Helicobacter pylori highlights from the first Canadian Digestive Disease Week

The first annual Canadian Digestive Disease Week (CDDW) took place in breath-taking Banff, Alberta from March 4 to 10, 1996. The CDDW meeting was attended by over 600 Canadian physicians, fellows and researchers, and included a Gastroenterology Fellows’ Program, and Practitioners’ and Scientific Programs. It was apparent from the posters and presentations that Helicobacter pylori research ongoing in Canada is at the leading edge. World leaders in H pylori research spoke on topics ranging from the genetics of H pylori and the pathogenesis of its associated diseases, to the clinical challenge of successful eradication of infection. This overview summarizes the information about H pylori presented at the 1996 CDDW. “WHY ALL THE FUSS?” “Why all the fuss?” was the ironically titled dynamic talk by Barry Marshall that started off the Practitioners’ Program with gusto (1). In 1983 Marshall co-identified the H pylori-gastritis association. In his talk Marshall paid tribute to those who have shed light over the past century on the association between the bacterium and peptic ulcer disease (PUD). The National Institutes of Health (NIH) treatment guidelines for treating H pylori in certain patients are the currently accepted recommendations (2). The findings presented at the 1996 CDDW clearly indicate that advances in H pylori research are happening faster than new treatment guidelines are formed. A MATISKO, ABR THOMSON. Helicobacter pylori highlights from the first Canadian Digestive Disease Week. Can J Gastroenterol 1997;11(1):71-76. The first annual Canadian Digestive Disease Week (CDDW) took place in Banff, Alberta from March 4 to 10, 1996. The CDDW meeting was attended by over 600 Canadian physicians, fellows and researchers, and included a Gastroenterology Fellows’ Program, as well as Practitioners’ and Scientific Programs. It was apparent from the posters and presentations that Helicobacter pylori research ongoing in Canada is at the leading edge. World leaders in H pylori research spoke on topics ranging from the genetics of H pylori and the pathogenesis of its associated diseases, to the clinical challenge of successful eradication of infection. This overview summarizes the information about H pylori presented at the 1996 CDDW.


EPIDEMIOLOGY
How H pylori infection is transmitted is unknown.Recent studies have demonstrated that the bacterium is present in biological fluids from the domestic cat (3).It has been suggested that cat owners may become infected through contact with their pet or by manipulation of the animal's feces.However, Renaud and co-workers (3) found that cat ownership did not correlate with H pylori status.They did, however, observe an association between the number of years a person had contact with a domestic cat before age 20 and current H pylori status, which is in accord with the theory that H pylori colonization is acquired during early childhood.
The prevalence of H pylori in patients with duodenal ulcer (DU) has been reported to be as high as 93%.Murat et al (4) examined H pylori in a small town and found that over a two-year period, 22 of 38 DU patients (58%) were H pylori-negative.The reasons for this low prevalence are unknown, and further work is needed in this important area.
Cigarette smoking has been recognized as an important cause of peptic ulcer relapse.Eradication of H pylori has been shown to reduce the relapse rate of both DU and gastric ulcer (GU) (5).In a study from Hong Kong presented at the CDDW, GU patients had no reinfection or ulcer relapse after H pylori eradication (6).For DU patients, the rates of reinfection and relapse were very low, and no difference was observed in reinfection and ulcer relapse rates between smokers and nonsmokers.This suggests that cigarette smoking may not increase the recurrence of peptic ulceration after successful H pylori eradication.

GENETICS AND MOLECULAR BIOLOGY OF
H PYLORI INFECTION Why do some H pylori-infected persons develop DU or GU while others do not?The answer may lie in both environmental and genetic factors, including the different expression of H pylori adaptive traits (7).There are a number of virulence gene clones from H pylori.For example, cagA (discovered in 1987 and cloned in 1993) is present in roughly 60% of H pylori strains (8).CagA is nontoxic and its function is unknown.Peptic ulcer patients have a greater rate of infection with cagA-positive strains than patients without ulcers (ie, DU is almost always associated with cagA positivity).However, some patients infected with cagA-positive strains of H pylori do not develop ulcers.The corollary also exists: some persons infected with cagA-negative H pylori develop ulcers and up to 28% of cytotoxin-negative strains are cagA-positive (8).However, ablation of the cagA locus has no effect on toxin production.Therefore, cagA appears to be only a marker of virulence, rather than a virulence factor (9). CagA is also believed to be a marker for picA, which encodes a protein with potential to induce cytokines, and picB.PicB triggers release of interleukin-8, a pro-inflammatory cytokine (9).
