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Prevalence of Helicobacter pylori vacA, cagA and iceA genotypes in Nigerian patients with duodenal ulcer disease

STELLA I. SMITH^*,, CHRISTIAN KIRSCH^*, KOLA S. OYEDEJI, ANTHONY O. ARIGBABU, AKITOYE O. COKER^||, EKKEHARD BAYERDÖFFER^* and STEPHAN MIEHLKE^*

*Medical Department I, Gastroenterology, Hematology, Oncology, Pulmonology and Infectious Diseases, Technical University Hospital, Dresden, Germany, Genetics Division and Microbiology Division, Nigerian Institute of Medical Research, Yaba, Lagos, Department of Surgery, Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife and ||Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos, Idi-Araba, Lagos, Nigeria

Corresponding author: Dr S. Miehlke (email: miehlke{at}mk1.med.tu-dresden.de).

Received 24 Jan. 2002; revised version received 3 April 2002; accepted 12 April 2002.

	Abstract

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Distinct virulence factors of Helicobacter pylori have been associated with clinical outcome of the infection; however, considerable variations have been reported from different geographic regions. Data on genotypes of African H. pylori isolates are sparse. The aim of this study was to determine the prevalence of specific genotypes of H. pylori in Nigerian patients with duodenal ulcer and non-ulcer dyspepsia. H. pylori was cultured from endoscopic biopsies obtained from 41 Nigerian patients (19 with duodenal ulcer, 22 with non-ulcer dyspepsia). The vacA alleles, cagA and iceA genotypes were determined by PCR. The vacA s1,m1 and s1,m2 genotypes were found in 26.3% and 22.7%, and in 73.7% and 72.7% of H. pylori isolates from patients with duodenal ulcer and non-ulcer dyspepsia, respectively. The iceA1 genotype was present in 94.7% and 86.4% of isolates from duodenal ulcer and non-ulcer dyspepsia patients, respectively. cagA⁺ infection was found predominantly (>90%) in Nigerian H. pylori isolates irrespective of the clinical diagnosis. In conclusion, vacA s1,m2, iceA1 and cagA⁺ are common genotypes of H. pylori isolated from Nigerian patients. As in several other developing countries there seems to be no association between these genotypes and duodenal ulcer disease.

	Introduction

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Helicobacter pylori is a common infection world-wide, the aetiological agent of chronic active gastritis and the major cause of peptic ulcer disease and primary gastric lymphoma of MALT type [1, 2]. H. pylori has also been recognised as a risk factor for gastric adenocarcinoma [3]. The reasons for these different outcomes of H. pylori infection remain unclear. Several H. pylori genes that are related to the risk of disease have been proposed. The cytotoxin-associated gene (cagA) is a marker for a genomic pathogenicity island of c. 40 kb and its presence is thought to be associated with a more severe clinical outcome of the infection [4, 5]. cagA is thought to be closely related to the vacuolating cytotoxin, encoded by the vacA gene. The vacA gene is present in virtually all H. pylori strains and contains at least two variable regions, the signal and the middle regions [6]. Strains harbouring the vacA s1 genotype show a high cytotoxic activity and have been linked to severe clinical disease such as peptic ulcer disease [7]. Another recently described putative virulence factor is iceA (induced by contact with the epithelium), which exists in at least two allelic forms, iceA1 and iceA2 [8]. iceA1 is upregulated upon contact of H. pylori with the gastric epithelium and has been also been suggested as a marker for peptic ulcer disease [9].

Subsequent studies have shown considerable inconsistencies of these associations, depending on the population or geographic origin of the isolates [10, 11]. There are few data available as regards the pattern of H. pylori genotypes in patients from African populations [12, 13]. Therefore, the aim of this study was to investigate the prevalence of the vacA, cagA and iceA genotypes of H. pylori isolates from Nigerian patients with duodenal ulcer or non-ulcer dyspepsia.

	Materials and methods

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Patients and H. pylori isolates

A total of 41 H. pylori isolates was obtained from 19 patients with active duodenal ulcer disease, and from 22 patients with dyspeptic symptoms, who had no current evidence and no history of peptic ulcer. All biopsies for primary H. pylori culture were obtained from the gastric antrum. The biopsies were smeared immediately on to Columbia agar base medium containing Dent's supplement and laked horse blood 7%. The plates were incubated at 37°C for 3–7 days under micro-aerobic conditions. H. pylori was identified by typical Gram's stain and colony morphology, and by positive biochemical tests for urease, catalase and oxidase.

