Viral Culture Confirmed SARS-CoV-2 Subgenomic RNA Value as a Good Surrogate Marker of Infectivity

Abstract

Determining SARS-CoV-2 viral infectivity is crucial for patient clinical assessment and isolation decisions. We assessed subgenomic RNA (sgRNA) as a surrogate marker of SARS-CoV-2 infectivity in SARS-CoV-2-positive reverse transcription PCR (RT-PCR) respiratory samples ( n = 105) in comparison with viral culture as the reference standard for virus replication. sgRNA and viral isolation results were concordant in 99/105 cases (94%), indicating highly significant agreement between the two techniques (Cohen’s kappa coefficient 0.88, 95% confidence interval [CI] 0.78 to 0.97, P < 0.001). sgRNA RT-PCR showed a sensitivity of 97% and a positive predictive value of 94% to detect replication-competent virus, further supporting sgRNA as a surrogate marker of SARS-CoV-2 infectivity. sgRNA RT-PCR is an accurate, rapid, and affordable technique that can overcome culture and cycle threshold ( C T ) value limitations and be routinely implemented in hospital laboratories to detect viral infectivity, which is essential for optimizing patient monitoring, the efficacy of treatments/vaccines, and work reincorporation policies, as well as for safely shortening isolation precautions.

SARS-CoV-2 Antigen Test Results to Infer Active or Non-Active Virus Replication Status in COVID-19 Patients

“…Previous studies’ findings supported us to adopt SARS-CoV-2 sgRNA RT-PCR as the reference method for our antigen tests’ assessment [ 6 , 15 , 16 , 17 ]. One study [ 15 ] using respiratory tract samples obtained from COVID-19 patients within 7 days of symptom onset showed the correlation between the SARS-CoV-2 antigen (using the Becton Dickinson BD Veritor System for Rapid Detection of SARS-CoV-2 test) and SARS-CoV-2 culture (using a VeroE6TMPRSS2 cell line-based high-sensitive culture test) positivity.…”

“…Another study [ 16 ] showed that three lateral flow assays and a (microfluidic) immunofluorescence assay correlated with cell-culture infectivity more significantly than SARS-CoV-2 gRNA RT-PCR. Finally, in Santos Bravo et al’s study [ 6 ], sgRNA RT-PCR proved to detect replication-competent SARS-CoV-2 in COVID-19 patients’ NPS or other respiratory samples with a sensitivity of 97% and a positive predictive value of 94% compared to SARS-CoV-2 culture. However, to comply with the assumption that culture may be suboptimal to detect the presence of the infectious virus due to poor sensitivity [ 2 ], we used a highly sensitive RT-PCR method (i.e., the sgRNA detection assay) to assess antigen assays in our study.…”

“…However, to comply with the assumption that culture may be suboptimal to detect the presence of the infectious virus due to poor sensitivity [ 2 ], we used a highly sensitive RT-PCR method (i.e., the sgRNA detection assay) to assess antigen assays in our study. Similar to us, Santos Bravo et al [ 6 ] chose to measure E-gene sgRNA, i.e., the sgRNA species known as the best option to detect infectivity so far [ 2 ].…”

“…Reverse transcription-PCR (RT-PCR)-based molecular assays offer a reliable means of detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic RNA (gRNA) in respiratory tract samples from patients who develop coronavirus disease 2019 (COVID-19) within 1 to 2 weeks of SARS-CoV-2 exposure [ 1 ]. Whereas there is no established RT-PCR cycle threshold (Ct) value at which individuals are likely to no longer be infectious [ 2 ], growing evidence shows that RT-PCR detection of SARS-CoV-2 subgenomic RNA (sgRNA), as with viral culture (i.e., the currently accepted reference standard for virus replication) [ 3 ], may be reliably used to measure infectivity [ 4 , 5 , 6 ]. Unlike diagnostic (gRNA-detecting) RT-PCR, detection of the SARS-CoV-2 antigen, which is per se an indicator of active infection, offers an accurate but less sensitive means of identifying acutely infected individuals [ 7 ] and, therefore, the possibility to identify individuals who are shedding the infectious virus and are likely to transmit SARS-CoV-2 [ 2 ].…”

SARS-CoV-2 Virus Culture, Genomic and Subgenomic RNA Load, and Rapid Antigen Test in Experimentally Infected Syrian Hamsters

“…Subgenomic RNA (sgRNA) is an intermediate product of SARS-CoV-2 replication and transcription, and its presence indicates active SARS-CoV-2 replication in patients. The abundance of sgRNA in samples is closely related to the infectivity of SARS-CoV-2 ( 8 ) and may be used as diagnostic index for ending COVID-19 patient isolation. Rapid antigen-based testing is widely used for SARS-CoV-2 screening and diagnostics because it is readily available, is inexpensive, and returns results quickly.…”