Prevalence and Genotypes of Helicobacter pylori in Gastric Biopsy Specimens from Patients with Gastroduodenal Pathologies in the Cukurova Region of Turkey

The effects of Helicobacter pylori genotypes on clinical prognosis in the Cukurova region of Turkey were investigated by PCR. The prevalence of type I strains carrying the s1c allele, unlike in neighboring regions and countries, was found to be significantly higher in patients with gastritis and/or gastric ulcers (P = 0.001), and that of type I strains carrying the s1a allele was found to be significantly higher in patients with duodenal ulcers (P < 0.001). The cagA gene was strongly associated with the more virulent vacA genotypes (P < 0.001).The effects of vacA allelic combinations of Helicobacter pylori on clinical prognosis show geographic differences in Western and Eastern populations. Although all strains of H. pylori contain the vacA gene, they vary in the ability to produce cytotoxin. Type m1 strains demonstrate more toxin activity than m2 strains, type s1a is more active than s1b, and type s2 produces no detectable activity (2, 3). The s1a alleles are frequently observed in strains from northern and eastern Europe, while the s1b allelic types are frequent in such regions as Central and South America, Spain, Portugal, and South Africa. The s1b types are rare in other regions and less virulent than other s1 subtypes (2, 3, 10, 19, 20, 22, 23). Furthermore, although the s1c allele has been stated to be more common in Asian strains and it may be associated with gastric cancers that are prevalent in that region, other studies have, in contrast, indicated that cancer development cannot be explained by s1c alleles alone (4, 12, 18-20, 23). Thus, the relationship between the genotypes of causative strains and clinical outcome should be considered in different geographic regions in order to make a true estimation of prognosis. There are very few studies on the relationship of vacA alleles with clinical outcomes in Turkey, and no such study has been carried out in our region (5, 6, 15). Therefore, we aimed to determine the prevalence of H. pylori, the effects of the cagA and vacA genes and vacA allelic types of the colonizing strains on clinical prognosis, and the relationship between the cagA and vacA genotypes in patients with gastritis and/or gastric ulcers (G/GU) and those with duodenal ulcers (DU) in the Cukurova region of Turkey.(Part of this study was presented as a poster [Determination of the Prevalence and Genotypes of H. pylori in Gastric Biopsy Specimens from Patients with Gastroduodenal Pathologies] at the XXI International Workshop on Helicobacter and Related Bacteria in Chronic Digestive Inflammation and Gastric Cancer in Riga, Latvia, 18 to 20 September 2008, and an abstract was published [Helicobacter 13:420, 2008].)The subjects of this study were 231 previously untreated patients seen at outpatient clinics of the gastroenterology departments of two regional hospitals between January 2005 and January 2006, 158 of whom had endoscopically diagnosed G/GU and 73 of whom had DU. Antral gastric biopsy specimens were transported to the laboratory in brain heart infusion broth (Oxoid, Basingstoke, Hampshire, England) within 2 h and stored at −20°C.Extraction of DNA was performed with a QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer''s instructions. The extracted DNA was measured in an Optimum-One UV-VIS spectrophotometer (Chebios, Rome, Italy) at a 260-nm wavelength for determination of DNA concentrations, which were confirmed to be sufficient for amplification (>3 μg/ml). Then, the DNA samples were stored at −20°C until the amplification of all of the gene regions to be investigated was completed.These samples were first examined by glmM (formerly ureC) gene PCR, the most sensitive, specific, and reliable method for determination of the prevalence of H. pylori (11, 13). H. pylori-positive DNA samples were analyzed for the cagA and vacA genes, and vacA-positive DNA samples were further investigated for the detection of vacA alleles (s1a, s1b, s1c, and s2 in the signal region and m1a, m1b, and m2 in the cytotoxin-encoding middle region) by the PCR method. The primers used in this study are shown in Table ​Table1.1. All amplification procedures were performed with a Thermal Cycler 2720 (Applied Biosystems, Foster City, CA) with a total volume of 50 μl of a PCR mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM each deoxynucleoside triphosphate, 25 pmol of each primer, 2.5 U of Taq polymerase, and 5 μl of each DNA sample. The PCR cycling conditions were as described previously (2, 13, 22, 23). Amplified PCR products were examined by electrophoresis on 2% agarose gels with ethidium bromide, and bands were analyzed under UV illumination.

TABLE 1.

