key: cord-0861944-yzllipq5
authors: Sukkar, SG; Cogorno, L; Pisciotta, L; Pasta, A; Vena, A; Gradaschi, R; Dentone, C; Guiddo, E; Martino, E; Beltramini, S; Donini, L.M.; Carmisciano, L; Sormani, MP; Bassetti, M
title: Clinical efficacy of eucaloric ketogenic nutrition in the Covid-19 cytokine storm (CSS): a retrospective analysis of mortality and Intensive Care Unit admission
date: 2021-03-07
journal: Nutrition
DOI: 10.1016/j.nut.2021.111236
sha: 47dafc66e8581624221388f9cdd1dc925f61a580
doc_id: 861944
cord_uid: yzllipq5

OBJECTIVE: : Primary objective is to explore the effect of eucaloric ketogenic diet (EKD) on mortality, admission to intensive care unit (ICU) and the need for non-invasive ventilation (NIV) in hospitalized patients affected by COVID-19 in comparison to eucaloric standard diet (ESD). Secondary objectives are: the verification of safety and feasibility of the diet and its impact on inflammatory parameters, in particular interleukin-6 (IL-6). RESULTS: : The preliminary multivariate analysis shows a statistical significance in survival (P=0,046) and need for ICU (P=0,049) with EKD compared with ESD. Considering EKD start day as a "time-dependent" variable, however, the results maintain a positive trend towards the application of the diet and it is not possible to reject the null hypothesis (p <0.05). IL-6 concentrations between t 0 and t 7 (seven days after the beginning of the diet) in the ketogenic nutrition group shows a trend almost significant (P = 0.062). EKD was safe and no adverse events were observed. CONCLUSIONS: : These results show a possible therapeutic role of EKD in the clinical management of COVID-19. Currently, a prospective controlled randomized trial is running out in order to confirm these preliminary data.

COVID-19 is a pandemic disease caused by SARS-CoV-2 virus that is characterized by respiratory and gastrointestinal symptoms 1 and by an all-cause in-hospital mortality of 43.6% (120/275) according to recent published experience, between 25 February 2020 and 25 March 2020 2 .

In a subgroup of patients, a cytokine storm syndrome (COVID-19 CSS) characterized by fulminant and fatal hypercytokinemia associated with multiorgan failure seems to be one of the most important precipitating factors of the disease 3 . Moreover, several risk factors, such as elderly, obesity and multiorgan comorbidity, have been associated to worst outcomes, and severe infection [4] [5] [6] .

Currently, there is no proven drug for the treatment of COVID-19 CSS. From the beginning of the pandemic, the approaches aimed at the control of hyperinflammation due to CSS are antiinflammatory therapies such as corticosteroids, interleukin-6 inhibitors, anti-GM-CSF, PD-1.

Recently, BB1 checkpoint inhibitors, hydroxychloroquine (HCQ), cytokine adsorption devices and intravenous immunoglobulin (IVIG) [7] [8] [9] have been proposed.

According to the WHO, systemic steroids are the only proven therapy in critical and severe COVID-19 10 , therefore, any possible alternative treatment could be investigated.

Recently, we proposed an immunometabolic hypothesis identifying a treatment capable of reducing the state of hyperinflammation associated with SARS-CoV-2 infection 11 .

There is an increasing evidence that macrophages, including resident alveolar macrophages (AMs) and macrophages recruited from blood, have a crucial role in ARDS pathogenesis 12 .

In a healthy state, AMs located in the interface between air and cellular tissue are the prevalent population in the alveolus. The M2 phenotype is the main represented form of these cells which have immunosuppressive functions and for which free fatty acids are an optimal energy fuel.

During an injury, as the exudative phase of ARDS, peripheral blood monocytes are recruited into the alveolar lumen, then turn into macrophages with M1 phenotype and finally release various potent proinflammatory mediators (macrophage inflammatory protein-2 and interleukin-8) which attract neutrophils into alveolar space and are responsible of the tissue damage as happens in ARDS.

Moreover, together with neutrophils, pulmonary activated platelets play a crucial thromboinflammatory role by forming platelet-neutrophil complexes (PNCs) and monocyte-platelet aggregates, causing the development of a procoagulant and pro-inflammatory environment 13 .

During the M1 phenotype activation there is a metabolic shift from oxidative phosphorylation (OXPHOS) to aerobic glycolysis (Warburg effect) with consequent increase in lactate production which 14 leads a decrease in type I interferon (IFN), a well-known defense mechanism against viruses 15 .

