Essentials of RT PCR and other molecular diagnostic for COVID-19. Infection spread by mass PCR-testing.

A short summary.

Adeel Afzal, an author associated with Cairo University and the University of Hafr Al Batin in Saudi Arabia, wrote a quality article on molecular diagnostic technologies for COVID-19. It was received by the journal on June 25 and published on August 2, 2020. There is a lot of informative data and figures on RT PCR. We publish here some of Adeel Afzal’s figures, tables, and a couple of our notes.

Also, we publish our hypothesis: People buy antigen test for SARS-CoV-2 in pharmacies, do not go to test centers, do not get infected there. The “Rapid spread” ends.

Adeel Afzal on molecular technologies and RT PCR for COVID-19.

The structure of SARS-CoV-2.

The genomic constitution of SARS-CoV-2 RNA. The viral genome consists of two large genes: ORF1a, and ORF1b, which encode non-structural proteins (NSP) including RdRp, whereas the smaller structural genomic region hosts S, E, M, and N genes.

Fig. 1. (c) The genomic constitution of SARS-CoV-2 RNA. The viral genome consists of two large genes: ORF1a, and ORF1b, which encode non-structural proteins (NSP) including RdRp, whereas the smaller structural genomic region hosts S, E, M, and N genes, which encode the structural proteins. CDC: Centers forDisease Control and Prevention; E gene: gene encoding envelop protein of SARS-CoV-2; HKU: Hong Kong University; M gene: gene encoding membrane protein of SARS-CoV-2; N gene: gene encoding nucleocapsid protein of SARS-CoV-2; NIH: National Institute of Health (Thailand); NIID: National Institute of Infectious Diseases (Japan); ORF1a/b: open reading frame 1a and b of SARS-CoV-2; RdRp: RNA-dependent RNA polymerase of SARS-CoV-2; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; S gene: gene encoding spike protein of SARS-CoV-2. Source: Afzal, 2020.

A molecular representation of the real-time RT-PCR principle.

A summary of commercially available (approved) and pre-commercial molecular diagnostics for SARS-CoV-2 detection.

Table 1 Figures of merit of the modern commercially available (approved) and pre-commercial (research use only) molecular diagnostics for SARS-CoV-2 detection. Source: Afzal, 2020.

Fig. 3.The evaluation of commercially available RT-PCR kits shows variations in the rate of detection and Ct values. (a) Experimental Ct values obtained for commercial RT-PCR assays (n = 13). The data points on top of the horizontal line (red, dotted) are negative, indicated with Ct = 42.5 for plotting purposes. The rate of detection of the RT-PCR kit is mentioned below the data points. (b) The data points for the clinical samples (n = 10) with the highest viral load that were positively identified by all RTPCR assays. The horizontal lines (blue) indicate the mean Ct value, triangles show the Ct values of the samples with the highest (sample 1) and lowest (sample 10) viral load according to the in-house E gene PCR. E: envelop protein of SARS-CoV-2; RdRp: RNA-dependent RNA polymerase of SARS-CoV-2; N: nucleocapsid protein of SARS-CoV-2; ORF1ab: open reading frame 1a and b of SARS-CoV-2; RT-PCR: reverse transcriptase-polymerase chain reaction; S: spike protein of SARS-CoV-2; SARSCoV-2: severe acute respiratory syndrome coronavirus 2 (reprinted with permission from[52]; copyright Elsevier, 2020). — Fig. 3.The evaluation of commercially available RT-PCR kits shows variations in the rate of detection and Ct values. (a) Experimental Ct values obtained for commercial RT-PCR assays (n = 13). The data points on top of the horizontal line (red, dotted) are negative, indicated with Ct = 42.5 for plotting purposes. The rate of detection of the RT-PCR kit is mentioned below the data points. (b) The data points for the clinical samples (n = 10) with the highest viral load that were positively identified by all RTPCR assays. The horizontal lines (blue) indicate the mean Ct value, triangles show the Ct values of the samples with the highest (sample 1) and lowest (sample 10) viral load according to the in-house E gene PCR. E: envelop protein of SARS-CoV-2; RdRp: RNA-dependent RNA polymerase of SARS-CoV-2; N: nucleocapsid protein of SARS-CoV-2; ORF1ab: open reading frame 1a and b of SARS-CoV-2; RT-PCR: reverse transcriptase-polymerase chain reaction; S: spike protein of SARS-CoV-2; SARSCoV-2: severe acute respiratory syndrome coronavirus 2. Source: Afzal, 2020.

“14% of SARS-CoV-2 variants were not detectable with at least one of the commercialized primers.”

