Unlike most bacterial pathogens, H. pylori typically colonizes the host for life unless specific treatment is given. H. pylori has co-evolved with humans for approximately 58000 years, and strain types that predominate within certain regions of the world correlate with human migration patterns[1,6]. Most infected individuals do not develop overt disease, leading to the hypothesis that some H. pylori strains are harmless or even beneficial[7]; however, the list of diseases potentially caused or worsened by H. pylori has been growing in recent years, making it premature to conclude that any strain is commensal.

Several unique properties contribute to H. pylori persistence. All H. pylori clinical isolates express urease. Urease converts urea to ammonia plus carbon dioxide, raising the pH of the surrounding area. This provides temporary protection against gastric acid, but H. pylori is not an acidophile. It requires the near-neutral pH found in the mucus layer directly adjacent to the gastric surface epithelium. The helical shape of H. pylori makes it easier for its polar flagella to propel H. pylori through viscous mucus. Chemotaxis systems direct H. pylori towards some amino acids, bicarbonate, and cholesterol, while acidic pH serves as a repellent[8]. This system keeps the organisms in the favorable milieu close to the surface epithelium.

The outer surface of H. pylori also has unique properties. H. pylori glycosylates host cholesterol and inserts it into its outer membrane. The function of glycosylated cholesterol is not entirely known, but H. pylori lacking cholesterol are extremely sensitive to environmental stress[9,10]. Lack of glucosylated cholesterol reduces CagA-mediated activities and interactions with T calls[10,11]. Another interesting membrane feature is the relatively non-toxic lipopolysaccharide (LPS) found in H. pylori, which may contribute to persistence by limiting the host inflammatory response. Unlike LPS from other species, H. pylori LPS is recognized by TLR-2 rather than TLR-4[12]. In some strains, side chains on the LPS O antigen mimic the Lewis blood group antigens Le^x and Le^y. The membrane extends to form a sheath covering the flagella. The combination of sheathed flagella, hypoinflammatory LPS, and molecular mimicry reduce the host response, allowing the organism to persist with minimal pathology. Furthermore, H. pylori forms outer membrane blebs that pinch off from the surface of the bacterium. These outer membrane vesicles contain both membrane-associated and cytoplasmic components, including CagA and other virulence factors[13]. In addition to being an alternative virulence factor-delivery mechanism, outer membrane vesicles may serve as “dummy targets” for the immune system, thus facilitating immune evasion.

H. pylori has a number of virulence factors which influence colonization and disease severity (Figure 1). CagA, encoded by cytotoxin-associated gene A (cagA) is the most important and best-studied virulence factor; however, it does not function in isolation. CagA-positive H. pylori strains are associated with greater inflammation and increased risk of ulcers and cancer in both humans and animals[14]. The cagA gene is part of a pathogenicity island that also encodes components of a type IV secretion apparatus. The secretion apparatus is a syringe-like structure that was once thought to have little importance other than its ability to inject CagA; however, recent studies suggest CagA-independent functions. For example, the structural protein CagL mimics fibronectin[15] and peptidoglycan is injected into host cells along with CagA[16]. Finally, it is important to note that the presence of cagA usually coincides with the presence of other virulence factors, including vacA, babA and oipA[17]. Thus, H. pylori pathogenesis is multifactorial and cannot be boiled down to one gene.

Open in New Tab   Full Size Figure   Download Figure

Figure 1 Major virulence factors of Helicobacter pylori. Type IV secretion system-mediated transport of CagA and autotransport of VacA monomers are depicted. Outer membrane vesicles can contain membrane-associated and soluble virulence factors. Representative outer membrane adhesins are shown. GGT: γ-Glutamyl transpeptidase.

CagA is responsible for myriad signaling alterations, which profoundly influence host cell physiology. When H. pylori makes contact with host cells, CagA is directly injected into the host cell cytoplasm where it becomes phosphorylated and binds the SH2 domain of host SHP-2[18]. SHP-2 is a phosphatase involved in transduction of signaling from receptor tyrosine kinases[19]. Mutations in SHP-2 are found in some human tumors, providing a mechanistic link between CagA activity and tumor development. CagA also interacts with other proteins involved in signal transduction, such as c-Met[19]. CagA colocalizes with tight junction proteins, causing decreased cell-cell adhesion and loss of cell polarity (Figure 2)[20]. This, along with cytoskeletal rearrangements, contributes to increased invasiveness of host cells through the extracellular matrix. CagA also drives the transition from an epithelial to a mesenchymal cell phenotype[12]. All of these phenotypes are associated with carcinogenesis[21]. CagA positive strains are often associated with increased IL-8 production; however H. pylori induces IL-8 secretion through multiple mechanisms, some of which are CagA-independent[22]. CagA induces genes encoding defensin 2 and other antimicrobial peptides, which could explain why CagA positive strains appear to be easier to eradicate than CagA negative strains[23]. Dendritic cells intoxicated by CagA display impaired activation, leading to decreased inflammatory cytokine production and decreased stimulation of a Th-1 response[24].

Open in New Tab   Full Size Figure   Download Figure

Figure 2 Effects of Helicobacter pylori on host cells. Helicobacter pylori virulence factors can lead to apoptosis, vacuolization, disruption of barrier function (leading to nutrient leakage), de-differentiation, and carcinogenesis.

Not all CagA proteins are created equal; some cagA alleles encode additional SH2 binding sites, further suppressing the function of SHP-2[18]. A five amino acid motif (EPIYA motif) in the carboxy-terminal portion of CagA is the phosphorylation site, and clinical isolates differ in the number of EPIYA motifs present[25]. EPIYA motifs are of particular interest because certain EPIYA polymorphisms are more strongly associated with cancer than others[26]. CagA lacking the EPIYA motif is not phosphorylated and activates the STAT3 pathway instead of the MAPK/ERK pathway, which is induced by SHP-2 binding[27]. STAT3 activation leads to increased cell migration, while MAPK/ERK activation leads to cell growth inhibition and morphological changes.Oddly, CagA reduces the cellular effects of VacA and vice versa. CagA reduces vacuolization and apoptosis caused by VacA and VacA reduces the cytoskeletal rearrangements caused by CagA[28]. These findings once again highlight the influence of H. pylori’s genetic variability and interactions between virulence factors on pathogenesis.

Vacuolating toxin A (VacA) was named for its ability to induce numerous large vacuoles in host cells. Unlike CagA, VacA forms an autotransporter structure to secrete itself without the need for host cell contact. VacA proteins oligomerize to form pore-like structures. VacA binds to receptor-like protein tyrosine phosphatases (RPTPα and RPTPβ) and other glycosylated transmembrane proteins on the host cell surface[29]. VacA is then endocytosed and forms anion-selective channels in vacuole membranes. The channels allow accumulation of chloride anions and weak bases, resulting in osmotic swelling[30]. VacA also inserts into mitochondrial membranes, causing mitochondrial dysfunction and subsequent apoptotic death of the cell[31]. Vacuolation is not the only effect of VacA intoxication. VacA disrupts the barrier function of epithelial cells, allowing leakage of crucial nutrients such as iron, nickel, and amino acids. This likely improves H. pylori growth[32]. In vitro, VacA inhibits antigen presentation and T cell activation, but it is not clear whether these activities occur in vivo[21].

All H. pylori strains contain vacA genes, but not all strains produce functional VacA. This is due to polymorphisms within the VacA gene, particularly at the amino terminus (s region), in the middle of the gene (m region), and in an intermediate region (i region). The s2 polymorphism yields an inactive toxin[28]. Thus, strains with the s2 allele are often termed “VacA negative”. The i region polymorphisms were discovered more recently and influence vacuolating activity; vacA containing the i1 allele produces the most active toxin[33]. Strains harboring the s1m1 allele have been more commonly associated with ulcers and gastric cancer, but it now appears that the i1 allele is more strongly associated with ulcers and cancer than the presence of the s1m1 genotype[28]. In spite of the dramatic effects of VacA on epithelial cells, it is unclear whether VacA plays a causative role in these diseases. More likely, VacA facilitates nutrient acquisition, improving the ability of H. pylori to colonize the gastric epithelium[34].

