The influence of Helicobacter pylori eradication therapy on the presence of H. pylori antigens in dental plaque and saliva

 

Namiot DB.1*A-D, Leszczyńska K.2B, Namiot A.3B,C,D, Kemona A.4B, Bucki R.5C-F,              Chilewicz M.6B, Namiot Z.7B,C,E,F

 

1.    Department of Prosthetic Dentistry, Medical University of Białystok, Poland

2.    Department of Diagnostic Microbiology, Medical University of Białystok, Poland

3.    Department of Human Anatomy, Medical University of Białystok, Poland

4.    Department of General Pathomorpholgy, Medical University of Białystok, Białystok 

5.    Department  of  Microbiological  and  Nanobiomedical Engineering, Medical University of   

   Bialystok, Poland;  The  Faculty  of  Human Sciences of the Jan Kochanowski University in

   Kielce, Poland

6.    Department of Internal Medicine and Gastroenterology, District Hospital, Poland

7.    Department of Physiology, Medical University of Białystok, Poland, Institute for Medicine, State College of Computer Science and Business Administration, Łomża, Poland

______________________________________________________________________________

A- Conception and study design ;  B - Collection of data;  C - Data analysis; D - Writing the paper; 

E- Review article; F - Approval of the final version of the article; G - Other

______________________________________________________________________________

 

 

ABSTRACT

______________________________________________________________________________

 


Purpose:The aim of this study was to evaluate the presence of H. pylori antigens in the oral cavity (dental plaque and saliva) of patients undergoing systemic eradication therapy.

Materials and methods:The study was conducted in 49 subjects with H. pylori stomach infection. H. pylori antigens in dental plaque and saliva were evaluated with immunological method.

Results:In subjects with initial H. pylori oral infection, the presence of H. pylori antigens in the oral cavity 6 weeks after successful or unsuccessful H. pylori eradication therapy in the stomach was 47.0% and 50.0%, respectively. In subjects without initial oral infection with H. pylori, the presence of H. pylori antigens in the oral cavity 6 weeks after successful and unsuccessful eradication therapy in the stomach was 30.0% and 20.0%, respectively.    

Conclusions:The immunological method detecting H. pylori antigens in the dental plaque and saliva cannot be recommended to evaluate the efficacy of H. pylori eradication in the oral cavity.

Key words: Helicobacter pylori treatment, immunoassay, gastric mucosa, oral cavity


______________________________________________________________________________________________

 

 

*Corresponding author:

Department of Prosthetic Dentistry, Medical University of Białystok

24A M. Skłodowskiej-Curie Str., 15-276 Białystok, Poland

Tel.: +48-85-748 57 69, e-mail: dorota.namiot@op.pl

 

Received: 08.01.2016

Accepted: 17.02.2016

Progress in Health Sciences

Vol. 6(1) 2016 pp 19-24

© Medical University of Białystok, Poland

INTRODUCTION


       Unsuccessful eradication of H. pylori results not only from increasing resistance of the bacteria against antibiotics, but also from the lack of an efficient method of bacterial elimination from extragastric locations [1-3]. The oral cavity is one of the sites where H. pylori may survive eradication therapy [1-3]. However, so far only a few studies have evaluated the presence of H. pylori in the oral cavity following eradication therapy [1,2,4,5].

       Studies on H. pylori oral infection are much more difficult than those concerning the stomach, mainly due to the much more abundant bacterial flora in the oral cavity. There are over a dozen different bacterial strains in the stomach, with H. pylori being dominant, while the bacterial flora of the oral cavity includes several hundred different strains, and the H. pylori population is relatively small [6]. Furthermore, most methods used for the detection of H. pylori in the stomach cannot be applied to the oral cavity [7]. In recent years, a test evaluating the presence of H. pylori antigens with immunoassay has been used for the assessment of H. pylori oral and stomach infection [8-11]. Earlier it had been successfully applied for the determination of H. pylori antigens in stool and recommended for the evaluation of efficacy of H. pylori eradication therapy in the stomach [12].

      The aim of this study was to evaluate the incidence of H. pylori antigens in dental plaque and saliva of patients undergoing a systemic eradication treatment.

 

MATERIALS AND METHODS

 

Study subjects

       Forty-nine patients, men and women aged 24-72 years, were included in the study (Table 1). The inclusion criteria were good general health, normal basic laboratory tests, and no history of antibiotic therapy or other medication influencing the presence of H. pylori in the oral cavity at least one month preceding study entry.

