Original Research Paper 2 Prevalence of Helicobacter ... · Tel.: +919823375529; fax:...

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*Original Research Paper 1 2

Prevalence of Helicobacter pylori detected by 3

PCR in saliva of male smokers and non 4

smokers with chronic periodontitis 5

6

Rajiv Kishor Saxena1, Abdul Samad Aziz2*, Madhav Govind Kalekar3, 7

Milsee Mol J.P.4, Adinath Narayan Suryakar5, Benjamin Tabita6, Ravi 8

Vasudev Shirahatti7, Raghavendra Shrishail Medikeri8 9

10 1MD (Microbiology), Professor,Dept. of Microbiology, Sinhgad Dental College, Pune 11

2M.Sc. (Biochemistry), Ph. D student, Dept. of Biochemistry, Grant Medical College and Sir 12

J. J. Group of Hospitals, Byculla, Mumbai 13

3Ph.D (Medical Biochemistry), Asso. Prof., Dept. of Biochemistry, Grant Medical College & 14

Sir J. J. Group of Hospitals, Byculla, Mumbai 15

4M.Sc (Biochemistry), Assi. Prof., Dept. of Biotechnology, Sinhgad College of Science, Pune 16

5Ph. D (Medical Biochemistry), FACBI, Registrar, Maharashtra University of Health 17

Sciences, Nashik 18

6M.D.S. Head, Department of Dentistry, Grant Medical College and Sir J. J. Group of 19

Hospitals, Byculla, Mumbai 20

7M.D.S. (Public Health Dentistry), PGDM( Biostatistics), Reader., Dept. of Public Health 21

Dentistry, Sinhgad Dental College, Pune 22

8M.D.S. (Periodontics),Reader., Dept. of Periodontics, Sinhgad Dental College, Pune 23

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Corresponding contributor: Mr. Abdul Samad Aziz 25

E mail address: [email protected] 26

Telephone: +919823375529 27

Fax: +91-020-26430962 28

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ABSTRACT 39

40 Aims: To assess the comparative prevalence of Helicobacter pylori (H pylori) in saliva of 41

smokers and non smokers with chronic periodontitis. 42

Study design: Male individuals diagnosed with chronic periodontitis with and without 43

smoking habits were enrolled in the study. The un-stimulated whole saliva was subjected to 44

H pylori DNA detection using real time PCR. The percent prevalence of H pylori DNA among 45

the groups, were statistically compared. 46

Place and Duration of the study: Department of Biochemistry and Department of Dentistry, 47

Grant Medical College and Sir JJ group of Hospitals, Mumbai and Department of 48

Microbiology, Sinhgad Dental College, Pune, between January 2010 and June 2010. 49

Materials and Method: A total of 48 males with chronic periodontitis were divided into two 50

groups, Group I (n=30, mean age=44.2±5.88 yrs) with smoking habit, Group II (n=18, mean 51

age 41.72±4.36yrs) without smoking habit. Healthy volunteers were enrolled as controls, 52

Group III (n=16, mean age 39.64±5.04 yrs). Periodontal status was evaluated by measuring 53

gingival index (GI), plaque index (PI), and clinical attachment loss (CAL). Salivary samples 54

were subjected to real time PCR for detection of H pylori DNA. 55

Result: Periodontal parameters were significantly changed between Group I and II 56

compared to Group III (P=.001). Overall, H pylori was not detected in Group III (100% 57

negative), whereas 5.5% of Group II and 13.3% in Group I patients have shown presence 58

of H pylori. Chi-square test have shown a significant change (P=.003) between Group I and 59

Group III however there is a non significant change between Group I and Group II (P=.312) 60

and between Group II and Group III (P=.186). 61

Conclusion: The smokers with chronic periodontitis may be at a relative higher risk of H 62

pylori infection in oral cavity, than non smokers. The study needs validation on a larger 63

sample size. 64

65 Keywords: H pylori, chronic periodontitis, smokers, PCR, saliva 66


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1. INTRODUCTION 69

H pylori is a Gram negative, spirally shaped bacterium, 0.5 – 0.9 µm wide by 2 – 4 µm long. 70