H. pylori encodes two variably expressed sialic acid-binding adhesins, BabA and SabA. BabA, encoded by babA2, binds to the Lewis b ABO blood group antigen (Le^b) and is present on red blood cells and certain epithelial cells. Expression of babA2 is variable, and can be turned “on” and “off” by slipped-strand mispairing mutations. BabA binding activity is most often present in CagA positive strains[34]. BabA-mediated binding to Le^b contributes to formation of double stranded DNA breaks in host cell lines and may promote cancer-associated gene mutations[35]. Adherence via BabA also enhances the ability of the type IV secretion apparatus to contact host cells, leading to a stronger inflammatory response[36]. These data suggest that BabA may indirectly influence pathology by improving adherence to host cells.

SabA is a sialic acid-binding adhesin that recognizes the sialyl-Lewis A antigens sLe^x and sLe^a[37,38]. SabA increases colonization density in patients lacking Leb, indicating its importance as an adhesin. Sialylated Lewis antigens are more prevalent on inflamed or cancerous gastric tissue, leading to the hypothesis that SabA is involved in carcinogenesis; however, not all studies demonstrate correlations between the presence of SabA and cancer[39].

H. pylori has about 30 other outer membrane proteins which may serve as adhesins, but only a few have known functions. AlpA and AlpB are now known to bind host laminin. Their presence modulates the inflammatory response in gerbils through unknown mechanisms[40]. It is not known whether variability in alpAB gene sequence or expression influence human disease. OipA (Outer Inflammatory Protein; a.k.a HopH) is an outer membrane protein that functions as an adhesin[41]. The gene can be turned on and off via slipped strand mispairing. Interestingly, most cagA positive strains possess an expressed form of oipA, making it more difficult to separate the clinical effects of oipA from those of cagA[17]. In vitro and in vivo animal experiments clearly show that OipA is associated with increased IL-8 production and carcinogenicity[42]. Inactivating oipA in a carcinogenic H. pylori strain eliminated tumorigenesis in gerbils[43]. This may be partially explained by the decreased nuclear translocation of beta-catenin induced by mutant strains lacking OipA. Beta-catenin is an important player in the gastric carcinogenesis process[44].

The neutrophil-activating protein (HP-NAP), encoded by napA, was first isolated from water extracts of H. pylori[45]. Although it is cytoplasmic, HP-NAP escapes via autolysis. Amino acid sequencing led to the napA gene, which is homologous to other bacterioferritins. Bacterioferritins are similar to mammalian ferritins in that they form multimeric, hollow structures that store iron and bind DNA. These proteins protect prevent iron-mediated chemical reactions from forming reactive oxygen species[46]. Further studies revealed that HP-NAP does not behave like other bacterioferritins. HP-NAP crosses both the epithelium and the endothelium, where it recruits neutrophils and stimulates both neutrophils and monocytes to produce IL-12 and IL-23[47,48]. These cytokines induce T cells to secrete interferon-γ and mediates the shift to a Th1 response[48]. HP-NAP binds to TLR-2, which typically recognizes bacterial cell wall components. Unlike other TLR-2 agonists, HP-NAP induces substantial IL-12 production[48]. HP-NAP also promotes clot formation and inhibits fibrin degradation by monocytes[49].

HP-NAP has been suggested as a tumor-fighting agent and an allergy modulator. Intratumoral injection of HP-NAP into mouse neuroendocrine or bladder tumors led to tumor regression and a shift towards cytotoxic T cells[50,51]. Since allergies are associated with a Th2-skewed immune response, inducing a shift towards a Th1 response could be beneficial[52]. Proof-of-principle has been demonstrated in a mouse model of ovalbumin-induced asthma. Injection of HP-NAP reduced eosinophilia and IgE concentrations in sensitized animals[53]. Enthusiasm for HP-NAP-based treatments is tempered by concerns regarding the potential for autoimmune reactions induced by homology between HP-NAP and human aquaporin in neural tissues[54].

The discovery that H. pylori gamma-glutamyl transpeptidase (GGT) functions as a virulence factor was surprising, since it hadn’t been identified as a virulence factor in other bacteria[55]. Moreover, it appears to be periplasmic rather than surface-associated or secreted. Its presence in outer membrane vesicles[13] could facilitate interactions with host cells. GGT increases IL-8 secretion and hydrogen peroxide production in epithelial cells and is associated with peptic ulcer development[56]. It also contributes to colonization, persistence, and tolerization of dendritic cells[57]. GGT-mediated degradation of glutathione produces pro-oxidant compounds that facilitate formation of reactive oxygen species. This mechanism could be responsible for host tissue damage[58].

In addition to H. pylori variability, several host factors also influence disease outcome. IL-1β is induced rapidly following infection. Polymorphisms in genes encoding IL-1β or its receptor antagonist increase the risk of gastric cancer in many populations; however this may not hold true for some Asian and white populations[21]. Polymorphisms in genes encoding TNFα, IL-10, and HLA are also implicated[21]. Reduced IL-10 expression results in a more inflammatory Th1-polarized host response. Smoking and a diet high in salty foods exacerbate the effects of H. pylori. All of these things could explain why some studies show clear correlations between H. pylori and a particular disease, while others do not.

Functional dyspepsia is defined as pain associated with the stomach or upper abdomen. Ulcers are one cause of dyspepsia, but many patients have no evidence of gastric damage. The role of H. pylori in non-ulcer dyspepsia has been somewhat controversial. The preponderance of evidence suggests that H. pylori infection contributes to some cases of dyspepsia. A randomized clinical trial involving 585 dyspepsia patients found that H. pylori eradication led to greater benefit in patients with epigastric pain syndrome than in postprandial distress syndrome[80]. These and other results suggest that the “test and treat” strategy is reasonable for non-ulcer dyspepsia patients[81], particularly those with epigastric pain. Dyspepsia symptoms do not always resolve following eradication therapy, but the risk of future ulcer and gastric cancer development are reduced.

Gastritis is universally present in H. pylori infected patients and is reproduced in various animal models. Acute gastritis, sometimes associated with dyspepsia or nausea, develops soon after initial infection. Acute gastritis affects the entire stomach and is accompanied by loss of acid secretion[82,83]. Neutrophils are recruited to the lamina propria and epithelium and damage results from reactive oxygen species and other neutrophil products. The acute phase gives way to chronic gastritis as lymphocytes replace neutrophils.

Chronic gastritis can be antrum-predominant, corpus-predominant, or diffuse (pangastritis or multifocal gastritis) (Figure 4). In antrum-predominant gastritis, acid secretion usually remains intact and H. pylori colonization is limited to the antrum[83]. Antral gastritis favors development of duodenal ulcers, while corpus-predominant gastritis favors gastric ulcer formation, sometimes progressing to metaplasia and adenocarcinoma[82,84]. Patients with diffuse gastritis typically have severely impaired acid secretion, which allows H. pylori to colonize the corpus. Chronic acid suppression mediated by proton pump inhibitors can lead to a switch from antral predominant to pangastritis[83]. Interestingly antrum-predominant gastritis and duodenal ulcers do not increase the risk of cancer development[85,86].

Open in New Tab   Full Size Figure   Download Figure

Figure 4 Typical Helicobacter pylori-associated pathology. Gastritis may be antral-predominant, corpus-predominant, or diffuse. The risk of developing adenocarcinoma or MALT lymphoma varies depending on the type of pathology present.

Historically, H. pylori has been responsible for 70%-85% of gastric ulcers and 90%-95% of duodenal ulcers[82,87]. The percentage of ulcers not associated with H. pylori is rising due to increased use of NSAIDs and decreasing prevalence of H. pylori infection; however H. pylori eradication is partially protective against ulcer development in NSAID users[87,88]. Also, the hypothesis that ulcers are caused by stress is reemerging. Several studies show that anxiety disorders and physical or emotional trauma correlate with ulcer development (reviewed in[89]). Studies testing the interaction between H. pylori and stress are lacking, so it is not clear whether those factors operate independently or whether there is an additive effect of infection and stress on ulcer formation.

