Virological assessment of hospitalized patients with COVID-2019

Relevance to Water

Are wastewater treatment plant (WWTP) operators at risk from SARS-CoV-2 infection through exposure to human urine or faeces?

Background information about SARS-CoV-2

The hormone angiotensin regulates fluid and blood pressure. In healthy humans angiotensin binds to its receptor (named angiotensin converting enzyme 2, or ACE2) which is expressed by cells that line the respiratory tract. Similar cells line the gastrointestinal tract and blood vessels hence the ACE2 receptor has been found in the throat, lungs, gut, heart and kidneys.

The spike protein on the outside of SARS-CoV-2 binds to ACE2. This sets off a train of events which end up with the virus inside the cell, where it ‘switches on’ genes, hijacks the cell and forces it to make more virus, which in turn break out and infect more cells.

Notes about laboratory methods used in this study

Quantitative real time polymerase chain reaction (qRTPCR) can detect the genomic RNA inside SARS-CoV-2 but cannot distinguish between genomic RNA molecules that have come from live or dead viruses.

Viruses collected from a person’s throat, urine or faeces samples can be transferred to a laboratory cell culture system. If the virus is dead, nothing happens. If the virus is alive, it can sometimes infect the cells in the enclosed culture dish and the amount of infection can be quantified.

When the virus infects the throat cells inside a patient, certain virus genes are ‘switched on’. Detection of this ‘subgenomic mRNA’ (SGmRNA) is another way to distinguish between live or dead virus. The presence of SGmRNA in a swab, sputem or faeces sample means that there was live, infectious virus inside cells lining the respiratory or gastrointestinal tracts, but it does not mean that there is live infectious virus in the sample. In this study only the cell culture infectivity test identified live infectious virus within a sample.

In this study throat swabs were preserved in a solution (viral transport medium) whereas sputem and faeces samples were ‘pure’, nothing was added to them until virus was extracted from all the samples in the laboratory.

What they did in this study (Methods)

Nine young to middle aged adults were infected with SARS-CoV-2 virus after a known contact with a COVID19 patient. Apart from being infected with SARS-CoV-2 virus they were otherwise healthy, although three did have other conditions; chronic obstructive pulmonary disease, hypothyroidism and hypercholestinemea.

Throat swabs, sputem, urine and blood samples were collected from all 9 patients for 12 to 28 days, but only thirteen faeces samples were collected from 4 patients on days 6 to 12. The viruses in these samples were assessed by qRTPCR of genomic RNA and SGmRNA, and by using a live cell culture infectivity test. The blood samples were used to examine the types and amounts of antibodies that the infected patients produced.

What they did not find (Results)

None of the nine patients were infected with other respiratory viruses such as influenza A or B. In this study the changes in blood antibodies and other symptoms were due to SARS-CoV-2.

There were no differences between two types of throat swabs; oropharyngeal and nasopharangeal.

What they found (Results)

The amount of live virus in each sample affected the ability of the research team to detect infectious virus. They needed more than a million copies of viral genomic RNA (roughly corresponding to a million virus particles) for the cell culture infectivity test to work.

It was easier to collect live virus from sputem and faeces than from throat swabs. This meant that when a swab and a sputem sample with similar viral loads of a million or more were collected from one patient at the same time, the swab sample often failed to yield any live infectious virus whereas the sputem sample did. Ideally both types of sample would have given the same result but in this study the different types of samples caused discrepancies between test results. To illustrate this, by day 3 of testing, 1 swab and 2 sputem samples had more than a million virus copies, but there were only 2 samples in which live infectious viruses were detected. It is also likely that sputem samples with lower viral loads contained infectious viruses that were not detected in the cell culture test.

Infectious viruses were detected in sputem samples that had high viral loads for 8 days. Sputem samples that contained more than a million virus copies were found for 14 days, but no infectious viruses were detected after 8 days. None of the 13 faecal samples yielded live infectious virus, even when the viral loads were higher than one million.

The other method for distinguishing between live and dead virus at the site of infection, ‘SGmRNA’, detected ‘switched on’ gene RNA from virus in sputem from days 4 to 11, and in 3 of 9 faeces samples collected days 6 to 9. The presence of SGmRNA is not evidence for the presence of live infectious SARS-CoV-2 virus in faeces.

Conclusions

The early day 1 and 2 samples had the highest viral loads, and viral loads decreased thereafter. When the samples with the highest viral loads were collected the patients either had no symptoms (prodromal) or very mild symptoms. This study did not examine the ability of early stage infectious people to shed virus or infect others but if prodromal people did not cough or sneeze then the high viral loads in their throats would be less likely to infect others.

Although viral RNA was detected by qRTPCR for up to a month, infectious viruses were only detected for 8 days, even when the viral load was so high that the infectivity test would have worked. The authors speculated that it is possible that the patients’ immune systems weakened the virus and made them less able to infect after 8 days.

Viral RNA found in faeces was probably from dead virus. The combination of tiny amounts of SGmRNA in 3 of 9 samples, and complete absence of any infectious virus detected in the cell culture test, leads to the conclusion that this study did not provide evidence for live infectious SARS-CoV-2 in faeces.

The authors note that although there is evidence for some SARS-CoV-2 infection of cells that line the gut, it is not known if the environment inside the lumen of the gut is hostile to virus, or if passage through the lower gut kills the virus, and more studies are needed to determine this.

Take Home Message

There was no evidence for infectious live SARS-CoV-2 in human urine or faeces in this small study of only four people (that faeces samples were collected from).

WWTP operators are unlikely to be at risk of SARS-CoV-2 infection from this source but larger studies are needed to increase the ‘weight of evidence’ to substantiate this conclusion.

REFERENCE:

Roman Wölfel, Victor M. Corman, Wolfgang Guggemos, Michael Seilmaier, Sabine Zange, Marcel A. Müller, Daniela Niemeyer, Terry C. Jones, Patrick Vollmar, Camilla Rothe, Michael Hoelscher, Tobias Bleicker, Sebastian Brünink, Julia Schneider, Rosina Ehmann, Katrin Zwirglmaier, Christian Drosten & Clemens Wendtner

https://doi.org/10.1038/s41586-020-2196-x

Published by Fiona Young

Adjunct Associate Professor, College of Medicine and Public Health, Flinders University, Adelaide, SA. She taught and researched in the disciplines of Medical and Environmental Biotechnology, with a focus on toxicology and mammalian cell culture using 3D systems to examine effects of toxins and pollutants on human cells.

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