helicobacter pylori: infection and new perspective for the...

Canadian Journal of Infectious Diseases and Medical Microbiology

Helicobacter pylori Infection and New Perspective for the Treatment

Lead Guest Editor Teresa FascianaGuest Editors Mara Di Giulio Paola Di Carlo and Ahlem Jouini

Helicobacter pylori Infection andNew Perspective for the Treatment

Canadian Journal of Infectious Diseasesand Medical Microbiology



Copyright copy 2019 Hindawi All rights reserved

This is a special issue published in ldquoCanadian Journal of Infectious Diseases and Medical Microbiologyrdquo All articles are open access ar-ticles distributed under the Creative Commons Attribution License which permits unrestricted use distribution and reproduction inany medium provided the original work is properly cited

Editorial Board

Sophie Alain FranceLuis C M Antunes BrazilChristian Bautista USAJoseacute Ramoacuten Blanco SpainFranco Maria Buonaguro ItalyElisabetta Caselli ItalyKapil Chousalkar AustraliaGianni Colotti ItalyMaria De Francesco ItalyMario DellrsquoAgli ItalyLouis DeTolla USAMarco Di Luca ItalyGiordano Dicuonzo ItalySong Z Ding ChinaAlessandra Falchi FranceNahuel Fittipaldi CanadaDaniele Focosi ItalyLetiacutecia Gastatildeo Franco SpainJorge Garbino SwazilandSandra Gemma ItalyCaroline Gilbert CanadaVladimir Gilca Canada

Massimo Girardis ItalyLaura Gragnani ItalyZitta Barrella Harboe DenmarkAim Hoepelman NetherlandsChristine A Hughes CanadaMatthias Karrasch GermanyYoav Keynan CanadaJohn E Kim CanadaPeter Kima USAKenneth Komatsu USABartheacuteleacutemy Kuate Defo CanadaLucia Lopalco ItalyCinzia Marianelli ItalyFrancesca Mariani ItalyAnandi Martin BelgiumFederico Martinoacuten-Torres SpainPietro Mastroeni UKClaudio M Mastroianni ItalyGabriele Messina ItalyJoseacute A Oteo SpainRosa Polo SpainBruno Pozzetto France

Leticia Reyes USAMaria Luisa Ricci ItalySigrid Roberts USANadine Rouphael USAMassimo Sartelli ItalyJoseacute Mariacutea Saugar SpainBassel E Sawaya USARoshanak Tolouei Semnani USALeigh Anne Shafer CanadaMichael Silverman CanadaTzanko Stantchev USAPascal Thibon FranceMaria Lina Tornesello ItalyAlice Tseng CanadaJulia Uhanova CanadaVidula Vachharajani USAMichel T Vaillant LuxembourgHendrik K Van Saene UKStefano Veraldi ItalyPeter J Weina USAWendy L Wobeser Canada

Contents

Helicobacter pylori Infection and New Perspective for the TreatmentTeresa Fasciana Paola Di Carlo Ahlem Jouini and Mara Di GiulioEditorial (2 pages) Article ID 9431369 Volume 2019 (2019)

Rifabutin and Furazolidone Could Be the Candidates of the Rescue Regimen for Antibiotic-ResistantH pylori in KoreaYoun I Choi Sang-Ho Jeong Jun-Won Chung Dong Kyun Park Kyoung Oh KimKwang An Kwon Yoon Jae Kim Seol So Jeong Hoon Lee Jin-Young Jeong and Sun-Mi LeeResearch Article (7 pages) Article ID 9351801 Volume 2019 (2019)

Immunization with a SyntheticHelicobacter pylori Peptide Induces Secretory IgA Antibodies andProtects Mice against InfectionDavid Espinosa-Ramos Diana Caballero-Hernaacutendez Ricardo Gomez-Flores Armando Trejo-Chaacutevez Luis Jeroacutenimo Peacuterez-Limoacuten Myriam Angeacutelica de la Garza-Ramos Reyes Tamez-Guerra Patricia Tamez-Guerra and Cristina Rodriguez-PadillaResearch Article (8 pages) Article ID 8595487 Volume 2019 (2019)

Application of PCR andMicroscopy to Detect Helicobacter pylori in Gastric Biopsy Specimen amongAcid Peptic Disorders at Tertiary Care Centre in Eastern NepalNayanum Pokhrel Basudha Khanal Keshav Rai Manish Subedi and Narayan Raj BhattaraiResearch Article (6 pages) Article ID 3695307 Volume 2019 (2019)

Estimating the Force of Infection withHelicobacter pylori in JapanTaishi Kayano Ki-Deok Lee and Hiroshi NishiuraResearch Article (7 pages) Article ID 1451490 Volume 2019 (2019)

Helicobacter pylori Infection and Its Risk Factors A Prospective Cross-Sectional Study inResource-Limited Settings of Northwest EthiopiaMarkos Negash Habtamu Wondifraw Baynes and Demeke GeremewResearch Article (7 pages) Article ID 9463710 Volume 2018 (2019)

EditorialHelicobacter pylori Infection and New Perspective forthe Treatment

Teresa Fasciana 1 Paola Di Carlo 1 Ahlem Jouini 2 and Mara Di Giulio 3

1Department of Health Promotion Mother and Child Care Internal Medicine and Medical Specialties University of PalermoPalermo Italy2Laboratory of Epidemiology and Veterinary Microbiology Group of Bacteriology and Biotechnology DevelopmentPasteur Institute of Tunis Universite de Tunis El Manar Tunis Tunisia3Department of Pharmacy ldquoG drsquoAnnunziordquo University Chieti-Pescara Chieti Italy

Correspondence should be addressed to Teresa Fasciana teresafascianavirgilioit

Received 17 July 2019 Accepted 17 July 2019 Published 29 July 2019

Copyright copy 2019 Teresa Fasciana et al is 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 isproperly cited

e alarming phenomenon of antibiotic resistance in Hel-icobacter pylori suggests to pay close attention in thetreatment In particular the clarithromycin resistance in Hpylori was designed by the World Health Organization(WHO) as high priority for antibiotic research in 2017 [1] InItaly the overall primary resistance to clarithromycin can bedetected in 352 of cases in France it can be detected in26 of cases while in Spain clarithromycin resistance ispresent in 272 of strains [2ndash4]

Naturalinnovative strategies as well as novel potenti-ators to restore the antibiotic susceptibility in resistantstrains could be used to improve the efficacy of H pylorieradication and they could be worthy to change attitude ofmedicine in dealing with the challenge known as H pylori[5 6]

Obviously these therapeutic strategies should be used inpatients infected by resistant H pylori and in presence ofcoinfection with other pathogens responsible to developsevere gastric diseases [7]

On the basis of this evidence the aim of this special issuewas to collect research manuscripts and review manuscriptscase reports and literature reviews with the objective toexpand our knowledge in this innovative field

In this special issue a total of seven manuscripts werereceived and five of these were accepted

is issue confirmed that the prevalence of H pylori indeveloping countries is high because the population lives inhouseholds with low socioeconomic status and hygiene

erefore in order to improve the diagnostic accuracyshould be recommended the combination of microscopyand PCR assay for effective monitoring ofH pylori infectionin these endemic areas PCR is a more sensitive assay todetect H pylori infection than microscopy and it is not yetconsidered as the gold standard assay

Using a mathematical model and the force of infection itis possible achieved to translate the decreasing pattern intothe time-dependent decline in the hazard rate of infectionand also permitted the future prediction of seroprevalence inareas with high prevalence of infection Moreover the modelcould be used to predict the future size of gastric cancer

In experimental studies it has been observed that the useof 52 kDa H pylori membrane peptide as a vaccine has beeneffective to immunize against the development of gastric ulcerwhen used in murine models However the isolation andpurification of such protein presents important challengestherefore the use of synthetic peptides designed from im-munogenic proteins has become an alternative for diagnosisand prophylaxis SinceH pylori causes superficial infection ofthe gastric tissue the main immunity mediators are secretoryIgA antibodies which are the objective of active oral vacci-nation Immunized animals produce specific serum IgG andIgA and intestinal and salivary IgAs and after challenge agastric cellular and antibody response can be observed Oneimmunogen-derived peptide antigen of 50ndash52 kDa with theamino-terminal end sequence Met-Val-r-Leu-Ile-Asn-Asn-Glu (MVTLINNE) produced by H pylori could be used

HindawiCanadian Journal of Infectious Diseases and Medical MicrobiologyVolume 2019 Article ID 9431369 2 pageshttpsdoiorg10115520199431369

to the prophylaxis of its infection e results showed thatimmunization with the MVTLINNE peptide stimulated thecellular immune response and increased proliferative re-sponse of thymus lymphocytes In addition MVTLINNEpeptide vaccination-mediated IgA production correlated withno alterations in the gastric mucosa and scarce presence ofbacilli after H pylori infection In conclusion these resultsindicated that prophylactic immunization significantly re-duced the number of colonizing bacteria which was asso-ciated with healthy gastric tissue

In the era with high percentage of resistance to clari-thromycin rifabutin furazolidone and tetracycline alone orin combination are promising candidates for rescue therapyof antibiotic-resistant H pylori strains as no definitiverescue therapy for H pylori eradication is available

In our opinion this first year of this special issueattracted interest in a field that is growing development andwe hope that the information contained in this special issuewill help to develop new strategies to preventtreat the Hpylori infection

Conflicts of Interest

e editors declare that they have no conflicts of interest

Teresa FascianaPaola Di CarloAhlem Jouini

Mara Di Giulio

References

[1] A Savoldi E Carrara D Y Graham M Conti andE Tacconelli ldquoPrevalence of antibiotic resistance in Heli-cobacter pylori a systematic review and meta-analysis inWorldHealth organization regionsrdquo Gastroenterology vol 155 no 5pp 1372ndash1382 2018

[2] T Fasciana G Scarpulla A Giammanco et al ldquoResistance toclarithromycin and genotypes in Helicobacter pylori strainsisolated in Sicilyrdquo Journal of Medical Microbiology vol 64no 11 pp 1408ndash1414 2015

[3] S Agudo G Perez-Perez T Alarcon andM Lopez-Brea ldquoHighprevalence of clarithromycin-resistantHelicobacter pylori strainsand risk factors associated with resistance in madrid SpainrdquoJournal of Clinical Microbiology vol 48 no 10 pp 3703ndash37072010

[4] J Raymond D Lamarque N Kalach S Chaussade andC Burucoa ldquoHigh level of antimicrobial resistance in FrenchHelicobacter pylori isolatesrdquo Helicobacter vol 15 no 1pp 21ndash27 2010

[5] A M Nasrat S A Nasrat R M Nasrat and M M Nasrat ldquoAcomparative study of natural eradication of Helicobacter pylorivs antibioticsrdquo General Medicine Open Access vol S1pp S1ndashS001 2015

[6] A F Ciccaglione M Di Giulio S Di Lodovico E Di CampliL Cellini and L Marzio ldquoBovine lactoferrin enhances theefficacy of levofloxacin-based triple therapy as first-linetreatment of Helicobacter pylori infection an in vitro and invivo studyrdquo Journal of Antimicrobial Chemotherapy vol 74no 4 pp 1069ndash1077 2019

[7] T Fasciana G Capra C Cala et al ldquoHelicobacter pylori andEpsteinndashBarr co-infection in gastric diseaserdquo PharmacologyOnline vol 1 pp 73ndash82 2017

2 Canadian Journal of Infectious Diseases and Medical Microbiology

Research ArticleRifabutin andFurazolidoneCouldBe theCandidates of theRescueRegimen for Antibiotic-Resistant H pylori in Korea

Youn I Choi 1 Sang-Ho Jeong1 Jun-Won Chung 1 Dong Kyun Park 1

Kyoung Oh Kim1 Kwang An Kwon1 Yoon Jae Kim1 Seol So2 Jeong Hoon Lee 2

Jin-Young Jeong3 and Sun-Mi Lee3

1Department of Gastroenterology Gil Medical Center Gachon University Incheon Republic of Korea2Department of Gastroenterology Asan Medical Center Ulsan University Seoul Republic of Korea3Asan Institute for Life Sciences Asan Medical Center University of Ulsan College of Medicine Seoul 138-736 Republic of Korea

Correspondence should be addressed to Jun-Won Chung drgreengilhospitalcom

Received 1 April 2019 Revised 20 June 2019 Accepted 24 June 2019 Published 10 July 2019

Guest Editor Teresa Fasciana

Copyright copy 2019 Youn I Choi et al is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

BackgroundAim In Korea the rate of Helicobacter pylori (H pylori) eradication has declined steadily as a result of increasingresistance to antibiotics especially dual resistance to clarithromycin and metronidazole However microbiological culture dataon drug-resistant H pylori is lacking is study evaluated the antimicrobial efficacy of candidate antibiotics against resistantH pylori strainsMethods After retrospectively reviewing the data from theHelicobacter Registry in Gil Medical Center (GMC)and Asan Medical Center (AMC) along with 4 reference strains we selected the 31 single- or multidrug-resistant strains esusceptibility of theH pylori strains to seven antibiotics (clarithromycin metronidazole levofloxacin amoxicillin tetracyclinerifabutin and furazolidone) and minimum inhibitory concentration were tested using the broth microdilution techniqueResults Among 31 antibiotic resistance strains for H pylori there were no strains resistant to rifabutin or furazolidone whichhad MICs of lt0008 and 05 μgmL respectively Only one tetracycline-resistant strain was found (MIC lt 2 μgmL)Amoxicillin and levofloxacin were relatively less effective against the H pylori strains compared to rifabutin or furazolidone(resistance rates 226 19 respectively) Tetracycline showed the relatively low resistance rates (32) for H pylori strainsConclusions erefore along with tetracycline which has already been used as a component for second-line eradicationregimen forHelicobacter rifabutin and furazolidone alone or in combination could be used to eradicate antibiotic-resistantHpylori strains where drug-resistant Helicobacter spp are increasing

1 Introduction

Helicobacter pylori infection is responsible for the devel-opment of chronic atrophic gastritis peptic ulcer diseaseand gastric malignant neoplasms such as gastric adeno-carcinoma and mucosa-associated lymphoid tissue lym-phoma [1 2] H pylori is recognized as a Class I carcinogenby the International Agency for Research on Cancer and theWorld Health Organization [1ndash3] e eradication of an-tibiotic-resistant H pylori is a global health issue [4]

However multidrug-resistant (MDR) strains of Hpylori have been increasing worldwide due to the increaseduse of antibiotics [1 2 4ndash9] In Korea the rate of H pylori

eradication has declined steadily in recent decades as aresult of increasing resistance to antibiotics especially dualresistance to clarithromycin and metronidazole [10ndash12]which has resulted from the increased clinical use ofmacrolides and metronidazole [13 14] In Korea includingIncheon and Seoul where this study was conducted the rateof resistance of H pylori to clarithromycin has surpassed15 [15ndash17] Several reports suggest that 96 of the strainsin Korea show dual resistance to clarithromycin andmetronidazole [7] Because the primary failure rate of Hpylori eradication has been increasing [12 13 18 19] real-world antimicrobial resistance data are needed to improvetherapeutic outcomes However there are little recent data


on in vitro antimicrobial effectiveness in Korea Indeedthere is no consensus on the optimal rescue therapy forsecond-line eradication failure Although the Maastricht Vconsensus recommended fluoroquinolone-containingtherapy as first- or second-line treatment after failure oftriple or nonbismuth quadruple therapy this cannot beapplied in Korea because of the increased rate of quinoloneresistance [20] erefore we conducted this real-worldupdated analysis of the in vitro antibacterial efficacy againstMDR H pylori

e 2013 revision of the Korean Clinical PracticeGuideline for H pylori recommends triple therapy with aproton pump inhibitor (PPI) amoxicillin and clari-thromycin or a bismuth-based quadruple regimen if clari-thromycin resistance is suspected [10 21]With the failure offirst-line therapy bismuth-based quadruple therapy or aregimen including two or more other antibiotics could beconsidered [21] Although levofloxacin- and rifabutin-basedtriple therapy have been suggested for rescue therapy thereis no consensus on their use in Korea erefore it isnecessary to identify antibiotics effective against antibiotic-resistantH pylori [22] eMaastricht VFlorence guidelinerecommends culturing H pylori testing for antimicrobialsusceptibility and selecting antibiotics based on the results ofresistance tests [23] However there are limited data onantimicrobial agents that are effective against antibiotic-resistant H pylori in Korea

erefore this study investigated the antimicrobial ac-tivity of rifabutin furazolidone and other antibacterialagents as candidates for treating antibiotic-resistantH pyloristrains especially focusing on the multidrug-resistantH pylori

2 Patients and Methods

21 Institutional Review Board Approval e InstitutionalReview Boards of Gil Medical Center (GMC) and AsanMedical Center (AMC) reviewed the study protocol (cer-tification number GAIRB2016-329)

22 Patient Characteristics is study examined 4 refer-ence strains and 31 strains isolated from patients at GMC(n 15) and AMC (n 16) in 2016 We retrospectivelyreviewed the data of cultures for Helicobacter pylori (Hpylori) up to 2016 from Helicobacter pylori Registry inGMC and AMC We analyzed and tested the candidatehelicobacter antibiotics including amoxicillin clari-thromycin metronidazole levofloxacin and tetracyclinefrom the strains ofH pylori Patientsrsquo clinical data such asinitial presentation of symptoms reasons for endoscopyantibiotics uses history and reasons for cultures of Hpylori were retrospectively reviewed in GMC and AMCCulture reasons for Helicobacter spp for patients were asfollows (1) patients who have reported several antibioticsexperiences or admission to hospital histories in 3 years(2) first-line or second-line treatment failures and (3)other clinically suspected medical condition of drugresistance such as patients with old age more than

65 years who have had more chance to exposure intoseveral antibiotics or patients with severe comorbidconditions such as congestive heart failure liver cir-rhosis renal failure autoimmune disorders pulmonarydisease and so on

23H pylori Strain Isolation Mucosal tissues collected fromthe gastric antrum of each patient were used to isolateH pylori To isolate the bacteria the tissues were placed in anaseptic Petri dish then crushed using a surgical knife andcultivated in Brucella broth agar and supplemented with5 sheep blood containing vancomycin (10 μgmL) tri-methoprim (5 μgmL) amphotericin B (5 μgmL) andpolymyxin B (25 IU) ese were cultured at 37degC undermicroventilation conditions (5O2 10 CO2 and 85N2)e colonies obtained from the initial cultures were con-firmed to be H pylori using Gram staining and biochemicalmethods Each strain identified as H pylori was stored atminus70degC in Brucella liquid medium (Difco LaboratoriesDetroit MI USA) containing 15 glycerol Shortly beforethe subsequent experiments they were melted multipliedand used

24 H pylori Antimicrobial Susceptibility Testing eminimum inhibitory concentrations (MICs) of the fol-lowing antimicrobial agents were tested clarithromycin(Abbott Laboratories Abbott Park IL USA) amoxicillinmetronidazole tetracycline levofloxacin rifabutin andfurazolidone (all from Sigma Chemical Co St Louis MOUSA)

25 Culture Conditions To test the MICs of H pylori weused the agar dilution method recommended by theClinical and Laboratory Standards Institute (CLSI) [24]an internationally recognized antimicrobial suscepti-bility testing laboratory using MuellerndashHinton agar(Difco Laboratories Detroit MI USA) supplementedwith 5 defibrinated sheep blood e medium wassterilized by autoclaving and each antimicrobial agentwas serially diluted in medium supplemented with 5sheep blood (Comed Seoul Korea) which was collectedwithin 2 weeks of birth and cooled to 80degC en thesuspension of H pylori strains (1 times 107 colony-formingunits) was cultured in blood culture medium for 72 hoursand inoculated on MuellerndashHinton agar containing anantimicrobial agent using a micropipette is was in-cubated at 37degC for 3 days under microventilation con-ditions and the presence of bacterial colonies wasobserved Each experiment was performed in triplicateand experiments were repeated at least three times perstrain

26 Antimicrobial Resistance Criteria e MIC was definedas the minimum dilution concentration of the antimicrobialagent that did not produce bacterial colonies e criterionfor resistance to each antimicrobial agent was set to MICgt1 μgmL as given in the CLSI for resistance to


clarithromycin [24 25] e resistance criteria for antimi-crobial agents were set to greater than 05 μgmL foramoxicillin [7] 8mgmL for metronidazole [7] 4 μgmL fortetracycline [7] 1 μgmL for levofloxacin antibiotics [26]025 μgmL for rifabutin [7 27] and 4 μgmL for furazoli-done [7 28] Resistance to two or more antimicrobials wasdefined as multidrug resistance (MDR) [29] For qualitycontrol H pylori strain (ATCC 43504) which is used as astandard strain in CLSI was selected [24]

3 Results

31 Characteristics of the Study Population e mean age ofthe study population was 582plusmn 103 years and 419(n 13) were more than 65 years Reasons for endoscopy ofstudy populations were as follows (1) patients who receivedendoscopy for routine health checkup (n 15) or (2) pa-tients with gastrointestinal symptoms such as dyspepsiaregurgitation or pain (n 16) e most common reasonfor eradication was peptic ulcer disease (n 15 484)(Table 1)