It is micro-aerophilic and requires carbon dioxide for growth. It produces an exceptionally 71

powerful urease which is vital to its survival in the stomach (Skirrow, M. B. 2002). The 72

association of H pylori with chronic type B gastritis and peptic ulcer disease has been 73

demonstrated (Fergusson, Jr. et al 1993). In the western world, average H pylori infection 74

rates in healthy adults are estimated to be between 10–40%, whereas patients with gastritis 75

and duodenal ulceration have infection rates of 80– 100%. Evidence exists for possible oral 76

transmission from person to person and fecal-oral transmission is thought to be particularly 77

prevalent in developing countries (Riggo, Lennon 1999). Recently H pylori, has been found 78

in association with dental plaque, suggesting the oral environment may be one of the many 79

potential pathways for transmission (Eskandari, A. et al 2010). Various studies (Umeda, M. 80

et al 2003, Gebara, E. C. et al 2004, Zaric, S. et al 2009) have associated presence of H 81

pylori in oral environment of subjects with periodontitis suggesting that progression of 82

periodontal pocket and inflammation may favor colonization by this species. Tobacco 83

smoking has been regarded as a true risk factor for periodontitis. Smokers have both 84

increased prevalence and more severe extent of periodontal disease, as well as higher 85

prevalence of tooth loss and endentulism compared to non-smokers (Tonetti, MS. 1998). A 86

reservoir of dental plaque exists in periodontal pockets in smokers with periodontitis and the 87

potential for re-infection of the stomach by H pylori is obvious (Watts, TLP. 2006). Further 88

(Suzuki T et al 2006) have demonstrated that smoking increases the treatment failure rate 89

for H pylori eradication. Saliva contains an abundance of biomolecules that reflects 90

physiological status. Salivary diagnostics offer an easy, inexpensive, safe and cost effective 91

approach for disease detection (Patil, Patil, 2011). Saliva based diagnosis has been reported 92

in periodontitis (Kaufman, Lamster 2000) and also in studies representing prevalence of H 93

pylori in periodontitis (Gebara, EC. et al 2004, Souto, Columbo 2008). Hence the present 94


study was undertaken to comparatively evaluate the prevalence of H pylori in saliva of 95

chronic periodontitis patients with and without smoking habits. 96

2. MATERIAL AND METHODS 97

98 The study was undertaken as per the approval of the Institutional Ethics Committee ( 99

registration number: 391/CPCSEA) of Grant Medical College and Sir J. J. Group of 100

Hospitals, Mumbai, following norms of the World Medical Association of Helsinki; ethical 101

principles for medical research involving human subjects (amended by 55th WMA General 102

Assembly, 2004). A written informed consent was obtained from all the subjects enrolled in 103

the study. The subjects had the right to refuse to participate in the study or to withdraw 104

consent to participate at any time without reprisal. 105

2.1 STUDY GROUPS 106

Individuals visiting the Department of Dentistry, Grant Medical College, Mumbai, were 107

constituted the study population and were divided into the following groups: 108

Group I: Smokers with chronic periodontitis; n=30 (mean age 44.2 ± 5.88 yrs) 109

Group II: Non-smoker with chronic periodontitis; n=18 (mean age 41.72 ± 4.36 yrs) 110

The patients in the study groups were clinically evaluated for chronic periodontitis according 111

to the criteria accepted by the American Academy of Periodontology in 1999 (Armitage, G C 112

2000). The patients were otherwise healthy, with no history of major illness and consumption 113

of antioxidants, antibiotics, anti inflammatory or any other drugs and had not received any 114

periodontal therapy for at least six months prior to the inception of the study. Subjects having 115

past gastric illness six months prior to the study and undergoing any treatment, diabetics and 116

alcoholics were excluded. The subjects in the smoker group were current smokers 117

(predominantly cigarette smokers) with smoking habit of ≥ 3 years and frequency of smoking 118

≥ 5 cigarette / day. Volunteers from the same geographical region as those of the patients 119

with apparently good oral and systemic health and without smoking habits were enrolled as 120

controls. Group III: Non-smoker healthy controls; n=16 (mean age 39.64 ± 5.04 yrs) 121


2.2 CLINICAL MEASUREMENTS 122

The periodontal status of all individuals was evaluated by measurement of gingival index 123

(GI) as developed by Loe H and Silness J (1963), plaque index (PI) as described by Silness 124

P and Loe H (1964), (Soben, P. 2003) and clinical attachment loss (CAL). CAL is measured 125

on six sites of each tooth (mesial, median and distal points at buccal and palatal aspects) 126

and the individual scores were compared on a scale for characterization of periodontitis as 127

slight, moderate or severe (John, MN. 2006). All clinical measurements were evaluated by a 128

single investigator using University of North Carolina (UNC-15) probe (Hu-Friedy, Chicago). 129

2.3 SAMPLE COLLECTION 130

After clinical evaluation the individuals were referred to the department of Biochemistry, 131

Grant Medical College, Mumbai, for saliva collection. Procedure for saliva collection was 132

accessed from Molmeth (URL: http://www.molmeth.org/protocols/1BXQD00) as per the 133

protocol derived from the WHO/IARC guideline “Common Minimal Technical Standards and 134

