A prospective, randomized, open-label trial of early versus late povidone-iodine gargling

Study design

This was a prospective, randomized, and open-label trial that looked at the efficacy and safety of gargling with PVP–I in asymptomatic-to-mild COVID-19 patients (aged ≥ 16 years) quarantined in three different locations. The protocol was approved by the Medical Center Clinical Research Review Board (CRB5200005), which has been establishe in Osaka General Medical Center, Osaka Prefectural Hospital Organization, Osaka, Japan. The first registration date is 24/11/2020 with the Japan Registry of Clinical Trials (number: jRCT1051200078). The study has been performed in accordance with the relevant guidelines and regulations and all study participants provided written informed consent.

Patients and randomization

The study has performed from 24/11/2020 to 22/4/2022. From 30/11/2020, to 17/3/2021, participants were recruited from three quarantine facilities in Osaka, Japan. On 22/3/2021, the follow-up was completed. The following inclusion criteria were used to select subjects: (1) age ≥ 16 years on the date of consent acquisition and with parental consent for those aged 16–19 years; (2) either sex; (3) quarantined at an accommodation facility but not inpatient status; (4) meet all of the following requirements: asymptomatic-to-mild COVID-19 patients with at least one (RT-PCR) test positive for SARS-CoV-2 (either pharyngeal swab, nasopharyngeal swab, or saliva test), capable of mouthwash and gargling, and willing to be quarantined for ≥ 6 days; and (5) voluntarily provided written consent to participate in the trial. The following were exclusion criteria: (1)being on receiving thyroid hormone, (2) having an iodine allergy, (3) being pregnant, (4) lactating female, and (5) already mouth washing or gargling with PVP–I prior enrollment.

Patients were randomly assigned at a 1:1 ratio with Viedoc4 EDC ststem to either the early interventional group (started mouthwash and gargling with PVP–I on day 2 after saliva sampling at the time of waking up) or the late interventional group (started mouthwash and gargling with PVP–I on day 5 after sampling of saliva at the time of waking up); accommodation facility, age, and gender were used as allocation adjusting factors. Because no placebos were available, the study was conducted in an open-label.

Intervention procedure

Table 1 depicts the saliva sampling and the intervention schedule. Mouth washing once and gargling twice with 20 mL of a 15-fold diluted solution containing PVP–I Gargle Solution 7% (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) or with the same volume of water constitute the intervention. From day 2 to day 6, the early interventional group was instructed to perform the PVP-1 intervention four times per day (before saliva sampling at the time of waking up, before lunch, before supper, and before sleeping). In the late interventional group, the intervention was performed with water from days 2 to 4, and then with PVP–I from days 5 following saliva sampling till day 6.

Measurement

The KANEKA Direct RT-qPCR Kit “SARS-CoV-2” was used for RT-qPCR testing at a centralized study laboratory, as directed by the manufacturer (Kaneka Corporation, Tokyo, Japan). A LightCycler 480 II was used to monitor amplified products (Roche Diagnostics, Basel, Switzerland). The SARS-CoV-2 virus was set to Ct = 40, as recommended by the manufacturer.

The saliva was tested for viral infectivity in a biosafety level 3 laboratory at the Osaka Habikino Medical Center. Vero E6 TMPRSS2 cells (JCRB1819) were obtained from JCRB bank at the National Institutes of Biomedical Innovation, Health, and Nutrition, Osaka, Japan. The cells were cultured according to the institution’s instruction and seeded into 96 well plates with 100 µL of medium and cultured for a day. Then, 10 µL of each salivary sample was added. The cells were monitored for cytopathic effect (CPE) after a 3-day incubation period, and SARS-CoV-2 infectivity was confirmed when CPE was detected in inoculated wells.

Observation schedule

The study’s observation schedule is shown in Table 1. Salivary samples were collected daily from days 2 to 6 upon waking up in the morning, before mouthwash and gargling; these were centrifuged to remove debris before being analyzed using RT-qPCR and CPE. During the study, the participants’ body temperature and physical conditions (presence or absence of changes, cough, suffocation, nasal congestion, stuffy nose, sore throat, nausea/vomiting, red-eye, headache, dullness, joint muscle pain, diarrhea, and light-headedness/loss of consciousness) were recorded twice a day.

The primary efficacy endpoint of this study was the clearance rate of SARS-CoV-2 viral load determined by RT-qPCR of saliva samples on day 5. On day 5, the clearance rate in each group was calculated by dividing the number of RT-qPCR-negative participants on day 5 by the number of RT-qPCR-positive participants on day 2. The 95% confidence interval (CI) was also computed. The chi-square test was used to compare the clearance rates of the early groups. The viral clearance rate of saliva samples at day 6, was calculated and analyzed using the same methods as for the primary endpoint. Safety was assessed based on adverse events that occurred during the study period by aggregating the number of cases and the number of cases by events in each group. The rate of SARS-CoV-2 viral infectivity to Vero E6 TMPRSS2 cells in saliva samples at day 5 was the investigational endpoint of efficacy. On day 5, the rate in each group was calculated by dividing the number of infectivity-positive participants on day 5 by the number of RT-qPCR-positive participants on day 2. The 95% CI was computed. The chi-square test was used to compare rates of the early and late groups.

In this study, datasets for intention-to-treat (ITT) and safety analysis (SAF) were determined and analyzed. In the efficacy evaluation, all subjects who used the PVP–I was included in the ITT, whereas all subjects who used the PVP–I were analyzed in the safety evaluation. The name of the adverse event was changed to MedDRA/J ver. 24.0 and used for analysis.

Statistical analysis

The sample size was calculated based on the assumption that viral clearance of saliva could be observed by day 5 in 60% of participants in the early interventional group with PVP–I and 40% in the late interventional group. With a power of ≥ 90% and a two-sided significance level of 0.05, this study required 203 participants in each group. To account for a 10% dropout rate, each group required 220 subjects. SAS software, version 9.4 was used for statistical analyses (SAS Institute, Cary, NC, USA).

Ethical approval

The study was centrally approved by the certified Medical Center Clinical Research Review Board (CRB5200005) which has been establishe in Osaka General Medical Center, Osaka Prefectural Hospital Organization, Osaka, Japan and was registered on 24/11/2020 with the Japan Registry of Clinical Trials (number jRCT1051200078).