A review on Helicobacter pylori Infection

Helicobacter pylori ( H. pylori ) was first identified in 1982 by the Australian physicians Barry Marshall and Robin Warren. Helicobacter pylori is also known as Campylobacter pylori . It is a gram-negative, micro-aerophilic, spiral (helical) bacterium usually found in the stomach. H. pylori infection is one of the most common and chronic bacterial infection, affecting approximately half of the world's population. Peptic ulcer disease, gastric ulcers, mucosa-associated lymphoid tissue lymphoma and gastric cancer all are linked to H. pylori. There are various diagnostic procedures to detect H. pylori infection and the choice of one approach over another is based on a number of considerations including accessibility, benefits and drawbacks of each method, cost, and the age of the patients. When H. pylori infection is diagnosed, doctor determines the therapy on the basis of patient's clinical status. Generally, eradication of H. pylori is recommended for treatment and prevention of the infection. In most of the cases H. pylori infections are cured with triple therapy. Moreover, quadruple therapies, sequential therapies and concomitant therapies have been developed as major alternative options to treat H. pylori infection. In this review pathophysiology, prevalence, transmission, clinical sign & symptoms, risk factors, and diagnostic techniques used to detect H. pylori infection as well as H. pylori eradication therapy regimens are discussed. Regulatory Aspects of : the topic of “Assessment of Drug Prescribing Pattern using W.H.O Prescribing Indicators”. Research and 2 case study PharmD Pharm and PharmD trust of include Drug Related Problem, Medication Error, Anti microbial resistance, Pharmacovigilance and Pharmacoepidemiology.


v Introduction
Helicobacter pylori (H. pylori) infection is the most frequent and persistent bacterial infection. This bacteria is adapted to live in the harsh and acidic environment of the stomach. In stomach H. pylori changes the environment by reducing the acidity, which creates a favourable condition to survive. According to a study, in 2015, over 50% of the world's population had H. pylori in their upper gastrointestinal tracts [1]. In developing countries almost 90% of people and in developed countries (excluding Japan) less than 40% of the populations are infected with this infection [2]. H. pylori can be transmitted from one person to another through direct contact with infected person's saliva, vomit or feces. Risk factors for H. pylori infection are related to living in crowded environment, living without a reliable supply of clean water, living with H. pylori infected person.
Diagnostic methods of H. pylori infection depends on several factors like availability of diagnostic tests, cost, accessibility, advantages and disadvantages of each method, age of patients etc. Triple therapy is the standard treatment option for H. pylori infection. Other treatment options like quadruple therapies (which involve diverse combinations of medicines), sequential therapies and concomitant therapies have been proposed as successful alternatives for H. pylori treatment, despite the fact that triple therapy delivers satisfactory cure rates.
v Pathophysiology • Adaptation to the stomach H. pylori uses its flagella to burrow into the mucus lining of the stomach to reach the epithelial cells underneath, where it is less acidic, in order to avoid the acidic environment of the stomach (lumen) [3]. H. pylori can detect the pH gradient in the mucus and then move towards the less acidic area (chemotaxis). This also keeps the bacteria from being swept away into the lumen with the bacteria's mucus environment, which is constantly moving from its site of creation at the epithelium to its dissolution at the lumen interface [4]. H. pylori is found in the mucus, on the inner surface of the epithelium, and sometimes inside the epithelial cells [5]. It adheres to the epithelial cells by producing adhesins, which bind to lipids and carbohydrates in the membrane of the epithelial cell. BabA is an adhesin that binds with Lewis b antigen, which is present on the surface of stomach epithelial cells [6]. BabA mediated H. pylori adhesion is acid sensitive and can be completely reversed by decreased pH. According to one theory, BabA's acid responsiveness promotes adherence which also facilitates an effective escape from an unfavourable environment at pH that is harmful for the organism [7]. SabA is an adhesin which binds to increased quantities of sialyl-Lewis x antigen expressed on gastric mucosa [8]. H. pylori not only uses chemotaxis to avoid low pH regions, but it also neutralises the acid in its surroundings by producing large amount of urease, which converts the urea present in the stomach to carbon dioxide and ammonia. These interact with the strong acids for creating a favourableneutralised area around H. pylori [9]. Mutants lacking urease are unable to colonise. Urease expression is necessary for both initial colonisation and the maintenance of chronic infection [10].
• Adaptation of H. pylori to high acidity of stomach H. pylori produces huge amount of urease, to produce ammonia to counteract stomach acidity. H. pylori arginase, a bimetallic enzyme binuclear Mn2-metalloenzyme arginase, crucial for pathogenesis of the bacterium in human stomach, a member of the ureohydrolase family, catalyzes the conversion of L-arginine to L-ornithine and urea, where ornithine is further converted into polyamines, which are necessary for several critical metabolic processes [11]. This provides acid resistance, which is necessary for the bacteria to colonise in the gastric epithelial cells. Arginase of H. pylori also plays a role in evasion of the pathogen from host immune system. Arginase competes with hostinducible nitric oxide (NO) synthase for the same substrate, L-arginine, which lowers the production of NO, a vital part of innate immunity and a powerful antimicrobial agent that can directly kill the pathogens [11]. Dysregulation of the host immunological response to H. pylori infection is mainly attributed to variations in the availability of Larginine and its conversion into polyamines [11].
