CCQM-P199b: Interlaboratory comparability study of SARS-CoV-2 RNA copy number quantification (preprint)

Nucleic acid amplification tests including reverse transcription-quantitative PCR (RT-qPCR) are used to detect RNA from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Standardized measurements of RNA can facilitate comparable performance of laboratory tests in the absence of existing reference measurement systems early on in a pandemic. Interlaboratory study CCQM-P199b 'SARS-CoV-2 RNA copy number quantification' was designed to test the fitness-for-purpose of developed candidate reference measurement procedures (RMPs) for SARS-CoV-2 genomic targets in purified RNA materials, and was conducted under the auspices of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) to evaluate the measurement comparability of national metrology institutes (NMIs) and designated institutes (DIs), thereby supporting international standardization. Twenty-one laboratories participated in CCQM-P199b and were requested to report the RNA copy number concentration, expressed in number of copies per microliter, of the SARS-CoV-2 nucleocapsid (N) gene partial region (NC_045512.2: 28274-29239) and envelope (E) gene (NC_045512.2: 26245-26472) (optional measurement) in samples consisting of in vitro transcribed RNA or purified RNA from lentiviral constructs. Materials were provided in two categories: lower concentration (approximately 10 x 1 - 10 x 4/uL in aqueous solution containing human RNA background) and high concentration (approximately 10 x 9/uL in aqueous solution without any other RNA background). For the measurement of N gene concentration in the lower concentration study materials, the majority of laboratories (n = 17) used one-step reverse transcription-digital PCR (RT-dPCR), with three laboratories applying two-step RT-dPCR and one laboratory RT-qPCR. Sixteen laboratories submitted results for E gene concentration. Reproducibility (% CV or equivalent) for RT-dPCR ranged from 19 % to 31 %. Measurements of the high concentration study material by orthogonal methods (isotope dilution-mass spectrometry and single molecule flow cytometry) and a gravimetrically linked lower concentration material were in a good agreement, suggesting a lack of overall bias in RT-dPCR measurements. However methodological factors such as primer and probe (assay) sequences, RT-dPCR reagents and dPCR partition volume were found to be potential sources of interlaboratory variation which need to be controlled when applying this technique. This study demonstrates that the accuracy of RT-dPCR is fit-for-purpose as a RMP for viral RNA target quantification in purified RNA materials and highlights where metrological approaches such as the use of in vitro transcribed controls, orthogonal methods and measurement uncertainty evaluation can support standardization of molecular methods.

SARS-CoV-2 Omicron Outbreak in a Dormitory in Saint-Petersburg, Russia (preprint)

The B.1.1.529 Omicron variant of SARS-CoV-2 is rapidly spreading, displacing the globally prevalent Delta variant. Before December 16, 2021, community transmission had already been observed in tens of countries globally. However, in Russia, all reported cases had been sporadic and associated with travel. Here, we report an Omicron outbreak at a students’ dormitory in Saint Petersburg, Russia. Out of the 462 sampled residents of the dormitory, 206 (44.6%) tested PCR positive, and 159 (77.1%) of these infections carried the S:ins214EPE insertion, indicating that they were of the Omicron strain. 104 (65%) of Omicron-positive patients have been vaccinated and/or reported previous covid-19. Whole genome sequencing confirmed that the outbreak is caused by the Omicron variant. Phylogenetic analysis showed that the outbreak has a single origin, and belongs to the S:346K sublineage of Omicron which may be characterized by an increased rate of spread, compared to other Omicron sublineages. The rapid spread of Omicron in a population with preexisting immunity to previous variants underlines its propensity for immune evasion.

Determining international spread of novel B.1.1.523 SARS-CoV-2 lineage (preprint)

Here we report the emergence of variant lineage B.1.1.523 that contains a set of mutations including 156_158del, E484K and S494P in Spike protein. E484K and S494P are known to significantly reduce SARS-CoV-2 neutralization by convalescent and vaccinee sera and are considered as mutations of concern. Lineage B.1.1.523 has presumably originated in Russian Federation and spread across European countries with the peak of transmission in April / May 2021. The B.1.1.523 lineage has now been reported from 27 countries.

