Clarithromycin Resistance and 23S rRNA Mutations in Helicobacter pylori

Helicobacter pylori infects about 50% of the world’s population and is a major cause of chronic gastritis, is strongly associated with the development of gastric and duodenal ulcers and has been linked with gastric adenocarcinoma and B-cell mucosa-associated lymphoid tissue lymphoma [1, 2]. H. pylori Infection results in a sequence of events, ultimately resulting in the development of some gastrointestinal disorders. The sequence was first suggested by Correa et al. in 1975 and has since been supported by many other studies. Colonization of the gastric mucosa by H. pylori first lead to the induction of an inflammatory response, predominantly by Th1 (T helper cells type 1). The initial acute gastritis is followed by active chronic gastritis, which lasts for life if the infection is not treated. Nevertheless, H. pylori-positive subjects are mostly unaware of this inflammation due to the lack of clinical symptoms. The Th1 response results in epithelial cell damage rather than in the removal of H. pylori because H. pylori is not an intracellular pathogen. The ongoing presence of H. pylori thus causes a lifelong proinflammatory response coupled to cellular damage and initiates the histological cascade. The continuous production of reactive oxygen species that results from the ongoing inflammation can result in DNA damage, thus inducing the multiple mutations thought to be required for initiation of the cancer cascade depicted in Figure 1 (3, 4). Among the new methods of magnifying endoscopy, a prototype of endocytoscopy developed by Olympus was used for ex vivo visualization of Helicobacter pylori on experimentally infected gastric biopsies. Moving bacteria were observed at 1100× magnification, giving hope for a possible direct detection during endoscopy. Kim et al. also used magnifying endoscopy on 103 patients to classify the gastric surface according to four patterns: flat, irregular, papillary or nonstructured, which were then compared to the updated Sydney System for histologic gastritis. Histologic gastritis was found in 91% of the biopsy sections with a nonflat type, and among them, 96% were confirmed to harbor H. pylori infection. In another study, the magnified endoscopic findings in the gastric body


Introduction
Helicobacter pylori infects about 50% of the world's population and is a major cause of chronic gastritis, is strongly associated with the development of gastric and duodenal ulcers and has been linked with gastric adenocarcinoma and B-cell mucosa-associated lymphoid tissue lymphoma [1,2]. H. pylori Infection results in a sequence of events, ultimately resulting in the development of some gastrointestinal disorders. The sequence was first suggested by Correa et al. in 1975 and has since been supported by many other studies. Colonization of the gastric mucosa by H. pylori first lead to the induction of an inflammatory response, predominantly by Th1 (T helper cells type 1). The initial acute gastritis is followed by active chronic gastritis, which lasts for life if the infection is not treated. Nevertheless, H. pylori-positive subjects are mostly unaware of this inflammation due to the lack of clinical symptoms. The Th1 response results in epithelial cell damage rather than in the removal of H. pylori because H. pylori is not an intracellular pathogen. The ongoing presence of H. pylori thus causes a lifelong proinflammatory response coupled to cellular damage and initiates the histological cascade. The continuous production of reactive oxygen species that results from the ongoing inflammation can result in DNA damage, thus inducing the multiple mutations thought to be required for initiation of the cancer cascade depicted in Figure 1 (3,4). Among the new methods of magnifying endoscopy, a prototype of endocytoscopy developed by Olympus was used for ex vivo visualization of Helicobacter pylori on experimentally infected gastric biopsies. Moving bacteria were observed at 1100× magnification, giving hope for a possible direct detection during endoscopy. Kim et al. also used magnifying endoscopy on 103 patients to classify the gastric surface according to four patterns: flat, irregular, papillary or nonstructured, which were then compared to the updated Sydney System for histologic gastritis. Histologic gastritis was found in 91% of the biopsy sections with a nonflat type, and among them, 96% were confirmed to harbor H. pylori infection. In another study, the magnified endoscopic findings in the gastric body were classified into four patterns and then correlated with histology results. Type 1 pattern corresponded to normal gastric mucosa, types 2 and 3 to H. pylori -infected mucosa and type 4 to atrophy. The sensitivity and specificity for these endoscopic findings were 92.7% and 100% for type 1, and 100% and 92.7% for types 2 and 3 together, respectively (5