The vacuolating cytotoxin gene vacA is present in all isolates of H pylori, and the encoded protein vacA (present in 50% to 60% of isolates) causes gastroduodenal damage in animal models (8).The vacuolating cytotoxin-producing strain is more prevalent in infected patients with peptic ulcer or gastric cancer than in controls.However, diversity exists between vacA products in different H pylori strains (ie, vacA genotypes have different cytotoxin activity).Prevalence of vacA genotypes (normally s1a, s1b and s2) varies in H pylori-infected symptomatic ulcer patients with either past or present ulcer (63% s1a, 30% s1b and 7% s2) (10).Conversely, variation also exists for H pylori-positive patients without ulcer: 16% s1a, 25% s1b and 59% s2.Ninety per cent of H pylori-positive dyspeptic patients are infected with the s2 strains and yet have no ulcers.
Urease and flagellar proteins are important in the acquisition of the infection and in colonization of H pylori within the stomach.One unique trait of H pylori is its ability to thrive in the gastric environment at a low pH.Urease activity raises the intragastric pH, especially in the alkaline microclimate of the bacterium adjacent to the gastric mucosa.The neutralizing effect of urease allows the bacterium to thrive in the highly acidic gastric environment.
Göttke and colleagues (7) isolated DNA from 33 strains of H pylori and sequenced the regions for vacA and flaA genes.No specific relationship in the pattern of inheritance was observed.The vacA gene encodes the 'vacuolating cytotoxin' that has been closely associated with tissue pathology in PUD.The flaA gene encodes a flagellar structural protein that is essential for viability.DNA sequence variation of the vacA and flaA genes of H pylori were determined in 37 clinical isolates from patients with PUD.The variability of vacA and flaA may not be as high as was reported previously, and the flaA gene was found to be less variable than vacA.Variability of both genes and their products was within the range of that of comparable genes from other gastrointestinal bacteria.These results indicate that H pylori population genetics are nonclonal.Implications of this finding for H pylori treatment are multiple: phenotypic markers are more reliable than genetic markers for pathogenicity; drug resistance genes can spread faster in clonal bacterial populations than in nonclonal; and it may be difficult to develop a vaccine for H pylori because recombination allows an increased antigenic variation (similar to the case with influenza).A high recombination rate complicates identification of reliable markers and targets.

GENOMIC MAPS, MOLECULAR DIVERSITY, GENETIC EXCHANGE AND ANTIBIOTIC RESISTANCE
Mapping of H pylori genes by pulsed field gel electrophoresis has allowed the study of genome size, making it possible to compare bacterial strains.The genome size for H pylori ranges from 1.6 to 1.73 megabases, about one-third the size of the Escherichia coli genome (9).Homologous recombination, intergenic rearrangement or silent mutation (a change in DNA sequence without change in protein amino acid sequence) in certain genes, ie, urease methylation, results in genetic diversity within H pylori. Evolution of strains within one bacterial family occurs over many generations.
It is speculated that rapid gene shuffling occurs within an individual strain at some point in the life of H pylori, perhaps when an organism goes through the coccoid phase or when it infects the host (9).The coccoid form of H pylori represents a temporary dormant state, and the conversion of the bacterium from the coccoid form could be an adaptation in response to unsuitable environmental conditions.Because about one in five Canadians infected with H pylori has an organism resistant to metronidazole, antibiotic resistance is important.This resistance to antimicrobial agents is a major determinant of the efficacy of H pylori eradication regimens.Resistance of H pylori has been observed for metronidazole, erythromycin, clarithromycin (including crossresistance to erythromycin or clarithromycin) and ciprol floxacin.No resistance has been observed for other agents used to treat H pylori, such as bismuth, penicillins, tetracycline or furazolidone.Plasmids in H pylori have not been shown to mediate genetic transfer, and there are no antibiotic-resistant plasmids (9).However, bacteriophage activity has been identified in one strain of H pylori.This bacteriophage is lysogenic and able to live over many generations.It is unknown whether transduction (genetic transfer from one organism to another by bacteriophage) is possible in H pylori. Natural transformation (transfer of a DNA fragment from donor to recipient cell, followed by recombination of recipient chromosome) is the most important gene exchange mechanism in H pylori strains.The frequency of natural transformation is measured in relation to the development of antibiotic resistance (ie, to metronidazole or clarithromycin).
