Lack of Association of Helicobacter pylori in Laryngeal Pathologies

Abstract

To study the association of Helicobacter pylori (H. pylori) in patients with laryngeal pathologies. Study design: prospective observational study. Tertiary care teaching hospital. One hundred consecutive patients with laryngeal lesions scheduled for microlaryngoscopy were enrolled in the study. Laryngopharyngeal reflux was assessed using the reflux symptom index and reflux finding score. Tissue samples from the laryngeal lesions were taken under general anaesthesia and were screened for the presence of H. pylori using real time polymerase chain reaction (PCR) for ureA genes and histopathological examination. Of the 100 patients, 14 had a significant reflux symptom index score and 35 had significant reflux finding score. The lesions in the study subjects included both benign and malignant laryngeal pathologies. Vocal cord polyps formed more than half of the laryngeal pathology (57%) studied. Our study could not detect H. pylori in any laryngeal lesions by PCR analysis and histopathological examination. H. pylori may not be associated with laryngeal pathologies.

Introduction

Helicobacter pylori is one of the most common infectious agent found in gastrointestinal tract worldwide [1]. Helicobacter pylori is a gram negative micro aerophilic spiral or helical shaped bacteria colonizing human gastric epithelial cells. This bacterium has a special tropism for the acidic environment in the stomach and hence colonises the mucosa of the stomach. H. pylori is responsible for a variety of gastric diseases ranging from peptic ulcer to gastric cancer and causes chronic bacterial infection in humans.

Recently, presence of H. pylori has been studied in extragastric sites like saliva, dental plaques and palatine tonsils by polymerase chain reactions (PCR) [2, 3]. Larynx is a region that could be directly exposed to the bacterium by means of laryngopharyngeal reflux which is a condition in which there is retrograde movement of the gastric contents into the laryngopharynx.

There are conflicting studies in the literature regarding the presence of H. pylori in laryngeal tissue. In 2011, Pajić-Penavić et al. conducted PCR analysis on cytobrush samples obtained from normal laryngeal mucosa and concluded that H. pylori was not present in normal laryngeal mucosa [4]. The study by Fellman et al. suggested that the upper aerodigestive tract was a reservoir in patients with H. pylori gastritis [5], while Cekin et al. detected H. pylori in 55.8% of subjects with laryngeal pathologies [6]. There is a paucity of studies from India looking at the prevalence of H. pylori in pathological conditions of larynx and so we conducted this study.

Material and Methods

Study Design

This prospective cross sectional study was conducted in the Department of Otorhinolaryngology of a tertiary care referral centre and teaching hospital located in Southern India. The study was approved by the Institutional Review Board (IRB Min No. 8449).

Study Population

Consecutive patients above the age of 18 years who presented with hoarseness of voice and with laryngeal lesions seen on flexible laryngoscopy were included. Informed consent for participation in the study was taken from each patient. Patients on treatment with proton pump inhibitors or H2 antagonists or antibiotics within a period of 4 weeks, patients with recent history of gastrointestinal bleeding and patients who received radiation therapy were excluded from the study.

Study Protocol

Patients were asked to fill a questionnaire which assessed the demographics and lifestyle factors which have an adverse impact on health like smoking, alcohol consumption etc. The symptoms and signs related to laryngopharyngeal reflux (LPR) were also noted. Reflux related symptoms were assessed by the reflux symptom index (RSI) questionnaire developed by Belafsky which analysed symptoms such as hoarseness, throat clearing, postnasal drip, difficulty in swallowing, cough after lying down, choking episodes, troublesome cough, and lump in throat, heart burn and indigestion [7]. These symptoms were scored from 0 to 5 depending on the severity (0-no problem, 5-severe problem). A RSI score of ≥ 13 was considered to be abnormal.

Reflux related findings were also assessed by flexible laryngoscopy and scored using reflux finding score (RFS) [8]. Presence of subglottic oedema, ventricular obliteration, vocal fold oedema, erythema, posterior commissure hypertrophy, vocal fold granuloma, diffuse laryngeal oedema and thick endolaryngeal mucus were looked for and scored depending on the severity. A RFS of ≥ 7 indicated the presence of laryngopharyngeal reflux.

All the studied patients underwent microlaryngoscopy and biopsy under general anaesthesia and a small bit of tissue was separately taken and transported immediately to the microbiology laboratory at 4 °C for detection of H. pylori using real time polymerase chain reaction (PCR) assay. The remaining tissue was sent in formalin to the Pathology laboratory for examination.

