Research Article Characteristics and Risk Factors of Helicobacter...

Research ArticleCharacteristics and Risk Factors of Helicobacter pyloriAssociated Gastritis A Prospective Cross-Sectional Study inNortheast Thailand

Taweesak Tongtawee12 Soraya Kaewpitoon23

Natthawut Kaewpitoon45 Chavaboon Dechsukhum26 Wilairat Leeanansaksiri7

Ryan A Loyd23 Likit Matrakool12 and Sukij Panpimanmas12

1Department of Surgery Institute of Medicine Suranaree University of Technology Nakhon Ratchasima 30000 Thailand2Suranaree University of Technology Hospital Nakhon Ratchasima 30000 Thailand3Family Medicine and Community Medicine Suranaree University of Technology Nakhon Ratchasima 30000 Thailand4Parasite Research Unit Suranaree University of Technology Nakhon Ratchasima 30000 Thailand5Faculty of Public Health Vongchavalitkul University Nakhon Ratchasima 30000 Thailand6School of Pathology Institute of Medicine Suranaree University of Technology Nakhon Ratchasima 30000 Thailand7School of Preclinic Institute of Science Suranaree University of Technology Nakhon Ratchasima 30000 Thailand

Correspondence should be addressed to Taweesak Tongtawee taweesaktsutacth

Received 22 October 2015 Revised 14 January 2016 Accepted 31 January 2016

Academic Editor Francesco Franceschi

Copyright copy 2016 Taweesak Tongtawee et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background and Aim Risk factors for Helicobacter pylori infection are genetic susceptibility and poor living conditions This studyaimed to investigate theMdm2 gene clarithromycin resistance and possible risk factors forHelicobacter pylori infectionMethodsRisk factors and clinical characteristics were analyzed including patient demographic data patient income personal historypossible source of transmission patient symptoms endoscopic findings patterns of clarithromycin resistance and patterns ofMdm2 SNIP309Results Ingestion of pickled fish (OR= 1127 95CI = 431ndash2945 119901 lt 00001) salt crab (OR = 883 95CI = 199ndash3914119901 lt 0001) and Papaya salad (OR= 873 95CI = 454ndash1679119901 lt 001)The prevalence of clarithromycin resistance was 56(wild type A21432142A is 238 mutation A21432142CG is 357 wild type + mutation is 405) The genetic polymorphismsof Mdm2 SNIP309 were SNIP309 TT homozygous in 78 SNIP309 GT heterozygous in 19 and SNIP309 GG homozygousin 3 Conclusion Pickled fish salt crab and Papaya salad are positive risk factors There was high prevalence of clarithromycinresistanceTheMdm2 SNIP309 GG homozygous genotype might be a risk factor for gastric cancer and the fact that it is infrequentin Thailand

1 Introduction

Since the discovery of Helicobacter pylori in 1983 strongevidence has indicated that H pylori infection plays animportant role in the pathogenesis of chronic gastritispeptic ulcer disease and gastric malignancy [1 2] Therisk factors for H pylori infection in both developing anddeveloped countries are closely related to poor living con-ditions and genetic susceptibility [3] Low socioeconomicstatus poor hygiene conditions overcrowding bed sharing

interfamilial clustering family history of parental gastricdisease and person-to-person contact through fecal-oral ororal-oral contamination may be the route of transmission[4ndash10] Among different Asian countriesH pylori infectionsare more frequent in developing countries such as IndiaBangladesh Pakistan and Thailand [11ndash13] In contrast inmore industrialized and developed regions of Asia like JapanChina and Singapore the frequency of H pylori infectionhas been reported to be somewhat lower [14]The prevalenceof H pylori infection varies between different geographic

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2016 Article ID 9130602 8 pageshttpdxdoiorg10115520169130602

2 Gastroenterology Research and Practice

locations including Thailand The prevalence of H pyloriinfection in the south region of Thailand (144) was thelowest compared with the northeast (606) north (469)and central (390) regions (all 119901 lt 0001) [15] Thenortheast region of Thailand shows the highest rate of Hpylori infection The primary resistance rate of H pylori toclarithromycin is different in each region of the world Theoverall global resistance from a systemic review in 2004was 99 (95 CI 83ndash117) [16] According to a nationwidesurvey ofH pylori antibiotic resistance inThailand antibioticresistance was present in 503 of cases including amoxicillin(52) tetracycline (17) clarithromycin (37) metron-idazole (36) ciprofloxacin (77) levofloxacin (72) andmultiple drugs (42) with unknown mutation patterns ofdrug resistance [17] Several methods have been proposedto increase the eradication rate including the extensionof the treatment duration to 14 days the use of a four-drug regimen (bismuth-containing quadruple sequentialand concomitant treatments) and the use of novel antibioticssuch as levofloxacin [18ndash21] The progressive loss of efficacyof standard eradication therapies has made the treatmentof H pylori more challenging than ever Endoscopic-guidedantibiotic susceptibility testing had previously been suggestedto guide treatment after failure of second-line therapiesHowever its role has expanded over the years in accordancewith the current Maastricht Guidelines Several authors havedealt with this topic developing both efficacy trials and cost-effectiveness trials against resistant H pylori infections aswell as infections in naıve patients However results are nothomogeneous enough to provide definitive advice becauseantibiotic resistance is not the only reason for treatmentfailureMoreover the culture-guided approach is surroundedby many practical issues such as the availability of bothendoscopy units and microbiology laboratories and the needfor a standard of quality that cannot be satisfied every-where Finally pretreatment susceptibility testing should bepart and not the only weapon of a targeted personalizedstrategy to overcome H pylori infection [22] The resultsof our previous study showed that adding a probiotic canimprove H pylori eradication rates [23 24] Furthermoreseveral studies and meta-analyses have reported that certainprobiotic strains can exhibit inhibitory activity against Hpylori bacteria In addition some probiotic strains can reducethe occurrence of side effects due to antibiotic therapy andconsequently increase the H pylori eradication rate [25ndash27] In addition geographical differences can also impairefficacy rates of different therapies as assessed in a recentmeta-analysis which showed that geographic weighting couldbe the main factor affecting the lack of differences betweensequential and 14-day triple therapy outcomes [28] H pyloriinfection plays an important role in gastric cancer but thereis a low incident of gastric cancer in the Thai populationin the setting of high prevalence of H pylori infectionThis unexpectedly low rate may be influenced by Thaigenetic predispositions to cancerMdm2 is themajor negativeregulator of p53 the key tumor suppressor involved in thetumorigenesis of the majority of human cancers Mdm2 isproposed to regulate p53 at the posttranslational level byenhancing p53 degradation throughE3 ligase activity [29ndash31]

