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Incidence of Helicobacter felis and the Effect of Coinfection with Helicobacter pylori on the Gastric Mucosa in the African Population
     Hepatology and Gastroenterology Research Unit, Department of Internal Medicine, University of Pretoria, Pretoria 0002, South Africa

    Ampath Pathology Laboratories, Pretoria, South Africa

    DST-NRF Centre of Excellence at the Percy FitzPatrick Institute, Molecular Ecology and Evolution Programme, Department of Genetics, University of Pretoria, Pretoria 0002, South Africa

    ABSTRACT

    Helicobacter pylori and Helicobacter felis are two of the Helicobacter spp. that infect humans. H. pylori has been linked to significant gastric pathology. Coinfection with Helicobacter spp. may influence infectious burden, pathogenesis, and antibiotic resistance; however, this has not been studied. The aims of this study were to identify the incidence of H. felis and to analyze the effects of coinfection with both organisms on gastric pathology in a well-characterized South African population. Biopsy samples from the gastric corpora and antra of volunteers (n = 90) were subjected to histological examination and PCR for the identification of H. pylori and H. felis. We further investigated the effect of global strain type on the occurrence of precursor lesions by assigning nucleotide sequences derived from PCR amplification of three genes to global groupings (ancestral Africa1, ancestral Africa2, ancestral Europe, ancestral Asia, and mixed). H. pylori was detected in 75 (83.3%), H. felis in 23 (25.6%), and coinfection in 21 (23.3%) of the volunteers by PCR. H. felis was randomly distributed among adults and children but clustered within families, suggesting intrafamilial transmission. Analysis of histopathology scores revealed no differences in atrophy, activity, and helicobacter density between H. felis-positive and H. felis-negative volunteers. H. pylori substrains common to southern Africa showed no differences in inflammation or atrophy scores. The incidences of H. felis and coinfection with H. pylori in the African population are high. H. felis infection, however, does not influence specific gastric pathology in this population.

    INTRODUCTION

    Helicobacter species infect the gastrointestinal tracts of several animals, including humans. Some of these bacteria have been linked to a range of human diseases (18, 20), including chronic gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma (4, 18). Two Helicobacter pathogens known to infect humans are Helicobacter felis and Helicobacter pylori. Primarily a pathogen of domestic animals (36), H. felis was first isolated in the stomachs of cats and was later found to infect humans (29, 41). H. felis is a gram-negative, spiral bacterium that measures approximately 0.4 by 5 to 10 μm (20). Reports of H. felis in humans are, however, rare, and recent studies failed to show the organism in human gastric biopsy specimens (7). The principal Helicobacter infection in humans is H. pylori, with infection rates in developing countries reaching 70 to 90% (2, 11); however, prevalence rates are decreasing in the developed world (3, 18, 40). Host immune reaction to H. pylori and genetic factors have been shown to play significant roles in pathogenesis (26, 33). The immune response to H. pylori in humans is predominantly Th1 cells, which may contribute to disease (43, 47). Host genetic polymorphisms that downregulate this immune response are protective against the development of gastric pathology (48, 52).

    Coinfection with other organisms is known to modulate the H. pylori immune reaction (21) and has thus been proposed to explain the "African enigma," in which high H. pylori infection rates are coupled with a low incidence of gastric carcinoma (5, 32). The degree to which coinfection of H. pylori with H. felis may result in altered immune reactions is not understood. Such coinfection may also allow the usually benign H. felis to cause disease (25), a factor especially relevant to immunocompromised individuals. Furthermore, coinfection may also result in H. pylori acquiring antibiotic resistance genes, given its competence for transformation (38). Although the incidence of H. felis infection in humans is low (20, 28), a recent study in Europe using a multiplex PCR found that more than 8.9% of the sample population were H. felis positive (53).