H. pylori is an accepted cause of gastric adenocarcinoma and MALT lymphoma (official name: extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue). Gastric adenocarcinoma is divided into intestinal subtype and diffuse subtype. Intestinal type adenocarcinoma is more common and has been well studied. The sequence of pathological changes leading to intestinal type cancer starts with gastritis, followed by gastric atrophy (loss of glandular structure) and progressing to intestinal metaplasia, dysplasia, and finally carcinoma[14]. This progression occurs over many years; consequently, most patients are middle aged or older. Environmental factors, such as smoking and diet, increase the risk of developing gastric adenocarcinoma. H. pylori-associated adenocarcinoma most often occurs in the distal (non-cardia) stomach. H. pylori-mediated adenocarcinoma has been replicated in animal models, particularly in gerbils[85,90]. The presence of cagA, vacAs1m1, babA2, and oipA increase the risk of cancer[91-94]. Cancer risk is further elevated by the presence of certain EPIYA motifs in CagA[26,95]. A number of good reviews discuss the molecular mechanisms in detail[85,96,97].

Diffuse type adenocarcinoma is characterized by scattered tumor cells and lack of glandular organization. Diffuse-type gastric cancer tends to occur in younger individuals and may have a strong genetic component, although H. pylori also plays a role[98]. Environmental factors contribute little to this type of cancer. Pangastritis and rugal hyperplasia confer higher risks of diffuse-type adenocarcinoma[99,100]. The mechanisms underlying development of diffuse-type cancer are poorly understood, but abnormal DNA methylation is likely involved. H. pylori induces hypermethylation of the gene encoding E-cadherin, which is implicated in hereditary diffuse-type cancer[99,101].

The association between MALT lymphoma and H. pylori was first published in 1991[102] and has since been corroborated by numerous studies. H. pylori is responsible for 92%-98% of gastric MALT lymphoma[3]. The formation of lymphoid follicles is necessary, but not sufficient for development of MALT lymphoma. CagA and other virulence factors contribute little to the risk of developing MALT lymphoma[103]; however the presence of CagA within tumor-associated B cells correlated with earlier remission following H. pylori eradication compared with patients whose tumors did not contain CagA[104]. Most gastric MALT lymphomas are low grade when discovered and are dependent upon continued antigen stimulation. Lymphocytes are prone to developing genetic mutations, and prolonged antigen-driven proliferation of B lymphocytes eventually leads to a population that is antigen independent[105]. Interestingly, the tumor-associated B cells do not recognize H. pylori. Some have suggested that B cells recognize autoantigens, but an in-depth study of antigen specificity did not uphold previous results[105,106]. Thus it is not clear whether antigen specificity plays a role in MALT lymphoma development.

The first line of treatment for H. pylori-positive MALT lymphoma is eradication therapy. H. pylori eradication leads to lasting remission in about 80% of patients[107]. Some H. pylori-negative MALT lymphomas, including a recurrent parotid lymphoma, have also responded to eradication therapy, suggesting that these were either false negatives or perhaps caused by another Helicobacter species[108,109].

Gastric adenocarcinoma and MALT lymphoma were the first cancers associated with H. pylori, but they may not be the last. Several other cancers have been recently associated with H. pylori, though the evidence is not ironclad. The most common extragastric cancer potentially influenced by H. pylori is colorectal adenocarcinoma. Gastric H. pylori routinely passes through the intestinal tract, so it is not unreasonable to suspect that H. pylori could influence development of colorectal cancer. Several studies support this hypothesis. Three recent meta-analyses found a significantly increased risk of both colon polyps and colon cancer in H. pylori-infected patients[110-112]. One study examined the prevalence of gastric pathology in patients with hyperplastic polyps, colon adenoma, advanced adenoma, and colon cancer[113]. All patient groups had higher prevalences of H. pylori-positive gastritis and intestinal metaplasia. The odds ratios were largest in patients with colon cancer. Those patients had significantly increased risks of gastritis, intestinal metaplasia, gastric adenoma, gastric cancer, and gastric lymphoma. Grouping patients according to whether or not the cancer was advanced revealed that the infection rate was higher in advanced cases (86%) than on non-advanced cases (51%). Forty eight percent of control patients were infected[114]. Oddly, cagA positive strains were not more prevalent in cancer patients. In summary, it appears that H. pylori could contribute to the risk or aggressiveness of colorectal cancer, but much additional research is needed to confirm the association and determine the underlying mechanism.

Pseudomyxoma peritonei (PMP) is a rare peritoneal cancer that most often originates in the appendix[115]. Appendiceal rupture allows the cancer to invade the peritoneal cavity. Bacteria are able to enter the peritoneum at the time of rupture, but the viscous mucus prevents leakage of fecal material into the peritoneum and peritonitis does not ensue. Instead, a subset of bacteria present within the mucus persistently colonizes the peritoneal cavity. The appendiceal rupture site heals, trapping the tumor, mucus, and bacteria within the peritoneal cavity. Subsequent mucin accumulates in the peritoneal cavity causing compression of abdominal organs. Pseudomyxoma peritonei originating from the appendix is essentially similar to the mucinous subtype of colorectal adenocarcinoma. In fact, mucinous colonic polyps sometimes cause PMP[116]. Live H. pylori have been found within tumor and mucinous material[117]. Patients who were treated with standard H. pylori eradication therapy prior to surgery had reduced nuclear β-catenin, indicating cellular normalization, and tended to survive longer than patients given only standard care[61,118].

H. pylori could increase the risk of other lymphoma types. Diffuse large B cell lymphoma (DLBCL) is an aggressive cancer that can occur in the stomach and elsewhere. It is considered separate from gastric MALT lymphoma, although the former can sometimes transform into the latter[119]. This type of cancer is currently not believed to be H. pylori-associated; however, there have been a number of case reports in which H. pylori eradication alone led to complete remission[120]. Inaba et al[121] found that primary gastric diffuse large B cell lymphoma increases the risk of developing gastric adenocarcinoma, often within 5 years of treatment. The author proposes that chemotherapy and radiation may hasten the development of gastric adenocarcinoma in H. pylori-positive patients. Testing DLBCL patients for H. pylori may therefore be prudent.

As yet, there is only scant evidence that H. pylori influences extragastric lymphomas. One patient with rectal MALT lymphoma had a complete response to H. pylori eradication therapy and remained disease-free after 34 mo[122]. Ocular adnexal lymphoma (OAL) is a most often a MALT lymphoma presenting on the orbit or conjunctiva[123]. A comparison of patients suffering from ocular adnexal lymphoma, extragastric lymphoma in other body sites, and controls found that OAL patients were 32% more likely to be infected with H. pylori than controls. Furthermore, patients with lymphoma at other sites were 13% more likely to be infected. Both of these findings were highly significant[124]. H. pylori DNA has been found in one OAL tumor out of 6 tested[125]. To date, there are no reports of H. pylori eradication therapy being tested in OAL patients.

A number of studies report that H. pylori infection protects against Barrett’s esophagus and subsequent laryngeal adenocarcinoma development (see section on laryngeal disease); however, some studies suggest a positive association with laryngeal squamous cell carcinoma[126-128]. Burdick[129] examined 75 patients with laryngeal squamous cell cancer. All samples were positive for ureA, and 47%-49% of ureA positive samples were also cagA positive. The cagA gene was much more prevalent in lymph node positive tumors than in lympth node negative tumors. Additionally, cagA was far more prevalent in supraglottic tumors than in glottic or subglottic tumors. The survival rate was also lower in cagA positive patients. Guilemany et al[130] found a significant correlation between hypopharyngeal squamous cell carcinoma and H. pylori infection (P < 0.001). It should be noted that not all studies have found a positive correlation[131,132].

The association between H. pylori infection and iron deficiency anemia (IDA) is well established[133,134], but the mechanisms underlying this association are unclear. The most obvious mechanism for H. pylori to cause IDA is through competition for dietary iron. H. pylori requires higher concentrations of inorganic iron and zinc for in vitro growth than other pathogens[59], yet there is no evidence that H. pylori has more iron- or zinc-dependent enzymes than other bacteria. H. pylori may have less efficient iron acquisition symptoms than other organisms, but this would suggest that H. pylori competes poorly for dietary iron. H. pylori is able to acquire iron from host transferrin and lactoferrin, yet it binds the iron-free forms of these proteins more avidly than the iron-loaded proteins[60]. All of this points towards an evolutionary advantage for H. pylori strains that do not overly tax the host iron supply. Nonetheless, H. pylori genomes vary considerably from strain to strain, as do virulence properties. Yokota et al[135] found that certain polymorphisms within the H. pylori neutrophil-activating protein were more prevalent in strains from IDA patients than from non-IDA patients. Moreover, IDA strains harboring these polymorphisms internalized iron more rapidly than other strains. Inorganic iron dissolves best in highly acidic conditions. Loss of gastric acid secretion due to H. pylori infection could therefore reduce bioavailability of dietary iron. Excess lactoferrin in gastric tissue could impair iron uptake, since transferrin is the normal route of host iron uptake[136]. In male patients with refractory IDA, H. pylori infection was associated with higher serum TNFα levels, particularly in those infected with CagA⁺H. pylori[137]. This is relevant because TNFα and other pro-inflammatory cytokines can induce anemia.

