Helicobacter pyloriBriann McGovern (microbiology spR)

Background1983-discovered by Warren and Marshall in Australia

Discovery revolutionised the treatment of duodenal and gastric ulcers

Earned them the Nobel Prize for Medicine in 2005

Formerly known as Campylobacter pyloridis

Nearly 20 species of Helicobacter are now recognised

The gastric helicobacters colonise the stomachs of animals. The monkey, cat, dog, cheetah all harbour their own species

H. cinaedi and H. fennelliae are associated with proctitis in homosexual men

H. pylori are found in the human stomach. Molecular studies suggest transmission from an animal source.

Helicobacter pyloriMcColl K. N Engl J Med 2010;362:1597-1604

DescriptionGram-negative spiral bacillus

Fastidious in terms of growth requirements:strictly micro-aerophilic :require C02 for growth:on charcoal medium

Has a tuft of sheathed unipolar flagella; specially adapted to colonise mucous membranes

Gram stain of H. pylori recovered from an individual without prior antimicrobial therapy. Organisms display typical seagull-like appearance.

Hallmark of the species is production of urease enzyme-urease breaks urea down to C02+NH3-amonia is a strong base-process helps H. pylori survivestrongly acidic stomach conditions

Very fragile (a point of importance when referring samples to the lab)

EpidemiologyH. pylori infection occurs worldwide

Prevalence varies greatly among countries and population groups

20 50% prevalence in middle age adults in industrialised countries

>80% prevalence in middle age adults in developing countries:may reflect poorer living conditions

TransmissionOral ingestion of bacterium within families (esp children)person-person contactfaecal-oral transmission?role of waterborne transmission

Usually contracted in the first 2 years of life

Site of infection Highly adapted organism that lives only on gastric mucosa

Gastric antrum is the most favoured site

Present in the mucus that overlies the mucosa

Gastric-biopsy specimen showing Helicobacter pylori adhering to gastric epithelium and underlying inflammationMcColl K. N Engl J Med 2010;362:1597-1604

Course of infectionAfter several days incubation period, patients suffer mild attack of acute gastritis-abdominal pain-nausea-flatulence-bad breath

Symptoms last about 2/52 but hypochlorhydria can last up to one year

Despite a substantial ab response, infxn and chronic gastritis persistAfter decades there may be progression to atrophic gastritis (conditions which are inhospitable for the bacteria) and numbers reduce

The outcome of infection by H. pylori reflects an interaction between:

H. pylori infection directly associated withPUD-lifetime risk 3% in US, 25% Japan-eradication provides long-term cureGastric carcinoma-strong evidence of increased risk 0.1-3%-unclear whether eradication reduces the risk of gastric cancerMALT lymphoma-72% 98% of MALT lymphoma infected with H. pylori

H. pylori may be implicated in:-

Non-ulcer dyspepsia -increased prevalence of H. pylori but data inconsistent (symptomatic improvement only 9%) -little evidence that chronic H. pylori infection in the absence of ulceration causes upper GI symptoms

GORD -recent meta-analysis : no significant association

Laboratory diagnosis:non-invasive testsSerology : detect an immune response by examining a blood sample for abs to the organism (ELISA): poor accuracy

Urea breath test : a urea solution labelled with C14 isotope is given to pt. The C02 subsequently exhaled by the pt contains the C14 isotope and this is measured. A high reading indicates presence of H. pylori

Faecal antigen test : detect H. pylori antigens in faecal specimens

Polymerase chain reaction (PCR) : can detect HP within a few hours. Not routine in clinical use.

Invasive testingHistological examination of biopsy specimens of gastric/duodenal mucosa take at endoscopyCLO-test : based again on urease-production by the organism->NH3 production->rise in pH=>change in the colour indicator of the kit-High sensitivity and specificity-Prompt result

Invasive testingCulture : -no more sensitive than skilled microscopy of histological sections-used for antibiotic resistance testing-requires selective agars and incubation periods

Tests for Helicobacter pylori InfectionMcColl K. N Engl J Med 2010;362:1597-1604

Indications for therapyStrongly recommended

Duodenal or gastric ulcer

MALT lymphoma

Atrophic gastritis

Recent resection of gastric cancer

Maastricht 2-2000 Consensus Report

Indications for therapyTreatment advised

Functional dyspepsia

Gastro-oesophageal reflux disease (patients requiring long-term acid suppressive therapy) Use of NSAIDsMaastricht 2-2000 Consensus Report

