**4. 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

assessment of the efficacy of clarithromycin in the triple-drug regimen. 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

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

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 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

Fig. 2. The structural formula of clarithromycin

effectively blocks bacterial protein synthesis.

be rare in peptidyltransferase [10, 11].

**4. Detection of clarithromycin resistance** 

the reference; however, these methods are time-consuming [16].

Fig. 3. Model of domain V and VI of 23S rRNA from *E. coli*, (12 , 13)

Clarithromycin Resistance and *23S rRNA* Mutations in *Helicobacter pylori* 105

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].

quenching dye (Figure 5) (20).

Fig. 5. Schematic model for TaqMan probe technology

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

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

in *H. pylori*, such as PCR-RFLP, PCR-OLA, PCR-DEIA, PCR- LiPA, PCR-PHFA, 3M-PCR, real-time PCR, FISH, FRET, DNA sequencing by conventional and real-time (pyrosequencing) techniques [2, 9-11]. 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].

Fig. 4. A view into the macrolide-binding pocket in *E. coli* highlighting the specific geometry of the 2611–2057 base pair. H bonds are shown as dotted lines. 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

in *H. pylori*, such as PCR-RFLP, PCR-OLA, PCR-DEIA, PCR- LiPA, PCR-PHFA, 3M-PCR, real-time PCR, FISH, FRET, DNA sequencing by conventional and real-time (pyrosequencing) techniques [2, 9-11]. 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

Fig. 4. A view into the macrolide-binding pocket in *E. coli* highlighting the specific geometry of the 2611–2057 base pair. H bonds are shown as dotted lines. 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

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

A2059 play a prominent role in binding, selectivity, and resistance. (14)

performed directly on gastric biopsies and gastric juice [9].

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

Clarithromycin Resistance and *23S rRNA* Mutations in *Helicobacter pylori* 107

**Country Year Technique Sample No. Resistance rete** Ref Australia 2004 Real-time PCR 92 24% 8 France 1994-1999 E-test 150 21% 32

France 2000 E-test 61 18% 34 France 2003 Real-time PCR 200 67% 11 France 2003 Real-time PCR 196 18.5% 7 France 2004-2008 Real-time PCR 126 20% 35 Germany 2001 FISH 109 31.2% 36 Germany 1999-2002 E-test 1233 20% 37 Netherland 2001 RAPD-PCR 976 5.2% 8 Italy 2003 PCR-RFLP 283 1.6% 12 Italy 2003 PCR-RFLP & E-test 230 14% 38 Ireland 2001 LiPA 50 26% 39 Spain 2008 E-test 118 35.6% 40 Brazil 2001 Agar Diliution 202 9.85% 41 Brazil 2003 Agar Dilution 155 16% 3 Mexico 1995-1997 E-test 195 24% 42 Peru 1995 Egg Yolk Agar 18 50% 43 Argentina 2000 PCR-RFLP 96 23.9% 44 USA 1996 PCR-OLA 72 55% 45 China 2001 Primer mismatch PCR 96 5% 16 China 200-2009 Agar Dilution 293 8.6%-20.7% 46 Japan 1998 Seminested PCR 85 9% 47 Japan 1999 PCR-RFLP 79 6.3% 22 Japan 2000 PCR-PHFA 412 22% 48 Japan 2001 Real-time PCR 186 21.2% 49 Japan 2001 PCR-RFLP 51 29% 50 Malaysia 2005-2007 E-test 187 2.1% 51 Indonesia 2006 Disk diffusion 126 27.8% 52 Korea 2004 PCR-RFLP 114 20.2% 53 Iran 2007 PCR-RFLP 263 22.6% 54 Iran 2009 PCR-RFLP 200 23.78% 55 Iran 2009 Real-time PCR 200 35.98% 55 Iran 2008-2009 E-test 121 5% 56 Tunisa 2005-2007 Real-time PCR 273 14.6% 57 Cuba 2005 E-test 46 10% 58

Table 2. Prevalence of clarithromycin resistance in the world. Comparison between date,

technique and region

Liquid Phase 41 56% 33

Immunoassay 61 37.7% 21

France 1998 Hybridization in

France 1999 PCR-DNA Enzyme

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].

