**7.4 Analysis 2**

110 Gastrointestinal Endoscopy

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% O2, 15% CO2, 80%N2) 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* 

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

A PCR assay targeted at the *ureC* (*glmM*) gene of *H. pylori* was performed with specific primer (Table 4) in an eppendorf mastercycler gradient (eppendorf, Germany). Briefly, the 23 µl PCR mixture, containing 1 µl of extracted DNA, 200 mM (each) deoxynucleoside triphosphates (dNTPs) (dNTP Mix, CinnaGen, Iran), 0.2 mM (each) primer (CinnaGen, Iran), 1.5 mM MgCl2, and 1 U of *Taq* polymerase (CinnaGen, Iran) in PCR buffer (CinnaGen, Iran), 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

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 MgCl2, 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

Fig. 5. Schematic diagram for detection of nucleotide alterations of *23S rRNA* by PCR-RFLP

(32). Resistance breakpoint for clarithromycin was defined as the >4 µg/liter.

after electrophoresis on a 1.5% agarose gel stained with ethidium bromide.

restriction products were analyzed by electrophoresis on a 2% agarose gel.

**Bacteria and culture conditions** 

**Antimicrobial susceptibility testing** 

**Conventional PCR assays** 

**PCR-RFLP analysis** 

assays

strain.

### **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 (DNPTM, 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 *Bsa*I or *Mbo*II. *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).

The real-time (TaqMan) PCR mixture was prepared until reaching a final solution of 25 µl containing 2 µl of extracted DNA, 200 mM dNTPs, 0.2 mM (each) primer HP23S-1 and HP23S-2, 0.1 mM probe Pwt, 0.2mM probe P44G, 0.1 mM probe P43G, 0.1 mM probe P43C and 0.75 mM probe P42G (Bioneer,Tokyo, Korea) (Table I), 1.5 mM MgCl2, and 1 U of *Taq*

Clarithromycin Resistance and *23S rRNA* Mutations in *Helicobacter pylori* 113

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%

*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

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].

2 2

A2143G A2142G A2142C unknown

1

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

1

female male

mutation

0 0

0 10 20 30 40

Gastritic Cancer

Duodenit Ulcer

Fig. 8. Prevalence of clinical signs in analysis 1

esophagitis, and 5% gastric cancer (Figure 7, 8).

**Culture, rapide urease test and PCR** 

and 29 of 62 (47%) with peptic ulcer (PU).

8

5

**8. Results 8.1 Analysis 1 Clinical features**  Peptic Ulcer

Gastritis

polymerase in PCR buffer 1X. Real-time PCR analysis was performed with an Rotor-Gene 6000 (Corbett Research, Australia). The conditions of PCR amplification were 95°C for 5 min and 45 cycles of 95°C for 30 s and 58°C for 40 s. All samples were repeated twice and positive and negative controls were enclosed in each assay. Data were analyzed with rotorgene 6000 software *ver1.7* (Corbett research). 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 5×102 to 5×107 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). *P*-values less than 0.05 were taken to indicate statistical significance.


Table 4. Oligonucleotides sequence used in this study

Fig. 7. Prevalence of patients reported symptoms in analysis 1

Fig. 8. Prevalence of clinical signs in analysis 1
