Assessment of nucleic acid extraction protocols for antibiotic resistance genes (ARGs) quantification in aircraft wastewater.

This study evaluated ten nucleic acid extraction protocols (EP1 to EP10) for measuring five endogenous antibiotic resistance genes (ARGs) in four aircraft wastewater samples (AWW1 to AWW4). The targeted ARGs, including blaCTX-M, blaNDM-1, ermB, qnrS, and tetA, encompassed highly and minimally abundant ARGs. TetA and ermB were consistently detected across four aircraft wastewater samples using the DNeasy Blood and Tissue Kit and the AllPrep PowerViral DNA/RNA kit. QnrS displayed high detection rates with specific extraction protocols and aliquot volumes. Concentrations of ARGs varied across aircraft wastewater samples, with differing extraction protocols influencing quantitative results. The concentrations of tetA, ermB, and qnrS in AWW1 were distinct, while AWW2 to AWW4 exhibited a broader range for tetA, ermB, qnrS, blaCTX-M, and blaNDM-1. EP1 consistently produced the highest concentrations for several ARGs. Collective data analysis revealed varying ARG concentrations across the ten extraction protocols, suggesting the importance of careful extraction protocol selection in ARG monitoring in aircraft wastewater samples. Based on the results, we suggest that a small sample volume (as low as 0.2 mL) may be sufficient for ARG characterization in aircraft wastewater samples. The findings also emphasize the need for considering toilet paper removal without compromising nucleic acid extraction efficiency. The study highlights promising prospects for aircraft wastewater monitoring of ARGs, calling for further investigation into the import and spread of unique ARGs through transport hubs.

The gut bacterium and pathobiont Bacteroides vulgatus activates NF-κB in a human gut epithelial cell line in a strain and growth phase dependent manner.

The gut microbiota is increasingly implicated in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC) although the identity of the bacteria that underpin these diseases has remained elusive. The pathobiont Bacteroides vulgatus has been associated with both diseases although relatively little is known about how its growth and functional activity might drive the host inflammatory response. We identified an ATP Binding Cassette (ABC) export system and lipoprotein in B. vulgatus ATCC 8482 and B. vulgatus PC510 that displayed significant sequence similarity to an NF-κB immunomodulatory regulon previously identified on a CD-derived metagenomic fosmid clone. Interestingly, the ABC export system was specifically enriched in CD subjects suggesting that it may be important for colonization and persistence in the CD gut environment. Both B. vulgatus ATCC 8482 and PC510 activated NF-κB in a strain and growth phase specific manner in a HT-29/kb-seap-25 enterocyte like cell line. B. vulgatus ATCC 8482 also activated NF-κB in a Caco-2-NF-κBluc enterocyte like and an LS174T-NF-κBluc goblet cell like cell lines, and induced NF-κB-p65 subunit nuclear translocation and IL-6, IL-8, CXCL-10 and MCP-1 gene expression. Despite this, NF-κB activation was not coincident with maximal expression of the ABC exporter or lipoprotein in B. vulgatus PC510 suggesting that the regulon may be necessary but not sufficient for the immunomodulatory effects.

Development of a triplex RT-qPCR assay for simultaneous quantification of Japanese encephalitis, Murray Valley encephalitis, and West Nile viruses for environmental surveillance.

The co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) has impacted human and animal health in multiple countries worldwide. To facilitate early warnings and surveillance of the presence of these viral infectious agents in the environment, a triplex reverse transcription-quantitative PCR (RT-qPCR) was developed for simultaneous quantification of JEV, MVEV, and WNV in potential hotspots such as piggery and urban wastewater and environmental water samples. The performance of the developed triplex RT-qPCR assay was compared with that of simplex counterparts, all using the same primer and probe sequences. The quantifiable results showed a concordance rate of 93.9%-100% (Cohen's kappa) between the triplex and simplex assays. The mean concentrations of exogenous JEV, MVEV, and WNV using the triplex and simplex RT-qPCR assays were remarkably similar in piggery/urban wastewater and environmental water samples. However, the impacts of the matrix effects (i.e., sample composition and PCR inhibition) of environmental water samples on the accurate quantification of these viruses need to be considered. Taken together, this newly developed triplex RT-qPCR assay of JEV, MVEV, and WNV will allow for a more rapid and cost-efficient sample analysis and data interpretation. The application of the triplex assay for environmental surveillance may be a valuable tool to complement the existing disease and mosquito surveillance approaches used to safeguard the health of both humans and animals.IMPORTANCEThe co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) poses significant threats to human and animal health globally. In this study, a triplex RT-qPCR assay was developed for simultaneous quantification of these viruses in wastewater and environmental water samples. Results demonstrated high concordance and sensitivity of the newly developed triplex RT-qPCR assay compared to simplex assays, indicating its efficacy for environmental surveillance. This cost-effective and rapid assay offers a vital tool for timely monitoring of mosquito-borne viruses in environmental samples, enhancing our ability to mitigate potential outbreaks and safeguard public health.