The clinical data concerning the role of Mdm2 SNIP309 ingastric cancer development is limited A case-control studyamong the Iranian gastric cancer population showed thatMdm2 SNIP309 is a risk factor for this cancer with anodds ratio of 208 (95 confidence interval = 137ndash434) Thesame trend was observed in the Chinese [32 33] This studyaimed to investigate the characteristics ofH pylori associatedgastritis clarithromycin resistance Mdm2 polymorphismsand significant risk factors of H pylori associated gastritisamong theThai population

2 Materials and Methods

21 Patients Three hundred patients undergoing esopha-gogastroduodenoscopy for investigation of dyspeptic symp-toms participated in this study from June 2014 to June 2015The following exclusion criteria were applied age below18 or above 70 years old previous H pylori eradicationtreatment prior to the previous 2 months significant medicalillnesses history of previous gastric surgery and the use ofantimicrobials or gastrointestinal medications like PPIs orbismuth compounds within the previous 2monthsThe studywas performed in accordance with good clinical practice andthe guidelines of theDeclaration ofHelsinki All patients pro-vided written informed consent and the study protocol wasapproved by the Ethics Committee for Research InvolvingHuman Subjects Suranaree University of Technology (EC-57-22 and EC-57-34)

22 Diagnosis of H pylori Associated Gastritis A diagnosis ofH pylori associated gastritis was made if H pylori were seenon histopathological examination and the rapid urease testwas positive Finally we proved bacterial infection by PCR

23 Biopsy Specimens Biopsy was done according to theUpdated Sydney classification [34] which indicates samplingfrom 5 biopsy sites Each specimen was obtained from eachof the following locations the lesser curvature of the corpusabout 4 cm proximal to the angularis (1) the lesser curvature(2) and the greater curvature of the antrum (3) both within2 to 3 cm of the pylorus the middle portion of the greatercurvature of the corpus approximately 8 cm from the cardia(4) and the incisura angularis (5)

24 Esophagogastroduodenoscopy (EGD) Local anesthesiawas the same as that for conventional gastroscopy Thegastroscopic procedures were performed using an upperGI video endoscope (Olympus EVIS EXERA III CV-190)The whole stomach was examined first with conventionalendoscopy and then biopsies were performed A symptomquestionnaire (abdominal pain vomiting diarrhea gastroin-testinal bleeding and iron deficiency anemia) was completedby the patient at the time of initial EGD in the endoscopyroom (timeout)

25 Histological Analysis Gastric tissue specimens for his-tological analysis were sent to the pathologists The hema-toxylin and eosin stain and Giemsa stain were used foridentification ofH pyloriThepathological analysis wasmade

Gastroenterology Research and Practice 3

by 5 pathologists at Bangkok Pathological Laboratory outsideSuranaree University of Technology

26 DNA Isolation Method The DNA of H pylori wasextracted from frozen gastric tissue biopsy specimens whichwere stored at a temperature of less than ndash20∘C using theQIAamp DNA FFPE tissue kit (Qiagen USA) DNA extrac-tion was performed according to the manufacturer protocolBriefly ten tissue sections of 5120583M thick were collected in15mL microcentrifuge tubes The tissue specimens wereplaced in amicrocentrifuge tube and bufferATL (180120583L) andproteinase K (20120583L) were addedThe samples were mixed byvortexing and incubated at 56∘C until the tissues were com-pletely lysed Buffer AL (200120583L) was added to the sampleswhich were subsequently incubated at 70∘C for 10 minutesNext 240 120583L of 100 ethanol was added to the sampleswhich were mixed by vortexing for 15 seconds Each samplewas placed in a QIAamp spin column and centrifuged at8000 rpm for 1 minute The columns were washed with AW1buffer (500120583L) and sampleswere centrifuged at 8000 rpm for1 minute AW2 buffer (500 120583L) was added to the column andsamples were centrifuged at 14000 rpm for 3 minutes BufferAE (200120583L) was added to each sample and samples wereincubated for 1 minute prior to centrifugation at 8000 rpmfor 1 minute Finally the DNA was extracted from thetissue

27 Real-Time PCR Hybridization Probe Methods for 23SrRNA Gene Point Mutation The mutation detection of the23S rRNA gene was performed by using the real-timePCR technique for template amplification A hybridizationfluorescent probe was utilized for PCR product detectionThe real-time PCR procedure was accomplished by using theLight Cyclerreg 480 instrument (Roche Diagnostics Neuillysur Seine France) The identification of target PCR productswas accomplished by melting curve analyses The target PCRproducts were amplified by using the primers HPYS andHPYA as reported in the previous literature PCR-RFLP canalso detect the point mutation A2142C of the 23S rRNA geneassociated with resistance of H pylori to clarithromycin Theamplified products have a size of 267 bp The hybridizationprobes include the one that is in the mutation sites of the23S rRNA gene of H pylori the sensor probe The sequenceis 5-GGCAAGACGGAAAGACC-3 nucleotides 2504 to2520 This sensor probe is labeled by LC-red 640 at 51015840 andphosphorylated at 31015840 The anchor probe hybridizes to thePCR product at the site 3 bp upstream to the sensor probeThe probe sequence is 5-TGTAGTGGAGGTGAAAATTCC-TCCTACCC-3 nucleotides 2473 to 2501 GenBank accessionnumber U27270 The probe is labeled with fluorescein at31015840 3 120583L DNA templates were subjected to PCR reaction inthe final volume of 20120583L The reaction mixture consists ofMgCl

