Three decades ago, pediatric gastritis and peptic ulcer disease lacked a putative microbial causative agent. Pediatric flexible upper endoscopy was becoming more available. In 1983, Warren and Marshall proposed that colonization of the human stomach with an organism, now known as Helicobacter pylori (H. pylori) , was associated with human disease, specifically peptic ulcer disease. The earliest reports of this bacterium in children noted its coincidence with gastritis and ulcer disease. In February 1994, the National Institutes of Health Consensus Development Conference concluded that H. pylori represented the major cause of peptic ulcer disease. Later that year the International Agency for Research on Cancer Working Group of the World Health Organization classified H. pylori as a group I (definite) human carcinogen.
H. pylori is a slow-growing gram-negative, microaerophilic bacterium that colonizes the gastric mucosa. H. pylori infection is acquired during the first decade of life and unless eradicated causes life-long infection. Large-scale analysis of H. pylori protein expression has been used to identify possible disease markers associated with the wide spectrum of putative Helicobacter -associated diseases, as well as being utilized in selecting potential vaccine candidates.
Many features of infection such as prevalence, clinical presentation and complications, diagnostic methods, and antibiotic resistance are age specific and differ between children and adults. Although H. pylori infects children worldwide, its role in certain pediatric diseases is a matter of some debate. Pediatricians and pediatric gastroenterologists must therefore avoid inappropriate investigation and potentially harmful therapies, while ensuring appropriate utilization of preventive medicine in minimizing future ill health.
Epidemiology
To date, humans are the only established natural reservoir of H. pylori in the animal kingdom. At least five different gastric non– H. pylori Helicobacter species have been isolated from patients at endoscopy. Nonhuman primates rapidly acquire H. pylori and can been used as animal models of disease.
Socioeconomic Risk Factors
Within populations where socioeconomic disparity exists, indices of lower socioeconomic status—including low household income, lower parental educational level, higher housing density, and poor sanitary conditions—continue to influence a higher H. pylori prevalence among children in comparison with their noninfected peers. Coinfection among parents (especially mothers), siblings, and nonsibling household contacts are established risk factors for childhood H. pylori acquisition. Child-rearing practices may also influence the transmission of infection. International improvements in social conditions and hygiene standards may contribute to reduce the risk of H. pylori infection in children.
Transmission of Infection
Direct person-to-person contact has been long thought of as a major mode of transmission. Rowland et al. showed that maternal infection was an independent risk factor for childhood H. pylori infection. Data from studies of a German birth cohort suggested an odds ratio (OR) of 13 for the effect on the index child of the mother being infected. The Pasitos Cohort Study reported that persistent H. pylori infection in older siblings increased the risks of H. pylori infection in younger siblings, and suggested a unidirectional transmission of infection from mother or older sibling to younger siblings. Biofilm formation by H. pylori strains is a possible mechanism of survival outside the body. H. pylori has been identified in water systems, by polymerase chain reaction (PCR) rather than by culture, but whether these strains are subsequently infectious remains uncertain.
Prevalence
H. pylori infection is declining worldwide, and may even be less than 10% in “westernized” countries. The rate of decline seems greatest in developing countries, in contrast to data from developed countries, which are less consistent. Declining rates in Belgium contrast with stable rates in The Netherlands and Portugal, for example. Studies from China, Tunisia, Turkey, The Netherlands, rural Alaska, and Israel have reported childhood H. pylori prevalence rates of 13.1%, 51.4%, 30.9%, 1.2%, 86%, and 32.5%, respectively. In a study from Uganda, H. pylori prevalence among HIV-infected children was lower (22.5%) compared to otherwise healthy Ugandan children (44.3%), most likely due to eradication with antibiotic therapy used for the treatment of their comorbid illnesses. Prevalence also differs between different ethnic groups living in the same geographic area, again reflecting differences in socioeconomic status, cultural habits, and family size. Tkachenko et al. described a dramatic example in their study of seroprevalence changes in St. Petersburg, Russia. Using two cross-sectional studies in children from 1995 and 2005 (and using the same enzyme-linked immunosorbent assay [ELISA] method for anti- H. pylori immunoglobulin G [IgG]) they showed that that 10 years later, the overall prevalence of H. pylori infection had decreased from 44% to 13%. Among children younger than 5 years of age, the prevalence decreased from 30% to 2%.
H. pylori is rapidly acquired early in our life cycle, generally during the early childhood years. Cross-sectional prevalence studies outnumber longitudinal studies examining H. pylori infection acquisition and loss. Many published studies have been of heterogeneous design and population mix, and used diverse methods of H. pylori detection, not all of which have been validated for use in pediatric populations. Higher rates of childhood H. pylori infection earlier in the 20th century likely account for the persisting higher disease prevalence with advancing age in developed countries. This “birth cohort effect” has been unmasked by cross-sectional seroprevalence studies from around the world.
Incidence
Beyond the first decade of life, the annual incidence of newly acquired infection is remarkably low. Rowland et al. described the incidence of infection in 327 Irish children between 2 and 4 years of age using the validated C urea breath test (UBT). Adopting strict criteria, they showed that acquisition of H. pylori infection occurred in early childhood years and that coinfection of household contacts and prolonged use of feeding bottles, but not pacifiers, beyond the age of 2 years were risk factors for infection. Over 4 years of follow-up, 279 index children not infected at baseline contributed 970 person-years of follow-up to the study. During this time, 20 children became infected with H. pylori . The authors did not find evidence of spontaneous infection clearance in their study children. The use of serologic, stool, and urine tests to detect H. pylori in other studies of infection incidence may potentially weaken the data, but the majority conclude that the incidence of H. pylori infection is highest in children younger than 5 years of age.
Many confounding factors that influence the epidemiology of H. pylori infection are difficult to control and must be borne in mind when interpreting published data. Given that H. pylori is acquired in childhood, environmental factors that influence infection most likely exert their greatest influence during childhood. These are among the most difficult factors to control in cross-sectional or retrospective studies.
Spontaneous Bacterial Clearance
Spontaneous clearance of H. pylori infection in early childhood has been reported. Clearance of IgG seen in very young infants is more likely to represent the detection and then clearance of maternal antibodies. However, more recent investigative data suggest that spontaneous elimination of H. pylori in childhood may be influenced by antibiotic use for systemic childhood infections, albeit an incomplete explanation.
Reinfection and Recrudescence
The risk of reinfection in the pediatric setting following successful eradication is low in developed countries. Generally, re-colonization of the same strain within 12 months of eradication (recrudescence) rather than reinfection (colonization with a new strain, more than 12 months after eradication) is considered responsible for most documented “cases.” Rowland et al. followed 52 children prospectively for a mean of 2 years following successful eradication therapy. Reinfection was identified by UBT; age younger than 5 years was the only risk factor for reinfection identified by logistic regression. Neither socioeconomic status nor number of infected family members influenced reinfection rates.
The risk of reinfection in developing countries is less clear. A recent meta-analysis of studies involving predominantly adult subjects suggested that reinfection rates might be higher in developing than developed countries. Continuing poor sanitary and water standards, and household density, may account in part for the differential reinfection rates between countries at economic extremes.
In a prospective longitudinal study of 136 children in Vietnam, the risk for recurrence of H. pylori was inversely proportional to age, with the youngest children running the greatest risk. The finding lends support to the observation that early childhood may be the main age of acquisition of H. pylori infection and for postponing attempts of eradication in high prevalence areas unless motivated for medical reasons.
Host Factors and Responses
H. pylori is a formidable pathogen. Its natural reservoir is the human population and it is carried and transmitted asymptomatically by the majority of infected hosts. H. pylori infection and disease outcomes are mediated by a complex interplay among bacterial, host, and environmental factors.
Host Genotype
Cytokine gene polymorphisms are important host factors that can alter H. pylori disease outcomes. Proinflammatory polymorphisms of the interleukin 1β ( IL-1 β) gene have been associated with the development of gastritis, predominantly involving the body of the stomach (corpus gastritis), hypochlorhydria, gastric atrophy, and gastric adenocarcinoma, but a reduced risk of duodenal ulceration. In the absence of these polymorphisms, H. pylori gastritis predominantly involves the antrum and is associated with normal to high acid secretion. Polymorphisms of the tumor necrosis factor α (TNFα) and IL-10 genes demonstrate a similar but less-pronounced association with the development of gastric cancer.
