# Prevalence and Correlation with Clinical Diseases ofHelicobacter pylori cagA and vacA Genotype among Gastric Patients from Northeast China

Authors: Faisal Aziz; Xin Chen; Xuesong Yang; Qiu Yan
Journal: BioMed Research International
 (2014)

---

## Abstract

Helicobacter pylori vacA and cagA genes have significant genetic heterogenicity, resulting in different clinical outcomes. Northeast part of China has reported high prevalence of H. pylori infections and gastric cancer. Hence, we investigated the H. pylori cagA and vacA genotypes with clinical outcomes in Northeast China. Gastric tissue samples (n
=
169), chronic gastritis (GIs), gastric ulcer (GU), and gastric cancer (GC) were analysed for 16S rRNA ureA, cagA, and cagA genotypes by PCR. A total of 141 (84%) cases were found positive for H. pylori by 16S rRNA and ureA. GC showed high H. pylori infection (93%) compared with GIs (72%) and GU (84%). The vacAs1am1 was highly found in GC (40%) and GU (36%), vacAs1am2 in GIs (33%), vacAs1bm1 (14%) and vacAs1bm2 (8%) in GU cases, and s2m1 in normal cases (33%), while vacAs1cm1 showed low frequency in GIs (2%) and GU (3%) and GC showed negative result. The East-Asian cagA strain was highly observed in GC (43%), as compared to GIs (41%) and GU (20%). The East-Asian cagA/vacAs1am1 was significantly higher in GC (23%) than in GU (22%) and GIs (145) patients. The East-Asian type cagA with vacAs1a and vacAm1 is the most predominant genotype in H. pylori strains of Northeast China.


---

## Body

## 1. Introduction

Helicobacter pylori is a causative agent of gastritis (GIs) and gastric ulcer (GU) diseases, which leads to the development of gastric cancer (GC). Hence it is classified as class I carcinogen by the World Health Organization. Developing countries haveH. pylori prevalence rate of 80% as compared to developed countries (20–50%) [1, 2]. Genetic diversity may play an important role in genotypic variation ofH. pylori, which makes it more virulent with diverse pathogenicity [3].H. pyloricarries different virulence factors, such as urease, flagellar, vacuolating cytotoxin A (VacA), and cytotoxin-associated gene A (CagA), that play an important role in invasion, colonization, and cell proliferation [4, 5]. High genetic variations ofcagA andvacA gene are associated with more severe infectionsofH. pylori [6].VacA toxin encoded byvacA gene induces cytoplasmic vacuoles and increases permeability, which leads to the damage of gastric epithelial cells [7]. ThevacA gene exhibits significant allelic variation in the signal (s) and middle (m) regions. The s-region consists of two major subtypes (s1 and s2) and s1-region has further three subtypes (s1a, s1b, and s1c), whereas m-region designates m1 and m2 subtypes [8, 9]. A pleomorphic combination of s and m regions affects the vacuolating activity ofvacA gene [10]. Different genotypic combination ofvacA region results in different pathogenicity level as follows: s1am1 and s1bm1 produce high amount of toxin and are considered the most virulent as compared to s1m2, which produces moderate vacuolating toxins [11, 12]. However, s2m1 and s2m2 are considered less toxic because of their inability to form vacuoles [9]. The s1am1 and s1bm1 subtypes are frequently reported in acute gastritis (GIs), peptic ulcer, and gastric cancer patients, while s2m1 and s2m2 have been reported in the gastric ulcer (GU) patients [9, 13].Cytotoxin-associated gene A (CagA) is a cytotoxin-associated protein, linked with peptic ulcer and gastric cancer [6].H. pyloristrains can be grouped as Western and East-Asian subtypes based on polymorphism in 3′ repeat region of thecagAgene, which results in the variation of clinical outcome in East-Asian and Western countries. 3′ region of thecagA gene consists of two types of repeat regions as 57 bp regions (first repeat region; FR region) and 102 bp regions (Western-type second repeat region; WSR region) [14]. The FR region of East-Asian strains has similar 57 bp region to Western type, but the repeat region of 162 bp was completely different [15]. East-Asian type ofcagAis more prevalent in East-Asian countries and is more commonly associated with gastric cancer mortality worldwide than Western subtypes [16].China has high rate ofH. pylori infections. Some studies reported high prevalence of thevacA andcagA genotypes from South China. There is little data present regarding theH. pylori vacA andcagA genotypes and their association with clinical outcomes in Northeast China, where the prevalence ofH. pylori infection and gastric cancer is much higher than in the south part of China [17]. ThecagAandvacAgenotyping is useful to determine molecular epidemiological status ofH. pyloristrain in the northeast part of China. Hence, we aim to evaluate the frequency ofvacA andcagA genotypes with their clinical outcomes in gastric patients from the northeast part of China. This study helps to characterize the more prevalent strain ofH. pylori in Northeast China, which is useful in diagnosis and treatment of gastric patients.


