Informative Use of Cycle-Threshold Values to Account for Sampling Variability in Pathogen Detection (preprint)

Nucleic acid amplification tests, like real-time polymerase chain reaction, are widely used for pathogen detection; however, their interpretation rarely accounts for sampling variability. Instead, cycle threshold values are often categorized reducing precision. We describe how pathogen cycle threshold values can be normalized to endogenous host gene expression to correct for sampling variability and compare the validity of this approach to standardization with a standard curve. Normalization serves as a valid alternative to standardization, does not require making a standard curve, increases precision, accounts for sampling variability, and can be easily applied to large clinical or surveillance datasets for informative interpretation.

Nasopharyngeal Angiotensin Converting Enzyme 2 (ACE2) Expression as a Risk-Factor for SARS-CoV-2 Transmission in Concurrent Hospital Associated Outbreaks (preprint)

Background: Widespread human-to-human transmission of the severe acute respiratory syndrome coronavirus two (SARS-CoV-2) stems from a strong affinity for the cellular receptor angiotensin converting enzyme two (ACE2). We investigate the relationship between a patient's nasopharyngeal ACE2 transcription and secondary transmission within a series of concurrent hospital associated SARS-CoV-2 outbreaks in British Columbia, Canada. Methods: Epidemiological case data from the outbreak investigations was merged with public health laboratory records and viral lineage calls, from whole genome sequencing, to reconstruct the concurrent outbreaks using infection tracing transmission network analysis. ACE2 transcription and RNA viral load were measured by quantitative real-time polymerase chain reaction. The transmission network was resolved to calculate the number of potential secondary cases. Bivariate and multivariable analyses using Poisson and Negative Binomial regression models was performed to estimate the association between ACE2 transcription the number of SARS-CoV-2 secondary cases. Results: The infection tracing transmission network provided n = 76 potential transmission events across n = 103 cases. Bivariate comparisons found that on average ACE2 transcription did not differ between patients and healthcare workers (P = 0.86). High ACE2 transcription was observed in 98.6% of transmission events, either the primary or secondary case had above average ACE2. Multivariable analysis found that the association between ACE2 transcription and the number of secondary transmission events differs between patients and healthcare workers. In health care workers Negative Binomial regression estimated that a one unit change in ACE2 transcription decreases the number of secondary cases (B = -0.132 (95%CI: -0.255 to -0.0181) adjusting for RNA viral load. Conversely, in patients a one unit change in ACE2 transcription increases the number of secondary cases (B = 0.187 (95% CI: 0.0101 to 0.370) adjusting for RNA viral load. Sensitivity analysis found no significant relationship between ACE2 and secondary transmission in health care workers and confirmed the positive association among patients. Conclusion: Our study suggests that ACE2 transcription has a positive association with SARS-CoV-2 secondary transmission in admitted inpatients, but not health care workers in concurrent hospital associated outbreaks, and it should be further investigated as a risk-factor for viral transmission.

Prediction of SARS-CoV-2 transmission dynamics based on population-level cycle threshold values: A Machine learning and mechanistic modeling study (preprint)

Background: Polymerase chain reaction (PCR) cycle threshold (Ct) values can be used to estimate the viral burden of Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) and predict population-level epidemic trends. We investigated the use of machine learning (ML) and epidemic transmission modeling based on Ct value distribution for SARS-CoV-2 incidence prediction during an Omicron-predominant period. Methods: Using simulated data, we developed a ML model to predict the reproductive number based on Ct value distribution, and validated it on out-of-sample province-level data. We also developed an epidemiological model and fitted it to province-level data to accurately predict incidence. Results: Based on simulated data, the ML model predicted the reproductive number with highest performance on out-of-sample province-level data. The epidemiological model was validated on outbreak data, and fitted to province-level data, and accurately predicted incidence. Conclusions: These modeling approaches can complement traditional surveillance, especially when diagnostic testing practices change over time. The models can be tailored to different epidemiological settings and used in real time to guide public health interventions.

