Patients were recruited both direct- and indirectly through social media and referred from urgent units and outpatient clinics when suspected or confirmed for COVID-19 through rtPCR-SARS-CoV-2.
Case-detection screening for COVID-19 was based on the presence of at least one symptom from the upper-respiratory tract, musculoskeletal, cardiovascular, gastrointestinal and neurological systems, or any unspecific symptom, not only those limited to fever, shortness of breath, dry cough, anosmia or ageusia. Candidates suspected for COVID-19 underwent rtPCR-SARS-CoV-2 (Abbott RealTime SARS-CoV-2 Assay, Abbott, USA; or Cobas SARS-CoV-2, Roche, Switzerland), and included in the study in case of confirmation. Patients previously confirmed for COVID-19 were also included if they fulfilled criteria for inclusion in the study.
To participate in the present prospective observational study, confirmed subjects had to fill the following inclusion criteria: 1. 18 years old and above; 2. Less than seven days since the beginning of symptoms; 3. Less than five days from the confirmation of COVID-19; 4. Lack of use or use in less than 72 hours of hydroxychloroquine, nitazoxanide and ivermectin; 5. Lack of previous use of glucocorticoids in the last seven days; and 5. Absence of clinical or radiological signs of progression to severe acute lung injury, including shortness of breath, oxygen saturation (SatO2) below 92%, and more than 25% of lungs affected in a chest computed tomography (CT) scan performed before entering in the study.
Patients included for the present analysis provided a written consent regarding the use of one of the three drugs: hydroxychloroquine, ivermectin or nitazoxanide, as experimental drugs for COVID-19, not officially approved nor having clinical evidence for COVID-19, following the approval of the Institutional Review Board (IRB) of the Ethics Committee of the National Board of Ethics Committee of the Ministry of Health, Brazil (CEP/CONEP: Parecer 4.173.074 / CAAE: 34110420.2.0000.0008), and as registered at ClinicalTrials.gov (Identifier: NCT04446429. Available at clinicaltrials.gov (https://clinicaltrials.gov/ct2/show/NCT04446429?term=NCT04446429&draw=2&rank=1).
Patients were characterized for basal characteristics, including sex, age (years old), weight (Kg), height (m) and body mass index (BMI) (kg/m2). Whether patients were married or had a partner living in the same room, and whether they had households or lived alone, were also questioned.
Patients were then actively questioned on personal history of myocardial infarction or cerebrovascular disease, and for existing diseases, including hypertension, congestive heart failure (CHF), lipid disorders (dyslipidemia, hypertriglyceridemia), type 2 diabetes mellitus (T2DM), prediabetes (self-reported, with exams checked), obesity (BMI > 30 kg/m2), asthma, chronic obstructive pulmonary disorder (COPD), chronic kidney disease (CKD), liver fibrosis or cirrhosis, depression clinically diagnosed, anxiety-related disorders, attention deficit hyperactivity disorder (ADHD), narcolepsy, and related disorders, insomnia clinically diagnosed , frank or subclinical hypothyroidism, Hashimoto’s disease, other autoimmune disorders (if any, these were specified), and previous and current cancer (except for prostate for males and breast for females).
For females, menopause, endometriosis, and current or previous breast, ovary, or endometrium cancer, and for males, hypogonadism, erectile dysfunction, benign prostate hyperplasia (BPH), and previous or current prostate cancer were also questioned.
Patients were also characterized for androgenic phenotypes. Females were analyzed for the existence of hyperandrogenic phenotypes, including polycystic ovary syndrome (PCOS), using at least two of the three Rotterdam criteria for PCOS (oligo- or amenorrhea, clinical or biochemical hyperandrogenism, and presence of more than 10 to 12 small cysts (2-9mm) peripherally distributed in the ovaries), idiopathic hirsutism (at least eight points in the Ferriman-Gallwey scale), androgenetic alopecia (AGA - confirmed by trichoscopy), or any state of evident hyperandrogenism, and were then classified as hyperandrogenic (HA) or non-hyperandrogenic (non-HA) females. Males were screened for the presence of male AGA, using the Norwood-Hamilton scale (stages I-VII), and classified as being AGA or non-AGA males.
