**8.2 Analysis 2**

#### **Clinical features**

164 patients with median age of 51.5 ranged from 15 to 88 years. Pain was present in 103 patient, nausea in 42, anorexia in 40, acid reflux in 33, heaviness after meal in 30, early satiety in 27, vomiting in 31 and flatulence in 42. The main endoscopic findings were 16 gastric ulcer, 3 gastric cancer, 55 non-ulcer disease, 50 gastric erosion, 34 nodularity, 37 gastritis and 3 duodenitis (Table 5).


Table 5. Distribution of *H. pylori* positive patients according to gender and age group (*n=164*)

#### **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.

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

164 patients with median age of 51.5 ranged from 15 to 88 years. Pain was present in 103 patient, nausea in 42, anorexia in 40, acid reflux in 33, heaviness after meal in 30, early satiety in 27, vomiting in 31 and flatulence in 42. The main endoscopic findings were 16 gastric ulcer, 3 gastric cancer, 55 non-ulcer disease, 50 gastric erosion, 34 nodularity, 37

> **Age group Male Female Total**  10-20 6 (3.66) 4 (2.44) 10 (6.10) 20-30 12 (7.32) 13 (7.93) 25 (15.24) 30-40 13 (7.93) 15 (9.15) 28 (17.07) 40-50 13 (7.93) 14 (8.54) 27 (16.46) 50-60 10 (6.10) 11 (6.71) 21 (12.80) 60-70 12 (7.32) 13 (7.93) 25 (15.25) 70-80 9 (5.49) 6 (3.66) 15 (9.15) 80-90 4 (2.44) 9 (5.49) 13 (7.93) Total 79 (48.17) 85 (51.83) 164 (100)

Table 5. Distribution of *H. pylori* positive patients according to gender and age group

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

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

**Detection of** *H. pylori* **directly in gastric biopsy samples** 

PCR-RFLP was compared with real-time PCR.

between the clarithromycin resistance, patient gender and clinical findings.

**Prevalence of clarithromycin resistance** 

gastritis and 3 duodenitis (Table 5).

**8.2 Analysis 2 Clinical features** 

(*n=164*)

**PCR-RFLP** 

PCR product of 249 bp.


Table 6. Frequency of *H. pylori* genotypes determined by real-time PCR and PCR-RFLP methods in analysis 2 (n=164)

Fig. 10. Graph obtained with real-time PCR for the susceptible and resistant strains isolated from different patients. *CT* for pure samples were less than 35 and *CT* of mix samples were between 35 and 40. (All graphs obtained by different probes are similar)

Clarithromycin Resistance and *23S rRNA* Mutations in *Helicobacter pylori* 117

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

Bacterial clarithromycin resistance was assessed on 164 consecutive, *H. pylori*-positive patients. Overall, a clarithromycin susceptible was detected in 105 (64.02%) patients and clarithromycin resistance was detected in 59 (35.98%) which were identified as 4 (6.78%) A2144G, 26 (44.07%) A2143G, 15 (25.42%) A2143C and 20 (33.9%) A2142G point mutations. Purely resistant strains were detected in 38 (64.41%), while a mixture of resistant and susceptible (heteroresistant) bacterial strains were found in the remaining 16 (27.12%) cases.

Quantification of different genotypes was directly performed on 159 DNA samples with defined genotype by using TaqMan real-time-PCR assay. The bacterial density by this technique could be evaluated for 159 *H. pylori*-positive patients and ranged from 1.53×104 to 5.89×1012. In order to evaluate relationship between bacterial concentration and point mutations, samples divided in 9 groups between 1×104 to 6×1012, including 104, 105, 106, 107, 108, 109,1010, 1011 and 1012 groups had 6, 11, 87, 59, 9, 2, 2, 2 and 2 samples respectively (Table

> 104 1 0 2 2 1 105 7 0 2 1 1 106 39 4 18 12 14 107 51 0 4 0 4 108 9 0 0 0 0 109 2 0 0 0 0 1010 2 0 0 0 0 1011 2 0 0 0 0 1012 2 0 0 0 0 Total 105 4 26 15 20

Chi-square analysis revealed more relationship between gastritis and age group 30-40 (*p*=0.007), NUD and 40-50 (*p*=0.001), early satiety and 10-20 (*p*=0.05), flatulence and 10-20

Table 9. Distribution of 5 different genotypes based on their concentrations

**type A2144G A2143G A2143C A2142G** 

significant difference between runs.

**Quantification of bacterial density** 

**Bacterial count** 

**Statistical analysis** 

Genotype of 5 (8.47%) strains were not detected (Table 6) [60].

**Wild** 

**Real-time PCR** 

7 and 8).


Table 7. Endoscopic finding and frequency of bacterial count of *H. pylori* positive patients (*n=164*)


Table 8. Interview data and bacterial count of *H. pylori* positive patients (*n=164*)

#### **Determination of the cut-off for the CT 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 *CT* values for mix genotypes. *CT* values ranged from 21.4 to 43.6. *CT* values less than 35 determined as pure sample DNA and *CT* 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 5×102 to 5×107 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*

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.

