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Of the 112 patients confirmed as positive for SARS-CoV-2 infection between January and March 2020, 87 (77.7%) were mild cases and 25 (22.3%) were severe. The demographic and clinical characteristics of each group are summarized in Table 1. The median age of the severe group was older than the mild group (median age, 65 vs 50 years, P < 0.001), and showed a tendency for higher male-to-female ratio (68.0% vs 51.7%, P = 0.176). Fever (76.8%) was the most common symptom among the 112 patients, followed by dry cough (54.5%), fatigue (33.0%), and shortness of breath (19.6%). Significantly higher proportions of patients in the severe disease group experienced fatigue (52.0% vs 27.6%, P = 0.030), chill (24.0% vs 5.8%, P = 0.015), and palpitation (20.0% vs 3.5%, P = 0.013), and showed greater abnormalities in biochemical indices at the time of admission than the milder patients goup, including higher levels of C-reactive protein (51.57 vs 6.27 mg/L, P < 0.001), D-dimer (1.09 vs 0.7 mg/L, P = 0.033), and lactate dehydrogenase (306 vs 226.5 U/L, P = 0.003). Leukocyte and neutrophil counts were higher in the severe group than in the mild group (7.26 × 109 vs 5.48 × 109/L, P = 0.016 and 5.72 × 109 vs 3.33 × 109/L, P < 0.001, respectively), while the lymphocyte count was lower (0.93 × 109 vs 1.35 × 109/L, P = 0.003) in patients with more severe disease.
Total (n = 112) Mild (n = 87) Severe (n = 25) P value Age, years 54 (40.25–66) 50 (38–64) 65 (52–75.5) < 0.001* Male 62 (55.36%) 45 (51.72%) 17 (68.00%) 0.176 Onset of symptoms to Hospital admission, days 7 (2.25–13) 9 (3–13) 6 (1–8) 0.064 RNA confirmation, days 7 (3–12) 9 (3–14) 5 (2–7.5) 0.004* Signs and symptoms Fever (temperature ≥ 37.3 ℃) 86 (76.79%) 66 (75.86%) 20 (80.00%) 0.792 Chest tightness 17 (15.18%) 11 (12.64%) 6 (24.00%) 0.205 Night sweats 2 (1.79%) 1 (1.15%) 1 (4.00%) 0.398 Shortness of breath 22 (19.64%) 15 (17.24%) 7 (28.00%) 0.259 Chill 11 (9.82%) 5 (5.75%) 6 (24.00%) 0.015* Fatigue 37 (33.04%) 24 (27.59%) 13 (52.00%) 0.030* Dry cough 61 (54.46%) 50 (57.47%) 11 (44.00%) 0.261 Vomiting 3 (2.68%) 2 (2.30%) 1 (4.00%) 0.535 Anorexia 17 (15.18%) 10 (11.49%) 7 (28.00%) 0.058 Palpitation 8 (7.14%) 3 (3.45%) 5 (20.00%) 0.013* Myalgia 3 (2.68%) 1 (1.15%) 2 (8.00%) 0.125 Dyspnoea 15 (13.39%) 11 (12.64%) 4 (16.00%) 0.740 Expectoration 16 (14.29%) 13 (14.94%) 3 (12.00%) 1.000 Pharyngalgia 9 (8.04%) 9 (10.34%) 0 (0.00%) 0.204 Diarrhoea 8 (7.14%) 7 (8.05%) 1 (4.00%) 0.681 Nausea 7 (6.25%) 6 (6.90%) 1 (4.00%) 1.000 Dizziness 5 (4.46%) 2 (2.30%) 3 (12.00%) 0.073 Headache 3 (2.68%) 1 (1.15%) 2 (8.00%) 0.125 Serologic test on admission (normal range) C-reactive protein, mg/L (< 8.2) 9.83 (2.03–47.71) 6.27 (1.45–22.62) 51.57 (23.99–113.50) < 0.001* D-Dimer, mg/L (< 0.05) 0.87 (0.36–1.57) 0.7 (0.31–1.46) 1.09 (0.82–2.38) 0.033* Lactate dehydrogenase, U/L (318–618) 232 (199–288) 226.5 (190–258) 306 (205–371) 0.003* White blood cell count, × 109/L (4–10) 5.65 (4.67–7.42) 5.48 (4.47–6.81) 7.26 (5.01–9.66) 0.016* Lymphocyte count, × 109/L (0.8–4) 1.27 (0.9–1.61) 1.35 (1.09–1.71) 0.93 (0.73–1.42) 0.003* Neutrophil count, × 109/L (2–7) 3.8 (2.85–5.23) 3.33 (2.75–4.83) 5.72 (3.76–7.87) < 0.001* Antibodies against NP, S/CO 443.09 (181.77–793.02) 400.12 (172.95–732.51) 548.26 (392.21–1031.98) 0.044* Peak viral load (Ct value) 35.63 (30.87–37) 36.39 (31.62–37) 31.48 (24.03–36.64) 0.002* SARS-CoV-2 RNA persistence, days 15 (9–27) 17.5 (10–27) 12 (7.5–32.5) 0.455 Data are presented as median (interquartile range) and n (%); *P < 0.05 (Student's t test, χ2 test, or Mann–Whitney U test); Ct, cycle threshold; NP, nucleocapsid protein; S/CO, signal-to-cutoff. Table 1. Clinical characteristics of COVID-19 patients.
