Virulence Genes of Helicobacter pylori in Gastritis , Peptic Ulcer and Gastric Cancer in Laos

BACKGROUND
Helicobacter pylori (H. pylori) infection is an established cause of peptic ulcers and gastric cancer. The aim of this study was to identify H. pylori genotypes and to examine their associations with geographical regions and gastritis, peptic ulcers and gastric cancer in Laos.


MATERIALS AND METHODS
A total of 329 Lao dyspeptic patients who underwent gastroscopy at Mahosot Hospital, Vientiane, Laos during December 2010--March 2012 were enrolled. Two biopsy specimens (one each from the antrum and corpus) were obtained for CLO testing and only CLO test-positive gastric tissue were used to extract DNA. PCR and sequencing were identified for variants of the cagA and vacA genotypes.


RESULTS
Some 119 Laos patients (36.2%) were found to be infected with H. pylori including 83 with gastritis, 13 with gastric ulcers (GU), 20 with duodenal ulcers (DU) and 3 with gastric cancer. cagA was detected in 99.2%. East-Asian-type cagA (62%) and vacA s1c (64.7%) were predominant genotypes in Laos. vacA s1c-m1b was significantly higher in GU than gastritis (53.8% vs. 24.1%; P-value=0.04) whereas vacA s1a-m2 was significantly higher in DU than gastritis (40.0% vs. 16.9%; P-value=0.03). East-Asian-type cagA and vacA s1c were significantly higher in highland than lowland Lao (100% vs. 55.8%; P-value=0.001 and 88.2% vs. 61.5%, P-value=0.03 respectively).


CONCLUSIONS
H. pylori is a common infection in Laos, as in other countries in Southeast Asia. The cagA gene was demonstrated in nearly all Laos patients, cagA and vacA genotypes being possible important factors in explaining H. pylori infection and disease outcomes in Laos.


Introduction
Helicobacter pylori (H.pylori) infection is now accepted as the major cause of chronic gastritis as well as severe diseases including peptic ulcers, gastric cancer and MALT lymphoma (Warren and Marshall., 1983;Dixon., 1991;Tytgat et al., 1993;Parsonnet et al., 1999;Wotherspoon et al., 1999;Sriamporn et al., 2002;Hamajima et al., 2004;Tokudome et al., 2006;Park et al., 2008;Pandey et al., 2011;Zheng et al., 2012).Although gastric cancer is one of the leading causes of cancer-related death, only a minority of individuals with H. pylori infection ever develops this particular cancer.The possible explanation might be related to differences in virulence factors of H. pylori strains in addition to host, environmental, and dietary factors.Human HLADQB1 genes might play important roles in H. pylori infection in Indonesian people (Zhao et al., 2012).Blood group O were likely to develop H. pylori infection related to peptic
cagA gene has been proposed as a marker for a genomic pathogenicity island (cag PAI) with approximately 40 kbp whose presence is associated with more severe clinical outcomes (Blaser et al., 1995;Atherton., 2000).Several studies in Western countries have demonstrated that cagA gene was associated with peptic ulcer and gastric cancer (Well et all., 1996;Parsonnet et al., 1997;Rudi et al., 1998).In contrast, the majority of studies from East Asian countries demonstrated high prevalence of cagA-positive H. pylori without diseases association (Maeda et al., 1998;Kim et al., 2001).The 3' repeat region of the cagA can be used as a marker to separate East Asia from those in Western countries.Interestingly, studies from Asian countries reported that this East-Asian cagA might be a subgenotype of cagA gene which plays important roles in gastroduodenal disease development (Yamaoka et al., 1998;Yamaoka et al., 1999, Yamaoka et al., 2000;Yamaoka et al., 2002).
The Lao People Democratic Republic (Laos) is located in Southeast Asia; the North is border with China and Myanmar, the South is border with Cambodia, the East is border with Vietnam and the West is border with Thailand.Laos has population of 6.28 million people in 2011 (World Development Indicators 2012, World Bank database), classified into 3 major difference ethnic groups: Lao Lum (lowland Lao), Lao Thoeng (midland Lao) and Lao Sung (highland Lao).The gastric cancer was one of the cancer related death in Laos with ASR 6.9 and 7.1 in male and female respectively (Nguyen et al., 2011).A previous study reported that the prevalence of H. pylori infection in Laos was 68.4% with no differences according to age and sex.However, H. pylori infection was more common in patients with duodenal ulcers (80.0%) than those of normal endoscopic findings (60.6%).In addition, there was no prior study of H. pylori genotype related with clinical disease outcomes (Rasachak and Sihavong., 2000).Therefore, this study was aimed to investigate the prevalence of cagA and vacA genotypes of H. pylori and to examine the association between the theses genotypes and gastroduodenal diseases in Laos' dyspeptic patients.