2(25mM) forward and reverse primers (20M each)

sensor and anchor probes (20M each) and 2 120583L of FastStartDNA Master Hybridization Probes (Roche Diagnostics)PCR amplification comprised an initial denaturation cycle at95∘C for 10min followed by 50 amplification cycles (witha temperature transition rate of 20∘Cs) consisting of 95∘Cfor 0 s annealing at 60∘C for 10 s and extension at 72∘C

for 17 s After amplification a melting step was performedconsisting of 95∘C for 0 s cooling to 45∘C for 30 s (with atemperature transition rate of 20∘Cs) and finally a slow risein the temperature to 85∘C at a rate of 01∘Cs with continuousacquisition of fluorescence decline According to previousreports using this real-time PCR protocol this melting curveanalysis can detect the possible three mutant genotypes alongwith thewild type according to different TmThe reportedTmof thewild type A2121C A2142G andA2143Gwere 615 58053 and 536∘C respectively

28 Real-Time PCR Hybridization Probe Methods for Mdm2SNIP309 Genotypes Mdm2 SNIP309 genotypes were ana-lyzed using real-time PCR The hybridization probes (light-cycler probe) were utilized for this analysis The real-time PCR procedure was accomplished by using the LightCyclerreg 480 instrument (Roche Diagnostics Neuilly surSeine France) The identification of target PCR productswas accomplished by melting curve analyses The target PCRproducts were amplified by using the primers as reported inthe previous literatureThe hybridization probes included theone that is in the SNIP309 (the sensor probe) This sensorprobe was labeled by LC-red 640 at 51015840 and phosphorylatedat 31015840 The anchor probe hybridizes to the PCR product at thesite 3 bp upstream to the sensor probe 3 120583L DNA templateswere subjected to PCR reaction in the final volume of 20120583LThe reaction mixture consisted of MgCl

2(25mM) forward

and reverse primers (20M each) sensor and anchor probes(20M each) and 2 120583L of FastStart DNAMasterHybridizationProbes (Roche Diagnostics) PCR amplification comprisedan initial denaturation cycle at 95∘C for 10min followedby 50 amplification cycles (with a temperature transitionrate of 20∘Cs) consisting of 95∘C for 0 s annealing at 60∘Cfor 10 s and extension at 72∘C for 17 s After amplificationa melting step was performed consisting of 95∘C for 0 scooling to 45∘C for 30 s (with a temperature transition rateof 20∘Cs) and finally a slow rise in the temperature to 85∘Cat a rate of 01∘Cswith continuous acquisition of fluorescencedeclineThe genotype of each patient was categorized into thethree genotypes SNIP309 GG homozygous TT homozy-gous and GT heterozygous based on the different meltingcurves

29 Risk Factors Endoscopic Finding Personal History andPossible Route of Transmission for H pylori Infection The fol-lowing variables collected in the questionnaire were analyzedas possible risk factors forH pylori infection age gender andpatientrsquos income The clinical symptoms experienced duringthe study included abdominal pain vomiting diarrhea GIbleeding and iron deficiency anemia Endoscopic findingsincluded gastric ulcer duodenal ulcer gastric and duodenalulcer inflammatory polyp nonulcer gastritis nonulcer duo-denitis and gastroesophageal reflux disease Possible sourcesof transmission included family history ingesting food fromstreet vendors being a farmer nonvegetarian food ingestingpickled fish ingesting salt crab ingesting Papaya salador ingesting Thai vermicelli with curry Personal historyincluded smoking alcohol ingestion high temperature foodintake and spicy food


Table 1 Association between patientrsquos demographic data and H pylori infection (univariate analysis)

Variable Positive for H pylori 119899 () Negative for H pylori 119899 ()119901 valuelowast

Total number 150 Total number 150Age group 091

17ndash30 41 (273) 28 (186)31ndash44 30 (20) 31 (206)45ndash58 41 (273) 43 (286)59ndash70 38 (253) 48 (32)

Sex (malefemale) 7278 8169 096Income 097lt5000 Bahtmonth 5 (33) 19 (127)5000ndash10000 Bahtmonth 86 (573) 69 (46)10000ndash15000 Bahtmonth 50 (333) 56 (373)gt15000 Bahtmonth 9 (6) 6 (4)

lowastSignificance is set at 119901 lt 005

210 Statistical Analysis The associations of patientrsquos demo-graphic data patientrsquos symptom endoscopic finding possiblesource of transmission and personal history with H pyloripositive results were examined through univariate analy-sis Backward stepwise procedures were used to build themultivariate analysis the final model included only thosevariables that were found to be statistically significant in theunivariate analysis The associations were expressed as oddsratios (OR)with their confidence intervals (95CI)The datawere analyzed with the SPSS software (SPSS for Windowsversion 16) Significance was set at 119901 lt 005

