Background
Helicobacter pylori is a microaerophilic spiral shaped bacteria colonizing gastric mucosa on approximately half of the global population and is related with severe gastroduodenal diseases including peptic ulcers and gastric cancer [
1]. The incidence of gastric cancer is highest in East Asia, whereas it is relatively low in Africa and South Asia although the prevalence of
H. pylori infection was highest in these area [
2]. Gastric cancer incidence has a tendency decreasing from North to South even within East Asia. This phenomenon could be explained partly by diversity of
H. pylori virulence [
3]. For example, most of
H. pylori isolates from East Asia are CagA-positive, while approximately 20 to 40% of isolates from Europe and Africa are CagA-negative [
4]. In addition, the classification of CagA into East-Asian-type and Western-type based on the differences of the repeat sequence (Glu-Pro-Ile-Tyr-Ala [EPIYA] segments) are likely associated with the incidence of gastric cancer [
5,
6] as well as a geographical distribution [
7]. The
vacA m1 type strains are common in areas of Northeast Asia, i.e., Japan and South Korea, whereas m2 type strains are predominant in areas of Southeast Asia, i.e., Taiwan and Vietnam [
8]. It will be interesting to analyze other virulence factors as geographical diversity marker such as duodenal ulcer promoting gene (
dupA), blood group antigen-binding adhesin (
babA), induced by contact with epithelium
(iceA) and
jhp0562 and β-
galT-
(jhp0563) which were also reported to have association with severe clinical outcomes [
9]. However, to our knowledge, there are no detailed studies investigating the status of these virulence genes in relation to the geographic/ethnic differences.
Previous study revealed a benefit utilization of multilocus sequence typing (MLST) analysis of
H. pylori which provided human population structure information in detail rather than other methods such as human microsatellite or mitochondrial DNA [
10]. MLST assesed seven
H. pylori populations based on seven housekeeping genes (
atpA, efp, mutY, ppa, trpC, ureI, and
yphC) including hpAfrica1, hpAfrica2, hpNEAfrica, hpEurope, hpEastAsia, hpAsia2, and hpSahul that could forecasting human migrations pattern [
11,
12]. The hpAfrica1, hpAfrica2 and hpNEAfrica strains are predominant in isolates from Africa, hpEurope strains are mainly isolated from ethnic Europeans, hpEastAsia strains are common in
H. pylori from East Asia, hpAsia2 strains are common in
H. pylori from South, Southeast, and Central Asia, and hpSahul strains are mainly isolated from aborigines of Australia and highlanders in New Guinea [
3].
Thailand is Southeast Asian country bordering with Myanmar in the North and West, Laos in the North and North-East, Cambodia in the East and with Malaysia in the South. Although Thai and Chinese are the major ethnic groups, Thailand is a multi-ethnic country which also consists of numerous minor tribes living in the mountain area of the North Thailand. Especially, Maesot, a district on Tak province in North region is a unique area with high culturally diverse due to several ethnic groups such as Thai, Burmese and minor ethnic people with unknown origin (Hmong and Karen) living together in the small town (120,569 people in 2008, Statistical yearbook Thailand 2013,
http://web.nso.go.th). Therefore MaeSot is an ideal area for focusing the roles of
H. pyori virulence factors among different ethnic groups as well as for mapping human migration patterns. In this study, we therefore aimed to clarify two important goals; (1)
H. pylori infection and the virulence genotypes, and (2) the origin of ethnics by using
H. pylori as a tracking tool. We hypothesized that different ethnics had different genes characteristics of
H. pylori, which would be associated with different ancestry.
Methods
Study population
We conducted a community-based endoscopic survey at Ban Chedi Ko School, Maesot city, Tak province during November 11–12, 2013. All the endoscopy equipments were brought from Bangkok and were set up at the gymnastic hall of the school. The project was announced by the local hospital officers 1 month before the survey. Four major ethnic groups including Thai, Thai-Chinese, Karen and Hmong who living in the same community were participated during the survey. Thai-Chinese were defined as Thai citizens who were the offspring of mixed marriages [
13]. Gastric biopsy specimens were collected from each patient from the antrum and corpus of the stomach; two samples from the lesser curvature of the antrum approximately 3 cm from pyloric ring, and one sample from the greater curvature of the corpus. Two specimens from the antrum were used for
H. pylori culture and histological examination. One specimen from the corpus was used for histological examination. The biopsy specimens for culture were immediately placed at − 20 °C, and stored at − 80 °C within a day of collection until used for culture. Peptic ulcers were diagnosed by endoscopic examinations. The normal gastric mucosa was defined as the absence of any activity and inflammation in both the antrum and corpus by histological examination. Blood samples were collected from all participants on the same day of endoscopy for measuring serum anti-
H. pylori antibody.
Written informed consent was obtained from all participants, and the study protocol was approved by Human Research Ethnics Committee of Faculty of Medicine, Thammasat University (Pathum Thani, Thailand) and Oita University Faculty of Medicine (Yufu, Japan).
