Prevalence of Helicobacter pylori vacA, cagA, and iceA Genotypes in Cuban Patients with Upper Gastrointestinal Diseases

Virulence factors of Helicobacter pylori can predict the development of different gastroduodenal diseases. There are scarce reports in Cuba about H. pylori isolates genotyping. The aim of the present investigation was to identify allelic variation of the virulence genes vacA, cagA, and iceA in sixty-eight patients diagnosed as H. pylori positive by culture. In seven out of 68 patients, strains from both gastric regions were obtained and considered independent. DNA was extracted from all the H. pylori strains and evaluated by PCR-genotyping. The vacA s1 allele, cagA gene, and iceA2 allele were the most prevalent (72.0%, 56.0%, and 57.3%, respectively). Alleles from m-region showed a similar frequency as s1a and s1b subtypes. The presence of multiple H. pylori genotypes in a single biopsy and two gastric region specimens were found. Significant statistical association was observed between iceA2 allele and patients with non-peptic ulcer dyspepsia (NUD) (P = 0.037) as well as virulence genotypes (s1, s1m2) and patients over 40 years old (P < 0.05). In conclusion, the results demonstrated a high prevalence of H. pylori virulent genotypes in Cuban patients over 40 years old while iceA2 alleles demonstrated a good specificity in patients with NUD.


Introduction
Helicobacter pylori is associated with the development of chronic gastritis, peptic ulcer disease (PUD), and gastric cancer (GC). Hence, since 1994, the World Health Organization has classified it as class I carcinogen [1]. Interestingly, despite the high prevalence of H. pylori infection in some countries, the frequency of severe diseases is much lower than other populations. In addition to host factors and diet, the varying outcomes of H. pylori infection could be related to the virulence of H. pylori strains differences [2]. Different virulence factors that play a role in the pathogenesis of the disease such as cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA), and iceA gene have been described [2,3]. The cagA gene, which encoded the CagA protein, is reported to be found in more than half of the H. pylori isolates. It is known that cagA is a marker for the cag pathogenicity island and is associated with increased IL-8 production, nuclear factor-kB activation, mucosal inflammation, and development of PUD and GC [3].
The protein VacA is responsible for the gastric epithelial erosion observed in infected hosts. The vacA gene encoding the vacuolating toxin comprises three variable parts, the sregion (encoding the signal peptide) and two alleles, s1 and s2. Within the s1 allele, several subtypes (s1a, s1b, and s1c) can be distinguished. For the m-region (middle), two alleles, m1 and m2, have been recognized [4]. The VacA activity level is defined by vacA s-and m-regions combination; s1m1 produces high amount of toxin and is considered the most virulent; however, s2m2 produces an inactive toxin [4,5]. In Western countries, infection with vacA s1m1 strain is more common in patients with PUD than those with chronic gastritis [5]. Recently, a third polymorphic determinant of vacuolating activity (located in the middle of s-and m-regions) has been described as an intermediate (i) region [6].
The iceA gene has two alleles: iceA1 and iceA2. The iceA1 allele, encoding a CATG-specific restriction endonuclease, is regulated by the contact of H. pylori with the human gastric cells [7]. In Western countries the presence of iceA1 allele is strongly associated with PUD [7,8].
In Cuba, there are scarce investigations regarding the pattern of virulence genes in H. pylori strains [9,10], but none have examined the s1 allele subtypes of vacA gene, the iceA gene nor the H. pylori strains genotypes isolated from younger or older patients. The aim of this study was to investigate the prevalence of cagA, vacA, and iceA genotypes of H. pylori isolates recovered from Cuban patients with dyspepsia.

Patients.
Gastric biopsies from 150 patients referred to gastroscopy at two Cuban hospitals in Havana from 2009 to 2010 were collected. Patients with a history of gastric surgery, active gastrointestinal bleeding or who had received antibiotics, proton pump inhibitors, or bismuth compounds in the last four weeks were excluded. Sixty-eight patients (35 male and 33 female) with a mean age of 39.2 (years range = 9 to 68) reported positive for H. pylori infection by culture were included. The protocol was approved by the Ethical Review Committee of the Tropical Medicine Institute "Pedro Kourí" (IPK) and all patients provided an informed consent.