Oligonucleotide primers used in this study

Gene and allele	Primer	Sequence (5′-3′)a	Size (bp) of PCR product (location)	Reference(s)
glmM	glmM1-F	AAG CTT TTA GGG GTG TTA GGG GTT T	294 (784-1077)b	13
	glmM2-R	AAG CTT ACT TTC TAA CAC TAA CGC
cagA	CagA-F1	GAT AAC AGG CAA GCT TTT GAG G	349 (1228-1576)c	6,22
	CagA-B1	CTG CAA AAG ATT GTT TGG CAG A
vacA	VA1-F	ATG GAA ATA CAA CAA ACA CAC	259 (797-1055)d or 286 (284-569)e	2,22
	VA1-R	CTG CTT GAA TGC GCC AAA C
vacA s1a	SS1-F	GTC AGC ATC ACA CCG CAA C	190 (866-1055)d	2
	VA1-R	CTG CTT GAA TGC GCC AAA C
vacA s1b	SS3-F	AGC GCC ATA CCG CAA GAG	187 (869-1055)f	2,23
	VA1-R	CTG CTT GAA TGC GCC AAA C
vacA s1c	SlC-F	CTC TCG CTT TAG TGG GGY T	213 (843-1055)f	23
	VA1-R	CTG CTT GAA TGC GCC AAA C
vacA s2	SS2-F	GCT AAC ACG CCA AAT GAT CC	199 (371-569)e	2,
	VA1-R	CTG CTT GAA TGC GCC AAA C
vacA m1a	VA3-F	GGT CAA AAT GCG GTC ATG G	290 (2741-3030)d	2,23
	VA3-R	CCA TTG GTA CCT GTA GAA AC
vacA m1b	VAm-F3	GGC CCC AAT GCA GTC ATG GAT	291 (2741-3031)f	23
	VAm-R3	GCT GTT AGT GCC TAA AGA AGC AT
vacA m2	VA4-F	GGA GCC CCA GGA AAC ATT G	352 (2284-2635)e	23
	VA4-R	CAT AAC TAG CGC CTT GCA C

Open in a separate window^aY = C or T.^bNucleotide positions in the glmM gene of H. pylori 26695.^cNucleotide positions in the cagA gene of H. pylori ATCC 53726 (GenBank accession no. L117714).^dNucleotide positions in the vacA gene of H. pylori 60190 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"U05676","term_id":"471727","term_text":"U05676"}}U05676).^eNucleotide positions in the vacA gene of H. pylori Tx30a (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"U29401","term_id":"984359","term_text":"U29401"}}U29401).^fCorresponding to nucleotide positions of H. pylori 60190.Fisher''s exact chi square test was used to analyze the data for the different patient groups. A P value of <0.05 was accepted as statistically significant.H. pylori was detected in 201 (87%) of a total of 231 biopsy specimens by the glmM PCR method. Our finding is similar to those reported by Saribasak et al. (15) and Bolek et al. (5), who also used the PCR method for diagnosis (89% and 84.6%, respectively), yet it is higher than the 75.4% found by Kantarceken et al. (8), who evaluated biopsy specimens histologically. This inconsistency is assumed to have been caused by the nature of PCR-based methods, which are accepted as being more sensitive and specific (11, 13).The effects of H. pylori genotypes on gastric colonization were studied by many researchers in large groups of cases, and type I (cagA⁺ vacA⁺) strains were shown to be more virulent than type II (cagA mutant) strains (1, 17, 21). The effects of the cagA and vacA alleles on prognosis were also intensely discussed in subsequent studies (2, 3, 9, 20, 22, 23, 24). In this study, we investigated the cagA and vacA genes and vacA gene alleles. However, we did not examine the cagA gene variants due to a lack of the means to perform sequence analysis during our study, although the effects of EPIYA motifs located at the 3′ end of the cagA gene on prognosis were shown in various studies.We identified 84.1% of our H. pylori strains as type I and 15.9% as type II. The prevalence of type I H. pylori in patients with DU was observed to be significantly higher than in patients with G/GU (94.3% and 78.6%, respectively; P = 0.004) (Table ​(Table2).2). In a great amount of research, the prevalence of type I H. pylori was reported to vary among different countries, regions, and patient groups, and it was reported as 68% in England (21), 78 to 80% in Turkey (5, 15), 80% in Belgium (11), 82% in Brazil (1), 82% in Japan (17), 93% in Nigeria (16), and 97% in China (24). The majority of those studies (1, 5, 11, 17, 21) indicated that in patients with DU, the cagA positivity rate is relatively higher than in patients with gastritis or GU and ranges from 80 to 100%. Our results revealed that, as in the rest of the world, cagA⁺ H. pylori strains were especially associated with DU in our country. In the another study performed in Turkey by Saribasak et al., type I H. pylori prevalences in patients with DU, GU, and gastritis were determined to be 89%, 100%, and 55%, respectively (15). In that study, the cagA positivity rate was accompanied by a severity of pathology similar to ours, but GU and gastritis were scored differently on the basis of clinical definitions. However, although in Nigeria, where a high cagA positivity rate was reported, this ratio was higher in patients with DU than in patients with nonulcer dyspepsia (95% and 91%, respectively), the overall evaluation makes the effects of the cagA profile on clinical outcome quite disputable (16).