Protocols”. Fasting un-stimulated whole saliva samples were obtained in the morning, during 135

which individuals were requested not to drink any beverages except water. They were given 136

drinking water (bottled) and asked to rinse their mouth out well (without drinking the water). 5 137

minutes after this oral rinse, the individuals were asked to spit whole saliva in a sterile 138

container. About 5ml of whole saliva was collected from each individual. They were asked to 139

refrain from talking and drop down the head and let the saliva run naturally to the front of the 140

mouth. They were also asked not to cough up mucus during the saliva collection. The 141

salivary samples were collected and stored at -40C until DNA extraction. The salivary 142

samples for PCR analysis were transported using cool bags to the Department of 143

Microbiology, Sinhgad Dental College, Pune. 144

2.4 PCR ANALYSIS 145

For PCR, DNA was extracted from saliva samples by using QIAGEN DNA extraction kit 146

(Germany) according to manufacturer’s instructions. Briefly, 200 µL saliva, 20 µL QIAGEN 147

Protease and 200 µL buffer AL (lysis buffer), were added, mixed and incubated at 56°C for 148


10 minutes. After brief centrifugation, 200 µL 96% ethanol was added and mixed. The 149

obtained mixture is applied to the QIAmp mini spin column and centrifuged at 8000 rpm for 1 150

minute. Pure DNA was eluted from the column using buffer AE (10 mM Tris.Cl; 0.5 mM 151

EDTA; pH 9.0) , and stored at -20°C till further analysis. The DNA samples obtained were 152

subjected to real –time PCR, performed using SYBR-Green PCR master mix (Applied 153

Biosystem). The RT-PCR was targeted at the 26 KDa Helicobacter species-specific antigen 154

(SSA) gene with primer sequence as (forward: 5’-TGGCGTGTCTATTGACAGCGAGC-3’, 155

reverse: 5’-CCTGCTG GCATACTTCACCATG-3’) (Lu, J.J. 1999, Ribeiro, M. 2007). The 156

reaction mixture was composed of follows: 20 µL of 2X SYBR Green PCR Master mix 157

(Qiagen), 50 nM of each primer and 1 µL of extracted DNA (200 ng).The reaction was cycled 158

with initial PCR activation step at 95°C for 10 min, followed by 40 cycles of denaturation at 159

95°C for 30 s, annealing at 60°C for 60 s and primer extension at 60°C for 60 s. Applied 160

Biosystems ABI 7500 SDS real-time thermal cycler was used for PCR data acquisition and 161

analysis. 162

2.5 STATISTICAL ANALYSIS 163

The measured values for the clinical parameters were subjected to statistical analysis using 164

Statistical Package for Social Sciences (SSPS software, version 11.5) for MS Windows. The 165

values were expressed as mean± SD. The underlying normality assumption was tested for 166

each clinical parameter using percentile to percentile (PP) plot technique before applying 167

any statistical test. Comparison of significance of difference of average of clinical parameters 168

across the three study groups was done using analysis of variance (ANOVA) technique with 169

Tukey’s correction for multiple group comparison. P – value <0.05 is considered to be 170

statistically significant. The values on H pylori are n (%) and the P – value on H pylori is 171

obtained using Chi – square test if cell frequencies were larger than 5, else Fisher’s exact 172

probability test were used. P – value <0.05 is considered to be statistically significant 173


3. RESULTS AND DISCUSSION 174

175 Chronic periodontitis is a chronic inflammatory disease of the periodontium and significantly 176

higher periodontal clinical parameters like GI, PI, CAL compared to healthy controls have 177

been documented (Akalin, F.A. 2005, Akalin, F.A. 2007, Wei, D. 2010.) Further smokers with 178

chronic periodontitis have shown altered clinical parameters than non smokers with chronic 179

periodontitis. (Erdemir, EO.et al 2004, Buduneli, N. 2006, Bulunet, K. et al 2007,). The 180

present study has observed significantly higher PI (3.0 ± 0.5 v/s 2.3 ± 0.6, P=.001) and CAL 181

(8.5 ± 1.0 v/s 7.7 ± 0.9, P=.021) in smokers with chronic periodontitis compared to non 182

smokers. However, smokers with chronic periodontitis showed significantly lower GI (1.9 ± 183

0.5 v/s2.4 ± 0.5, P=.001) compared to non smokers. (Table-1.) The lowered GI observed in 184

smokers could be attributed to the smoking habit. Smoking associated periodontitis is 185

characterized by limited gingival redness. Smoking leads to sustained peripheral 186

vasoconstriction caused by chronic low dose of nicotine. This leads to reduced gingival 187

bleeding. (Heasman, L. 2006). 188

189

Table 1: Mean values for the clinical parameters and the percent prevalence for H pylori in 190

various study groups 191

Parameters Group I (n=30)

Group II (n=18)

Group III (n=16)

P value†

Group I vs.