TreatmentGoal of treatment to eradicate infection

Triple therapy regimens consist of one anti-secretory agent and two antimicrobial agents for 7 to 14 days

Triple therapy regimens musthave cure rate of approximately 80%be without major side effects- minimal induction of resistance

First line treatmentCombination of two or more antimicrobial agents increases rates of cure and reduces the risk of selecting for resistant H. pylori

Many factors may result in failure of treatment microbial factors patient compliance geographical differences

First line therapyPPI b.d. + clarithromycin 500mg b.d. + amoxicillin 1000mg b.d. or metronidazole 400mg BD minimum of 7 days

In case of failure

Second line therapyPPI b.d. + bismuth subsalicylate/subcitrate 120mg QDS + metronidazole 500mg t.d.s. + tetracycline 500mg q.d.s.for a minimum of 7 daysIf bismuth is not available, PPI based triple therapies should be used

Subsequent failures should be handled on a case-case basis. Patients failing second-line therapy in primary care should be referred

Maastricht 2-2000 Consensus Report

NEJM 2002;347:1175-86

Guidelines for Evaluation and Management of Helicobacter pylori InfectionMcColl K. N Engl J Med 2010;362:1597-1604

ReinfectionReinfection following successful bacterial cure is unusual

Commonly represents recrudescence of the original bacterial strain

In adults, reacquisition of the bacteria occurs in

Failure of treatmentFailure of initial course occurs in 1 in 5

2nd-line Tx : either an alternative regimenquadruple Tx (PPI+bismuth+2 antibx)

Key points->H. pylori is a flagellated spiral micro-aerobe->Infection is a risk factor for gastric cancer->Causes PUD and gastritis->Produces a cell-damaging toxin->Transmission route is unclear->Dz rates are falling in industrialised countries->Tx is by eradication using combination therapy

RecommendationsThe noninvasive test-and-treat strategy for H. pylori infection is reasonable for younger patients who have upper gastrointestinal symptoms but not alarm symptoms

Noninvasive testing can be performed with the use of the urea breath test, fecal antigen test, or serologic test; the serologic test is the least accurate

Triple therapy with a proton-pump inhibitor, clarithromycin, and amoxicillin or metronidazole remains an appropriate first-line therapy

Recurrence or persistence of symptoms after eradication therapy for uninvestigated dyspepsia is much less likely to indicate that treatment has failed than to indicate that the symptoms are unrelated to H. pylori infection.

RecommendationsFurther eradication therapy should not be considered unless persistent H. pylori infection is confirmed

Data are lacking to inform the optimal management of recurrent or persistent dyspepsia after noninvasive testing and treatment of H. pylori infection

Options include symptomatic acid-inhibitory therapy, endoscopy to check for underlying ulcer or another cause of symptoms, and repeat of the H. pylori test-and-treat strategy; other potential reasons for the symptoms should also be reconsidered

Clinical Practice Helicobacter pylori InfectionKenneth E.L. McColl, M.D.N Engl J MedVolume 362(17):1597-1604April 29, 2010Thanks for your attention!

Amoxicillin resistanceAmoxicillin resistance rare (0.3%-1.4% generally, rates as high as 38% have been reported)

Stable resistance has been reported

Because H. pylori can exchange DNA through natural transformation and also through conjugation, spread of amoxicillin resistance among H. pylori is potentially a major threat

?Restriction of indications for treatment rather than indiscriminate eradication of H. pylori

Risk factors for resistanceRisk factors for metronidazole resistanceSex female > maleEthnicity Asian > EuropeanTest method use of E test vs. agar dilution

Risk factors for clarithromycin resistanceGeographical region prescribing trends/genotypeAge increasing ageSex female >maleUlcer status inactive >active

Consumption & ResistancePerez Aldana et al. 593 Japanese patients dyspepsia, no previous triple therapyClarithromycin resistance 7% in 1997/8 to 15% in 1999/2000Metronidazole resistance 6.6% in 1997/8 to 12% in 1999/2000(Amoxicillin resistance reported 0.3%)

