*Helicobacter pylori* **Infection, Gastric Physiology and Micronutrient deficiency (Iron and Vitamin C) in Children in Developing Countries**

Shafiqul Alam Sarker

[105] Parsonnet J*. Helicobacter pylori*. *Infect Dis Clin North Am*. Mar 1998;12(1):185-97.

lymphoma. *N Engl J Med*. May 5 1994;330(18):1267-71.

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204 Trends in Helicobacter pylori Infection

*biol*. Jan 27 2004;4:5.

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[106] Parsonnet J, Hansen S, Rodriguez L, et al. *Helicobacter pylori* infection and gastric

[107] Raguza D, Machado RS, Ogata SK, et al. Validation of a monoclonal stool antigen test for diagnosing *Helicobacter pylori* infection in young children. *J Pediatr Gastroenterol*

[108] Siberry GK, Iannone R, eds. Formulary: drug doses. In: *The Harriet Lane Handbook*. 15th ed. St Louis, MO: Mosby; 2000:622, 630, 645-6, 674-5, 772-3, 795, 837.

[109] Vinette KM, Gibney KM, Proujansky R, Fawcett PT. Comparison of PCR and clinical laboratory tests for diagnosing *H. pylori* infection in pediatric patients. *BMC Micro‐*

[110] Wewer V, Andersen LP, Paerregaard A, et al. Treatment of *Helicobacter pylori* in chil‐

[111] Windle HJ, Kelleher D, Crabtree JE. Childhood *Helicobacter pylori* infection and growth impairment in developing countries: a vicious cycle?. *Pediatrics*. Mar

dren with recurrent abdominal pain. *Helicobacter*. Sep 2001;6(3):244-8.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58375

### **1. Introduction**

At least half the worldʹs population are infected by *Helicobacter pylori (H. pylori),* making it the most widespread infection in the world [1]. Although infection occurs worldwide, there are significant differences in the prevalence of infection both within and between countries [2-4]. The overall prevalence of *H. pylori* infection in developed countries is lower than that in developing countries [3, 5]. The increased infection rate in developing countries is likely due to poor sanitary and/or living conditions. In such communities the incidence of *H pylori* infection in infancy is also high [6, 7], and has also been associated with malnutrition and growth faltering [8]. Epidemiological data suggest the prevalence of *H. pylori* infection in children under 10 years resident in developed countries to be 0 to 5% compared to 13 to 60% in their developing country counterparts [9]. The age at which this bacterium is acquired seems to influence the possible pathologic outcome of the infection - people infected at an early age are likely to develop more intense inflammation that may be followed by atrophic gastritis with a higher risk of gastric ulcer, gastric cancer or both. Acquisition at an older age brings different gastric changes that are more likely to result in duodenal ulcer. Individuals infected with *H. pylori* have a 10 to 20% lifetime risk of developing peptic ulcers and a 1 to 2% risk of acquiring stomach cancer [10]

*H. pylori* infection also exerts diverse effects of gastric physiology - it may increase or reduce gastric acid secretion or result in no overall change in the acid output [11]. It is important to know why *H. pylori* infection produces different aberrations in gastric physiology, and consequently gastric or duodenal ulcer or gastric cancer. *H. pylori* infection has also been suggested to be associated with a variety of conditions outside of the alimentary tract. The list

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of the proposed 'extragastric' association continues to grow despite the fact that *H. pylori* is a non-invasive organism and as such the infections are essentially confined to the surface of gastric-type mucosa. In an initial overview of the non-gastrointestinal manifestations of *H. pylori* [12], some biological plausibility has been suggested that underlie its association with iron deficiency anemia (IDA); cross sectional studies have demonstrated a relatively strong association. [13]

**2.1.** *H. pylori* **infection and gastric acid perturbation**

Acid secretion by the gastric parietal cell is regulated by paracrine, endocrine, and neural pathways. The physiological stimuli for acid secretion include histamine, acetylcholine, and gastrin, each of which binds to receptors located on the basolateral plasma membranes of the cells. The antral region of stomach contains G cells which release hormone gastrin. When meal is ingested, the protein component stimulates G cell to release Gastrin, which travels through the bloodstream to parietal cells in the body region (fundus) to secret acid [25]. Gastrin directly does not stimulate parietal cells but stimulates the adjacent enterochromafin-like cell (ECL cells) to release histamine, which in turn stimulates the parietal cells. Stimulation of acid secretion typically involves an activation of a cAMP-dependent protein kinase cascade that triggers the translocation and insertion of the proton pump enzyme, H,K-ATPase, into the apical plasma membrane parietal cells [26]. As the acid accumulates and overcome the buffering effects of the food, the fall in intragastric pH inhibits further release of gastrin and

*Helicobacter pylori* Infection, Gastric Physiology and Micronutrient deficiency (Iron and Vitamin C) in…

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207

thus prevents secretion of excessive amount of acids (negative feedback control).

when gastritis is mixed antral or body, *H. pylori* may have no effect on acid secretion.

*H. pylori* infection in the antral region of the stomach disrupts the negative feedback control of gastrin release, resulting inappropriately high and sustained levels of gastrin following meal [31, 32]. In those subjects, the gastritis is non-atrophic and, therefore, the increased gastrin release stimulates the healthy body region of stomach to secret excessive amounts of acid [33, 34]. The increased amount of acid output produced by this pattern of gastritis results in an increased duodenal acid loads damaging the duodenal mucosa, which may eventually result in ulcers formation [35]. Eradicating *H. pylori* infection in subjects with this type of gastritis

In subjects with atrophic gastritis or body predominant gastritis, there also is increased gastrin release, but that is not accompanied with increased acid secretion. In such subjects, acid secretion is reduced or completely absent (achlorhydria) [36, 37]. The low acid secretion,

**2.2.** *H. pylori* **infection and increased acid output (hyperchlorhydria)**

leads to lowering of serum gastrin with concomitant reductions in acid output.

**2.3.** *H. pylori* **infection and low acid output (hypochlorhydria)**

*Helicobacter pylori* infection exerts diverse effects on gastric physiology. In acute *H pylori* infection transient hypochlorhydria in adults is well documented [27, 28]. However, the relationship between chronic *H. pylori* infection and gastric acid secretion is not fully under‐ stood. It may increase gastric acid secretion, reduce it or results no overall changes in acid output. [11]. These alterations in acid secretion depend on the degree and distribution of gastritis caused by the infection [29, 30]. In subjects with an antral predominant gastritis, without atrophy, acid secretion is normal or increased. This is the pattern of gastritis seen in patients who develops duodenal ulceration. When gastritis is body predominant, a situation leading to gastric atrophy, *H. pylori* infection lead to markedly reduced acid secretion or achlorhydria which is also seen in patients who develops non-cardiac gastric cancer. Finally,

In this chapter, we will review and update the consequence of *H. pylori* infection, its role on the gastric acid secretion and some other conditions, notably IDA and iron absorption in children. The relationship between *H. pylori* infection and vitamin C levels in the blood and gastric juice will also be reviewed.

### **2. Consequences of initial** *H. pylori* **infection in children**

There are not enough studies on the natural history of gastric infections in childhood years. In older children and adolescents, and adults it appears that *H. pylori* infection and the accom‐ panying gastritis are lifelong unless specific eradication therapies are employed. However, several epidemiological studies, using serology [14, [15] and breath tests ( [16, 17] as indirect markers of gastric infection reported spontaneous clearance in the pre-school aged children. Current evidence suggests that the overwhelming majority of *H. pylori* infected children and adolescents develop a chronic-active, antral- predominant gastritis) [18]. There is a single report suggesting the potential for *H. pylori* colonization of stomach of children without mucosal inflammation in antrum or gastric fundus [19]. Although, infection with the Cag Apositive strain was associated with more pronounced changes in the gastric physiology, limited studies in children reported no association between Cag A status and the severity of gastritis [20].

Some studies reported pan-gastritis involving both body and antrum in children infected with *H. plyori* [21, 22]. However, a formal mapping study to delineate the extent and the severity of bacterial colonization of the stomach, as well as the accompanying host cells mucosal inflam‐ matory response to infection is lacking. To date there are not enough studies in children evaluating the healing of mucosal inflammation following eradication therapy. There are indirect evidences to suggest that resolution of inflammatory response may occur more rapidly in children than had been reported for adults [23]. In France, Ganga-Zandzou and colleagues prospectively monitored the consequences of untreated *H. pylori* infection in a group of asymptomatic children [24]. Although the density of bacterial colonization was not changed, there was both marked antral nodularity and more severe mucosal inflammation in the antrum over the 2-year follow-up of the children. However, there is the lack for comparable studies in other pediatric population, e.g. among those residing in developing countries and a greater length of follow-up. Further studies are needed to delineate the inflammatory and immune responses during development in order to provide additional insights into the interactions between the *H. pylori* and the host in such populations.

### **2.1.** *H. pylori* **infection and gastric acid perturbation**

of the proposed 'extragastric' association continues to grow despite the fact that *H. pylori* is a non-invasive organism and as such the infections are essentially confined to the surface of gastric-type mucosa. In an initial overview of the non-gastrointestinal manifestations of *H. pylori* [12], some biological plausibility has been suggested that underlie its association with iron deficiency anemia (IDA); cross sectional studies have demonstrated a relatively strong

In this chapter, we will review and update the consequence of *H. pylori* infection, its role on the gastric acid secretion and some other conditions, notably IDA and iron absorption in children. The relationship between *H. pylori* infection and vitamin C levels in the blood and

There are not enough studies on the natural history of gastric infections in childhood years. In older children and adolescents, and adults it appears that *H. pylori* infection and the accom‐ panying gastritis are lifelong unless specific eradication therapies are employed. However, several epidemiological studies, using serology [14, [15] and breath tests ( [16, 17] as indirect markers of gastric infection reported spontaneous clearance in the pre-school aged children. Current evidence suggests that the overwhelming majority of *H. pylori* infected children and adolescents develop a chronic-active, antral- predominant gastritis) [18]. There is a single report suggesting the potential for *H. pylori* colonization of stomach of children without mucosal inflammation in antrum or gastric fundus [19]. Although, infection with the Cag Apositive strain was associated with more pronounced changes in the gastric physiology, limited studies in children reported no association between Cag A status and the severity of

Some studies reported pan-gastritis involving both body and antrum in children infected with *H. plyori* [21, 22]. However, a formal mapping study to delineate the extent and the severity of bacterial colonization of the stomach, as well as the accompanying host cells mucosal inflam‐ matory response to infection is lacking. To date there are not enough studies in children evaluating the healing of mucosal inflammation following eradication therapy. There are indirect evidences to suggest that resolution of inflammatory response may occur more rapidly in children than had been reported for adults [23]. In France, Ganga-Zandzou and colleagues prospectively monitored the consequences of untreated *H. pylori* infection in a group of asymptomatic children [24]. Although the density of bacterial colonization was not changed, there was both marked antral nodularity and more severe mucosal inflammation in the antrum over the 2-year follow-up of the children. However, there is the lack for comparable studies in other pediatric population, e.g. among those residing in developing countries and a greater length of follow-up. Further studies are needed to delineate the inflammatory and immune responses during development in order to provide additional insights into the interactions

**2. Consequences of initial** *H. pylori* **infection in children**

between the *H. pylori* and the host in such populations.

association. [13]

206 Trends in Helicobacter pylori Infection

gastritis [20].

gastric juice will also be reviewed.

Acid secretion by the gastric parietal cell is regulated by paracrine, endocrine, and neural pathways. The physiological stimuli for acid secretion include histamine, acetylcholine, and gastrin, each of which binds to receptors located on the basolateral plasma membranes of the cells. The antral region of stomach contains G cells which release hormone gastrin. When meal is ingested, the protein component stimulates G cell to release Gastrin, which travels through the bloodstream to parietal cells in the body region (fundus) to secret acid [25]. Gastrin directly does not stimulate parietal cells but stimulates the adjacent enterochromafin-like cell (ECL cells) to release histamine, which in turn stimulates the parietal cells. Stimulation of acid secretion typically involves an activation of a cAMP-dependent protein kinase cascade that triggers the translocation and insertion of the proton pump enzyme, H,K-ATPase, into the apical plasma membrane parietal cells [26]. As the acid accumulates and overcome the buffering effects of the food, the fall in intragastric pH inhibits further release of gastrin and thus prevents secretion of excessive amount of acids (negative feedback control).