3 Results

A total of 300 patients were enrolled in this study [153 males(51) and 149 females (49)] from the northeast regions ofThailand The total number of H pylori infected individualswas 150 and the total number of noninfected individualswas also 150 Looking at the patientsrsquo demographic dataand H pylori infection by univariate analysis showed noassociation between them (Table 1) This study showed ahigh rate of 23S ribosomal RNA point mutations (562)Among the mutations group the rates of cases which had thewild type genotype mutant strain and mixed wild type andmutant genotype were 238 357 and 405 respectively(Table 2 and Figure 1) The incidence of Mdm2 SNIP309TT homozygous was 78 and that of Mdm2 SNIP309GT heterozygous was 19 and that of Mdm2 SNIP309GG homozygous was 3 The results show that the Mdm2SNIP309 TT and Mdm2 SNIP309 GT genotypes are ratherhigh in this Thai population (Table 3 and Figure 2)

31 Patientrsquos Symptom Endoscopic Finding and H pyloriInfection The associations between patientrsquos symptomsendoscopic findings andH pylori infection were analyzed byusing univariate analysis The results showed an associationonly between the patientrsquos symptoms and H pylori infection(119901 lt 001) but no association was found by using thebinary logistic regression model Abdominal pain and iron

Table 2 Mutation patterns of 23S ribosomal RNA point mutations

Test susceptibleresistant to clarithromycin 119899 = 168

Wild type A21432142A (susceptible) 238Mutation A21432142CG (resistant) 357Wild type + mutation (susceptible + resistant) 405

Table 3Mdm2 SNIP309 polymorphism

Mdm2 SNIP309 polymorphism 119899 = 300

SINP309 TT homozygous 78SNIP309 GT heterozygous 19Mdm2 GG homozygous 3

0448

0398

0348

0298

0248

0198

0148

0098

0048

minus0002

minus0052

46 48 50 52 54 56 58 60 62 64 66 68 70 72 74

Temperature (∘C)

minus(ddT)

fluor

esce

nce (

498

ndash640

)

mutationMix wild type and

(A2143A2142GC)

A2143A2142GCmutation Wild

type

Melting peaks

Figure 1 Pattern of clarithromycin resistance by using the real-timePCR hybridization probe method

deficiency anemia had this relative association withH pyloriinfection (Table 4)

32 Possible Source of Transmission Personal History and Hpylori Infection Theassociations between the possible sourceof transmission personal history andH pylori infectionwereanalyzed by using univariate analysis The results showed anassociation only between the source of transmission and H


Table 4 Association between patientrsquos symptoms endoscopic findings and H pylori infection (univariate analysis)


Total number 150 Total number 150Patientrsquos symptom lt001lowast

Abdominal pain 73 (487) 68 (453)Gastrointestinal bleeding 12 (8) 12 (8)Vomiting 8 (53) 11 (73)Diarrhea 3 (2) 10 (67)Iron deficiency anemia 54 (36) 49 (327)

Endoscopic finding 017Gastric ulcer 7 (47) 12 (8)Duodenal ulcer 22 (147) 13 (87)Gastric and duodenal ulcer 3 (2) 12 (8)Inflammatory polyp 8 (53) 9 (6)Nonulcer gastritis 95 (633) 84 (56)Nonulcer duodenitis 1 (07) 11 (73)Gastroesophageal reflux disease 14 (93) 9 (6)


Table 5 Predictive value of patientrsquos symptoms for H pyloriinfection (multivariate analysis)

Variable 95 confidenceOdds ratio Interval 119901 value

Abdominal pain 111 057ndash141 064Gastrointestinal bleeding 091 040ndash208 083Vomiting 064 025ndash163 035Diarrhea 028 007ndash106 006Iron deficiency anemia 115 072ndash186 054Significance is set at 119901 lt 005

1500016000

1300014000

1100010000

12000

9000

70008000

50006000

30004000

10002000Fl

uore

scen

ce (4

65

ndash510

)

Fluorescence (533ndash580)

Endpoint fluorescence scatter plot

Mdm2 SNIP309 Allele G

Mdm2 SNIP309 Allele TG

Mdm2 SNIP309 Allele T

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000

Figure 2 Pattern of genetic Mdm2 SNIP309 polymorphism usingreal-time PCR hybridization probes (light-cycler probe)

pylori infection (119901 lt 001) (Table 5) The results showedingestion of pickled fish salt crab or Papaya salad to besignificant predictors for H pylori infection (119901 lt 0001)(Table 6) Subjects who ingested pickled fish were 1127 timesmore likely to haveH pylori infection comparedwith subjectswho did not Those who ingested salt crab were 883 times

more likely to have H pylori infection compared with thosewho did not and those who ingested Papaya salad were 873times more likely to have H pylori infection compared withthose who did not (Table 7)

4 Discussion

The prevalence of H pylori infection varies between differ-ent geographic locations including Thailand The highestprevalence of H pylori infection is found in the northeast(606) [15]The reasons are unclear and limited studies werereported fromThailand Some studies reported that risk fac-tors forH pylori infection are generally considered to includelower education level and low annual income [35 36] Thefecal-oral and oral-oral routes are important transmissionroutes of H pylori and the oral cavity is a potential extragas-tric reservoir forH pylori [37] Some popular foods found inthe northeast region of Thailand including pickled fish saltcrab and Papaya salad were found to be significant predictorsofH pylori infection in our area by both univariate andbinarylogistic regression model analysis To answer this questionwe plan to try to cultureH pylori from these suspected foodsAge group patientrsquos income and personal history were notassociated with H pylori infection in our area Abdominalpain and iron deficiency anemia were the most commonsymptoms in the patients with H pylori infection whereasduodenal ulcer and nonulcer gastritis were themost commonendoscopic findings in our patient population fromnortheastThailand but neither was statistically associatedwithH pyloriinfection in northeastThailand According to our results themajority of histologically provenH pylori infected cases havemutant genotype which confers clarithromycin resistancethe clinical data indicates that most of these cases have apoor response to treatment with standard regimen Thisobservation indicates that in the cases that have resistantstrains this treatment protocol (clarithromycin base tripletherapy) is ineffective to eradicate the bacteria in our area and