Comparing the decay of human wastewater-associated markers and enteric viruses in laboratory microcosms simulating estuarine waters in a temperate climatic zone using qPCR/RT-qPCR assays.

This study investigated the decay rates of wastewater-associated markers and enteric viruses in laboratory microcosms mimicking estuarine water environments in temperate Sydney, NSW, Australia using qPCR and RT-qPCR assays. The results demonstrated the reduction in concentrations of Bacteroides HF183, Lachnospiraceae Lachno3, cross-assembly phage (crAssphage), pepper mild mottle virus (PMMoV), human adenovirus (HAdV 40/41), and enterovirus (EV) over a span of 42 days under spring/summer temperatures, presence/absence of microbiota, and different light conditions. The study found that HF183, Lachno3, crAssphage, PMMoV, HAdV 40/41, and EV exhibited varying decay rates depending on the experimental conditions. The average T90 values ranged from a few days to several months, indicating the rapid decay or prolonged persistence of these markers and enteric viruses in the estuarine environment. Furthermore, the study examined the effects of indigenous microbiota and spring/summer temperatures on wastewater-associated markers and enteric viruses decay rates. It was found that the presence of microbiota and temperature significantly influenced the decay rates of HF183 and PMMoV. Additionally, the study compared the effects of artificial sunlight and spring/summer temperatures on marker decay rates. Bacterial markers decayed faster than viral markers, although among viral markers crAssphage decay rates were relatively faster when compared to PMMoV. The exposure to artificial sunlight significantly accelerated the decay rates of bacterial markers, viral markers, and enteric viruses. Temperature also had an impact on the decay rates of Lachno3, crAssphage, and HAdV 40/41. In conclusion, this study provides valuable insights into the decay rates of wastewater-associated markers and enteric viruses under different experimental conditions that mimicked temperate environmental conditions. The findings contribute to our understanding of the fate and persistence of these markers in the environment which is crucial for assessing and managing risks from contamination by untreated human wastewater.

Development and comparative assessment of RT-qPCR and duplex RT-LAMP assays for the monitoring of Aichi Virus A (AiV-A) in untreated wastewater samples.

Diverse enteric pathogens, transmitted through human and animal feces, can cause gastroenteritis. Enteric viruses, such as human Aichi virus, specifically A (AiV-A), are emerging pathogens that cause illnesses even at low doses and are spreading globally. This research developed a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the 3CD junction and a reverse transcription colorimetric loop-mediated isothermal amplification (RT-cLAMP) duplex assay targeting junctions 2BC and 3CD of the AiV-A genome for rapid and sensitive detection of AiV-A in metropolitan and regional wastewater samples. The performance of these assays was evaluated using control materials and by analyzing wastewater samples. In serially diluted control materials, RT-qPCR provided quantifiable data (mean 1.51 log10 GC/2 µL of nucleic acid) down to a dilution of 1 × 10-5 pg/µL. In comparison, the duplex RT-cLAMP assay detected down to 1 × 10-4 pg/µL, indicating that its sensitivity was one order of magnitude less than that of RT-qPCR. Of the 38 wastewater samples from 38 metropolitan and regional wastewater treatment plants (WWTPs) in Queensland, Australia, 21 (55.3 %) tested positive by RT-qPCR with concentrations in ranging from 3.60 to 6.23 log10 GC/L. In contrast, only 15 (39.5 %) of 38 wastewater samples were positive using the duplex RT-cLAMP assay. The methods demonstrated substantial qualitative agreement (κ = 0.730), with a concordance of 86.5 %, demonstrating the reliability of RT-cLAMP for detecting AiV-A in wastewater samples. The duplex RT-cLAMP assay, despite demonstrating reduced detection sensitivity, has proven effective and holds promise as a supplementary approach, especially in settings with limited resources where rapid and affordable testing is crucial.

