Omicron Subvariants: Clinical, Laboratory, and Cell Culture Characterization

BackgroundThe variant of concern, Omicron, has become the sole circulating SARS-CoV-2 variant for the past several months. Omicron subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 evolved over the time, with BA.1 causing the largest wave of infections globally in December 2021- January 2022. In this study, we compare the clinical outcomes in patients infected with different Omicron subvariants and compare the relative viral loads, and recovery of infectious virus from upper respiratory specimens. MethodsSARS-CoV-2 positive remnant clinical specimens, diagnosed at the Johns Hopkins Microbiology Laboratory between December 2021 and July 2022, were used for whole genome sequencing. The clinical outcomes of infections with Omicron subvariants were compared to infections with BA.1. Cycle threshold values (Ct) and the recovery of infectious virus on VeroTMPRSS2 cell line from clinical specimens were compared. ResultsThe BA.1 was associated with the largest increase in SARS-CoV-2 positivity rate and COVID-19 related hospitalizations at the Johns Hopkins system. After a peak in January cases fell in the spring, but the emergence of BA.2.12.1 followed by BA.5 in May 2022 led to an increase in case positivity and admissions. BA.1 infections had a lower mean Ct when compared to other Omicron subvariants. BA.5 samples had a greater likelihood of having infectious virus at Ct values less than 20. ConclusionsOmicron subvariants continue to associate with a relatively high positivity and admissions. The BA.5 infections are more while BA.2 infections are less likely to have infectious virus, suggesting potential differences in infectibility during the Omicron waves. FundingCenters for Disease Control and Prevention contract 75D30121C11061, NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, and The Modeling Infectious Diseases in Healthcare Network (MInD) under awards U01CK000589.

Within-host evolutionary dynamics and tissue compartmentalization during acute SARS-CoV-2 infection

The global evolution of SARS-CoV-2 depends in part upon the evolutionary dynamics within individual hosts with varying immune histories. To characterize the within-host evolution of acute SARS-CoV-2 infection, we deep sequenced saliva and nasal samples collected daily from immune and unvaccinated individuals early during infection. We show that longitudinal sampling facilitates high-confidence genetic variant detection and reveals evolutionary dynamics missed by less-frequent sampling strategies. Within-host dynamics in both naive and immune individuals appeared largely stochastic; however, we identified clear mutational hotspots within the viral genome, consistent with selection and differing between naive and immune individuals. In rare cases, minor genetic variants emerged to frequencies sufficient for forward transmission. Finally, we detected significant genetic compartmentalization of virus between saliva and nasal swab sample sites in many individuals. Altogether, these data provide a high-resolution profile of within-host SARS-CoV-2 evolutionary dynamics.

Sequence Proven Reinfections with SARS-CoV-2 at a Large Academic Center

BackgroundIncreased reinfection rates with SARS-CoV-2 have recently been reported, with some locations basing reinfection on a second positive PCR test at least 90 days after initial infection. MethodsWe identified cases where patients had two positive tests for SARS-CoV-2 and evaluated which of these had been sequenced as part of our surveillance efforts, and evaluated sequencing and clinical data. Results750 patients (920 samples) had a positive test at least 90 days after the initial test. The median time between tests was 377 days, and 724 (79%) of the post 90-day positives were collected after the emergence of the Omicron variant in November 2021. Sequencing was attempted on 231 samples and successful in 127. Successful sequencing spiked during the Omicron surge and showed higher median days from initial infection compared to failed sequences (median 398 days compared to 276 days, p<0.0005). A total of 122 (98%) patients showed evidence of reinfection, 45 of which had sequence proven reinfection and 77 had inferred reinfections (later sequence showed a clade that was not circulating when the patient was initially infected). Children accounted for only 4% of reinfections. 43 (96%) of 45 infections with sequence proven reinfection were caused by the Omicron variant, 41 (91%) were symptomatic, 32 (71%), were vaccinated prior to the second infection, and 6 (13%) were Immunosuppressed. Only 2 (4%) were hospitalized, and both had underlying conditions. ConclusionSequence proven reinfections increased with the Omicron variant but generally caused mild infections.