Postulated that increase in antibiotic resistance was associated with a huge increase in clarithromycin use in Japan between 1993 and 2000Consumption of metronidazole gradually increased during that timeRate of resistance to metronidazole low, compared to worldwide rates, due to low consumption of metronidazole in Japan

Secondary resistanceDevelopment of secondary resistance for clarithromycin and/or metronidazole may occur in 50% of cases

AMNCH study (1997): secondary resistance to clarithromycin acquired in 58.3% of strains in cases of treatment failure

Using most effective regimen will help to minimise developed of secondary resistance

Resistance primary & secondaryAMNCH (1997)Clarithromycin, metronidazole, omeprazole x 1/52Primary resistance MET 35.6%, CLA 3.4%Eradication rate 81.6% overall: 98.2% fully sensitive isolates; 57.1% metronidazole resistant strains; 0% dual resistant strainsSecondary resistance to clarithromycin acquired in 58.3% of cases of treatment failure

Antibiotic sensitivity testingExcellent eradication rates have been achieved when prior resistance to one particular antibiotic was known

Methodological problems:- slow growing microaerophilic organism (10 14 days) and difficulty interpreting susceptibility data

More than 50% of the population may carry a mixture of sensitive and resistant strains in non-uniform distribution

May achieve better results if multiple biopsies from several sites are used

Molecular testingClarithromycinMolecular assays detecting 23S rRNA mutation esp. PCR-RFLP

Can be performed on DNA from gastric aspirate or biopsy - ?same day results

Others e.g. DNA enzyme immunoassay (DEIA), PCR line probe assay (LiPA)

LiPA detects seven distinct resistance mutations suitable for testing large numbers of people

LiPA assay H. pylori in West of Ireland clarithromycin resistance 27% (not correlated with conventional C&S)

First line therapyPPI (RBC) b.d. + clarithromycin 500mg b.d. + amoxicillin 1000mg b.d. or metrodiazole 500md BD minimum of 7 days

In case of failure

Second line therapyPPI b.d. + bismuth subsalicylate/subcitrate 120mg QDS + metronidazole 500mg t.d.s. + tetracycline 500mg q.d.s.for a minimum of 7 daysIf bismuth is not available, PPI based triple therapies should be used

Subsequent failures should be handled on a case-case basis. Patients failing second-line therapy in primary care should be referred

Maastricht 2-2000 Consensus Report

2nd line treatmentQuadruple therapies with bismuth, PPI + 2 antibiotics

Success rate of eradication variable -

Other 2nd line optionsRescue therapies with rifabutin - promising with rates of 78.6% despite multiple therapies (resistance H. pylori rpoB gene)

Stepwise therapies combining triple therapy, quadruple therapy, high doses of clarithromycin and proton pump inhibitor in a stepwise fashion (100% eradication in one series)

C&S testing in St.JamessReinitiated C&S testing of biopsy samples in selected cases of treatment failure

18 patients biopsies cultures to date12 patients specimens did not grow sufficiently for sensitivity testing9 patients had curved GNB on gram stain but failed to culture1 patients specimens were negative on gram and culture2 patients had differences in gram stain depending on biopsy location, neither grew on culture.3 patients with successful culture & sensitivity results

C&S testing in St.JamessC.B. - Triple therapy received

April 2001July 2002October 2002UnknownClarithromycn x 1/52Clarithromycin x 2/52UnknownMetronidazole x 152Amoxicillin x 2/52UnknownOmeprazpole x 1/52Lansoprazole x 2/52

C&S testing in St.JamessC.B. Culture & sensitivity results

Bx. Site GramCultureCLAMETAMOXTETAntralCurved GNBH. pyloriRMIC>256RMIC>256RMIC>256RMIC>256FundalCurved GNBH. pyloriRMIC>256RMIC>256RMIC>256RMIC>256

Gram stain of H. pylori recovered from patient, C.B. following 3 courses of antimicrobial therapy.Organisms appear enlarged and of abnormal morphology.