32 Characteristics of the Isolated Strains of H pylori Of the31 isolated strains 9 were resistant to one antimicrobialagent and 22 (710) were resistant to two or more anti-microbial agents including 13 strains resistant to twoantibiotics (419) seven strains resistant to three anti-biotics (226) and two strains resistant to four antibiotics(64) e most common combination of drug resistancewas clarithromycin +metronidazole (16 strains 516)(Table 2)

Of the 31 strains 1 (32) was resistant to tetracyclineand none were resistant to rifabutin or furazolidone(Table 3)

33 Clarithromycin MIC and Resistance in H pylori Strainse range of MICs for clarithromycin was very broad from003 to gt128 μgmL Overall the MICs of the 31 strains hadtwo distinct peaks (Figure 1) with MIClt 00625 μgmL in371 of the isolates and very high MICs in others (eg 1632 64 and 128 μgmL) e CLSI criterion for clari-thromycin resistance is MIC gt1 μgmL [24] Of the 31 strainstested 22 were resistant (711) according to this criterion(Table 3)

34 Metronidazole MIC and Resistance in H pylori Strainse MIC for metronidazole ranged from 1 to 128 μgmL(Figure 1) e resistance standard for metronidazole wasestablished to exceed 8 μgmL which is normally usedwithout established criteria [24] e resistance rateaccording to this standard was 677 (2131)

35 Simultaneous Clarithromycin and Metronidazole Re-sistance in H pylori Strains Of the 31 strains studied 22showed MDR and 16 strains (516) were resistant to bothclarithromycin and metronidazole accounting for 64 of all

MDR strains Rifabutin and furazolidone had excellentantibacterial activity with no resistant strains

36 Quinolone MIC and Resistance in H pylori StrainseMIC for levofloxacin ranged from 025 to 64 μgmLecriterion for bacterial resistance to quinolone antibiotics is

Table 1 e demographic characteristics of the patient withH pylori strain (N 31)

Characteristics N ()Age meanplusmn SD (years) 582plusmn 103

Age gt65 years N () 13 (419)Men N () 16 (516)Smoking N () 6 (193)Drinking N () 12 (387)Comorbidity

Diabetes mellitus type 2 2 (64)Hypertension 8 (258)Liver cirrhosis 1 (32)Cerebrovascular disorders 1 (32)yroid cancer 1 (32)Idiopathic pulmonary fibrosis 1 (32)Pulmonary tuberculosis 1 (32)

Reasons for Helicobacter spp culturessect

First-line or second-line empirical treatmentfailure 9 (290)

Patientsrsquo reported antibiotic uses history in 3 years 15 (484)Other clinically suspected medical condition of

drug resistancepara 13 (419)

Reason for eradication for H pyloriPeptic ulcer disease 15 (484)Early gastric cancer 2 (65)MALToma 4 (129)Atrophic gastritis 10 (323)

paraOther clinically suspected medical conditions of drug resistance patientswith old age more than 65 years who have hadmore chance to exposure intoseveral antibiotics or patients with severe comorbid conditions such ascongestive heart failure liver cirrhosis renal failure autoimmune disorderspulmonary disease and so on sectTotal sum of population is not 100 sinceduplication cases NSAID nonsteroidal anti-inflammatory drug HTNhypertension PUD peptic ulcer disease EGC early gastric cancerMALToma mucosa-associated lymphoid tissue lymphoma

Table 2 Drug resistance of H pylori isolates (N 31)

No of resistantantibiotics

Types of drugresistance

No of strains()

1CLR 3 (97)MT 3 (97)LVX 3 (97)

2AMX+CLM 2 (65)AMX+LVX 1 (32)CLM+MET 10 (323)

3CLM+MET+LVX 4 (129)AMX+MET+LVX 2 (65)AMX+CLM+LVX 1 (32)

4 CLM+MET+TET+LVX 1 (32)AMX+CLM+MET+LVX 1 (32)

Total mdash 31AMX amoxicillin CLM clarithromycin MET metronidazole TET tet-racycline LVX levofloxacin

Canadian Journal of Infectious Diseases and Medical Microbiology 3

MIC gt1 μgmL [24] Using this standard 419 (1331) ofthe strains were resistant to levofloxacin (Table 3)

37 Tetracycline MIC and Resistance in H pylori Strainse MIC for tetracycline ranged from lt003 to 2 μgmL(Figure 1) Only 1 of the 31 strains was resistant to tetracycline

38 Rifabutin and Furazolidone MIC and Resistance inH pylori Strains e MIC for rifabutin ranged fromlt000098 to 00078 μgmL and that of furazolidone fromlt003 to 05 μgmL (Figure 1) e criteria for resistance areMIC gt025 μgmL for rifabutin and MIC gt4 μgmL for fu-razolidone None of the 31 strains was resistant to eitherdrug

Table 3 Prevalence of antibiotic resistance of H pylori isolates

Resistant breakpoint of MIC (μgmL) No of resistant strainsTotal strains Resistance rate ()Clarithromycin gt1 2231 711Metronidazole gt8 2131 677Levofloxacin gt1 1331 419Amoxicillin gt05 731 226Tetracycline gt4 131 32Rifabutin gt025 031 0Furazolidone gt4 031 0MIC minimum inhibitory concentration

0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128

MIC of clarithromycin (μgmL)

(a)

0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128

MIC of metronidazole (μgmL)

(b)

o

f H p

ylor

i iso

late

s

0

20

40

60

80

100

120

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128

MIC of rifabutin (μgmL)

(c)

o

f H p

ylor

i iso

late

s

0

10

20

30

40

50

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128

MIC of furazolidone (μgmL)

(d)

Figure 1 MIC distribution of (a) clarithromycin (b) metronidazole (c) rifabutin and (d) furazolidone for H pylori MIC minimuminhibitory concentration


4 Discussion

In this in vitro analysis of antimicrobial effectiveness weaimed to investigate which of the antibiotics were effectivefor multidrug resistance H pylori and found that rifabutinand furazolidone had excellent potential for eradicating notonly single-drug-resistant H pylori but also MDR H pylorithrough culture-based data in Korea No strains were re-sistant to rifabutin or furazolidone which had very lowMICs of lt000098 and 05 μgmL respectively for allstrains Tetracycline also had low MICs which were lt2 μgmL for all but one resistant strain erefore along withtetracycline which has already been used as a componentfor second-line eradication regimen for Helicobacterrifabutin and furazolidone alone or in combination couldbe used to eradicate antibiotic-resistantH pylori strains Incomparison amoxicillin and levofloxacin were only par-tially effective against the H pylori strains in this in vitrostudy

To our knowledge this is the first in vitro antimicrobialanalysis of antibiotics candidate rifabutin and furazolidonein MDR H pylori in Korea where clarithromycin resistancerate exceeds 15

Studies have explored several antibiotics as rescuetherapy following the failure of first- and second-linetreatment in Korea Sung et al evaluated the efficacy ofrifabutin-based rescue therapy among patients with third- fourth- or fifth-line eradication failure [30] Rifabutin-based rescue therapy had an approximately 55 eradi-cation rate with few side effects Jeong et al reported thatrifabutin-based therapy eradicated over 70 of H pyloriin third-line rescue therapy in 21 patients [31] eremight be several reasons for the variation in the eradi-cation rate of rifabutin-based therapy in Korea First thesample sizes of these studies were small Second becauseH pylori eradication failure is diagnosed using the rapidurease test after treatment the causes of eradicationfailure were unclear Major causes of eradication failureapart from drug-resistant H pylori are loss of compliancewith treatment the density of H pylori in the stomachwall presence of CagA and smoking In our in vitroantibacterial efficacy analysis of drug-resistant H pyloririfabutin showed excellent antimicrobial activity in MDRH pylori To our knowledge this is the first in vitroanalysis of rifabutin in MDR H pylori Given the highrates of tuberculosis infection and antituberculosismedication use in Korea the low rate of rifabutin-resistantHelicobacter spp is interesting Before selecting rifabutinas rescue therapy in Korea clinicians should carefullymonitor its major side effects including rare myelosup-pressive events [27 30 32ndash34] strictly limit its use toconfirmed eradication cases and monitor patient com-pliance closely to avoid the development of rifamycin-resistant tuberculosis [32]

Another option for third-line rescue therapy in Koreais furazolidone In a meta-analysis Mohammadi et alreported an H pylori eradication rate exceeding 80 and alow rate of side effects in Iran where MDR H pylori iscommon [35] In a multicenter randomized controlled

trial in China where MDR H pylori is also common Xieet al reported an eradication rate with furazolidone of upto 90 in 720 patients with H pylori [36] Despite limiteddata on furazolidone as a Helicobacter spp treatment inKorea Kim et al reported a 15 resistance rate in first-line treatment failure patients [37] However their studywas conducted in 2001 and recent data on furazolidone forHelicobacter spp eradication in Korea are not availableBecause the antibacterial resistance rate differs amongcountries large multicenter population-based studies areneeded in Korea Our study showed that furazolidone hasan extremely low rate of resistance in drug-resistant Hpylori in vitro

Tetracycline is one component of bismuth-based qua-druple therapy (PPI bismuth metronidazole and tetracy-cline) which is effective for Helicobacter spp eradicationespecially in areas with high levels of clarithromycin re-sistance [13 38] In our in vitro study only one strain ofdrug-resistant H pylori was resistant to tetracycline sug-gesting that tetracycline is still effective for drug-resistant Hpylori eradication in Korea

is study had several limitations First it did notconfirm the eradication rate by actually treating the patientsso it is impossible to know how the in vitro results willcorrespond to in vivo effects Further studies need to confirmthe eradication rate of furazolidone and rifabutin and safetyin actual patients Second because we studied antibiotic-resistant H pylori strains that were selected randomly ourresult might not reflect the general prevalence of antibiotic-resistant H pylori in Korea selection bias could be an issueNevertheless this was the first study of the effectiveness ofrifabutin and furazolidone in Korea thoughH pylori culturedata and almost all of the antibiotics used in clinical practicefor H pylori eradication in Korean were covered irdbecause the antibiotic resistance data for H pylori wererelatively small in this study it should be cautious forphysicians to generalize these results to a general populationor other ethics Given that the cost and time to obtain resultsof MIC for each antibiotic from culture data of Helicobacterspp it might be important to invent and use molecularmethods to evaluate the resistance of drugs directly in bi-opsies samples when it is impossible to isolate the strains[39]

In conclusion this study showed that rifabutin fura-zolidone and tetracycline alone or in combination arepromising candidates for rescue therapy of antibiotic-re-sistant H pylori strains as no definitive rescue therapy forH pylori eradication is available A future eradicationregimen could potentially be designed based on theseresults

Data Availability

e data used to support the findings of this study areavailable from the corresponding author upon request


e authors declare that they have no conflicts of interest


Authorsrsquo Contributions

Youn I Choi and Sang-Ho Jeong contributed equally to thiswork Dr Youn I Choi Sang-Ho Jeong Dong Kyun Parkand Jun Won Chung contributed to the study concept anddesign Dr Youn I Choi Seol So Jeong Hoon Lee Jin-Young Jeong and Sun-Mi Lee analyzed and interpreted thedata Youn I Choi drafted the manuscript and Kyoung OhKim Kwang An Kwon and Yoon Jae Kim critically revisedthe manuscript for important intellectual content All au-thors approved the final version of the manuscript

References

[1] A Savoldi E Carrara D Y Graham M Conti andE Tacconelli ldquoPrevalence of antibiotic resistance in Heli-cobacter pylori a systematic review and meta-analysis inworld health organization regionsrdquoGastroenterology vol 155no 5 pp 1372e17ndash1382e17 2018

[2] M Kekilli I Onal S Ocal Z Dogan and A Tanoglu ldquoIn-efficacy of triple therapy and comparison of two differentbismuth-containing quadruple regimens as a firstline treat-ment option for Helicobacter pylorirdquo Saudi Journal of Gas-troenterology vol 22 no 5 pp 366ndash369 2016

[3] T C G Fasciana C Capra S Zambuto et al ldquoHelicobacterpylori and epstein-barr co-infection in gastric diseaserdquoPharmacologyonline vol 1 pp 73ndash82 2017

[4] R Pellicano R M Zagari S Zhang G M Saracco andS F Moss ldquoPharmacological considerations and step-by-stepproposal for the treatment of Helicobacter pylori infection inthe year 2018rdquo Minerva Gastroenterologica e Dietologicavol 64 no 3 pp 310ndash321 2018

[5] Y-T Kuo J-M Liou E M El-Omar et al ldquoPrimary anti-biotic resistance in Helicobacter pylori in the asia-pacific re-gion a systematic review and meta-analysisrdquo LancetGastroenterology amp Hepatology vol 2 no 10 pp 707ndash7152017

[6] Y Hu Y Zhu and N-H Lu ldquoPrimary antibiotic resistance ofHelicobacter pylori in Chinardquo Digestive Diseases and Sciencesvol 62 no 5 pp 1146ndash1154 2017

[7] JW Chung S Y Kim H J Park C S Chung HW Lee andS M Lee ldquoIn vitro activity of diphenyleneiodonium towardmultidrug-resistant Helicobacter pylori strainsrdquo Gut andLiver vol 11 no 5 pp 648ndash654 2017

[8] P Malfertheiner F Megraud C A OrsquoMorain et al ldquoMan-agement of Helicobacter pylori infection-the maastricht vflorence consensus reportrdquo Gut vol 66 no 1 pp 6ndash30 2017

[9] T Fasciana C Cala C Bonura E Di Carlo D Matranga andG Scarpulla ldquoResistance to clarithromycin and genotypes inHelicobacter pylori strains isolated in Sicilyrdquo Journal ofMedical Microbiology vol 64 no 11 pp 1408ndash1414 2015

[10] SW Lee H J Kim and J G Kim ldquoTreatment ofHelicobacterpylori infection in Korea a systematic review and meta-analysisrdquo Journal of Korean Medical Science vol 30 no 8pp 1001ndash1009 2015

[11] C Auesomwang M Maneerattanaporn W D CheyP Kiratisin S Leelakusolwong and T Tanwandee ldquoTen-dayhigh-dose proton pump inhibitor triple therapy versus se-quential therapy for Helicobacter pylori eradicationrdquo Journalof Gastroenterology and Hepatology vol 33 no 11pp 1822ndash1828 2018

[12] J S Kim B W Kim S J Hong J I Kim K N Shim andJ H Kim ldquoSequential therapy versus triple therapy for the

first line treatment of Helicobacter pylori in Korea a na-tionwide randomized trialrdquo Gut and Liver vol 10 no 4pp 556ndash561 2016

[13] J P Gisbert ldquoReview second-line rescue therapy of heli-cobacter pylori infectionrdquo 8erapeutic Advances in Gastro-enterology vol 2 no 6 pp 331ndash356 2009

[14] S W Ko Y J Kim W C Chung and S J Lee ldquoBismuthsupplements as the first-line regimen for Helicobacter pylorieradication therapy systemic review and meta-analysisrdquoHelicobacter vol 24 no 2 article e12565 2019

[15] J W Lee N Kim J M Kim R H Nam H Chang andJ Y Kim ldquoPrevalence of primary and secondary antimicrobialresistance of Helicobacter pylori in Korea from 2003 through2012rdquo Helicobacter vol 18 no 3 pp 206ndash214 2013

[16] J W Chung Y K Jung Y J Kim et al ldquoTen-day sequentialversus triple therapy for Helicobacter pylori eradication aprospective open-label randomized trialrdquo Journal of Gastro-enterology and Hepatology vol 27 no 11 pp 1675ndash1680 2012

[17] J W Chung G H Lee J Y Jeong S M Lee J H Jung andK D Choi ldquoResistance of Helicobacter pylori strains to an-tibiotics in Korea with a focus on fluoroquinolone resistancerdquoJournal of Gastroenterology and Hepatology vol 27 no 3pp 493ndash497 2012

[18] T Fujioka N Aoyama K Sakai Y Miwa M Kudo andJ Kawashima ldquoA large-scale nationwide multicenter pro-spective observational study of triple therapy using rabe-prazole amoxicillin and clarithromycin for Helicobacterpylori eradication in Japanrdquo Journal of Gastroenterologyvol 47 no 3 pp 276ndash283 2012

[19] W D Chey G I Leontiadis C W Howden and S F MossldquoACG clinical guideline treatment of Helicobacter pyloriinfectionrdquo American Journal of Gastroenterology vol 112no 2 pp 212ndash239 2017

[20] H Lee S N Hong B H Min J H Lee P L Rhee andY C Lee ldquoComparison of efficacy and safety of levofloxacin-containing versus standard sequential therapy in eradicationof Helicobacter pylori infection in Koreardquo Digestive and LiverDisease vol 47 no 2 pp 114ndash118 2015

[21] S G Kim H K Jung H L Lee J Y Jang H Lee andC G Kim ldquoGuidelines for the diagnosis and treatment ofHelicobacter pylori infection in Korea 2013 revised editionrdquoJournal of Gastroenterology and Hepatology vol 29 no 7pp 1371ndash1386 2014

[22] L Jonaitis R Pellicano and L Kupcinskas ldquoHelicobacterpylori and nonmalignant upper gastrointestinal diseasesrdquoHelicobacter vol 23 article e12522 2018

[23] H Suzuki and J Matsuzaki ldquoHelicobacter pylori eradicationfailure may have confounded the recent large-scale healthdatabase study that showed proton pump inhibitors increasegastric cancer riskrdquo Gut vol 67 no 11 pp 2071-2072 2018

[24] P D Midolo J M Bell J R Lambert J D Turnidge andM L Grayson ldquoAntimicrobial resistance testing of Heli-cobacter pylori a comparison of etest and disk diffusionmethodsrdquo Pathology vol 29 no 4 pp 411ndash414 1997

[25] M Heep D Beck E Bayerdorffer and N Lehn ldquoRifampinand rifabutin resistance mechanism in Helicobacter pylorirdquoAntimicrobial Agents and Chemotherapy vol 43 no 6pp 1497ndash1499 1999

[26] J K Akada M Shirai K Fujii K Okita and T Nakazawa ldquoInvitro anti-Helicobacter pylori activities of new rifamycin de-rivatives KRM-1648 and KRM-1657rdquo Antimicrobial Agentsand Chemotherapy vol 43 no 5 pp 1072ndash1076 1999

[27] G Fiorini A Zullo N Vakil I M Saracino C Ricci andV Castelli ldquoRifabutin triple therapy is effective in patients


with multidrug-resistant strains of Helicobacter pylorirdquoJournal of Clinical Gastroenterology vol 52 no 2 pp 137ndash140 2018

[28] L Zhuge Y Wang S Wu R L Zhao Z Li and Y XieldquoFurazolidone treatment for Helicobacter Pylori infection asystematic review and meta-analysisrdquo Helicobacter vol 23no 2 article e12468 2018

[29] D Bravo A Hoare C Soto M A Valenzuela andA F Quest ldquoHelicobacter pylori in human health and diseasemechanisms for local gastric and systemic effectsrdquo WorldJournal of Gastroenterology vol 24 no 28 pp 3071ndash30892018

[30] J Sung N Kim Y H Park et al ldquoRifabutin-based fourth andfifth-line rescue therapy in patients with for Helicobacterpylori eradication failurerdquoKorean Journal of Gastroenterologyvol 69 no 2 pp 109ndash118 2017

[31] M H Jeong J-W Chung S J Lee et al ldquoComparison ofrifabutin- and levofloxacin-based third-line rescue therapiesfor Helicobacter pylorirdquo Korean Journal of Gastroenterologyvol 59 no 6 pp 401ndash406 2012

[32] A F Ciccaglione R Tavani L Grossi L Cellini L Manzoliand L Marzio ldquoRifabutin containing triple therapy andrifabutin with bismuth containing quadruple therapy forthird-line treatment of Helicobacter pylori infection two pilotstudiesrdquo Helicobacter vol 21 no 5 pp 375ndash381 2016

[33] H Mori H Suzuki J Matsuzaki et al ldquoRifabutin-based 10-day and 14-day triple therapy as a third-line and fourth-lineregimen for Helicobacter pylori eradication a pilot studyrdquoUnited European Gastroenterology Journal vol 4 no 3pp 380ndash387 2016

[34] H C Lim Y J Lee B An S W Lee Y C Lee andB S Moon ldquoRifabutin-based high-dose proton-pump in-hibitor and amoxicillin triple regimen as the rescue treatmentfor Helicobacter pylorirdquo Helicobacter vol 19 no 6pp 455ndash461 2014

[35] MMohammadi B Attaran RMalekzadeh andD Y GrahamldquoFurazolidone an underutilized drug for H pylori eradicationlessons from Iranrdquo Digestive Diseases and Sciences vol 62no 8 pp 1890ndash1896 2017

[36] Y Xie Y Zhu H Zhou Z F Lu Z Yang and X ShuldquoFurazolidone-based triple and quadruple eradication therapyfor Helicobacter pylori infectionrdquo World Journal of Gastro-enterology vol 20 no 32 pp 11415ndash11421 2014

[37] J J Kim R Reddy M Lee J G Kim F A El-Zaatari andM S Osato ldquoAnalysis of metronidazole clarithromycin andtetracycline resistance of Helicobacter pylori isolates fromKoreardquo Journal of Antimicrobial Chemotherapy vol 47 no 4pp 459ndash461 2001

[38] N Almeida J M Romaozinho M M Donato C LuxoO Cardoso and M A Cipriano ldquoTriple therapy with high-dose proton-pump inhibitor amoxicillin and doxycycline isuseless for Helicobacter pylori eradication a proof-of-conceptstudyrdquo Helicobacter vol 19 no 2 pp 90ndash97 2014

