Detection and distribution of virulence genes
cagA
Overall, 263 (97.8%) patients were infected with cagA-positive strains (Table 1). Of these cagA-positive strains, 66 (89.2%) were isolated from Shandong, 74 (94.9%) from Guangxi, 100% from Hunan, Qinghai and Heilongjiang. The prevalence of cagA-positive strains was lower in Shandong and Guangxi than in other three regions, but the difference was not statistically significant (χ2 = 4.635, P > 0.05). The sequencing results showed that CagA type was mainly East Asian type, accounting for 91.8% (236/257), whereas only 9 strains were Western type, and 55.6% (5/9) were isolated from Heilongjiang. Among 236 East Asian type CagA, 234 were of the ABD subtype and 2 of the AABD subtype. The distribution of CagA sequence types is shown in Table 2. The CagA type was mainly the ABD subtype, accounting for 91.1% and the ABD subtypes show a significant association with different regional areas (χ2 = 19.772, P < 0.01). Most of the Western strains were from Heilongjiang and there was a significant difference between the ABC subtype and Heilongjiang isolates (χ2 = 15.512, P < 0.01).
Table 2
Distribution of CagA types in different regions of Chinaa
Region
|
EPIYA motif typesb
|
Total
|
AB
|
ABDc
|
AABD
|
ABCc
|
ABCC
|
SD
|
9
|
55
|
1
|
1
|
0
|
66
|
GX
|
0
|
71
|
1
|
1
|
1
|
74
|
HN
|
0
|
46
|
0
|
0
|
0
|
46
|
QH
|
0
|
32
|
0
|
1
|
0
|
33
|
HL
|
3
|
30
|
0
|
5
|
0
|
38
|
Total
|
12(4.7)
|
234(91.1)
|
2(0.8)
|
8(3.1)
|
1(0.4)
|
257
|
acagA-positive strains were from five regions of China: Shandong (SD), Guangxi (GX), Hunan (HN), Qinghai (QH) and Heilongjiang (HL).
bvalues in parentheses are percentages.
cindicating a significant difference between different regional areas(p < 0.01).
vacA
The geographic distribution of vacA s- and m-alleles is shown in Table 1. In the 269 isolates, multiple vacA genotypes were detected in either the s-region (80.7%), the m-region (25.6%), or both (17.1%). The prevalence of vacA s- and m- region genotypes varied between the five regions of China. The most common vacA s-region genotype was s1 (93.3%): 93.2% of isolates from Shandong, 91% from Guangxi, 95.7% from Hunan, 87.9% from Qinghai and 100% from Heilongjiang. For the vacA m-region, 198 patients (73.6%) were infected with m1 H. pylori strains. The prevalence of vacA m1 genotype from Heilongjiang was the highest and that of Shandong was the lowest (92.1% and 60.8%). In the combination of vacA s and m region genotypes, s1m2 was predominant and found in 182 (67.7%) of the H. pylori strains and the s1/m1 was detected in 180 (66.9%).
For the vacA s subtype, the dominant genotype in 269 isolates was s1a (88.1%). The vacA s1b was not detected in our study. Among the 74 vacA s1 strains from Shandong, the prevalence of s1a (86.5%) and s1c (82.4%) was nearly similar. The distribution of s1a (85.9%) and s1c (89.7%) in Guangxi resembled that of Shandong. Of the 46 s1 strains from Hunan, 40 (87%) were s1a and 43 (93.5%) were s1c. In Qinghai, 87.9% of the s1 strains were s1a and 69.7% were s1c. Among strains from Heilongjiang, 37 (97.4%) of the 38 s1 strains were s1a, 33 (86.8%) was s1c. Of all the cultures, 252 (93.7%) could be genotyped as either m1, m2a or m2b. Among the 152 s1 strains from Guangxi, Hunan and Heilongjiang, m1 (76.5%) was more prevalent than m2a (48.1%). Of the 69 s1 strains from Shandong, the prevalence of m1 (55.4%) and m2a (59.6%) was about similar. Among the 29 vacA s1 strains from Qinghai, 14 (42.4%) contained m1 and 11 (33.3%) were m2a (Table 3 and Fig. 1).