Idiopathic thrombocytopenic purpura (ITP) is characterized by autoimmune destruction of platelets, which leads to bruising. Significant evidence points to H. pylori as a causative agent in some cases of ITP. For example, H. pylori eradication therapy increases platelet counts only in those who were infected with H. pylori, not those who were uninfected ruling out an off-target effect of antibiotics[138]. Among ITP patients treated for H. pylori infection, patients who had a lower serum pepsinogen I/II ratio responded better (i.e., greater improvement in platelet counts) than those with higher pepsinogen I/II ratios. This correlated with more severe atrophic gastritis in responders than in non-responders[139]. Host factors likely play a role as well. H. pylori-infected patients who lack a particular single nucleotide polymorphism in IL-β, the IL-β (-511) T allele, were more likely to develop ITP before age 50 years than uninfected ITP patients[140].

H. pylori appears contribute to several inflammatory skin diseases. Rosacea is the most common skin disease potentially associated with H. pylori. In one study, H. pylori was present in 81% of rosacea patients who also had gastric complaints, and almost all of those patients harbored cagA+ strains[141]. A similar Egyptian study found that both cagA and the s1m1 allele of vacA were more prevalent in papular rosacea patients. The papular rosacea patients responded better to eradication therapy than the erythematous rosacea patients[142]. A limited study of ocular rosacea patients reports that the seven ocular rosacea patients responded better to H. pylori eradication therapy than did rosacea patients without ocular symptoms[143].

Chronic prurigo consists of intensely itchy nodules. There are several variants, including prurigo pigmentosa and prurigo chronica multiformis. These disease are most common in young women of Japanese or Turkish descent[144]. Limited studies suggest higher prevalence of H. pylori or clinical improvement following H. pylori eradication therapy[145]. H. pylori-associated gastric diseases are also more prevalent in these patients[145-147]. Immunohistochemical evidence of H. pylori in a prurigo pigmentosa patient strengthens the case for a link and suggests that H. pylori acts directly within the skin, rather than indirectly through inflammatory mediators[148]. These mechanisms are not mutually exclusive, but much more research is needed to clarify etiology and pathogenic mechanisms related to H. pylori.

Chronic idiopathic urticaria consists of itchy wheals that are present on most days with no known cause. A meta-analysis of 10 chronic urticaria studies completed prior to 2003 found that the overall remission rate following H. pylori eradication was 30.9%. This was statistically significant with an odds ratio of 2.9[149]. Chiu et al[150] found no difference in the H. pylori infection rates in chronic spontaneous urticaria (CSU) patients, but 63.6% of patients had complete remission after H. pylori eradication. Likewise, Campanati et al[151] found no difference in H. pylori prevalence, but found significant improvement following H. pylori eradication therapy. In the same study, the authors found an unusually high rate of small intestinal bacterial overgrowth in CSU patients. Those patients were treated with the recommended antibiotic, but their CSU symptoms did not improve. Yoshimasu and Furukawa[152] grouped patients according to anti-H. pylori titer. None of the patients with high titers was cured with antihistamine treatment alone. Eradication therapy raised the cure rate to 56%. Patients with low or absent anti-H. pylori titers were more likely to respond to antihistamine treatment alone. These studies point to the conclusion that H. pylori is one of several causes of CSU.

Case reports suggested an association between psoriasis and H. pylori[153,154], providing impetus for a larger study by Onsun et al[155]. Stratifying a group of 300 plaque psoriasis patients by symptom score revealed that 100% of patients with moderate or severe psoriasis were H. pylori positive, while only 37% of mild psoriasis patients were infected. H. pylori positive patients were treated with acitretin alone, H. pylori eradication therapy alone, or both. All treatment groups showed improvement after 8 wk, but improvement was more rapid when H. pylori eradication was included. H. pylori treatment alone was just as effective as both eradication therapy and acitretin[155].

H. pylori may contribute to diseases of pregnancy. Hyperemesis gravidarum consists of severe and prolonged vomiting that goes far beyond typical “morning sickness”. A meta-analysis of clinical studies found that the majority support the hypothesis that H. pylori contributes to hyperemesis gravidarum, although further studies are needed[156]. One study found that 80% of hyperemesis gravidarum patients were H. pylori-positive compared with 35% of controls[157]. Mansour and Nashaat[158] report improvements in H. pylori-positive hyperemesis gravidarum patients following eradication therapy[158].

A study comparing women with normal pregnancies to those with pre-eclampsia (PE) yielded striking results[159]. The correlation between H. pylori seropositivity and PE was highly significant, with an odds ratio of 9.22. The relationship was even stronger for pregnancies with both PE and fetal growth retardation (OR = 35.56). CagA⁺ strains were overrepresented in both PE and PE with fetal growth retardation, yielding odds ratios of 17.66 (PE) and 54.97 (PE with fetal growth retardation). In contrast, pregnancies with only fetal growth retardation were not associated with greater than expected H. pylori seropositivity. A more recent study found that antibodies against CagA cross-react with beta-actin of cytotrophoblast cells[160]. This may impair placental invasion.

Since H. pylori is frequently present in the oral cavity, perhaps it is not surprising that it is occasionally found in the ear, nose and throat. Aside from gastroesophageal reflux disease, too few studies have been done to determine whether H. pylori causes disease in those locations. Nonetheless, some of the data are tantalizing. In one study, H. pylori was cultured from 40% of adults with otitis media with effusion[161]. Other studies have used the Campylobacter-like organism test (CLO test) to identify H. pylori infections in the middle ears of patients with otitis medium or tympanosclerosis[162,163]. Melake et al[164] compared children undergoing myringotomy for chronic otitis media with children who were undergoing tonsillectomy/adenoidectomy, but did not have a history of otitis media. The study group was roughly three times more likely to have evidence of H. pylori in the stomach, adenoids, or tonsils than the control group. Almost all of the study patients with evidence of gastric H. pylori were also either culture- or PCR-positive for H. pylori in middle ear fluid. Interestingly, another study found that 48 of 49 biopsies taken from 37 children with adenotonsillar hypertrophy contained H. pylori[165], raising the possibility that the association between H. pylori and chronic otitis media could be stronger than it appeared to be in the Melake study. H. pylori worsened inflammation in a rabbit model of histamine-induced otitis media with effusion[166]. This suggests that H. pylori may not be the initial cause of otitis media, but these bacteria may exacerbate pre-existing infections.

Several studies have demonstrated the presence of H. pylori in nasal polyps[167-169]. One study failed to find a difference in gastric colonization between patients with nasal polyps vs patients with concha bullosa, but H. pylori was only found in nasal biopsies from nasal polyposis patients[170]. In a study of nasal polyps vs benign laryngeal disease, H. pylori was detected in all 60 patients with either nasal polyps or benign laryngeal disease. Interestingly, cagA-positive H. pylori was detected in 23.3% of patients with laryngeal disease, but in none of the nasal polyps[171]. CagA is often associated with more severe gastric disease, but this toxin may put H. pylori at a disadvantage in certain anatomical sites.

The relationship between H. pylori infection and esophageal diseases has been a controversial topic for years. According to the dogma, eradicating H. pylori increases gastric acid output. This leads to exacerbation of gastroesophageal acid reflux disease (GERD) and an increased risk for developing Barrett’s esophagus and esophageal adenocarcinoma. The decreasing prevalence of H. pylori infection is therefore predicted to increase the prevalence of esophageal diseases. Indeed, there has been an increase in the incidence of esophageal adenocarcinoma[172]. Some studies and meta-analyses have concluded that H. pylori eradication worsens gastroesophageal reflux disease, while others report no effect[173-176]. There is also an inverse correlation between H. pylori infection and Barrett’s esophagus[177-179]. A recent meta-analysis confirms the inverse correlation between H. pylori infection and esophageal adenocarcinoma in Eastern and Western populations. The analysis also revealed a significantly reduced risk of esophageal squamous cell carcinoma in Eastern populations, but not Western populations[173].

