Race, African Ancestry, and Helicobacter pylori Infection in a Low-Income United States Population

This article is featured in Highlights of This Issue, p. 723

Although distal gastric cancer rates have been declining over the past century, gastric cancer remains second only to lung cancer as the most common cause of death from cancer worldwide (1). In the United States, gastric cancer incidence shows a large racial disparity, with an incidence in African Americans almost twice that in whites, and mortality differentials even greater (2). African Americans are also more likely to be infected with Helicobacter pylori (H. pylori), a gram-negative spiral bacterium that colonizes the stomach of approximately half of the world's population. H. pylori is generally acquired in childhood, and is currently the strongest known risk factor for gastric cancer (3). Overall H. pylori prevalence in the United States is estimated to be approximately 30%, but African Americans as a high-risk group are thought to have a prevalence around 50% to 60% (4–6).

H. pylori was officially classified as a human carcinogen in 1994 (7). However, only a fraction of persons infected with H. pylori ever develop neoplasia, and cancer risk is dependent upon strain-specific factors as well as host characteristics (8). These observations, in conjunction with evidence that carriage of certain H. pylori strains is inversely related to risk of esophageal adenocarcinoma, a usually fatal malignancy currently increasing in incidence (9–12), as well as possibly asthma, allergies, and gastroesophageal reflux disease (13–17) underscore the importance of understanding the heterogeneous nature of the bacterium.

Because of the large variation in H. pylori isolates and associated variability in risk profile, characterization of this diversity is crucial to identify high-risk populations for cost-effective disease prevention and potential new risk markers to further classify H. pylori into high- and low-risk groups. The most well-studied marker to date is the H. pylori protein cytotoxin-associated antigen (CagA), present in approximately 60% of United States H. pylori strains (18). CagA is a component of a type IV bacterial secretion system termed the cag island, and cagA-positive strains of H. pylori inject CagA into host cells, altering host cell physiology and the adaptive immune response in a manner that permits H. pylori persistence (19, 20). Emerging immunoproteomics studies have identified additional H. pylori antigens (21), and new epidemiologic research on 15 distinct human H. pylori antibodies has revealed important implications for gastric cancer risk (22, 23). Specifically, Gao and colleagues have reported that the simultaneous presence of the H. pylori vacuolating toxin (VacA), Helicobacter cysteine-rich protein C (HcpC), and the chaperonin GroEL, in addition to CagA, increased the risk of chronic atrophic gastritis (a precursor lesion to gastric cancer) 18-fold (23), and that GroEL may be a new independent risk marker for gastric cancer (22).

The current investigation seeks to characterize overall H. pylori prevalence and sero-positivity for 15 H. pylori proteins in the primarily low-income population as captured in the Southern Community Cohort Study (SCCS), a study designed to investigate cancer disparities among African Americans and whites. In addition, given the excess burden of gastric cancer among African Americans, an important aim was to evaluate, for the first time, the association between level of African ancestry and H. pylori biomarkers of gastric cancer risk.

Between 2002 and 2009, the SCCS, a prospective cohort study, recruited approximately 86,000 men and women aged 40 to 79 from 12 southeastern states at community health centers (CHCs, ∼86%) and by mail (∼14%; ref. 24). All participants completed a baseline survey, which for those enrolled at a CHC involved a comprehensive computer-assisted in-person interview. A validated food frequency questionnaire was used to collect information on regular diet (25, 26). Participants self-reported their race using a printed card with instructions to choose all applicable racial/ethnic categories. At the time of the baseline interview at the CHCs, venous blood samples (20 mL) were collected, refrigerated, and shipped overnight to Vanderbilt University to be centrifuged the next day and stored at −80°C. Among participants who enrolled in the SCCS from March 2002 to October 2004 and donated a blood sample at baseline (N = 12,162), 792 were randomly selected using a 2 × 2 × 3 × 3 factorial design, with 22 individuals selected within each of the 36 strata defined by self-reported race (African American/white), sex, smoking status (current/former/never), and body mass index (18–24.9/25–29.9/30–45 kg/m²). This design provided a balanced distribution across these factors in consideration of other blood biomarkers being measured in addition to H. pylori. Fifty microliters of serum samples were aliquoted for H. pylori assays.

H. pylori multiplex serology, a new antibody detection technology based on fluorescent polystyrene beads (Luminex) and recombinant glutathione S-transferase (GST) fusion protein capture (27, 28) was performed as recently described (29). Fifteen H. pylori proteins (UreA, Catalase, GroEL, NapA, CagA, CagM, Cagδ, HP0231, VacA, HpaA, Cad, HyuA, Omp, HcpC, HPO0305) were used as antigens. All sera were analyzed once within a single assay day. For all 15 antigens, antigen-specific cut-point values previously determined in a validation study (29) were applied using a bridging panel of 78 previously characterized sera containing 38 H. pylori negative sera and 40 H. pylori positive sera. H. pylori sero-positivity was defined as sero-positivity to >3 proteins, which has shown good agreement (κ = 0.70) with commercial serological assay classification (29). To test the reliability of the assay within our population, 2 individuals were randomly selected to have 5 replicate samples sent to the lab; the determination of sero-positivity for all of the H. pylori proteins detected was strongly consistent [only 1 (0.3%) replicate of 30 was not identical with the others].

