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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.
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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.
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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.
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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.
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BackgroundPrior observation has shown differences in COVID-19 hospitalization rates between SARS-CoV-2 variants, but limited information describes differences in hospitalization outcomes. MethodsPatients admitted to 5 hospitals with COVID-19 were included if they had hypoxia, tachypnea, tachycardia, or fever, and data to describe SARS-CoV-2 variant, either from whole genome sequencing, or inference when local surveillance showed [≥]95% dominance of a single variant. The average effect of SARS-CoV-2 variant on 14-day risk of severe disease, defined by need for advanced respiratory support, or death was evaluated using models weighted on propensity scores derived from baseline clinical features. ResultsSevere disease or death within 14 days occurred for 950 of 3,365 (28%) unvaccinated patients and 178 of 808 (22%) patients with history of vaccination or prior COVID-19. Among unvaccinated patients, the relative risk of 14-day severe disease or death for Delta variant compared to ancestral lineages was 1.34 (95% confidence interval [CI] 1.13-1.55). Compared to Delta variant, this risk for Omicron patients was 0.78 (95% CI 0.62-0.97) and compared to ancestral lineages was 1.04 (95% CI 0.84-1.24). Among Omicron and Delta infections, patients with history of vaccination or prior COVID-19 had one-half the 14-day risk of severe disease or death (adjusted hazard ratio 0.46, IQR 0.34-0.62) but no significant outcome difference between Delta and Omicron infections. ConclusionsAlthough the risk of severe disease or death for unvaccinated patients with Omicron was lower than Delta, it was similar to ancestral lineages. Severe outcomes were less common in vaccinated patients, but there was no difference between Delta and Omicron infections.
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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.
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BackgroundThe emerging SARS-CoV-2 variant of concern (VOC) B.1.6.17.2 (Delta) quickly displaced the B.1.1.7 (Alpha) and is associated with increases in COVID-19 cases nationally. The Delta variant has been associated with greater transmissibility and higher viral RNA loads in both unvaccinated and fully vaccinated individuals. Data is lacking regarding the infectious virus load in Delta infected individuals and how that compares to individuals infected with other SARS-CoV-2 lineages. MethodsWhole genome sequencing of 2,785 clinical isolates was used to characterize the prevalence of SARS-CoV-2 lineages circulating in the National Capital Region between January and July 2021. Clinical chart reviews were performed for the Delta, Alpha, and B.1.2 (a control predominant lineage prior to both VOCs) variants to evaluate disease severity and outcome and Cycle threshold values (Cts) were compared. The presence of infectious virus was determined using Vero-TMPRSS2 cells and anti-SARS-CoV-2 IgG levels were determined from upper respiratory specimen. An analysis of infection in unvaccinated and fully vaccinated populations was performed. ResultsThe Delta variant displaced the Alpha variant to constitute 88.2% of the circulating lineages in the National Capital Region by July, 2021. The Delta variant associated with increased breakthrough infections in fully vaccinated individuals that were mostly symptomatic when compared to the Alpha breakthrough infections, though it is important to note there was a significantly longer period of time between vaccination and infection with Delta infections. The recovery of infectious virus on cell culture was significantly higher with the Delta variant compared to Alpha in both vaccinated and unvaccinated groups. The impact of vaccination on reducing the recovery of infectious virus from clinical samples was only observed with Alpha variant infections but was strongly associated with low localized SARS-CoV-2 IgG for both variants. A comparison of Ct values showed a significant decrease in the Delta compared to Alpha with no significant differences between unvaccinated and vaccinated groups. ConclusionsOur data indicate that the Delta variant is associated with increased infectious virus loads when compared to the Alpha variant and decreased upper respiratory antiviral IgG levels. Measures to reduce transmission in addition to increasing vaccinations rates have to be implemented to reduce Delta variant spread. 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.
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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.
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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.