Was SARS-CoV-2 actually present in March 2019 wastewater samples?

You can also read this post on Lesswrong.

The initial Reuters headline read Coronavirus traces found in March 2019 sewage sample, Spanish study shows. This claim would have significant implications if true. However, it doesn’t make sense that SARS-CoV-2 could have been circulating in Spain in early 2019 given the known transmissibility and disease characteristics of COVID-19. The most likely explanation is a false positive, either due to sample contamination or a PCR match on a different sequence, such as another coronavirus. However, given the uncertainty of the origin of the virus and the scale of the pandemic, a rigorous investigation seems justified. It would be worth screening other early and late 2019 samples and trying to sequence any coronavirus viral RNA.

The following preprint is the origin of the claim, and I have pasted the relevant passages from the paper.


Relevant paragraph is:

Most COVID-19 cases show mild influenza-like symptoms (14) and it has been suggested that some uncharacterized influenza cases may have masked COVID-19 cases in the 2019-2020 season (11). This possibility prompted us to analyze some 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.

Methods seem fairly normal:

Eight hundred-milliliter samples of wastewater were concentrated through precipitation with 20% polyethylene-glycol 6000 and resuspended in 3 mL of PBS, pH 7.4 (9). Nucleic acid extraction was performed from 1mL of the concentrate and eluted in 50 µL using the NucliSENS® miniMAG® extraction system (bioMérieux)

Five one-step RT-qPCR assays (RNA UltraSense™ One-Step Quantitative RTPCR System, Invitrogen, Life Technologies) targeting the RNA-dependent RNA polymerase (RdRp) gene, IP2 and IP4 fragments, from Institute Pasteur, Paris (Institut Pasteur. Protocol: Real-time RT-PCR assays for the detection of SARS-CoV-2. 2020 https://www.who.int/docs/default-source/coronaviruse/realtime-rt-pcr-assays-for-thedetection-of-sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2), the envelope protein (E) gene, E fragment, from Charité, Berlin (10), and the nucleoprotein (N), N1 and N2 fragments, from CDC, Atlanta (Centers for Disease Control and Prevention. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. 2020 https://www.fda.gov/media/134922/download)..?fbclid=IwAR1uJPUjXuDzq9gNLFr_jiS6na5EVExX4eTYMDwgsINvcG6GDHRtlqNjClE) The standard curve were constructed using the Twist Synthetic SARS-CoV-2 RNA Control 2 (MN908947.3) (Twist Bioscience). Technical details are included in the Appendix.”

Outstanding questions

What could the authors do to increase their confidence in their results?

  • Rerun PCR using different assays targeting different regions of SARS-CoV-2?
  • Sequence every bit of RNA in the sample they can and look for matches?

How many wastewater centers around the world have samples they can check? Are they checking them now?

Were the IP2 and IP4 samples handled and tested independently? How hard would it have been for them both to be contaminated in the lab?

How good are the IP2 and IP4 target assays in particular? Can we test them against other coronavirus / other samples to look for false positive matches?