Group II

Group I vs.

Group III

Group II vs.

Group III

Clinical GI PI

CAL (mm)

1.9 ± 0.5 3.0 ± 0.5 8.5 ± 1.0

2.4 ± 0.5 2.3 ± 0.6 7.7 ± 0.9

0.7± 0.08 0.5 ± 0.2 1.9 ± 0.3

.001

.001

.021

.001

.001

.001

.001

.001

.001

H pylori (% prevalence)

+ve 13.3% 5.5% 0.0% .186‡ .003

‡ .132

‡

† P values for mean ± SD are obtained using one way analysis of variance (ANOVA) with Tukey’s correction for multiple group comparisons. P 192

vaule < .05 is considered to be statistically significant. 193

‡P – value for H pylori is obtained using Chi – square test if cell frequencies are larger than 5, else Fisher’s exact probability test was used. P 194

vaule < .05 is considered to be statistically significant. 195

The infection by H pylori is widely accepted as an important cause of gastritis and is strongly 196

associated with peptic ulcer disease and gastric cancer. The human stomach was 197


considered to be the only reservoir for H pylori until this bacterium was discovered in the 198

human oral cavity, saliva, dental plaque and in oral lesions or ulcers. (Hu, W. et al 2002, 199

Eskandari, A. et al 2010, Gao, J. et al 2011). Various studies (Dye, BA. et al 2002, Souto, 200

Columbo 2008, Rajendran, R.et al 2009, Fernando, N. et al 2009) have observed the 201

possible role of oral cavity as a reservoir for H pylori and have correlated H pylori 202

colonization and periodontal disease. Asqah,M.Al, et al (2009) have suggested that patients 203

with poor oral hygiene have a higher prevalence of H pylori in dental plaque and the oral 204

cavity may be a reservoir for H pylori. Dionf et al (2011) have stated that periodonto-205

pathogens like F nucleatum and Eikenella corrodens could co aggregate with H pylori in the 206

subgingival dental plaque. Saliva plays a significant role in maintaining a healthy oral 207

environment (Javed, F. et al 2009). It has been suggested that microorganism in dental 208

plaque can survive in saliva (Patil, Patil 2011). Studies (Gebara, EC. et al 2004, Souto, 209

Columbo 2008,) have detected H pylori in saliva and gingival plaques of periodontitis 210

patients. According to Tiwari SK et al (2005) PCR amplification of the DNA of the ulcer 211

causing bacterium H pylori in saliva samples has been shown to have comparable sensitivity 212

to DNA tests using gastric biopsy samples or histopathological analysis of gastric tissue. 213

Thus detection of H pylori in saliva using sensitive methods like PCR may reveal the 214

presence of pathogen in the oral cavity. The present study has not detected H pylori DNA in 215

healthy controls (100% negative), whereas 5.5% of non smokers and 13.3% smokers with 216

chronic periodontitis have shown presence of H pylori DNA. Smokers with chronic 217

periodontitis showed significant percent prevalence (P = .003) than healthy controls, 218

however the percent prevalence has not reached significance (P = .186) compared to non 219

smokers with chronic periodontitis, though the percent prevalence values are relatively 220

higher. The non significance observed may be due to smaller sample size of the study 221

population. 222

It is well accepted that persistent H pylori infection results in an inflammatory response in the 223

stomach leading to high induction of pro inflammatory cytokines, such as TNF α, IL1 and IL8 224


(Suganum, M. et al 2008). H pylori directly interact with host cells, induce pro inflammatory 225

cytokines and stimulates production of free radicals leading to inflammatory alterations in 226

GIT (Tsuji, T. et al 2003). On a similar paradigm, these inflammatory markers, which may be 227

produced by H pylori in oral cavity, may add an additional burden on the pre existing 228

inflammation due to chronic periodontitis in oral cavity. Thus H pylori in oral cavity may 229

contribute to the inflammatory destruction of peridontium in chronic periodontitis. 230