Characterizing Longitudinal Antibody Responses in Recovered Individuals Following COVID-19 Infection and Single-Dose Vaccination in British Columbia, Canada: a Prospective Cohort Study (preprint)

Background: Investigating antibody titres in individuals who have been both naturally infected with SARS-CoV-2 and vaccinated can provide insight into antibody dynamics and correlates of protection over time. Methods: Human coronavirus (HCoV) IgG antibodies were measured longitudinally in a prospective cohort of PCR-confirmed, COVID-19 recovered individuals (k=57) in British Columbia pre- and post-vaccination. SARS-CoV-2 and endemic HCoV antibodies were measured in serum collected between Nov. 2020 and Sept. 2021 (n=341). Primary analysis used a linear mixed-effects model to understand the effect of single dose vaccination on antibody concentrations adjusting for biological sex, age, time from infection and vaccination. Secondary analysis investigated the cumulative incidence of high SARS-CoV-2 anti-spike IgG seroreactivity equal to or greater than 5.5 log10 AU/mL up to 105 days post-vaccination. No re-infections were detected in vaccinated participants, post-vaccination by qRT-PCR performed on self-collected nasopharyngeal specimens. Results: Bivariate analysis (complete data for 42 participants, 270 samples over 472 days) found SARS-CoV-2 spike and RBD antibodies increased 14-56 days post-vaccination (p<0.001) and vaccination prevented waning (B=1.66 [95%CI: 1.45-3.46]); while decline of nucleocapsid antibodies over time was observed (B=-0.24 [95%CI: -1.2-(-0.12)]). A non-significant trend towards higher spike antibodies against endemic beta-HCoVs was also noted. On average, SARS-CoV-2 anti-spike IgG concentration increased in participants who received one vaccine dose by 2.06 log10 AU/mL (95%CI: 1.45-3.46) adjusting for age, biological sex, and time. Cumulative incidence of high SARS-CoV-2 spike antibodies (>5.5 log10 AU/mL) was 83% greater in vaccinated compared to unvaccinated individuals. Conclusions: Our study confirms that vaccination post-SARS-CoV-2 infection provides multiple benefits, such as increasing anti-spike IgG titers and preventing decay up to 85 days post-vaccination.

Determining the optimal SARS-CoV-2 mRNA vaccine dosing interval for maximum immunogenicity (preprint)

Objective: Emerging evidence indicates that longer SARS-CoV-2 vaccine dosing intervals results in an enhanced immune response. However, the optimal vaccine dosing interval for achieving maximum immunogenicity is unclear. Methods: This study included samples from adult paramedics in Canada who received two doses of either BNT162b2 or mRNA-1273 vaccines and provided blood samples 6 months (170 to 190 days) after the first vaccine dose.The main exposure variable was vaccine dosing interval (days), categorized as "short" (first quartile), "moderate" (second quartile), "long" (third quartile), and "longest" interval (fourth quartile). The primary outcome was total spike antibody concentrations, measured using the Elecsys SARS-CoV-2 total antibody assay. Secondary outcomes included: spike and RBD IgG antibody concentrations, and inhibition of angiotensin-converting enzyme 2 (ACE-2) binding to wild-type spike protein and several different Delta variant spike proteins. We fit a multiple log-linear regression model to investigate the association between vaccine dosing intervals and the antibody concentrations. Results: A total of 564 adult paramedics (mean age 40 years, SD=10) were included. Compared to "short interval" ([≤]30 days), higher dosing interval quartiles (moderate: 31-38 days; long: 39-73 days and longest: [≥]74 days) were all associated with increased Elescys spike total antibody concentration. Compared to the short interval, "long" and "longest" interval quartiles were associated with higher spike and RBD IgG antibody concentrations. Similarly, increasing dosing intervals increased inhibition of ACE-2 binding to viral spike protein, regardless of the vaccine type. Conclusion: Increased mRNA vaccine dosing intervals longer than 30 days result in higher levels of circulating antibodies and viral neutralization when assessed at 6 months.

Performance of Immunoglobulin G Serology on Finger Prick Capillary Dried Blood Spot Samples to Measure SARS-CoV-2 Humoral Immunogenicity (preprint)

