Background:
After submitting our review report on Corman et al. (referred hereinafter as CD-report) and republishing it on a scientific preprint server [50] and Researchgate.net [51] we offered the report for public discussion at cormandrostenreview.com on 27th November 2020. The scientific community provided additional literature, references, and analyses concerning the CD-report and the Corman et al. manuscript. Several “advocatus diaboli” confronted us with correct or assumed problems in our report. The most common critique of the CD-report was the lack of “wet lab” experiments to support our concerns over the technical flaws in the PCR protocol.
Aim:
This vibrant debate on our CD report has provided additional information worthy of further public documentation to address these critiques. We summarize the current published knowledge of “wet lab testing”, routine diagnostic use and validation of the original PCR-Protocol described by Corman et al. Further, this addendum highlights that independent research groups (some of them with Corman and/or Drosten as author) also pointed out important concerns with the original manuscript and Corman PCR protocol distributed by the WHO. Many of these references were already provided by the authors of the original CD-report but it is worth underscoring their relevance to the formation of our critiques of the CD manuscript.
Methods:
We searched the literature for ‘SARS-CoV-2 qPCR’ and ‘Corman’ or ‘Charité’. Then we combined these references with those provided by other scientists working in relevant Life Sciences/data analysis fields.
In the first section of the addendum, the publications will be discussed point by point, highlighting their findings in relation to the CD-report. In a second section, additional aspects about the Corman et al. publication are discussed. This spans a meta-analysis of the unusual peer-review process, timeframes, and further technical vulnerabilities of the Corman et al. PCR-protocol.
An additional concern was raised about the CD-report regarding the discussion of appropriate controls. We cite several studies that underscore the importance of internal controls in assessing viral load and the lack of such internal controls in the Corman qPCR method. These internal controls are required for normalizing swab sampling variance and they are critical for interpreting viral load. They are notably absent from the Corman PCR protocol. Several people also expressed confusion regarding the NCBI submissions provided by Corman et al. The sequences provided lack two of the target gene sequences Corman et al. claim to target. The only sequences referenced in the manuscript are listed (KC633203, KC633204, KC633201, GU190221, GU190222, GU190223) and none of these have sequences that match their N and E gene primers. This not only brings their validation into question but also prevents others from reproducing the work presented in Corman et al.
Results:
We present 20 scientific publications providing ‘wet lab’ evidence of the performance of the Corman et al. PCR protocol. Of those, 17 found problems with incorrect primer design (mismatches, dimer formation, melting temperature) in the SARS-CoV-2 specific “confirmatory” test named RdRp-PCR for “RNA-dependent RNA-polymerase” or the E-gene assay.
These documented problems include:
Documented primer dimers and False Positives in non-template controls (NTCs)
Documented poor sensitivity and False Negatives compared to other assays
No internal control to normalize the sample preparation variability and its impact on viral load estimation
No defined Ct for calling samples “Positive cases”
Poorly documented positive controls and sequences used in their study
Conclusion:
We believe the references provided in this addendum itemize the scientific consensus evident in the literature regarding the flaws in the original PCR detection method for SARs-CoV-2 published by Corman et al.. Further, since several important flaws were published in peer-reviewed journals, the lack of correction of the original PCR protocol by either Eurosurveillance or as an update in the Charité-WHO protocol brings into question the scientific integrity of the authors of Corman et al. These references settle any remaining debate that the Corman et al. manuscript should be retracted on technical grounds alone. The rapidity of the peer-review and conflicts of interest are even more troubling.
Index
Section 1
A. Wet lab evidence of primer design flaws
a) Background and Pinollel et al. (Letter to the editor of Eurosurveillance)
b) Review of the literature
a. Background
1. Muenchhoff et al.
2. Jung et al.
3. Etievant et al.
4. Gand et al.
5. Konrad et al.
6. Sethuraman et al.
7. Nalla et al.
8. Vogels et al.
9. Kuchinski et al.
10. Ratcliff et al.
11. Jaeger et al.
12. Khan et al.
13. Opota et al.
14. Bara et al.
15. Santos et al.
16. Anantharajah et al.
17. Dahdouh et al.
18. Poljak et al.
19. Boutin et al.
20. Pfefferle et al.
b. Summary wet lab evidence of primer design flaws
Section 2: Additional Aspects:
B. Meta-data analysis on EuroSurveillance.org (peer review timeframes)
C. Missing positive controls for PCR test validation
D. In silico analysis / Primer homology to human DNA
E. Further Discussion – The Consequences of False Positives / False Negatives
Section 3 References
Note: sentences written in italics are original citations from the respective publications
Zitat von carstenm
Mit Zitat antworten
(Ich habe auch erstmal danach gegooglet! 
)
