Ivermectin compared with placebo in the clinical evolution of Mexican patients with asymptomatic and mild COVID-19: a randomized clinical trial

Despite the development and application of against SARS-CoV-2 the the to �nd some therapies to avoid or ameliorate the fatal evolution of the COVID-19. Since the publication of the potential use of Ivermectin as a treatment against the a pleiad of information about it has been published. However, the evidence is not strong or weak enough to conclude its usefulness in the clinical evolution of patients infected with SARS-CoV-2. We evaluate the e�cacy and safety of ivermectin in the treatment of Mexican patients with asymptomatic and mild COVID-19 in a three-day administration in comparison to placebo.

Despite the development and application of vaccines against SARS-CoV-2 around the world, the scienti c community is still trying to nd therapies to avoid or ameliorate the fatal evolution of COVID-19. Since the publication of the potential use of ivermectin as a treatment against the disease, 1 much information about it has been published. However, the evidence is not strong or weak enough to conclude its usefulness in the clinical evolution of patients infected with SARS-CoV-2. [2][3][4] Ivermectin has been described as a broad-spectrum antiviral, inhibiting nuclear import due to its ability to inactivate host nuclear transport proteins, such as integrase and NS5, limiting the ability of West Nile virus to infect at low concentrations. 5 It also inhibits the replication of yellow fever virus and other viruses, such as dengue, likely by attacking nonstructural helicase 3 activity. 4,6 The aim of the study was to evaluate the e cacy and safety of ivermectin in the treatment of patients with asymptomatic and mild COVID-19 in a three-day administration in comparison to placebo.

Study design and participants
The protocol was approved by the local ethics, biosafety and investigation committees of the Investigación Biomédica para el Desarrollo de Fármacos S.A de C.V. and the Mexican health ministry COFEPRIS: 203301410A0055. The procedures were conducted in compliance with the Helsinki Declaration and Good Clinical Practice guidelines. Written informed consent was obtained from all patients. The study is registered on ClinicalTrials.gov: NCT04407507. The study adheres to CONSORT guidelines and include a completed CONSORT checklist as an additional le.
A randomized, double-blind, placebo-controlled trial was conducted to determine the e cacy and safety of ivermectin among subjects with asymptomatic and mild COVID-19. Subjects were included in two different sites in Guadalajara and Zapopan, Mexico: Hospital Hispano and Investigacion Biomedica para el Desarrollo de Farmacos (Ibiomed).

Participants
Eligible participants were > 18-year-old men and women diagnosed with SARS-CoV-2 infection by realtime polymerase chain reaction (RT-PCR) testing of nasopharyngeal swab samples. We considering viral load undetectable if the inferior limit was ≥ 40 copies/µL. Patients with moderate or severe COVID-19, 7 diagnosis of other respiratory infections, impaired liver function tests (> 5 times above the normal level of alanine aminotransferase or aspartate aminotransferase), history of recurrent urinary tract infections, pregnancy or nursing women, active participation in other clinical trials, and use of antibiotics, verapamil, cyclosporine A, tri uoperazine or antiparasitic treatment for a concomitant disease were excluded. Moreover, subjects with a reported allergy or sensitivity to ivermectin, or acetaminophen or its use during the protocol were also excluded.

Randomization
Participants were randomly allocated 1:1 to receive oral ivermectin or placebo using a macro in Microsoft Excel (version 16.38; Microsoft Corporation, Redmond, WA, USA) with random numbers. The investigators or study coordinators enrolled and assigned participants to intervention. An unblinded pharmacist provided masked intervention according to permuted blocks of 2 in the randomization sequence. The rest of the clinical staff, investigators, and participants were blinded to the assignment.

Interventions
Patients received 12 mg per day ivermectin tablets or placebo for 3 days. Both groups receiving 500 mg acetaminophen tablets four times a day for 14 days to eliminate symptom bias. Ivermectin (Ivexterm) and placebo were provided by the drug manufacturer in Mexico (Grossman Laboratories).

Outcomes measures
The primary endpoint was the proportion of subjects without disease progression to severity according to COVID-19 guidelines by the NIH 7 from randomization to 14 days. Severity was considered if the participants had an oxygen saturation (SpO 2 ) < 94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO 2 /FiO 2 ) < 300 mm Hg, a respiratory rate > 30 breaths/min, or lung in ltrates > 50%.
Secondary endpoints were the indirect analysis of the viral load using the threshold cycle (Ct) value of the nucleocapsid (N) gene from SARS-CoV-2, and the presence and frequency of COVID-19 symptoms were measured on Days 1, 5 and 14. Subjects were asked to complete a diary of symptoms and adverse events for 14 days. They recorded the presence of the following symptoms: fever, cough, muscular pain, fatigue, breath di culty, headache, diarrhoea, palpitations, expectoration and "other". In the "others" eld question, several subjects answered hypogeusia/ageusia, hyposmia/anosmia and backpain.
Vital signs (temperature, blood pressure, pulse rate, oxygen saturation, and respiratory rate) and RT-PCR were measured on Days 1, 5, and 14. Laboratory tests were performed at baseline and Day 14. A security follow-up phone call was performed on Day 21.

