Helicobacter pylori Virulence Factor Genotypes in Children in the United States: Clues about Genotype and Outcome Relationships

The most common Helicobacter pylori genotype among 37 U.S. children was cagA positive, vacA s1m1, and oipA “on” (n = 17, 45.9%), followed by cagA negative, vacA s2m2, and oipA “off” (n = 8, 21.6%), similar to the pattern in adults. cagA positivity was more common in blacks than in whites (i.e., 100% versus 56.5%, P = 0.032).Infection with Helicobacter pylori is etiologically associated with gastritis, peptic ulcer disease, gastric atrophy, and gastric cancer. H. pylori is thought to be typically acquired in childhood, with infection continuing for decades if not lifelong. This long bacterial host association involves countless generations of bacteria which are thought to continually evolve as the intragastric conditions change, such that those best suited for the local conditions outgrow and replace less suited neighbors. There has been considerable interest in the molecular epidemiology of H. pylori''s putative virulence factors, especially CagA, VacA, and OipA (outer inflammatory protein A) (8, 11, 12). However, there are few studies of children and only one previous study investigating the relationship between H. pylori virulence factors and ethnic groups of children in the United States (4-6, 11). This study reports the patterns of H. pylori virulence factor genotypes in children of different ethnic groups in the United States.The biopsy specimens and cultures were obtained as part of a multicenter study from 5 widely dispersed sites in the United States. The study was designed to validate the [¹³C]urea breath test for the diagnosis of H. pylori infection in children aged between 2 years and 17 years and 11 months (2). Symptomatic children scheduled for endoscopy were enrolled. Gastric biopsy specimens were evaluated by histology, rapid urease testing, and culture of H. pylori using established techniques (2). H. pylori culture isolates were evaluated for cagA and vacA genotypes using established PCR assays as previously described (9). The number of EPIYA (Glu-Pro-Ile-Tyr-Ala) repeat motifs in the 3′ region of the cagA gene was evaluated using PCR as previously described (7). OipA is a member of the large outer membrane protein family whose functional status is regulated by slipped-strand mispairing based on the number of CT dinucleotide repeats in the 5′ region of the gene (a switch status of “on” indicates the gene is functional, and a switch status of “off” indicates it is nonfunctional) (10). The 5′ region of the oipA gene was amplified using previously described primers (10), and the PCR fragments were purified and directly sequenced at Macrogen, Ltd., in Seoul, South Korea.Forty-eight of 176 children enrolled were H. pylori infected, based on two positive tests or a positive H. pylori culture. The mean age was 11.5 years (range, 3.2 to 17.9 years). Thirty-seven were H. pylori culture positive. None had atrophic gastritis. Only one patient had a significant endoscopic abnormality, a duodenal ulcer (cagA positive, vacA s1m2, and oipA on). The most common H. pylori genotype was cagA positive, vacA s1m1, and oipA on (n = 17, 45.9%), followed by cagA negative, vacA s2m2, and oipA off (n = 8, 21.6%), cagA positive, vacA s1m2, and oipA on (n = 5), cagA positive, vacA s1m1, and oipA on (n = 3), cagA negative, vacA s2m2, and oipA on (n = 2), cagA negative, vacA s1m1, and oipA on (n = 1), and cagA positive, vacA s1m2, and oipA off (n = 1) (Table ​(Table1).1). Overall, 70% of strains were cagA positive, which is similar to is the proportion found in U.S. adults (6).

TABLE 1.

Relationship between H. pylori genotype and ethnic group

Race	No. of children with indicated results for cagA, vacA, and oipAa							Total
	Pos, s1m1, on	Neg, s2m2, off	Pos, s1m2, on	Pos, s1m1, off	Neg, s2m2, on	Neg, s1m1, on	Pos, s1m2, off
White	8	8	3	1	1	1	1	23
Black	8	0	1	2	0	0	0	11
Asian	1	0	1	0	1	0	0	3
Total	17	8	5	3	2	1	1	37

Open in a separate window^aPos, positive; Neg, negative.The frequency of cagA positivity was significantly higher in blacks than in whites (i.e., 100% versus 56.5%, P = 0.032 by Fisher''s exact test). Three prior studies have examined the relationship between cagA status and ethnic groups (black versus white) in U.S. adults (4, 6, 11). Two of the three studies reported that the prevalence of the cagA gene was significantly higher in blacks than in whites (4, 11), which is in agreement with the current study of children.It is currently thought that the basic genotype acquired in childhood remains throughout life. This notion is supported by studies of migrants, who typically show the same pattern as those remaining in the home country, and studies of families, where siblings tend to have similar strains. This concept has also been the basis for the use of H. pylori genotyping to trace the migration of humans throughout the world (e.g., out of Africa) (3). However, as noted above, strains are expected to evolve based on the changing environment of the stomach (i.e., development of atrophic gastritis). The number of EPIYA repeat motifs in the 3′ region of the cagA gene has been related to virulence (i.e., risk of gastric cancer) (7). It was proposed that this change occurred in response to atrophy rather than being responsible for atrophy, as the development of atrophy allowed the more acid-susceptible multi-EPIYA repeat-containing strains to survive and become dominant (7). A recent report in which the number of EPIYA repeat motifs was evaluated longitudinally in three families confirmed that this can occur (1). The results in children are also consistent with this notion, as all cagA genes studied contained three EPIYA motifs (n = 26), which differs from the results in the U.S. adult patients, where four or more EPIYA motifs were found in 21% of those studied (17/81) (7). Future studies comparing cagA gene structure between children and adults in the same population would be useful to address whether the strains thought to be more virulent in relation to the development of gastric cancer predated the development of atrophic gastritis or outcompeted other bacteria as a consequence of the changes in the intragastric environment.In summary, a study of the relationship between H. pylori virulence factor-associated genotypes in relation to ethnic groups of children in the United States found that the prevalence of the cagA gene was significantly higher in blacks than in whites. Conclusions drawn from characterization of strains from patients after the development of a clinical H. pylori outcome (e.g., gastric cancer) may be misleading, as they and the outcome may both reflect changes in the intragastric environment rather than a cause and effect relationship.