Diagnosis of H. pylori infection and gastritis stage determination
The H. pylori infection status was diagnosed by serum anti-H. pylori antibody by enzyme-linked immunosorbent assay kit (Eiken Co., Ltd., Tokyo, Japan), culture, histology including immunohistochemistry (IHC). For H. pylori culture, the biopsy specimen was homogenized in normal saline and was inoculated onto H. pylori selective media (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). The plates were incubated for up to 7 days at 37 °C under microaerophilic conditions (10% O2, 5% CO2, and 85% N2). The H. pylori like colony were sub-cultured onto Mueller–Hinton II Agar medium (Becton–Dickinson, Sparks, MD, USA) supplemented with 7% horse blood (Nippon Bio-test, Tokyo, Japan) without antibiotics. H. pylori was identified on the basis of colony morphology, gram staining and positive reactions for oxidase, catalase, and urease. Isolated strains were stored at − 80 °C in Brucella Broth (Becton–Dickinson, Sparks, MD, USA) containing 10% dimethyl sulfoxide and 10% horse serum.
All biopsy specimens for histological testing were fixed in 10% buffered formalin and embedded in paraffin. Serial sections were stained with hematoxylin and eosin as well as May–Giemsa stains. The degree of inflammation (monocyte infiltration), activity (neutrophil infiltration), atrophy, intestinal metaplasia, and bacterial density were classified into four grades according to the updated Sydney system: 0, ‘normal’; 1, ‘mild’; 2, ‘moderate’; and 3, ‘marked’ [
14]. Samples with grade 1 or more atrophy were considered atrophy-positive [
15]. In addition, gastritis stage was assessed based on topographic locations (antrum and corpus), according to the Operative Link on Gastritis Assessment (OLGA) system [
16].
IHC was performed as previously described [
17]. Briefly, after antigen retrieval and inactivation of endogenous peroxidase activity, tissue sections were incubated with anti-
H. pylori antibody (DAKO, Glostrup, Denmark), anti-CagA antibody (b-300 Santa Cruz, CA, USA) or anti-East-Asian-type CagA-specific antibody (α-EAS Ab) diluted 1:2000 with diluting solution (DAKO) overnight at 4 °C. The α-EAS Ab was immunoreactivity with only the East-Asian-type CagA, and not with the Western-type-CagA [
18]. IHC using the α-EAS Ab was proven to be useful tool for typing East-Asian-type CagA immunohistochemically in Japan [
17], Vietnam and Thailand [
19]. After washing, the sections were incubated with biotinylated goat anti-rabbit or anti-rat IgG (Nichirei Co., Tokyo, Japan), followed by incubation with a solution of avidin-conjugated horseradish peroxidase (Vectastain Elite ABC kit; Vector Laboratories Inc., Burlingame, CA, USA). Peroxidase activity was detected using H
2O
2/diaminobenzidine substrate solution.
H. pylori were identified by Giemsa staining and positively immunostained with anti-
H. pylori antibody. The bacterial density scores evaluated by the updated Sydney system equal or greater than grade 1 were considered positive for
H. pylori infection.
H. pylori infection was clarified when histology confirmed by IHC and/or culture tests showed positive.
H. pylori genotyping
DNA was extracted from
H. pylori culture using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s directions. The DNA was kept at − 20 °C until used for genotyping study. The
cagA genotypes (positivity, EPIYA repeat region and pre-EPIYA) were determined by polymerase chain reaction (PCR) amplification-based direct sequencing using the ABI 3100 Genetic Analyzer DNA Sequencer. The
vacA genotypes (s1 or s2, m1 or m2, i1 or i2, d1 or d2 and c1 or c2 regions),
iceA genotype (
iceA1 or
iceA2), and the presence of
dupA, babA, jhp0562, and
β-
(1,3)galT genes were determined based on PCR product size (Additional file
1: Tables S1.) as described previously [
20‐
27] by Microchip Electrophoresis system for DNA/RNA Analysis MCE
® -202 Multina (Shimadzu, Japan).
