Prevalence of tipα gene in the Indian population
267 clinical H. pylori isolates are included in our study were isolated from - -Gastritis (n=80), Duodenal Ulcer (DU) (n=77), Non Ulcer Dyspepsia (NUD) (n=51), Gastric Ulcer (n=7), Gastro-Esophageal Reflux Disease (GERD) (n=32) and Controls (n=20). The Male:Female (M:F) was 59:30 and mean age group was 43.03 ± 16.19.
The genotyping of tipα was done using primer as shown in Table 4 which gave an amplicon size of 168 bp (Figure 1). The prevalence rate of tipα gene was found to be 59.9% (160 out of 267) (95% CI: 54.36% to 100%) in the Indian population. However, 100% (27/27) of the H. pylori strains isolated from East India are tipα positive followed by 90% (9/10) in South India, 75.3% (49/65) in North India and only 45.4% (75/165) in North-East India (Table 1).
Genotypic status of cagA and vacA gene
Multiplex PCR was done for detection of cagA and vacA gene in all H. pylori isolates. (Figure 2). The cagA gene was detected in 79.8% isolates (213/267) (95% CI: 53.7% to 97.6%) and rest 20.3% isolates (54/267) showed absence of cagA gene which was further confirmed using empty site PCR (Table 1). Region wise more than 90% of the H. pylori isolates are cagA positive in North East India 93.9% (155/165) and South India 90% (9/10) followed by East India showing prevalence of 74% (20/27). However surprisingly only 44.6% (29/65) of the North Indian strains are cagA positive (Table 1).
The genotype of different vacA alleles examined in overall isolates were found to be s1m1 in 75.3% (201/267), s2m2 in 16.8% (45/267) and s1m2 in 7.9% (21/267) isolates (Table 1). vacA s1m1 allele is found to be positive in more than 90% of the H. pylori isolates from North East and South India. The H. pylori isolates from East India shows that 55.5% (15/27) strains are s1m1, 18.5% (5/27) are s1m2 and only 25.9% (7/27) of the strains are s2m2. However, the strains from North India shows that 40% (26/65) are s1m1, 16.9% (11/65) are s1m2 while 43% (28/65) are s2m2 (Table 1).
Table 1: Prevalence of tipα, cagA and vacA gene in H. pylori isolates from different regions of India.
Isolates from different states
|
Total no. of isolates
|
tipα positive isolates (%)
|
tipα
negative isolates
(%)
|
cagA
positive isolates
(%)
|
cagA
negative isolates
(%)
|
vacA s1m1
(%)
|
vacA s2m2
(%)
|
vacA s1m2
(%)
|
North East India
|
165
|
75 (45.4)
|
90
(54.5)
|
155
(93.9)
|
10
(6.1)
|
151
(91.5)
|
10
(6.06)
|
4
(2.42)
|
North India
|
65
|
49 (75.3)
|
16
(24.6)
|
29
(44.6)
|
36
(55.4)
|
26
(40)
|
28
(43)
|
11
(16.9)
|
South India
|
10
|
9
(90)
|
1
(10)
|
9
(90)
|
1
(10)
|
9
(90)
|
0
(0)
|
1
(10)
|
East India
|
27
|
27
(100)
|
0
(0)
|
20
(74)
|
7
(26)
|
15
(55.5)
|
7
(25.9)
|
5
(18.51)
|
Total
|
267
|
160
(59.9)
|
107
(40.1)
|
213
(79.8)
|
54
(20.3)
|
201
(75.3)
|
45
(16.8)
|
21
(7.9)
|
Association of tipα, cagA, vacA with different disease outcomes
Table 2 shows association of tipα, cagA and vacA with different disease outcomes. Our study shows the presence of tipα gene in H. pylori isolated from patients suffering from various upper gastrointestinal diseases – 41.3% Gastritis (33/80), 84.38% Non Ulcer Dyspepsia (43/51), 58.4% Duodenal Ulcer (45/77), 42.9% Gastric Ulcer (3/7), 75% GERD (24/32). The association of tipα gene was found significant only in Non Ulcer Dyspepsia (NUD) with respect to the controls (p < 0.05) (Table 2). Significant association of cagA virulent gene was found in Duodenal Ulcer (p < 0.01) and Gastritis (p < 0.001). vacAs1m1 allele was also found to be significantly associated with Duodenal Ulcer (p < 0.05) and Gastritis (p < 0.05).
Table 2: Detection and distribution of H. pylori and virulent genes in patients with various gastrointestinal disease outcomes.
|
Control (%)
n = 20 (7.5)
|
Gastritis
(%)
n = 80
(29.9)
|
GERD (%)
n = 32
(12)
|
NUD (%)
n = 51
(19.1)
|
DU (%)
n = 77
(28.8)
|
Gastric Ulcer (%)
n = 7
(2.6)
|
Total (%)
n = 267
(100)
|
cagA positive
|
13 (65)
|
74 (92.5)* (*=0.001)
|
16 (50)
|
37 (72.5)
|
68 (88.3) * (*= 0.01)
|
5 (71.4)
|
213 (79.7)
|
cagA negative
|
7 (35)
|
6 (7.5)
|
16 (50)
|
14 (27.5)
|
9 (11.7)
|
2 (28.6)
|
54 (20.3)
|
tipα positive
|
12 (60)
|
33 (41.3)
|
24 (75)
|
43 (84.3) *
(*=0.02)
|
45 (58.4)
|
3 (42.9)
|
160 (59.9)
|
tipα
negative
|
8 (40)
|
47 (58.8)
|
8 (25)
|
8 (15.7)
|
32 (41.6)
|
4 (57.1)
|
107 (40.1)
|
vacAs1m1
|
12 (60)
|
74 (92.5) * (*= 0.00)
|
14 (43.8)
|
31 (60.8)
|
65 (84.4) * (*= 0.05)
|
5 (71.4)
|
201 (75.2)
|
vacAs2m2
|
4 (20)
|
6 (7.5)
|
14 (43.8)
|
12 (23.5)
|
7 (9.1)
|
2 (28.6)
|
45 (16.9)
|
vacAs1m2
|
4 (20)
|
0 (0)
|
4 (12.5)
|
8 (15.7)
|
5 (6.5)
|
0 (0)
|
21 (7.9)
|
*p < 0.05
Association of tipα with cagA and vacA gene
In our study we found 56.8% (121/213) cagA positive and 72.2% (39/54) cagA negative showed presence of tipα gene but the association was found to be not significant (Table 3). We found that 95.2% of vacAs1m2 also shows the presence of tipα and the association is found to be significant (p < 0.05). 71.1% of vacAs2m2 and 53.7% of vacAs1m1 shows the presence of tipα but the association was not significant.
Table 3: Association of vacA, cagA and tipα in H. pylori strains
|
Total H.pylori isolates (%)
n = 267
|
cagA positive (%)
n = 213
|
cagA negative (%)
n = 54
|
vacAs1m1
(%)
n = 201
|
vacAs2m2
(%)
n = 45
|
vacAs1m2
(%)
n = 21
|
tipα Positive
|
160 (59.9)
|
121 (56.8)
|
39 (72.2)
|
108 (53.7)
|
32 (71.1)
|
20 (95.2) * (*= 0.001)
|
tipα negative
|
107 (40.1)
|
92 (43.1)
|
15 (27.7)
|
93 (46.2)
|
13 (28.9)
|
1 (4.7)
|
*p < 0.05