Table 3
Geographic distribution of vacA subtypes of s- and m-region in Chinaa,b
Region
|
Total
|
s1a
|
s1c
|
s2
|
Multiple genotypes
|
m1
|
m2a
|
m2b
|
m1
|
m2a
|
m2b
|
m2a
|
m2b
|
s
|
m
|
s + m
|
SD
|
74
|
39(52.7)
|
39(52.7)
|
31(41.9)
|
35(47.3)
|
38(51.4)
|
30(40.5)
|
2(2.7)
|
2(2.7)
|
50(67.6)
|
5(6.8)
|
4(5.4)
|
GX
|
78
|
46(60)
|
28(35.9)
|
14(18)
|
49(62.8)
|
32(41)
|
16(20.5)
|
0
|
0
|
65(83.3)
|
9(11.5)
|
7(9)
|
HN
|
46
|
36(78.3)
|
22(47.8)
|
20(43.5)
|
39(84.8)
|
23(50)
|
21(45.7)
|
1(2.2)
|
0
|
39(84.8)
|
9(19.6)
|
8(17.4)
|
QH
|
33
|
14(42.4)
|
10(30.3)
|
7(21.2)
|
11(33.3)
|
9(27.3)
|
6(18.2)
|
3(9.1)
|
2(6)
|
26(78.8)
|
8(24.2)
|
6(18.2)
|
HL
|
38
|
34(89.5)
|
22(57.9)
|
17(44.7)
|
30(79)
|
18(47.4)
|
15(39.5)
|
2(5.3)
|
2(5.3)
|
30(79)
|
27(71.1)
|
21(55.3)
|
Total
|
269
|
169(62.8)
|
121(45)
|
89(33.1)
|
164(61)
|
120(44.6)
|
88(32.7)
|
8(3)
|
6(2.2)
|
210(78.1)
|
58(25.6)
|
46(17.1)
|
aH. pylori isolates were from five regions of China: Shandong (SD), Guangxi (GX), Hunan (HN), Qinghai (QH) and Heilongjiang (HL).
bValues in parentheses are percentages.
The geographic distribution of vacA s and m subtypes was different in the five regions of China (Table 3 and Fig. 1). The prevalence of s1c in the isolates from Hunan was 93.5%, significantly higher (χ2 = 10.760, P < 0.05) than that in Shandong (82.4%), Guangxi (89.7%), Qinghai (69.7%) and Heilongjiang (86.8%). In contrast, s1a was more frequent in isolates from Heilongjiang (97.4%) than the other four regions, but no statistical significance was noted (χ2 = 3.718, P > 0.05). The prevalence of m1 strains in Heilongjiang (92.1%) was significantly higher (χ2 = 15.668, P < 0.001) than in Shandong (60.8%) and Qinghai (51.5%). The frequency of the m2b subtype in Hunan (50%) was significantly higher (χ2 = 10.714, P < 0.01) than in Guangxi (23.1%) and Qinghai (24.2%) and there was no significant difference in prevalence of vacA m2a (χ2 = 5.450, P > 0.05). We also examined s1a/m1, s1c/m1, s1/m2a, s1/m2b different combinations in patients according to analysis of the vacA subtypes. The dominant vacA subtype combination in the five regions was s1a/m1 (62.8%) and detection of vacA s1a/m1 was significantly high 34 (89.5%) in Heilongjiang strains (χ2 = 32.218, P < 0.001).
iceA
Of the 269 isolates, iceA1 was found in 212 isolates (78.8%) and iceA2 in 214 isolates (79.6%). In these isolates 160 (59.5%) contained both iceA1 and iceA2, which indicating mixed infection. The distribution of iceA varied considerably for isolates from five different geographic regions of China. The iceA1 was present in 95.7% and 84.8% of H. pylori strains isolated from Hunan and Qinghai, respectively, whereas only 66.7% of isolates from Guangxi were infected with iceA1 positive strains. This difference was statistically significant (χ2 = 15.510, P < 0.001). The iceA2 frequency was significantly more prevalent in Hunan (100%) and Qinghai (97%) strains than in Shandong (52.7%) strains (χ2 = 42.147, P < 0.001). The prevalence rates of mixed iceA genotypes in Shandong, Guangxi, Hunan, Qinghai and Heilongjiang were 36.5%, 51.3%, 95.7%, 78.8% and 60.5%, respectively and the mixed iceA was more prevalent in Hunan strains than in the other four regions strains (χ2 = 48.502, P < 0.001) (Table 1).
oipA
258 (95.9%) patients were infected with oipA-positive strains (Table 1). The oipA-positive isolates were present in 100% of Guangxi and Hunan isolates and the prevalence rates in Shandong, Qinghai and Heilongjiang were 98.6%, 78.8% and 92.1%, respectively. The oipA gene was more prevalent in Guangxi and Hunan strains than in Qinghai strains (χ2 = 27.531, P < 0.001).
dupA
122 (45.4%) patients were infected with dupA-positive strains (Table 1). The dupA-positive isolates were present in 73.9% of Hunan, 81.8% of Qinghai and 65.8% of Heilongjiang. In contrast, only 32.4% of Shandong and 15.4% of Guangxi isolates were infected with dupA-positive H. pylori (χ2 = 72.497, P < 0.001).
