Victor Vusi Mabasa

Inferring epidemic trajectories of viral infections from wastewater data can be a useful addition to clinical-based surveillance, as it provides low cost, population-level data that includes both symptomatic and asymptomatic individuals who contribute to the sewer system. However, methods for analyzing wastewater data have been primarily applied to high-resource settings. It remains an open question to what extent epidemic dynamics can also be estimated from wastewater data in low-resource settings, where measurements are less frequent and the underlying catchment population is not clearly characterized. We used SARS-CoV-2 wastewater data from the Gauteng Province in South Africa (June 2021 to March 2022). We used the R packages EpiSewer and EpiNow2 to estimate the effective reproduction number (Rt) from wastewater data and geographically matched clinical surveillance data, respectively. The comparison between wastewater and clinical Rt showed that the observed trends are not perfectly aligned. Despite these differences, wastewater and clinical Rt estimates identified similar transmission patterns, which were similar to the trend seen directly from the recorded data. Maximum wastewater Rt of 1.38 (95% CI: 1.17–1.44) was observed in early November 2021, whilst clinical Rt was 1.18 (90% CI: 0.85–1.27) in June 2021. The change in Rt aligned with an increase or decrease in recorded cases. Our findings demonstrate that, even with limited data, estimating epidemic trajectories is feasible, providing valuable insights for informing public health recommendations. However, whenever possible, we recommend using wastewater surveillance as a complementary tool for clinical surveillance.

Measles outbreaks have surged globally in recent years, but current surveillance systems have limited capacity to monitor measles virus (MeV) transmission and evolution at population scale. Although MeV can be detected in wastewater, the public health potential of wastewater genomic surveillance for MeV remains largely unexplored. Here, we deploy sensitive, low-cost MeV wastewater genomic surveillance combining virus concentration, whole-genome amplicon sequencing, and bioinformatic analysis alongside routine clinical genomic surveillance during the 2024-25 outbreak in South Africa. Integrated phylogenetic analyses of wastewater and clinical MeV genomes revealed previously undetected interprovincial spread and transmission links not captured by standard N450 sequencing. Our findings demonstrate that wastewater-integrated whole-genome surveillance expands the coverage and resolution of routine MeV monitoring and provides a scalable tool to advance measles control and elimination efforts.

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)

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.

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.

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.

Norovirus (NoV) GII.4 is the predominant genotype associated with gastroenteritis pandemics and new strains emerge every 2–3 years. Between 2008 and 2011, environmental studies in South Africa (SA) reported NoVs in 63% of the sewage-polluted river water samples. The aim of this study was to assess whether wastewater samples could be used for routine surveillance of NoVs, including GII.4 variants. From April 2015 to March 2016, raw sewage and effluent water samples were collected monthly from five wastewater treatment plants in SA. A total of 108 samples were screened for NoV GI and GII using real-time RT-qPCR. Overall 72.2% (78/108) of samples tested positive for NoVs with 4.6% (5/108) GI, 31.5% (34/108) GII and 36.1% (39/108) GI + GII strains being detected. Norovirus concentrations ranged from 1.02 × 10² to 3.41 × 10⁶ genome copies/litre for GI and 5.00 × 10³ to 1.31 × 10⁶ genome copies/litre for GII. Sixteen NoV genotypes (GI.2, GI.3, GI.4, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GII.9, GII.10, GII.14, GII.16, GII.17, GII.20, and GII.21) were identified. Norovirus GII.2 and GII.17 co-dominated and the majority of GII.17 strains clustered with the novel Kawasaki 2014 variant. Sewage surveillance facilitated detection of Kawasaki 2014 in SA, which to date has not been detected with surveillance in children with gastroenteritis <5 years of age. Combined surveillance in the clinical setting and environment appears to be a valuable strategy to monitor emergence of NoV strains in countries that lack NoV outbreak surveillance.