On the other hand, some studies suggest a more complex relationship between the incidence of esophageal disease and the prevalence of H. pylori. Studies of gastric pH and manometric values have not found a difference between H. pylori infected and uninfected patients or in patients before and after H. pylori eradication[175,180]. McColl reports that H. pylori-induced gastric atrophy reduces the risk of esophageal adenocarcinoma, but antral-predominant gastritis with increased acid production increases the risk[181]. In contrast, Derakshan et al[182] report that atrophy does not impact the risk of adenocarcinoma. While univariate analysis showed a protective effect of H. pylori infection on esophageal adenocarcinoma, this effect was lost upon multivariate analysis and only smoking and GERD symptoms > 2 times per week remained significant[182]. Obesity is one of the strongest risk factors for esophageal adenocarcinoma, and the worsening obesity epidemic could be partially responsible for the rise in esophageal adenocarcinoma cases[179].

If the dogma holds true, one would expect that regions of the world with lower H. pylori prevalence should have higher rates of esophageal adenocarcinoma and squamous cell carcinoma. Ethnic Malays in the Northeastern peninsular region of Malaysia have only a 4%-5% rate of H. pylori infection. The rates of esophageal adenocarcinoma and Barrett’s esophagus in this population were lower than in populations with greater H. pylori prevalence[183]. Therefore, lack of H. pylori alone does not explain the increasing prevalence of esophageal disease.

A few studies have implicated H. pylori and other Helicobacter species human liver and gallbladder disease. H. pylori has been detected in livers from hepatocellular carcinoma patients by PCR and histology[184]. Chronic cholecystitis patients were tested for the presence of H. pylori in the gallbladder mucosa. Patients with H. pylori positive gallbladders had increased levels of inducible nitric oxide synthase and higher incidences of adenomyomatosis and metaplasia[74]. Following the discovery of H. pylori, H. hepaticus was found in many colonies of research mice. This infection causes chronic active hepatitis and laboratory rodents are now routinely tested for Helicobacter infections[185]. While H. pylori predominantly colonizes the intestines, other Helicobacter species, such as H. hepaticus, H. bilis, and H. cinaedi infect the intestines and biliary tract in humans. H. bilis appears to be highly prevalent in patients with pancreaticobiliary maljunction[186]. The non-pylori helicobacters have been largely overlooked and more studies are needed to determine their prevalence and capacity to cause disease.

A handful of studies have linked H. pylori infection with bronchiectasis and chronic obstructive pulmonary disease (COPD)[187-189]. Tsang et al[188] compared the seroprevalence of H. pylori among bronchiectasis, tubercoulosis, and control groups. Bronchiectasis patients were 76.0% seropositive, while tuberculosis and control patients were 52.9% and 54.3% seropositive for H. pylori. Interestingly, one study aimed at determining whether H. pylori seroprevalence differs between inflammatory bowel disease patients and controls used COPD patients for comparison[190]. They reasoned that COPD patients would have similar degrees of antibiotic exposure to inflammatory bowel disease patients. Separate age-matched control groups were used for IBD and COPD patients because COPD patients tend to be older. Seroprevalence of H. pylori is clearly lower among IBD patients than among controls. On the other hand, H. pylori seroprevalence was higher in COPD patients than in that control group. They do not mention whether the difference reached statistical significance.

Conversely, one very large study failed to find an association between COPD risk and H. pylori infections in nonsmokers[191]. A small study of Turkish soldiers also failed to find a correlation between bronchiectasis and H. pylori[187]. Oddly, patients with a history of gastric or cardiovascular disease were excluded. Gastric and extragastric diseases related to H. pylori often coexist in patients and may indicate the presence of more virulent H. pylori strains and/or a genetic predisposition to disease. Thus, there is insufficient evidence to draw conclusions regarding the role of H. pylori and lung disease.

Credible evidence suggests a link between H. pylori infection and certain eye diseases, particularly open angle glaucoma. Kountouras et al[192] have been the primary proponents of this association, but studies have thus far been small; no study has involved patient groups larger than 100 subjects. In one study, the rate of H. pylori infection in glaucoma patients was almost double that of a control group. When these patients were followed for two years, intraocular pressure and visual field parameters were improved in patients who had undergone successful H. pylori eradication therapy, but not in patients who were H. pylori-negative or whose eradication therapy failed. Histological evidence has been found in eye tissue from 5 out of 43 H. pylori-positive patients, but in none of the control patients[75]. Cresyl violet staining was used to detect H. pylori. This stain reacts with Helicobacter and Campylobacter, but few other bacterial species. Therefore, the strength of this evidence is stronger than histology using less specific stains, but not as strong as that obtained from immunohistochemistry. Two studies, one in Canada and one in Israel, have failed to find a higher prevalence of H. pylori infection in glaucoma patients[193,194]. More studies are clearly warranted.

Central serous retinopathy (CSR) results when the retinal pigment epithelial barrier breaks down, causing fluid to leak into the subretinal space; this leads to visual impairment[195]. Permanent damage can result if the fluid is not reabsorbed quickly. To date, only a handful of clinical trials have been performed to explore the potential relationship between CSR and H. pylori; however, the results of these studies have been impressive. No published study has failed to find a correlation between CSR and H. pylori evidence. Three studies have measured the effects of H. pylori eradication on the course of the disease[195-197]. All three found some evidence of improvement. In the study by Rahbani-Nobar et al[196] one group of H. pylori-infected patients received eradication therapy, while another group did not. Reabsorbtion was significantly more rapid in the eradicated group than in the non-eradicated group.

There is tantalizing, but far from conclusive, evidence suggesting a link between H. pylori infection and development of Alzheimer’s and Parkinson’s diseases. In Parkinson’s disease, H. pylori clearly interacts with L-dopa. The bacteria can bind L-dopa and impact the absorption of L-dopa treatment[198-200]. Some studies indicate that H. pylori eradication reduces motor fluctuations, but one study found that motor fluctuations were lower in infected patients than in uninfected patients[199,201,202]. A large Danish study found an association between Parkinson’s disease diagnosis and H. pylori eradication treatment 5 or more years prior to Parkinson’s disease diagnosis[203]. This suggests that past H. pylori infection may be just as relevant to Parkinson’s disease as current infection. A double-blind, placebo-controlled trial found improvements in stride length, but worsening of rigidity, following eradication therapy in anti-nuclear antibody-negative patients[204]. Anti-nuclear antibody-positive patients did not benefit from eradication. Alarmingly, patients who remained H. pylori-positive following treatment (eradication failure) experienced rapid declines in motor functions. One interesting study examined the prevalence of H. suis, which frequently infects livestock, in Parkinson’s disease patients. H. suis colonization was found in 12% of patients, but only 0.6% of controls. Many of the H. suis-infected patients had previously undergone H. pylori eradication therapy[205]. They hypothesize that H. suis infection could explain the higher prevalence of Parkinson’s disease among farmers. At present, treatment of H. pylori in Parkinson’s disease patients is not recommended due to the potential for deterioration of motor function associated with eradication failure.

With respect to Alzheimer’s disease or general cognitive function, some studies have found correlations with H. pylori infection and some have not[206-208]. A very large, cross sectional study using data from the National Health and Nutrition Examination III, phase 1 found that H. pylori seropositivity was strongly associated with poorer cognition among adults aged 60-90 years[209]. A small study of Alzheimer’s patients and age-matched controls found that H. pylori eradication significantly improved cognitive status, as measured two years after treatment[210]. The five-year survival rate was improved in H. pylori-eradicated Alzheimer’s patients, but again, this was a small study[211]. An H. pylori histidine-rich, nickel binding protein, Hpn, forms amyloid structures in vitro and has been postulated to play a role in Alzheimer’s disease[212], but in vivo data are lacking.