Real Time PCR Analysis

Biopsy samples were stored at − 70 °C until the DNA extraction was done. DNA was extracted from the biopsy homogenate using the QIAamp DNA minikit (Qiagen, Hilden, Germany). DNA extracts from the biopsy samples were subjected to real-time PCR, which was performed in a step-one real time PCR (Applied Biosystem, Fostor City, CA, USA). Real-time PCR targeting ureA gene of H. pylori was performed using the H. pylori Genesig Advanced Kit.

The kit is provided with 2× qPCR master mix and primer/probe mix specific for the detection of urease encoding ureA gene subunit.

For UreA detection, the 20 µl reaction mixture was prepared as follows: 10 µl of qPCR master mix, 1 µl of primer/probe mix, 4 µl of RNAse/DNAse free water and 5 µl of DNA extract with the concentration of at least 5 ng/µl. The amplification reaction was performed with the enzyme activation at 95 °C for 15 min, followed by 50 cycles consisting of denaturation at 95 °C for 10 s and annealing at 60 °C for 1 min. Samples were run in duplicates and were considered positive if at least one of the reactions was positive.

The sample is considered to be positive for the target showing cycle threshold (Ct) value of 26 ± 3 with corresponding sigmoid curve. Absence of target in the sample will be indicated as showing either no amplification curve or curve with lower Ct value.

Histopathology Examination

Helicobacter pylori is seen as comma or S-shaped bacilli on HE stain [9]. In addition to hematoxylin and eosin (HE) staining, an additional modified Giemsa staining was done on the sample sent for histopathological examination. 2–4 μm thickness paraffin sections were prepared and stained by modified Giemsa technique. The organism appears purple in colour on Giemsa stain.

Modified Giemsa Staining

The slide was kept in xylene for 15 min and then washed with graded alcohol followed by water. Then equal amounts of Geimsa stain and working buffer solution was added and the slide was kept covered for 5 min. The slide was then washed with water for 2 min, dried and then mounted.

Sample Size

From literature review done, the average prevalence of Helicobacter pylori infection in laryngeal pathologies was about 40% [4]. By applying the formula n = Z²PQ/d², where n = sample size, Z = 95% confidence limits, P = prevalence, Q = 100-P, d = precision (10%). The sample size was calculated to be 100.

Statistical Analysis

The qualitative variables were expressed using frequencies and percentages. The prevalence of H. pylori in laryngeal pathologies was calculated using frequencies and percentages with 95% confidence limits. Chi-square test was done on Reflux symptom index questionnaire with Reflux Finding Score to determine any association between the two. The analysis was performed using SPSS Version 21.0 (SPSS Inc., Chicago, Illinois).

Results

Study Group Characteristics

A total of 100 patients with laryngeal pathologies in the age group 20–76 years (mean 48 years) were enrolled in the study and the majority were men (89%) (Table 1). The most common presenting symptom was hoarseness followed by throat clearing, foreign body sensation of throat and belching. Of them, 14 had a significant RSI score and 35 had significant RFS (Tables 1 and 2). The correlation between RSI and RFS (Table 3) was found to be statistically significant (p = 0.002).

Table 1.

Baseline demographics

Parameter		N
Study subjects		100
Sex	Male	89
	Female	11
Side	Right
	Left
Level of voice use	1	0
	2	27
	3	44
	4	29
Risk factors for vocal pathology	Voice abuse/misuse	59
	Smoking	57
	Alcohol	20
Reflux symptom index ≥ 13		14
Reflux finding score ≥ 7		35

Table 2.

Presenting complaints

Symptoms	Number of patients
Hoarseness	97
Throat clearing	59
Foreign body sensation throat	34
Belching	29
Throat pain	8
Cough	13
Difficulty in breathing	11
Difficulty in swallowing	5
Neck swelling	0

Table 3.

Reflux symptom index versus reflux finding score

Reflux finding score	Reflux symptom index
	Negative	Positive
Negative	65	4
Positive	25	10

The correlation between RSI and RFS was found to be statistically significant (p = 0.002)

Laryngeal Lesion

The lesions in the study subjects included both benign pathologies like vocal cord polyps, vocal cord keratosis, vocal cord papilloma, chronic laryngitis and malignant conditions like laryngeal carcinoma (Fig. 1). Subjects with vocal cord polyps formed more than half of the study population (57%). Out of the 57 patients with vocal cord polyps, 36.8% (n = 21) had significant reflux finding score. We studied the significance of reflux in patients with laryngeal cancer by Reflux finding score and found out that 11 (36.7%) out of 30 patients had reflux which was not statistically significant.

