The effect of modulation of the nasal microbiome in managing chronic inflammatory conditions of the nose and paranasal sinuses

Introduction:

Chronic inflammatory conditions of the nose and paranasal sinuses affect the quality of life of the patients adversely.

Chronic rhinosinusitis (CRS) is the inflammatory condition of the nose characterized by nasal discharge or post-nasal drip, nasal obstruction, nasal congestion, facial pain, pressure or fullness and decreased sense of smell for a duration of 12 or more weeks.It is complemented with objective findings like the Computed tomography and nasal endoscopy.1 Globally, this condition is quite prevalent, with more than 10% in Europe and the United States. It interferes with work, leisure and sleep, thereby disrupting the patient’s daily life, thereby impacting the health-related quality of life of these patients.2,3

Atrophic Rhinitis is a well-known disease for ages first described by Fraenkel in the late 19th century.4 It is aprogressive degenerative condition of the nasal cavity characterized by a foul odour (ozena), nasal obstruction, dryness, and crusting, predominant among women.5 Its prevalence varies in different regions of the world, commoner in tropical countries such as India. It has a prevalence rate of 1% in tropical countries.6

These two chronic nasal inflammatory nasal conditions disrupt the patient’s daily activities, thereby impeding his/her quality of life. We aim to formulate a new treatment strategy based on a novel nasal test solution that can bring about a change in Microbiome and help in patient care. However, since the product is under Investigational procedure, the composition will be revealed later on.

Review of Literature

Chronic rhinosinusitis

Chronic rhinosinusitis is an inflammatory disorder of the nose and paranasal sinuses, more than 12 weeks. It is suggested that CRS is an inflammatory disease rather than an infectious one. The bacterial infection, however, may be contributing by the initiation of inflammation. 7,8,9 It has been hypothesized that inflammatory response is triggered in the sinus cavity by a bacterial infection which results in chronic changes and symptoms.10,11

Studies suggest a polymicrobial process to be causing CRS. Decreased microbial diversity, evenness and richness in chronic inflammatory diseases elsewhere have also been found in CRS patients.12-16 The decrease in diversity maybe because of the increased presence of anaerobic bacteria growing in biofilms. 16,17 Whereas the overall bacterial burden and the phylum level abundance are constant, the relative abundance of specific genera of bacteria is found altered in CRS. 16-19

A depleted signature of Corynebacterium, Anaerococcus, Peptoniphilus, Finegoldia, Propionibacterium, Peptoniphilus are found in CRS. All of these bacteria are healthy bacteria of the URT. 20,15 The shift from the beneficial microbial community may be the reason for the increased inflammatory response (Toll-like receptor responses) and the clinical severity.13,21

The CRS patients have a sinus microbiome signature dominated by Pseudomonadaceae, Corynebacteriaceae, Streptococcaceae and Staphylococcaceae, in a study.21 126 Other studies have shown an overgrowth of Corynebacterium tuberculostearicum and Staphylococcus in sinuses.12,14 along with Curtobacteria, Corynebacterium, Staphylococcus, H. influenza and Pseudomonas in the middle meatal area.22,23 The microbiome of CRS patients have Pseudomonas aeruginosa, Staph aureus and coagulase-negative Staphylococcus, as per some studies.14, 24-28

A recent study by Lal et al. compared the microbiota of middle and inferior meatus of CRS patients with healthy controls. They found that the CRS patients without nasal polyps had an abundance of Haemophilus, Streptococcus and Fusobacterium sp., but a loss of bacterial diversity compared to controls. In patients with CRS without polyp, the study found the abundance of Alloiococcus, Staphylococcus, and *Corynebacterium.*29 Copeland et al. in their research found that CRS has a negative correlation with six OTUs, affiliated to Corynebacterium, Dolosigranulum and Staphylococcus. Furthermore, the Corynebacterium OTU410908 signature correlated negatively with the SNOT-22 score, giving an idea about the severity of the disease.30

Usually, the genera, which are anaerobic like the Anerococcus, Finegoldia, Lactobacillus and Peptoniphilus, are more abundant in patients with CRS than healthy individuals.30

CRS is generally categorized into CRS with polyp (CRSwNP) and CRS without polyp (CRSsNP).12,13,30 CRSwNP is usually associated with aspirin intolerance and asthma.23  When compared, it was noted that CRSwNP had abundant signatures of Staphylococcus, Corynebacterium and Alloiococcus. In contrast, CRSsNP had an abundance of anaerobes Haemophilus, Fusobacteria and Streptococcus, and depleted Alloiococcus, Rothia, Corynebacterium and Finegoldia.