Population structure analysis of H. pylori strains
The
H. pylori population was constructed by MLST sequence datasets comprised of seven housekeeping genes (
atpA, efp, mutY, ppa, trpC ureI, and
yphC) from 430 strains with different genotype obtained from the pubMLST data base (
http://pubmlst.org/). These sequence datasets were integrated with Maesot sequence data. A neighbor-joining tree was constructed based on the sequence alignment using MEGA v.6.06. We analyzed bacterial population structure using STRUCTURE (v.2.3.2) software. Markov chain Monte Carlo simulations of STRUCTURE were run in the non admixture model with burn-in of 20,000 followed by 30,000 iterations for each run. To run STRUCTURE, a hypothetical number of bacterial populations, K, must be input. We set K as 8–15 and performed five runs for each K.
Data analysis
Data were analyzed by using SPSS, version 16 (SPSS Inc., Chicago, IL, USA). The discrete variables were tested by using Chi square test while continuous variables were tested by Mann–Whitney U test and t test. The P- value < 0.05 was considered statistically significant.
Nucleotide sequencing
Nucleotide sequence data of cagA-positive strains are available under the DDBJ Accession Numbers LC092222 to LC092367. The sequencing data for the seven housekeeping genes of the 89 strains are available under the DDBJ Accession Numbers LC092368 to LC092829 and LC271796 to LC271956.
Discussion
We revealed
H. pylori infection in Maesot was 55.5%, an intermediate rate when compared to the previous reports about the prevalence of
H. pylori infection in Thailand that showed in a big range from 17.5–88.5% [
13,
18,
30‐
45]. To our knowledge, this is the first report about
H. pylori prevalence in minor ethnics of Thailand. Interestingly, our results showed that both of Hmong and Karen ethnics who called as “minor mountain peoples” had a higher prevalence of
H. pylori infection than predominant ethnics. Although environmental factors such as foods, habit and sanitary could influence the prevalence of
H. pylori, our analysis showed that it is associated with the difference source of ancestry.
Totally,
H. pylori with East-Asian-type
cagA and 18-bp or 39-bp deletion types was the majority genotype in Maesot strains. Histological results confirmed that East-Asian-type
cagA strains induced severer histological scores than those infected with Western-type
cagA, concordance with the current consensus [
46‐
48]. In contrast with our expectation, strains with pre-EPIYA 18-bp deletion also induced greater score in histology rather than other genotypes. In the current consensus 39-bp deletion types strains (mostly in East Asian countries) could induce severer histological scores than 18-bp deletion strains (predominant in Vietnam) since the fact that gastric cancer incidence in East Asian countries such as Japan, Korea and China are higher than that in Vietnam. It is suggested that pre-EPIYA region may only become marker of geographical diversity in each population rather than associated with clinical outcomes. Maesot strains also had virulence genotypes of
vacA including s1, m1, i1, d1 and c1. Moreover, they also contained predominant of virulence genotype such as
babA positive,
iceA1 type and
jhp0562 positive. Therefore, Maesot
H. pylori theoretically have enough ability as an agent for inducing severe gastrointestinal diseases. However, Age standardize incidence rate (ASR) for gastric cancer in North Thailand (4.1, 5.4/100,000 in female and male) is still much lower than other countries (e.g., lower than Myanmar [11.2/100,000] or Vietnam [16.3/100,000]) suggesting host and environmental factors are dominant as denominator for clinical outcomes in Thailand rather than bacterial factors (data available from the International Agency for Research on Cancer; GLOBOCAN2012,
http://globocan.iarc.fr/). Interestingly, we found high proportion of
dupA-negative strains in Maesot that probably become as a specific marker for Maesot population as similar as in Indonesian population [
49].
It is still questionable why the genotypes in Maesot strains were relatively consistent with minimal mixture between ethnics although living in the same area. Previously we found a mixed genotype in Thai-Chinese [
13]. The virulence genes analysis showed that majority of Hmong, Thai-Chinese and Thai ethnics were infected with East-Asian-type
cagA strains, whereas most of Karen strains were Western-type
cagA. In addition, pre-EPIYA no deletion type, which is reported as the major type in Western population was also predominant in Karen strains. Finally, based on STRUCTURE, majority of Karen strains belonged to hpAsia2 population type. These results supported that Karen ancestry seems to be related with West Asia, a region with similar genotypes and population type as Karen strains [
3]. Based on the history within Karen peoples, they arrived as refugee to Myanmar from North and are establishing themselves along the border between Thailand and Myanmar [
50]. A mitochondrial DNA study showed that origin of Bamar as a predominant ethnic in Myanmar (68%) was different with Karen, even though they shared Tibeto-Burma origin and same source ancestor from Northwestern of China [
51,
52].
