Corman-Drosten Review Report – 27/11/20

CONCISE REVIEW REPORT – Main Author Dr. Pieter Borger (MSc, PhD), Molecular Genetics, W+W Research Associate, Lörrach, Germany

Brief synopsis of this brilliant and insightful report- full report link at the bottom of the page.

This paper will show numerous serious flaws in the Corman-Drosten paper, the significance of which has led to worldwide misdiagnosis of infections attributed to SARS-CoV-2 and associated with the disease COVID-19. We are confronted with stringent lockdowns which have destroyed many people’s lives and livelihoods, limited access to education and these imposed restrictions by governments around the world are a direct attack on people’s basic rights and their personal freedoms, resulting in collateral damage for entire economies on a global scale.

There are ten fatal problems with the Corman-Drosten paper which we will outline and explain in greater detail in the following sections.

The first and major issue is that the novel Coronavirus SARS-CoV-2 (in the publication named 2019-nCoV and in February 2020 named SARS-CoV-2 by an international consortium of virus experts) is based on in silico (theoretical) sequences, supplied by a laboratory in China [1], because at the time neither control material of infectious (“live”) or inactivated SARS-CoV-2 nor isolated genomic RNA of the virus was available to the authors. To date no validation has been performed by the authorship based on isolated SARS-CoV-2 viruses or full length RNA thereof. According to Corman et al.:

“We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.” [1]

The focus here should be placed upon the two stated aims: a) development and b) deployment of a diagnostic test for use in public health laboratory settings. These aims are not achievable without having any actual virus material available (e.g. for determining the infectious viral load). In any case, only a protocol with maximal accuracy can be the mandatory and primary goal in any scenario-outcome of this magnitude. Critical viral load determination is mandatory information, and it is in Christian Drosten’s group responsibility to perform these experiments and provide the crucial data.

Nevertheless these in silico sequences were used to develop a RT-PCR test methodology to identify the aforesaid virus. This model was based on the assumption that the novel virus is very similar to SARS-CoV from 2003 as both are beta-coronaviruses.

The PCR test was therefore designed using the genomic sequence of SARS-CoV as a control material for the Sarbeco component; we know this from our personal email-communication with [2] one of the co-authors of the Corman-Drosten paper. This method to model SARS-CoV-2 was described in the Corman-Drosten paper as follows:

“the establishment and validation of a diagnostic workflow for 2019-nCoV screening and specific confirmation, designed in absence of available virus isolates or original patient specimens. Design and validation were enabled by the close genetic relatedness to the 2003 SARS-CoV, and aided by the use of synthetic nucleic acid technology.”

The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is an important biomolecular technology to rapidly detect rare RNA fragments, which are known in advance. In the first step, RNA molecules present in the sample are reverse transcribed to yield cDNA. The cDNA is then amplified in the polymerase chain reaction using a specific primer pair and a thermostable DNA polymerase enzyme. The technology is highly sensitive and its detection limit is theoretically 1 molecule of cDNA. The specificity of the PCR is highly influenced by biomolecular design errors.

What is important when designing an RT-PCR Test and the quantitative RT-qPCR test described in the Corman-Drosten publication?

1. The primers and probes:

a) the concentration of primers and probes must be of optimal range
(100-200 nM)
b) must be specific to the target-gene you want to amplify
c) must have an optimal percentage of GC content relative to the total nitrogenous bases (minimum 40%, maximum 60%)
d) for virus diagnostics at least 3 primer pairs must detect 3 viral genes (preferably as far apart as possible in the viral genome)

2. The temperature at which all reactions take place:

a) DNA melting temperature (>92°)
b) DNA amplification temperature (TaqPol specific)
c) Tm; the annealing temperature (the temperature at which the primers and probes reach the target binding/detachment, not to exceed 2 ̊C per primer pair). Tm heavily depends on GC content of the primers

3. The number of amplification cycles (less than 35; preferably 25-30 cycles);

In case of virus detection, >35 cycles only detects signals which do not correlate with infectious virus as determined by isolation in cell culture [reviewed in 2]; if someone is tested by PCR as positive when a threshold of 35 cycles or higher is used (as is the case in most laboratories in Europe & the US), the probability that said person is actually infected is less than 3%, the probability that said result is a false positive is 97% [reviewed in 3]

4. Molecular biological validations; amplified PCR products must be validated either by running the products in a gel with a DNA ruler, or by direct DNA sequencing

5. Positive and negative controls should be specified to confirm/refute specific virus detection

6. There should be a Standard Operational Procedure (SOP) available

SOP unequivocally specifies the above parameters, so that all laboratories are able to set up the exact same test conditions. To have a validated universal SOP is essential, because it enables the comparison of data within and between countries.