Genomic DNA was extracted from buffy coat using QIAamp DNA kits (Qiagen) according to manufacturer's instructions, and genotyping was carried out using the Illumina GoldenGate genotyping platform (Illumina Inc.). Laboratory personnel were blinded to the status of the samples. Blinded quality control samples (N = 29) and another 171 pairs of duplicate samples were included and the consistency rate was 99.9%. As described previously (30), a set of 276 single nucleotide polymorphisms were selected to estimate African and European ancestry levels, using a Bayesian clustering approach implemented using STRUCTURE software (version 2.2.3; ref. 31). STRUCTURE identifies groups of individuals with similar allele frequency profiles and estimates the shared ancestry of individuals based solely on their genotypes under an assumption of Hardy–Weinberg equilibrium and linkage equilibrium in ancestral populations. It identifies a specified number (K) of ancestry population clusters (K = 2 in this study) and assigns individual admixture estimates for each, with the estimates summing to 1 across these clusters. An admixture estimate (from 0.00 to 1.00) for both African ancestry and European ancestry was thus generated for each participant.

This study includes 686 (86.6%) of the 792 originally sampled participants, as the available serum was depleted from other assays performed on this group for 77 (9.7%), samples for 3 (0.4%) individuals were unusable because of serum handling issues, 3 individuals (0.4%) were missing information on antibiotic use, and the ancestry estimates for 23 (2.9%) were highly discordant with self-reported race, implying potential data entry errors.

Participants were classified by race based on their self-identification as African American only or white only. Ancestry estimates were modeled both as continuous variables for trend and as the categorical variables “white” (all whites), and “low,” “medium,” and “high” African ancestry based on previously utilized cut-points of <85%, 85% to 94.99%, and ≥95% African ancestry level (30). The outcomes of sero-positivity for H. pylori as well as for the 15 individual H. pylori proteins were modeled as dichotomous variables (yes/no).

Unconditional logistic regression was used to calculate odds ratios (ORs) and 95% CI for the prevalence of H. pylori infection and for the presence of antibodies to specific H. pylori proteins by race and ancestry level. Statistical adjustment was made for age at enrollment, sex, smoking status, and body mass index, and also education (<high school education/high school or GED/>high school education) and antibiotic prescriptions in the past year (0/1 or more), chosen because they were associated with both race and H. pylori sero-positivity in the data. Crude and adjusted polytomous logistic regression models were also created to explore the association between race and ancestry level and the 3 outcomes of sero-negativity, sero-positivity to H. pylori but not to CagA (H. pylori+, CagA−), and sero-positivity to both H. pylori and CagA (H. pylori+, CagA+). For these analyses, the 5 (0.7% of the population) individuals (2 African American and 3 white) who were found to be sero-negative to H. pylori but sero-positive to CagA were excluded. The associations of African ancestry levels and sero-positivity to each of the 15 individual H. pylori proteins were also explored using multivariable logistic regression. All statistical analyses were performed using SAS 9.2 (SAS Institute).

Because of the stratified study design, the percentage of individuals in each category of sex, cigarette smoking status, and body mass index are comparable between the self-identified African Americans and whites in this study (Table 1). Other demographic and lifestyle characteristics were also similar in most respects, although African Americans were more likely to have a lower level of education, be in the highest category of fruit and vegetable intake, be hypertensive, and less likely to have asthma, emphysema, duodenal or gastric ulcer, arthritis, or to have been given a prescription for antibiotics in the past year. The median estimated percentage of African ancestry among self-identified African Americans was 97.0% (range 50.5%–99.9%); among self-identified whites the median percentage African ancestry was 0.4% (range 0.1%–17.1%).

In this sample of low-income African Americans and whites, 89% of African Americans (88% of men and 90% of women) and 69% of whites (69% of both men and women) were sero-positive for H. pylori. African Americans, compared to whites, had a 2- to 6-fold increased odds of sero-positivity to 8 of the 15 H. pylori proteins examined, including CagA (OR, 6.4; 95% CI, 4.5–9.1), and VacA (OR, 2.3; 1.5–3.5), the 2 previously established markers for gastric cancer risk, as well as GroEL, HcpC, Omp, Cad, HP 0305, and HpaA (Table 2). None of the 15 H. pylori proteins was recognized as significantly more common among whites.