## 2. Materials and Methods

### 2.1. Patients and Gastroendoscopy

All gastric patients underwent gastroendoscopy and examination at Dalian 1st & 2nd Affiliated Hospital, Dalian, China. Gastric tissues were obtained from the antral and corpus part of the stomach during gastrointestinal endoscopy. Gastric patient’s tissue samples were characterized as chronic gastritis (GIs), gastric ulcer (GU), and gastric cancer (GC). Clinical diseases were diagnosed by endoscopic appearance as “normal” in cases of intact mucosa, chronic gastritis, gastric ulcer, and gastric cancer. All the samples were processed and frozen at −20°C until tested. Fresh surgical gastric cancer tissues were collected in transport media and analyzed in laboratory. All gastric samples collected from the patients and the research protocols were in accordance with the Institutional Review Board of Dalian Medical University.


### 2.2. DNA Extraction

The SDS-PK method was adopted to extract DNA from gastric patient’s tissues. Gastric tissues were crushed manually and resuspended in 20μL of 10% SDS, 80 μL of proteinase K buffer (0.5 M EDTA and 4 M NaCl, pH 7.5), 40 μL of proteinase K (10 mg/mL) and make final volume up to 380 μL with sterile water. The mixture was incubated at 55°C overnight. The following day, 100 μL of 6 M NaCl was added, followed by centrifugation at 14,000 rpm for 5 min and the supernatant was separated in new tube. To precipitate the DNA, 500 μL of absolute isopropanol was added, mixed well and centrifuged for 5 minutes at 9000 rpm. DNA pellet was washed with 70% ethanol and air dried. The pellet was resuspended in 50 μL of TE buffer (10 mM Tris and 1 mM EDTA; pH 8.0). Samples were stored at −20°C until used.


### 2.3. Polymerase Chain Reaction (PCR)

PCR was performed to detectH. pyloriby using specific primers. Target gene, amplicon size, primer names, and sequences are shown in Table 1. For PCR amplification, 1-2 μg of DNA samples was added to a PCR mixture containing 20 pmol forward and reverse primers, 1.5 mM MgCl2, 1.5 U of Taq polymerase (Takara, Japan), 2.5 μL PCR buffer, and 200 μM of dNTPs to the total volume of 25 μL. PCR amplification was performed under the following conditions: initial denaturation at 95°C for 3 min followed by 30 cycles of denaturation at 95°C for 30 s, annealing for 30 s (Table 1), polymerization at 72°C for 30 s, and final polymerization at 72°C for 5 min (Bio-Rad Thermocycler). The PCR reaction products were electrophoresed on 1.5% agarose gel with 2000 bp DNA ladder (Takara, Japan) and the bands were visualized by ethidium bromide staining, followed by analysis with Quantity One software (Bio-Rad, USA).H. pylori strain was detected by using specific primers targeting 16S rRNA andureAgenes (Table 1). ThecagA andvacA statuses were determined fromH. pylori positive samples by PCR using their respective primers as described in Table 1.Table 1
Primer sets used for genotypingH. pylori by PCR.
Target site
Amplicon size (bp)
Primer names and sequences
Annealing temperature
References
16S rRNA
138
HP-F (5-GCGACCTGCTGGAACATTAC-3)HP-R (5-CGTTAGCTGCATTACTGGAGA-3)
60°C
Gramley et al., 1999 [18]
UreA
411
HPU1-F (5-GCCAATGGTAAATTAGTT-3)HPU1-R (5-CTCCTTAATTGTTTTTAC-3)
45°C
Smith et al., 2004 [19]
vacAs1a
190
AA1-F (5-GTCAGCATCACACCGCAAC-3)AA1-R (5-TGCTTGAATGCGCCAAAC-3)
56°C
Atherton et al., 1995 [9]
vacAs1b
187
SS3-F (5-AGCGCCATACCGCAAGAG-3)SS3-R (5-CTGCTTGAATGCGCCAAAC-3)
56°C
Atherton et al., 1995 [9]
vacAs1c
213
S1C-F (5-CTCGCTTTAGTGGGGCTA-3)S1C-R (5-CTGCTTGAATGCGCCAAAC-3)
56°C
Yamaoka et al., 1999 [20]
vacAs2
199
SS2-F (5-GCTAACACGCCAAATGATCC-3)SS2-R (5-CTGCTTGAATGCGCCAAAC-3)
56°C
Atherton et al., 1995 [9]
vacAm1/m2
570/645
VAG-F (5-CAATCTGTCCAATCAAGCGAG-3)VAG-R (5-GCGTCTAAATAATTCCAAGG-3)
57°C
Yamaoka et al., 1999 [20]
cagA
189
CAGA-F (5-TTGACCAACAACCACAAACCGAAG-3)CAGA-R (5-CTTCCCTTAATTGCGAGATTCC-3)
62°C
van Doorn et al., 1998 [21]
Western typecagA
Variable
CAGT-F (5-ACCCTAGTCGGTAATGGG-3)CAGW-R (5-TGCCCTACAMCACCSAAACCAC-3)CAGW-F (5-AAAAATTGACCRACTCAATC-3) CAGT-R (5-GCTTTAGCTTCTGAYACYGC-3)
61°C
Yamaoka et al., 1999 [22]
East-Asian typecagA
Variable
CAGT-F (5-ACCCTAGTCGGTAATGGG-3)CAGJ-R (5-GCAATTTTGTTAATCCGGTC-3) CAGJ-F (5-GCATCAGCAGGTAAAGGAGT-3)CAGT-R (5-GCTTTAGCTTCTGAYACYGC-3)
52°C
Yamaoka et al., 2000 [14]


