key: cord-1028549-ruh3sb7q
authors: Raghavan, Kadalraja; Dedeepiya, Vidyasagar Devaprasad; Suryaprakash, Vaddi; Rao, Kosagi-Sharaf; Ikewaki, Nobunao; Sonoda, Tohru; Levy, Gary A.; Iwasaki, Masaru; Senthilkumar, Rajappa; Preethy, Senthilkumar; Abraham, Samuel JK
title: Beneficial Effects of novel Aureobasidium Pullulans strains produced Beta-1,3-1,6 Glucans on Interleukin-6 and D-Dimer levels in COVID-19 patients; results of a randomized multiple-arm pilot clinical study
date: 2021-09-25
journal: Biomed Pharmacother
DOI: 10.1016/j.biopha.2021.112243
sha: 271a4f454d698363458d848eae658c2f5f37f7a8
doc_id: 1028549
cord_uid: ruh3sb7q

OBJECTIVE: In this pilot clinical study, we report the beneficial effects of beta glucans derived from two strains AFO-202 and N-163 of a black yeast Aureobasidium pullulans on the biomarkers for cytokine storm and coagulopathy in COVID-19 patients. METHODS: A total of 24 RT-PCR positive COVID-19 patients were recruited and randomly divided into three groups (Gr): Gr. 1 control (n=8) – Standard treatment; Gr. 2: Standard treatment + AFO-202 beta glucan (n=8); and Gr. 3, Standard treatment + combination of AFO-202 and N-163 beta glucans (n=8) for 30 days. RESULTS: There was no mortality or requirement of ventilation of the subjects in any of the groups. There was a decrease in D-Dimer values (751 ng/ml to 143.89 ng/ml) and IL-6 values (7.395 pg/ml to 3.16 pg/ml) in Gr. 1 in 15 days but the levels increased to abnormal levels on day 30 (D-Dimer: 202.5 ng/ml; IL-6 55.37 pg/ml); which steadily decreased up to day 30 in groups 2 (D-dimer: 560.99 ng/dl to 79.615; IL-6: 26.18 pg/ml to 3.41 pg/ml) and 3 (D-dimer: 1614 ng/dl to 164.25 ng/dl; IL-6: 6.25 pg/ml to 0.5 pg/ml). The same trend was observed with ESR. LCR and LeCR increased while NLR decreased significantly in Gr. 3. CD4+ and CD8+ T cell count showed relatively higher increase in Gr.3. There was no difference in CRP within the groups. CONCLUSION: As these beta glucans are well known food supplements with a track record for safety, larger multi-centric clinical studies are recommended to validate their use as an adjunct in the management of COVID-19 and the ensuing long COVID-19 syndrome.

COVID-19 is caused by the novel strain of enveloped RNA virus, SARS-CoV-2 (Levy and Talbot, 1995) with symptoms including fever, cough, shortness of breath, fatigue, body aches, headache, loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting and diarrhoea. The COVID-19 is an ongoing pandemic with nearly 200 million people affected worldwide and more than four million deaths to date (WHO Coronavirus (COVID-19) Dashboard). Many patients are hospitalized 10 to 12 days after a positive RT-PCR test (Thomas et al., 2021) . Recovery occurs within 14 days in most of the affected patients, but it may take more than 25 days in some patients (Sabico et al., 2021) . Even patients with mild symptoms may progress to requiring oxygen therapy and mechanical ventilation and/or leading to death. The progression to J o u r n a l P r e -p r o o f 8 severe disease is attributed to the hyperactive inflammatory response leading to a cytokine storm, thereby causing organ damage (Soni et al., 2020) . A dysfunctional immune-coagulation response activating the coagulation cascade leading to a hypercoagulable state may cause adverse clinical outcomes including death. Patients with co-morbidities such as diabetes, dyslipidemia and risk of coagulation are prone to a rapid progression of the disease and an immune-inflammatory-coagulation dysregulation-related adverse outcome (Soni et al., 2020) . Several interventions are part of the standard treatment regimen, including pharmacological agents such as anti-virals, IL6 inhibitors, anti-coagulants, steroids and supplements such as vitamins or zinc (Solidarity clinical trial). The solidarity trial, an international clinical trial to help find an effective treatment for COVID-19 continues to generate evidence for the use of these interventions, and the search for effective COVID-19 therapeutics continues (Solidarity clinical trial). Many of the pharmacological interventions have associated side effects, and in regard to the supplements, zinc or ascorbic acid have not been able to significantly decrease the duration of symptoms compared with standard care (Thomas et al., 2021) .