The overall prevalence of periodontal disease is high in India because of several factors like 231

huge population (app 1000 million), of which 72% reside in the rural. Poor oral health care 232

system and para dental infrastructure, with the dentist: population ration of 1:2 lac are 233

important factors leading to periodontitis. Also habits like smoking, pan( betel leaves) with 234

tobacco chewing was shown to be significant risk factor for development of periodontal 235

disease (Agarwal, V. et al 2010). 236

Tobacco smoke exposure increases susceptibility to respiratory tract infections, sexually 237

transmitted diseases, periodontitis, H pylori infection, meningitis, otitis media and post 238

surgical and nosocomial infections (Bagaitkar, J. et al 2008). Tobacco smoking is the main 239

risk factor associated with chronic destructive periodontal disease which results in bone loss, 240

pocket formation and premature tooth loss. There is an emerging evidence to suggest that 241

sub-gingival calculus formation is more prevalent and severe in smokers compared to non-242

smokers (Bergstrom, J. 2004). Various factors contribute to the deleterious periodontal 243

effects of smoking, including alterations in both microbial and host response factors. The 244

proposed mechanisms for the negative effects of smoking on periodontium may include 245

vascular alterations, altered neutrophill functions (chemotaxis, phagocytosis and oxidative 246

burst), increased prevalence of periopathogens and altered fibroblast attachment and 247

functions. It can also increase secretion of TNF α, prostaglandin E2, neutrophil elastase and 248

collaginase in gingival cervicular fluid (Georgia, Margaret 2004, Agarwal, V. et al 2010,). 249

The present study has observed a relative increase in the percent prevalence of H pylori in 250

smokers than non smokers with chronic periodontitis. Smokers appear to be at higher risk of 251


becoming infected with H pylori and this increased risk may be due to the adverse effects of 252

smoking on antioxidants or the immune system that may interfere with the normal protection 253

against H pylori, also they have elevated levels of circulating inflammatory mediators, which 254

may exacerbate the detrimental effects of H pylori associated gastric inflammation 255

(Parasher, Eastwood 2000, Kim, J. et al 2012). Peleterio B et al (2008) in the review article 256

have showed that tobacco availability was positively associated with the prevalence of 257

intestinal mataplasia among H pylori –infected subject at the area level. In India prevalence 258

of intestinal mataplasia in H pylori –infected subjects is 8.2% and tobacco availability 259

(cigarettes/day/adult) is 3.75. Shikata K et al (2008) have observed that the combination of 260

cigarette smoking and H pylori infection increased the risk of gastric cancer more than 261

smoking alone or H pylori infection alone. And when their independent and joint influences 262

on gastric cancer are taken into account, the impact is large, and a large part is due to their 263

co-occurrence. Also, the risk of intestinal mataplasia was higher in subjects infected with 264

high virulence H pylori strains and among smokers, and was further increased when both 265

factors were simultaneously present (Peleterio B et al 2007). Similar to the above studies it 266

may be possible that co occurrence of periodontitis and smoking habit may favor H pylori 267

infection in oral cavity. The significantly increased (P = .001) plaque formation among 268

smokers may become a favorable niche for colonization of perio-pathogens and may help co 269

aggregation of H pylori, as observed in our study. 270

The present study has enrolled only male subjects, considering urban Indian social customs 271

with respect to smoking habits. Also their smoking status was based on the verbal autopsy. 272

The non-recruitment of healthy smokers (smokers without periodontitis) in the study puts a 273

limitation for better inter and intra group comparison. It should be pointed out that detection 274

by PCR does not implicate cell viability or pathogenicity (Gebara, E.C.E. et al 2006), still 275

detection of H pylori DNA in salivary samples confirms its presence in the oral cavity of 276

periodontitis cases and smoking may contribute to the higher prevalence of H pylori in 277

smokers. Further investigations based on larger sample size are recommended for validation 278


of the present observations and also to elucidate if basic periodontal therapies would be 279

employed to eradicate this pathogen from the oral cavity and prevent its reinfection in higher 280

risk groups like smokers. 281

4. CONCLUSION 282

283 The detection of salivary H pylori DNA in chronic periodontitis and further relative increase in 284

its presence in smokers with chronic periodontitis indicates that smoking may provide 285

suitable environment for colonization of H pylori in the oral cavity. Smokers with chronic 286

periodontitis are at a higher risk of infection by H pylori. This information may help in 287

understanding the deleterious effects of smoking with respect to oral health and may 288

contribute in designing and implementation of preventive programs for smokers. 289

ACKNOWLEDGEMENTS 290

291 Authors are thankful to Prof. Salma Aziz for financial assistance, Ms. Preeti for technical 292

assistance, Mr. Mehmood S for helping in statistical analysis and Dr. Mendanha RE, Mrs. 293