### 2.4. Statistical Analysis

Categorical data were analysed by using chi-square test.Pvalue of less than 0.05 was regarded as significant. The statistical software GraphPad prism 5.03 was used for analyzing the data.


## 2.1. Patients and Gastroendoscopy

All gastric patients underwent gastroendoscopy and examination at Dalian 1st & 2nd Affiliated Hospital, Dalian, China. Gastric tissues were obtained from the antral and corpus part of the stomach during gastrointestinal endoscopy. Gastric patient’s tissue samples were characterized as chronic gastritis (GIs), gastric ulcer (GU), and gastric cancer (GC). Clinical diseases were diagnosed by endoscopic appearance as “normal” in cases of intact mucosa, chronic gastritis, gastric ulcer, and gastric cancer. All the samples were processed and frozen at −20°C until tested. Fresh surgical gastric cancer tissues were collected in transport media and analyzed in laboratory. All gastric samples collected from the patients and the research protocols were in accordance with the Institutional Review Board of Dalian Medical University.


## 2.2. DNA Extraction

The SDS-PK method was adopted to extract DNA from gastric patient’s tissues. Gastric tissues were crushed manually and resuspended in 20μL of 10% SDS, 80 μL of proteinase K buffer (0.5 M EDTA and 4 M NaCl, pH 7.5), 40 μL of proteinase K (10 mg/mL) and make final volume up to 380 μL with sterile water. The mixture was incubated at 55°C overnight. The following day, 100 μL of 6 M NaCl was added, followed by centrifugation at 14,000 rpm for 5 min and the supernatant was separated in new tube. To precipitate the DNA, 500 μL of absolute isopropanol was added, mixed well and centrifuged for 5 minutes at 9000 rpm. DNA pellet was washed with 70% ethanol and air dried. The pellet was resuspended in 50 μL of TE buffer (10 mM Tris and 1 mM EDTA; pH 8.0). Samples were stored at −20°C until used.


