Clinical endoscopic findings improved: We found improvement of clinical endoscopic parameters, almost in all cases on the test side following the intervention. Using the Fischer exact test, we found that the improvement in nasal crusting, nasal discharge, and nasal mucosal condition in the test side to be statistically significant. However, the improvement in the size of the nasal cavity and atrophic turbinates was not statistically significant. This is evident from the endoscopic scores assigned to the test and control sides before and after the intervention (Table 2). The pre-intervention scores between the two sides of the nasal cavity were not found to be statistically significant, reflecting the fact that the pre-treatment condition of the test side and the control side of the participants were similar. However, the post-intervention scores on 3 features of crusting, mucosal congestion, and discharge were found to be statistically significant improvement on the test side. However, the features of atrophic turbinates and nasal cavity roominess though different was not statistically significant (Table 2).
Nasal obstruction, crust formation, and nasal discharge improved: There was an improvement in the patient symptom as well, following the intervention. Patients presented with the symptoms a wide range of symptoms which were: nasal obstruction, foul smell, impaired smell sensation, crusting, nasal discharge, epistaxis, headache, epiphora, and myiasis in the nasal cavity. Following the intervention, the patients were enquired about the improvement of their symptoms in the sides of the nose. The symptom of epiphora and myiasis were excluded as only one patient presented with those. A 2X2 contingency table was made and Fischer exact test was applied for each symptom whether improved or not improved in the test side and the control side. It was found that except for headache there was an improvement of symptoms in all the cases. However, the improvement was statistically significant in the test side for nasal obstruction, crust formation, and nasal discharge (Table 3).
Each symptom was given a score of 1 and the cumulative score was noted in each patient before and after the intervention and compared between the test and the control side. A maximum possible score was 9 for a patient who would be having all the 9 symptoms, and a minimum of 0 who would not have any of the symptoms. There was no difference in symptom scores between the test and control side pre-intervention. But a statistically significant difference in the cumulative symptom score was found between the test and control side following the intervention. This clearly was indicative of the improvement in the test side following the intervention (Table 4).
Restoration of mucosal glands: Specimen from the nasal cavity was examined histopathologically for the following features; basement membrane thickening, presence of granulation tissue, fibrosis, mucus gland concentration, bacterial colonies, and glandular hypertrophy. All the features in the post-treatment specimens had improved. However, only three features were statistically significant. The pre-treatment features were similar in both test and control specimens, indicating the improvement was due to the specific intervention made during the study (Table 5).
Emphasis was given on the presence of mucus-secreting glands,as the dryness (due to thinning) of the mucosa and glandular atrophy are the hallmark of this disease. Median and interquartile range was used to compare the histological feature of glandular hypertrophy. The pre-treatment values showed no statistically significant difference between the test and the control side. The post-treatment values showed improvement on the test side, which was statistically significant (Table 6). Figure 2 shows the decrease in inflammation in the test side with the restoration of mucus glands following treatment compared to the pre-treatment slide.
Alteration in Nasal microbiome: We propose that the marked clinical and histological improvement in the test side be due to the change in the microbiome of the nasal cavity on the test side. The nasal microbiome was analyzed to understand the differences in detail. Microbiome analysis was performed on Twenty samples,ten each from the test (right) side and control (left) side, of which five samples each were from before treatment and after treatment.The genus composition of the nasal microbiome pre-and post-treatment in the case and control group is in Figure 3. Actinobacteria and Proteobacteria are the most abundant microbes, with a marginally higher amount in the test side, followed by Bacteroides. The least abundant in either sample before or after treatment is the Vibrionaceae. The alpha diversity is found to be increased post-treatment on the test (right) side compared to the control side. The real observed diversity of species between the test (right) and control side, more evident with the Shannon index, as shown in figure 4.
The beta diversity of the samples collected before and after treatment in the control (Left) side cluster closely. In contrast, the groups cluster distinctly before and after on the test (right) side, suggesting an effect on the microbial composition following the intervention in the test side, Figure5a. The Venn diagram plotted, filtering OTUs that are 80% abundant at the species level for each of the study group, About 78 OTUS were identified to be common among all four groups, while 0, 2,1, and 15 OTUs were found specific to the Left (untreated), Left (Treated), Right (untreated) and Right (treated) groups respectively, Figure 5b.
Two types of analysis for the bacterial groups at all bacterial levels were done, and a statistical difference was observed.The Kruskal Wallis test was used to test all four groups together, Figure 6b. However, for this strategy, first, it is crucial to have the same microbiota composition before the treatment in the right and the left sides. The risk is not to detect some taxa that are influenced by the treatment, and, some taxa might be present that are not stable over time. For these reasons, a second strategy was adopted. Where, the difference between ‘before’ and ‘after’ intervention was calculated (additionally, convert the difference by log2). And, the difference between the test (right) and the control (left) side was compared using (Wilcoxon test). In the data, some taxa increased on the test (right) side and decreased on the control (left) side, Figure 6a.
Certain bacteria have increased and others were decreased in proportion in the test side compared to the control side following the intervention. These are Rhodobacter spheroides, Plancomycete sp., Sphingomonas asaccharolytica, Bacteroides caccae, Methylotenera mobilis, Stapia indica, Comamonas terrigena, Flexithrix dorotheae and Roseicyclus mahoneyensis, Figure 6b.
Increased expression of SCFA receptors: There was a significant difference in microbiome pattern in the test and control side post-intervention. There was clinical improvement in between the test and control side following the intervention. Gut microbiome studies have found that Short-chain fatty acids (SCFAs), are generated by the healthy microbiome16. The SCFAs play an anti-inflammatory role through activation of the GPR43 receptors. These GPR43 receptors are detected by immunohistochemistry.
GPR43 receptors was found to be increased in the post-intervention test specimen (Fig 7). In view of the improvement with honey therapy, the subjects were prescribed to use honey on both sides following the study. They are being followed up clinically following the study for a period of 8 months and above, and 18 patients were found to be doing well with relief from symptoms. One patient did not follow up after completion of the study.