Regular use of medications were also actively searched for hypertension and other heart- or vascular-related conditions, including selective and non-selective beta-blocker (carvedilol, nebivolol, metoprolol, propranolol, atenolol), angiotensin converting enzyme inhibitors (ACEi) (captopril, enalapril), angiotensin-2 receptor blockers (ARB) (valsartan, olmisartan, losartan…), loop diuretics (furosemide, as the only loop diuretic available in Brazil), thiazide diuretics (hydrochlorothiazide, indapamide), calcium channel blocker (CCB) (amlodipine, felodipine, nifedipine, nicardipine, verapamil, ditialzem), potassium-sparing diuretic (spironolactone, as the only K-sparing diuretic available in Brazil), statins (pitavastatin, rosuvastatin, atorvastatin, simvastatin, pravastatin), other lipid-lowering drugs (fibrates, ezetimibe, PCSK-9 inhibitors), aspirin, clopidogrel, direct Xa factor inhibitors (apibaxan, rivaroxaban), thrombin inhibitors (dabigatran), and enoxaparin; for T2DM and T1DM, obesity, and used as off-label therapies for prediabetes, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), insulin resistance and overweight, including biguanides (metformin), glucagon-like peptide-1 receptor analogues (GLP1Ra) (liraglutide, semaglutide, dulaglutide, exenatide), sodium-glucose cotransporter-2 inhibitors (SGLT2i) (dapagliflozin, canagliflozin, empagliflozin), dipeptidyl-peptidase 4 inhibitors (DPP4i) (vildagliptin, sitagliptin, saxagliptin, linagliptin), sulfolnylureas (glicazide, glipizide, glimepiride, glibenclazide), glitazones (pioglitazone), alpha-glucosidase inhibitor (acarbose), insulin (of any type, fast, regular, or long duration), and lipase inhibitor (orlistat); use of hormonal replacement or treatment regimens, including levothyroxine for hypothyroidism, growth hormone (GH) for GH deficiency of the adult, testosterone for males, progesterone (P) alone, estradiol (E) alone,combined (E+P) therapy, oral contraceptives and other hormonal regimens for females, and aromatase inhibitors (anastrozole, letrozole) or selective estrogen receptor modulators (SERM) (tamoxifen) for hypogonadotrophic (central) hypogonadism in males) or hormone-sensitive breast cancer in females; central acting drugs, including hypnotics (zolpidem (“Ambien”), zopiclone, eszopiclone, ramelteon (“Rozerem”)), selective serotonin reuptake inhibitors (SSRIs) (sertraline, fluoxetine, duloxetine, paroxetine, venlafaxine, desvenlafaxine, vortioxetine (“Brintellix”), escitalopram, citalopram), other anti-depressant and humor stabilizer drugs (bupropion, trazodone, agomelatine, amitriptyline, nortriptyline, topiramate, oxcarbamazepine), benzodiazepines (lorazepam, alprazolam, bromazepam, midazolam, diazepam, clonazepam), atypical Antipsychotics (olanzapine (Zyprexa), quetiapine (Seroquel), risperidone (Risperdal), clozapine, aripiprazole) and central nervous system (CNS) stimulants (lisdexamphetamine, methylphenidate, modafinile); and other drugs, including oral minoxidil, finasteride, dutasteride, and proton-pump inhibitors (PPI) (dexlansoprazole, esomeprazole, pantoprazole, omeprazole), not limited to these drugs, and supplementation with omega-3 (> 3g/day), vitamin D (> 1,000iu/day), zinc (> 15mg/day), biotin (> 500mcg/day), and vitamin C (> 500mg/day).
Bacillus Calmette-Guérin (BCG) vaccine for tuberculosis, pneumococcal vaccine received since 2017 and influenza vaccine received in 2020, current smoking ( > 2 packs/week and > 10 pack-year history) and significant regular physical activity (> 150 minutes/week, moderate-to-vigorous - > 3.0 METs for > 1y) were questioned.
For the characterization of the clinical manifestations of COVID-19, patients were actively questioned for the presence, beginning, duration and intensity of one or more symptoms among the following: fever, ‘feverish’, dry cough, self-reported perception of ‘sinusitis’, self-reported perception of ‘sore throat’, rhinorrhea, hipo- or anosmia, dis- or ageusia, weakness, dizziness, fatigue, myalgia, arthralgia, upper back pain, lower back pain, respiratory-dependent thoracic pain, shortness of breath, diarrhea, nauseas, vomiting, abdominal pain, conjunctival hyperemia, pre-orbital pain, dry eyes and dry mouth. Oxygen saturation (%), heart rate (bpm), and in high-risk patients, systolic and diastolic blood pressure (SBP and DBP, respectively) were measured.
According to clinical manifestations, included participants were clustered into one of the following types of clinical presentation: 1. Anosmia-Ageusia dominance, with at least one of anosmia and ageusia, and less than two symptoms of dengue fever-like, URTI-like or GI infection-like clusters; 2. Dengue fever-like, with at least three of myalgia, arthralgia, upper back pain, conjunctival hyperemia or pre-orbital pain; 3. Upper respiratory tract infection (URTI) URTI-like, with at least two of nasal congestion or rhinorrhea, dry cough, self-reported perception of “sinusitis”, or self-reported perception of “sore throat”; 4. Gastrointestinal (GI) infection-like, with at least two of diarrhea, nauseas, vomiting, or abdominal pain; 5. Mixed between types, when there are sufficient number of symptoms to fulfill criteria for at least two clusters; 6. Unspecific clinical presentation, when symptoms do not fulfill criteria for any cluster; or 7. Asymptomatic.
In addition, patients were grouped according to the use or non-use of drugs with antiandrogenic (AA) activity, including spironolactone, cyproterone and other androgen receptor (AR) antagonists, dutasteride, finasteride, GnRH analogues, and androgen-deprivation therapies (ADT).
Hydroxychloroquine was given at a dose of 400mg/day for five days, nitazoxanide was given at a dose of 500mg, twice a day, for six days, and ivermectin was given at a dose of 0.2mg/kg/day for three days. One of these drugs (exceptionally, two of them were used, except for the combination of ivermectin and nitazoxanide) was associated with azithromycin 500mg/day for five days.