[39] F Lorusso M P Caleca C Bellavia D Pistoia S Gallina andR Speciale ldquoe EBV-DNA can be used as a diagnostic andfollow-up parameter of the rhinopharyngeal tumors in thenon-endemic population of the western sicilyrdquo Indian Journalof Otolaryngology and Head amp Neck Surgery pp 1ndash5 2018


Research ArticleImmunization with a Synthetic Helicobacter pylori PeptideInduces Secretory IgA Antibodies and ProtectsMice against Infection

David Espinosa-Ramos1 Diana Caballero-Hernandez 1 Ricardo Gomez-Flores 1

Armando Trejo-Chavez 2 Luis Jeronimo Perez-Limon 1

Myriam Angelica de la Garza-Ramos 3 Reyes Tamez-Guerra1 Patricia Tamez-Guerra1

and Cristina Rodriguez-Padilla1

1Universidad Autonoma de Nuevo Leon Facultad de Ciencias Biologicas Departamento de Microbiologıa e InmunologıaSan Nicolas de los Garza NL CP 66450 Mexico2Universidad Autonoma de Nuevo Leon Facultad de Medicina Veterinaria y Zootecnia Departamento de PatobiologıaCampus de Ciencias Agropecuarias Escobedo NL CP 66050 Mexico3Universidad Autonoma de Nuevo Leon Facultad de Odontologıa y Centro de Investigacion y Desarrollo en Ciencias de la SaludUnidad de Odontologıa Integral y Especialidades Av Dr Aguirre Pequentildeo y Silao SN Monterrey NL CP 64460 Mexico

Correspondence should be addressed to Ricardo Gomez-Flores rgomez60hotmailcom

Received 5 November 2018 Revised 5 February 2019 Accepted 11 February 2019 Published 1 April 2019


Copyright copy 2019David Espinosa-Ramos et al+is 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 isproperly cited

Helicobacter pylori is a spiral Gram-negative bacterium associated with inflammation of the gastric mucosa peptic ulcer andgastric adenocarcinoma whose treatment has failed due to antibiotic resistance and side effects Furthermore because there are novaccines effective against H pylori an appropriate vaccine design targeting conservedessential genes must be identified In thepresent study a H pylori 50ndash52 kDa immunogen-derived peptide antigen with the sequence Met-Val-+r-Leu-Ile-Asn-Asn-Glu(MVTLINNE) was used to immunize against H pylori infection For this mice received an intraperitoneal injection of 100 μg ofH pylori peptide on the first week followed by two weekly subcutaneous reinforcements and further 109 bacteria administrationin the drinking water for 3weeks+ymic cells proliferative responses to concanavalin A serum levels of IL-2 IL-4 IL-6 IL-10 IL-17 IFN-c and TNF-α cytokines and IgG1 IgG2a IgG2b IgG3 IgM and IgA immunoglobulins were evaluated Significant(plt 005) increases on lymphoproliferation and spleen weights after immunization were observed In contrast infection sig-nificantly (plt 005) decreased lymphoproliferation which was recovered in immunized mice In addition levels of serum TH1and TH2 cytokines were not altered after immunization except for the significant increase in IL-6 production in immunized andor infected animals Moreover immunization correlated with plasma secretory IgA and IgG whereas infection alone only elicitedIgM antibodies Peptide immunization protected 100 of mice against virulent H pylori MVTLINNE peptide deserves furtherresearch as an approach to the prophylaxis of H pylori infection

1 Introduction

H pylori is a Gram-negative spiral-shaped bacterium thatrepresents the main factor for the development of humanchronic gastritis duodenal ulcer and gastric adenocarci-noma [1ndash3] Despite the decrease in the incidence of gastriccarcinoma due toH pylori in recent years this disease is still

the most common cause of death from cancer worldwide Infact it is the fourth cause of cancer cases per year accordingto a 2000 report with 945000 new cases [1] In developedcountries 70 to 90 of the population acquires the infectionbefore 10 years of age and its routes of transmission are oral-oral or fecal-oral but iatrogenia may be also involved whenperforming endoscopy with a contaminated tube [4 5] In


addition to surgery which includes partial gastrectomy awide variety of antibiotics have been proposed for thetreatment of gastric ulcer accepted by the Food and DrugAdministration namely the use of bismuth subsalicylatemetronidazole and tetracycline along with an antacid agenthowever this regimen can cause systemic damage such aspseudomembranous colitis in 11 and vaginal candidiasis inexcess of 10 in women under treatment [4] Commonlythe first-line treatment consists of a 7 to 10 days regimenwith a proton-pump inhibitor plus amoxicillin and clari-thromycin [6] However antibiotics are affected by in-creasing levels of resistance [7 8] For instanceclarithromycin resistance has recently been reported in 26272 and 25 of patients infected by H pylori in FranceSpain and Italy respectively [9] However in developingcountries particularly in Mexico resistance reaches 282[10] It has recognized that infection is strongly associatedwith the socioeconomic and sociodemographic conditionsof the population where the variation of H pylori virulence-associated genotypes could favor the development of gas-trointestinal tract pathologies in infected patients [11] Be-cause of this there has been an increasing interest in thedevelopment of vaccines as a prophylaxis to H pylori in-fection [12]

In experimental studies it has been observed that the useof 52 kDa H pylorimembrane peptide as a vaccine has beeneffective to immunize against the development of gastriculcer when used in murine models However the isolationand purification of such a protein presents importantchallenges therefore the use of synthetic peptides designedfrom immunogenic proteins has become an alternative fordiagnosis and prophylaxis Since H pylori causes a super-ficial infection of the gastric tissue the main immunitymediators are secretory lgA antibodies which are the ob-jectives of active oral vaccination [13] Immunized animalsproduce specific serum IgG and IgA and intestinal andsalivary IgA and after challenge a gastric cellular andantibody response can be observed [14ndash16]

+e aim of the present study was to evaluate the pre-ventive effectiveness of vaccination with the MVTLINNEpeptide designed from a 52 kDa H pylori immunogenicprotein in a murine model

2 Materials and Methods

21 Reagents Cell Line and Culture MediaPenicillin-streptomycin solution L-glutamine phosphate-buffered saline (PBS) and RPMI 1640 medium were ob-tained from Life Technologies (Grand Island NY) Fetalbovine serum (FBS) sodium dodecyl sulfate (SDS) NN-dimethylformamide (DMF) 3-[45-dimethylthiazol-2-yl]-25-diphenyltetrazolium bromide (MTT) and concanava-lin A (Con A) were purchased from Sigma-Aldrich (StLouis MO) RPMI 1640 medium supplemented with 10FBS 1 L-glutamine and 05 penicillin-streptomycinsolution was referred as complete RPMI 1640 mediumH pylori strain ATCC 700824 was purchased from theAmerican Type Culture Collection (Rockville MD) andgrown on Brucella broth at 37degC+e strain was identified by

Gram staining morphology and biochemical positive testsfor catalase and urease Extraction buffer was prepared bydissolving 20 (wtvol) SDS at 37degC in a solution of 50each DMF and demineralized water and the pH was ad-justed to 47

22 Animals Female BALBc mice (20ndash25 g) were providedby the Bioterium of Facultad de Ciencias Biologicas atUniversidad Autonoma de Nuevo Leon +ey were kept ina pathogen- and stress-free environment at 24degC under alight-dark cycle (light phase 06 00ndash18 00 h) and givenwater and food ad libitum All animal treatments andsurgical procedures were performed in accordance with theGuide for Care and Use of Laboratory Animals by theNational Institute of Health (Bethesda MD) and approvedby the University Ethics and Animal Care Committee

23 Immunization Procedure +e immunizing peptidemethionine-valine-threonine-leucine-isoleucine-asparagine-asparagine-glutamic acid (MVTLINNE) was synthesized byGenscript (Nanjing China) with gt85 purity and twomodifications acetylation at the amino terminus and ami-dation at the carboxyl terminus +e lyophilized peptide wasstored at minus20degC until immunization for this peptide solutionwas prepared at a concentration of 1mgmL in sterile salineMice were immunized by sc injection with the H pyloripeptide (100 μg in 500 μl of distilled water) in Sigma adjuvant(Sigma-Aldrich) (1 1) on day 0 and in incomplete Freundadjuvant (1 1) on days 21 and 28 Mice were bled and spleensand thymuses were removed on day 91

24 H pylori Challenge H pylori was cultured on Brucellaagar under microaerophilic conditions at 37degC in 5 O210 CO2 and 85 N2 H pylori concentration was deter-mined by CFU counts Seventy days after the last immu-nization mice were challenged with a H pylori suspension(109 CFUL) in the drinking water [17] for 21 days afterwhich blood was obtained by terminal cardiac punctureand the spleen thymus and stomach were asepticallyremoved

25 T-Cell Proliferation Assay T-cell proliferation was de-termined by a colorimetric technique usingMTT [18] 100 μlthymus cell suspensions (1times 107 cellsml) from immunizedimmunized plus infected infected and control animals wereadded to flat-bottomed 96-well plates (Costar CorningNY) containing triplicate cultures (100 μl) of RPMI 1640medium supplemented with 5 fetal bovine serum (unsti-mulated control) in the presence or absence of Con A(625 μgml) or MVTLINNE peptide (10 μgml) for 48 h at37degC in 95 air-5 CO2 atmosphere After incubation for44 h at 37degC with 5 CO2 MTT (05mgml final concen-tration) was added and cultures were additionally incubatedfor 4 h Cell cultures were then incubated for 16 h withextraction buffer (100 μl) and optical densities resultingfrom dissolved formazan crystals were then read in amicroplate reader (Bio-Tek Instruments Inc Winooski


VT) at 540 nm +e lymphocyte proliferation index (LPI)was calculated as follows LPIA540 in resident or ConA-treated cellsA540 in untreated cells

26 Plasma Cytokine and Antibody Responses Plasmasamples were evaluated for IL-2 IL-4 IL-6 IL-10 IFN-cand TNF-α levels using the mouse +1+2+17 kit (BDBiosciences San Jose CA) and IgG1 IgG2a IgG2b IgG3IgM and IgA using the BDTM cytometric bead array (CBA)mouse immunoglobulin isotyping kit by flow cytometry(Accuri C6 BD Biosciences)

27 Mouse Gastric Tissue Histopathology Histopathologicalanalysis of gastric biopsies from experimental mice 21 daysafter infection was performed After sacrificing infected orimmunized + infected mouse stomachs were removed andwashed with sterile water A longitudinal segment along thegreater curvature from the esophagus to the stomach wasused for histological examination To determine histologicalalterations tissue segments were fixed in 4 formalin so-lution dehydrated paraffin-embedded and 3 μm sectionsstained with hematoxylin-eosin by using routine proceduresSections were observed in an Olympus IX71 microscopeand the image acquisition was performed in an Infinity Icamera with the Infinity Capture Software (Lumenera CoOttawa ON) Sections were also stained with WarthinndashStarry stain for H pylori detection All histological analyseswere performed blinded by an experienced veterinarianpathologist [19]

28 Statistical Analysis +e results were expressed asmeanplusmn SEM of the response of 5 animals per treatmentgroup from three independent experiments Level of sig-nificance was assessed by Studentrsquos t-test and ANOVA

3 Results

31 Effect of Immunization and Infection on Spleen WeightsSpleens were excised and weighed after animalrsquos death Asseen in Figure 1 immunization significantly (plt 005) in-creased 37 spleen weights in contrast to 12 nonsignif-icant increase in infected mice as compared with untreatedcontrols

32 Effect of Immunization on Aymic LymphocyteProliferation +ymus cell lymphocytes from immunizedimmunized plus infected infected and control animalswere incubated in the presence or absence of Con A orMVTLINNE peptide and lymphoproliferative responsesdetermined as explained above Con A significantly(plt 005) induced 12-fold increase 18-fold decrease and133-fold increase in thymus lymphoproliferation fromimmunized immunized plus infected and infected re-spectively as compared with untreated control

33 Plasma Cytokine Levels and Antibody Production Asshown in Figure 2 immunization andor infection did not

alter cytokines IL-2 IL-4 IL-10 IL-17 IFN-c and TNF-αlevels however IL-6 significantly (plt 005) increased ascompared with untreated control In addition antibodyisotype observed in infected and untreated control groups wasIgM whereas immunization induced IgM IgA IgG1 andIgG2a antibodies immunization and infection induced IgMIgA IgG1 IgG2a IgG2b and IgG3 antibodies (Table 1)

34 Gastric TissueHistopathology In infected animals at thelevel of the mucosa adjacent to the esophagus a wide ul-cerative area which is composed of cellular detritus elon-gated bacteria and some spores of unicellular parasites isobserved in addition at the level of the submucosa there arediscrete foci of inflammatory infiltrate of mononuclear cellsmainly constituted by lymphocytes and some plasma cells(Figure 3(a)) When performing the WarthinndashStarry stainelongated H pylori bacteria and spores are also observed(Figure 3(a)) +e diagnosis was ulcerative gastritis withpresence of bacteria in gastric epithelium In regard to im-munized and infected animals no ulcerative inflammatorydegenerative or neoplastic changes nor the presence ofbacteria were observed at the mucosal level only someparasitic ovoid structures on the edge of some areas of thegastric epithelium were formed When performingWarthinndashStarry staining these unicellular parasites were alsoobserved +e diagnosis showed presence of few ovoid par-asitic structures in the gastric epithelium In both groups ofanimals esophagus did not show pathological changes

4 Discussion

It is estimated that 50 of the world population has beeninfected by H pylori a disease that although in the earlystages is not considered deadly in the long term it leads tomore serious diseases such as cancer In recent years in-fections related to bacteria and viruses have been associatedwith the development of gastric diseases including cancerchronic gastritis and MALT lymphoma In particular therole of H pylori and EpsteinndashBarr virus (EBV) in gastric

Immunized mice Infected mice0

20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)

Figure 1 Spleen weights spleens were removed and weighed aftermice death following immunization orH pylori infection protocolas detailed in the text Data represent meanplusmn SEM of 5 animals perexperimental group from 3 independent experiments lowastplt 005


carcinogenesis has been evaluated +e relevance of theinflammatory response is hypothesized by recent studiesshowing how coinfection with H pylori and EBV can causetissue damage through inflammatory reactions or throughincreased contact between the CagA protein ofH pylori andEBV which supports the increased activation of B cells intransit through the gastric mucosa [20] Fasciana et aldemonstrated that the correlation of H pylori and EBV ishighly frequent [20] +erefore it is important to find newalternatives either for treatment or prophylaxis +e use ofsynthetic peptides designed from immunogenic proteins isconsidered an alternative for diagnosis and prophylaxis butrequires homogeneity in the antigenic preparation as

described by Giammanco et al [21] In the present study amurine model was used to determine the preventive po-tential of the MVTLINNE peptide which is part of theterminal portion of a 52ndash55 kDa protein identified as ahomologue of citrate synthase and has previously beendescribed and patented for its usefulness as a diagnostic toolfor H pylori infection [22]

InH pylori preclinical models the evaluation focuses onthe proliferation of lesions in the stomach mucosa as a resultof infection and other methods such as molecular diagnosisare also useful in the evaluation of the success of an in-tervention therapy [23 24] however its use requires ex-pensive equipment and is not always available Both the lowmortality rate and the chronic nature of the disease limitpreclinical models of the disease Another important aspectin a preclinical model is the selection of the animal to beused the most frequent is the mouse and in some studies therat However the strain of animals used is also importantwhen interpreting the studies since there are importantintrinsic physiological and immunological variations thatcan determine the treatment outcome In the present study

Untreated control Immunized Infected Immunized + infected0

1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)

Figure 2 MVTLINNE peptide immunization increases plasma IL-6 levels Plasma IL-2 IL-4 IL-6 IL-10 IL-17 IFN-c and TNF-α levelswere measured in immunized immunized and infected infected and untreated control animals as explained in the text Data representmeanplusmn SEM of 5 animals per experimental group from 3 independent experiments plt 005 as compared with untreated control

Table 1 Plasma immunoglobulins

Experimental group Antibody isotypeUntreated control IgMImmunized IgM IgA IgG1 IgG2aInfected IgMImmunized + infected IgM IgA IgG1 IgG2a IgG2b IgG3


an in vivo model of H pylori was established in the BALBcmouse a noninvasive method of infection administering thebacteria in the drinking water was developed [17] +ismodel was used to evaluate the preventive efficacy of theadministration of a synthetic peptide from a H pyloriprotein the MVTLINNE immunogenic peptide was ad-ministered twice first intraperitoneally on week 1 and thensubcutaneously as reinforcement on week 4 in order toenhance the animalrsquos immune response against infection Inthis concern the most used method for in vivo infection isthe oral administration with a cannula of a known quantityof the bacteria [25] However this method requires con-siderable skill and carries risks for the welfare of the ex-perimental animals thus an alternative method to reducethese risks was selected in which bacteria are administeredin the drinking water [17]+ere is controversy regarding thetime that the viability of the bacteria in water is maintainedhowever several reports indicated that it is sufficient for oralinfection [26 27] In our study the data indicated thatinfected mice have immunological and histopathologicalresponse parameters consistent with infection (Figures 1ndash4)

Cellular immune response plays a critical role against Hpylori infection which has been shown in immunodeficientmice models [28 29] In the present study results showedthat immunization with the MVTLINNE peptide stimulatedthe cellular immune response as shown by the larger size ofthe spleen of immunized mice (Figure 1) and increased ConA-mediated proliferative response of thymus lymphocytes(Figure 4)H pylori-mediated gastritis involves the release ofcytokines from inflammatory cells which contributes tomaintain and amplify the local inflammation processHowever in this work the analysis of TH1TH2 cytokineprofiles did not allow us to reach a conclusion about thesuccess of the vaccination strategy since no statisticallysignificant differences were found between the experimentalgroups (Figure 2) However there was an indication of anIL-6-mediated inflammatory response during infection andor immunization (Figure 2) IL-6 plays an relevant role ininnate and adaptive host defense by inducing IFN-c

production immunoglobulin secretion and neutrophilactivation [30] and hence its involvement in protectionagainst microbial infection in vivo [31] In contrast it wasshown that IFN-c may be involved in induction ofH pylori-mediated gastric inflammation [32]

It has been reported that H pylori infection is associatedwith overexpression of IL-6 at the margin of gastric ulcer bymacrophages [33 34] Furthermore it was shown that

(a)

(b)

Figure 3 MVTLINNE peptide immunization protects mice from infection Histological alterations and H pylori presence in gastric tissuesegments of infected or immunized + infected animals were determined as explained in the text (a) Infected animals and (b) immu-nized + infected animals (40x magnification)

Concanavalin A MVTLINNE peptide 00

04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I

Untreated controlImmunization

InfectionImmunization + infection

Figure 4 MVTLINNE peptide immunization stimulates Con A-mediated thymic lymphocyte proliferation Lymphoproliferationwas determined in thymus cell suspensions from immunizedimmunized and infected infected and untreated control animals+ymuses were surgically excised and mechanically dissociatedinto single-cell suspensions Lymphocyte suspensions were thenincubated in the presence or absence of Con A (625 μgml) andorMVTLINNE peptide (10 μgml) and lymphoproliferation wasmeasured by the MTT reduction assay as explained in the textData represent LPI meansplusmn SEM of triplicates from three in-dependent experiments n 5 in each group Untreated controloptical density was 048plusmn 0004 lowastplt 005 as compared with theuntreated control


gastric epithelium significantly contributed to the antral IL-1β and IL-6 response from H pylori-infected duodenal ulcerpatients and asymptomatic carriers [26] In addition in-creased production of IL-6 and TNF-α in human antralmucosa cultures from H pylori-infected chronic gastritispatients has been observed by others [35]

Macrophage cytokine upregulation in gastric tissuesduring H pylori infection has been proven [34] particularlyincreased expression levels of IL-1 TNF-α and IL-6 IL-6mRNA expression in gastritis tissues was shown to correlatewith H pylori-mediated infection and inflammation [34ndash37] and serum IL-6 concentrations were related toH pylori-induced gastric cancer [38] Since inflammation plays asignificant role in gastric carcinogenesis it has been sug-gested that polymorphisms in genes involved in inflam-matory response may partly explain why only a subgroup ofpatients infected with H pylori develop gastric cancerProinflammatory cytokine genetic background is believed toplay a pathogenic role in age-related diseases converselygenetic variations determining increased production of anti-inflammatory cytokines or decreased production of proin-flammatory cytokines have been shown to be associated withsuccessful aging It has been reported that polymorphisms inthe IL-1 and IL-10 genes could contribute to determining thebackground for inflammation in which H pylori infectionmight facilitate cancer development [39]

A potential mechanism by which H pylori induces IL-6production by macrophages in chronic gastritis patients wasreported to be related to heat shock protein 60 stimulation[40] Furthermore in the present study serum IL-17 was notaltered by immunization andor infection although othershave reported its upregulated expression in H pylori-in-fected human gastric mucosa [41]