Given this background, the biological-response modifier glucans (BRMG) such as the Aureobasidium pullulans produced beta glucans have been suggested as an alternative This BRMG has been reported to reduce the levels of IL-1β, IL-2, IL-4, IL-6, IL-12, TNF-α, IFN-γ, and sFasL while increasing IL8 and sFAS, thereby exerting an effective optimal defence against viral infection without hyperinflammation (Ikewaki et al., 2007) .

The metabolic effects of the A. pullulans beta glucan in normalizing blood glucose and lipid levels add to their benefits for use in COVID-19 as fasting blood glucose is an independent outcome of COVID-19 severity and mortality ( a . The potential of this BRMG in acting as a prophylactic supplement to help combat coagulopathy associated with COVID-19 has also been described ( b Ikewaki et al.,

In an animal study using healthy SD rats, AFO-202 beta glucan was beneficial in decreasing neutrophil to lymphocyte ratio (NLR) while increasing lymphocyte to CRP ratio (LCR) and the leukocyte-CRP ratio (LeCR 

The present study was an open label, prospective, randomized, comparative, multiple arm pilot clinical study to evaluate the immune enhancement and immunomodulatory efficacy of supplementation with A. pullulans' novel strains produced beta glucans compared to those who undergo a conventional therapeutic regimen alone in adult subjects with COVID-19 caused by SARS-CoV2(B-CoV).

Adult subjects between 18 and 65 years (both ages and sexes inclusive) who were confirmed to be positive for SARS-CoV2 by way of RT-PCR with or without comorbidities, and with mild to moderate COVID-19 symptoms (ICMR COVID-19 management protocol) but who required hospitalization were included in the study.

Severe COVID-19-affected patients requiring intensive care, children and pregnant women were excluded.

There were three groups (Gr.) comprised of eight subjects each (Total n =24). Results are presented as mean or mean ± standard deviation for the continuous normal J o u r n a l P r e -p r o o f variables. Independent sample T-test and Kruskal-Wallis Test was used for comparison between the groups. A p-value < 0.05 was considered significant.

The baseline characteristics of the patients are presented in Table 1 The oxygen saturation increased in all the groups in the 15-and 30-day follow-up but the difference was not significant. The temperature and heart rate also showed no significant difference among the groups (figure 1).

In the control group, the mean fasting blood glucose (FBG) levels on day 0 were 210.14 mg/dl which decreased to 121.5 mg/dl on day 30. In Gr. 2, the mean FBG decreased from 154.14 mg/dl to 103 mg/dl in 30 days. In Gr. or post-COVID sequalae, especially in those with co-morbid conditions, having a safe track record for the past two decades. 

The intestinal microbiota as a therapeutic target in hospitalized patients with COVID-19 infection

Nutraceutical functions of beta-glucans in human nutrition

Interleukin-6 in Covid-19: A systematic review and meta-analysis. Reviews in medical virology

Current Concepts in Molecular Biology and Pathogenesis

Covid-19: Has India's deadly second wave peaked?

Gut microbiota and Covid-19-possible link and implications

Laboratory features of severe vs. non-severe COVID-19 patients in Asian populations: a systematic review and meta-analysis

How Delta variant is causing new spikes across the world?

How Dangerous Is the Delta Variant (B.1.617.2)?