Nimi S. for manuscript proof reading. 294

COMPETING INTERESTS 295

296 Authors have declared that no competing interest exists. 297

AUTHORS’ CONTRIBUTIONS 298

299 Dr. Rajiv Kishor Saxena: Performed PCR analysis and helped to wrote the first draft of 300

manuscript 301

Mr. Abdul Samad Aziz: Managed literature searches, wrote protocol, data acquisition and 302

manuscript preparation. 303

Dr. Madhav Govind Kalekar : Designed, guided and supervised the study 304

Ms. Milsee J. P.: Helped in PCR analysis, manuscript editing and manuscript review 305

Dr. Adinath Narayan Suryakar : Designed and guided the study 306

Dr. Tabita Benjamin: Managed the evaluation of the clinical parameters 307

Dr. Ravi Vasudev Shirahatti: Helped in manuscript editing and manuscript review 308

Dr. Raghuvendra Shrishail Medikeri: Helped in manuscript editing and manuscript review 309


All authors read and approved the final manuscript 310

CONSENT 311

All authors declare that ‘written informed consent was obtained from the patient for 312

publication of this study. A copy of the written consent may be made available for review by 313

the editorial office/chief editor/editorial board members of this journal 314

ETHICAL APPROVAL 315

316 All authors hereby declare that all experiments have been examined and approved by the 317

appropriate ethics committee and have therefore been performed in accordance with the 318

ethical standards laid down in the 1964 Declaration of Helsinki. 319

REFERENCES 320

321 Agarwal, V., Khatri, M., Singh, G., Gupta, G., Marya, C.M., Kumar, V (2010) Prevalence of 322

periodontal disease in India. J. Oral Health and Community Dentistry.4(spl)7-16. 323

Akalin, F. A., Baltacioglu, E., Alver, A., Karabulut,E. (2007) Lipid peroxidation level and total 324

oxidant status in serum, saliva and gingival cervicular fluid in patients with chronic 325

periodontitis. J Clinical Periodontol.34:558-565. 326

Akalin, F. A., Toklu, E., Renda, N. (2005) Analysis of superoxide dismutase activity levels in 327

gingival and gingival cervicular fluid in patients with chronic periodontitis and periodontally 328

healthy subjects. J Clinical Periodontol.32:238-243. 329

Armitage, G.C. (2000) Development of a classification system for periodontal disease and 330

conditions. Northwest Dent 79(6):31-35. 331

Asqah,M,Al., Hamoudi,N,Al., Anil,S., Jebreen,A.Al., Hamoudi,W.K.Al. (2009) Is the 332

presence of Helicobacter pylori in the dental plaque of patients with chronic periodontitis a 333

risk factor for gastric infection? Can J Gastroenterol.23(3):177-179 334

Bagaitkar, J., Demuth, D. R., Scott, D. A. (2008) Tobacco use increases susceptibility to 335

bacterial infection. Tobacco Induced Diseases.4(12):doi:10.1186/1617-9625-4-12. 336

Bergstrom, J. (2004) Tobacco smoking and chronic destructive periodontal disease. 337

Odontology.92:1-8 338


Buduneli, N., Kardesler, L., Isik, H., Willis, C. S., Hawkins, S. I., Kinane, D. F., Scott, D. A. 339

(2006) Effect of smoking and gingival inflammation on salivary antioxidant capacity. J. of 340

Clin. Periodontol.33:159-164. 341

Bulunet, K., Gulay, T., Muhittin, S., Selin, P., Ilkim, D., Utku, T. (2007) Gingival cervicular 342

fluid prostaglandin E2 and TBARS levels in smokers and non smokers with chronic 343

periodontitis following phase I periodontal therapy and adjunctive use of flurbiprofen. J. 344

Periodontol.78:104-111. 345

Diouf, A., Seck-Diallo, A. M., Faye, M., Benoist, H. M., Sembene, M., Diallo, P. D., Martinez-346

Gomis, J., Sixou, M. (2011) Prevalance of Helocobacter pylori detected by real time PCR 347

in the subgingival plaque of patients with chronic periodontitis. Odontostomatol 348

Trop.34(133):5-12. 349

Dye, B. A., Kruszon-Moran. D., McQuallan, G. (2002) The relationship between periodontal 350

disease attributes and Helicobacter pylori infection among adults in the United States. 351

American Journal of Public Health.92(11):1809-1815. 352

Erdemir, E. O., Duran, I., Haliloglu, S. (2004) Effect of smoking on clinical parameters and 353

the gingival cervicular fluid levels of IL-6 and TNF α in patients with chronic periodontitis. 354

J. Periodontol,31:99-104. 355

Eskandari, A., Mahmoudpour, A., Abolfarli, N., Lafzi, A. (2010) Detection of Helicobacter 356

pylori using PCR in dental plaque of patients with and without gastritis. Med. Oral. Patol 357

