Do Zinc Supplements Enhance the Clinical Efficacy of Hydroxychloroquine?: a Randomized, Multicenter Trial.

No specific treatment for COVID-19 infection is available up till now, and there is a great urge for effective treatment to reduce morbidity and mortality during this pandemic. We aimed to evaluate the effect of combining chloroquine/hydroxychloroquine (CQ/HCQ) and zinc in the treatment of COVID-19 patients. This was a randomized clinical trial conducted at three major University hospitals in Egypt. One hundred ninety-one patients with a confirmed diagnosis of COVID-19 infection were randomized into two groups: group I (96) patients received both HCQ and zinc, and group II (95) received HCQ only. The primary endpoints were the recovery within 28 days, the need for mechanical ventilation, and death. The two groups were matched for age and gender. They had no significant difference regarding any of the baseline laboratory parameters or clinical severity grading. Clinical recovery after 28 days was achieved by 79.2% in the zinc group and 77.9% in zinc-free treatment group, without any significant difference (p = 0.969). The need for mechanical ventilation and the overall mortality rates did not show any significant difference between the 2 groups either (p = 0.537 and 0.986, respectively). The age of the patient and the need for mechanical ventilation were the only risk factors associated with the patients' mortality by the univariate regression analysis (p = 0.001 and < 0.001, respectively). Zinc supplements did not enhance the clinical efficacy of HCQ. More randomized studies are needed to evaluate the value of adding zinc to other therapies for COVID 19. ClinicalTrials.gov Identifier: NCT04447534.

Introduction

The world has witnessed an increasing number of cases with COVID-19 infection since December 2019, reaching more than 20 million infected people worldwide [1-7]. COVID-19 infection is caused by a highly contagious single-stranded RNA virus called the SARS-CoV-2 virus [1], which is transmitted mainly through droplets, aerosol, and close contact [2-4]. Despite the fact that the respiratory system is the primarily affected organ, SARS-CoV-2 can affect many other organs [5-7].

No specific treatment for COVID-19 infection is available up till now, and there is a great urge for effective treatment to reduce morbidity and mortality during this pandemic. Chloroquine (CQ) and hydroxychloroquine (HCQ) can prevent SARS-CoV-2 infection by changing endosomal pH required for virus/cell fusion, together with altering glycosylation of cellular receptors of SARS-CoV [8]. There is a debate in the literature regarding the efficacy of CQ and HCQ in the treatment of COVID-19 infection [9-12]. Adding azithromycin to CQ and HCQ was reported to reduce the complications and fatality rates, and reduce the viral load in COVID-19 patients [7-22]. These reports resulted in emerged ideas calling for combining CQ and HCQ with other drugs in the treatment of COVID-19.

Zinc is essential for different cellular and enzymatic activities, as well as being a necessary cofactor for many viral proteins [15-25]. Zinc was also proved to inhibit RNA-dependent RNA polymerase of SARS-CoV in cell culture [17]. Chloroquine is known to increase the intracellular concentrations of zinc, and thus enhance its effect [18].

Dietary plant polyphenols such as the flavonoids quercetin (QCT) and epigallocatechin-gallate act as antioxidants and as signaling molecules. Remarkably, the activities of numerous enzymes that are targeted by polyphenols are dependent on zinc. Husam and his colleagues have previously shown that these polyphenols chelate zinc cations, and they hypothesized that these flavonoids might also be acting as zinc ionophores, transporting zinc cations through the plasma membrane [19].

Eight studies are registered on clinicaltrials.gov to evaluate the efficacy of zinc with hydroxychloroquine either in the treatment of or prophylaxis against COVID-19 infection. Only one study is completed, 4 studies are still recruiting patients, and 2 studies did not start recruitment yet, while one study was withdrawn. None of these registered studies has shown the results up till now.

We aimed to evaluate the effect of combining CQ/HCQ and zinc in the treatment of COVID-19 patients.

Methods

This was a randomized controlled study conducted in three Egyptian tertiary care centers in Assiut, Tanta, and Cairo. A written informed consent was taken from each participant in this trial. Approval of the Institutional ethical committee was taken before starting the trial. The trial was registered on clinicaltrials.gov (ClinicalTrials.gov Identifier: NCT04447534).

This study included patients with a confirmed diagnosis of COVID-19 infection by real-time PCR test during the period between 23 June and 23 August 2020. All the included patients were classified into mild, moderate, severe, and critical according to the WHO case severity classification for COVID-19 infection. Mild cases constituted of patients with symptoms for COVID-19 infection but not complicated with pneumonia or hypoxia. Moderate cases included patients with mild viral pneumonia and SpO2 > 90% on room air. Severe cases involved patients with signs of severe pneumonia such as respiratory rate > 30 breaths/min, severe respiratory distress, or SpO2 < 90% on room air. Finally, critical cases included patients with acute respiratory distress syndrome sepsis and septic shock [26].

