Helicobacter pylori Virulence Genes Detected by String PCR in Children from an Urban Community in Northeastern Brazil

Abstract

The accuracy of a nested PCR in gastric DNA obtained by a string test for the diagnosis of Helicobacter pylori infection in asymptomatic children was 94.0%. The cagA-positive toxigenic vacAs1m1 strains were the most prevalent strains, indicating that this population is colonized early by the strains associated with gastric cancer.

TEXT

Helicobacter pylori infection significantly increases the risk of development of peptic ulcer disease, distal gastric carcinoma, and gastric lymphoma (1). Infection of the general population with virulent strains, especially those carrying the cagA gene and vacAs1 genotype, is a predictor of increased risk for development of severe H. pylori-associated diseases. However, the majority of the methods used for genotyping H. pylori strains require an invasive procedure, endoscopy, for tissue sample collection and are not indicated in epidemiological studies evaluating asymptomatic individuals, especially children. The string test, a minimally invasive nonendoscopic procedure, seems to be an accurate method to obtain gastric specimens in order to investigate H. pylori virulence genes. It has been demonstrated that the genotypes of H. pylori strains in DNA from the gastric juice or tissue samples are identical (2).

Previously we have shown a high prevalence of infection by H. pylori strains carrying the cagA gene and the vacAs1 allele in dyspeptic adult patients who underwent endoscopy in northeastern Brazil (3). Furthermore, we have demonstrated that in this population, the infection is acquired earlier in childhood (4), another predictor of gastric cancer. However, we are unaware of studies evaluating the profile of the circulating strains in children of the general population living in areas at increased risk of gastric cancer. Therefore, our aim was to investigate whether the most virulent strains of H. pylori circulate in asymptomatic children from the population, by obtaining H. pylori DNA in gastric juice or mucus by the string test. We also aimed to evaluate the accuracy of an H. pylori-specific nested PCR for the diagnosis of the infection in asymptomatic children.

The study was approved by the Ethics and Research Committee of the Federal University of Ceará. All children and their parents signed the informed consent. Individuals who had participated in previous H. pylori epidemiological studies in Parque Universitário, a low-income urban community in Fortaleza, Brazil, were invited to participate (5). Children who had taken antibiotics potentially active against H. pylori were not included. Fifty children (24 females and 26 males) 8 to 18 years old with a mean age of 14.3 years were evaluated. After a 6-h fast, the children were submitted to the [¹³C]urea breath test (¹³C-UBT) (6) and immediately after to the string test. We used a homemade string test with a small capsule, which increased the adherence of the participants, following the protocol previously described, with minor modifications (7). A gelatin capsule containing a 90-cm-long absorbent cotton string was swallowed with up to 200 ml of water. A 20-cm-long portion of the string was pulled out from the capsule and taped to the subject's cheek. After an hour, the string was retrieved orally. The proximal 30 cm of the string was discarded. The distal gastric mucus/juice-impregnated string was flushed with 5 ml of saline to reduce contamination by oropharyngeal organisms and then placed into a sterile bottle containing 3 ml of brain heart infusion broth and immediately sent for processing. The liquid from the vial containing the string was centrifuged at 13,000 × g for 10 min. The DNA was extracted from the pellet using the QIAamp (QIAgen, Hilden, Germany) kit according to the manufacturer's recommendations. For H. pylori DNA detection, a nested PCR specific for H. pylori ureA was employed (8). PCR amplification of the vacA signal sequence and midregion was performed by using the primers described by Atherton and colleagues (9), and the s1 genotype was further characterized into s1a, s1b, or s1c variants (10). The cagA gene was amplified as described previously (11). Negative and positive controls were included in all reactions. Data were analyzed by two-tailed χ² or Fisher test. All individuals swallowed the capsule without inconvenience. Among 43 children H. pylori positive by ¹³C-UBT, 40 were also positive for the ureA gene by the nested PCR. In the 7 ¹³C-UBT-negative children, the ureA nested PCR was also negative. The string ureA nested PCR had a sensitivity of 93.0% and a specificity of 100% compared with the ¹³C-UBT. The agreement between the two tests was of 94.0%, higher than that reported in the literature in different countries when conventional PCRs for detecting H. pylori specific genes were used (12–16). The accuracy of the string nested PCR was excellent, because we compared its results with those of a noninvasive test (¹³C-UBT) that has high sensitivity and specificity for the diagnosis of H. pylori infection in children older than 6 years (17).

The vacA gene was detected by conventional PCR in 82.5% of the ureA nested-PCR-positive samples. Among them, the most virulent vacAs1 genotype was the most prevalent, being observed in 27 (81.8%) samples, a higher frequency than we demonstrated in symptomatic children from the southeast region of Brazil (18). Otherwise, the vacAs2 nontoxigenic genotype was observed in 6 (18.2%) samples. All 27 vacAs1 strains were s1b. cagA was detected in 22 (66.7%) of 33 vacA-positive strains. Neither vacA nor cagA was amplified in the samples from ¹³C-UBT-negative children. vacAm1 and -m2 alleles were detected in 17 (60.7%) and 11 (39.3%) samples, respectively. The vacAs1 vacAm1 genotype, considered the higher cytotoxin producer, was the most frequent vacA allelic combination and was associated with cagA positivity (P = 0.005) (Table 1). cagA-positive status and vacAs1 genotype were associated neither with the age (P ≥ 0.55) nor with the gender (P ≥ 0.32) of the children.

Table 1.

Distribution of vacA genotypes according to the cagA status of the H. pylori strains in this study

vacA genotype	No. of samples:
	cagA positive	cagA negative	Total
vacAs1 vacAm1	14	3	17
vacAs1 vacAm2	3	2	5
vacAs2 vacAm2	0	6	6
s1a	5	0	5

^aOnly the s region was detected.

Infection by multiple vacA genotypes was not observed, in agreement with the knowledge that it occurs more frequently in adults with the H. pylori-associated severe diseases, probably due to the microevolution that may represent intrahost diversification during long-term colonization (19).

In conclusion, we found that the string test is a safe and simple method to obtain gastric DNA in children, which allowed using nested and conventional simple, accurate, and inexpensive PCR the detection of H. pylori virulennce genes. This approach may be of particular value in H. pylori molecular epidemiological studies. Of note, asymptomatic children from the community we studied are frequently colonized by the most virulent H. pylori strains.