## 2.3. Polymerase Chain Reaction (PCR)

PCR was performed to detectH. pyloriby using specific primers. Target gene, amplicon size, primer names, and sequences are shown in Table 1. For PCR amplification, 1-2 μg of DNA samples was added to a PCR mixture containing 20 pmol forward and reverse primers, 1.5 mM MgCl2, 1.5 U of Taq polymerase (Takara, Japan), 2.5 μL PCR buffer, and 200 μM of dNTPs to the total volume of 25 μL. PCR amplification was performed under the following conditions: initial denaturation at 95°C for 3 min followed by 30 cycles of denaturation at 95°C for 30 s, annealing for 30 s (Table 1), polymerization at 72°C for 30 s, and final polymerization at 72°C for 5 min (Bio-Rad Thermocycler). The PCR reaction products were electrophoresed on 1.5% agarose gel with 2000 bp DNA ladder (Takara, Japan) and the bands were visualized by ethidium bromide staining, followed by analysis with Quantity One software (Bio-Rad, USA).H. pylori strain was detected by using specific primers targeting 16S rRNA andureAgenes (Table 1). ThecagA andvacA statuses were determined fromH. pylori positive samples by PCR using their respective primers as described in Table 1.Table 1
Primer sets used for genotypingH. pylori by PCR.
Target site
Amplicon size (bp)
Primer names and sequences
Annealing temperature
References
16S rRNA
138
HP-F (5-GCGACCTGCTGGAACATTAC-3)HP-R (5-CGTTAGCTGCATTACTGGAGA-3)
60°C
Gramley et al., 1999 [18]
UreA
411
HPU1-F (5-GCCAATGGTAAATTAGTT-3)HPU1-R (5-CTCCTTAATTGTTTTTAC-3)
45°C
Smith et al., 2004 [19]
vacAs1a
190
AA1-F (5-GTCAGCATCACACCGCAAC-3)AA1-R (5-TGCTTGAATGCGCCAAAC-3)
56°C
Atherton et al., 1995 [9]
vacAs1b
187
SS3-F (5-AGCGCCATACCGCAAGAG-3)SS3-R (5-CTGCTTGAATGCGCCAAAC-3)
56°C
Atherton et al., 1995 [9]
vacAs1c
213
S1C-F (5-CTCGCTTTAGTGGGGCTA-3)S1C-R (5-CTGCTTGAATGCGCCAAAC-3)
56°C
Yamaoka et al., 1999 [20]
vacAs2
199
SS2-F (5-GCTAACACGCCAAATGATCC-3)SS2-R (5-CTGCTTGAATGCGCCAAAC-3)
56°C
Atherton et al., 1995 [9]
vacAm1/m2
570/645
VAG-F (5-CAATCTGTCCAATCAAGCGAG-3)VAG-R (5-GCGTCTAAATAATTCCAAGG-3)
57°C
Yamaoka et al., 1999 [20]
cagA
189
CAGA-F (5-TTGACCAACAACCACAAACCGAAG-3)CAGA-R (5-CTTCCCTTAATTGCGAGATTCC-3)
62°C
van Doorn et al., 1998 [21]
Western typecagA
Variable
CAGT-F (5-ACCCTAGTCGGTAATGGG-3)CAGW-R (5-TGCCCTACAMCACCSAAACCAC-3)CAGW-F (5-AAAAATTGACCRACTCAATC-3) CAGT-R (5-GCTTTAGCTTCTGAYACYGC-3)
61°C
Yamaoka et al., 1999 [22]
East-Asian typecagA
Variable
CAGT-F (5-ACCCTAGTCGGTAATGGG-3)CAGJ-R (5-GCAATTTTGTTAATCCGGTC-3) CAGJ-F (5-GCATCAGCAGGTAAAGGAGT-3)CAGT-R (5-GCTTTAGCTTCTGAYACYGC-3)
52°C
Yamaoka et al., 2000 [14]


## 2.4. Statistical Analysis

Categorical data were analysed by using chi-square test.Pvalue of less than 0.05 was regarded as significant. The statistical software GraphPad prism 5.03 was used for analyzing the data.


## 3. Results

### 3.1. Gastric Patients History

This study was designed to determine the frequency ofcagA andvacA genotypes in gastritis (GIs), gastric ulcer (GU), and gastric cancer (GC) patients from the northeast part of China. A total of 169 gastric patients, 63 males and 106 females ranging in age from 10 to 80 years (average age of 56.32
±
2.394), were included in the study. Among 169 cases, 51 (30.17%) were diagnosed as GIs, 36 (21.30%) were diagnosed as GU, and 73 (43.19%) samples were regarded as GC, while 9 (5.32%) were normal cases.


### 3.2. Detection ofH. pylori Infection

Helicobacter pylori specific genes (ureAand 16S rRNA) revealed that (141) 84% of samples wereH. pyloripositive. These included 6 (54%) normal, 37 (72%) GIs cases, 30 (84%) GU and 68 (93%) of theH. pylori positive GC cases (Table 2).Table 2
Distribution ofcagA and vacA genotypes with diseases outcomes.
Description
Totaln
=
169
Normaln
=
9
Chronic gastritisn
=
51
Gastric ulcern
=
36
Gastric cancern
=
73
N
%
N
%
N
%
N
%
N
%
H. pylori positive
141
84
6
54
37
72
30
84
68
93
cagA positive
86
61
1
11
21
41
18
50
46
63
(i) Western typecagA
27
19
1
11
2
4
11
31
13
18
(ii) East-Asian typecagA
59
42
0
0
21
41
7
20
31
43
vacAs1am1
51
36
0
0
9
18
13
36
29
40
vacAs1am2
24
17
1
11
17
33
2
6
4
6
vacAs1bm1
7
5
0
11
1
2
5
14
1
1
vacAs1bm2
7
5
0
0
2
4
3
8
2
3
vacAs1cm1
2
2
0
0
1
2
1
3
0
0
vacAs2m1
12
6
3
33
9
18
0
0
0
0