*Helicobacter pylori* infection exerts diverse effects on gastric physiology. In acute *H pylori* infection transient hypochlorhydria in adults is well documented [27, 28]. However, the relationship between chronic *H. pylori* infection and gastric acid secretion is not fully under‐ stood. It may increase gastric acid secretion, reduce it or results no overall changes in acid output. [11]. These alterations in acid secretion depend on the degree and distribution of gastritis caused by the infection [29, 30]. In subjects with an antral predominant gastritis, without atrophy, acid secretion is normal or increased. This is the pattern of gastritis seen in patients who develops duodenal ulceration. When gastritis is body predominant, a situation leading to gastric atrophy, *H. pylori* infection lead to markedly reduced acid secretion or achlorhydria which is also seen in patients who develops non-cardiac gastric cancer. Finally, when gastritis is mixed antral or body, *H. pylori* may have no effect on acid secretion.

### **2.2.** *H. pylori* **infection and increased acid output (hyperchlorhydria)**

*H. pylori* infection in the antral region of the stomach disrupts the negative feedback control of gastrin release, resulting inappropriately high and sustained levels of gastrin following meal [31, 32]. In those subjects, the gastritis is non-atrophic and, therefore, the increased gastrin release stimulates the healthy body region of stomach to secret excessive amounts of acid [33, 34]. The increased amount of acid output produced by this pattern of gastritis results in an increased duodenal acid loads damaging the duodenal mucosa, which may eventually result in ulcers formation [35]. Eradicating *H. pylori* infection in subjects with this type of gastritis leads to lowering of serum gastrin with concomitant reductions in acid output.

#### **2.3.** *H. pylori* **infection and low acid output (hypochlorhydria)**

In subjects with atrophic gastritis or body predominant gastritis, there also is increased gastrin release, but that is not accompanied with increased acid secretion. In such subjects, acid secretion is reduced or completely absent (achlorhydria) [36, 37]. The low acid secretion, despite increased gastrin levels, indicates markedly impaired ability of oxyntic mucosa to secret acid in response to gastrin. Following eradication of *H. pylori* infection in patients with this pattern of gastritis, there is recovery in acid secretion [36, 37]. However, the degree of recovery in acid outputs is variable – acid output resumes to normal level in some patients while very small increase occur in others [36]. The recovery in acid outputs following eradi‐ cation of infection coincides with the disappearance of organism as well as resolution of inflammation of the body mucosa. However, there is little evidence of resolution of the atrophy of body mucosa. Capurso *et al. (*23) observed that both pangastritis and pangastritis-induced hypochlorhydria were more prevalent in adult patients with *H. pylori* who had anemia than in those who did not have anemia.

**Non-infected**

*H. pylori* therapy

factor.

**(n=30) <sup>p</sup>**

**4. Potential mechanisms of** *H. pylori***-induced hypochlorhydria**

Basal Acid Output (BAO) 0.23 ± 0.30 0.06 0.62 ±0.9 NS 0.65 ± 0.65 Stimulated Acid Output 2.04 ±1.4 0.001 3.4 ±2.5 NS 3.3 ±2.1

**Table 1.** Comparison of acid outputs (mMol/h between infected and non-infected children along with effect of anti-

*H pylori* induced hypochlorhydria might be due to the bacterium releasing some substances that can directly inhibit acid secretion. Several candidate substances have been identified, which inhibits parietal cell function *in vitro*, but the evidence for involvement of these sub‐ stances for the *in-vivo* effects remains weak. *H. pylori* infection also produces ammonia, which may uncouple the proton pump [42]. But the ammonia produced by *H. pylori* infection in hypochlorhdric subjects is relatively small [43]. Another problem in attributing the impairment of oxyntic mucosal function to the presence of *H. pylori* organisms is that density of colonization of the gastric mucosa with the organism is similar or lower in subjects with hypocholorhydria than in subjects with normal or high acid secretion [36]. Therefore, current knowledge precludes attributing impaired function of oxyntic mucosa as a direct effect of some bacterial

An alternative explanation for the impaired acid secretory function is infection-induced inflammation of the oxyntic mucosa, since the severity of inflammation of the body mucosa is more marked in subjects with *H. pylori* associated hypochlorhydria than in subjects with *H. pylori* infection with normal or increased acid secretion [44]. This raises the possibility that a

The molecular mechanisms underlying *H. pylori*-induced hypochlorhydria is not completely understood. However, it has been shown that *H. pylori*–induced pro-inflammatory factors, such as interleukin-1β, may contribute to hypochlorhydria [26, 45].The increased production of this cytokines may be important because it is very potent inhibitor of acid secretion [46] and and may play a role in chronic *H. pylori-*induced hypochlorhydria. Polymorphisms in IL-1β gene cluster may control the extent and the duration of hypochlorhydria with initial *H. pylori infection* [47], which has been noted to be linked to increased risk for atrophy and consequently gastric cancer [48]. It is possible that inflammatory factors, such as IL-1β cause an inhibition of acid secretion from parietal cells via paracrine pathways. Using freshly isolated rabbit gastric glands and culture parietal cells, Fang and colleague observed that Vac-A toxin treatments inhibits gastric acid secretion by preventing the recruitment of gastric H, K-adenosine triphosphatase (H, K-ATPase), the parietal cell enzyme mediating acid secretion. [49]. This was the first evidence that *H. pylori* Vac-A toxin impairs gastric parietal cell physiology by disrupting the apical membrane cycloskeletal linkers of the gastric parietal cells. Studies in animal models as well as epidemiologic studies of *H. pylori* isolates from humans have

product of inflammatory response following infection might inhibit acid secretion.

**Infected before treatment (n=30) <sup>p</sup>**

*Helicobacter pylori* Infection, Gastric Physiology and Micronutrient deficiency (Iron and Vitamin C) in…

**Infected after treatment (n=28)** 209

http://dx.doi.org/10.5772/58375

### **3. Role of** *H. pylori* **infection in gastric acid perturbation in children**

There are only a few pediatric case reports on gastric acid secretion in *H. pylori* infection. Several studies in the pre-*H. pylori* era [38, 39] and very recently [40] studies observed the maximal acid output higher in children with duodenal ulcer than in the children without peptic-ulcer disease. However, no study has examined the relationship between gastric acid secretion and *H. pylori* infection in asymptomatic young children living in developing countries. The effect of *H. pylori* on gastric acid production can be examined by studying individuals with and without infection or, more directly by examining before and after eradication of *H. pylori* [41] In an attempt to see if *H. pylori* infection is associated with gastric acid perturbation in Bangladeshi children, basal gastric acid output (GAO-B) and stimulated gastric acid output (GAO-S) just before and after pentagastrin stimulation in age matched *H. pylori*-infected and non-infected children were measured. Experiments were repeated in infected children 8 weeks after completing a 2-week course of anti-*H. pylori* therapy to evaluate the influence of *H. pylori* on gastric acid secretion. Comparison of acid output between infected and non-infected children both before and after eradication therapy is shown in Table 1. Both the basal acid output (GAO-B) and the stimulated gastric acid outputs (GAO-S) were significantly lower in *H. pylori* infected children compared to *H. pylori* negative group. The mean GAO-B and GAO-S of the infected children were estimated to be 30% and 50% respectively of that of non-infected children. Successful eradication therapy was associated with a significant rise of both the basal and the stimulated acid output values reaching equivalence to those in the *H. pylori*-negative children. Improvement of GAO following anti- *H. pylori* therapy suggests a causal link of *H. pylori* infection and depressed GAO in this population. Whether the observed reversibility of acid secretion to normal level within a relatively short term period of eradication therapy was associated with recovery from corpus gastritis is not known as gastric biopsy for histological examination was not performed. It is also important to know the acid secretory status after a long term period of eradication in settings with possibilities of having re-infection by the organism as a consequence of poor hygiene and environmental contamination.


**Table 1.** Comparison of acid outputs (mMol/h between infected and non-infected children along with effect of anti-*H. pylori* therapy

### **4. Potential mechanisms of** *H. pylori***-induced hypochlorhydria**

despite increased gastrin levels, indicates markedly impaired ability of oxyntic mucosa to secret acid in response to gastrin. Following eradication of *H. pylori* infection in patients with this pattern of gastritis, there is recovery in acid secretion [36, 37]. However, the degree of recovery in acid outputs is variable – acid output resumes to normal level in some patients while very small increase occur in others [36]. The recovery in acid outputs following eradi‐ cation of infection coincides with the disappearance of organism as well as resolution of inflammation of the body mucosa. However, there is little evidence of resolution of the atrophy of body mucosa. Capurso *et al. (*23) observed that both pangastritis and pangastritis-induced hypochlorhydria were more prevalent in adult patients with *H. pylori* who had anemia than

**3. Role of** *H. pylori* **infection in gastric acid perturbation in children**

organism as a consequence of poor hygiene and environmental contamination.

There are only a few pediatric case reports on gastric acid secretion in *H. pylori* infection. Several studies in the pre-*H. pylori* era [38, 39] and very recently [40] studies observed the maximal acid output higher in children with duodenal ulcer than in the children without peptic-ulcer disease. However, no study has examined the relationship between gastric acid secretion and *H. pylori* infection in asymptomatic young children living in developing countries. The effect of *H. pylori* on gastric acid production can be examined by studying individuals with and without infection or, more directly by examining before and after eradication of *H. pylori* [41] In an attempt to see if *H. pylori* infection is associated with gastric acid perturbation in Bangladeshi children, basal gastric acid output (GAO-B) and stimulated gastric acid output (GAO-S) just before and after pentagastrin stimulation in age matched *H. pylori*-infected and non-infected children were measured. Experiments were repeated in infected children 8 weeks after completing a 2-week course of anti-*H. pylori* therapy to evaluate the influence of *H. pylori* on gastric acid secretion. Comparison of acid output between infected and non-infected children both before and after eradication therapy is shown in Table 1. Both the basal acid output (GAO-B) and the stimulated gastric acid outputs (GAO-S) were significantly lower in *H. pylori* infected children compared to *H. pylori* negative group. The mean GAO-B and GAO-S of the infected children were estimated to be 30% and 50% respectively of that of non-infected children. Successful eradication therapy was associated with a significant rise of both the basal and the stimulated acid output values reaching equivalence to those in the *H. pylori*-negative children. Improvement of GAO following anti- *H. pylori* therapy suggests a causal link of *H. pylori* infection and depressed GAO in this population. Whether the observed reversibility of acid secretion to normal level within a relatively short term period of eradication therapy was associated with recovery from corpus gastritis is not known as gastric biopsy for histological examination was not performed. It is also important to know the acid secretory status after a long term period of eradication in settings with possibilities of having re-infection by the

in those who did not have anemia.

208 Trends in Helicobacter pylori Infection

*H pylori* induced hypochlorhydria might be due to the bacterium releasing some substances that can directly inhibit acid secretion. Several candidate substances have been identified, which inhibits parietal cell function *in vitro*, but the evidence for involvement of these sub‐ stances for the *in-vivo* effects remains weak. *H. pylori* infection also produces ammonia, which may uncouple the proton pump [42]. But the ammonia produced by *H. pylori* infection in hypochlorhdric subjects is relatively small [43]. Another problem in attributing the impairment of oxyntic mucosal function to the presence of *H. pylori* organisms is that density of colonization of the gastric mucosa with the organism is similar or lower in subjects with hypocholorhydria than in subjects with normal or high acid secretion [36]. Therefore, current knowledge precludes attributing impaired function of oxyntic mucosa as a direct effect of some bacterial factor.

An alternative explanation for the impaired acid secretory function is infection-induced inflammation of the oxyntic mucosa, since the severity of inflammation of the body mucosa is more marked in subjects with *H. pylori* associated hypochlorhydria than in subjects with *H. pylori* infection with normal or increased acid secretion [44]. This raises the possibility that a product of inflammatory response following infection might inhibit acid secretion.

