Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

Abstract

Background: Coronavirus disease 2019 (COVID-19) causes severe community and nosocomial outbreaks. Comprehensive data for serial respiratory viral load and serum antibody responses from patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not yet available. Nasopharyngeal and throat swabs are usually obtained for serial viral load monitoring of respiratory infections but gathering these specimens can cause discomfort for patients and put health-care workers at risk. We aimed to ascertain the serial respiratory viral load of SARS-CoV-2 in posterior oropharyngeal (deep throat) saliva samples from patients with COVID-19, and serum antibody responses.

Methods: We did a cohort study at two hospitals in Hong Kong. We included patients with laboratory-confirmed COVID-19. We obtained samples of blood, urine, posterior oropharyngeal saliva, and rectal swabs. Serial viral load was ascertained by reverse transcriptase quantitative PCR (RT-qPCR). Antibody levels against the SARS-CoV-2 internal nucleoprotein (NP) and surface spike protein receptor binding domain (RBD) were measured using EIA. Whole-genome sequencing was done to identify possible mutations arising during infection.

Findings: Between Jan 22, 2020, and Feb 12, 2020, 30 patients were screened for inclusion, of whom 23 were included (median age 62 years [range 37-75]). The median viral load in posterior oropharyngeal saliva or other respiratory specimens at presentation was 5·2 log10 copies per mL (IQR 4·1-7·0). Salivary viral load was highest during the first week after symptom onset and subsequently declined with time (slope -0·15, 95% CI -0·19 to -0·11; R2=0·71). In one patient, viral RNA was detected 25 days after symptom onset. Older age was correlated with higher viral load (Spearman's ρ=0·48, 95% CI 0·074-0·75; p=0·020). For 16 patients with serum samples available 14 days or longer after symptom onset, rates of seropositivity were 94% for anti-NP IgG (n=15), 88% for anti-NP IgM (n=14), 100% for anti-RBD IgG (n=16), and 94% for anti-RBD IgM (n=15). Anti-SARS-CoV-2-NP or anti-SARS-CoV-2-RBD IgG levels correlated with virus neutralisation titre (R2>0·9). No genome mutations were detected on serial samples.

Interpretation: Posterior oropharyngeal saliva samples are a non-invasive specimen more acceptable to patients and health-care workers. Unlike severe acute respiratory syndrome, patients with COVID-19 had the highest viral load near presentation, which could account for the fast-spreading nature of this epidemic. This finding emphasises the importance of stringent infection control and early use of potent antiviral agents, alone or in combination, for high-risk individuals. Serological assay can complement RT-qPCR for diagnosis.

Funding: Richard and Carol Yu, May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Michael Tong, Marina Lee, Government Consultancy Service, and Sanming Project of Medicine.

Copyright © 2020 Elsevier Ltd. All rights reserved.

Figures

Figure 1

Recombinant NP and RBD of spike protein used for EIA (A) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showing purity of His-tagged RBD of spike protein (lane 1) and His-tagged NP (lane 3). Lane 2 is protein molecular weight marker. (B) Western-blot analysis of RBD of spike protein (lane 1) and NP (lane 2) using anti-His monoclonal antibody. Positive control (NP of severe fever with thrombocytopenia syndrome virus) is in lane 3 and negative control (GST-tagged protein) is in lane 4. (C) Western-blot confirmatory assay of NP using patient's serum. Anti-His monoclonal antibody in lane 1, negative patient serum in lane 2, serum samples from a patient with COVID-19 obtained during the acute phase of illness (5 days after symptom onset) in lane 3 (dilution 1 part to 100 parts) and during the convalescent phase (18 days after symptom onset) in lane 4 (dilution 1 part to 3200 parts), lane 5 (1 part to 1600 parts), and lane 6 (1 part to 800 parts). (D) Western-blot confirmatory assay with spike protein RBD using serum samples from patients with COVID-19. Anti-His monoclonal antibody in lane 1, negative patient serum in lane 2, serum from two patients with COVID-19 in lanes 3 and 4 (dilution 1 part to 100 parts). NP=nucleoprotein. RBD=receptor-binding domain. His=polyhistidine. GST=glutathione S-transferase. COVID-19=coronavirus disease 2019.

Figure 2

Temporal profile of serial viral load from all patients (n=23) Most viral load data are from posterior oropharyngeal saliva samples, except for three patients who were intubated, in whom viral load data from endotracheal aspirates are shown separately. Datapoints denote the mean; error bars indicate SD; slope represents best fit line. The number of patients who provided a sample on each day is shown in the table below the plot. D=days after symptom onset. S=saliva. E=endotracheal aspirate.

Figure 3

Relation between viral load and age or disease severity Correlation between age and peak viral load (A). Comparison of initial (B) and peak (C) viral load between severe and mild cases. Comparison of initial (D) and peak (E) viral load between patients with comorbidities and those without comorbidities.

Figure 4

Temporal profiles of serum IgM and IgG against NP and spike protein RBD, as ascertained by EIA Each line represents an individual patient. NP=nucleoprotein. RBD=receptor-binding domain. OD450–620=optical density at 450–620 nm.

Figure 5

Correlation between MN antibody titres and anti-NP or anti-RBD IgG or IgM OD450–620=optical density at 450–620 nm. MN=microneutralisation. NP=nucleoprotein. RBD=receptor-binding domain.