Development and evaluation of a colorimetric LAMP based-assay targeting the Bacteroides HF183 marker for tracking sewage pollution in environmental waters.

Surface waters are vulnerable to contamination by human and animal feces, posing risks to human health due to potential exposure to enteric pathogens. This research developed a colorimetric loop-mediated isothermal amplification (cLAMP) assay to detect sewage associated Bacteroides dorei HF183/BacR287 (HF183) marker in wastewater and environmental water samples. The host sensitivity and host specificity of the assay were evaluated, and their performance was compared to the Bacteroides HF183 qPCR assay using control materials (gBlocks), environmental water samples seeded with untreated sewage, and ambient environmental water samples. In serial dilutions of control materials, qPCR produced quantifiable data across all dilutions, while cLAMP detected the marker down to 0.001 pg/µL of control materials, which was two orders of magnitude less sensitive than qPCR. All untreated sewage samples (n = 12) tested positive for HF183 by both the qPCR and cLAMP assays, demonstrating a host sensitivity value of 1.00 (maximum value of 1.00). The host specificity by analysing 70 non-human fecal nucleic acid samples revealed cLAMP's specificity value of 0.81 compared to qPCR's 0.64. When testing sewage-seeded environmental water samples, both methods detected HF183 for the lowest amount of sewage, indicating similar detection sensitivity. The application of cLAMP for tracking sewage pollution in environmental waters showed promising results, with moderate agreement between cLAMP and qPCR (κ = 0.510). However, cLAMP occasionally missed detections compared to qPCR, particularly in low-concentration samples. Overall, the cLAMP HF183 assay demonstrated promising potential as a rapid and sensitive method for detecting sewage pollution, offering a viable alternative to qPCR in certain environmental monitoring scenarios.

Comparison of adsorption-extraction (AE) workflows for improved measurements of viral and bacterial nucleic acid in untreated wastewater.

The lack of standardized methods and large differences in virus concentration and extraction workflows have hampered Severe Acute Respiratory Syndrome (SARS-CoV-2) wastewater surveillance and data reporting practices. Numerous studies have shown that adsorption-extraction (AE) method holds promise, yet several uncertainties remain regarding the optimal AE workflow. Several procedural components may influence the recovered concentrations of target nucleic acid, including membrane types, homogenization instruments, speed and duration, and lysis buffer. In this study, 42 different AE workflows that varied these components were compared to determine the optimal workflow by quantifying endogenous SARS-CoV-2, human adenovirus 40/41 (HAdV 40/41), and a bacterial marker gene of fecal contamination (Bacteroides HF183). Our findings suggest that the workflow chosen had a significant impact on SARS-CoV-2 concentrations, whereas it had minimal impact on HF183 and no effect on HAdV 40/41 concentrations. When comparing individual components in a workflow, such as membrane type (MF-Millipore™ 0.45 µm MCE vs. Isopore™ 0.40 µm), we found that they had no impact on SARS-CoV-2, HAdV 40/41, and HF183 concentrations. This suggests that at least some consumables and equipment are interchangeable. Buffer PM1 + TRIzol-based workflows yielded higher concentrations of SARS-CoV-2 than other workflows. HF183 concentrations were higher in workflows without chloroform. Similarly, higher homogenization speeds (5000-10,000 rpm) led to increased concentrations of SARS-CoV-2 and HF183 but had no effect on HAdV 40/41. Our findings indicate that minor enhancements to the AE workflow can improve the recovery of viruses and bacteria from the wastewater, leading to improved outcomes from wastewater surveillance efforts.

Vitamins C and E to prevent complications of pregnancy-associated hypertension.

