Quantitative detection of SARS‐CoV‐2 RNA in nasopharyngeal samples from infected patients with mild disease

Abstract

Diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS‐COV‐2) cases is based on the count of real‐time reverse transcription‐plymerase chain reaction (RT‐PCR) positive people. Viral load by real‐time RT‐PCR has been suggested as a biomarker of the SARS‐CoV‐2 infection. However, the association of viral load and severity of the disease is not yet resolved. Nasopharyngeal samples from 458 patients were tested by RT‐PCR for SARS‐CoV‐2 diagnosis. Relative quantitation was made by the comparative threshold cycle (ΔΔCt) formula between ORF1ab viral and RNase P housekeeping genes. Absolute viral load was calculate using a reference positive control. Most prevalent clinical signs were cough (75.8%), myalgia (66.7%), and fever (48.5%). Hypertension (18.2%), neurological diseases (15.1%), and asthma and hypothyroidism (12.1%) were most frequent comorbidities. Fever, either as an exclusive symptom or combined with others, was associated with high viral loads ( 2 ‐ ∆ ∆ C t range, 35.65–155.16; 4.25–4.89 log10 RNA copies/test]). During the first week after onset of symptoms in mild patients up to 60 years‐old was detected the peak of viral load. Children under 10 years old have a high viral load (313.84; 2.50) in the first 2 days postinfection with a sharp decline thereafter. Cases between 10 and 49 years old mostly showed low and moderate viral load during the first 2 days postinfection (range, 0.03 to 17.24; −1.50 to 1.24). Patients over 60 years old have high viral load up to the second week after the onset of symptoms (range, 25.32–155.42; 1.40–2.19), indicating the longer presence of the virus in them. These findings suggest the viral load in nasopharyngeal swabs would help to monitor the SARS‐CoV‐2 infection in mild coronavirus disease 2019 cases.

Lower nasopharyngeal viral loads in pediatric population. The missing piece to understand SARS-CoV-2 infection in children?

“…Understanding the role of children in the transmission of the virus would help designing appropriate control measures, planning the reopening of schools and restoring intergenerational contacts 3 . Several studies have addressed the viral loads of children, some have concluded that viral loads are not higher than adults 1 , 4 but some of them found the opposite 5 , 6 . Yet determination of viral loads is complicated by the lack of standardization and the nature of nasopharyngeal samples that are taken by surface swabbing 7 .…”

“…Some studies use standard curves to translate Ct values into RNA copies/ml 1 , one used the weight of collected secretions to give RNA copies/g of secretion 4 but these approaches do not address the variation in the amount of sample collected by the swabs. An accurate normalization assay should be based on a marker of collected cell mass like the human RNAseP 6 , 7 .…”

Performance and Usefulness of a Novel Automated Immunoassay HISCL SARS-CoV-2 Antigen Assay Kit for the Diagnosis of COVID-19

“…Since the early phase of this pandemic, RT-qPCR tests have been used for diagnoses, epidemiological studies, and political decision making [27][28][29]. Severe cases with high infectivity usually show high viral loads (i.e., low Ct values) and vice versa [30]. However, the interpretation of positive results needs extra caution because Ct values can vary due to differences in PCR conditions [31].…”

Molecular diagnostics in the era of COVID-19

“…This study also suggests that older individuals over the age of 60 have prolonged infections (viral loads of 1.40-2.19 log 10 RNA cp/ test) up to 2 weeks post symptom onset, whilst children under 10 experienced peak viral loads (2.50 log RNA cp/ test) during the first 2 days following symptom onset declining rapidly thereafter. 133 Recent reports published by bodies such as the AACC (American Association for Clinical Chemistry) and PHE have outlined the challenges with using Ct values for estimating viral loads thereby clearly highlighting the need for quantitative molecular technologies for viral load monitoring, which would help to support both clinical and public health decisions. 113,114,129,133,134…”

“…133 Recent reports published by bodies such as the AACC (American Association for Clinical Chemistry) and PHE have outlined the challenges with using Ct values for estimating viral loads thereby clearly highlighting the need for quantitative molecular technologies for viral load monitoring, which would help to support both clinical and public health decisions. 113,114,129,133,134…”