 


 

 Table 1. Characteristics of the 49 patients qualified for eradication therapy

 

Age (median, range)

51 (24-72)

Gender (M/F)

27 / 22

Smokers

17 (34.7%)

Alcohol users

14 (28.6%)

Diagnosis

   dyspepsia

   duodenal ulcer disease

   gastric ulcer disease

 

18 (36.7%)

26 (53.1%)

5 (10.2%)

H. pylorieradication (first / consecutive)

39 / 10

Number of natural teeth (mean ± SD)

17.2 ± 8.6

Denture users

   fixed

   removable

   fixed  + removable

27 (55.1%)

8 (16.3%)

15 (30.6%)

4 (8.2%)


 

Samples collection and processing

      Each patient had a gastroscopy twice, i.e. prior to and 6 weeks after the completion of eradication therapy. During the gastroscopy, biopsy specimens of the gastric mucosa from the prepyloric and body regions were taken: one for rapid urease test (Campylobacter- like organism test - CLO test), two for  histological examination and one for bacterial culture from each site. The CLO test was prepared in the Physiology Department of the Medical University of Białystok according to the method described by Marshall et al. [13]. The sensitivity and specificity of the test in relation to the histological examination, culture, and stool test were 84.3% and 88.4%, 87.5%

 

and 83.5%, and 75.4% and 87.5%, respectively [8].

            The biopsy specimens for the histological examination were placed in buffered formalin, processed using a standard method and assessed by experienced pathologists. The specimens taken for the bacterial culture were placed into transport medium (Portagerm pylori, bioMerieux), delivered immediately to the microbiology laboratory, inoculated into culture medium and cultivated in microaerophilic conditions for at least 7 days. The subject was considered infected if the results of at least two of the three tests used for detection of infection in endoscopically taken slices of the gastric mucosa (CLO test, histology, culture) were positive (Table 2).


Table 2.Gastric and oral cavity H. pylori infections in the population qualified for eradication therapy (n=49)

 

Saliva

Dental plaque

Gastric mucosa specimens

        H. pylori antigens

Culture

CLO-test

Histology

 n

-

-

+

+

+

15

-

+

+

+

+

9*

+

+

+

+

+

9*

+

-

+

+

+

6

-

+

+

+

-

2

+

+

+

+

-

1

+

-

+

-

+

1

-

-

-

+

+

1

-

-

+

-

+

1

-

A

-

+

+

1

-

A

+

+

+

1

-

B

+

+

-

1

+

B

-

+

+

1

 A – too small mass of dental plaque to perform examination; B – toothlessness; *- one subject did not complete the    

 eradication therapy

 


 

Helicobacter pyloriantigen test

           The saliva and dental plaque were collected before each gastroscopic examination; i.e. prior to the initial gastroscopy and 6 weeks after the completion of  eradication treatment. Each patient was asked to abstain from any oral hygiene procedures on the day of the study. The freshly collected plaque was placed into 0.15 mol/L NaCl solution and initial incubation in microaerophilic conditions for a 72-hour period was performed [9]; the aim of this procedure was to increase the number of bacteria in the studied samples. Each subject provided 5 mL of unstimulated saliva, which was subsequently centrifuged. The saliva sediment was then used to determine the presence of H. pylori antigens [10]. The oral cavity of the examined subject was defined as infected if the test for the presence of H. pylori antigens in dental plaque or saliva was positive. The detection of H. pylori antigens in dental plaque and saliva was performed according to the manufacturer’s instructions provided with each kit, which was originally designed for detecting H. pylori antigens in stool. In brief, the prepared supernatant of the dental plaque or saliva was transferred to a well plate coated by the manufacturer with monoclonal antibodies against H. pylori. The solution containing monoclonal antibodies against H. pylori conjugated with horseradish peroxidase was also added to the wells.

         After a 60-minute incubation, unbound anti-bodies were washed away and tetramethylbenzidine, a substrate for horseradish peroxidase, was added. The reaction was stopped by the addition of sulphuric acid. The yellow colour intensity was measured spectrophotometrically at 450 nm.