This study is a retrospective analysis of patients suffering from SARS-CoV-2 disease who were admitted to IRCCS San Martino Hospital between February and July 2020, with a peak in hospital admission in March 2020, and who underwent a ketogenic diet. In this regard, in the Infectious Disease Unit, a ketogenic diet entered the routine protocol of the ward in the absence of indications or contraindications for a specific diet in COVID-19, according to its anti-inflammatory role.

All patients signed consent for the use of personal treatment data and informed consent to undergo any type of therapy during their hospitalization.

They were also informed that a ketogenic diet entered the routine protocol of the ward, in the absence of contraindications, and had the possibility of accepting or refusing immediately or thereafter, in case of poor palatability or taste. The pharmacological protocol was not conditioned by the choice of the diet.

The exclusion criteria for EKD were type I diabetes mellitus; insulin-dependent type II diabetes or type II diabetes in treatment with sulfonylureas, repaglinide, GLP-1 analogs, SGLT2 inhibitors, or recent ASCVD (within one month); food allergies to the diet components; any metabolic disorder that can affect gluconeogenesis; clinical history of severe hypertriglyceridemia with or without pancreatitis; and pregnancy or lactation.

Meanwhile, an RCT, with the purpose of studying the EKD in a larger sample of subjects in the whole hospital, randomizing the nutritional treatment, was submitted and approved in June by the ethics committee (KETOCOV-1 Register number CER Liguria: 198/2020 -DB id 10517; ClinicalTrials.gov identifier (NCT number:04492228), and it was started at the end of September 2020 with the recrudescence of the infection in Italy.

Considering the approval of the RCT by the Ethics Committee, at the end of the first wave of the pandemic in July, the data of the patients treated in the Infectious Disease Unit who followed the routine diet protocol with the EKD for a minimum period of 2 weeks were analyzed.

To avoid the confounding effect of interfering variables between the two diet groups, a one-to-two propensity score-matching analysis was performed with patients treated in other facilities and made available to a single management software to have adequate controls for a valid statistical analysis (see paragraph 2.4 Statistical Analysis).

Inclusion criteria to enter in the analysis of the data were as follows: documented diagnosis of COVID-19 defined by a positive RT-PCR assay result of a respiratory sample, P/F >100 (arterial oxygen concentration to the fraction of inspired oxygen) or mild-moderate ARDS according to "The ARDS Definition Task Force, Acute Respiratory Distress Syndrome: The Berlin Definition" 16 and age older than 18 years.

The initial sample of 669 patients was reduced to 479 patients (297 males and 182 females) because 190 patients had missing data (118) or did not meet the inclusion criteria (72). After the propensity score-matching analysis, 34 EKD patients were included in the study and compared with 68 SD patients.

All patients were investigated for demographic data and the presence of the following comorbidities: diabetes, hypertension, ASCVD, heart failure, chronic pulmonary disease, solid and hematological neoplasia, ulcerative disease, moderate/severe liver disease, dementia, collagen diseases, metastatic neoplasia, and hemiplegia. The Charlson Comorbidity Index (CCI) has been used as a measure of 1-year mortality risk [12] and was calculated for all subjects. The laboratory data and P/F ratio (arterial oxygen concentration to the fraction of inspired oxygen) were taken into account on the day of hospital admittance for the patients considered the control group (fed a standard diet) and the day before the administration of the ketogenic diet for patients in the studied group.

Laboratory data and the P/F ratio (arterial oxygen concentration to the fraction of inspired oxygen) were taken into account on the day of hospital admittance for the patients considered the control group (fed a standard diet) and the day before the administration of EKD for patients in the studied group.

Laboratory data required by the ward doctor included routine blood tests (blood cell count, azotemia, creatinine, AST/GOT, CPK, LDH, albumin, triglycerides, IL-6, PCR, ferritin, lipid profile, fibrinogen, blood sugar, HbA1c (basal), vitamin D (basal)), urine test (basal) and complete urine analysis.

The study was conducted in accordance with the Declaration of Helsinki. 

Patients included in the analysis belonged to two different dietary groups: the standard diet group, including subjects fed the ESD, and the ketogenic diet group, including subjects fed the EKD.

According to the LARN (Reference Intake of Nutrients and Energy for Italian Population) and to the Italian guidelines for a healthy diet 17,18, the standard oral diet was based on the Mediterranean style 19 and was characterized by 30 kcal/kg/day (ranging from 1800 to 2100 kcal), protein intake of 16-20%, lipid intake of 26-30% and carbohydrate intake of 42-50%.

The EKD ranged from 1800 to 2100 kcal, it was characterized by a very low carbohydrate amount (< 30 g, 5-6% of total energy) to induce ketosis, with a polyunsaturated/unsaturated/saturated fat ratio of 3:2:1. The protein content of the ketogenic diet was similar to that of an average Mediterranean diet (17-18% of the total calories). The average protein intake for ESD was about of 86,5 ± 5,5 grams meanwhile the average protein intake for EKD was of 89,4 ± 2,5 grams.