The sinuses are not bereft of oxygen, and anaerobic organisms are generally not found in abundance.  Therefore, the abundance of anaerobic microorganisms may result from the pathology and disease progression in these patients.31 Fusobacteria, which is associated with suppuration, may cause the anaerobic condition in the paranasal sinuses. 22,29

The severity of inflammation in CRS has a positive correlation with phylum Proteobacteria (Pseudomonas) and phylum *Bacteroidetes (Prevotella).*32 It has also found that CRS patients have altered taste molecule response, with less sensitive to bitter taste and more sensitive to sweet.33 The bitter receptors play a role in bacterial detection and defence; hence it is evident that the CRS patients would have decreased bitter sensation. Due to these changes, CRS patients have reduced ciliary beating stimulation in Upper Respiratory tract (URT), showing altered NO levels.34,35

Nasal washes, corticosteroids, and sinus surgery are the most common treatments for CRS and may significantly influence the URT microbiome.

Atrophic Rhinitis:

Atrophic Rhinitis is a chronic, gradually progressing degenerative condition of the nasal cavity characterized by a foul odour (ozena), nasal obstruction, dryness, and crusting.36

The aetiology of primary atrophic Rhinitis is primarily unknown. Progressive metaplasia and atrophy of all mucosal components (epithelium, vessels, and glands) occur because of increased osteoclastic activity. Which results in a volumetric decrease of sinonasal structures. The turbinate show shrinkage, and the inferior turbinate is affected the most. Endoscopic inspection reveals a large, wide nasal cavity with dried mucosa. 5

Classically, the management of patients with primary atrophic Rhinitis aims at alleviating symptoms using nasal irrigation douche or ointment application. In 1971, Young37 proposed closure of the nostril (Young’s operation). Saunders38 suggested endonasal microplasty (intranasal implant insertion) for the surgical management of primary atrophic Rhinitis. However, Young’s operation has its demerit on the possibility of decreased patient’s quality of life.

A study done by Friji et al. in 2014 using autologous lipoaspirate transfer and platelet-rich plasma in five patients with atrophic Rhinitis showed good result. The study had a promising impact, with normal-looking mucosa and symptomatic improvement in all five patients in follow up. 39

A study was done in 2018 by B Altas et al. to see the effect of ozone treatment in experimental animals. They found that only one of the pathological parameters of atrophic Rhinitis (vascular ectasia) improved significantly. This leads them to conclude that ozone may have limited or no effect on atrophic Rhinitis at the histopathological level. 40

Osama G et al. in 2019, compared two groups using mitomycin c douching and sodium bicarbonate. Both the groups received rifampicin oral daily for three months. This study found a significant improvement in the group in which Mitomycin C was used in the douche compared to bicarbonate douche. 41

Jaswal A et al., in their study in 2008 conducted in atrophic rhinitis patients, found rifampicin to be the most effective treatment in controlling the signs and symptoms of atrophic Rhinitis. They divided the participants into three groups, one group receiving oral rifampicin, one group receiving local placentrex injection and the control group receiving no oral or injectable intervention. All three groups received bicarbonate nasal douching. 42

A case report published by Namburi SK et al. in 2008, where they used a nasal prosthesis and found improvement of signs and symptoms in the patient after ten months follow up.43

Studies by Borgstein 1993 and Nielsen 1995 suggested using rifampicin, cotrimoxazole and ciprofloxacin for the disease. Johnsen 2001 suggested using sesame oil for combatting nasal dryness.44,45,46 None, however, could establish an ideal management protocol for the disease.

A study done by Killera S et al. honey used in the treatment of atrophic Rhinitis showed significant improvement compared to the group in which 25% anhydrous glucose in glycerin was used. They noted an improvement of symptoms in the test group compared to the control group. 47

Gaps identified/The rationale of the study:

The is a need to address the change in the microbial pattern of the nose and paranasal sinuses in these disease conditions. The change can be brought about by promoting healthy bacteria in this ecological niche which will help the host to fight against the pathogens, thereby getting a cure from these disease conditions. This will help to improve the quality of life of these patients.

Moreover, there are no Indian data on nasal microbiome or the changes in disease conditions.

Hypothesis:

There will be an improvement in the quality of life in patients suffering from chronic inflammatory conditions of the nose and paranasal sinuses who will administer the nasal spray of the test solution

Objectives:

Primary:

1.     To test whether daily nasal irrigation with the test solution improves sinus symptoms and quality of life of patients with chronic inflammation of the nose and Paranasal sinuses - PNS (in CRS and Atrophic Rhinitis) using SNOT(Sinonasal Outcome test) 22 score and or RSDI (Rhinosinusitis Disability Index) score.