## **5. 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. Rates of clarithromycin resistance in *H. pylori* strains have been reported in France 21%, Spain 28.3%, Portugal 44.8%, Poland 23.5%, Mexico 21.6%, China 5%, Korea 16.7%, Italy 26.7% and Germany 47% [5, 15, 22-24]. A similar geographical distribution has been observed in Iran, with clarithromycin resistance values ranging from 21% [25] to 35.98% .This relevant discrepancy could be explained not only by the different geographic distribution of resistances, but also by the use of different methodologies to assess clarithromycin resistant status. For example, a study recently performed in the same geographic area (Shahrekord, Iran) has reported clarithromycin resistance rate 22.62% [26] distinctly lower compared with our present experience (35.98%). 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, 27-29]. 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 and et al. suggests that acquisition of antibiotic resistance could result from the horizontal transfer of clarithromycin resistance determinants from resistant cells to susceptible cells of the same strain, probably increasing the population of resistant strains. In addition, such genetic transfer could occur between different strains, since mixed infections with different *H. pylori* strains does occur in some individuals [31].

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

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

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. Rates of clarithromycin resistance in *H. pylori* strains have been reported in France 21%, Spain 28.3%, Portugal 44.8%, Poland 23.5%, Mexico 21.6%, China 5%, Korea 16.7%, Italy 26.7% and Germany 47% [5, 15, 22-24]. A similar geographical distribution has been observed in Iran, with clarithromycin resistance values ranging from 21% [25] to 35.98% .This relevant discrepancy could be explained not only by the different geographic distribution of resistances, but also by the use of different methodologies to assess clarithromycin resistant status. For example, a study recently performed in the same geographic area (Shahrekord, Iran) has reported clarithromycin resistance rate 22.62% [26] distinctly lower compared with our present experience (35.98%). 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

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, 27-29]. 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 and et al. suggests that acquisition of antibiotic resistance could result from the horizontal transfer of clarithromycin resistance determinants from resistant cells to susceptible cells of the same strain, probably increasing the population of resistant strains. In addition, such genetic transfer could occur between different strains, since mixed infections with different *H. pylori* strains does occur in

been shown to be uncorrelated to culture-determined bacterial density [10].

**5. Epidemiology of clarithromycin resistance** 

limitations, such as a low sensitivity, even in expert-hands [22].

rapidly.

some individuals [31].


Table 2. Prevalence of clarithromycin resistance in the world. Comparison between date, technique and region

Clarithromycin Resistance and *23S rRNA* Mutations in *Helicobacter pylori* 109

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 clarithromycin-

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

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

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

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

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 (DNPTM, CinnaGen, Iran) according to the manufacturer's instruction and

resistant *H. pylori* will facilitate the choice of an appropriate eradication regimen

patients in whom a first eradication attempt has failed.

ineligible or declined participation in the study were not recorded.

application of their anonymous data for research purpose.

immediately used for molecular analysis.

**7. Problem statement** 

**7.1 Aplication area** 

**7.2 Material and methods** 

**Study design** 

were performed.

**7.3 Analysis 1** 

**Patients** 

Currently, many research aims to evaluation of clarithromycin resistance in *H. pylori* were performed which showed variety of resistance rate around the world, due to 3 main reasons including differences in i: methods, ii: region and iii: date (Table 2).

Many investigators were assessed clarithromycin resistance rate in Europe in 1994 to 2009. In these studies, clarithromycin resistance was more prevalent in France. Also, Fontana et al by PCR-RFLP method showed that clarithromycin resistance was more divers between north (1.6%) and south (14%) of Italy in 2003.

In continent of America, clarithromycin resistance was varies between 9.8% in Brazil to 55% in USA. Less variety were observed in eastern Asia, by 20% to 27% resistance rate. In Iran (Middle East) with variety of customs, Kargar et al showed resistant rate of 23% and 36% in same geographic region in 2009 due to different methods including PCR-RFLP and real-time PCR assays respectively but, PCR-RFLP showed 22% and 23%resistance rate in 2007 and 2009 respectively. Also more variation was observed in 2 near region with different customs and economic level, with 36% in low economic level and 5% in high economic level.