Fig. 1.

Distribution of laryngeal pathologies

Polymerase Chain Reaction

In real-time PCR analysis, all the samples were found to be negative for the ureA gene of H. pylori.

Histopathological Examination

Neither the HE stain nor the Modified Giemsa staining could detect H. pylori in the sampled tissue of the benign and malignant laryngeal lesions.

Discussion

This probably is the first Indian study which looked at the presence of H. pylori in subjects with various benign and malignant laryngeal pathologies as a review of literature did not show any studies from India. Thirty percent of the studied subjects had malignancy and over half of them had vocal cord polyps. H. pylori was not detected in any of the study samples.

Helicobacter pylori a common coloniser of human gastric mucosa, reaches the stomach which is its primary reservoir, via the oral cavity. The transmission can be either oro-oral or faeco-oral. Whether the bacteria can colonise in the upper aerodigestivetract involving oral cavity, pharynx and larynx are still under debate. Studies that detected the presence of H. pylori in the dental plaques, saliva of patients, in tonsillectomy specimen, however failed to culture the bacteria [2, 3].

It was noted that H. pylori was present in 82.5% of patients with GERD [10]. Repeated exposure of the laryngeal tissue to the refluxate results in laryngopharyngeal reflux disease. As the inflammation continues symptoms increase and signs persists. The prevalence of reflux in laryngeal disorders was around 50% in a study by Koufman et al. [11]. In this study, the presence of laryngopharyngeal reflux was assessed using RSIand RFS score similar to the study by Cekin et al. [6]. Significant RFS was seen in only 35% of patients in the present study. Congested arytenoids was the most common finding observed followed by posterior commissure hypertrophy. Helicobacter pylori seen in the area of laryngeal carcinoma could be due to migration from the oral cavity or the stomach as suggested by Pajić Matić et al. [12].

Different methods are available to detect H. pylori infection as it is difficult to culture the bacteria [13]. Various studies using different methods to determine whether this bacterium could be responsible for pathogenesis of laryngeal diseases or associated with it, have carried out (Table 4). A positive association between H. pylori and laryngeal diseases (benign and/or malignant) was seen using serum antibodies for H. pylori [14], rapid urease testing [15–17], PCR [5,6,17] also a negative association was seen using histopathology and immunohistochemical methods [18] and rapid urease testing [19, 20].

Table 4.

Various studies conducted to determine the association between H. pylori and laryngeal pathology

References	Study population (country)	Number of patients	Diseased/normal laryngeal tissue	Tissue sampled	Method used to detect H. pylori microbiology	Method used to detect H. pylori pathology	Test for H. pylori positive/negative
Borowski et al. [15]	Germany	35	Chronic laryngitis	Laryngeal tissue biopsy	Rapid urease testing	–	Positive
Jaspersen et al. [20]	Germany	38	Chronic laryngitis	Laryngeal tissue biopsy	Rapid urease test	Histology (negative)	Negative
Rubin et al. [14]	UK	101	Benign	Serum	Antibodies	–	Positive
Akbayir et al. [18]	Turkey	100	Benign Laryngeal lesions and cancer	Laryngeal tissuebiopsy	–	Histopatholgy (HE) Imunohistochemical examination	Negative
Masoud et al. [19]	Iran	74	Benign Laryngeal lesions and cancer	Laryngeal tissuebiopsy	Rapid urease testing	Histopathological examination	Negative
Ozyurt et al. [17]	Turkey	27	Benign Laryngeal lesions and cancer	Laryngeal tissuebiopsy	PCR for bacterial DNA ureC and by real-time PCR for cagA	Histopathological examination	Positive
Pajić-Penavić et al. [4]	Croatia	20	Normal laryngeal mucosa	Cytobrush sample	C urea breath test and PCR analysis	–	Negative
Cekin et al. [6]	Turkey	43	Benign Laryngeal lesions &cancer	Laryngeal tissuebiopsy	PCR-ureC gene of the H. pylori genome	–	Positive
Siupsinskiene et al. [16]	Lithuania	78	Benign Laryngeal lesions &cancer	Laryngeal tissuebiopsy	Rapid urease testing	Histopathological examination (Giemsa)	Positive
Fellman et al. [5]	Switzerland	8	Benign Laryngeal lesions and cancer	Laryngeal tissuebiopsy	PCR		Positive
Pajić-Penavić et al. [12]	Croatia	51	Carcinoma larynx	Laryngeal biopsy	DNA were analyzed using the standardized fluorescent ABI Helicobacter plus-minus PCR assay	Histopathology	Positive
Present study	India	100	B&M	Laryngeal biopsy	PCR targeting ureA gene	Histopathological examination (HE & Giemsa)	Negative

