Treatment of Atrophic Rhinitis with Manuka Honey– a non-randomized control trial, and a new theory of etiopathogenesis for the disease.

Exact etiology of Atrophic rhinitis (AtR) is yet unknown. Polygenic and polybacterial causes have been implicated in the onset and progression of this disease. AtR doesn’t respond to any particular modality of treatment that targets specic etiology, this describes its multifactorial nature. In this study, we report on a non-randomized control trial on the use of a nasal spray of 10% Manuka honey in patients with AtR attending the out-patient unit of the Department of Otorhinolaryngology and Head Neck Surgery. In this study, we show signicant observations: 1. Decreased fetid smell, 2. Thickening of the mucosa, 3. Decreased inammation with healed mucosal ulcers, 4. Increased concentration of the mucosal glands, 5. Alteration in Nasal microbiome, and 6. Increased expression of SCFA receptors. These changes occurred in response to honey therapy, and are consequent to the resetting of the Nasal microbiome.


Introduction
Atrophic Rhinitis (AtR) is a chronic in ammatory condition of the nose with unknown etiology. The incidence of Primary AtR is uncommon in developed countries. It has declined markedly in North America, Britain, and some parts of Europe, with no such decline in Asia and Africa. Primary AtR is high in developing countries, especially in countries with a warm climate; with the highest prevalence seen in the arid areas bordering the great deserts 1,2 . Primary AtR affects about 1% of the Indian population and is more frequent in young and middle-aged individuals, with a female preponderance. It affects people with low-socioeconomic status residing in conditions of poor hygiene. The disease has a colossal impact on the social life of the patient. Social ostracization is common in patients due to the obnoxious smell, emitted from the nose of these patients [3][4] .
Over the years, many factors have been thought to contribute to the development of this disease.The factors include nutritional de ciency (iron, fat-soluble vitamins, and proteins, phospholipid), autonomic dysfunction leading to excessive vasoconstriction, re ex sympathetic dystrophic syndrome, endocrinal de ciency with imbalances due to estrogen de ciency, immune dysfunction, and bio lm formation [5][6][7][8][9] , but none has been proven to be the direct cause. AtR is essentially diagnosed clinically, based on the triad of fetor, greenish crusts, and roomy nasal cavities, typically seen in full-blown cases in later stages. In the early stages, the patient may only complain of altered smell with the presence of thick crusts. Early in the disease, the turbinates may look normal, but in the later stages, the turbinates are atrophied. None of the studies done so far has been able to establish an ideal management protocol for the disease 9-10 . However, they have suggested methods to control nasal dryness by using lubricants or methods to decrease evaporation from the mucosal surface. Some studies have tried antibiotics with limited success 11 . The variable responses to different treatment regimes are due to the multifactorial nature of this disease 12,13 .It does not respond to any particular modality of treatment targeting a speci c etiology.
Throughout the history of the human race, honey has been used as a medicine, the most persistent use being dressing of wounds. Manuka honey is a mono oral honey produced by certain Leptospermum species native to New Zealand and Australia. This has been registered by appropriate medical regulatory bodies as a wound care product 14,15 . In this study,we proposed to treat AtR by using honey locally to heal the ulcers and to regenerate the mucosa by replenishing the commensal micro ora of the nose. Honey is a product that has been used by the Asian Indian population for time immemorial in various forms it is well accepted culturally and ethnically. The acceptance and adherence to the honey treatment strategy were thereby not doubted by the AtR patient population under treatment.

Materials And Methods
We conducted this study in Odisha a state in the eastern region of India, which typically has hot and humid conditions throughout the year. Tewnty ve patients were screened for the study and six participants were rejected of whom two were diabetic, four had a history of some nasal surgery.
Investigations were done to rule out secondary causes for the condition. Following the inclusion and exclusion criteria for the study, 19-candidates were enrolled for the study (Fig 1 -CONSORT).
Procedure: Nasal endoscopy was done crusts were removed and a score was assigned depending on clinical feature which could be noted by doing nasal endoscopy. The score was assigned as per the following clinical features: Crusting: Gross -2; Minimal -1; Nil -0; Discharge: Thick -2; Thin -1; Absent -0; Nasal mucosa: Congested -2; Not congested -1; Atrophic turbinate: Present -1; Not Present -0; Size of Nasal cavity: Roomy -2; Not roomy -1.A nasal mucosal biopsy was taken from both sides on day-1 of the study. The right side of the nasal cavity of each patient was considered as the test (as the rule for all tests) and the left side was considered as control. After taking the biopsy the right nasal cavity (test) was sprayed with freshly prepared manuka honey solution in normal saline (10%) and the left nasal cavity (control) was sprayed with normal saline only, using separate applicators. The spraying of honey preparation was done twice a week for 8 weeks. During this period, they were instructed not to apply anything else in their nose or take any other medications without information from the Principal Investigator. At the end of the study duration following the intervention (at the end of 8 weeks), a repeat endoscopic score was assigned to the nasal cavities (both test and control sides), and the nasal mucosa was again biopsied, from nasal cavities (both test and control sides) from all the patients.
Of the 19-participants with primary AtR who were enrolled for the study, 9-participants were males and 10 were females.The average age of the study group was 33.8 ±10.7 years. The participants were predominantly rural and semi-urban dwellers with an average hemoglobin percentage of 11.8 ±1.7 mg%, average ESR 18.6 ±16.2 mm at the end of 1 st hour, and an average leucocyte count of 9,439±1680 per Cumm.
AtR patients presented with a wide range of symptoms, nasal obstruction was the commonest presenting symptom (100%) to epiphora which was the least common presenting symptom (5.26%) ( Table 1). Epiphora as a presenting symptom has not been reported before in other studies on AtR.