Host innate and adaptive immune responses are ordinarily under genetic influence. Harboring of genetic polymorphisms in key immunity-related genes offers an attractive explanation in part for diverse host phenotypic responses to this bacterium within given ethnic populations. Polymorphisms in IL-1?? and its receptor have been linked to gastric cancer susceptibility. Individuals with the IL-1B _31*C or _511*T and IL-1RN *2/*2 genotypes are shown to be at increased risk of developing hypochlorhydria and gastric atrophy in response to H. pylori infection. In children, polymorphisms in the IL-1 receptor antagonist gene, IL-1RN , but not the IL-1B gene cluster, were associated with susceptibility to duodenal ulcer. This polymorphism, along with cytotoxin-associated gene A (CagA)+ H. pylori strain infection, is independently associated with duodenal ulcer. Polymorphisms in cytokine genes including TNFα , IL-8 , and IL-10 , which result in a proinflammatory phenotype in the host, have been linked to increased severity of H. pylori disease in children and cancer susceptibility in adulthood.
Pattern-recognition receptors (PRRs) are germ-line–encoded proteins that detect conserved microbial molecular motifs and danger signals. They include the membrane-associated Toll-like receptor (TLR) family and cytosolic nucleotide-binding oligomerization domain (Nod)-like receptor (NLR) family. Polymorphisms in TLR4, which senses bacterial lipopolysaccharide (LPS), have been linked with susceptibility to H. pylori –induced gastritis and gastric cancer. However, these findings have not been consistently replicated, albeit in populations of different ethnicity. A study of 486 Brazilian children undergoing gastroscopy for abdominal pain failed to show an association between polymorphisms in TLR2, TLR4 , and TLR5 and H. pylori infection or duodenal ulceration. However, children with TLR4 polymorphisms were more likely to have infection with CagA+ H. pylori strains and enhanced mucosal IL-8 and IL-10 levels as measured by enzyme-linked immunosorbent assay (ELISA). Of interest, human leukocyte antigen (HLA) class I and II allele studies have not shown a predisposition to H. pylori infection, although they may affect susceptibility to gastric cancer.
These limited data suggest that in the genetically susceptible host, failure to attenuate the inflammatory response to H. pylori infection locally leads to chronic inflammation and reduced acidity. This milieu may favor subsequent colonization with non– H. pylori species and/or production of genotoxic effects. It is likely that in this era of genome-wide association studies, more elaborate research methodologies may uncover more clues to the influence of host genotype on H. pylori infection and disease.
The Host Immune Response
The seemingly robust, complex acute host immune response to H. pylori infection is surprisingly ineffective at clearing this bacterium and preventing H. pylori from establishing a life-long bacterial niche. Host and bacterial factors are clearly at play in moderating infection and disease spectrum and severity.
Antral gastritis is associated with increased stimulated acid production and predisposes to duodenal ulceration, whereas corpus-predominant or pan-gastritis is associated with reduced acid production and predisposes to gastric ulcer and gastric adenocarcinoma. The degree of gastric infiltration by neutrophils also correlates with the development of gastroduodenal ulcerations, and this is in part dependent on the release of damaging inflammatory mediators such as reactive oxygen species (ROS).
Innate Immune Receptors
H. pylori is a predominantly extracellular organism that generally adheres to the gastric mucosal surface. Gastric epithelial cells, and to a lesser extent, intraepithelial myeloid cells, affect the first host responses to infection. TLRs and NLRs constitute the majority of our innate receptor arsenal. Cell-surface TLRs, including TLR2, TLR4, and TLR5, do not seem to mediate significant initial immune responses to H. pylori . Indeed, H. pylori LPS is less potent than that of Salmonella spp. or Escherichia coli, and modification of H. pylori LPS facilitates immune evasion.
Given that H. pylori is primarily an extracellular pathogen, it may seem intriguing that it engages NLRs at all. This interaction is in part mediated by its gene-encoded type IV secretion system (TFSS), a molecular syringe that facilitates translocation of bacterial effector proteins and products into cells. H. pylori peptidoglycan is translocated into host cells where it engages with its receptor, NOD1. Downstream effects of NOD1 activation include instigation of NF-kB–mediated inflammatory cascades and subsequent IL-8 production.
Autophagy
Autophagy is an evolutionarily conserved process that results in the sequestration of cytosolic components within autophagosomes that then fuse with lysosomes, resulting in the degradation of the lysosomal contents ( Figure 27-1 ).
Autophagy is induced in host cells in response to vacuolating cytotoxin A (VacA) to mitigate the effects of the toxin. However, persistence of VacA induces the formation of defective autophagosomes with attenuated ability to eliminate bacteria and potentially genotoxic material. In addition, a polymorphism in the autophagy gene, ATG16LI , results in both inefficient induction of autophagy in response to the toxin and increased susceptibility to infection in humans. Together these findings provide a novel mechanism for chronic host H. pylori infection and have important implications for H. pylori– mediated infection, inflammation, and carcinogenesis ( Figure 27-2A,B ).
Myeloid Cells—Macrophages
H. pylori induces chronic gastritis in almost all hosts. Initial neutrophil recruitment gives way to mononuclear cell infiltrates with lymphocytes and macrophages and the attendant epithelial cell damage. Following H. pylori infection, enhanced levels of IL-1β, IL-2, IL-6, IL-8, and TNFα are detected in the gastric epithelium. Phagocytosis of H. pylori by macrophages results in marked IL-6 production. The precise role of TLRs in macrophage chemokine and cytokine responses to H. pylori remains unclear. As effector cells, macrophages generate nitric oxide (NO), catalyzed by the enzyme inducible NO synthase (iNOS), which is upregulated in macrophages following H. pylori infection in vitro. Through its arginase enzyme, encoded by the gene rocF, H. pylori competes with the eukaryotic macrophage for the iNOS substrate L-arginine (L-Arg) to enhance its survival. The enzyme generates urea from L-Arg, which is then utilized by urease to synthesize ammonia and neutralize the gastric luminal Hydrogen Chloride (HCl). Reduced macrophage NO generation therefore confers an immune evasion advantage.
Myeloid Cells—Dendritic Cells
Mucosal dendritic cells (DCs) are an important component of our antigen-presenting cell repertoire. They express an array of innate immune receptors and their cytokine responses influence the differentiation of T helper (Th) cells. Transepithelial and lamina propria DCs identified in patients with chronic gastritis respond to both live bacteria and H. pylori antigens, releasing varying quantities of IL-6, IL-8, IL-10, and IL-12. In contrast to macrophages, TLR and myeloid differentiation factor (MyD88) expression in DCs is not dispensable for complete H. pylori recognition and DC activation. Endosomal TLRs that recognize microbial nucleic acids were recently identified as important components of DC responses to H. pylori . C-type lectin receptors (CLRs) expressed by DCs are crucial for directing immune responses to pathogens. DC-specific intercellular adhesion molecule 3 (ICAM3)–grabbing nonintegrin (DC-SIGN) recognizes H. pylori fructose residues, and following pathogen binding, it triggers the expression of specific cytokines that influence T-cell differentiation. DC-SIGN binding of H. pylor i was recently shown to result in enhanced IL-10 and reduced IL-12 and IL-6 expression, which could subsequently favor type 2 Th (Th2) cell polarization.
Myeloid Cells—B Lymphocytes
H. pylori also induces a vigorous mucosal humoral response that does not lead to eradication but does contribute to tissue damage. Infiltrating B lymphocytes and plasma cells give rise to H. pylori –specific IgA and IgG antibodies. CD4+ T cells help B lymphocytes to produce antibodies, and also contribute to inflammation in the gastric mucosa by producing high amounts of interferon γ (IFN γ ). H. pylori -specific IgG or IgA antibodies can be detected in peripheral blood from early stages of infection. The IgG and IgA responses to H. pylori in the serum and gastric mucosa have been well described and may be involved in protective immunity, but these B cell–mediated antibody responses could also be counterproductive. The development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma stems from activated B cells. Recently it has been reported that chronic gastric infection with H. pylori protected splenic B cells from apoptosis, indicating a B-cell activation/survival phenotype that may have implications for MALT lymphoma. In addition to producing antigen-specific antibodies, B cells can also produce potentially harmful autoantibodies. The implications of T-cell—B-cell interactions in the pathology of the immune response remain to be fully explained.
Myeloid Cells—T Lymphocytes
A variety of T-cell responses have been characterized, in both the gastric mucosa and the periphery. Immature Th cells can differentiate into Th1, Th2, and Th17 functional subtypes. The data on H. pylori –induced effects on T-cell differentiation, both locally and systemically, have been difficult to reconcile. In the human gastric mucosa, H. pylori induces recruitment of CD4+ and CD8+ T cells, and murine studies have shown that the gastric inflammation is T-cell dependent, as experimentally H. pylori does not induce gastritis in T-cell–deficient mice. H. pylori –specific T cells with a Th1 phenotype (i.e., secreting IFNγ) have been cloned from H. pylori –infected gastric mucosa and, through the production of IFNγ, were cytotoxic to gastric epithelial cells. Conversely, it has been suggested that activation of a Th2 cell response, and production of Th2 cell cytokines like IL-4, is protective against severe pathology, and curbs potentially detrimental effects of Th1-related cytokines.