junctions, and induces apoptosis, and certain phospholipases, are toxic to epithelial cells and the ammonia produced to control pH [12]. CagA is a gene related with cytotoxins, has the potential to be carcinogenic and can also lead to inflammation [13]. Colonization of the stomach by H. pylori can lead to chronic gastritis (an inflammation of the stomach lining), at the site of infection. Helicobacter cysteine-rich proteins (Hcp), particularly HcpA (hp0211), are known to provoke an inflammatory immunological response [14]. H. pylori can increase the levels of COX-2 in H. pylori positive gastritis [15]. Chronic gastritis is likely to underlie H. pylori related diseases [16]. When the effects of inflammation allow stomach acid and the digesting enzyme pepsin to surpass the defences that protect the stomach and duodenal mucous membranes, ulcers develop in the stomach and duodenum. The location of H. pyloricolonisation, which affects the where the ulcer develops, is influenced by the stomach's acidity [17]. In people producing large amounts of acid, H. pylori colonizes near the pyloric antrum (exit to the duodenum) to avoid the acid-secreting parietal cells at the fundus (near the entrance to the stomach). H. pylori can also colonise in the stomach for persons who produce normal or less acid. The inflammatory response caused by bacteria colonizing near the pyloric antrum induces G cells in the antrum to secrete the hormone gastrin, which travels through the bloodstream to parietal cells in the fundus [18]. In addition to increasing the amount of parietal cells over time, gastrin induces the parietal cells to produce more acid into the stomach lumen [19]. The increased acid load damages the duodenum, which can cause duodenal ulcer. When H. pylori colonize other areas of the stomach, the inflammatory reaction can result in atrophy of the stomach lining and ulcer formation in the stomach. This can increase the risk of stomach cancer.

• Cag Pathogenicity Island
About 50 -70% of H. pylori strains in Western countries possess the cag pathogenicity island genes, which may increase the pathogenicity of H. pylori [20]. The risk of developing peptic ulcers or stomach cancer is higher in the people of Western countries, who infected with strains carrying the cag PAI than those infected with strains lacking the island. Following attachment of H. pylori to stomach epithelial cells, the type IV secretion system expressed by the cag PAI "injects" the inflammationinducing agent, peptidoglycan, from their own cell walls into the epithelial cells. The immunological sensor Nod1 in the cytoplasm recognizes the injected peptidoglycan, which stimulates the release of cytokines that cause inflammation [21]. The cag PAI-encoded protein CagA is also delivered through the type-IV secretion system into the stomach's epithelial cells, where it interferes with the cytoskeleton, cell adhesion, intracellular signaling, cell polarity, and other cellular processes [22]. A host cell membraneassociated tyrosine kinase (TK) phosphorylates the CagA protein on tyrosine residues after it has entered the cell. Then, protein tyrosine phosphatase or protooncogene Shp2 is allosterically activated by CagA [23]. Pathogenic strains of H. pylori activate the membrane protein with a tyrosine kinase (TK) domain known as the epidermal growth factor receptor (EGFR). The pathogenesis of H. pylori is associated with the activation of the EGFR, which may be facilitated by altered signal transduction and gene expression in host epithelial cells. It has also been proposed that the CagA protein's Cterminal domain (amino acids 873 -1002) functions independently of protein tyrosine phosphorylation to control host cell gene transcription [24,25].

• Cancer
Researchers are looking into two connected processes through which H. pylori could encourage cancer. One method entails an increased rate of host cell mutation and greater free radical generation close to H. pylori. The other hypothesised process, known as a "perigenetic pathway", entails improving the phenotypic of the transformed host cell through changes to cell proteins, such as adhesion proteins [26]. It has been proposed that H. pylori causes inflammation and elevated levels of TNF-α and / or interleukin-6 (IL-6). According to the proposed perigenetic mechanism, inflammation-associated signalling molecules, such as TNF-α, can alter gastric epithelial cell adhesion and result in the dispersion and migration of mutant epithelial cells without the requirement of additional mutations in tumour suppressor genes, such as genes encoding cell adhesion proteins [27]. The strain of H. pylori to which an individual is exposed may have an impact on their chance of getting stomach cancer. The cytotoxinassociated gene A (CagA) and the vacuolating toxin A (VacA), two proteins produced at high levels by H. pylori strains, appear to cause greater tissue damage than those that produce lower levels or that lack those genes completely [28]. These proteins are toxic to the cells lining the stomach and signal strongly to the immune system that an invasion is occurring. As a result of the bacterial presence, neutrophils and macrophages set up residence in the tissue to fight the bacteria assault [ [36]. Infection with H. pylori also results in DNA double-strand breaks and other distinctive DNA damages, such as the oxidative DNA damage 8-OHdG [37]. Numerous epigenetic changes brought on by H. pylori are also connected to the development of cancer [38,39]. These epigenetic changes are brought on by H. pyloriinduced CpG site methylation in gene promoters and H. pylori-induced altered expression of multiple microRNAs [38,39]. H. pylori infection is linked to epigenetically decreased DNA repair machinery efficiency, which promotes the accumulation of mutations and genomic instability as well as gastric carcinogenesis, according to a review by Santos and Ribeiro [40]. Raza et al. [41] shown in particular that expression of two DNA repair proteins, ERCC1 and PMS2, was significantly decreased once H. pylori infection had progressed to cause dyspepsia. 20 % of infected people get dyspepsia [42]. The human gastric infection with H. pylori also results in epigenetically decreased protein expression of the DNA repair proteins MLH1, MGMT, and MRE11, according to a review by Raza et al. Reduced DNA repair in the presence of increased DNA damage boosts carcinogenic mutations and is probably a major factor in the development of H. pylori carcinogenesis.
• Survival of Helicobacter pylori H. pylori's pathogenesis depends on its capacity to endure in the harsh gastric environment, which is characterized by acidity, peristalsis, and phagocyte assault accompanied by release of reactive oxygen species [43]. During host colonisation, H. pylori causes an oxidative stress reaction. The H. pylori genome develops potentially fatal and carcinogenic oxidative DNA adducts as a result of this oxidative stress response [44]. Many bacterial pathogens, such as Neisseria gonorrhoeae, Hemophilus influenzae, Streptococcus pneumoniae, S. mutans, and H. pylori are susceptible to oxidative stress and oxidative DNA damage [45]. Each of these pathogens appears to rely on transformation-mediated recombinational repair to survive the DNA damage brought on by oxidative stress. Therefore, it would seem that transformation and recombinational repair help an infection succeed. DNA transfer between bacterial cells via an intermediary medium, known as transformation, appears to be a component of an adaptation for DNA repair. H. pylori is naturally competent for transformation. While many organisms are competent only under certain environmental conditions, such as starvation, H. pylori is competent throughout logarithmic growth [46]. All living organisms have genetic code for responses to stressful situations including those that result in DNA damage [46]. In H. pylori to fix DNA doublestrand breaks (DSBs), homologous recombination is necessary. RecA is loaded onto single-strand DNA (ssDNA) by the AddAB helicase-nuclease complex, which resects DSBs and promotes strand exchange that results in homologous recombination and repair. RecA and AddAB are necessary for effective gastric colonisation, which implies that H. pylori either experiences double-strand DNA damage in the stomach that must be repaired or has to undergo another recombination-mediated process. In particular, natural transformation is increased by DNA damage in H. pylori, and there is a link between the DNA damage response and DNA uptake in H. pylori [46]. This suggests that natural competence aids in the persistence of H. pylori in its human host and explains why most clinical isolates still retain competence. Since it resolves Holliday junctions, an intermediary in the recombinational repair process, RuvCprotein is crucial. Reduced macrophage survival, increased vulnerability to oxidative stress and DNA-damaging agents, and an inability to establish successful infection in a mouse model are all characteristics of H. pylori mutants that are defective in RuvC [47]. Similar to this, RecN protein is crucial for DSB repair in H. pylori [48]. The decreased capacity of an H. pylorirecN mutant to colonise mice stomachs emphasises the significance of recombinational DNA repair in H. pylori survival inside its host [48].