Oral Cir Bucal.1:15(1):e28-31. 358

Ferguson, Jr. D. A., Li, Chuanfu., Patel, N. R., Mayberry, W. R., Chi, D. S., Thomas, E. 359

(1993). Isolation of Helicobacter pylori from saliva. J. Clin. Microbiology.31(10):2802-04. 360

Fernando, N., Jaykumar, G., Perara, N., Amarasingha, I., Meedin, F., Holton, J. (2009) 361

Presence of Helicobacter pylori in betel chewers and non betel chewers with and without 362

oral cancers. BMC Oral Health.9:23 doi:10.1186/1472-6831-9-23. 363


Gao, J., Li, Y., Wang, Q., Qi, C., Zhu, S. (2011) Correlation between distribution of 364

Helicobacter pylori in oral cavity and chronic stomach conditions. J Hiazhong Uni Sci 365

Technology Med Sci. 31 (3):409-12. 366

Gebara, E. C. E., Faria, C. M., Pannuti, C., Mayer, M. P. A., Lima, L. A. P. A. (2006) 367

Persistence of Helicobacter pylori in the oral cavity after systemic eradication therapy. J 368

Clinical Periodontol.33:329-333. 369

Gebara, E. C., Pannuti, C., Faria, C. M., Chehter, L., Mayer, M. P., Lima, L. A. (2004) 370

Prevalence of Helicobacter pylori detected by polymerase chain reaction in the oral cavity 371

of periodontitis patients. Oral Microbiol Immunol.19(4):227-80. 372

Georgia, K. J., Margaret, H. (2004) Cigarette smoking and the periodontal patients. J. 373

Periodontol.75:196-209. 374

Heasman,L., Stacey,F., Pershaw,P.M.,McCracken,G.I.,Hepburn,S.,Heasman,P.A. (2006) 375

The effect of smoking on periodontal treatment response: a review of clinical evidence. J. 376

Clinical Periodontol.33: 241-253. 377

Hu, W., Cao, C., Meng, H., Zhang, J., Ma, D., Zhang, L. (2002) Detection and analysis of 378

Helicobacter pylori in oral cavity and stomach from chronic gastritis patients. Zhonghoa Yi 379

Xue Za Zhi. 10;82(15):1037-41. 380

Javed, F., Klingsper, L., Sundin, V., Altamash, M., Klinge, B., Engstrom, P. E. (2009) 381

Periodontal conditions, oral Candida albicans and salivary proteins in type 2 diabetes 382

subjects with emphasis on gender. BMC oral health.9:12doi:10.1186/1472-6831-9-12. 383

John, M.N. (2006) Classification of disease and condition affecting the periodontium, in : 384

Newman, M. G., Takei, H. H., Klokkevold, P. R., Carranza, F. A. (Eds.), Carranza’s Clinical 385

Periodontology 10th ed. Saunders/Elsivier, St Louis-Missouri.pp.100-109. 386

Kaufman, E., Lamster, I. B. (2000) Analysis of saliva for periodontal diagnosis. J Clin 387

Periodontol 27:453-465. 388


Kim, J., Cho, Y. A., Choi, I. J., Lee, Y. S., Kim, S. Y., Shin, A., Cho, S. J., et al. (2012) 389

Effects of Interleukin-10 polymorphism, Helicobacter pylori infection and smoking on the 390

risk of non cardia gastric cancer. PlosOne.7(1)e-29643. 391

Lu, J. J., Perng, C. L., Shyu, R. Y., Chen, C. H., Lou, K., Chong, S. K. F., Lee, C.H. (1999) 392

Comparison of five PCR methods for detection of Helicobacter pylori DNA in gastric tissue. 393

J of Clinical Microbiology. 37(3):772-774. 394

Parasher, G., Eastwood, G.L. (2000) Smoking and peptic ulcer in the Helicobacter pylori era. 395

Eur J Gastroenterol Hepatol.12(8): 843-853. 396

Patil, P.B., Patil, B.R. (2011) Saliva: a diagnostic biomarker of periodontal disease. J. Ind 397

Soc Periodontol, 15(4):310-317. 398

Peleteiro, B., Lunet, N., Figueiredo, C., Carneiro, F., David, L., Barros, H. (2007) Smoking 399

Helicobacter pylori virulence, and type of intestinal mataplasia in Portuguese males. 400

Cancer Epidemiol Biomarkers Prev. 16(2):322-26. 401

Peleterio, B., Bastos, J., Barros, H., Lunet, N. (2008) Systemic review of the prevalence of 402

gastric intestinal metaplasia and its area-level association with smoking. Gac Sanit. 22(3): 403