The association between H. pylori infection and diabetes was only recently suggested[213], but the preponderance of studies have shown positive associations[214]. Metabolic syndrome consists of obesity, particularly central obesity combined with two or more other factors, such as insulin resistance, hypertension, dyslipidemia, or elevated fasting glucose. Metabolic syndrome is accepted as a precursor to diabetes[215]. A large, cross sectional study of Japanese patients revealed a significant relationship between H. pylori infection and metabolic syndrome (OR = 1.39, P < 0.001)[216]. Microalbinuria, neuropathy, and heart disease are common complications of diabetes. Patients infected with H. pylori, particularly cagA⁺H. pylori, were found to be at higher risk of microalbuminuria, which indicates endothelial leakage and is a risk factor for cardiovascular disease and diabetic nephropathy[217,218]. Studies also suggest at higher rate of diabetic complications, such as nephropathy, neuropathy, and retinopathy, in H. pylori-positive diabetics[219,220]. Coronary heart disease is also more prevalent in H. pylori-positive diabetics than H. pylori-negative diabetics[221].

The mechanisms underlying the association between H. pylori and diabetes are unclear. Chronic inflammation due to H. pylori infection is believed to contribute to many H. pylori-associated diseases, and diabetics have higher levels of inflammatory markers than non-diabetics[222]. Nonetheless, Jeon et al[223] did not find that the inflammatory markers IL-6 and C-reactive protein were elevated in H. pylori-positive diabetics compared to H. pylori-negative diabetics. Therefore, it appears that other causes of inflammation, perhaps including adiposity, trump H. pylori-mediated inflammation in diabetics.

Does H. pylori eradication benefit patients who have already developed diabetes? Very few studies have sought to answer this important question. CagA⁺H. pylori strains are strongly associated with poor glycemic control[218,224], so one would naturally think that these patients would benefit from eradication therapy. In a study by Akanuma et al[225] found that H. pylori eradication did not improve glycemic control. Experiments in mice actually suggest that H. pylori infection improves glucose homeostasis[226]. H. pylori eradication regimens are not 100% effective in any population, but type 2 diabetics are at higher risk for eradication failure[227].

The impact of H. pylori infection on ghrelin production could complicate the interpretation of diabetes studies. H. pylori is known to suppress gastric ghrelin, and ghrelin levels in the stomach rise following H. pylori eradication[228]. Ghrelin stimulates appetite, which could explain why some studies have found that patients gain weight following H. pylori eradication[225,229]. It is also possible that resolution of gastric symptoms leads to weight gain, but regardless of the mechanism, weight gain is not beneficial to diabetic patients. Clearly, more studies are needed to clarify the impact of H. pylori eradication on diabetic patients, but at this point in time, treatment of H. pylori infection in diabetic patients cannot be recommended.

An association between H. pylori and coronary artery disease was first suggested in 1994, twelve years after Barry Marshall and Robin Warren discovered H. pylori[230]. The methodologies and endpoints used in studies are diverse, making it difficult to compare studies. The presence of CagA seropositivity tends to strengthen associations with cardiac diseases including atherosclerosis, unstable angina, and cardiac X syndrome[231-237]. A meta-analysis found that cagA-positive H. pylori increases the risk of both ischemic stroke and coronary heart disease[62]. There is ample evidence demonstrating that H. pylori infection alters biomarkers that are associated with CVD. For example, several studies have linked H. pylori infection to alterations in lipid profiles[238-240]. Moreover, the amount of LDL cholesterol elevation correlated with the degree of gastric inflammation[241] and H. pylori eradication normalized lipid profiles[242,243]. H. pylori infection has also been found to correlate with increased carotid intimal thickness, reduced serum paraoxonase-1 activity, and increased risk of peripheral artery disease[244,245].

The role of H. pylori in promoting atherosclerosis or other manifestations of cardiovascular disease remains controversial because a number of studies have failed to find any association between H. pylori infection and the disease parameters studied[246,247]. Studies of healthy adults and children failed to find associations between C-reactive protein levels and H. pylori infection[247]. H. pylori infection was also not found to correlate with fibrinogen or von Willebrand factor levels in coronary angiography patients[248]. McDonagh et al[246] failed to find an increased odds ratio for coronary heart disease in H. pylori-infected patients. With so many conflicting studies, it is impossible to recommend testing for or treatment of H. pylori in any CVD patient population. Moreover, few in vivo studies have been carried out in animal models. Nonetheless, the number of studies positively correlating H. pylori infection with various aspects of CVD warrants continued research on the potential for H. pylori to influence various types of cardiovascular disease.

A recent meta-analysis of studies revealed that CagA seropositivity increased the risk of autoimmune thyroid disease by 2.24 fold (95%CI: 1.06-4.75). Infection with any H. pylori strain significantly increased the risk for Grave’s disease (OR = 4.35), but not for Hashimoto’s thyroiditis[249]. While the overall H. pylori infection rate was not elevated in Hashimoto’s thyroiditis, these patients did have a significantly higher rate of cagA⁺H. pylori infection than controls[250]. An exploration of both host genetics and H. pylori virulence factors revealed that CagA⁺ strains were more strongly associated with Grave’s disease than cagA^- strains and certain HLA-DQA1 alleles altered the risk of developing Grave’s disease[251]. H. pylori infection, especially cagA⁺, was found to be more prevalent in patients with Grave’s disease than controls; CagA has some homology with thyroid peroxidase, which could trigger an autoimmune response[252]. Autoantibodies against thyroid peroxidase dropped significantly in all 5 H. pylori-positive patients who accepted eradication therapy, but did not drop in 5 who refused to be treated. Anti-thyroglobin titers also dropped in 4/5 treated patients[253]. H. pylori infection may also decrease the efficacy of thyroxine therapy in patients with hypothyroidism[254]. In support of the potential for H. pylori to cause thyroid disease, euthyroid patients with thyroid nodules were significantly more likely to be infected with H. pylori than patients without thyroid nodules (P = 0.002)[255].

Systemic sclerosis is an autoimmune disorder caused by autoantibodies Information regarding the potential for H. pylori to cause or worsen systemic sclerosis is scant, but intriguing. A few studies have found positive correlations between systemic sclerosis and H. pylori infection[256-258]. The presence of H. pylori infection is strongly associated with worse gastrointestinal, skin, and joint symptoms, as well as increased erythrocyte sedimentation rate in systemic sclerosis patients[259]. Danese et al[256] found that 90% of H. pylori strains infecting systemic sclerosis patients were cagA⁺, compared with only 37% in controls. Reinauer et al[260] demonstrated that H. pylori could be eradicated in systemic sclerosis patients, but did not follow up to determine whether symptom scores improved. Further studies are needed to confirm the association between H. pylori and systemic sclerosis and determine whether H. pylori eradication is beneficial.

In contrast to systemic sclerosis, several studies indicate a protective effect of H. pylori infection on multiple sclerosis[261,262], but the picture becomes more complicated when the presence of neuromyelitis optica (NMO) is included. NMO is an autoimmune disease in which antibodies against aquaporin lead to demyelination of the spinal cord and optic nerve, leading to paralysis and blindness. It has been considered to be a variant of multiple sclerosis, but is now proposed to be a separate disease[263]. Li et al[264] attempted to dissect the possible relationship between MS and H. pylori by correlating antibodies against HP-NAP with autoantibodies against aquaporin-4 (AQP4) and with circulating myeloperoxidase. Patients with MS and neuromyelitis optica were more likely than controls to be infected with H. pylori than those with conventional MS. There was a significant correlation between seropositivity for AQP4 and seropositivity for HP-NAP. HP-NAP seropositive patients also had higher myeloperoxidase levels and worse disability. H. pylori and Chlamydia pneumoniae seropositivity were found to be more prevalent in NMO patients who also have anti-AQP4 antibodies, but not in those lacking that autoimmune marker[265]. These data clearly indicate that MS and NMO patients must be studied separately with regard to the potential for H. pylori involvement. H. pylori antigens have been proposed as a treatment for multiple sclerosis[266], but the potential for causing or exacerbating other diseases makes this a risky proposition.

