Evaluating and optimizing Acid-pH and Direct Lysis RNA extraction for SARS-CoV-2 RNA detection in whole saliva.

COVID-19 has been a global public health and economic challenge. Screening for the SARS-CoV-2 virus has been a key part of disease mitigation while the world continues to move forward, and lessons learned will benefit disease detection beyond COVID-19. Saliva specimen collection offers a less invasive, time- and cost-effective alternative to standard nasopharyngeal swabs. We optimized two different methods of saliva sample processing for RT-qPCR testing. Two methods were optimized to provide two cost-efficient ways to do testing for a minimum of four samples by pooling in a 2.0 mL tube and decrease the need for more highly trained personnel. Acid-pH-based RNA extraction method can be done without the need for expensive kits. Direct Lysis is a quick one-step reaction that can be applied quickly. Our optimized Acid-pH and Direct Lysis protocols are reliable and reproducible, detecting the beta-2 microglobulin (B2M) mRNA in saliva as an internal control from 97 to 96.7% of samples, respectively. The cycle threshold (Ct) values for B2M were significantly higher in the Direct Lysis protocol than in the Acid-pH protocol. The limit of detection for N1 gene was higher in Direct Lysis at ≤ 5 copies/µL than Acid-pH. Saliva samples collected over the course of several days from two COVID-positive individuals demonstrated Ct values for N1 that were consistently higher from Direct Lysis compared to Acid-pH. Collectively, this work supports that each of these techniques can be used to screen for SARS-CoV-2 in saliva for a cost-effective screening platform.

Targeting a free viral fraction enhances the early alert potential of wastewater surveillance for SARS-CoV-2: a methods comparison spanning the transition between delta and omicron variants in a large urban center.

Introduction: Wastewater surveillance has proven to be a valuable approach to monitoring the spread of SARS-CoV-2, the virus that causes Coronavirus disease 2019 (COVID-19). Recognizing the benefits of wastewater surveillance as a tool to support public health in tracking SARS-CoV-2 and other respiratory pathogens, numerous wastewater virus sampling and concentration methods have been tested for appropriate applications as well as their significance for actionability by public health practices. Methods: Here, we present a 34-week long wastewater surveillance study that covers nearly 4 million residents of the Detroit (MI, United States) metropolitan area. Three primary concentration methods were compared with respect to recovery of SARS-CoV-2 from wastewater: Virus Adsorption-Elution (VIRADEL), polyethylene glycol precipitation (PEG), and polysulfone (PES) filtration. Wastewater viral concentrations were normalized using various parameters (flow rate, population, total suspended solids) to account for variations in flow. Three analytical approaches were implemented to compare wastewater viral concentrations across the three primary concentration methods to COVID-19 clinical data for both normalized and non-normalized data: Pearson and Spearman correlations, Dynamic Time Warping (DTW), and Time Lagged Cross Correlation (TLCC) and peak synchrony. Results: It was found that VIRADEL, which captures free and suspended virus from supernatant wastewater, was a leading indicator of COVID-19 cases within the region, whereas PEG and PES filtration, which target particle-associated virus, each lagged behind the early alert potential of VIRADEL. PEG and PES methods may potentially capture previously shed and accumulated SARS-CoV-2 resuspended from sediments in the interceptors. Discussion: These results indicate that the VIRADEL method can be used to enhance the early-warning potential of wastewater surveillance applications although drawbacks include the need to process large volumes of wastewater to concentrate sufficiently free and suspended virus for detection. While lagging the VIRADEL method for early-alert potential, both PEG and PES filtration can be used for routine COVID-19 wastewater monitoring since they allow a large number of samples to be processed concurrently while being more cost-effective and with rapid turn-around yielding results same day as collection.

Actionable wastewater surveillance: application to a university residence hall during the transition between Delta and Omicron resurgences of COVID-19.

