Introduction

Helicobacter pylori is a gram-negative, microaerophilic, rod-shaped bacterium that infects the gastric mucosa in more than half of the human population worldwide [1, 2]. The prevalence of H. pylori is higher in developing countries owing to the generally lower basic sanitation and hygiene conditions brought on by low socioeconomic infrastructures [3, 4]. Infections with this bacterium can lead to the development of several gastropathies, such as chronic gastritis, peptic ulcer, atrophy, and metaplasia [5, 6]. Additionally, H. pylori infection is considered one of the main risk factors for the development of gastric adenocarcinoma. As a result, the International Agency for Research on Cancer, subsidized by the World Health Organization, has classified H. pylori as a type I carcinogen [7].

Helicobacter pylori infections have different clinical outcomes depending on the virulence of the bacterial strains, environmental factors, and the genetic characteristics and lifestyles of the hosts [8]. The cagA gene, located in the chromosomal region called the chromosomal pathogenicity island (cagPAI), encodes the CagA protein (cytotoxin-associated gene A), a highly immunogenic antigen that stimulates the production of chemotactic factors in the host gastric epithelium, inducing an intense inflammatory response. This gene is considered an important marker of virulence and is associated with an increased risk for the development of gastritis, peptic ulcer, atrophy, and gastric carcinoma [8, 9].

The vacA gene encodes vacuolating cytotoxin A (VacA), considered an important virulence factor in the pathogenesis of gastric cancer and peptic ulcer. VacA induces vacuoles in gastric epithelial cells. In addition, it induces the release of cytochrome C from mitochondria, leading to apoptosis, the inhibition of T cell proliferation, and the induction of pro-inflammatory responses [10,11,12].

In 2005, Lu et al. [13] described a gene located in the plasticity region of the H. pylori genome, which they named the duodenal ulcer-promoting gene (dupA). This gene was found to be homologous to the virulence gene virB which encodes a type IV secretion protein in Agrobacterium tumefaciens. The dupA gene is also related to neutrophil infiltration in the mucosa and the induction of elevated IL-8 levels by gastric epithelial cells [14].

According to Lu et al. [13], infection with H. pylori dupA-positive strains was associated with the development of duodenal ulcers, and the presence of the dupA gene in the infecting strain was considered a protective factor against gastric atrophy, intestinal metaplasia, and gastric cancer. As a result, this gene was characterized as the first disease-specific virulence gene of H. pylori [13]. However, similar to other H. pylori virulence genes, the role of this gene in the development of gastropathies varies according to geographical regions [15,16,17].

Although the pathogenic mechanism of the protein encoded by the dupA gene (i.e., DupA) is not yet well-understood, it may be involved in the induction of interleukin-8 (IL-8) production by the gastric epithelium [13]. Strains carrying the dupA gene also appear to be more resistant to stomach acid, which may partly explain the association of this virulence factor with the development of ulcers [18, 19].

Although several bacterial virulence factors have been suggested as markers for digestive diseases [20,21,22,23,24], there is still a lack of information on the biomarkers which are characteristic of the clinical conditions present in infected individuals, especially in South America. Some studies have indicated the usefulness of the dupA gene as a virulence biomarker [11, 19, 25,26,27]. However, the role of this gene in the etiology and pathogenesis of different gastropathies is not fully understood. Bearing in mind that identification of predictive biomarkers for a given clinical condition is a promising strategy for personalized medicine, the aim of this study was to verify the association between the presence of the H. pylori dupA gene and clinical outcomes in dyspeptic patients in the center-west of Brazil. In addition, a phylogenetic analysis was performed to assess the origin of circulating strains.