Campylobacter jejuni infection is known to be a risk factor for development of Guillain Barré syndrome, and H. pylori may contribute as well. Guillain Barré syndrome is an autoimmune attack on peripheral nerves that leads to paralysis, which is usually temporary. A few small studies have found higher H. pylori titers of H. pylori antibodies in serum or CSF in Guillain Barré patients than in controls[267-269]. Associations were particularly strong in patients with the acute inflammatory demyelinating polyradiculoneuropathy (AIDP) type of Guillain Barré syndrome. Another study found anti-VacA in all AIDP patients[270]. Homology between VacA and [Na(+) + K(+)]-ATPase is suggested as a potential cause.

The hygiene hypothesis proposes that inadequate exposure to certain bacteria during the first one or two years of life results in an immune system that is hypersensitive to environmental agents, leading to various allergic diseases. A Th2-skewed immune system favors allergies, while Th1 responses, which are induced by H. pylori, favor autoimmune and inflammatory diseases. The incidence of allergies has risen dramatically in recent decades, while the prevalence of H. pylori infection has fallen. This led investigators to search for a link between the two phenomena. Several studies suggested an inverse relationship between H. pylori infection and asthma. A meta-analysis confirmed this association[271]. Proof-of-principle has been established in a mouse model of asthma induced by ovalbumin or house dust mite allergen[272]. In accordance with the hygiene hypothesis, neonatal infection of mice proved most effective in preventing asthma. Protection correlated with infiltration of regulatory T cells into the lungs. Adoptive transfer of regulatory T cells from H. pylori-infected mice into allergic mice offered protection. Some evidence also suggests that H. pylori may be protective against food allergies, celiac disease, and allergic rhinitis as well[273-275]. CagA⁺ strains do not appear to be relevant to asthma, but purified HP-NAP reduced lung eosinophilia and Th2-associated cytokines in ovalbumin-induced asthma in mice[53]. Certainly, H. pylori is not the only organism implicated in allergy prevention. Actinobacteria, Bifidobacterium, and certain Clostridia and Bacteroides spp., are associated with a lower risk of allergic disease, as well as a higher overall diversity of organisms[276,277]. It is not known which organisms can most safely and effectively prevent or treat allergic disease.

Considering that H. pylori has numerous direct and indirect effects on host physiology, one should keep an open mind regarding the possibility that H. pylori contributes to a variety of extragastric diseases. H. pylori has now been detected by culture, PCR, or histology in several epithelial tissues, including the eyes, ears, nose, and skin. While it is not yet possible to draw firm conclusions regarding the role of H. pylori in most extragastric diseases, the evidence cannot be ignored. Studies with contradictory results could have several explanations. Regional variations in both human and H. pylori genotypes contribute to established H. pylori-associated diseases. Inconsistencies may also indicate that H. pylori is a minor contributor or serves to exacerbate, rather than cause, a particular disease. Just as H. pylori is not responsible for all cases of gastric cancer or ulcers, H. pylori is unlikely to be the sole cause of other disorders. Future studies should measure the presence of H. pylori virulence factors, in addition to overall seropositivity, since several diseases are associated with only some H. pylori genotypes. Studies designed to elucidate mechanisms through which H. pylori contributes to disease would also improve credibility.

The urea breath test or fecal antigen testing are the preferred methods for establishment of active infection[279]; however these recommendations come with caveats. The urea breath test consists of drinking carbon-13 labeled or carbon-14 labeled urea. The ingested radiolabeled carbon is converted to carbon dioxide and ammonia by the urease created by the H. pylori. The carbon dioxide is absorbed in the bloodstream and a sample of expired air is analyzed for the presence of the labeled carbon. The test also has the advantage of identifying active infection, which cannot be categorized by serology. The sensitivity and specificity for the urea breath test is 99% and 98%, respectively[284]. Since the urea breath test requires specialized equipment, it is not always feasible in small clinics.

Stool also may be submitted for analysis for H. pylori antigens using monoclonal or polyclonal antibodies. The test initially used an ELISA process, but has been modified into an immunocard, making it suitable for small clinics and field-testing in third world countries. The sensitivity and specificity for the test is 94.1% and 91.8%, respectively. Stool testing also is more economical than endoscopy for confirmation of eradication. Patient submission of stool samples may be a limitation of this test for some patients, but it is highly suitable for infants and children[285,286].

Proton pump inhibitors and H₂ receptor antagonists need to be withdrawn for several days prior to testing using the urea breath test or stool antigen test due to the potential for false negatives. In addition, antibiotics taken during the four weeks prior to testing can suppress the infection and lead to a false negative. Some studies suggest that waiting until 8 wk post-treatment improves test accuracy[285].

Serology testing for H. pylori is widely available and relatively inexpensive. Microtiter plates coated with H. pylori antigens, combined with a secondary antibody, and are used to detect H. pylori-specific IgG. Serology results are not affected by acid suppression therapy or recent antibiotic use; however, seropositivity is not a confirmation of current H. pylori infection. Likewise, serology cannot be used to confirm cure following an anti-microbial regimen. These antibodies persist for an extended period of time, often greater than half a year. Due to the often-patchy distribution of H. pylori, serum samples are more consistent than other tissue samples. The sensitivity and specificity of serology for detection of initial infection compares to the gold standard of histology with sensitivity of 85% and specificity of 79%[287].

Endoscopy is required to obtain biopsy material and to assess the degree of pathology in the stomach. Giemsa, Warthin-Starry, and Diff-Quik stains in addition to standard hematoxylin and eosin staining can be used to identify H. pylori. Stains such as Giemsa provide identification of the bacteria, assessment of inflammation, and also provide evidence for intestinal metaplasia. No other tests allow for the identification of intestinal metaplasia. Multiple biopsies are needed for increased diagnostic yield targeting at least three areas including the angularis, the greater curvature of the corpus, and from the greater curvature of the antrum. The high cost of infrastructure, variability in pathologist review, and training of personnel are limitations that prevent this method from being considered as the gold standard for diagnosis[278,288]. Biopsies from the corpus, in addition to the antrum, increases the diagnostic yield by 10%[289].

Rapid Urease testing involves utilization of the H. pylori urease to identify active organisms. Samples of gastric tissue are placed on a reactive strip or agar gel. A pH sensitive color indicator, buffer, and urea are added to the sample. Urea is converted to ammonia causing a pH increase and subsequent color change in the sample area of the test device. Readings can be taken between 1 h and 24 h based on the specific commercial test kit used. Sensitivity greater than 93% and specificity of 98% is reported for rapid urease testing[290].

Culture provides a high specificity for the diagnosis of H. pylori; however, it often lacks the sensitivity seen with other invasive methods. From a practical standpoint, culture techniques are technically challenging and costly. H. pylori is a fastidious, microaerophilic organism requiring 5-7 d to form visible colonies on solid media. Laboratory personnel must culture specimens promptly and avoid prolonged exposure to ambient oxygen levels to maximize the organism’s survival. Culture options for detection are primarily recommended for situations requiring specific antimicrobial sensitivity testing. The tissue may be placed directly on culture media, dragged across the agar surface, or gently homogenized and plated.

PCR techniques can be used to detect H. pylori DNA most reliably in biopsy samples, but saliva and feces have also been used. PCR sensitivity nearing 100% and specificity of 100% can be obtained[291]. Contamination from improperly cleaned endoscopes may create false positive results. False negative results may occur due to PCR inhibitors within gastric tissue or feces. Wide acceptance of PCR techniques in the clinical setting is limited and PCR is primarily used in research settings[292]. Fluorescence in situ hybridization (FISH) permits sensitive and specific detection of H. pylori in gastric biopsies without the need for DNA extraction. A fluorescently labeled DNA probe hybridizes with the complementary sequence within the bacterial chromosome. FISH can detect coccoid H. pylori and is more sensitive than standard staining procedures[293]. The use of different fluorescent dyes can permit simultaneous detection of H. pylori 16S rRNA and gene sequences associated with antibiotic resistance[294]. A commercially available kit has proven to be reliable and far more rapid than culture-based methods for simultaneous detection of H. pylori and clarithromycin resistance[295,296].