H pylori resistance to metronidazole also varies from one country to another.Sung et al (11) from Hong Kong studied the resistance of 1015 strains of H pylori to metronidazole, ampicillin and tetracycline after triple therapy.No resistance to ampicillin or tetracycline was found.However, metronidazole resistance was found to increase over five years, from 22% to 73% of strains.This rapid increase corresponded to use of metronidazole in triple therapy.Clarithromycin is widely used in H pylori eradication combination therapies.Clarithromycin resistance is related to a mutation in the 23S rRNA subunit.This mutation prevents the binding of the drug and therefore prevents antimicrobial action against the bacterium.The prevalence of H pylori strain resistance to clarithromycin varies, from 10% in France to 4% in the United States.In a Montreal study of adult DU patients, metronidazole and clarithromycin resistance was shown to be 18% and 0%, respectively (12).In a study by Best and colleagues (13), primary resistance was 2.1% in Nova Scotia.Clarithromycin is currently a suitable antibiotic for treatment of H pylori in Nova Scotia; however, regional differences in antimicrobial resistance rates may exist.

HOST FACTORS
Host genetics may play a role in the pathogenesis of PUD.PUD was known to be associated with genetics before the discovery of H pylori; evidence includes familial aggregation, association with ABO blood group, association with genetic syndromes, elevated pepsinogen levels in afflicted families and human leukocyte antigen markers (especially B12 and B5) associated with a higher rate of PUD (8).Genetic associations are also observed with H pylori: there is familial aggregation of the H pylori infection; H pylori binds to fucosylated blood group antigen associated with type O blood; and H pylori is associated with increased prostaglandins that normalize with H pylori eradication.Blood groups may serve as receptors for H pylori, as found in a twin study (14).The correlation coefficient for H pylori for monozygote twins reared apart was 0.66.Thus, genetics of the host may also influence H pylori-associated disease outcomes.

CANCER CONNECTION WITH H PYLORI H pylori has been classified by the International Agency for
Research on Cancer of the World Health Organization as a group 1 carcinogen, ie, a definite cause of gastric adenocarcinoma in humans (15).The precise mechanisms by which the bacteria contribute to carcinogenesis are unclear.H pylori infection may lead to an alteration in the epithelial cell cycle, which may explain the association with gastric cancers.Jones et al ( 16) investigated the degree of apoptosis in a pediatric population by using a TUNEL (Td T-mediated dUTPbiotin nick end-labelling) assay, which identifies preprogrammed cell death.The surface epithelium and lamina propria were tested in both H pylori-positive and -negative gastritis patients.They found that infection with H pylori causes a reversible increase in gastric epithelial cell apoptosis, compared with both secondary gastritis and noninflamed gastric mucosa.These findings suggest that induction of apoptosis is a potential virulence factor of H pylori. Thus, chronic infection with H pylori may increase the target cell population for tumourigenic events and may explain the association between chronic infection and gastric cancer.
Gastric mucosa-associated lymphoid tissue (MALToma) is the most common type of extranodal lymphoma.To ascertain whether MALTomas are malignant, clonality via polymerase chain reaction H pylori in fixed tissue must be demonstrated (17).There is strong evidence that H pylori is associated with MALToma: MALToma regresses after H pylori eradication; in vitro, H pylori stimulates isolated MALToma cells; and there is as high an association of H pylori to MALToma as H pylori to gastric carcinoma (17).

H PYLORI DIAGNOSIS
Only about one-third of H pylori-positive patients have antral nodularity, but if this nodularity is present, then there is more than a 95% chance that the patient has an H pylori infection (18).The specificity and sensitivity of histological tests for H pylori depend on the expertise of the pathologist and the quality of the biopsy (19).Histological diagnosis of H pylori can be done using the following stains: hematoxylin and eosin, Giemsa or Warthin-Starry silver.
Microbiological culture, the only diagnostic tool that allows testing for antimicrobial sensitivity, has the highest specificity of all H pylori tests (19).This method is laborious and time-consuming, although sensitivity and specificity can be high.In a study comparing two selection media for H py-H pylori highlights CAN J GASTROENTEROL VOL 11 NO 1 JANUARY/FEBRUARY 1997 lori, Gaudreau and co-workers (20) found that Skirrow medium (Difco) recovered significantly more isolates of H pylori than H pylori medium (Oxoid), with less bacterial overgrowth at three and seven days.They also found that culture and histopathology had the same sensitivity to diagnose H pylori infection, and that a simple medium was adequate to transport H pylori.