Recently, a significant expansion of γδ-TCR+ T lymphocytes and high concentrations of IL-10 were observed in the peripheral blood of H. pylori –infected subjects in comparison to healthy controls. No differences were detected between infected and noninfected subjects with regard to the frequencies of CD3+, CD19+, CD4+, and CD8 + T cells and their subsets. A mixed T-cell response, although favoring a Th2 cell profile, was reported in a study of gastric T cells from subjects with and without H. pylori infection. Using flow cytometry and real-time (RT)-PCR, mixed H. pylori –specific T regulatory (T reg ) and Th cell subsets with a predominant CD4+ IL.10 + response were found in the gastric antrum. This more “tolerant” response could in part explain incomplete clearance of H. pylori and chronicity of infection. Previous investigations had reported predominant Th1 cell responses to H. pylori , which could be partly explained by methodologic differences. Of interest, patients in this study with ulceration had reduced T reg and increased Th1 and Th2 cell response profiles compared to those without ulceration.
A specific subset of CD4+ T cells termed T helper 17 (Th17) cells, which are distinct from and antagonized by the classical Th1 or Th2 cells, has been described recently. Th17 cells produce IL-17 and play a prominent role in the development of chronic inflammation associated with inflammatory and autoimmune disorders. T-cell production of IL-17 stimulates the production of IL-1, IL-6, TNFα, and matrix metalloproteinases by fibroblasts, endothelial cells, epithelial cells, and macrophages. IL-17 upregulation occurs at both RNA and protein levels in H. pylori -infection. IL-17 activates extracellular signal-related kinase 1 and 2/mitogen-activated protein (ERK1/2 MAP) in gastric epithelial cells and IL-17 expression levels correlate with the IL-8 content and number of infiltrating neutrophils in H. pylori –infected mucosa. Furthermore, IL-23 is overexpressed in H. pylori –infected gastric mucosa, which could contribute to sustaining IL-17 production. The exact molecular mechanism by which IL-23 regulates IL-17 in H. pylori –infected mucosa remains to be ascertained, but the signal transducer and activator of transcription 3 (STAT3) likely plays a key role in IL-23–driven IL-17 production during H. pylori infection. A comparison of gastric levels of Th17- and T reg -associated cytokines between children and adults showed that in children, T reg -cell differentiation was more predominant and might help to explain the increased susceptibility of pediatric patients to infection.
Apoptosis and Neoplasia
H. pylori induces apoptosis both in vitro and in vivo by several mechanisms. H. pylori or its products can induce apoptosis directly. For example, VacA induces the release of cytochrome c from mitochondria. Alternatively, the bacterium induces host immune responses, which then mediate apoptosis. For instance, Th1 cell cytokines (TNFα and IFNγ) markedly potentiate H. pylori –induced epithelial cell apoptosis. H. pylori also upregulates expression of the Fas death receptor. The absence of Fas signaling has been associated with less apoptosis and enhanced premalignant gastric mucosal changes.
The mechanisms of H. pylori –related carcinogenesis are unclear and are likely the result of both bacterial and host factors, and environmental factors such as smoking, high-salt diet, and antioxidant ingestion. H. pylori disrupts the DNA mismatch repair system (DMRS). By leading to gastric atrophy, H. pylori may be permitting its own replacement by more genotoxic bacteria.
Acid Homeostasis
To promote chronic infection, H. pylori has developed an array of mechanisms to survive in the harsh acidic environment of the gastric mucosa. One of these is an “acid acclimation mechanism” that promotes adjustment of periplasmic pH in the acidic environment of the stomach by regulating activity of urease, UreI, and α-carbonic anhydrase. Previous studies indicated that the ArsS two-component system regulated transcription of urease. A recent study using ArsS mutants extended these findings to demonstrate that ArsS also mediated trafficking of urease to the inner membrane upon acute but not prolonged acid exposure, signaling another method by which the ArsS two-component system regulates acid acclimation. H. pylori infection can cause hypergastrinemia by both reducing D-cell somatostatin production and increasing G-cell gastrin production; removal of H. pylori reverses these effects. However, the ultimate effect of infection on acid homeostasis depends on the topographic distribution of H. pylori –induced inflammation within the stomach. In antral-predominant gastritis, gastrin release leads to higher acid levels, and persistently high gastrin levels increase the parietal cell mass. This in turn results in increased acid delivery to the duodenum, inducing gastric metaplasia. H. pylori can colonize gastric metaplasia, resulting in inflammation and, possibly, ulceration.
With pangastritis or corpus-predominant gastritis, H. pylori infection suppresses acid production, both directly and indirectly. Inflammatory mediators inhibit parietal cell acid secretion and enterochromaffin-like cell histamine production. Reduced acid secretion further increases gastrin levels, thereby promoting gastric epithelial cell proliferation. Epithelial cell characteristics become altered, leading to progressive gastric gland loss, and thus gastric atrophy. Gastric atrophy increases the risk of gastric ulceration and noncardia gastric adenocarcinoma
Putative Hormonal Effects
H. pylori infection can affect the expression of the appetite- and satiety-controlling hormones leptin and ghrelin. In children, an inverse relationship between serum ghrelin concentration and the severity of H. pylori –induced gastritis has been demonstrated. H. pylori infection leads to a reduction of the density of gastric ghrelin–positive cells. The decrease in ghrelin is associated with neutrophil activity, chronic inflammation, glandular atrophy, and low serum pepsinogen level. H. pylori infection decreases gastric D cells that produce somatostatin and the subsequent loss of gastric G-cell inhibition is a mechanism for hypergastrinemia in H. pylori –infected individuals. H. pylori eradication has been shown to lead to an increase in body weight. The underlying mechanism has been suggested to be of gastric hormonal origin, with both ghrelin and leptin contributing to this effect. Although the prevalence of H. pylori infection was previously shown to be significantly higher in lean rather than obese patients, there is neither a sound scientific basis nor robust data to support the hypothesis that H. pylori is a protective factor against obesity.
Bacterial Factors and Adaptations
Genetic Diversity
The H. pylori genome (1.65 million base pairs [bp]) codes for more than 1500 proteins, including enzymes that modify the antigenic structure of surface molecules, control the entry of foreign DNA into the bacterium, and influence bacterial motility. These factors are essential for H. pylori to colonize humans effectively. More than100 bacterial genes are required for gastric colonization, and their expression can be upregulated within the stomach. H. pylori lacks a genetic mismatch repair system to control the confidentiality of replication, which results in a high mutation rate. Genetic diversity of H. pylori has likely resulted from endogenous mutations and recombination. Many H. pylori isolates have a hyper-mutator phenotype, which favors the emergence of variants after selective pressure; the rapid development of high-level resistance to commonly used antibiotics such as clarithromycin is one such example. H. pylori are also highly competent for uptake of DNA; thus, the H. pylori genome continuously changes during chronic colonization by acquiring fragments of DNA from other H. pylori strains. In essence, each host is colonized not by a single clone, but by a variety of usually closely related organisms.
Colonization and Environmental Adaptation
After ingestion, H. pylori colonizes the gastric mucous layer, and can adhere to and invade gastric epithelial cells. Gastric acidity, motility, nutrient availability, and host immune responses are but some of the barriers to colonization. H. pylori has adapted remarkably to many of these barriers. Motility of H. pylori depends on the presence of up to six functional unipolar flagella. Recent studies indicate that proper assembly of flagella requires peptidoglycan-degrading enzymes that promote the correct localization and function of the flagella motor. H. pylori regulates cell motility by responding to chemotactic cues, which then alter flagellar activity. Chemotactic (Che-) mutants have altered colonization patterns . H. pylori senses environmental chemical cues via four chemoreceptors: Tlp A, B, C, and D. Using isogenic chemoreceptor mutants, Rolig et al. demonstrated that T1pD is necessary for H. pylori to survive and grow in the infected and inflamed antrum but not elsewhere in the murine stomach. H. pylori can hydrolyze urea to generate ammonia and modify the local pH. Its urease activity is governed by a unique pH-gated urea channel. H. pylori urease binds substrate with a much higher affinity than that of other bacterial species.
