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Wastewater-based surveillance (WBS) of SARS-CoV-2 offers a complementary tool for clinical surveillance to detect and monitor Coronavirus Disease 2019 (COVID-19). Since both symptomatic and asymptomatic individuals infected with SARS-CoV-2 can shed the virus through the fecal route, WBS has the potential to measure community prevalence of COVID-19 without restrictions from healthcare-seeking behaviors and clinical testing capacity. During the Omicron wave, the limited capacity of clinical testing to identify COVID-19 cases in many jurisdictions highlighted the utility of WBS to estimate disease prevalence and inform public health strategies. However, there is a plethora of in-sewage, environmental and laboratory factors that can influence WBS outputs. The implementation of WBS therefore requires a comprehensive framework to outline an analysis pipeline that accounts for these complex and nuanced factors. This article reviews the framework of the national WBS conducted at the Public Health Agency of Canada to present WBS methods used in Canada to track and monitor SARS-CoV-2. In particular, we focus on five Canadian cities – Vancouver, Edmonton, Toronto, Montreal and Halifax – whose wastewater signals are analyzed by a mathematical model to provide case forecasts and reproduction number estimates. This work provides insights on approaches to implement WBS at the national scale in an accurate and efficient manner. Importantly, the national WBS system has implications beyond COVID-19, as a similar framework can be applied to monitor other infectious disease pathogens or antimicrobial resistance in the community.

The spread of SARS-CoV-2 has been studied at unprecedented levels worldwide. In jurisdictions where molecular analysis was performed on large scales, the emergence and competition of numerous SARS-CoV-2 lineages has been observed in near real-time. Lineage identification, traditionally performed from clinical samples, can also be determined by sampling wastewater from sewersheds serving populations of interest. Of particular interest are variants of concern (VOCs), SARS-CoV-2 lineages that are associated with increased transmissibility and/or severity. Here, we consider clinical and wastewater data sources to retrospectively assess the emergence and spread of different VOCs in Canada. We show that, overall, wastewater-based VOC identification provides similar in-sights to the surveillance based on clinical samples. Based on clinical data, we observed a synchrony in VOC introduction as well as similar emergence speeds across most Canadian provinces despite the large geographical size of the country and differences in provincial public health measures. In particular, it took approximately four months for VOC Alpha and Delta to contribute to half of the incidence, whereas VOC Omicron achieved the same contribution in less than one month. By quantifying the timing and rapidity of SARS-CoV-2 VOCs invasion in Canada, this study provides important benchmarks to support preparedness for future VOCs, and to some extent, for future pandemics caused by other pathogens.

Background The incidence of antimicrobial-resistant Neisseria gonorrhoeae (GC) is rising in Canada; however, antimicrobial resistance (AMR) surveillance data are unavailable for infections diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs), representing over 80% of diagnoses. We developed a set of 10 improved molecular assays for surveillance of GC-AMR and prediction of susceptibilities in NAAT specimens. Methods Multiplex real-time PCR (RT–PCR) assays were developed to detect SNPs associated with cephalosporin (ponA, porB, mtrR −35delA, penA A311V, penA A501, N513Y, G545S), ciprofloxacin (gyrA S91, parC D86/S87/S88) and azithromycin [23S (A2059G, C2611T), mtrR meningitidis-like promoter] resistance. The assays were validated on 127 gonococcal isolates, 51 non-gonococcal isolates and 50 NAATs with matched culture isolates. SNPs determined from the assay were compared with SNPs determined from in silico analysis of WGS data. MICs were determined for culture isolates using the agar dilution method. Results SNP analysis of the 50 NAAT specimens had 96% agreement with the matched culture RT–PCR analysis. When compared with MICs, presence of penA A311V or penA A501 and two or more other SNPs correlated with decreased susceptibility and presence of three or more other SNPs correlated with intermediate susceptibility to cephalosporins; presence of any associated SNP correlated with ciprofloxacin or azithromycin resistance. NAAT-AMR predictions correlated with matched-culture cephalosporin, ciprofloxacin and azithromycin MICs at 94%, 100% and 98%, respectively. Conclusions We expanded molecular tests for N. gonorrhoeae AMR prediction by adding new loci and multiplexing reactions to improve surveillance where culture isolates are unavailable.

