Prevalence of H. pylori Isolates and Genotypes and CagA Gene

Prevalence of H. pylori Isolates and Genotypes and CagA Gene 1. Introduction The best-known gastric Helicobacter species is Helicobacter pylori which is a slightly curved, microaerophilic Gram-negative. This microorganism has established a niche in the stomach of more than half of the world’s human population and become a dominant species of the human gastric microbiome. H. pylori colonization causes persistent inflammatory responses.H. pylori-induced gastritis is the strongest singular risk factor for cancers of the stomach however only a small proportion of infected individuals proceed to the development of malignancy. It is still unknown what factors are responsible for the different outcomes ranging from asymptomatic gastritis, peptic ulcer disease (PUD) to gastric cancer (GC). The changes in the epidemiology of these different demonstration of H. pylori infection suggest an environmental factor, an interaction between environmental factor and the host, or a change in prevalence of strains differing in virulence H. pylori has two phenotypic charact eristics among strains are the vacuolating cytotoxin (vac) and cytotoxic associated gene A (vacA) protein which encoded by the and cagA genes respectively. cagA and genes are considered as candidates for the identification of strains with enhanced virulence. For instance, gastric mucosa from patients infected with cagApositive H. pylori strains which containing a 40-kb cag pathogenicity island (which includes cagA gene) typically exhibits more severe inflammation than that of gastric mucosa infected with cagA-negative strains (Jafari et al., 2008). Despite the gene is present in most H. pylori, but the product may not be expressed in all cases. They have important attempt to described and classify distinction in the gene and to associate specific genotypes with different H. pylori-associated diseases The protein contains three distinct segments that exhibit variation within the amino-terminus. These areas of variation are broadly defined as the signal (s), intermediate (i), and midd le (m) regions. Two primary variants have been described for signal region (s1 or s2) and middle region (m1 or m2) while three variants has been reported for intermediate region (i1, i2, or i3) (Jones et al., 2010). The m region affects host cell tropism of toxins. Encoding the m1 region are toxic to a broader range of host cells compared to that of m2 region. The i region which determine the vacuolating capabilities is positioned between the s and m region and is the most recent region to be described. The i1 variants of have been shown to have stronger vacuolating activity than i2 variants (Jones et al., 2011). Atherton et al. (1995) reported three of the four possible subtypes with the exeption of s2-m1 variant. The presence of the s2-m1 variant was later reported but with very rare prevalence (0 to 3%). Despite variants have been described, but is still remains unclear whether the genotypes are useful markers for the prediction of clinical outcomes. Initial reports indicated tha t the s1 and i2 genotype would be found in close association with clinical outcomes in Western countries (Jafari et al., 2008); however the prevalence of this genotype was extremely high (almost 100%) in East Asian countries irrespective of the clinical outcomes (Jafari et al., 2008). The prevalence of H. pylori infection is about 50% of the world’s population and has been reported to be 40 to 60% in Malaysian (Goh et al., 2009). The purpose of this study is to identify the prevalence of H. pylori isolates in patients with a full spectrum of clinical demonstration of H. pylori infection to evaluate whether the assumed associations with genotypes and cagA gene would be applicable to patients with H. pylori infection in Malaysian populations. 2. Material and methods Isolation and culture of H. pylori Gastric biopsies were obtained from each patient by using endoscopy. Each specimen was placed in a transport medium and sent to the laboratory immediately. The specimens were then incubated onto chocolate (selective/non-selective) agar containing 10% sheep blood for 3 days at 37 ºC under microaerobic conditions (10% CO2, 5% O2, 85% N2). All H. pylori strains were positive for urease, oxidase, catalase and were identified by Gram staining under light microscopy. Colonies directly harvested from the plates were used for DNA extraction. Endoscopic findings were also recorded and based on the information patients were divided into two groups. Group I patients had duodenal or gastric ulcer (Peptic Ulcer Disease, PUD) and group II patients had Non-Ulcer Dyspepsia (NUD). Population studied A total of 209 gastric biopsies were collected from patients presenting with symptoms of gastroduodenal diseases during July 2011 to August 2012 at the University of Malaya Medical Centre (UMMC). Four biopsy specimens were taken from the greater curve of the antrum, body, cardia and Esophagous, one was used for histological examination and one for H. pylori culture. Genomic DNA extraction Bacteria from both the 10 single colonies and the sweeps were resuspended in 1.2 ml Elution buffer (EB). Bacterial pellets were obtained by centrifugation at 5,233 Ãâ€"gfor 5 min, and genomic DNA was extracted by using a Qiagen kit. The amount of DNA was calculated by spectrophotometry. Detection of H. pylori and genotyping of 16sRNA, cag A, E, L and vac genes and EPIYA motifs by PCR In this study PCR was used to detect the H. pylori specific 16sRNA gene for confirmation of H. pylori isolates, the virulence-associated vacA mosaic structure and the presence of cag A, E, L genes. All primer sets were selected from the published literatures (Table 1). The PCR was carried out in a volume of 25  µl including100 ng of genomic DNA, 20 pmol of each of primers (Table 1), 5  µl of i-PCR 5X Master Mix (iDNA, Singapore) which contain 0.1 unit/ µl 5  µl of i-PCR 5X Master Mix (iDNA, Singapore) which contain 0.1 unit/ µl of Taq DNA Polymerase, 1 mM of dNTP’s, 10% of glycerol and 7.5 mM of MgCl2. Negative control tubes included all materials except template DNA. The sequences of the primers are listed in Table 1. PCR products were checked on 2.0% agarose gels with 100 bp ladder size markers. Polymerase chain reaction (PCR) for the EPIYA motifs was performed in separate reactions according to the method described by Argent et al. (Mitchell et al., 2009). using the common forward primer cag2 with one of three reverse primers, cagAP1C, cagAP2TA, and cagAP3E, for the amplià ¯Ã‚ ¬Ã‚ cation of EPIYA-A, -B, and -C and -D, respectively. Table 1. Oligonucleotide primers used for cagA/E and L, vacA alleles and EPIYA motifs