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Effects of hypertonic alkaline nasal irrigation on COVID‐19

Abstract Objective The causative agent of COVID‐19 is a novel member of coronaviridaes, SARS‐CoV‐2. It has been reported that the spike (S) protein of SARS‐CoV‐2 is responsible of infectivity. The S protein is demonstrated to be inactivated under environmental condition, such as hypertonicity and alkaline pH. The aim of the study was to investigate the effect of hypertonic alkaline nasal irrigation (HANI) on SARS‐CoV‐2. Methods Sixty patients divided into two groups. The patients in Group 1 used hydroxychloroquine (HCQ), and the patients in Group 2 used HCQ and HANI. Nasopharyngeal samples were collected at the beginning, on 3rd and 7th day of the PCR test positivity. The nasopharyngeal viral load (NVL) changes analyzed with quantitative PCR. Results NVL decrease in weekly period was statistically significant for both groups, when the difference between NVL day 0 and 3rd in Group 1 and NVL difference between day 0 and 3rd in Group 2 were compared. The difference between Groups 1 and 2 in terms of NVL change was statistically significant (P < 0.05). Conclusion We demonstrated a significant decrease in nasopharyngeal SARS‐CoV‐2 load with HANI solution and suggest that HANI may be promising modality for the COVID‐19 treatment. Level of evidence IB

3 434 058 deaths related to COVID-19 were reported. 2 The causative agent of COVID-19 is a member of the family Coronaviridae, named SARS-CoV-2. SARS-CoV-2 is a pleomorphic, enveloped, singlestranded RNA virus. 3,4 SARS-CoV-2, like SARS-CoV, has four structural proteins: Spike (S), nucleocapsid (N), membrane "matrix" (M), and envelope (E) proteins. [5][6][7] COVID-19 is transmitted by inhalation and/or to contact of infected droplets to the mucosa. 8,9 Dominant infection site of SARS-CoV-2 in the respiratory tract is nose. 10,11 SARS-CoV-2 is primarily replicated in nasal cells, which supports the hypothesis that viral replication develops in the proximal airways and these infective particles are transported to the distal via micro-aspirates. 10 S protein binds to the angiotensin-converting enzyme-2 (ACE-2) receptor and initiates intracellular entry. Binding of the S protein with ACE-2 receptor provides proteolytic cleavage and exposes the fusion protein, which is thought to be responsible for the onset of viral infectivity. 12,13 The S protein is affected by the environment changes such as pH and tonicity. In vitro pH level changes affect SARS-CoV-2, the structural proteins of the virus changes conformationally, and its infectivity. 8 Another study demonstrated the inactivation of SARS-CoV-2, in vitro, under hypertonic conditions. 14 Nasal irrigation (NI) is a routine part of the treatment of rhinosinusitis and allergic rhinitis and nasal postoperative care. Sprays used for NI provide removal of debris, mucus, and purulent secretion and regulate mucociliary clearance (MCC). 15,16 Hypertonic alkalinebuffered fluids also been improving MCC. 17 In light of this information, the aim of this study was to investigate the possible positive in vivo effects of hypertonic alkaline nasal irrigation (HANI) prepared based on pure Lake Van water, the Earth's largest soda lake in Turkey, with high salt (23 g/L) and pH (about 10) levels, 18 on nasopharyngeal SARS-CoV-2 viral load (NVL).

| MATERIAL AND METHODS
This study is a clinic-randomized controlled trial. It was approved by the Istanbul University-Cerrahpasa Ethics Committee (83045809-604.01.02).

| Populations, inclusion, and exclusion criteria
This study was conducted on patients who applied to Cerrahpasa Medical Faculty Hospital Between July 2020 and September 2020.
Patients, who applied to the hospital on the day the symptoms started, were diagnosed with COVID-19 and with no pulmonary involvement in thorax computer tomography (CT) imaging or limited lung involvement in CT (with the involvement of less than 25% of the lungs quantitatively in thorax CT imaging, without symptoms such as dyspnea, tachypnea, and with sO2 > 95% without oxygen support) were included.
Patients under 18 years of age, pregnant and lactating patients, patients with additional neurological or psychiatric diseases, patients with known cancer who have received or are receiving chemotherapy, radiotherapy or chemoradiotherapy, patients with chronic hepatitis and/or chronic kidney disease, patients allergic to hydroxychloroquine (HCQ) and its derivatives, or with allergic reactions during treatment, and patients who had intolerable side effects due to HCQ treatment were excluded from the study.
According to these criteria, 60 patients whose oro-nasopharyngeal swab qualitative PCR tests were positive for COVID-19 included.
Informed consent forms were obtained from patients.

| Randomization and blinding
The participants were randomized in two equal groups via a computer program. The clinic and laboratory staff are blind to randomization status. The laboratory analysis was done blindly. The study outcomes were evaluated blindly.

