On 26 November 2021 WHO designated a new variant of concern B.1.1.529 named Omicron. This variant has a large number of mutations, some of which are concerning. Preliminary evidence suggests an increased risk of reinfection with this variant and reduced neutralization by convalescent and vaccinated sera, as compared to other VOCs. Implementation of the high-throughput rRT-PCR screening for Omicron is of great importance for monitoring the spread of this VOC in the population, especially in resource-limited countries lacking sufficient sequencing capacity. Omicron lineage B.1.1.529 (BA.1) has some indels that turned out to be a good target for its detection. In the current protocol, we use ins214EPE for this purpose. Here we describe the 1-step quantitative multiplex RT-qPCR assay consisting of the newly developed Ins214EPE detection set and widely used Hong Kong University N gene assay for SARS-CoV-2 detection (Chu et al., 2020). The assay was validated on the Omicron variant RNA kindly provided by the Pathogenic Microorganisms Variability Laboratory (Dr. Vladimir Guschin, Gamaleya Institute, Moscow, Russia) and RNA from the collection of Smorodintsev Research Institute of Influenza. Omicron RNA specimens were positive in the assay as expected. Negative controls were found negative. 10-fold serial dilutions of Omicron RNA were used to assess ins214EPE assay amplification efficiency. The amplification efficiency was 98,9% (R2 = 0,99). The developed rRT-PCR assay demonstrates high specificity. It was tested on 26 clinical samples (RNA extracted from oropharyngeal swabs) with previously characterized viruses belonging to 8 different SARS-CoV-2 lineages (including Delta B.1.617.2+AY.*) Specific signal was detected only in samples with SARS-CoV-2 Omicron lineage RNA (confirmed by whole-genome sequencing). Specificity was additionally tested on clinical samples positive for other respiratory viruses from the collection of Smorodintsev Research Institute of Influenza - influenza, parainfluenza, human seasonal coronaviruses (OC43, NL63, 229E, HKU1), hRSV, rhinoviruses, bocaviruses, metapneumovirus (33 in total) - with no false-positive results. Ins214EPE Cq 6x B.1.1.7 2x B.1.351 5x AT.1 6x B.1.617.2 4x AY.122 P.1 B.1.1.529 28,72 B.1.1.529 26,29 virus RP Cq SARS Cq Ins214 Cq RSV A 28,76 RSV A 30,56 RSV A 27,70 RSV B 31,49 RSV B 30,98 RSV B 32,33 NL63 32,20 NL63 30,42 NL63 24,95 Oc43 30,34 Oc43 30,69 Oc43 28,64 HKU1 30,06 HKU1 28,30 HKU1 30,73 229E 29,11 229E 32,52 229E 29,37 BoV 32,26 BoV 30,75 BoV 27,25 Rv 32,85 Rv 33,76 Rv 27,75 Piv1 28,63 Piv2 24,72 Piv3 27,01 Piv4 23,90 Adv 29,47 MPV 30,12 HIV A 29,13 HIV A 28,45 HIV A 28,16 39,06 c+ 34,15 26,61 28,44 Analytical sensitivity determination is underway. We consider developed assay to be useful in wide populational RT-PCR screening to assess the spread of Omicron variant.
Omicron lineage (B.1.1.529) – SARS-CoV-2 variant of concern designated on 26 November 2021 by WHO. This variant has a large number of mutations, some of which are concerning. There is already enough evidence that Omicron lineage connected with increased risk of reinfection with this variant and reduced neutralization by convalescent and vaccinated sera, as compared to other VOCs. Omicron subvariants includes several subvariants with strikingly different genetic characteristics: BA.1, BA.2, BA.3. We developed RT-qPCR specific to omicron lineage with detemination of BA.1 and BA.2/BA.3 for epidemiological reasons. Omicron lineage B.1.1.529 and its sublineages BA.1 and BA.2/BA.3 have some indels that turned out to be a good target for its detection. Using annotations of amino acid substitutions and indels for more than 5 million samples in 1513 Pango lineages from the GISAID database available on 2021-12-03, relative frequencies of substitutions and indels by each lineage were calculated with Python v3.9.2 programming language, Pandas v1.3.5 and Numpy v1.3.5 tools. Using this data we found indels highly specific for BA.1 and BA.2/BA.3: ERS31del for Omicron (B.1.1.529) lineage, including BA.1, BA.2 and BA.3; and Ins214EPE specific only to BA.1. For these targets have been developed assays, that were then multiplexed. Assay for detection BA.1 have been already published at protocols.io. Here we publish multiplex RT-qPCR detecting all Omicron subvariants with the opportunity to differentiate BA.1 and BA.2/BA.3. Characteristics and analysis of specificity of Ins214EPE were already published in previous protocol. We only can add that limit of detection of the test is 1000 copies/ml Here we describe features of newly introduced ERS31del assay. 10-fold serial dilutions of SARS-CoV-2 RNA of Omicron lineage were used to assess ERS31del assay amplification efficiency. The amplification efficiency was 105% (R2 = 0,99). Developed RT-qPCR assay demonstrates high specificity. It was tested on 27 clinical samples (RNA extracted from oropharyngeal swabs) with previously characterized viruses belonging to 10 different SARS-CoV-2 lineages. RP 2-SARS-ORF NdeI Ins214 1 c- 2 P.1 35,35 15,45 3 B.1.351 39,91 4 B.1 15,69 5 B.1 33,84 26,69 6 B.1 24,26 26,98 7 B.1.1.7 25,27 20,64 8 B.1.1.7 30,54 28,05 9 B.1.1.7 26,99 31,25 10 AT.1 30,30 33,07 11 AT.1 30,02 31,33 12 AT.1 28,00 33,00 13 B.1.1.523 29,12 27,09 14 B.1.1.523 27,39 23,46 15 B.1.1.523 30,02 27,09 16 B.1.1.317 25,22 25,31 17 B.1.1.317 27,58 22,71 18 B.1.1.317 28,70 23,91 19 B.1.617.2 26,66 34,03 20 B.1.617.2 25,72 28,43 21 B.1.617.2 26,78 25,42 22 AY.122 23,96 28,95 23 AY.122 31,16 35,05 24 AY.122 24,19 34,93 25 BA.1 27,98 22,20 23,57 22,08 26 BA.1 27,08 24,93 27,16 24,65 27 BA.1 25,19 27,48 29,83 25,57 28 BA.2 26,50 19,64 21,07 29 BA.2 24,70 20,81 22,70 Specific signal was detected only in samples with SARS-CoV-2 Omicron lineage RNA: BA.1 and BA.2 (confirmed by whole-genome sequencing). Specificity was additionally tested on clinical samples positive for other respiratory viruses from the collection of Smorodintsev Research Institute of Influenza - influenza A and B, human seasonal coronaviruses, hRSV, rhinoviruses - with no false-positive results. Analytical sensitivity Limit of detection of the test is 1000 copies/ml