Mucosal Adherence
The adherence of H. pylori to the gastric mucosa is important for initial colonization and subsequent persistence in the human gastric mucosa. H. pylori employs genetic diversification to adapt to the changing environment to promote colonization and persistent infection. The H. pylori genome encodes five major outer membrane protein (OMP) families, which can bind to antigens (receptors) on gastric epithelial cells, thereby anchoring bacteria to counteract mechanical clearance. The 5′ and 3′ end regions of the omp genes (encoding OMPs) are highly conserved, which could allow for recombination, thereby switching loci and bacterial phenotype. The Hop ( Helicobacter outer membrane protein) family can act as adhesion molecules and include the blood-group antigen-binding adhesin (BabA), sialic acid–binding adhesin (SabA), adherence-associated lipoprotein A/B (AlpA and AlpB), outer membrane inflammatory protein (OipA), and HopZ. Lewis b and related blood-group antigens are recognized by BabA, whereas sialyl-Lewis x and sialyl-Lewis a antigens are recognized by SabA Lewis (Le) blood-group epitopes on the surface of H. pylori mimic structures present on human gastric surfaces and could be implicated in adverse autoimmune reactions of the host. Most studies in adults found that the majority of the H. pylori strains express type 2 Le x and/or Le y antigens, whereas pediatric isolates have the tendency to express also type 1 Le b antigen. The corresponding receptors for AlpA/B, OipA, and HopZ remain unknown. H. pylori binds tightly and specifically to gastric epithelial cells using these adhesins. Clinical isolates obtained from pediatric subjects showed variability in the copy number and locus of the omp genes sabA and sabB , implicating intragenomic recombination among strains. The adhesin BabA2 is associated with higher levels of bacterial colonization, neutrophil infiltration, and IL-8 secretion in the gastric mucosa, suggesting that BabA2 facilitates colonization and augments host immune responses. Infections with babA2 -positive strains are associated with peptic ulcer disease and pre-neoplastic gastric lesions. Sialyl-Lewis x expression on the gastric epithelium is promoted by both gastric inflammation and malignant transformation. In vitro studies demonstrated that sabA gene duplication increases SabA protein production and adherence. Using binding assays to a panel of glycosphingolipids, the structural requirements for binding of BabA to the host cell adhesion receptor were further assessed. BabA was found to bind blood group determinants on both the type 1 and type 4 core chains in these in vitro assays.
The dimeric form of trefoil factor 1 (TFF1) can interact with H. pylori . This small protein is coexpressed with the gastric mucin, MUC5AC. TFF1 acts as a linker molecule, binding to both MUC5AC and H. pylori LPS in a pH-dependent manner, and may be a factor in determining the tropism of this organism for gastric tissue.
cag Pathogenicity Island
Type IV secretion systems (T4SS) are biologic “molecular syringes” produced by many gram-negative bacteria, which transport proteins or DNA–protein complexes into other cells. The H. pylori T4SS is a filamentous, sheathed organelle with a rigid, needle-like pilus protruding from the bacterial surface. These pili are often located at one bacterial pole and are induced on cell contact. CagF is a chaperone-like protein that is crucial for the translocation of CagA. CagL is a pilus-covering protein that anchors and targets the T4SS to the host transmembrane cell adhesion molecule, integrin α 5 β 1 , where CagA is then injected. CagL was recently linked with hypergastrinemia, a major risk factor for the development of gastric adenocarcinoma. This CagL-mediated effect was independent of α5β1 but dependent on αVβ5 integrin signaling. Thus, CagL may constitute a novel target in the treatment of precancerous conditions triggered by H. pylori– induced hypergastrinemia.
One distinguishing feature of H. pylori strains is the presence or absence of the cag pathogenicity island (PAI). The cytotoxin-associated gene A ( cagA ) encodes for a 120 to 140 kDa immune-dominant protein, CagA, a marker for the cag PAI. This 37 kb fragment of chromosomal DNA is acquired by horizontal transfer and encodes for components of the T4SS that transports CagA and peptidoglycan into host cells. The predicted size of CagA is larger than the channel of T4SS. Several proteins including CagL, CagY, and CagA that are present on the T4SS use beta 1 integrin as a receptor to deliver CagA into the host cell. The crystal structure of the N-terminal region of CagA identified a single layer beta sheet (SLB) region that acts as the functional binding domain for β1 integrin as determined by yeast two hybrid protein-interaction screens. CagA can modulate cell growth, motility, alter tight junctions, disturb cell polarity, and activate STAT3 in vitro and in vivo. Translocated CagA can also induce IL-8 release in infected cells and activate NF-κB in a strain- and time-dependent manner.
As shown in Figure 27-3 , CagA modulates several signal transduction cascades in host cells. The role of CagA in disease pathogenesis is not completely understood. Numerous studies, particularly in developed countries, have shown that cag PAI–positive H. pylori strains confer an increased risk for peptic ulcer disease and gastric cancer over strains that lack the cag PAI. In children, infection with a CagA+ strain has been associated with peptic ulcer disease in some populations but not others. The 3′ region of the cagA gene in clinical isolates can vary with respect to EPIYA and CM motifs, and a variety of studies continue to elucidate the association of these variations with disease outcome in different populations. CM is a 16 amino acid sequence responsible for CagA multimerization. In a Portuguese population, the infection with strains with higher numbers of EPIYA C was not associated with duodenal ulcer (DU), but was associated with gastric precancerous lesions and increased risk of gastric cancer. However, in isolates obtained from patients in three New York hospitals, heterogeneity in EPIYA was not identified, but a great heterogeneity in CM located before and after the EPIYA C was detected.
Yamaoka et al. reported that the prevalence of cagA gene was significantly higher in African American children than in white American children (100 vs. 56.5%, p = 0.032). Such large differences have not been reported in adult studies.
Heme oxygenase 1, which exhibits anti-inflammatory and anti-oxidant effects, reduced CagA phosphorylation during H. pylori infection of gastric epithelial cells in vitro. Of interest, hmox 1 expression and HO 1 protein levels were diminished in gastric epithelial cells of CagA+ H. pylori– infected patients, suggesting that the bacterium may have developed a strategy to counteract hmox 1 expression.
A second T4SS expressed by H. pylori involves ComB proteins, encoded by comB genes. These proteins have all the characteristics of a pore-forming transmembrane complex suitable for DNA translocation through the cellular envelope. ComB is functionally unrelated to the cag PAI system, such that mutations within comB do not affect the function of cag PAI, and vice versa.
The Plasticity Region
A comparison of two fully sequenced genomes of H. pylori identified a large single hypervariable region that contained almost one-half of the strain-specific genes. This region, called the plasticity region, has an altered G and C content and contains several insertion sequences, suggesting it may be acquired from horizontal transfer. A study of H. pylori strains isolated from 200 Brazilian patients, identified an association between the presence of a gene located in the plasticity region, JHP947 , and the presence of DU and gastric carcinoma. A further study also indicated that JHP947 was associated with secretion of IL-12 and DU disease. The gene JHP0562 encodes for the cell envelope protein, glycosyltransferase, which may be essential for the survival of H. pylori and may contribute to the persistence of infection. This gene has been shown to increase susceptibility to peptic ulcer disease in children, and the virulence factors cagPAI, vacA s1 allele, babA homB, oipA “on” and hopQ 1 allele were also associated with jhp0562. In addition, a cluster of genes with homology to genes of the T4SS has been located in the plasticity region.
Vacuolating Cytotoxin A, VacA
Vacuolating cytotoxin A (VacA) is an 88 kDa pore-forming protein produced by approximately 50% of H. pylori strains. vacA is a polymorphic gene that appears to be conserved among all H. pylori strains. As shown in Figure 27-4 , the vacA gene possesses variable signal regions (s1 or s2) and midregions (m1 or m2). Strains of H. pylori that express active forms of the toxin are associated with more severe cases of disease. A wide range of cellular effects have been attributed to VacA, as illustrated in Figure 27-5 , including induction of apoptosis, alteration of the process of antigen presentation, and autophagy induction. The role of VacA in mediating disease in humans is unclear. In certain populations, specific vacA alleles are associated with the presence of disease such as gastric adenocarcinoma. Association between vacA alleles and disease presentation and outcome in children has been variably reported.
Pathoepigenetics
Pathoepigenetics, which refers to epigenetic changes that occur during infection, is an area of increasing interest in H. pylori infection. H. pylori infection is now known to mediate epigenetic changes, including hypermethylation of promotor regions in H. pylori –infected murine tissue and human gastric cancer specimens. Hypermethylation of the transcription factor FOXD3 (forkhead box D3) was identified in H. pylori –infected murine gastric tissue and correlated with decreased survival in patients with gastric cancer. Although not previously known to be a tumor suppressor, in vitro assays indicated that FOXD3 exhibited tumor suppressor function supporting a role for deregulation of FOXD3 in tumorigenesis.
MicroRNAs (miRNAs) regulate gene transcription, and many miRNAs have been implicated in tumorigenesis. In a study of cells expressing CagA, miR-26a and miR-101 expression was attenuated.
Host Immune Evasion and Manipulation
Despite the presence of a vigorous immune response, H. pylori eradication is not usually observed unless specific antibiotic therapy is provided. This demonstrates the effectiveness of H. pylori ‘s strategies in evading host immunity. Although not completely resistant to host immune activation, H. pylori has evolved a variety of mechanisms to reduce recognition by immune sensors, downregulate activation of immune cells, and escape immune effectors.