Sample size and statistical analysis
The sample size was calculated according to the study by Wölfel et al. 8 We considering the viral RNA concentrations isolated in throat and nasopharyngeal samples at the begin of the infection adn the difference of 10000 copies at 10 days. A sample size of 54 patients provided 80% power to detect a 0.10 absolute difference in the proportions of placebo group using a 2-sided test with a signi cance level of 0.05. The sample size was in ated to a total of 66 participants to allow for 20% dropouts.
An exploratory analysis was carried out to identify the nature of the variables and their distribution. All tests were 2-tailed. For the quantitative variables, Kolmogorov-Smirnov tests were used to identify whether they adjusted to the normality assumptions with a 95% con dence interval. The statistical analysis to compare the difference of means between groups was calculated with t-test, and the ordinal variables were analyzed with the Chi 2 test. Values of p ≤ 0.05 were considered signi cant. We used IBM SPSS software (version 26, IBM Corporation, Armonk, NY, USA) for all statistical analyses.

Patients' characteristics
From 2020 July 21 to 2021 January 9, 104 subjects were screened, and 66 subjects were enrolled to receive either ivermectin (n = 33) or placebo (n = 33). Of these subjects, 10 were excluded from the e cacy analysis because nine of them were enrolled without a positive SARS-CoV-2 test, and one withdrew consent after the rst visit ( Fig. 1).
However, they were included in the safety analysis. The demographic and baseline characteristics of the group are listed in Table 1. The comparative analysis of the baseline measurements showed no differences between study groups.  (Fig. 2B).

Symptoms
Compliance to the diary was 96% and 92.9% in the placebo and ivermectin groups, respectively (X 2 = 2.23; p = 0.525). The data are presented as the percentage of days answered by group.
Cough, fatigue, myalgia, and headache were the most frequent symptoms. Regarding asymptomatic and symptomatic subjects (Fig. 3), on day one, 13.8% of the subjects who received ivermectin were asymptomatic, and 4% received placebo. At day ve, asymptomatic patients accounted for 12% and 18% on placebo and ivermectin, respectively. On Day 14, these values reached 45.8% in those who received placebo and 37% in those who received ivermectin, and no signi cant difference was observed between groups regarding this distribution.
We also classi ed symptoms according to the number of symptoms per subject (1 to 3, 4 to 5, and ≥ 7 symptoms reported per day), showing the expected progressive decrease in the number of symptoms reported per day over time for both arms.
Regarding vital signs, the participants in the ivermectin group had a higher heart rate on day 5 (72.78 ± 15.7 vs 83.46 ± 10.5 bpm; p = 0.007), but without signi cant difference in comparison to baseline (p = 0.873) (Supplementary Table 1).

Safety
Overall, 30 adverse events were reported, 17 in the placebo group and 13 in those treated with ivermectin. The majority of events was associated with COVID-19, but no differences were observed in the proportion of events that occurred between groups (Table 2). In the ivermectin group, a serious adverse event occurred; encephalitis secondary to SARS-CoV-2 occurred on study Day 10, and the subject made a full recovery. Regarding clinical laboratory tests, some differences were observed between baseline and Day 14 posttreatment: cholesterol and platelets increased in both the placebo and ivermectin groups. A decrease in blood sodium was observed in the placebo subjects from 139 ± 2.48 mEq/L to 129 ± 38.2 mEq/L (p = 0.007), and an increment in erythrocytes was observed from 5.05 ± 0.52 M/µL to 6.84 ± 2.60 M/µL (p = 0.002) from baseline to Day 14 (Supplementary Table 2).