H. pylori detection
Various diagnostic assays for the detection of an H. pylori infection are available. Histological detection and culturing of the pathogen are gold standard, which require invasive gastroduodenoscopy to obtain gastric biopsy specimens. In the last decade, noninvasive approaches, such as serological detections, the [ 13 C] and [ 14 C]urea breath test (UBT), and detection H. pylori antigen or DNA in feces, helped and improved the evaluation of H. pylori infection status in patients. Because of low sensitivities of most serological assays for younger than 12 years of age patients, they are not suitable for pediatric. The UBT is a well-established noninvasive diagnostic assay and gives excellent performance for both adults and children, but its specificity decreases for infants and young children and need. In addition, the performance of UBT with infants and young children requires trained staff for air sampling with a face mask, and the test also requires expensive instruments, such as an isotope ratio mass spectrometer or an infrared isotope ratio spectrometer. Enzyme immunoassays (EIAs) for the identification of H. pylori antigens in fecal specimens circumvent these difficulties. EIAs based on monoclonal antibodies have shown consistent excellent results, with very high sensitivities and specificities for both adults and children. A major disadvantage of all the noninvasive tests described above is their inability to provide information on the susceptibility or resistance of H. pylori to antibiotics [2].

Macrolide resistance
H. pylori infection can be cured by antibiotics, however the ideal anti-H. pylori treatment has y e t t o b e f o u n d . M a n y f a c t o r s h a v e b e e n implicated in treatment failure, including ineffective penetration of antibiotics into the gastric mucosa, antibiotic inactivation by low gastric pH, lack of compliance, and emergence of acquired antibiotic resistance by H. pylori. Despite the success of the current anti-Helicobacter therapies, it is suggested that eradication rates among patients with gastritis are lower than among patients with peptic ulcer disease, with the causes of this phenomenon still being the subject of speculation [6]. The macrolide class of antimicrobial agents is over 30 years old and is still at the forefront of antimicrobial therapy as well as drug discovery and development. Clarithromycin is a recently approved 14-membered macrolide with increased stability in acid and improved pharmacokinetics, including the appearance of a microbiologically active metabolite in humans. Clarithromycin possesses broad-spectrum antimicrobial activity, inhibiting a range of gram-positive and gram-negative organisms, some anaerobes, and atypical pathogens, in many cases with greater in vitro activity than erythromycin [7]. Clarithromycin is a semi-synthetic macrolide antibiotic. Chemically, it is 6-0-methylerythromycin. The molecular formula is C 38 H 69 NO 13 , and the molecular weight is 747.96 ( Figure 2). Clarithromycin is a white to off-white crystalline powder. It is soluble in acetone, slightly soluble in methanol, ethanol, and acetonitrile, and practically insoluble in water.
Currently, a seven-day, triple-drug regimen has been recommended as one of the first-line therapies for H. pylori management. This treatment includes omeprazole (a proton-pump inhibitor), clarithromycin, and amoxicillin or metronidazole [2,8]. However, this therapy is being investigated because of increased eradication failures due to the prevalence of clarithromycin resistant H. pylori infections. Many studies have shown that between 0-50% of H. pylori isolates were clarithromycin resistant, which leads to a need for long term It is well known that the abuse of macrolide antibiotics including clarithromycin might lead to clarithromycin resistant forms of H. pylori [8]. Clarithromycin is a bacteriostatic antibiotic, which belongs to a group of macrolides bound to peptidyltransferase loop of domain V and VI of the 23S rRNA molecule (Figure 3 and 4). This binding interferes with protein elongation, and thus effectively blocks bacterial protein synthesis. The antibacterial activity of clarithromycin is similar to that of other macrolides, but clarithromycin is better absorbed in the gastric mucus layer, more acid-stable, and therefore more effective against H. pylori. Resistance to clarithromycin is thought to develop when substitutions in one nucleic acid at or near this binding site on the ribosome prevent the drug from binding, thereby making it ineffective [9]. Mutations A2144G, A2143G, A2142G and A2143C are the most often observed and reported by investigators, other mutations such as A2142C, A2115G, G2141A, A2142T and T2717C have been described but appear to be rare in peptidyltransferase [10,11].