Association among the genotypes in H. pylori strains
The prevalence of cagA gene is relatively high in China and it was independent of iceA and dupA gene. The cagA was present in 204 out of 212 iceA1 genes (96.2%), 209 out of 214 iceA2 genes (97.7%) and 117 out of 122 dupA genes (95.9%). Table 4 shows the association of vacA with dupA and iceA genotypes in H. pylori strains. It was found that the infection rate of vacA s1c was higher than vacA s1a in dupA and iceA positives strains. Similarly, vacA m1 positive strains detected a significantly high dupA 91 (74.6%), iceA1 154(72.6%) and iceA2 162 (75.7%) genotypes. Furthermore, there was a high prevalence rate of dupA 71.3% (87/122), iceA1 66.5% (141/212) and iceA2 77.1% (165/214) in the vacA s1a/m1 positive strains.
Table 4
Association of vacA with dupA and iceA genotypes
|
SD n (%)
|
GX n (%)
|
HN n (%)
|
QH n (%)
|
HL n (%)
|
vacA
|
dupA
|
iceA1
|
iceA2
|
dupA
|
iceA1
|
iceA2
|
dupA
|
iceA1
|
iceA2
|
dupA
|
iceA1
|
iceA2
|
dupA
|
iceA1
|
iceA2
|
s-region
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
s1a
|
19(25.7)
|
52(70.3)
|
33(44.6)
|
10(12.8)
|
42(53.8)
|
58(74.4)
|
28(60.9)
|
39(84.8)
|
40(87)
|
23(70)
|
23(70)
|
25(75.8)
|
24(63.2)
|
28(73.7)
|
31(81.6)
|
s1c
|
19(25.7)
|
48(64.9)
|
28(37.8)
|
9(11.5)
|
47(60.2)
|
60(76.9)
|
32(70)
|
41(89.1)
|
43(93.5)
|
20(60.6)
|
18(54.5)
|
21(63.6)
|
23(60.5)
|
28(73.7)
|
29(76.3)
|
m-region
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
m1
|
17(23)
|
32(43.2)
|
28(37.8)
|
7(9)
|
41(52.6)
|
47(60.3)
|
30(65.2)
|
39(84.8)
|
41(89.1)
|
14(42.4)
|
15(45.5)
|
17(51.5)
|
23(60.5)
|
27(71.1)
|
29(76.3)
|
m2a
|
17(23)
|
31(41.9)
|
24(32.4)
|
3(3.8)
|
21(26.9)
|
28(35.9)
|
16(34.8)
|
23(50)
|
25(54.3)
|
6(18.2)
|
10(30.3)
|
12(36.4)
|
16(42.1)
|
18(47.4)
|
17(44.7)
|
m2b
|
9(12.2)
|
29(39.2)
|
21(28.4)
|
3(3.8)
|
12(15.4)
|
17(21.8)
|
18(39.1)
|
23(50)
|
23(50)
|
7(21.2)
|
8(24.2)
|
7(21.2)
|
13(34.2)
|
13(34.2)
|
16(42.1)
|
s/m region
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
s1am1
|
15(20.3)
|
26(35.1)
|
26(35.1)
|
9(11.5)
|
35(44.9)
|
40(51.3)
|
24(52.2)
|
35(76.1)
|
40(87)
|
11(33.3)
|
11(33.3)
|
13(39.4)
|
22(57.9)
|
26(68.4)
|
28(73.7)
|
s1am2a
|
13(17.6)
|
28(37.8)
|
21(28.4)
|
4(5.1)
|
17(21.8)
|
25(32.1)
|
13(28.3)
|
20(43.5)
|
22(47.8)
|
6(18.2)
|
7(21.2)
|
9(27.3)
|
15(39.5)
|
18(47.4)
|
17(44.7)
|
s1am2b
|
5(6.8)
|
27(36.5)
|
17(23)
|
3(3.8)
|
9(11.5)
|
13(16.7)
|
14(30.4)
|
20(43.5)