Characterizing SARS-CoV-2 transcription of subgenomic and genomic RNAs during early human infection using multiplexed ddPCR

Control of SARS-CoV-2 (SCV-2) transmission is a major priority that requires understanding SCV-2 replication dynamics. We developed and validated novel droplet digital PCR (ddPCR) assays to quantify SCV-2 subgenomic RNAs (sgRNAs), which are only produced during active viral replication, and discriminate them from full-length genomic RNAs (gRNAs) in a multiplexed format. We applied this multiplex ddPCR assay to 144 cross-sectional nasopharyngeal samples. sgRNAs were quantifiable across a range of qPCR cycle threshold (Ct) values and correlated with Ct values. The ratio of sgRNA:gRNA was remarkably stable across a wide range of Ct values, whereas adjusted amounts of N sgRNA to a human housekeeping gene declined with higher Ct values. Interestingly, adjusted sgRNA and gRNA amounts were quantifiable in culture-negative samples, although levels were significantly lower than in culture-positive samples. Longitudinal daily testing of 6 persons for up to 14 days revealed that sgRNA is concordant with culture results during the first week of infection but may be discordant with culture later in infection. Further, sgRNA:gRNA is constant during infection despite changes in viral culture. These data indicate stable viral transcription during infection. More work is needed to understand why cultures are negative despite persistence of viral RNAs.

Limitations of molecular and antigen test performance for SARS-CoV-2 in symptomatic and asymptomatic COVID-19 contacts

ObjectivesCOVID-19 has brought unprecedented attention to the crucial role of diagnostics in pandemic control. We compared SARS-CoV-2 test performance by sample type and modality in close contacts of SARS-CoV-2 cases. MethodsClose contacts of SARS-CoV-2 positive individuals were enrolled after informed consent. Clinician-collected nasopharyngeal (NP) swabs in viral transport media (VTM) were tested with a nucleic acid test (NAT). NP VTM and self-collected passive drool were tested using the PerkinElmer real-time reverse transcription PCR (RT-PCR) assay. For the first 4 months of study, mid-turbinate swabs were tested using the BD Veritor rapid antigen test. NAT positive NP samples were tested for infectivity using a VeroE6TMPRSS2 cell culture model. ResultsBetween November 17, 2020, and October 1, 2021, 235 close contacts of SARS-CoV-2 cases were recruited, including 95 with symptoms (82% symptomatic for <5 days) and 140 asymptomatic individuals. NP swab reference tests were positive for 53 (22.6%) participants; 24/50 (48%) were culture positive. PerkinElmer testing of NP and saliva samples identified an additional 28 (11.9%) SARS-CoV-2 cases who tested negative by clinical NAT. Antigen tests performed for 99 close contacts showed 83% positive percent agreement (PPA) with reference NAT among early symptomatic persons, but 18% PPA in others; antigen tests in 8 of 11 (72.7%) culture-positive participants were positive. ConclusionsContacts of SARS-CoV-2 cases may be falsely negative early after contact, which more sensitive platforms may identify. Repeat or serial SARS-CoV-2 testing with both antigen and molecular assays may be warranted for individuals with high pretest probability for infection.

A Quick Displacement of the SARS-CoV-2 variant Delta with Omicron: Unprecedented Spike in COVID-19 Cases Associated with Fewer Admissions and Comparable Upper Respiratory Viral Loads

