Delayed Rise of Oral Fluid Antibodies, Elevated BMI, and Absence of Early Fever Correlate With Longer Time to SARS-CoV-2 RNA Clearance in a Longitudinally Sampled Cohort of COVID-19 Outpatients

Abstract

Background: Sustained molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the upper respiratory tract (URT) in mild to moderate coronavirus disease 2019 (COVID-19) is common. We sought to identify host and immune determinants of prolonged SARS-CoV-2 RNA detection.

Methods: Ninety-five symptomatic outpatients self-collected midturbinate nasal, oropharyngeal (OP), and gingival crevicular fluid (oral fluid) samples at home and in a research clinic a median of 6 times over 1-3 months. Samples were tested for viral RNA, virus culture, and SARS-CoV-2 and other human coronavirus antibodies, and associations were estimated using Cox proportional hazards models.

Results: Viral RNA clearance, as measured by SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR), in 507 URT samples occurred a median (interquartile range) 33.5 (17-63.5) days post-symptom onset. Sixteen nasal-OP samples collected 2-11 days post-symptom onset were virus culture positive out of 183 RT-PCR-positive samples tested. All participants but 1 with positive virus culture were negative for concomitant oral fluid anti-SARS-CoV-2 antibodies. The mean time to first antibody detection in oral fluid was 8-13 days post-symptom onset. A longer time to first detection of oral fluid anti-SARS-CoV-2 S antibodies (adjusted hazard ratio [aHR], 0.96; 95% CI, 0.92-0.99; P = .020) and body mass index (BMI) ≥25 kg/m² (aHR, 0.37; 95% CI, 0.18-0.78; P = .009) were independently associated with a longer time to SARS-CoV-2 viral RNA clearance. Fever as 1 of first 3 COVID-19 symptoms correlated with shorter time to viral RNA clearance (aHR, 2.06; 95% CI, 1.02-4.18; P = .044).

Conclusions: We demonstrate that delayed rise of oral fluid SARS-CoV-2-specific antibodies, elevated BMI, and absence of early fever are independently associated with delayed URT viral RNA clearance.

Keywords: COVID-19; RT-PCR; SARS-CoV-2; antibody; viral RNA.

Figure 1.

CONSORT flow diagram and sampling schedule. A, A convenience sample of 475 adults with recent positive SARS-CoV-2 RT-PCR tests from an outpatient testing site of the Johns Hopkins Health System was assessed for eligibility between April 21, 2020, and July 23, 2020. Preference was given to participants 40 years of age and older. Study kits were sent to 118 participants, and data and samples from 95 participants were included in the analyses presented here. B, Study sampling schedule. Clinical RT-PCR results from the medical record are included in these analyses. Samples on study days 0–14 were self-collected at home with telephone or video guidance by trained study staff. Participants presented to a research clinic for collection of samples a median (range) of 45 (27–88) days after study day 0. Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 2.

Longitudinal SARS-CoV-2 RT-PCR sampling in outpatients. SARS-CoV-2 RT-PCR test results by day from symptom onset. Each row represents 1 participant. Shown here are nasal samples. Stars indicate a sample time point at which the nasal sample was negative and the oral fluid sample was positive. Included in the top grouping are participants whose last RT-PCR test was negative, and in the bottom group are those whose last RT-PCR test was positive. Shaded gray lines indicate the number of days from symptom onset until the midpoint between the last positive sample and the next negative sample (top grouping) or until the last positive sample (bottom grouping). Nasopharyngeal (NP) swabs collected by a health care worker are indicated by triangles; self-collected midturbinate nasal-oropharyngeal (OP) swab combined in viral transport media are indicated by circles. Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 3.

Kinetics of RT-PCR cycle thresholds, virus culture, and oral fluid SARS-CoV-2 antibodies in mild–moderate COVID-19. A, Longitudinal Abbott m2000 SARS-CoV-2 RT-PCR cycle threshold values from nasal-OP swab study samples (blue circles and triangles in Figure 2) by day from symptom onset are shown in gray. Longitudinal oral fluid antispike (Mt. Sinai) IgG mean fluorescence intensity shown in purple. Lines connect values from the same participant. Each sample with a positive RT-PCR result was assessed for propagation of SARS-CoV-2 on VeroE6-TMPRSS2 cells. Samples with positive virus culture are indicated by red circles. The cutoff for positive anti-S IgG is shown with a purple dotted line (see the “Methods” for cutoff calculation). B, The subset of 59 participants with a nasopharyngeal swab collected within 7 days of symptom onset and run on the NeuMoDx SARS-CoV-2 RT-PCR platform is shown by day from symptom onset. The line connects cycle threshold means. C, The subset of nasal-OP swab samples with a cycle threshold <20 is grouped according to whether virus culture of the sample was positive or negative. Means with 95% CIs are shown. *P < .00001 by 1-tailed Student t test. D, The subset of samples shown in (B) that had a simultaneous adequate oral fluid sample is shown here. Filled diamonds represent study time points at which oral fluid contained detectable IgG antibodies to Mt Sinai S-RBD, and open diamonds represent study time points at which oral fluid was negative for anti-S-RBD IgG. Abbreviations: Ab, antibody; COVID-19, coronavirus disease 2019; Cult, culture; IgG, immunoglobulin G; OP, oropharyngeal; RT-PCR, reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; S-RBD, spike receptor binding domain.

Figure 4.

Kaplan-Meier plots SARS-CoV-2 RT-PCR positivity by day from symptom onset in mild–moderate COVID-19. A, Kaplan-Meier survival curve with 95% CIs for positive upper respiratory tract SARS-CoV-2 RT-PCR by day from symptom onset. B, Kaplan-Meier survival curve by BMI category. Abbreviations: BMI, body mass index; COVID-19, coronavirus disease 2019; RT-PCR, reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 5.

Cox proportional hazards model for viral RNA clearance. An aHR <1 denotes longer time to SARS-CoV-2 viral RNA clearance in the upper respiratory tract as measured by RT-PCR. To account for missing data points, 20 data sets were imputed using PPM MICE. Model selection using LASSO was performed on each of the 20 data sets. The top 9 variables of 35—selected 15 or more times out of 20 (Supplementary Table 2)—were chosen for inclusion in the Cox proportional hazards model, which was performed using the pooled imputed data set. ^aStandardized log mean fluorescence intensity and treated as a time-varying variable. Abbreviations: aHR, adjusted hazard ratio; ECD, spike ectodomain; hCoV, human coronavirus; LASSO, least absolute shrinkage and selection operator; MICE, multiple imputation by chained equation; PPM, predictive mean matching; RT-PCR, reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; S-RBD, spike receptor binding domain.