One-shot immunization with Sputnik Light (the first component of Sputnik V vaccine) is effective against SARS-CoV-2 Delta variant: efficacy data on the use of the vaccine in civil circulation in Moscow (preprint)

ObjectivesVaccination remains the most effective response to the COVID-19 pandemic. Most vaccines use two-dose regimens. In turn, single-dose vaccines also have high potential, since, on the one hand, they simplify the vaccination program, make it more accessible and convenient for more people around the world, and on the other hand, they are better suited for subsequent revaccination. However, there is not enough data on the effectiveness of single-dose vaccine variants against new genetic lines to assess their current potential. It is not clear how much a single dose of immunization protects against the globally dominant delta variant. In this work, we investigated the effectiveness of a single dose vaccine (Sputnik Light, the first component of Sputnik V vaccine) against the Delta variant in Moscow. MethodsTo assess the effectiveness of one dose of viral vector vaccine based on rAd26 against the delta variant in Moscow, we used data from the Moscow registries of vaccination against COVID-19 and the incidence of COVID-19. The availability of data on the number of seropositive residents of Moscow made it possible to consider the size of the immune layer formed because of a previous COVID-19 disease or vaccination. To calculate the effectiveness, the proportion of COVID-19 cases among those vaccinated with a single dose and the proportion of cases among those who were not vaccinated in July 2021. ResultsOur data indicate that throughout July 2021, the dominant variant of the coronavirus at the level of 99.5% in Moscow was the SARS-CoV-2 delta variant and its subsidiary lines. Considering the immune layer of 46% allowed us to calculate the effectiveness of a one-shot vaccine against the delta variant in Moscow during the first three months after vaccination at the level of 69.85% (95% confidence interval [CI], 64.08 to 74.70). In the 18-29-year-old group, the overall vaccine efficacy against the delta variant was 88.61%, in the 18-59 group - 75.28%. Sputnik Light demonstrates higher efficacy against Delta variant than many two-shot vaccines. ConclusionThe results indicate a high efficacy of a single immunization first component of Sputnik V vaccine against delta variant among young and middle-aged people, at least during the first 3 months after receiving the one-shot vaccine.

Spread of Endemic SARS-CoV-2 Lineages in Russia Before April 2021 (preprint)

In 2021, the COVID-19 pandemic is characterized by global spread of several lineages with evidence for increased transmissibility. Russia is among the countries with the highest number of confirmed COVID-19 cases, making it a potential hotspot for emergence of novel variants. Here, we show that among the globally significant variants of concern, alpha (B.1.1.7), beta (B.1.351) or gamma (P.1), none have been sampled in Russia before January 2021. Instead, between summer 2020 and spring 2021, the epidemic in Russia has been characterized by the spread of two lineages that are rare elsewhere: B.1.1.317 and a sublineage of B.1.1 including B.1.1.397 (hereafter, B.1.1.397+). Their frequency has increased in different parts of Russia. Mutational composition and frequency dynamics suggest that B.1.1.317 and B.1.1.397+ may be more transmissible than the previously predominant B.1.1. On top of these lineages, in January 2021, B.1.1.7 emerged in Russia, reaching the frequency of 17.4% (95% C.I.: 12.0%-24.4%) in March 2021. Additionally, we identify three novel distinct lineages, AT.1, B.1.1.524 and B.1.1.525, that have started to spread, together reaching the frequency of 11.8% (95% C.I.: 7.5%-18.1%) in March 2021. These lineages carry combinations of several notable mutations, including the S:E484K mutation of concern, deletions at a recurrent deletion region of the spike glycoprotein (S:Δ140-142, S:Δ144 or S:Δ136-144), and nsp6:Δ106-108 (also known as ORF1a:Δ3675-3677). Community-based PCR testing indicates that these variants have continued to spread in April 2021, with the frequency of B.1.1.7 reaching 21.7% (95% C.I.: 12.3%-35.6%), and the joint frequency of B.1.1.524 and B.1.1.525, 15.2% (95% C.I.: 7.6%-28.2%). Although these variants have been displaced by the onset of delta variant in May-June 2021, the frequency increase of lineages B.1.1.317, B.1.1.397+, AT.1, B.1.1.524 and B.1.1.525 suggest that the combinations of mutations observed in them could have increased the rate of their spread.

