BREAKING! COVID-19 Diagnostics: Study Warns Two Viral Gene Targets Needed In RT-PCR Tests Due To Emerging Strains With Mutations In N Genes Such As G29140T

COVID-19 Diagnostics: Genomic and biotech researchers from the Chan Zuckerberg Biohub-San Francisco, and collaborators have warned against only targeting one viral gene when carrying out reverse transcriptase-polymerase chain reaction (RT-PCR) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).


 
The study team conducted a study showing that a mutation had arisen in the N gene of the virus that impaired the annealing of a commonly used RT-PCR primer. This implies that most current RT-PCR testing platforms being used are non-sensitive enough and will most probably be giving inaccurate readings.
 
The study findings are published on a preprint server and are currently being peer-reviewed. https://www.biorxiv.org/content/10.1101/2020.08.25.265074v1
 
Dr Manu Vanaerschot and colleagues say the World Health Organization (WHO) recommends that in some areas where community spread of SARS-CoV-2 is high, RT-PCR screening of just one discriminatory target is considered sufficient.
 
Alarmingly however, the current study identified a nucleotide change in an N gene primer sequence that disrupted annealing and amplification, thereby reducing diagnostic sensitivity.
 
The study says the findings strongly support the routine use of at least two targets when testing for SARS-CoV-2 using RT-PCR, even in areas where transmission rates are high.
 
Standard routine testing for SARS-CoV-2 involves using RT-PCR to test for the viral genome in respiratory samples, and this is usually carried out using primer pairs that target more than one viral gene.
 
Shockingly, the WHO recommends that “in areas where the COVID-19 virus is widely spread, a simpler algorithm might be adopted in which, for example, screening by RT-PCR of a single discriminatory target is considered sufficient.”
 
Furthermore of the US FDA-approved Emergency Use Authorizations that currently exist, more than 36 of these testing platforms only screen for one target in RT-PCR assays.
 
The implications are that a lot of COVID-19 cases are either going undetected or misdiagnosed.
 
To date, the COVID-19 diagnostic laboratory at the Chan Zuckerberg Biohub and the University of California San Francisco (CLIAHUB) has been receiving samples for no-cost testing from various counties in California since April 7th, 2020.
 
The testing protocol involves assays that target the N gene and the E gene in SAR-VoV-2, and other groups commonly use these assays.
 
In a major shocking discovery in In July, the researchers identified clustering of 35 samples from Madera County that showed poor assay performance for the N gene, but not the E gene, and the study team has now set out to identify potential reasons why.
 
The study team assessed the concordance of the cycle threshold (Ct) values for the two assays in 3,957 SARS-CoV-2-positive tests that were conducted between May 27th and August 7th. The Ct value is a relative measure of the concentration of a target during PCR.
 
It was found that of 3,629 samples that were positive for the E gene and N gene, the differ ence in Ct value between the N and E gene (ΔCt[N-E]) was 0.40.
 
The study team also defined two potential mutant sample sets.
 
The initial set (set A), which comprised 42 samples collected from Madera County between June 30th and August 4th, included samples that had an ΔCt(N-E) value of 2.96 or more and an E gene Ct value 30 or less.
 
The team reports that in sample set A, the ΔCt(N-E) was an average of 5.35, representing an approximate 41-fold impairment of N gene amplification.
 
The subsequent sample set (Set B), which comprised 19 samples collected from Madera County over the same time period, included samples that had an E gene Ct value of more than 30 and an ΔCt(N-E) of more than 2.96 or an E gene Ct value of more than 30 and no N gene Ct value detected.
 
Subsequent sequencing of the detected N gene fragment in samples from set A, identified 34 samples that had a single mutation in the forward primer binding site in the N gene, which corresponds to G29140T in the SARS-CoV-2 genome.
 
It was found that the other eight samples had a wild-type N gene fragment, indicating that the higher average ΔCt(N-E) of 5.99 that was observed in these samples was an artifact.
 
From the 17 randomly-selected control samples from Madera county that had an ΔCt(N-E) value of less than 2.96, none contained the G29140T mutation.
 
Interestingly, sequencing of the detected gene fragment in samples from set B identified twelve samples that had the G29140T mutation.
 
Significantly, RT-PCR targeting the N gene did not detect SARS-COV-2 in five of ten 10 samples, says the team.  
 
In order to assess the effect the G29140T mutation had on PCR targeting of the N gene, the researchers synthesized a new N gene forward primer that had complete complementarity to the mutated sequence and compared the performance of this primer to the original primer among 16 mutant and 14 wild- type samples.
 
Also it was observed among  the mutant samples, the ΔCt(N-E) fell from 5.44 with the original primer to 0.19 with the mutated primer. Among wild-type samples, the ΔCt(N-E) increased from 0.46 with the original primer to 7.34 with the mutated primer.
 
The study team commented, “These data validate that the G29140T mutation is causative for the observed aberrant N gene Ct values.”
 
Dr Vanaerschot and colleagues warns the findings demonstrate that even in areas where community spread of SARS-CoV-2 is high, mutations can arise that impair recognition of RT-PCR primers and decrease diagnostic sensitivity. This could lead to under-diagnosis if laboratories only used one target for SARS-CoV-2 detection.
 
Dr Vanaerschot  concluded, “Our findings strongly support the routine use of at least two targets for SARS-CoV-2 detection by RT-PCR.”

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