Helicobacter pylori is a bacterial pathogen that colonizes exclusively the mucous layer of the human stomach and classified as carcinogenic to humans by the  International Agency for Research on Cancers (IARC, 1994). However, H. pylori organisms vary in their carcinogenic potential  and  the  common  type  of  gastric  cancers   is adenocarcinoma which occurs most commonly in East Asian countries than western countries (Higashi et al., 2002; Suzuki and Mori, 2016). Since the discovery of H. pylori by Marshall and Warren (1984), it has been confirmed that H. pylori infection results in chronic active gastritis and can lead to gastric ulcer, duodenal ulcer and gastric cancer (Malfertheiner et al., 2007; Yamaoka, 2010). Infection with H. pylori is more common and acquired at an earlier age (30 to 50%) in children and attain over 90% during adulthood in developing countries (Salih, 2009). In South-west, Nigeria, studies reported high prevalence rates (60.5 to 73%) of H. pylori among dyspeptic patients (Ndububa et al., 2001; Adesanya et al., 2002; Ola et al., 2006).  Cytotoxin associated gene A (cagA) is one of the important virulence factors of H. pylori. The CagPAI contains 31 genes, including CagA gene, which is one of the markers for the cag pathogenecity island (CagPAI). CagA codes for 120 to 145 kDa protein that is associated with cytotoxin production and more severe clinical outcomes such as peptic ulcer and gastric cancer (Arents et al., 2001; Hatakeyama, 2006). 

Previous studies have reported the prevalence of H. pylori and the virulence genes in relation to disease outcome among dyspeptic patients in Nigeria (Smith et al., 2002; Olokoba et al., 2013). However, data relating to the information on the association of the cagA gene with the disease outcome is still controversial and there is dearth of information on protein expression of putative cagA genes in relationship to clinical outcomes. This information is important as CagA protein is one of the major virulence factors of H. pylori. Hence, this study was aimed at confirming whether any correlation exists between the CagA protein of these strains and the endoscopic findings among dyspeptic patients from South-western, Nigeria and also to investigate the evolutionary relationships among H. pylori cagA gene with the GenBank strains.

A total number of one hundred and twenty-four isolates  of  H. pylori from dyspetic patients were obtained from the laboratory of Dr. S. I. Smith of the Nigeria Institute of Medical Research, Yaba. Medical history and the endoscopic findings of the patients were obtained from four different hospitals based on the records provided by the gastroenterologist between April 2012 - December 2015, by which approval to conduct the study was obtained from Ethics Committee of the Nigerian Institute of Medical Research Yaba, Lagos.

Culturing of H. pylori

The isolates were sub-cultured on Columbia blood agar base (Oxoid), containing laked horse serum (7%) and Dent’s antibiotic supplement (Oxoid) in a microaerophilic atmosphere conditions at 37°C as described previously (Toledo and Lopez-Solis, 2010). Colonies were identified by Gram staining and biochemical tests such as oxidase, catalase and urease reactions.

DNA extraction and PCR amplification

Genomic DNA of the isolates were extracted with the QIAamp® DNA kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted DNA was stored at -20°C until use. The PCR amplification of H. pylori 16s rRNA was carried out in 25 µl reaction mixture and PCR conditions were: 35 cycles at 94°C for 1 min, annealing 60°C for 1 min, 72°C for 1 min, and a final elongation at 72°C for 5 min. The DNAs of J99 and P12 were used as positive controls. H. pylori 16S rRNA gene-positive samples were subjected to PCR to detect cagA gene using the primers and PCR conditions as described previously by Smith et al. (2002) and the PCR primers are listed in Table 1.

Sequencing and bioinformatic analysis

The amplified PCR products of cagA gene from twelve selected strains based on each centre used were purified and subjected to sequencing reactions by inqaba™ biotech, South Africa    (BigDye® Terminator v3.1 Cycle Sequencing). Blasting and Genebee (http://www.genebee.msu.su/services/phtree_reduced.html) were used for phylogenetic prediction.

Statistical analysis

The data obtained were subjected to descriptive statistical analysis using EPI info 7. Chi squared test was used to determine associations between the protein status of the isolates and the endoscopic findings and the differences were considered to be statistically significant when the p value obtained was less than 0.05.

The phylogenetic relationships among the 12 twelve selected strains and four cagA genes of H. pylori strains (PHL 121, LSU 2003-1, CAM23 and 01-1086) obtained from GenBank based on high sequence similarity with blasting, revealed that cag A genes were divided into two major groups: Strains LU 14 to 01-1086 and strains LA2 to LA4 (Figure 2), having four sub groups: LU 14 – IF9; LA1-IB6; CAM23-10-1086 and LA2-LA4 (Figure 2)

From these findings, none of the patients  studied  were diagnosed with gastric cancer, more so, with the presence of cagA positive isolates. Abdulkareem et al. (2015) reported that, there is usually low incidence of gastric cancer in Africa (Africa enigma) despite a high prevalence of H. pylori with most strains positive for the virulent genes such as cagA gene and vacA gene. A lot of factors may contribute to this, and one of which could be mixed infection of cagA negative and cagA positive strains which could lower disease burden, and diets rich in anti-oxidants which reduces risk of gastric cancer (Abdulkareem et al., 2015). A study from Soweto, South Africa indicated that host response to H. pylori among Africans may be protective against virulent form of H. pylori  (Segal  et  al.,  2001).  Also,  the  ability   of   CagA secreted by different strains of H. pylori to perturb host-cell functions and to trigger gastric cancer is determined by the binding potential of Src homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) which is high in H. pylori strains from East Asian countries than those from Western countries (Higashi et al., 2002).

The phylogenetic tree of cagA genes revealed that the strains were clearly delineated into 2 major phylogenetic clades with strains LA2 and LA4 distantly separated from others. However, blasting of the edited sequences of strains LA2 and LA4 in NCBI platform, revealed that they were 89 and 87% respectively, most similar to the western type of cytotoxin associated protein A (cagA) gene, partial cds of H. pylori strain 01-1086 from Colombia (Accession no: JQ318034.1). The other strains clustered in the second clade but with significant genetic variability. This clade further subdivided into 3 minor clades with strain LU15 most distant and stands as a bridge linking the clade with strains LA 2 and LA4.  Strains LA1, IB5 and IB6 clustered to form a minor clade with IB 5 and IB6 more related. Also, the results from blasting indicated that IB 5 and IB6 were 95 and 96%, respectively, most similar to H. pylori cag Pathogenicity Island, strain LSU2003-1 originated from African with Accession number of FR666843.1 (Olbermann et al., 2010). Strains from Ife (IF 9, IF 10 and IF 11) clustered forming a minor clade and were all related (96, 95 and 95%). While cluster from LU 14, LU 16 and LU 17 were related to Western cagA gene of H. pylori strain PHL121 (accession no: GU173869) obtained primarily from gastritis patients in philippines (Cortes et al., 2010).

This study demonstrated a high prevalence of H. pylori infection of the cagA genotype and the phylogenetic analysis revealed that cagA gene was related to the western cagA. However, there was no significant association between CagA protein and the clinical outcomes. 

The authors have not declared any conflict of interests.