Future?Targetted novel therapies role of functional genomics

H. pylori virulence factors use in clinical practice

Vaccination prophylactic & therapeutic vaccination (animal models)

ReferencesSuerbaum S, Michetti P. Helibacter pylori infection. NEnglJMed 2002;347:1175-86Malfertheiner P, Megraud F et al. Current concepts in the management of Helicobacter pylori infectionThe Maastricht 2-2000 Consensus Report. Aliment Pharmacol Ther 2002; 16:167-80Hjalmarsson S, Sjolund M, Engstrand L. Determining antibiotic resistance in Helicobacter pylori. Expert Rev Mol Diagn. 2(3):267-72, 2002 May.Vaira D, Vakil N, Menegatti M et al. The stool antigen test for detection of Helicobacter pylori after eradication therapy. Ann Intern Med 2002;136:280-7Qasim A, OMorain CA. Review article: treatment of Helicobacter pylori and factors influencing eradication. Aliment Pharmacol Ther 2002; 16 (Seppl. 1):24-30.vanZwet AA, Vandenbrouke-Grauls CMJ et al. Stable amoxicillin resistance in Helicobacter pylori. Lancet 1998;352:1595Perez Aldana et al. The relationship between consumption of antimicrobial agents and the prevalence of primary Helicobacter pylori resistance. Helicobacter 2002;7(5):306-309Xia HX, Buckley M, Keane CT. Clarithromycin resistance in Helicobacter pylori: prevalence in untreated dyspeptic patients and stability in vitro. J Antimicrob Chemother 1996;37:473-81Jenks PJ. Causes of failure of eradication of Helicobacter pylori. BMJ 2002;325:3-4Buckley MJ, Xia HX et al. Metronidazole resistance reduces efficacy of triple therapy and leads to secondary clarithromycin resistance. Dig Dis Sci 1997;42:2111-5

ReferencesHouben MHM, Van De Beek D. A systematic review of Helicobacter pylori eradication therapy the impact of antimicrobial resistance on eradication rates. Aliment Pharamcol Ther 1999;13:1047-55Owen RJ. Molecular testing for antibiotic resitance in Helicobacter pylori. Gut 2002;50:285-9Meyer JM, Siliman N. Risk factors for Helicobacter pylori resistance in the United States: The Surveillance of H. pylori Antimicrobial resistance partnership (SHARP) study, 1993 1999. Ann Intern Med 2002;136:13-24Wen M, Zhang Y et al. An evaluative system for the response of antibacterial therapy: Based on the morphological change of Helicobacter pylori and mucosal infllammation. Pathology International April 99 49:4;332-341

OutlineThe Clinical ProblemGastric and Duodenal UlcersGastric CancerGastric Malt LymphomaOther Gastrointestinal ConditionsStrategies and EvidenceCandidates for Testing for H. pylori InfectionTests for H. pylori InfectionNonendoscopic TestsEndoscopic TestsTreatment of H. pylori InfectionConfirmation of EradicationManagement of Persistent Infection after TreatmentAreas of UncertaintyGuidelinesConclusions and Recommendations

Case history C.B. 40 y.o. maleApril 04 - OPD epigastric discomfort aggravated by certain foods, relieved by PPI

OGD (4/04) Diffuse non-erosive gastritis. CLO +ve

Triple therapy (4/04) drugs, duration not documented

August 04 OPD occasional symptoms, ranitidine PRN, urea breath test (UBT) arranged

Case historyNovember 04 OPD UBT negative, off PPI and discharged from clinic December 2004.

July 2005 GP UBT +ve symptomatic

July 2005 triple therapy - clarithromycin, metronidazole & omeprazole x 1/52 (GP)

October 2005 triple therapy clarirthromycin, amoxicillin, lansoprazole x 2/52 (OPD)

Case historyNovember 2005 UBT +ve - OGD arranged

Februrary 2006 OGD normal oesophagus, stomach + duodenum

CLO positive

Antral & fundal biopsies sent for culture & sensitivity

NEJM 2002;347:1175-86

Host response to infectionH. pylori attaches to gastric epithelial cells triggering host inflammatory response

HP infected patients gastric epithelium have enhanced levels of IL-1, IL-2, IL-6, IL-8, TNF.