Table 6 Association between possible source of transmission personal history and H pylori infection (univariate analysis)


Total number 150 Total number 150Possible root of transmission lt001lowast

Family history 2 (13) 14 (93)Ingesting food from street vendor 15 (10) 23 (153)Being a farmer 4 (27) 35 (233)Nonvegetarian food mdash 28 (187)Ingesting pickled fish 42 (28) 5 (53)Ingesting salt crab 16 (107) 2 (13)Ingesting Papaya salad 68 (453) 13 (87)Thai vermicelli eaten with curry 3 (2) 30 (20)

Personal history 005Smoking 49 (327) 20 (133)Alcohol drinking 89 (593) 30 (20)High temperature food intake 5 (33) 65 (433)Spicy food 7 (47) 35 (233)


Table 7 Predictive value of possible source of transmission for Hpylori infection

Variable 95 confidenceOdds ratio Interval 119901 valuelowast

Family history 013 002ndash058 008Ingesting food from street vendor 058 029ndash116 012Being a farmer 009 003ndash026 lt001lowast

Ingesting pickled fish 1127 431ndash2945 lt001lowast

Ingesting salt crab 883 199ndash3914 lt001lowast

Ingesting Papaya salad 873 454ndash1679 lt001lowast

Thai vermicelli eaten with curry 008 002ndash027 lt001lowastlowastSignificance is set at 119901 lt 005

provide clinical practice outcomes The possible reasons thatunderlie the mixed genotypes are multiple infections of thesame patient by two strains or the occurrence of a mutationafter infection by a single strain Further genotypic analysesare necessary to confirm these possible mechanisms andlargermulticenter studies aboutmutation patterns are neededto test this hypothesis Physicians should be concerned aboutthe local resistance prevalence in their area and choose themost appropriate regimen for H pylori eradication

Cancer of the stomach is the fifth most common humancancer worldwide Intriguingly marked variation in thegastric cancer incidence is observed The highest incidenceof gastric cancer is found in Asia such as Korea (418100000)whereas the lowest incidence is found in Africa andNorthernAmerica In the Thai population the incidence of gastriccancer is only 35100000 As chronic gastritis is the majorpredisposing condition to gastric cancer study of the associa-tion between these conditions has become an area of interestHowever in Thailand where the gastric cancer incidencefalls into the low-risk country group the prevalence of H

pylori associated gastritis is intriguingly high especially inthe northeast region Thailand is an area of the enigmaticsituation that is also reported in Africa [38] Many studieshave investigated the association between H pylori infectionand gastric cancer in Asia A study from India failed todemonstrate an association between H pylori infection andgastric cancer [39ndash42] whereas studies fromChina and Japandemonstrated an association betweenH pylori infection andgastric cancer [43 44] Our study suggests that the frequencyof Mdm2 SNIP309 GG is very low among the Thai popu-lation which can explain to some extent the low incidenceof gastric cancer changes Mdm2 SNIP309 TT homozygousand Mdm2 SNIP309 GT heterozygous are both rather highin prevalence According to previous data these genotypesconfer a protective effect for certain human cancers includinggastric cancer and may provide the answer to the ldquoThailandenigmardquo This study reported several lifestyle-related riskfactors for stomach cancer in northeast Thailand [45] Hpylori infection was associated with variables indicative ofingestion of some popular foods such as pickled fish saltcrab and Papaya salad These are favorite foods in thenortheast and north regions ofThailand including Laos PDRUnfortunately there is lack of data on the clarithromycinresistance gastric cancer and H pylori infection prevalencein Laos PDR which has a similar lifestyle to the northeastregion of Thailand In conclusion H pylori infection wasassociated with variables indicative of ingestion of somepopular foods There was high prevalence of clarithromycinresistance Therefore the use of clarithromycin-based tripletherapy is not recommended as an empiric first-line regimenfor H pylori eradication in our area The Mdm2 SNIP309GG homozygous genotype might be a risk factor for gastriccancer and the fact that it is infrequent in Thailand couldexplain to some extent the low incidence of gastric cancer intheThai population Larger multicenter studies are needed totest this hypothesis


Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by a grant for medical investigationfrom Suranaree University of Technology and was approvedby the Ethics Committee for Research Involving HumanSubjects Suranaree University of Technology (EC-57-22 andEC-57-34)

References

[1] M Mihara K Haruma T Kamada et al ldquoThe role of endo-scopic findings for the diagnosis ofHelicobacter pylori infectionevaluation in a country with high prevalence of atrophicgastritisrdquo Helicobacter vol 4 no 1 pp 40ndash48 1999

[2] M Asaka M Kato T Sugiyama et al ldquoFollow-up survey ofa large-scale multicenter double-blind study of triple therapywith lansoprazole amoxicillin and clarithromycin for eradi-cation of Helicobacter pylori in Japanese peptic ulcer patientsrdquoJournal of Gastroenterology vol 38 no 4 pp 339ndash347 2003

[3] V Misra R Pandey S P Misra and M Dwivedi ldquoHelicobacterpylori and gastric cancer Indian enigmardquo World Journal ofGastroenterology vol 20 no 6 pp 1503ndash1509 2014

[4] EUROGAST Study Group ldquoEpidemiology of and risk factorsfor Helicobacter pylori infection among 3194 asymptomaticsubjects in 17 populationsrdquo Gut vol 34 no 12 pp 1672ndash16761993

[5] H M Malaty and D Y Graham ldquoImportance of childhoodsocioeconomic status on the current prevalence ofHelicobacterpylori infectionrdquo Gut vol 35 no 6 pp 742ndash745 1994