The molecular mechanisms underlying *H. pylori*-induced hypochlorhydria is not completely understood. However, it has been shown that *H. pylori*–induced pro-inflammatory factors, such as interleukin-1β, may contribute to hypochlorhydria [26, 45].The increased production of this cytokines may be important because it is very potent inhibitor of acid secretion [46] and and may play a role in chronic *H. pylori-*induced hypochlorhydria. Polymorphisms in IL-1β gene cluster may control the extent and the duration of hypochlorhydria with initial *H. pylori infection* [47], which has been noted to be linked to increased risk for atrophy and consequently gastric cancer [48]. It is possible that inflammatory factors, such as IL-1β cause an inhibition of acid secretion from parietal cells via paracrine pathways. Using freshly isolated rabbit gastric glands and culture parietal cells, Fang and colleague observed that Vac-A toxin treatments inhibits gastric acid secretion by preventing the recruitment of gastric H, K-adenosine triphosphatase (H, K-ATPase), the parietal cell enzyme mediating acid secretion. [49]. This was the first evidence that *H. pylori* Vac-A toxin impairs gastric parietal cell physiology by disrupting the apical membrane cycloskeletal linkers of the gastric parietal cells. Studies in animal models as well as epidemiologic studies of *H. pylori* isolates from humans have suggested that VacA toxin enhances the ability of *H. pylori* to colonize in the stomach and contributes to the development of symptomatic diseases (15).

individuals with unexplained IDA [68]. However, methodological limitations, including small sample sizes and lacks of control groups, among others, do not allow conclusive interpretation of the results. No previous study has examined whether or not active *H. pylori* infection is causally linked to IDA in young children living in less developed countries. Although the mechanisms for *H. pylori*-associated IDA is not fully understood, *H. pylori*-induced chronic pan gastritis with resultant achlor-or hypo-chlorhydria [41, 69] and reduced ascorbic *acid* secretion in the *gastric* mucosa [70, 71] may lead to reduced iron absorption since they are essential for alimentary iron absorption; they not only convert ferric iron to the ferrous form, which maintains solubility at the alkaline pH of the duodenum, but also chelates with ferric chloride that is also stable at a pH >3. Uptake of iron by *H. pylori* is also suggested [72]. Whether *H.*

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In Bangladesh, a randomized controlled community based study was conducted to deter‐ mine whether or not *H. pylori* is a cause of IDA or a reason for treatment failure of iron supple‐ mentation in children with IDA [73]. The population consisted of 260 children, 2-5 years (200 *H. pylori* infected,detectedbypositiveurea breath test[UBT], and60uninfected) with IDA.IDA was defined as a combination of low hemoglobin (Hb <110 g/L) and a low serum ferritin (SF <12 μg/L) plus elevated serum transferrin receptor (sTfR >8.3 mg/L) [74]. ID was defined as Hb <110 g/LandSF <12μg/L, or sTfR>8.3mg/L.They were randomly assignedto one of 4 regimens: (i)2-week course of anti-*H. pylori* (anti- *H. pylori*) triple therapy (amoxicillin 15mg/kg.dose and clarithromycin 7.5mg/kg.dose, both administered twice daily; and a single 20 mg dose of omeprazole per day) plus a 90-day course of oral ferrous sulfate in elixir (3 mg/kg elemental iron daily) (anti- *H. pylori* therapy plus iron);(ii) a 2-week's anti- *H. pylori* therapy plus place‐ bo for iron for 90 days (anti- *H. pylori* alone);(iii) 2-week's course of placebo for anti- *H. pylori* therapy but ferrous sulfate (3 mg/kg for 90 days) (Fe alone); and (iv) 2-week's placebos for anti-*H. pylori* therapy and a 90-day course of placebo for iron (positive control). For precisely determining the role of *H. pylori* infection in the treatment failure of iron supplementation, the study included a fifth group of children with IDA but without *H. pylori* infection (negative control), who were treated with open iron therapy alone for 90 days. Iron status was reas‐ sessed after 90 days in all children; those with continued IDA/ID were given a 60-day course of

The results of the study indicated that iron status, as reevaluated on day-90, improved in all groups. However, the improvement was significantly higher among 3 groups receiving iron (anti- *H. pylori* plus iron, iron alone or negative control receiving iron). A greater proportion of infected children receiving iron experienced correction of IDA than those receiving placebo or anti- *H. pylori* alone (68% for anti- *H. pylori* plus iron, 76%)for iron alone, 25% for placebo and 36% for anti- *H. pylori* alone, F=49, p <0.0001 (Figure 1). The results suggest no role of anti-*H. pylori* in IDA. Regarding ID, iron therapy had the most pronounced effect - correction occurred in 100% of children receiving iron compared to only 50% of children receiving anti-*H. pylori* alone or placebo. It is important to note that compared to placebo or iron therapy, anti- *H. pylori* therapy did not improve iron status or decrease IDA and ID prevalence. Therefore, the study concluded that *H. pylori* is neither causally linked with IDA nor is a reason for treatment failure of iron supplementation in children. The findings were in contrast with

*pylori* is a cause or associated with ID or IDA is not fully elucidated.

ferrous sulfate.

### **4.1. Potential consequences of** *H. pylori* **induced hypochlorhydria**

*H. pylori*, by producing hypochlorhydria or impaired gastric barrier may contribute to childhood malnutrition in developing countries through malabsorption or increased suscept‐ ibility to enteric infections [50]. Several observations demonstrated a correlation between *H. pylori* and malabsorption of essential nutrients; epidemiological studies have shown an association between *H. pylori* infection and iron deficiency anemia, while the absorption of some vitamins such as vitamin B12, vitamin A, vitamin C, folic *acid* and Vitamin E may also be affected by the infection [51]. The main mechanism related to malabsorption of these components is the modified intragastric pH (hypo or achlorhydria) due to *H. pylori* infection. On the other hand, *H. pylori* eradication has been shown to improve serum level of iron and vitamin B12, and some effects on Vitamin A and Vitamin E absorption as well as late effects on ghrelin levels [51].

### **5.** *H. pylori* **infection and iron deficiency anemia**

#### **5.1. Iron deficiency and iron deficiency anemia**

Iron deficiency (ID) and iron deficiency anemia (IDA) are major public health problems, especially in children and women of childbearing age in developing countries [52], and is considered one of the ten leading global risks factors in terms of its attributable disease burden [53]. It has been estimated that globally approximately 1.6 billion people, representing 25% of the total population are anemic [54]. ID is considered to account for 50% of identified anemia, and 800,000 deaths worldwide can be attributed to IDA. Deficiency of this trace element has adverse implications on health at all stages of life. When iron deficiency occurs during critical windows of brain development, the resultant cognitive deficits may be irreversible and unresponsive to subsequent improvements in the iron status [55]. In adults, ID and IDA can adversely impact physical work capacity and work productivity - variables that may have a detrimental impact on their economic potential [56].

#### **5.2.** *H. pylori* **and iron deficiency anemia**

Several reports have demonstrated an association between *H. pylori* infection and anemia, ID, and IDA, although the mechanisms of the interactions have not been well defined [13, 57-60]. A few case reports indicate that successful eradication of *H. pylori* results in improving iron status and anemia [61-63]. Other studies implicate *H. pylori* as a cause of IDA, refractory to oral iron treatment (refractory iron deficiency or sideropenic anemia); similarly eradication of *H. pylori* has resulted in improved iron status in children [58, 64-67]. Overall, these findings suggest that a substantial proportion of global ID and IDA might be attributed to *H. pylori* infection, leading to a recommendation by some for *H. pylori* eradication therapy in infected

individuals with unexplained IDA [68]. However, methodological limitations, including small sample sizes and lacks of control groups, among others, do not allow conclusive interpretation of the results. No previous study has examined whether or not active *H. pylori* infection is causally linked to IDA in young children living in less developed countries. Although the mechanisms for *H. pylori*-associated IDA is not fully understood, *H. pylori*-induced chronic pan gastritis with resultant achlor-or hypo-chlorhydria [41, 69] and reduced ascorbic *acid* secretion in the *gastric* mucosa [70, 71] may lead to reduced iron absorption since they are essential for alimentary iron absorption; they not only convert ferric iron to the ferrous form, which maintains solubility at the alkaline pH of the duodenum, but also chelates with ferric chloride that is also stable at a pH >3. Uptake of iron by *H. pylori* is also suggested [72]. Whether *H. pylori* is a cause or associated with ID or IDA is not fully elucidated.

suggested that VacA toxin enhances the ability of *H. pylori* to colonize in the stomach and

*H. pylori*, by producing hypochlorhydria or impaired gastric barrier may contribute to childhood malnutrition in developing countries through malabsorption or increased suscept‐ ibility to enteric infections [50]. Several observations demonstrated a correlation between *H. pylori* and malabsorption of essential nutrients; epidemiological studies have shown an association between *H. pylori* infection and iron deficiency anemia, while the absorption of some vitamins such as vitamin B12, vitamin A, vitamin C, folic *acid* and Vitamin E may also be affected by the infection [51]. The main mechanism related to malabsorption of these components is the modified intragastric pH (hypo or achlorhydria) due to *H. pylori* infection. On the other hand, *H. pylori* eradication has been shown to improve serum level of iron and vitamin B12, and some effects on Vitamin A and Vitamin E absorption as well as late effects

Iron deficiency (ID) and iron deficiency anemia (IDA) are major public health problems, especially in children and women of childbearing age in developing countries [52], and is considered one of the ten leading global risks factors in terms of its attributable disease burden [53]. It has been estimated that globally approximately 1.6 billion people, representing 25% of the total population are anemic [54]. ID is considered to account for 50% of identified anemia, and 800,000 deaths worldwide can be attributed to IDA. Deficiency of this trace element has adverse implications on health at all stages of life. When iron deficiency occurs during critical windows of brain development, the resultant cognitive deficits may be irreversible and unresponsive to subsequent improvements in the iron status [55]. In adults, ID and IDA can adversely impact physical work capacity and work productivity - variables that may have a

Several reports have demonstrated an association between *H. pylori* infection and anemia, ID, and IDA, although the mechanisms of the interactions have not been well defined [13, 57-60]. A few case reports indicate that successful eradication of *H. pylori* results in improving iron status and anemia [61-63]. Other studies implicate *H. pylori* as a cause of IDA, refractory to oral iron treatment (refractory iron deficiency or sideropenic anemia); similarly eradication of *H. pylori* has resulted in improved iron status in children [58, 64-67]. Overall, these findings suggest that a substantial proportion of global ID and IDA might be attributed to *H. pylori* infection, leading to a recommendation by some for *H. pylori* eradication therapy in infected

contributes to the development of symptomatic diseases (15).

**5.** *H. pylori* **infection and iron deficiency anemia**

**5.1. Iron deficiency and iron deficiency anemia**

detrimental impact on their economic potential [56].

**5.2.** *H. pylori* **and iron deficiency anemia**

on ghrelin levels [51].

210 Trends in Helicobacter pylori Infection

**4.1. Potential consequences of** *H. pylori* **induced hypochlorhydria**

In Bangladesh, a randomized controlled community based study was conducted to deter‐ mine whether or not *H. pylori* is a cause of IDA or a reason for treatment failure of iron supple‐ mentation in children with IDA [73]. The population consisted of 260 children, 2-5 years (200 *H. pylori* infected,detectedbypositiveurea breath test[UBT], and60uninfected) with IDA.IDA was defined as a combination of low hemoglobin (Hb <110 g/L) and a low serum ferritin (SF <12 μg/L) plus elevated serum transferrin receptor (sTfR >8.3 mg/L) [74]. ID was defined as Hb <110 g/LandSF <12μg/L, or sTfR>8.3mg/L.They were randomly assignedto one of 4 regimens: (i)2-week course of anti-*H. pylori* (anti- *H. pylori*) triple therapy (amoxicillin 15mg/kg.dose and clarithromycin 7.5mg/kg.dose, both administered twice daily; and a single 20 mg dose of omeprazole per day) plus a 90-day course of oral ferrous sulfate in elixir (3 mg/kg elemental iron daily) (anti- *H. pylori* therapy plus iron);(ii) a 2-week's anti- *H. pylori* therapy plus place‐ bo for iron for 90 days (anti- *H. pylori* alone);(iii) 2-week's course of placebo for anti- *H. pylori* therapy but ferrous sulfate (3 mg/kg for 90 days) (Fe alone); and (iv) 2-week's placebos for anti-*H. pylori* therapy and a 90-day course of placebo for iron (positive control). For precisely determining the role of *H. pylori* infection in the treatment failure of iron supplementation, the study included a fifth group of children with IDA but without *H. pylori* infection (negative control), who were treated with open iron therapy alone for 90 days. Iron status was reas‐ sessed after 90 days in all children; those with continued IDA/ID were given a 60-day course of ferrous sulfate.

