3.1 Characteristics of COVID-19 patients
Fifty-two patients with COVID-19 and 33 controls matched for age and sex were finally included in the analysis (Fig. 1). Patients who presented only with symptoms such as fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, and loss of taste and/or smell, were classified as mild disease (n=30), while patients presenting with additional clinical and/or radiographic evidence of lower respiratory tract infection with SpO2 ≥94% on room air were classified as having moderate disease (n=22). None had severe disease status at the time of first evaluation. The majority of patients had a favorable clinical course during the next 4 weeks (mild-stable or moderate-stable), while 8 patients of the 22 who were classified as moderately ill progressed to severe clinical status and required prolonged hospitalization (moderate-progressed).
Age and BMI were similar between patients and controls (44.8 ± 14.2 years vs. 47.6 ± 12.8 years, p=0.359 and 25.9 ± 5.1 kg/m2 vs. 24.8 ± 3.8 kg/m2). Diabetes mellitus type 2 (5.8%), hypertension (13.5%), and hypothyroidism (13.5%) were the most common comorbidities in COVID-19 patients. A total of 31% of patients and 19% of controls were current smokers (p=0.397). Table 1 summarizes the demographic and clinical characteristics of COVID-19 patients stratified by disease status and course as mild-stable (n=30), moderate-stable (n=14), and moderate-progressed (n=8), and controls. As expected, patients with a mild-stable disease course were significantly younger and had lower body mass index (BMI) than the remaining patients (Table 1). Also as expected, the median serum measurements of CRP were about 6-fold higher in COVID-19 patients than controls [5.6 (0.05, 90.1) mg/L vs. 0.9 (0.1, 9.0) mg/L, p<0.001). Serum IL-6 levels were detectable in 7 (21%) controls, and in 46 (88%) patients and the median serum measurements were also about 6-fold higher in COVID-19 patients than controls [4.5 (0.1, 46.6) pg/mL vs. 0.1 (0.1, 15.1) pg/mL p<0.001, respectively), being much higher in patients with pneumonia than in those without (Table 1). Individual serum CRP levels were strongly correlated with the corresponding serum IL-6 levels in COVID-19 patients (rho= 0.564, p<0.001 (Sup. Fig. 1).
Table 1
Demographic, clinical and laboratory characteristics of COVID-19 Patients and Controls
Parameters
|
COVID-19 Patients (Cases)#
|
Healthy Controls
(n=33)
|
p value
|
Mild -Stable
(n=30)
|
Moderate-Stable
(n=14)
|
Moderate- Progressed
(n=8)
|
Age (yrs),
|
39.0±14.1
|
53.4 ±10.3
|
51.4 ± 10.1
|
47.6 ± 12.8
|
0.002 a,b,c
|
Males (n, %)
|
17 (56.7%)
|
7 (50%)
|
4 (50%)
|
19 (57.6%)
|
0.952
|
Days from positive PCR test
|
4 (2, 13)
|
4 (2, 15)
|
3 (2,9)
|
n/a
|
0.743
|
BMI (kg/m2),
|
24.7± 5.0
|
29.6 ± 4.5
|
25.0 ± 3.7
|
24.8 ± 3.8
|
0.014 a,d,e
|
DMT2 (n, %)
|
1 (3.3%)
|
2 (14.3%)
|
0
|
1 (4.0%)
|
0.381
|
Smoking (n, %)
|
8 (36.4%)
|
3 (25%)
|
2 (28.6%)
|
5 (19.2%)
|
0.612
|
Hypertension (n,%)
|
2 (6.7%)
|
4 (28.6%)
|
1 (12.5%)
|
1 (4.0%)
|
0.088
|
*Serum CRP levels
RV of the assay:<8mg/L
|
3.0 (0.1, 17.6)
|
17.0 (0.5, 90.2)
|
18.3 (0.3, 51.0)
|
0.9 (0.1, 9.1)
|
<0.001 a,b,e,f
|
*Serum Il-6 levels
RV of the assay:<3.13 pg/ml
|
1.8 (0.1, 30.9)
|
7.0 (1.6, 32.2)
|
14.7 (3.0, 46.6)
|
0.1 (0.1, 15.1)
|
<0.001 a,b,c,e,f
|
*Plasma ACTH levels
RV of the assay: morning 9-52pg/ml; afternoon 5-30 pg/ml
|
16.1 (5, 56)
|
19.1 (5, 31)
|
14.0 (5, 40)
|
16.3 (5, 47)
|
0.642
|
*Plasma Aldosterone levels
RV of the assay: 40-310 pg/ml
|
127 (60, 367)
|
85 (32, 523)
|
206 (33, 275)
|
140 (42.4, 438)
|
0.037
|
Morning (8am) cortisol measurement in the saliva **
RV of the assay: <0.78 mcg/dl
|
0.493 (0.09, 1.122)
|
0.506 (0.281, 1.170)
|
0.828 (0.199, 1.060)
|
0.533 (0.285, 1.020)
|
0.573
|
Mid-Day (12pm) cortisol measurement in the saliva**
RV of the assay: <0.78 mcg/dl
|
0.313 (0.076, 1.164)
|
0.321 (0.095, 0.421)
|
0.310 (0.054, 0.663)
|
0.188 (0.083, 0.453)
|
0.002
|
Evening (6pm) cortisol
measurement in the saliva**
RV of the assay: <0.24 mcg/dl
|
0.149 (0.062, 0.906)
|
0.228 (0.121, 0.892)
|
0.391 (0.054, 1.010)
|
0.081 (0.054, 0.243)
|
<0.001
|
Nocturnal (10pm) cortisol measurement in the saliva**
RV of the assay: <0.21 mcg/dl
|
0.116 (0.054, 1.010)
|
0.234 (0.054, 1.410)
|
0.183 (0.090, 0.834)
|
0.054 (0.054, 0.332)
|
<0.001
|
#: Classification based on disease severity (https://www.covid19treatmentguidelines.nih.gov/overview/clinical-spectrum/), and the clinical outcome of patients in the 4 weeks follow-up period of the study.