[6] O PGalpin C JWhitaker andA J Dubiel ldquoHelicobacter pyloriinfection and overcrowding in childhoodrdquoThe Lancet vol 339no 8793 p 619 1992

[7] F Perri M Pastore G Leandro et al ldquoHelicobacter pyloriinfection and growth delay in older childrenrdquo Archives ofDisease in Childhood vol 77 no 1 pp 46ndash49 1997

[8] D Vaira J Holton M Menegatti et al ldquoRoutes of transmissionofHelicobacter pylori infectionrdquo Italian Journal of Gastroenterol-ogy and Hepatology vol 30 no 3 pp 279ndash285 1998

[9] H Brenner D Rothenbacher G Bode and G Adler ldquoParentalhistory of gastric or duodenal ulcer and prevalence ofHelicobac-ter pylori infection in preschool children population basedstudyrdquo British Medical Journal vol 316 no 7132 pp 655ndash6651998

[10] B A Al-Knawy M-E K Ahmed S Mirdad A ElMekki andO Al-Ammari ldquoIntrafamilial clustering of Helicobacter pyloriinfection in Saudi Arabiardquo Canadian Journal of Gastroenterol-ogy vol 14 no 9 pp 772ndash774 2000

[11] C Thiede A Morgner B Alpen et al ldquoWhat role doesHelicobacter pylori eradication play in gastric malt and gastricmalt lymphomardquo Gastroenterology vol 113 no 6 pp 61ndash641997

[12] N Lunet and H Barros ldquoHelicobacter pylori infection andgastric cancer facing the enigmasrdquo International Journal ofCancer vol 106 no 6 pp 953ndash960 2003

[13] D Y Graham E Adam G T Reddy et al ldquoSeroepidemiologyof Helicobacter pylori infection in Indiamdashcomparison of devel-oping and developed countriesrdquoDigestive Diseases and Sciencesvol 36 no 8 pp 1084ndash1088 1991

[14] H Miwa M F Go and N Sato ldquoH pylori and gastric cancerthe Asian enigmardquo The American Journal of Gastroenterologyvol 97 no 5 pp 1106ndash1112 2002

[15] T Uchida M Miftahussurur R Pittayanon et al ldquoHelicobacterpylori infection in Thailand a nationwide study of the CagAphenotyperdquo PLoS ONE vol 10 no 9 Article ID e0136775 2015

[16] F Megraud ldquoH pylori antibiotic resistance prevalence impor-tance and advances in testingrdquoGut vol 53 no 9 pp 1374ndash13842004

[17] R-K Vilaichone P Gumnarai T Ratanachu-ek and VMahachai ldquoNationwide survey of Helicobacter pylori antibioticresistance in Thailandrdquo Diagnostic Microbiology and InfectiousDisease vol 77 no 4 pp 346ndash349 2013

[18] L Fuccio M E Minardi R M Zagari D Grilli N Magriniand F Bazzoli ldquoMeta-analysis duration of first-line proton-pump inhibitor-based triple therapy for Helicobacter pylorieradicationrdquoAnnals of InternalMedicine vol 147 no 8 pp 553ndash562 2007

[19] D Vaira A Zullo N Vakil et al ldquoSequential therapy versusstandard triple-drug therapy forHelicobacter pylori eradicationa randomized trialrdquo Annals of Internal Medicine vol 146 no 8pp 556ndash563 2007

[20] A Zullo V De Francesco C Hassan S Morini and DVaira ldquoThe sequential therapy regimen for Helicobacter pylorieradication a pooled-data analysisrdquo Gut vol 56 no 10 pp1353ndash1357 2007

[21] E C NistaM Candelli M A Zocco et al ldquoLevofloxacin-basedtriple therapy in first-line treatment for Helicobacter pylorieradicationrdquoTheAmerican Journal of Gastroenterology vol 101no 9 pp 1985ndash1990 2006

[22] T Tongtawee C Dechsukhum W Leeanansaksiri et al ldquoEffectof pretreatment with Lactobacillus delbrueckii and Streptococcusthermophillus on tailored triple therapy for Helicobacter pylorieradication a prospective randomized controlled clinical trialrdquoAsian Pacific Journal of Cancer Prevention vol 16 no 12 pp4885ndash4890 2015

[23] T Tongtawee C Dechsukhum W Leeanansaksiri et alldquoImproved Helicobacter pylori eradication rate of tailored tripletherapy by adding Lactobacillus delbrueckii and Streptococcusthermophilus in Northeast region of Thailand a prospectiverandomized controlled clinical trialrdquoGastroenterology Researchand Practice vol 2015 Article ID 518018 7 pages 2015

[24] G Cammarota G Ianiro S Bibbo et al ldquoCulture-guidedtreatment approach for Helicobacter pylori infection review ofthe literaturerdquoWorld Journal of Gastroenterology vol 20 no 18pp 5205ndash5211 2014

[25] R Zhu K Chen Y-Y Zheng et al ldquoMeta-analysis of the efficacyof probiotics in Helicobacter pylori eradication therapyrdquo WorldJournal of Gastroenterology vol 20 no 47 pp 18013ndash18021 2014

[26] M Homan and R Orel ldquoAre probiotics useful in Helicobacterpylori eradicationrdquo World Journal of Gastroenterology vol 21no 37 pp 10644ndash10653 2015

[27] H Szajewska and M Kołodziej ldquoSystematic review with meta-analysis Lactobacillus rhamnosus GG in the prevention ofantibiotic-associated diarrhoea in children and adultsrdquoAlimen-tary Pharmacology ampTherapeutics vol 42 no 10 pp 1149ndash11572015