Discussion
In this clinical trial of patients with asymptomatic and mild COVID-19, those who were randomized to receive ivermectin for 3 days in the early stages of diagnosis at a dose of 12 mg/day presented a reduction on viral load within the ve days after starting treatment in relation to the group that received placebo.
These results agree with those observed by Ahmed et al., 9 who evaluated the effect of ivermectin alone and in combination with doxycycline compared to placebo in hospitalized COVID-19 patients. They observed that treatment with ivermectin for 5 days resulted in earlier viral clearance (9.7 days) than the group treated in combination (11.5 days) or with placebo (12.7 days). A signi cant difference was observed against placebo at 7 and 14 days. In concordance, Pott-Junior et al. 10  This effect could be associated in a directly proportional way to the dose, since a preliminary report indicated that providing ivermectin two times the dose that we administered (24 mg/day) for a single day translated into a higher proportion of subjects negative for COVID-19 at day ve compared to 12 mg/day or placebo. In this trial, no statistical signi cance was observed; thus, it could be assumed that an indication of ivermectin for more days is required to reach such signi cance. 13 However, our study did not show a correlation between the calculated dose (12 mg between weight in kilograms) and the Ct value of the N gene (data not shown). According to Schmith et al. 14 report, single repeated doses of 200 mcg/kg, 120 mg weekly or 60 mg every 72 hours were not enough to reach the concentrations relative to the 50% inhibition (IC 50 ) established by Caly et al., 1 but they did not intend consecutive daily doses as we did. The diverse results with similar or different posology could be explained considering the pharmacokinetics (PK) of ivermectin. 15 Absorption is intestinal, and diarrhea is a common clinical manifestation of COVID-19. This nding decreases the absorption rate and bioavailability and therefore the effect. Moreover, the elimination is principally in feces. However, there is no report of individual results of the effect or presence of diarrhea, and our data did not show any differences in Ct value between those who presented diarrhea and those who did not on any of the evaluated days (data not shown).
Similarly, a randomized study in hospitalized patients showed a bene t in the Ct value of patients treated with ivermectin for seven days (100, 200 or 400 mcg/kg) in relation to those who did not receive ivermectin, with no-dose behavior.
Our ndings and the others previously mentioned can potentially be translated into avoiding progression to severe disease. According to Liu's and Samaha's report, there is a relationship between the Ct value and the intensity of the disease. 12,16 Furthermore, other studies have shown that receiving ivermectin as part of COVID-19 treatment is associated with a lower mortality rate, accompanied by lower levels of in ammatory biomarkers, such as C-reactive protein, ferritin and D-dimer. [16][17][18] In this study, an evaluation of the presence of symptoms associated with COVID-19 was carried out through the delivery of a diary; in general, the participants had an adherence to the completion of the symptom diary greater than 80%. The analysis revealed that the subjects who received ivermectin presented a higher frequency of symptoms from basal evaluation, of which fatigue and diarrhea were reported as adverse events expected from receiving ivermectin in the Food and Drug Administration (FDA) technical data sheet. In addition, in the same document, muscle pain and headache have also been observed in clinical trials. 19 Therefore, we could assume that these may be due to the drug and not the disease.
These ndings do not correspond to the results by Chaccour et al. 11 in his pilot study, since they observed a lower frequency of the following symptoms in those treated with ivermectin: cough, anosmia, hyposmia and shortness of breath. They did not observe any differences in the frequency of fever or an increase in the frequency of gastrointestinal symptoms by 3.5 times. 11 Recently, a randomized clinical trial by Shahbaznejad et al. 20 reported that patients who used ivermectin at a single dose, calculated according to weight, decreased the hospital stay and duration of symptoms compared to the control group.
On the other hand, a randomized trial with 400 patients, in which the time to resolution of symptoms was evaluated in subjects who received ivermectin (300 mcg/kg) for 5 days, did not observe a signi cant difference between groups, with a time to resolution slightly lower in the placebo arm (10 vs. 12 days). 21 The evidence available to date seems to indicate that there is little or no bene t in the resolution of symptoms associated with the consumption of ivermectin at a standard dose. 22 Regarding safety issues, no relationship was found between the drug or any adverse event. The encephalitis event that occurred in the subject who received ivermectin was diagnosed as secondary to SARS-CoV-2 infection, a phenomenon that has been previously documented. 23,24 Taken together, these data do not show that ivermectin is effective in the treatment of COVID-19 by "accelerating" viral clearance in the rst week, which may translate into a lower rate of complications.

Declarations
Ethics approval and consent to participate The protocol was approved by the local ethics, biosafety and investigation committees and the Mexican health ministry COFEPRIS: 203301410A0055. The study is registered on ClinicalTrials.gov: NCT04407507.

Consent for publication
Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
Investigación Biomédica para el Desarrollo de Fármacos S.A. de C.V. covered all the expenses generated during the study.
This trial was supported Investigación Biomédica para el Desarrollo de Fármacos S.A. de C.V. The funding source participated in study design, data collection, data analysis, data interpretation, or writing of the report, since most of the team are a liated, but no commercial purposes are intended with this trial results.