A Sensitive and Rapid Wastewater Test for SARS-COV-2 and Its Use for the Early Detection of a Cluster of Cases in a Remote Community.

Throughout the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has been used to monitor trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence in the community. A major challenge in establishing wastewater surveillance programs, especially in remote areas, is the need for a well-equipped laboratory for sample analysis. Currently, no options exist for rapid, sensitive, mobile, and easy-to-use wastewater tests for SARS-CoV-2. The performance of the GeneXpert system, which offers cartridge-based, rapid molecular clinical testing for SARS-CoV-2 in a portable platform, was evaluated using wastewater as the input. The GeneXpert demonstrated a SARS-CoV-2 limit of detection in wastewater below 32 copies/mL with a sample processing time of less than an hour. Using wastewater samples collected from multiple sites across Canada during February and March 2021, a high overall agreement (97.8%) was observed between the GeneXpert assay and laboratory-developed tests regarding the presence or absence of SARS-CoV-2. Additionally, with the use of centrifugal filters, the detection threshold of the GeneXpert system was improved to <10 copies/mL in wastewater. Finally, to support on-site wastewater surveillance, GeneXpert testing was implemented in Yellowknife, a remote community in Northern Canada, where its use successfully alerted public health authorities to undetected transmission of COVID-19. The identification of SARS-CoV-2 in wastewater triggered clinical testing of recent travelers and identification of new COVID-19 cases/clusters. Taken together, these results suggest that GeneXpert is a viable option for surveillance of SARS-CoV-2 in wastewater in locations that do not have access to established testing laboratories. IMPORTANCE Wastewater-based surveillance is a powerful tool that provides an unbiased measure of COVID-19 prevalence in a community. This work describes a sensitive wastewater rapid test for SARS-CoV-2 based on a widely distributed technology, the GeneXpert. The advantages of an easy-to-use wastewater test for SARS-CoV-2 are clear: it supports surveillance in remote communities, improves access to testing, and provides faster results allowing for an immediate public health response. The application of wastewater rapid testing in a remote community facilitated the detection of a COVID-19 cluster and triggered public health action, clearly demonstrating the utility of this technology. Wastewater surveillance will become increasingly important in the postvaccination pandemic landscape as individuals with asymptomatic/mild infections continue transmitting SARS-CoV-2 but are unlikely to be tested.

Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin.

Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of beta-lactamase when the latter was fused to a C-terminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the beta-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.

Creation of a fully functional cysteine-less variant of osmosensor and proton-osmoprotectant symporter ProP from Escherichia coli and its application to assess the transporter's membrane orientation.

Transporter ProP of Escherichia coli is an osmosensor and an osmoprotectant transporter. Previous results suggest that medium osmolality determines the proportions of ProP in active and inactive conformations. A cysteine-less (Cys-less) variant was created and characterized as a basis for structural and functional analyses based on site-directed Cys substitution and chemical labeling of ProP. Parameters describing the osmosensory and osmoprotectant transport activities of Cys-less ProP-(His)(6) variants were examined, including the threshold for osmotic activation and the absolute transporter activity at high osmolality (in both cells and proteoliposomes), the dependence of K(M) and V(max) for proline uptake on osmolality, and the rate constant for transporter activation in response to an osmotic upshift (in cells only). Variant ProP-(His)(6)-C112A-C133A-C264V-C367A (designated ProP) retained similar activities to ProP-(His)(6) in both cells and proteoliposomes. The bulky Val residue was favored over Ala or Ser at position 264, whereas Val strongly impaired function when placed at position 367, highlighting the importance of residues at those positions for osmosensing. In the ProP* background, variants with a single Cys residue at positions 112, 133, 241, 264, 293, or 367 retained full function. The native Cys at positions 112, 133, 264, and 367, predicted to be within transmembrane segments of ProP, were poorly reactive with membrane-impermeant thiol reagents. The reactivities of Cys at positions 241 and 293 were consistent with exposure of those residues on the cytoplasmic and periplasmic surfaces of the cytoplasmic membrane, respectively. These observations are consistent with the topology and orientation of ProP predicted by hydropathy analysis.