2.2.
Culture and Genomic DNA Isolation. Antrum and corpus biopsy specimens from each patient were kept in sterile saline solution (0.9%) at 4 ∘ C. The endoscopic biopsy specimens were smeared on the surface of Columbia chocolate agar plates enriched with Dent supplement (Oxoid, England) and 1% of fetal calf serum (Gibco, USA) and incubated under microaerophilic conditions (Campy Packs, Oxoid) for up to 3 days. H. pylori isolates were identified by typical Gram-staining morphology and positive biochemical urease, oxidase, and catalase [11]. Biopsies of 68 patients yielded 75 H. pylori isolates; those obtained from two gastric biopsy sites of seven patients were considered independent. Primary cultures of H. pylori were conserved at −80 ∘ C in brain heart infusion with 20% glycerol and later on were subcultured as it was described above.
All colonies from the subculture were used for chromosomal DNA extraction by Wizard Genomic DNA Purification Kit (Promega, USA) according to the manufacturer's instructions. DNA content and purity were determined by measuring the absorbance at 260-280 nm (Spectrophotometer MRC, Spain) and by amplification of ureA gene [12]. Samples were stored at −20 ∘ C before polymerase chain reaction (PCR) amplification was performed. Table 1. Amplification of ureA, cagA, vacA, and iceA genes by PCR was made in a volume of 50 L containing 1X PCR buffer (pH = 7), 3 mM MgCl 2 , 0.2 mM deoxynucleoside triphosphate, 0.5U Taq polymerase (Sigma, USA), 25 pmol of each primer, 2 L of chromosomal DNA, and sterile distilled water (Sigma). PCR amplifications were performed in an automated thermal cycler (Techne, Belgium). All runs included a negative DNA control consisting of PCR grade water and two positive DNA controls from H. pylori reference strains, ATCC43504 (genotype vacAs1am1/cagA+/iceA1) and 26695  The cagA gene was detected in 42 (56.0%) strains. Overall 68 (90.7%) isolates had a single iceA allele; iceA2 was detected in 43 (57.3%) and iceA1 in 25 (33.3%) strains. Seven strains were identified with both iceA alleles (7 strains, 9.3%).

H. pylori Genotypes and Clinical Association.
Regarding endoscopy aspects of the mucosa, patients were distributed Table 3: H. pylori strains recovered from gastric antrum and corpus of seven patients.

Patients
Stomach region Endoscopic diagnostic vacA gene subtypes cagA gene presence * iceA gene subtypes Despite the fact that s1 allele of vacA gene and cagA+ H. pylori strains were more frequently identified in patients with NUD (31 patients, 70.5%) and with PUD (16 patients, 66.7%), respectively, no statistical association was observed. However, the presence of iceA2 allele in 63.6% (28 strains) of isolated strains from patients having NUD was associated statistically ( = 0.037) ( Table 2).

Association between Age of Patients and H. pylori Geno-
types. The 50.7% (38 strains) of H. pylori strains were isolated from patients under 40 years old (group 1) while the 49% (37 strains) were recovered from patients over 40 years old (group 2). Genotypes s1 and s1m2 were more frequently found in patients belonging to group 2 while the genotypes s2 and s2m2 were more often detected in group 1, both with a significant statistical association ( < 0.05). H. pylori strains isolated from patients over 40 years old were more frequent in those with PUD; between these two variables a statistical association was observed ( = 0.007) ( Table 2).