One hundred ninety-one patients with a confirmed diagnosis of COVID-19 infection were equally randomized into two groups: group I: zinc group, included 96 patients who received both HCQ and zinc and group II (without zinc group), included 95 who received HCQ only. Patient with hypokalemia or hypomagnesemia, porphyria, neutrophilia, myasthenia gravis, maculopathy or changes in the visual field, heart failure, prolonged QT interval in ECG, liver cirrhosis, psoriasis, epilepsy, anemia from pyruvate kinase and G6PD deficiencies, chronic kidney disease, and pregnant or lactating females were excluded from this study. Collected data included history, clinical examination, laboratory investigation at admission, and follow-up during the hospitalization period. Primary outcome measures included recovery within 28 days, need for mechanical ventilation, and death.

Hydroxychloroquine was given in a dose of 400 mg twice daily on the first day, then 200 mg twice daily for 5 days, while zinc was given in a dose of zinc sulfate 220 mg (50 mg of elemental zinc) twice daily as many clinical trials did. Both groups received the standard of care treatment for COVID-19 infection, according to the Egyptian Ministry of Health guidelines for 15 days. This study was approved by the Ethics Committee of the Faculty of Medicine, Tanta University. The privacy and confidentiality of the data for participated patients were guaranteed.

Statistical Analysis

The normality of the variables was tested by the Shapiro-Wilk test. Statistical Package for Social Sciences (SPSS) V. 23 was used for data analysis. Data were expressed in number (No.), percentage (%), mean (x̅), and standard deviation (SD). Student’s t test was used for normally distributed continuous variables and Mann-Whitney’s test for not normally distributed ones. Chi-square test (χ2) was used to study the association between qualitative variables, and whenever any of the expected cells were less than five, Fischer’s exact test was used. Binary logistic regression was used to ascertain the effect of the potential risk factors on the patients’ mortality. Two-sided p value < 0.05 was considered statistically significant.

Sample Size Calculation

The sample size calculation was based on a previous study by Shah et al. 2012, who studied the effect of zinc on severe pneumonia. The required sample was 86 patients in each group with 0.8 as the probability (power), 0.05 as type I error probability, 3 as the difference in the mean duration of pneumonia between the two groups, and 1:1 ratio of experimental to control subjects. The sample size was inflated by 10% to compensate for the dropouts [27].

Results

The two groups were matched for age and gender (p = 0.940 and 0.062, respectively). They had no significant difference regarding smoking, associated comorbidities, or clinical severity grading (p 0.706, 0.384, 0.781, respectively) (Table 1).

Table 1

Baseline patients’ characteristics in the two groups

Character	Zinc group 1 (n = 96)No. (%)	Without zinc group (n = 95)No. (%)	p value
Age in years (mean ± SD)	43.48 ± 14.62	43.64 ± 13.17	0.940
Gender
Male	52 (54.2)	64 (67.4)	0.062
Female	44 (45.8)	31 (32.6)
Smoking	42 (44.2)	39 (41.05)	0.706
Comorbidities
No	35 (36.5)	42 (44.2)	0.076
HTN	21 (21.9)	16 (16.8)
Diabetes	19 (19.8)	7 (7.4)
Hepatic	6 (6.25)	9 (9.5)
Multiple	15 (15.6)	21(22.1)
Clinical severity grading
Mild	9 (9.4)	12 (12.6)
Moderate	58 (60.4)	55 (57.9)	0.781
Severe	18 (18.8)	20 (21.1)
Critical	11 (11.6)	8 (8.4)

*n, number; HTN, hypertension; SD, standard deviation

The laboratory investigations did not show any significant difference between the two groups. This is detailed in Table 2.

Table 2

Laboratory investigations in the two groups

Investigation	Zinc group 1 (n = 96)Mean ± SDMedian	Without zinc group (n = 95)Mean ± SDMedian	p value
Hemoglobin	13.38 ± 1.95 13.65	13.77 ± 1.70 13.50	0.143
Platelets	228.48 ± 80.06 219	249.62 ± 82.50 244.0	0.052
WBCs	5.55 ± 3.13 4.55	5.63 ± 2.92 4.50	0.599
Direct bilirubin	0.24 ± 0.09 0.20	0.25 ± 0.09 0.20	0.280
Indirect bilirubin	0.49 ± 0.21 0.40	0.53 ± 0.22 0.50	0.087
Albumin	4.18 ± 0.27 4.10	4.19 ± 0.29 4.10	0.861
ALT	46.64 ± 38.92 36.50	36.58 ± 20.93 28.0	0.057
AST	37.49 ± 40.51 28.50	39.58 ± 26.62 30.0	0.053
D-dimer	1.49 ± 4.55 0.360	0.88 ± 2.56 0.40	0.346
Ferritin	373.31 ± 414.00 262.90	317.01 ± 220.50 234.00	0.390
Creatinine	1.01 ± 0.14 0.92	0.96 ± 0.28 0.90	0.119
CRP	28.85 ± 62.90 12.10	14.92 ± 17.16 9.00	0.967