Many bacteria have unmethylated cytosine-guanine–rich DNA (CpG DNA) that is recognized by TLR9. However, H. pylori DNA is highly methylated, which likely minimizes detection by TLR9, at least in certain cell types. Bacterial flagella are usually recognized by TLR5, but H. pylori flagellin does not strongly activate TLR5 signaling pathway. TLR4 recognizes bacterial LPS. Owing to modifications within the lipid A core, H. pylori LPS is relatively anergic, stimulating TLR4 on macrophages but not on gastric epithelial cells. H. pylori counteracts macrophage function by inhibiting phagocytosis, disrupting phagosome maturation, and promoting apoptosis. H. pylori VacA interferes with both the uptake and processing of antigens, suppresses T-cell proliferation and activation, and induces selective T-cell apoptosis. In addition, H. pylori can disrupt cytokine-signaling pathways in vitro. Furthermore, by mimicking gastric epithelial Lewis antigens and by antigenic variation of surface proteins, the bacterium evades host adaptive responses. The relative importance of each of these strategies is not yet established, and may well vary from host to host.
Antibiotic Resistance—The Bacterial Perspective
Antimicrobial drug resistance is a major cause of treatment failure and has led to declining eradication rates. H. pylori antibiotic resistance mechanisms are based mainly on point mutations located on the bacterial chromosome. Given its lack of DNA mismatch repair mechanisms, it is not surprising that antibiotic resistance easily develops de novo , although horizontal gene transfer is another possible resistantoce mechanism. Resistance of H. pylori to commonly used antibiotic groups—including nitroimidazoles, macrolides, penicillins, tetracyclines, and fluoroquinolones—has been characterized and provides the basis for designing current and future treatment regimens.
Metronidazole and tinidazole are bactericidal antibiotics administered in prodrug form that need to be activated within the target cell by electron transfer processes. This leads to the formation of nitro-anion radicals and imidazole intermediates that cause lethal damage to subcellular structures and DNA. In H. pylori , resistance is mediated by reduced or abolished activity of any of several putative electron acceptors, such as ferredoxin (FdxA), flavodoxin (FldA), and oxygen-insensitive NAD(P)H nitroreductase (RdxA). Regardless of whether mutations in these factors alter the expression of the corresponding protein, or result in a truncated protein or changed amino acid sequence, the finding that mutations in multiple proteins all result in an increased minimum inhibitory concentration (MIC) of antibiotic might explain the wide range in levels of resistance.
Clarithromycin is a bacteriostatic antibiotic that binds reversibly to 23S ribosomal RNA (rRNA) to interfere with protein synthesis. In H. pylori , resistance to clarithromycin and other related macrolides is mostly because of point mutations in one of two adjacent 23S rRNA nucleotides. These substitutions decrease ribosomal affinity for macrolides, resulting in increased resistance.
Amoxicillin binds to penicillin-binding proteins (PBPs) and interferes with bacterial cell wall synthesis, resulting in lysis of replicating bacteria. In H pylori , resistance is mediated primarily by alterations to PBPs. Many H. pylori isolates described as being amoxicillin resistant are often just tolerant of penicillins—that is, their resistance is transient and not stable. Stable amoxicillin resistance in H. pylori is rare and is mediated by mutational changes in PBP1A. Amoxicillin resistance in H. pylori also ensues from alterations in OMP composition, resulting in an increased diffusion barrier effect.
Tetracycline is a bacteriostatic antibiotic that binds to the 16S rRNA, resulting in inhibition of protein synthesis and bacterial growth. The main mechanism of tetracycline resistance in H. pylori arises from base-pair substitutions in the 16S rRNA primary binding site of tetracycline, although reduced membrane permeability could contribute to tetracycline resistance.
Fluoroquinolones are bactericidal antibiotics that inhibit DNA gyrase, an enzyme that catalyses the negative supercoiling of DNA. In H. pylori , resistance to fluoroquinolones is caused by point mutations in the DNA gyrase-encoding gene gyrA .
H. pylori –infected children and children who experience a failure of eradication therapy differ significantly in the expression of adhesion and activation molecules on circulating monocytes. A decrease, both in the proportion of CD11c- and CD14-bearing monocytes and the expression of CD11c and CD14 molecules on circulating monocytes, was found in children in whom the eradication therapy failed. H. pylori eradication therapy in children leads to a reduction in CD11b, CD11c, and CD18beta2 integrin monocyte expression.
Disease Associations
Various diseases are purported to be associated with H. pylori infection. The spectrum of H. pylori– related disease encountered in childhood varies somewhat from that recognized in the adult population.
Gastrointestinal Manifestations
Gastritis
Infection with H. pylori is associated with chronic gastritis in both children and adults. The first reports of this came from Marshall and Warren, who ingested the organism and subsequently developed gastritis. Eradication of H. pylori results in the healing of gastritis. Whether gastritis, in the absence of ulcer disease, has any manifest symptoms, including recurrent abdominal pain or nonulcer dyspepsia, has been a source of controversy. The best available evidence from multiple geographic areas of the world suggests that H. pylori gastritis remains largely asymptomatic in children and is not a causal factor in recurrent abdominal pain.
Ulcer Disease
H. pylori infection plays a causal role in the development of duodenal and gastric ulcers, and should be eradicated when detected in such settings. The lifetime risk of peptic ulcer disease in the setting of H. pylori– positive gastritis in adults varies geographically. In the United States, this risk is reported to be as low as 3%, whereas in Japan the rate is 25%.
In children, ulcer disease is thought to be less common than adults, with reported rates in symptomatic children of 2% to 22.5%. The recent pediatric European treatment registry report noted gastritis in 87% of 518 children, with the remainder having duodenal or gastric ulceration. The burden of non -H. pylori– associated peptic ulcer disease in children also appears to be increasing. A recent European multicenter study reported ulcers and/or erosions in 8.5% of children, of whom 27% had H. pylori infection. Similarly, in a single-center retrospective study from Taiwan, of the 1234 children who had an upper endoscopy, only 67 (5.4%) had peptic ulcer disease, of whom 32 (48%) of 67 were infected with H. pylori, whereas 36% of children had no identified risk factors associated with peptic ulcer disease.
The pattern of H. pylori gastritis and the age at H. pylori acquisition both appear to be important determinants of future sequelae. Antral-predominant gastritis has a higher risk of duodenal ulcer, whereas subjects with corpus-predominant gastritis are more prone to gastric ulcers and gastric malignancy. A combination of bacterial factors and host genotype and immune responses are also likely determine whether children proceed to develop ulceration. A duodenal ulcer promoting gene ( dupA ), located in the plasticity region of the H. pylori genome, may be one such virulence marker. A recent meta-analysis of 50 studies has confirmed the role of the iceA allele as a virulence factor for the development of peptic ulcer disease (PUD), especially DU.
Gastroesophageal Reflux and Reflux Disease
Currently there is no convincing clinical data that H. pylori status or its eradication affects gastroesophageal reflux disease (GERD) in children. Various epidemiologic studies have demonstrated an inverse relationship between rates of H. pylori infection and the prevalence of GERD and/or the aggravation of esophagitis with H. pylori eradication. Conversely, many studies have found no relationship between reflux symptoms and H. pylori eradication. The first prospective evaluations of the effect of H. pylori eradication on GER symptoms in pediatric patients were published in 2004. Both symptoms of GER and epigastric pain were unrelated to H. pylori status and eradication outcome. Similar results have been found in adult and pediatric studies.
Gastric Carcinoma
Gastric cancer is the second most frequent cause of cancer-related death in the world and the fourth most common cause of cancer-related death in Europe. H. pylori infection is mostly associated with noncardia adenocarcinomas. The risk attributed with positive serology ranges from 2.1 to 8.7. Several subsequent studies have placed the relative risk of gastric cancer in infected individuals between two- and sixfold. One study, which subsequently controlled for bias, even suggested that risk is much higher. A meta-analysis of H. pylori association with early gastric cancer suggested an OR of 3 compared with noncancer controls. Several studies have examined the role of H. pylori eradication in cancer prevention. A recent meta-analysis suggests a beneficial role in Asian countries with a high prevalence of gastric cancer, whereas benefits in the West were less clear. A prospective study of 1526 Japanese subjects conducted over an 8-year period reported that, during follow-up, gastric cancer developed in infected patients only; no cases were detected in eradicated or uninfected patients. Of interest, in the western world, around 60% of H. pylori isolates possess CagA compared with virtually all isolates in Japan. A randomized primary prevention trial from China appeared to show no effect of H. pylori treatment on gastric cancer, but on sub-group analysis, subjects without endoscopic precancerous lesions at baseline had significantly less gastric cancer. However, in a recent study by Kim et al. in the adult population, it was shown that successful H. pylori eradication may reduce the occurrence of metachronous gastric cancer after endoscopic resection in patients with early gastric cancer.