v Prevalence of Helicobacter pylori Infection
In Europe, Northern countries have a lower frequency of H. pylori infection than in Southern and Eastern countries. In Netherlands, the existence of antibodies against H. pylori and the CagA antigen was examined in a randomly chosen sample of 1550 blood donors from four different areas [49]. In this study, non-European immigrants were not examined; only native Dutch respondents were tested. According to this study, the prevalence of H. pylori infection was 32%, with 28% of participants who tested positive for the infection carrying CagApositive strain. Due to a birth cohort effect, the seroprevalence of H. pylori decreased from 48% in persons born between 1935-1946, and 16% in those born between 1977-1987. Additionally, in the same age cohorts, the percentage of CagA-positive participants dropped from 38% to 14 %. According to these findings, it is possible to anticipate a further decline in H. pylori prevalence in Netherlands in future decades.
A population-based prospective analysis of a cohort of more than 6500 pregnant women from the Netherlands was also published [50]. According to this study, 24 % of Dutch women have H. pylori. The most significant conclusion was that non-Dutch women had a much greater prevalence of H. pylori, with 64% of them being seropositive. However, in the latter group, first-generation immigrants who were infected and born abroad had a higher risk of H. pylori infection than secondgeneration immigrants. Therefore, in this study, ethnicity was a significant predictor of H. pylori.
Portugal reported to have a greater frequency of H. pylori infection than Northern European countries, with an infection rate of 84.2 %, with 61.7 % of strains also positive for CagA [51]. Another study, based on a proportion of included participants, defined an incidence rate of infection of 3.6/100 person-years, confirming that Portugal's H. pylori infection rate remains the highest in Europe.
Similarly, Eastern Europe also reported to have high infection rates. More than 4600 individuals were examined across the country, as part of a population-based cross-sectional survey in Turkey, resulting in a weighted overall infection prevalence of 82.5 % [52]. It's interesting to note that the incidence was lowest among residents of the southern region of the country, where people usually consume a diet rich in citrus fruits. However, vitamin C is successful in preventing the most of the infections, but few researches shown that it may also be involved in the H. pylori infection. According to several studies, age and gender usually don't have any link with an increased risk of H. pylori infection. In fact, most of the studies reported no difference of H. pylori infection between men and women in both adults and children [59]. In the adult population, no significant correlation was found between infection and age [50,54]. The birth cohort effect appears to be responsible for the age-specific gradient in H. pylori H. pylori infection has been linked with certain socioeconomic factors. Particularly, people with a low socioeconomic status were more likely to carry H. pylori infection [50,63] [52]. Few researches claimed that smoking and drinking alcohol is the main risk factors for this infection [52,63]. On the other hand, one research shows that frequent alcohol use was found to be protective against H. pylori infection [52]. But more severe gastritis is observed in the antrum than in corpus (P < 0.05). The topographic distribution of inflammation was pangastritis (61.9%), followed by antral (32.1%) and corpus (5.9%). The antrum had a greater H. pylori density than in the corpus. Fluorescent in situ hybridization (FISH) is a new method used in histological preparations. It is used for detecting a specific bacterial component or factor, such as clarithromycin resistance [101, 102]. FISH employs a series of fluorescent protein-labeled oligonucleotide probes to target a specific gene. For example -16S and 23S ribosomal RNA genes are the commonly used probes. This test takes around 3 hours to complete this assay and probe for both H. pylori and clarithromycin resistance. The ability to evaluate both probes in a short time adds value to the H. pylori diagnosis. The specific location of the bacteria in the stomach mucosa has been determined using in situ hybridization and immunochemical methods [103]. Despite its benefits, FISH is timeconsuming, expensive method. So, it is not used in clinical practice.