### 3.3. Detection of theH. pylori vacA Genotyping and Clinical Manifestations

Among 141H.pyloripositive gastric tissues, we determined six different allelic variants of thevacAgene inH. pylori positive gastric tissues. AmongH.pyloripositive gastric tissues, 51 (36%) were positive forvacAs1am1, 24 (17%) forvacAs1am2, and 7 (5%) forvacAs1bm1, whereas 7 (5%) werevacAs1bm2, 2 (2%) were vacAs1cm1, and 12 (6%) were positive forvacAs2 m1. As shown in Table 2, detailed analysis revealed that 29 (40%) of the GC cases had s1am1 genotype (P
<
0.05), which was significantly higher than GIs (9) (18%), GU (13) (36%), and normal cases (P
<
0.05). Respective percentages in GIs, GU, and GC tissue samples of s1am2 were high (33%, 6%, and 6%) as compared to s1mb1 (2%, 14%, and 1%) and s1bm2 (4%, 8%, and 3%)vacA genotype. s1cm1 was observed in GIs and GU, while s2m1 was found only in normal and GIs cases (Table 2).


### 3.4.H. pylori cagA Genotyping with Clinical Association

We found that the prevalence ofcagA gene was 86 (61%), out of which 27 (19%) were Western types and 59 (42%) were East-Asian types (Table 2). All negative tissues for 16S rRNA gene were also negative forcagAandvacAgenes. In detailed analysis, Western-typecagAgene was observed in 4% of the patients with GIs, 31% with GU, and 18% with GC (P
<
0.05), whereas 11% of the normal cases were also positive. East-Asian type strains were observed in 41% of the patients with GIs, 20% with GU, and 43% with GC (P
<
0.05) (Table 2).


### 3.5. Combinational Study ofH. pylori vacA withcagA Genotype

In combinational analysis ofvacA strain with Western type ofcagA, as shown in Table 3, we found that 17 (20%) of theH. pylori strains havevacAs1am1/cagAcombination, while 7 (8%) ofH. pylori strains havevacAs1am2/cagA. In contrast,vacAs1bm1/cagA andvacAs1bm2/cagA showed low number ofH. pylori strains of 2 (2%) and 1 (1%), respectively. ThevacAs1cm1/cagA andvacAs2cm1/cagA were not found inH. pylori positive strains. In detailed analysis, 6 (17%) of GU patients havevacAs1am1, 2 (6%)vacAs1am2, 1 (3%)vacAs1bm1, and 1 (3%)vacAs1bm2. Gastric cancer patients showed a number ofH. pylori strains as follows: 9 (12%) havevacAs1am1, 5 (7%)vacAs1am2, and 1 (1%)vacAs1bm1, andvacAs1bm2 has no case reported. GIs patients showed only 1 (2%)vacAs1am1 combinationalH. pylori strain (Table 3). In contrast, combinational analysis of East-Asian type ofcagA withvacA genes demonstrated more frequentlyH. pylori strains ofvacAs1am1/cagA 32 (37%) andvacAs1bm2/cagA 19 (22%) thanvacAs1bm1/cagA 6 (7%) andvacAs1bm2/cagA 2 (2%) (P
<
0.05). However,vacAs1cm1/cagA andvacAs2cm1/cagA were not found inH. pylori positive strains. Distribution analysis further showed that GIs patients have 7 (14%) s1am1/cagA, 4 (18%)vacAs1am2/cagA, and 3 (6%)vacAs1bm1/cagA H. pylori positive strains. In GU patients, we found 8 (22%)vacAs1am1/cagA and 2 (6%)vacAs1am2/cagA combinations inH. pyloripositive strain. In contrast, GC patients showed high number ofvacAs1am1/cagA 17 (23%) andvacAs1am2/cagA 13 (18%)H. pylori positive strains. However, combinations ofvacAs1bm1/cagA andvacAs1bm2/cagA in GC were found in low percentages as 3 (4%) and 2 (3%), respectively (P
<
0.05) (Table 3).Table 3
Combinational study betweencagA and vacA genotypes with diseases outcomes.
Description
Totaln
=
169
Normaln
=
9
Chronic gastritisn
=
51
Gastric ulcern
=
36
Gastric cancern
=
73
N
%
N
%
N
%
N
%
N
%
TotalH. pylori positive
141
84
6
67
37
73
30
83
68
93
TotalcagA positive
86
61
1
11
21
41
18
50
46
63
WesterncagA/vacAs1am1
17
20
1
11
1
2
6
17
9
12
WesterncagA/vacAs1am2
7
8
0
0
0
0
2
6
5
7
WesterncagA/vacAs1bm1
2
2
0
0
0
0
1
3
1
1
WesterncagA/vacAs1bm2
1
1
0
0
0
0
1
3
0
0
WesterncagA/vacAs1cm1
0
0
0
0
0
0
0
0
0
0
WesterncagA/vacAs2m1
0
0
0
0
0
0
0
0
0
0
East-AsiancagA/vacAs1am1
32
37
0
0
7
14
8
22
17
23
East-AsiancagA/vacAs1am2
19
22
0
0
4
18
2
6
13
18
East-AsiancagA/vacAs1bm1
6
7
0
0
3
6
0
0
3
4
East-AsiancagA/vacAs1bm2
2
2
0
0
0
0
0
0
2
3
East-AsiancagA/vacAs1cm1
0
0
0
0
0
0
0
0
0
0
East-AsiancagA/vacAs2m1
0
0
0
0
0
0
0
0
0
0