*ALT, alanine transaminase; AST, aspartate transaminase; CRP, C-reactive protein; WBCs, white blood cells; n, number; SD, standard deviation

There was no significant difference between the two groups regarding the clinical course or any of the different outcomes. The mean duration of hospital stay was 13.51 ± 5.34 days in the zinc group and 14.01 ± 6.26 days in the zinc-free group (p = 0.553). Seventy-six patients (79.2%) in the zinc group and 74 patients in the zinc-free group showed complete recovery after 28 days (p = 0.969). Four patients in the zinc group and 6 patients in the zinc-free group needed mechanical ventilation (p = 0.537). The overall mortality did not significantly differ in the two groups either, as 5 patients died in each group with p = 0.986 (Table 3).

Table 3

Clinical course and outcomes of the two groups

Clinical course	Zinc group 1 (n = 96)Mean ± SD	Without zinc group (n = 95)No. (%)Mean ± SD	p value
Duration of hospital stay in days	13.51 ± 5.34	14.01 ± 6.26	0.553
	No. (%)	No. (%)
Recovery after 28 days	76 (79.2)	74 (77.9)	0.969
Need for mechanical ventilation	4 (4.2)	6 (6.3)	0.537
Fate
Survived	91 (94.8)	90 (94.7)	0.986
Died	5 (5.2)	5 (5.3)

*n, number; SD, standard deviation

The univariate analysis revealed that the patients’ age and the need for mechanical ventilation were the only risk factors significantly associated with mortality (p = 0.001 and < 0.001, respectively). The addition of zinc to HCQ did not considerably affect the overall COVID-19 mortality in this study (p = 0.986) (Table 4).

Table 4

Regression analysis of the effect of potential risk factors on the patients’ mortality

Variable	Univariate
	p value	OR	95% CI
			Lower	Upper
Age	0.001	1.097	1.040	1.157
Gender	0.180	0.411	0.112	1.507
Smoking
ALT	0.842	0.998	0.976	1.020
Albumin	0.370	0.303	0.022	4.127
Creatinine	0.277	0.856	0.646	1.133
Ferritin	0.393	1.001	0.999	1.002
CRP	0.785	0.996	0.967	1.026
Need for mechanical ventilation	< 0.001	138.44	23.592	812.427
DM	0.785	0.996	0.967	1.026
Zinc treatment	0.986	0.056	0.277	3.534

*ALT, alanine transaminase; CRP, C-reactive protein; n, number; SD, standard deviation; OR, odds ratio; DM, diabetes mellitus

Discussion

In Egypt, there have been an increasing number of cases with COVID-19 infection since March 2020. Many treatment protocols were updated to treat the coronavirus infection based on the evidence available at this time. The initial protocols were primarily dependent on hydroxychloroquine. In the Egyptian leading university hospitals, we aimed to evaluate the effect of combining CQ/HCQ and zinc in treating COVID-19 patients.

The treatment teams in the Egyptian universities, which incorporated infectious diseases consultants and clinical pharmacists, adopted the importance of integration of zinc into the treatment protocol of treatment. To the best of our knowledge, this is the first clinical trial investigating the role of the addition of zinc to hydroxychloroquine in the treatment of COVID 19 patients.

The main hypothesis behind this approach was the fact that zinc was proven to have an inhibitory effect on the RNA-dependent RNA polymerase of SARS-CoV in cell culture [17, 18]. Moreover, CQ and HCQ are known to increase the intracellular concentrations of zinc and thus enhance its effect [18].

Despite these proved benefits of zinc in the literature, this study found that zinc supplements did not enhance the clinical efficacy of HCQ.

There are a lot of questions now about the efficacy of CQ or HCQ in the treatment of COVID 19 patients. A recent randomized study found that adding HCQ to standard care did not add significant benefit, did not decrease the need for ventilation, and did not reduce mortality rates in COVID-19 patients [11]. A recent meta-analysis found that hydroxychloroquine alone did not reduce mortality in hospitalized COVID-19 patients, and even when added to azithromycin, this was significantly associated with increased mortality [28].

This study’s major strength is that being the first randomized study to evaluate the effect of combining hydroxychloroquine (HCQ) and zinc in the treatment of COVID-19 patients.

On the other hand, the study’s limitations may be depending mainly on the patients’ clinical outcomes and not the viremic response. However, this is due to the limited resources in such a developing country. Another limitation is that zinc absorption may be limited with high phytate diet, and other medications and serum zinc were not measured before, during, or after treatment in tis clinical trial.

In conclusion, zinc supplements did not add value or enhance the clinical efficacy of HCQ. Zinc supplementation may be studied further with other drug regimens for COVID 19, but it did not add any clinical values when added to HCQ.