SARS-CoV-2: What is its fate in urban water cycles and how can the water research community respond?

This paper is an “Editorial Perspective” published in the Royal Society of Chemistry (RSC) journal “Environmental Science: Water Research & Technology”*. The authors are Vincenzo Naddeo (University of Salerno Italy) and Haizhou Liu (University of California at Riverside, USA). The paper is made free to all readers as part of the RSC collection on corona virus research.

The authors provide a perspective on the fate of SARS-CoV-2 in urban water cycles and associated risks to public health. They remind us that previous studies have shown that coronaviruses (including SARS-CoV) can exist and maintain viability in sewage and hospital wastewater, originating from the faecal excretions of infected patients. Previous studies have also highlighted the persistence of (other surrogate) coronaviruses in aquatic environments and wastewater treatment plants.

The authors speculate that if a coronavirus did contaminate a drinking water supply system (perhaps one with low disinfection residual), survival might be enhanced by colonising bacteria in biofilms. If this were to occur, there could be exposure risks from the production of aerosols during activities such as showering.

The theoretical possibility of such exposure risks highlights the need to properly manage water supply systems to ensure that such risks are maintained at safe levels. Current water and wastewater disinfection strategies, using chlorine and UV irradiation are anticipated to be very effective for SARS-CoV-2 inactivation, but proper operational control is always essential.

The authors point out that some particular wastewater treatment processes, such as membrane bioreactors may also play an important role since they have been shown to be effective for virus removal, including for some enveloped viruses (such as coronaviruses) and some non-enveloped viruses (e.g. norovirus) that are known to be more resistant than enveloped viruses.

This Editorial Perspective highlights a number of water-cycle related research needs. These include:

  • Decentralised virus inactivation treatment for wastewater discharged from potential hot-spots, such as hospitals, community clinics and nursing homes. This could reduce the environmental loading of viruses and risks of secondary transmission.
  • Portable point-of-use disinfection devices for drinking water in individual households. For example, LED-based UV systems for decentralised disinfection. Such systems could be of value in countries where water treatment and/or distribution integrity is not reliable.
  • Improved understanding of the efficacy of emerging disinfection technologies for coronavirus inactivation, especially treatment steps that are integrated into potable water reuse, including UV-based advanced oxidation processes (UV/AOPs) and ozone/biologically activated carbon (O3/BAC).
  • Review of regulatory guidelines for virus removal in potable reuse systems for possible more stringent requirements in the event of a major coronavirus outbreak.
  • Understanding the role of drinking water distribution systems in potentially harbouring coronaviruses, including the potential role of bacterial colonies and biofilm growth.
  • An improved understanding of relationships between measures to control pandemic viral outbreaks (e.g., widespread use of disinfectants) and impacts on chemical pollution and the proliferation of antibiotic resistant bacteria.

In concluding comments, the authors consider how increasing globalisation enhances the significance of shared global health risks. They argue that the governments of developed countries must support and finance water and sanitation systems in developing countries, in order to also protect the citizens of their own countries.

REFERENCE:

Naddeo V and Liu H (2020) Editorial Perspectives: 2019 novel coronavirus (SARS-CoV-2): what is its fate in urban water cycle and how can the water research community respond? Environmental Science: Water Research & Technology*.

https://doi.org/10.1039/D0EW90015J

*The author of this blog post (Stuart Khan) is an Associate Editor of this journal and thus declares and interest in the promotion of work published in it.

Modes of transmission of virus causing COVID-19: implications for IPC precaution recommendations

The WHO has recently released a scientific briefing on the modes of transmission of the COVID-19 virus, which can be accessed here.

What does the briefing cover?

The briefing defines the difference between droplets, which are larger than 5 microns and generated by coughing or sneezing, and droplet nuclei, which are less than 5 microns and may be generated during certain medical procedures such as intubation. The briefing also describes the difference between droplet transmission, which requires close contact within 1 m, and airborne transmission of droplet nuclei, which can stay suspended in the air and be transmitted over distances larger than 1 m. Significantly, analysis of a large number of COVID-19 cases in China did not find evidence for airborne transmission. The briefing also provides a summary of the implications of recent studies reporting the detection of COVID-19 virus from air sampling.

Of note, the briefing indicates that there have been no reports of faecal-oral transmission of the COVID-19 virus. Although RNA of the COVID-19 virus has been detected in the stools from COVID-19 patients in multiple studies, there has been only one report claiming the detection of culturable virus from a single stool specimen.