## 3.1. Gastric Patients History

This study was designed to determine the frequency ofcagA andvacA genotypes in gastritis (GIs), gastric ulcer (GU), and gastric cancer (GC) patients from the northeast part of China. A total of 169 gastric patients, 63 males and 106 females ranging in age from 10 to 80 years (average age of 56.32
±
2.394), were included in the study. Among 169 cases, 51 (30.17%) were diagnosed as GIs, 36 (21.30%) were diagnosed as GU, and 73 (43.19%) samples were regarded as GC, while 9 (5.32%) were normal cases.


## 3.2. Detection ofH. pylori Infection

Helicobacter pylori specific genes (ureAand 16S rRNA) revealed that (141) 84% of samples wereH. pyloripositive. These included 6 (54%) normal, 37 (72%) GIs cases, 30 (84%) GU and 68 (93%) of theH. pylori positive GC cases (Table 2).Table 2
Distribution ofcagA and vacA genotypes with diseases outcomes.
Description
Totaln
=
169
Normaln
=
9
Chronic gastritisn
=
51
Gastric ulcern
=
36
Gastric cancern
=
73
N
%
N
%
N
%
N
%
N
%
H. pylori positive
141
84
6
54
37
72
30
84
68
93
cagA positive
86
61
1
11
21
41
18
50
46
63
(i) Western typecagA
27
19
1
11
2
4
11
31
13
18
(ii) East-Asian typecagA
59
42
0
0
21
41
7
20
31
43
vacAs1am1
51
36
0
0
9
18
13
36
29
40
vacAs1am2
24
17
1
11
17
33
2
6
4
6
vacAs1bm1
7
5
0
11
1
2
5
14
1
1
vacAs1bm2
7
5
0
0
2
4
3
8
2
3
vacAs1cm1
2
2
0
0
1
2
1
3
0
0
vacAs2m1
12
6
3
33
9
18
0
0
0
0


## 3.3. Detection of theH. pylori vacA Genotyping and Clinical Manifestations

Among 141H.pyloripositive gastric tissues, we determined six different allelic variants of thevacAgene inH. pylori positive gastric tissues. AmongH.pyloripositive gastric tissues, 51 (36%) were positive forvacAs1am1, 24 (17%) forvacAs1am2, and 7 (5%) forvacAs1bm1, whereas 7 (5%) werevacAs1bm2, 2 (2%) were vacAs1cm1, and 12 (6%) were positive forvacAs2 m1. As shown in Table 2, detailed analysis revealed that 29 (40%) of the GC cases had s1am1 genotype (P
<
0.05), which was significantly higher than GIs (9) (18%), GU (13) (36%), and normal cases (P
<
0.05). Respective percentages in GIs, GU, and GC tissue samples of s1am2 were high (33%, 6%, and 6%) as compared to s1mb1 (2%, 14%, and 1%) and s1bm2 (4%, 8%, and 3%)vacA genotype. s1cm1 was observed in GIs and GU, while s2m1 was found only in normal and GIs cases (Table 2).


## 3.4.H. pylori cagA Genotyping with Clinical Association

We found that the prevalence ofcagA gene was 86 (61%), out of which 27 (19%) were Western types and 59 (42%) were East-Asian types (Table 2). All negative tissues for 16S rRNA gene were also negative forcagAandvacAgenes. In detailed analysis, Western-typecagAgene was observed in 4% of the patients with GIs, 31% with GU, and 18% with GC (P
<
0.05), whereas 11% of the normal cases were also positive. East-Asian type strains were observed in 41% of the patients with GIs, 20% with GU, and 43% with GC (P
<
0.05) (Table 2).