The results of the study indicated that iron status, as reevaluated on day-90, improved in all groups. However, the improvement was significantly higher among 3 groups receiving iron (anti- *H. pylori* plus iron, iron alone or negative control receiving iron). A greater proportion of infected children receiving iron experienced correction of IDA than those receiving placebo or anti- *H. pylori* alone (68% for anti- *H. pylori* plus iron, 76%)for iron alone, 25% for placebo and 36% for anti- *H. pylori* alone, F=49, p <0.0001 (Figure 1). The results suggest no role of anti-*H. pylori* in IDA. Regarding ID, iron therapy had the most pronounced effect - correction occurred in 100% of children receiving iron compared to only 50% of children receiving anti-*H. pylori* alone or placebo. It is important to note that compared to placebo or iron therapy, anti- *H. pylori* therapy did not improve iron status or decrease IDA and ID prevalence. Therefore, the study concluded that *H. pylori* is neither causally linked with IDA nor is a reason for treatment failure of iron supplementation in children. The findings were in contrast with a randomized controlled trial by Choe and colleague who showed that treatment of the infection was associated with a more rapid response to oral iron compared with iron supple‐ mentation alone, and that *H. pylori* eradication led to enhanced iron metabolism even in those not receiving oral iron therapy, suggesting a causal relationship between *H. pylori* infection and IDA [62]. A recent meta-analysis of 12 case reports and series, 19 observational epidemio‐ logic studies and 6 interventional trials, concluded *H. pylori* infection to be a major risk factor for iron deficiency or IDA especially in high-risk groups [75]. Several other meta-analyses of randomized controlled clinical trials suggested that treatment of *H. pylori* infection could be effective in improving anemia and iron statue in IDA patients infected by *H. pylori*, particularly in patients with moderate or severe anemia [76], [77], [78]. Although an association between the pathogenesis of IDA and *H. pylori* infection has been well recognized, a causal link is yet to be established.

**5.3.** *H. pylori* **and iron absorption**

children.

*5.3.1. Mechanism of IDA*

Non-heme iron absorption requires an acidic milieu. Non-water-soluble iron compounds, e.g. ferrous and ferric pyrophosphate are often used in the fortification programs because they cause no unacceptable organoleptic changes in the fortified food. However, these compounds need gastric acid for their solubilization and absorption. Therefore, if gastric acid output is compromised as a consequence of *H. pylori* infection in a large proportion of the target population, the effect of food fortification programs using ferrous fumarate might be less than expected due to reduced absorption of iron from fortified foods. Keeping this in mind, a study was conducted in 12 Bangladeshi children to measure iron absorption from a non-watersoluble iron compound (ferrous fumarate) and from a water-soluble iron compound (ferrous sulfate) before and after treatment of their *H. pylori infections*. For comparison, 12 uninfected age matched children were studied in parallel; all children had IDA [79]. Iron absorption from ferrous fumarate was compared with that from a highly bioavailable, water-soluble iron compound (ferrous sulfate) in a randomized, crossover study using a double stable-isotope technique. Incorporation of 57Fe and 58Fe into erythrocytes 14 days after administration was used as an index of iron absorption [80]. The study noted geometric mean of iron absorption from ferrous sulfate and ferrous fumarate to be 19.7% and 5.3% respectively (*P* < 0.0001; *n* = 12) before treatment and 22.5% and 6.4% respectively after treatment (*P* < 0.0001; *n* = 11) of *H. pylori*-infected children (Table 2). The corresponding values for uninfected children were 15.6% and 5.4% (*P* < 0.001; *n* = 12). Geometric mean relative absorption (absorption of ferrous fumarate compared to ferrous sulfate) was 26.9% and 34.8% in *H. pylori*-infected and uninfected children respectively, and 28.3% in *H. pylori*-infected children after treatment. The results clearly indicate that iron absorption from ferrous fumarate was significantly lower than that from ferrous sulfate in both *H. pylori-*infected and uninfected Bangladeshi children and also that *H. pylori* infection, *per se,* does not influence iron absorption in young children. The efficacy of ferrous fumarate in iron fortification programs to prevent iron deficiency in young children should, therefore, be further evaluated. The results of iron absorption tests may rule out the possibility of *H. pylori* induced hypochlorhydria in interfering iron absorption in Bangladeshi

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The interaction between *H. pylori* and iron metabolism, based on clinical, ferrokinetic and microbiological evidences has generated increasing interest. Iron is an essential micronutrient for virtually all organisms, and *H. pylorus is* no exception. *H. pylori* may cause iron deficiency anemia by competing with the host for the acquisition of alimentary iron. In the stomach, ingested food provides iron in heme and nonheme forms. The low pH and the digestive enzymes in the stomach release iron from ligands to the gastric lumen. *H. pylori* and the host both compete for the free iron by deploying mechanisms specifically devised to sequester and facilitate the acquisition of iron as well as other essential metals. *H. pylori* seems particularly adept at competing for iron [81]; it has been established that *H. pylori* competes for iron in murine hosts to an extent so as to cause iron deficiency when the dietary iron intake is poor [82]. In order to ensure a sufficient supply of iron from the environment, *H. pylori* cells display

**Figure 1.** Treatment failure (%) of anemia, IDA, and ID in children receiving different therapies. \*Value of zero for the Fe-alone group (Adapted from Sarker *et. al*. 2008)

### **5.3.** *H. pylori* **and iron absorption**

a randomized controlled trial by Choe and colleague who showed that treatment of the infection was associated with a more rapid response to oral iron compared with iron supple‐ mentation alone, and that *H. pylori* eradication led to enhanced iron metabolism even in those not receiving oral iron therapy, suggesting a causal relationship between *H. pylori* infection and IDA [62]. A recent meta-analysis of 12 case reports and series, 19 observational epidemio‐ logic studies and 6 interventional trials, concluded *H. pylori* infection to be a major risk factor for iron deficiency or IDA especially in high-risk groups [75]. Several other meta-analyses of randomized controlled clinical trials suggested that treatment of *H. pylori* infection could be effective in improving anemia and iron statue in IDA patients infected by *H. pylori*, particularly in patients with moderate or severe anemia [76], [77], [78]. Although an association between the pathogenesis of IDA and *H. pylori* infection has been well recognized, a causal link is yet

**Figure 1.** Treatment failure (%) of anemia, IDA, and ID in children receiving different therapies. \*Value of zero for the

Fe-alone group (Adapted from Sarker *et. al*. 2008)

to be established.

212 Trends in Helicobacter pylori Infection

Non-heme iron absorption requires an acidic milieu. Non-water-soluble iron compounds, e.g. ferrous and ferric pyrophosphate are often used in the fortification programs because they cause no unacceptable organoleptic changes in the fortified food. However, these compounds need gastric acid for their solubilization and absorption. Therefore, if gastric acid output is compromised as a consequence of *H. pylori* infection in a large proportion of the target population, the effect of food fortification programs using ferrous fumarate might be less than expected due to reduced absorption of iron from fortified foods. Keeping this in mind, a study was conducted in 12 Bangladeshi children to measure iron absorption from a non-watersoluble iron compound (ferrous fumarate) and from a water-soluble iron compound (ferrous sulfate) before and after treatment of their *H. pylori infections*. For comparison, 12 uninfected age matched children were studied in parallel; all children had IDA [79]. Iron absorption from ferrous fumarate was compared with that from a highly bioavailable, water-soluble iron compound (ferrous sulfate) in a randomized, crossover study using a double stable-isotope technique. Incorporation of 57Fe and 58Fe into erythrocytes 14 days after administration was used as an index of iron absorption [80]. The study noted geometric mean of iron absorption from ferrous sulfate and ferrous fumarate to be 19.7% and 5.3% respectively (*P* < 0.0001; *n* = 12) before treatment and 22.5% and 6.4% respectively after treatment (*P* < 0.0001; *n* = 11) of *H. pylori*-infected children (Table 2). The corresponding values for uninfected children were 15.6% and 5.4% (*P* < 0.001; *n* = 12). Geometric mean relative absorption (absorption of ferrous fumarate compared to ferrous sulfate) was 26.9% and 34.8% in *H. pylori*-infected and uninfected children respectively, and 28.3% in *H. pylori*-infected children after treatment. The results clearly indicate that iron absorption from ferrous fumarate was significantly lower than that from ferrous sulfate in both *H. pylori-*infected and uninfected Bangladeshi children and also that *H. pylori* infection, *per se,* does not influence iron absorption in young children. The efficacy of ferrous fumarate in iron fortification programs to prevent iron deficiency in young children should, therefore, be further evaluated. The results of iron absorption tests may rule out the possibility of *H. pylori* induced hypochlorhydria in interfering iron absorption in Bangladeshi children.

#### *5.3.1. Mechanism of IDA*

The interaction between *H. pylori* and iron metabolism, based on clinical, ferrokinetic and microbiological evidences has generated increasing interest. Iron is an essential micronutrient for virtually all organisms, and *H. pylorus is* no exception. *H. pylori* may cause iron deficiency anemia by competing with the host for the acquisition of alimentary iron. In the stomach, ingested food provides iron in heme and nonheme forms. The low pH and the digestive enzymes in the stomach release iron from ligands to the gastric lumen. *H. pylori* and the host both compete for the free iron by deploying mechanisms specifically devised to sequester and facilitate the acquisition of iron as well as other essential metals. *H. pylori* seems particularly adept at competing for iron [81]; it has been established that *H. pylori* competes for iron in murine hosts to an extent so as to cause iron deficiency when the dietary iron intake is poor [82]. In order to ensure a sufficient supply of iron from the environment, *H. pylori* cells display


iron source is well documented [87]. Several iron-repressible outer membrane proteins from

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Ascorbic acid is the reduced form of the vitamin, which can act as a potent antioxidant for neutralizing nitrite-derived mutagens protecting against gastric carcinogenesis [90]. Vitamin C is first absorbed and then is actively secreted, mainly in the antral mucosa, from plasma into gastric juice. Once there, it is able to react with nitrosating agents preventing N-nitroso compounds formation; however, vitamin C in the stomach interacts with iron improving its absorption. In children*, H. pylori* infection was associated with reduced gastric juice ascorbic acid concentration, and the effect was more pronounced in patients with the CagA positive strain [20]. In adults, *H. pylori* infection is also recognized to lower the concentration of vitamin C in gastric juice as evident from a study involving randomly chosen 25-74 years old men and women of north Glasgow, UK. Compared to the non-infected, the *H. pylori* infected had 20% lower concentration of vitamin C in their plasma. [91]. The mechanism whereby *H. pylori* infection lowers vitamin C concentration in gastric juice is unclear, but there are several possibilities. Infection has been associated with significant reduction of gastric juice vitamin C concentration due to chronic gastritis and/or *H pylori* oxidase activity [92]. Study in Korean children also demonstrated significant negative correlation between vitamin C level in gastric juice and the degree of active and chronic inflammation in the antral mucosa. Vitamin C levels in whole blood, plasma, and gastric juice and the gastric juice pH were also closely related to the severity of *H. pylori* infection and the histologic changes in the stomach in those children [93].*H. pylori* has been noted to potentiate the polymorphonuclear leukocyte oxidative burst [94], accompanied by a considerable production of reactive oxygen metabolites. Within the microcirculation of the gastric mucosa ascorbic acid may be consumed during scavenging of these reactive oxygen metabolites as vitamin C is the first line of defense against the oxygen free radical damage in the human body [95]. Low level of Vitamin C may be a consequence of an irreversible inactivation of the ingested vitamin C in the intestinal lumen prior to its absorption. Studies have demonstrated that *H. pylori* produces reductions in stomach vitamin C due to its degradation to dehydroascorbic acid (DHAA) - a metabolite that may be oxidized afterwards irreversibly to 2,3- Diketo- 1-gluconic acid. DHAA is unstable at high pH values, and thus hypochlorhydria or achlorhydria may reduce the stability even further and thus the

In developing countries, low intake of vitamin C-enriched food is associated with higher prevalence of *H. pylori* infection, and together will lead to significantly reduced systemic availability of this vitamin. Therefore, prolonged *H. pylori* infection, as it is frequent in developing countries, may impact absorption of several micronutrients including vitamin C. The impact of *H. pylori* infection on the prevalence of micronutrient malnutrition is not currently known, but it is known that there is a strong correlation of both high prevalence of *H. pylori* infection and micronutrient deficiency in developing regions. Various fortification programs are being carried out in developing regions using iron and/or zinc sources e.g. electrolytic iron, ferric pyrophosphate, and zinc oxide. They need secretion of an adequate

*H. pylori*, including FrpB1, seem to be responsible for heme utilization [88]

**5.4.** *H. pylori* **and vitamin C**

bioavailability of this vitamin.