COVID-19 pandemic in Saint Petersburg, Russia: combining surveillance and population-based serological study data in May, 2020 - April, 2021 (preprint)

Background: The COVID-19 pandemic in Russia has already resulted in 500,000 excess deaths, with more than 5.6 million cases registered officially by July 2021. Surveillance based on case reporting has become the core pandemic monitoring method in the country and globally. However, population-based seroprevalence studies may provide an unbiased estimate of the actual disease spread and, in combination with multiple surveillance tools, help to define the pandemic course. This study summarises results from four consecutive serological surveys conducted between May 2020 and April 2021 at St.Petersburg, Russia and combines them with other SARS-CoV-2 surveillance data. Methods: We conducted four serological surveys of two random samples (May-June, July-August, October--December 2020, and February-April 2021) from adults residing in St.Petersburg recruited with the random digit dialing (RDD), accompanied by a telephone interview to collect information on both individuals who accepted and declined the invitation for testing and account for non-response. We have used enzyme-linked immunosorbent assay CoronaPass total antibodies test (Genetico, Moscow, Russia) to report seroprevalence. We corrected the estimates for non-response using the bivariate probit model and also accounted the test performance characteristics, obtained from independent assay evaluation. In addition, we have summarised the official registered cases statistics, the number of hospitalised patients, the number of COVID-19 deaths, excess deaths, tests performed, data from the ongoing SARS-CoV-2 variants of concern (VOC) surveillance, the vaccination uptake, and St. Petersburg search and mobility trends. The infection fatality ratios (IFR) have been calculated using the Bayesian evidence synthesis model. Findings: After calling 113,017 random mobile phones we have reached 14,118 individuals who responded to computer-assisted telephone interviewing (CATI) and 2,413 provided blood samples at least once through the seroprevalence study. The adjusted seroprevalence in May-June, 2020 was 9.7% (95%: 7.7-11.7), 13.3% (95% 9.9-16.6) in July-August, 2020, 22.9% (95%: 20.3-25.5) in October-December, 2021 and 43.9% (95%: 39.7-48.0) in February-April, 2021. History of any symptoms, history of COVID-19 tests, and non-smoking status were significant predictors for higher seroprevalence. Most individuals remained seropositive with a maximum 10 months follow-up. 92.7% (95% CI 87.9-95.7) of participants who have reported at least one vaccine dose were seropositive. Hospitalisation and COVID-19 death statistics and search terms trends reflected the pandemic course better than the official case count, especially during the spring 2020. SARS-CoV-2 circulation showed rather low genetic SARS-CoV-2 lineages diversity that increased in the spring 2021. Local VOC (AT.1) was spreading till April 2021, but B.1.617.2 substituted all other lineages by June 2021. The IFR based on the excess deaths was equal to 1.04 (95% CI 0.80-1.31) for the adult population and 0.86% (95% CI 0.66-1.08) for the entire population. Conclusion: Approximately one year after the COVID-19 pandemic about 45% of St. Petersburg, Russia residents contracted the SARS-CoV-2 infection in, or 2.2 mln people. Combined with vaccination uptake of about 10% it was enough to slow the pandemic until the Delta VOC started to spread. Combination of several surveillance tools provides a comprehensive pandemic picture. Funding: Polymetal International plc.

SARS-CoV-2 escape from cytotoxic T cells during long-term COVID-19 (preprint)

Evolution of SARS-CoV-2 in immunocompromised hosts may result in novel variants with changed properties, but the mode of selection underlying this process remains unclear. While escape from humoral immunity certainly plays a role in intra-host evolution, escape from cellular immunity is poorly understood. Here, we report a case of long-term COVID-19 in an immunocompromised patient with non-Hodgkin’s lymphoma who received treatment with rituximab and lacked neutralizing antibodies. Over the 318 days of the disease, the SARS-CoV-2 genome gained a total of 40 changes, 34 of which were present by the end of the study period. Among the acquired mutations, 12 reduced or prevented binding of known immunogenic SARS-CoV-2 HLA class I antigens, suggesting that virus immunoediting is largely driven by cytotoxic CD8 T cell clones. The two changes with the strongest effect, nsp3:T504A and nsp3:T504P, were experimentally assessed in a cytotoxic assay of the patient's CD8 T cells. Both these changes were associated with immune escape, with a stronger effect observed for nsp3:T504P, the change which ultimately got fixed. Together, these results suggest that CD8 T cell escape may be an underappreciated contributor to SARS-CoV-2 evolution in humans.