Detection of clarithromycin resistance
Many diagnostic assays have been developed for Helicobacter pylori: culture, histology, rapid urease test, urea breath test, serology, stool antigen test, and molecular-based tests [2,5,10,11,15]. Culture has the great advantage of permitting subsequent determination of the antimicrobial susceptibility of the strain isolated, in particular to macrolides. However, disadvantages of culture include special conditions for specimen transportation, the use of complicated media with special conditions for maintenance, the need for special incubation conditions, and the length of time necessary to obtain a result [10]. In routine practice the detection of clarithromycin resistance is mainly based on phenotypic methods performed after culture: agar diffusion for the E-test or the agar dilution method, which is considered the reference; however, these methods are time-consuming [16]. The association between point mutations in the 23S rRNA gene and macrolide resistance in H. pylori potentially provides a new approach for diagnosing macrolide resistant H. pylori strains [9]. Numerous molecular-based methods are now available to assess clarithromycin  [2,[9][10][11]. Most assays are polymerase chain reaction (PCR)based using different methods to study the amplicons. The PCR-based molecular techniques are quicker than microbiological susceptibility testing, and more importantly, they can be performed directly on gastric biopsies and gastric juice [9]. The macrolide-binding pocket is located at the upper part of the ribosomal peptide exit tunnel, at a distance from the peptidyl transferase center that allows the accommodation of polypeptides of 5-6 residues. It is formed mainly by 23S rRNA domain V nucleotides, among which A2058 and A2059 play a prominent role in binding, selectivity, and resistance. (14) A PCR-based approach to diagnostic testing has the advantage over existing non-culturebased tests in that it is simple to perform and can provide additional genotypic information about the infecting strain, including markers associated with antibiotic susceptibilities. In addition, although the initial cost of equipment is high, the cost of reagents and consumables for each test is extremely low in comparison with corresponding costs for other methods such as the urea breath test and stool antigen test [17]. Great advantage of PCR is that it does not require viable bacteria. The transport conditions are thus not as critical as they are for culture, and shipment costs are cheaper. The cost of the reagents necessary for our technique is reasonable: $6.20 for H. pylori detection only and $8.60 for detection and clarithromycin susceptibility testing. By microbiological techniques, detection is cheaper, $1.70, but the price of detection with clarithromycin susceptibility testing by E-test, $6.90, is comparable to that of PCR [10]. Nested PCR generally increases sensitivity but also has a high risk of contamination. Real-time PCR has several advantages over conventional PCR, such as short working time, high specificity, and low risk of contamination. Thus far, some studies used real-time PCR targeting either ureC or 16S rRNA and 23S rRNA for the www.intechopen.com quantitative detection of H. pylori in gastric biopsies [11]. FISH allowed simultaneous detection of H pylori and the point mutations. Also, FISH could be directly applied to formalin fixed tissue sections without extensive preparation of nucleic acid. Another advantage of this technique compared with PCR based systems became obvious when mixed strains and tissue sections harboring more than one H. pylori strain were examined. A mixed culture cannot be unequivocally differentiated from a strain carrying two different rRNA operons by restriction enzyme analysis or filter hybridization; this can be done easily by whole cell hybridization technology [18]. PCR-RFLP has some disadvantage than other PCR base techniques including: i: when multiple strains are present in one sample, PCR-RFLP technique can not able to identify genotypes that has low concentration, ii: PCR-RFLP technique can not able to detect two mutations A2144G and A2143C, iii: Mixed populations containing a resistant strain associated with a susceptible strain are difficult to detect by PCR-RFLP. The resistant strain is often well detected because the mutation conferring resistance produces a restriction site that results in a new band on the electrophoresis profile. However, the uncut fragment corresponding to the susceptible clone is usually not taken into consideration. The uncut fragment is indeed more often attributed to an incomplete restriction than to a mixed population. Even for pure resistant strains, a band corresponding to the uncut fragment of DNA is sometimes still present on the electrophoresis profile due to partial restriction. Thus, a mixed population containing a susceptible strain associated with a resistant strain is detected as a single resistant strain [19]. A TaqMan probe is a short oligonucleotide (DNA) that contains a 5' fluorescent dye and 3' quenching dye. To generate a light signal (i.e., remove the effects of the quenching dye on the fluorescent dye), two events must occur. First, the probe must bind to a complementary strand of DNA. Second, Taq polymerase, the same enzyme used for the PCR, must cleave the 5' end of the TaqMan probe (5' nuclease activity), separating the fluorescent dye from the quenching dye ( Figure 5) (20).

