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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater.

There is currently a clear benefit for many countries to utilize wastewater-based epidemiology (WBE) as part of ongoing measures to manage the coronavirus disease 2019 (COVID-19) global pandemic. Since most wastewater virus concentration methods were developed and validated for nonenveloped viruses, it is imperative to determine the efficiency of the most commonly used methods for the enveloped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Municipal wastewater seeded with a human coronavirus (CoV) surrogate, murine hepatitis virus (MHV), was used to test the efficiency of seven wastewater virus concentration methods: (A-C) adsorption-extraction with three different pre-treatment options, (D-E) centrifugal filter device methods with two different devices, (F) polyethylene glycol (PEG 8000) precipitation, and (G) ultracentrifugation. MHV was quantified by reverse-transcription quantitative polymerase chain reaction and the recovery efficiency was calculated for each method. The mean MHV recoveries ranged from 26.7 to 65.7%. The most efficient methods were adsorption-extraction methods with MgCl2 pre-treatment (Method C), and without pre-treatment (Method B). The third most efficient method used the Amicon® Ultra-15 centrifugal filter device (Method D) and its recovery efficiency was not statistically different from the most efficient methods. The methods with the worst recovery efficiency included the adsorption-extraction method with acidification (A), followed by PEG precipitation (F). Our results suggest that absorption-extraction methods with minimal or without pre-treatment can provide suitably rapid, cost-effective and relatively straightforward recovery of enveloped viruses in wastewater. The MHV is a promising process control for SARS-CoV-2 surveillance and can be used as a quality control measure to support community-level epidemic mitigation and risk assessment.

Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater: a surveillance tool for assessing the presence of COVID-19 infected travellers.

BACKGROUND: Wastewater-based epidemiology (WBE) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be an important source of information for coronavirus disease 2019 (COVID-19) management during and after the pandemic. Currently, governments and transportation industries around the world are developing strategies to minimize SARS-CoV-2 transmission associated with resuming activity. This study investigated the possible use of SARS-CoV-2 RNA wastewater surveillance from airline and cruise ship sanitation systems and its potential use as a COVID-19 public health management tool. METHODS: Aircraft and cruise ship wastewater samples (n = 21) were tested for SARS-CoV-2 using two virus concentration methods, adsorption-extraction by electronegative membrane (n = 13) and ultrafiltration by Amicon (n = 8), and five assays using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR). Representative qPCR amplicons from positive samples were sequenced to confirm assay specificity. RESULTS: SARS-CoV-2 RNA was detected in samples from both aircraft and cruise ship wastewater; however concentrations were near the assay limit of detection. The analysis of multiple replicate samples and use of multiple RT-qPCR and/or RT-ddPCR assays increased detection sensitivity and minimized false-negative results. Representative qPCR amplicons were confirmed for the correct PCR product by sequencing. However, differences in sensitivity were observed among molecular assays and concentration methods. CONCLUSIONS: The study indicates that surveillance of wastewater from large transport vessels with their own sanitation systems has potential as a complementary data source to prioritize clinical testing and contact tracing among disembarking passengers. Importantly, sampling methods and molecular assays must be further optimized to maximize detection sensitivity. The potential for false negatives by both wastewater testing and clinical swab testing suggests that the two strategies could be employed together to maximize the probability of detecting SARS-CoV-2 infections amongst passengers.

Pregnant trauma patients may be at increased risk of mortality compared to nonpregnant women of reproductive age: trends and outcomes over 10 years at a level I trauma center.