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We collected 1065 throat swabs from 112 COVID-19 patients for viral load monitoring (mean of 9.5 throat specimens per patient). The median of viral RNA following-up time was 57 days (interquartile range [IQR], 49-63.75 days), and the longest was 80 days. The temporal profile of the viral load for all patients is shown (Fig. 1A, 1B). The peak viral load in throat swabs was significantly higher in the severe group than in the mild group but did not differ by sex and showed no correlation with age (Fig. 1C-1E). The median duration of viral shedding was 15 days (IQR, 9-27 days). In five patients (4.5%; four mild and one severe case), viral RNA had turn negative for two consecutive tests and then recurrence occurred 45 days after the initial symptom onset. In another patient with mild disease, viral RNA was detected by RT-qPCR up to 45 days after symptom onset.
Figure 1. SARS-CoV-2 RNA profiles in throat swabs of COVID-19 patients. A Temporal profile of viral load (inversely related to cycle threshold [Ct] value) in all patients (n = 112). Each line represents an individual patient. The thick line shows the trend in viral load using smoothing splines. B SARS-CoV-2 RNA positive rate at different time intervals. C Comparison of peak viral load according to sex. D Relationship between age and peak viral load. E Comparison of peak viral load between mild and severe patients. Results are shown as median and interquartile range; P values were derived from Mann-Whitney U test, or Spearman's test; r values were derived from Spearman's test.
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Longitudinal antibody responses were evaluated in our cohort. A total of 474 serum samples were obtained from the 112 patients (mean of 4.23 serum specimens per patient). These patients were followed up biweekly up to 80-days post disease onset. IgM and IgG antibodies against SARS-CoV-2 NP were detected by ELISA. To further understand the correlation between antibody responses to SARS-CoV-2 and clinical parameters, the antibody responses were stratified according to sex, age, and disease severity (Fig. 2A). Overall, the levels of IgM antibodies varied in the two groups, while there was no significant difference in IgG levels when patients were stratified according to sex, disease severity, and age. Anti-NP IgM levels peaked at the first 2 weeks after symptom onset and decreased steadily thereafter, and were undetectable in most patients after 4-6 weeks. Compared to females, male patients had significantly higher serum anti-NP IgM level; and the anti-NP IgM level was higher in older patients and the severe patients group compared to the younger group and the mild disease group, respectively, but the differences were not statistically significant. Anti-RBD IgM level reached its peak at 3-4 weeks post disease onset, which occurred later than anti-NP IgM level.
Figure 2. Profiles of antibodies against SARS-CoV-2 NP and RBD in COVID-19 patients (n = 112). A Antibodies against SARS-CoV-2 NP and RBD in patients according to sex, age, and disease severity. Results are shown as median and interquartile range; P values were derived from the Mann-Whitney U test. B Seropositive rates of IgM and IgG against SARS-CoV-2 NP at different intervals. C, D Relationship between levels of antibodies against SARS-CoV-2 NP (C) and RBD (D) measured by ELISA. The P and r values were derived from Spearman's test. *P < 0.05; **P < 0.01.
We then evaluated the seropositive rates of anti-NP IgM and IgG antibodies based on the number of days after symptom onset (Fig. 2B; Supplementary Table S1). The positivity rate for IgM peaked in the second week with 86.49% and declined steadily afterward. The seroconversion rate of anti-NP IgG was 69.2% in the first week after symptom onset, reached 100% in the fifth week, and then remained 100% up to 80 days post disease onset.
