Comparative analysis of adsorption-extraction (AE) and nanotrap® magnetic virus particles (NMVP) workflows for the recovery of endogenous enveloped and non-enveloped viruses in wastewater.

In this study, two virus concentration methods, namely Adsorption-extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) along with commercially available extraction kits were used quantify endogenous pepper mild mottle virus (PMMoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in nucleic acid extracted from 48 wastewater samples collected over six events from eight wastewater treatment plants (WWTPs). The main aim was to determine which workflow (i.e., concentration and extraction methods) produces greater concentrations of PMMoV and SARS-CoV-2 gene copies (GC) in comparison with each other. Turbidity and total suspended solids (TSS) of wastewater samples within and among the eight WWTPs were highly variable (41-385 NTU and 77-668 mg/L TSS). In 58 % of individual wastewater samples the log10 GC concentrations of PMMoV were greater by NMVP workflow compared to AE workflow. Paired measurements of PMMoV GC/10 mL from AE and NMVP across all 48 wastewater samples were weakly correlated (r = 0.455, p = 0.001) and demonstrated a poor linear relationship (r2 = 0.207). The log10 GC concentrations of SARS-CoV-2 in 69 % of individual samples were greater by AE workflow compared to NMVP workflow. In contrast to PMMoV, the AE and NMVP derived SARS-CoV-2 GC counts were strongly correlated (r = 0.859, p < 0.001) and demonstrated a strong linear relationship (r2 = 0.738). In general, the PMMoV GC achieved by the NMVP workflow decreased with increasing turbidity, but the PMMoV GC by the AE workflow did not appear to be sensitive to either turbidity or TSS levels. These findings suggest that suspended solids concentration, and the intended target for analysis should be considered when validating an optimal workflow for wastewater surveillance.

Occurrence of multiple respiratory viruses in wastewater in Queensland, Australia: Potential for community disease surveillance.

The early warning and tracking of COVID-19 prevalence in the community provided by wastewater surveillance has highlighted its potential for much broader viral disease surveillance. In this proof-of-concept study, 46 wastewater samples from four wastewater treatment plants (WWTPs) in Queensland, Australia, were analyzed for the presence and abundance of 13 respiratory viruses, and the results were compared with reported clinical cases. The viruses were concentrated using the adsorption-extraction (AE) method, and extracted nucleic acids were analyzed using qPCR and RT-qPCR. Among the viruses tested, bocavirus (BoV), parechovirus (PeV), rhinovirus A (RhV A) and rhinovirus B (RhV B) were detected in all wastewater samples. All the tested viruses except influenza B virus (IBV) were detected in wastewater sample from at least one WWTP. BoV was detected with the greatest concentration (4.96-7.22 log10 GC/L), followed by Epstein-Barr virus (EBV) (4.08-6.46 log10 GC/L), RhV A (3.95-5.63 log10 GC/L), RhV B (3.74-5.61 log10 GC/L), and PeV (3.17-5.32 log10 GC/L). Influenza viruses and respiratory syncytial virus (RSV) are notifiable conditions in Queensland, allowing the gene copy (GC) concentrations to be compared with reported clinical cases. Significant correlations (ρ = 0.60, p < 0.01 for IAV and ρ = 0.53, p < 0.01 for RSV) were observed when pooled wastewater influenza A virus (IAV) and RSV log10 GC/L concentrations were compared to log10 clinical cases among the four WWTP catchments. The positive predictive value for the presence of IAV and RSV in wastewater was 97 % for both IAV and RSV clinical cases within the four WWTP catchments. The overall accuracy of wastewater analysis for predicting clinical cases of IAV and RSV was 97 and 90 %, respectively. This paper lends credibility to the application of wastewater surveillance to monitor respiratory viruses of various genomic characteristics, with potential uses for increased surveillance capabilities and as a tool in understanding the dynamics of disease circulation in the communities.

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater.

We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2-5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.

RT-qPCR and ATOPlex sequencing for the sensitive detection of SARS-CoV-2 RNA for wastewater surveillance.

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to detect and quantify SARS-CoV-2 RNA in wastewater, while monitoring viral genome mutations requires separate approaches such as deep sequencing. A high throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique has shown promise in detecting variants of concern (VOC) while also providing virus quantitation data. However, detection sensitivities of both RT-qPCR and sequencing can be impacted through losses occurring during sample handling, virus concentration, nucleic acid extraction, and RT-qPCR. Therefore, process limit of detection (PLOD) assessments are required to estimate the gene copies of target molecule to attain specific probability of detection. In this study, we compare the PLOD of four RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab) for detection of SARS-CoV-2 to that of ATOPlex sequencing by seeding known concentrations of gamma-irradiated SARS-CoV-2 into wastewater. Results suggest that among the RT-qPCR assays, US CDC N1 was the most sensitive, especially at lower SARS-CoV-2 seed levels. However, when results from all RT-qPCR assays were combined, it resulted in greater detection rates than individual assays, suggesting that application of multiple assays is better suited for the trace detection of SARS-CoV-2 from wastewater samples. Furthermore, while ATOPlex offers a promising approach to SARS-CoV-2 wastewater surveillance, this approach appears to be less sensitive compared to RT-qPCR under the experimental conditions of this study, and may require further refinements. Nonetheless, the combination of RT-qPCR and ATOPlex may be a powerful tool to simultaneously detect/quantify SARS-CoV-2 RNA and monitor emerging VOC in wastewater samples.

Monitoring of SARS-CoV-2 in sewersheds with low COVID-19 cases using a passive sampling technique.