| Sample size and sampling technique
In our study, we planned to use parametric tests to obtain more statistically significant results. At least 30 patients were planned to be included in each group, since the minimum sample size for parametric tests was 30. 19 Sixty patients were separated in two groups as a control group (Group1) and a study group (Group 2). In addition, a posthoc test analysis was planned to calculate the sampling power of valid equations.

| Procedures and data collection
The patients in Group 1 were given only COVID-19 treatment and in Group 2 were given a HANI solution (Na + : 7747 mg/L, K + : 508 mg/L,

| Collection of swab samples and viral RNA isolation
Nasopharyngeal swab samples were collected from patients whose RT-qPCR test resulted positive for COVID-19, on the first day of diagnosis (day 0), the 3rd and 7th days. RT-qPCR kit (Bio Speedy, Turkey) targeting RNA-dependent RNA polymerase (RdRp) gene was used for the qualitative detection of SARS-CoV-2 (for initial diagnosis). Six hours after NI, the swab samples were taken first from the oropharynx and then from the nasopharynx with the same swab stick by an otorhinolaryngologist at the clinic and initially stored at À20 C.
Then, the samples were transferred to the and viral RNAs were isolated by using a RTA Viral RNA Isolation Kit (RTA, Turkey) in the first 24 hours and the samples were stored À80 C.

| Real-time PCR
The PCR master mixture was prepared using a SentiFAST Probe No-

| Statistical analysis
Normal distribution of data was analyzed with the Kolmogorov-Smirnov test and Levene's tests were used to assess homogeneity.
The Mann-Whitney U test and the Friedman test were used (for continuous variables) and Chi-square test (for categorical variables) were used to comparing the groups. The statistically significant level was accepted as a P value < 0.05. The SPSS 21.0 was used for statistical analysis Normal distribution of data were analyzed with the Kolmogorov-Smirnov test and Levene's tests were used to assess homogeneity. The comparisons of two independent groups were made using the Mann-Whitney U test. The dependent group analyzes were performed using the Friedman test. The subgroup analyzes were performed using the Wilcoxon analysis and interpreted with the Bonferroni correction, statistical significance level of alpha was accepted as P adj < 0.017.
The rates in independent groups were compared using the Chisquare test. The significance level of alpha was accepted as P < 0.05 for other tests. The calculation of the sample power was performed by using the G * Power software version 3.1. 20

| RESULTS
The demographic data of patients are shown in Table 1. Three patients in Group 1 were hospitalized (10%) due to development of respiratory distress, dyspnea, tachypnea, and a decrease in blood oxygen saturation in room air (sO 2 < 95%) ( Table 1).
Patients' blood samples that collected on day 0 were analyzed. In  There were not any adverse events reported in regards to using the nasal spray such as epistaxis, smell loss, and burning. There was no statistically significant difference in the evaluations of the patients in the groups according to the initial (0th), 3rd day, and 7th day virus density (P = 0.243; P = 0.165; P = 0.067, respectively). During the study period, the decreases in the NVL observed in the groups were statistically significant (P < 0.001). In Group 1, the decrease in NVL in the first 3 days was not statistically significant (P adj = 0.027). The first 3-day NVL decrease in Group 2 patients was found to be statistically significantly higher than Group 1 (P adj = 0.019). There was no statistically significant difference in the groups according to the 7-day NVL decrease (P adj = 0.152). No significant difference was found in other statistical evaluations (Figure 2) (Tables 3 and 4).
Three patients (10%) in Group 1 had to be hospitalized due to deterioration in their condition. The mean NVL of outpatients (27 patients) of Group 1 on day 0, 3rd, and 7th were 76 400, 2686, and 50.02/μL while the mean NVL of inpatients (3 patients) of Group 1 on day 0, 3rd, and 7th were 50 100, 111 000, and 23/μL according to this, there was no statistically significant difference. However, the increase of NVL on third day was thought to be valuable.
The posthoc test, which was used to determine the sampling power of the present study, was performed with a 5% error probability according to the sample size used. The sample power (1 À β err prob) was 0.8.