 

Antibacterial therapy

      Eradication therapy was performed in 49 patients with stomach infection (a positive result in at least two of three tests used for evaluation of H. pylori infection in endoscopically taken samples) using a set of drugs: (1) Controloc (pantoprazole) 40 mg b.i.d., (2) Duomox (amoxicillin) 1000 mg b.i.d., and (3) Klacid (clarithromycin) 500 mg b.i.d. The therapy was continued for 7 days. The drugs were taken half an hour before meals with a half glass of water. The therapy was accepted as performed according to the study protocol if the patient had taken all medications designed for the therapy. During treatment and the subsequent 6 weeks, the patients did not change or modify oral hygiene procedures. Eradication therapy in the stomach was considered successful if the result of none of the three tests evaluating the presence of H. pylori in the endoscopically taken specimens 6 weeks after the treatment was positive. The eradication of H. pylori in the oral cavity was defined successful, if 6 weeks after the treatment, no H. pylori antigens were found in the dental plaque or saliva.

 

Statistical analysis

          The results were analysed statistically using Fisher’s exact test (Statistica 8.0). Statistical significance was accepted at p<0.05.

 

RESULTS

 

      Of the 49 subjects with H. pylori stomach infection (a positive result in at least 2 of 3 tests) 2 subjects did not  take all medications and were excluded from final analysis. In the remaining 47 subjects with an infected stomach who completed the eradication therapy according to the study protocol, 27(57.4%) had at baseline the presence of H. pylori antigens in the oral cavity (dental plaque or saliva) (Table 2). Among the 27 subjects with H. pylori oral infection, H. pylori antigens were found in the saliva of 8 subjects, in the dental plaque of 10 subjects, and in both the dental plaque and saliva of 9 subjects (Table 3). The therapy led to H. pylori eradication in the stomach and oral cavity in 55.3% (26/47) and 55.6% (15/27) of subjects, respectively, but only in 29.6% (8/27) of subjects in both.

 


 

Table 3.Presence of H. pylori antigens in the oral cavity before and after eradication therapy; patients with no antigens in the oral cavity prior to eradication therapy but with such antigens  after treatment are also included

 

 

Before treatment

Post treatment

 

Eradication successful in the stomach

Eradication unsuccessful in the stomach

Dental plaque

10

7 **

3*

Saliva

8

3*

2

Dental plaque and saliva

9

0

3*

Total

27 / 47 (57.4%)

10 / 27 (37.0%)

8 / 20 (40.0%)

         

 *- one subject with no H. pylori antigens prior to eradication therapy; **- two subjects with no H. pylori antigens    

  prior to eradication therapy

 


      After successful gastric eradication, the presence

of H. pylori antigens in the oral cavity (dental plaque and/or saliva) was found in 37.0% (10/27) of subjects; in 3 subjects H. pylori antigens were not found prior to the treatment. After  unsuccessful gastric eradication, the presence of H. pylori antigens in the oral cavity was found in 40.0% (8/20) of subjects; 2 subjects had no antigens in the oral cavity before the treatment. The efficacy of gastric eradication in subjects with and without H. pylori antigens in the oral cavity at baseline was 63.0% (17/27) and 50.0% (10/20) (p>0.05), respectively. In subjects with initial H. pylori oral infection, the presence of H. pylori antigens in the oral cavity after successful and unsuccessful eradication in the stomach was 47.1% (8/17) and 50.0% (5/10) (p>0.05), respectively. In subjects without initial oral infection with H. pylori, the presence of H. pylori antigens in the oral cavity after successful and unsuccessful eradication therapy in the stomach was 30.0% (3/10) and 20.0% (2/10) (p>0.05), respectively. The efficacy of H. pylori eradication therapy in the dental plaque, saliva or both was 30.0%, 50.0%, and 33.3%, respectively (Table 4). 

 


 

 

Table 4.The efficacy of H. pylori eradication therapy in the dental plaque, saliva or both

 

Dental plaque

3 / 10 (30.0%)*

Saliva

4 / 8 (50.0%)

Dental plaque and saliva

3 / 9 (33.3%)*

* -two subjects, in whom the gastric eradication was successful, but too little dental plaque was collected for the post-treatment evaluation, are not included.


 

DISCUSSION

 

      Determination of H. pylori antigens in the oral cavity is likely encumbered with certain error. A positive result is obtained probably both for living and already dead bacteria, as well as for spiral and coccoid forms [9,14,15]. It can, therefore, be speculated that this factor was the fundamental reason for the lack of relationship between bacterial eradication in the stomach and oral cavity. Nonetheless, the different

tests for H. pylori stomach and oral infection were used in the study; this could be a source of some

 

divergences in the results between these two locations.