236-47. 404

Rajendran, R., Rajeev, R., Anil, S., Mohammad Alasqah., Rabi, A.G. (2009) Helicobacter 405

pylori infection is a confounder, modulating mucosal inflammation in oral submucous 406

fibrosis. Indian J Dent Res. 20(2):206-211. 407

Ribeiro, M., Ecclissato, C., Mattos, R., Mendonca, S., Pedrazzoli, J.Jr. (2007) Quantitative 408

real-time PCR for the clinical detection of Helicobacter pylori. Genetics and Molecular 409

Biology. 30(2):431-434. 410

Riggo, M. P., Lennon, A. (1999) Identification by PCR of Helicobacter pylori in subgingival 411

plaque of adult periodontitis patients. J. Med. Microbiol. 48:317-322 412

Shikata, K., Doi, Y., Yonemoto, K., Arima, H., Ninomiya, T., Kubo, M., Tanizaki, Y., 413

Matsumoto, T., Lida, M., Kiyohara, Y. (2008) Population based prospective study of the 414


combined influence of Cigarette smoking and Helicobacter pylori infection on gastric 415

cancer incidence. Am J Epidemiol. 168(12):1409-1415. 416

Skirrow, M. B., (2002). Campylobacter and Helicobacter, in: Greenwood D., Slack R. C. B., 417

Pentherer, J. F. (Eds.), Medical Microbiology. 16thedition. Churchill Livingstone, USA, pp. 418

288 -295. 419

Soben, P. (2003) Indices in Dental Epidemiology, in: Essentials of Preventive and 420

Community Dentistry. 2nd

ed. Arya Med Pubc, New Delhi, pp.127-240. 421

Souto, R., Colombo, A. P. V. (2008) Detection of Helicobacter pylori by polymerase chain 422

reaction in the subgingival biofilm and saliva of non-dyspeptic periodontal patients. J. 423

Periodontol. 79:97-103. 424

Suganum, M., Yamaguchi, K., Ono, Y., Matsumoto, H., Hayashi, T., Ogawa, T. (2008) TNF α 425

inducing protein, a carcinogen factor secreted from H pylori, enters gastric cancer cells. 426

Int. J. Cancer. 23:117-122. 427

Suzuki, T., Matsuo, K., Ito, H., Sawaki, A., Hirose, K., Wakaki, K., et al (2006) Smoking 428

increases the treatment failure for Helicobacter pylori eradication. Am J Med. 119(3):217-429

24. 430

Tiwari, S. K., Khan, A. A., Ahmed, K. S., Ahmed I., Kauser, F., Hussain, M. A., et al. (2005) 431

Rapid diagnosis of H pylori infection in dyspeptic patients using salivary secretions- a 432

noninvasive approach. Singapore Med J. 46(5):224-228. 433

Tonetti, M.S. (1998) Cigarette smoking and periodontal diseases: Etiology and management 434

of diseases. Annals of Periodontol. 3(1):88-101 435

Tsuji, S., Kawai, N., Tsujii, M., Kawano, S., Hori, M. (2003) Inflammation related promotion 436

of gastrointestinal carcinogenesis – a perigenetic pathway. Ailment Pharmacol Ther. 18 437

sup1:82-89. 438

Umeda, M., Kobayashi, H., Takeyuchi, Y., Hayashi, J., Morotome-Hayashi, Y., Yano, K., et 439

al. (2003) High prevalence of Helicobacter pylori detected by PCR in the oral cavities of 440

Periodontitis patients. J Periodontol. 74(1):129-34. 441


Watts, T. L. P. (2006) Smoking, Helicobacter pylori and Periodontitis (letters to editor) BMJ. 442

332:1513 443

Wei, D., Zhang, X. L., Wang, Y. Z., Yang, C. X., Chen, G. (2010) Lipid peroxidation levels, 444

total oxidant status and superoxide dismutase in serum,saliva and gingival cervicular fluid 445

in chronic periodontitis patients before and after periodontal therapy. Australian Dental 446

Journal. 55:70-78 447

Zaric, S., Bojic, B., Jankovic, L. J., Dapcevic, B., Popovic, B., Cakic, S., et al (2009) 448

Periodontal therapy improves gastric Helicobacter pylori eradication. J Dent Res. 449

88(10):946-50. 450

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Original Research Paper 2 Prevalence of Helicobacter ... · Tel.: +919823375529; fax:...

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Transcript of *Original Research Paper 2 Prevalence of Helicobacter ... · * Tel.: +919823375529; fax:...

Original Research Paper 2 Prevalence of Helicobacter ... · Tel.: +919823375529; fax:...

Transcript of Original Research Paper 2 Prevalence of Helicobacter ... · Tel.: +919823375529; fax:...