Fig. 5. Schematic model for TaqMan probe technology
Culture has the great advantage of permitting subsequent determination of the antimicrobial susceptibility of the strain isolated, in particular to macrolides. However, disadvantages of culture include special conditions for specimen transportation, the use of complicated media with special conditions for maintenance, the need for special incubation conditions, and the www.intechopen.com length of time necessary to obtain a result [10]. Low bacterial density and a patchy distribution of the pathogen may have been the reasons for the negative culture and histology results. This may also be true for the patient determined to be negative by the rapid urease test, histology, and culture but positive by both PCR assays in the biopsy and the stool specimen, thus resulting in a reduction of the PCR specificity to 98% [11]. Culture is only semiquantitative and is time consuming. Histology is also semiquantitative, but its accurateness is relatively weak because of great interobserver variation. Urea breath test has been shown to be uncorrelated to culture-determined bacterial density [10].

Epidemiology of clarithromycin resistance
Resistance to clarithromycin is the main predictor of failure of eradication treatments including this compound. Because of an increased use of these macrolides, not only for H. pylori eradication, but also for the treatment of respiratory tract infections, the prevalence rate of resistant strains is increasing, and the detection of resistance is becoming of major importance. To render this detection process more effective, results must be available rapidly.
Several studies have shown that the eradication of H. pylori plays an important role in the treatment of some gastrointestinal diseases and the results of all currently used tests for bacterial detection can be affected by the treatment [21]. Therefore, a growing attention has been assigned to H. pylori antibiotic susceptibility monitoring, due to the increasing prevalence of antibiotic resistance worldwide. Nevertheless, it is possible that the different methods used (antibiogram vs. real-time PCR) could have played a role in these discordant data as bacterial culture is hampered by limitations, such as a low sensitivity, even in expert-hands [22]. Although H. pylori has two copy of 23S rRNA gene in own genome, but many authors suggested that more than one strain can present in one patient [11,[27][28][29]. Also many authors displayed multiple genotypes in one sample because: 1) A troubling aspect of resistance to some antibiotics by H. pylori is a phenomenon that has been given the name heteroresistance. This phenomenon has been previously observed for resistance to metronidazole [30]; 2) H. pylori is transformable bacterium and the transformation frequency for clarithromycin resistance of H. pylori was found to be approximately 2×10 -6 transformants per viable cell [31]; 3) More importantly, Taylor

Epidemiology of clarithromycin resistance mutations
Many studies aim to assessment of clarithromycin resistance rate were preformed around the world. This studies showed A2144G > A2142G > A2143G > A2142C > A2143C order for clarithromycin resistance point mutations (Table 3).

Problem statement
Following the recognition of the important pathogenic role of H. pylori infection in the development of gastroduodenal diseases, there has been a continuous search for improved eradication therapy (Occhilini et al., 1997). H. pylori culture, as well as antimicrobial susceptibility studies is difficult to perform as well as labor intensive. Moreover, although the culture method allows antimicrobial susceptibility testing for several antibiotics, only the susceptibilities of macrolides and, in particular, of clarithromycin are really useful since the last is a major predictor of treatment failure. Therefore, detection of clarithromycinresistant H. pylori will facilitate the choice of an appropriate eradication regimen

Aplication area
The characterization of resistance mechanisms in H. pylori and their easy detection will facilitate the choice of appropriate treatment regimens and ultimately the control of infection. PCR-RFLP can be used directly with biopsy specimens, thereby avoiding the requirement for time-consuming culture-based methods. This is particularly important for patients in whom a first eradication attempt has failed.

Study design
In order to assess the clarithromycin resistance rate, 23S rRNA point mutations responsible for clarithromycin resistance and effect of technique, region and date, 2 separate analyses were performed.