The evidence presented to demonstrate the presence of culturable virus was not comprehensive. The authors did not state how many stool samples were screened that were negative. The conclusion of cell infection was based on the observation by electron microscopy of virus particles on/in Vero cells. However, the authors did not report if there was cytopathic effect or other evidence of cell infection, such as an increase in virus particle number compared with the number in the original stool sample.

Conclusions

Based on their review of available data, the WHO advice regarding transmission modes and disease prevention remain unchanged.

References

https://www.who.int/news-room/commentaries/detail/modes-of-transmission-of-virus-causing-covid-19-implications-for-ipc-precaution-recommendations

http://weekly.chinacdc.cn/en/article/id/ffa97a96-db2a-4715-9dfb-ef662660e89d

Chemicals and microbes in bioaerosols from reaction tanks of six wastewater treatment plants: survival factors, generation sources, and mechanisms

Wastewater treatment commonly includes the production of air bubbles (aeration) at the bottom of activated sludge or during Dissolved Air Flocculation and Floatation. When the bubbles rise to the surface they burst, form droplets and ‘aerosolise’. This is how pathogenic microorganisms in the wastewater can become ‘airborne’. 

The Experiment: Bioaerosol samples were collected from 0.1, 1.5 or 3m above the surface of six WWTP that use biochemical reaction tanks, or an aeration basin, or an aeration pool. Total Organic Carbon, insoluble, soluble, and total suspended particles were quantified. The particles in the samples were examined using Scanning Electron Microscopy, the composition was determined using Energy Dispersive X-Ray Spectroscopy (EDX), and bacteria and fungi were cultured.

Results: The highest numbers of large heavy particles were found near the water surface whereas 3m above the water surface there were fewer particles but 75% were small particles <2.5μm. There was a reduction in WW-derived chemicals to 5.36% at 3m, but still 76% of culturable bacteria and fungi. They did not examine virus.

The concentration of bioaerosols was highly influenced by wind speed, temperature and relative humidity.

Conclusion: The authors noted that ‘effective measures should be adopted to protect on-site workers’, not because of SARS-CoV-2 but because of general pathogen load. Surgical masks can prevent a worker breathing in approximately 66% of aerosolised pathogens (see Noti et al., 2012).

REFERENCE:

Yanjie Wang1,2, Huachun Lan3, Lin Li 1,2, Kaixiong Yang1,2, Jiuhui Qu3 & Junxin Liu1,2 (2018) Scientific Reports 8:9362  https://DOI:10.1038/s41598-018-27652-2

Detection of Infectious Influenza Virus in Cough Aerosols Generated in a Simulated Patient Examination Room

Bioaerosols are generated during wastewater treatment (eg Wang et al., 2018) and personnel working in public are also at risk of infection through inhaling aerosolised virus. This study describes the working efficacy of a N95 respirator mask (left image) or a surgical mask (right image).

The Experiment: A mechanical dummy which coughed infectious influenza virus was placed in a room (humidity 44-63%) with another ‘breathing’ dummy which contained aerosol samplers in its ‘mouth’. Health, clinical and other workers do not seal the masks to their faces (right image), and the dummy modelled this . Virus was extracted from the samplers, surgical gloves, masks and respirator masks, then cultured to identify infectious virus. Extracts of the viral DNA were subjected to qRTPCR to facilitate quantification of the infectious virus.

Results: the N95 mask prevented 67% of the infectious virus entering the dummy’s mouth, whereas the surgical mask blocked entry to 57%. Infectious virus was found in the outer water-repellent and middle filtering layers of both types of mask, and on the surfaces of surgical gloves attached to the head of the ‘breathing’ dummy.

Conclusions: Wearing surgical masks reduces but does not remove risk of infection. This is because the way these masks are worn leaves gaps between the wearers face and the mask which allow entry of aerosol particles, even when the masks are tied tightly to the face.

REFERENCE:

John D. Noti, William G. Lindsley, Francoise M. Blachere, Gang Cao, Michael L. Kashon, Robert E. Thewlis, Cynthia M. McMillen, William P. King, Jonathan V. Szalajda, and Donald H. Beezhold (2012) Clinical Infectious Diseases 54(11):1569–77.

https://DOI: 10.1093/cid/cis237

Presence of SARS-Coronavirus-2 in sewage

Authors: Gertjan Medema and colleagues at KWR in The Netherlands

In a non peer reviewed preprint (preprint pdf) report on their use of rt-PCR to detect RNA from SARS-COV-2 at multiple locations.  Their sampling started in February, before COVID19 cases were detected in The Netherlands, and extended when cases were detected at all sites.

