István Jankovics^1*, Mariann Borsos², Sara Mirani³, Levente Lükő³, Dávid Kuti⁴, Béla Dénes⁵

¹Complex Medical Centre Déli klinika, Hungary.

²Adware Research Ltd., Hungary.

³Faculty of Pharmacy, University of Debrecen, Hungary

⁴National Public Health Center, Budapest, Hungary

⁵Veterinary Diagnostic Directorate, National Food Chain Safety Office, Budapest, Hungary

^*Corresponding Author: István Jankovics, Complex Medical Centre Déli klinika.

Abstract
Reducing the risk of acquiring upper respiratory tract infections is a significant health issue with severe ecological consequences. Vaccinations against respiratory tract infections (influenza, COVID-19, pertussis, pneumococcus) provide an unreplaceable role in the prevention. However, other personal and social measurements are promoted to reduce symptoms and decrease transmission and unfavorable outcomes. Polyphenol-containing Cistus creticus extract, alongside other polyphenol-rich ingredients, was shown to be beneficial in treating upper respiratory tract infections. However, clinical data about the prophylactic impact of the nasopharyngeal application of the polyphenol-rich cistus extract is scarce. A retrospective study was designed to evaluate its potential role in preventing disease and decreasing the burden of symptoms.

Keywords: Cistus creticus, polyphenols, URTI, prevention, quality of life

Introduction
Upper respiratory tract infections constitute one of the major global health problems resulting in high morbidity and mortality in developed and developing countries [1]. In recent years, comprehensive scientific research aimed to clarify the pathomechanism of upper respiratory tract infections, focusing on COVID-19 diseases caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to find and develop optimized prophylactic and therapeutic strategies for this emerging disease-causing pandemic. Antiviral treatments like redeliver, ritonavir-boosted nirmatrelvir, molnupiravir, and immunomodulatory therapies including corticosteroids,   tocilizumab, and baricitinib form cornerstones of the risk-stratified strategy of the management. [2,3] Several studies focused on the paramedical and holistic additional adjuvant treatment options of upper respiratory tract infections, such as vitamins, essential mineral nutrients, and recently polyphenol-rich fitotherapeutical compounds, with controversial results. However, in terms of prophylaxis, there needs to be more data and a lack of studies with good quality. Therefore, validation of these adjuvant compounds is challenging [4-6].

With its high concentration of polyphenols, a native medicinal plant from the Mediterranean Basin, Cistus creticus, was shown to have a pleiotropic beneficial effect both in vitro and in vivo for treating upper respiratory tract infections, including mild COVID-19 involving any upper respiratory tract. Additionally, polyphenols, the most critical component of the cistus creticus complex, can bind to the polybasic region of proteins. In this way, it inhibits the area responsible for the un—fusion activity of the hemagglutinin of the influenza virus and the S protein of the SARS-CoV-2 virus. Binding to the given protein region prevents the virus from entering the host cell and prevents infection. [7-11]. This study was designed to assess the beneficial effects of cistus extract-containing products in the clinical course of upper respiratory tract infections.

Methods
Study design
A single-center retrospective comparative study was conducted among consecutive adult (age ≥ 18 years) asymptomatic, healthy, at baseline SARS-CoV-2 PCR hostile persons who were defined as close contact according to whom definitions of SARS-CoV-2 infected patients receiving diagnostic and medical care in the Complex Medical Centre private clinic in Budapest, Hungary. Verbal and written consent was obtained from each patient before participation in standard care procedures offered by the clinic. The study period was between February 2021 and May 2021. The private clinic has provided care for COVID-19 outpatients since the start of the pandemic. Close contacts of SARS-CoV-2 PCR-positive patients were offered medication, including receiving Cistus creticus extract containing nasopharyngeal spray (patented name Virostop) to use three times a day added to standard of care (symptomatic treatment). A Group of close contacts who did not opt to use the spray received standard care according to the physician's decision.

The study was performed under ethical and IRB approval (IV/7423- 3/2021/EKU; Medical Research Council, Hungary).

Cistus creticus extract composition
Cistus creticus Acrocephalus extra., 100 mg/ml; Echinacea purpura extra., 240 mg/ml; Salvia officinalis extra., 45 mg/ml; Citrus bioflavonoid complex, 20 mg/ml; Acerola dried ext. 120 mg/ml (the patented name is Virostop).

In vitro tissue culture studies with Cistus creticus extra., Echinaceae purpureae extra., Salvia officinalis extra., Citrus bioflavonoid complex herbal preparations gave a virus inhibitory selectivity index (SI) of 8-16 (significant values) against seasonal influenza A(H1N1) PDM, A(H3N2), B, and against SARS CoV-2 virus. The investigated combination of Cistus creticus Acrocephalus extra., Echinaceae purpureae extra., Salvia officialis extra., Citrus bioflavonoid complex (patented name Virostop) resulted in two times higher antiviral in vivo inhibition effect than Cistus creticus extra. Alone. [14]

Subject eligibility, data collection, and outcomes
All adult (> 18 years of age at the time of examination) outpatients attending the clinic with upper respiratory tract symptoms were offered to invite their close contact with negative SARS-CoV-2 PCR (case definition as per WHO) in the study. Collected data were basic demographics, comorbidities, comedication, lifestyle factors, the severity of symptoms, and quality of life scores based on the standardized Wisconsin Upper Respiratory Symptom Survey (WURSS24) test a validated questionnaire designed to assess the quality of life and symptoms of acute viral respiratory tract infections. All close contacts of SARS-CoV-2 positive patients receiving care at the clinic were offered to participate in the study and were asked to complete the test battery every second day (day 2, 4, 6, 8, 10, and final evaluation of day 12). Detection of SARS-CoV-2 in pharyngeal wash samples was performed by RT-PCR amplification of SARS-CoV-2 N-gene fragments, based on WHO guidelines on days 0 and 10 (±2). SARS-CoV-2 immunoglobulins (Ig), including IgA, were also determined to identify seroconversion, particularly in asymptomatic persons. Data were collected from the institute's electronic patient management framework program.

The primary outcome was the SARS-CoV-2 positivity in the two groups (cistus extract users and standard of care). The secondary endpoint was a composite of standardized symptomatic scores.

Statistics
Descriptive statistics included the number of cases (n) with percentages (%) for categorical variables, whereas, for continuous variables, mean, standard deviation, minimum, median, and maximum were described. Age was compared by a two-sample Wilcoxon test, while a chi-square test between treatment groups reached the distribution of gender. The number and frequency of concomitant medication use were calculated by synchronous medication category and treatment group. The ratio of patients who used the given concomitant medication was compared by chi-square test for each concurrent medication category. The effect of treatment on symptoms and derived scores were analysed using repeated measures ANOVA. Fisher's exact test compared the ratio of patients with positive PCR results on Day 10. Day (time point), treatment group, time point, treatment group interaction, and use of NSAID were included in the model as fixed factors. Residual plots investigated goodness-of-fit. For all statistical tests, a 2-tailed p-value of < 0.05 determines statistical significance. The electronic patient management framework program carried out data collection, and with Microsoft Office Excel 2016, tests were calculated using SAS 9.4, and graphical representations were prepared using the R 4.1.1 version.

Results
There were 182 subjects involved in the study, 108 (59.3 %) in the standard-of-care plus cistus extract group and 74 (40.7 %) in the standard-of-care group. There were no statistically significant differences between the treatment groups in gender distribution and age (Table 1).

At the time of the second PCR (day 10±2 of treatment), none of the patients using cistus extract as prophylaxis became PCR positive, whereas 4 in the standard of care group (p=0.026, Table 2).