MINOR CONCERNS WITH THE CORMAN-DROSTEN PAPER

1. In Table 1 of the Corman-Drosten paper, different abbreviations are stated – “nM” is specified, “nm” isn’t. Further in regards to correct nomenclature, nm means “nanometer” therefore nm should read nM here.

2. It is the general consensus to write genetic sequences always in the 5’-3’ direction, including the reverse primers. It is highly unusual to do alignment with reverse complementary writing of the primer sequence as the authors did in figure 2 of the Corman-Drosten paper. Here, in addition, a wobble base is marked as “y” without description of the bases the Y stands for.

3. Two misleading pitfalls in the Corman-Drosten paper are that their Table 1 does not include Tm-values (annealing-temperature values), neither does it show GC-values (number of G and C in the sequences as %-value of total bases).

MAJOR CONCERNS WITH THE CORMAN-DROSTEN PAPER

A) BACKGROUND

The authors introduce the background for their scientific work as: “The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travelers does already occur”.

According to BBC News [4] and Google Statistics [5] there were 6 deaths world-wide on January 21st 2020 – the day when the manuscript was submitted. Why did the authors assume a challenge for public health laboratories while there was no substantial evidence at that time to indicate that the outbreak was more widespread than initially thought?

As an aim the authors declared to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Further, they acknowledge that “The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.”

“In four individual test reactions, weak initial reactivity was seen however they were negative upon retesting with the same assay. These signals were not associated with any particular virus, and for each virus with which initial positive reactivity occurred, there were other samples that contained the same virus at a higher concentration but did not test positive. Given the results from the extensive technical qualification described above, it was concluded that this initial reactivity was not due to chemical instability of real-time PCR probes and most probably to handling issues caused by the rapid introduction of new diagnostic tests and controls during this evaluation study.” [1]

The first sentence of this excerpt is clear evidence that the PCR test described in the Corman-Drosten paper generates false positives. Even under the well-controlled conditions of the state-of-the-art Charité-laboratory, 4 out of 310 primary-tests are false positives per definition. Four negative samples initially tested positive, then were negative upon retesting. This is the classical example of a false positive. In this case the authors do not identify them as false positives, which is intellectually dishonest.

Another telltale observation in the excerpt above is that the authors explain the false positives away as “handling issues caused by the rapid introduction of new diagnostic tests”. Imagine the laboratories that have to introduce the test without all the necessary information normally described in an SOP.

8. The Corman-Drosten paper was not peer-reviewed

Before formal publication in a scholarly journal, scientific and medical articles are traditionally certified by “peer review.” In this process, the journal’s editors take advice from various experts (“referees”) who have assessed the paper and may identify weaknesses in its assumptions, methods, and conclusions. Typically a journal will only publish an article once the editors are satisfied that the authors have addressed referees’ concerns and that the data presented supports the conclusions drawn in the paper.” This process is as well described for Eurosurveillance [16].

The Corman-Drosten paper was submitted to Eurosurveillance on January 21st 2020 and accepted for publication on January 22nd 2020. On January 23rd 2020 the paper was online. On January 13th 2020 version 1-0 of the protocol was published at the official WHO website [17], updated on January 17th 2020 as document version 2-1 [18], even before the Corman-Drosten paper was published on January 23rd at Eurosurveillance.