The February samples were negative at all sewage sites examined (incoming wastewater at treatment plants).  In early March, 5 of 7 sites were positive (while only 4 of 7 of the corresponding cities had reported positive COVID cases).  By their March 15/16 samples, 6 of 7 sites were positive for RNA from SARS-COV-2.

While no inference about the presence of viable virus can be made from the data, the authors conclude “The detection of the virus in sewage, even when the COVID- 19 prevalence is low, indicates that sewage surveillance could be used to monitor the circulation of the virus in the population and as early warning tool for increased circulation in the coming winter or unaffected populations.”

I agree.

SARS-CoV-2–Positive Sputum and Feces After Conversion of Pharyngeal Samples in Patients With COVID-19

This study was reported as a letter to the scientific journal Annals of Internal Medicine on 30 March 2020.

The authors report the outcomes of testing for SARS-CoV-2 RNA in sputum and faecal samples from COVID-19 patients after their apparent recovery, which was indicated by throat swabs no longer testing positive.

The key finding was that virus RNA was detected in sputum and faeces up to 39 and 13 days, respectively, after the throat swabs were negative.

This is a significant finding since it may indicate that the patients still harbour the disease and/or that testing these alternative bodily excretions may provide a more sensitive or reliable test than the current reliance on throat swabs.

However, there is a risk that this study may be misreported, or misinterpreted, by some as indicating that these samples (particularly faeces) contain viable, infectious virus.

This study was undertaken by testing for SARS-CoV-2 RNA by real-time quantitative fluorescence polymerase chain reaction (RT-qPCR). This method can detect the presence of parts of the genetic material (RNA) of the virus, but it cannot indicate that infectious virus is present.

It is possible that infectious virus is present in these samples, but alternative culture-based methods would be required to ascertain that it is.

REFERENCE

Chen C, Gao G, Xu Y, Pu L, Wang Q, Wang L, Wang W, Song Y, Chen M, Wang L, Yu F, Yang S, Tang Y, Zhao L, Wang H, Wang Y, Zeng H and Zhang F (2020) SARS-CoV-2–Positive Sputum and Feces After Conversion of Pharyngeal Samples in Patients With COVID-19. Annals of Internal Medicine

https://doi.org/10.7326/M20-0991

Exhaled Air Dispersion during Coughing with and without Wearing a Surgical or N95 Mask

This study gives information about surgical mask containment of particles coughed out by an infected person and is relevant to personnel who have to work amongst potentially infectious members of the public who may or may not be wearing masks.

In this study a dummy of a 70kg man enclosing a mechanical lung model for human respiration was sat at a 45°C angle in a negative pressure room. Smoke was added to the ‘lungs’, and oxygen flows adjusted to simulate a normal, healthy cough. The dummy was bare-faced, or fitted with a surgical mask, or a N95 respirator mask (see images). A laser light sheet and video were used to capture images of smoke dispersal during 20 coughs for each of surgical mask, N95 mask or no-mask conditions. Multiple frames from each of the cough videos were subjected to image analysis using purpose-built software to construct contours of smoke particle concentration (roughly equivalent to viral particles) around the dummy.

RESULTS: Without a mask the dummy’s cough formed a turbulent jet, some of which was downwards but most projected forwards to a maximum of 75cm at 7.39m/s. The surgical mask did not prevent the forwards projection of smoke particles but did reduce the distance to a maximum of 34cm, as well as 31cm out to each side. The N95 mask reduced forwards projection to 18cm and sideways projection to 17cm each side.

Small particles, which may include infectious viral particles, can escape from behind masks, but masks still reduce transmission risk. In a negative pressure room the unmasked cough plume reached 75cm, whereas a similar study (Tang et al., 2012) found a normal healthy cough plume in normal atmospheric pressure reached 64cm. Recommendations for 1.5 – 2m distancing allow for additional dispersal and reduction of infectious dose, and further reduce risk of COVID19 infection for personnel.

REFERENCE: David S. Hui, Benny K. Chow, Leo Chu, Susanna S. Ng, Nelson Lee, Tony Gin, Matthew T. V. Chan (2012) PLoS ONE 7(12): e50845.

https://doi:10.1371/journal.pone.0050845

Survival of Severe Acute Respiratory Syndrome Coronavirus (2005)

This research paper was published in 2005 and relates to SARS-CoV (responsible for the 2002/03 SARS outbreak), not SARS-CoV-2 (responsible for COVID-19). However, since these two viruses are closely related, it is insightful to observe what is known about the former, in order to consider what may be possible (or even likely) for the latter.