Next, we estimated Spearman's correlation among the antibody titres against NP and RBD (Fig. 2C, 2D). ELISA OD values for anti-RBD IgM showed a weak but significant positive correlation with anti-NP IgM levels (r = 0.285; P < 0.001; Fig. 2C), and anti-RBD IgG levels showed a significant positive association with anti-NP IgG levels (r = 0.623; P < 0.001; Fig. 2D). While there was no correlation between the persistence of SARS-CoV-2 RNA and antibodies levels (Supplement Fig. S1).
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In late July, we conducted another round of follow-up in which included 54 recovered patients. The follow-up time ranged from 158 to 194 post onset of symptoms. All the samples collected in July tested positive (S/CO > 1) for antibodies against NP; the temporal profile is shown in Fig. 3A. At 158-194 days post onset of symptoms, the antibody levels decreased approximately 46.17% overall (median, 151.61 S/CO, IQR, 287.11-453.01 S/CO; P = 0.128; Fig. 3B) compared with those at the acute phase (the period when the viral RNA can be found in a respiratory specimen). Moreover, the median percentage of decrease was 46.31% (IQR 5.37%-68.96%) for antibodies against NP in the mild group, whereas it was 46.02% (IQR 11.01%-65.75%) in the severe group.
Figure 3. Profile of antibodies against SARS-CoV-2 NP measured by CIMA. A Temporal pattern of antibodies to NP in all patients (n = 112). Each line represents an individual patient. The thick line shows the trend using smoothing splines. B Dynamic changes in antibodies against NP in the acute phase (the period when the viral RNA can be found in a respiratory specimen) and 158-194 days post symptoms onset (n = 54). The P and r values were derived from Spearman's test.
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We compared SARS-CoV-2 RNA dynamics and antibody responses between 18 asymptomatic individuals and 17 symptomatic COVID-19 patients, and the asymptomatic individuals were the close contacts of these symptomatic cases. The clinical characteristics of these individuals are summarized in Table 2. The symptomatic group tended to have a higher median age and included more males than the asymptomatic group.
Asymptomatic (n = 18) Symptomatic (n = 17) P value Age, years 38 (23–55.75) 52 (37–63) 0.06 Male 7 (38.89%) 11 (64.71%) 0.181 IgM and IgG against NP peak titre, S/CO 60.79 (9.99–194.54) 302.44 (169.29–567.85) < 0.001* Peak viral load (Ct value) 35.26 (32.1–37) 34.3 (29.82–37) 0.725 RNA persistence, days 2.5 (1–8.25) 4 (1–20.5) 0.508 Data are presented as median (interquartile range) and n (%); *P < 0.05 (χ2 test or Mann–Whitney U test); Ct, cycle threshold; NP, nucleocapsid protein; S/CO, signal-to-cutoff. Table 2. Characteristics of asymptomatic individuals and the related symptomatic cases.
In total, 276 throat swabs (127 from 18 asymptomatic individuals and 149 from 17 symptomatic cases) were collected for viral RNA load monitoring. There were no statistically significant differences in the peak Ct value and the persistence of viral RNA between the asymptomatic individuals and the symptomatic cases (Fig. 4A-4C). A further 116 serum samples (2-5 per patient) were obtained at different time points and tested for antibodies against NP. The date of diagnosis was set as the starting point to compare the antibody dynamics between the asymptomatic individuals and symptomatic cases. The temporal profile of antibodies is shown in Supplementary Figure S2. Antibody levels against NP were significantly lower in the asymptomatic group (Fig. 4D). The serologic conversion of IgG occurred earlier in the symptomatic cases than in the asymptomatic carriers, and the IgM seroconversion rate in the asymptomatic group was much lower (16.7% vs 70.6%, P = 0.002; Fig. 4E).
Figure 4. SARS-CoV-2 RNA and antibody dynamics in asymptomatic patients (n = 18) and symptomatic cases (n = 17). A Temporal profile of viral RNA load (inversely related to cycle threshold [Ct] value). Each line represents an individual patient. The thick line shows the trend in viral load using smoothing splines. B, C Peak viral RNA load (B) and persistence of viral RNA (C) in asymptomatic patients and symptomatic cases. D Antibodies against NP measured by CIMA. E Seropositivity of antibodies against NP at different intervals measured by ELISA. Results are shown as median and interquartile range; P values were derived from the Mann-Whitney U test.