Materials and methods

Ethical Considerations and Participants

This study was approved by the Research Ethics Committee of Hospital das Clínicas of the Universidade Federal de Goiás (UFG) (Approval Numbers 2.519.032 and Certificate of Presentation of Ethical Appreciation 83,422,017.7.0000.5078). Patients with dyspepsia (age: > 18 years) who had undergone an upper gastrointestinal tract (GI) endoscopy at the Hospital das Clínicas of UFG were recruited. The exclusion criteria were applied: patients who had used proton pump inhibitors in the 2 weeks preceding sample collection, or blocking agents in the week preceding the procedure, patients who had used immunosuppressants or antibiotics in the past 8 weeks, patients with active gastrointestinal bleeding, and pregnant and lactating women. All participants provided signed informed consent.

Screening for H. pylori infection and detection of the dupA gene

Two samples of the gastric antrum and gastric body were collected from the 117 patients during the upper GI endoscopy. DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The extracted DNA was used for the molecular screening of H. pylori infection by amplifying a fragment of the constitutive ribosomal 16S rRNA gene (hpx) using the polymerase chain reaction (PCR).

The hpx-positive samples were then subjected to dupA gene detection and sequencing. The PCRs were performed using primers previously described in the literature [28] (Table 1). The PCR mixture (final volume: 50 μL) comprised 33.5 µL of Milli-Q water, 5 µL of 10 × PCR buffer containing MgCl2 (1.5 mM), 2 µL of dNTP (2.5 mM), 2 µL of each oligonucleotide (10 pmol each), 5 µL of DNA sample (50 ng), and 0.5 µL of Taq DNA polymerase (2.5 units). For each reaction, positive and negative controls were also tested.

Table 1 Primer sequences, PCR amplification conditions, and gene fragment sizes
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After the PCR-amplified fragments had been electrophoresed on a 2% agarose gel, the fragment bands were visualized by nucleic acid staining with Blue Green Loading dye (Lac Biotecnologia, Sao Paulo, Brazil) and exposure under ultraviolet light. The size of the amplified product was determined by comparison against a 100-bp DNA ladder molecular weight marker (Cellco, Sao Paulo, Brazil).

Diagnosis of gastroduodenal pathologies

The endoscopic examination of the patients was performed by the Department of Gastroenterology, while histopathological analyses of the biopsied gastric mucosal samples were done by the Clinical Pathology Laboratory of Hospital das Clínicas (UFG). Endoscopic examination was performed according to recommendations of the Brazilian Society of Digestive Endoscopy. The procedure was performed by a gastroenterologist using an Olympus CV endoscope (Olympus Medical Europe, Germany). Videoendoscopic images were viewed on a high-definition, flat-panel monitor. Alfentanil (50 mcg) and propofol (40 mg) were administered intravenously before the procedure [29].

For the histopathological examination, the gastric mucosal samples were fixed in 10% buffered formalin and stained with Giemsa and hematoxylin–eosin [30]. Grading of the inflammatory infiltrate was performed according to the Sidney system [31].

DNA sequencing and phylogenetic analysis

All dupA gene amplification products were purified with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison WI, USA). DNA sequencing was performed using an ABI-PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Carlsbad, CA, USA) in an ABI-PRISM 3100 genetic analyzer (Applied Biosystems). Sequencing was performed at least twice, using the same forward and reverse primers used in the PCR assays. All the sequences were compared with those listed on the GenBank nucleotide and protein databases (National Center for Biotechnology Information, Bethesda, MD, USA), using the Blastn and Blastx algorithms, respectively.

To establish the phylogenetic origin of the Brazilian strains, a phylogenetic tree based on dupA was built using the gene sequences from the following 17 H. pylori strains from different parts of the world (available from GenBank): three sequences from Portugal (PORT: KT290607, KT290572, and KT290619); two from Brazil (BRA: HQ228198 and JF951131); three from Argentina (ARG: FJ763612, FJ763614, and AB649939); four from Japan (JAP: AB739514, AB739579, AB739507, and KC707841); two from China (CHI: KC707837 and KM245039); and three from India (IND: KC894690, KC894688, and KM245039). Additionally, three dupA sequences (NEHP04, NEHP05, and NEHP06) which belonged to H. pylori–infected patients in our study who had duodenitis, esophagitis, and gastritis, respectively, were included in the phyogenetic tree.