#### **Real-time PCR**

116 Gastrointestinal Endoscopy

**Disease 104 105 106 107 108 109 1010 1011 1012 Total Peptic Ulcer** 1 (0.6) 0 (0) 7 (4.27) 4 (2.44) 2 (1.22) 1 (0.6) 0 (0) 0 (0) 0 (0) 15 (9.15) **Gastric Cancer** 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0.6) 0 (0) 2 (1.22) 3 (1.83) **Gastric Erosion** 1 (0.6) 2 (1.22) 20 (12.19) 18 (10.98) 5 (3.05) 1 (0.6) 0 (0) 2 (1.22) 0 (0) 49 (29.88) **Nodularity** 2 (1.22) 4 (2.44) 13 (7.93) 11 (6.71) 3 (1.83) 0 (0) 0 (0) 0 (0) 0 (0) 33 (20.12) **Gastritis** 3 (1.83) 3 (1.83) 15 (9.15) 13 (7.93) 1 (0.6) 0 (0) 1 (0.6) 0 (0) 0 (0) 36 (21.95) **Duodenitis** 0 (0) 1 (0.6) 1 (0.6) 1 (0.6) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 3 (1.83) **Atrrophy** 0 (0) 0 (0) 0 (0) 2 (1.22) 1 (0.6) 0 (0) 0 (0) 0 (0) 0 (0) 3 (1.83) **Duodenit Ulcer** 0 (0) 0 (0) 13 (7.93) 8 (4.88) 1 (0.6) 0 (0) 0 (0) 0 (0) 0 (0) 22 (13.41)

Table 7. Endoscopic finding and frequency of bacterial count of *H. pylori* positive patients

**Symptoms 104 105 106 107 108 109 1010 1011 1012 Total Pain** 3 (1.83) 9 (5.49) 42 (25.61) 32 (19.51) 6 (3.66) 2 (1.22) 2 (1.22) 2 (1.22) 1 (0.6) 99 (60.36) **Nausea** 3 (1.83) 1 (0.6) 20 (12.19) 13 (7.93) 3 (1.83) 0 (0) 0 (0) 1 (0.6) 1 (0.6) 42 (25.61) **Anorexia** 3 (1.83) 3 (1.83) 18 (10.98) 14 (8.54) 0 (0) 1 (0.6) 0 (0) 0 (0) 0 (0) 39 (23.78) **Acid reflux** 4 (2.44) 1 (0.6) 12 (7.32) 9 (5.49) 4 (2.44) 0 (0) 0 (0) 0 (0) 1 (0.6) 31 (18.90)

**after meal** 1 (0.6) 3 (1.83) 9 (5.49) 12 (7.32) 3 (1.83) 0 (0) 1 (0.6) 0 (0) 0 (0) 29 (17.68) **Early satiety** 2 (1.22) 1 (0.6) 13 (7.93) 8 (4.88) 1 (0.6) 0 (0) 1 (0.6) 0 (0) 0 (0) 26 (15.85) **Vomiting** 3 (1.83) 2 (1.22) 12 (7.32) 10 (6.10) 1 (0.6) 0 (0) 0 (0) 0 (0) 1 (0.6) 29 (17.68) **Flatulence** 3 (1.83) 4 (2.44) 19 (11.58) 11 (6.71) 1 (0.6) 0 (0) 1 (0.6) 0 (0) 1 (0.6) 40 (24.39)

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 *CT* values for mix genotypes. *CT* values ranged from 21.4 to 43.6. *CT* values less than 35 determined as pure sample DNA and *CT* between 35 and 40 indicate as mix DNA sample. Detection of fluorescence over 40 cycles

Concerning amplification of *H. pylori* DNA obtained from a pure culture, linearity was achieved over a 5-log range of input DNA amounts, from 5×102 to 5×107 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*

Table 8. Interview data and bacterial count of *H. pylori* positive patients (*n=164*)

**Determination of the cut-off for the CT values** 

might be false positive (Figure 10).

**Development of TaqMan real-time PCR** 

(*n=164*)

**Heaviness** 

Bacterial clarithromycin resistance was assessed on 164 consecutive, *H. pylori*-positive patients. Overall, a clarithromycin susceptible was detected in 105 (64.02%) patients and clarithromycin resistance was detected in 59 (35.98%) which were identified as 4 (6.78%) A2144G, 26 (44.07%) A2143G, 15 (25.42%) A2143C and 20 (33.9%) A2142G point mutations. Purely resistant strains were detected in 38 (64.41%), while a mixture of resistant and susceptible (heteroresistant) bacterial strains were found in the remaining 16 (27.12%) cases. Genotype of 5 (8.47%) strains were not detected (Table 6) [60].
