Development and Validation of the Skimmed Milk Pellet Extraction Protocol for SARS-CoV-2 Wastewater Surveillance.

Wastewater surveillance for SARS-CoV-2 may serve as a useful source of data for public health departments as the virus is shed in the stool of infected individuals. However, for wastewater data to be actionable, wastewater must be collected, concentrated, and analyzed in a timely manner. This manuscript presents modifications on a skimmed milk concentration protocol to reduce processing time, increase the number of samples that can be processed at once, and enable use in resource-limited settings. Wastewater seeded with Human coronavirus OC43 (OC43) was concentrated using a skimmed milk flocculation protocol, and then pellets were directly extracted with the QIAamp Viral RNA Mini kit. This protocol has a higher average effective volume assayed (6.35 mL) than skimmed milk concentration methods, with and without Vertrel XF™, which involve resuspension of the pellets in PBS extraction prior to nucleic acid extraction (1.28 mL, 1.44 mL, respectively). OC43 was selected as a recovery control organism because both it and SARS-CoV-2 are enveloped respiratory viruses that primarily infect humans resulting in respiratory symptoms. The OC43 percent recovery for the direct extraction protocol (3.4%) is comparable to that of skimmed milk concentration with and without Vertrel XF™ extraction (4.0%, 2.6%, respectively). When comparing SARS-CoV-2 detection using McNemar's chi-square test, the pellet extraction method is not statistically different from skimmed milk concentration, with and without Vertrel XF™ extraction. This suggests that the method performs equally as well as existing methods. Added benefits include reduced time spent per sample and the ability to process more samples at a single time. Direct extraction of skimmed milk pellets is a viable method for quick turnaround of wastewater data for public health interventions.

Modifications of IL-6 by Hypochlorous Acids: Effects on Receptor Binding.

Interleukin-6 (IL-6) has been implicated in the pathogenesis of inflammatory events including those seen with COVID-19 patients. Positive clinical responses to monoclonal antibodies directed against IL-6 receptors (IL-6Rs) suggest that interference with IL-6-dependent activation of pro-inflammatory pathways offers a useful approach to therapy. We exposed IL-6 to hypochlorous acid (HOCl) in vitro at concentrations reported to develop in vivo. After HOCl treatment, binding of IL-6 to IL-6R was reduced in a dose-dependent manner using a bioassay with human cells engineered to provide a luminescence response to signal transduction upon receptor activation. Similar results followed the exposure of IL-6 to N-chlorotaurine (NCT) and hypobromous acid (HOBr), two other reactive species produced in vivo. SDS-PAGE analysis of HOCl-treated IL-6 showed little to no fragmentation or aggregation up to 1.75 mM HOCl, suggesting that the modifications induced at concentrations below 1.75 mM took place on the intact protein. Mass spectrometry of trypsin-digested fragments identified oxidative changes to two amino acid residues, methionine 161 and tryptophan 157, both of which have been implicated in receptor binding of the cytokine. Our findings suggest that exogenous HOCl and NCT might bring about beneficial effects in the treatment of COVID-19. Further studies on how HOCl and HOBr and their halogenated amine derivatives interact with IL-6 and related cytokines in vivo may open up alternative therapeutic interventions with these compounds in COVID-19 and other hyperinflammatory diseases.

A comparison of SARS-CoV-2 wastewater concentration methods for environmental surveillance.

Wastewater1 surveillance of SARS-CoV-2 may be a useful supplement to clinical surveillance as it is shed in feces, there are many asymptomatic cases, and diagnostic testing can have capacity limitations and extended time to results. Although numerous studies have utilized wastewater surveillance for SARS-CoV-2, the methods used were developed and/or standardized for other pathogens. This study evaluates multiple methods for concentration and recovery of SARS-CoV-2 and seeded human coronavirus OC43 from municipal primary wastewater and/or sludge from the Greater Seattle Area (March-July 2020). Methods evaluated include the bag-mediated filtration system (BMFS), with and without Vertrel™ extraction, skimmed milk flocculation, with and without Vertrel™ extraction, polyethylene glycol (PEG) precipitation, ultrafiltration, and sludge extraction. Total RNA was extracted from wastewater concentrates and analyzed for SARS-CoV-2 and OC43 with RT-qPCR. Skimmed milk flocculation without Vertrel™ extraction performed consistently over time and between treatment plants in Seattle-area wastewater with the lowest average OC43 Cq value and smallest variability (24.3; 95% CI: 23.8-24.9), most frequent SARS-CoV-2 detection (48.8% of sampling events), and highest average OC43 percent recovery (9.1%; 95% CI: 6.2-11.9%). Skimmed milk flocculation is also beneficial because it is feasible in low-resource settings. While the BMFS had the highest average volume assayed of 11.9 mL (95% CI: 10.7-13.1 mL), the average OC43 percent recovery was low (0.7%; 95% CI: 0.4-1.0%). Ultrafiltration and PEG precipitation had low average OC43 percent recoveries of 1.0% (95% CI: 0.5-1.6%) and 3.2% (95% CI: 1.3-5.1%), respectively. The slopes and efficiency for the SARS-CoV-2 standard curves were not consistent over time, confirming the need to include a standard curve each run rather than using a single curve for multiple plates. Results suggest that the concentration and detection methods used must be validated for the specific water matrix using a recovery control to assess performance over time.

Modification of Major Contributors Responsible for Latrine Malodor on Exposure to Hypochlorous Acid: The Potential for Simultaneously Impacting Odor and Infection Hazards to Encourage Latrine Use.

Open defecation remains a common practice in developing countries and leads to high incidence and prevalence of acute gastroenteritis, which is most often caused by human noroviruses (human NoV). Encouraging the use of toilets and pit latrines is one method of improving sanitation; however, it is often hindered by not only cultural traditions but also from a reluctance to use latrines and toilets due to their odor and impression of uncleanliness. In an effort to establish new means to encourage toilet and latrine use, laboratory experiments tested the ability of hypochlorous acid (HOCl) to modify the malodorous compounds identified in the air in latrines in developing countries (indole, p-cresol, dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and butyric acid) and inactivate MS2 bacteriophage, a surrogate for human NoV. After 5 minutes, > 94% of indole, p-cresol, DMDS, and DMTS was modified as determined by high-pressure liquid chromatography in the presence of 100 ppm HOCl. A log10 reduction value (LRV) greater than 6 was seen for MS2 bacteriophage after 5 minutes of exposure to 100 ppm HOCl in solution. Sensory studies indicated that there was a significant difference (P ≤ 0.05) between the untreated and HOCl-treated samples for all five malodorous compounds tested. The findings suggest that introduction of HOCl into the headspace air could encourage latrine and toilet use. Optimization of HOCl dosing in air to accomplish both odor control and reduction of infectious hazards is worthy of further study.