Insufficient Fibrinolysis in COVID-19: A Systematic Review of Thrombolysis Based on Meta-Analysis and Meta-Regression (preprint)

Background: How aberrant fibrinolysis influences the clinical progression of COVID-19 presents a clinicopathological dilemma challenging intensivists. To investigate whether abnormal fibrinolysis is a culprit or protector or both, we associated elevated plasma D-dimer with clinical variables to identify a panoramic view of the derangements of fibrinolysis that contribute to the pathogenesis of COVID-19 based on studies available in the literature.Methods: We performed this systematic review based on both meta-analysis and meta-regression to compute the correlation of D-dimer at admission with clinical features of COVID-19 patients in retrospective studies or case series. We searched the databases until Aug 18, 2020, with no limitations by language. The first hits were screened, data extracted, and analyzed in duplicate. We did the random-effects meta-analyses and meta-regressions (both univariate and multivariate). D-dimer associated clinical variables and potential mechanisms were schematically reasoned and graphed.Findings: Our search identified 42 observational, or retrospective, or case series from six countries (n=14,862 patients) with all races and ages from 1 to 98-year-old. The weighted mean difference of D-dimer was 0.97 ug/mL (95% CI 0.65, 1.29) between relatively mild (or healthy control) and severely affected groups with significant publication bias. Univariate meta-regression identified 58 of 106 clinical variables were associated with plasma D-dimer levels, including 3 demographics, 5 comorbidities, 22 laboratory tests, 18 organ injury biomarkers, 8 severe complications, and 2 outcomes (discharge and death). Of these, 11 readouts were negatively associated with the level of plasma D-dimer. Further, age and gender were confounding factors for the identified D-dimer associated variables. There were 22 variables independently correlated with the D-dimer level, including respiratory rate, dyspnea plasma K+, glucose, SpO2, BUN, bilirubin, ALT, AST, systolic blood pressure, and CK. We thus propose that insufficient hyperfibrinolysis (fibrinolysis is accelerated but unable to prevent adverse clinical impact for clinical deterioration COVID-19) as a peculiar mechanism.Interpretation: The findings of this meta-analysis- and meta-regression-based systematic review supports elevated D-dimer as an independent predictor for mortality and severe complications. D-dimer-associated clinical variables draw a landscape integrating the aggregate effects of systemically suppressive and locally (i.e., in the lung) hyperactive derangements of fibrinolysis. D-dimer and associated clinical biomarkers and conceptually parameters could be combined for risk stratification, potentially for tracking thrombolytic therapy or alternative interventions.Funding: NIH grants: HL87017 (HLJ), HL095435 (MAM), HL134828 (MAM), HL130402 (AK&SI), AI112381(SLL), AI150473 (SLL), HL154103 (SI &AK), and HL14285301 (SI)Declaration of Interests: None of the authors have any conflict of interest to declare. Unrelated to the subject of this review, Dr. Idell serves as a Founder and Chief Scientific Officer of Lung Therapeutics Inc., (LTI) and a member of the Board of Directors of LTI, which has conducted Phase 1 trial and and is now conducting a Phase 2 clinical trial of urokinase plasminogen activator for patients with infectious pleural loculation and failed drainage. He has an equity position (first tier COI) in the company, as does the University of Texas Horizon Fund and The University of Texas Health Science Center at Tyler (UTHSCT). He has a conflict of interest plan acknowledging and managing these declared conflicts of interest through The University of Texas Health Science Center at Tyler (UTHSCT) and UT System. Dr. Komissariv has received funding from LTI and has a COI management plan at UTHSCT accordingly.