Analysis 1
Patients 263 consecutive patients with dyspeptic symptoms attending the endoscopy center of the gastroenterology department of the Hajar Hospital, Shahrekord, Iran, were enrolled in this study from July to December 2008. Patient-reported symptoms and endoscopic findings of pathologist were recorded at the time of the consultation by the pathologist help, and these data were obtained retrospectively for analysis. The number of participants who were ineligible or declined participation in the study were not recorded. For the purpose of analysis, three global variables were created: 1) patient-reported included age, gender and symptoms, 2) clinical signs, and 3) clarithromycin resistance data. Patientreported symptoms included pain, anorexia, heaviness after meal, early satiety, nausea, vomiting and flatulence. Clinical signs were included gastric ulcer, gastric cancer, non-ulcer disease, gastic erosion, nodularity, gastritis and duodenit. All patients read and signed an 'informed consent' form at the beginning of endoscopy declared their satisfaction for application of their anonymous data for research purpose. Three biopsy specimens were taken from antrum and corpus of each patient, using a disinfected endoscope. Biopsy samples were placed in 0.1 ml of sterile saline solution and sent to the Biotechnology research center of Shahrekord Azad University. A rapid test for the detection of urease activity was performed by Gastro Urease kit (Bahar-afshan, Iran) according to manufacturer's instructions. DNA was isolated from each tissue with a DNA extraction kit (DNP TM , CinnaGen, Iran) according to the manufacturer's instruction and immediately used for molecular analysis.

Bacteria and culture conditions
Biopsy samples were cultured on Brucella agar (Merck) supplemented with 7% fresh horse blood, vancomycin (6mg/L, Merck), trimethoprim (5mg/L, Merck) and amphotericin (2mg/L, Merck). For primary culture, plates were incubated at 37°C in a microaerophilic atmosphere (5% O 2 , 15% CO 2 , 80%N 2 ) for 3 to 5 days. Strains were identified according to colony morphology, Gram staining and positive reactions with urease, catalase, and oxidase. The ureC gene (glmM) which encodes urease was used as a DNA target to confirm H. pylori strain.

Antimicrobial susceptibility testing
The susceptibilities of the H. pylori isolates to clarithromycin were examined by an agar dilution method according to CLSI (Clinical and Laboratory Standard Institute) protocol (32). Resistance breakpoint for clarithromycin was defined as the >4 µg/liter.

Conventional PCR assays
A PCR assay targeted at the ureC (glmM) gene of H. pylori was performed with specific primer ( , was held for 5 min at a denaturation temperature of 95°C, followed by 30 cycles of 1 min each at a denaturation temperature of 95°C, an annealing temperature of 58°C, and an elongation temperature of 72°C and by 5 min at 72°C. The amplified fragment was visualized after electrophoresis on a 1.5% agarose gel stained with ethidium bromide.

PCR-RFLP analysis
A 1,400-bp fragment of the 23S rRNA gene was amplified with primers Cla-18 and Cla-21 [44] (Table 3). PCR amplification of DNA was performed in a final volume of 24 µl PCR mixture, containing 2 µl of extracted DNA, 200 mM dNTPs, 0.2 mM (each) primer, 1.5 mM MgCl 2 , and 1 U of Taq polymerase in PCR buffer. Amplification was carried out in an eppendorf mastercycler gradient over 30 cycles, each for 1 min at 95°C, 1 min at 62°C, and 1 min at 72°C. These cycles were performed after a denaturation of 5 min at 95°C and a final elongation step at 72°C for 5 min. The amplicon was digested with BsaI for 1 h at 37°C and MboII (Fermentas GMBH, Germany) for 1 h at 37°C to detect the restriction site occurring when the mutation was A to G transition at 2143 or 2142, respectively ( Figure 5). The restriction products were analyzed by electrophoresis on a 2% agarose gel.

