Sensitive tracking of circulating viral RNA through all stages of SARS-CoV-2 infection

Abstract

BACKGROUNDCirculating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA may represent a more reliable indicator of infection than nasal RNA, but quantitative reverse transcription PCR (RT-qPCR) lacks diagnostic sensitivity for blood samples.METHODSA CRISPR-augmented RT-PCR assay that sensitively detects SARS-CoV-2 RNA was employed to analyze viral RNA kinetics in longitudinal plasma samples from nonhuman primates (NHPs) after virus exposure; to evaluate the utility of blood SARS-CoV-2 RNA detection for coronavirus disease 2019 (COVID-19) diagnosis in adults cases confirmed by nasal/nasopharyngeal swab RT-PCR results; and to identify suspected COVID-19 cases in pediatric and at-risk adult populations with negative nasal swab RT-qPCR results. All blood samples were analyzed by RT-qPCR to allow direct comparisons.RESULTSCRISPR-augmented RT-PCR consistently detected SARS-CoV-2 RNA in the plasma of experimentally infected NHPs from 1 to 28 days after infection, and these increases preceded and correlated with rectal swab viral RNA increases. In a patient cohort (n = 159), this blood-based assay demonstrated 91.2% diagnostic sensitivity and 99.2% diagnostic specificity versus a comparator RT-qPCR nasal/nasopharyngeal test, whereas RT-qPCR exhibited 44.1% diagnostic sensitivity and 100% specificity for the same blood samples. This CRISPR-augmented RT-PCR assay also accurately identified patients with COVID-19 using one or more negative nasal swab RT-qPCR results.CONCLUSIONResults of this study indicate that sensitive detection of SARS-CoV-2 RNA in blood by CRISPR-augmented RT-PCR permits accurate COVID-19 diagnosis, and can detect COVID-19 cases with transient or negative nasal swab RT-qPCR results, suggesting that this approach could improve COVID-19 diagnosis and the evaluation of SARS-CoV-2 infection clearance, and predict the severity of infection.TRIAL REGISTRATIONClinicalTrials.gov. NCT04358211.FUNDINGDepartment of Defense, National Institute of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the National Center for Research Resources.

Keywords: COVID-19; Diagnostics; Molecular diagnosis.

Figure 1. Flow diagram describing the numbers and disposition of the study subjects.

Figure 2. Analytical validation of the CRISPR-ABC assay.

(A) CRISPR-ABC assay schematic. A SARS-CoV-2 ORF1ab target amplified from plasma RNA is quantified by comparing target- and CRISPR-mediated probe cleavage against that produced by a standard curve generated by RT-PCR of SARS-CoV-2 ORF1ab RNA samples of known concentration. (B) CRISPR-ABC signal in positive control (PC; 10⁴ copy/μL) and no template control (NTC; nuclease-free water) samples. (C) CRISPR-ABC specificity with healthy human plasma spiked with or without indicated virus RNA or virions. (D) Limit of detection and (E) linear range of the assay. Shading denotes the 95% confidence interval of the fitted line. (F) CRISPR-ABC reproducibility for replicate plasma samples spiked with 0 to 1 copy/μL of inactivated SARS-CoV-2 virus. Graphs present the mean ± SD of 3 technical replicates for each sample. ****P < 0.0001 for a difference between the zero concentration sample and all other groups by 1-way ANOVA adjusted for multiple comparisons.

Figure 3. CRISPR-ABC analysis of samples from SARS-CoV-2-infected NHPs.

(A) Sample collection timeline (plasma and nasal and rectal swabs) versus SARS-CoV-2 infection. (B) CRISPR-ABC signal at the indicated sample time points. Shading indicates the 95% confidence interval of the fitted line. (C) CRISPR-ABC signal for samples from individual NHPs at indicated time points. SARS-CoV-2 RNA abundance is expressed as the relative photoluminescence (PL) intensity of the sample, since most samples had values below the LoQ of the CRISPR-ABC assay (Supplemental Data 1). Dotted lines indicate the positive result threshold. Data represent mean ± SD of 3 technical replicates for each sample.

Figure 4. Blood CRISPR-ABC results of adult COVID-19 cases.

(A) CRISPR-ABC signal in baseline blood samples of 34 adults with COVID-19 diagnosed by nasal or nasopharyngeal RT-qPCR and 125 archived blood samples collected before the COVID-19 pandemic. (B) SARS-CoV-2 RNA copy number in the 34 COVID-19 subjects. (C) Comparison of CRISPR-ABC signal values of blood samples from hospitalized (n = 25) and nonhospitalized (n = 9) patients with COVID-19 by a general linear model analysis adjusted for age. A and C are box plots with maximum, Q3, median, Q1, and minimum value of PL intensity of different groups. Dotted lines indicate the positive result threshold. Dashed lines in B indicate the linear range and LoQ and LoD of the CRISPR-ABC assay. All samples were analyzed in triplicate. ****P < 0.0001 by Mann-Whitney U test; ***P < 0.001 by general linear model analysis, adjusting for age and symptom duration differences between these groups.

Figure 5. Plasma CRISPR-ABC results of pediatric cases.

(A) Positive (red) and negative (blue) results for paired nasal swab RT-qPCR and plasma CRISPR-ABC assays of 32 children screened for COVID-19. (B–D) Positive (red) and negative (blue) results for COVID-19 plasma CRISPR-ABC, nasal swab RT-qPCR, and serological results at the indicated time points after first evaluation. Dashed lines indicate the positive result threshold. Data indicate the mean ± SD of 3 technical replicates.

Figure 6. CRISPR-ABC plasma results for symptomatic adults with negative RT-qPCR results.

Case history summaries for 2 of 5 patients with 1 or more negative nasal swab RT-qPCR result. (A) Case history for a symptomatic patient with CT scan results consistent with COVID-19 who had multiple RT-qPCR negative results by nasal swab, but had a CRISPR-ABC positive plasma sample upon retroactive testing and improved after receiving COVID-19 convalescent plasma, consistent with a COVID-19 diagnosis. (B) Case history for a patient with symptoms and CT scan results consistent with COVID-19 who had negative RT-qPCR and CRISPR-ABC test results but subsequently improved after receiving enhanced antibiotic and antifungal treatment and was determined not to have had COVID-19. Red arrows on CT scan images denote COVID-19-associated “ground glass” opacity regions. The CRISPR-ABC results present the mean ± SD of 3 technical replicates for each sample.