Serology tests for H pylori include physician office tests (qualitative: yes/no) and ELISA tests (quantitative: antibody level).Many available commercial tests are quick, simple and inexpensive.One major downfall of this detection method is the risk of false positive or false negative results.Serological tests may remain positive for some time after H pylori has been eradicated.However, there is a slight decrease in titres that can be observed six weeks after eradication.Fallone and co-workers (21) compared antibody titres of H pylori in patients in whom eradication was successful with those in whom it failed.Although either immunoglobulin (Ig) A or IgG serology can be useful in confirming eradication as early as one month after therapy, using titres from three, six or 12 months after therapy is more accurate.Therefore, it is possible to use paired sera to assess H pylori eradication, one pre-treatment and one at six to 12 months after treatment.A reduction in titres can then be used to indicate whether the H pylori infection is cured.The degree of change in titres needed to indicate eradication of H pylori will vary depending on the methodology used.However, while serological testing is more applicable for initial diagnosis of H pylori, using serological tests is not a practical approach in prospective trials or in a physician's practice to determine H pylori eradication.
Burak et al (22) investigated the accuracy of the 14 C urea breath test compared with culture, histology (Giemsa staining) and rapid urease test.H pylori infection was defined as positive results in at least two of the three tests.Agreement among tests occurred in 88% of subjects.Tests were not statistically different in their ability to detect H pylori.The 14 C urea breath test is a sensitive and specific test for the detection of H pylori; its accuracy is comparable with saliva, serology, rapid urease tests, histology and microbiology.Saliva-based tests (using IgG antibodies) have a specificity less than 60% and a sensitivity of about 80% (22).

TREATMENT OF H PYLORI INFECTION
The NIH has recommended treatment for H pylori eradication in patients with a current ulcer on initial presentation or recurrence, and those on continued acid suppression treatment for a previously diagnosed peptic ulcer (2).Treatment for H pylori eradication is now in the hands of primary care physicians (23).Chiba et al (23) surveyed Canadian physicians regarding the management and treatment of dyspepsia.They found that 7.2% of a family physician's practice is devoted to dyspepsia patients, and 23% of these patients present for the first time.The majority of patients are treated with lifestyle changes and/or antacids, or empirical drug therapy.If empirical therapy is used for dyspepsia, an H 2 blocker was the physician's drug of choice for ulcer and reflux-like dyspepsia, while prokinetics were often used for reflux and dysmotility-like dyspepsia.For patients known to be H pylori-positive, treatment was usually as above, whereas for H pylori-negative patients, treatment was directed at the symptom subgroup.Family physicians surveyed judged that of those who initially responded to empirical therapy, about 50% require continued therapy for a mean of six months.Family physicians estimated that the mean time to obtain a gastroenterologist's consultation was five weeks, and 70% indicated that this time delay influenced their decision not to refer a patient.If this time was reduced to, eg, less than two weeks, then family physicians would consider referring all dyspeptic patients.Most newly diagnosed dyspeptic patients in Canada are treated with empirical therapy, according to symptom classification, and referred for endoscopy after about two empirical treatment courses have failed.Dyspeptic patients with confirmed H pylori infection tend to be referred to a gastroenterologist earlier than those who are H pylori-negative (23).
When a patient presents with H pylori and PUD, H pylori must be treated as the primary disease (24).Because not all published studies are randomized controlled clinical trialsmany studies are only published in abstract form and are not peer-reviewed -the literature is complex, fragmented and confusing (25).It is clear that H pylori infection does not disappear spontaneously and that antimicrobial therapy is required to cure patients.Factors influencing eradication rates with triple therapy include the patient's compliance to drug therapy (26), smoking status, age and the duration of therapy (27).
Until 1995, triple therapy (bismuth subsalicylate, metronidazole, and either amoxicillin or tetracycline) was considered to be the best treatment for eradication of H pylori. 'Standard' triple therapy consists of bismuth 120 mg qid (in Canada we only have bismuth subsalicylate), tetracycline 500 mg qid and metronidazole 400 mg tid.In the metaanalysis by Chiba et al (28), bismuth plus one antibiotic achieved an eradication rate of 50%, whereas bismuth plus two antibiotics achieved an eradication rate of 82%.This regimen achieved eradication rates of 90% if the H pylori strain was sensitive to metronidazole (29).However, the downfalls of this combination therapy include a high risk of side effects (30% to 50%); lowered compliance (up to 16 tablets per day must be taken for 14 days), which in turn reduces efficacy; and resistance to metronidazole (30).