|
20(43.5)
|
6(18.2)
|
7(21.2)
|
5(15.2)
|
12(31.6)
|
12(31.6)
|
14(36.8)
|
s1cm1
|
14(18.9)
|
26(35.1)
|
23(31.1)
|
9(11.5)
|
36(46.2)
|
41(52.6)
|
28(60.9)
|
36(78.3)
|
39(84.8)
|
9(27.3)
|
8(24.2)
|
11(33.3)
|
21(55.3)
|
24(63.2)
|
25(65.8)
|
s1cm2a
|
16(21.6)
|
24(32.4)
|
20(27)
|
4(5.1)
|
19(24.4)
|
28(35.9)
|
14(30.4)
|
26(56.5)
|
23(50)
|
6(18.2)
|
4(12.1)
|
8(24.2)
|
15(39.5)
|
16(42.1)
|
14(36.8)
|
s1cm2b
|
7(9.5)
|
24(32.4)
|
16(21.6)
|
3(3.8)
|
10(12.8)
|
15(19.2)
|
16(34.8)
|
21(45.7)
|
21(45.7)
|
6(18.2)
|
6(18.2)
|
5(15.2)
|
12(31.6)
|
11(28.9)
|
14(36.8)
|
We examined different combinations based on the analysis of the vacA subtypes (s1a, s1c, m1, m2a, m2b), cagA and iceA (iceA1, iceA2) in patients. The most prevalent combination s1cm1/cagA+/iceA2 was present in 51.7% (139/269) including 25.7% (19/74) of Shandong, 56.4% (44/78) of Guangxi, 84.8% (39/46) of Hunan, 33.3% (11/33) of Qinghai and 68.4% (26/38) of Heilongjiang. The predominant common combination genotypes in Guangxi and Hunan was s1cm1/cagA+/iceA2 (56.4% and 84.8%, χ2 = 5.409, P < 0.05) and that was s1am1/cagA+/iceA2 (42.4% and 73.7%, χ2 = 7.143, P < 0.01) in Qinghai and Heilongjiang, while the most common in Shandong was s1am2a/cagA+/iceA1 (36.5%) (Table 5).
Table 5
Combination genotypes of cagA, vacA and iceA in China
Combination
|
No. of isolates
|
|
SD n (%)
|
GX n (%)
|
HN n (%)
|
QH n (%)
|
HL n (%)
|
Total n (%)
|
s1am1/cagA+/iceA1
|
26(35.1)
|
33(42.3)
|
35(76.1)
|
12(36.4)
|
26(68.4)
|
132(49.1)
|
s1am1/cagA+/iceA2
|
22(29.7)
|
37(47.4)
|
36(78.3)
|
14(42.4)
|
28(73.7)
|
137(50.9)
|
s1am2a/cagA+/iceA1
|
27(36.5)
|
19(24.4)
|
21(45.7)
|
8(24.2)
|
18(47.4)
|
93(34.6)
|
s1am2a/cagA+/iceA2
|
19(25.7)
|
26(33.3)
|
22(47.8)
|
10(30.3)
|
17(44.7)
|
94(34.9)
|
s1am2b/cagA+/iceA1
|
23(31.1)
|
8(10.3)
|
20(43.5)
|
7(21.2)
|
11(28.9)
|
69(25.7)
|
s1am2b/cagA+/iceA2
|
15(20.3)
|
10(12.8)
|
20(43.5)
|
6(18.2)
|
14(36.8)
|
65(24.2)
|
s1cm1/cagA+/iceA1
|
24(32.4)
|
39(50)
|
37(80.4)
|
9(27.3)
|
24(63.2)
|
133(49.4)
|
s1cm1/cagA+/iceA2
|
19(25.7)
|
44(56.4)
|
39(84.8)
|
11(33.3)
|
26(68.4)
|
139(51.7)
|
s1cm2a/cagA+/iceA1
|
26(35.1)
|
23(29.5)
|
21(45.7)
|
6(18.2)
|
16(42.1)
|
92(34.2)
|
s1cm2a/cagA+/iceA2
|
19(25.7)
|
30(38.5)
|
23(50)
|
8(24.2)
|
15(39.5)
|
95(35.3)
|
s1cm2b/cagA+/iceA1
|
21(28.4)
|
11(14.1)
|
21(45.7)
|
6(18.2)
|
11(28.9)
|
70(26)
|
s1cm2b/cagA+/iceA2
|
13(17.6)
|
14(17.9)
|
21(45.7)
|
5(15.2)
|
14(36.8)
|
67(24.9)
|