TABLE 2.

Distribution of cagA and vacA genotypes in 201 H. pylori-positive samples by glmM PCR

Genotype	No. (%) of samples
	G/GUa (n = 131)	DUb (n = 70)	Overall (n= 201)
cagA+vacA+	103 (78.6)	66 (94.3)	169 (84.1)
cagA mutant vacA+	26 (19.9)	4 (5.7)	30 (14.9)
cagA mutant vacA mutant	2 (1.5)		2 (1.0)

Open in a separate window^aSpecimens obtained from patients with G/GU via endoscopy.^bSpecimens obtained from patients with DU via endoscopy.We also analyzed a total of 199 vacA-positive samples in order to determine predominant vacA alleles in the Cukurova region and to detect the effects of the alleles on prognosis and their relationship with cagA. The most frequent vacA allelic combination was s1a/m1b in 49 (24.6%) strains, followed by s1c/m1b in 45 (22.6%) and s1a/m1a in 40 (20.1%) strains. No sign of the s2/m1 genotype was detected in any of the strains. The most prevalent genotypes in patients with G/GU and DU were s1c/m1b (25.6%) and s1a/m1b (38.6%), respectively (Table ​(Table3).3). The relatively higher prevalence of the s1a/m1b genotype in patients with DU than in those with G/GU was determined to be statistically significant (P = 0.001). The predominant vacA genotype was s1a/m1b (27.8%) in type I strains and s1/m2 (36.7%) in type II strains (Table ​(Table33).

TABLE 3.

Distribution of vacA allelic combinations in all of 199 vacA⁺ and 169 type I (cagA⁺ vacA⁺) H. pylori strains from patients with G/GU and DU

vacA allelic combination	No. (%) of samples
	G/GUa		DUb		Overall
	All vacA+ strains	Type I strainsc	All vacA+ strains	Type I stains	All vacA+ strains	Type I strains	Type II strainsd
s1a/m1a	21 (16.3)	20 (19.4)	19 (27.2)	19 (28.8)	40 (20.1)	39 (23.1)	1 (3.3)
s1a/m1b	22 (17.0)	20 (19.4)	27 (38.6)	27 (40.9)	49 (24.6)	47 (27.8)	2 (6.6)
s1b/m1a	8 (6.2)	3 (2.9)	1 (1.4)		9 (4.5)	3 (1.8)	6 (20.0)
s1b/m1b	5 (3.9)				5 (2.5)		5 (16.7)
s1c/m1a	26 (20.1)	26 (25.3)	8 (11.4)	8 (12.1)	34 (17.1)	34 (20.1)
s1c/m1b	33 (25.6)	33 (32.0)	12 (17.1)	12 (18.2)	45 (22.6)	45 (26.6)
s1/m2	9 (7.0)	1 (1.0)	3 (4.3)		12 (6.1)	1 (0.6)	11 (36.7)
s2/m2	5 (3.9)				5 (2.5)		5 (16.7)
Total	129	103	70	66	199	169	30