Wastewater surveillance has gained traction during the COVID-19 pandemic as an effective and non-biased means to track community infection. While most surveillance relies on samples collected at municipal wastewater treatment plants, surveillance is more actionable when samples are collected "upstream" where mitigation of transmission is tractable. This report describes the results of wastewater surveillance for SARS-CoV-2 at residence halls on a university campus aimed at preventing outbreak escalation by mitigating community spread. Another goal was to estimate fecal shedding rates of SARS-CoV-2 in a non-clinical setting. Passive sampling devices were deployed in sewer laterals originating from residence halls at a frequency of twice weekly during fall 2021 as the Delta variant of concern continued to circulate across North America. A positive detection as part of routine sampling in late November 2021 triggered daily monitoring and further isolated the signal to a single wing of one residence hall. Detection of SARS-CoV-2 within the wastewater over a period of 3 consecutive days led to a coordinated rapid antigen testing campaign targeting the residence hall occupants and the identification and isolation of infected individuals. With knowledge of the number of individuals testing positive for COVID-19, fecal shedding rates were estimated to range from 3.70 log10 gc ‧ g feces-1 to 5.94 log10 gc ‧ g feces-1. These results reinforce the efficacy of wastewater surveillance as an early indicator of infection in congregate living settings. Detections can trigger public health measures ranging from enhanced communications to targeted coordinated testing and quarantine.

Averting an Outbreak of SARS-CoV-2 in a University Residence Hall through Wastewater Surveillance.

A wastewater surveillance program targeting a university residence hall was implemented during the spring semester 2021 as a proactive measure to avoid an outbreak of COVID-19 on campus. Over a period of 7 weeks from early February through late March 2021, wastewater originating from the residence hall was collected as grab samples 3 times per week. During this time, there was no detection of SARS-CoV-2 by reverse transcriptase quantitative PCR (RT-qPCR) in the residence hall wastewater stream. Aiming to obtain a sample more representative of the residence hall community, a decision was made to use passive samplers beginning in late March onwards. Adopting a Moore swab approach, SARS-CoV-2 was detected in wastewater samples just 2 days after passive samplers were deployed. These samples also tested positive for the B.1.1.7 (Alpha) variant of concern (VOC) using RT-qPCR. The positive result triggered a public health case-finding response, including a mobile testing unit deployed to the residence hall the following day, with testing of nearly 200 students and staff, which identified two laboratory-confirmed cases of Alpha variant COVID-19. These individuals were relocated to a separate quarantine facility, averting an outbreak on campus. Aggregating wastewater and clinical data, the campus wastewater surveillance program has yielded the first estimates of fecal shedding rates of the Alpha VOC of SARS-CoV-2 in individuals from a nonclinical setting. IMPORTANCE Among early adopters of wastewater monitoring for SARS-CoV-2 have been colleges and universities throughout North America, many of whom are using this approach to monitor congregate living facilities for early evidence of COVID-19 infection as an integral component of campus screening programs. Yet, while there have been numerous examples where wastewater monitoring on a university campus has detected evidence for infection among community members, there are few examples where this monitoring triggered a public health response that may have averted an actual outbreak. This report details a wastewater-testing program targeting a residence hall on a university campus during spring 2021, when there was mounting concern globally over the emergence of SARS-CoV-2 variants of concern, reported to be more transmissible than the wild-type Wuhan strain. In this communication, we present a clear example of how wastewater monitoring resulted in actionable responses by university administration and public health, which averted an outbreak of COVID-19 on a university campus.

Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada.