Dunne and van Zanten (31) retrospectively reviewed charts of all patients treated with bismuth triple therapy at one gastrointestinal unit from 1991 to 1995.Overall eradication rate was 79% with tetracycline, which was more effective than triple therapies with amoxicillin (28).Patient age was an influential factor in the success of H pylori eradication: patients older than 50 years had a higher eradication rate.
Proton pump inhibitors (PPIs) have a small antimicrobial effect in vitro and may inhibit urease and bacterial ATPase activity (32).The increased intragastric pH achieved with PPI treatment is optimal for increased antimicrobial activity, which may be due to an increased concentration of the anti-microbial in the presence of PPIs, decreased immunoglobulin denaturation and optimal leukocyte function at an increased intragastric pH (32).
Dual combination therapies such as omeprazole and amoxicillin are simpler than 'standard' triple therapy and have been recommended by the NIH (2).This is an approved regimen in Canada.This omeprazole-amoxicillin combination is affordable and safe, with only 10% of patients reporting adverse effects (33).The reported eradication rates with omeprazole-amoxicillin range from 30% to 85%.The high variability is partially due to the wide variety of doses and durations that have been tested using this combination (34).
Metronidazole diffuses from the serum into the gastric lumen (35).In the acidic environment of gastric juice (pH less than 2) metronidazole is rapidly converted to the ionized form.Despite decreased total metronidazole concentrations in gastric juice following omeprazole administration, the concentration of nonionized metronidazole remains the same.This is because nonionized metronidazole is the form that is in equilibrium with serum metronidazole, and the same concentrations of nonionized metronidazole accumulate at any level of gastric pH.At low pH 'trapping' of metronidazole in ionized form causes a high total metronidazole concentration (35).After therapy with omeprazole at gastric pH greater than 4 all metronidazole crossing the mucosa remains uncharged and there is no 'trapping'.Thus, omeprazole does not adversely affect nonionized metronidazole concentrations to which H pylori is exposed, which is likely important with metronidazole-omeprazole combination therapy (35).
Clarithromycin is the most stable macrolide in gastric juice.Monotherapy with clarithromycin can achieve an H pylori eradication rate of up to 54%.Both clarithromycin and its metabolite demonstrate synergy with omeprazole.In a study by Hunt and colleagues (36), these three regimens (omeprazole 40 mg qid + clarithromycin 500 mg tid versus omeprazole 40 mg qid versus clarithromycin 500 mg tid) were compared in terms of DU healing, H pylori eradication and ulcer relapse.Ninety-four per cent of DU healed at six months and a 72% H pylori eradication rate was attained.More consistent eradication rates have been observed with omeprazole-clarithromycin than with omeprazole-amoxicillin.
After the general use of standard triple therapy and the subsequent omeprazole-based dual combinations (omeprazole-amoxicillin and omeprazole-clarithromycin), H pylori eradication research investigated triple therapies using omeprazole plus two different antibiotics instead of just one (such as omeprazole plus metronidazole plus clarithromycin) given twice a day for one week.The results achieved in the landmark MACH1 study demonstrate the high efficacy and safety profile of five combinations of omeprazole plus two antibiotics (37).Each of the five active treatment arms achieved H pylori eradication rates ranging from 79% to 96%.Three additional weeks of omeprazole therapy was given to patients to ensure ulcer healing.In the 'placebo' (omeprazole-only) arm, the H pylori eradication rate was only 1%.Two treatments achieved the desired eradication rate above 90%: omeprazole-amoxicillin-clarithromycin 500 mg bid (95% CI 93% to 100%) and omeprazole-metronidazole-clarithromycin 250 mg bid (95% CI 90% to 99%).The eradication treatments were well tolerated.For these reasons, the MACH1 regimens are rapidly replacing standard triple therapy as the treatment of choice.
Lansoprazole triple therapies have shown H pylori eradication rates of 70% to 100%.'Classic' bismuth triple therapy plus omeprazole or lansoprazole (seven to 10 days or longer) -also called 'PPI-based quadruple therapy' -have been tested in a few small studies.Eradication rates are 95% to 98% with 'classic' bismuth triple therapy plus omeprazole (38).The use of a seven-day omeprazole-based triple therapy to manage H pylori-positive PUD has been endorsed in new practice guidelines from the American College of Gastroenterology (39).They recommend three treatment regimens that involve an acid pump inhibitor.However, those guidelines state that "there are too few data for other PPIs to establish equivalency with omeprazole" (39).