**Clinical features**

Abdominal pain was present in 76% of patients (200/263), dyspepsia in 8%(20/263), vomiting in 6%(16/263), heartburn in 6%(17/263), and weakness 4%(10/263). The main endoscopic findings were 36% gastritis, 13% duodenal ulcer, 10% gastric ulcer, 9% esophagitis, and 5% gastric cancer (Figure 7, 8).

#### **Culture, rapide urease test and PCR**

*H. pylori* could be cultured from 84 of 263 (32%) patients, while a positive RUT or PCR band was observed in 54%(143 of 263) and 85%(223 of 263) of patients, respectively. Of the 84 patients with positive *H. pylori* culture 35/84 (41.7%) were males and 49/84 (58.3%) females. *H. pylori* was successfully cultured from 55 of 135 (41%) patients with non-ulcer dyspepsia and 29 of 62 (47%) with peptic ulcer (PU).

When the PCR was regarded as the "gold standard" of *H. pylori* identification, the sensitivity, specificity, PPV and NPV of RUT were 61%, 87%, 96% and 29% respectively. But for culture method were 36.77%, 95%, 97% and 21.22% respectively [59].

Fig. 9. Frequency of *23S rRNA* mutations in analysis 1 (n=19)

Clarithromycin Resistance and *23S rRNA* Mutations in *Helicobacter pylori* 115

105 (64.01) Wild Wild 3 (1.83) A2144G Wild 13(7.93) A2143G A2143G 7(4.27) A2143C Wild

15(9.15) A2142G A2142G 6(3.66) A2143G/Wild A2143G/Wild 1(0.61) A2143C/Wild Wild 1(0.61) A2142G/Wild A2142G/Wild 1(0.61) A2143G/A2143C/Wild Wild

1(0.61) A2143G/A2143C A2143G 1(0.61) A2143G/A2143C Wild 1(0.61) A2143G/A2142G A2143G/A2142G 1(0.61) A2143C/A2142G Wild 1(0.61) A2143G/A2143C/A2142G A2143G/A2142G 1(0.61) A2143G/A2143C/A2142G Wild

5(3.05) Not Determined Wild

**0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44**

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)

1(0.61) A2144G/A2143G/A2143C/

Table 6. Frequency of *H. pylori* genotypes determined by real-time PCR and PCR-RFLP

**Real-time-PCR PCR-RFLP** 

Wild A2143G

**Cycle Number**

**Genotype Outcome sample (%)**

Single genotype 143 (87.19)

Heteroresis tance 21 (12.81)

methods in analysis 2 (n=164)

 **5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5**

**Fluorescence**

#### **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.
