SARS-CoV-2 Subgenomic N (sgN) Transcripts in Oro-Nasopharyngeal Swabs Correlate with the Highest Viral Load, as Evaluated by Five Different Molecular Methods

Abstract

The COVID-19 pandemic has forced diagnostic laboratories to focus on the early diagnostics of SARS-CoV-2. The positivity of a molecular test cannot respond to the question regarding the viral capability to replicate, spread, and give different clinical effects. Despite the fact that some targets are covered by commercially-available assays, the identification of new biomarkers is desired in order to improve the quality of the information given by these assays. Therefore, since the subgenomic transcripts (sgN and sgE) are considered markers of viral activity, we evaluated these subgenomic transcripts in relation to the genomic amplification obtained using five different commercial CE-IVD tools. Methods: Five CE-IVD kits were compared in terms of their capability to detect both synthetic SARS-CoV-2 viral constructs (spiked in TMB or PBS medium) and targets (N, E, RdRp and Orf1ab genes) in twenty COVID-19–positive patients’ swabs. The sgN and sgE were assayed by real-time RT-qPCR and digital PCR. Results: None of the diagnostic kits missed the viral target genes when they were applied to targets spiked in TMB or PBS (at dilutions ranging from 100 pg to 0.1 pg). Nevertheless, once they were applied to RNA extracted from the patients’ swabs, the superimposability ranged from 50% to 100%, regardless of the extraction procedure. The sgN RNA transcript was detected only in samples with a higher viral load (Ct ≤ 22.5), while sgE was within all of the Ct ranges. Conclusions: The five kits show variable performances depending on the assay layout. It is worthy of note that the detection of the sgN transcript is associated with a higher viral load, thus representing a new marker of early and more severe infection.

Impact of Full Vaccination with mRNA BNT162b2 on SARS-CoV-2 Infection: Genomic and Subgenomic Viral RNAs Detection in Nasopharyngeal Swab and Saliva of Health Care Workers

“…Whether persistent levels of sgRNA are actual proof of infectivity is still unclear; however, negative sgRNA can indicate that subjects are not infectious [ 22 ]. To date, previous studies reported evidence regarding the detection of viral sgRNAs using sequencing, RT-PCR and ddPCR, and all these findings were based on non-vaccinated cases [ 8 , 23 , 24 ]. The detection of viral sgRNA coupled with an increased viral load has been shown to be indicative of an aggravation of the disease [ 11 ].…”

“…In the context of COVID-19 vaccination campaigns, it is highly important to understand the base of evidence regarding vaccine effectiveness in preventing symptomatic and asymptomatic infections and transmission from infected individuals to susceptible contacts. Investigating both viral genomic and subgenomic RNAs in subjects infected with SARS-CoV-2 might be relevant for epidemiology purposes and infection control, with potential for clinical significance mainly related to the duration of the disease [ 24 ]. The detection of sgRNAs can provide useful data when combined with routine assays based on the detection of viral gRNA, particularly to assess the phase (early vs. late) of infection.…”

Long-chain polyphosphates impair SARS-CoV-2 infection and replication

“…Note also that transcription of the main SARS-CoV-2 sgRNAs was also impaired (e.g., sgN ). Thus, we suggest that polyPs reduce active viral replication ( 34 ). This antiviral action was also seen with mutations in the S gene, which enhanced viral infectivity and fitness; this further supports the potential for the use of polyPs to inhibit different emerging variants of SARS-CoV-2.…”

Case Report: Discovery a Novel SARS-CoV-2 Variant in a Six-Months Long-Term Swab Positive Female Suffering From Non-Hodgkin Lymphoma

“…All tests were performed within Lab n. 777777 of CEINGE Biotecnologie Avanzate S.C.a R.L., belonging to the CORONET Campania Regional network. Briefly, Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed by Allplex SARS-CoV-2 assay (Seegene, Seoul, Korea) as already reported by our group ( 9 ), according to manufacturer’s instructions, on the NSF deriving RNA of our patient. The study was approved by the Ethical Committee of the University Federico II of Naples (authorization n.180/20/ES1 on 25.05.2020) and was performed in compliance with the Declaration of Helsinki.…”