Adapted from Sarker et al. 2004

**Table 2.** Fractional iron absorption from ferrous fumarate and ferrous sulfate in uninfected children with iron deficiency anemia (IDA) and in *Helicobacter pylori* infected children with IDA before and after treatment

a repertoire of high-affinity iron-uptake systems. It seems that *H. pylori* strains isolated from patients with IDA demonstrates enhanced iron-uptake activity and may be more adept at competing with the host for iron [83]. So far, little is known about how *H. pylori* cells acquire iron bound to host-binding proteins. *In-vitro* studies indicate that human lactoferrin (LF) supports full growth of *H. pylori* in media lacking other iron sources [72]. LF is released from neutrophil, which captures iron from transferrin in conditions with iron-poor state (hypofer‐ rimia) and has been observed to be abundant in human stomach resection specimens from patients with superficial or atrophic gastritis [84], [84]. The iron uptake by *H. pylori* via a specific human LF receptor may thus play a major role in the virulence of *H. pylori* infection in its uptake of iron. A 70-kDa LF-binding protein from the outer membrane proteins of *H. pylori* was identified in bacterium grown in an iron-starved medium, implicating the protein in iron uptake [85]. Comparative binding experiments with bovine or human LF, and with transferrin of horse, bovine or human origin indicated that this protein is highly specific for human LF. By means of this LF-binding protein, *H. pylori* is able to by-pass the human hypoferremic defensive response -a phenomenon when total extracellular iron is reduced in the host limiting bacterial growth. Further *in vivo* studies demonstrated increased concentration of LF in the biopsy specimen [86] and in the gastric juice [86] of patients with *H. pylori*-related gastritis, and also that LF tissue levels correlate significantly with the degree of inflammation of the gastric mucosa. Two outer membrane proteins, FrpB1 and FrpB2 have also been implicated in hemoglobin binding [81]. In keeping with this, the ability of *H. pylori* to use hemoglobin as an iron source is well documented [87]. Several iron-repressible outer membrane proteins from *H. pylori*, including FrpB1, seem to be responsible for heme utilization [88]

### **5.4.** *H. pylori* **and vitamin C**

a repertoire of high-affinity iron-uptake systems. It seems that *H. pylori* strains isolated from patients with IDA demonstrates enhanced iron-uptake activity and may be more adept at competing with the host for iron [83]. So far, little is known about how *H. pylori* cells acquire iron bound to host-binding proteins. *In-vitro* studies indicate that human lactoferrin (LF) supports full growth of *H. pylori* in media lacking other iron sources [72]. LF is released from neutrophil, which captures iron from transferrin in conditions with iron-poor state (hypofer‐ rimia) and has been observed to be abundant in human stomach resection specimens from patients with superficial or atrophic gastritis [84], [84]. The iron uptake by *H. pylori* via a specific human LF receptor may thus play a major role in the virulence of *H. pylori* infection in its uptake of iron. A 70-kDa LF-binding protein from the outer membrane proteins of *H. pylori* was identified in bacterium grown in an iron-starved medium, implicating the protein in iron uptake [85]. Comparative binding experiments with bovine or human LF, and with transferrin of horse, bovine or human origin indicated that this protein is highly specific for human LF. By means of this LF-binding protein, *H. pylori* is able to by-pass the human hypoferremic defensive response -a phenomenon when total extracellular iron is reduced in the host limiting bacterial growth. Further *in vivo* studies demonstrated increased concentration of LF in the biopsy specimen [86] and in the gastric juice [86] of patients with *H. pylori*-related gastritis, and also that LF tissue levels correlate significantly with the degree of inflammation of the gastric mucosa. Two outer membrane proteins, FrpB1 and FrpB2 have also been implicated in hemoglobin binding [81]. In keeping with this, the ability of *H. pylori* to use hemoglobin as an

*H. pylori -infected children after*

**Relative absorption**

**Ferrous sulfate**

mean 5.3 19.7 26.9 6.4 22.5 28.3 5.4 15.6 34.8 +1SD 13.5 32.9 49.0 12.9 33.0 47.7 12.7 30.1 64.3 -1SD 2.1 11.8 14.8 3.2 15.4 16.8 2.3 8.1 18.8

*P*<sup>1</sup> <0.0001 <0.0001 <0.001 1P value for iron absorption from ferrous fumarate compared with that from ferrous sulfate within each group.

**Table 2.** Fractional iron absorption from ferrous fumarate and ferrous sulfate in uninfected children with iron deficiency anemia (IDA) and in *Helicobacter pylori* infected children with IDA before and after treatment

% % %

*treatment (n=11)* **Uninfected children (n=12)**

**Ferrous fumarate**

**Ferrous sulfate**

**Relative absorption**

*H. pylori -infected children before treatment (n=12)*

**Relative absorption**

**Ferrous fumarate**

**Ferrous sulfate**

**Ferrous fumarate**

214 Trends in Helicobacter pylori Infection

Adapted from Sarker et al. 2004

Geometric

Ascorbic acid is the reduced form of the vitamin, which can act as a potent antioxidant for neutralizing nitrite-derived mutagens protecting against gastric carcinogenesis [90]. Vitamin C is first absorbed and then is actively secreted, mainly in the antral mucosa, from plasma into gastric juice. Once there, it is able to react with nitrosating agents preventing N-nitroso compounds formation; however, vitamin C in the stomach interacts with iron improving its absorption. In children*, H. pylori* infection was associated with reduced gastric juice ascorbic acid concentration, and the effect was more pronounced in patients with the CagA positive strain [20]. In adults, *H. pylori* infection is also recognized to lower the concentration of vitamin C in gastric juice as evident from a study involving randomly chosen 25-74 years old men and women of north Glasgow, UK. Compared to the non-infected, the *H. pylori* infected had 20% lower concentration of vitamin C in their plasma. [91]. The mechanism whereby *H. pylori* infection lowers vitamin C concentration in gastric juice is unclear, but there are several possibilities. Infection has been associated with significant reduction of gastric juice vitamin C concentration due to chronic gastritis and/or *H pylori* oxidase activity [92]. Study in Korean children also demonstrated significant negative correlation between vitamin C level in gastric juice and the degree of active and chronic inflammation in the antral mucosa. Vitamin C levels in whole blood, plasma, and gastric juice and the gastric juice pH were also closely related to the severity of *H. pylori* infection and the histologic changes in the stomach in those children [93].*H. pylori* has been noted to potentiate the polymorphonuclear leukocyte oxidative burst [94], accompanied by a considerable production of reactive oxygen metabolites. Within the microcirculation of the gastric mucosa ascorbic acid may be consumed during scavenging of these reactive oxygen metabolites as vitamin C is the first line of defense against the oxygen free radical damage in the human body [95]. Low level of Vitamin C may be a consequence of an irreversible inactivation of the ingested vitamin C in the intestinal lumen prior to its absorption. Studies have demonstrated that *H. pylori* produces reductions in stomach vitamin C due to its degradation to dehydroascorbic acid (DHAA) - a metabolite that may be oxidized afterwards irreversibly to 2,3- Diketo- 1-gluconic acid. DHAA is unstable at high pH values, and thus hypochlorhydria or achlorhydria may reduce the stability even further and thus the bioavailability of this vitamin.

In developing countries, low intake of vitamin C-enriched food is associated with higher prevalence of *H. pylori* infection, and together will lead to significantly reduced systemic availability of this vitamin. Therefore, prolonged *H. pylori* infection, as it is frequent in developing countries, may impact absorption of several micronutrients including vitamin C. The impact of *H. pylori* infection on the prevalence of micronutrient malnutrition is not currently known, but it is known that there is a strong correlation of both high prevalence of *H. pylori* infection and micronutrient deficiency in developing regions. Various fortification programs are being carried out in developing regions using iron and/or zinc sources e.g. electrolytic iron, ferric pyrophosphate, and zinc oxide. They need secretion of an adequate amount of hydrochloric acid for optimal absorption. Higher prevalence of *H. pylori* infection is associated with low levels of vitamin C in serum and in gastric juice in children [20]; however, there is no consensus about the usefulness of vitamin C supplementation in the management of *H. pylori* infection. In review of the current literature, it may be concluded that high concentration of vitamin C in gastric juice might inactivate *H. pylori* urease [98], the key enzyme for survival of the pathogen and its colonization into acidic stomach. However, it is not certain if vitamin C will be useful in regions with high prevalence of iron and/or zinc deficiency as well as high *H. pylori* contamination rates.

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279-82.

### **6. Conclusion**

The combination of micronutrient deficiency and more frequent enteric infections consequent to *H pylori*–induced hypochlorhydria is likely to have a profound impact on health of children in developing countries with high prevalence of *H pylori* and lower intake of reliable nutritional sources of bioavailable iron and ascorbic acid. Thus, prevention of *H pylori* infection could potentially have an important impact on iron deficiency anemia or other micronutrient deficiencies in the developing world.

### **Acknowledgements**

I gratefully acknowledge Dr. Mohammed Abdus Salam, Director, Research & Clinial Admin‐ istration and Strategy, icddr,b for his review and valuable comments.

### **Author details**

Shafiqul Alam Sarker

Centre for Nutrition and Food Security (CNFS), Gastroenterology Unit, Dhaka Hospital, In‐ ternational Centre for Diarrhoeal Diseases Research, Bangladesh

### **References**


[3] Mitchell, H.M., et al., Epidemiology of Helicobacter pylori in southern China: identi‐ fication of early childhood as the critical period for acquisition. J Infect Dis, 1992. 166(1): p. 149-53.

amount of hydrochloric acid for optimal absorption. Higher prevalence of *H. pylori* infection is associated with low levels of vitamin C in serum and in gastric juice in children [20]; however, there is no consensus about the usefulness of vitamin C supplementation in the management of *H. pylori* infection. In review of the current literature, it may be concluded that high concentration of vitamin C in gastric juice might inactivate *H. pylori* urease [98], the key enzyme for survival of the pathogen and its colonization into acidic stomach. However, it is not certain if vitamin C will be useful in regions with high prevalence of iron and/or zinc deficiency as

The combination of micronutrient deficiency and more frequent enteric infections consequent to *H pylori*–induced hypochlorhydria is likely to have a profound impact on health of children in developing countries with high prevalence of *H pylori* and lower intake of reliable nutritional sources of bioavailable iron and ascorbic acid. Thus, prevention of *H pylori* infection could potentially have an important impact on iron deficiency anemia or other micronutrient

I gratefully acknowledge Dr. Mohammed Abdus Salam, Director, Research & Clinial Admin‐

Centre for Nutrition and Food Security (CNFS), Gastroenterology Unit, Dhaka Hospital, In‐

[1] Pounder, R.E. and D. Ng, The prevalence of Helicobacter pylori infection in different

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istration and Strategy, icddr,b for his review and valuable comments.

ternational Centre for Diarrhoeal Diseases Research, Bangladesh

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well as high *H. pylori* contamination rates.

deficiencies in the developing world.

