- A recent Spanish study claims to have found traces of SARS-CoV-2 in a March 2019 sewage sample
- This challenges current consensus that the virus originated in China in December 2019
- Local and international experts have expressed scepticism about the study, and call for further research
A research team from the University of Barcelona, one of Spain's most prestigious universities, released a study last week noting that they had detected traces of the new coronavirus on January 15, 2020 – 41 days before the first official case was declared in February 2020. All the samples prior to this date tested negative, except for a sample from March 2019 – nine months before Covid-19 was first identified in China.
The results were published in the preprint server medRxiv and have yet to be peer-reviewed.
‘Covid-19 cases masked with influenza’
“SARS-CoV-2 was detected in Barcelona sewage long before the declaration of the first Covid-19 case, indicating that the infection was present in the population before the first imported case was reported,” the researchers wrote, adding: “Sentinel surveillance of SARS-CoV-2 in wastewater would enable adoption of immediate measures in the event of future Covid-19 waves.”
According to a news release by the university, lead author of the study, Albert Bosch, a professor in the Department of Microbiology of the University of Barcelona and president of the Spanish Society of Virology, people infected with Covid-19 (the disease caused by SARS-CoV-2) may have been mistakenly diagnosed with flu in primary care, causing this to contribute to community transmission before public health authorities took measures.
"In the specific case of Barcelona," the virologist continues, "having detected the spread of SARS-CoV-2 a month in advance would have allowed a better response to the pandemic."
The team’s work is part of a larger sentinel monitoring project and they have analysed weekly samples obtained at two large wastewater treatment plants in Barcelona since 13 April. However, the study has met with criticism and scepticism among some scientists.
SARS-CoV-2 in wastewater?
Firstly, we will take a look at existing research on wastewater and the coronavirus.
Since the outbreak of the pandemic, scientists have been working hard to understand the potentially deadly viral disease. Although key transmission routes appear to be through inhalation via person-to-person aerosol/droplet transmission, as well as fomite-to-hand contamination, there is a need for ongoing research so that we can better understand how the virus operates.
Scientists have, therefore, started to look at the role of wastewater as a risk factor.
Health24 reported on a preliminary study in May, involving UK and Israeli scientists who surveyed sewage and warned about the risk of SARS-CoV-2 spreading through wastewater. Another study published in Science of the Total Environment notes that:
“Both viable SARS-CoV-2 and viral RNA are shed in bodily excreta, including saliva, sputum, and faeces, which are subsequently disposed of in wastewater.” The researchers also discuss the findings of previous studies on SARS-CoV and MERS-CoV that detected their viral RNA in faeces.
Findings question current consensus
If, as the researchers of the new study believe, the new coronavirus was present in wastewater in Barcelona in March 2019, this would mean that the virus was circulating much earlier than previously believed. It would also challenge consensus that it originated in China in December 2019. However, some scientists are doubtful about the findings, among them, Professor Francois Balloux from University College London, who has labelled the evidence as “shaky”.
Other experts not involved in the study have suggested that there is a possibility the wastewater tests might have produced false positives due to contamination or improper storage of the samples.
“I don’t trust the results,” Dr Irene Xagoraraki, an environmental engineer at Michigan State University told the New York Times.
What local experts say
Professor Wolfgang Preiser, the head of the division of medical virology at the Department of Pathology at the University of Stellenbosch said that the study as a whole seems well done and that the authors are respectable and their methods seem sound. Their results, Preiser added, appear entirely plausible, in light of what is known from other affected areas.
Preiser’s “bone of contention”, however, is that the majority of the published paper describes research on wastewater treatments from early 2020, but that the March 2019 finding was only briefly mentioned in the very last paragraph of the paper.
“It is striking how ‘in passing’ they [researchers] drop what would be a veritable bomb,” Preiser said, continuing:
“However, for me it is not a bomb until proven as such. This experiment needs to be repeated; the finding should also be confirmed by testing other samples from around the same time to exclude contamination etc.
“A simple first question of mine would be: Where did the virus go in between? To be picked up in sewage would mean there need to be quite a substantial number of people infected. And it then disappeared after they all recovered (or fell ill, perhaps even died, undiagnosed?) to be introduced again almost a year later?”
The study’s results defy what we know about how this virus behaves, Preiser added, and explained that there most likely would have been infections among healthcare staff, for example, and that, in the absence of preventive measures, it would have spread. Preiser, therefore, believes the findings to be premature and suggests further confirmation:
“Repeating the test of the positive sample, testing of other samples around that time point, and ideally sequencing of the virus then and now would all help. I personally would be extremely surprised if this stood up. I regard it as a lab artifact until proven otherwise.”
Dr Edward Archer, postdoctoral fellow at the Department of Microbiology and the Stellenbosch University Water Research Institute, also questions how exactly the team managed to carry out such a retrospective study on "old" samples.
“We are not sure yet how stable such genetic markers will be in wastewater, even if some preservation was done prior to storage, but the international community cautioned that merely freezing samples may not be suitable.”
Other international experts take on the study
Coronavirus testing typically involves screening for more than one gene, explains Claire Crossan, research fellow, virology, Glasgow Caledonian University in an article for The Conversation. In this case, Crossan explains that the researchers tested for three genes and found a positive result for the March 2019 sample in one of these three genes, called the RdRp gene. They then further screened for two regions of the RDRp gene, and both regions were only detected around the 39th cycle of amplification.
Apart from the possibility that the sample could have accidentally been contaminated with SARS-CoV-2 in the laboratory, Crossan explains another possible reason for the positive result:
“Another explanation is that there is other RNA or DNA in the sample that resembles the test target site enough for it to give a positive result at the 39th cycle of amplification.” According to Crossan, scientists usually use up to 40–45 rounds of amplification and PCR tests become less "specific" with increasing rounds of amplification.
Xagoraraki also told the New York Times that the researchers of the study used tests that search for bits of three different genes, and said that the only tests that turned up positive were for the RdRp gene – and that one of the other tests, for a gene called N, is known to be more sensitive.
“It should have shown a signal as well,” Xagoraraki said. Another point that put Xagoraraki on the fence about the results is whether the virus could have truly survived for over a year without the sample being put in a deep freeze of -80 degrees, noting that this questions the credibility of the results.
Like Preiser and Archer, Crossan suggests further tests be done in order to conclude that the sample contains SARS-CoV-2, and that a finding of that magnitude would need to be replicated separately by independent laboratories. However, Bosch said repeating experiments on the positive sample from March 2019 would not be possible as it was depleted during the first test:
“We proved it from this sample but we cannot repeat it. But contamination was unlikely, he said. “The way we work, when there is contamination, we notice it,” he said, further pointing to another way to confirm the finding: searching through stored samples of blood from patients in Barcelona hospitals in March 2019.
This is because, he explained, if the virus were circulating in Spain in March 2019, some people would most likely have been hospitalised due to flu-like symptoms.