The sequences were first aligned with ClustalX v2.1.0.0 and then manually edited using BioEdit v7.1.7.0. Phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis (MEGA) v10.1. The tree was built using maximum-likelihood analysis, with 1000 bootstraps. The evolutionary model chosen by the algorithm for the set of sequences used was General Time Reversible + Invariant Sites (GTR + I) [32].

Statistical analysis

Descriptive statistics were performed to calculate the absolute frequencies and relative percentages for the categorical variables. Subsequently, inferential statistics were performed using Fisher’s exact test to assess possible associations between the variables. Additionally, the relative risk for the development of gastric diseases in relation to the bacterial genetics was calculated. For the inferential statistics, a significance level of 5% (p < 0.05) was adopted. For statistical calculations, the BioEstat® 5.3 software was used.

Results

In this study, gastric biopsies were obtained from 117 patients with dyspepsia, of which 64.1% (75/117) were considered positive for H. pylori (mean age: 42 years; standard deviation: 10.8). Among the patients infected by the bacterium, 27 (36.0%) were men and 48 (64.0%) women. The dupA gene was detected in 70.7% (53/75) of H. pylori–positive samples.

In total, four gastropathies were diagnosed in the study population, with some patients having more than one gastric disorder. Of the individuals infected with dupA-positive strains, 67.9%, 15.1%, 11.3%, and 5.7% had a diagnosis of gastritis, duodenitis, oesophagitis, and atrophy respectively, and 18.9% had a gastric mucosa without changes (normal). Of the individuals infected with dupA-negative strains, 68.2%, 13.6%, 22.7%, and 4.5% had gastritis, duodenitis, oesophagitis, and atrophy respectively, and 22.7% had a normal mucosa (Fig. 1). These data suggested that the presence of the dupA gene in infectious H. pylori strains was not associated with the development of these gastropathies in the population studied (p-value = 0.8059).

Fig. 1
figure 1

Gastric disease profile of patients infected with dupA-positive and dupA-negative Helicobacter pylori strains

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The most prevalent gastropathy among individuals infected with a dupA-positive strain was gastritis. Stratification by sex revealed that 88.6% of the women infected with dupA-positive strains had gastritis, whereas only 44.4% of the men likewise infected had this disease, with the difference between the two groups being statistically significant (p = 0.0020) (Table 2). Additionally, the relative risk analysis showed that for women, a dupA-positive H. pylori infection increased the chance of developing gastritis by twofold.

Table 2 Helicobacter pylori dupA positivity in patients with gastritis
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To evaluate the phylogenetic relationships of the H. pylori strains analyzed in the present work with H. pylori strains from other geographical regions, partial sequences (~ 200 bp) of the dupA gene from the Brazilian strains were used for the construction of the phylogenetic tree. These partial sequences amplified from the infectious strains showed 100% homology with one another (data not shown).

The phylogenetic tree consisted of two main clades, one of which grouped the strains from the eastern countries (except for the KT290619 sequence) and the other of which grouped the strains from the western countries. The sequences obtained in this study were grouped with those from the western countries (Fig. 2). The KT290619 sequence was positioned in an intermediate zone between the two main clades and was therefore used to root the phylogenetic tree.

Fig. 2
figure 2

Phylogenetic tree based on the partial sequences of the dupA gene of Helicobacter pylori strains from Brazil (local strains) and sequences from strains reported in different parts of the world (available in public databases). Triangles: strains from eastern countries; squares: strains from western countries

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Discussion

Helicobacter pylori plays a critical role in development of various digestive diseases. However, there is no consensus regarding the main virulence factor involved in the etiology and pathogenesis of each disease [33]. Several studies have been carried out to elucidate the role of dupA in the development of gastric disorders [14].