Ø Culture
Because no significant amount of commensal bacterial flora is expected, a newly acquired gastric biopsy specimen is the ideal specimen for culturing H. pylori (except in patients with reduced gastric acid production, in whom an overabundance of commensal bacteria is possible). Gastric juice sample or the string test is the less invasive alternatives for biopsy collection. Culture may also be done using gastric juice samples or the string test, although the sensitivity is lower than when biopsy specimens are used [104 -106]. When performed under optimal conditions, culturing generally has a sensitivity of greater than 90% and a specificity of 100% H. pylori is very sensitive organism. So, it must be cultured as soon as possible after being collected. Biopsies can be stored at 4 °C for up to 24 hours in a transport medium (e.g., Stuart's transport medium). H. pylori can be kept frozen at -80 °C after isolated, ideally in broth with 15% to 20% glycerol. Various types of medium can be used for H. pylori culture like selective agars (e.g., Pylori-agar, Skirrow agar, Wang media), which contain specific antibiotics to suppress commensal bacteria, and nonselective agars (e.g., blood agar, Columbia blood agar). Before discarding cultures as negative, these should be cultured for at least 7 days under microaerobic conditions (85% N2, 10% CO2, 5% O2) at 35 °C to 37 °C. H. pylori is identified by morphological traits as well as positive catalase, oxidase, and urease reactions [112]. Culturing is the most specific approach for identifying H. pylori. But the results depend on the microbiologist's skill, specimen quality, and the transport media which is used for culture [113]. From a long time, the use of H. pylori culture to diagnose infection was limited in research and epidemiology studies. Culturing is mostly used to confirm the antibiotic sensitivity of H. pylori after 2 treatment failure in patients. However, few laboratories are performing the culture and susceptibility test before 2 treatment failures and it would be preferable because of the increase in antibiotic resistance rates, particularly to clarithromycin and metronidazole. Because standard therapies are failing at an increasing rate, bacterial culture may become an essential tool for assessing antibiotic resistance in patients and managing the antibiotic failure. According to a recent study, antibiotic resistance is high in the pediatric population of Brazilian children and

Ø Polymerase chain reaction
In clinical practice, PCR is used to detect H. pylori in small samples that have few bacteria present. PCR does not require any specific processing equipment or transportation, and it may be used on samples collected using both invasive and noninvasive methods. Furthermore, PCR is faster than many other diagnostic procedures, and it may be used to identify a wide range of bacterial genotypes and in epidemiological investigations. A significant disadvantage of PCR is that it can identify DNA segments of dead bacterium in the stomach mucosa of patients after treatment; as a result, it can yield false-positive results [116,117]. H. pylori may be detected by PCR in materials collected using non-invasive or minimally invasive techniques, such as gastric juice, stomach content, saliva, feces etc. So, molecular approaches can be applied to the specimens acquired from string tests or orogastric brushes. Molecular testing may be useful for the materials that cannot be properly cultured due to prolonged transport or in cases where H. pylori isolation is impossible due to contamination. In gastric biopsy specimens, molecular approaches such as PCR have been shown to be effective in identifying infections and testing for clarithromycin resistance, which is caused by changes in the 23S rRNA gene [118]. Because of the increasing frequency of antibiotic resistance in some countries with a high prevalence of H. pylori, molecular tests may be useful as H. pylori diagnostic options. Efflux pumps play a key role in antibiotic resistance in other Gram-negative bacteria, and treatment options caused by multidrugresistant bacteria are limited [119].

Ø Rapid urease test (RUT)
The RUT utilizes the ability of H. pylori to create significant amounts of urea as the basis criteria for diagnosing infection. Biopsies obtained during endoscopy are put in a solution that contains urea and a pH indicator. If urease is present, then urea will break down into carbon dioxide and ammonia. That causes the raising of medium's pH and colour change in pH indicator. Depending on the amount of bacteria in biopsy sample, the RUT can give results in minutes to 24 hours. The RUT is a low-cost, widely accessible, and highly specific test. Although few members of the oropharyngeal microbiota produce urease, which is swallowed in the saliva, the stomach's strong acid rapidly denatures this weaker enzyme. However, due to reduced urease activity, which might be induced by a recent intake of antibiotics, bismuth compounds, or proton pump inhibitors (PPIs), there is a substantial risk of false-negative results with RUT [120]. In patients with achlorhydria, a false-negative urease test can be obtained. The number of bacteria in the biopsy affects RUT sensitivity; a positive result requires at least 10000 cells. In the presence of blood, the RUT has been found to have low sensitivity and specificity. By increasing the incubation time, RUT specificity decreases and hence the risk of a false positive rises. When RUT findings from individual gastric antrum and corpus tissue specimens were compared to histology results (as the gold standard), combining the tissues increased H. pylori identification from 64 % in separate specimens to 69.2 % [121]. Commercial RUTs have specificities ranging from 95% to 100%; although their sensitivity is slightly lower (approximately 85 % -95 %). Gel-based (CLOtest, HpFast) and paper-based (PyloriTek, ProntoDryHpOne) RUTs are commercially available [122,123].
• Noninvasive methods to detect an infection Ø Serology Several types of tests are available to detect antibodies against H. pylori. The enzyme immunoassay (EIA) test is mostly used. The majority of commercial EIA tests work by detecting IgG, with sensitivity and specificity ranging from 60% to 100%. The incidence of infection, changes in location, and features of the study populations are all relevant elements to consider when evaluating the quality of serology tests for the identification of active H. pylori infection. Local validation of a serology test is required, and it is important to make modifications to cut-off levels for particular populations are critical. The maximum sensitivity is shown in tests comprising complicated antigen combinations of multiple strains [124]. When deciding whether a serology test should be used as the method of choice, there are a number of factors to consider. Serology testing should be considered in patients who have recently taken antibiotics or PPIs, have bleeding ulcers, or have gastric atrophy [125].
Whole-blood tests, as well as antibody detection in urine or saliva, do not show the same level of reliability as laboratory-based testing, and they are not recommended for diagnosing of H. pylori infection [125]. Serology tests are inexpensive and widely available. In fact, the accessibility of these tests can result in their use by laboratories lacking in expertise in the diagnosis of H. pylori, which may lead to incorrect data interpretation. Another disadvantage is the antibodies persist in the host after eradication therapy [126]. As a result, it may take some time after the start of eradication therapy and the confirmation of a significant decline in antibody titers. Due to this circumstance, serology can only be used to establish an infection that has been completely eradicated. On the other hand, serology findings are unaffected by recent antibiotic or PPI therapy. In general, the serology test is very useful alternative to the UBT test for properly identifying individuals with a negative result [127]. Both the full bacterial cell and particular H. pylori proteins have been detected by using serology. Not all individuals who infected with H. pylori gets develop disease, and the vast range of disease linked to H. pylori infection may be due to the heterogeneity of H. pylori strains [128]. The presence or lack of virulence factors, some of which have been utilised as serological markers, can cause between-strain variability. The cagA and vacA genes have been associated to enhanced pathogenicity of H. pylori. CagA is a 120-kDa to 140-kDa protein that is encoded by the cagA gene [129]. Strains expressing the CagA protein induce a greater frequency of duodenal ulcer and gastric adenocarcinoma, as well as more severe inflammation and gastric atrophy [130]. Although the vacA gene is found in all H. pylori strains, it is only expressed in 50 % to 65 % of the strains. VacA is an 81-kDa to 91-kDa protein that is encoded by the vacA gene, causes vacuole formation in gastric epithelial cells [129]. CagA and VacA are both immunogenic proteins, and serologic assays that have been develop to identify and diagnose H. pylori infection and seroprevalence of virulence factor [131 -133]. Currently, CagA, VacA, GroEL, gGT, HcpC, and UreA are the six highly immunogenic virulence factors, which are expressed in Escherichia coli, purified, and immobilised on nitrocellulose membranes to detect serologic immune responses against these virulence factors in the patients. The sensitivity and specificity of this novel assay were 97.6 % and 96.2 %, respectively [134].

