Quantification of SARS-CoV-2 viral load in inpatients

Due to the rapid spread of Severe Acute Respiratory Syndrome Virus Type 2 (SARS-CoV-2) worldwide, sensitive and specific diagnostic kits are being rapidly developed. Primarily, these test kits are based on the reverse transcriptase-polymerase chain reaction (RT-PCR) for detecting total viral ribonucleic acid (RNA). One limitation of RT-PCR testing is that as the infection progresses over time, the relationship between positive RT-PCR and viral infectivity becomes less pronounced.

Transmission electron micrograph of SARS-CoV-2 virus particles (UK B.1.1.7 variant) isolated from patient samples and cultured in cell culture. Image taken at the NIAID Integrated Research Facility (IRF) in Fort Detrick, Maryland. Credit: NIAID

Previous studies have shown that SARS-CoV-2 remains in the upper respiratory tract for approximately 14.5 days after the onset of disease symptoms. However, many studies have reported the presence of viral RNA weeks, long before most people were infected. For this reason, the US Centers for Disease Control and Prevention (CDC) has created new guidelines for isolation prevention measures.

According to previous guidelines, the quarantine period for mild to moderately infected individuals was 10 days after the onset of symptoms, based on studies of viral infectivity in clinical samples. For severely infected patients or immunocompromised conditions, quarantine for up to 20 days was recommended. However, current studies show that some immunosuppressed patients may remain contagious for several weeks, regardless of their symptoms. This observation makes it difficult to formulate a strategy for isolation based on the symptoms of the disease.

It is difficult to stop the spread of the infection because there is no fast, high-throughput testing system to distinguish between sick and healthy people. The methods available to determine infectivity are time consuming and impractical. As a result, scientists are interested in discovering molecular markers that may correlate with infectivity. With this excuse, they used SARS-CoV-2 total RNA (positive and negative strands) and subgenomic RNA (nucleocapsid (N), spike (S), envelope (E), membrane (M), and six accessory proteins). I studied. ). Subgenomic RNA can be distinguished from total RNA by placing alternative PCR primers. Evaluation of subgenomic RNA dynamics during infection revealed the absence of subgenomic RNA in the throat of SARS-CoV-2 patients up to 5 days after the onset of symptoms. Little information is available on subgenomic RNA kinetics in inpatients and immunosuppressed patients with severe COVID-19 infection.

New paper published in medRxiv* Preprint server, investigating the role of subgenomic RNA transcripts in SARS-CoV-2 infection. Previously, scientists believed that subgenomic RNA could be a traceable infectious marker for monitoring infectivity. In the current study, RT-PCR was used to amplify whole and subgenome nucleoprotein genes (N) and envelope genes (E). Nasopharyngeal samples were collected from 190 patients admitted in Michigan between March 13, 2020 and June 10, 2020.

Researchers have observed a strong correlation between total RNA and subgenomic RNA. They found that the time points after the onset of symptoms were the same, and that the reduction in infection rate was the same for both total RNA and subgenomic RNA. The predictability of subgenomic copy numbers from total copy numbers indicates that recognition of subgenomic RNA does not add additional information about infectivity beyond that obtained from total copy numbers alone. As a result, scientists conclude that there is no additional benefit in assessing subgenomic RNA.

In this study, scientists found that negative subgenome E RT-PCR was positively correlated with the time the patient was uninfected. Therefore, subgenome E is not a valid marker for predicting the infectivity of the virus. The study also found that the median number of days from the onset of symptoms to the negative subgenome NRT-PCR was 25 days. Therefore, the use of subgenome N as a marker of infectivity significantly prolongs quarantine and is unpredictable. This difference may be due to the higher expression levels of subgenome N compared to subgenome E.

Comparison of cycle thresholds and days since symptom onset of clinical samples from 185 inpatients. Total N (Panel A), Subgenome N (Panel B), Total E (Panel C), and Subgenome E (Panel D). Red dots on panels A and C represent negative samples of the subgenome, and black dots represent positive samples of the subgenome. Of the 185 patients, 56 were negative for sgE and 28 were negative for sgN (shown on the y-axis). Pearson correlation coefficient: N = -0.404 p <0.0001; SgN = -0.466, p <0.0001; E = -0.456 p <0.0001; sgE = -0.427 <0.0001. The linear regression linear equation is shown in each panel.

Previous studies have shown that subgenomic RNA is a good marker for active infection because it degrades more rapidly than total RNA without replication. This result was inconsistent with the results obtained in the current study. The difference in this result is not due to the difference in degradation of the subgenome transcript and the whole transcript, but due to the difference in the detection of the transcript.

Studies conclude that determination of infectivity by evaluation of subgenomic RNA is not recommended. Similarly, for non-immunosuppressed patients, the total RNA copy number threshold provides the same information that can be obtained from total RNA copy number. One limitation of current research is that scientists have not isolated the virus from hospital samples and have not correlated it with total copy number or subgenome copy number. Only the correlation between transcripts and symptom duration has been used as a determinant of infectivity.

*Important Notices

medRxiv Publish preliminary scientific reports that should not be considered definitive as they are not peer-reviewed, guide clinical practice / health-related behaviors, and should not be treated as established information.