BACKGROUND: Oxidative stress has been proposed as a mechanism linking the poor placental perfusion characteristic of preeclampsia with the clinical manifestations of the disorder. We assessed the effects of antioxidant supplementation with vitamins C and E, initiated early in pregnancy, on the risk of serious adverse maternal, fetal, and neonatal outcomes related to pregnancy-associated hypertension. METHODS: We conducted a multicenter, randomized, double-blind trial involving nulliparous women who were at low risk for preeclampsia. Women were randomly assigned to begin daily supplementation with 1000 mg of vitamin C and 400 IU of vitamin E or matching placebo between the 9th and 16th weeks of pregnancy. The primary outcome was severe pregnancy-associated hypertension alone or severe or mild hypertension with elevated liver-enzyme levels, thrombocytopenia, elevated serum creatinine levels, eclamptic seizure, medically indicated preterm birth, fetal-growth restriction, or perinatal death. RESULTS: A total of 10,154 women underwent randomization. The two groups were similar with respect to baseline characteristics and adherence to the study drug. Outcome data were available for 9969 women. There was no significant difference between the vitamin and placebo groups in the rates of the primary outcome (6.1% and 5.7%, respectively; relative risk in the vitamin group, 1.07; 95% confidence interval [CI], 0.91 to 1.25) or in the rates of preeclampsia (7.2% and 6.7%, respectively; relative risk, 1.07; 95% CI, 0.93 to 1.24). Rates of adverse perinatal outcomes did not differ significantly between the groups. CONCLUSIONS: Vitamin C and E supplementation initiated in the 9th to 16th week of pregnancy in an unselected cohort of low-risk, nulliparous women did not reduce the rate of adverse maternal or perinatal outcomes related to pregnancy-associated hypertension (ClinicalTrials.gov number, NCT00135707).

Influence of membrane pore-size on the recovery of endogenous viruses from wastewater using an adsorption-extraction method.

The ongoing COVID-19 pandemic has emphasized the significance of wastewater surveillance in monitoring and tracking the spread of infectious diseases, including SARS-CoV-2. The wastewater surveillance approach detects genetic fragments from viruses in wastewater, which could provide an early warning of outbreaks in communities. In this study, we determined the concentrations of four types of endogenous viruses, including non-enveloped DNA (crAssphage and human adenovirus 40/41), non-enveloped RNA (enterovirus), and enveloped RNA (SARS-CoV-2) viruses, from wastewater samples using the adsorption-extraction (AE) method with electronegative HA membranes of different pore sizes (0.22, 0.45, and 0.80 µm). Our findings showed that the membrane with a pore size of 0.80 µm performed comparably to the membrane with a pore size of 0.45 µm for virus detection/quantitation (repeated measurement one-way ANOVA; p > 0.05). We also determined the recovery efficiencies of indigenous crAssphage and pepper mild mottle virus, which showed recovery efficiencies ranging from 50% to 94% and from 20% to 62%, respectively. Our results suggest that the use of larger pore size membranes may be beneficial for processing larger sample volumes, particularly for environmental waters containing low concentrations of viruses. This study offers valuable insights into the application of the AE method for virus recovery from wastewater, which is essential for monitoring and tracking infectious diseases in communities.

Unveiling indicator, enteric, and respiratory viruses in aircraft lavatory wastewater using adsorption-extraction and Nanotrap® Microbiome A Particles workflows.