Monitoring SARS-CoV-2 RNA in sewer systems, upstream of a wastewater treatment plant, is an effective approach for understanding potential COVID-19 transmission in communities with higher spatial resolutions. Passive sampling devices provide a practical solution for frequent sampling within sewer networks where the use of autosamplers is not feasible. Currently, the design of upstream sampling is impeded by limited understanding of the fate of SARS-CoV-2 RNA in sewers and the sensitivity of passive samplers for the number of infected individuals in a catchment. In this study, passive samplers containing electronegative membranes were applied for at least 24-h continuous sampling in sewer systems. When monitoring SARS-CoV-2 along a trunk sewer pipe, we found RNA signals decreased proportionally to increasing dilutions, with non-detects occurring at the end of pipe. The passive sampling membranes were able to detect SARS-CoV-2 shed by >2 COVID-19 infection cases in 10,000 people. Moreover, upstream monitoring in multiple sewersheds using passive samplers identified the emergence of SARS-CoV-2 in wastewater one week ahead of clinical reporting and reflected the spatiotemporal spread of a COVID-19 cluster within a city. This study provides important information to guide the development of wastewater surveillance strategies at catchment and subcatchment levels using different sampling techniques.

Detection of the Omicron (B.1.1.529) variant of SARS-CoV-2 in aircraft wastewater.

On the 26th of November 2021, the World Health Organization (WHO) designated the newly detected B.1.1.529 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) the Omicron Variant of Concern (VOC). The genome of the Omicron VOC contains more than 50 mutations, many of which have been associated with increased transmissibility, differing disease severity, and potential to evade immune responses developed for previous VOCs such as Alpha and Delta. In the days since the designation of B.1.1.529 as a VOC, infections with the lineage have been reported in countries around the globe and many countries have implemented travel restrictions and increased border controls in response. We putatively detected the Omicron variant in an aircraft wastewater sample from a flight arriving to Darwin, Australia from Johannesburg, South Africa on the 25th of November 2021 via positive results on the CDC N1, CDC N2, and del(69-70) RT-qPCR assays per guidance from the WHO. The Australian Northern Territory Health Department detected one passenger onboard the flight who was infected with SARS-CoV-2, which was determined to be the Omicron VOC by sequencing of a nasopharyngeal swab sample. Subsequent sequencing of the aircraft wastewater sample using the ARTIC V3 protocol with Nanopore and ATOPlex confirmed the presence of the Omicron variant with a consensus genome that clustered with the B.1.1.529 BA.1 sub-lineage. Our detection and confirmation of a single onboard Omicron infection via aircraft wastewater further bolsters the important role that aircraft wastewater can play as an independent and unintrusive surveillance point for infectious diseases, particularly coronavirus disease 2019.

Comparative analysis of rapid concentration methods for the recovery of SARS-CoV-2 and quantification of human enteric viruses and a sewage-associated marker gene in untreated wastewater.

To support public-health-related disease surveillance and monitoring, it is crucial to concentrate both enveloped and non-enveloped viruses from domestic wastewater. To date, most concentration methods were developed for non-enveloped viruses, and limited studies have directly compared the recovery efficiency of both types of viruses. In this study, the effectiveness of two different concentration methods (Concentrating pipette (CP) method and an adsorption-extraction (AE) method amended with MgCl2) were evaluated for untreated wastewater matrices using three different viruses (SARS-CoV-2 (seeded), human adenovirus 40/41 (HAdV 40/41), and enterovirus (EV)) and a wastewater-associated bacterial marker gene targeting Lachnospiraceae (Lachno3). For SARS-CoV-2, the estimated mean recovery efficiencies were significantly greater by as much as 5.46 times, using the CP method than the AE method amended with MgCl2. SARS-CoV-2 RNA recovery was greater for samples with higher titer seeds regardless of the method, and the estimated mean recovery efficiencies using the CP method were 25.1 ± 11% across ten WWTPs when wastewater samples were seeded with 5 × 104 gene copies (GC) of SARS-CoV-2. Meanwhile, the AE method yielded significantly greater concentrations of indigenous HAdV 40/41 and Lachno3 from wastewater compared to the CP method. Finally, no significant differences in indigenous EV concentrations were identified in comparing the AE and CP methods. These data indicate that the most effective concentration method varies by microbial analyte and that the priorities of the surveillance or monitoring program should be considered when choosing the concentration method.

Wastewater surveillance demonstrates high predictive value for COVID-19 infection on board repatriation flights to Australia.

Controlling importation and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from overseas travelers is essential for countries, such as Australia, New Zealand, and other island nations, that have adopted a suppression strategy to manage very low community transmission. Wastewater surveillance of SARS-CoV-2 RNA has emerged as a promising tool employed in public health response in many countries globally. This study aimed to establish whether the surveillance of aircraft wastewater can be used to provide an additional layer of information to augment individual clinical testing. Wastewater from 37 long-haul flights chartered to repatriate Australians was tested for the presence of SARS-CoV-2 RNA. Children 5 years or older on these flights tested negative for coronavirus disease 19 (COVID-19) (deep nasal and oropharyngeal reverse-transcription (RT)-PCR swab) 48 h before departure. All passengers underwent mandatory quarantine for 14-day post arrival in Howard Springs, NT, Australia. Wastewater from 24 (64.9 %) of the 37 flights tested positive for SARS-CoV-2 RNA. During the 14 day mandatory quarantine, clinical testing identified 112 cases of COVID-19. Surveillance for SARS-CoV-2 RNA in repatriation flight wastewater using pooled results from three RT-qPCR assays demonstrated a positive predictive value (PPV) of 87.5 %, a negative predictive value (NPV) of 76.9 % and 83.7% accuracy for COVID-19 cases during the post-arrival 14-day quarantine period. The study successfully demonstrates that the surveillance of wastewater from aircraft for SARS-CoV-2 can provide an additional and effective tool for informing the management of returning overseas travelers and for monitoring the importation of SARS CoV-2 and other clinically significant pathogens.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.