The beneficial effects of vitamin D in COVID-19 were attributed to be mediated through its multiple actions on the immune system.
Vit. D is known to enhance the production of various anti-microbial peptides by the immune cells and vit. D modulates the immune system according to the internal milieu. It reduces the dysregulated production of self-damaging pro-inflammatory cytokines and promotes the expression of anti-inflammatory cytokines by immune cells. The dynamic role of vit. D can be of immense value in the context of immune dysfunction observed in COVID-19 patients with cytokine storm and acute respiratory distress syndrome 2–6.
Tough the protective immuno-modulatory effects of vit. D were explored in many autoimmune diseases and respiratory tract infections, there is a dearth of information from the randomized clinical trials in COVID-19. Pulse D therapy is a targeted approach to increase the serum vit. D level by using high dose (60,000 IUs) oral supplementation of vit. D daily for a specific period of time determined by the individual’s BMI, initial level of vit. D and the formulation 19. This study aims to objectively investigate the role of vit. D and the impact of Pulse D therapy in reducing the inflammatory biomarkers of COVID-19.
MATERIALS AND METHODS
This is a randomized prospective open label parallel assignment interventional clinical trial. Confirmed COVID-19 patients above the age of 18 years with hypovitaminosis D (vit.D level below 30 ng/ml) and mild to moderate illness (SpO2>90%) as per the revised guidelines for COVID-19 were included.
Patients with severe illness and patients who have taken high dose vit. D (60,000 IUs) in the last 3 months, patients with active malignancy, chronic renal disease and HIV, pregnant and breastfeeding mothers were excluded.
After admission, mild to moderately ill patients were allotted the serial numbers and were screened for serum vit. D level along with inflammatory markers of COVID-19. Haemogram with neutrophil/lymphocyte (N/L) ratio was performed on Mindray BC-6200 using scatter fuorescence cube method. Vit.D, Serum Ferritin and Interleukin 6 (IL6) were estimated on Advia Centaur XPT machine using direct chemiluminometric antibody competitive immunoassay method, direct chemiluminometric two-site sandwich immunoassay method and direct chemiluminometric one step immunoassay method respectively. Lactate dehydrogenase (LDH) and C reactive protein (CRP) were estimated on AU5800 Beckman Coulter machine using photometric kinetic UV-IFCC and photometric immunoturbimetric methods respectively.
Patients with hypovitaminosis D were randomized into two groups vis a vis Experimental group/vit. D group (VD Group) and Active comparator/control group (NVD group) alternatively as per their pre allotment serial numbers. Subjects of VD group received adjunctive Pulse D therapy (60,000 IUs of vit. D in the form of aqueol nano solution (Deksel) per day for 8 days for subjects with body mass index (BMI) of 18–25 and 10 days for subjects with BMI>25) along with the routine standard treatment for COVID-19. Subjects of NVD group received standard treatment for COVID-19 alone. After the completion of treatment with vit. D, repeat serum samples for vit. D and the inflammatory markers were collected on 9th or 11th day respectively for VD group. Similarly, samples were collected on 9th day for patients with BMI of 16–25 and 11th day for patients with BMI>25 in NVD group. Subjects in both the groups (VD and NVD) who have not received the drugs like Remdesivir, Favipiravir, Ivermectin or Dexamethasone were sub categorized into eVD and eNVD sub groups. Exclusive role of vit. D (without the influence of antiviral drugs or corticosteroids) in reducing the inflammatory markers of COVID-19 was studied in these subgroups.
Differences in the serum parameters between the two groups were analysed for statistical significance using MedCalc (Ver.19.5.1). Descriptive statistics of parametric variables were represented by Mean ± SD and significance analysis by t test.
p value<0.05 was considered statistically significant and p<0.01 as highly significant.
One hundred and thirty confirmed COVID-19 subjects were included and 87 subjects could complete the study. Details are enumerated in the flow diagram (Figure 1.).