Detection of SARS-CoV-2 RNA in wastewater is a promising tool for informing public health decisions during the COVID-19 pandemic. However, approaches for its analysis by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) are still far from standardized globally. To characterize inter- and intra-laboratory variability among results when using various methods deployed across Canada, aliquots from a real wastewater sample were spiked with surrogates of SARS-CoV-2 (gamma-radiation inactivated SARS-CoV-2 and human coronavirus strain 229E [HCoV-229E]) at low and high levels then provided "blind" to eight laboratories. Concentration estimates reported by individual laboratories were consistently within a 1.0-log10 range for aliquots of the same spiked condition. All laboratories distinguished between low- and high-spikes for both surrogates. As expected, greater variability was observed in the results amongst laboratories than within individual laboratories, but SARS-CoV-2 RNA concentration estimates for each spiked condition remained mostly within 1.0-log10 ranges. The no-spike wastewater aliquots provided yielded non-detects or trace levels (<20 gene copies/mL) of SARS-CoV-2 RNA. Detections appear linked to methods that included or focused on the solids fraction of the wastewater matrix and might represent in-situ SARS-CoV-2 to the wastewater sample. HCoV-229E RNA was not detected in the no-spike aliquots. Overall, all methods yielded comparable results at the conditions tested. Partitioning behavior of SARS-CoV-2 and spiked surrogates in wastewater should be considered to evaluate method effectiveness. A consistent method and laboratory to explore wastewater SARS-CoV-2 temporal trends for a given system, with appropriate quality control protocols and documented in adequate detail should succeed.

The Atypical Dual Specificity Phosphatase hYVH1 Associates with Multiple Ribonucleoprotein Particles.

Human YVH1 (hYVH1), also known as dual specificity phosphatase 12 (DUSP12), is a poorly characterized atypical dual specificity phosphatase widely conserved throughout evolution. Recent findings have demonstrated that hYVH1 expression affects cellular DNA content and is a novel cell survival phosphatase preventing both thermal and oxidative stress-induced cell death, whereas studies in yeast have established YVH1 as a novel 60S ribosome biogenesis factor. In this study, we have isolated novel hYVH1-associating proteins from human U2OS osteosarcoma cells using affinity chromatography coupled to mass spectrometry employing ion mobility separation. Numerous ribosomal proteins were identified, confirming the work done in yeast. Furthermore, proteins known to be present on additional RNP particles were identified, including Y box-binding protein 1 (YB-1) and fragile X mental retardation protein, proteins that function in translational repression and stress granule regulation. Follow-up studies demonstrated that hYVH1 co-localizes with YB-1 and fragile X mental retardation protein on stress granules in response to arsenic treatment. Interestingly, hYVH1-positive stress granules were significantly smaller, whereas knocking down hYVH1 expression attenuated stress granule breakdown during recovery from arsenite stress, indicating a possible role for hYVH1 in stress granule disassembly. These results propagate a role for dual specificity phosphatases at RNP particles and suggest that hYVH1 may affect a variety of fundamental cellular processes by regulating messenger ribonucleoprotein (mRNP) dynamics.

Investigating redox regulation of protein tyrosine phosphatases using low pH thiol labeling and enrichment strategies coupled to MALDI-TOF mass spectrometry.

A central feature of the protein tyrosine phosphatase (PTP) catalytic mechanism is an attack of the substrate's phosphate moiety by a thiolate ion in the signature CX5R motif. In addition to being an effective nucleophile in this form, the thiolate ion is also susceptible to reversible redox regulation. This attribute permits temporal inhibition of PTP activities, which affects numerous cellular processes utilizing kinase-mediated signal propagation. Accumulating evidence has revealed diverse mechanisms adopted by PTPs to avoid irreversible thiol oxidation of the active site Cys residue, often involving structurally proximal thiols within the active site region. Therefore, there has been a significant effort made to develop thiol labeling strategies coupled to mass spectrometry to identify and characterize redox sensitive thiols within PTPs as a necessary step in understanding how a particular PTP is regulated by redox signaling. A common drawback to many current methods is the use of neutral pH labeling techniques, requiring special attention with regards to non-specific thiol oxidation during sample preparation. This study describes the use of rapid, low pH thiol labeling methods to overcome this issue. Mercury immobilized metal affinity chromatography (Hg-IMAC) demonstrated high selectivity and specificity while enriching for thiol-containing peptides from the atypical dual specificity phosphatase hYVH1 (also known as DUSP12). This approach revealed several reversibly oxidized thiols within the catalytic domain of hYVH1. Subsequently, use of another low pH labeling reagent, 4,4-dithiopyridine (4-DTP) helped identify novel disulfide linkages providing evidence that hYVH1 utilizes a disulfide exchange mechanism to prevent irreversible oxidation of the catalytic Cys residue in the active site.