Sample collection
200 biopsy samples were obtained over a 6-month period (June 2009 to November 2009) from 200 dyspeptic patients referred for endoscopy at Hajar hospital in Iran. All patients read and signed an 'informed consent' form at the beginning of endoscopy declared they are satisfied with application of their anonymous data for research purpose. Every patient history sheet was examined in detail and clinical findings including demographic data were recorded. The mean age of the patients was 52.5 years (range, 17 to 88 years), and 48.1% of the patients were men. Rapid urease test was performed with a Gastro urease kit (baharafshan, Iran). DNA was isolated from each tissue with a DNA extraction kit (DNP TM , CinnaGen, Iran) according to the manufacturer's instruction and immediately used for molecular analysis. Conventional PCR for detection of ureC gene and PCR-RFLP analysis for detection of point mutations were performed as in analysis 1. Fig. 6. PCR-RFLP patterns obtained after digestion with BsaI or MboII. BsaI cuts the PCR product of the wild-type sequence into two fragments of 1,000 and 400bp and that of the A2143G sequence into three fragments of 700, 400, and 300bp. MboII cuts the PCR product into two fragments of 700bp only when A2142G is present in the sequence: 1)A2142G positive control, 2)A2142G negative control, 3)A2142G positive strain, 4)A2143G positive control, 5)A2143G negative control, 6)A2143G positive strain

Real-time (TaqMan) PCR assay
The real-time PCR was performed by using primer pair HP23S-1 and HP23S-2 and modified probes Pwt, P44G, P43G and P43C that previously reported by Pina et al [33] and newly designed probe P42G according to 23S rRNA gene sequence (GenBank accession no. U27270) for identification of wild type, A2144G, A2143G, A2143C and A2142G genotypes respectively ( Table 3).  . In order to test the specificity of the primers purified DNA of non-H. pylori strains was also used as a template for PCR.

Quantification real-time PCR
In order to made standard serial dilution, standard density of bacteria was prepared. After DNA extraction, one 10-fold serial dilution of H. pylori DNA was made, with bacterial concentrations ranging from 5×10 2 to 5×10 7 bacteria per 1 µl. Bacterial quantification was performed by TaqMan probe technology of real-time PCR as described above.

Statistical analysis
Statistical analyses were conducted with chi-square using the SPSS for Windows (version 17; SPSS, Inc., Chicago, Illinois, USA

Prevalence of clarithromycin resistance
According to the agar dilution method, 19 of 84 (22.62%) cultured strains were resistant to clarithromycin. A 1.4kb fragment of the 23S rRNA gene could be amplified in all 19 strains. Furthermore, MboII cuts PCR products were obtained from 3 and BsaI cuts another 13 amplified fragments of the resistant strains. In one strain, neither MboII nor BsaI was able to digest the amplicon. However, specially primed mismatched PCR yielded an amplicon, indicating that this strain had the A2142C mutation ( Figure 9). No correlation was observed between the clarithromycin resistance, patient gender and clinical findings.

Analysis 2
Clinical features

Detection of H. pylori directly in gastric biopsy samples
RUT results showed that the 164 (82%) of the patients were H. pylori-positive. DNA samples derived from gastric biopsy samples of confirmed H. pylori-positive patients were positive by the diagnostic PCR assays for the ureC targets. The ureC PCR assay confirmed the presence of H. pylori in all of these 164 biopsy samples (100%) and generated the expected PCR product of 249 bp.

PCR-RFLP
Thirty-nine of 59 resistant strains detected by real-time PCR methods were detected by PCR-RFLP and were distributed as follows: 15 A2143G and 15 A2142G single mutation, 6 wild type/A2143G, one wild type/A2142G and 2 A2143G/A2142G genotypes (Table 6). BsaI cuts the PCR product of the wild-type sequence into two fragments of 1,000 and 400bp and that of the A2143G sequence into three fragments of 700, 400, and 300bp. MboII cuts the PCR product into two fragments of 700bp only when A2142G is present in the sequence ( Figure  6). sensitivity and specificity of PCR-RFLP were 74.68% and 100% respectively when we PCR-RFLP was compared with real-time PCR.

Genotype Outcome sample (%)
Real-time-PCR PCR-RFLP   Table 8. Interview data and bacterial count of H. pylori positive patients (n=164)

Determination of the cut-off for the C T values
The detection of fluorescence was realized in the different channels of the rotor-gene thermocycler during 45 cycles of amplification. Among the 164 biopsy specimens, a detection of fluorescence was positive in one of the different channels in 143 (87.2%) cases. Sixteen biopsy specimens displayed more than one C T values for mix genotypes. C T values ranged from 21.4 to 43.6. C T values less than 35 determined as pure sample DNA and C T between 35 and 40 indicate as mix DNA sample. Detection of fluorescence over 40 cycles might be false positive ( Figure 10).