Whole genome sequencing (WGS) is considered the best instrument to track both virus evolution and the spread of new, emerging variants. However, WGS still does not allow the analysis of as many samples as qPCR does. Epidemiological and clinical research needs to develop advanced qPCR methods to identify emerging variants of SARS-CoV-2 while collecting data on their spreading in a faster and cheaper way, which is critical for introducing public health measures. This study aimed at designing a one-step RT-qPCR assay for multiplex detection of the Omicron lineage and providing additional data on its subvariants in clinical samples. The RT-qPCR assay demonstrated high sensitivity and specificity on multiple SARS-CoV-2 variants and was cross-validated by WGS.
Under the COVID-19 pandemic, healthcare workers were at the highest risk of getting infected with the disease; this necessitates specialized studies in this occupational group. The aim of the study was to identify non-occupational risk factors and laboratory markers indicating that severe clinical forms of new coronavirus infection would probably develop in healthcare workers in the initial period of the pandemic. The study included 366 workers who suffered COVID-19 in 2020–2021. The disease was confirmed by examining smears from the pharynx and nose with PCR. Some of the samples were examined using the SARS-CoV-2 whole genome sequencing technology. To determine laboratory prognostic indicators evidencing the development of more severe forms of the disease (pneumonia), a number of healthcare workers underwent laboratory examination during the acute period of the disease, namely: general clinical and biochemical blood tests, immunophenotyping of lymphocytes, analysis of the hemostasis system and cytokine levels. To study non-occupational risk factors of pneumonia, all healthcare workers after recovery were asked to fill in a Google form developed by the authors. The most severe clinical forms of COVID-19 were registered in healthcare workers who were older than 40 years, with low physical activity and a body mass index higher than 25.0, had diabetes mellitus and chronic diseases of the genitourinary system. When analyzing the results of laboratory tests, markers indicating development of pneumonia were identified and their critical values (cut-off points) were determined: the level of lymphocytes (below 1.955•109/l), T-cytotoxic lymphocytes (below 0.455•109/l), T-helpers (below 0.855•109/L), natural killers (below 0.205•109/l), platelets (below 239•109/L), erythrocyte sedimentation rate (above 11.5 mm/h), D-dimer (above 0.325 mcg/ml), total protein (below 71.55 g/L), lactate dehydrogenase (above 196 U/L), C-reactive protein (above 4.17 mg/l), and interleukin-6 (above 3.63 pg/l). The study identified non-occupational risk factors causing development of severe COVID-19 and established laboratory prognostic indicators.
Under the COVID-19 pandemic, healthcare workers were at the highest risk of getting infected with the disease; this necessitates specialized studies in this occupational group. The aim of the study was to identify non-occupational risk factors and laboratory markers indicating that severe clinical forms of new coronavirus infection would probably develop in healthcare workers in the initial period of the pandemic. The study included 366 workers who suffered COVID-19 in 2020–2021. The disease was confirmed by examining smears from the pharynx and nose with PCR. Some of the samples were examined using the SARS-CoV-2 whole genome sequencing technology. To determine laboratory prognostic indicators evidencing the development of more severe forms of the disease (pneumonia), a number of healthcare workers underwent laboratory examination during the acute period of the disease, namely: general clinical and biochemical blood tests, immunophenotyping of lymphocytes, analysis of the hemostasis system and cytokine levels. To study non-occupational risk factors of pneumonia, all healthcare workers after recovery were asked to fill in a Google form developed by the authors. The most severe clinical forms of COVID-19 were registered in healthcare workers who were older than 40 years, with low physical activity and a body mass index higher than 25.0, had diabetes mellitus and chronic diseases of the genitourinary system. When analyzing the results of laboratory tests, markers indicating development of pneumonia were identified and their critical values (cut-off points) were determined: the level of lymphocytes (below 1.955•109/l), T-cytotoxic lymphocytes (below 0.455•109/l), T-helpers (below 0.855•109/L), natural killers (below 0.205•109/l), platelets (below 239•109/L), erythrocyte sedimentation rate (above 11.5 mm/h), D-dimer (above 0.325 mcg/ml), total protein (below 71.55 g/L), lactate dehydrogenase (above 196 U/L), C-reactive protein (above 4.17 mg/l), and interleukin-6 (above 3.63 pg/l). The study identified non-occupational risk factors causing development of severe COVID-19 and established laboratory prognostic indicators.
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