Because of the marginal efficacy and antibiotic resistancein the clinics and eradication of H pylori protects fromdamaging gastric tissues the development of a safe andeffective vaccine for humans continues to be an active re-search issue [25] +e use of whole bacteria may be po-tentially harmful whereas recombinant vaccines became analternative for prophylaxis however additional immuno-genic antigens must be tested [14] In the present study oralvaccination with the MVTLINNE peptide induced pro-tective IgA and IgG antibodies as shown in Table 1 SinceHpylori produces an intraluminal infection immunity may bemediated at least in part by secretory IgA antibodies Forinstance human breast milk IgA protects children againstHpylori infection [42] Oral immunization with killedH pyloriwas reported to induce specific IgA and IgG antibodies inmice gastrointestinal secretions and sera [25] It is recog-nized that oral vaccination induces an IgA-dependentmucosal immune response that eradicates long-term in-fection withH pylori in mice [43 44] Oral administration ofH pylori recombinant urease plus adjuvant was reported toinduce protective and long-lasting protective specific IgAimmunity against challenge with virulentH felis [15 16 45]

In our study MVTLINNE peptide vaccination-mediatedIgA production correlated with no alterations in the gastricmucosa and scarce presence of bacilli after H pylori in-fection (Figure 3(b)) as compared with untreated control

(Figure 3(a)) Taken together these results indicated thatprophylactic immunization significantly reduced the num-ber of colonizing bacteria which was associated with healthygastric tissue [46]

Data Availability

+e experimental data used to support the findings of thisstudy are included within the article


+e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

We would like to thank Laboratorio de Inmunologıa yVirologıa of Facultad de Ciencias Biologicas at UniversidadAutonoma de Nuevo Leon for supporting this study +iswork was financially supported by Programa de Inves-tigacion Cientıfica y Tecnologica (PAICYT) (grant numberCN-553) of Universidad Autonoma de Nuevo Leon Mexicoto Ricardo Gomez-Flores We are also grateful to Dr JoseAlberto Ramos Silva for his technical assistance duringimage acquisition and Dr Erika Coronado Cerda for hertechnical advice during cytometry analysis

Supplementary Materials

Spleen weights Studentrsquos t-test analysis a Studentrsquos t-testanalysis of spleen weights between immunized and controlgroups is depicted One-way ANOVA test for peptide-immunization lymphoproliferation data an ANOVA testfor data related to the effect of peptide immunization onthymus lymphocyte proliferation as compared with controlswas performed One-way ANOVA test for peptide-immunization and ConA-mediated lymphoproliferationdata an ANOVA test for data related to the effect of peptideimmunization on concanavalin A-mediated thymus lym-phocyte proliferation as compared with controls was per-formed One-way ANOVA test for IL-6 production anANOVA test for data related to the effect of peptide im-munization on IL-6 production as compared with controlswas developed (Supplementary Materials)

References

[1] J C Arana and A Corona ldquoCancer gastricordquo Revista de laFacultad de Medicina vol 47 no 5 pp 204ndash209 2009

[2] M J Blaser ldquoHelicobacter pylori and the pathogenesis ofgastroduodenal inflammationrdquo Journal of Infectious Diseasesvol 161 no 4 pp 626ndash633 1990

[3] T L Cover and M J Blaser ldquoHelicobacter pylori and gas-troduodenal diseaserdquo Annual Review of Medicine vol 43no 1 pp 135ndash145 1992

[4] B E Dunn H Cohen and M J Blaser ldquoHelicobacter pylorirdquoClinical Microbiology Reviews vol 10 no 4 pp 720ndash7411997

[5] B A Dye D Kruszon-Moran and G McQuillan ldquo+e re-lationship between periodontal disease attributes and


Helicobacter pylori Infection among adults in the UnitedStatesrdquo American Journal of Public Health vol 92 no 11pp 1809ndash1815 2002

[6] Y O Alahdab and C Kalayci ldquoHelicobacter pylori man-agement in 2013rdquo World Journal of Gastroenterology vol 20no 18 pp 5302ndash5307 2014

[7] V De Francesco F Giorgio C Hassan et al ldquoWorldwide Hpylori antibiotic resistance a systematic reviewrdquo Journal ofGastrointestinal amp Liver Diseases vol 19 no 4 pp 400ndash4142010

[8] J-Y Wu S Wang Y Lee et al ldquoDetection of genotypicclarithromycin-resistant Helicobacter pylori by string testsrdquoWorld Journal of Gastroenterology vol 20 no 12 pp 3343ndash3349 2014


[10] L Ladron-de-Guevara L Bornstein-Quevedo S Gonzalez-Huezo B Castantildeeda-Romero F G Costa and M di Silvio-Lopez ldquoErradicacion de Helicobacter pylori en Mexico con unesquema basado en levofloxacina versus la triple terapiaestandar resultados de un estudio clınico de fase iiib abiertoaleatorizado de no inferioridadrdquo Revista de Gastroenterologıade Mexico vol 84 2019

[11] A Chiarini C Cala C Bonura et al ldquoPrevalence of virulence-associated genotypes of Helicobacter pylori and correlationwith severity of gastric pathology in patients from westernSicily Italyrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 28 no 5 pp 437ndash446 2009

[12] T G Blanchard and J G Nedrud ldquoHelicobacter pylori vac-cines Helicobacter pylori in the 21st centuryrdquo Vaccines for the21st Century A Tool for Decisionmaking pp 167ndash189 NationalAcademies Press Washington DC USA 2010

[13] C Leppold T Tanimoto A Ozaki T Morita and P V SarildquoHelicobacter pylori vaccinationrdquo Ae Lancet vol 387no 10020 pp 748-749 2016

[14] H Kleanthous G A Myers K M Georgokopoulos et alldquoRectal and intranasal immunization with recombinanturease induce distinct local and serum immune responsesin mice and protect against Helicobacter pylori infectionrdquoInfection and Immunity vol 66 pp 2879ndash2886 1998

[15] C K Lee R Weltzin W D +omas Jr et al ldquoOralimmunization with recombinant Helicobacter pylori ureaseinduces secretory IgA antibodies and protects mice fromchallenge with Helicobacter felisrdquo Journal of Infectious Dis-eases vol 172 no 1 pp 161ndash172 1995

[16] J Pappo W D +omas Jr Z Kabok N S TaylorJ C Murphy and J G Fox ldquoEffect of oral immunization withrecombinant urease on murine Helicobacter felis gastritisrdquoInfection and Immunity vol 63 pp 1246ndash1252 1995

[17] K F Boehnke K A Eaton M Valdivieso L H Baker andC Xi ldquoAnimal model reveals potential waterborne trans-mission ofHelicobacter pylori infectionrdquoHelicobacter vol 20no 5 pp 326ndash333 2015

[18] M Franco-Molina R Gomez-Flores P Tamez-GuerraR Tamez-Guerra L Castillo-Leon and C Rodrıguez-PadillaldquoIn vitro immunopotentiating properties and tumour celltoxicity induced by Lophophora williamsii (peyote) cactusmethanolic extractrdquo Phytotherapy Research vol 17 no 9pp 1076ndash1081 2003

[19] T Ermak R Ding B Ekstein et al ldquoGastritis in urease-immunized mice after Helicobacter felis challenge may be due

to residual bacteriardquo Gastroenterology vol 113 no 4pp 1118ndash1128 1997


[21] A Giammanco A Nardone R Pebody et al ldquoEuropean sero-epidemiology network 2 standardization of immunoassayresults for pertussis requires homogeneity in the antigenicpreparationsrdquo Vaccine vol 26 no 35 pp 4486ndash4493 2008

[22] M Dunkley S J Harris R J McCoy et al ldquoProtection againstHelicobacter pylori infection by intestinal immunisation witha 5052-kDa subunit proteinrdquo FEMS Immunology andMedical Microbiology vol 24 no 2 pp 221ndash225 1999

[23] T Horemans M Deschacht S Clais et al ldquoAn alternativesensitive method to detect Helicobacter pylori DNA in fecesrdquoHelicobacter vol 16 no 2 pp 113ndash118 2011

[24] F Lorusso M P Caleca C Bellavi et al ldquo+e EBV-DNA canbe used as a diagnostic and follow-up parameter of the rhi-nopharyngeal tumors in the non-endemic population of theWestern Sicilyrdquo Indian Journal of Otolaryngology and Head ampNeck Surgery vol 72 pp 1ndash5 2019

[25] S J Czinn and J G Nedrud ldquoOral immunization againstHelicobacter pylorirdquo Infection and Immunity vol 59 no 7pp 2359ndash2363 1991

[26] L Beneduce D Tarantino G Spano M LibergoliM Labonia and S Massa ldquoSurvival of Helicobacter pylori inwell waterrdquoWorld Journal of Microbiology and Biotechnologyvol 19 no 5 pp 505ndash508 2003

[27] R K Aziz M M Khalifa and R R Sharaf ldquoContaminatedwater as a source of Helicobacter pylori infection a reviewrdquoJournal of Advanced Research vol 6 no 4 pp 539ndash547 2015

[28] K A Eaton and M E Mefford ldquoCure of Helicobacter pyloriinfection and resolution of gastritis by adoptive transfer ofsplenocytes in micerdquo Infection and Immunity vol 69 no 2pp 1025ndash1031 2001

[29] C Lindholm M Quiding-Jarbrink H Lonroth A Hamletand A M Svennerholm ldquoLocal cytokine response in Heli-cobacter pylori-infected subjectsrdquo Infection and Immunityvol 66 no 12 pp 5964ndash5971 1998

[30] J H Curfs J F Meis and J A Hoogkamp-Korstanje ldquoAprimer on cytokines sources receptors effects and inducersrdquoClinical Microbiology Reviews vol 10 no 4 pp 742ndash7801997

[31] M Kopf H Baumann G Freer et al ldquoImpaired immune andacute-phase responses in interleukin-6-deficient micerdquo Na-ture vol 368 no 6469 pp 339ndash342 1994

[32] N Sawai M Kita T Kodama et al ldquoRole of gamma in-terferon in Helicobacter pylori-induced gastric inflammatoryresponses in a mouse modelrdquo Infection and Immunity vol 67no 1 pp 279ndash285 1999

[33] S K Pathak S Basu A Bhattacharyya et al ldquoTLR4-Dependent NF- B activation and mitogen- and stress-activated protein kinase 1-triggered phosphorylation eventsare central to Helicobacter pylori peptidyl prolyl cis- trans-isomerase (HP0175)-mediated induction of IL-6 release frommacrophagesrdquo Ae Journal of Immunology vol 177 no 11pp 7950ndash7958 2006

[34] P R Harris L E Smythies P D Smith and A DuboisldquoInflammatory cytokine mRNA expression during early andPersistent Helicobacter pylori Infection in nonhuman pri-matesrdquo Ae Journal of Infectious Diseases vol 181 no 2pp 783ndash786 2000

[35] J E Crabtree T M Shallcross R V Heatley and J I WyattldquoMucosal tumour necrosis factor alpha and interleukin-6 in


patients with Helicobacter pylori associated gastritisrdquo Gutvol 32 no 12 pp 1473ndash1477 1991

[36] P Gionchetti D Vaira M Campieri et al ldquoEnhanced mu-cosal interleukin-6 and-8 in Helicobacter pylori-positivedyspeptic patientsrdquo American Journal of Gastroenterologyvol 89 no 6 pp 883ndash887 1994

[37] Y Yamaoka M Kita T Kodama N Sawai and J ImanishildquoHelicobacter pylori cagA gene and expression of cytokinemessenger RNA in gastric mucosardquo Gastroenterologyvol 110 no 6 pp 1744ndash1752 1996

[38] CWWu S R Wang M F Chao et al ldquoSerum interleukin-6levels reflect disease status of gastric cancerrdquo AmericanJournal of Gastroenterology vol 91 no 7 pp 1417ndash14221996

[39] G Forte C Cala L Scola et al ldquoRole of environmental andgenetic factor interaction in age-related disease developmentthe gastric cancer paradigmrdquo Rjuvenation Research vol 11no 2 pp 509ndash512 2008

[40] A P Gobert J-C Bambou C Werts et al ldquoHelicobacterpylori heat shock protein 60 mediates interleukin-6 pro-duction by macrophages via a toll-like receptor (TLR)-2-TLR-4- and myeloid differentiation factor 88-independentmechanismrdquo Journal of Biological Chemistry vol 279 no 1pp 245ndash250 2004

[41] F Luzza T Parrello G Monteleone et al ldquoUp-regulation ofIL-17 is associated with bioactive IL-8 expression in Heli-cobacter pylori-infected human gastric mucosardquo Ae Journalof Immunology vol 165 no 9 pp 5332ndash5337 2000

[42] J E +omas S Austin S A Dale et al ldquoProtection by humanmilk IgA against Helicobacter pylori infection in infancyrdquoLancet vol 342 no 8862 p 121 1993

[43] T Goto A Nishizono T Fujioka J Ikewaki K Mifune andM Nasu ldquoLocal secretory immunoglobulin A and post-immunization gastritis correlate with protection againstHelicobacter pylori infection after oral vaccination of micerdquoInfection and Immunity vol 67 no 5 pp 2531ndash2539 1999

[44] J Ikewaki A Nishizono T Goto T Fujioka and K Mifuneldquo+erapeutic oral vaccination induces mucosal immune re-sponse sufficient to eliminate long-term helicobacter pyloriinfectionrdquo Microbiology and Immunology vol 44 no 1pp 29ndash39 2000

[45] G A Myers T H Ermak K Georgakopoulos et al ldquoOralimmunization with recombinant Helicobacter pylori ureaseconfers long-lasting immunity against Helicobacter felis in-fectionrdquo Vaccine vol 17 no 11-12 pp 1394ndash1403 1999

[46] C A Garhart R W Redline J G Nedrud and S J CzinnldquoClearance of Helicobacter pylori infection and resolutionof postimmunization gastritis in a kinetic study of prophy-lactically immunized micerdquo Infection and Immunity vol 70no 7 pp 3529ndash3538 2002


Research ArticleApplication of PCR and Microscopy to Detect Helicobacterpylori in Gastric Biopsy Specimen among Acid PepticDisorders at Tertiary Care Centre in Eastern Nepal

Nayanum Pokhrel1 Basudha Khanal1 Keshav Rai1 Manish Subedi2

and Narayan Raj Bhattarai 1

1Department of Microbiology B P Koirala Institute of Health Sciences Dharan Nepal2Department of Internal Medicine B P Koirala Institute of Health Sciences Dharan Nepal

Correspondence should be addressed to Narayan Raj Bhattarai bhattarai03yahoocom

Received 26 September 2018 Revised 27 December 2018 Accepted 20 January 2019 Published 5 February 2019


Copyright copy 2019NayanumPokhrel et al0is is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

BackgroundHelicobacter pylori infection is most prevalent in developing countries It is an etiological agent of peptic ulcer gastricadenocarcinoma and mucosal-associated lymphoid tissue (MALT) lymphoma Despite the development of different assays toconfirm H pylori infection the diagnosis of infection is challenged by precision of the applied assay Hence the aim of this studywas to understand the diagnostic accuracy of PCR and microscopy to detect the H pylori in the gastric antrum biopsy specimenfrom gastric disorder patientsMethods A total of 52 patients with gastric disorders underwent upper gastrointestinal endoscopywith biopsy0eH pylori infection in gastric biopsies was identified after examination bymicroscopy and 23S rRNA specific PCR0e agreement between two test results were analysed by McNemarrsquos test and Kappa coefficient Result H pylori infection wasconfirmed in 9 (1730) patients by both assays 625 in antral gastritis 2222 in gastric ulcer 100 in gastric ulcer withduodenitis 50 in gastric ulcer with duodenal ulcer and 3333 in severe erosive duodenitis with antral gastritis Out of nine Hpylori infection confirmed patients 3 patients were confirmed by microscopy and 8 patients by PCR In case of two patients bothmicroscopy and PCR assay confirmed theH pylori infection0e agreement between two test results was 8654 and disagreed by1346 (p valuegt 005) Conclusion We found that PCR assay to detectH pylori is more sensitive than microscopy However weadvocate for the combination of both assays to increase the strength of diagnostic accuracy due to the absence of the gold standardassay for H pylori infection

1 Introduction

Helicobacter pylori (H pylori) is a Gram-negative bacteriumthat plays a remarkable role in the causation of gastroin-testinal diseases such as peptic ulcers low-grade B-celllymphoma (MALT lymphoma) and gastric cancer [1 2]Several epidemiological studies also evidenced thatH pylori-infected individuals showed the incidence of gastric carci-noma [3] 0e discrepancy of H pylori prevalence has beenshown among different population as well as in differentcountries In fact the transmission of the infection isinfluenced by the socioeconomic conditions About 90prevalence have been reported in developing nations incomparison with 50 occurrence in developed countries

[4 5] Moreover both gastric cancer and peptic ulcer causemore than a million deaths per year globally thus making itan important health issue [6 7]

Diagnostic tests for H pylori include invasive andnoninvasive methods with the involved techniques beingeither direct or indirect Microscopy detection of the bacteriaand culture is a direct method whereas demonstration ofurease production and detection of stool antigen or anantibody is considered an indirect method which is used asa response marker of infectious diseases Advancement inmolecular methods is now used as a reliable tool for di-agnosis of infectious diseases due to its increasing sensi-tivity and specificity [8] Due to resource constraintsdiagnosis by noninvasive tests such as urea breath test or


invasive approach by bacterial culture of the biopsied tissueis not performed in our setting Likewise the reliability ofimmunological tests is always a matter of debate In recentyears application of molecular method such as polymerasechain reaction (PCR) has revolutionized the diagnosticapproaches for the detection of H pylori In addition it alsotracks the several genetic alteration in bacilli for un-derstanding the drugs resistance characteristics [9] andcoinfection of pathogens in gastric disease [10] 0e mo-lecular approach has also helped in comparative analysisbetween conventional methods such as microscopy andrapid urease test with PCR in resource-limited settings foreffective diagnosis and treatment In our setup with theadvantage of the availability of molecular methods wecompared microscopy with PCR to see the effectiveness ofeach method for further evaluation of the study

It is utmost important to identify H pylori infection ingastroduodenal diseases so that the probable gastrointestinalmalignancy can be prevented on time In developingcountries such as Nepal the prevalence of H pylori is no-tably higher in number of duodenal ulcer gastric ulcer andgastritis but a few data on burden of infections are available[11] 0erefore this study has the aim to detect H pylori inupper gastrointestinal endoscopic biopsy specimens bydifferent diagnostic tools and evaluate the accuracy of Hpylori detecting tools in acid peptic disorder patients at-tending B P Koirala Institute of Health Sciences Dharan


21 Patients and Samples 0is study was performed at B PKoirala Institute of Health Sciences (BPKIHS) DharanNepal from January 2017 to December 2017 Ethicalclearance was obtained from the Institutional ReviewCommittee (IRC-321073074) at BPKIHS A written con-sent from 52 patients with symptoms of dyspepsia was takenbefore the biopsy specimen was collected for the study 0epatient with age less than 14 years was excluded in this studyLikewise the patients with history of long-term drugsknown to cause gastritis such as steroids anticoagulants andlesions suggestive of malignancy on endoscopy were ex-cluded from the study

About 4mm biopsy specimen from either the infectedsite or normal mucosa of the gastric antrum was collected0e tissue biopsy was cut with a sterile scalpel blade in asterile Petri dish into two pieces First specimen was pre-served in normal saline and kept in a freezer at minus80degC forPCR Second tissue biopsy was processed for microscopicassessment [12] In this study storage of the biopsy speci-mens was done at minus80degC which prevents the deterioration ofDNA before the PCR analysis In order to confirm the PCRinhibition PCR-negative samples were diluted in 1 10 PCRgrade water and PCR was repeated

22 Microscopy A smear was prepared by picking the bi-opsy specimen with a sterile swab and smeared onto twoclean microscopic glass slides After air-drying the smearwas fixed with uppermost flame of the Bunsen burner and

allowed to cool 0e smear was stained with the modifiedGram-staining technique using carbol fuchsin as thecounterstain In the second glass slide smear was fixed withmethanol and Giemsa staining was performed [12]

23 PCR 0e biopsy sample was taken out from the freezerand thawed at 37degC prior to processing the sample0eDNAextraction from the biopsy specimen was performed byusing the Wizard Genomic DNA purification kit (PromegaCat no A1125) [13] In brief the biopsy was homogenizedby a glass rod in nucleic lysis solution and lysate was in-cubated at 65degC for 30minutes and at 80degC for 5minutesAfter removal of protein precipitates from the lysate thesupernatant containing DNA was further precipitated inisopropanol and 70 ethanol separately Ethanol was gentlyremoved and pellet was air-dried Finally 100 microL of DNArehydration solution was added to rehydrate the DNA byincubating overnight at 4degC and stored at minus20degC

0e H pylori-specific PCR was performed to detect 23SrRNA gene [14] PCR master mix was prepared in 25 μl finalvolume which constituted 2mM of MgCl2 01mgml ofBSA and 0175 microM of primer HP-23S-F (5prime-AGATGG-GAGCTGTCTCAACCAG-3prime) 025 microM of primer HP-23S-R (5prime-TCCTGCGCATGATATTCCC-3prime) and 02mMDNTPmix 05 unit of Hotstar Taq polymerase (Qiagen Catnr 203605) and 25 microL of DNA template PCR water wasused as a negative control and the DNA from the biopsyspecimen with H pylori PCR positive result was consideredas a positive control Mastercycler ProS (Eppendorf Ger-many) thermocycler was used to amplify the target DNA inthe samples After the electrophoresis of the PCR product in2 agarose gel at 5Vcm and ethidium bromide staining theDNA band was visualized with UV exposure 0e samplewas determined as H pylori-positive PCR result if DNAband of length 137 bp was seen in gel