BackgroundThe increase in SARS-CoV-2 infections in December 2021 in the United States was driven primarily by the Omicron variant which largely displaced the Delta over a three week span. Outcomes from infection with the Omicron remain uncertain. We evaluate whether clinical outcomes and viral loads differ between Delta and Omicron infections during the period when both variants were co-circulating. MethodsRemnant clinical specimens from patients that tested positive for SARS-CoV-2 after standard of care testing between the last week of November and the end of December 2021were used for whole viral genome sequencing. Cycle threshold values (Ct) for viral RNA, the presence of infectious virus, and levels of respiratory IgG were measured, and clinical outcomes were obtained. Differences in each measure were compared between variants stratified by vaccination status. ResultsThe Omicron variant displaced the Delta during the study period and constituted 95% of the circulating lineages by the end of December 2021. Patients with Omicron infections (N= 1121) were more likely to be vaccinated compared to patients with Delta (N = 910), but were less likely to be admitted, require ICU level care, or succumb to infection regardless of vaccination status. There was no significant difference in Ct values based on the lineage regardless of the vaccination status. Recovery of infectious virus in cell culture was reduced in boosted patients compared to fully vaccinated without a booster and unvaccinated when infected with the Delta lineage. However, in patients with Omicron infections, recovery of infectious virus was not affected by vaccination. ConclusionsOmicron infections of vaccinated individuals are expected, yet admissions are less frequent. Admitted patients might develop severe disease comparable to Delta. Efforts for reducing the Omicron transmission are required as even though the admission risk is lower, the numbers of infections continue to be high. Research in context Evidence before this studyThe unprecedented increase in COVID-19 cases in the month of December 2021, associated with the displacement of the Delta variant with the Omicron, triggered a lot of concerns. An understanding of the disease severity associated with infections with Omicron is essential as well as the virological determinants that contributed to its widespread predominance. We searched PubMed for articles published up to January 23, 2022, using the search terms ("Omicron") AND ("Disease severity") as well as ("Omicron") AND ("Viral load") And/ or ("Cell culture"). Our search yielded 3 main studies that directly assessed the omicrons clinical severity in South Africa, its infectious viral load compared to Delta, and the dynamics of viral RNA shedding. In South Africa, compared to Delta, Omicron infected patients showed a significant reduction in severe disease. In this study, Omicron and non-Omicron variants were characterized based on S gene target failure using the TaqPath COVID-19 PCR (Thermo Fisher Scientific). In the study from Switzerland that assessed the infectious viral load in Omicron versus Delta, the authors analyzed only 18 Omicron samples that were all from vaccinated individuals to show that compared to Delta, Omicron had equivalent infectious viral titers. The third study that assessed the Omicron viral dynamics showed that the peak viral RNA in Omicron infections is lower than Delta. No published studies assessed the clinical discrepancies of Omicron and Delta infected patients from the US, nor comprehensively assessed, by viral load and cell culture studies, the characteristics of both variants stratified by vaccination status. Added value of this studyTo the best of our knowledge, this is the only study to date to compare the clinical characteristics and outcomes after infection with the Omicron variant compared to Delta in the US using variants characterized by whole genome sequencing and a selective time frame when both variant co-circulated. It is also the first study to stratify the analysis based on the vaccination status and to compare fully vaccinated patients who didnt receive a booster vaccination to patients who received a booster vaccination. In addition, we provide a unique viral RNA and infectious virus load analyses to compare Delta and Omicron samples from unvaccinated, fully vaccinated, and patients with booster vaccination. Implications of all the available evidenceOmicron associated with a significant increase in infections in fully and booster vaccinated individuals but with less admissions and ICU level care. Admitted patients showed similar requirements for supplemental oxygen and ICU level care when compared to Delta admitted patients. Viral loads were similar in samples from Omicron and Delta infected patients regardless of the vaccination status. The recovery of infectious virus on cell culture was reduced in samples from patients infected with Delta who received a booster dose, but this was not the case with Omicron. The recovery of infectious virus was equivalent in Omicron infected unvaccinated, fully vaccinated, and samples from patients who received booster vaccination. FundingNIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, Centers for Disease Control and Prevention contract 75D30121C11061.

Longitudinal analysis of SARS-CoV-2 vaccine breakthrough infections reveal limited infectious virus shedding and restricted tissue distribution

The global effort to vaccinate people against SARS-CoV-2 in the midst of an ongoing pandemic has raised questions about the nature of vaccine breakthrough infections and the potential for vaccinated individuals to transmit the virus. These questions have become even more urgent as new variants of concern with enhanced transmissibility, such as Delta, continue to emerge. To shed light on how vaccine breakthrough infections compare with infections in immunologically naive individuals, we examined viral dynamics and infectious virus shedding through daily longitudinal sampling in a small cohort of adults infected with SARS-CoV-2 at varying stages of vaccination. The durations of both infectious virus shedding and symptoms were significantly reduced in vaccinated individuals compared with unvaccinated individuals. We also observed that breakthrough infections are associated with strong tissue compartmentalization and are only detectable in saliva in some cases. These data indicate that vaccination shortens the duration of time of high transmission potential, minimizes symptom duration, and may restrict tissue dissemination.

Mitigation of SARS-CoV-2 Transmission at a Large Public University

In the Fall of 2020, many universities saw extensive transmission of SARS-CoV-2 among their populations, threatening the health of students, faculty and staff, the viability of in-person instruction, and the health of surrounding communities.1, 2 Here we report that a multimodal "SHIELD: Target, Test, and Tell" program mitigated the spread of SARS-CoV-2 at a large public university, prevented community transmission, and allowed continuation of in-person classes amidst the pandemic. The program combines epidemiological modelling and surveillance (Target); fast and frequent testing using a novel and FDA Emergency Use Authorized low-cost and scalable saliva-based RT-qPCR assay for SARS-CoV-2 that bypasses RNA extraction, called covidSHIELD (Test); and digital tools that communicate test results, notify of potential exposures, and promote compliance with public health mandates (Tell). These elements were combined with masks, social distancing, and robust education efforts. In Fall 2020, we performed more than 1,000,000 covidSHIELD tests while keeping classrooms, laboratories, and many other university activities open. Generally, our case positivity rates remained less than 0.5%, we prevented transmission from our students to our faculty and staff, and data indicate that we had no spread in our classrooms or research laboratories. During this fall semester, we had zero COVID-19-related hospitalizations or deaths amongst our university community. We also prevented transmission from our university community to the surrounding Champaign County community. Our experience demonstrates that multimodal transmission mitigation programs can enable university communities to achieve such outcomes until widespread vaccination against COVID-19 is achieved, and provides a roadmap for how future pandemics can be addressed.