2.     To study the microbiome profile of patients with chronic inflammatory diseases of the nose and PNS

Secondary:

1.     To study the normal nasal microbiome profile across age and gender

2.     To test whether there is the improvement of CT scan picture of the patients following intervention using Lund Mackay (LM) score

3.     To test the histopathological changes following intervention

4.     To assess the change in the microbiome in these patients’ following intervention

Material and Methods:

***Study Design:***Randomized control trial (RCT) – Stratified randomization using randomization software

***Study place and Duration:***Bhubaneswar, for a period of 3 years

Sample size calculation and Participant recruitment:

To detect a Minimal clinical important difference (MCID) of 10 points in SNOT22 score with a standard deviation of 9 and level of significance of 0.05 with a power of 85% the sample size would be 15 per group, with the allocation ratio 1:1.

OR

(From the article by Gallo S et al. in April 2020 using the mean and standard deviation of the outcome, the required sale size per group is 12 considering attrition of 20% the sample size would be 15 per group)

Level of Significance - 0.05

Power - 0.85

**Two Sample, Continuous Outcome (**1-2)

Mean in Group 1 (1) 48.9

Mean in Group 2 (2) 22.9

Difference in Means under H1 (μ1-μ2) 26

Standard deviation of outcome (s) 20.8

Effect Size (ES) 1.25

Sample Size Required Per Group 12

Attrition - Percentage Expected to be Lost/Missing Data 20%

Sample Size Required Per Group, Accounting for Attrition 15

Total Sample size: 30 + 60 = 90

Inclusion criteria:

Adults (more than 18 years);

Diagnosis of CRS based on American Academy of Otolaryngology-Head Neck Surgery guidelines, refractory CRS,

Atrophic Rhinitis based on clinical examination

Exclusion criteria:

Pregnancy, comorbidities that preclude travel for follow up, declining to be included in the study, a SNOT22 score of less than 10, LM score of less than 4 in cases of CRS.

Methods:

Atrophic Rhinitis:

Patients will be diagnosed based on history and clinical examination. Patients would have a foul odour (ozena), nasal obstruction, dryness, and crusting.

After diagnosis and allocation into the study, they will be randomized using randomization software. Endoscopy will be done in both the groups, and a nasal mucosal biopsy taken on day zero. The test group will be administered with the test solution nasal spray, and the control group will be administered with the normal saline nasal spray.

The nasal sprays would be given to the patients, which can last for two weeks. The compliance will be checked by calling them each week on a particular day. They would be called at intervals of 2 weeks to replenish the nasal spray and perform endoscopy and clear crusts from the nose.

Chronic Rhinosinusitis (CRS):

Patients would be selected as per criteria by the American Academy of Otolaryngology and Head and Neck Surgery.

Twelve weeks or longer of two or more of the following signs and symptoms:

• mucopurulent drainage (anterior, posterior, or both),

• nasal obstruction (congestion),

• facial pain-pressure-fullness, or

• decreased sense of smell.

AND inflammation is documented by one or more of the following findings:

• purulent (not clear) mucus or edema in the middle meatus or anterior

ethmoid region,

• polyps in nasal cavity or the middle meatus, and/or

• radiographic imaging showing inflammation of the paranasal sinuses

The patients would be randomized into test and control group. The groups would get their usual treatment as per protocol, but the test group would get the test nasal spray, and the control group would get normal saline nasal spray. If surgery is done, then a surgery score would be given depending on the type and extent of surgery.

The nasal sprays would be given to each patient to carry home, which can last for 2 weeks. The compliance would be checked by calling them each week on a particular day. They would be called at intervals of 2 weeks to replenish the nasal spray and perform endoscopy of the nose. The compliance would be checked by calling them each week. They would be called at intervals of 2 weeks to replenish the nasal spray and perform endoscopy and clear crusts from the nose.

Dosage and application of nasal spray solution

The sprays would be administered two puffs, both nostrils thrice daily for a period of 3 months.

Composition of nasal spray:

A fixed dose mixture of prebiotics and postbiotics in normal saline. Composition of the nasal spray: mixture of prebiotics and postbiotics in normal saline. The composition comprises Zymosan A, Laminarin, Cellulose, Lactose N fucopentose, Cellutriose, Acetate, Propionate, in Normal Saline.