v Tests used to detect the eradication of Helicobacter pylori Infection Ø Urea breath test (UBT)
The UBT is based on H. pylori's capacity to break down orally absorbed 13 C-or 14 C-labeled urea into CO2 and ammonia if it is present in the stomach environment. 13 CO2 or 14 CO2 diffuses into the bloodstream, is expelled through the lungs, and can be measured in the exhaled air. The test is simple to carry out and does not need an endoscopy. 13 C is a nonradioactive, non-harmful isotope that may be safely used in children and pregnant women. 13 C is often measured in breath samples using an isotope ratio mass spectrometer, although the instrument is expensive [135]. On the other hand, 14 C-urea is affordable, but it must be used in a nuclear medicine department that is permitted to store and dispose of radioactive reagents [136,137]. Majority of the studies show that the UBT's sensitivity and specificity are more than 90% [138]. The UBT's high sensitivity, especially after eradication therapy, may be explained by the fact that in situations of moderate colonization or patchy H. pylori distribution, the UBT is more likely to provide positive results than biopsy-based tests. Other ureaseforming pathogens rarely cause false-positive results. In contrast to serology, the UBT can provide false-negative findings if it is conducted after the use of medications such as PPIs and antibiotics. The UBT may have limited value if the patient has recently taken PPIs, antibiotics, or bismuth compounds [137]. False-negative 13 C-UBT results can be caused by corpus-predominant gastritis. The misdiagnosis of corpus-predominant gastritis may result in a significant misassessment of patients who require endoscopy and eradication therapy [139]. Post-treatment UBT is usually performed in 4 to 6 weeks after eradication therapy [140]. According to a meta-analysis, the 13 C-UBT test was found to be accurate for all ages. The authors achieved 95.9% sensitivity, 95.7 % specificity, likelihood-ratio (LR) + 17.4, LR -0.06 and diagnostic odds ratio (OR) of 424.9. The 13 C-UBT demonstrated a high sensitivity of 96.6 %, specificity of 97.7%, LR+ 42.6, LR-0.04, and diagnostic odds ratio of 1042.7 in children older than 6 years. Children less than 6 years old showed greater variability in accuracy in this study, with the test showing a sensitivity of 95%, specificity of 93.5 %, LR+ 11.7, LR-0.12, and diagnostic odds ratio of 224.8 [141].

Ø Stool antigen test (SAT)
To identify antigens against H. pylori in stool samples, the SAT employs an enzyme immunoassay. It is a reliable method for diagnosing an active infection and confirming that the infection has been well treated. Stool samples can be kept for 24 hours at room temperature or 72 hours at 4 °C. Without refrigeration, the SAT's sensitivity drops significantly in 2 to 3 days [142]. Results of the SAT may be influenced by the disorders of digestive problems, PPI therapy, or the presence of a bleeding ulcer. SAT's diagnostic accuracy in identifying H. pylori infection eradication has been evaluated. A recent research compared the ability of 2 SAT approaches to identify H. pylori after eradication therapy in dyspeptic patients: the enzyme immunoassay (Premier Platinum HpSA) and the immunochromatographic method (ImmunoCardHpSA STAT). For ImmunoCardHpSA STAT, the sensitivity, specificity, PPV, NPV, and accuracy were 100 %, 91.0 %, 84.6 %, 100 %, and 94.0 % respectively. On the other hand, for Premier Platinum HpSA, the sensitivity, specificity, PPV, NPV, and accuracy were 84.9 %, 92.5 %, 84.8 %, 92.5 %, and 90.0 % respectively. In another investigation, the capacity of 5 SATs, including 2 monoclonal EIAs and 3 rapid immunochromatographic assay tests, to identify H. pylori infection in adult patients exhibiting dyspeptic symptoms prior to eradication therapy was assessed. The sensitivity and specificity for the two EIAs were 92.2 % and 94.4 % and sensitivity and specificity for the Premier Platinum HpSA Plus test was 48.9 % and 88.9 % for the H. pylori antigen test. The One Step HpSA test had a sensitivity and specificity of 86.7 % and 88.9 % respectively. The ImmunoCard STAT HpSA test had a sensitivity and specificity of 68.9 % and 92.6 % respectively. In H. pylori fecal antigen test had a sensitivity and specificity of 78.9 % and 87 % respectively. In adult dyspeptic patients, the Premier Platinum HpSAPlus EIA test was the best reliable test for diagnosing H. pylori infection [143]. The SAT's sensitivity and specificity depends on the different clinical environment and whether the test is done before or after treatment [144,145]. The SAT is equivalent to the UBT in detecting H. pylori infection in untreated individuals. However, the UBT is superior to the SAT in post eradication, particularly in populations with low H. pylori prevalence. The polyclonal SAT was the first introduced SAT (Premier Platinum HpSA, Meridian Bioscience Inc., OH, United States). This test was followed by a monoclonal test (FemtolabH. pylori, Connex, Germany), which was found to be better than the polyclonal test in both testing untreated patients and monitoring with treated patients [146 -148]. The monoclonal SAT has been shown to be superior to the polyclonal test for both early diagnosis of infection and confirmation of H. pylori eradication [144]. The monoclonal test and the UBT are the only 2 noninvasive tests approved by European Guidelines for determining the effectiveness or failure of eradication therapy. The use of a monoclonal enzyme-linked immunosorbent assay to detect H. pylori antigen in feces is one of the most effective non-invasive techniques for diagnosing infection in children [149]. The H. pylori SAT seems to work effectively in children, independent of their age [150 -152]. The sensitivity, specificity, PPV, and NPV of 20 investigations of the SAT in a total of 2789 patients before they received therapy for H. pylori were 90 %, 96 %, 93 %, and 93 % respectively. The confirmation of H. pylori eradication after therapy with the SAT yielded good findings in 8 investigations examining a total of 307 children, with a sensitivity of 97 %, specificity of 97 %, PPV of 88 %, and NPV of 99 % [153].