Resistance of H. pylori to commonly used antibiotics is on the rise worldwide. Overall, H. pylori resistance to metronidazole is prevalent, while resistance to amoxicillin and tetracycline are low; however the picture is far more complex at the regional level. For example, amoxicillin resistance is below 3% in America and Europe, but over 60% in Africa[300]. Africa also has the highest rates of resistance to metronidazole (92.4%) and tetracycline (43.9%)[300]. Metronidazole resistance is above 50% in much of the world but there are indications that metronidazole resistance may be dropping in northern Europe[301]. Within Europe, resistance patterns vary by country and even within a country. For example, the reported clarithromycin resistance rate is 1.5% in Sweden, but 7.5% in Germany and clarithromycin resistance in Italy is lower in the north than in the south[301]. Increasing resistance to clarithromycin and levofloxacin has been attributed to widespread use of these antibiotics for respiratory tract and urinary tract infections, respectively[300]. In the (Helicobacter pylori Antimicrobial Resistance Monitoring Program), a resistance pattern showed 29.1% of United States isolates were resistant to one antimicrobial agent and 5% were resistant to two or more antimicrobial agents[302]. Multidrug resistance remains low worldwide[300], offering hope that rescue therapy will work in most patients. Previous treatment for H. pylori is the single largest risk factor for drug resistance[302]. Success rates of antimicrobial therapy do not always mirror in vitro susceptibility data. This could be partially due to variability in antibiotic resistance testing protocols and on poor patient compliance[301]. In summary, local antimicrobial resistance data should be consulted to increase the chances of eradication success following the first treatment.

Specific H. pylori mutations responsible for antibiotic resistance have been identified. Inactivation of the rdxA gene is well known to contribute to metronidazole resistance, though other mechanisms likely exist[303]. The point mutations A2143G, A2142G, and A2142C are responsible for clarithromycin resistance in H. pylori. Although the test is not yet available in the clinical setting, the A2143G mutation was associated with a lower eradication rate. In a clinical trial, sequential therapy was found to be more successful in eradication than triple therapy[300]. Certain gyrA mutations are associated with fluoroquinolone resistance[304]. For individuals who have failed eradication with the primary regimen, use of alternative antibiotics must be considered. In the future, it may be feasible to determine likely antimicrobial resistance patterns using H. pylori DNA isolated from stool without the need for endoscopy[305].

Recently, the concept of sequential therapy has been proposed with a 10-14 d regimen of a PPI twice daily and amoxicillin twice daily for five days, followed by a PPI plus clarithromycin and tinidazole twice daily[306]. Zullo et al[307] reported in a 2007 pooled-data analysis, an eradication rate greater than 90% with an intention to treat analysis thus revealing a higher eradication rate than standard therapy. A recent meta-analysis revealed an odds ratio (OR) for eradication of 2.99 (95%CI: 2.7-3.62), yielding a number needed to treat (NNT) of 6 in favor of sequential therapy compared to triple therapy. Additionally, the OR for eradication with sequential therapy compared with 10 d of triple therapy was 2.92 (95%CI: 1.95-4.38) with a NNT of 8 positive for sequential therapy. Factoring for clarithromycin resistance, the OR for eradication with sequential therapy was 10.21 (95%CI: 2.01-34.58) compared with triple therapy; however, the number of patients was small[299]. Another large meta-analysis found superiority compared to triple therapy with 93.5% eradication (95%CI: 91.3 to 95.5) compared to 76.9% (95%CI: 71.0-82.8) for standard triple therapy[308]. Sequential therapy appeared to be superior in subgroup analyses involving trial quality, smoking status, ulcer disease or non-ulcer dyspepsia, clarithromycin or imidazole resistance or both, duration of therapy and diagnostic method[308].

A novel regimen for treatment of H. pylori was proposed in an open label prospective trial of 653 patients using levofloxacin, omeprazole, nitazoxanide, and doxycycline (LOAD) for 7 or 10 d compared to triple therapy with lansoprazole, amoxicillin, and clarithromycin (LAC). In this intention-to treat analysis, the rate of eradication for LOAD-7 d regimen was 90% and 88.9% for LOAD-10 d regimen and combination eradication rate of 89.4% vs 73.3% eradication rate with LAC. No adverse event differences were noted in the two groups[309]. The pill burden was limited to twice daily with both regimens, however, the current cost of the LOAD regimen, in particular nitazoxanide, may limit its widespread usage. Additionally, more comprehensive multi-center trials will be needed to confirm these results.

In a pooled analysis of several refereed studies and abstracts, retreatment with triple and quadruple therapies were found to achieve eradication rates in the range of 70%-76%. Additionally, the substitution of two antibiotics compared to one antibiotic in the re-treatment regimen was found to be superior for eradication[310]. Attempts should be made to avoid antibiotics previously utilized for H. pylori eradication. As clarithromycin is utilized for most triple therapy regimens, quadruple therapy is often utilized as the salvage therapy. A levofloxacin 500 mg daily, PPI, and amoxicillin 1 g twice daily regimen is suggested in the American College of Gastroenterology guidelines with an eradication rate possibly higher than quadruple therapy[278]. In a multicenter, prospective trial, rifabutin-based rescue therapy with amoxicillin and PPI for ten days after three previous treatment failures was found to have an approximate 50% eradication rate providing an additional strategy for refractory infections[311]. After the second attempt at treatment and confirmation of failure of eradication, antibiotic sensitivity testing should be implemented with tissue acquisition and culture for guidance in selecting the appropriate antimicrobial regimen.

A number of studies have investigated the roles of probiotics in improving eradication rates and decreasing antibiotic side effects, but there is not yet a consensus regarding their utility. A recent meta-analysis evaluating probiotics including both Lactobacillus and Bifidobacterium species reports increased eradication in adults, but no effect in children. Total side effects were decreased with the supplementation of probiotics. Individual side effects were not assessed in this meta-analysis; however, the effects of confounders could not be assessed due to the small trial sizes[312]. Another recent meta-analysis differentiated between studies using Lactobacillus alone vs combinations containing other genera[313]. They found that the eradication rates were raised significantly by 17% when Lactobacillus was used alone, compared with only 2.8% in patients treated with a combination of species. Contrary to the findings of the aforementioned study, they did not detect a reduction of overall side effects. Diarrhea may be reduced with the usage of Saccharomyces boulardii, but other adverse effects (epigastric pain, taste disturbances, nausea, and gas/bloating) were not affected. Trial numbers are limited with this organism[314]. A few trials have evaluated lactoferrin as adjuvant therapy. A meta-analysis in 2009 found benefit, but small sizes of the trials and lack of robust data necessitate additional clinical trials[315]. A recent study compared the effects of probiotics or a combination of probiotics and lactoferrin on eradication and severity of antibiotic-related side effects[316]. Neither treatment improved eradication, but both reduced side effects and increased patient compliance. The addition of lactoferrin to probiotics did not confer any additional benefit compared to probiotics alone. In summary, there is some evidence that probiotics improve eradication rates, but evidence that probiotics can reduce the side effects of eradication therapy is not conclusive. Given the excellent safety profile of probiotics, it may be reasonable to suggest that patients eat yogurt or take an over-the-counter supplement containing Lactobacillus and/or other probiotic species.

Simvastatin as a supplement to clarithromycin, amoxicillin, and omeprazole has been shown to increase the eradication rate of H. pylori when used as an adjuvant in a randomized controlled trial. No differences were noted in compliance or side effects. The proposed mechanism for augmented eradication is the anti-inflammatory effects of Simvastatin, which are unrelated to its cholesterol-lowering activity (Nseir, 22646167). Further studies are needed for confirmation before advocating this strategy.

Generally, bismuth compounds, tetracycline, and flouroquinolones regimens should be avoided in pregnancy due to potential teratogenic effects. Metallic taste is a common side effect with metronidazole and clarithromycin. Photosensitivity with doxycycline and tetracycline is possible. Additionally, metronidazole may cause an adverse reaction with alcohol and with prolonged usage may create a peripheral neuropathy. Other less common side effects are noted with each of these medications.

Based on Maastricht IV/Florence Consensus Report, eradication of H. pylori should be confirmed with a urea breath test or a laboratory-based, validated monoclonal stool test 4 wk after the conclusion of treatment. In cases of a gastric ulcer or gastric MALT lymphoma, follow-up is suggested with an esophagogastroduodenoscopy and biopsy of gastric tissue[279]. The ACG Guidelines also suggests testing with individuals with persistent dyspepsia symptoms following resection with an early gastric cancer[278].