2.1 STUDY DESIGN
This randomized, double-blind, placebo-controlled clinical trial (IGZ-2) was carried out in 5 co-participating research centers, distributed in 4 municipalities in the state of Rio de Janeiro, Brazil, as a strategic decision due to the need to concentrate molecular biology analyzes to maintain their standardization and quality, remembering that each RT-PCR equipment has its own sensitivity and different kits of reagents for RT-PCR have different performances. Two research centers were located in the city of Campos dos Goytacazes, namely Hospital São José and Santa Casa de Miseriocordia de Campos. In the municipality of São fidelis, the third research center was at Hospital Armando Vidal. In the municipality of Itaocara, the fourth research center was located at the Hospital de Itaocara. And the fifth research center was located in the municipality of Cambuci at Hospital Moacyr Gomes de Azevedo. However, the research centers served as an outpatient clinic for the evaluation of the participants, since this study treated mild patients contaminated with SARS-COV-2, all of whom were followed in their homes. This trial was approved by the institutional review board of the National Health Surveillance Agency CE 0937457/21-4. The study was approved by the National Council for Research Ethics, CAAE 52176421.8.0000.5244. The study was also published in clinical trials (NCT05033145). All enrolled participants provided written informed consent. The methods are described in detail in the supplementary material.
Patients in the FNC group were treated with oral AZVUDINE tablets 5 mg (five tablets once a night) and standard treatment. For the 5mg dose of AZVUDINE, the mean half-life is 13.8h, with the intact drug and metabolites being excreted in the urine within 24h. Patients in the control group were treated with placebo added standard treatment.
Patients: Patients meeting the following criteria were enrolled in the study: 1) age 18 and over, regardless of gender; 2) respiratory or blood samples that were tested positive for SARS-CoV-2 nucleic acid by RT-PCR, or respiratory or blood samples that were tested highly homologous with the known SARS-CoV-2 by viral gene sequencing; 3) the confirmation of COVID-19 according to the diagnostic criteria of “the latest Clinical guide-lines for novel coronavirus” issued by the World Health Organization (WHO) on 2020 January 28. All enrolled patients signed informed consent forms.
Exclusion criteria included 1) known or suspected allergy to the com- position of AZVUDINE tablets; 2) patients with malabsorption syndrome or any other condition that affects gastrointestinal absorption, the need for intravenous nutrition or an inability to take oral medication; 3) patients on anti-HIV treatment; 4) patients with one of the following conditions: respiratory failure and the need for mechanical ventilation; shock; intensive care unit (ICU) monitoring and treatment for other organ failures; 5) pregnant women or those who were lactating or may have a birth plan during the trial period and within 6 months after the end of the trial; 6) patients participating in other clinical trials or using experimental drugs within 12 weeks before administration; and 7) patients with other conditions that were not suitable for participating in this experiment according to the judgment of the researcher.
The definition of mild COVID-19 the definition of common COVID-19 was patients with fever, respiratory and characteristic symptoms such as loss of smell, loss of taste, diarrhea, dizziness, fever, chill, sore throat, dyspnea, tachypnea, nausea and abdominal pain.
Enrollment: Volunteers were approached at health posts and testing points for SARS-COV-2. Patients with fever, cough or other symptoms related to COVID-19 were approached by the study team, who provided an explanation of all pertinent information. After this process the ICF was applied and samples were collected for the nucleic acid test by RT-PCR for laboratory tests. The following day, the researcher evaluated the results of the exams and verified whether the volunteer met the inclusion criteria, thus proceeding to the randomization stage.
Randomization: Patients were randomly assigned in a 1:1 ratio to the FNC group or control group. Randomization was accomplished by using a random table that was generated in Researcher IGZ v2.0 Software at 1:1. Each enrolled subject was given a number, randomly assigned to the FNC group and control group according to a predetermined random table and received treatment according to the corresponding treatment regimen.
Procedures: Patients in the FNC group were treated with oral AZVUDINE tablets 5 mg d-1 (five tablets once a night) and symptomatic treatment. The FNC dose was determined due COVID-19 clinical trials preliminary results, considering that the maximum safety dose study of AZVUDINE was performed for 5mg, a daily dose of 5mg may meet clinical treatment. In addition, for the 5mg dose of AZVUDINE, the mean half-life is 13.8h, with the intact drug and metabolites being excreted in the urine within 24h. Patients in the control group were treated with placebo added standard treatment.
The patient’s vital signs, oxygen (via finger pulse oximetry), and respiratory symptoms and signs were monitored every day. On odd days and the discharge day, the patient’s routine blood, erythrocyte sedimentation rate (ESR), C-reactive protein, blood biochemistry, blood coagulation, myocardial markers, procalcitonin, myocardial zymogram, and arterial blood gas were monitored. SARS-CoV-2 nucleic acids were tested by RT-PCR after the patients began taking their drugs. Nucleic acid detection analyzes were performed every 48hs days throughout the treatment period for optimal measurement of participant's viral load. Two consecutive negative results configured clinical discharge. These tests were used to obtain the mean times of the nucleic acid negative conversion (NANC).