**Acknowledgements**

**Author details**

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Shafiqul Alam Sarker

**6. Conclusion**

216 Trends in Helicobacter pylori Infection


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**Section 5**

**Modern Methods of Bacterial DNA Recovering**

**Modern Methods of Bacterial DNA Recovering**

**Chapter 8**

*Helicobacter pylori* **and Liver – Detection of Bacteria in**

Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver in humans [1]. It is the fifth most common cancer in men (523,000 cases per year, 7.9% of all cancers) and the seventh among women (226,000 cases per year, 3.7% of all cancers) [2], with over a half of million new cases diagnosed annually. It is the second leading cause of the cancer related mortality in the world [3,4,5] and its prevalence differs according to geographic location,

In general, the distribution of HCC cases presents great geographic variation, with higher incidence in Easter Asia and sub-Saharan Africa where infection with hepatitis B virus (HBV) is endemic with rates of over 20 per 100,000 individuals. Mediterranean countries such as Italy, Spain and Greece have intermediate rates of 10 to 20 per 100,000 individu‐ als. The North and South America have a relatively low incidence (< 5 per 100,000 individ‐ uals) [4], although a rising incidence was observed in the USA, probably associated with the rise in hepatitis C virus (HCV) infection. Recent decreases in the incidence of HCC were reported among Chinese populations in Hong Kong, Shangai and Singapore; the inci‐

In Brazil, there are few reports on the prevalence of HCC [7]. It was suggested that the prevalence was considered low according to epidemiologic and retrospective studies [8]. In

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Liver Tissue from Patients with Hepatocellular**

**Technique (LCM)**

Bruna Maria Röesler and

http://dx.doi.org/10.5772/57080

gender, age and ethnicity [6].

dence in Japan is also decreasing [3].

**1. Introduction**

José Murilo Robilotta Zeitune

Elizabeth Maria Afonso Rabelo-Gonçalves,

Additional information is available at the end of the chapter

**Carcinoma Using Laser Capture Microdissection**

*Helicobacter pylori* **and Liver – Detection of Bacteria in Liver Tissue from Patients with Hepatocellular Carcinoma Using Laser Capture Microdissection Technique (LCM)**

Elizabeth Maria Afonso Rabelo-Gonçalves, Bruna Maria Röesler and José Murilo Robilotta Zeitune

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57080

### **1. Introduction**

Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver in humans [1]. It is the fifth most common cancer in men (523,000 cases per year, 7.9% of all cancers) and the seventh among women (226,000 cases per year, 3.7% of all cancers) [2], with over a half of million new cases diagnosed annually. It is the second leading cause of the cancer related mortality in the world [3,4,5] and its prevalence differs according to geographic location, gender, age and ethnicity [6].

In general, the distribution of HCC cases presents great geographic variation, with higher incidence in Easter Asia and sub-Saharan Africa where infection with hepatitis B virus (HBV) is endemic with rates of over 20 per 100,000 individuals. Mediterranean countries such as Italy, Spain and Greece have intermediate rates of 10 to 20 per 100,000 individu‐ als. The North and South America have a relatively low incidence (< 5 per 100,000 individ‐ uals) [4], although a rising incidence was observed in the USA, probably associated with the rise in hepatitis C virus (HCV) infection. Recent decreases in the incidence of HCC were reported among Chinese populations in Hong Kong, Shangai and Singapore; the inci‐ dence in Japan is also decreasing [3].

In Brazil, there are few reports on the prevalence of HCC [7]. It was suggested that the prevalence was considered low according to epidemiologic and retrospective studies [8]. In

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1997, Brazilian researchers showed that HVB was the most common cause of liver disease in patients with HCC [9]. After that, another survey demonstrated that in Southeastern and Southern Brazil, HCV accounted for over 55% of cases. In the Northeast and North, HCV accounted for less than 50% and HBV accounted for 22-25% of cases; hepatitis B was more prevalent in the Northern than in the Southern regions [10].

analysis of human hepatic cell line (HepG2) co-cultured with *H. pylori* have revealed that bacteria may exert the pathological effect on HepG2 cells by up-regulating the expression of some proteins enrolled in transcription regulation, signal transduction and metabolism [38].

*Helicobacter pylori* and Liver – Detection of Bacteria in Liver Tissue from Patients with Hepatocellular...

http://dx.doi.org/10.5772/57080

229

In most pathology laboratories, archives of formalin-fixed paraffin-embedded (FFPE) tissues represent the only tissue specimens available for routine diagnostics. A major advantage of such archives is that long-term clinical data is often available [39]. Furthermore, another benefit of using FFPE tissues is that they are the easiest to store and transport [40]. Because of this, FFPE tissues have been used in PCR-based studies related to cancer research, genetics, infectious diseases and molecular epidemiology [41]. Additionally, the use of FFPE tissue also allows employing modern transcriptomic and epigenomic methods with nucleic acids [39].

However, isolating high-quality genomic DNA from FFPE sections remains a challenge for researchers. Formalin is the most commonly used tissue fixative worldwide because it offers the best compromise between cost, practicality and morphological fixative properties [39, 42]. However, the fixation of tissue in formalin leads to extensive protein-DNA cross-linking of all tissue components and nucleic acids isolated from these specimens are highly fragmented [43]. This is particularly troublesome when long DNA regions are amplified, old paraffin blocks are used or fixation time is over three days [41, 44]. Because of this, FFPE tissue requires special protocols in order to extract small amounts of DNA suitable for amplification [45]. Neverthe‐ less, methods of DNA extraction from FFPE tissue are generally laborious and time consuming.

Although studies on the role of the *H. pylori* in the development of HCC were more frequent in the last decade [46], most of them presents a prospective nature. This probably occurs because the retrospective studies frequently employ FFPE liver tissue and DNA extraction is a limiting factor in this type of sample. However, several researchers have detected *H. pylori* and its virulence factors (vacA genes and 26 kDa) in paraffin embedded liver samples [21, 27,

Laser capture microdissection (LCM) is a technique that has recently become available for isolation of individual or groups of cells from a heterogeneous tissue sections by microscopic visualization. The technique was first described in 1996 by researchers of the National Institutes of Health (NIH) in Bethesda, MD [49] and allows the isolation of cells reducing the interference from nontarget cell population. The method allows selection of unmixed starting

The LCM system is based on an inverted light microscope fitted with a laser device to facilitate the visualization and procurement of cells [51]. The PALM MicroBeam System (Carl Zeiss, MicroImaging GmbH, Göottingen, Germany) was used in this study and it is based on the Laser Microdissection and Pressure Catapulting technology. This system consists of an inverted microscope with a motorized stage and a pulsed "cold" nitrogen ultra-violet (UV) laser. The laser is focused through the objective lenses to a micron-sized spot diameter. The narrow laser focal spot allows the ablation of the material while the surrounding tissue remains fully intact. The microscope stage and UV laser are controlled by a PC, and a video camera allows for tissue sections to be displayed on the PC screen. Cells or regions of interest are then identified and manually delineated on the computer screen using the software program. The

material for DNA, RNA or protein extraction for further downstream analyses [50].

34, 47, 48].

The development of HCC has been attributed to several risk factors. In general, chronic viral infection with HBV and HCV is considered the major cause of HCC in 75-80% of cases [1], although HBV is globally considered the leading risk factor responsible for 50% of cases [11]. Furthermore, cirrhosis [12], exposure to the carcinogenic fungal aflatoxin B1 [13], inherited diseases [14], Wilson's disease [15] and heavy alcohol consumption [16] are also risk factors attributed to its development. Recently, upcoming risk factors for HCC include obesity, diabetes and related nonalcoholic fatty liver disease [3].

In 1994, researchers described the infectious agent *Helicobacter* (*H.*) *hepaticus* and its role in causing active hepatitis and associated liver tumors in mice [17]. Since then, several studies related to *H. hepaticus* experimental infection have demonstrated that this bacterium may induce a strong inflammatory change in the liver leading to HCC. Considering that *H. pylori* was classified as a class I carcinogen [18] and *Helicobacter* spp. DNA was detected in hepatic tissue from patients with different hepatobiliary diseases, it has been proposed that in humans, as in animals, *Helicobacter* spp. may also colonize and induce chronic hepatic diseases mainly HCC.

In fact, studies related to the possible association between *H. pylori* and hepatobiliary diseases have been developed since 1998, when Helicobacter DNA was identified in Chilean patients with chronic cholecystitis [19]. After that, a variety of researches have been conducted to verify the role of *H. pylori* in the development of HCC [20, 21, 22, 23, 24, 25, 26 e 27]. Considering the role of chronic inflammation and infection in the development of cancer, in the case of HCC, future studies should be performed to verify the role of *Helicobacter* infection in the liver pathophisiology [28]. However, whether this bacterium causes liver tumor or acts as a cofactor in the process of carcinogenesis needs to be confirmed.

The mechanism by which *H. pylori* colonizes the human liver is not totally enlightened. The *H. pylori* DNA detected in the liver tissue may result from bacterial translocation from the stomach into the blood through the portal system, especially in the later stages of chronic liver disease when portal hypertension occurs [1, 29, 30]. In addition, the bacteria may reach the liver by phagocytes and macrophages or circulating retrograde transfer from the duodenum [31]. However, the studies involving the growth of *H. pylori* from the HCC liver reinforce the bacterial colonization ruling out the possibility of retrograde contamination [32, 33]. Addi‐ tionally, no other bacteria from the digestive tract are associated with human hepatocarcino‐ genesis [23, 34].

Several researchers have suggested that *H. pylori* may damage hepatocytes in vitro by a cytopathic effect in a liver and HCC cell lines [35, 36]. Furthermore, it was demonstrated in a HCC cell line (Huh7) that an inoculum of *H. pylori* was able to adhere and internalize into hepatocytes and this result was also dependent on virulence factors of bacteria [37]. Proteomic analysis of human hepatic cell line (HepG2) co-cultured with *H. pylori* have revealed that bacteria may exert the pathological effect on HepG2 cells by up-regulating the expression of some proteins enrolled in transcription regulation, signal transduction and metabolism [38].

1997, Brazilian researchers showed that HVB was the most common cause of liver disease in patients with HCC [9]. After that, another survey demonstrated that in Southeastern and Southern Brazil, HCV accounted for over 55% of cases. In the Northeast and North, HCV accounted for less than 50% and HBV accounted for 22-25% of cases; hepatitis B was more

The development of HCC has been attributed to several risk factors. In general, chronic viral infection with HBV and HCV is considered the major cause of HCC in 75-80% of cases [1], although HBV is globally considered the leading risk factor responsible for 50% of cases [11]. Furthermore, cirrhosis [12], exposure to the carcinogenic fungal aflatoxin B1 [13], inherited diseases [14], Wilson's disease [15] and heavy alcohol consumption [16] are also risk factors attributed to its development. Recently, upcoming risk factors for HCC include obesity,

In 1994, researchers described the infectious agent *Helicobacter* (*H.*) *hepaticus* and its role in causing active hepatitis and associated liver tumors in mice [17]. Since then, several studies related to *H. hepaticus* experimental infection have demonstrated that this bacterium may induce a strong inflammatory change in the liver leading to HCC. Considering that *H. pylori* was classified as a class I carcinogen [18] and *Helicobacter* spp. DNA was detected in hepatic tissue from patients with different hepatobiliary diseases, it has been proposed that in humans, as in animals, *Helicobacter* spp. may also colonize and induce chronic hepatic

In fact, studies related to the possible association between *H. pylori* and hepatobiliary diseases have been developed since 1998, when Helicobacter DNA was identified in Chilean patients with chronic cholecystitis [19]. After that, a variety of researches have been conducted to verify the role of *H. pylori* in the development of HCC [20, 21, 22, 23, 24, 25, 26 e 27]. Considering the role of chronic inflammation and infection in the development of cancer, in the case of HCC, future studies should be performed to verify the role of *Helicobacter* infection in the liver pathophisiology [28]. However, whether this bacterium causes liver tumor or acts as a cofactor

The mechanism by which *H. pylori* colonizes the human liver is not totally enlightened. The *H. pylori* DNA detected in the liver tissue may result from bacterial translocation from the stomach into the blood through the portal system, especially in the later stages of chronic liver disease when portal hypertension occurs [1, 29, 30]. In addition, the bacteria may reach the liver by phagocytes and macrophages or circulating retrograde transfer from the duodenum [31]. However, the studies involving the growth of *H. pylori* from the HCC liver reinforce the bacterial colonization ruling out the possibility of retrograde contamination [32, 33]. Addi‐ tionally, no other bacteria from the digestive tract are associated with human hepatocarcino‐

Several researchers have suggested that *H. pylori* may damage hepatocytes in vitro by a cytopathic effect in a liver and HCC cell lines [35, 36]. Furthermore, it was demonstrated in a HCC cell line (Huh7) that an inoculum of *H. pylori* was able to adhere and internalize into hepatocytes and this result was also dependent on virulence factors of bacteria [37]. Proteomic

prevalent in the Northern than in the Southern regions [10].

diabetes and related nonalcoholic fatty liver disease [3].

in the process of carcinogenesis needs to be confirmed.

diseases mainly HCC.