Gastric cancer has been more strongly linked to cagA + strains and host gene polymorphisms including IL-1, TNFα , and IL-10 . Environmental factors such as smoking and dietary intake play an important role in determining disease risk.
Acquiring H. pylori infection at a very early age has been related to a much greater gastric cancer risk, especially in the setting of a positive family history of gastric cancer. The familial aggregation of stomach cancer may, in part, be explained by familial aggregation of H. pylori infection.
Although the development of low-grade gastric MALT lymphoma associated with chronic H. pylori gastritis has been reported in children, to date, there have been no reports of gastric adenocarcinoma in childhood.
Mucosa-Associated Lymphoid Tissue Lymphoma
Primary malignant tumors of the stomach are uncommon in children and usually consist of lymphoma and sarcoma. Primary gastric lymphoma can be divided histologically into mucosa-associated lymphoid tissue (MALT) lymphoma and non-MALT lymphoma. Primary gastric lymphoma is a very rare malignancy in children. The risk of gastric MALT lymphoma is significantly increased with H. pylori infection. Some 72% to 98% of patients with gastric MALT lymphoma are infected with H. pylori . The eradication of H. pylori alone induces regression (and remission) of gastric MALT lymphoma in 70% to 80% of cases. Failure of the lymphoma to respond to eradication therapy has been associated with certain genetic abnormalities within the host, including the presence of the specific genetic translocations t(11;18)(q21;q21). Such cases usually progress to high-grade tumors. Most subjects who respond to eradication therapy remain in remission for many years.
Extraintestinal Manifestations
Iron Deficiency Anemia
Most of the published studies describing anemia in the setting of H. pylori infection have been in pediatric populations. In general, within developed countries, iron deficiency anemia (IDA) is seen more frequently in children and adolescents than in adults. Thus, if H. pylori infection does lead to IDA, children are more likely to be affected than adults are.
Epidemiologic studies have indicated an association between H. pylori seropositivity and low serum ferritin and hemoglobin levels. A seroepidemiologic study of 937 children found iron deficiency to be twice as common in H. pylori– positive children than in uninfected children. A number of case reports have demonstrated IDA, previously resistant to iron replacement therapy, responding to the eradication of H. pylori , with a few reports of H. pylori eradication resulting in improvement of anemia even without iron supplementation. Recent studies have reported conflicting evidence regarding the influence of H. pylori status and eradication on iron deficiency and on iron treatment failure. Whether iron deficiency, like H. pylori , is an index of dietary or socioeconomic status rather than a cause of anemia per se remains to be fully elucidated.
Age may be an important factor in the relationship between H. pylori infection and iron deficiency, with a stronger association in older age groups. In one study, when stratified by age group, H. pylori was associated with a twofold increased prevalence of anemia in school-aged children compared with infants younger than 18 months, independent of socioeconomic variables. However, the diagnosis of H. pylori is not straightforward in children younger than 18 months of age. In this study, the difference in prevalence between the school-aged children (54%) and infants (15%) needs to be evaluated, as the two age groups may be from very different populations and have very different risks of both anemia and H. pylori infection.
Several mechanisms responsible for H. pylori– mediated iron-deficiency anemia have been postulated. Chronic gastrointestinal bleeding due to gastritis, erosions, or ulceration may be to blame, although most studies have not detected occult gastrointestinal blood loss in infected patients with anemia. The levels of gastric acidity and gastric ascorbic acid are important for the absorption of dietary iron, and hypochlorhydria could affect iron bioavailability. In most children, the mucosal and glandular structure within the gastric body remains completely normal during chronic H. pylori infection, with atrophy being an unusual, later phenomenon. Another suggested mechanism is related to the sequestration of iron by antral H. pylori infection. H. pylori is known to possess genes with an iron-scavenging function, thereby enabling the bacterium to extract iron from its host. Children with low iron intake or increased iron requirements form an obvious risk group for IDA. In this vulnerable group, it is possible that even minor disturbances in the iron absorption mechanisms that may occur due to H. pylori infection might quickly lead to a deficiency state. Guidelines for H. pylori infection in children suggest that children presenting with unexplained iron deficiency anemia that is refractory to therapy may warrant investigation for H. pylori infection.
Short Stature
A number of studies have suggested that H. pylori infection may have a negative effect on growth, although reports are conflicting. In 2001, Richter et al. conducted a cross-sectional population-based study in Germany involving 3315 children aged 5 to 7 years. The overall prevalence of H. pylori infection was about 7%. A small, but statistically significant, difference in height (before and after age and sex adjustment) was detected between H. pylori– positive and H. pylori– negative children, and was more pronounced in boys. In contrast to other studies, no significant difference in socioeconomic status was found between the two groups. It has been suggested that growth retardation could be a result of either gastritis or comorbidity, such as anemia. Alternatively, H. pylori infection and growth retardation may be caused coincidentally by the same confounding factors, such as socioeconomic factors.
Ozen et al. investigated the effect of H. pylori and economic status on growth parameters leptin, ghrelin, and insulin-like growth factor 1 (IGF-1) concentrations in children. Evidence that H. pylori may impair growth significantly was confounded by socioeconomic factors, as the effect was seen only in children living in unfavorable socioeconomic conditions.
Notwithstanding the considerable literature on an association between H. pylori and short stature, no study to date has demonstrated a causal relationship between H. pylori and short stature by showing increase in growth velocity in children following eradication of H. pylori .
Other Suggested Associations
A wide variety of extraintestinal manifestations has been suggested to have an association with H. pylori infection. These range in diversity from dermatologic and autoimmune problems such as chronic urticaria and atopy to idiopathic thrombocytopenia (ITP). Current data suggest that a role for H. pylori in adult ITP is plausible and that treatment is justifiable and beneficial. This has now been reflected in the latest international treatment guidelines. Data from pediatric studies to date are less convincing, however. A multicenter, prospective, controlled study evaluating the effect of eradication of H. pylori infection in Italian children with ITP did show a significantly higher platelet response among those who underwent successful eradication of H. pylori infection. A critical assessment of the evidence on a relationship with sudden infant death syndrome (SIDS) has concluded that a causal association is very unlikely. A recent analysis of data from the National Health and Nutrition Examination Survey (NHANES) suggested an inverse correlation with asthma. Current evidence for other putative associations in children is not compelling.
Benefits of Disease
It is postulated that there may be potential benefits to H. pylori infection. H. pylori may stimulate specific local and systemic immunoglobulin secretion and thus participate in host defense against exogenous pathogens. H. pylori may synthesize antibacterial peptides, which would prevent other faster-growing bacteria from colonizing the gastric mucosa and gastrointestinal tract. Some, but not all, studies suggest that H. pylori may be associated with protection from diarrheal diseases in both children and adults in developed countries. The potential positive final consequences of H. pylori elimination for human health should not be overlooked.
It has been recently suggested that an increased prevalence of allergic diseases could be, at least partially, explained by the decreased incidence of H. pylori infection, although the literature is not definitive. A potential mechanism involves an effect of H. pylori on induction of T-cell effectors. A large retrospective study that used UBT and not serology to determine the H. pylori infection status cited being H. pylori negative as an independent risk factor for pediatric asthma. Studies from Chile and Ethiopia have also shown an inverse relationship between pediatric atopy, allergy, and H. pylori infection. In contrast, a large Dutch study did not confirm any association between H. pylori seropositivity and wheezing (OR 0.52; 95% confidence interval [CI] 0.25–1.06), allergic rhinitis (OR 0.96; 95% CI 0.51–1.81), atopic dermatitis (OR 1.05; 95% CI 0.56–1.98), or physician-diagnosed asthma (OR 0.87; 95% CI 0.37–2.08).
Diagnosis
The ideal test for H. pylori would be noninvasive, highly accurate, inexpensive, and readily available. The test would differentiate between active and past infection, and discriminate between H. pylori infection and H. pylori– associated disease. No such test currently exists. Thus, it is important to appraise the advantages and disadvantages of the tests that are available and to assess their suitability for use in children.
The primary indication for investigation in children remains to diagnose the cause of significant symptoms and not simply to detect the presence of H. pylori . Testing for H. pylori is not helpful unless it alters clinical management. There remain certain scenarios in which screening for the presence of H. pylori is not absolutely indicated but may be considered by clinicians (see Box 27-1 ).
- 1.
Clinical investigation of gastrointestinal symptoms should aim to determine their underlying cause and not merely the presence of H. pylori infection.
- 2.
Diagnostic testing for H. pylori infection is not indicated in children with functional abdominal pain.
- 3.
