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

Abstract

Background: Circulating SARS-CoV-2 RNA may represent a more reliable indicator of infection than nasal RNA, but RT-qPCR lacks diagnostic sensitivity for blood samples. Methods: A 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 (NHP) after virus exposure; to evaluate the utility of blood SARS-CoV-2 RNA detection for 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. Results: CRISPR-augmented RT-PCR consistently detected SARS-CoV-2 RNA in the plasma of experimentally infected NHPs from 1 to 28 days post-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, while RT-qPCR exhibited 44.1% diagnostic sensitivity and 100% specificity for the same blood samples. This CRISPR-augmented RT-PCR assay also accurately identified COVID-19 patients with one or more negative nasal swab RT-qPCR result. Conclusion: Results 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.

Biologic correlates of beneficial convalescent plasma therapy in a COVID-19 patient reveal disease resolution mechanisms

“…Care of the patient required no organ biopsies, and we found no viral RNA in peripheral blood cells (not shown). Nevertheless, our data strongly point to RNAemia as a proxy for severe infection, as also proposed by others 22 , and perhaps as a tool to monitor viral clearance systemically to identify cases of post-acute viral persistence, which may potentially lead to protracted symptoms 55 .…”

Study of the Plasma and Buffy Coat in Patients with SARS-CoV-2 Infection—A Preliminary Report

“…In our study, we demonstrated the presence of viral components in 13 out of 24 patients (54.17%) by plasma RT-PCR. However, RT-qPCR has been reported to exhibit poor and highly variable diagnostic sensitivity (1–40%) when employed to detect SARS-CoV-2 RNA in blood samples from confirmed COVID-19 cases, with most positive samples exhibiting high Ct values indicative of a low viral RNA concentration [ 18 ]. More research is needed to shed light on the prognostic significance of plasma viral RNA in COVID-19 patients and its relationship with predictive factors.…”

The SARS-CoV-2/Receptor Axis in Heart and Blood Vessels: A Crisp Update on COVID-19 Disease with Cardiovascular Complications

“…The FXIIa is one of the main proteins of the contact system (CS), which is an innate inflammatory defense mechanism orchestrated against any negatively charged foreign or misfolded proteins, or nucleic acids such as DNA or RNA or microorganisms such as viruses, found in the intravascular niche [ 105 , 106 ]. This speculates that either the whole coronaviruses, which contains conserved negatively charged amino acids located within domain III in the carboxyl end of nucleocapsid proteins [ 107 ] or the SARS-CoV-2 RNA that are circulating in the bloodstream [ 108 ] are readily capable of inducing FXII-associated intrinsic coagulation pathway to form a pathological thrombus [ 104 ]. In fact, a pulmonary vessel wall isolated from COVID-19 positive lung tissues exhibited increased expression and activity of FXIIa [ 109 ], showing the commencement of the pathogenic pro-coagulant chain.…”