PCR scores over the other methods used to identifying H. pylori in that it allows researchers and clinicians to identify H. pylori in small samples that have few bacteria present. DNA present below the estimated threshold hinders the detection of target. The targets routinely used to detect H. pylori by PCR methods include the 16S rRNA gene, the random chromosome sequence, the 26-kDa species-specific antigen (SSA) genes, the urease (ureA) gene and the glmM (ureC) gene. However the opinions are divided regarding the most appropriate target. Espinoza et alcompared the efficacy of ureA gene and ureC gene in detecting H. pylori by PCR method and concluded that PCR assay using ureC gene could detect all those strains which were not picked up by ureA analysis [21]. Another study conducted by Smith et alto evaluate the most appropriate method of PCR to detect H. pylori suggested that using 26 kDa SSA gene was the best method [22]. In our study we used the Genesig kit which detects the target ureA gene in H. pylori. The presence of bacterium could not be detected in any of the benign or malignant laryngeal lesions by PCR analysis.

In the histopathological examination of the laryngeal specimen by modified Giemsa technique, Siupsinskiene et al. identified the bacterium in 45.5% of patients with chronic laryngitis and 46.2% of patients with laryngeal cancer [22]. However in the present study we couldn’t identify H. pylori using either HE stain or modified Giemsa technique. The routine histopathological studies using hematoxylin and eosin (HE) stain can detect H. pylori in tissue samples. Various stains like Warthin-Starry, Giemsa, toluidine blue, acridine orange, Genta, etc. can be used to detect H. pylori when HE stain is inconclusive. At least 2 different stain techniques are to be used on biopsy samples as per recent guidelines: HE to evaluate inflammatory cells, and Giemsa or Genta stain to detect H. pylori. Giemsa stain being simple, highly sensitive, and inexpensive, it is the preferred method in clinical practice [23].

Strength and Limitations of the Study

Strength of the Study

Real time PCR testing and histopathological examination using two stains were used to detect H. pylori in our study.

Limitations of the Study

The amount of DNA in the test specimen was variable and H. pylori is known to have a patchy distribution in the stomach [22]. The tissue specimen had a mixture of both host DNA and bacterial DNA and all the bits were at good concentration but it is difficult to distinguish host cell concentration from the original bacterial concentration.

Since our study used only one target to detect H. pylori, we could have possibly missed out the organism by PCR analysis.

Conclusion

Helicobacter pylori (H. pylori) may not be associated with laryngeal pathology. The heterogeneity in the methodology used in the various studies could be a possible reason for the conflicting results reported. More studies are required in this field which targets all possible genes so that the bacteria is not missed out.

Authors Contribution

The following is the contribution of the authors towards submission of this manuscript: NAJ: study design, acquisition of data, drafting the manuscript, final approval of the version to be submitted. SSM: concept and study design, analysis and interpretation of data, critical revision of the manuscript and final approval of the version to be submitted. SA: study design, analysis and interpretation of data, critical revision of the manuscript and final approval of the version to be submitted. BV: study design, analysis and interpretation of data, critical revision of the manuscript and final approval of the version to be submitted. YDB: analysis and interpretation of data, critical revision of the manuscript and final approval of the manuscript. ABP: analysis and interpretation of data, critical revision of the manuscript and final approval of the manuscript.

Funding

FLUID RESEARCH GRANT (Institutional grant for research). This work received financial support from Fluid Research Grants, Christian Medical College, Vellore. (IRB Min No. 8449).

Availability of Data and Material

Yes.

Code Availability

Not applicable.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics Approval

The study was approved by instituitional review board and ethics committee and was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki.

Consent to Participate

Yes.

Consent for Publication

Yes.