Abstract: Importance: Measuring humoral immunogenicity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines and finding population-level correlates of protection against coronavirus disease (COVID-19) presents an immediate challenge to public health practitioners. Objective: To study the diagnostic accuracy and predictive value of finger prick capillary dried blood spot (DBS) samples tested using an anti-immunoglobulin G (IgG) serology assay to measure SARS-CoV-2 seropositivity and the humoral immunogenicity of COVID-19 vaccination. Design, Setting and Participants: This cross-sectional study enrolled participants (n= 644) who had paired DBS and serum samples collected by finger prick and venipuncture, respectively, in British Columbia, Canada between January 12th, 2020 and May 21st, 2021. Samples were tested by a multiplex electrochemiluminescence assay for SARS-CoV-2 anti-Spike (S), -Nucleocapsid (N) and -receptor binding domain (RBD) IgG reactivity using a Meso Scale Discovery (MSD) platform. Additionally, unpaired DBS samples (n= 6,706) that were collected in the province during the same time period were included for analysis of SARS-CoV-2 anti-N IgG reactivity. Exposure: Collection of a capillary DBS by finger prick alone or paired with serum by venipuncture. Outcome: Humoral immune response to SARS-CoV-2 measured by detection of anti-S, -N or -RBD IgG. Results: In comparison to a paired-serum reference, DBS samples possessed a sensitivity of 80% (95% CI: 61%-91%) and specificity of 97% (95% CI: 95%-98%). Receiver operator characteristic curve analysis (ROC) found that participant DBS samples tested for anti-SARS-CoV-2 IgG by MSD V-PLEX COVID-19 Coronavirus Panel 2 assay accurately classify SARS-CoV-2 seroconversion at an 88% percent rate, AUC= 88% (95% CI: 81%-96%). Modelling found that a DBS-based testing approach has a high positive predictive value (PPV) (98% [95% CI: 98%-99%]) in a theoretical population with seventy-five percent COVID-19 vaccine coverage. At lower vaccine coverages of fifteen and forty-five percent, the test's PPV decreased and the negative predictive value increased. Conclusion: We demonstrate that DBS samples, when tested using an electrochemiluminescence assay, provide a valid alternative to traditional venipuncture and should be considered to reliably detect SARS-CoV-2 seropositivity.

Single-dose mRNA vaccine effectiveness against SARS-CoV-2, including P.1 and B.1.1.7 variants: a test-negative design in adults 70 years and older in British Columbia, Canada (preprint)

Introduction: Randomized-controlled trials of mRNA vaccine protection against SARS-CoV-2 included relatively few elderly participants. We assess singe-dose mRNA vaccine effectiveness (VE) in adults [≥]70-years-old in British Columbia (BC), Canada where the second dose was deferred by up to 16 weeks and where a spring 2021 wave uniquely included co-dominant circulation of B.1.1.7 and P.1 variants of concern (VOC). Methods: Analyses included community-dwelling adults [≥]70-years-old with specimen collection between April 4 (epidemiological week 14) and May 1 (week 17). Adjusted VE was estimated by test-negative design through provincial laboratory and immunization data linkage. Cases were RT-PCR test-positive for SARS-CoV-2 and controls were test-negative. Vaccine status was defined by receipt of a single-dose [≥]21 days before specimen collection, but a range of intervals was assessed. In variant-specific analyses, test-positive cases were restricted to those genetically-characterized as B.1.1.7, P.1 or non-VOC. Results: VE analyses included 16,993 specimens: 1,226 (7.2%) test-positive cases and 15,767 test-negative controls. Of 1,131 (92%) viruses genetically categorized, 509 (45%), 314 (28%) and 276 (24%) were B.1.1.7, P.1 and non-VOC lineages, respectively. VE was negligible at 14% (95% CI 0-26) during the period 0-13 days post-vaccination but increased from 43% (95% CI 30-53) at 14-20 days to 75% (95% CI 63-83) at 35-41 days post-vaccination. VE at [≥]21 days was 65% (95% CI 58-71) overall: 72% (95% CI 58-81), 67% (95% CI 57-75) and 61% (95% CI 45-72) for non-VOC, B.1.1.7 and P.1, respectively. Conclusions: A single dose of mRNA vaccine reduced the risk of SARS-CoV-2 in adults [≥]70-years-old by about two-thirds, with protection only minimally reduced against B.1.1.7 and P.1 variants. Substantial single-dose protection in older adults reinforces the option to defer the second dose when vaccine supply is scarce and broader first-dose coverage is needed.

Mutations in emerging variant of concern lineages disrupt genomic sequencing of SARS-CoV-2 clinical specimens (preprint)

Mutations in emerging SARS-CoV-2 lineages can interfere with the laboratory methods used to generate high-quality genome sequences for COVID-19 surveillance. Here, we identify 46 mutations in current variant of concern lineages affecting the widely used laboratory protocols for SARS-CoV-2 genomic sequencing by Freed et al. and the ARTIC network. We provide laboratory data showing how three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada, and we also demonstrate how we modified the Freed et al. protocol to restore performance.