228 Trends in Helicobacter pylori Infection

genesis [23, 34].

In most pathology laboratories, archives of formalin-fixed paraffin-embedded (FFPE) tissues represent the only tissue specimens available for routine diagnostics. A major advantage of such archives is that long-term clinical data is often available [39]. Furthermore, another benefit of using FFPE tissues is that they are the easiest to store and transport [40]. Because of this, FFPE tissues have been used in PCR-based studies related to cancer research, genetics, infectious diseases and molecular epidemiology [41]. Additionally, the use of FFPE tissue also allows employing modern transcriptomic and epigenomic methods with nucleic acids [39].

However, isolating high-quality genomic DNA from FFPE sections remains a challenge for researchers. Formalin is the most commonly used tissue fixative worldwide because it offers the best compromise between cost, practicality and morphological fixative properties [39, 42]. However, the fixation of tissue in formalin leads to extensive protein-DNA cross-linking of all tissue components and nucleic acids isolated from these specimens are highly fragmented [43]. This is particularly troublesome when long DNA regions are amplified, old paraffin blocks are used or fixation time is over three days [41, 44]. Because of this, FFPE tissue requires special protocols in order to extract small amounts of DNA suitable for amplification [45]. Neverthe‐ less, methods of DNA extraction from FFPE tissue are generally laborious and time consuming.

Although studies on the role of the *H. pylori* in the development of HCC were more frequent in the last decade [46], most of them presents a prospective nature. This probably occurs because the retrospective studies frequently employ FFPE liver tissue and DNA extraction is a limiting factor in this type of sample. However, several researchers have detected *H. pylori* and its virulence factors (vacA genes and 26 kDa) in paraffin embedded liver samples [21, 27, 34, 47, 48].

Laser capture microdissection (LCM) is a technique that has recently become available for isolation of individual or groups of cells from a heterogeneous tissue sections by microscopic visualization. The technique was first described in 1996 by researchers of the National Institutes of Health (NIH) in Bethesda, MD [49] and allows the isolation of cells reducing the interference from nontarget cell population. The method allows selection of unmixed starting material for DNA, RNA or protein extraction for further downstream analyses [50].

The LCM system is based on an inverted light microscope fitted with a laser device to facilitate the visualization and procurement of cells [51]. The PALM MicroBeam System (Carl Zeiss, MicroImaging GmbH, Göottingen, Germany) was used in this study and it is based on the Laser Microdissection and Pressure Catapulting technology. This system consists of an inverted microscope with a motorized stage and a pulsed "cold" nitrogen ultra-violet (UV) laser. The laser is focused through the objective lenses to a micron-sized spot diameter. The narrow laser focal spot allows the ablation of the material while the surrounding tissue remains fully intact. The microscope stage and UV laser are controlled by a PC, and a video camera allows for tissue sections to be displayed on the PC screen. Cells or regions of interest are then identified and manually delineated on the computer screen using the software program. The

microscope is then instructed to collect delineated regions. The noncontact capture of homo‐ geneous tissue samples or individual cells is achieved by means of catapulting using PALM's patented Laser Pressure Catapulting technology. With the same laser, the separated cells, or the selected tissue area, can be directly catapulted into the eppendorf cap containing a depressed lid [52, 53]. The Figure 1 shows the LCM technology employed in this study.

**2.2. Methods**

*2.2.1. Tissue preparation for LCM*

*2.2.2. Bacterial microdissection*

*2.2.3. DNA extraction*

*2.2.4. PCR amplification*

negative control for PCR assays.

stored at -80°C until DNA extraction.

Six *H. pylori* positive samples were cut into 10μm-thick sections and mounted on 0.17mm PEN membrane-covered slides (Carl Zeiss, MicroImaging GmbH, Göttingen, Germany). After slicing, the sections were placed at 60°C for 30 minutes, then deparaffinized in 3 xylene baths (3x1min), rehydrated in decreasing alcohols (100%, 95% e 70%, each for 30 seconds) and washed for 30 seconds in tap water. Further, the routine staining with carbol fuchsin was performed [66]. At this step the sections have remained in the dye for at most 15 seconds. The stained sections were observed under the microscope for the identification of bacteria.

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231

Stained bacteria were microdissected using a PALM MicroBeam system (Carl Zeiss, MicroI‐ maging GmbH, Göttingen, Germany) (Figure 1A). The areas with target bacteria were traced around and microdissected together with the pieces of thin membrane by laser microbeam and then ejected into the Eppendorf tube cap by a single laser shot (Figure 1B). The tube was

After microdissection, the cap was inserted into an Eppendorf tube containing 100μl digestion buffer, prepared with 10mM Tris-HCl (pH 8.0), 1mM EDTA, 1% Tween 20 and 0.3% proteinase K. After that, samples remained in a water bath at 56°C for 3 hours and the tube was heated to 95°C for 5 min to inactivate proteinase K. The crude lysate was directly employed as template for PCR. All of these procedures were previously described with minor modifications [48].

The samples were further amplified by PCR using *H. pylori* 16S rRNA primers. The sequence of the sense primer (JW21) was 5'-GCGACCTGCTGGAACATTAC-3'(position 691-710) and the antisense primer (JW22) was 5'-CGTTAGCTCCATTACTGGAGA-3' (position 829-809) and they amplified a product of approximately 129bp [27]. Briefly, 1μl of DNA extracted was added to 25μl PCR mix containing deoxynucleoside triphosphates (dNTPs) at concen‐ trations of 200 μM each, 2.0μl of 25mM MgCl2, 0.25 μl of GoTaq Hot Start Polymerase (Promega Corp., Madison, WI, USA), 4.0 μl of 5X GoTaq Flexi Buffer (supplied with the enzyme) and 20 pmol each primer (Life Technologies, Carlsbed, CA, USA). Amplification reactions included an initial 2-minute denaturation step at 94ºC, followed by 40 cycles of 30 seconds at 94ºC, 30 seconds at 55ºC and 45 seconds at 72ºC. A final extension step for 7 minutes at 72ºC was performed. The DNA extracted from *H. pylori* from FFPE gastric tissue was used as positive control and distilled water in place of the DNA samples was used as

LCM has been used in a wide variety of applications, including pathology [54, 55], organ transplantation [56, 57], gene expression [52, 58] and molecular characterization of cancer cells [59, 60, 61]. LCM is compatible with most stains and tissue preservation techniques including frozen sections, FFPE tissues, cytology preparations and cultured cells [52, 62]. Because of its high precision and accuracy LCM has been successfully employed to isolation of bacterial cells in FFPE tissues including *H. pylori* [48, 61, 62, 64, 65].

**Figure 1.** Images of LCM technology used in the study. In A is represented the complete PALM MicroBeam system. In B is illustrated the path of the laser beam passing through the objective lens to reach the tissue slice and the catapulting process for capturing the cells of interest into the Eppendorf cap. Source of the images: www.zeiss.de.

### **2. Clinical samples, methods and results**

### **2.1. Clinical samples**

This study was carried out utilizing six cases of FFPE liver tissue from patients with HCC from Department of Anatomic Pathology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. The mean ages of patients was 56.0 years, with 4 male cases (66,6%) and 2 female cases (33,4%). All the fragments of liver were obtained during hepatic surgery (either transplantation or partial hepatectomy). These samples were *H. pylori* positive previously detected by polymerase chain reaction (PCR) with *H. pylori* specific 16S rRNA primers [27]. The selection criteria for the paraffin blocks included specimens archived for 5 years (2008 to 2012). The present study was approved by the Ethics Committee of the Faculty of Medical Sciences, UNICAMP (CEP 616/2009).

### **2.2. Methods**

microscope is then instructed to collect delineated regions. The noncontact capture of homo‐ geneous tissue samples or individual cells is achieved by means of catapulting using PALM's patented Laser Pressure Catapulting technology. With the same laser, the separated cells, or the selected tissue area, can be directly catapulted into the eppendorf cap containing a depressed lid [52, 53]. The Figure 1 shows the LCM technology employed in this study.

LCM has been used in a wide variety of applications, including pathology [54, 55], organ transplantation [56, 57], gene expression [52, 58] and molecular characterization of cancer cells [59, 60, 61]. LCM is compatible with most stains and tissue preservation techniques including frozen sections, FFPE tissues, cytology preparations and cultured cells [52, 62]. Because of its high precision and accuracy LCM has been successfully employed to isolation of bacterial cells

)

**Figure 1.** Images of LCM technology used in the study. In A is represented the complete PALM MicroBeam system. In B is illustrated the path of the laser beam passing through the objective lens to reach the tissue slice and the catapulting

This study was carried out utilizing six cases of FFPE liver tissue from patients with HCC from Department of Anatomic Pathology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. The mean ages of patients was 56.0 years, with 4 male cases (66,6%) and 2 female cases (33,4%). All the fragments of liver were obtained during hepatic surgery (either transplantation or partial hepatectomy). These samples were *H. pylori* positive previously detected by polymerase chain reaction (PCR) with *H. pylori* specific 16S rRNA primers [27]. The selection criteria for the paraffin blocks included specimens archived for 5 years (2008 to 2012). The present study was approved by the Ethics Committee of the Faculty of Medical Sciences, UNICAMP (CEP 616/2009).

process for capturing the cells of interest into the Eppendorf cap. Source of the images: www.zeiss.de.

in FFPE tissues including *H. pylori* [48, 61, 62, 64, 65].

230 Trends in Helicobacter pylori Infection

**2. Clinical samples, methods and results**

**2.1. Clinical samples**

(a) (b)

### *2.2.1. Tissue preparation for LCM*

Six *H. pylori* positive samples were cut into 10μm-thick sections and mounted on 0.17mm PEN membrane-covered slides (Carl Zeiss, MicroImaging GmbH, Göttingen, Germany). After slicing, the sections were placed at 60°C for 30 minutes, then deparaffinized in 3 xylene baths (3x1min), rehydrated in decreasing alcohols (100%, 95% e 70%, each for 30 seconds) and washed for 30 seconds in tap water. Further, the routine staining with carbol fuchsin was performed [66]. At this step the sections have remained in the dye for at most 15 seconds. The stained sections were observed under the microscope for the identification of bacteria.

### *2.2.2. Bacterial microdissection*

Stained bacteria were microdissected using a PALM MicroBeam system (Carl Zeiss, MicroI‐ maging GmbH, Göttingen, Germany) (Figure 1A). The areas with target bacteria were traced around and microdissected together with the pieces of thin membrane by laser microbeam and then ejected into the Eppendorf tube cap by a single laser shot (Figure 1B). The tube was stored at -80°C until DNA extraction.