## 3.5. Combinational Study ofH. pylori vacA withcagA Genotype

In combinational analysis ofvacA strain with Western type ofcagA, as shown in Table 3, we found that 17 (20%) of theH. pylori strains havevacAs1am1/cagAcombination, while 7 (8%) ofH. pylori strains havevacAs1am2/cagA. In contrast,vacAs1bm1/cagA andvacAs1bm2/cagA showed low number ofH. pylori strains of 2 (2%) and 1 (1%), respectively. ThevacAs1cm1/cagA andvacAs2cm1/cagA were not found inH. pylori positive strains. In detailed analysis, 6 (17%) of GU patients havevacAs1am1, 2 (6%)vacAs1am2, 1 (3%)vacAs1bm1, and 1 (3%)vacAs1bm2. Gastric cancer patients showed a number ofH. pylori strains as follows: 9 (12%) havevacAs1am1, 5 (7%)vacAs1am2, and 1 (1%)vacAs1bm1, andvacAs1bm2 has no case reported. GIs patients showed only 1 (2%)vacAs1am1 combinationalH. pylori strain (Table 3). In contrast, combinational analysis of East-Asian type ofcagA withvacA genes demonstrated more frequentlyH. pylori strains ofvacAs1am1/cagA 32 (37%) andvacAs1bm2/cagA 19 (22%) thanvacAs1bm1/cagA 6 (7%) andvacAs1bm2/cagA 2 (2%) (P
<
0.05). However,vacAs1cm1/cagA andvacAs2cm1/cagA were not found inH. pylori positive strains. Distribution analysis further showed that GIs patients have 7 (14%) s1am1/cagA, 4 (18%)vacAs1am2/cagA, and 3 (6%)vacAs1bm1/cagA H. pylori positive strains. In GU patients, we found 8 (22%)vacAs1am1/cagA and 2 (6%)vacAs1am2/cagA combinations inH. pyloripositive strain. In contrast, GC patients showed high number ofvacAs1am1/cagA 17 (23%) andvacAs1am2/cagA 13 (18%)H. pylori positive strains. However, combinations ofvacAs1bm1/cagA andvacAs1bm2/cagA in GC were found in low percentages as 3 (4%) and 2 (3%), respectively (P
<
0.05) (Table 3).Table 3
Combinational study betweencagA and vacA genotypes with diseases outcomes.
Description
Totaln
=
169
Normaln
=
9
Chronic gastritisn
=
51
Gastric ulcern
=
36
Gastric cancern
=
73
N
%
N
%
N
%
N
%
N
%
TotalH. pylori positive
141
84
6
67
37
73
30
83
68
93
TotalcagA positive
86
61
1
11
21
41
18
50
46
63
WesterncagA/vacAs1am1
17
20
1
11
1
2
6
17
9
12
WesterncagA/vacAs1am2
7
8
0
0
0
0
2
6
5
7
WesterncagA/vacAs1bm1
2
2
0
0
0
0
1
3
1
1
WesterncagA/vacAs1bm2
1
1
0
0
0
0
1
3
0
0
WesterncagA/vacAs1cm1
0
0
0
0
0
0
0
0
0
0
WesterncagA/vacAs2m1
0
0
0
0
0
0
0
0
0
0
East-AsiancagA/vacAs1am1
32
37
0
0
7
14
8
22
17
23
East-AsiancagA/vacAs1am2
19
22
0
0
4
18
2
6
13
18
East-AsiancagA/vacAs1bm1
6
7
0
0
3
6
0
0
3
4
East-AsiancagA/vacAs1bm2
2
2
0
0
0
0
0
0
2
3
East-AsiancagA/vacAs1cm1
0
0
0
0
0
0
0
0
0
0
East-AsiancagA/vacAs2m1
0
0
0
0
0
0
0
0
0
0