Overall, compared to whites, African Americans had more than 3 times the odds (adjusted OR, 3.5; 95% CI, 2.3–5.4) of being H. pylori-positive (Table 3). Odds ratios significantly increased with increasing percentage of African ancestry, with ORs of 1.6 (95% CI, 0.8–3.0), 4.1 (95% CI, 2.0–8.6), and 5.2 (95% CI, 2.8–9.5) for low, medium, and high African ancestry, respectively. Examining the association among African Americans alone, compared to African Americans of low African ancestry, African Americans of medium African ancestry had an over 2-fold increased risk (OR, 2.5; 95% CI, 1.0–6.6) and African Americans of high African ancestry had an over 3-fold increased risk (OR, 3.4; 95% CI, 1.4–8.0). When separating outcomes using joint H. pylori and CagA status, the increased risk among African Americans was primarily related to CagA sero-positivity, for which African Americans were at 8.1-fold (95% CI, 5.0–13.1) increased odds, and African Americans of low, medium, and high African ancestry had ORs of 2.5 (95% CI, 1.2–5.3), 9.6 (95% CI, 4.3–21.5), and 13.1 (95% CI, 6.8–25.3), respectively. Again, when examining these associations among African Americans only, African Americans of medium and high African ancestry were of significantly greater risk of CagA-positive H. pylori strains than African Americans of low African ancestry (OR. 3.5; 95% CI, 1.3–10.0 and OR, 4.9; 95% CI,2.0–12.4, respectively).

Among the generally low-income individuals in the southeastern United States included in this study, 79% were sero-positive for H. pylori. Self-reported African American race and increasing percentage of genetic estimation of African ancestry were strongly associated with prevalence of H. pylori infection, particularly with CagA sero-positivity. The racial difference is noteworthy because socioeconomic differences by race were minimized by study design (both blacks and whites tended to be of low income and education level) and by statistical adjustment for residual differences. Furthermore, the strong gradient in risk of infection, especially for CagA+ strains, with percent African ancestry is a new finding with potentially major implications regarding susceptibility to this common infection.

Another new finding arising from this research concerns information on the distribution of African ancestry in an African American population spanning rural as well as urban areas of 12 southern states. We found that the levels of African ancestry among African Americans in our population (median, 97%; mean, 92%) tended to be higher than those seen generally in previous studies among African Americans (32–35), but comparable to estimates among Gullah-speakers in South Carolina (36). Although beyond the scope of this report, we observed notable geographic variation in percent African ancestry suggesting that clustering of black populations with very high percent African ancestry many exist in multiple areas of the South.

The estimates of H. pylori prevalence in this study (89% for African Americans and 69% for whites) are much higher than the previously reported prevalence of 30% for the United States overall, and 50% to 60% for African Americans (5). Worldwide, H. pylori prevalence has generally been reported to be higher in developing than developed countries, with estimates of 25% in Australia (37); 28% to 65% in Europe (lower in Western European than Eastern European countries; refs. 38 and 39); 64% to 72% in the Middle East, China, and Japan (5, 39–41); and around 80% to 90% in many countries in South America and Africa (5, 42). Of note, in this study only 2 (0.6%) of the African Americans and 9 (2.7%) of the whites were born outside of the United States.

In line with our findings, the few studies that have specifically examined United States racial differences in H. pylori prevalence have consistently found higher rates among African Americans, but none have shown the exceptionally high rates of infection, especially for CagA+ strains, reported herein. In the Third National Health and Nutrition Examination Survey (NHANES) reflective of the entire adult US population in 1988 to 1991, 54% of African Americans versus 28% of non-Hispanic whites were H. pylori positive (6); among those with less than 12 years education and considered at “high risk,” the subset closest to our sample, 60% of African Americans and 41% of non-Hispanic whites were sero-positive. In NHANES 1999 to 2000, the prevalence remained nearly the same among blacks (53%) but declined to 22% among whites (4). Other surveys have shown that the racial disparity tends to begin early in life; for example, African American children aged 5 to 9 years have been observed to have an H. pylori infection prevalence of about 30%, compared to 12% for the overall child infection rate in the United States (43). Although an inverse association between socioeconomic status and H. pylori infection has been one of the most consistent findings (44), even among African American and white children attending the same schools, a longitudinal study in Louisiana observed that over the 12-year follow-up period, African American children had a 4-fold greater acquisition rate than whites, and only 4% of African American children proceeded to lose the infection, compared to 50% of the white children (45).

In terms of strain-specific H. pylori infection, the NHANES data revealed that approximately 60% of adults that were H. pylori positive were also CagA+ (46). In a small series of H. pylori-positive dyspepsia patients undergoing upper endoscopy in Nashville, Tennessee, 61% of non-Hispanic whites versus 90% of African Americans were sero-positive for CagA (47). Higher prevalences of CagA sero-positivity among blacks have also been reported in other series (48, 49). In the current study, among H. pylori-positive individuals, 36% of whites and 75% of African Americans were CagA+. Although this difference is substantial, the trend in CagA sero-positivity was even more striking when analyzed within the context of African ancestry level.