Testing for H. pylori may be considered in the following circumstances:
- •
those with first-degree relatives with gastric cancer
- •
those with refractory iron-deficiency anemia, where other causes have been ruled out
- •
- 4.
There is currently insufficient evidence supporting testing in the following conditions: otitis media, upper respiratory tract infections, periodontal disease, food allergy, idiopathic thrombocytopenic purpura, or short stature.
- 5.
“Test and treat” strategies are not recommended in children.
- 6.
The initial diagnosis of H. pylori infection should be either based on a positive biopsy culture or based on positive histopathology (antrum and corpus biopsies) plus a positive rapid urease test.
- 7.
Both urea breath tests and ELISA-based H. pylori stool antigen tests are reliable noninvasive tests for determining H. pylori eradication.
- 8.
Tests based on the detection of H. pylori antibodies (IgG, IgA) in serum, whole blood, urine, and saliva are not reliable for use in the clinical setting.
- 9.
Initial testing for H. pylori should wait a minimum of 2 weeks after stopping PPI therapy and 4 weeks after stopping antibiotics.
- 10.
H. pylori eradication is recommended in H. pylori– positive peptic ulcer disease.
- 11.
H. pylori treatment may be considered in the following situations:
- •
biopsy-proven infection in the absence of peptic ulcer disease
- •
H. pylori– infected children whose first-degree relative has gastric cancer
- •
- 12.
Reliable noninvasive tests for eradication are recommended no sooner than 4 weeks following completion of therapy.
Invasive (requiring endoscopy) and noninvasive tests are available to diagnose H. pylori infection, and clinicians must consider their suitability and validity for use in children and the likely implications of a positive or negative result before they are requested ( Tables 27-1 and 27-2 ).
| Invasive | Noninvasive |
|---|---|
|
|
|
|
|
|
|
| Method | Positive Predictive Value (%) | Negative Predictive Value (%) | Sensitivity (%) | Specificity (%) |
|---|---|---|---|---|
| Invasive: | ||||
| Histopathology | 82 | 95-99 | ||
| Rapid urease test (RUT) | 85 | 99 | ||
| Culture | 85 | 100 | ||
| Noninvasive: | ||||
| Serum H. pylori IgG | 72 | 90 | 86 | 80 |
| Serum cytotoxin-associated gene product A IgG | 71 | 89 | 83 | 80 |
| Salivary H. pylori IgG | 82 | 79 | 66 | 91 |
| H. pylori fecal antigen | 97 | 98 | 97 | 98 |
| Urea breath test | 90 to 100 | 90 to 100 | 75 to 100 | 78 to 100 |
Standardized test methodology is also necessary to obtain reliable, comparable results. Any local changes in methodology or lack of local validation could have a strong negative impact on the reliability of the test.
Invasive Tests
Endoscopy and Biopsy
The reference standard for investigating H. pylori infection and its consequences in childhood continues to be a combination of upper gastrointestinal endoscopy and accompanying biopsies for histologic and microbial detection and/or culture. It allows visualization of the upper gastrointestinal tract and also facilitates the diagnosis of diseases other than those related to H. pylori infection. Gastric inflammation caused by H. pylori is not always observed macroscopically. Nodularity within the stomach, resembling a cobblestone pavement, is seen more frequently in children than in adults, but although associated with, it is not specific for H. pylori gastritis. Nodules measure 1 to 4 mm in diameter, have a smooth surface, are the same color as the surrounding mucosa, and are seen most often within the gastric antrum. Antral nodularity correlates with the severity of histologic gastritis.
Endoscopy facilitates the collection of mucosal biopsies upon which a variety of direct tests can be performed. Two broad patterns of H. pylori gastritis have been described—antral predominant gastritis (linked to increased peptic ulcer risk) and pangastritis (linked to increased risks of gastric atrophy). Therefore, biopsy samples of the gastric antrum and body should be obtained at endoscopy. Biopsy specimens obtained in the pre-pyloric antrum have the highest yield for detecting H. pylori infection. Children with endoscopically documented peptic ulcer disease should have multisite biopsies.
Endoscopy is not without its disadvantages, including procedural risk, anesthetic/sedation requirements, relative expense, and limited access to appropriate pediatric specialist centers. This reinforces the importance of having a clear and valid clinical indication to perform an endoscopy in children.
Culture
Culture is a potential reference standard for the diagnosis of suspected H. pylori infection. However, the bacterium has fastidious growth requirements and its morphology transforms under adverse conditions. Cultivation requires a microaerophilic environment and complex media and, although specific, its sensitivity can vary greatly between laboratories. Diagnostic yield is improved when multiple biopsy specimens are collected. It is important that specimens for culture are processed within 2 to 3 hours of collection, as H. pylori lacks regulatory genes, thereby rendering its survival for long periods outside the gastric environment poor.
A major advantage of culture is the ability to perform antibiotic sensitivity testing on the isolates, which can influence the outcome of therapy. Both phenotypic and genotypic methods are available for antimicrobial susceptibility testing.
Rapid Urease Test
The urease activity of H. pylori enabled the development of a variety of diagnostic tests, including the rapid urease test (RUT). Urease catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The production of ammonia leads to an increase in the local pH. Samples are placed within a gel containing urea and a pH indicator. A color change occurs as urea is broken down by the bacteria. The use of RUTs in pediatrics is limited by a significantly lower sensitivity compared with that of histologic evaluation; this is possibly related to a lower mucosal bacterial load. A variety of studies have validated this method in children. Elitsur and Neace examined a group of 94 children in West Virginia. Using histology as the reference standard, 19% of their study population was H. pylori positive. When utilizing a combination rather than a single investigation as the comparative standard, the sensitivity of the RUT in childhood is significantly improved. In a study of 59 Mexican children with a disease prevalence of 37%, of the three invasive techniques (culture, histologic examination of antrum, and corpus biopsies with hematoxylin and eosin [H&E] and Giemsa staining and RUT), RUT was the most sensitive and had the best negative predictive value (NPV) (100%).
New RUTs are emerging, but as with all RUTs, their interpretation is influenced by the number of tissue specimens tested, the location of biopsy sites, bacterial load, and previous use of antibiotics or proton pump inhibitors (PPIs).
Histopathology
On H&E staining of gastric mucosal biopsies obtained from H. pylori –infected patients, a superficial infiltrate is usually seen with substantial numbers of plasma cells and lymphocytes within the mucosa. Biopsies obtained from children infected with H. pylori generally have less neutrophil infiltration compared with tissue obtained from infected adults.
Methods of H. pylori detection include routinely stained H&E slides, modified Romanovsky methods (Giemsa and Diff-3), and Sayeed stains or silver stains (Dieterle, Warthin-Starry, Steiner, and Genta). Genta stains have shown 82% concordance with immunohistochemistry. In children, H&E and Giemsa stains have shown a sensitivity of 82% and specificity of 95%.
Although H. pylori colonization results in chronic gastritis, not all chronic gastritis is caused by H. pylori . Eshun et al. retrospectively reviewed 37 patients and 12 controls and found that higher grades of gastritis were associated with greater numbers of organisms. When biopsies in children display chronic inflammation and a negative urease test result, immunohistochemical stains should be utilized to further investigate for H. pylori infection.
The site from which the biopsy specimen is obtained can affect the accuracy of diagnosis. Elitzur et al. studied 206 children to determine the optimal biopsy location. After sampling six different sites, they concluded that the mid-antrum at the lesser curvature was the best location for detecting H. pylori histologically in children. More recently, of biopsies from 89 consecutive children undergoing upper gastrointestinal endoscopy for symptoms suggestive of acid peptic disease, 25% were H. pylori positive. All H. pylori cases had a positive RUT and positive histology ( H. pylori demonstrated with Giemsa staining) on biopsies from the cardia, but not the antrum. Adult studies have demonstrated the cardia to be second only to the antrum in yield of H. pylori on biopsy.
Recently, techniques such as fluorescence in situ hybridization (FISH) and PCR analysis have been used to detect H. pylori in biopsy specimens and even identify those with particular antibiotic resistance properties. However, these are neither routinely used nor available widely.
Noninvasive Tests
Although endoscopy with histopathology remains the standard of care to investigate consequences of H. pylori infection, noninvasive tests are well performing, validated posttreatment tests in children.
Urea Breath Test
The urea breath test (UBT) is still considered the most accurate noninvasive method for detecting H. pylori infection. Urea is labeled with the nonradioactive C isotope and then ingested. C is a naturally occurring nonradioactive isotope. The UBT can be used safely, even in very young infants, and can be repeated without risk to the child. Urea hydrolysis by H. pylori produces ammonia and labeled carbon dioxide. Urea passes rapidly down its concentration gradient into the epithelial blood supply, and within minutes, it appears in the breath ( Figure 27-6 ). Labeled urea (50 to 100 mg) is usually given with a test meal to delay gastric emptying. Breath samples are collected at variable times postingestion. For optimal results, the gastric environment should be acidic. Detection requires a mass spectrometer, and results are reported as delta over baseline (DOB) values for the measured ratio of CO 2 / 12 CO 2 . DOB values exceeding a fixed cut-off value are considered indicative of H. pylori infection.