H. pylori genotyping

Bacterial chromosomal DNA was extracted from freshly harvested H. pylori cultures with phenol/chloroform. For PCR analysis of the targeted genes 2 µl of DNA were added to 50 µl of reaction mixture containing 1xPCR buffer, 1.5 mM MgCl₂, 0.2 mM (each) deoxynucleotide (Gibco BRL, Germany) and 0.5 µM of respective oligonucleotide primers. AmpliTaq Gold (Perkin Elmer, Germany) 2.5 U was added to each tube and overlaid with mineral oil. PCR was performed with a thermal cycler (GeneAmp 2400; Perkin Elmer). The amplification cycles (cagA, vacA s1/s2 and iceA) consisted of an initial denaturation of target DNA at 94°C for 9 min and then denaturation at 94°C for 1 min, primer annealing at 60°C or 56°C (vacA m1, m2) for 1 min and extension at 72°C for 1 min (35 cycles). The final cycle included an extension step for 5 min. The primers used to amplify the targeted genes are summarised in Table 1 [6, 9, 14]. Negative controls were added to each PCR run including all reagents except template DNA. H. pylori ATCC 49503 was used as the positive control. PCR products were visualised by agarose 1.5% gel electrophoresis.

	Results

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Fig. 1 shows the results of electrophoresis of PCR products. The predicted 259-bp PCR product for vacA s1 was identified in 40 H. pylori strains (98%). As shown in Table 2, the majority of isolates harboured the vacA s1,m2 genotype, followed by the s1,m1 genotype. A vacA s2,m1 genotype has not been found. There were no significant differences between the two patient groups.

View larger version (44K): [in this window] [in a new window]   Fig. 1. Agarose 1.5% gel electrophoresis of PCR products for H. pylori genotyping. Lanes: M, 100-bp ladder; 1 and 2, vacA m1 and m2; 3 and 4, iceA1 and iceA2; 5 and 6, vacA s1 and s2; 7, cagA.

The 183-bp PCR product indicating the presence of the cagA gene was obtained with 38 isolates (93%). cagA⁺ infection was present in 95% of duodenal ulcer patients and in 91% of patients with non-ulcer dyspepsia (p >0.05).

All H. pylori isolates possessed the iceA gene. In all, 37 isolates (90.2%) were positive for iceA1, of which 18 (94.7%) were from duodenal ulcer patients and 19 (86.4%) from non-ulcer dyspepsia patients (Table 2). A PCR product for both iceA1 and iceA2 was obtained from only one isolate from a non-ulcer dyspepsia patient.

The combined vacA s1/cagA⁺/iceA1 genotype was present in 82.5% of all H. pylori isolates (Table 3).

	Discussion

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The study demonstrated a high prevalence of H. pylori infection of the vacA s1, cagA⁺/iceA1⁺ genotype in patients from Nigeria with duodenal ulcer disease or non-ulcer dyspepsia. Therefore, this study is consistent with previous reports from the USA, Europe and Asia suggesting that cagA status and the vacA genotype of H. pylori may not predict the clinical outcome of infection [10, 15]. In contrast, another study from South Africa has shown that the vacA s1 allele accounted for 100% of ulcer strains and 67% of gastritis strains [12]. In that study, the vacA s2 type was found exclusively in gastritis patients, whereas in the present study the s2 allele was detected in only one patient with non-ulcer dyspepsia. In the present study, the vacA middle region types were not independently associated with the occurrence of peptic ulceration, which is in agreement with the study by Kidd et al. [12]. Another report by Letley et al. [13] supports these results, in that the vacA middle region polymorphism is not related to peptic ulceration. Kidd et al. also described the first case of a vacA s2,m1 H. pylori isolate from a duodenal ulcer patient [12]. The present study did not find such a genotype among a central African population. The prevalence of cagA⁺ strains in Nigerian peptic ulcer patients and those with non-ulcer dyspepsia was similar to that shown in the study from South Africa. To our knowledge, this is the first study on alleles of the iceA gene in H. pylori isolates from Africa. In contrast to a previous report by van Doorn et al. [9], no association was found between the iceA1 allele and peptic ulcer disease. In another study by Yamaoka et al. [10], the iceA1 allele was predominant in Japanese and Korean populations, whereas iceA2 was prevalent in the USA and Colombia. An association between iceA1 and severity of clinical disease could not be shown in either population. This is also consistent with other studies from Asia suggesting that cagA, vacA and the iceA genotype were not associated with peptic ulcer disease [16].

In conclusion, this study demonstrated that vacA s1, iceA1 and cagA⁺ are common genotypes of H. pylori in Nigeria. As in other developing countries, there seems to be no association between these genotypes and duodenal ulcer disease in this particular population.

	Acknowledgments

 
S.I.S. is supported by the Alexander von Humboldt Stiftung, Bonn, Germany (IV NRI 1067290 STP). The study was presented at the XIVth International Workshop on Gastroduodenal Pathology and Helicobacter pylori, Strasbourg, France, September 2001.

	References

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