Digital Tbucket Tank (DTT)

External peer review of the RT-PCR test for the detection of SARS-CoV-2 reveals 10 essential scientific shortcomings on a molecular and methodological level: Consequences for false positive results.

Dear reader, today we will start with a quote from Prof. Albert Einstein, (from a radio broadcast for United Jewish Appeal, April 11, 1943):

"The pursuit of truth and knowledge is one of the most beautiful things that a person is capable of, even if pride in this pursuit is mostly on the lips of those who are least filled with such pursuits."

The world has been with the for almost a year Corona pandemic employed. A great many measures have been taken to control the situation. We at the DTT Science-Tank Team have never addressed the subject of the corona pandemic. The reason is quite simple. In order to form a scientific opinion on a topic, you need a healthy and respectful dispute, especially in science. Good scientists have always done that. If you just look at the history of quantum physics and the various legendary feuds between scientists, you learn how science works. Unfortunately took place in the present Corona crisis Hardly tired of a proper dispute, until now.

It goes without saying that the basis for various measures by governments is the PCR tests represent.

The work published in early 2019: Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR - to be found here (https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.3.2000045)

Authors:

Victor M Corman, Olfert Landt, Marco Kaiser, Richard Molenkamp4, Adam Meijer, Daniel KW Chu6, Tobias Bleicker1, Sebastian Brünink, Julia Schneider, Marie Luisa Schmidt1, Daphne GJC Mulders4, Bart L Haagmans, Bas van der Veer, Sharon van den Brink , Lisa Wijsman, Gabriel Goderski, Jean-Louis Romette, Joanna Ellis, Maria Zambon, Malik Peiris, Herman Goossens, Chantal Reusken, Marion PG Koopmans, Christian Drosten   

is known. It was and is perhaps still considered the main trigger for the implementation of PCR tests.

Without going into the details of the work, we'd like to go into one Review report to draw attention:

Image source: Pixabay

External peer review of the RTPCR test to detect SARS-CoV-2 reveals 10 major scientific flaws at the molecular and methodological level: consequences for false positive results - to be found here (https://cormandrostenreview.com/report/)      

Autoren:

Pieter Borger, Bobby Rajesh Malhotra, Michael Yeadon, Clare Crai, Kevin McKernan, Klaus Steger, Paul McSheehy, Lidiya Angelova, Fabio Franchi, Thomas Binder, Henrik Ullrich, Makoto Ohashi, Stefano Scoglio, Marjolein Doesburg-van Kleffens, Dorothea Gilbert, Rainer Klement, Ruth Schruefer, Berber W Pieksma, Jan Bonte, Bruno H Dalle Carbonare, Kevin P Corbett, Ulrike Kämmerer             

      
The article is the translation of the essential points of the review into German: Source (https://cormandrostenreview.com/report/):

External peer review of the RT-PCR test for the detection of SARS-CoV-2 reveals 10 essential scientific shortcomings on a molecular and methodological level: Consequences for false positive results.

In the publication entitled "Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR"(Eurosurveillance 25 (8) 2020) the authors provide a diagnostic workflow and RT-qPCR protocol for the detection and diagnosis of 2019-nCoV (now known as SARS-CoV2) which they claim is validated and provides a robust diagnostic methodology for use in public health laboratories. In view of the consequences of this publication for societies worldwide, a group of independent researchers carried out a point-by-point review of the above publication in which

1) all components of the presented test design have been cross-checked,

2) the RT-qPCR protocol recommendations have been assessed for good laboratory practice

and

3) the parameters have been examined using the relevant scientific literature in the field.

That published RT-qPCR protocol for the detection and diagnosis of 2019-nCoV and the manuscript have numerous technical and scientific errors, including an inadequate primer design, a problematic one and an inadequate one RT-qPCR protocol and the lack of an exact Test validation. Neither the presented test nor the manuscript itself meet the requirements for an acceptable scientific publication. Furthermore, serious conflicts of interest of the authors are not mentioned. Finally, the very short period of time between submission and acceptance of the publication (24 hours) suggests that a systematic Peer review process either was not performed here or is of problematically poor quality. We provide compelling evidence of several scientific inadequacies, Flaws and defects.

In view of the scientific and methodological shortcomings presented here, we are confident that the editorial staff of Eurosurveillance has no choice but to publish to withdraw.