Although Hmong contained strains with proportion of
vacA m1 as similar as Thai, they had higher proportion of East-Asian-type
cagA and
iceA1 positive strains. On the other hand,
vacA m1 predominant was a differentiator with Thai-Chinese. Additionally, although most of Hmong strains belonged to hspEAsia population as same as Thai-Chinese, pre-EPIYA 18-bp deletion type was predominant. The 18-bp deletion type had been reported as the majority type in Vietnamese population [
24]. Thus, there are two implications to explain these data. First, Hmong may become a high risk population in Thailand associated with higher prevalence and severer virulence genotypes of
H. pylori (East-Asian-type
cagA with 18-bp deletion). Second, Hmong ancestry source will be probably different from Thai or Thai-Chinese and assumed to have some connection with Vietnamese based on similarity of pre-EPIYA genotype. When we only compared
H. pylori with East-Asian-type
cagA and categorized onto hspEAsia from Bhutan, Hmong in Maesot (this study) and Hmong in Vietnam (unpublished data), they closed each other and make one group in the phylogenic tree (Additional file
5: Figure S4.), which indicated a possibility of similar origin. It is believed that second half of the 19th century, the large number of Hmong settlers migration from Sichuan, Guizhou and Yunnan, penetrated the Peninsula and went as far south as the 17th parallel near Tak in Thailand [
53].
Our results confirmed our previous study [
13] that Thai-Chinese strains contained East-Asian-type
cagA and
vacA m2 as the major genotypes. The
vacA m2 genotype is common among ethnic Chinese in the Southern parts of East Asia [
8,
28]. We completed our previous study with confirmation that
H. pylori from Thai-Chinese ethnic contained pre-EPIYA 39-bp deletion type which is also predominantly in East Asian countries [
24]. Additionally, STRUCTURE also confirmed the strains belonged to hspEAsia. The influences of Chinese in Thailand started from 27th century before present when people from Guangxi, China settled in Northeast Thailand. During Ayutthaya period (13th century) many Chinese intermarried with local Thai that while infusing of Chinese culture in Thailand and continuously until the late of 18th century which mostly from Cháozhōu prefecture [
54]. The latest, they have immigrated to Thailand from South-West China (e.g. Yunnan) [
13].
In contrast with our previous study [
13] that majority of
cagA genotype in strains isolated from Thai in Bangkok (capital city) was Western-type, Thai strains in Maesot contained East-Asian-type
cagA. It is indicated that generally Thai in Maesot had higher risk for gastric cancer than Thai who living in the capital that may also partly explained the difference of gastric cancer risk between two regions (ASR female and male in North vs Central were 4.1, 5.4 vs. 1.8, 3.8, respectively) [
55]. STRUCTURE showed that Thai strains belonged to three populations; hpAsia2, hpEurope and hspEAsia. hpAsia2 and hpEurope population may reflect cultural influences from India into Thailand including Buddhism (∼ 2300 years ago). India population also belonged to hpAsia2 and hpEurope [
56], however the more recent importation also should be considered. Started 600 years ago Thailand contacted with European countries including Portugal, Spain, French, Denmark, Netherlands and England [
57].
Previous study showed that α-EAS Ab could detect in the most of Japanese East-Asian-type
cagA strains [
17,
29]. In contrast, we found that only 71.4% of East-Asian-type CagA of Maesot strains could detect by same antibody due to an unidentical sequences for the α-EAS Ab designed amino acid sequence. These differences could explained with several reasons. First, previous study recruited 166 patients from center area of Thailand that 44 of them (83.0%) were Western-type CagA and only 9 (17.0%) were East-Asian type
cagA. Thus, it may influenced the sensitivity rate of the test. Importantly, the kit showed a consistency to exclude all Western-type
cagA in Thai strains in both of study. Second, Thailand is a cultural cross roads between East and South Asia which contained a lot of recombination genotype among strains including CagA. It is supported by the big diversity of CagA epitope in this study that could not detect by α-EAS Ab (3–14 amino acid differences). Therefore, the kit could not use in the entire of Thailand region and a validation in each region is a critical importance. Our group also reported a low immunoreactivity of α-EAS Ab in East-Asian-type CagA Bhutanese strains [
58], suggesting that the α-EAS antibody might specific for each country.
Finally, we had some limitations in this study. Since we conducted a community-based endoscopic survey for only 2 days, we could not get the similar sample size of patients in each ethnic group. Although we could not find any difference in the prevalence/genotypes of virulence factors such as babA, iceA, babA, jhp0562 and gastric mucosal status, further studies with larger number of patients, especially for Karen ethnic, will be necessary to confirm our current data.
Authors’ contributions
Conception and design: VM, TR, RV and YY. Experiment: PS, UT. Data analysis: PS, UT, RS, JAand MM. Drafting the manuscript: PS, MM and YY. Sample collection: VM, TR, RV, UT and YY. Obtain funding and final approve submit: YY. All authors read and approved the final manuscript.