Histopathology examination:

A biopsy of nasal mucosa would be taken at the initiation of the study, and at the end of 3 months, a repeat biopsy would be taken from the nasal mucosa of the patients. A SNOT 22 and/or RSDI score would be taken at the beginning of the study, which would be repeated after a period of 3 months. A CT scan LM score would also be taken at the beginning of the study and at the end of 3 months.

Growth factors and their receptors play essential roles in cell proliferation,   migration,       tissue injury repair and ulcer healing. In gastric mucosa, transforming   growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) activates their common receptor, controlling cell proliferation. TGF-alpha predominantly plays this role under normal conditions and after acute injury, while EGF exerts its actions mainly             during chronic ulcers’ healing.

During regeneration of injured gastric mucosa, these growth factors serve predominantly to restore the epithelial component. Other growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) serve to promote restoration of the connective tissue and microvessels (angiogenesis) in injured mucosa.

The growth factor involvement will be explored in samples with histopathological improvement and where there would be healed ulcers with an increased mucosal layer of the nasal mucosa. TGF-alpha and EGF receptor expression would be checked in the histopathology samples as they are the major growth factors.

Blinding: Double-blind: patient will not know which group they will be allocated to,    and the investigator will not know which arm the patient is assigned.

DNA extraction

DNA will be extracted using QIAGEN’s QIAamp DNA FFPE Tissue kit following the manufacturer’s guidelines for DNA Purification from Tissue

Sequencing Methodology

25 ng of DNA will be used to amplify 16S rRNA hypervariable region V3-V4.

Sequence Data QC

The sequence data will be generated using Illumina MiSeq. Data quality will be checked using FastQC and MultiQC software. The data will be checked for base call quality distribution, % bases above Q20, Q30, %GC, and sequencing adapter contamination. All the samples have to pass QC threshold (Q20>95%).

Data Analysis

The reads will be trimmed (20bp) from 5’ end to remove the degenerate primers. The trimmed reads will be processed to remove adapter sequences and low-quality bases using Trimgalore. The QC passed reads will be imported into mothur, and the pairs will be aligned with each other to form contigs. The contigs will be screened for errors, and only those between 300bp and 532bp will be retained. Any contig with ambiguous base calls will be rejected. The high-quality contigs will be checked for identical sequences, and duplicates will be merged.

Although the primers for the experiment are designed for 16s bacterial rRNA, there is a good chance for nonspecific amplification of other regions. To correct for this, we will align the contigs to a known database for 16s rRNA. Depending on the variable region being amplified, most of the contigs will align to its respective region on the database. Any ambiguous contigs aligning to other regions on the database will be discarded. After this process, the gaps and the overhang at the ends from the contigs will be removed and processed for chimaera removal, which may have formed due to pcr errors. UCHIME algorithm will be used to flag contigs with chimeric regions. A known reference of all the chimeric sequences will be used to identify and remove possible chimeric sequences. The filtered contigs will be processed and classified into taxonomical outlines based on the GREENGENES v.13.8-99 database. The contigs will then be clustered into OTUs (Operational Taxonomic Unit). After the classification, OTU abundance will be estimated. PICRUSt will be used to predict gene family abundance. PICRUSt program was designed to estimate the gene families contributing to a metagenome by bacteria or archaea identified using 16S rRNA sequencing. The 16s RNA copy numbers will be normalized by PICRUSt’s precalculated files. The metagenomes will be predicted using predict_metagenomes.py script. The predicted pathways will be collapsed into higher categories. OTU contributions for the particular functions will be estimated by metagenome_contributions.py script.

Statistical analysis:

Continuous variables will be presented as Mean ± S.D. and categorical variables as a percentage. Comparison of means of continuous variables (parametric) between two groups will be done using two-sided unpaired t -test. Fisher’s exact test will be used for categorical variables. Statistical analyses will be performed using statistical software considering a significance level of P < 0 ·05.

 Expected outcomes:

1.               Improved quality of life of patients with chronic inflammation of the nose and PNS (in CRS and Atrophic Rhinitis)

2.               Microbiome profile of patients with chronic inflammatory diseases of the nose and PNS

3.               Normal nasal microbiome profile across age and gender

4.               The CT scan picture of the patients with the disease and following intervention

5.               The histopathological changes following intervention in Chronic inflammatory diseases

6.               To assess the change in the microbiome in these patients following intervention

Timelines:

Q1

Q2

Q3

Q4

Q5

Q6

Q7

Q8

Q9

Q10

Q11

Q12

|Patient recruitment

             |Collection of data

             |Interim data analysis

             |Data analysis and presentation

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