[28] G Losurdo G Leandro M Principi et al ldquoSequential vsprolonged 14-day triple therapy for Helicobacter pylori eradi-cation the meta-analysis may be influenced by lsquogeographicalweightingrsquordquo International Journal of Clinical Practice vol 69 no10 pp 1112ndash1120 2015

[29] YHaupt RMayaAKazaz andMOren ldquoMdm2promotes therapid degradation of p53rdquo Nature vol 387 no 6630 pp 296ndash299 1997

[30] R Honda H Tanaka and H Yasuda ldquoOncoproteinMDM2 is aubiquitin ligase E3 for tumor suppressor p53rdquo FEBS Letters vol420 no 1 pp 25ndash27 1997

[31] M H G Kubbutat S N Jones and K H Vousden ldquoRegulationof p53 stability by Mdm2rdquo Nature vol 387 no 6630 pp 299ndash303 1997

[32] M-TMoradi Z Salehi KAminian andAYazdanbod ldquoEffectsof p53 codon 72 andMDM2 SNP309 polymorphisms on gastriccancer risk among the Iranian populationrdquoAsian Pacific Journalof Cancer Prevention vol 15 no 17 pp 7413ndash7417 2014

[33] XWang J Yang B Ho et al ldquoInteraction ofHelicobacter pyloriwith genetic variants in the MDM2 promoter is associated withgastric cancer susceptibility in Chinese patientsrdquo Helicobactervol 14 no 5 pp 114ndash119 2009

[34] M F Dixon R M Genta J H Yardley and P Correa ldquoClassi-fication and grading of gastritis The updated Sydney SystemInternational Workshop on the Histopathology of GastritisHouston 1994rdquoThe American Journal of Surgical Pathology vol20 no 10 pp 1161ndash1181 1996

[35] J Raymond V B Nguyen G Vidal-Trecan and N KalachldquoHelicobacter pylori infection in children of developing coun-triesrdquoMedecine Tropicale vol 65 no 4 pp 383ndash388 2005

[36] N Kim ldquoEpidemiology and transmission route of Helicobacterpylori infectionrdquoKorean Journal of Gastroenterology vol 46 no3 pp 153ndash158 2005

[37] Q-H Zou and R-Q Li ldquoHelicobacter pylori in the oral cavityand gastric mucosa a meta-analysisrdquo Journal of Oral Pathologyand Medicine vol 40 no 4 pp 317ndash324 2011

[38] C Holcombe ldquoHelicobacter pylori the African enigmardquo Gutvol 33 no 4 pp 429ndash431 1992

[39] V Kate and N Ananthakrishnan ldquoHelicobacter pylori andgastric carcinoma evidence for the linkrdquo National MedicalJournal of India vol 13 no 6 p 329 2000

[40] V Kate N Ananthakrishnan S Badrinath and C RatnakarldquoPrevalence of Helicobacter pylori infection in disorders of theupper gastrointestinal tract in south Indiardquo National MedicalJournal of India vol 11 no 1 pp 5ndash8 1998

[41] A K Khanna P Seth G Nath V K Dixit and M KumarldquoCorrelation of Helicobacter pylori and gastric carcinomardquoJournal of Postgraduate Medicine vol 48 no 1 pp 27ndash28 2002

[42] R Sivaprakash U A Rao S P Thyagarajan B RamathilakamandV Jayanthi ldquoInvestigation for the prevalence ofHelicobacterpylori infection in patients with gastric carcinoma in MadrasIndiardquo Japanese Journal of Medical Science and Biology vol 49no 1 pp 49ndash56 1996

[43] M Asaka M Kato M Kudo et al ldquoRelationship betweenHelicobacter pylori infection atrophic gastritis and gastriccarcinoma in a Japanese populationrdquo European Journal ofGastroenterology and Hepatology vol 7 no 1 pp S7ndashS10 1995

[44] L Cai S Z Yu and Z F Zhang ldquoHelicobacter pylori infectionand risk of gastric cancer in Changle County Fujian ProvinceChinardquoWorld Journal of Gastroenterology vol 6 no 3 pp 374ndash376 2000

[45] K Suwanrungruang S Sriamporn S Wiangnon et alldquoLifestyle-related risk factors for stomach cancer in northeastThailandrdquo Asian Pacific Journal of Cancer Prevention vol 9 no1 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


locations including Thailand The prevalence of H pyloriinfection in the south region of Thailand (144) was thelowest compared with the northeast (606) north (469)and central (390) regions (all 119901 lt 0001) [15] Thenortheast region of Thailand shows the highest rate of Hpylori infection The primary resistance rate of H pylori toclarithromycin is different in each region of the world Theoverall global resistance from a systemic review in 2004was 99 (95 CI 83ndash117) [16] According to a nationwidesurvey ofH pylori antibiotic resistance inThailand antibioticresistance was present in 503 of cases including amoxicillin(52) tetracycline (17) clarithromycin (37) metron-idazole (36) ciprofloxacin (77) levofloxacin (72) andmultiple drugs (42) with unknown mutation patterns ofdrug resistance [17] Several methods have been proposedto increase the eradication rate including the extensionof the treatment duration to 14 days the use of a four-drug regimen (bismuth-containing quadruple sequentialand concomitant treatments) and the use of novel antibioticssuch as levofloxacin [18ndash21] The progressive loss of efficacyof standard eradication therapies has made the treatmentof H pylori more challenging than ever Endoscopic-guidedantibiotic susceptibility testing had previously been suggestedto guide treatment after failure of second-line therapiesHowever its role has expanded over the years in accordancewith the current Maastricht Guidelines Several authors havedealt with this topic developing both efficacy trials and cost-effectiveness trials against resistant H pylori infections aswell as infections in naıve patients However results are nothomogeneous enough to provide definitive advice becauseantibiotic resistance is not the only reason for treatmentfailureMoreover the culture-guided approach is surroundedby many practical issues such as the availability of bothendoscopy units and microbiology laboratories and the needfor a standard of quality that cannot be satisfied every-where Finally pretreatment susceptibility testing should bepart and not the only weapon of a targeted personalizedstrategy to overcome H pylori infection [22] The resultsof our previous study showed that adding a probiotic canimprove H pylori eradication rates [23 24] Furthermoreseveral studies and meta-analyses have reported that certainprobiotic strains can exhibit inhibitory activity against Hpylori bacteria In addition some probiotic strains can reducethe occurrence of side effects due to antibiotic therapy andconsequently increase the H pylori eradication rate [25ndash27] In addition geographical differences can also impairefficacy rates of different therapies as assessed in a recentmeta-analysis which showed that geographic weighting couldbe the main factor affecting the lack of differences betweensequential and 14-day triple therapy outcomes [28] H pyloriinfection plays an important role in gastric cancer but thereis a low incident of gastric cancer in the Thai populationin the setting of high prevalence of H pylori infectionThis unexpectedly low rate may be influenced by Thaigenetic predispositions to cancerMdm2 is themajor negativeregulator of p53 the key tumor suppressor involved in thetumorigenesis of the majority of human cancers Mdm2 isproposed to regulate p53 at the posttranslational level byenhancing p53 degradation throughE3 ligase activity [29ndash31]