Effects Of Intervention
Clinical endoscopic ndings 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 signi cant. However, the improvement in the size of the nasal cavity and atrophic turbinates was not statistically signi cant. 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 signi cant, re ecting the fact that the pretreatment 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 signi cant improvement on the test side. However, the features of atrophic turbinates and nasal cavity roominess though different was not statistically signi cant ( 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 signi cant 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 preintervention. But a statistically signi cant 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, brosis, mucus gland concentration, bacterial colonies, and glandular hypertrophy. All the features in the post-treatment specimens had improved. However, only three features were statistically signi cant. The pre-treatment features were similar in both test and control specimens, indicating the improvement was due to the speci c 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 signi cant difference between the test and the control side. The post-treatment values showed improvement on the test side, which was statistically signi cant (Table 6). Figure 2 shows the decrease in in ammation 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 ve 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 gure 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, ltering OTUs that are 80% abundant at the species level for each of the study group, About 78 OTUS were identi ed to be common among all four groups, while 0, 2,1, and 15 OTUs were found speci c 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, rst, 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 in uenced 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 signi cant 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 microbiome 16 . The SCFAs play an anti-in ammatory 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.

Discussion
AtR is a chronic, progressive degenerative condition of the nasal cavity characterized by a foul odour (ozena), nasal obstruction, dryness, and crusting. Endoscopic inspection reveals a large, wide nasal cavity with dried mucosa. Classically, the management of patients with primary AtR aims to alleviate symptoms using nasal irrigation douche or ointment application. Attempts of treating this nasal condition started as early as 1971, closure of the nostril (Young's operation 17 ) and endonasal microplasty (intranasal implant insertion),were the surgical management of primary AtR 18 , these options had its demerit on the possibility of decreased quality of life of the patient. Studies using rifampicin, cotrimoxazole and cipro oxacin for the disease 11,19,20 and found rifampicin to be the most effective treatment. In addition the use of lubricants like sesame oil in controlling the signs and symptoms of AtR was also attempted 21 . None, however, could establish an ideal or de nitive management protocol for the disease. Even tough rifampicin shows promise in the treatment of AtR, but in our opinion, its use should be reserved for Tuberculosis, especially in developing countries like India.
Honey formulations have been tried for the treatment of various sinus and nasal in ammations. It was found that thyme honey spray reduces in ammation and polyp formation in chronic rhinosinusitis patients, their research also revealed accelerated mucosal healing in those patients 13 .We designed a study with mono oral Manuka honey as it has a speci c carbohydrate concentration containing compound (data Provided in SI). Manuka honey disrupts cellular aggregates, it prevents the formation of bio lms by a wide range of problematic pathogens 22 , including Streptococcus and Staphylococcus species, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Acinetobacter baumannii, and Klebsiella pneumonia 23,24 . This has clear clinical implications for using honey on wounds containing bio lms. In this study, following the intervention, we found signi cant improvement in the symptom score of the patients suffering from AtR. We observed a decrease in fetid smell and the histopathological study showed thickening of the mucosa, decreased in ammation with healed mucosal ulcers, and increased concentration of the mucosal glands. The mucosal sparing effect occurred due to honey therapy. In a similar study honey was used in the treatment of AtR showed signi cant improvement compared to the group in which 25% anhydrous glucose in glycerin was used 25 .
However, in their study, the type of honey used was not speci ed and the cause for improvement was not evaluated.
We hypothesized that the Manuka honey would have an therapeutic effect on AtR and propose, Evidences from this study indicate a new theory of aetiology of causation of the AtR : In AtR, nasal obstruction, fetid smell, impaired smell sensation, crusting, nasal discharge are caused due to 'dysbiosis'. The bacteria which harbour the nose in this disease, feed on the mucosal layer causing erosion and produces fetid smell due to the release of sulfur-containing metabolic end products. This progressive damage may momemtarily respond to antibiotics or other therapy that alter microbes or provide lubrication locally. Manuka honey as opposed to antibiotics, is a concoction of complex carbohydrate, these carbohydrates promote microbial diversity.The complex carbohydrates of Honey provide the microbes (source of energy), altering the microenvironment and alters the pH of the nasal cavity. The altered microenvironment and pH, along with ambient nutrition,changes the unhealthy ambient microbiome. The healthy microbiome is allowed to ourish in the nose. Correction of 'dysbiosis' has mucosal sparing effect in these patients is due to the 'colonization resistance' which is offered by the stabilizing bacteria. Improved mucosa provides required lubrication and the healing of the ulcers is enhanced by the Short chain fatty acids produced by the renewed nasal microbiome.