The effective detection of viruses in aircraft wastewater is crucial to establish surveillance programs for monitoring virus spread via aircraft passengers. This study aimed to compare the performance of two virus concentration workflows, adsorption-extraction (AE) and Nanotrap® Microbiome A Particles (NMAP), in detecting the prevalence and concentrations of 15 endogenous viruses including ssDNA, dsDNA, ssRNA in 24 aircraft lavatory wastewater samples. The viruses tested included two indicator viruses, four enteric viruses, and nine respiratory viruses. The results showed that cross-assembly phage (crAssphage), human polyomavirus (HPyV), rhinovirus A (RhV A), and rhinovirus B (RhV B) were detected in all wastewater samples using both workflows. However, enterovirus (EV), human norovirus GII (HNoV GII), human adenovirus (HAdV), bocavirus (BoV), parechovirus (PeV), epstein-barr virus (EBV). Influenza A virus (IAV), and respiratory syncytial virus B (RsV B) were infrequently detected by both workflows, and hepatitis A virus (HAV), influenza B virus (IBV), and respiratory syncytial virus B (RsV A) were not detected in any samples. The NMAP workflow had greater detection rates of RNA viruses (EV, PeV, and RsV B) than the AE workflow, while the AE workflow had greater detection rates of DNA viruses (HAdV, BoV, and EBV) than the NMAP workflow. The concentration of each virus was also analyzed, and the results showed that crAssphage had the highest mean concentration (6.76 log10 GC/12.5 mL) followed by HPyV (5.46 log10 GC/12.5 mL using the AE workflow, while the mean concentrations of enteric and respiratory viruses ranged from 2.48 to 3.63 log10 GC/12.5 mL. Using the NMAP workflow, the mean concentration of crAssphage was 5.18 log10 GC/12.5 mL and the mean concentration of HPyV was 4.20 log10 GC/12.5 mL, while mean concentrations of enteric and respiratory viruses ranged from 2.55 to 3.74 log10 GC/12.5 mL. Significantly higher (p < 0.05) mean concentrations of crAssphage and HPyV were observed when employing the AE workflow in comparison to the NMAP workflow. Conversely, the NMAP workflow yielded significantly greater (p < 0.05) concentrations of RhV A, and RhV B compared to the AE workflow. The findings of this study can aid in the selection of an appropriate concentration workflow for virus surveillance studies and contribute to the development of efficient virus detection methods.

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 to 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 endogenous 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 as sensitive to either turbidity or TSS levels. These findings suggest that wastewater sample turbidity or suspended solids concentration, and the intended target for analysis should be considered when validating an optimal workflow for wastewater surveillance of viruses.

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.

Draft genome sequence of Treponema sp. strain JC4, a novel spirochete isolated from the bovine rumen.

Morphologically and biochemically diverse members of the Treponema genus are present in the gastrointestinal tract of ruminants, yet very little is understood about their functional importance to this microbiome. Here we describe the annotated draft genome sequence of Treponema sp. strain JC4, a novel spirochete isolated from a bovine rumen sample.

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.

Evaluation of process limit of detection and quantification variation of SARS-CoV-2 RT-qPCR and RT-dPCR assays for wastewater surveillance.

Effective wastewater surveillance of SARS-CoV-2 RNA requires the rigorous characterization of the limit of detection resulting from the entire sampling process - the process limit of detection (PLOD). Yet to date, no studies have gone beyond quantifying the assay limit of detection (ALOD) for RT-qPCR or RT-dPCR assays. While the ALOD is the lowest number of gene copies (GC) associated with a 95% probability of detection in a single PCR reaction, the PLOD represents the sensitivity of the method after considering the efficiency of all processing steps (e.g., sample handling, concentration, nucleic acid extraction, and PCR assays) to determine the number of GC in the wastewater sample matrix with a specific probability of detection. The primary objective of this study was to estimate the PLOD resulting from the combination of primary concentration and extraction with six SARS-CoV-2 assays: five RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab (CCDC N and CCDC ORF1ab), and E_Sarbeco RT-qPCR, and one RT-dPCR assay (US CDC N1 RT-dPCR) using two models (exponential survival and cumulative Gaussian). An adsorption extraction (AE) concentration method (i.e., virus adsorption on membrane and the RNA extraction from the membrane) was used to concentrate gamma-irradiated SARS-CoV-2 seeded into 36 wastewater samples. Overall, the US CDC N1 RT-dPCR and RT-qPCR assays had the lowest ALODs (< 10 GC/reaction) and PLODs (<3,954 GC/50 mL; 95% probability of detection) regardless of the seeding level and model used. Nevertheless, consistent amplification and detection rates decreased when seeding levels were < 2.32 × 103 GC/50 mL even for US CDC N1 RT-qPCR and RT-dPCR assays. Consequently, when SARS-CoV-2 RNA concentrations are expected to be low, it may be necessary to improve the positive detection rates of wastewater surveillance by analyzing additional field and RT-PCR replicates. To the best of our knowledge, this is the first study to assess the SARS-CoV-2 PLOD for wastewater and provides important insights on the analytical limitations for trace detection of SARS-CoV-2 RNA in wastewater.

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.

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.

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.