| DISCUSSION
COVID-19 has affected the whole world in the past year. In the medical field, the most effort in the past year has been invested in the treatment and prevention of this disease that affects the health care system in an unprecedented way. In this study, we showed that the use of HANI reduces NVL in the early period of the disease, as the first in the literature.
In COVID-19, most patients exhibit mild clinical symptoms. Mild symptoms are influenza-like fever, cough, fatigue, muscle pain, and taste, and smell disorders. Progressive pneumonia and/or ARDS can be seen in severe cases. 21,22 In our study, the most common symptom in both groups was fatigue, similar to the literature.
The time between the onset of COVID-19 symptoms has been reported to be between 2 and 14 days (average of 5.2 days) after viral exposure. 8 There are two hypotheses regarding the spread of SARS-CoV-2 infection: Inhalation of infected droplets into the lung 23,24 or the presence of a viral inoculum in the nose, which is the first site of infection, and the aspiration of micro-aspirates from the naso-oropharynx into the lungs. 11,25 In viral cultures taken 24, 48, and 96 hours after SARS-CoV-2 infection, higher viral replication rate was detected in the samples taken from nose than the viral load obtained in the epithelium of the large airways. These findings suggested that the primary infection site is the nasal cavity. 10 S protein is the part that binds to ACE-2 receptor and initiates intracellular entry. 12,13 It was also determined that the distribution of ACE-2 expression in the respiratory tract was diverse and that it was most intensively found in the nasal region. This suggests that the nasal region is more frequently affected in the early stages of COVID-19. The nasal cavity is exposed to environmental factors in high and variable doses and develops varying degrees of innate immunity. This situation is thought to be responsible for different clinical responses to COVID-19. 10,26 Any procedure in the nose that relieves infection and viral load will prevent the disease from progressing and spreading to the lungs and the whole body.
NI provides mechanical cleaning and helps to remove contaminants such as mucus, cell debris, pathogens, allergens, and particles.
While increasing MCC, it reduces the contact of particles present in the inhaled air with the mucosa. 15, 16 Ramalingam et al reported that the need for drug use, domestic transmission, and the spread of the viruses, that infect the upper respiratory tract, were reduced with NI and mouth washing performed with hypertonic solution. 27 The viral replication of SARS-CoV-2 was inhibited with 210 mM NaCl by 90% and completely inhibited with 260 mM NaCl solutions, in vitro. 14 High salt concentration in the extracellular space creates a hyperosmotic medium. It has been suggested that this will inhibit the entry of the SARS-CoV-2 into cell. 13 Previous studies reported that the SARS-CoV-2 is affected by pH changes and hypertonic medium in vitro. 14,28 It has been reported that the S protein of the SARS-CoV exhibits cell fusion at neutral pH and was affected by the medium, such as pH and tonicity, and undergo conformational changes. 28 We prepared a solution to achieve the effects of both hypertonic and alkaline solutions reported previously in the literature. The HANI solution used in the study was prepared by based on the water of lake Van. It contains bicarbonate, sulfate, sodium, chloride, potassium, and its pH is 9.3. The HANI solution creates a hyperosmolar medium in the nasal region. In our hypothesis, the interaction of the S protein with ACE-2 will be impaired, as a result of the conformational changes, due to a hypertonic and alkaline environment and the unbinding virus is mechanically removed from the nasopharynx due to NI and MCC which increases with NI.
In previous clinical studies, it was reported that COVID-19 disease had a more severe course in men. 29 In order to show the possible positive effect on patients who are likely to have a more severe clinical course, the study groups were formed with a predominance of male patients. In our study, in the first 3 days, the decrease in NVL in the Group 2 was statistically significant (P < 0.001) but not in the Group 1 (P adj > 0.017). The first 3-day NVL decrease in Group 2 patients was found to be statistically significantly higher than Group 1 (P < 0.05). It means that the use of HANI may help in the early viral T A B L E 3 The viral load density on Days 0, 3, 7, and viral load differences between Day 0-3 and Day 0-7 in Group 1 and Group 2 response. COVID-19 is a biphasic disease, with a viral response phase in the early period and an inflammatory phase in the later period. 30 There was a statistically significant decrease in NVL in both groups on seventh day. It is noteworthy that this NVL decrease in Group 2 was almost twice as much as that in Group 1. These findings suggest that HANI may have a contribution to viral clearance in upper airways.
However, there was no significant difference between in groups regarding NVL decrease between 3rd-7th and 0th-7th days. Previous studies showed that the significant NVL decrease in COVID-19 patients was observed within the first 4 days after the onset of symptoms. 31 Accordingly, the absence of a significant decrease in NVL in the present study, except in the early period, may be due to late inflammatory response.  36 Therefore, the results we obtained in the group with a worse prognosis are more clinically significant.
In addition, since the patients we included in the study were in home isolation, we did not have objective information about their compatibility with drug use. However, there are studies in the literature showing that patients adapt better to the use of their medicines in the period of COVID-19. 37 Therefore, we think patients were followed our instructions. In our study, we used NVL values to determine treatment efficacy and determined these values with RT-qPCR test, similar to other studies in the literature. 38,39 However, the most important limitation of our methodology is that it is not certain whether the values determined by the RT-qPCR test belong to live viruses. 40

| CONCLUSION
A significant decrease in NVL due to HANI and the presence of patients who has severe disease in the group who did not perform NI suggest that HANI may be effective in the treatment of COVID- 19. The fact that it reduces NVL significantly in the early stage has the potential to prevent the spread of the disease by reducing intracommunity transmission. The repetition of data that can be obtained with larger numbers is required to support the present findings.