In order to evaluate the quality of DNA extraction thesecond human-specific PCR was done to assure the presenceof human DNA in each sample Human β-globin gene wasthe targeted to amplify using the primers KM29 and KM38following the protocol developed by Saiki et al [15]

24 Statistical Analysis Data were entered in MS Excel 2007worksheet and further analysed by using SPSS softwareversion 115 [16] and R package [17] Kappa coefficient (κ)was used for qualitative analysis of categorical dataMcNemarrsquos test was applied to analyse the disagreementbetween the tests 0e chi-squared test was used to analysethe p value between the categorical data

3 Results

Out of 52 patients enrolled in this study majority of thepatients were young adults between ages of 20 and 30 years(25) followed by 60 to 70 years (2115) which has beendepicted in Table 1 Female patients were 25 (4807)found lesser than male patients 27 (5193) Laboratoryanalysis of biopsy demonstrated that 9 (1730) patientswere confirmed H pylori infection as shown in Table 2


Amongst them 3 (577) cases of H pylori infection wereconfirmed by microscopy and 8 (1538) cases were con-firmed by PCR assay

0e spiral- or curved-shaped morphology resemblingHpylori was confirmed in 2 (384) Gram-stained biopsiesand 1 (19) Giemsa-stained biopsy We found microscopypositivity in 576 patients which could be due to small size ofthe biopsy Out of three microscopy-positive cases the en-doscopic examination showed one case of severe erosiveduodenitis + antral gastritis and one gastric ulcer +duodenalulcer However one microscopy-positive case had normalmucosa in endoscopic examination Among microscopy-positive cases one had consumed PPI in less than twoweeks prior to endoscopywith no history of antibiotic intake inany of the patients 0e forty-nine microscopy-negative caseshave history of PPI consumption in 30 (6122) patients and5 (1020) patients had taken antibiotic in less than 2weeks0e alteration in the morphology of bacteria from the spiralto coccoid form due to consumption of PPI and antibioticscan be responsible for false negativity leading to the possibilityof misdiagnosis by the microscopic technique [18 19]

Among 9 cases ofH pylori confirmed by biopsy analysisendoscopic investigation showed that 32 patients hadconfirmed gastritis 6 had duodenitis and 13 had ulcers asshown in Table 2 But one patient with confirmed H pyloriunder biopsy analysis had no abnormality in endoscopicobservation

In this study 44 out of 52 cases had PCR-negative resultsIn order to rule out the false-negative H pylori PCR resultshuman β-haemoglobin PCR was performed to assure thequality of DNA extraction Out of three microscopicallypositive cases H pylori PCR was also positive in two casesAmong the PCR-positive cases endoscopy examinationshowed two antral gastritis cases two gastric ulcer cases one

gastric ulcer + duodenal ulcer case and one severe erosiveduodenitis + antral gastritis case and two gastric ulcer +duodenitis cases But one case of normal mucosa in en-doscopy had also showed PCR-positive test result

Overall 9 (1730) patients of acid peptic disease (APD)were tested positive by either of the two methods As yetreported elsewhere none of the diagnostic assay is stand-alone and universal for disease diagnosis because of severalextrinsic and intrinsic limitations [20] On comparing thedifferent laboratory methods used in detecting H pyloricombination methods using both conventional and mo-lecular techniques have been recommended [21] Out of 52patients in the present study H pylori is confirmed by PCRalone in 6 (1153) cases microscopy alone in 1 (192)case and combination of PCR and microscopy in 2 (384)cases Combination of diagnostic assays has proven to bepromising in detecting H pylori In this study the combi-nation of diagnostic assays microscopy and PCR increasedthe test positivity from 577 (352) to 1731 (952)

Two (952) in three microscopy positives and 5 (238)in 8 PCR confirmed H pylori positive had no history of PPIintake In case of patient with history of antibiotic intakeboth diagnostic tests had shown the H pylori-negative re-sults but H pylori-positive test results were demonstrated inpatients without antibiotic intake as shown in Table 30eHpylori microscopy results were 8654 in agreement withPCR results whereas 1346 results between both diagnostictests were in disagreement but kappa statistical analysisshowed that the disagreement was not significant (p val-ue 014) as depicted in Table 4 0e PCR is superior indiagnosing the presence of the bacteria in gastric biopsy thanthe microscopy McNemarrsquos analysis between two assays hadshown an agreement of 8654 (Kappa test p 014) whichmeans diagnostic efficiency of both assays was not signifi-cantly different

4 Discussion

Several assays have been proposed to detect the H pyloriinfection up to date none of the assay is considered as goldstandard for diagnosis of H pylori due to the question indiagnostic precision and feasibility of the available assays[21] In this study microscopy and PCR were used as thediagnostic assay in detecting the presence of the H pyloribacterium in patients with acid peptic disorders using gastricmucosal biopsy Moreover bacterial distribution is mostlyirregular andor decreased bacterial load in the available cutspecimen In contrast to our study Khalifehgholi et al usedGiemsa staining assay and identifiedH pylori in 778 of thespecimen [22] Likewise Siavoshi et al found 479H pyloripositive by Gram staining [23] and Roy et al found 6583H pylori positive by using modified Giemsa along withhematoxylin + eosin [24] 0e results were undoubtedlyhigher than our findings which could be due to variation instaining techniques and sampling population Moreover thestudy conducted in Nepalese population showed that 675of stomach carcinoma cases were found H pylori positivityby histopathology and rapid urease analysis [11] 0econtrasting results in the aforementioned study could be

Table 2 Comparison of endoscopy findings and positive H pyloritests (n 52)

Endoscopy finding No of cases () H pylori-positive case ()Normal 1 (192) 1 (1111)Gastritis 32 (6154) 2 (2222)Duodenitis 6 (1154) 1 (1111)Ulcers 13 (250) 5 (5556)Total 52 9

Table 1 Demographic and laboratory analysis (n 52)

Age (years) Frequency20ndash30 13 (25)30ndash40 8 (1538)40ndash50 8 (1538)50ndash60 7 (1346)60ndash70 11 (2115)70ndash80 5 (963)GenderFemale 25 (4807)Male 27 (5193)Laboratory testMicroscopy positive 3 (577)PCR positive 8 (1538)


differences in study population enrolled and the applicationof diagnostic methods such as histopathological examina-tion using hematoxylin and eosin However several reportson advantages of staining by other methods had been re-ported and it was not used in the present study [25 26]

PCR diagnosis by amplifying the conserved gene 23SrRNA has highest performance than other PCR assays [27]Hence 23S rRNA PCR was used to identify H pylori in-fection in this study In addition it is efficient to rule out theother neighbouring species which were close to phylogeneticcluster of Helicobacter bacteria 0e advent of molecularmethods for diagnosis of H pylori infection has proven tobe a reliable tool as it amplifies the target gene by more than106 fold thereby increasing the diagnostic sensitivity andspecificity enabling better clinical management [21] Inaddition it is also capable to detect clarithromycin resistancegenotype due to point mutations in the H pylori 23S rRNAgene [28] Eight (1538) out of 52 biopsy specimens de-tected 23S rRNA which was much lower than that reportedby Archampong et al in Ghana (484) with cagA gene [29]Ruparelia et al in Brazil (50) with ureA+ ureC gene [30]and Sugimoto et al (44) with 16S rRNA [31] Hundredpercent diagnostic accuracy cannot be achieved by the ap-plication of single PCR assay [31] since the genomic flexi-bility between strains of H pylori complicates the choice oftarget genes [20] Discrepancy in PCR resulting among thestudies could be due to the difference in the type of targetgenes In other studies at least two types of genes were usedeither a combination of two virulent genes or a conservedvirulent gene Despite advantages with application ofmultiple PCR this study had only single target to amplifyHpylori 23S rRNA Furthermore other factors such as storageconditions presence of PCR inhibitor and repeated thawingand freezing leads to the loss of DNA in the biopsy material[32 33] Indeed low bacterial load patchy distribution ofbacteria in the mucosa and intake of PPI and antibiotics

have been found to negatively influence the outcome ofdiagnostic tests including PCR 0e PCR results were foundexactly same as the previous and confirmed the absence ofPCR inhibitors However none of the patients who had PCRpositive took antibiotics for any major or minor sicknessesprior to endoscopy and 3 (375) PCR-positive cases weretaking PPI in less than two weeks In case of PCR negativePPI was consumed by 28 (6363) patients and antibioticsby 5 patients (1136) in less than two weeks 0is indicatesthat the growth of H pylori could be inhibited by uptake ofPPI less than two week but more study with large samplesare required to show the significant association

Shetty et al showed that diagnostic the sensitivity ofmicroscopy was the highest (547) followed by PCR(545) and rapid urease test (RUT) (489) whereas theculture had sensitivity (291) Among different assays thePCR had shown the highest sensitivity and specificity [34]Due to resource constraints the culture could not be per-formed and the rate of false positivity in RUT refrained usfrom performing this test Moreover Lim et al showed thatrpoB PCR also showed highest positive rate (537) followedby glmM PCR (488) [35] Ruparelia et al showed that thecombination of serology and PCR had the highest sensitivity(100) rather than RUT (8181) in the Indian population[30] Moreover recently published report on PCR diagnosisof rhinopharyngeal tumor also had the consistent resultswith this work [36] 0erefore in absence of the goldstandard assay for identifying H pylori the combination ofdiagnostic assays could be applied in order to reduce thefalse-negative H pylori infection

5 Conclusion

Although PCR is more sensitive assay to detect H pyloriinfection than microscopy it is not yet considered as thegold standard assay 0erefore in order to improve the

Table 4 Comparison of diagnostic results for H pylori (n 52)

Microscopy PCR Frequency Agreementlowast Disagreement$ Kappa test (p value)Positive Positive 2

8654 1346 014Positive Negative 1Negative Positive 6Negative Negative 43lowastAgreement between microscopy and PCR $Disagreement between microscopy and PCR

Table 3 History of PPI and antibiotic intake in comparison with diagnostic test positive for H pylori

History of PPI intake Microscopy positive PCR positiveYes (n 31) 1 (322) 3 (967)No (n 21) 2 (952) 5 (238)Total (n 52) 3 (577) 8 (153)p value 002lowast 032History of antibiotic intakeYes (n 5) mdash mdashNo (n 47) 3 (638) 8 (170)Total (n 52) 3 (576) 8 (153)p value 1 073lowastStatistical significant at 005


diagnostic accuracy we recommend the combination ofmicroscopy and PCR assay for effective monitoring of Hpylori infection in endemic sites

Data Availability

0is is a hospital-based study Samples were collected duringthe routine diagnostic procedure and the samples were usedto evaluate the performance of PCR to find their role in theimplementation of diagnostics in hospital 0erefore thedata of the analysis are available upon the request from thecorresponding author or head department of Microbiology(hodmicrobiologybpkihsedu) BP Koirala Institute ofHealth Sciences Dharan Nepal


0e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

0e authors would like to thank all patients who gave theirconsent for this study and the Molecular Biology Laboratoryat the Department of Microbiology BPKIHS

References

[1] S Maeda and A F Mentis ldquoPathogenesis of Helicobacterpylori infectionrdquo Helicobacter vol 12 no 1 pp 10ndash14 2007

[2] S F Moss and P Malfertheiner ldquoHelicobacter and gastricmalignanciesrdquo Helicobacter vol 12 no 1 pp 23ndash30 2007

[3] D J McGee and H L T Mobley ldquoPathogenesis of Heli-cobacter pylori infectionrdquo Current Opinion in Gastroenter-ology vol 16 no 1 pp 24ndash31 2000

[4] H Cheng F Hu L Zhang et al ldquoPrevalence of Helicobacterpylori infection and identification of risk factors in ruraland urban Beijing Chinardquo Helicobacter vol 14 no 2pp 128ndash133 2009

[5] B Salih ldquoHelicobacter pylori infection in developing coun-tries the burden for how longrdquo Saudi Journal of Gastro-enterology vol 15 no 3 pp 201ndash207 2009

[6] T L Testerman and J Morris ldquoBeyond the stomach anupdated view of Helicobacter pylori pathogenesis diagnosisand treatmentrdquo World Journal of Gastroenterology vol 20no 36 pp 12781ndash12808 2014

[7] A Axon ldquoHelicobacter pyloriand public healthrdquo Heli-cobacter vol 19 no 1 pp 68ndash73 2014

[8] R H Hunt S D Xiao F Megraud et al ldquoHelicobacter pyloriin developing countries World gastroenterology organisationglobal guidelinerdquo Journal of Gastrointestinal and Liver Dis-eases vol 20 pp 299ndash304 2011


[10] T Fasciana G Capra C Cala et al ldquoHelicobacter pylori andepstein-barr co-infection in gastric diseaserdquo Pharmacolo-gyonline vol 1 pp 73ndash82 2017

[11] N 0apa K B Shah B B Bhandari et al ldquoAssociation ofHelicobacter pylori infection and stomach cancerrdquo MedicalJournal of Shree Birendra Hospital vol 12 no 2 pp 36ndash412013

[12] Public Health England ldquoStandards for microbiology in-vestigationsrdquo Investigation of Gastric Biopsies for Helicobacterpylori vol B55 no 6 pp 1ndash21 2014

[13] Promega Wizardreg Genomic DNA Purification Kit PromegaMadison WI USA 2017 httpsworldwidepromegacom

[14] C Schabereiter-Gurtner A M Hirschl B Dragosics et alldquoNovel real-time PCR assay for detection of Helicobacterpylori infection and simultaneous clarithromycin suscepti-bility testing of stool and biopsy specimensrdquo Journal ofClinical Microbiology vol 42 pp 4512ndash4518 2004

[15] R K Saiki S Scharf F Faloona et al ldquoEnzymatic amplifi-cation of beta-globin genomic sequences and restriction siteanalysis for diagnosis of sickle cell anemiardquo Science vol 230no 4732 pp 1350ndash1354 1992

[16] SPSS Inc SPSS 115 for Windows SPSS Chicago IL USA2002

[17] R Core Team R A Language and Environment for StatisticalComputing R Foundation for Statistical Computing ViennaAustria 2018

[18] H Russmann V A Kempf S Koletzko et al ldquoComparison offluorescent in situ hybridization and conventional culturingfor detection of Helicobacter pylori in gastric biopsy speci-mensrdquo Journal of Clinical Microbiology vol 39 no 1pp 304ndash308 2001

[19] K Trebesius K Panthel S Strobel et al ldquoRapid and specificdetection ofHelicobacter pylorimacrolide resistance in gastrictissue by fluorescent in situ hybridisationrdquo Gut vol 46 no 5pp 608ndash614 2000

[20] I B Ramis E P de Moraes M S Fernandes et al ldquoEval-uation of diagnostic methods for the detection ofHelicobacterpylori in gastric biopsy specimens of dyspeptic patientsrdquoBrazilian Journal of Microbiology vol 43 pp 903ndash908 2012

[21] F Jalalypour S Farajnia M H Somi et al ldquoComparativeevaluation of RUT PCR and ELISA tests for detection ofinfection with cytotoxigenic H pylorirdquo Advanced Pharma-ceutical Bulletin vol 6 pp 261ndash266 2016

[22] M Khalifehgholi F Shamsipour H Ajhdarkosh et alldquoComparison of five diagnostic methods for Helicobacterpylorirdquo Iranian Journal of Microbiology vol 5 pp 396ndash4012013

[23] F Siavoshi P Saniee S Khalili-Samani et al ldquoEvaluation ofmethods for H pylori detection in PPI consumption usingculture rapid urease test and smear examinationrdquo Annals ofTranslational Medicine vol 3 p 11 2015

[24] A D Roy S Deuri and U C Dutta ldquo0e diagnostic accuracyof rapid urease biopsy test compared to histopathology inimplementing ldquotest and treatrdquo policy for Helicobacter pylorirdquoInternational Journal of Applied and Basic Medical Researchvol 6 no 1 pp 18ndash22 2016

[25] O Rotimi A Cairns S Gray et al ldquoHistological identificationof Helicobacter pylori comparison of staining methodsrdquoJournal of Clinical Pathology vol 53 no 10 pp 756ndash7592000

[26] L Laine D N Lewin W Naritoku et al ldquoProspectivecomparison of HampE Giemsa and Genta stains for the di-agnosis of Helicobacter pylorirdquo Gastrointestinal Endoscopyvol 45 no 6 pp 463ndash467 1997

[27] F Khadangi M Yassi and M A Kerachian ldquoReview di-agnostic accuracy of PCR-based detection tests for Heli-cobacter Pylori in stool samplesrdquo Helicobacter vol 22 no 62017

[28] Z Zhen-Hua H De-Qiang X Yong et al ldquoCharacterizationof 23S rRNA gene mutation in primary and secondaryclarithromycin-resistant Helicobacter pylori strains from East


Chinardquo Turkish Journal of Gastroenterology vol 24 no 1pp 5ndash9 2013

[29] T N Archampong R H Asmah E K Aidoo et al ldquoHeli-cobacter pylori cagA and vacA genes in dyspeptic Ghanaianpatientsrdquo BMC Research Notes vol 10 no 1 p 231 2017

[30] J R Ruparelia N R Sodagar J S Patel et al ldquoComparison ofconventional diagnostic modalities with PCR for detection ofHelicobcter pylori infection in symptomatic patientsrdquo AsianJournal of Medical and Pharmaceutical Researches vol 3pp 105ndash110 2013

[31] M Sugimoto J Y Wu S Abudayyeh et al ldquoUnreliability ofresults of PCR detection of Helicobacter pylori in clinical orenvironmental samplesrdquo Journal of Clinical Microbiologyvol 47 no 3 pp 738ndash742 2009

[32] S A Chisholm R J Owen E L Teare et al ldquoPCR-baseddiagnosis of Helicobacter pylori infection and real-time de-termination of clarithromycin resistance directly from humangastric biopsy samplesrdquo Journal of Clinical Microbiologyvol 39 no 4 pp 1217ndash1220 2001

[33] S K Patel C B Pratap A K Jain et al ldquoDiagnosis ofHelicobacter pylori what should be the gold standardrdquoWorldJournal of Gastroenterology vol 20 no 36 pp 12847ndash128592014

[34] V Shetty M Ballal G Balaraju et al ldquoHelicobacter pylori indyspepsia phenotypic and genotypic methods of diagnosisrdquoJournal of Global Infectious Diseases vol 9 no 4 pp 131ndash1342017

[35] C Y Lim K H Lee M J Cho et al ldquoDetection of Heli-cobacter pylori in gastric mucosa of patients with gastrodu-odenal diseases by PCR-restriction analysis using the RNApolymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 41 no 7 pp 3387ndash3391 2003

[36] F Lorusso M P Caleca C Bellavia et al ldquo0e EBV-DNA canbe used as a diagnostic and follow-up parameter of theRhinopharyngeal tumors in the non-endemic population ofwestern Sicilyrdquo Indian Journal of Otolaryngology and Head ampNeck Surgery 2018 In press


Research ArticleEstimating the Force of Infection withHelicobacter pylori in Japan

Taishi Kayano1 Ki-Deok Lee12 and Hiroshi Nishiura 1

1Graduate School of Medicine Hokkaido University Kita 15 Jo Nishi 7 Chome Kitaku Sapporo 0608638 Japan2Department of Infectious Diseases School of Medicine Eulji University Seoul 01830 Republic of Korea

Correspondence should be addressed to Hiroshi Nishiura nishiurahgmailcom

Received 23 November 2018 Accepted 27 December 2018 Published 30 January 2019

Academic Editor Paola Di Carlo

Copyright copy 2019 Taishi Kayano et alis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Although the seroprevalence against Helicobacter pylori (H pylori) in Japan has declined over the birth year Japanesepeople have yet exhibited a relatively high risk of gastric cancer e present study employed mathematical models to estimate thetime- and age-dependent force of infection withH pylori in Japan predicting the future seroprevalence by time and ageMethods Weinvestigated the published seroprevalence data againstH pylori in Japan from 1980ndash2018 Solving theMcKendrick partial differentialequationmodel the seroprevalence wasmodeled as a function of survey year and ageMaximum likelihood estimation was conductedto estimate parameters governing the time- and age-dependent force of infection Results Among all fitted models the time-dependent and age-independent model with an exponentially decaying force of infection over years was most favored Fitted modelsindicated that the force of infection started to decrease during andor shortly after theWorldWar II Using the parameterized modelthe predicted fraction seropositive at the age of 40 years in 2018 was 022 but it is expected to decrease to 013 in 2030 and 005 in 2050respectively Conclusion e time dependence was consistent with the decline in the force of infection as a function of the birth yeare force of infection has continuously and greatly declined over time implying the diminished transmission ofH pylori through thetime course and small chance of persistence ese findings are critical to anticipate the future decline in gastric cancer incidence

1 Introduction

Helicobacter pylori (H pylori) is a bacterium known as themost important cause of gastric ulcer and cancer [1] ebacterium is a helix-shaped gram-negative microaerophiliccurved rod with four to six flagella at the same locationwhich is most commonly found in the stomach [2] Al-though the exact mode of transmission has yet to beclarified it is believed that the fecal-oral andor oral-oralroute are the most likely routes of transmission A majorityof people infected with H pylori do not exhibit any clinicalsigns or symptoms During the acute phase of infection thesymptoms associated with acute gastritis may occurMoreover chronic gastritis can lead to clinical symptomsthat are associated with nonulcer dyspepsia e long-lasting natural history of inflammation caused bychronic and atrophic gastritis is thought to be followed bycarcinogenesis and thus the gastric cancere pathogenicfactors of carcinogenesis such as VacA and CagA have been

identified and host genetic factors and several cytokinenetworks are proposed as the pathophysiological mecha-nism of cancer [3]

