Tracking SARS-CoV-2 and its variants in wastewater in Tunisia.

Wastewater-based genomic surveillance can improve community prevalence estimates and identify emerging variants of pathogens. Wastewater influents and treated effluents from six wastewater treatment plants (WWTPs) in Tunisia were analyzed between December 2021 and July 2022. Wastewater samples were analyzed with reverse transcription solid digital PCR (RT-sdPCR) and whole-genome sequencing to determine the amount of SARS-CoV-2 RNA and assign SARS-CoV-2 lineages. The virus variants detected in wastewater samples were compared with COVID-19 prevalence data. The quantitative results in wastewater influents revealed that viral RNA concentrations at the treatment plants corroborate with locally reported clinical cases and show an increase before the increment of clinically diagnosed new COVID-19 cases between April and July 2022. Delta and Omicron variants were identified in the Tunisian wastewater. Interestingly, the presence of variant BA.5 was detected in samples prior to its inclusion as a variant of concern (VOC) by the Tunisian National Health Authorities. SARS-CoV-2 was detected in wastewater effluents, indicating that the wastewater treatment techniques used in the majority of Tunisian WWTPs are inefficient in removing the virus traces. This study reports the first identification of SARS-CoV-2 VOCs in Tunisian wastewater samples.

Evaluation of three viral concentration methods for detection and quantification of SARS-CoV-2 in wastewater.

Wastewater-based epidemiology (WBE) could be useful as an early warning system for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic spread. Viruses are highly diluted in wastewater. Therefore, a virus concentration step is needed for SARS-CoV-2 wastewater detection. We tested the efficiency of three wastewater viral concentration methods: ultrafiltration (UF), electronegative membrane filtration and aluminum hydroxide adsorption-elution. We spiked wastewater with inactivated SARS-CoV-2 and we collected 20 other wastewater samples from five sites in Tunisia. Samples were concentrated by the three methods and SARS-CoV-2 was quantified by reverse transcription digital PCR (RT-dPCR). The most efficient method was UF with a mean SARS-CoV-2 recovery of 54.03 ± 8.25. Moreover, this method provided significantly greater mean concentration and virus detection ability (95%) than the two other methods. The second-most efficient method used electronegative membrane filtration with a mean SARS-CoV-2 recovery of 25.59 ± 5.04% and the least efficient method was aluminum hydroxide adsorption-elution. This study suggests that the UF method provides rapid and straightforward recovery of SARS-CoV-2 in wastewater.

Enterovirus Migration Patterns between France and Tunisia.

The enterovirus (EV) types echovirus (E-) 5, E-9, and E-18, and coxsackievirus (CV-) A9 are infrequently reported in human diseases and their epidemiologic features are poorly defined. Virus transmission patterns between countries have been estimated with phylogenetic data derived from the 1D/VP1 and 3CD gene sequences of a sample of 74 strains obtained in France (2000-2012) and Tunisia (2011-2013) and from the publicly available sequences. The EV types (E-5, E-9, and E-18) exhibited a lower worldwide genetic diversity (respective number of genogroups: 4, 5, and 3) in comparison to CV-A9 (n = 10). The phylogenetic trees estimated with both 1D/VP1 and 3CD sequence data showed variations in the number of co-circulating lineages over the last 20 years among the four EV types. Despite the low number of genogroups in E-18, the virus exhibited the highest number of recombinant 3CD lineages (n = 10) versus 4 (E-5) to 8 (E-9). The phylogenies provided evidence of multiple transportation events between France and Tunisia involving E-5, E-9, E-18, and CV-A9 strains. Virus spread events between France and 17 other countries in five continents had high probabilities of occurrence as those between Tunisia and two European countries other than France. All transportation events were supported by BF values > 10. Inferring the source of virus transmission from phylogenetic data may provide insights into the patterns of sporadic and epidemic diseases caused by EVs.