Open in a separate window^aSpecimens obtained from patients with G/GU via endoscopy.^bSpecimens obtained from patients with DU via endoscopy.^ccagA⁺ vacA⁺ strains.^dcagA mutant strains.In 59 (57.3%) and 40 (38.8%) of the 103 type I H. pylori strains from patients with G/GU, the s1c and s1a alleles were detected, respectively, while in 46 (69.7%) and 20 (30.3%) of the 66 type I H. pylori strains from patients with DU, the s1a and s1c alleles were found, respectively (Table ​(Table3).3). The relatively higher prevalence of the s1c allele in patients with G/GU and of the s1a allele in patients with DU was determined to be statistically significant (P = 0.001 and P < 0.001, respectively).The findings of related studies on the distribution of vacA alleles in patient groups seem to be different, yet it is the common opinion that while the s1a, s1b, and m1a alleles have higher prevalences in European, African, and American strains, the s1c and m1b allelic types are endemic in East Asia (1, 9, 10, 19, 23). In a study conducted in Istanbul, Turkey, Bolek et al. reported that among type I strains, the most frequent vacA genotypes were s1/m1 in patients with DU and GU and s1/m2 in patients with gastritis, while among type II strains the most frequent vacA genotype was s1/m2 in all groups (5). Nevertheless, those researchers did not investigate the subtypes of s and m alleles. We found that s1/m2 was the most common genotype among type II strains in both groups, similar to those of Bolek et al., but except for the s1/m2 genotype detected in one patient with G/GU, all type I strains were s1/m1.In addition, we observed that the prevalences of the s1a/m1a genotype, known as the Western type (23), the s1c/m1b and s1a/m1b genotypes, known as the East Asian type (23), and the s1c/m1a genotype, which may be called the mixed type, which was not previously reported, were close to each other. However, in two studies of s allelic subtypes in Turkey, Erzin et al. (6) and Saribasak et al. (15) reported that the prevalence of the s1a allele was substantially high (83% in both studies), there was no s1c allele and the m2 allele was more prevalent than the m1 allele in the Istanbul region. The high prevalence of the s1c allelic types observed in our study but not in the above-mentioned two studies is noteworthy and is assumed be due to geographic and ethnic differences. In addition, this difference may be attributable to some technical discrepancies in the methodologies used, such as differences in the primers used and the researchers'' levels of experience. The findings on the s1b and s2 alleles in the above-mentioned studies, on the other hand, seem to be consistent with ours. Besides, Erzin et al. correlated type I strains with DU and the s1a allele (6) and Saribasak et al. correlated them with peptic ulcer disease and the s1a allele (15).Hussein et al. have reported the s1/m2 allelic type, unlike the ones in our study, to be the most frequent type in the neighboring countries Iran and Iraq, and they also indicated that the prevalence of the s2/m2 genotype was higher in Iran (27%) and close to ours in Iraq (8%) (7). In a great number of studies conducted in different parts of the world, s2 allelic types have been found to be less frequent and to be associated with nonulcerative dyspepsia or asymptomatic carriage (14, 16, 23). Thus, s2 allelic types which we marked only in type II strains are consistent with those reported in related studies.The s2/m2 genotype was detected only in five (2.5%) patients with G/GU, and all of these strains were cagA negative. While 165 (98.2%) of a total of 168 strains carrying the more virulent s1a/m1a, s1a/m1b, s1c/m1a, and s1c/m1b genotypes were cagA positive, only 4 (12.9%) of a total of 31 strains carrying the nonvirulent s2/m2 genotype, the less virulent s1/m2 genotype, or the s1b/m1a or s1b/m1b genotype, supposedly less virulent in our region, were cagA positive (Table ​(Table3).3). The association between the more virulent vacA genotypes and the cagA gene was found to be statistically significant (P < 0.001). Therefore, it is important that whether the association between the more virulent vacA genotypes and the cagA gene is a coincidence or the cagA gene prefers strains with more virulent vacA genotypes be discussed. We assume that our results strengthen the idea that the cag pathogenicity island, an unstable gene island carrying the cagA gene, might prefer the virulent vacA allelic types during insertion into the genome.In conclusion, the prevalences of type I H. pylori strains in our region were determined to be 78.6% and 94.3% in patients with G/GU and DU, respectively. The most prevalent vacA genotype was s1c/m1b in patients with G/GU and s1a/m1b in patients with DU, and more-virulent vacA genotypes were strongly associated with the cagA gene. The results imply that gastric colonization caused by strains carrying the s1c allele, which is supposed to be a risk factor for gastric cancer in Asia, is also important in our region. Thus, it is suggested that not only H. pylori colonization of antral biopsy specimens obtained from patients with gastroduodenal complaints during endoscopic examination be demonstrated, but also the detection of genotypic and allelic combinations which can help in the diagnosis and eradication of infections caused by strains carrying the s1c allele, which may lead to gastric cancer, be considered in the Cukurova region of Turkey.