Community Surveillance of Omicron in Ontario: Wastewater-based Epidemiology Comes of Age

Wastewater-based surveillance of SARS-CoV-2 RNA has been implemented at building, neighbourhood, and city levels throughout the world. Implementation strategies and analysis methods differ, but they all aim to provide rapid and reliable information about community COVID-19 health states. A viable and sustainable SARS-CoV-2 surveillance network must not only provide reliable and timely information about COVID-19 trends, but also provide for scalability as well as accurate detection of known or unknown emerging variants. Emergence of the SARS-CoV-2 variant of concern Omicron in late Fall 2021 presented an excellent opportunity to benchmark individual and aggregated data outputs of the Ontario Wastewater Surveillance Initiative in Canada; this public health-integrated surveillance network monitors wastewaters from over 10 million people across major population centres of the province. We demonstrate that this coordinated approach provides excellent situational awareness, comparing favourably with traditional clinical surveillance measures. Thus, aggregated datasets compiled from multiple wastewater-based surveillance nodes can provide sufficient sensitivity (i.e., early indication of increasing and decreasing incidence of SARS-CoV-2) and specificity (i.e., allele frequency estimation of emerging variants) with which to make informed public health decisions at regional- and state-levels.Ontario Ministry of the Environment, Conservation and Parks|| Genome Canada and Ontario Genomics (OGI-209)||NSERC (ALLRP 555041-20 to C.O.)||Ontario Clean Water Agenc

Interactions between flagellar and type III secretion proteins in <it>Chlamydia pneumoniae</it>

Abstract Background Flagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments. Chlamydia pneumoniae is an obligate, intracellular pathogen whose genome contains at least three orthologs of flagellar proteins, namely FliI, FlhA and FliF, but the role of these proteins remains unknown. Results Full length FliI, and fragments of FlhA, FliF, and FliI, were cloned and expressed as either GST or His tagged proteins in E. coli. The GST-tagged full length FliI protein was shown to possess ATPase activity, hydrolyzing ATP at a rate of 0.15 ± .02 μmol min-1 mg-1 in a time- and dose-dependant manner. Using bacterial-2-hybrid and GST pull-down assays, the N-terminal domain of FliI was shown to interact with the cytoplasmic domain of FlhA, but not with FliF, and the cytoplasmic domain of FlhA was shown to interact with the C-terminus of FliF. The absence of other flagellar orthologs led us to explore cross-reaction of flagellar proteins with type III secretion proteins, and we found that FliI interacted with CdsL and CopN, while FlhA interacted with CdsL and Cpn0322 (YscU ortholog CdsU). Conclusions The specific interaction of the four orthologous flagellar proteins in C. pneumoniae suggests that they interact in vivo and, taken together with their conservation across members of the chlamydiae sps., and their interaction with T3S components, suggests a role in bacterial replication and/or intracellular survival.</p

Characterization of the Putative Type III Secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae▿

Type III secretion (T3S) is utilized by a wide range of gram-negative bacterial pathogens to allow the efficient delivery of effector proteins into the host cell cytoplasm through the use of a syringe-like injectisome. Chlamydophila pneumoniae is a gram-negative, obligate intracellular pathogen that has the structural genes coding for a T3S system, but the functionality of the system has not yet been demonstrated. T3S is dependent on ATPase activity, which catalyzes the unfolding of proteins and the secretion of effector proteins through the injectisome. CdsN (Cpn0707) is predicted to be the T3S ATPase of C. pneumoniae based on sequence similarity to other T3S ATPases. Full-length CdsN and a C-terminal truncation of CdsN were cloned as glutathione S-transferase (GST)-tagged constructs and expressed in Escherichia coli. The GST-tagged C-terminal truncation of CdsN possessed ATPase activity, catalyzing the release of ADP and Pi from ATP at a rate of 0.55 ± 0.07 μmol min−1 mg−1 in a time- and dose-dependent manner. CdsN formed oligomers and high-molecular-weight multimers, as assessed by formaldehyde fixation and nondenaturing polyacrylamide gel electrophoresis. Using bacterial two-hybrid and GST pull-down assays, CdsN was shown to interact with CdsD, CdsL, CdsQ, and CopN, four putative structural components of the C. pneumoniae T3S system. CdsN also interacted with an unannotated protein, Cpn0706, a putative CdsN chaperone. Interactions between CdsN, CdsD, and CopN represent novel interactions not previously reported for other bacterial T3S systems and may be important in the localization and/or function of the ATPase at the inner membrane of C. pneumoniae

Detection of cervical precancerous lesions with Aptima HPV assays using SurePath preservative fluid specimens

SurePath specimens from women referred to colposcopy were treated with Aptima Transfer Solution (ATS) before testing in Aptima HPV (AHPV) and Aptima HPV 16, 18/45 (AHPV-GT) assays. Untreated SurePath specimens were tested with the cobas HPV test. PreservCyt specimens were assessed for cytology and tested with AHPV. High-grade cervical intraepithelial neoplasia lesions served as the reference standard. Excellent agreement (95.5%; k=0.91) was observed for ATS-treated SurePath specimens between Tigris and Panther systems and between the PreservCyt and ATS-treated SurePath specimens (91.1%, k=0.81) with the AHPV assay on Tigris. Agreement between the AHPV and cobas assays with SurePath specimens was substantial (89.9%, k=0.80). AHPV sensitivity for CIN2+(n=147) was 91.2% for SurePath and PreservCyt. Cobas HPV sensitivity was 93.9% for SurePath specimens. AHPV testing of SurePath specimens was more specific (59.4%) than cobas (54.7%) (p<0.001). Detection and genotyping showed similar absolute and relative risks. ATS-treated SurePath specimens tested with AHPV and AHPV-GT assays showed similar performance with greater specificity than cobas HPV on SurePath specimens. Similar overall results were seen using a CIN3 disease endpoint. Keywords: Human papillomavirus, SurePath, PreservCyt, Cervical intraepithelial neoplasia, CIN2+, Aptima transfer solution (ATS