The journal “Clinical Infectious Diseases” appears to be a well-respected journal (ranked by InCites Journal Citation Reports in Quartile 1 for each of “Immunology”, “Infectious Diseases”, and “Microbiology”) [I checked this since the misspelling of the important word “fomites” in the very first sentence raised a red flag about authenticity/quality].

The paper reinforces up-front that the primary modes of transmission of SARS-CoV appear to be direct mucus membrane contact with infectious droplets and through exposure to contaminated surfaces (fomites). Due particularly to the latter, knowledge of the survival characteristics of the virus is essential for identifying appropriate infection-control measures.

This study reports the results of experiments designed to investigate the survival of SARS-CoV strain GVU6109 in faecal and respiratory specimens. The focus is on potential transmission in a clinical setting, which is clear by the nature of the surfaces in which the viral survival tests were conducted, including a laboratory request form, an impervious disposable gown, and a cotton nondisposable gown. The disinfection performances of sodium hypochlorite, household detergent, and a commercial peroxygen compound were also investigated.

The experiments described in this paper did not test faecal samples from SARS-infected patients. Instead, four uninfected faecal samples were used and a 10% suspension of each was prepared in phosphate-buffered saline solution. After centrifugation, the supernatant was collected and a total of 1.8 mL of each 10% stool suspension was spiked with 0.2 mL of virus stock. These samples were incubated in closed containers at room temperature (20°C) for periods of between 30 min and 7 days.

The SARS-CoV strain (GVU6109) used in this study had been isolated from a lung tissue specimen obtained from a patient during the SARS outbreak in 2003. To test “survival” in these experiments, the virus was inoculated into the Vero E6 cell line, which was grown in minimum essential medium (MEM) with 2% fetal calf serum at 37°C.

Surviving SARS-CoV was not recoverable within 1 day after incubation in normal adult faecal specimens or within 3 hours after incubation in a baby faecal specimen. However, it survived for 4 days in a diarrheal faecal specimen. The duration of survival for SARS-CoV in this faecal suspension was retested in another 2 diarrheal specimens, with the same results.

SARS-CoV was also reported to survive in respiratory specimens, such as a nasopharyngeal aspirate specimen, as well as throat and nasal swab specimens, for >7 days at room temperature and for >20 days at 4°C.

Even at a relatively high concentration, the virus could not be recovered after drying of a paper request form, and its infectivity was shown to last longer on the disposable gown than on the cotton gown. All disinfectants tested were shown to achieve >3 Log10 inactivation within 5 min.

The authors concluded that faecal and respiratory samples can remain infectious for numerous days at room temperature. They also surmised that the risk of infection via contact with droplet-contaminated paper is small; and that and that absorbent material, such as cotton, is preferred to nonabsorptive material for PPE in some circumstances.

REFERENCE:

Lai MYY, Cheng PKC and Lim WWL (2005) Survival of Severe Acute Respiratory Syndrome Coronavirus. Clinical Infectious Diseases, 41(7), e67-e71.

https://doi.org/10.1086/433186

An Imperative Need for Research on the Role of Environmental Factors in Transmission of Novel Coronavirus (COVID-19)

On this blog, we have previously highlighted the recently produced open access Environmental Science and Technology ‘virtual issue’, collating numerous years of research on the fate and behaviour of enveloped viruses in the environment.

This virtual issue includes a current “scientific opinion” (non-peer reviewed) piece by four researchers, Guangbo Qu (Chinese Academy of Sciences), Xiangdong Li (The Hong Kong Polytechnic University), Ligang Hu (Chinese Academy of Sciences), and Guibin Jiang (Chinese Academy of Sciences). The focus of this scientific opinion is on the need for research on the role of environmental factors in transmission of SARS-CoV-2.

Although certainly not a dominant transmission pathway, these authors argue that faecal transmission routes should be considered based on reports of SARS-CoV-2 detection in faecal samples of infected patients. They describe how studies have shown that SARS-CoV can survive in [somewhat synthetic] stool samples for four days. They write that in one study, surrogate coronaviruses were reported to remain infectious in water and sewage for periods of days to weeks. At room temperature, in pure water, or pasteurised settled sewage, these researchers reported that the time required for 2 Log10 (99%) reduction of virus infectivity (for surrogate coronaviruses) was several days.

