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 (preprint)

Background: The World Health Organisation recommends wastewater based epidemiology (WBE) for SARS-CoV-2 as a complementary tool for monitoring population-level epidemiological features of the COVID-19 pandemic. Yet, uptake of WBE in low-to-middle income countries (LMIC) is low. We report on findings from SARS-CoV-2 WBE surveillance network in South Africa, and make recommendations regarding implementation of WBE in LMICs Methods: Seven laboratories using different test methodology, quantified influent wastewater collected from 87 wastewater treatment plants (WWTPs) located in all nine South African provinces for SARS-CoV-2 from 01 June 2021 to 31 May 2022 inclusive, during the 3rd and 4th waves of COVID-19. Regression analysis with district laboratory confirmed SARS-CoV-2 case loads, controlling for district, size of plant and testing frequency was determined. The sensitivity and specificity of rules based on WBE data to predict an epidemic wave based on SARS-CoV-2 wastewater levels were determined. Results: Among 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, R squared=0.59), but ranged 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 SARS-CoV-2 compared with a rolling mean over the previous 5 weeks was the most sensitive predictive rule (58%) to predict a new wave. Conclusion: Variation in the strength of correlation across testing laboratories, and redundancy of findings across co-located testing plants, suggests that test methodology should be standardised and that surveillance networks may utilise a sentinel site model without compromising the value of WBE findings for public health decision-making. Further research is needed to identify optimal test frequency and the need for normalisation to population size, so as to identify predictive and interpretive rules to support early warning and public health action. Our findings support investment in WBE for SARS-CoV-2 surveillance in low and middle-income countries.

Molecular identification of SARS-CoV-2 variants of concern at urban wastewater treatment plants across South Africa (preprint)

The use of wastewater for SARS-CoV-2 surveillance is a useful complementary tool to clinical surveillance. The aims of this study were to characterize SARS-CoV-2 from wastewater samples, and to identify variants of concern present in samples collected from wastewater treatment plants in South African urban metros from April 2021 to January 2022. A total of 325 samples were collected from 15 wastewater treatment plants. Nucleic acids were extracted from concentrated samples, and subjected to amplicon-based whole genome sequencing. To identify variants of concerns and lineages, we used the Freyja tool (https://github.com/andersen-lab/Freyja), which assigns each sample with the prevalence of each variant present. We also used signature mutation analysis to identify variants in each wastewater treatment site. A heatmap was generated to identify patterns of emerging mutations in the spike gene using Excel conditional formatting. Using the Freyja tool, the Beta variant was detected and became predominate from April to June 2021 followed by the Delta variant and lastly the Omicron variant. Our heatmap approach was able to identify a pattern during the changes of predominate variant in wastewater with the emergence of mutations and the loss of others. In conclusion, sequencing of SARS-CoV-2 from wastewater largely corresponded with sequencing from clinical specimens. Our heatmap has the potential to detect new variants prior to emergence in clinical samples and this may be particularly useful during times of low disease incidence between waves, when few numbers of positive clinical samples are collected and submitted for testing. A limitation of wastewater sequencing is that it is not possible to identify new variants, as variants are classified based on known mutations in clinical strains.