To the best of our knowledge, this study is the first to examine the association between African ancestry level (characterized using ancestry informative genetic markers) and H. pylori infection. The environmental factor most consistently associated with H. pylori infection is low socioeconomic status (40, 50), but we could eliminate all but very minor influences of education, income and other demographic factors in the observed black/white differences because of the fairly homogenous population studied, and because we were able to adjust in detail for education level. Educational level was the only socioeconomic status variable adjusted for, as it was the only one significantly associated with both race/African ancestry and H. pylori status. Additional adjustment for household income and household size was examined as well, but these factors had little impact on the main results. Furthermore, when examining the associations between race and African ancestry and H. pylori prevalence among only those without a high school education (n = 210), the strong association between increasing African ancestry and increasing likelihood of H. pylori prevalence seen in the larger study was replicated in this subgroup defined by low educational status (data not shown). However, we cannot rule out the possibility that unmeasured lifestyle factors associated with African ancestry may influence H. pylori infection. Although we did not have information on history of treatment for H. pylori, we did adjust for antibiotic use in the previous year, a factor that while different by race (African American versus white), was not associated with level of African ancestry among African Americans. Our results raise the possibility of an increased genetic susceptibility to H. pylori infection that may operate in conjunction with greater opportunities for environmental exposure in low-income populations to yield higher H. pylori infection rates.

The associations we observed could reflect the genetics of H. pylori itself. A study comparing a polymorphic H. pylori DNA sequence found a 180-bp insertion in 100% of West African isolates, 45% of South African isolates, 23% of Spanish isolates and 10% of North American isolates, and 56% of isolates from African Americans compared to 17% of isolates from Caucasian Americans (P < 0.05), suggesting that H. pylori strains colonizing African Americans today retain residual characteristics of those in Africa (51). Unfortunately, we had no endoscopy specimens from which we could isolate the bacteria, and are not aware of other investigations which have classified H. pylori origins among African Americans.

A German study of the association of the 15 H. pylori proteins measured here with gastric outcomes found that all 15 were positively associated with chronic atrophic gastritis (23), a precursor to intestinal-type gastric cancer, and 7 were significantly associated with gastric cancer (22). However, the few other population-based epidemiologic studies examining an assortment of H. pylori antigens, using different methods for detection, have not found the same associations (52, 53). In our study, antibodies to 12 of the 15 H. pylori proteins were more common among African Americans than whites, including 6 of the 7 proteins identified as risk factors for gastric cancer. Furthermore, all of these associations were heightened when examining prevalence by increasing African ancestry levels (see Supplementary Table). In addition, the proteins GroEL, Cad, HP 0305, and HpaA were significantly associated with African American race and African ancestry level independent of the known gastric cancer virulence factor CagA (data not shown).

Our findings that low-income African Americans and whites have developing country levels of H. pylori infection, and that African Americans, especially those of higher percentage of African ancestry, have a particularly high prevalence of antibodies against the H. pylori virulence factor CagA, now provides a framework for further research into better detection and prevention of gastric cancer in this population. Additional research may help delineate those at highest risk for increased surveillance and candidacy for H. pylori eradication. Examination of bacterial samples may help clarify the ancestral origins of the H. pylori strains and their links to cancer risks. H. pylori strain-specific research in high-risk populations such as the one in this study also may help to inform as well as contribute to ongoing efforts at vaccine development. Ultimately, studies such as the current investigation may help identify determinants, and means of amelioration, of the long-standing higher rates of gastric cancer among African Americans.

No potential conflicts of interest were disclosed.

We would like to thank Shumei Wang for effort in developing some of the RHM and p58 fragments of VacA, Regina Courtney and Rodica Cal-Chris for serum and DNA sample preparations, and Heather Munro and Sarah Cohen for statistical review.

The Southern Community Cohort Study is funded by a grant from the National Cancer Institute (R01 CA092447; L.B. Signorello, W. Zheng, W.J. Blot). The development of Helicobacter pylori multiplex serology was funded in part by the Joint Initiative for Innovation and Research of the German Helmholtz Association (A. Michel, M. Pawlita). Analysis of the samples in this study was funded by an American Cancer Society—Institutional Research Grant to Vanderbilt University (ACS-IRG-58-009-50; M. Epplein). Genotyping work was partially supported by a grant from the Komen for the Cure Foundation (OP05-0927-DR1; S.M. Williams). The serum/plasma sample preparations were performed at the Survey and Biospecimen Core, which is supported in part by the Vanderbilt-Ingram Cancer Center (P30 CA68485; Q. Cai).

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