Primary Outcome: The primary outcome was the proportion of patients hospitalized during the study through day 28, according to the WHO Ordinary Clinical Progression Scale (Jun/2020), Score 4 to 10.
Secondary outcomes: Proportion of participants with a clinical outcome of CURE during the study; Improvement in clinical status in at least one category compared to screening on the Ordinal Scale of Clinical Improvement (WHO, Jun/2020); Severity and duration of symptoms: fever, cough, fatigue or tiredness, breathlessness, myalgia, nasal congestion or runny nose, sore throat, headache, chills, nausea, vomiting, anosmia, ageusia; Baseline changes in liver and kidney function; Time of use of AZVUDINE until the second negative conversion of RT-PCR; Evaluation of SARS-CoV-2 viral load negative conversion time by RT-PCR between AZVUDINE group (FNC) and control group; Evaluation of the number of cycles for the detection of SARS-CoV-2 viral load by RT-PCR and application of the standard curve to calculate the viral load; Analysis of the relationship between the calculated and/or quantified viral load and the clinical evolution of the participants in the AZVUDINE group (FNC) in relation to the control group; Frequency and intensity of adverse events, unexpected adverse events, and serious adverse events.
Safety was regularly assessed by monitoring vital signs, changes in laboratory values (liver function, renal function), and adverse events (including type, incidence, severity, time and drug correlation, and assessment of severity. Previous studies have already shown that individuals who used FNC did not experience any type of serious adverse event drug related (Ren et al., 2020).
2.2 QUANTIFICATION OF SARS-COV-2 VIRAL LOAD BY REVERSE TRANSCRIPTION–POLYMERASE CHAIN REACTION (RT-PCR)
Total RNA was extracted using the MagMAXTM Viral/Pathogen Nucleic Acid Isolation kit (Applied Biosystems) according to the manufacturer's instructions, using nasal and throat swabs from the clinical study participants. Once total RNA was extracted, RT-PCRs were performed using the TaqPathTM COVID-19 CE-IVD RT-PCR kit (ANVISA Reg.: 10358940107) according to the manufacturer's instructions, using the QuantStudio5 RT-PCR equipment, Applied Biosystems, (ANVISA Reg: 10358940069). The primers and probes targeted the ORF1ab and N genes.
A standard curve was constructed using serial dilutions of the positive control (TaqPath COVID-19 Control), which is SARS-CoV-2 viral RNA at a known concentration of 1 x 104 copies/µL. The CTs obtained from each sample by RT-PCR were plotted on the standard curve to estimate the viral load of each sample.
A positive RT-PCR result occurs in CTs ≤37. In this case, when viral RNA is present, the specific probe used to detect SARS-CoV-2 is broken by DNA polymerase, emitting fluorescence. High copy number of viral RNA generates high levels of fluorescence, therefore, the CT value appears earlier during the reaction. Low copy number of viral RNA generates low level of fluorescence, and consequently, the CT value appears later. Values of CTs>37 are considered negative. By establishing a viral RNA concentration curve (present in the positive control), we will obtain a curve of CTs, from lower values (higher copies of viral RNA) to higher values (lower copies of viral RNA).
2.3 QUANTIFICATION OF SARS-COV-2 VIRAL LOAD BY DROPLET DIGITAL POLYMERASE CHAIN REACTION (DDPCR)
Total RNA was extracted using the MagMAXTM Viral/Pathogen Nucleic Acid Isolation kit (Applied Biosystems) according to the manufacturer's instructions, using nasal and throat swabs from the clinical study participants. Once total RNA was extracted, the ddPCR were performed subsequently.
Reverse transcription–PCR was conducted with primers and probes targeting the ORF1ab and N genes and a positive reference gene. Reaction system and amplification conditions were performed according to the manufacturer’s specifications (Shanghai BioGerm Medical Technology Co Ltd, China). The result was considered valid only when the cycle threshold (Ct) value of the reference gene was 38 or less. The result was considered positive when the Ct values of both target genes were 38 or less and negative when they were both greater than 38. If only one of the target genes had a Ct value of 38 or less and the other was more than 38, it was interpreted as a single-gene positive.
Digital droplet PCR analyzes were performed by the Targeting One Digital PCR System; COVID-19 digital PCR detection kit; droplet generation Kit; Droplet detection kit. The kits allow detection of the ORF1ab gene, N gene and a positive reference gene. The detection limit was 10 copies/test. (Targeting OneTechnology is licensed by the China Food and Drug Administration).
2.4 STATISTICAL ANALYSIS:
For the analysis of demographic information and baseline eigenvalues, the mean value, standard deviation, quartiles, minimum and maximum values for numerical variables were calculated. For categorical data, frequency and percentage were calculated. The comparison of the two groups under general conditions was analyzed with appropriate methods according to the types of indicators. The Mann-Whitney test was used to compare the groups regarding quantitative data. Fisher's exact test was used for categorical data. Statistical analyzes were performed using the R-studio software.