    The effect of coinfection with other Helicobacter spp. has not been systematically studied. Therefore, we investigate whether coinfection of H. pylori with H. felis influences gastric pathology. Furthermore, we identify the prevalence of H. felis infection in a well-studied (11, 37) rural African community and report patterns of transmission as inferred from detailed family pedigrees.

    MATERIALS AND METHODS

    Study population. The study population comprised 90 healthy individuals, for whom extensive pedigree information was available, from a rural, black, South African community (Ogies, Mpumalanga) that have been followed as part of a long-term surveillance program on the epidemiology of H. pylori (11, 23). This population had many of the risk factors that are associated with a high prevalence of H. pylori infection (6, 49) and thus served as an ideal study population to investigate the effects of H. pylori-H. felis coinfection. Thirty-three volunteers were children aged between 2 and 15 years, and 57 volunteers were adults 16 years and older. Ethical approval for the study was obtained from the University of Pretoria Ethics Committee and the Unitas Hospital Board.

    Histopathology. Four formalin-fixed endoscopic biopsy specimens, two each from the gastric corpus and antrum, were processed and embedded in paraffin wax. Three-micrometer-thick sections were cut from all biopsy specimens and stained with hematoxylin and eosin and methylene blue. Sections were examined by an experienced gastrointestinal pathologist blind to sample identity and graded according to the updated Sydney classification (13). Five variables (chronic inflammation, neutrophil polymorph activity, Helicobacter density, glandular atrophy, and intestinal metaplasia) were graded as absent (0), mild (1), moderate (2), or severe (3). The sections were also evaluated for the presence of atrophy and intestinal metaplasia.

    Molecular methods. DNA was extracted from biopsy samples by using a QIAamp DNA mini kit from QIAGEN (Hilden, Germany) according to the manufacturer's protocol. Extracted DNA was stored at –70°C until required. PCR on the DNA samples was performed to identify H. felis and H. pylori and for subsequent gene sequencing using previously published primers (forward, 5'-GTG AAG CGA CTA AAG ATA AAC AAT-3', and reverse, 5'-GCA CCA AAT CTA ATT CAT AAG AGC-3') spanning the ureA and ureB genes of H. felis (22). A Roche PCR master kit (Roche Applied Science, Johannesburg, South Africa) was used with a 50-μl reaction tube containing 5 μl of template DNA and 0.5 μM of each primer. An initial denaturation at 94°C for 5 min was followed by 40 cycles of denaturation at 94°C, annealing at 62°C, and extension at 72°C with an Applied Biosystems GeneAmp 9700 thermal cycler. All negative samples were retested with an internal control to eliminate the possibility of PCR inhibitors. Helicobacter felis DNA from strain ATCC 49179 was used as a positive control and sterile deionized water as the negative control. To confirm the specificity of the amplicons obtained in PCR-positive samples, the product was purified using a Roche HighPure PCR product purification kit according to the manufacturer's protocol. Nineteen of the 23 H. felis-positive samples were sequenced using an Applied Biosystems automated sequencer, and a BLAST search was done on the NCBI website.

    H. pylori was identified using primers targeting the ureB gene (HpureF113, 5'-GCT CAC ACT TCC ATT TCT-3', and HpureR113, 5'-GCC AGG TTC AAA CCT TAC-3'). In addition, we used a heminested PCR directed at the phosphoglucosamine mutase gene (glmM) developed in our laboratory and reported elsewhere (23) (primers glmMF, 5'-CGC GAG CCA CAA CCC TTT TGA AG-3', and glmMR, 5'-CGC GCT CAC TTG CAA AGC GCA CAC-3') to test for the presence of H. pylori. Briefly, the heminested PCR included an initial round of 10 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min. The product was reamplified with an internal primer (glmMI, 5'-GCT TAT CCC CAT GCA CGA TAT TC-3') and glmMF in 20 cycles. All PCR products were analyzed in a 2% agarose gel stained with ethidium bromide. A 1-kb ladder from Promega (Madison, Wis.) was used as a molecular size marker.