Patients
Total of 329 Lao dypeptic patients who underwent gastroscopy at Mahosot Hospital, Vientiane, Laos during December 2010 -March 2012 were enrolled in this study.Inclusion criteria consisted of (1) age more than 15 years old, (2) had indication for upper gastrointestinal endoscopy, (3) no prior history of chronic severe medical illness such as chronic renal failure, congestive heart failure or HIV infection (4) no history of bleeding diathesis and (5) providing informed consent.Exclusions were (1) patient had received antibiotic or proton pump inhibitor within 1 month prior to the study, (2) had history of previous H. pylori eradication, and (3) had contraindication for gastric biopsy.Gastric biopsy specimens were taken each from antrum and corpus.Gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer were identified by endoscopy, and gastric cancer was further confirmed by histopathology.Gastritis was defined as histological gastritis in the absence of peptic ulcer or gastric malignancy.The status of H. pylori infection was determined based on the result of rapid urease test (CLO test).Informed consent was obtained from all patients and the protocol was approved by the Ethics Committee of Lao National Ethics Committee for Health Research.

H. pylori genotyping
H. pylori DNA was extracted from gastric tissue in case the CLO-test yielded positive results.DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, Inc.Santa Clarita, CA, USA) according to the manufacturer's instructions.The cagA genotypes (East-Asian type or Western type) and vacA genotypes (s region: s1a, s1b and s1c and m regions: m1a, m1b, m1c and m2) were evaluated by polymerase chain reaction (PCR) as previously described (Atherton et al., 1995;Doorn et al., 1998;Yamaoka et al., 1998;Mukhopadhyay et al., 2000;Kersulyte et al., 2000;Ghose et al., 2002;Yamaoka et al., 2002).To confirm the results obtained by PCR and to determine the status of vacA m1c in case of vacA m1a, m1b and m2 were negative, PCR-based sequencing was also performed as previously described (Yamaoka et al., 2002).The PCR condition was 35 cycles consisting of 1 minute at 95°C, 1 minute at 52°C and 1 minute at 72°C.The final cycle included a 7-minutes extension step to ensure full extension of the PCR products.The amplified fragment was detected by a 1.5% agarose gel electrophoresis using an ultraviolet transilluminator.DNA direct sequencing was performed using AB 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.

Data analyses
All results are expressed as frequency and percentage as appropriate.Fisher's exact test or the Chi-square test was used for analyzing of categorical data.A p-value<0.05 was considered statistically significant.The data analysis was performed using SPSS version 19 (SPSS Inc., Chicago, IL, USA).The study was conducted according to the good clinical practice guideline as well as the Declaration of Helsinki and was approved by our local ethics committee.All subjects signed informed consent to participate in this study.

Relationship between H. pylori virulence factors and clinical outcomes
Among 119 H. pylori positive patients, 118 patients had positive for cagA gene (99.2%)(Table 1).The majority of cagA genotype was East-Asian-type cagA (74/118, 62.7 %), and Western type cagA was found only 37.3 % (44/118).The East-Asian-type cagA was more commonly found in gastric ulcer (66.7%) than duodenal ulcer patients (50%) but the differences could not reach statistically significant (Table 1).Although, there were only 3 patients with gastric cancer but all showed East-Asian-type cagA.