The authors argue that infected faecal material in wastewater can generate further transmission routes, including through aerosols produced by toilet flushing. They remind us that a contaminated faulty sewage system in a high-rise housing estate was previously linked to a SARS outbreak in Hong Kong.

A further potential transmission route proposed in this opinion article is via airborne dust. Poor air pollution, with high levels of particular matter, occurs frequently in some developing countries, and the potential roles of dust and particulate matter in the transmission (and possible long-range transfer) of SARS-CoV-2 remain uninvestigated.

The concluding message from this opinion piece is that the survival of SARS-CoV-2 in various environmental media, including water, airborne particulate matter, dust, and sewage, under a variety of environmental conditions, warrant systematic investigation.

REFERENCE:

Qu G, Li X, Hu L and Jiang G (2020) An Imperative Need for Research on the Role of Environmental Factors in Transmission of Novel Coronavirus (COVID-19). Environmental Science & Technology.

https://doi.org/10.1021/acs.est.0c01102

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV

One thing that is well understood about SARS-CoV-2 is that it is closely related to SARS-CoV. The limited amount of research that is available (see previous posts on this blog) indicate that the inactivation of SARS-CoV in the environment, and in response to various disinfection practices is a reasonable surrogate for the inactivation of SARS-CoV-2. This is a valuable insight since it means that previous studies investigating the inactivation of SARS-CoV can be used to provide realistic insights to the expected inactivation of SARS-CoV-2.

This paper, published in 2004 is a good example of the types of insights from SARS-Cov that are available. It describes how SARS-CoV can be inactivated by ultraviolet light (UV) at 254 nm, heat treatment of 65 °C or greater, alkaline (pH > 12) or acidic (pH < 3) conditions, formalin and glutaraldehyde treatments.

UV radiation

UV light at wavelength 254 nm (UVC) emitted at 4016 μW/cm2 (where μW = 10−6 J/s) was shown to achieve significant inactivation of SARS-CoV after 6 minutes. The text states that this resulted in a “400-fold decrease in infectious virus”, which equates to 2.6 Log10 inactivation. [Note that Figure 1 from the paper appears to indicate around 4 Log10 inactivation, with the reason for the apparent discrepancy unclear, at least to me]. The approximate UV “dose” from this lamp after 6 minutes would be 1450 mJ/cm2.

Unsurprisingly, UV radiation in the UVA spectrum (365 nm) was shown to be ineffective.

Gamma irradiation

Gamma radiation (3000, 5000, 10,000, and 15,000 rad) from a 60Co source was shown to be ineffective.

Heat treatment

Heating at 56 °C achieved significant inactivation after 20 min. However, the virus remained infectious at a level close to the limit of detection for the assay, for at least 60 min, suggesting that some virus particles were stable at 56 °C. At 65 °C, most of the virus was inactivated after 4 min, but again, some infectious virus could still be detected after 20 min. However, complete inactivation (to below the limit of detection of the assay) was observed at 75 °C in 45 min. The authors report that “these results suggest that viral inactivation by pasteurization may be very effective”.

Formaldehyde and glutaraldehyde

The authors examined formalin and glutaraldehyde inactivation by incubating virus samples with formalin or glutaraldehyde at two different dilutions (1:1000 and 1:4000). Neither formalin nor glutaraldehyde, at a 1:4000 dilution, was able to completely inactivate virus at 4 °C, even after exposure for 3 days. At 25 and 37 °C, formalin inactivated most of the virus, close to the limit of detection of the assay, after 1 day, yet some virus still remained infectious on day 3. Glutaraldehyde completely inactivated the virus by day 2 at 25 °C and by day 1 at 37 °C. The authors conclude that “both formalin and glutaraldehyde inactivation maybe effective, if proper conditions are met”.

pH

After exposing SARS-CoV to extreme alkaline conditions of pH 12 and 14 for 1 h, and subsequently reversing the conditions to a neutralised, buffered solution, the virus was observed to be completely inactivated (to below the limit of detection of the assay). However, moderate variations of pH conditions from 5 to 9 had little or no effect on virus infectivity, regardless of the temperature. Highly acidic pH conditions of 1 and 3 also completely inactivated the virus at 25 and 37 °C. However, at cooler temperatures (4 °C), a pH of 3 was not fully effective.

REFERENCE:

Darnell, M. E. R., Subbarao, K., Feinstone, S. M. and Taylor, D. R. (2004) Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV. Journal of Virological Methods, 121(1), 85-91.

https://doi.org/10.1016/j.jviromet.2004.06.006