SUMMARY CATALOG OF DEFECTS FOUND IN THE PAPER


The Corman-Drosten paper contains the following specific errors:


1. There is no specified reason for using these extremely high concentrations of primers in this protocol. The concentrations described lead to increased unspecific binding and PCR product amplifications, which makes the test unsuitable as a specific diagnostic tool for the detection of the SARS-CoV-2 virus.


2. Six unspecified wobbly positions result in tremendous variability in real laboratory implementations of this test; the confusing unspecific description in the Corman-Drosten paper is not suitable as a standard working protocol, which makes the test unsuitable as a specific diagnostic tool for identifying the SARS-CoV-2 virus.


3. The test cannot distinguish between whole virus and viral fragments. Therefore, the test cannot be used as a diagnostic tool for intact (infectious) viruses, making the test unsuitable as a specific diagnostic tool for identifying the SARS-CoV-2 virus and allowing conclusions to be drawn about the presence of an infection.


4. A difference of 10 ° C in relation to the annealing temperature Tm for primer pair 1 (RdRp_SARSr_F and RdRp_SARSr_R) also makes the test unsuitable as a specific diagnostic tool for identifying the SARS-CoV-2 virus.


5. A fatal error is the lack of a Ct value at which a sample is considered positive and negative. This Ct value is also not found in the subsequent applications, which makes the test unsuitable as a specific diagnostic tool for detecting the SARS-CoV-2 virus.


6. The PCR products have not been validated at the molecular level. This fact makes the protocol unusable as a specific diagnostic tool for the detection of the SARS-CoV-2 virus.


7. The PCR test does not contain an unambiguous positive control to assess its specificity for SARS-CoV-2, nor a negative control to exclude the presence of other coronaviruses, which makes the test a specific diagnostic tool for identifying SARS-CoV-2. Virus is unsuitable.


8. The test design in the Corman-Drosten paper is so vague and flawed that one can go in dozens of different directions; nothing is standardized and there is no SOP. This strongly questions the scientific validity of the test and makes it unsuitable as a specific diagnostic tool for identifying the SARS-CoV-2 virus.


9. Most likely, the Corman-Drosten paper was not peer-reviewed, which makes the test unsuitable as a specific diagnostic tool for identifying the SARS-CoV-2 virus.


10. We find serious conflicts of interest in at least four authors, in addition to the fact that two of the authors of the Corman-Drosten paper (Christian Drosten and Chantal Reusken) are members of the Eurosurveillance editorial board. A conflict of interest was added on July 29, 2020 (Olfert Landt is Managing Director of TIB-Molbiol; Marco Kaiser is Senior Researcher at GenExpress and serves as scientific advisor for TIB-Molbiol), which was not declared in the original version (and in PubMed -Version is still missing); TIB-Molbiol is the company that was "the first" to produce PCR kits (Light Mix) on the basis of the protocol published in the Corman-Drosten manuscript and, in its own words, sold these PCR test kits even before publication [ 20]; in addition, Victor Corman & Christian Drosten did not mention their second affiliation: the commercial test laboratory "Labor Berlin". Both are responsible for virus diagnostics there [21] and the company is active in the field of real-time PCR tests.



CONCLUSION


The decision as to which test protocols will be published and made generally accessible rests in the hands of Eurosurveillance. A decision to recognize the errors that are evident in the Corman-Drosten study has the advantage that the costs and suffering of people will be greatly minimized in the future.
Isn't it in Eurosurveillance's best interest to withdraw this paper? Our conclusion is clear. Given all of the tremendous PCR protocol design flaws and flaws described here, we conclude that there is not much choice left within the framework of scientific integrity and responsibility.



REFERENCES


[1] Corman Victor M, Landt Olfert, Kaiser Marco, Molenkamp Richard, Meijer Adam, Chu Daniel KW, Bleicker Tobias, Brünink Sebastian, Schneider Julia, Schmidt Marie Luisa, Mulders Daphne GJC, Haagmans Bart L, van der Veer Bas, van den Brink Sharon, Wijsman Lisa, Goderski Gabriel, Romette Jean-Louis, Ellis Joanna, Zambon Maria, Peiris Malik, Goossens Herman, Reusken Chantal, Koopmans Marion PG, Drosten Christian. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020; 25 (3): pii = 2000045. https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045


[2] Email communication between Dr. Peter Borger & Dr. Adam Meijer: Supplementary Material


[3] Jafaar et al., Correlation Between 3790 Quantitative Polymerase Chain Reaction – Positive Samples and Positive Cell Cultures, Including 1941 Severe Acute Respiratory Syndrome Coronavirus 2 Isolates. https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1491/5912603