v Treatment options for Helicobacter pylori Infection
H. pylori causes serious infections in human body [154]. H. pylori's involvement in active chronic gastritis is related to gastroduodenal ulcers, which plays a risk factor for stomach cancer development [155].
There are a variety of treatment options available for H. pylori infection; however, there is not a single antibiotic treatment that can completely cure the infection. Historically, the infection was treated with a combination of antibiotics like clarithromycin, amoxicillin, metronidazole, tetracycline, fluoroquinolones, tinidazole etc. These antibiotics are frequently used in conjunction with antisecretory drugs like PPIs or bismuth salts. Different combinations of these drugs have been demonstrated to be successful, with varying eradication rates and tolerability. The fast rise of antibiotic-resistant strains of H. pylori, as well as poor patient adherence to therapy, has harmed the efficiency of the most routinely used medicines. In several geographic locations, these variables have lowered treatment efficacy to unsatisfactory levels (≤ 80%). As a result, alternative treatment procedures have just been validated and are already being employed in place of the standard triple therapy. These methods have been employed in locations where there is a high level of clarithromycin resistance, which is a key risk factor for failure of treatment regimen [156]. Amoxicillin resistance has remained relatively stable, but metronidazole and clarithromycin resistance rates have been progressively increasing [157 -161]. The prevalence of antibiotic resistance varies greatly by area and it is linked with the use of antibiotics such as clarithromycin and metronidazole, for respiratory or gastrointestinal infections. The test-and-treat technique was one of the first strategies to be established for the treatment of H. pylori infection. This treatment technique is mainly recommended for the patients younger than 45 years old with chronic dyspepsia, peptic ulcer disease, low-grade MALT, and atrophic gastritis. The test-andtreat technique is based on detecting the existence of H. pylori and eradicating it once it is found. In dyspeptic patients who reside in populations with a moderate-to-high prevalence of H. pylori infection (≥ 10% to 20%), an alternative to the test-and-treat technique is needed, whereas the empirical PPI strategy may be preferred in populations with a low incidence of H. pylori infection [162 -164]. Because diagnostic tests are less accurate in populations with a low prevalence of H. pylori, the test-and-treat technique must be carefully used in these populations [165]. Only a small percentage of patients with functional dyspepsia experience long-term relief from their symptoms once H. pylori is eradicated [166 -168].
It is advised to divide first-line empiric therapy into two large groups: populations with low clarithromycin resistance and populations with high clarithromycin resistance. The acceptable resistance values are set as < 15% to 20% [164]. The following information summarizes current advice and treatment options for H. pylori eradication. According to clarithromycin resistance, they are listed as first line, second line, and third line treatments.
ü First-line treatment in areas with low clarithromycin resistance Triple treatment is the most commonly used approach. In this therapy PPI (lansoprazole 30 mg/12 h, omeprazole 20 mg/12 h, pantoprazole 40 mg/12 h, rabeprazole 20 mg/12 h, or esomeprazole 40 mg/24 h), clarithromycin (500 mg/12 h), and amoxicillin (1 g/12 h) are used for 7 to 14 days. The duration of treatment is controversial, while a meta-analysis found that 14 days yields eradication rates that are 5% greater than 7 days. In case of penicillin allergy, metronidazole can be used instead of amoxicillin because it is equally effective and regarded equivalent [169]. There are several factors involved for why clarithromycin susceptibility decreases the success rate of therapy. These factors include the patient's poor adherence to the drug regimen, stomach acidity, bacterial strain concentration, bacterial mutations, and clarithromycin resistance. There is also a lot of variation in these figures. For example, in the Netherlands, where clarithromycin resistance is not prevalent, it is estimated to be between 1% and 5% [164]. The efficacy rates of triple therapy has been demonstrated to be dependent on PPIs; as a result, several strategies have been tried to increase the success of triple therapy, such as increasing the dose of PPIs and increasing the period of treatment. When compared with standard PPI dosages, the eradication rate increased from 6% to 10%, according to a meta-analysis. A double dosage of esomeprazole exhibited a larger positive impact, according to a study. Because PPI activity is dependent on the cytochrome (CYP) 450 2C19 and MDR polymorphisms, the existence of certain polymorphisms in the host's metabolism can alter the effectiveness of PPIs. According to a recent meta-analysis, hosts who are extensive PPI metabolizers (depending on their CYP2C19 status) had lower cure rates. Furthermore, as compared to the T/C and C/C genotypes, the MDR T/T genotype had a lower cure rate [170]. Adjuvant therapy is sometimes used in conjunction with the standard treatment for H. pylori infection. For example, lactoferrin has been used as adjuvant therapy. Two metaanalyses that examined at the use of lactoferrin found that it reduce the adverse effects of standard treatment, while the latest consensus of Maastricht (IV) states that more evidence and better-designed research are needed before final conclusions can be drawn [171,172]. Saccharomyces boulardii is another adjuvant that has been used, with positive outcomes in some studies [173].
ü First-line treatment in areas with high clarithromycin resistance Ø Quadruple therapy A quadruple therapy can be used in the areas where clarithromycin resistance is high. In this therapy, for 10 to 14 days, patients are given a combination of a PPI, bismuth subsalicylate (525 mg × 4 times daily), and 2 antibiotics, metronidazole (250 mg × 4 times daily) and tetracycline (500 mg × 4 times daily). This regimen is well tolerated, and patients tend to adhere to the schedule. But this therapy is not available in all places. Sometimes, it is recommended that doctors have alternatives in mind, such as sequential therapy or quadruple therapy without bismuth [164].