BACKGROUND: Pregnancy has been identified as a risk factor for poor outcomes after traumatic injury, but prior outcome analyses are conflicting and dated. We sought to examine outcomes in a contemporary cohort. METHODS: Retrospective cohort analysis at a level I trauma center's institutional registry from 2009 to 2018, with comparison to population-level demographic trends in women of reproductive age and pregnancy prevalence. Unadjusted cohorts of pregnant versus nonpregnant trauma patients were compared. Pregnant patients then were matched on age, mechanism of injury, year, and injury severity score with nonpregnant controls for adjusted analysis with a primary outcome of maternal mortality. RESULTS: Despite declining birth and pregnancy rates in the population, pregnant women comprised a stable 5.3% of female trauma patients of reproductive age without decline over the study period (p = 0.53). Compared with nonpregnant women, pregnant trauma patients had a lower injury severity score (1 [1-5] vs 5 [1-10] p < 0.0001) and a shorter length of stay (1 [1-2] vs 1 [1-4] p = 0.04), were less likely to have CT imaging (48.8% vs 67.4%, p < 0.0001) and more likely to be admitted (89.3% vs 79.2%, p = 0.003). Positive toxicology screens were less prevalent in pregnant women, but only for ethanol (5.4% vs 31.4%, p < 0.0001); there was no difference in rates of cannabis, opiates, or cocaine. After matching to adjust for age, year, mechanism of injury, and injury severity score, mortality occurred significantly more frequently in the pregnant cohort (2.1% vs 0.2%, OR = 13.5 [1.39-130.9], p = 0.02). CONCLUSION: Pregnant trauma patients have not declined in our population despite population-level declines in pregnancy. After adjusting for lower injury severity, pregnant women were at substantially greater risk of mortality. This supports ongoing concern for pregnant trauma patients as a vulnerable population. Further efforts should optimize systems of care to maximize the chances of rescue for both mother and fetus.

First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community.

Infection with SARS-CoV-2, the etiologic agent of the ongoing COVID-19 pandemic, is accompanied by the shedding of the virus in stool. Therefore, the quantification of SARS-CoV-2 in wastewater affords the ability to monitor the prevalence of infections among the population via wastewater-based epidemiology (WBE). In the current work, SARS-CoV-2 RNA was concentrated from wastewater in a catchment in Australia and viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) resulting in two positive detections within a six day period from the same wastewater treatment plant (WWTP). The estimated viral RNA copy numbers observed in the wastewater were then used to estimate the number of infected individuals in the catchment via Monte Carlo simulation. Given the uncertainty and variation in the input parameters, the model estimated a median range of 171 to 1,090 infected persons in the catchment, which is in reasonable agreement with clinical observations. This work highlights the viability of WBE for monitoring infectious diseases, such as COVID-19, in communities. The work also draws attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.

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.

Differences down-under: alcohol-fueled methanogenesis by archaea present in Australian macropodids.

The Australian macropodids (kangaroos and wallabies) possess a distinctive foregut microbiota that contributes to their reduced methane emissions. However, methanogenic archaea are present within the macropodid foregut, although there is scant understanding of these microbes. Here, an isolate taxonomically assigned to the Methanosphaera genus (Methanosphaera sp. WGK6) was recovered from the anterior sacciform forestomach contents of a Western grey kangaroo (Macropus fuliginosus). Like the human gut isolate Methanosphaera stadtmanae DSMZ 3091(T), strain WGK6 is a methylotroph with no capacity for autotrophic growth. In contrast, though with the human isolate, strain WGK6 was found to utilize ethanol to support growth, but principally as a source of reducing power. Both the WGK6 and DSMZ 3091(T) genomes are very similar in terms of their size, synteny and G:C content. However, the WGK6 genome was found to encode contiguous genes encoding putative alcohol and aldehyde dehydrogenases, which are absent from the DSMZ 3091(T) genome. Interestingly, homologs of these genes are present in the genomes for several other members of the Methanobacteriales. In WGK6, these genes are cotranscribed under both growth conditions, and we propose the two genes provide a plausible explanation for the ability of WGK6 to utilize ethanol for methanol reduction to methane. Furthermore, our in vitro studies suggest that ethanol supports a greater cell yield per mol of methane formed compared to hydrogen-dependent growth. Taken together, this expansion in metabolic versatility can explain the persistence of these archaea in the kangaroo foregut, and their abundance in these 'low-methane-emitting' herbivores.