After characterization and clinical clustering, in case participant had not started on any of the following drugs, one of them, between hydroxychloroquine, azithromycin or ivermectin was given. The drug was chosen in a quasi-random manner, i.e., hydroxychloroquine tended to be avoided in patients at higher risk for heart complications, drugs with previous history of intolerance were avoided, and also according to clinical judgement, availability, and individual medical history. Drugs were given in the following regimens: hydroxychloroquine - 400mg/day in a single daily dose for five days, nitazoxanide - 500mg twice a day for six days, and ivermectin -0.2mg/kg/day in a single daily dose for three days. In some patients, more than one of the three drugs were used (combination of ivermectin and nitazoxanide was avoided for all patients). In these cases, patients were considered as taking the two of the three drugs prescribed. All these drugs were associated with azithromycin 500mg/day in a single daily dose for five days, independently of other combinations.
In case patients had already started on one of the three drugs, it was maintained, and adjustments in doses and inclusion of azithromycin were performed.
Optionally, vitamin D, vitamin C, zinc, Xa factor inhibitors (apibaxan or rivaroxaban), enoxaparin, glucocorticoids, colchicine, N-acetyl-cysteine and bromhexine were prescribed, according to clinical, biochemical or radiological abnormalities, including the addition of enoxaparin or a Xa factor inhibitor in patients with high risk for thrombosis and increased D-dimer, and glucocorticoids for increased ultrasensitive C-reactive protein (usCRP) or radiologically diagnosed lung injury affecting 25% or more of lungs. Dutasteride or spironolactone were openly prescribed in some cases as per the evidence of anti-COVID action, mechanistic plausibility, and in fully accordance with the approval of the IRB.
Patients had virtual medical visit in the Day 0 and 14, and in case of any adverse effect. They were followed up by the research team on a daily basis, with active questions regarding clinical manifestations, speed of recovery, and occurrence of new symptoms, on Days 1, 2, 3, 7, 14, 30 and 60.
Selected patients underwent commercially available automatized and standardized biochemical tests (COBAS, Roche, Switzerland) ultrasensitive C-reactive protein (usCRP) (serum; Latex-intensified immunoturbidimetry;, erythrocyte sedimentation rate (ESR) (mm/1h) (blood; automatized spontaneous sedimentation), ferritin (ng/mL) (serum; chemoiluminescence – CLIA; ) - and D-dimer (ng/mL; plasma; immunoturbidimetry), with intra- and inter-assay below 3.5 and 4.5%, respectively, and chest computed tomography (CT) scan.
Clinical outcomes were analyzed for overall patients and those not using AA. Multiple disease course and disease progression scales were employed. Time-to-treat, duration of positive rtPCR-SARS-CoV-2, duration of symptoms including and not including anosmia and ageusia, and loss of ability for everyday activities in Days 0, 3, 7 and 14 were the primary clinical course outcomes. Brescia COVID-19 Respiratory Severity Scale (from 0 to 4), hospitalization, mechanical ventilation, use of noradrenaline or dopamine, and death were the clinical progression outcomes.
Patients were also actively followed for 60 days, whenever any residual or relapsing symptom appeared, for the persistence or new-onset symptoms after COVID-19 rtPCR cure, for physical, mental, or both post-COVID symptoms. Assessed physical symptoms include easy tiredness, loss of physical performance not fully justified by the disease, lack of any progressive recovery in physical capacity, loss of libido, unjustified muscle pain, prolonged muscle recovery, arthralgia, development of autoimmune diseases, persistence of menstrual irregularity, decrease of male fertility (when compared to previously documented fertility rate), and new-onset gastrointestinal symptoms, not limited to these, when not justifiable by any other cause. Mental symptoms include brain fog, attention deficit hyperactive disorder (ADHD) -like manifestations, changes in daily activity patterns and cognitive abilities, and other mental manifestations unrelated to post-traumatic stress disorder (PTSD) or any anxiety state related to the process of COVID-19.
Full raw data is publicly available at a repository (https://osf.io/cm4f8/).
Sample size was determined based on the assumptions that the sample size estimate for the chi-squared test will require 80% power to detect the difference in proportions at p = 0.05, that 95% of subjects would complete the study, and based on the hospitalization and death rates between 3 and 20%, and 0.3 and 2.5%, respectively (5,29,30). Based on these assumptions, the minimum sample size was 354 subjects. The study would terminate earlier in case of unexpected outcomes and adverse effects that could justify its termination.
Data was provided for absolute number and percentages, and for both mean and median, and standard deviation (SD) and 95% confidence interval (95%CI), respectively. In order to avoid overestimation of our findings, we assumed that all data was non-normally distributed, and performed non parametric-based analyses. Nonparametric ANOVA (Kruskal-Wallis) was performed for all parameters and post-hoc adjusted Dunn’s test was performed for subgroup analyses, whenever p < 0.2. All statistical tests were performed using XLSTAT version 22.4.1 (Microsoft, USA).