H. pylori Strains Recovered from Different Gastric Regions.
Fourteen H. pylori strains were isolated from two stomach regions, antrum and corpus of seven patients. In only one of these patients, a strain with identical genotype (s1am1/cagA+/iceA2) was found. However, in the majority of them, at least a variation in one of the investigated genes was observed. The variation percentages of the following genes, cagA+, vacA (s-region), and iceA1, were 57.1% (8/14 strains), 71.4% (10/14 strains), and 42.9% (6/14 strains), respectively (Table 3).
Our results are in agreement with other developed studies in Cuba, Europe, and East Africa where a higher prevalence (70% or more) of the s1 subtype had been reported [9,15,16]. In contrast, an elevated frequency of strains belonging to s2 subtype was described in Jordan [17]. These data demonstrated the high genetic variability of strains in different countries. A low percentage of the strains (5%) harbouring both alleles s1 and s2 has been reported previously [18]. The current research is the first study developed in Cuba to analyze both the s1 variants of vacA gene and the gene iceA in H. pylori strains. Concerning previous research, the dominant vacA gene subtype in North and South America, Central Europe, and Australia was s1a [19] but in Portugal and Brazil was s1b [20,21]. The same frequency of s1a and s1b subtypes, as it was observed in our study, is similar to reports from industrialised countries as France, Italy, USA, and Canada [22].
The existence of nontypeable H. pylori strains has been described previously, using similar primers [23]. In our investigation, two of the three sequenced strains were identified as s1a-s1b subtypes. Similar results have been informed by other authors, defining the consensus sequence as a recombinant of two originals with different subtypes each [24]. For mregion of the H. pylori strains, m1 and m2 subtypes were approximately equally prevalent as in Europe and Latin America [25].
The percentage of vacA genotype s1b/m1 in this study is similar to research developed in Mexico, Brazil, and Costa Rica [26]. However, in Japan and China, the most prevalent genotypes are s1c/m1 and s1c/m2, respectively [27].
In European, Venezuelan, and North American populations, only 60% of H. pylori isolates harboured cagA gene [16,28], as what occurred in the current study. However, in Japan and Korea, the proportion of cagA + strains is usually over 90% [29]. In our investigation, the prevalence of cagA + strains is similar to the report of Torres [9] but is lower in comparison with another Cuban study (70%) [10].
The predominance of iceA2 subtype is in agreement with reports from Colombia and USA [30]. Both subtypes of the iceA gene have been identified in Brazil, Malaysia, and Korea [31,32].
Patients with H. pylori strains recovered from gastric antrum and corpus were infected with different cagA, vacA, and iceA genotypes. Also, multiple H. pylori genotypes were observed in a single biopsy specimen. Previous detailed molecular analysis has shown that each of the H. pylori strains contains only one cagA allele and each one of the s-and m-region subtypes of vacA gene. Therefore, it is an exact indicator for the presence of multiple strains of this organism if different genotypes are found [33]. The coexistence of more than one strain in the same individual may reflect the capacity of H. pylori to evolve genetic variations during the long-term colonization from childhood [3].
The absence of association between virulence genes explored and PUD could be influenced by the small number of patients studied with this pathology. Similar results have been described previously [3,9,10]. Moreover, the predominance of high virulence Cuban H. pylori strains in the group of patients with benign gastric diseases has unusual results in Western population. As it was reported in previous Cuban studies [9,10], despite the presence of highly virulent H. pylori, the incidence of gastric cancer is lower in dyspeptic patients (gastric cancer death rate in Cuba: 7.5/100 000, http://files.sld.cu/dne/files/2014/05/anuario-2013-esp-e.pdf). Although this behavior has been observed in several previous studies [3,7], it is probable that these findings suggest the action of environmental and host factors in Cuban patients. Further research studies to examine the role of host immunological factors might help to explain the different outcomes of H. pylori-induced disease in Cuban individuals.
We found an association between H. pylori strains harbouring the iceA2 allele in patients with NUD. This behaviour has also been described in Europe, Saudi Arabia, and Turkey [34]. Several studies suggest an association of the iceA1 variant and PUD and between iceA2 variants with gastritis [34,35]. However, this association varies among populations; in Brazil, for instance, iceA1 allele is associated with gastritis [31]. A recent meta-analysis confirms the relationship between the iceA allelic types and clinical outcomes [7].
The current investigation also showed statistical association of more virulent variants of H. pylori (s1 and s1m2) strains in the group of older patients. In Portugal and Tunisia, virulent strains have been detected in adult patients more frequently than children [36]. It has been described that H. pylori strains experiment recombination with others more virulent and better adapted strains to host changes, resulting in genotypes variable distribution between age groups [37,38].
In summary, our results show a high prevalence of main virulence factors in Cuban isolates similar to that observed in other Western populations. In addition, we found strains with multiple genotypes, as it has been observed in countries with a high prevalence of H. pylori infection. Notably, a significant association was found among iceA2 allele and NUD as well as strains with more virulent types and older patients. The iceA gene may be considered a useful marker in patients with gastroduodenal diseases. The relationship between H. pylori virulence factors and clinical outcomes in Cuban population is still unclear; therefore, further studies are required to determine the role of environmental and immunological factors.

Ethical Approval
The project was approved by the Ethical Review Committee of the IPK, Cuba.