IL-8 appears to have central role, acting as a potent neutrophil activating chemokine

Host response to infectionH. pylori attaches to gastric epithelial cells triggering host inflammatory response

H. pylori bind to class II MHC molecules on surface of gastric epithelial cells inducing apoptosis

HP urease & porins may contribute to extravasation & chemotaxis of neutrophils

HP infected patients gastric epithelium have enhanced levels of IL-1, IL-2, IL-6, IL-8, TNF

IL-8 appears to have a central role

Host response to infectionMarked systemic & mucosal humoral response antibodies do not lead to eradication of the infection but may contribute to tissue damage

Th1 response predominates and Th1 cytokines promote gastritis may occur due to increased IL-8 production[Th2 response expected (extracellular pathogen) does not occur, would protect gastric mucosa]

NEJM 2002;347:1175-86

Host responseMost disease causing HP strains are type I containing the cag-pathogenicity island (PAI)

cag-PAI carrying strains induce a much stronger IL-8 response than cag-negative

Proteins encoded by cag-PAIinduction of IL-8 production of gastric epithelial cells translocation of CagA protein from bacteria to host

PathogenesisHP genome studies have shown that the genome changes continuously during chronic colonisation importing small pieces of foreign DNA from other HP strains

Phase variable genes encode proteins including enzymes modifiying surface antigens, control the entry of foreign DNA into the bacteria and influence motility

PathogenesisUrease production essential for survival urease hydolyses urea into carbon dioxide & ammonia permitting survival in acidic milieu

Motility essential for colonisation HP flagella have adapted to gastric niche.

Vacuolating cytotoxin (VacA) expressed by majority of strains of HP, appears to increase bacterial fitness

Huge variation in VacA strains

DiagnosisMinimally invasive testingUrea breath test (UBT) - initial diagnosis & follow up for eradication [sensitivity/specificity 90%]

Serological testing pre-treatment diagnosis of HP infection. AMNCH study (2000): 82.4% sensitive and 85% specific

Stool antigen test 89-98% sensitivity & >90% specificity

Stool immunoassay & PCR under investigation

DiagnosisEndoscopy & biopsy indicated:-Severe symptoms

Anaemia

Weight loss

Patients >50 years

DiagnosisUrease test (CLO) on biopsy specimen rapid detection (sensitivity 79 100%, specificity 92 100%)

Sensitivity of urease test can be improved by additional biopsies

False negatives active or recent bleeding, antibiotic or antisecretory therapy

Histological examination can be performed if urease test negative (>90% sensitive and specific)

Treatment failureTreatment failure may result from:-Bacterial resistance in theory the most important factor in failure of primary treatment

Poor adherence to antibiotic guidelines

Patient compliance positive role shown for compliance enhancing programmes

Geographical region bacterial/patient genotype, prevelance of resistance, local antibiotic useCost & duration of therapyMinors:- drug metabolism pathways (e.g. CYP2C19 genotype), intragastric pH, antimicrobial stability

**Later reclassified as HP*****Rate of acquisition has fallen sharply but continued increase in prevalence seen due to cohort effect, reflecting more intense transmission at the time when earlier birth cohorts were children.

Epidemiological monitoring suggest that marked improvements in sanitation have initiated a decline in HP infection that will ultimately lead to its elimination from the US ppulation. However, without intervention it is predicted to remain endemic for at least another century.

**Spontaneous elimination in children probably aided by administration of antibiotics for other reasons.GASTRIC ANTRUM****Eradication of H. pylori dramatically reduces the recurrence rates of HP associated peptic ulcers.

Strong evidence links H. pylori to increased risk of gastric cancer. In 1994, H. pylori was classed as a type I (definite carcinogen)

In a recent prospective study of Japanese subjects (over 1500), 2.9% of HP infected patients developed gastric carcinoma. No carcinoma was identified in 208 non-infected control patients. No carcinoma was detected in a subgroup of 253 patients who received eradication therapy early in follow up.

Studies also suggest that eradication of Helicobacter prevents recurrence of endoscopically resected early gastric carcinomas.

72 98% of patients with MALT lymphoma are infected with H. pylori.**Cochrane review suggests symptomatic relief may be achieved in only 9% of NUD patients. However, endpoint may miss the other benefits of H. pylori eradication.

In GORD, several case-control and cohort studies H. pylori has been shown to have a protective role against GORD. However. Long term use of PPIs in these patients may aggravate HP mediated corpus gastritis increasing the risk of gastric carcinoma.