Ø Sequential therapy A group of Italian researchers proposed the sequential treatment. It involves a 5-day course of a PPI and amoxicillin (1g × 2 times daily), followed by a 5-day course of a PPI and tinidazole clarithromycin / metronidazole (500 mg × 2 times daily). Sequential therapy and bismuth-based quadruple therapy have demonstrated to be equally effective in firstline therapy in most studies [169]. Sequential therapy was studied in a pediatric population with iron deficiency [174]. Children aged 12 to 15 years with active H. pylori infection were evaluated for serum ferritin, and then were randomly assigned into 2 groups to receive either standard or sequential eradication therapy. The UBT confirmed eradication after six weeks when the treatment was completed, and serum ferritin levels were assessed. H. pylori eradication rates differed between sequential and standard therapy, however serum ferritin levels did not differ substantially between the two therapy groups or between the same group before and after treatment.
H. pylori eradication rates in children after sequential therapy compared to triple therapy were evaluated in a recent meta-analysis. This study included 857 children between the ages of 3 to 18 who matched the inclusion criteria. 318 (78 %, 95 % CI: 73 % -82 %) of the 409 patients who got sequential therapy were free of infection, compared to 314 (71 %, 95 % CI: 66 % -75 %) of the 444 patients who received standard triple therapy (RR = 1.14, 95 % CI: 1.06 -1.23). Sequential therapy is preferable to 7 day standard triple therapy, but not substantially better than 10 or 14 day triple therapy. Furthermore, no significant variations in the risk of adverse effects were detected between groups who are receiving various therapies.

Ø Concomitant therapy
In places where clarithromycin resistance is more than 20% and bismuth-based quadruple treatment is not avaible, concurrent therapy is used instead of sequential therapy. Concomitant treatment involves the simultaneous administrationof 3 antibiotics (metronidazole, clarithromycin, and amoxicillin) and a PPI for 10 days. In comparison to conventional triple treatment, this concomitant therapy is more effective and well tolerated [175]. In Greece, a recent controlled trial compared concomitant therapy with triple therapy. There high resistance to clarithromycin (25%) and metronidazole (40%) is mostly seen. Concurrent therapy yielded a 90 % eradication rate, but triple therapy only had a 73.8 % eradication rate [176]. Unfortunately, concurrent treatment had a significant rate of adverse effects, with 30.9 % of individuals reporting at least one adverse effect. On the other hand, the adverse effects were minor, and patients were able to complete their therapy despite them [177]. Another research looked at a combination of PPI, amoxicillin, rifabutin, and ciprofloxacin, and found H. pylori eradication rates of 95.2 %. Amoxicillin was substituted by bismuth in patients with a penicillin allergy, with no significant effect on the eradication rate (94.2 %) [178]. A clinical study compared concomitant and sequential therapy and showed no difference in H. pylori eradication rates after treatment with these therapies. H. pylori infected patients from 11 Spanish hospitals took part in this study. Patients were randomly assigned to undergo either sequential or concurrent therapy. In sequential therapy Omeprazole (20 mg / 12 h) and amoxicillin (1 g / 12 h) were given for 5 days, followed by 5 days of omeprazole (20 mg / 12 h), clarithromycin (500 mg / 12 h), and metronidazole (500 mg / 12 h). Same medicines were used in concomitant therapy. But in concomitant therapy these medicines were given for 10 days. Four weeks after therapy ceased, H.pylori eradication was confirmed with 13 C-UBT or histology. The concomitant and sequential eradication rates were 87% and 81%, respectively, by intentionto-treat (P = 0.15) and 91% and 86%, respectively, per protocol (P = 0.131). They came to the conclusion that concomitant therapy had no substantial advantages over sequential therapy [179]. A second study, which compared the sequential and concurrent therapies included 164 H. pylori infected patients. Patients were given either sequential (n = 86) or concurrent (n = 78) treatments for 14 days. In the sequential treatment group, patients were given rabeprazole (20 mg) and amoxicillin (1 g) in the first week, followed by rabeprazole (20 mg), clarithromycin (500 mg), and metronidazole (500 mg) in the second week. Rabeprazole (20 mg), amoxicillin (1 g), clarithromycin (500 mg), and metronidazole (500 mg) were given to patients in the concurrent treatment group for 2 weeks. Four weeks after the completetion of therapy, 13 C-UBT confirmed the H.pylori eradication. The intention-to-treat and per protocol eradication rates were 75.6% (95% CI: 66.3% -84.9%) and 76.8% (95% CI: 67.1% -85.5%) in the sequential therapy group, and 80.8% (95% CI: 71.8% -88.5%) and 81.3% (95% CI: 71.6% -90.7%) in the concomitant therapy group, respectively. The researchers came to the conclusion that the 2-week concurrent and sequential therapies showed suboptimal efficacy. Furthermore, there were no significant differences in eradication rates, compliance, or adverse effects between the two therapies in this study.

Ø Hybrid therapy
A recently described treatment called hybrid therapy involves two steps: a PPI and amoxicillin (1 g / 12 h) for 7 days, followed by a PPI and three antibiotics -amoxicillin (1 g / 12 h), metronidazole (500 mg / 12 h), and clarithromycin (500 mg / 12 h) for 7 days. The eradication rates in a research contrasting hybrid and sequential therapy were 89.5% and 76.7% (P = 0.001) respectively. Both therapy groups' patients experienced similar serious adverse effects. Particularly, 3.8% of patients receiving sequential therapy and 2.4% of patients receiving hybrid therapy both reported adverse effects [180]. Patients with non-ulcer dyspepsia who were infected with H. pylori were included in a recent trial that examined concurrent, sequential, and hybrid therapies [181]. The patients were randomly assigned to one of three treatments: 1) Concomitant therapy with omeprazole (20 mg), amoxicillin (1 g), clarithromycin (500 mg), and tinidazole (500 mg) for 5 days; 2) Sequential therapy with omeprazole (20 mg) and amoxicillin (1 g) for 5 days, followed by omeprazole (20 mg), clarithromycin (500 mg), and tinidazole (500 mg) for 5 days; 3) hybrid therapy with omeprazole (20 mg) and amoxicillin (1 g) for 7 days, followed by omeprazole (20 mg), amoxicillin (1 g), clarithromycin (500 mg), and tinidazole (500 mg) for 7 days. H. pylori eradication was detected by 13 C-UBT six weeks after treatment. In this study, the intention-to-treat and per protocol analyses revealed eradication rates of 85.5% and 91.6%, respectively, with the concomitant therapy regimen; 91.1% and 92.1%, respectively, with the sequential therapy; and 80% and 85.7%, respectively, with the hybrid therapy regimen.