Development of TaqMan real-time PCR
Concerning amplification of H. pylori DNA obtained from a pure culture, linearity was achieved over a 5-log range of input DNA amounts, from 5×10 2 to 5×10 7 bacteria, with 500 bacteria corresponding to 1000 23S rRNA gene copies given that there are two gene copies in the genome of H. pylori. PCR was also positive for 40 bacteria or 80 copies of the 23S rRNA www.intechopen.com gene fragment. The amplification efficiencies obtained with DNA prepared from gastric biopsy specimens were identical to those obtained with DNA samples prepared from bacterial colonies. Thus, the sensitivity of our assay, for DNA samples prepared either from cultures or from gastric biopsy specimens, could quantitatively reach 400 bacteria or 800 copies of the amplicon and qualitatively reach 40 bacteria or 80 copies. The reproducibility of our assay was evaluated with 10-fold serial dilutions of purified H. pylori DNA, with no significant difference between runs.

Conclusion
In conclusion, we have developed a TaqMan real-time PCR assay that permits accurate, fast, and cost-effective detection of H. pylori directly from gastric biopsy specimens as well as detection of clarithromycin resistance. This PCR technique is a good candidate for automated real-time PCR methods allowing simple and rapid detection of H. pylori and its resistance to clarithromycin by clinical laboratories which do not practice H. pylori culture. Identification of sensitive and resistant strains by using PCR-RFLP method was based on the determination of resistant strains. Therefore, other resistant strains, which mutations cannot be identified by PCR-RFLP method, are considered as sensitive. But, Identification of sensitive and resistant strains by using real-time PCR method was based on the determination of sensitive strains. In the first step, determination of sensitive, resistance and mix of sensitive and resistance strains were done and in the next step, determination of mutations types in resistance and mix strains were done, that leading to accurate diagnosis of the resistant and sensitive strains. Based on the patterns of competitive growth as well as the individual growth of different clarithromycin resistance mutant strains, Wang and colleagues conclude that the order of preference of competitive accumulation is A2142G A2143G A2142C A2143C (A2142T). If the same is true in vivo, once an A-to-G transient mutation occurs (spontaneously or drug induced), the other types of mutation that exist in the same environment, if any, are likely to be overgrown after a period of time. A-to-C or A-to-T mutants could be isolated only when an A-to-G mutant has not appeared at that particular gastric niche. Their results provide a rational explanation for the mutation pattern observed in clinical isolates [13]. These results are confirmed by Van der Ende and coworkers and discussed that the growth rates of H. pylori isolates with the A21423G, A21423C, or A21433G mutation did not differ from that of the wild type, but H. pylori isolates with other 23S rDNA mutations grew more slowly [11]. Based on two opinions, we used real-time PCR for simultaneous identification of H. pylori, Clarithromycin resistance point mutations and direct quantification of gastric mucosal density in order to answer to this question; why some clarithromycin resistance point mutations are more prevalent than other mutations. At first; Wang described an additional possible mechanism yet to be identified, by which the A-to-G mutations are preferentially produced in H. pylori, may also contribute to the observed predominance of A-to-G mutations [13]. Second; Van der Ende [11] described that; If only a single colony from the primary H. pylori populations is used to test for clarithromycin susceptibility, the results can be misinterpreted. Assessment of 23S rRNA mutations in H. pylori directly from biopsy specimens by molecular biological techniques has the advantage that infection with a mixed H. pylori population is easily detected. In addition, knowledge of the type of 23S rRNA www.intechopen.com mutation may be important since clarithromycin MICs are associated with the type of 23S rRNA mutation in H. pylori [11]. Several studies which done by Pina [34], Marais [28] and Russmann [36] in Europe revealed that A2144G, A2143G and A2143C were more prevalent in Europe respectively. However, the current study showed that A2143G, A2142G, A2143C and A2144G were more prevalent mutation in Iran respectively. Our data showed that wild type strains more related to 10 6 to 10 7 bacteria in gastric biopsy specimens of patients. Also, strains with clarithromycin resistance mutation assessed in this study are significantly related to 10 6 in gastric biopsy which are equal to density of wild type strain. This data revealed that these mutated strains has a same growth rate to wild type, preferentially produced in populations and selected by natural selection force and they can exist and distributed in H. pylori population