Relationship between H. pylori virulence factors and ethnic group
All 17 ethnic highland Lao were infected with H. pylori possessing East-Asian-type cagA (Table 2).On the other hand, East-Asian-type cagA and Western-type cagA were found in ethnic lowland Lao as 55.8% and 44.2%, respectively.The prevalence of East-Asian-type cagA was significantly higher in ethnic highland Lao than that of lowland Lao (p=0.001).Among 6 cases from mildland Lao, 4 possessed East-Asian type cagA and 2 were Western type cagA.Overall, the prevalence of East-Asian-cagA was varies, decreasing at increasingly southerly latitudes.Majority of vacA s genotype in ethnic highland Lao were s1c and significantly higher than lowland Lao   (88.2% vs. 61.5%, p=0.03).In contrast, s1a genotype was significantly lower in ethnic highland Lao compared with ethnic lowland Lao (11.8% vs. 36.5%,p=0.04).Among 6 cases from mildland Lao, s1a and s1c were found in 3 and 3 cases, respectively.As for vacA m region, m2 is predominant in highland (58.8%) and lowland (58.3%)Lao.Only 1 case in midland Lao showed m2 genotype.In contrast, m1b is predominant in midland Lao.However, the differences were not statistically significance.

Discussion
H. pylori cagA-positive strains have been reported to be associated with severe diseases such as atrophic gastritis and gastric cancer (Hamlet et al., 1999;Andreson et al., 2002;Podzorski et al., 2003).In this study, cagA gene was found in nearly all patients (99.2%) and the majority of cagA genes were East-Asian-type (62.7%).This prevalence is similar to the previous studies that have shown positive cagA gene more than 90% in Asian countries, and had no association with disease outcome (Maeda et al., 1998;Kim et al., 2001;Zhou et al., 2004;Nguyen et al., 2011).The East-Asian-type cagA strain was sometimes considered to be more virulent than Western-type cagA strain (Azuma et al., 2004;Vilaichone et al., 2004).Interestingly, all 3 cases of gastric cancers in this study were East-Asian-type cagA.The possible explanation might be due to small number of gastric cancer patients.A large future study of gastric cancer patients in Lao might be performed to identify this relationship.
The s1/m1 strains are reported to be more virulence than s1/m2 and s2/m2 (Atherton et al., 1995).Compatible with the prior study, our study demonstrated that m1b was significantly higher in gastric ulcer than those in chronic gastritis and m2 was significantly lower in gastric ulcer than chronic gastritis.When we combined vacA s and m regions together, we found that s1c-m1b were significantly associated with gastric ulcer which had similar result to Taiwan study (Wang et al., 1998).
In Laos there are 3 major ethnic groups, ethnic highland Lao who live in North and East part that have been sharing the border with China and Vietnam.Lowland Lao who live in Central and South part and share the border with Thailand and Cambodia.These findings might explain that East-Asian-type cagA type was demonstrated in all strain from highland Lao as similar with China and Vietnam (Zhou et al., 2004;Nguyen et al., 2011;Uchida et al., 2009), and Western-type cagA which mainly found in ethnic lowland were similar to Thailand and Cambodia (Vilaichone et al., 2004;Breurec et al., 2011).For the same explanation, majority of vacA subtype were s1c, and m2 which found in strains from ethnic highland and similar to China and Vietnam (Zhou et al., 2004;Uchida et al., 2009;Nguyen et al., 2011), whereas vacA s1a and m2 genes were commonly found in lowland Lao which similar to Thailand and Cambodia (Vilaichone et al., 2004, Breurec et al., 2011) as sharing border together.
In conclusion H. pylori was a common infection in Laos same as other countries in Southeast Asia.cagA gene was demonstrated in nearly all Laos patients.Interestingly, cagA and vacA genotype might be important factors to explain H. pylori infection and disease outcomes in Laos.
The great diversity of H. pylori genotype in Laos suggests that future examination of H. pylori from different area and ethnic groups of other Southeast Asian countries as well as in the surrounding regions will provide new evidences both in diversity of H. pylori and migration of human hosts.

Table 1 . Virulence Factors of Lao H. pylori and Clinical Outcomes
*P value <0.05 when compared with chronic gastritis; ** P value < 0.05 when compared with gastric ulcer

Table 2 . Virulence Factors of Lao H. pylori and Ethnic Group
*P value =0.001 when compared with lowland Lao; **P value=0.001when compared with highland Lao; ***P value < 0.05 when compared with lowland Lao