Validation of this technique in childhood is ongoing. A multicenter trial in 2000 children demonstrated that the DOB cut-off value varied with changes in 13 C urea dose, type of test meal, and time of breath collection. Test meals that contain citric acid result in higher DOB values in H. pylori– positive patients and thus may improve the sensitivity of the test in children. A study by Kindermann et al. of 1499 German children confirmed UBT utility in children older than 6 years of age, with 149 cases being validated histologically. The positive predictive value (PPV) and NPV for children older than 6 years of age were 98% and 100%, but for children younger than the age of 6 years, they were 69% and 100%, respectively. Koletzko et al. have demonstrated a significant inverse relationship between DOB values and age in both infected and noninfected children. The analysis of Kindermann et al. demonstrates a false-positive rate of about 8%. These findings support the concept that the DOB cut-off value needs to be calculated by the receiver-operating characteristic (ROC) curve for each protocol in each patient population.
The accuracy of noninvasive tests remains challenging in young toddlers and infants. One difficulty posed by this patient group is the lack of sufficient numbers of study patients. Recent attempts to address this by modifying the analysis protocol suggest improvements in the false-positive rate in children younger than the age of 6 years. The optimal cut-off for a positive test in children younger than 5 years is higher than in adults. Increasing the cut-off from a DOB of 5 to 8 improved UBT specificity from 95.5% to 98.1%. Another study reported a sensitivity and specificity of the UBT of just 83% and 91%, respectively, in comparison to histology and rapid urease testing in 40 children. Such heterogeneous results suggest that UBT in children should be interpreted qualitatively and cautiously, and underscores the importance of consistency in UBT protocol and analysis.
Serologic Tests
H. pylori infection induces both cellular and humoral immune responses, resulting in an early increase in specific IgM level, and a later and persistent increase in specific IgA and IgG level. In general, serologic assays cannot be used on their own in children for diagnosis or monitoring of H. pylori infection because of widely variable sensitivity and specificity for detection of antibodies (IgG or IgA) against H. pylori in children. IgA-based tests detect only 20% to 50% of H. pylori– infected patients. Tests based on the detection of specific anti –H. pylori IgG antibodies in the serum offer a better sensitivity than IgA-based tests, but cannot distinguish active from past infection.
The results of serologic tests are influenced by the duration of infection and the ability of the host to mount an immune response. In some children, the duration and degree of infection may not have been present for a sufficient duration to generate an immune response. Given the low test sensitivities in infants and toddlers, epidemiologic studies based on antibody testing may underestimate the prevalence and transmission of H. pylori infection in the very young. Office-based serology tests, although technically simple to perform and attractively convenient, are not recommended for diagnosis. Their inaccuracy was such that 33% of positive tests in dyspeptic patients in a primary care setting were false positives.
The identification and validation of a panel of biomarkers of H. pylori infection in children including pepsinogen I and II and gastrin 17 is awaited. Data from adult studies, although promising, have failed to produce convincing results to date to advocate their routine use.
Results of salivary antibody tests have been disappointing. Urine antibody results have been variable. A pilot study of 132 adult patients demonstrated a sensitivity of 86% and a specificity of 91%. A follow-up European multicenter trial using the same assay had a sensitivity and specificity, respectively, of 89% and 69%. Validation of these alternative noninvasive methods in children is hampered by inconsistent use of an acceptable reference standard, and therefore they are not currently recommended for clinical use.
Stool Antigen Test
Stool testing for H. pylori antigen is an inexpensive, noninvasive method for determining H. pylori infection. When comparing the cost-effectiveness of this method against UBT, the stool test requires an optical spectrophotometer, usually present in any laboratory, has negligible maintenance costs, and does not require dedicated personnel.
Both polyclonal and monoclonal assays have been developed and tested in children. A large review of studies from 1999 to 2001, evaluating 3419 patients, suggested that the polyclonal test is not as reliable as the UBT. Kato et al. studied 264 children aged 2 to 17 years and demonstrated an overall sensitivity of 96%, specificity of 96.8%, PPV of 93.2%, and NPV of 98.4%. Results were independent of age.
In contrast, monoclonal antibody testing has shown greater promise. A multicenter study evaluated the test in 302 symptomatic children and compared it with UBT, RUT, histologic examination, and biopsy. The sensitivity, specificity, PPV, and NPV were 98%, 99%, 98%, and 99%, respectively. Similar findings were demonstrated previously in a pediatric group. A more recent meta-analysis of monoclonal stool antigen detection reported a pooled sensitivity of 94% and specificity of 97%. It is likely that in the near future, monoclonal stool antigen testing will be sanctioned as an alternative to the currently recommended UBT in the posttherapy setting.
Commercially available stool immune-chromatography kits are also available, but interobserver variability and equivocal results are problematic. Two studies found high sensitivities (94.6% and 100%) and specificities (98.4% and 100%) for this test, whereas another study reported far less accurate results (pre- and posttreatment: specificity 92.3% and 100%, respectively; sensitivity 65% and 60%, respectively).
Sophisticated molecular technologies have recently become more widely available and have been used to detect H. pylori in stool. Whereas bi-probe RT-PCR assays in adults have shown excellent results, testing in children to date has been somewhat disappointing. Reports by Falsafi et al. and Lottspeich et al. found a reasonable specificity but a poor sensitivity in children. An association between higher scores of H. pylori on histologic evaluation and more severe gastritis with positivity of stool on PCR was also observed and may explain the insufficient sensitivity in children who, for the most part, have less severe gastritis.
Summary
WHEN is TESTING INDICATED?
At present, the primary goal of testing is to diagnose the cause of clinical symptoms and not simply to detect the presence of H. pylori . As in many clinical scenarios, testing is not helpful unless it will alter the management of the disease. There remain certain scenarios in which screening for the presence of H. pylori is not absolutely indicated but may be considered by clinicians (see Box 27-1 ). As clinical and epidemiologic data on the manifestations of H. pylori improve over time, remaining ambiguity should diminish. Box 27-1 summarizes the current recommended indications for testing developed by the recent joint North American Society for Pediatric Gastroenterology, Hepatology and Nutrition/European Society for Paediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN/ESPGHAN) consensus conference.
Treatment
Children with peptic ulcer disease and H. pylori infection should receive treatment; the treatment endpoint should be the eradication of infection. However, as discussed previously, the majority of children infected with H. pylori do not have peptic ulcer disease. For many, the diagnosis of H. pylori infection is incidental and their management is controversial.
Few randomized placebo-controlled treatment trials are available for the different outcomes, often with only small numbers of cases included. These and other differences explain why some of the recommendations for adults may not apply in children. As a first-line therapy, the joint NASPGHAN/ESPGHAN guidelines proposed one of three different regimens: triple therapy with a PPI and amoxicillin and imidazole or clarithromycin; or with a bismuth salts, amoxicillin, and imidazole; or sequential therapy. Box 27-2 summarizes the existing H. pylori treatment guidelines recommended by the NASPGHAN.
Treatment Indications
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In the presence of H. pylori –positive peptic ulcer disease (PUD), eradication of the organism is recommended.
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When H. pylori infection is detected by biopsy-based methods in the absence of PUD, H. pylori treatment may be considered.
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A “test and treat” strategy is not recommended in children.
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Treatment may be offered in children who are infected with H. pylori and whose first-degree relative has gastric cancer.
Treatment Recommendations
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First-line eradication regimens are the following: triple therapy with a PPI + amoxicillin + clarithromycin, or an imidazole or bismuth salts + amoxicillin + an imidazole, or sequential therapy.
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Antibiotic susceptibility testing for clarithromycin is recommended before initial clarithromycin-based triple therapy in areas/populations with a known high resistance rate (>20%) of H. pylori to clarithromycin (see Figure 27-1 ).
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It is recommended that the duration of triple therapy be 7 to 14 days. Costs, compliance, and adverse effects should be taken into account.
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A reliable noninvasive test for eradication is recommended at least 4 to 8 weeks following completion of therapy.
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If treatment has failed, three options are recommended: esophagogastroduodenoscopy (EGD), with culture and susceptibility testing including alternative antibiotics, if not performed before guide therapy; fluorescence in situ hybridization (FISH) on previous paraffin-embedded biopsies if clarithromycin susceptibility testing has not been performed before guide therapy; modification of therapy by adding an antibiotic, using different antibiotics, adding bismuth, and/or increasing the dose and/or duration of therapy.
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