Detection of Helicobacter pylori in stool samples of young children using real-time polymerase chain reaction.

BACKGROUND: The aims of this study were to develop and validate a multiplex real-time polymerase chain reaction (q-PCR) assay of Helicobacter pylori in stool samples of healthy children. Additionally, we determined the prevalence of clarithromycin resistance and cagA gene in H. pylori-positive samples. MATERIALS AND METHODS: Archived stool samples from 188 children aged 6-9 years and 272 samples of 92 infants aged 2-18 months were tested for H. pylori antigens using enzyme immunoassay (EIA). A multiplex q-PCR assay was designed to detect H. pylori 16S rRNA and urease and the human RNase P gene as an internal control. Kappa coefficient was calculated to assess the agreement between q-PCR and EIA. RESULTS: Laboratory validation of the q-PCR assay using quantitated H. pylori ATCC 43504 extracted DNA showed S-shaped amplification curves for all genes; the limit of detection was 1 CFU/reaction. No cross-reactivity with other bacterial pathogens was noted. Applying the multiplex q-PCR to DNA extracted from fecal samples showed clear amplification curves for urease gene, but not for 16S rRNA. The prevalence of H. pylori infection was 50% (95% CI 43%-57%) by q-PCR (urease cycle threshold <44) vs 59% (95% CI 52%-66%) by EIA. Kappa coefficient was .80 (P < .001) and .44 (P < .001) for children aged 6-9 years and 2-18 months, respectively. Sixteen samples were positive for cagA and three were positive for clarithromycin resistance mutation (A2143G) as confirmed by sequencing. CONCLUSIONS: The developed q-PCR can be used as a cotechnique to enhance the accuracy of H. pylori detection in epidemiological studies and in clinical settings.

Helicobacter pylori infection, serum pepsinogens, and pediatric abdominal pain: a pilot study.

The significance of Helicobacter pylori (H. pylori) infection in pediatric abdominal pain remains poorly recognized. We examined associations of H. pylori infection and serum pepsinogens (PGs), as non-invasive markers of gastritis, with pediatric abdominal pain. A case-control study was conducted among 99 children aged 5-17 years admitted to one hospital for abdominal pain (cases) without an apparent organic reason. Using enzyme-linked immunosorbent assays, sera were tested and compared with 179 controls for anti-H. pylori immunoglobulin G (IgG) antibodies and PGI and PGII levels. Multivariable analysis was performed to adjust for potential confounders. H. pylori IgG sero-positivity was 34.3 and 36.3% in cases and controls, respectively, P = 0.7. H. pylori-infected children had higher median PGI and PGII levels and a lower PGI/PGII ratio than uninfected children. Cases infected with H. pylori had a higher median PGII level (P < 0.001) and lower PGI/PGII ratio (P = 0.036) than controls infected with H. pylori. The percentage of cases with PGII ≥7.5 µg/L, as indication for antral inflammation, was higher than in controls: 58.6 versus 44.7%, P = 0.027. Children with PGII levels ≥7.5 µg/L had increased risk for abdominal pain: adjusted prevalence ratio 1.73 [95% confidence intervals 1.02, 2.93], P = 0.039. CONCLUSION: Children with increased serum PGII levels, as an indication of gastritis, are more likely to have abdominal pain. Serum PGs can be a useful non-invasive marker for gastritis, in evaluating children with severe abdominal pain with no apparent organic reason. What is Known: • The significance of Helicobacter pylori infection in pediatric abdominal pain remains debated. • Serum pepsinogens (PGs), non-invasive markers of gastric inflammation, were rarely utilized in assessing the association between H. pylori in pediatric abdominal pain of unknown origin. What is New: • High serum PGII level, as an indication of gastritis, rather than H. pylori infection itself, was associated with increased risk for abdominal pain. • Serum PGs can be a useful biomarker for gastritis in evaluating children with severe abdominal pain with no apparent organic reason.

Prevalence and determinants of serological evidence of atrophic gastritis among Arab and Jewish residents of Jerusalem: a cross-sectional study.

OBJECTIVE: Understanding the correlates of premalignant gastric lesions is essential for gastric cancer prevention. We examined the prevalence and correlates of serological evidence of atrophic gastritis, a premalignant gastric condition, using serum pepsinogens (PGs) in two populations with differing trends in gastric cancer incidence. METHODS: In a cross-sectional study, using ELISA we measured serum PGI and PGII concentrations (Biohit, Finland), Helicobacter pylori serum IgG and cytotoxin-associated gene A (CagA) antigen IgG antibodies in archived sera of 692 Jews and 952 Arabs aged 25-78 years, randomly selected from Israel's population registry in age-sex and population strata. Multivariable logistic regression analyses were performed. RESULTS: Using cut-offs of PGI <30µg/L or PGI:PGII <3.0, the prevalence of atrophic gastritis was higher among Arab than Jewish participants: 8.8% (95% CIs 7.2% to 10.8%) vs 5.9% (95% CI 4.4% to 7.9%), increasing with age in both groups (p<0.001 for trend). Among Jewish participants, infection with H. pylori CagA phenotype was positively related to atrophic gastritis: adjusted OR (aOR) 2.16 (95% CI 0.94 to 4.97), but not to non-CagA infections aOR 1.17 (95% CI 0.53 to 2.55). The opposite was found among Arabs: aOR 0.09 (95% CI 0.03 to 0.24) for CagA positive and aOR 0.15 (95% CI 0.06 to 0.41) for Cag A negative phenotypes (p<0.001 for interaction). Women had a higher atrophic gastritis prevalence than men. Obesity and smoking were not significantly related to atrophic gastritis; physical activity tended to be inversely associated in Arabs (p=0.08 for interaction). CONCLUSIONS: The prevalence of atrophic gastritis was higher among Arabs than Jews and was differently associated with the CagA phenotype.

Sero-prevalence of Helicobacter pylori CagA immunoglobulin G antibody, serum pepsinogens and haemoglobin levels in adults.

Associations observed of Helicobacter pylori infection with haemoglobin levels are inconsistent. We examined associations of H. pylori sero-prevalence and serum pepsinogens (PGs), as non-invasive markers of atrophic gastritis, with haemoglobin levels. A cross-sectional study was undertaken among 654 Jewish and 937 Arab residents of Jerusalem, aged 25-78 years, randomly selected from Israel's national population registry in age-sex and population strata. Sera were tested for H. pylori IgG, cytotoxin-associated gene A (CagA) antigen IgG antibody and PGs levels. Multivariable models were fitted to account for confounders. Participants with atrophic gastritis (PGI < 30 µg/L or a PGI: PGII < 3.0) had lower haemoglobin levels than those without: beta-coefficient -0.34 (95% CI -0.59, -0.09); in men -0.27 (95% CI -0.67, 0.12), and in women -0.43 (95% CI -0.74, -0.12). Lower haemoglobin levels were noted in persons with CagA antibody than in those H. pylori sero-negative or H. pylori-CagA sero-negative: beta-coefficient -0.14 (95% CI -0.29, 0.01). Anaemia was more common among women and men with than without atrophic gastritis: adjusted OR 2.58 (95% CI 1.48, 4.48) and 1.52 (95% CI 0.59, 3.95), respectively. In conclusion, independent of known correlates, atrophic gastritis and apparently CagA sero-positivity, a marker of H. pylori virulent strains, are associated with lower haemoglobin levels.