Daily sampling of early SARS-CoV-2 infection reveals substantial heterogeneity in infectiousness

The dynamics of SARS-CoV-2 replication and shedding in humans remain poorly understood. We captured the dynamics of infectious virus and viral RNA shedding during acute infection through daily longitudinal sampling of 60 individuals for up to 14 days. By fitting mechanistic models, we directly estimate viral reproduction and clearance rates, and overall infectiousness for each individual. Significant person-to-person variation in infectious virus shedding suggests that individual-level heterogeneity in viral dynamics contributes to superspreading. Viral genome load often peaked days earlier in saliva than in nasal swabs, indicating strong compartmentalization and suggesting that saliva may serve as a superior sampling site for early detection of infection. Viral loads and clearance kinetics of B.1.1.7 and non-B.1.1.7 viruses in nasal swabs were indistinguishable, however B.1.1.7 exhibited a significantly slower pre-peak growth rate in saliva. These results provide a high-resolution portrait of SARS-CoV-2 infection dynamics and implicate individual-level heterogeneity in infectiousness in superspreading.

SARS-CoV-2 Infections in mRNA Vaccinated Individuals are Biased for Viruses Encoding Spike E484K and Associated with Reduced Infectious Virus Loads that Correlate with Respiratory Antiviral IgG levels.

IntroductionCOVID-19 large scale immunization in the US has been associated with infrequent breakthrough positive molecular testing. Whether a positive test is associated with a high viral RNA load, specific viral variant, recovery of infectious virus, or symptomatic infection is largely not known. MethodsIn this study, we identified 133 SARS-CoV-2 positive patients who had received two doses of either Pfizer-BioNTech (BNT162b2) or Moderna (mRNA-1273) vaccines, the 2nd of which was received between January and April of 2021. The positive samples were collected between January and May of 2021 with a time that extended from 2 to 100 days after the second dose. Samples were sequenced to characterize the whole genome and Spike protein changes and cycle thresholds that reflect viral loads were determined using a single molecular assay. Local SARS-CoV-2 IgG antibodies were examined using ELISA and specimens were grown on cell culture to assess the recovery of infectious virus as compared to a control unvaccinated cohort from a matched time frame. ResultsOf 133 specimens, 24 failed sequencing and yielded a negative or very low viral load on the repeat PCR. Of 109 specimens that were used for further genome analysis, 68 (62.4%) were from symptomatic infections, 11 (10.1%) were admitted for COVID-19, and 2 (1.8%) required ICU admission with no associated mortality. The predominant virus variant was the alpha (B.1.1.7), however a significant association between lineage B.1.526 and amino acid change S: E484K with positives after vaccination was noted when genomes were compared to a large control cohort from a matched time frame. A significant reduction of the recovery of infectious virus on cell culture as well as delayed time to the first appearance of cytopathic effect was accompanied by an increase in local IgG levels in respiratory samples of vaccinated individuals but upper respiratory tract IgG levels were not different between symptomatic or asymptomatic infections. ConclusionsVaccination reduces the recovery of infectious virus in breakthrough infections accompanied by an increase in upper respiratory tract local immune responses. FundingNational Institute of Health (The Johns Hopkins Center of Excellence in Influenza Research and Surveillance, HHSN272201400007C), Johns Hopkins University, Maryland Department of Health, Centers for Disease Control and Prevention.

Longitudinal assessment of diagnostic test performance over the course of acute SARS-CoV-2 infection

What is already known about this topic?Diagnostic tests and sample types for SARS-CoV-2 vary in sensitivity across the infection period. What is added by this report?We show that both RTqPCR (from nasal swab and saliva) and the Quidel SARS Sofia FIA rapid antigen tests peak in sensitivity during the period in which live virus can be detected in nasal swabs, but that the sensitivity of RTqPCR tests rises more rapidly in the pre-infectious period. We also use empirical data to estimate the sensitivities of RTqPCR and antigen tests as a function of testing frequency. What are the implications for public health practice?RTqPCR tests will be more effective than rapid antigen tests at identifying infected individuals prior to or early during the infectious period and thus for minimizing forward transmission (provided results reporting is timely). All modalities, including rapid antigen tests, showed >94% sensitivity to detect infection if used at least twice per week. Regular surveillance/screening using rapid antigen tests 2-3 times per week can be an effective strategy to achieve high sensitivity (>95%) for identifying infected individuals.