HP has been implicated in many other diseases but documentation to date is poor.CLO-TEST*Culture plate******Long-term PPI therapy in GORD may aggravate HP-mediated corpus gastritis, thus increasing the risk of gastric carcinoma.

HP infection, use of NSAIDs & aspirin are independent risk factors for PUD.**Once eradication is achieved, reinfection rates are low so benefits of treatment are durable.

***********No significant risk factors for amoxicillin resistance.*******Resistance difficult to interpret as laboratory breakpoints, i.e. the in vitro concentrations that define the cut off between sensitive and resistant strains, do not correlate with levels required for eradication of infection from the gastric mucosa.**PCR-RFLP = PCR-Restriction fragment length polymorphism

Same day PCR efficacy may be affected by presence of inhibitors from the clinical specimens,and specificity reduced by presence of high levels of non-H pylori DNA

DEIA enzyme linked immunoabsorbent assay with a labelled anti-dsDNA monoclonal antibody

Molecular mechanisms for metronidazole resistance poorly understood so not possible to design straight-forward lab assays for detection of resistance*********H.pylori seen on gram in C.B. appear enlarged with abnormal morphology.

We aim on future specimens to examine the Helicobacter pylori with electron microscopy.

One Japanese study has reported thickening of the cell wall of H.pylori in patients previously given triple therapy. These abnormal bacteria were also present in patients who had follow up negative urea breath tests. **************H. pylori usually acquired in childhood.

Acute infection causes transient hypochlorhydria and is rarely diagnosed.

Chronic gastritis will develop in virtually all persistently colonised patients, but 80 90% will never have symptoms.

Patients with high acid output are likely to have antal predominant gastritis, predisposing them to duodenal ulcers,

Patients with lower acid output are more likely to have corpus gastritis, prediposing them to gastric ulcers and predispose to gastric carcinoma.*IL-8 appaears to have a central role, acting as a potent neutrophil activating chemokine.

**IL-8 appaears to have a central role, acting as a potent neutrophil activating chemokine.

**Th2 cells stimulate B cells in response to extracellular pathogens. As H. pylori is non-invasive and induces a strong humoral response a Th2 response would be expected.

Studies in gene targetted mice have shown that Th2 cytokines are protective against gastric inflammation. **The cag-pathogenicity island is a chromosal region within the H. pylori gene containing 29 genes and 37,000 bp.

Most of the cag genes are probably involved in assembly of secretory machinery that translocate the protein cagA into the cytoplasm of the gastric epithelium.

Within the cell cagA is phosphorylated leading to a growth factor like cellular response and cytokine production.

***Phase-variation describes a process of reversible switching between phenotypes, mediated by a genetic re-organisation, mutation or modification, which is not associated with a loss of coding potential, and which occurs at a comparatively high frequency. Phase-variation results in the continuous generation of alternative phenotypes within a population, which facilitate adaptation to changing environmental conditions. Genes that undergo phase variation have been called 'contingency genes' which emphasises the evolutionary and functional implications of the variability of this subset of hypermutable genes. The switching of these genes occurs stochastically within the variable population. **Gene mutations in cheW have been shown in vitro to abolish chemotaxis completely. May be a possible target for future therapy.

VacA inserts itself into the epitlial membrane creating an anion selective voltage dependant channel which may provide nutrients to the bacteria.

VacA also targetted at mitochondial membrane releasing cytochrome c and inducing apoptosis

VacA is not essential for colonisation as VacA negative strains can colonise in animal models, and strains with inactive VacA genes hace been isolated from patients.

In Western countries, certain VacA variants were associated with more severe disease. However this has not been replicated in Asian studies. **For follow up of eradication therapy, the UBT is indicated at an interval of greater than 4 weeks to avoid false negative results.

Sensitivity & specificity testing of serological testing similar to that of UBT but inconsistent results have been reported. Because H. pylori strains differ among geographical locations, knowledge of locally prevalent strains would optimise accuracy. Is of limited used in determining the success of treatment.

Stool antigen testing can be used for follow up of eradication, an interval of 8 weeks should be allowed. One prospective, multicenter study showed a positive result perform 7 days after completion of theraoy identified patients in whom eradication has been unsuccessful.Stool antigen testing may be of particular use in children under 6 years.

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