According to PANGOlin phylogeny, the SARS-CoV-2 Omicron variant of concern (VOC) consists of B.1.1.529 sublineages BA.1, in which there are 36 further sublineages; BA.2, sometimes called stealth Omicron or Omicron 2, with 3 further sublineages), and BA.3. As of April 2, 2022, among the ≈2 million Omicron sequences deposited in GISAID, spike mutation L452R (S:L452R) had been reported at <0.5% prevalence in BA.1 (425 cases), BA.1.1 (1,441), BA.1.17 (630), BA.1.15 (1,848), BA.1.1.15 (67), BA.1.15.1 (571), BA.1.14 (123), BA.1.1.1 (150), BA.1.1.2 (30), BA.1.16 (116), BA.1.1.14 (138), BA.1.1.13 (107), BA.1.1.16 (40), BA.1.1.11 (117), BA.1.13 (38), BA.1.1.12 (11), BA.1.9 (430), BA.1.12 (73), BA.1.9 (430), BA.1.12 (73), BA.1.1.10 (11), B.1.13.1 (5), BA.1.1.4 (1), BA.1.1.8 (24), BA.1.1.3 (6), BA.1.3 (3), BA.1.5 (1), BA.1.1.7 (8), BA.1.6 (4), BA.1.1.5 (1), BA.1.2 (3), BA.1.4 (1), BA.1.16.1 (1), BA.2 (125), and BA.2.3 (22).

The microbiology laboratory at Azienda Socio Sanitaria Territoriale (ASST), Sette Laghi (Territorial Social Health Authority of the Seven Lakes; Varese, Italy), which serves a wide area of the northern Lombardy region of Italy, has started a whole-genome next-generation sequencing (NGS) program for SARS-CoV-2­­–positive patients who seek care at emergency departments, as well as healthcare workers and patients in selected wards at 5 referral hospitals. During December 3, 2021–January 27, 2022, we identified 301 patients who tested positive by qualitative real-time reverse transcription PCR, then had blood samples undergo NGS; 220 samples were positive for Delta VOC and 81 for Omicron VOC. Among the Omicron cases, 25 were positive for spike mutation L452R (S: L452R) (Table). Of the sequences that we deposited in the GISAID database (https://www.gisaid.org), 17 belonged to PANGOlin sublineage BA.1, 3 to BA.1.1, 2 to BA.3, and 3 to undetermined sublineages. This proportion corresponded to a L452R prevalence of 31% (25 of 81 Omicron-positive participants in our study), compared with <0.5% worldwide. The wide heterogeneity in viral sequences excluded the likelihood of local transmission chains and supported the hypothesis of multiple introductions and convergent gene evolution.

The Omicron VOC leads to lower hospitalization and intensive care unit admission rates than some other VOCs, although it is resistant to most spike monoclonal antibodies (A. Peralta-Santos, unpub. data, https://doi.org/10.1101/2022.01.20.477163). To be effective, sotrovimab and small chemical antivirals have to be administered in the first days after onset of symptoms (D.K. Rai, unpub. data, https://www.biorxiv.org/content/10.1101/2022.01.17.476644v1), a timeframe not compatible with that needed to perform and receive results from spike or whole-genome NGS.

Attempts are underway to develop Omicron-specific PCRs, but in the interim, many laboratories are exploiting previously developed, commercially available, variant-specific PCRs to more promptly discriminate Omicron from Delta variants, an approach that relies on identifying the S: L452R mutation. Convergent evolution has led S: L452 mutations to occur in time across many different variants of interest (e.g., L452R in SARS-CoV-2 Iota and Epsilon and L452Q in Lambda), accounting for an overall 60% prevalence among SARS-CoV-2 isolates deposited in GISAID as of January 30, 2022. However, under the current simplified understanding of the variant landscape, it had been supposed that L452R mutations occurred in nearly all Delta samples across hundreds of AY sublineages, but not in Omicron samples. Our data clearly show that the approaches now being use currently are at risk of becoming unreliable for identifying variants because of gaps in knowledge.

Of note, L452R has been associated not only with resistance to some monoclonal antibodies (1) but also with T-cell immunity escape (N. Le Bert, unpub. data, https://doi.org/10.1101/2022.01.20.477163). The population of the northern Lombardy area has 80% coverage for 2-dose/single vaccine and 56% of residents have received an additional booster (2). Under such heavy selective pressure from vaccine-elicited immunity, it is not surprising that Omicron mutations leading to T-cell escape have a fitness advantage, and hence their prevalence should continue to increase.

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