In the 21st century the incidence of gastric cancer inJapan has continuously declined over time in all age groupsHowever recent global estimates have indicated thatJapanese people have yet exhibited a relatively high risk ofgastric cancer [4] while the incidence of gastric cancer indeveloped regions have steadily declined As an underlyingexplanation of the high incidence of gastric cancer a highprevalence of H pylori in the elderly in Japan has beenconsidered as consistent with the natural history [1] On theother hand in many countries with decreased gastriccancer incidence over time the seroprevalence of H pylorihas abruptly declined in young age cohorts [5]

A multi-institutional study across Japan conducted byUeda and his colleagues [6] has demonstrated a monotonicdecline of H pylori seroprevalence by birth cohorts Simi-larly analyzing the annual health check-up data Hirayama


et al [7] have shown a continuous decline in H pyloriseroprevalence in Japan eg a decline from 46 amongthose born in 1940s to 18 among those born in 1970sMoreover another published clinical epidemiologic studyindicated that the seroprevalence in Japanese children wasless than 2 [8] Considering that the route of transmissionwith H pylori is likely associated with direct contact andhygienic conditions during the childhood decreased contactwith environment in early ages (eg reduced chance to swimin the pond and reduced chance of parent-to-child trans-mission via bathroom) may have occurred leading to thedecreased seroprevalence of H pylori even among adults

While numerous studies have already reported thedecreased seroprevalence of H pylori and also decreasedincidence of gastric cancer the hazard rate or the time-and age-dependent risk of infection with H pylori has yetto be explicitly reconstructed from the seroepidemio-logical data Published series of cross-sectional seroepi-demiological studies in Japan offer a unique opportunityto estimate the so-called force of infection ie the rate atwhich susceptible individuals are infected e presentstudy aims to devise mathematical models to estimate thetime- and age-dependent force of infection with H pyloriin Japan elucidating the transmission dynamics in thepast and predicting the future seroprevalence by time andage

2 Method

21 Epidemiological Data We investigated the seropre-valence data against H pylori in Japan from 1980ndash2018Although the present study does not strictly adhere to thestatement and officially acknowledged methodologicaldetails of the systematic review the following literaturereview was systematically conducted searching theMEDLINE and Web of Science databases using the fol-lowing search terms

ldquoSeroprevalence OR Seroepidemic OR Seropositive ORSerological OR Serosurvey ORIgGAND Helicobacter OR H pyloriAND Japanrdquo

All titles identified by the search strategy were in-dependently screened by two authors (TK and HN)Abstracts of potentially relevant titles were then reviewedfor eligibility and articles were selected for closer eval-uation if a description of the seroepidemiological study ofH pylori among the Japanese was available Clinical andepidemiological studies that rested on laboratory methodsother than serology and that offered nontractable seroe-pidemiological data over time or age were excludedBefore the present study there was a narrative reviewarticle by Inoue [1] and we added to our literature those

cited in the review but were missed by the above-mentioned systematic search

22 Time and Age Elements From each included paper weextracted the information over the number of positivenegative samples by survey year and age When the originaldata yielded the age information as only discrete agegroups we used the midpoint of age for modeling purposee birth year was calculated as survey year minus age Asthe total number of samples and the count of positivesamples are available we computed the 95 confidenceintervals (CI) of observed seroprevalence using a binomialdistribution

23 Mathematical Model e observed seropositive frac-tion represents the time- and age-specific history of thepast exposure We employ a mathematical model tocapture the time- and age-dependent transmission dy-namics of H pylori from the seroprevalence data and inparticular the present study jointly explores the time atwhich the rate of infection t0 started to decrease Lets(a t) be the fraction of susceptible individuals at age a

and year t Assuming that everyone is born susceptible toH pylori and discarding maternal antibodies theboundary condition would be s(0 t) 1 for any t Letλ(a t) be the force of infection ie the rate at whichsusceptible individuals experience infection which de-pends on age a and year t the susceptible individuals aredepleted by

z

za+

z

zt1113888 1113889s(a t) minusλ(a t)s(a t) (1)

We assume that the force of infection is separable to age-and time-components ie

λ(a t) f(a)g(t) (2)

Integrating both sides of equation (1) along the char-acteristic line we obtain

s(a t) s(0 tminus a)exp minus1113946t

tminusaλ(yminus t + a y) dy1113888 1113889

exp minus1113946t

tminusaf(yminus t + a)g(y) dy1113888 1113889

(3)

for tgt a Using the susceptible fraction s(a t) theseroprevalence at age a and in year t is obtained from1minus s(a t) To quantify the force of infection we imposethree different parametric assumptions First we assumethat g(t) was initially a constant λ0 but from the year t0has been exponentially decreasing with year tie g(t) λ0 exp(minusδ(tminus t0)) and also that the force ofinfection is age-independent We have the seroprevalencep(a t) parameterized as


p(a t)

1minus exp minus1113938t

tminusa λ0 exp minusδ sminus t0( 1113857( 1113857 ds1113872 1113873 for tminus age t0

1minus exp minusλ0 t0 minus t + a( 1113857minus 1113938t

t0λ0 exp minusδ sminus t0( 1113857( 1113857 ds1113874 1113875 for tminus alt t0

⎧⎪⎪⎨

⎪⎪⎩(4)

Hereafter we identify this model as model 1Alternatively following the year t0 we assume that g(t)

has experienced a time-dependent decay that follows the

Gompertz law with year t ie g(t) λ0 exp(minusβ(exp(c(tminus t0))minus 1)) and also that the force of infection is age-independent We identify it as model 2 and we have

p(a t)


tminusa λ0 exp minusβ exp c sminus t0( 1113857( 1113857minus 1( 1113857( 1113857 ds1113872 1113873 for tminus age t0


t0λ0 exp minusβ exp c sminus t0( 1113857( 1113857minus 1( 1113857( 1113857 ds1113874 1113875 for tminus alt t0

⎧⎪⎪⎨

⎪⎪⎩(5)

As model 3 we assume that g(t) has experienced atime-dependent exponential decay following t0 g(t) λ0exp(minusδ(tminus t0)) and throughout the course of time we also

assume an age-dependent exponential decayie f(a) exp(minusρa) e seroprevalence of model 3 isdescribed as

p(a t)


tminus aλ0 exp minusδ sminus t0( 1113857( 1113857exp(minusρ(sminus t + a)) ds1113872 1113873 for tminus age t0

1minus exp minus1113938t0

tminus aλ0 exp(minusρ(sminus t + a)) ds minus 1113938

t

t0λ0 exp minusδ sminus t0( 1113857( 1113857exp(minusρ(sminus t + a)) ds1113874 1113875 for tminus alt t0

⎧⎪⎪⎨

⎪⎪⎩(6)

In the above-mentioned models λ0 δ t0 β and ρ aredealt with as parameters to be estimated To quantify the forceof infection by estimating those parameters we employed alikelihood-based approach Given that there were mat pos-itive individuals among the total of serum samples drawnfrom nat individuals in age a and year t the likelihoodfunction to estimate parameter θ was modeled as

L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)

e 95CI of parameters were computed using the profilelikelihood Once all parameters are estimated we calculated thepredicted seroprevalence as a function of time and age es-pecially in years 2018 2030 and 2050 for the exposition of theadvantage of our approach to estimate the force of infection

3 Results

In total 10 seroprevalence studies were identified and usedin the following analyses (Figure 1) [1 5 7ndash17] Excludedseroepidemiological and clinical studies from the followinganalyses are a cohort study with variable timing of sero-prevalence surveys that cannot be traced back from theliterature [5] a clinical study that used urinary samples fortesting H pylori [17] and a study that missed the in-formation of age grouping in summarizing seroepidemio-logical datasets [15] All included publications measured theH pylori-IgG antibody at the population level in a cross-sectional manner by age groups e identified oldest survey

took place in 1974 while the latest study was conducted in2011 A small number of studies focused on particular agegroups especially on children recruiting those aged from0ndash11 years [9] and those at high school [14] Figure 1 revealsthat the seroprevalence drastically declined with birth yearand thus most likely with timee figure also indicated thatthe sample size was not large enough to distinguish theseroprevalence in one study from others in nearby birthcohorts involving many overlapping confidence intervals ofseroprevalence Moreover for each birth year the expectedvalue of the seroprevalence did not evidently increaseaccording to the order of survey year implying a limited ageeffect

Fitting three different models to the identified data wecompared the goodness of fit as informed by the Akaikeinformation criterion (AIC) (Table 1) Among all threemodels including those employing the time-dependent andtime- and age-dependent forces of infection the model 1with an exponential decay of the force of infection with timeand without age dependence yielded the minimum AIC andwas considered as the best fit model Figure 2 compares theobserved and predicted seroprevalence data by birth yearconfirming that the observed patterns were overall wellcaptured by the model 1 Due to the absence of age de-pendence the predicted value did not vary over the verticalaxis in Figure 2

Figure 3 shows the estimated force of infection with Hpylori as a function of calendar time in Japan using models 1and 2 Qualitatively the predicted values of models 1 and 2were close to each othere estimated year in which the forceof infection was considered to have started to decrease was


estimated at 1937 in model 1 while that of model 2 was 1945the year corresponding to the end of the World War II Es-timated parameters of the best fittedmodel 1 λ0 δ and t0 were0056 (95 CI 0048 0065) per year 0047 (95 CI 00450050) per year and 1937 (95 CI 1933 1940) respectively

Figure 4 shows the seroprevalence against H pylori as afunction of age in the past and the future in Japan In

Figure 4(a) the seroprevalence has exhibited a sigmoidalshape to increase as a function of age While a part of the pastobserved data look not well aligned with the predicted values(eg those in 1984) the observed points with small serologicalsamples suffered from broad uncertainty (ie wide confidenceintervals) and the predicted values in general well capturedthe observed patterns of the seroprevalence data by time

00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion

Figure 1 Seroprevalence of anti-Helicobacter pylori antibody in Japan by birth year Antibody positive fraction is reviewed as a function ofbirth year Same marks represent the dataset arising from an identical publication in the same survey year Whiskers extend to lower andupper 95 confidence intervals

Table 1 Model comparison of the time- and age-dependent force of infection to capture the transmission dynamics of Helicobacterpylori in Japan

Model identity Functional assumption Number of parameters AICModel 1 Time-dependent FOI with an exponential decay 3 9372Model 2 Time-dependent FOI with a Gompertz-type decay 4 38563

Model 3 Time- and age-dependent FOI with an exponentialtime-decay and exponential age-decay 4 27505

AIC Akaike information criterion FOI force of infection

0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)

Figure 2 Comparison between observed and predicted seroprevalence against Helicobacter pylori in Japan by birth year Observed data(filled marks) in various surveys are plotted by birth year and compared against model prediction (unfilled marks) that assumes timedependence in the force of infection with an exponential decay Predictions were made as a function of survey year and age


and age When the future seroprevalence was predicted(Figure 4(b)) the seroprevalence revealed a clear pattern ofright shift over age by the year of prediction (ie the elevationof age at infection over future years is anticipated) For in-stance the predicted fraction seropositive at the age of40 years in 2018 would be 022 but it is expected to decrease to013 in 2030 and 005 in 2050 respectively

4 Discussion

e present study explored the long-term dynamics of Hpylori infection in Japan estimating the force of infection

from a total of 10 different seroepidemiological surveydatasets Fitting time-dependent and time- and age-dependent models the time-dependent force of infectionwith an exponential decline was selected as the best fitmodel Fitted models indicated that the force of infectionstarted to decrease during andor shortly after the WorldWar II Subsequently the force of infection was consideredto have steadily declined over time Using the parameterizedmodel the age of seropositive individuals was predicted tobe greatly shifted to older groups in the future which wouldbe more evident than in the past To our knowledge thepresent study is the first to elucidate the time-dependent

0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)

Figure 3 Estimated force of infection of Helicobacter pylori as a function of calendar time in Japan Model 1 (bold straight line) is theestimate of time-dependent force of infection with an exponential decay Model 2 (dashed line) is the estimate of time-dependent force ofinfection with Gompertz-type decay

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)

Observed (1984)Observed (1991)Observed (1994)Observed (2008)

Predicted (1984)Predicted (1991)Predicted (1994)Predicted (2008)

Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)

Prediction (2018)Prediction (2030)Prediction (2050)

Sero

posit

ive f

ract

ion

(b)

Figure 4 Prediction of the seroprevalence againstHelicobacter pylori in the past and the future in Japan (a) Comparison between observedand predicted seroprevalence by age and survey year Marks represent observed data while lines are the expected values derived from thetime-dependent force of infection with an exponential decay (b) Prediction of the future seroprevalence againstHelicobacter pylori in JapanGradual right shift in the seroprevalence is captured by our time-dependent force of infection with an exponential decay


dynamics of H pylori in Japan offering predictions of se-ropositivity in 2030 and 2050

e time dependence was consistent with the decline inthe force of infection as a function of birth year (Figure 1) andwithout many variations over vertical axis reflecting limitedvariations over age Such decline with birth year has also beenseen in other settings eg China [18] Using themathematicalmodel and the force of infection the present study achieved totranslate the decreasing pattern into the time-dependentdecline in the hazard rate of infection and also permittedthe future prediction of seroprevalence e present studyendorses the long-lasting notion of expert on this subject(eg a hypothesis by Blaser [19]) the seroprevalence con-tinuously and greatly declines over time implying the di-minished transmission over time and small chance ofpersistence ese findings are critical to anticipate the futuredecline in gastric cancer which have already been studiedusing mathematical models [20 21]

While the time- and age-dependent model was notselected as the best model it should be noted that thepresent study did not exclude the possibility that there isstrong age dependence in the force of infection especiallyamong young children In fact the age effect was stronglyindicated in the literature [22ndash26] and as we noted fromFigure 1 the uncertainty bound of seroprevalence in eachbirth year was very broad and the sampling error was notavoidable Both empirical and theoretical studies clearlydemonstrate that the infection mostly occurs in children[22 23] and this notion is consistent with our age-dependent term of model 3 ie as a function of ageand the force of infection was considered to have decreasedexponentially with the rate of 057 per year indicating thatthe average age at infection was about 2 years e in-fections would therefore perhaps be mostly seen amongchildren and additional studies with more precise obser-vation with greater sample size would be essential to betteridentify the age dependence

Apart from the predicted future course of the sero-prevalence it is natural to wonder how the epidemiology ofgastric cancer will behave in the future Not only theseroprevalence but also the future demographic dynamicseg aging and decreasing population would be a highlyinfluential factor in regulating the incidence of cancer inthe future Estimating the induction period ie the timefrom exposure to H pylori to the cancer developmentthe future incidence of gastric cancer will become pre-dictable by employing a mathematical modeling approachIn line with this intent another interesting direction is toexplore the epidemiological impact of eradication therapyof H pylori on the gastric cancer incidence in an explicitmanner

Four limitations must be noted First the present studyrested on the review of collected seroprevalence studies indifferent geographic locations Depending on the geographicregion in Japan environmental conditions that can lead toinfection can be different (eg urban vs rural) but we col-lectively analyzed all the datasets as serial cross-sectional datain a single population Second even though we performed thesystematic search of literature and minimized the sampling

error the sample size was small for each age group in eachindividual study and the sampling error was substantial inquantifying the age effect ird fixed cutoffs used by a se-rological assay might have resulted in underascertainment ofseropositive individuals and it can sacrifice specificity whilesensitivity is ensured to be high [27] Fourth the force ofinfection is sometimes modeled as a function of the preva-lence of infectious individuals but the present study did notfully disentangle the underlying transmission dynamics bydecomposing the force of infection into multiple mathe-matical functions

Besides without proper use of mathematical models onecannot clarify the hazard rate of infection and predict thefuture seroprevalence [28ndash33] In many countries the declinein seroprevalence has been observed over birth cohort[1 4 5 22 34] and the present study uniquely and suc-cessfully identified the strong signature of the time de-pendence in the force of infection in Japan To further predictthe future size of gastric cancer we trust that the present studysubstantially contributes to building the foundation for suchsophisticated exercise

Data Availability

e data used to support the findings of this study are availablefrom the corresponding author upon request

Disclosure

e funders had no role in the study design data collectionand analysis decision to publish or preparation of themanuscript


e authors declare that there are no conflicts of interest

Acknowledgments

HN received funding support from the Japan Agency forMedical Research and Development (JP18fk0108050) theJapan Society for the Promotion of Science KAKENHI (grantnos 16KT0130 17H04701 17H05808 and 18H04895) theInamori Foundation the Telecommunication AdvancementFoundation and the Japan Science and Technology Agency(JST) CREST Program (JPMJCR1413)

References

[1] M Inoue ldquoChanging epidemiology of Helicobacter pylori inJapanrdquo Gastric Cancer vol 20 no 1 pp 3ndash7 2016

[2] B Marshall and J R Warren ldquoUnidentified curved bacilli inthe stomach of patients with gastritis and peptic ulcerationrdquo6e Lancet vol 323 pp 1311ndash1315 1984

[3] T Chiba H Marusawa H Seno and N WatanabeldquoMechanism for gastric cancer development by Helicobacterpyloriinfectionrdquo Journal of Gastroenterology and Hepatologyvol 23 no 8 pp 1175ndash1181 2008

[4] J K Y Hooi W Y Lai W K Ng et al ldquoGlobal prevalence ofHelicobacter pylori infection systematic review and meta-


analysisrdquo Gastroenterology vol 153 no 2 pp 420ndash4292017

[5] M Watanabe H Ito S Hosono et al ldquoDeclining trends inprevalence of Helicobacter pyloriinfection by birth-year in aJapanese populationrdquo Cancer Science vol 106 no 12pp 1738ndash1743 2015

[6] International Agency for Research on Cancer (IARC) ldquoIARCHelicobacter pylori Working Group Helicobacter pyloriEradication as a strategy for preventing gastric cancerrdquo inIARC Working Group Reports vol 8 pp 1ndash181 InternationalAgency for Research on Cancer Lyon France 2014

[7] J Ueda M Gosho Y Inui et al ldquoPrevalence of HelicobacterpyloriInfection by birth year and geographic area in JapanrdquoHelicobacter vol 19 no 2 pp 105ndash110 2014

[8] Y Hirayama T Kawai J Otaki K Kawakami and Y HaradaldquoPrevalence of Helicobacter pyloriinfection with healthysubjects in Japanrdquo Journal of Gastroenterology and Hepatol-ogy vol 29 no 4 pp 16ndash19 2014

[9] M Okuda T Osaki Y Lin et al ldquoLow prevalence and in-cidence of Helicobacter pyloriInfection in children apopulation-based study in Japanrdquo Helicobacter vol 20 no 2pp 133ndash138 2014

[10] T Fujisawa T Kumagai T Akamatsu K Kiyosawa andY Matsunaga ldquoChanges in seroepidemiological pattern ofHelicobacter pylori and hepatitis A virus over the last 20 yearsin Japanrdquo 6e American Journal of Gastroenterology vol 94no 8 pp 2094ndash2099 1999

[11] H M Malaty E Tanaka T Kumagai et al ldquoSeroepidemi-ology of Helicobacter pylori and hepatitis A virus and themode of transmission of infection a 9-year cohort study inrural Japanrdquo Clinical Infectious Diseases vol 37 no 8pp 1067ndash1072 2003

[12] T Kumagai H M Malaty D Y Graham et al ldquoAcquisitionversus loss of Helicobacter pyloriInfection in Japan resultsfrom an 8-year birth cohort studyrdquo Journal of InfectiousDiseases vol 178 no 3 pp 717ndash21 1998

[13] M Asaka T Kimura M Kudo et al ldquoRelationship of Hel-icobacter pylori to serum pepsinogens in an asymptomaticJapanese populationrdquo Gastroenterology vol 102 no 3pp 760ndash766 1992

[14] T Akamatsu S Ichikawa S Okudaira et al ldquoIntroduction ofan examination and treatment for Helicobacter pylori in-fection in high school health screeningrdquo Journal of Gastro-enterology vol 46 no 12 pp 1353ndash1360 2011


[16] S Shiota K Murakami T Fujioka and Y YamaokaldquoPopulation-based strategies for Helicobacter pylori-associ-ated disease management a Japanese perspectiverdquo ExpertReview of Gastroenterology and Hepatology vol 4 no 2pp 149ndash156 2014

[17] T Tamura E Morita T Kondo et al ldquoPrevalence of Heli-cobacter pylori infectionmeasured with urinary antibody in anurban area of Japan 2008-2010rdquo Nagoya Journal of MedicalScience vol 74 no 1-2 pp 63ndash70 2012

[18] X Yu X Yang T Yang Q Dong L Wang and L FengldquoDecreasing prevalence of Helicobacter pylori according tobirth cohorts in urban Chinardquo 6e Turkish Journal of Gas-troenterology vol 28 no 2 pp 94ndash97 2017

[19] M J Blaser ldquoHypothesis the changing relationships ofHelicobacter pyloriand humans implications for health anddiseaserdquo Journal of Infectious Diseases vol 179 no 6pp 1523ndash1530 1999