*: Blood sampling was performed at the day of recruitment (for both patients and controls) at unscheduled timepoints.
**: Saliva sampling was performed at the next day at pre-scheduled timepoints within the day (namely, 8am; 12pm; 6pm; 10pm).
Values are shown as mean ± SD, or median (min, max), as applicable. Categorical variables are shown as absolute number (valid percentage).
One way ANOVA or Kruskal–Wallis H test with Bonferroni or Dunn’s Post Hoc Tests, respectively were performed for multiple comparisons between groups, as applicable.
a: p<0.05 for comparisons between patients with mild-stable and moderate stable disease
b: p<0.05 for comparisons between patients with mild-stable and moderate-progressed disease
c: p<0.05 for comparisons between patients with mild-stable disease and healthy controls
d: p<0.05 for comparisons between patients with moderate -stable and moderate -progressed disease
e: p<0.05 for comparisons between patients with moderate-stable disease and healthy controls
f: p<0.05 for comparisons between patients with moderate - progressed disease and healthy controls
PCR, polymerase chain reaction; yrs, years; BMI, body mass index; DMT2, diabetes mellitus type 2; RV, reference values; CRP, C- reactive protein; IL-6, interleukin 6; ACTH, Adrenocorticotropin
|
3.2 Diurnal cortisol secretion in COVID-19 patients
Salivary cortisol levels in the morning (8am) were similar in COVID-19 patients and controls, whereas no significant differences were evident between patient subgroups (Table 1). All controls and most of the COVID-19 patients (46/52, 88%) exhibited the expected gradual decrease of salivary cortisol during the day, with the lowest measurements obtained during evening and nocturnal sampling (Fig. 2a, b). However, as shown in Fig. 2c daily cortisol secretion was clearly increased in patients with either mild-stable, moderate-stable, or moderate-progressed COVID-19 compared to that of controls.
Except for the morning measurements, all other cortisol measurements during the day were significantly higher in COVID-19 patients than in controls (Table 1). While evening and nocturnal cortisol levels decreased by 80% and 83%, respectively, in controls (Fig. 2b), COVID-19 patients displayed much lower mean respective decreases, respectively 37% and 54% (both p<0.001). Overall, time-integrated daily cortisol secretion, as assessed by the AUC, was almost 2-fold higher in COVID-19 patients than controls (AUC: 4.81± 2.46 vs. 2.75 ± 0.810, respectively, p<0.001), being lower, albeit non-significantly, in patients with mild disease than in the remaining patients (Fig. 2c). Thus, the presence of pneumonia was not related to the increased daily cortisol secretion in these patients (β = 0.132, 95% CI; -0.8, 2). Notably, the highest AUC of cortisol was observed in a patient with moderate-stable COVID-19 (individual value 10.86), which was almost double compared to the highest AUC observed among controls (individual value 4.73).
3.3 ACTH and aldosterone levels and diurnal DHEA secretion in COVID 19.
Plasma measurements of both ACTH and aldosterone upon presentation of COVID-19 patients were similar to those of controls, irrespectively of disease status (Table 1). Similarly, next morning salivary DHEA measurements were comparable in 30 representative COVID-19 patients and their matched controls (Sup. Fig. 2a). In contrast to the increased time-integrated daily cortisol secretion, the sequential measurements of DHEA in saliva were similar in COVID-19 patients, irrespectively of disease status, and controls (Sup. Fig. 2a). As a result, daily DHEA secretion did not differ between COVID 19 patients and controls [AUC for DHEA: 1956 (328, 6210) vs. 1546 (116, 6512), p=0.736) (Sup. Fig. 2b).
3.4 IL-6 serum levels correlate with daily cortisol secretion in COVID-19
Serum IL-6 levels measured upon presentation were strongly correlated with the next day evening (rho=0.412, p=0.004) and nocturnal (rho=0.555, p<0.001), but not with morning (rho=0.219, p=0.126) or midday (rho=0.127, p=0.374) salivary cortisol levels (Fig. 3). In 20 representative COVID-19 patients, we also estimated the parallel diurnal IL-6 levels in saliva and found that increased rates of IL-6 secretion were sustained in all COVID-19 patients tested from morning to night (Sup. Fig. 3).
Collectively, individual serum IL-6 levels upon COVID-19 patients’ presentation correlated with next-day time-integrated daily cortisol secretion (rho=0.373, p=0.011).
No associations were noted between individual serum IL-6 levels and concomitant plasma ACTH (rho=0.167, p=0.242) or plasma aldosterone (rho=0.091, p=0.529) concentrations in COVID-19 patients. Likewise, serum IL-6 levels did not correlate with the next-day time-integrated daily DHEA secretion measured in saliva (rho=-0.025, p=0.904).
Finally, no correlations were observed between plasma ACTH levels and the next-day salivary cortisol measurements (Fig. 4), the time-integrated daily cortisol (rho=-0.048, p=0.749) and DHEA secretion (rho=-0.009, p=0.964), or with the concomitantly measured plasma aldosterone levels (rho=0.171, p=0.235) in COVID-19 patients.