    Fragments from three housekeeping genes (ureI, ureC, and mutY) were PCR amplified (with ureIF, 5' CAA AAG TTA TTC GTA AGG TGC 3'; ureIR, 5' ATT GCC CAT TAA ACG CTC 3'; mutYF, 5' AAA GGG CAA ATC GCA CAT TTG GG 3'; mutYR, AGC GAA GTG ATG AGC CAA CAA AC 3'; and glmM primers as described above) and sequenced. Sequences from the three genes were analyzed to determine genetic diversity and H. pylori subtypes, based on nucleotide identity, compared to the subtypes identified in global H. pylori samples (16). Sequences aligned using ClustalX and MEGA2 (27) were used to construct unrooted neighbor-joining phylograms based on uncorrected p distances for each of the three genes. Structure2 (42) was used to assign the generated sequences to previously recognized H. pylori subtypes. An admixture model that incorporates recombination between nucleotides was used, and thus individual nucleotides could be assigned to ancestral populations based on their linkage with adjacent sites. Falush et al. (16) identified five ancestral population clusters, namely, ancestral Africa1, ancestral Africa2, ancestral Europe1, ancestral Europe2, and ancestral East Asia. Samples generated in this study were assigned to ancestral populations on the basis of nucleotide proportions, where a cutoff of 80% was used to assign individuals to their respective ancestral populations.

    Statistical analysis. A chi-square test, with expected values corrected for overall rate of infection and sampling effort, was performed to determine whether H. felis was asymmetrically distributed among children and adults. A Mann-Whitney nonparametric test was used to compare differences in activity, atrophy, and Helicobacter density between positive samples and negative samples and between alternate strains. A chi-square test was done to determine whether H. felis infections were significantly clustered among family groups. Statistical tests were done using the SPSS 13.0 statistical package for Windows.

    RESULTS

    The specificity of ureB and glmM oligonucleotide primers has been demonstrated previously (11, 23). We analyzed gastric pathology for 90 individuals (360 biopsy specimens) by means of the updated Sydney classification system. These 90 individuals were further tested, using PCR, for the presence of H. felis and H. pylori infection. Of the 90 individuals sampled, 23 were positive for H. felis and 75 tested positive for H. pylori (Fig. 1; Table 1). All sequences obtained from PCR products were confirmed to be of H. felis origin. The frequencies of H. felis among adults and children indicated that infections were randomly distributed among these groups (2 = 0.06, df = 1, P > 0.05). Combining the gastric pathology and PCR results, it is clear that atrophy, the precursor lesion of gastric cancer, is not significantly more prevalent among H. felis-positive individuals (Table 2). This is evident with both corpus and antrum samples (Table 2). In addition, no significant differences in activity and atrophy were detected between positive and negative samples from either the corpus or the antrum. H. felis infections are clustered within families (Fig. 2; Table 3); however, the distribution of infections among families was not significant (2 = 13.95, df = 8, P < 0.10). Assignment of sequences to H. pylori subtypes by using Bayesian clustering methods indicated that samples from the current study were comprised predominantly of ancestral Africa1 and ancestral Africa2 nucleotides, as is evident in the ureI unrooted neighbor-joining tree (Fig. 3). Mann-Whitney nonparametric tests showed no significant difference in gastric inflammation (corpus, U = 97.5; antrum, U = 82.5; P > 0.05), atrophy (corpus, U = 67; antrum, U = 67; P > 0.05), or activity (corpus, U = 90; antrum, U = 102; P > 0.05) between these predominant groups.

    DISCUSSION

    Various studies have demonstrated a high incidence of H. pylori in the African population (8, 9, 46, 49). Helicobacter species infections other than H. pylori have also been reported to occur in the human stomach (12, 20, 22, 39, 53). In this study, we found an overall H. felis infection rate of 25.56%. Consistent with previous studies, H. pylori prevalence was established to be 83.3% in the population studied (Table 1). Coinfection with both organisms was common (Table 1), with only two H. felis samples being negative for H. pylori. Infection with both organisms did not significantly increase the Helicobacter infectious burden (Table 1). This result suggests that coinfection with other Helicobacter species does not significantly influence H. pylori's propensity to colonize the gastric environment. These results show that there is a higher incidence of H. felis in the African population than in Europe (53) or South America (7). Whether this is due to specific host factors or environmental and social factors remains to be elucidated.