The mean age of patients who have completed the study (n=87) was 45±13 years, range 20–83 years and the mean age of patients in VD group (n=44) was 47±12 years, range 20–70 years and in NVD group (n=43) was 44±14 years, range 20–83 years.
There was no significant difference:
Out of the 87 subjects who have completed the study, 75% (n=65) were men and 25% (n=22) were women.
Owing to randomization n=37, 28 men and n=7, 15 women got allotted to VD and NVD groups respectively. The difference in the inflammatory markers before treatment between the genders in VD and NVD groups was not significant (p>0.05) except for IL6 (p=0.02) in VD group and Ferritin (p=0.002) in NVD group with men having higher levels. The difference in the inflammatory markers after treatment between the genders in VD and NVD groups was not significant (p>0.05) except for higher CRP (p=0.02) in women and higher Ferritin (p=0.002) in men in NVD group. In spite of matching various independent parameters, significant difference (p<0.05) in all the inflammatory markers between VD and NVD groups was noted before treatment with all the markers being high in VD group. Analysis of inflammatory markers and vit. D in the VD group before versus after treatment has shown highly significant reduction (p ><0.01) in all the measured inflammatory markers and a significant increase (p><0.01) in vit. D.
Unlike the VD group, analysis of inflammatory markers in the NVD group before and after treatment has not shown significant reduction (p>0.05) except CRP. On the contrary levels of IL6 and Ferritin have increased though they were not significant statistically.
The difference in the reduction of inflammatory markers between the two groups (NVD vs VD) was highly significant (p<0.01) with the reduction in VD group being markedly higher than the NVD group (Table 3). Fifteen cases each in VD and NVD group have not received any drugs like Remdesivir, Favipiravir, Ivermectin or Dexamethasone. Analysis of inflammatory markers in the eVD sub group (before and after treatment) has shown highly significant reduction (p><0.01) in all the measured inflammatory markers after Pulse D therapy. Significant increase in vit. D level was noted in all the measured inflammatory markers after Pulse D therapy. Significant increase in vit. D level was noted (p<0.01).
Analysis of inflammatory markers in the eNVD sub group (before and after treatment) has not shown any significant reduction (p>0.05). The levels of Ferritin and N/L ratio on the contrary have increased in the post samples when compared to the pre samples.
The difference in the reduction of inflammatory markers between the two sub groups (eNVD vs eVD) was significant with the reduction in eVD sub group being markedly higher than the eNVD sub group except for Ferritin. Tough the reduction of median Ferritin levels after Pulse D therapy was quite high in the VD group, it was not statistically significant.
Difference in the mean hospital stay between VD vs NVD groups (13±5 days vs 14±5 days) was not significant (p=0.9). Intensive care support was required for 9 subjects (VD group: n=4, NVD group: n=5) and 7 of them died (VD group: n=2, NVD group: n=5). 6 out of these 7 subjects (VD group: n=2, NVD group: n=4) died after 5±1 day of enrollment without completing the study. One subject in NVD group died after 21 days of enrollment. All of them had very high levels of inflammatory markers at admission when compared to the survivors. The difference was highly significant (p<0.01) ) for IL6, CRP, Ferritin and significant (p=0.02) for N/L ratio and LDH. 2 of the 7 non survivor subjects (28.5%) had either diabetes or hypertension as co-morbidity. No adverse reactions attributable to vit. D toxicity were noted in any of the patients studied. Serum calcium level in VD group after treatment was within normal limits (9±0.5 mg/dl).
Immune dysregulation in COVID-19 is marked by increased inflammatory biomarkers such as N/L ratio, CRP, LDH, IL6 and Ferritin. Vitamin D is a potential immunomodulator and its adjunctive role in the treatment of COVID-19 is established by this study. Improvement of serum vit. D level to 80–100ng/ml has significantly reduced the inflammatory markers without any side effects. Hence, adjunctive Pulse D therapy can be added safely to the existing treatment protocols of COVID-19.