In this study, carried out in the mid-western region of Brazil, the dupA gene was detected in 70.7% of the infectious H. pylori strains but was not associated with gastropathies. Similarly, in another study carried out in the southeastern region of Brazil, the dupA gene was found in 92.3% of infectious H. pylori strains but was also not associated with the development of gastric diseases, despite its high frequency in the region [34]. Similar results were found in the study by Pacheco et al. [35], carried out on patients with dyspepsia in the state of São Paulo, Brazil, where the dupA gene was detected in 92.4% of samples positive for H. pylori and was not associated with gastric diseases. Other studies in Belgium, China, South Africa, and the USA have also found no association between the gene and gastric diseases [36].

Talebi et al. [19] demonstrated that infection by dupA-positive strains in fact protected against the development of gastric ulcers. Additionally, Lu et al. [13] proposed the use of the dupA gene as a protective biomarker for gastric atrophy, intestinal metaplasia, and gastric cancer. By contrast, that same study revealed a positive association between the presence of the dupA gene in the infecting strain and the development of duodenal ulcer. In our study, it was not possible to assess the association of the dupA gene with ulcers, atrophy, and gastric cancer owing to the low sample size of infected individuals with these diseases.

In a study by Schmidt et al. [37], the presence of the dupA gene was found to be associated with chronic gastritis. This result corroborates our findings, where the presence of dupA in the infecting strain showed a positive correlation with the development of gastritis in women. In 2017, Paredes-Osses et al. [11] demonstrated that the severity of gastric diseases was significantly decreased in women infected with dupA-positive H. pylori strains. By contrast, dupA did not have any significant effect on the disease severity in male patients.

The pathogenic mechanisms of DupA in women have not yet been clarified and deserve further investigation. Given that the establishment of gastric diseases is due to virulence factors of the pathogen as well as the genetic characteristics of the host and environmental factors, it would be necessary to validate the host factors that predispose patients to gastroduodenal diseases. A study by Yeh et al. [38] provides evidence that polymorphisms in the matrix metalloproteinase-3 promoter contribute to the increased susceptibility of H. pylori–infected women to duodenal ulcers.

The phylogenetic analysis showed that the dupA sequences from this study were grouped in the same clade as sequences from strains isolated from patients residing in western countries (Argentina, Brazil, and Portugal). This grouping may have been influenced by geographical proximity or by the migration or transit history of the peoples of those countries. It is important to consider that Brazil was once colonized by Portugal and for many years was dependent on this country, and the intense movement of people between the two countries could have increased the chances of transmission of H. pylori strains.

The grouping of the sequences from the eastern countries (China, India, and Japan) had strong statistical support, indicating their total separation from the sequences from the western countries. This genetic separation between western and eastern countries has also been described by Chiurillo et al. [21], who used H. pylori iceA1 sequences (encoding a virulence factor) from Venezuelan strains. Although the number of dupA sequences from western strains is limited in the GenBank database, our phylogenetic tree was similar to that reported for other H. pylori virulence genes [39].

The dupA sequences isolated in this study revealed a high rate of homology, suggesting that the gene is highly conserved in circulating strains in mid-western Brazil. This is likely related to the fact that isolates from a single individual, a family member, and a specific geographical region are often clonal over short periods of time, despite that the general population structure of H. pylori is panmictic [40].

This study was limited by the small number of samples screened. Therefore, a more comprehensive phylogeographic analysis with a larger number of samples is needed to elucidate the impact of the genetic heterogeneity of H. pylori on patients with dyspepsia in Brazil. Notwithstanding, this is the first study that has used partial sequences of dupA from H. pylori strains in Brazil to determine their phylogenetic relationships and origin.

Conclusion

Presence of dupA in the infecting strain cannot be considered a biomarker of gastropathies in the study population. However, in women, infection with dupA-positive H. pylori increases the risk of developing gastritis. The dupA gene is highly conserved among circulating strains in mid-western Brazil. Our data provide perspectives for future studies aiming to evaluate parasite-host relationship factors, which can influence development of gastropathies.