“The manufacturers need to regularly optimize the oligonucleotides through frequent analysis of the updated genomic sequences,”

Afzal, 2020 (1):

Owing to the rapid pandemic-scale spread of SARS-CoV-2, it shows very high genomic diversity in gene position and nucleotide sequences [83,84]. The outcomes of the genomic sequencing of SARS-CoV-2 are regularly shared on the GISAID database [27]. Pachetti et al.[85]performed an analysis of 220 SARS-CoV-2 genomic sequences placed on GISAID and found eight novel recurrent genomic mutations, which suggested that SARS-CoV-2 was evolving and different strains of SARS-CoV-2 might coexist. The evolution of SARS-CoV-2 and genetic diversity present a colossal new challenge to molecular diagnostic manufacturers. Osório and Correia-Neves [86] analyzed 1825 high-coverage genomic sequences from the GASAID database. Their analysis revealed that~79% (26/33) primer binding sequences in at least one gene used in RT-PCR were mutated compared to Wuhan-Hu-1 (NC_045512) [86]. Therefore, they concluded that 14% of SARS-CoV-2 variants were not detectable with at least one of the commercialized primers. Consequently, the manufacturers need to regularly optimize the oligonucleotides through frequent analysis of the updated genomic sequences on GASAID to enhance their accuracy and reliability.

RT-PCR results are potentially erratic, and clinical diagnosis of symptomatic individuals should not rely on RT-PCR only.

In a recent study, Liu et al., 2020 (2), wrote that RT-PCR results were potentially erratic, and clinical diagnosis of symptomatic individuals or those with epidemiological history should not rely on RT-PCR only. RT-PCR must be combined with clinical examination and computed tomography (CT) to medically judge the suspected individuals.

Test centers, where infected and healthy gather together, may be the key contributors to the spread of SARS-CoV-2.

We do NOT recommend engaging in assymptomatic testing. Test centers, where infected and healthy gather together, may be the key contributors to the spread of SARS-CoV-2.

The New Neander’s Medical on December 21, 2020:

COVID-19 essentials. 1. In a study, the average Ct in the Uk was around 30, for asymptomatic, symptomatic, severe. Rapid antigen tests at this levels shows too many false positives. 2. Do not engage in asymptomatic testing. Test centers is the place where healthy get infected.
… an important study in the UK found average Ct in the Uk was around 30 for asymptomatic, symptomatic, severe.

COVID-19 essentials.
1. In a study, the average Ct in the Uk was around 30, for asymptomatic, symptomatic, severe. Rapid antigen tests at this levels shows too many false positives.
2. Do not engage in asymptomatic testing. Test centers is the place where healthy get infected.

Balloux, you were surprised by these results because you read too much propaganda and were throwing dirt at Prof. Raoult. Also, you missed the imporant study that found average Ct in the Uk was around 30, for asymptomatic, symptomatic, severe.
Raoult: Abbott rapid test is better. pic.twitter.com/DpA8vHYTgJ
— The New Neander's Medical (@NNeanderMedical) December 21, 2020

Hypothesis: People buy antigen test for SARS-CoV-2 in pharmacies, do not go to test centers, do not get infected there. The “Rapid spread” ends.

The New Neander’s Medical on December 29, 2020:

2/n Ukraine: “The Minister noted a decrease in the number of PCR tests, he explained this by an increase in rapid tests for antigens.”
Hypothesis: People buy antigen test for SARS-CoV-2 in pharmacies, do not go to test centers, do not get infected there. The “Rapid spread” ends.

Asymptomatic undetected infections are 40-45% of all infections with SARS-CoV-2. False negatives on PCR-tests are around 30%. Undetected and false negative cancel each other.

From another article on our website:

Currently, between 1 and 2 percent of all the reported COVID-19 cases die.

Currently, there is so much testing, that the reported cases reflect almost ALL infected with SARS-CoV-2. The Effective Case fatality rate (CFR) is, therefore, equal to The Effective Infection fatality rate (IFR). And CFR=IFR=1-2%. This means that between 1 and 2 percent of all the reported COVID-19 cases die. This is a lot if all the population had to get infected with SARS-CoV-2.

The New Neander’s Medical on November 16, 2020:

COVID-19 epidemiology. There is a lot of testing in most of the countries. Test positivity rates differ from 10% in France and Spain, to 30% in Switzerland, Belgium, Ukraine. But mortality converges in all countries towards 2%(1-2%) which hints that most of the cases are detected.

COVID-19. A “Certificate of Civil Protection Aptitude During Epidemics”: learn to avoid pathogens.

Selected references:

1. A. Afzal. Journal of Advanced Research 26 (2020) 149–159.

2. Li Y, Yao L, Li J, Chen L, Song Y, Cai Z, et al. Stability issues of RT-PCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19. J Med Virol 2020;n/a.

2 Comments

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I absolutely agree with you that many of the cases are transmitted at test centers (and now, vacc centers). However I have no hard evidence of this (perhaps- contact tracing could determine some cases that could not have possibly been acquired elsewhere?). Please update if you find any hard evidence of this other than mechanistic plausibility because yes, I wholeheartedly agree with your hypothesis. (Also: all tests should be done drive thru or outdoors).