The Contrasting Role of Nasopharyngeal Angiotensin Converting Enzyme 2 (ACE2) Expression in SARS-CoV-2 Infection: A Cross-Sectional Study of People Tested for COVID-19 in British Columbia (preprint)

BackgroundAngiotensin converting enzyme 2 (ACE2) serves as the host receptor for SARS-CoV-2, with a critical role in viral infection. We aim to understand population level variation of nasopharyngeal ACE2 expression in people tested for COVID-19 and the relationship between ACE2 expression and SARS-CoV-2 viral RNA load, while adjusting for expression of the complementary protease, Transmembrane serine protease 2 (TMPRSS2), soluble ACE2, age, and biological sex. MethodsA cross-sectional study of n=424 participants aged 1-104 years referred for COVID-19 testing was performed in British Columbia, Canada. Participants who tested negative or positive for COVID-19 were matched by age and biological sex. Viral and host gene expression was measured by quantitative reverse-transcriptase polymerase chain reaction. Bivariate analysis and multiple linear regression were performed to understand the role of nasopharyngeal ACE2 expression in SARS-CoV-2 infection. The ACE2 gene was targeted to measure expression of transmembrane and soluble transcripts. FindingsAnalysis shows no association between age and nasopharyngeal ACE2 expression in those who tested negative for COVID-19 (P=0{middle dot}092). Mean expression of transmembrane (P=1{middle dot}2e-4), soluble ACE2 (P<0{middle dot}0001) and TMPRSS2 (P<0{middle dot}0001) differed between COVID-19-negative and -positive groups. In bivariate analysis of COVID-19-positive participants, expression of transmembrane ACE2 positively correlated with SARS-CoV-2 RNA viral load (P<0{middle dot}0001), expression of soluble ACE2 negatively correlated (P<0{middle dot}0001), and no correlation was found with TMPRSS2 (P=0{middle dot}694). Multivariable analysis showed that the greatest viral RNA loads were observed in participants with high transmembrane ACE2 expression (B=0{middle dot}886, 95%CI:[0{middle dot}596 to 1{middle dot}18]), while expression of soluble ACE2 may protect against high viral RNA load in the upper respiratory tract (B= -0{middle dot}0990, 95%CI:[-0{middle dot}176 to -0{middle dot}0224]). InterpretationNasopharyngeal ACE2 expression plays a dual, contrasting role in SARS-CoV-2 infection of the upper respiratory tract. Transmembrane ACE2 positively correlates, while soluble ACE2 negatively correlates with viral RNA load after adjusting for age, biological sex and expression of TMPRSS2. FundingThis project (COV-55) was funded by Genome British Columbia as part of their COVID-19 rapid response initiative.

Antibody reactivity to SARS-CoV-2 in adults from the Vancouver metropolitan area, Canada (preprint)

Background: Quantifying antibody reactivity to SARS-CoV-2 antigens may help understand its effect on COVID-19 severity at the population level. This antibody reactivity may be particularly prevalent among childcare providers, including pediatric health care workers (HCW) who may be more exposed to circulating coronaviruses. Methods: Cross-sectional study that included adults in the Vancouver area in British Columbia (BC), Canada, between May 17 and June 19, 2020. A novel 10-plex antibody assay (IgG) was used to measure antibody reactivity against the spike protein from circulating coronaviruses (229E, NL63, OC43, and HKU1), SARS-CoV, and four SARS-CoV-2 antigens. Seroreactivity from previous viral exposure was ascertained using this assay, and by measuring total SARS-CoV-2 IgG/M/A antibodies against a recombinant spike (S1) protein using a commercial CLIA assay. Findings: Among 276 participants (71% HCW), three showed evidence of direct viral exposure, yielding an adjusted seroprevalence of 0.6% [95%CI 0.2 to 3.1%], with no difference between HCW and non-HCW, or between paediatric and adult HCW. Among the remaining 273 unexposed individuals, 7.3% [95%CI 4.5% to 11.1%], 48.7 [95%CI 42.7% to 54.8%] and 82.4% [95%CI 77.4% to 86.7%] showed antibody reactivity against SARS-CoV-2 RBD, N or Spike proteins, respectively. This reactivity was evenly distributed as a function of age, sex or between paediatric and adult HCW, and partly correlated with reactivity to circulating coronaviruses (Spearman; range: 0.147 to 0.513 for significant correlation after false-discovery rate adjustment at 5%). Interpretation: A substantial proportion of individuals in this population showed antibody reactivity against SARS-CoV-2 antigens despite low serological evidence of SARS-CoV-2 exposure.