The clinical data concerning the role of Mdm2 SNIP309 ingastric cancer development is limited A case-control studyamong the Iranian gastric cancer population showed thatMdm2 SNIP309 is a risk factor for this cancer with anodds ratio of 208 (95 confidence interval = 137ndash434) Thesame trend was observed in the Chinese [32 33] This studyaimed to investigate the characteristics ofH pylori associatedgastritis clarithromycin resistance Mdm2 polymorphismsand significant risk factors of H pylori associated gastritisamong theThai population

2 Materials and Methods

21 Patients Three hundred patients undergoing esopha-gogastroduodenoscopy for investigation of dyspeptic symp-toms participated in this study from June 2014 to June 2015The following exclusion criteria were applied age below18 or above 70 years old previous H pylori eradicationtreatment prior to the previous 2 months significant medicalillnesses history of previous gastric surgery and the use ofantimicrobials or gastrointestinal medications like PPIs orbismuth compounds within the previous 2monthsThe studywas performed in accordance with good clinical practice andthe guidelines of theDeclaration ofHelsinki All patients pro-vided written informed consent and the study protocol wasapproved by the Ethics Committee for Research InvolvingHuman Subjects Suranaree University of Technology (EC-57-22 and EC-57-34)

22 Diagnosis of H pylori Associated Gastritis A diagnosis ofH pylori associated gastritis was made if H pylori were seenon histopathological examination and the rapid urease testwas positive Finally we proved bacterial infection by PCR

23 Biopsy Specimens Biopsy was done according to theUpdated Sydney classification [34] which indicates samplingfrom 5 biopsy sites Each specimen was obtained from eachof the following locations the lesser curvature of the corpusabout 4 cm proximal to the angularis (1) the lesser curvature(2) and the greater curvature of the antrum (3) both within2 to 3 cm of the pylorus the middle portion of the greatercurvature of the corpus approximately 8 cm from the cardia(4) and the incisura angularis (5)

24 Esophagogastroduodenoscopy (EGD) Local anesthesiawas the same as that for conventional gastroscopy Thegastroscopic procedures were performed using an upperGI video endoscope (Olympus EVIS EXERA III CV-190)The whole stomach was examined first with conventionalendoscopy and then biopsies were performed A symptomquestionnaire (abdominal pain vomiting diarrhea gastroin-testinal bleeding and iron deficiency anemia) was completedby the patient at the time of initial EGD in the endoscopyroom (timeout)

25 Histological Analysis Gastric tissue specimens for his-tological analysis were sent to the pathologists The hema-toxylin and eosin stain and Giemsa stain were used foridentification ofH pyloriThepathological analysis wasmade









2(25mM) forward











3 Results









0448

0398

0348

0298

0248

0198

0148

0098

0048

minus0002

minus0052

46 48 50 52 54 56 58 60 62 64 66 68 70 72 74

Temperature (∘C)

minus(ddT)

fluor

esce

nce (

498

ndash640

)


(A2143A2142GC)


type

Melting peaks














1500016000

1300014000

1100010000

12000

9000

70008000

50006000

30004000

10002000Fl

uore

scen

ce (4

65

ndash510

)






1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000




4 Discussion






















Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























2(25mM) forward











3 Results









0448

0398

0348

0298

0248

0198

0148

0098

0048

minus0002

minus0052

46 48 50 52 54 56 58 60 62 64 66 68 70 72 74

Temperature (∘C)

minus(ddT)

fluor

esce

nce (

498

ndash640

)


(A2143A2142GC)


type

Melting peaks














1500016000

1300014000

1100010000

12000

9000

70008000

50006000

30004000

10002000Fl

uore

scen

ce (4

65

ndash510

)






1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000




4 Discussion






















Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results









0448

0398

0348

0298

0248

0198

0148

0098

0048

minus0002

minus0052

46 48 50 52 54 56 58 60 62 64 66 68 70 72 74

Temperature (∘C)

minus(ddT)

fluor

esce

nce (

498

ndash640

)


(A2143A2142GC)


type

Melting peaks














1500016000

1300014000

1100010000

12000

9000

70008000

50006000

30004000

10002000Fl

uore

scen

ce (4

65

ndash510

)






1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000




4 Discussion






















Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























1500016000

1300014000

1100010000

12000

9000

70008000

50006000

30004000

10002000Fl

uore

scen

ce (4

65

ndash510

)






1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000




4 Discussion






















Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Characteristics and Risk Factors of Helicobacter...

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