[4] BBC, January 21st 2020: https://www.bbc.com/news/world-asia-china-51185836;
archives: https://archive.is/0qRmZ


[5] Google Analytics - COVID19-deaths worldwide: https://bit.ly/3fndemJ
archives: https://archive.is/PpqEE


[6] Laboratory testing for COVID-19 Emergency Response Technical Center, NIVD under
China CDC March 15th, 2020: http://www.chinacdc.cn/en/COVID19/202003/P020200323390321297894.pdf


[7] Real-Time PCR Handbook Life Technologies: https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/real-time-pcr-
handbook.pdf Nolan T, Huggett J, Sanchez E. Good practice guide for the application of quantitative PCR (qPCR) First Edition 2013


[8] Trestan Pillonel et al, Letter to the editor: SARS-CoV-2 detection by real-time RT-PCR: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268274/


[9] Kurkela, Satu, and David WG Brown. "Molecular-diagnostic techniques." Medicine 38.10
(2009): 535-540.


[10] Wolfel et al., Virological assessment of hospitalized patients with COVID-2019
https://www.nature.com/articles/s41586-020-2196-x


[11] Thermofischer Primer Dimer Web Tool: https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library /thermo-scientific-web-tools/multiple-primer-analyzer.html

[12] Primer-BLAST, NCBI - National Center for Biotechnology Information: https://www.ncbi.nlm.nih.gov/tools/primer-blast/


[13] Marra MA, Steven JMJ, Caroline RA, Robert AH, Angela BW et al. (2003) Science. The Genome sequence of the SARS-associated coronavirus. Science 300 (5624): 1399-1404.


[14] Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome: https://www.ncbi.nlm.nih.gov/nuccore/MN908947


[15] Borger P. A SARS-like Coronavirus was expected but nothing was done to be prepared. At J Biomed Sci Res 2020. https://biomedgrid.com/pdf/AJBSR.MS.ID.001312.pdf
https://www.researchgate.net/publication/341120750_A_SARS-like_Coronavirus_was_Expected_but_nothing_was_done_to_be_Prepared;  Archives: https://archive.is/i76Hu


[16] Eurosurveillance paper evaluation / review process: https://www.eurosurveillance.org/evaluation

[17] Official recommendation of the Corman-Drosten protocol & manuscript by the WHO, published on January 13th 2020 as version 1.0 of the document: https://www.who.int/docs/default-source/coronaviruse/wuhan-virus -assay-
v1991527e5122341d99287a1b17c111902.pdf; archive: https://bit.ly/3m3jXVH

[18] Official WHO recommendation for the Corman / Drosten RT-qPCR-protocol, which directly derives from the Eurosurveillance-publication, document-version 2-1, published on
17th January 2020: https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf?sfvrsn=a9ef618c_2

[19] Eurosurveillance Editorial Board, 2020: https://www.eurosurveillance.org/upload/site-assets/imgs/2020-09-Editorial%20Board%20PDF.pdf; Archives: https://bit.ly/2TqXBjX

[20] Instructions For Use LightMix SarbecoV E-gene plus EAV Control, TIB-Molbiol & Roche Molecular Solutions, January 11th 2020: https://www.roche-as.es/lm_pdf/MDx_40-0776_96_Sarbeco-E-gene_V200204_09164154001 (1 ) .pdf
Archive, timestamp - January 11th 2020: https://archive.is/Vulo5;  archives: https://bit.ly/3fm9bXH [21] Christian Drosten & Victor Corman, responsible for viral diagnostics at Labor Berlin:
https://www.laborberlin.com/fachbereiche/virologie/  Archives: https://archive.is/CDEUG

[22] Tom Jefferson, Elizabeth Spencer, Jon Brassey, Carl Heneghan Viral cultures for COVID-19 infectivity assessment. Systematic review. Systematic review doi: https://doi.org/10.1101/2020.08.04.20167932-XNUMX-XNUMX-XNUMX https://www.medrxiv.org/content/10.1101/2020.08.04.20167932v4

[23] Kim et al., The Architecture of SARS-CoV-2 Transcriptome: https://www.sciencedirect.com/science/article/pii/S0092867420304062

[24] ECDC reply to Dr. Peter Borger, 18th November 2020: Supplementary Material

[25] Prof. Dr. Ulrike Kämmerer & team, survey & Primer-BLAST table