[20] H B Gershengorn and S M Blower ldquoModeling cancer as aninfectious disease the epidemiology ofHelicobacter pylorirdquo inMathematical Approaches for Emerging and Reemerging In-fectious Diseases C Castillo-Chavez Ed pp 325ndash339Springer New York NY USA 2001

[21] M Rupnow R D Shachter D K Owens and J Parsonnet ldquoAdynamic transmission model for predicting trends in Heli-cobacter pylori and associated diseases in the United StatesrdquoEmerging Infectious Diseases vol 6 no 3 pp 228ndash237 2000

[22] H M Malaty A El-Kasabany D Y Graham et al ldquoAge atacquisition of Helicobacter pylori infection a follow-up studyfrom infancy to adulthoodrdquo 6e Lancet vol 359 no 9310pp 931ndash935 2002

[23] F Alarid-Escudero E A Enns R F MacLehose J ParsonnetJ Torres and K M Kuntz ldquoForce of infection of Helicobacterpylori in Mexico evidence from a national survey using ahierarchical Bayesian modelrdquo Epidemiology and Infectionvol 146 no 8 pp 961ndash969 2018

[24] S J O Veldhuyzen van Zanten P T Pollak L M BestG S Bezanson and T Marrie ldquoIncreasing prevalence ofHelicobacter pylori infection with age continuous risk ofinfection in adults rather than cohort effectrdquo Journal of In-fectious Diseases vol 169 no 2 pp 434ndash437 1994

[25] M Rowland L Daly M Vaughan A Higgins B Bourke andB Drumm ldquoAge-specific incidence of Helicobacter pylorirdquoGastroenterology vol 130 no 1 pp 65ndash72 2006

[26] B Zabala Torrres Y Lucero A J Lagomarcino et al ldquoReviewprevalence and dynamics of Helicobacter pylori infectionduring childhoodrdquo Helicobacter vol 22 no 5 article e123992017

[27] G Kafatos N J Andrews K J McConway P A C MapleK Brown and C P Farrington ldquoIs it appropriate to use fixedassay cut-offs for estimating seroprevalencerdquo Epidemiologyand Infection vol 144 no 4 pp 887ndash895 2015

[28] Y Hamaguchi T Yamaguchi and H Nishiura ldquoEstimatingthe annual risk of tuberculosis infection in Japan frominterferon-gamma release assay datardquo Journal of 6eoreticalBiology vol 460 pp 125ndash133 2019

[29] B Yuan and H Nishiura ldquoEstimating the actual importationrisk of dengue virus infection among Japanese travelersrdquo PLoSOne vol 13 no 6 Article ID e0198734 2018

[30] K Nabae H Satoh H Nishiura et al ldquoEstimating the risk ofparvovirus B19 infection in blood donors and pregnantwomen in Japanrdquo PLoS One vol 9 no 3 Article ID e925192014

[31] K Yoshii R Kojima and H Nishiura ldquoUnrecognized sub-clinical infection with tickborne encephalitis virus JapanrdquoEmerging Infectious Diseases vol 23 no 10 pp 1753-17542017

[32] R Kinoshita and H Nishiura ldquoAssessing age-dependentsusceptibility to measles in Japanrdquo Vaccine vol 35 no 25pp 3309ndash3317 2017

[33] R Kinoshita and H Nishiura ldquoAssessing herd immunityagainst rubella in Japan a retrospective seroepidemiologicalanalysis of age-dependent transmission dynamicsrdquo BMJOpen vol 6 no 1 article e009928 2016

[34] S E Roberts S Morrison-Rees D G Samuel K orneA Akbari and J G Williams ldquoReview article the prevalenceof Helicobacter pyloriand the incidence of gastric canceracross Europerdquo Alimentary Pharmacology and 6erapeuticsvol 43 no 3 pp 334ndash345 2015


Research ArticleHelicobacter pylori Infection and Its Risk Factors A ProspectiveCross-Sectional Study in Resource-Limited Settings ofNorthwest Ethiopia

Markos Negash 1 Habtamu Wondifraw Baynes 2 and Demeke Geremew1

1Department of Immunology and Molecular Biology School of Biomedical and Laboratory SciencesCollege of Medicine and Health Sciences University of Gondar Gondar Ethiopia2Department of Clinical Chemistry School of Biomedical and Laboratory Sciences College of Medicine and Health SciencesUniversity of Gondar Gondar Ethiopia

Correspondence should be addressed to Markos Negash markosnegashyahoocom

Received 19 July 2018 Accepted 19 September 2018 Published 18 October 2018


Copyright copy 2018 Markos Negash et al is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Helicobacter pylori (H pylori) is implicated for the causation of gastrointestinal tract infections including gastriccancer Although the infection is prevalent globally the impact is immense in countries with poor environmental and socio-economic status including Ethiopia Epidemiological study on the magnitude of H pylori and possible risk factors has pricelessimplication erefore in this study we determined the prevalence and risk factors of H pylori infection in the resource-limitedarea of northwest Ethiopia Methods A prospective cross-sectional study was conducted on northwest Ethiopia among 201systematically selected dyspeptic patients Data were collected using a structured and pretested questionnaire and stool and serumsamples were collected and analyzed by SD BIOLINEH pylori Ag and dBestH pyloriDisk tests respectively Chi-square test wasperformed to see association between variables and binary and multinomial regression tests were performed to identify potentialrisk factors P values lt005 were taken statistically significant Result Prevalence of H pylori was found to be 711 (143201) and373 (75201) using the dBest H pylori Test Disk and SD BIOLINE H pylori Ag test respectively H pylori seropositivity usingdBestH pyloriDisk tests is significantly associated in age groups lt10 years (P 0044) and married patients (P 0016) In thosepatients with H pylori (a positive result with either the Ab or Ag test) drinking water from well sources had 223 times risk ofgetting H pylori infection (P 0017) and drinking coffee (151 (079ndash296 P 0025)) and chat chewing (178 (102ndash346P 0008) are the common risk factors Conclusion e present study discovered considerable magnitude ofH pylori among thedyspeptic patients in the study area H pylori infection is frequent in individuals drinking water from well sources and thus poorsanitation and unhygienic water supply are contributing factors Policies aiming at improving the socioeconomic status willreduce potential sources of infection transmission and ultimately the prevalence and incidence of H pylori

1 Background

Helicobacter pylori (H pylori) was the first formally rec-ognized bacterial carcinogen It has been etiologically as-sociated with gastritis peptic ulcer disease gastricadenocarcinoma and primary gastric lymphoma [1 2]

Helicobacter pylori (H pylori) colonizes 70ndash90 of thepopulation in developing countries whereas it is around50 in developed countries [3ndash5] In developing countriesan early childhood acquisition of H pylori (30ndash50)

reaching over 90 during adulthood is the pattern of in-fection Unless treated colonization persists lifelong Hpylori infection has been attributed to poor socioeconomicstatus poor hygienic practice and overcrowding condition[6 7] a whole mark in developing countries

e bacterium differs genetically survives in harsh acidicgastric environment and currently develops resistance forseveral antibiotics Although epidemiological distribution ofH pylori varies globally the magnitude ofH pylori has beenshown to be 701 (Africa) 694 (South America) 666


(Western Asia) 343 (Western Europe) and 371 (NorthAmerica) [8ndash10]

e prevalence of H pylori in the Ethiopian dyspepticpatients is similarly high to other developing countriesbecause most Ethiopian population live in households withlow socioeconomic status and hygiene [7 11 12] Magnitudeof H pylori among the outpatient department (based ona test kit detecting Immunoglobulin G (IgG) antibodies) atthe University of Gondar Hospital (UOG Hospital) wasranged between 657 and 856 [13 14] Besides it isa common reason to seek primary healthcare service andaccounts for 10 of hospital admissions [15 16]

All previous prevalence researches in the study area wereconducted using IgG andor IgM antibody rapid tests whichhave questionable performance in detecting acute infectionand distinguishing active infection from previous exposureHence the current study was conducted with an aim todetermine the prevalence of H pylori infection among thedyspeptic patients attending the UOG hospital in northwestEthiopia using stool antigen as well as serum antibodiestechnique and assessing potential risk factors

2 Methods

21 Study Design Period and Area is is a facility-basedcross-sectional study which was conducted on patients withdyspepsia from February to March 2016 at the University ofGondar Hospital Gondar Ethiopia e University ofGondar Hospital is one of the pioneer teaching hospitals inEthiopia conducting community-based researches pro-viding teaching and diagnostic services for more than 5million inhabitants

22 Study Participants and Clinical Data Collection Afterinformed consent was taken from the dyspeptic patientswho visited the hospital outpatient department suspected ofH pylori infection all relevant clinical and sociodemo-graphic data were collected using a structured and pretestedquestionnaire by trained data collectors

23 Specimen Collection and Processing Stool and bloodspecimens were collected from each patient for H pyloriantigen and antibody tests respectively e blood wascentrifuged until serum is separated and stored in minus20degCe stool specimens were also stored in minus20degC until the testswere performed For this study we followed the methods ofNegash et al [17] which has been evaluated four H pyloridiagnostic tests in the study area

231 SD Bioline H pylori Ag Test (Standard Diagnostic IncKorea) Principle the SD BIOLINE H pylori Ag rapid testkit result window has 2 precoated lines ldquoTrdquo (Test Line) andldquoCrdquo (Control Line) Both the Test Line and the Control Linein the result window are not visible before applying anysamples e ldquoTrsquordquo window coated with monoclonal anti-Hpyloriwill form a line after the addition of the stool specimen(if there isH pylori antigen)e Control window is used for

the procedural control and a line should always appear if thetest procedure is performed correctly and the test reagentsare working [17]

232 dBest H pylori Test Disk (Ameritech DiagnosticReagent Co Ltd Tongxiang Zhejiang China) Principlethis test contains a membrane strip which is precoated withH pylori capture antigen on the test band region e Hpylori antigen-colloid gold conjugate and serum samplemoves along the membrane chromatographically to the testregion (T) and forms a visible line as the antigen-antibody-antigen gold particle complex forms is test device hasa letter of T and C as ldquoTest Linerdquo and ldquoControl Linerdquo on thesurface of the case Both the test line and control line in theresult window are not visible before applying any samplese control line is used for the procedural control Controlline should always appear if the test procedure is performedproperly and the test reagents of the control line are working[17]

24 Statistical Analysis e data were cleaned and doubleentered on the excel spread sheet and transported to Sta-tistical Package for Social Sciences (SPSS) e chi-squaretest was performed to see association between dependentand independent variables Binary logistic regression andmultinomial regression tests were performed to identifypotential risk factors of H pylori infection P value less than005 were considered statistically significant

3 Result

31 Demographic Characteristics A total of 201 dyspepticpatients were included in the study and serum and stoolsamples were analyzed by dBest H pylori Test Disk and SDBIOLINE H pylori Ag tests respectively e mean plusmn SD(range) age of the participants was 295 plusmn 1485 (7ndash85) yearswith a median of 23 years e majority (140) of the studyparticipants were male (69) study subjects from the urbanarea (141) accounted 70 and 69 (343) of the participantswere married Of 201 participants 104 (51) were students38 (189) were farmers and 23 (114) were house wives(Table 1) In this study participants who were diagnosed aspositive to the H pylori stool antigen test were immediatelycommenced appropriate therapy

32 Prevalence of H pylori with respect to Sociodemography ofParticipants Accordingly the prevalence of H pylori wasfound to be 711 (143201) and 373 (75201) using thedBest H pylori Ab Test Disk (95 CI 642ndash776) and SDBIOLINE H pylori Ag test (95 CI 303ndash443) respectively(Table 2) e highest prevalence of H pylori infection wasseen among the males than the females (98 vs 45 by Ab testand 79 vs 27 by Ag test) and H pylori is more frequent inindividuals living from the urban area than rural (101 vs 42using the Ab test and 76 vs 30 using the Ag test) respectivelyRegarding the occupational status the students are themajority groups who come up positive for H pylori (both in


the Ab and Ag tests) than others and meanwhile H pyloriseropositivity using the dBest H pylori Disk tests is signif-icantly associated with the age groups lt10 years (P value

0044) and married patients (P value 0016) (Table 1)

33 H pylori Infection across Clinical Parameters and Asso-ciated Risk Factors Clinically the patients with heartburnabdominal fullness and belching had come up with positivefor the H pylori tests and likewise belching is significantlyassociated (P 0038) in logistic regression with the an-tibody test In those patients with H pylori (a positive resultwith either a Ab or Ag test) drinking water from wellsources had 223 times risk of getting H pylori infection(P 0017) and drinking coffee (151 (079ndash296 P 0025)and chat chewing (178 (102ndash346 P 0008) are the mostcommon risk factors (Tables 3 and 4)

4 Discussion

A recent study demonstrated that 653 of the patients werepositive for H pylori IgG using the immunochromato-graphic method [13] is shows that the current prevalenceofH pylori infection based on antibodies is much lowerecurrent 373 magnitude of H pylori using the SDBIOLINEH pylori Ag test is lower than a 523 and 53 ofreport from Ethiopia [18 19] and studies from African andAsian countries [20ndash22] e variation for these findingsmight be the difference in the socioeconomic factors ex-posure for risk factors study settings and essentially thevariability in the diagnostic methods

e present study revealed that H pylori seropositivityhas been associated with age In developing nations wherethe majority of children are infected before the age of 10 theprevalence in adults peaks at more than 80 before age 50[23ndash25] While in developed countries evidence of infectionin children is unusual but becomes more common later onadulthood In this study the increment in serological pos-itivity of H pylori is seen starting from children throughadulthood which reaches the peak on 18ndash30 age groups (68(557)) but cases are becoming lower as the age gets olderand older Within any age group infection appears to bemore common in blacks and Hispanics compared to thewhite population these differences are probably in partrelated to socioeconomic factors [26 27]

Table 1 Prevalence ofH pylori infection among the dyspeptic patients across sociodemographic characteristics at the University of GondarHospital Outpatient Department N 201

Sociodemographic characteristics Positive for the Ab test N () Positive for the Ag test N () Total N ()

Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)

lt10 1 (50)lowast 1 (50) 2 (1)10ndash19 10 (556) 12 (667) 18 (9)20ndash29 82 (672) 68 (557) 122 (606)30ndash39 12 (667) 6 (333) 18 (9)40ndash49 13 (929) 9 (643) 14 (7)50ndash59 12 (923) 5 (385) 13 (64)ge60 13 (929) 5 (357) 14 (7)

Residence Urban 101 (716) 76 (539) 141 (701)Rural 42 (70) 30 (50) 60 (299)

Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)

Government 14 (636) 8 (364) 22 (11)House wife 17 (739) 9 (391) 23 (114)Merchant 7 (875) 4 (50) 8 (4)No jobs 2 (333) 2 (333) 6 (3)

Education

Illiterate 40 (80) 24 (48) 50 (249)Primary 12 (632) 8 (421) 19 (95)Secondary 15 (625) 10 (417) 24 (119)College 76 (704) 64 (493) 108 (537)

Marital status Married 56 (812)lowastlowast 34 (493) 69 (343)Single 87 (659) 72 (545) 132 (657)

Number of siblings0 94 (671) 80 (571) 140 (697)1ndash4 29 (763) 15 (395) 38 (189)5ndash10 20 (87) 11 (478) 23 (114)

N number Ag antigen Ab antibody lowastP value 0044 lowastlowastP value 0016

Table 2 Prevalence of H pylori infection among the dyspepticpatients attending the University of Gondar Hospital OutpatientDepartment N 201

Serologic testsPrevalence of H pylori

N Percent () SE 95 CIdBest H pylori Ab rapid test 143 711 32 642ndash776SD BIOLINE H pylori Ag test 75 373 35 303ndash443N number SE standard error CI confidence interval Ag antigenAb Antibody


e increased prevalence of infection with age wasinitially thought to represent a continuing rate of bacterialacquisition throughout onersquos lifetime However epidemio-logic evidence now indicates most infections are acquiredduring childhood even in developed countries [24 28] Mostinfections were acquired before five years of age with a de-clining incidence thereafter in one report from Ireland [29]us the frequency of H pylori infection for any age groupin any locality reflects that particular cohortrsquos rate of bac-terial acquisition during childhood years [28] e organ-isms can be cultured from vomitus or diarrheal stoolssuggesting the potential for transmission among familymembers during periods of illness [30 31]

e risk of acquiring H pylori infection is related to thesocioeconomic status and living conditions early in lifeFactors such as density of housing overcrowding number ofsiblings sharing a bed and lack of running water have allbeen linked to a higher acquisition rate ofH pylori infection[32ndash34] Our study proved that risk factors for acquiring Hpylori infection are most prevalent in the patients with Hpylori infection Moreover studies in the developingcountries continue to show that childhood hygiene practicesand family education determines the prevalence of H pyloriinfection [35] In this study illiterate individual accounts themajority (40143 were positive for H pylori Ab and 24106were positive for H pylori Ag tests) of H pylori cases next tothose who visited college e association of H pylori in-fection with the level of education income andraceethnicity is not unique to H pylori since similar as-sociations have been described with other chronic infectionsincluding cytomegalovirus herpes simplex virus-1 and

hepatitis B [36] Studies indicated that declination of Hpylori infection has been attributed to economic progressand improvement in sanitation [37]is study revealed thatmost (101143 (antibody) 76106 (antigen)) H pylori pos-itive cases are from the urban areas indicating that urban-ization accompanied with poor sanitation

e route by which infection occurs remains unknownbut multiple ways of transmission are reported [38 39]Person-to-person transmission of H pylori through eitherfecaloral or oraloral seems most likely [31 39] Humansappear to be the major reservoir of infection however Hpylorus has been isolated from primates in captivity andfrom domestic cats [40 41] One report described theidentification of H pylori in milk and gastric tissue of sheepsuggesting that sheep may be a natural host for the organism[42]is may explain the higher infection rate that has beenobserved among shepherds compared to their siblings [43]Similarly in our study form the totalH pylori cases farmersaccounted the second highest proportion showing that closecontact with domestic cattle may potentially result H pyloritransmission

In addition to fecaloral transmission of bacteria con-taminated water supplies in developing countries may serveas an environmental source of bacteria In this study ma-jority (111143 (antibody) 86106 (antigen)) of H pyloripositive individuals use water sources from pipeline eorganism remains viable in water for several days and usingthe polymerase chain reaction techniques evidence of Hpylori can be found inmost samples of municipal water fromthe endemic areas of infection [44ndash46] Children who reg-ularly swim in rivers streams and pools drink stream water

Table 3 Prevalence of H pylori infection among the dyspeptic patients across risk factors at the University of Gondar Hospital OutpatientDepartment N 201

Risk factors Positive for the Ab test N () Positive for the Ag test N () Total N () MultivariateP valueOR (95 CI)

Water sourcePipeline 111 (694) 86 (538) 160 (796)

223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)

Washing handswith soap 87 (731) 63 (529) 119 (592) 104 (039ndash29) 0743

Using toilet 69 (734) 49 (521) 94 (468) 180 (062ndash648) 0496Drinking alcohol 40 (656) 34 (557) 61 (303) 102 (048ndash29) 0949Drinking coffee 69 (742) 51 (548) 93 (463) 151 (079ndash296) 0025Chat chewing 4 (80) 4 (80) 5 (25) 178 (102ndash346) 0008

Table 4 Prevalence of H pylori infection among the dyspeptic patients across clinical parameters at the University of Gondar HospitalOutpatient Department N 201

Clinical parameters Positive for the Ab test N () Positive for the Ag test N () Total N ()Heartburn 139 (706) 104 (472) 197 (98)Epigastric pain 139 (709) 103 (526) 196 (975)Abdominal fullness 133 (704) 101 (534) 189 (94)Vomiting 51 (729) 41 (586) 70 (348)Nausea 106 (711) 83 (557) 149 (741)Belching 110 (714) 75 (487)lowast 154 (766)Melena 43 (717) 27 (45) 60 (299)Bloody vomiting 14 (875) 12 (75) 16 (8)N number Ab antibody Ag antigen lowastP value 0038


or eat uncooked vegetables are more likely to be infected[47] H pylori have been cultured from diarrheal stools ofchildren in Gambia West Africa where almost all in-habitants are infected by five years of age [48]

Intrafamilial clustering of infection further supportsperson-to-person transmission Infected individuals aremore likely to have infected spouses and children thanuninfected individuals [34 49] A study of children inColumbia found that the risk of infection correlated directlywith the number of children aged 2 to 9 in the householdwhile younger children were more likely to be infected ifolder siblings were also infected [50] Isolation of geneticallyidentical strains of H pylori from multiple family members[51] and custodial patients in the same institution [52] andfurther studies support transmission among persons sharingthe same living environment In addition to the familial typeof transmission that occurs in developed and other nationshorizontal transmission between persons who do not belongto a core family also appears to take place in countries wherethe prevalence of infection is high [49] As revealed bystudies conducted on Ethiopia and ailand [14 53 54] Hpylori infection is higher in married individuals demon-strating that cluster living environment has an impact on Hpylori transmission

At last it should be considered that the dyspepticpatients other than the present serum antibody and stoolantigen tests did not undergo further confirmatory tests(endoscopy with biopsy for the histology culture andorthe very least urea breath test) due to economicconstraints