The primary outcomes were 30-day mortality, ICU admission and the need for CPAP, and they were also considered together in a combined outcome.

Secondary outcomes were the effects of the EKD on biological and inflammatory parameters and, in particular, on IL-6.

The To adjust for baseline differences that are intrinsic in nonrandomized studies, patients on the EKD diet (34 patients) were matched 1:2 to patents following a standard diet (68 patients) by a propensity score (PS) ( Table 1) .

Preliminarily, covariates or factors entered into the model were identified by univariate statistical analysis, and the probability value for inclusion was p ≤ 0.05 between 445 and 34 subjects fed the ESD and EKD, respectively. Table 1 .

The median age (IQR) was 67 (53-77) years, and no significant differences in demographics, comorbidity history, laboratory measures, concomitant pharmacotherapy or P/F values were detected between the two groups.

In our experience, the overall COVID 19 mortality was 21.6% (n. 22 out of 102 patients), while it was 27.9% (n.19 out of 68 patients) and 8.8% (n. 3 out of 34 patients) in patients fed ESD and EKD, respectively. A total of 14 out of 102 patients (13.7%) were admitted in ICU, but 19.1% (n. 13 out of 68 patients) and 2.9% (n. 1 out of 34 patients) of the patients fed with EKD and ESD, respectively, were admitted in ICU.

The explorative Cox regression analysis showed a significance association of both survival (P<0,027) and the need for ICU (P<0,025) with the different diets proposed ( Table 2 ).

The findings of the main Cox regression analysis are reported in Table 3 .

The 30-day mortality in the survival analysis showed a trend of lower risk in patients fed a ketogenic diet (HR 0.416, 95% CI 0.122 -1.413) than in subjects fed a standard diet, although this result did not reach statistical significance (P = 0.160) (Figure 2A) .

Moreover, the ketogenic diet had a trend of association with lower admission in the ICU (HR 0.357, 95% CI 0.045 -2.847, P = 0.331) in contrast to the standard diet ( Figure 2B ). were detectable between the two groups of dietary patterns. EKD-treated patients had a median IL-6 difference of -51.8 ρg/mL or a mean IL-6 difference of -169 ρg/mL (data from 22 of the 34 pairs) compared to controls.

After IL-6 imputation into delta "lower than" or "higher than or equal to" 0, the Binning IL-6 trend* (chi sq = 3.698, df = 1, p-value = 0.05447) ( Table 4, figure 3 ). EKD was safe and no adverse events were observed in patients fed an EKD. In particular acidosis was never observed and the arterial pH at baseline is similar in controls and EKD patients (p = 0.100).

At t7, compared to t0, arterial pH falls to a similar percentage in EKD patients compared to controls (57% vs 42%, p = 0.484).

The median variation of arterial pH is about +0.0075 in the controls vs -0.0100 in the EKD patients (p = 0.342).

Nutritional status appears to be a relevant factor influencing the outcome of patients with COVID-19, but little information has emerged about the impact of early nutritional support in pre-ICU patients on the course of the disease 20. Surely nutrition has a pivotal role in the prevention of the comorbidities most frequently associated with COVID-19, such as hypertension, cardiovascular and cerebrovascular disease and diabetes, which have been noted as twofold, threefold and twofold, respectively, higher in ICU/severe cases than in their non-ICU/severe counterparts 21 .

Nevertheless, obesity is associated with a worse prognosis in patients affected by COVID-19, especially among the young 22,4-5 .

A hyperinflammatory response to COVID-19 has been recognized as the main cause of morbidity and mortality in these patients 23 .

The genetic substrate of CSS has been recently suggested and alpha-1 antitrypsin deficiency alleles may contribute to national differences in COVID-19 infection.

The association between alpha-1 antitrypsin deficiency and severity and mortality rates has not yet been defined, and the exact pathophysiological mechanism that determines this process is unknown 24 .

However, COVID-19 CSS appears 8-10 days after the onset of symptoms of the disease and is characterized by high fever, dyspnea, bilateral pulmonary infiltrates that can evolve into ARDS and multisystemic organ failure 25 .

Effective treatments for COVID-19 and COVID-19 CSS are needed immediately. Patient timing and selection seem to be particularly crucial in managing the acute phase of COVID-19.

There is a consensus that ketosis protects healthy tissues against oxidative stress by simultaneously decreasing ROS production and increasing endogenous antioxidant capacity 22 . It is well known that the ketogenic diet can inhibit inflammation. Studies have shown that a KD reduces circulating inflammatory markers in humans 26 . The KD, via hydroxybutyrate (HB), is capable of activating hydroxycarboxylic acid receptor 2 (HCA2), a G protein-coupled receptor, which inhibits NF-kB in macrophages, dendritic cells and microglia and reduces neuroinflammation 27 .

