The role of wastewater-based epidemiology for SARS-CoV-2 in developing countries: Cumulative evidence from South Africa supports sentinel site surveillance to guide public health decision-making.

The uptake of wastewater-based epidemiology (WBE) for SARS-CoV-2 as a complementary tool for monitoring population-level epidemiological features of the COVID-19 pandemic in low-and-middle-income countries (LMICs) is low. We report on the findings from the South African SARS-CoV-2 WBE surveillance network and make recommendations regarding the implementation of WBE in LMICs. Eight laboratories quantified influent wastewater collected from 87 wastewater treatment plants in all nine South African provinces from 01 June 2021 to 31 May 2022 inclusive, during the 3rd and 4th waves of COVID-19. Correlation and regression analyses between wastewater levels of SARS-CoV-2 and district laboratory-confirmed caseloads were conducted. The sensitivity and specificity of novel 'rules' based on WBE data to predict an epidemic wave were determined. Amongst 2158 wastewater samples, 543/648 (85 %) samples taken during a wave tested positive for SARS-CoV-2 compared with 842 positive tests from 1512 (55 %) samples taken during the interwave period. Overall, the regression-co-efficient was 0,66 (95 % confidence interval = 0,6-0,72, R2 = 0.59), ranging from 0.14 to 0.87 by testing laboratory. Early warning of the 4th wave of SARS-CoV-2 in Gauteng Province in November-December 2021 was demonstrated. A 50 % increase in log copies of SARS-CoV-2 compared with a rolling mean over the previous five weeks was the most sensitive predictive rule (58 %) to predict a new wave. Our findings support investment in WBE for SARS-CoV-2 surveillance in LMICs as an early warning tool. Standardising test methodology is necessary due to varying correlation strengths across laboratories and redundancy across testing plants. A sentinel site model can be used for surveillance networks without affecting WBE finding for decision-making. Further research is needed to identify optimal test frequency and the need for normalisation to population size to identify predictive and interpretive rules to support early warning and public health action.

CrAssphage May Be Viable Markers of Contamination in Pristine and Contaminated River Water.

Viruses are the most biologically abundant entities and may be ideal indicators of fecal pollutants in water. Anthropogenic activities have triggered drastic ecosystem changes in rivers, leading to substantial shifts in chemical and biological attributes. Here, we evaluate the viability of using the presence of crAssphage as indicators of fecal contamination in South African rivers. Shotgun analysis revealed diverse crAssphage viruses in these rivers, which are impacted by chemical and biological pollution. Overall, the diversity and relative abundances of these viruses was higher in contaminated sites compared to pristine locations. In contrast to fecal coliform counts, crAssphage sequences were detected in pristine rivers, supporting the assertion that the afore mentioned marker may be a more accurate indicator of fecal contamination. Our data demonstrate the presence of diverse putative hosts which includes members of the phyla Bacteroidota, Pseudomonadota, Verrucomicrobiota, and Bacillota. Phylogenetic analysis revealed novel subfamilies, suggesting that rivers potentially harbor distinct and uncharacterized clades of crAssphage. These data provide the first insights regarding the diversity, distribution, and functional roles of crAssphage in rivers. Taken together, the results support the potential application of crAssphage as viable markers for water quality monitoring. IMPORTANCE Rivers support substantial populations and provide important ecosystem services. Despite the application of fecal coliform tests and other markers, we lack rapid and reproducible approaches for determining fecal contamination in rivers. Waterborne viral outbreaks have been reported even after fecal indicator bacteria (FIB) were suggested to be absent or below regulated levels of coliforms. This indicates a need to develop and apply improved indicators of pollutants in aquatic ecosystems. Here, we evaluate the viability of crAssphage as indicators of fecal contamination in two South African rivers. We assess the abundance, distribution, and diversity of these viruses in sites that had been predicted pristine or contaminated by FIB analysis. We show that crAssphage are ideal and sensitive markers for fecal contamination and describe novel clades of crAss-like phages. Known crAss-like subfamilies were unrepresented in our data, suggesting that the diversity of these viruses may reflect geographic locality and dependence.

Diarrhoeal Disease in Relation to Possible Household Risk Factors in South African Villages.