## 4. Discussion

Several studies have focused on the diversity of theH. pylori vacA andcagA virulence genotypes. However, there is little information available related to the frequency ofH. pylori vacA andcagA genotypes in Northeast China. In the present study, we determined thevacA andcagA statuses among gastritis (GIs), gastric ulcer (GU), and gastric cancer (GC) patients’ samples. The prevalence ofH. pylori was found to be considerably high in the Pacific Asian countries: for example, China has high prevalence ofH. pylori infection and gastric cancer compared to the rest of the world [23]. In this study, we observed high prevalence ofH. pylori infection (84%) in gastric patients from 2011 to 2013 (Table 2). Previous studies from the northern and central part of China reportedH. pylori prevalence rate of 58% from 1990 to 2002 [24]. In comparison, other countries in the same region such as Singapore, Malaysia, Taiwan, and Vietnam showed lowH. pyloriprevalence [23]. These results indicate thatH. pylori infections have epidemiological diversity, which show different prevalence in different geographical locations.Genetic variations of thevacAgenotype make the requirement to investigate clinical outcomes, which is directly linked with the virulence status ofH. pylori.vacA gene shows variation in the signal and midregion that determineH. pylori cytotoxin activity. In our study, we predominantly foundvacA subtypes of s1a, m1, and m2, while s1b and s1c were found in low frequency (Table 2). Our results showed comparablevacA frequency rates in China with those from other reported data; for example, Hou et al. reported that s1a and m2 were more prevalentvacAgenes in China [25], while Mishra et al. found thatvacAs1a andvacAm1 were major subtypes in India [26]. Previously, studies carried out on the Chinese population have reported high prevalence of s1a subtype, while s1b subtype is rarely found in East-Asian countries [27–29]. In another study, s1b subtype was not detected in Hong KongvacA positiveH. pylori infected patients, while m2 subtype was found in high frequency of 67%. Similarly, we also rarely observed s1c subtype, while s1c was exclusively found from East-Asian isolates [30]. In our study, we found thatvacAs1am1 (36%) is a more prevalent genotype as compared to thevacAs1am2 (17%), while slbm1 (5%), s1bm2 (5%), s1 cm1 (2%), and s2 m1 (6%) genotypes were found in low frequency (Table 2). Previous study showed high prevalence ofvacAgenotype; Ahmad et al. reported the prevalence of s1b/m2 (54.5%) in adult dyspeptic patients from Pakistan [8]. According to Mishra et al.,vacAs1a/m1 (53.2%) has predominant genotype in India [26]. However, Hou et al. reported high (90.5%) frequency ofvacAgenotype in China [25].vacAs1a and s1b genotypes are prominently associated with high toxin activity and linked to clinical outcome of the diseases. In a detailed analysis ofvacA genotype, we found high frequency of s1am1 in GU (36%) and GC (40%) patients, while s1am2 was highly found in GIs (33%) and normal cases (11%). s1bm1 and s1bm2 showed high frequency in GU (14%) and (8%), respectively, while s2m1 was found only in normal (33%) and GIs patients (18%) (Table 2). Our results indicate diverse epidemiology of gastritis patients containingvacAs2 m1 genotype in the northeast part of China. ThevacAs1 andvacAm1 bearingH. pylori strains have been associated with increased virulence capability and higher gastric epithelial damage and ulceration than s2 and m2 strains [31].H. pylori CagA induces pathological alterations, which is closely associated with development of gastritis, gastric ulcer, and gastric cancer.H. pylori cagA-positive strains are more virulent causing higher levels of gastric mucosal inflammation in gastritis and gastric cancer [32, 33]. In present study, about 61% of strains werecagApositive, which comprises 19% Western-typecagA and 42% East-Asian typecagA strain. Conversely, our results showed low prevalence ofcagA gene in gastric patients from Northeast China (Table 2). Previous studies reported high prevalence (93.9%) ofcagA-positiveH. pylori strain in China [34–36]. According to Hou et al.,H. pylori cagA has high prevalence of 93.2% in Shanghai (the southern part of China) [25]. The neighboring countries of China were also reported to have high prevalence ofcagA; for example, India has highcagA prevalence of 96.2% [26]. Rasheed et al. reported that 52% ofH. pyloristrains carriedcagAgene with the positivity rate of 80% in GC, 74% in GU, 63% in duodenal ulcer (DU), and 11% in normal cases from Pakistan [37]. Conversely, in Western Europe,cagA-positive strains are less prevalent and more frequently found in GU or GC patients [38].In combinational analysis ofvacA andcagAgenotypes, we found thatH. pylori strains have high frequency ofvacAs1am1 (37%) orvacAs1am2 (22%) with East-Asian typecagA genotype (Table 3). Rasheed et al. reported high frequency ofcagA(61.9%) with predominantvacAs1a/m2 genotype inH. pyloriinfected gastric tissues of Pakistani children [37]. A previous study reported significantly higher percentages ofcagA-positive andvacAs1 andvacAm1 genotypes with high risk for GC [39]. A recent study conducted in China found no differences in the distribution ofcagA-positive andvacAm genotypes [40]. Our results showed high prevalence ofvacAs1 m1 and East-Asian typecagA-positiveH. pyloristrain in gastric cancer patients.These results might have useful roles in clinical appreciation so as to categorize the specific and most prevalent biomarkers ofH. pylori strains in the northeast part of China that will be helpful to precisely diagnose large population of gastric cancer patients.H. pylorihas high capability of developing antibiotic resistance, which make difficult to treatH. pylori by conventional antibiotics; however, targeted marker therapy leads to effectiveH. pylori treatment and helps reduce antibiotic resistance.In conclusion, the present study showed high diversity of theH. pylori vacAandcagA genotyping. We have reported high frequency of East-Asian typecagA with predominantvacAs1am1 andvacAs1am2 genotypes in gastric ulcer, gastritis, and gastric cancer patients. This study helps to effectively diagnose and treat gastric patients by understanding the trend ofH. pylori infection in Northeast China.



---

*Source: 142980-2014-05-13.xml*