Wastewater-based surveillance (WBS) has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Quantities of viral fragments of SARS-CoV-2 in wastewater are related to numbers of clinical cases of COVID-19 reported within the corresponding sewershed. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) or sequencing. A multiplex RT-qPCR assay to detect and estimate the prevalence of multiple VOCs, including Omicron/Alpha, Beta, Gamma, and Delta, in wastewater RNA extracts was developed and validated. The probe-based multiplex assay, named “N200” focuses on amino acids 199-202, a region of the N gene that contains several mutations that are associated with variants of SARS- CoV-2 within a single amplicon. Each of the probes in the N200 assay are specific to the targeted mutations and worked equally well in single- and multi-plex modes. To estimate prevalence of each VOC, the abundance of the targeted mutation was compared with a non- mutated region within the same amplified region. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from six sewersheds in Ontario, Canada collected between December 1, 2021, and January 4, 2022. Using the N200 assay, the replacement of the Delta variant along with the introduction and rapid dominance of the Omicron variant were monitored in near real-time, as they occurred nearly simultaneously at all six locations. The N200 assay is robust and efficient for wastewater surveillance can be adopted into VOC monitoring programs or replace more laborious assays currently being used to monitor SARS- CoV-2 and its VOCs.

SARS-CoV-2 variants of concern (VoC) have been increasingly detected in clinical surveillance in Canada and internationally. These VoC are associated with higher transmissibility rates and in some cases, increased mortality. In this work we present a national wastewater survey of the distribution of three SARS-CoV-2 mutations found in the B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma) VoC, namely the S-gene 69–70 deletion, N501Y mutation, and N-gene D3L. RT-qPCR allelic discrimination assays were sufficiently sensitive and specific for detection and relative quantitation of SARS-CoV-2 variants in wastewater to allow for rapid population-level screening and surveillance. We tested 261 samples collected from 5 Canadian cities (Vancouver, Edmonton, Toronto, Montreal, and Halifax) and 6 communities in the Northwest Territories from February 16th to March 28th, 2021. VoC were not detected in the Territorial communities, suggesting the absence of VoC SARS-CoV-2 cases in those communities. Percentage of variant remained low throughout the study period in the majority of the sites tested, however the Toronto sites showed a marked increase from ~25% to ~75% over the study period. The results of this study highlight the utility of population level molecular surveillance of SARS-CoV-2 VoC using wastewater. Wastewater monitoring for VoC can be a powerful tool in informing public health responses, including monitoring trends independent of clinical surveillance and providing early warning to communities., Graphical abstract Unlabelled Image

1AbstractThe COVID-19 pandemic has stimulated wastewater-based surveillance, allowing public health to track the epidemic by monitoring the concentration of the genetic fingerprints of SARS-CoV-2 shed in wastewater by infected individuals. Wastewater-based surveillance for COVID-19 is still in its infancy. In particular, the quantitative link between clinical cases observed through traditional surveillance and the signals from viral concentrations in wastewater is still developing and hampers interpretation of the data and actionable public-health decisions.We present a modelling framework that includes both SARS-CoV-2 transmission at the population level and the fate of SARS-CoV-2 RNA particles in the sewage system after faecal shedding by infected persons in the population.Using our mechanistic representation of the combined clinical/wastewater system, we perform exploratory simulations to quantify the effect of surveillance effectiveness, public-health interventions and vaccination on the discordance between clinical and wastewater signals. We also apply our model to surveillance data from three Canadian cities to provide wastewater-informed estimates for the actual prevalence, the effective reproduction number and incidence forecasts. We find that wastewater-based surveillance, paired with this model, can complement clinical surveillance by supporting the estimation of key epidemiological metrics and hence better triangulate the state of an epidemic using this alternative data source.

Objective Both HIV infection and identifying as MSM have been linked to altered rectal microbiota composition, but few studies have studied sexual behavioural associations with rectal microbiota within MSM. In addition, most rectal microbiota studies in MSM have been limited geographically to Europe and North America, and replication of findings in lower and middle-income countries is lacking. Design A cross-sectional study. Methods We enrolled MSM from Nairobi, Kenya, and determined their HIV/sexually transmitted infection status. Rectal specimens were obtained for 16s rRNA sequencing of the rectal microbiota, and sexual behaviour was characterized using a standardized questionnaire. Microbiome differences were modelled using nonparametric statistics, Bray-Curtis ecological distance metrics and analyses of differential taxa abundance. Multivariable linear regression was used to model HIV status and recent sexual activity as predictors of alpha diversity, controlling for a range of covariates. Results Alpha diversity was consistently lower in Kenyan HIV-infected MSM (n = 80), including those on antiretroviral therapy (ART) compared with HIV-uninfected MSM. A statistical trend was observed for clustering of HIV status by Prevotella or Bacteroides dominance (P = 0.13). Several taxa were enriched in HIV-positive men, including Roseburia, Lachnospira, Streptococcus and Granulicatella. Receptive anal sex with several types of sexual partners (paying, regular, casual) was associated with lower Chao1 and Simpson diversity, independent of HIV status, while HIV infection was associated lower Chao1 (P = 0.030) but not Simpson diversity (P = 0.049). Conclusion Both HIV infection and sexual behaviour were associated with rectal microflora alpha diversity, in particular richness, but not Prevotella spp. dominance, in Kenyan MSM. Associations were more robust for sexual behaviour.