     A relatively low gastric eradication rate may be attributed to the short treatment period, increasing resistance of H. pylori against clarithromycin, smoking habit, and consecutive but not the first attempt of eradication [16-18]. A low oral eradication rate results not only from bacterial resistance against clarithromycin, but also trace concentrations of amoxicillin in saliva and poor antibiotic penetration into the dental plaque [19-21]. Moreover, in subjects who do not brush their teeth or perform it carelessly, the bacteria that were killed  or transformed into  the

 

coccoid form during antibacterial therapy may remain in the dental plaque for many weeks, giving a positive result in the antigen test. It is of note that coccoid forms present the same set of antigens as the spiral forms [14].

       The case of 5 subjects who presented with H. pylori antigens in the oral cavity 6 weeks after  treatment, despite their absence in the pre-eradication assessment, indicates that the baseline evaluation could have been imprecise. It is less probable, yet still possible, that it was a result of new oral infection, which took place after the completion of eradication therapy. It should also be stressed that H. pylori may reside not only in the dental plaque and saliva, from which it is isolated most frequently, but also on the oral mucosa, periodontal pockets and palatine tonsils [15,22,23]; within these locations H. pylori can likely also survive eradication therapy. In the studied population with stomach infection, some subjects presented with bacterial antigens only in the saliva, some in the dental plaque and 30.0% both in the dental plaque and saliva. Large differences may result from the fact that some subjects were completely edentulous. In some, due to the lack of molars and premolars, the dental plaque was collected from the anterior teeth, which are less frequently the residence of H. pylori [24].

       In some subjects, especially those not brushing their teeth after the evening meal, the dental plaque could have been contaminated with food. In 4 subjects, who presented with bacterial antigens in dental plaque or saliva prior to eradication therapy, the evaluation of plaque infection after treatment was impossible due to insufficient mass of the collected plaque; only the results obtained from plaques weighing over 2 mg may be considered reliable [9].

 

CONCLUSIONS

 

       The immunological method detecting H. pylori antigens in the dental plaque and saliva cannot be recommended to evaluate the efficacy of H. pylori eradication in the oral cavity.

 

Conflicts of interest

None declared.

 

Funding

The study was supported by the Medical University of Białystok, grant no. 3 – 18608 L.

The study was approved by ethics committee of the Medical University of Białystok and each subject provided written informed consent before entry to the study.

 

REFERENCES

 

1.     Song H-Y, Li Y. Can eradication rate of gastric Helicobacter pylori be improved by killing oral Helicobacter pylori ? World J Gastroenterol. 2013 Oct 21;19(39):6645-50.

2.     Miyabayashi H, Furihata K,  Shimizu T, Ueno I, Akamatsu T. Influence of oral Helicobacter pylori on the success of eradication therapy against gastric Helicobacter pylori. Helicobacter 2000 Mar;5(1): 30-7.

3.     Namiot DB, Namiot Z, Kemona A, Bucki R, Gołębiewska M. Oral health status and oral hygiene practices of patients with peptic ulcer and how these affect Helicobacter pylori eradication from the stomach. Helicobacter 2007 Feb;12(1): 63-7.

4.     Cześnikiewicz-Guzik M, Loster B, Bielanski W, Guzik TJ, Konturek PC, Zapala J, Konturek SJ. Implications of oral Helicobacter pylori for the outcome of its gastric eradication therapy. J Clin Gastroenterol. 2007 Feb;41(2):145-51.

5.     Zou QH, Li RQ. Helicobacter pylori in the oral cavity and gastric mucosa: a meta-analysis. Oral Pathol Med. 2011 Apr;40(4):317-24.

6.     Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005 Nov; 43(11): 5721-32. 

7.     Nguyen AMH, El-Zaatari FAK, Graham DY. Helicobacter pylori in the oral cavity. A critical review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1995;79(6):705-9.

8.     Namiot A, Leszczyńska K, Namiot DB, Chilewicz M, Bucki R, Kemona A, Namiot Z. Application of Helicobacter pylori antigen test to evaluate gastric mucosa specimens. Prog Health Sci. 2014; 4(2): 52-7.

