I've been getting many questions about pre-Wuhan covid cases detected around the world. In particular, 2 studies: Barcelona (March 2019) and Italy (Sep 2019); as well as the widely disseminated USA (Dec 2019) study.

I think best to make a single thread discussing all of these.
Important for readers to understand that if you can detect a pathogen in wastewater at a city level, the outbreak has to be quite obvious. It's not like 1 in 100,000 people has covid and you see it in the wastewater.
The study looked at March 2019 wastewater from Barcelona - why? Because it was originally supposed to be their negative control.

Negative control, meaning that it was not expected to have had any SARS2 virus in it.
The study also checked for multiple targets - in other words checking for the presence of several parts of the SARS2 genomes in each wastewater sample.

If you only see 1 or 2 targets, it's not as confident of a result as if all or most of the targets show positive results.
For negative controls, they analyzed "archival WWTP samples from January 2018 to December 2019 (Figure 2). All samples came out to be negative for the presence of SARS-CoV-2 genomes with the exception of March 12, 2019, in which both IP2 and IP4 target assays were positive."
If you look at their actual data, the "positives" for March 2019 shown in Fig 2B and C are close to Ct 40, and the other 3 (out of 5) targets were negative.
When you see this kind of anomaly, one thing to do is to immediately amplify that target, check the size of the product, and/or send it for sequencing. All of this can happen within 2 days or less depending on resources. But the authors did not do it.
The manuscript was posted in June 2020. If I had found such an astounding result, I would've done the test immediately and gotten myself a @ScienceMagazine paper if it could be confirmed that SARS2 (precursor) was circulating in Barcelona at such a detectable level in March '19.
Furthermore, Barcelona is one of the most touristed cities in the world. If COVID was so widespread in March 2019 that it could be detected in the wastewater of the city, it would've exploded worldwide. We would see such a diversity of SARS2 virus genomes.
On to the Italy Sep 2019 study. I did a very gif-filled thread back when it was first released because I didn't think people would take this that seriously.

I was wrong.

Dr. Shi from the WIV even cited it in her most recent @ScienceMagazine article. https://twitter.com/Ayjchan/status/1327815340563320834
Although this article technically passed peer review. I'd like to impress on readers that the editorial team of the journal are from the same institute as the (first and senior) authors of the paper.

I point this out for journals of any impact factor. https://journals.sagepub.com/doi/full/10.1177/0300891620974755?journalCode=tmja
The authors used an in-house test that had not been peer-reviewed at the time to claim that 14% of cancer patients in Italy in September 2019 tested positive for COVID.

There was no control for common cold (coronavirus) antibodies or actually any antibodies for that matter.
I want readers to understand what this study is claiming.

Somehow, 14% of ASYMPTOMATIC people who just went for lung cancer screening - according to this study - turned out to have antibodies for COVID-19 in September 2019. Dropping down to only 3% in Jan 2020...
We're literally looking at America post-Christmas in January 2021. If 14% of asymptomatic Americans had COVID antibodies in September 2020, best believe there would not only be 3% asymptomatic Americans in Jan 2021 with COVID antibodies.

Those are coronavirus sprinkles.
The issues with this study were best explained by @trvrb
tldr the study's positive rate was very close to the predicted false positive rate. Meaning that it was likely that this study had likely just picked up false positives (not real covid cases). https://twitter.com/trvrb/status/1333647437869633537
Does this analysis mean that 0 Americans had covid in December 2019? No.

But certainly 2% of Americans in those West Coast cities DID NOT have COVID by Dec 2019.

Please imagine if that were true. This pandemic would've exploded much earlier.
As far as we can tell, only ~124 people in Wuhan (population 11 million) - ~0.001% - were confirmed COVID cases by December 2019 and the city had to be locked down in late January.
Even with the lockdown, those 100s of Nov/Dec cases in Wuhan had unfortunately resulted in a pandemic.

If 2% of people in West Coast USA cities had covid in Dec 2019, I don't even know what the pandemic would look like right now.
In Wuhan, a study looking at 640 throat swabs from patients who actually had influenza-like illness (not asymptomatic!), only found positive results starting in Jan 2020. https://twitter.com/Ayjchan/status/1333819548114575362
If you propose that SARS-CoV-2 originated far OUTSIDE of China, I'd like you to please consider that the closest relatives of this virus have been found in Yunnan (Southern China).

If your hypothesis is Barcelona, Milan, or Washington - how did it get there in the first place?
If your hypothesis is that somehow SARS2 virus got into Wuhan on frozen food from outside of China - what's the story here?

No covid cases have been determined to come from frozen (imported) food.
Even at the very first market in Wuhan associated with the covid outbreak, none of hundreds of animal samples tested positive for the virus. https://www.wsj.com/articles/china-stalls-global-search-for-coronavirus-origins-wuhan-markets-investigation-11589300842
If we take their word, no animals in Wuhan city or even the province of Hubei have been found to be the source of the virus.
https://www.sciencemag.org/sites/default/files/Shi%20Zhengli%20Q%26A.pdf

Also from the Chinese CDC director: https://www.bloomberg.com/news/features/2020-12-30/china-is-making-it-harder-to-solve-the-mystery-of-how-covid-began
An analogy would be proposing that there's been a WILD panda population in California all this time that we weren't aware of.
I'm getting questions about singular cases detected outside China in late 2019. I'll try to provide analysis of these too but these reports are tough to match up to what has been observed wrt the rest of the pandemic. Will explain why...
This reports a suspect Nov '19 case: "a 4-year-old boy who lived in the surrounding area of Milan and had no reported travel history"
Evidence: sequencing and "amplification of a 470-bp fragment of the gene encoding the SARS-CoV-2 spike" from throat swab.
https://wwwnc.cdc.gov/eid/article/27/2/20-4632_article
The short spike sequence was identical to the reference SARS2 sequence from a Dec patient in Wuhan.

Problematically, the Milan sample was too degraded and prevented "sequencing of longer genomic regions that could have been helpful in determining the origin of the strain."
In this situation, it's difficult to understand how a child in Milan got SARS2 (a virus whose closest relatives frm South China) without travel history, why it didn't lead to a detectable outbreak in his town if it was already very similar/identical to the virus seen in Wuhan...
... and how, if there was some early, very sparse transmission of SARS2 in the outskirts of Milan, that this would have led to the first detected outbreak being not in Milan or anywhere else in Italy or Europe, but in Wuhan, China, half a world away.
Looking at their actual figure, I'm not convinced that this is a real positive. Sorry, Gianotti et al.

So I really advise people not to use this study to say that there were sparse covid cases in Milan in November 2019.
https://onlinelibrary.wiley.com/doi/epdf/10.1111/bjd.19804
Lastly, this study claims late Dec '19 covid case in France. I think there's a bit more of a possibility that this could be true because covid outbreak was already detected in Wuhan, likely starting back in Nov '19 or maybe earlier. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196402/pdf/main.pdf
This one has some possibility because despite no recent travel to China, "One of his children presented with ILI (influenza-like illness) prior to the onset of his symptoms."

But again, the only evidence was a single amplicon target (yellow line, Ct ~31). Not sequence verified.
If anyone (scientist, journalist, or friend) cites these papers to say that SARS2 was circulating outside of China before it broke out in Wuhan, please free feel to share this thread with them.
Here's another (different kind of) panda gif as a reward.
Thanks @HL3133 for shedding light on the late Dec French case. I was also asked to try @threadreaderapp unroll this... let’s give it a go. https://twitter.com/hl3133/status/1349430497445863424
You can follow @Ayjchan.
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