Real-time polymerase chain reaction (PCR) assays to detect antimicrobial resistance-associated mutations were tested on Neisseria gonorrhoeae-positive clinical samples with matched isolates. Of the nucleic acid amplification tests/cultures, 87.7% (64/73), 98.6% (72/73), and 98.4% (62/63) predicted cephalosporin, ciprofloxacin, and azithromycin susceptibilities, respectively. N. gonorrhoeae multiantigen sequence type was correctly predicted for 98.7% (79/80), and 13 of 58 N. gonorrhoeae-negative specimens showed false-positive results.

Background Bacterial STIs increase mucosal inflammation and HIV acquisition risk. However, most data are limited to symptomatic STIs, and more data are required regarding the prevalence and correlates of asymptomatic bacterial STIs among high-risk young women, and how these vary by exposure to sex work. Methods We estimated the prevalence of 6 STIs in urine collected during a cross-sectional study of women aged 14–24 years in Mombasa, Kenya (n=870). Participants were recruited from sex work hotspots, and self-identified as engaged or not engaged in formal sex work. STIs including Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), Mycoplasma hominis (MH), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG) and Neisseria gonorrhea (NG) were detected using the Seegene Anyplex qPCR panel. The cervicovaginal microbiome and cytokines were also characterized in a subset of 168 participants. Results The prevalence of any STI was 25.1% (95% CI, 22.3–28.0%). The most common STIs were UU (13.2%) and MH (11.3%), followed by CT (6.6%), TV (3.2%), NG (0.3%), and MG (0.5%). U. urealyticum was more prevalent among sex workers compared to non-sex workers (19.7 vs 10.4%, p=0.001). Number of partners positively correlated with UU (P=0.004), while duration of sexual activity was positively correlated with MH (P Conclusion U. urealyticum and M. hominis were the most prevalent STIs among young women from Mombasa, Kenya, and these organisms were associated with sexual exposure and an inflammatory mucosal milieu that has been linked to increased HIV acquisition. Disclosure No significant relationships.

We developed a real-time PCR assay to detect single nucleotide polymorphisms associated with ciprofloxacin resistance in specimens submitted for nucleic acid amplification testing (NAAT). All three single nucleotide polymorphism (SNP) targets produced high sensitivity and specificity values. The presence of ≥2 SNPs was sufficient to predict ciprofloxacin resistance in an organism.

Whereas the infant gut microbiome is the subject of intense study, relatively little is known regarding the nares microbiome in newborns and during early life. This study aimed to survey the typical composition and diversity of human anterior nare microflora for developing infants over time, and to explore how these correlate to their primary caregivers. Single nare swabs were collected at five time points over a one-year period for each subject from infant-caregiver pairs. Our study comprised of 50 infants (recruited at 2 weeks, post delivery) and their 50 primary caregivers. Applying the chaperonin-60 (cpn60) universal target (UT) amplicon as our molecular barcoding marker to census survey the microbial communities, we longitudinally surveyed infant nares microbiota at 5 time points over the course of the first year of life. The inter- and intra-subject diversity was catalogued and compared, both longitudinally and relative to their adult primary caregivers. Although within-subject variability over time and inter-subject variability were both observed, the assessment detected only one or two predominant genera for individual infant samples, belonging mainly to phyla Actinobacteria, Firmicutes, and Proteobacteria. Consistent with previously observed microbial population dynamics in other body sites, the diversity of nares microflora increased over the first year of life and infants showed differential operational taxonomic units (OTUs) relative to their matched primary caregiver. The collected evidence also support that both temporal and seasonal changes occur with respect to carriage of potentially pathogenic bacteria (PPBs), which may influence host predisposition to infection. This pilot study surveying paired infant/caregiver nare microbiomes provides novel longitudinal diversity information that is pertinent to better understanding nare microbiome development in infants.

The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA , mtrR , penA , porB , and one N. gonorrhoeae -specific marker ( porA ). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae -negative NAAT specimens, and 34 N. gonorrhoeae -positive NAAT specimens. Twenty-four of the N. gonorrhoeae -positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA , ponA , and penA , 99.6% for mtrR , and 95.2% for porB . The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB , 95.8% for ponA and mtrR , and 91.7% for penA . We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae . Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.