3.1 Clinical and Radiological Characteristics of COVID-19 patients
All 129 patients had chest CT findings and/or concordant symptoms for SARS- CoV-2 infection, in addition to laboratory confirmation with a positive PCR (n=97, 75.2%) and/or antibody tests (n=111, 86%). The mean age of the patients was 46.4 ± 15.8 years, and 54.3% of them were male. The median length of hospital stay was 7 days (min-max; 1 – 24days). Dry cough, fever and dyspnea were the most relevant symptoms of remained patients, with the frequency of 64.8%, 45.7% and 27.1%, respectively. Of these, fever and dyspnea were statistically more frequent in patients producing Nabs (52.6% vs 35.3%, p=0.05 and 33.3% vs 17.6%, p=0.05, respectively).
Serum samples were obtained from all for antibodies against SARS-CoV-2. Given that the period of hospitalization varied based on the clinical status and treatment modalities used for each patient, hospitalization periods were grouped for analyses into three, as 5-9, 10-14 and 15-28 days. The median time from onset of symptoms to antibody test was 11 days (ranging from 5 to 28 days). The incident rate of developing NAbs were found to be 5.6 per 100 person-days among patients tested for NAbs between 5 to 9 days of the initiation of symptoms; 6.5 per 100 person-days among patients tested for NAbs between 10 to 14 days; and, 3.7 per 100 person-days among patients tested for NAbs between 15 to 28 days. Abnormalities on chest CT were significantly higher in patients who elicited NAbs (94.9% vs 72.5%, p<0.001). Bilateral multi-lobar ground glass opacities and consolidation, i.e., advanced stage CT findings, were significantly more frequent in those eliciting NAbs at hospital discharge(p=0.012) (Table 1).
The distribution of NAb titers which analysed in discharge serum of the patients were plotted based on age, gender, disease severity and chest CT findings at the time of admission (Figure 2). NAb titers were not significantly different according to age or gender (Figure 2A, 2B). The median NAb titer was significantly higher(p=0.008) in severe patients (SN50; 1:25, IQR 1:42.5) when compared non-severe counterparts (SN50; 1:7.5, IQR 1:25) (Figure 2C). The median NAb titers were 1:17.5(Interquartile range (IQR); 1:20) and 1:7.5(IQR; 1:17.5) in the patients with advanced and early stage findings on thorax CT, respectively. There was a statistically significant different between these groups(p=0.006) (Figure 2D
Severe patients consisted of 17.8% (n=23), of which nine (39.1%) required ICU support on the follow up. They were older than their counterparts in the non-severe group (mean ages were 60.9 ± 13.5 vs 43.4 ± 14.7 years, respectively, p< 0.001 counterparts). Hypertension was determined significantly common in severe group (p= 0.007) and those who developed dyspnea and needed oxygen support with nasal cannula more frequently(p<0.001) (Supplemantary Table 1). Significantly higher AST, LDH, CK, PT, INR, D-dimer, CRP, ferritin, neutrophil lymphocyte ratio (NLR), lower lymphocyte levels, and eGFR were more prevalent on admission in the severe group compared to their counterparts in the non-severe group (all p<0.05, Supplemantary Table 2).
3. 2 Seroconversion Characteristics of NAbs, lgM, and IgG against SARS-CoV-2
The seroconversion rate at the time of hospital discharge were 60.5%, 30.2%, and 51.9% for NAb, lgM, and IgG, respectively. Using VNA as the reference test, the sensitivity of the IgG was 85.9% and the specificity was 72.5%. The median time to testing(discharge) were longer (12 versus 8 days, p<0.001) in the group with NAb positivity at discharge. NAb positivity was significantly higher (75% vs 42.4%, p<0.001) after 10 days from symptoms onset; NAb positivity was the highest (79.5%) among those who were tested 10-14 days after their symptoms’ onset(p<0.001) (Figure 3). It is remarkable that, 34 patients (26.4% of total patients) who remained seronegative for NAbs, were discharged in less than 10 days.
IgM and IgG antibody response were 20.3% and 44.1% among patients within 5-9 days since onset, and increased to 34.1% and 47.7% within 10- 14 days; the highest levels 50% and 83.3% were detected respectively, among patients tested 15 days (max follow-up was 28 days) after the onset of symptoms (Figure 3).
Seroconversion rates of IgG response was statistically significantly (82.6% vs 45.3%, p=0.006) and frequency of NAb positivity was slightly higher in the severe group (78.3% vs 56.6% p=0.05). Eight of the nine patients (88.9%) who needed ICU support over hospitalization elicited NAbs. Additionally, median NAb titers (SN50 level) were significantly higher in patients with severe infection (median 25 vs 7.5, p=0.009). Out of 23 severe patients, 52.2% (n=12) had higher NAb titers (SN50 ≥ 1:25); the odds of high NAb were 2.89 times among severe patients compared to non-severe patients (95% CI=1.15 – 7.28, p= 0.021) (Supplemantary Table 1).
3.3 Comparison of the VNA and ELISA
A total of 129 discharged serum samples of the patients were analyzed with both a in-house ELISA kit, and VNA assay. We found that, 67 (51.9%) samples were positive in IgG ELISA and 78(60.5%) samples were positive in VNA. In addition, 53 (41.1%) samples were positive and 37(28.7%) were negative in both assays. Fourteen samples were found to be VNA negative but ELISA positive, whilst 22 samples were VNA positive but ELISA negative. Three samples were indeterminate in ELISA while positive with VNA (Table 1). Accordingly, sensitivity and specificity values of ELISA were 67.9% and 72.5%, respectively, in determining immunity in patients recovered from COVID-19.
3.4 The relation of NAbs with laboratory parameters in COVID-19 patients
The distribution of laboratory parameters of the patients by NAbs positivity revealed that CRP, AST, LDH, CK were all significantly higher in patients with NAbs positivity (Table 2). The correlation analyses were performed to determine the predictors of higher NAb titers (SN50 levels). There was a weak, positive correlation between SN50 levels and antibody testing time (i.e., the length of days after symptoms’ onset), NLR, AST, LDH, CK, ferritin, PT and INR; whilst lymphocyte level had a weak, negative correlation (Supplemantary Table 3).
3.5 Association between disease severity and presence and higher titers of NAb
The rate ratios for the presence of NAbs at hospital discharge were 1.16 and 0.66, comparing patients with discharge at days 10-14 and 15-28, respectively, with patients discharged before 10 days after symptoms onset. Kaplan-Meier analyses revealed that both NAb production started earlier and was more prominent among non-severe cases than severe cases, if followed over time. A similar course was observed for higher NAb titers, comparing severe and non-severe cases. The significant difference obtained for higher NAb titers favoring severe cases(p=0.02) in binary analyses disappeared when time-to-event was controlled for. Survival analysis revealed a negative association between severity and presence of NAb at hospital discharge (Figure 4).