses using a one-step real-time reverse-transcription PCR (RT-PCR). The norovirus BC genotyping region (570–579 bp) was amplified from detected norovirus strains and subjected to Illumina MiSeq NGS. Noroviruses were detected in 81% (162/200) of samples. The majority (89%, 89/100) of raw sewage samples were positive for at least one norovirus, compared with 73% (73/100) of final effluent samples. Overall, a total of 89 different GI and GII RdRp-capsid combinations were identified, including 51 putative novel recombinants, 34 previously reported RdRp-capsid combinations, one emerging novel recombinant and three Sanger-sequencing confirmed novel recombinants.

Timely pathogen surveillance and reporting is essential for effective public health guidance. Web dashboards have become a key tool for communicating public health information to stakeholders, health care workers, and the broader community. Over the SARS-CoV-2 pandemic, wastewater surveillance has increasingly been incorporated into public health workflows for outbreak monitoring and response, enabling community-representative and low-cost monitoring to supplement clinical surveillance. However, the methods used for visualization and dissemination of clinical and wastewater surveillance data differ across programs, and best practices are yet to be defined. In this work, we demonstrate data workflows and dashboards used to perform wastewater-based public health surveillance in tandem with clinical data across local and national scales, leveraging custom-built, reproducible, and open-source software. Using a centralized data aggregation and analysis hub approach, we establish multiple data pipelines for data storage, wrangling, and standardized analyses, and deploy custom-built web dashboards that allow for immediate public release. We find that our approach is effective across scales, computing architectures, and dissemination strategies, and provides an adaptable model to incorporate additional pathogens and epidemiological data.

Background Sensitive clinical surveillance and high vaccination coverage are required to meet the WHO 2030 measles elimination target. Whilst wastewater and environmental surveillance (WES) has proven usefulness in polio surveillance, it has not been applied to measles control. We describe the development of digital RT-PCR (RT-dPCR) for detection and quantification of measles virus (MeV) in wastewater, and application to retained concentrated samples obtained from 28 national sentinel SARS-CoV-2 wastewater surveillance sites and 19 localised collection points in a single province before, during and after a measles outbreak of over 2,000 laboratory-confirmed cases. Methods We validated a RT-dPCR assay incorporating WHO-recommended primers for detection and differentiation of wild-type and vaccine genotypes. We applied this to retained wastewater samples concentrated by ultrafiltration and stored at −20°C. We compared wastewater findings by district and epidemiological weeks with laboratory-confirmed measles case data obtained from national fever-rash surveillance for districts with adequate surveillance indicators (>2/100,000 non-measles cases) Findings Amongst 2,149 wastewater concentrates obtained between 16 February 2021 and 08 March 2024, we identified 43 (2%) samples containing MeV RNA in concentrations ranging from 2,04-6,11 genome copies/mL. MeV genotype A (vaccine strain) was co-detected in wastewater in 6 instances along with non-A (wild-type) genotypes. Comparison of wastewater and clinical fever-rash surveillance data by epidemiological week and district identified 27 district-time instances where at least one positive wastewater sample was identified, of which 14 (52%) districts reported at least one laboratory-confirmed clinical case in that same epidemiological week. Amongst districts with positive wastewater samples, wastewater surveillance detected MeV in 13/27 (48%) time-district pairs when clinical surveillance failed to detect cases. Amongst district-time instances where at least one laboratory-confirmed measles case was detected (N=127), MeV was detected in at least one wastewater sample collected in that same epidemiological week in 14 (11%) districts Interpretation Wastewater surveillance may be a useful complementary surveillance tool for identification of districts with ongoing measles circulation. MeV detection rates may be improved by real-time testing to limit RNA degradation and improved concentration and nucleic acid extraction processes. Ongoing wastewater surveillance for MeV should be conducted in order to understand the role of WES in measles control and elimination. Funding This work was funded by BMGF (INV-049271)

Norovirus (NoV) is the second most important cause of viral diarrheal disease in children worldwide after rotavirus and is estimated to be responsible for 17% of acute diarrhea in low-income countries. This study aimed to identify and report NoV genotypes in Mozambican children under the age of five years with acute diarrhea. Between May 2014 and December 2015, stool specimens were collected within the Mozambique Diarrhea National Surveillance (ViNaDia) and tested for NoV genogroups I (GI) and II (GII) using conventional reverse transcriptase-polymerase chain reaction (RT-PCR). Partial capsid and RNA-dependent RNA polymerase (RdRp) nucleotide sequences were aligned using the Muscle tool, and phylogenetic analyses were performed using MEGA X. A total of 204 stool specimens were tested for NoV. The detection rate of NoV was 14.2% (29/204). The presence of NoV was confirmed, by real-time RT-PCR (RT-qPCR), in 24/29 (82.8%) specimens, and NoV GII predominated (70.8%; 17/24). NoV GII.4 Sydney 2012[P31] was the predominant genotype/P-type combination detected (30.4%; 7/23). This is the first study which highlights the high genetic diversity of NoV in Mozambican children and the need to establish a continuous NoV surveillance system.

Growing global concerns over water scarcity, worsened by climate change, drive wastewater reclamation efforts. Inadequately treated wastewater presents significant public health risks. Previous studies in South Africa (SA) have reported high norovirus levels in final effluent and sewage-polluted surface water, indicating pathogen removal inefficiency. However, the viability of these virions was not explored. This study assessed human norovirus viability in final effluent from wastewater treatment works (WWTWs) in Pretoria, SA. Between June 2018 and August 2020, 200 samples were collected from two WWTWs, including raw sewage and final effluent. Norovirus concentrations were determined using in-house RNA standards. Viability of noroviruses in final effluent was assessed using viability RT-qPCR (vPCR) with PMAxx™-Triton X-100. There was no significant difference in GI concentrations between raw sewage (p = 0.5663) and final effluent (p = 0.4035) samples at WWTW1 and WWTW2. WWTW1 had significantly higher GII concentrations in raw sewage (p < 0.001) compared to WWTW2. No clear seasonal pattern was observed in norovirus concentrations. At WWTW1, 50% (7/14) of GI- and 64.9% (24/37) of GII-positive final effluent samples had no quantifiable RNA after vPCR. At WWTW2, the majority (92.6%, 25/27) of GII-positive final effluent samples showed a 100% RNA reduction post vPCR. PMAxx™-Triton X-100 vPCR provides a more accurate reflection of discharge of potentially viable noroviruses in the environment than standard RT-qPCR. Despite significant reductions in potentially viable noroviruses after wastewater treatment, the levels of potentially viable viruses in final effluent are still of concern due to the high initial load and low infectious dose of noroviruses.