To support our theory, we showed an improvement in all the clinical symptoms in all our patients. Mucosal thickening, Altered nasal microbiome, increased GPR43 expression are proofs for healthier resetting nasal micro-environment.
Microbiome, is the entire population of microorganisms that are present in an ecologic niche, irrespective of their cultivability.In human body, these ecological niches are sites like the gut, skin or nasal cavity 26 . Studies suggest that each body habitat has its own characteristic bacterial community which changes throughout the life of the person depending on the age 22,27,28 . The commensal bacteria which is present in the human nasal cavity suppress opportunistic pathogen colonization. The competition they offer to the pathogens for the limited space, nutrients and may also be due to metabolites they produce that may not be favourable to the competitors 29 . Studies suggest that environmental factors have a role in the modulation of the nasal microbiome composition 30,31 . Some organisms, like the Staphylococcus aureus, can be both commensal as well as an opportunistic pathogen 32,33 .
An imbalance in the ambient microbial community or the microbiome dysbiosis can impact human health signi cantly and has been implicated for various diseases of the gut as per studies 34 . Nasal microbiome dysbiosis has been associated with chronic rhinosinusitis; neurological diseases like to Parkinson's disease, allergic rhinitis, asthma and otitis media.
We observed a signi cant difference in nasal microbial pro le post-intervention, in the test side compared to the control side. There is an altered abundance of bacterial species Rhodobacter spheroides, Plancomycete sp., Sphingomonas asaccharolytica, Bacteroides caccae, Methylotenera mobilis, Stapia indica, Comamonas terrigena, Flexithrix dorotheae and Roseicyclus mahoneyensis. There was an improvement, in clinical and histological parameters in all patients following intervention in the test side. We deduce that these changes are due to the resetting of the microbiome.
During health and in the absence of antibiotic exposure the microbiome can effectively inhibit colonization and overgrowth by invading microbes such as pathogens. This phenomenon is called 'colonization resistance' and is associated with a stable and diverse microbiome in tandem with a controlled lack of in ammation and involves speci c interactions between the mucosal immune system and the microbiome. It is the mechanism whereby the intestinal microbiota protects itself against incursion by new and often harmful microorganisms. In our opinion a similar mechanism is occurring in the nasal cavity as well.
The change in microbiome changes the in ammatory process by acting on the G protein-coupled receptor 43 (GPR43 receptors 16 ). The new, healthy microbiome in nose produces short-chain fatty acids (SCFAs) following metabolism of complex carbohydrates in honey. The SCFAs targets several receptors G proteincoupled receptors on the cell surface of which the most notable is the GPR43. Role of GPR43 in regulating the role of intestinal in ammatory responses have been studied. It has been found that mice which are GPR43-de cient have exacerbated disease symptoms. The results suggest that GPR43 mediates the protective effects of SCFA in intestinal in ammation. Studies have shown that dietary bers with long-chain carbohydrates with high solubility act as a source of energy for selected groups of bacteria, possessing the ability to promote the growth of bene cial microorganisms. These prebiotics have been identi ed as potential Nasal spray in modifying Nasal microbiome for health.
Limitations of the study: The limitation of the study lies in the fact that it is not a randomized control study and is conducted in a single institution. Post-surgical secondary AtR could have been included to increase the ambit of treatment. A randomized control study which would be multi-institutional would have yielded a much diverse and more valid data. Having said that the present study does have its advantage. The case and control being the same person eliminates selection bias and matching bias.
The study does conclude with the improvement following the intervention. It tried to search for the reasons for the improvement. By doing so, the study has come up with a new theory of etiology for the disease.

Conclusion
Manuka honey having a concoction of complex carbohydrates acts as a prebiotic promoting bene cial microbiome of the nasal cavity. The healthy microbiome provides colonization resistance and produces SCFAs, which play an anti-in ammatory role decreasing the existing in ammation. The disease process halts, mucosa heals, and normalcy returns. The cure for the disease provided by Manuka honey helps us establish the microbiome theory, for the Primary AtR, which can be extrapolated for secondary AtR too, especially those caused after extensive nasal surgery.

Declarations
Contribitions :The author's responsibilities were as follows-SS and BR: were responsible for conception, analysis of data, and writing the manuscript; SS, SP, PS and RM: were responsible for sample, Histopathology, and clinical data collection; FM, GV, NM and BR: were responsible for the Sequencing and data analyses. None of the authors had a personal or nancial con ict of interest.