9.     Namiot DB,Leszczyńska K, Namiot Z, Chilewicz M, Bucki R, Kemona A. The occurrence of Helicobacter pylori antigens in dental plaque; an association with oral health status and oral hygiene practices. Adv Med Sci. 2010;55(2):167-71.

10.Namiot DB, Leszczyńska K, Namiot A, Leszczyńska UM, Bucki R, Milewski R, Namiot Z. The influence of oral health status and dental plaque removal practices on the occurrence of Helicobacter pylori antigens in saliva. Dent Med Prob. 2013;50(3):275-81.

11.Namiot A, Leszczyńska K, Namiot DB, Bucki R, Kemona A, Chilewicz M, Namiot Z. The clinical importance of Helicobacter pylori antigens detected in the dental plaque and feces. Prog Health Sci. 2015;5(2):24-9.   

 

 

12.Malfertheiner P, Megraud F, O’Morain C, Bazzoli F, El-Omar E, Graham D, Hunt R, Rokkas T, Vakil N, Kuipers EJ, The European Helicobacter Study Group (EHSG). Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut 2007 Jun; 56(6):772-81.

13.Marshall BJ, Warren JR, Francis GJ, Langton SR, Goodwin CS, Blincow ED. Rapid urease test for the management of Campylobacter pyloridis-associated gastritis. Am J Gastroenterol. 1987 Mar; 82(3):200-10.

14.Saito N, Konishi K, Sato F, Kato M, Takeda H, Sugiyama T, Asaka M. Plural transformation-processes from spiral to coccoid Helicobacter pylori and its viability. J Infect. 2003 Jan;46(1):49-55.

15.Kusano K, Inokuchi A, Fujimoto K, Miyamoto H, Tokunaga O, Kuratomi Y, Shimazu R, Mori D, Yamasaki F, Kidera K, Tsunetomi K, Miyazaki J. Coccoid Helicobacter pylori exists in the palatine tonsils of patients with IgA nephropathy. J Gastroenterol. 2010 Apr;45(4):406-12.

16.Rożynek E, Dzierżanowska D, Celińska-Cedro D, Jeliaszewicz J. Primary resistance to metronidazole and other antibiotics of Helicobacter pylori isolated from children in Poland. Eur J Clin Microbiol Infect Dis. 1997 Dec;16(12):943-94.  

17.Andrzejewska E, Szkaradkiewicz A, Karpiński T. Antimicrobial resistance of Helicobacter pylori clinical strains in the last 10 years. Pol J Microbiol. 2009;58(40):301-5.  

18.Namiot DB, Leszczyńska K, Namiot Z, Kurylonek AJ, Kemona A. Smoking and drinking habits are important predictors of Helicobacter pylori eradication. Adv Med Sci. 2008;53(2):310-15.

19.Goddard  AF, Jessa MJ, Barrett DA, Shaw PN, Idstrom JP, Cederberg C, Spiller RC. Effect of omeprazole on the distribution of metronidazole, amoxicillin, and clarithromycin in human gastric juice. Gastroenterology 1996 Aug; 111(2):358-67.

20.Wust J, Hardegger  U. Penetration of clarithro-mycin into human saliva. Chemotherapy 1993 Sep-Oct; 39(5):293-96.

21.Sedlacek MJ, Walker C. Antibiotic resistance in an in vitro subgingival biofilm model. Oral Microbiol Immunol. 2007 Oct; 22(5):333-9.

22.Umeda M, Kobayashi H, Takeuchi Y, Hayashi J, Morotome-Hayashi Y, Yano K, Aoki A, Ohkusa T, Ishikawa I. High prevalence of Helicobacter pylori detected by PCR in the oral cavities of periodontitis patients. J Periodontol. 2003 Jan;74(1):129-34.

23.Dowsett SA, Archila I, Segreto VA, Gonzalez CR, Silva A, Vastola KA, Bartizek RD, Kowolik MJ. Helicobacter pylori infection in indigenous families of Central America: serostatus and oral and fingernail carriage. J Clin Microbiol. 1999 Aug;37(8):2456-60.

24.Song Q, Lange T, Spahr A, Adler G, Bode G. Characteristic distribution pattern of Helicobacter pylori in dental plaque and saliva detected with nested PCR. J Med Microbiol. 2000 Apr;49(4): 349-53.