Study population
The study population included 72 patients with COVID-19, confirmed using a positive RT-PCR, with mild to moderate symptoms, within 72 h of a positive result or onset of symptoms. This study was approved by the Vajira Institutional Review Board no. 171/64 and was conducted in compliance with the Declaration of Helsinki and Good Clinical Practice Guidelines. Written informed consent was obtained from all patients. More details of the trial can be found in the protocol (Supplement 1). The inclusion criteria comprised adult men and women aged 18 to 80 years, nonpregnant or breast-feeding women, and mild to moderate symptoms as defined by the World Health Organization (WHO) severity score for COVID-19 [13]. Mild disease was defined as cough, runny nose, anosmia, fever, and diarrhea without dyspnea or tachypnea, and moderate disease was defined as pneumonia with oxygen saturation >90%. All patients were admitted to the hospital.
The patients were excluded if they were allergic to ivermectin, had the potential for a drug-to-drug interaction with ivermectin, such as tamoxifen or warfarin; were previously treated with ivermectin in the last 7 days; had received any herbal medicine; had severe chronic illness (severe congestive heart failure, chronic kidney disease stages 4 to 5, chronic liver disease or had hepatic dysfunction or liver function test results more than 1.5 times the normal level, terminal cancer); had concurrent bacterial infection; or were unwilling to participate in the trial. Patients with severe symptoms, likely due to cytokine release syndrome, uncontrolled comorbidities, and immunocompromised status were also excluded. No important changes were made to methods or trial outcomes after trial commencement. Also, no interim analyses or discontinued rules applied to the trial.
Sample size calculation
The sample size calculation was based on a related reference study [14]. The number of 25 patients per group in the comparison of two proportions was calculated to have 90% power at a two-sided significance level of 0.05, allocation ratio 1:1 using continuity calculation. We used Stata, Version 16.0 to detect the proportion of patients with positive PCR at day 7 in the intervention and control groups of 9.8% (4/41; p1 = 0.098) and 55.6% (25/45; p2 = 0.556), respectively. The sample size was inflated to 36 participants per group (72 in total) to account for a possible 30% loss-to-follow-up, noncompliance, and drop-out.
Study design and intervention
This study constituted a randomized, double-blind, placebo-controlled trial. This randomized, single-center, parallel-arm, superiority trial among adults was conducted at the Faculty of Medicine, Vajira Hospital, Navamindradhiraj University, from September 2021 to November 2021.
The patients were randomized in a permuted block of four in a randomized sequence prepared by a pharmacist, who was unblinded, in Microsoft Excel [15]. Allocation assignment was concealed from the investigators and patients. The patients were allocated in one of the two groups: group A (ivermectin arm) or group B (control arm), as shown in Fig. 1. The patients were randomized in a 1:1 ratio. Group A received 12 mg ivermectin daily for 5 days, as recommended by related studies [14], along with standard care. Group B received standard care alone, including favipiravir or andrographolide, corticosteroids, cetrizine, and paracetamol. No changes were made in the protocol after recruitment. Ivermectin was provided by the pharmacist by bottle. Patients were asked to take the investigational product on an empty stomach, except on the first study day, when administered after the postitive test result was confirmed.
Clinical, laboratory, and virological monitoring
The study coordinator reviewed the patient’s history to screen for eligibility. The potential study participants were contacted by telephone to obtain informed consent. Eligible patients underwent physical examination by the doctor in the ward. Baseline characteristics, such as age, sex, comorbidities, duration of symptoms, and disease severity on admission, were recorded at the time of enrollment. All patients were confirmed as having COVID-19 using a baseline nasopharyngeal swab for RT-PCR. A follow-up RT-PCR was performed on days 7 and 14 following drug intervention to estimate the change in viral load. Complete blood count, renal and liver function tests, C-reactive protein, D dimer, and chest radiography were performed the day of enrollment and day 14. Patients were contacted via telephone by the research team every day through day 14. On day 28, a telephonic interview was performed for the final questions pertaining to general health, well-being, and the possible development of side effects after treating with ivermectin.
Processing and analysis of respiratory samples
Nasopharyngeal swabs were collected from suspected COVID-19 cases by trained medical technologists. The swabs were stored in 2 mL of viral transport media (VTM) (Dewei Medical Equipment Co., Ltd., China), transported at 4 °C, and processed within 4 h at the Biomolecular Unit, central laboratory of Vajira Hospital. Viral RNA extraction was performed on each VTM sample using the commercial kit (Zybio Nucleic Acid Extraction Kit) on automated nucleic acid extraction system (magnetic bead method) (Zybio Inc., China), according to the manufacturer’s instructions. RT-PCR tests were run on a Slan 96P Real-Time PCR System using a 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech Inc.). The kit is designed to detect N and ORF1 ab genes of SARS-CoV-2, along with one housekeeping gene as the internal amplification control. A 40 μL reaction contained 26 μl of reaction buffer, 4 μl of 2019-nCoV-PCR-Enzyme Mix, and 10 μl of RNA. Thermal cycling was performed at 50 °C for 30 min for reverse transcription and one cycle at 95 °C for 1 min. Then 45 cycles at 95 °C for 15 s and at 60 °C for 31 s were performed and analyzed using ABI 7500 Software. A positive RT-PCR result was defined when both target genes reached a cycle threshold (Ct) of <40.
Outcome measurement
The primary outcome was to evaluate the efficacy of ivermectin in viral clearance of SARS-CoV-2 on days 7 and 14 after intervention, and compare that to placebo. The secondary outcomes were duration of hospitalization, frequency of clinical worsening, need for mechanical ventilation, all-cause mortality in both groups, survival on day 28, and adverse events in the study group.
Statistical analysis
Data were analyzed according to the intention-to-treat principle. All descriptive data were expressed as mean (standard deviation) and frequency (percentage). Comparisons between the treatment group were determined using the Student t test for parametric continuous variables or the Mann–Whitney U for nonparametric continuous variables, as appropriate, and by the Pearson X2 test for categorial variables. Comparisons between the mean duration of viral clearance and duration of hospitalization were evaluated by the independent t-test or Mann–Whitney U test, as appropriate. Univariate analysis of the primary mortality outcome and comparisons between the treatment groups were determined using the chi-squared test. The primary end point of time from randomization to day 28 with ivermectin versus placebo was assessed using a Kaplan–Meier plot and compared with a long rank test. The hazard ratio and 95% confidence interval for the cumulative incidence of clinical worsening in both the treatment groups were estimated using the Cox proportional hazards model. Statistical significance was set as P<0.05, and all tests were two-tailed. Statistical analysews were performed using STATA, Version 18.1 (stata group).