Finally, from a clinical point of view, previous experiences show a clinical improvement in respiratory function following a ketogenic diet. After ten days of modified protein-saving fasting (a ketogenic diet with very low calorie content), a statistically significant improvement in functional residual capacity (FRC) and expiratory reserve volume (ERV) was observed 28 . In addition, a 20-day ketogenic diet shows a significant decrease in end-tidal carbon dioxide tension (PETCO2) 29 .

As reported in the introduction, the use of corticosteroids is currently suggested by the World Health Organization guidelines, and it is actually the first approach in severe disease 9 .

In our study, corticosteroid treatment was present in 75% of patients treated with the ESD and 85.3% of patients treated with the EKD.

Tocilizumab was present in 54.4% of patients treated with the ESD and in 67.6% of patients treated with EKD..

The anti-inflammatory efficacy of the EKD was almost significant and therefore seems independent from steroid or anticytokine treatment with tocilizumab. In fact, the analysis of the course of interleukin-6 in the first week of therapy highlights the almost significant variation in IL-6 from the beginning to seven days after the start of the EKD. IL-6 did not increase but rather tended to be slightly reduced in the group treated with an EKD (table 4; Figure 3 ). This trend underlines the fact that patients after one week were, at that time, at increased risk of COVID-19 CSS.

Dietary treatment is, in this regard, a possible immunomodulation mainly aimed at the activity of macrophages without interfering with antiviral clinical efficacy.

During the treatment, the patients did not present any adverse events related to diet.

A limitation of the study is the lack of controlled randomization, and the study was conducted in a single hospital facility. A further limitation is the number of cases included (34 patients in EKD vs 68 in SD) , since the cases were discontinued due to the absence of cases admitted from the end of July; therefore, it is possible that the study was underpowered.

The strength of the study is the usefulness of the propensity score, which, with the large hospital COVID-19 database (of approximately 669 cases), allowed a proper 1:2 matching, overcoming the absence of controlled randomization.

The current study provides preliminary data for all patients who were on a EKD during the epidemic period that ended at the end of July.

The preliminary multivariate analysis (table 2) demonstrated a significance of both survival (P=0,046) and the need for ICU (P=0,049) with EKD compared with standard diet (table 2) . Indeed, as reported, the rigorous statistical strategy used for the analysis of the present work data, even if retrospective, was based on the use of a Cox regression with time dependent variables and was developed to minimize the possibility of bias derived from the administration of the ketogenic diet (compared to standard hospital food) at different time distances from patients' hospitalization. By using the day of start of ketogenic diet as a variable "time-dependent" patients are all considered at zero time as controls and only when they start the ketogenic diet they are considered as cases (patients fed with ketogenic diet). Through this strategy, the effect of an adverse event/early outcome (in the first days) on a patient in the ketogenic group impacts more because the group is less numerous, conversely, if such an early occurrence affects a patient control its statistical effect is greatly diluted. In scientific work this strategy is not always applied and there may be a statistical distortion known as "Immortality Bias" where the potential distortion described above is not considered and data are analysed on static patient groups over the study period. In fact, not considering the ketogenic diet variable as "time-dependent" in our work all the results would reach the statistical significance (p <0.05). On the contrary, by eliminating the described Immortality Bias and applying a rigorous statistical analysis it was not possible to reject the null hypothesis (p <0.05). However the results maintain a positive trend towards the application of the ketogenic diet.

The reported data are therefore an exact picture of the actual state, and the trends of reduced mortality, reduced need for ICU admission and reduced IL-6 between 0 and 7 days, although not statistically significant, suggest a possible alternative treatment to the disease, in the absence of side effects, additional costs or risks for the patient.

Differently from other studies in which nutrition is considered a support to drug therapy, this study is the first to underline the role of clinical nutrition therapy as a pathophysiological support to drug therapy in improving the prognosis of not only COVID-19 but also in other infectious diseases in which immunomodulation could have a role in reducing hyperinflammation syndromes.

In conclusion, this retrospective pilot study provides valuable preliminary information regarding the possible role of an EKD in controlling mortality and ICU admission by means of the immunomodulation of COVID-19 CSS.

These data must necessarily be supported by further evidence from a larger sample, and the randomized controlled prospective clinical trial, currently started in September, with the recrudescence of COVID-19 infection in Italy, could be particularly useful.

No conflicts of interest exist.

The paper has been submitted to the Ligurian Ethical committee that approved its publication on November, 24 2020. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Table 1