Diarrhoeal disease is a significant contributor to child morbidity and mortality, particularly in the developing world. Poor sanitation, a lack of personal hygiene and inadequate water supplies are known risk factors for diarrhoeal disease. Since risk factors may vary by population or setting, we evaluated the prevalence of diarrhoeal disease at the household level using a questionnaire to better understand household-level risk factors for diarrhoea in selected rural areas in South Africa. In a sub-sample of dwellings, we measured the microbial quality of drinking water. One in five households had at least one case of diarrhoea during the previous summer. The most widespread source of drinking water was a stand-pipe (inside yard) (45%) followed by an indoor tap inside the dwelling (29%). Storage of water was common (97%) with around half of households storing water in plastic containers with an opening large enough to fit a hand through. After adjusting for confounders, the occurrence of diarrhoea was statistically significantly associated with sourcing water from an indoor tap (Adjusted Odds Ratio (AOR): 2.73, 95% CI: 2.73, 1.14⁻6.56) and storing cooked/perishable food in non-refrigerated conditions (AOR: 2.17, 95% CI: 2.17, 1.44⁻3.26). The highest total coliform counts were found in water samples from kitchen containers followed by stand-pipes. Escherichia coli were most often detected in samples from stand-pipes and kitchen containers. One in four households were at risk of exposure to contaminated drinking water, increasing the susceptibility of the study participants to episodes of diarrhoea. It is imperative that water quality meets guideline values and routine monitoring of quality of drinking water is done to minimise diarrhoea risk in relevant rural communities. The security of water supply in rural areas should be addressed as a matter of public health urgency to avoid the need for water storage.

The reach of human health risks associated with metals/metalloids in water and vegetables along a contaminated river catchment: South Africa and Mozambique.

BACKGROUND: Anthropogenic pollution was identified as an environmental problem of concern when, in 2008, dozens of crocodiles died in the Olifants River catchment near the border of South Africa and Mozambique. Given the close proximity of households to the river and their making use of river water, we aimed to determine to what extent water pollution has an impact on health of indigent communities in South Africa and Mozambique in the catchment area. METHODS: Water and vegetable samples were collected from the study areas. Biota samples were washed with double de-ionized Milli-Q water and freeze-dried. Heavy metal analyses in water and vegetables were done by means of Inductively Coupled Plasma Optical Emission Spectroscopy. Metal concentrations were applied in a human health risk assessment to estimate health risks. RESULTS: Mean concentrations of antimony, arsenic, cadmium, chromium, mercury, molybdenum, nickel and selenium in water samples from South Africa exceeded the World Health Organization guidelines for safe levels of intake. Only iron exceeded the recommended guidelines in water samples from Mozambique. Metals/metalloids were found in lower concentrations at Mozambique sites downstream of South African sites. In vegetables, uranium was between 10 and 20 times above safe guidelines in South Africa and between 3 and 6 times in Mozambique. Arsenic in water samples posed the highest cancer risk. CONCLUSIONS: Even with a reduction in the metal concentrations in river water from South Africa to Mozambique, the potential to cause adverse human health impacts from direct use of polluted river water is evident in both countries.

Abundance of Pathogenic Escherichia coli Virulence-Associated Genes in Well and Borehole Water Used for Domestic Purposes in a Peri-Urban Community of South Africa.

In the absence of pipe-borne water, many people in Africa, especially in rural communities, depend on alternative water sources such as wells, boreholes and rivers for household and personal hygiene. Poor maintenance and nearby pit latrines, however, lead to microbial pollution of these sources. We evaluated the abundance of Escherichia coli and the prevalence of pathogenic E. coli virulence genes in water from wells, boreholes and a river in a South African peri-urban community. Monthly samples were collected between August 2015 and November 2016. In all, 144 water samples were analysed for E. coli using the Colilert 18 system. Virulence genes (eagg, eaeA, stx1, stx2, flichH7, ST, ipaH, ibeA) were investigated using real-time polymerase chain reaction. Mean E. coli counts ranged between 0 and 443.1 Most Probable Number (MPN)/100 mL of water sample. Overall, 99.3% of samples were positive for at least one virulence gene studied, with flicH7 being the most detected gene (81/140; 57.6%) and the stx2 gene the least detected gene (8/140; 5.7%). Both intestinal and extraintestinal pathogenic E. coli genes were detected. The detection of virulence genes in these water sources suggests the presence of potentially pathogenic E. coli strains and is a public health concern.

Environmental health outcomes and exposure risks among at-risk communities living in the Upper Olifants River Catchment, South Africa.

Potential exposure to water and air pollution and associated health impacts of three low-income communities in the Upper Olifants River Catchment, South Africa, was investigated through a cross-sectional epidemiological study comprising a household survey. Water samples were collected and analysed for microbial indicators and pathogens. Ambient air-monitoring included some of the criteria pollutants, as well as mercury and manganese. Associations between environmental exposure and health outcomes were analysed by means of logistic regression. Despite poor water and air quality episodes, the communities' self-perceived health was good with relatively low prevalence of reported health outcomes. Hygiene practices with respect to water collection and storage were often poor, and most likely contributed to the regularly contaminated water storage containers. Community proximity to the polluted stream was associated with increased prevalence in adverse health outcomes. This paper reports on preliminary results and additional multivariate analyses are necessary to further understand study results.

Genome Sequence of Vibrio cholerae G4222, a South African Clinical Isolate.

Vibrio cholerae, a Gram-negative pathogen autochthonous to the aquatic environment, is the causative agent of cholera. Here, we report the complete genome sequence of V. cholerae G4222, a clinical isolate from South Africa.