### *2.2.3. DNA extraction*

After microdissection, the cap was inserted into an Eppendorf tube containing 100μl digestion buffer, prepared with 10mM Tris-HCl (pH 8.0), 1mM EDTA, 1% Tween 20 and 0.3% proteinase K. After that, samples remained in a water bath at 56°C for 3 hours and the tube was heated to 95°C for 5 min to inactivate proteinase K. The crude lysate was directly employed as template for PCR. All of these procedures were previously described with minor modifications [48].

#### *2.2.4. PCR amplification*

The samples were further amplified by PCR using *H. pylori* 16S rRNA primers. The sequence of the sense primer (JW21) was 5'-GCGACCTGCTGGAACATTAC-3'(position 691-710) and the antisense primer (JW22) was 5'-CGTTAGCTCCATTACTGGAGA-3' (position 829-809) and they amplified a product of approximately 129bp [27]. Briefly, 1μl of DNA extracted was added to 25μl PCR mix containing deoxynucleoside triphosphates (dNTPs) at concen‐ trations of 200 μM each, 2.0μl of 25mM MgCl2, 0.25 μl of GoTaq Hot Start Polymerase (Promega Corp., Madison, WI, USA), 4.0 μl of 5X GoTaq Flexi Buffer (supplied with the enzyme) and 20 pmol each primer (Life Technologies, Carlsbed, CA, USA). Amplification reactions included an initial 2-minute denaturation step at 94ºC, followed by 40 cycles of 30 seconds at 94ºC, 30 seconds at 55ºC and 45 seconds at 72ºC. A final extension step for 7 minutes at 72ºC was performed. The DNA extracted from *H. pylori* from FFPE gastric tissue was used as positive control and distilled water in place of the DNA samples was used as negative control for PCR assays.

### *2.2.5. Detection of PCR products*

For analysis of the amplified products, 5μl of the amplicons were put on 1,5% agarose gels containing 1μg of ethidium bromide per ml. The amplicons were visualized by UV transillu‐ mination.

### *2.2.6. Sequence analysis*

The 16S rRNA amplicons were further identified by sequence analysis using ABI Prism Dye Terminator sequencing kit with AmpliTaq DNA polymerase and the ABI 3500xL Sequencer (Applied Biosystems, Foster City, CA, USA). Sequence comparison was then carried out using the Blast program and GenBank databases.

### **2.3. Results**

Analyzing the tissue sections stained with carbol fuchsin, we visualized microorganisms resembling *H. pylori* mainly in hepatic sinus from HCC samples. The number of cocci was greater than of bacilli (Figure 3).

Our PCR results showed that all six microdissected samples were positive for 16S rRNA gene (Figure 2) and showed 98% similarity to *H. pylori* 16S rRNA gene by sequence analysis (GeneBank accession number CP003419.1) (Figure 4).

**Figure 3.** Optical microscopy of FFPE liver fragments of HCC patients with PCR-positive *H. pylori* 16S rRNA and stained with carbol fuchsin. In (A) and (C) bacteria are represented within sinusoid (arrows) before microdissection (magnifica‐ tion: 610X). In (B) and (D) the same samples are represented after bacterial microdissection (magnification: 610X).

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**Figure 4.** Electropherogram sequence of *H. pylori* 16S rRNA gene and alignment results of the BLAST databases.

**Figure 2.** Results of amplification of *H. pylori* 16S rRNA gene. 1, 2 and 5 are positive samples; 3 and 4 are negative samples. MM: molecular marker, PC: positive control and NC: negative control.

*Helicobacter pylori* and Liver – Detection of Bacteria in Liver Tissue from Patients with Hepatocellular... http://dx.doi.org/10.5772/57080 233

*2.2.5. Detection of PCR products*

232 Trends in Helicobacter pylori Infection

the Blast program and GenBank databases.

(GeneBank accession number CP003419.1) (Figure 4).

greater than of bacilli (Figure 3).

mination.

**2.3. Results**

*2.2.6. Sequence analysis*

For analysis of the amplified products, 5μl of the amplicons were put on 1,5% agarose gels containing 1μg of ethidium bromide per ml. The amplicons were visualized by UV transillu‐

The 16S rRNA amplicons were further identified by sequence analysis using ABI Prism Dye Terminator sequencing kit with AmpliTaq DNA polymerase and the ABI 3500xL Sequencer (Applied Biosystems, Foster City, CA, USA). Sequence comparison was then carried out using

Analyzing the tissue sections stained with carbol fuchsin, we visualized microorganisms resembling *H. pylori* mainly in hepatic sinus from HCC samples. The number of cocci was

Our PCR results showed that all six microdissected samples were positive for 16S rRNA gene (Figure 2) and showed 98% similarity to *H. pylori* 16S rRNA gene by sequence analysis

MM 1 2 3 4 5 PC NC

**Figure 2.** Results of amplification of *H. pylori* 16S rRNA gene. 1, 2 and 5 are positive samples; 3 and 4 are negative

samples. MM: molecular marker, PC: positive control and NC: negative control.

**Figure 3.** Optical microscopy of FFPE liver fragments of HCC patients with PCR-positive *H. pylori* 16S rRNA and stained with carbol fuchsin. In (A) and (C) bacteria are represented within sinusoid (arrows) before microdissection (magnifica‐ tion: 610X). In (B) and (D) the same samples are represented after bacterial microdissection (magnification: 610X).

**Figure 4.** Electropherogram sequence of *H. pylori* 16S rRNA gene and alignment results of the BLAST databases.

### **3. Conclusion**

The results described here confirm the identification of *H. pylori* in FFPE liver tissue from patients with HCC. Although specific primers were used for amplification of the *H. pylori* 16S rRNA gene, we cannot exclude the possibility of cross-reaction of these primers with other *Helicobacter* spp [65].

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Considering the difficulty of DNA extraction from paraffin embedded samples, the use of LCM simplified the achievement of specific DNA because the DNA extraction process was reduced to a single digestion step of bacterial cells without further purification. Consequently, the crude lysate was used directly as template for PCR amplification. This is particularly advantageous when compared to traditional methods of DNA extraction that are generally laborious, toxic and time consuming [67].

Another advantage of using LCM is that it allowed the exact location of *H. pylori* in the liver, since bacteria were mainly found in the peritumoral tissue. Considering that our samples presented tumoral and peritumoral tissue in the same paraffin block, the technique was highly effective for obtaining a target bacterial population within a selected area in the HCC tissue. This is very important when we consider that the necrotic state and nuclease content of tissues may influence in recovering intact DNA specially when performing traditional methods for DNA extraction [68].

Furthermore, LCM was useful to reduce the interference from nontarget cell population considering that bacteria were found in small quantities in the liver tissue (Figure 3). In relation to nontarget cell population, it is important to consider that the major obstacle in the analysis of tumoral tissue is that it is composed by different cell types including stroma and inflam‐ matory cells [69, 70, 71] and there is a potential dilution effect of the larger quantities of nontarget DNA found in whole tissue sections [64]. Thus, the employment of LCM was very efficient in isolating *H. pylori* despite of the reduced bacterial quantity in the HCC tissue.

In summary, we suggest that LCM can be extensively applied for identification of *H. pylori* in FFPE liver tissue. Further studies will be performed in order to amplify virulence genes of bacteria as well as to isolate *H. pylori* from other tissues using LCM technique.

### **Acknowledgements**

This work was supported by grants from FAPESP (2009/09889-5) and FAEPEX 101/2011.

### **Author details**

Elizabeth Maria Afonso Rabelo-Gonçalves\* , Bruna Maria Röesler and José Murilo Robilotta Zeitune

\*Address all correspondence to: elizabeth.goncalves@gc.unicamp.br

Department of Internal Medicine, Center of Diagnosis of Digestive Diseases, Faculty of Medical Sciences, State University of Campinas, Campinas, São Paulo, Brazil

### **References**

**3. Conclusion**

234 Trends in Helicobacter pylori Infection

other *Helicobacter* spp [65].

and time consuming [67].

DNA extraction [68].

**Acknowledgements**

José Murilo Robilotta Zeitune

Elizabeth Maria Afonso Rabelo-Gonçalves\*

**Author details**

The results described here confirm the identification of *H. pylori* in FFPE liver tissue from patients with HCC. Although specific primers were used for amplification of the *H. pylori* 16S rRNA gene, we cannot exclude the possibility of cross-reaction of these primers with

Considering the difficulty of DNA extraction from paraffin embedded samples, the use of LCM simplified the achievement of specific DNA because the DNA extraction process was reduced to a single digestion step of bacterial cells without further purification. Consequently, the crude lysate was used directly as template for PCR amplification. This is particularly advantageous when compared to traditional methods of DNA extraction that are generally laborious, toxic

Another advantage of using LCM is that it allowed the exact location of *H. pylori* in the liver, since bacteria were mainly found in the peritumoral tissue. Considering that our samples presented tumoral and peritumoral tissue in the same paraffin block, the technique was highly effective for obtaining a target bacterial population within a selected area in the HCC tissue. This is very important when we consider that the necrotic state and nuclease content of tissues may influence in recovering intact DNA specially when performing traditional methods for

Furthermore, LCM was useful to reduce the interference from nontarget cell population considering that bacteria were found in small quantities in the liver tissue (Figure 3). In relation to nontarget cell population, it is important to consider that the major obstacle in the analysis of tumoral tissue is that it is composed by different cell types including stroma and inflam‐ matory cells [69, 70, 71] and there is a potential dilution effect of the larger quantities of nontarget DNA found in whole tissue sections [64]. Thus, the employment of LCM was very efficient in isolating *H. pylori* despite of the reduced bacterial quantity in the HCC tissue. In summary, we suggest that LCM can be extensively applied for identification of *H. pylori* in FFPE liver tissue. Further studies will be performed in order to amplify virulence genes of

bacteria as well as to isolate *H. pylori* from other tissues using LCM technique.

\*Address all correspondence to: elizabeth.goncalves@gc.unicamp.br

Medical Sciences, State University of Campinas, Campinas, São Paulo, Brazil

This work was supported by grants from FAPESP (2009/09889-5) and FAEPEX 101/2011.

Department of Internal Medicine, Center of Diagnosis of Digestive Diseases, Faculty of

, Bruna Maria Röesler and


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**Section 6**

**Eradication Therapy of H. pylori Infection: New**

**Strategies**


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**Chapter 9**

*Helicobacter pylori* **Infection — Challenges of**

**Alternative Treatments**

Roland N. Ndip

**1. Introduction**

http://dx.doi.org/10.5772/57462

Amidou Samie, Nicoline F. Tanih and

Additional information is available at the end of the chapter

**Antimicrobial Chemotherapy and Emergence of**

Classified as a class one carcinogen, *Helicobacter pylori* is a gram-negative coccobacillus (0.5 μm wide by 2 - 4 μm long), microaerophilic, flagellated organism that has chronically infected more than 50% of the world's population [1, 2, 3, 4]. Significant evidence exist that links the bacterium to the pathogenesis and development of certain diseases such as gastric ulcers, chronic gastritis and stomach cancers, although most of the people harboring this organism are asymptomatic [5, 6]. The prevalence of infection caused by this organism increases with advancing age and is reported to be higher in developing countries and among low socioeconomic populations, probably owing to conditions that favor the infection such as poor hygiene, crowded living conditions, and inadequate or no sanitation. The prevalence of this infection in human varies with geographical location and socio-demographic characteristics of the population; however does not parallel the incidence of morbidity caused by the infection [7, 8]. Studies have highlighted inconsistencies in the prevalence rates for *Helicobacter* and disease. In industrialized countries there is generally a low prevalence of *H. pylori* infection and yet a relatively high prevalence of gastric cancer. On the other hand, some countries with

Over the years, different treatment regimens have been proposed for eradication of *H. pylori.* Eradication of the organism has proven to be the first therapeutic approach and constitutes a reliable long-term prophylaxis of peptic ulcer relapse, accelerating ulcer healing and reducing the rate of ulcer complications [10]. Successful regimens generally require two or more antibiotics coupled with a proton pump inhibitor [11]. A proton pump inhibitor (PPI) or

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high *Helicobacter* prevalence rates have low gastric cancer prevalence [9].