ü Second-line treatment in areas that have a low clarithromycin resistance Bismuth-based quadruple treatment and regimens combining a PPI and levofloxacin / amoxicillin are available in places where clarithromycin resistance is low [173,182]. However, due to a rise in levofloxacin resistance, the usage of levofloxacin has been questioned [183]. As a result, susceptibility studies should be done prior to commencing therapy.
ü Second-line treatment in areas that have high clarithromycin resistance If bismuth-based quadruple therapy fails, a triple therapy comprising a PPI, levofloxacin, and amoxicillin is recommended. The rise in levofloxacin resistance should be considered once more [164].
ü Third-line treatment It is not recommended to provide further antibiotic treatments after 2 unsuccessful treatments in areas with low or high clarithromycin resistance. Biopsy specimens should be acquired whenever feasible to culture and test for susceptibility [168]. In some circumstances, "rescue" or "salvage" therapy have shown positive effects. For 14 days, rifabutin (150 mg × 2 times daily), amoxicillin (1 g × 2 times daily), and ciprofloxacin (500 mg × 2 times daily) are used as a rescue treatment. Despite the fact that this therapy produces outstanding results, serious adverse effects have been reported [184]. In addition to a base therapy of furazolidone (200 mg × 2 daily), bismuth subcitrate (120 mg × 4 daily), and tetracycline (500 mg × 4 daily), other rescue therapies include a double dose of PPIs plus azithromycin (500 mg / day for 3 days), followed by a double dose of PPIs plus furazolidone (200 mg × 3 times daily) for 10 days. The risk of H. pylori infection recurrence after effective eradication has been calculated at 11.5 % for this therapy [177].
ü Eradication of H. pylori in pregnancy If a patient develops a peptic ulcer while pregnant or breastfeeding, the illness should be managed only with acid suppression. Eradication of H. pylori should be accomplished after childbirth. In pregnancy bismuth, quinolones, and tetracyclines are contraindicated and metronidazole should be avoided [176]. Only one published report has examined H. pylori eradication in pregnant women, especially in pregnant women with iron deficiency anemia [185]. The researchers conducted a randomised placebo-controlled experiment on 40 women aged between 14 to 30 weeks who had H. pylori infection as identified by the SAT. Women were randomly assigned into two groups: group I (n = 20) was treated with amoxicillin, clarithromycin, and omeprazole for two weeks, whereas group II (n = 20) was treated with placebo. Iron and folic acid were given to both groups at therapeutic dosages. The rise in haemoglobin, packed cell volume, serum iron, and percentage of transferrin saturation was substantially larger (P < 0.05) in the group receiving H. pylori eradication medication than the placebo group after 6 weeks of iron and folic acid supplementation therapy. The researchers found that H. pylori infection is common in pregnant women with iron deficiency anaemia, and that eradication therapy improved the response to oral iron supplementation in H. pylori infected pregnant women with iron deficiency anaemia.
ü Use of probiotics in treating H. pylori infection Probiotics may compete directly with H. pylori by interfering with H. pylori adherence or by generating antimicrobial compounds, according to certain theories. Lactobacillus reuteri (L. reuteri) has been studied for its effectiveness in H. pylori eradication treatment. Gastric histology and 13 C-UBT were used to identify H. pylori infection in this investigation. For 8 weeks, intervention consisted of 10 8 colony-forming units of L. reuteri (DSM 17938) with pantoprazole (20 mg × 2 times daily). Patients were assessed 4 to 6 weeks after receiving 13 C-UBT treatment for H. pylori eradication. They revealed that L. reuteri with pantoprazole twice daily cured 13.6% (3/22; 95% CI: 2.9% -34.9%) of patients with H. pylori infection by intentionto-treat analysis and 14.2% (3/21; 95% CI: 3.0% -36%) by per protocol analysis. If the cure rate can be increased by changing the dosage, dosing interval, or duration of therapy, researchers believe L. reuteri might play a role in H. pylori eradication therapy [164]. A recent meta-analysis looked into whether a probiotic product comprising Lactobacillus and Bifidobacterium may increase H. pylori eradication rates while reducing adverse effects [186]. The study comprised 1469 patients in 10 clinical trials, including 708 patients in the probiotic supplementation group and 761 patients in the control group. Pooled ORs by intention-to-treat and per-protocol analyses in the probiotic supplementation group vs the control group were 2.066 (95% CI: 1.398 -3.055) and 2.321 (95% CI: 1.715 -3.142) respectively. The pooled OR of overall side effect incidence was considerably lower in the probiotics supplementation group (OR = 0.305; 95 % CI: 0.117 -0.793). They concluded that adding a probiotic comprising Lactobacillus and Bifidobacterium with initial H. pylori eradication medication in adults may improve eradication rates and reduce overall side effects. Another study aimed to determine whether adding probiotics to a standard anti-H. pylori regimen could minimize the prevalence of gastrointestinal side effects and improve the eradication rate. In a double blind, randomized, placebo-controlled study, 66 H. pylori-positive children were diagnosed by RUT or histology then treated with a triple medication treatment protocol (omeprazole, amoxicillin, and furazolidon) and randomly assigned to receive either a probiotic or a placebo [187]. Esophagogastroduodendoscopy was performed on all of the patients. The SAT was used to determine H. pylori status in 4 to 8 weeks after therapy was completed. The group that got probiotics had a considerably greater percentage of H. pylori eradication (P = 0.04). Furthermore, the probiotic-supplemented children had a lower rate of nausea / vomiting (P = 0.02) and diarrhoea (P = 0.039) throughout therapy than the placebo-treated children. Probiotics