With input from Dr Monica Vasudev

1278:   How to diagnose UK strain by existing RT PCR test

1. Just use three-target assay (N, ORF1ab, S).
2. Look for S gene target failures (positive N, ORF and negative S gene).
3. The VOC includes a deletion of six nucleotides in the S gene,causing loss of two amino acids at positions 69 and 70 (Δ69-70) and has been previously reported by another group to result in S gene dropout in commercial assays (Bal A, et al. medRxiv, 2020).
4. Δ69-70 is present in over 99% of sequenced S gene dropouts, but less than 0.1% of sequenced S gene positives.
5. This is termed as S-gene target failure (SGTF).
6. The new SARS-CoV-2 variant, first detected in southern England, has a transmission advantage of 0.4 to 0.7 points higher in reproduction number, or R0, in comparison with the initial strain, reported British researchers.
7. This variant has a considerable transmission advantage, and its reproduction numbers could range from 1.4 to 1.8, suggest researchers at Imperial College London (ICL).
8. Researchers noted a larger proportion of individuals below 20 years of age among reported cases of the variant versus non-variant cases, calling it ‘a shift in age composition.’ The variants R0 is estimated to be 40%-80% higher than for the wild-type virus.
9. For context in the U.S., previous research noted that seasonal influenza had a median reproduction number of 1.28, while the median reproduction number for the 1918 flu pandemic was 1.80.
10. It does not seem likely to impact COVID-19 vaccine effectiveness, though it may render certain treatments less effective, such as convalescent plasma.
11. A preliminary report from the U.K.s Centre for Mathematical Modelling of Infectious Diseases on December 23 had estimated that the transmission may be at least 56% and up to 70% higher. The findings were updated on December 31, noting that the frequency of the variant had increased considerably in all regions of England, with a 50% or greater frequency in all National Health System regions.
12. The Imperial College team assessed both epidemiological and genetic data, including 1,904 whole genomes from October and December 5 with a genetic background of 48,128 genomes collected over the same period. There was a high correlation between S-gene target failure (SGTF) during COVID-19 PCR testing and frequency of the variant, thus suggesting that S-gene target failure could be a biomarker to detect the variant in the community.
13. From monitoring patterns in which genes are found to be present in the PCR test, it can be identified where cases of the new variant are likely to be increasing. The new variant has genetic changes in the S gene, hence, it is no longer detected by the current test. Samples that would previously have been positive on all three genes are now found positive only on the ORF1ab and N genes.
14. Re-infection:  Lab data were used to detect possible reinfections, defined as an episode of polymerase chain reaction (PCR) positivity at least 90 days before a recent PCR positive detection. Two reinfections were detected in the variant case group (1.13/1000 cases) in comparison with 3 reinfections in the comparator group (1.70/1000 cases, Fisher’s exact P=1.00). The same definition was applied to SGTF cases. These SGTF cases comprised of samples with ORF and N gene Ct values of < 31, S gene defined as negative or positive on the basis of presence or absence regardless of Ct value. The rate of detected re-infections in national SGTF cases was 0.60/1000 compared to 0.61/1000 in non-SGTF cases (P=0.94). When restricting to the Kent area, the rate of detected reinfections was 0.51/1000 compared to 0/1000 in non-SGTF cases (P=0.69).
15. Only a small fraction of all new cases of VOC 202012/01 are identified using whole genome sequencing, and this data typically lags test date by about two weeks. A proxy SGTF is, therefore, used to indicate VOC carriage.
16. One of the S gene mutations in the VOC, which deletes amino acids 69 and 70 (Δ69-70), causes a reproducible SGTF in the Thermopath TaqPath assay, which is used in three UK lighthouse laboratories.
17. This coincidental occurrence yields a good proxy for monitoring trends in VOC 202012/01. SGTF is almost perfectly correlated with the presence of Δ69-70. Considering 14,950 tested samples where both the sequence and the SGTF status are known, 99.3% of Δ69-70 sequences (1,831 of 1,843) are SGTF, compared to 0.05% of sequences without the deletion (7 of 13107).
18. The UK has a high throughput national testing system for community cases based in a small number of large laboratories.
19. Three of these laboratories employ a three-target assay (N, ORF1ab, S).
20. S gene target failures (in otherwise positive samples) started escalating considerably from late November. The VOC has a deletion of six nucleotides in the S gene,with loss of two amino acids at positions 69 and 70 (Δ69-70).
21. Researchers found Δ69-70 in >99% of sequenced S gene dropouts, but less than 0.1% of sequenced S gene positives from the same labs. This is S-gene target failure (SGTF). Further confirmation came from molecular analysis. Sequencing of diagnostic PCR amplicon products from S gene dropout samples revealed S gene target to contain the Δ69-70 deletion in the middle of the amplicon. Since S gene is successfully amplified, the S gene dropout must be due to a failure of the qPCR probe to bind as a result of the Δ69-70 deletion. Some other variants also have Δ69-70, but as of late November, the VOC represents nearly all observed sequences with that mutation.

[Excerpts from: Public Health England. Investigation of novel SARS-COV-2 variant. Variant of Concern 202012/01; Public Health England. Investigation of novel SARS-CoV-2 variant. Variant of Concern 202012/01. Technical briefing 2; Medpage Today]

Dr KK Aggarwal

President CMAAO, HCFI and Past National President IMA