    High infection rates of H. felis in an African population are of importance since H. pylori infection rates are also significantly higher in Africa than in other parts of the world (35). Given that H. pylori has the capacity for horizontal genetic exchange (34, 38, 50), genetic variability in H. pylori strains is higher than in any other bacterium. Understanding this horizontal gene exchange is crucial given that H. felis strains resistant to metronidazole, a major component of H. pylori treatment (10, 30), have recently been reported (54). Transfer of antibiotic resistance genes could lead to the emergence of H. pylori strains that are clinically difficult to eradicate. Such a decrease in the efficacy of eradication therapy due to antibiotic resistance would be extremely taxing on already-stretched health budgets in resource-poor African countries.

    Despite the high prevalence of both Helicobacter spp. in this study, significant gastric pathology was not evident. Only six volunteers (all H. pylori positive) showed any signs of atrophy, and one volunteer had mild intestinal metaplasia, the precursor lesions for gastric cancer (24, 44, 45). Specifically, no differences in gastric atrophy or inflammation scores were seen between the H. pylori subtypes (ancestral Africa1 and 2) most commonly found in southern Africa. All volunteers that showed significant gastric pathology were H. pylori positive, and none of the H. felis-positive biopsy specimens showed atrophy or intestinal metaplasia. This result is interesting in that it suggests, contrary to previous reports (12, 28, 29), that H. felis does not cause gastric pathology. Rather, it would appear that the presence of H. felis may be protective against atrophy. It is possible that coinfection may alter the immune response and protect against progression to atrophy. This may in part explain the African enigma. However, this finding was not statistically significant (Table 2), given the low gastric pathology in the population. Biopsy specimens from multiple gastric sites in a large cross-sectional study may shed more light on this finding. Activity, a marker of inflammation, was not significantly (Table 2) different in H. felis-positive biopsy samples compared to H. felis-negative biopsy samples. Unlike H. pylori (31), H. felis does not appear to influence typical immune responses, as reflected by chronic activity scores.

    The frequency of H. felis-positive biopsy samples was not significantly different for children compared to adults. Some studies have shown high infection rates of H. pylori among children whose parents are also infected (1, 14, 55). Fiedorek et al. (19) showed that the rate of acquisition of H. pylori increases with age. A horizontal mode of transmission is the most probable route of transmission since H. felis is primarily a domestic animal pathogen (12, 36). However, given that there is clustering, although not significant at the level of H. felis infection (P > 0.05) within families, a vertical transmission route could be inferred from the results of the current study. Previously, familial clustering has been used as evidence of mother-to-child transmission of H. pylori (15). On the other hand, high-resolution genetic analysis has demonstrated that H. pylori transmission is predominantly horizontal and that familial clustering is most likely the result of social interaction within family groups (11). Similarly, familial clustering of H. felis infections may be the result of social interactions (17, 51); however, high-resolution genetic studies would confirm this.

    In conclusion, H. felis infection rates are high in the African population and coinfection with H. pylori is common. H. felis infection seems to cluster within families, suggesting intrafamilial acquisition. H. felis infection does not influence specific gastric pathology in this population. H. pylori may acquire genes from H. felis that may enhance colonization, pathogenicity, and drug resistance.

    ACKNOWLEDGMENTS

    The Hepatology Research Fund, University of Pretoria, funded this work.

    We thank Paul Rheeder for assistance with data analysis. Robert Bond and Martin Nieuwoudt reviewed the manuscript. We acknowledge Gezina Kies for sample collection and diligent cataloging.

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