Normally, peer review is a time-consuming process since at least two experts from the field have to critically read and comment on the submitted paper. In our opinion, this paper was not peer-reviewed. Twenty-four hours are simply not enough to carry out a thorough peer review. Our conclusion is supported by the fact that a tremendous number of very serious design flaws were found by us, which make the PCR test completely unsuitable as a diagnostic tool to identify the SARS-CoV-2 virus. Any molecular biologist familiar with RT-PCR design would have easily observed the grave errors present in the Corman-Drosten paper before the actual review process. We asked Eurosurveillance on October 26th 2020 to send us a copy of the peer review report. To date, we have not received this report and in a letter dated November 18th 2020, the ECDC as host for Eurosurveillance declined to provide access without providing substantial scientific reasons for their decision. On the contrary, they write that “disclosure would undermine the purpose of scientific investigations.” [24].

9. Authors as the editors

A final point is one of major concern. It turns out that two authors of the Corman-Drosten paper, Christian Drosten and Chantal Reusken, are also members of the editorial board of this journal [19]. Hence there is a severe conflict of interest which strengthens suspicions that the paper was not peer-reviewed. It has the appearance that the rapid publication was possible simply because the authors were also part of the editorial board at Eurosurveillance. This practice is categorized as compromising scientific integrity.

SUMMARY CATALOGUE OF ERRORS FOUND IN THE PAPER

The Corman-Drosten paper contains the following specific errors:

1. There exists no specified reason to use these extremely high concentrations of primers in this protocol. The described concentrations lead to increased nonspecific bindings and PCR product amplifications, making the test unsuitable as a specific diagnostic tool to identify the SARS-CoV-2 virus.

2. Six unspecified wobbly positions will introduce an enormous variability in the real world laboratory implementations of this test; the confusing nonspecific description in the Corman-Drosten paper is not suitable as a Standard Operational Protocol making the test unsuitable as a specific diagnostic tool to identify the SARS-CoV-2 virus.

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

4. A difference of 10° C with respect to the annealing temperature Tm for primer pair1 (RdRp_SARSr_F and RdRp_SARSr_R) also makes the test unsuitable as a specific diagnostic tool to identify the SARS-CoV-2 virus.

5. A severe error is the omission of a Ct value at which a sample is considered positive and negative. This Ct value is also not found in follow-up submissions making the test unsuitable as a specific diagnostic tool to identify the SARS-CoV-2 virus.

6. The PCR products have not been validated at the molecular level. This fact makes the protocol useless as a specific diagnostic tool to identify the SARS-CoV-2 virus.

7. The PCR test contains neither a unique positive control to evaluate its specificity for SARS-CoV-2 nor a negative control to exclude the presence of other coronaviruses, making the test unsuitable as a specific diagnostic tool to identify the SARS-CoV-2 virus.

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 highly questions the scientific validity of the test and makes it unsuitable as a specific diagnostic tool to identify the SARS-CoV-2 virus.

9. Most likely, the Corman-Drosten paper was not peer-reviewed making the test unsuitable as a specific diagnostic tool to identify the SARS-CoV-2 virus.

10. We find severe conflicts of interest for 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 editorial board of Eurosurveillance. A conflict of interest was added on July 29 2020 (Olfert Landt is CEO of TIB-Molbiol; Marco Kaiser is senior researcher at GenExpress and serves as scientific advisor for TIB-Molbiol), that was not declared in the original version (and still is missing in the PubMed version); TIB-Molbiol is the company which was “the first” to produce PCR kits (Light Mix) based on the protocol published in the Corman-Drosten manuscript, and according to their own words, they distributed these PCR-test kits before the publication was even submitted [20]; further, Victor Corman & Christian Drosten failed to mention their second affiliation: the commercial test laboratory “Labor Berlin”. Both are responsible for the virus diagnostics there [21] and the company operates in the realm of real time PCR-testing.

In light of our re-examination of the test protocol to identify SARS-CoV-2 described in the Corman-Drosten paper we have identified concerning errors and inherent fallacies which render the SARS-CoV-2 PCR test useless.

CONCLUSION

The decision as to which test protocols are published and made widely available lies squarely in the hands of Eurosurveillance. A decision to recognise the errors apparent in the Corman-Drosten paper has the benefit to greatly minimise human cost and suffering going forward.

Is it not in the best interest of Eurosurveillance to retract this paper? Our conclusion is clear. In the face of all the tremendous PCR-protocol design flaws and errors described here, we conclude: There is not much of a choice left in the framework of scientific integrity and responsibility.