5 Conclusion

e present study discovered considerable magnitude of Hpylori in the study area H pylori infection is frequent inindividuals drinking water from well sources and thus poorsanitation and unhygienic water supply are contributingfactors Policies aiming at improving the socioeconomicstatus will reduce potential sources of infection trans-mission and ultimately the prevalence and incidence of Hpylori infection

Abbreviations

Ab AntibodyAg AntigenIgG Immunoglobulin GIgM Immunoglobulin MIRB Institutional Review BoardSD Standard deviationSPSS Statistical Package for Social SciencesUOG University of Gondar Hospital

Data Availability

e dataset supporting the conclusions of this article isincluded within the article

Ethical Approval

is project was ethically cleared by the Institutional ReviewBoard (IRB) of the University of Gondar Participation wasvoluntary and informed verbal consent was taken from alladult participants and from the next of kin caretakers orguardians on behalf of the minorschildren before inclusionto the study Initially the participants were briefly explainedabout the objectives of the study risks and benefits of theprocedures and on voluntary participation and the right towithdraw at any stage of the study using their local languageParticipants were then asked if they understood what hasbeen explained to them If and only if they understand thefacts implications and future consequences of their actionon themselves or their children they would like to be part ofthe study Written consent was not acquired because all theparticipants were recruited from the outpatient departmentlaboratory of the Gondar University Hospital where all theparticipant patients were sent to undergo the H pyloriantibody test e additional stool antigen test was a non-invasive procedure with minimal or no risk associated withit Besides the patients were benefited from the stool antigentest as it added further information on whether to com-mence eradication therapy by the attending physician eresult from the antibody test was collected from the labo-ratory record book Official permission was also obtainedfrom the University of Gondar Hospital before access to therecord book and the conduct of the study ereforeconsidering all these facts only the verbal agreement wasacquired to be included in the study e IRB has alsoevaluated the consent procedure and cleared it as sufficientParticipants who were diagnosed as positive to the H pyloristool antigen test were immediately linked to the medicaloutpatient department of the University of Gondar Hospitalfor appropriate treatment and follow-up


e authors declare that they have no conflicts of interestwith regard to the present study


MN HWB and DG conceived the study concept anddesigned the study MN and DG carried out data collectionand laboratory analysis MN HWB and DG supervised thedata collection and laboratory analysis MN HWB and DGanalyzed the data and prepared the first manuscript draftMN and DG reviewed the draft All authors read and ap-proved the final manuscript All the authors are currentlyworking at the University of Gondar

Acknowledgments

Our gratitude goes to the technical staffs the University ofGondar Hospital and the staff for the unreserved supportduring the study and all the study participants


References

[1] J G Kusters A H M Van Vliet and E J Kuipers ldquoPath-ogenesis of Helicobacter pylori infectionrdquo Clinical Microbi-ology Reviews vol 19 no 3 pp 449ndash490 2006

[2] M Safavi R Sabourian and A Foroumadi ldquoTreatment ofHelicobacter pylori infection current and future insightsrdquoWorld Journal of Clinical Cases vol 4 no 1 pp 5ndash19 2016

[3] B A Salih ldquoHelicobacter pylori infection in developingcountries the burden for how longrdquo Saudi Journal of Gas-troenterology vol 15 no 3 pp 201ndash207 2009

[4] R M Patrick S R Kenn and A P Michel MedicalMicrobiology Elsevier New York NY USA 5th edition 2005

[5] Y G David Y C Lee and M S Wu ldquoRational Helicobacterpylori therapy evidence-based medicine rather thanmedicine-based evidencerdquo Clinical Gastroenterology andHepatology vol 12 no 2 pp 177e3ndash186e3 2014

[6] R S Mhaskar I Ricardo A Azliyati et al ldquoAssessment of riskfactors of Helicobacter pylori infection and peptic ulcer dis-easerdquo Journal of Global Infectious Diseases vol 5 no 2pp 60ndash67 2013

[7] D Asrat I Nilsson Y Mengistu et al ldquoPrevalence of Heli-cobacter pylori infection among adult dyspeptic patients inEthiopiardquo Annals of Tropical Medicine amp Parasitology vol 98no 2 pp 181ndash189 2004

[8] T N A Archampong R H Asmah E KWiredu R K GyasiK N Nkrumah and K Rajakumar ldquoEpidemiology of Heli-cobacter pylori infection in dyspeptic Ghanian patientsrdquo PanAfrican Medical Journal vol 20 p 178 2015

[9] T Fasciana C Cala C Bonura et al ldquoResistance to clari-thromycin and genotypes in Helicobacter pylori strains iso-lated in Sicilyrdquo Journal of Medical Microbiology vol 64no 11 pp 1408ndash1414 2015

[10] J K Y Hooi W Y Lai W K Ng et al ldquoGlobal prevalence ofHelicobacter pylori infection systematic review and meta-analysisrdquo Gastroenterology vol 153 no 2 pp 420ndash429 2017

[11] K Desta D Asrat and F Derbie ldquoSeroprevalence of Heli-cobacter pylori infection among healthy blood donors inAddis Ababa Ethiopiardquo Ethiopian Journal of Health Sciencesvol 12 pp 109ndash115 2002

[12] T Tesfahun M Yohannes D Kassu and A Daniel ldquoSero-prevalence of Helicobacter pylori infection in and its re-lationship with ABO blood groupsrdquo Ethiopian Journal ofHealth Development vol 19 no 1 pp 55ndash59 2005

[13] M Biniam M Beyene and D Mulat ldquoSeroprevalence andtrend of Helicobacter pylori infection in Gondar Universityhospital among dyspeptic patients Gondar North WestEthiopiardquo BMC Research Notes vol 6 no 1 p 346 2013

[14] M Feleke K Afework M Getahun et al ldquoSeroprevalence ofHelicobacter pylori in dyspeptic patients and its relationshipwith HIV infection ABO blood groups and life style ina university hospital Northwest Ethiopiardquo World Journal ofGastroenterology vol 12 no 12 pp 1957ndash1961 2006

[15] Y Niv and T Rokkas ldquoRecent advances inHelicobacter pylorieradicationrdquo Annals of Gastroenterology vol 28 no 4pp 415-416 2015

[16] A S Doffou K A Attia M F Yao Bathaix et al ldquoeHelicobacter pylori eradication rate in a high prevalence area(West Africa) three triple therapy comparative studyrdquo OpenJournal of Gastroenterology vol 5 no 12 pp 200ndash206 2015

[17] M Negash A Kassu B Amare G Yismaw and B MogesldquoEvaluation of SD BIOLINEH pylori Ag rapid test againstdouble ELISA with SD H pylori Ag ELISA and EZ-STEP

H pylori Ag ELISA testsrdquo BMC Clinical Pathology vol 18no 1 p 4 2018

[18] K Dargaze G Baye A Agersew and A Zelalem ldquoHeli-cobacter pylori infection and its association with anemiaamong adult dyspeptic patients attending Butajira HospitalEthiopiardquo BMC Infectious Diseases vol 14 no 1 p 656 2014

[19] G Taddesse A Habteselassie K Desta S Esayas andA Bane ldquoAssociation of dyspepsia symptoms and Heli-cobacter pylori infections in private higher clinic AddisAbaba Ethiopiardquo Ethiopian Medical Journal vol 49pp 109ndash116 2011

[20] H Shmuely S Obure J D Passaro et al ldquoDyspepsiasymptoms and Helicobacter pylori infection Nakuru KenyardquoEmerging Infectious Diseases vol 9 no 9 pp 1103ndash11072003

[21] B V Oti R G Pennap O Dennis S A Ajegena andPM Adoga ldquoPrevalence and predictors ofHelicobacter pyloriinfection among patients attending a healthcare facility inNorth-Central Nigeriardquo Asian Pacific Journal of TropicalDisease vol 7 no 6 pp 352ndash355 2017

[22] L Shokrzadeh K Baghaei Y Yamaoka et al ldquoPrevalence ofHelicobacter pylori infection in dyspeptic patients in IranrdquoGastroenterology Insights vol 4 no 1 p 8 2012

[23] M Selgrad A Kandulski and P Malfertheiner ldquoDyspepsiaand Helicobacter pylorirdquo Digestive Disease vol 26 no 3pp 210ndash214 2008

[24] M S Pearce D I Campbell K D Mann L Parker andJ Eomas ldquoDeprivation timing of preschool infections andH pylori seropositivity at age 49ndash51 years the Newcastlethousand familiesrsquo birth cohortrdquo BMC Infectious Diseasesvol 13 no 1 p 422 2013

[25] W Jafri J Yakoob S Abid S Siddiqui S Awan andS Q Nizami ldquoHelicobacter pylori infection in childrenpopulation-based age-specific prevalence and risk factors ina developing countryrdquo Acta Paediatrica vol 99 no 2pp 279ndash282 2010

[26] U C Ghoshal R Chaturvedi and P Correa ldquoe enigma ofHelicobacter pylori infection and gastric cancerrdquo IndianJournal of Gastroenterology vol 29 no 3 pp 95ndash100 2010

[27] A C Hernando-Harder N Booken S Goerdt M V Singerand H Harder ldquoHelicobacter pylori infection and dermato-logic diseasesrdquo European Journal of Dermatology vol 19no 5 pp 431ndash444 2009

[28] E Chak G W Rutherford and C Steinmaus ldquoe role ofbreast-feeding in the prevention of Helicobacter pylori in-fection a systematic reviewrdquo Clinical Infectious Diseasesvol 48 no 4 pp 430ndash437 2009


[30] A Vega H Silva and T Cortintildeas ldquoEvaluation of a serum-freetransport medium supplemented with cyanobacterial extractfor the optimal survival of Helicobacter pylori from biopsysamples and strainsrdquo European Journal of Clinical Microbi-ology amp Infectious Diseases vol 31 no 2 pp 135ndash139 2012

[31] S Perry M De La Luz Sanchez S Yang et al ldquoGastroenteritisand transmission of Helicobacter pylori infection in house-holdsrdquo Emerging Infectious Diseases vol 12 no 11pp 1701ndash1708 2006

[32] S I Yokota M Konno S I Fujiwara et al ldquoIntrafamilialpreferentially mother-to-child and intraspousal Helicobacterpylori infection in Japan determined by mutilocus sequencetyping and random amplified polymorphic DNA finger-printingrdquo Helicobacter vol 20 no 5 pp 334ndash342 2015


[33] R Shi S Xu H Zhang et al ldquoPrevalence and risk factors forHelicobacter pylori infection in Chinese populationsrdquo Heli-cobacter vol 13 no 2 pp 157ndash165 2008

[34] M Kivi A L Johansson M Reilly and Y Tindberg ldquoHel-icobacter pylori status in family members as risk factors forinfection in childrenrdquo Epidemiology and Infection vol 133no 4 pp 645ndash652 2005

[35] M Nouraie S Latifi-Navid H Rezvan et al ldquoChildhoodhygienic practice and family education status determine theprevalence of Helicobacter pylori infection in Iranrdquo Heli-cobacter vol 14 no 1 pp 40ndash46 2009

[36] A Zajacova J B Dowd and A E Aiello ldquoSocioeconomic andraceethnic patterns in persistent infection burden among US adultsrdquo Journals of Gerontology Series A Biological Sciencesand Medical Sciences vol 64A no 2 pp 272ndash279 2009

[37] F Rasheed T Ahmad and R Bilal ldquoPrevalence and riskfactors of Helicobacter pylori infection among Pakistanipopulationrdquo Pakistan Journal of Medical Sciences vol 28no 4 pp 661ndash665 2012

[38] C Dube T C Nkosi A M Clarke N Mkwetshana E Greenand R N Ndip ldquoHelicobacter pylori in an asymptomaticpopulation of eastern Cape Province South Africa publichealth implicationrdquo Reviews on Environmental Health vol 24no 3 pp 249ndash255 2009

[39] B Stanstrom A Mendis and B Marshall ldquoHelicobacter pylorithe latest in diagnosis and treatmentrdquo Australian FamilyPhysician vol 37 no 8 pp 608ndash612 2008

[40] A A Mohamed and A H El-Gohari ldquoEpidemiological as-pects of Helicobacter pylori infections as an emergence zoo-notic disease animal reservoirs and public health implications(a review article)rdquo in Proceedings of 7th International ScientificConference pp 17ndash25 Mansoura Egypt 2012

[41] M Tabatabaei ldquoApplication of molecular and central culturalmethods for identification of Helicobacter spp in differentanimal sourcesrdquo Global Veterinaria vol 8 pp 292ndash297 2012

[42] M P Dore A R Sepulveda H El-Zimaity et al ldquoIsolation ofHelicobacter pylori from sheep-implications for transmissionto humansrdquo American Journal of Gastroenterology vol 96no 5 pp 1396ndash1401 2001

[43] M P Dore M Bilotta D Vaira et al ldquoHigh prevalence ofHelicobacter pylori infection in shepherdsrdquo Digestive Diseasesand Sciences vol 44 no 6 pp 1161ndash1164 1999

[44] N C Quaglia A Dambrosio G Normanno et al ldquoHighoccurrence of Helicobacter pylori in raw goat sheep and cowmilk inferred by glmM gene a risk of food-borne infectionrdquoInternational Journal of Food Microbiology vol 124 no 1pp 43ndash47 2008

[45] N R Bellack M W Koehoorn Y C MacNab andM G Morshed ldquoA conceptual model of waterrsquos role asa reservoir in Helicobacter pylori transmission a review of theevidencerdquo Epidemiology and Infection vol 134 no 3 p 4392006

[46] N Queralt R Bartolome and R Araujo ldquoDetection ofHelicobacter pylori DNA in human faeces and water withdifferent levels of faecal pollution in the north-east of SpainrdquoJournal of Applied Microbiology vol 98 no 4 pp 889ndash8952005

[47] T L Cover andM J Blaser ldquoHelicobacter pylori in health anddiseaserdquo Gastroenterology vol 136 no 6 pp 1863ndash18732009

[48] O Secka Y Moodley M Antonio et al ldquoPopulation geneticanalyses of Helicobacter pylori isolates from Gambian adultsand childrenrdquo PLoS One vol 9 no 10 Article ID e1094662014

[49] S Schwarz G Morelli B Kusecek et al ldquoHorizontal versusfamilial transmission of Helicobacter pylorirdquo PLoS Pathogensvol 4 no 10 article e1000180 2008

[50] K J Goodman and P Correa ldquoTransmission of Helicobacterpylori among siblingsrdquo =e Lancet vol 355 no 9201pp 358ndash361 2000

[51] T Falsafi N Sotoudeh M M Feizabadi and F MahjoubldquoAnalysis of genomic diversity among Helicobacter pyloristrains isolated from iranian children by pulsed field gelelectrophoresisrdquo Iranian Journal of Pediatrics vol 24 no 6pp 703ndash709 2014

[52] B Linz C R R Vololonantenainab A Seck et al ldquoPop-ulation genetic structure and isolation by distance of Heli-cobacter pylori in Senegal and Madagascarrdquo PLoS One vol 9no 1 Article ID e87355 2014

[53] H L Chen M J Chen S C Shih H Y Wang I T Lin andM J Bair ldquoSocioeconomic status personal habits andprevalence of Helicobacter pylori infection in the inhabitantsof Lanyurdquo Journal of the Formosan Medical Associationvol 113 no 5 pp 278ndash283 2014

[54] T Uchida M Miftahussurur R Pittayanon et al ldquoHeli-cobacter pylori infection inailand a nationwide study of theCagA phenotyperdquo PLoS One vol 10 no 9 Article IDe0136775 2015








Editorial Board




Contents





























Mara Di Giulio

References



















1 Introduction


















3 Results






















No of strains()

1CLR 3 (97)MT 3 (97)LVX 3 (97)











0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(a)

0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(b)

o

f H p

ylor

i iso

late

s

0

20

40

60

80

100

120

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(c)

o

f H p

ylor

i iso

late

s

0

10

20

30

40

50

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(d)



4 Discussion









Data Availability







References























































1 Introduction



















3 Results






4 Discussion



20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)







1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References


























































Contents





























Mara Di Giulio

References



















1 Introduction


















3 Results






















No of strains()

1CLR 3 (97)MT 3 (97)LVX 3 (97)











0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(a)

0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(b)

o

f H p

ylor

i iso

late

s

0

20

40

60

80

100

120

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(c)

o

f H p

ylor

i iso

late

s

0

10

20

30

40

50

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(d)



4 Discussion









Data Availability







References























































1 Introduction



















3 Results






4 Discussion



20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)







1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References
















































































Mara Di Giulio

References



















1 Introduction


















3 Results






















No of strains()

1CLR 3 (97)MT 3 (97)LVX 3 (97)











0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(a)

0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(b)

o

f H p

ylor

i iso

late

s

0

20

40

60

80

100

120

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(c)

o

f H p

ylor

i iso

late

s

0

10

20

30

40

50

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(d)



4 Discussion









Data Availability







References























































1 Introduction



















3 Results






4 Discussion



20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)







1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References




































































1 Introduction


















3 Results






















No of strains()

1CLR 3 (97)MT 3 (97)LVX 3 (97)











0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(a)

0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(b)

o

f H p

ylor

i iso

late

s

0

20

40

60

80

100

120

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(c)

o

f H p

ylor

i iso

late

s

0

10

20

30

40

50

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(d)



4 Discussion









Data Availability







References























































1 Introduction



















3 Results






4 Discussion



20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)







1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References







































































3 Results






















No of strains()

1CLR 3 (97)MT 3 (97)LVX 3 (97)











0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(a)

0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(b)

o

f H p

ylor

i iso

late

s

0

20

40

60

80

100

120

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(c)

o

f H p

ylor

i iso

late

s

0

10

20

30

40

50

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(d)



4 Discussion









Data Availability







References























































1 Introduction



















3 Results






4 Discussion



20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)







1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References































































0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(a)

0

5

10

15

20

25

30

35

40

o

f H p

ylor

i iso

late

s

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(b)

o

f H p

ylor

i iso

late

s

0

20

40

60

80

100

120

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(c)

o

f H p

ylor

i iso

late

s

0

10

20

30

40

50

006

25

012

5

025 0

5 1 2 4 8 16 32 64 128


(d)



4 Discussion









Data Availability







References























































1 Introduction



















3 Results






4 Discussion



20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)







1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References


























































4 Discussion









Data Availability







References























































1 Introduction



















3 Results






4 Discussion



20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)







1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References




























































References























































1 Introduction



















3 Results






4 Discussion



20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)







1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References



















































































1 Introduction



















3 Results






4 Discussion



20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)







1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References









































































3 Results






4 Discussion



20

40

60

80

100

120

140

160lowast

Sple

en w

eigh

ts (m

g)







1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References






























































1

2

3

4

5

6TH

1 cy

toki

nes (

pgm

l)

IL-2IFN-γTNF-α

(a)


05

10

15

20

lowast

lowast

lowast

TH2

cyto

kine

s (pg

ml)

IL-4IL-6IL-10

(b)


01

02

03

04

05

06

07

08

IL-1

7 (p

gm

l)

(c)









(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References






























































(a)

(b)



04

08

12

16

20

24p lt 005

p lt 005

lowast

lowastLP

I











Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References
































































Data Availability




Acknowledgments




References






























































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References

















































































































1 Introduction
















3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References





































































3 Results










4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References

































































4 Discussion











5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References






























































5 Conclusion









Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References



























































Data Availability




Acknowledgments


References
















































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References












































































1 Introduction








2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References




























































2 Method








z

za+

z



λ(a t) f(a)g(t) (2)




exp minus1113946t


(3)



p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References


























































p(a t)





⎧⎪⎪⎨

⎪⎪⎩(4)




p(a t)





⎧⎪⎪⎨

⎪⎪⎩(5)



p(a t)





t


⎧⎪⎪⎨

⎪⎪⎩(6)


L(θ n m) 1113945a

1113945t

nat

mat

1113888 1113889p(a t)mat (1minusp(a t))

natminusmat

(7)


3 Results









00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References





























































00

02

04

06

08

10

1900 1920 1940 1960 1980 2000 2020Birth year

A 1974B 1984C 1986D 1991

E 1994F 1994G 2007 8 and 9

H 2008I 2010J 2011

Survey year

Sero

posit

ive f

ract

ion






0102030405060708090

100

1900 1920 1940 1960 1980 2000 2020Birth year

ObservedPredicted

Prop

ortio

n se

ropo

sitiv

e (

)




4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References



























































4 Discussion



0

001

002

003

004

005

006

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010Year

Model 1Model 2

Forc

e of i

nfec

tion

(per

yea

r)


00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)



Ser

opos

itive

frac

tion

(a)

00

02

04

06

08

10

0 10 20 30 40 50 60 70 80 90Age (years)


Sero

posit

ive f

ract

ion

(b)










Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References

































































Data Availability


Disclosure




Acknowledgments


References














































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References


































































































1 Background









2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References





























































2 Methods








3 Result







4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References





























































4 Discussion





Sex Male 98 (70) 79 (567) 140 (697)Female 45 (738) 27 (443) 61 (303)

Age (years)



Occupation

Farmer 30 (789) 19 (50) 38 (189)Student 73 (702) 64 (615) 104 (517)


Education

















223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References


































































223 (126ndash446) 0017River 27 (771) 15 (429) 35 (174)Well 5 (833) 5 (833) 6 (3)









5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References





























































5 Conclusion


Abbreviations


Data Availability


Ethical Approval






Acknowledgments



References


























































References


























































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