ICMR COVID-19 Management protocol

Immunological actions of Sophy beta-glucan (beta-1,3-1,6 glucan), currently available commercially as a health food supplement

Biological response modifier glucan through balancing of blood glucose may have a prophylactic potential in COVID-19 patients

Coagulopathy associated with COVID-19 -Perspectives & Preventive strategies using a biological response modifier Glucan

beta glucans: wide-spectrum immune-balancing food-supplement-based enteric (β-WIFE) vaccine adjuvant approach to COVID-19

Beneficial immuneregulatory effects of novel strains of Aureobasidium pullulans AFO-202 and N

PREPRINT (Version 1) available at Research Square

Hepatoprotective effects of Aureobasidium pullulans derived Beta 1,3-1,6 biological response modifier glucans in a STAM-animal model of non-alcoholic steatohepatitis

Immune and metabolic beneficial effects of Beta 1,3-1,6 glucans produced by two novel strains of Aureobasidium pullulans in healthy middle-aged Japanese men: An exploratory study

Commentary: Beyond "TRIM" Benefits of β-Glucan by Blood Glucose and Lipid Balancing Potentials in Its Defense Against COVID-19

Vitamin D3 to Treat COVID-19: Different Disease, Same Answer

Oxygen saturation as a predictor of mortality in hospitalized adult patients with COVID-19 in a

Anticoagulation and bleeding risk in patients with COVID-19

Unexplained elevation of erythrocyte sedimentation rate in a patient recovering from COVID-19: A case report

Role of Immune Dysregulation in Increased Mortality Among a Specific Subset of COVID-19 Patients and Immune-Enhancement Strategies for Combatting Through Nutritional Supplements

Effects of a 2-Week 5000 IU versus 1000 IU Vitamin D3 Supplementation on Recovery of Symptoms in Patients with Mild to Moderate COVID-19: A Randomized Clinical Trial

Role of interleukin 6 as a predictive factor for a severe course of Covid-19: retrospective data analysis of patients from a long-term care facility during Covid-19 outbreak

Vitamin D: A simpler alternative to tocilizumab for trial in COVID-19?

D-dimer level is a useful predictor for mortality in patients with COVID-19: Analysis of 483 cases

Solidarity" clinical trial for COVID-19 treatments

Vitamin D supplementation for the treatment of COVID-19: a living systematic review. The Cochrane database of systematic reviews

Effect of High-Dose Zinc and Ascorbic Acid Supplementation vs Usual Care on Symptom Length and Reduction Among Ambulatory Patients With SARS-CoV-2 Infection: The COVID A to Z Randomized Clinical Trial

WHO Coronavirus (COVID-19) Dashboard

To immunosuppress: whom, when and how? That is the question with COVID-19

May omega-3 fatty acid dietary supplementation help reduce severe complications in Covid-19 patients

Clinical characteristics and predictive value of lower CD4+T cell level in patients with moderate and severe COVID-19: a multicenter retrospective study

Unique immunological profile in patients with COVID-19

D-dimer level is associated with the severity of COVID-19

The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes

Standard treatment + AFO-202 beta glucan supplementation); and D. Gr. 3: (Standard treatment + AFO-202 and N-163 beta glucan supplementation) at baseline (day 0), day 15 and day 30

A-C; Serum Interleukin (IL)-6 levels in subjects with COVID-19

3: (Standard treatment + AFO-202 and N-163 beta glucan supplementation) at baseline (day 1), day 15 and day 30 wherein the decrease was statistically significant in Gr

AFO-202 beta glucan supplementation)

N-163 beta glucan supplementation) at baseline (day 0), day 15 and day 30 wherein the decrease was statistically significant in Gr

Standard treatment + AFO-202 beta glucan supplementation) and I. Gr. 3: (Standard treatment + AFO-202 and N-163 beta glucan supplementation

Decrease in neutrophil to lymphocyte ratio (NLR) values in subjects with COVID-19 A. Gr. 1: Control

N-163 beta glucan supplementation) at baseline (day 0), day 15 and day 30. D-F: Increase in Lymphocyte to c-reactive protein (LCR) values in subjects with

Standard treatment + AFO-202 beta glucan supplementation)

3: (Standard treatment + AFO-202 and N-163 beta glucan supplementation)

Leukocyte to c-reactive protein (LeCR) values in subjects with COVID-19

Standard treatment + AFO-202 beta glucan supplementation); and I

CD8+ T cell counts at baseline (Day 0), day 15 and day 30 in the different treatment arms of COVID-19 subjects showing increase

Reviewing and editing, Rajappa Senthilkumar -Formal analysis, Gary A. Levy and Senthilkumar Preethy -Writing original draft, Samuel JK Abraham -Conceptualization and writing original draft Highlights:  Multiple-arm clinical trial of beta 1,3-1,6 glucans in COVID-19 patients