Analysis of Clarithromycin Resistance and CagA Status in Helicobacter pylori by Use of Feces from Children in Thailand

The clarithromycin resistance and CagA status of Helicobacter pylori in Thai children were investigated using fecal samples. Of the 284 samples, H. pylori was detected in 120 samples, and the clarithromycin resistance rate was 29.2%. The cagA gene was detected in 59 samples, and only 6.8% of these samples contained the East Asian CagA type.Helicobacter pylori is a pathogenic bacterium that colonizes the human stomach. The prevalence of antibiotic-resistant H. pylori, especially clarithromycin-resistant H. pylori, has been increasing worldwide and makes it difficult to successfully eradicate H. pylori. Clarithromycin resistance in H. pylori has been shown to be due to mutations at positions 2142 and 2143 of the 23S rRNA gene (7, 9).Although H. pylori is closely associated with gastric cancer, the rate of mortality due to gastric cancer is relatively low in Thailand, even though the rate of H. pylori infection in Thailand has been reported to be over 80% (6, 8). A difference in pathogenicity between H. pylori strains may explain the lack of the expected correlation between the rate of mortality due to gastric cancer and the rate of H. pylori infection. The CagA protein, which is one of the most important pathogenicity factors of H. pylori, has been classified into two types: the East Asian CagA type, found in H. pylori isolates from Japan, South Korea, and China, and the Western CagA type, found in H. pylori isolates from Europe, North America, and Australia. Each CagA type has tyrosine phosphorylation segments characterized by a Glu-Pro-Ile-Tyr-Ala (EPIYA) motif in the C-terminal region (3). However, the Western CagA type contains the EPIYA-A and EPIYA-B segments, followed by a variable number of EPIYA-C segments, while the East Asian CagA type contains the EPIYA-A, EPIYA-B, and EPIYA-D segments. Furthermore, the East Asian CagA type has been reported to induce more-severe cellular changes than the Western CagA type (2).Recently, we identified a noninvasive method for detecting clarithromycin-resistant H. pylori isolates from feces with high sensitivity and specificity (5). In this study, we used this previously developed method to investigate the clarithromycin resistance and CagA status of H. pylori in feces of Thai children.Fecal samples were obtained from 284 children (116 males/116 females; mean age, 6.60 years range, 1 to 12 years]) from three schools in Chiang Mai in August 2006. The ages and genders of 52 children of ethnic minorities could not be obtained. The study protocol and the informed-consent document were reviewed and approved by Research Ethics Committee, Faculty of Medicine, Chiang Mai University.DNA was extracted from the feces, and the 23S rRNA gene of H. pylori was amplified as previously described (5). Samples were considered to contain clarithromycin-resistant H. pylori if mutations at positions 2142 and 2143 of the 23S rRNA gene were detected.For the amplification of the cagA gene, we designed new primers targeting the region containing the EPIYA-A and EPIYA-B segments by comparing 81 of the cagA genes registered in the DNA Data Bank of Japan (data not shown). Amplification was performed using primer pairs comprising primers 2553F (5′-AACCCTAGTCGGTAATGGGTTRTCT-3′) and 3222R (5′-ATTGCTATTAATGCGTGTGTGGC-3′) for the first-round PCR and 2612F (5′-CGGACATCAGGAAAGAATTGAA-3′), 2609F (5′-TTTCGGATATCAAGAAGAATTGAA-3′), and 2998R (5′-TTGAAAGCCCTACTTTACTGAGATCA-3′) for the second-round PCR.Samples were designated cagA positive when a PCR product of 180 bp was detected after the third-round PCR, which was performed using Go Taq Green master mix (Promega, Madison, WI) and the 2609F, 2612F, 2779R (5′-CACTCACCTTTTTTAGCAACTTGAG-3′), and 2780R (5′-GCTTTTACCTTTTTAGCAACTTGAG-3′) primers. The cagA-typing PCR was performed using an East Asian CagA-specific primer pair (East-Asian-F 5′-AAAGGAGTGGGCGGTTTCA-3′] and East-Asian-R 5′-CCTGCTTGATTTGCCTCATCA-3′]) and a Western CagA-specific primer pair (Western-F 5′-GGCATGATAAAGTTGATGATCTCAGT-3′] and Western-R 5′-AAAGGTCCGCCGAGATCAT-3′]), which targeted the EPIYA-D and EPIYA-C segments, respectively. Each typing PCR was performed using 1.5 μl of the second PCR product. For each PCR amplification, a PCR mixture that contained ultrapure water as the template was included to rule out false-positive results.Of the 284 fecal samples obtained from Thai children, H. pylori was detected in 120 (42.3%). Of the 120 H. pylori-positive samples, clarithromycin-resistant H. pylori was detected in 35 (29.2%) samples, and both clarithromycin-susceptible and -resistant H. pylori isolates were detected simultaneously in 5 samples. The incidence of clarithromycin-resistant H. pylori in this study was slightly higher than the rate reported for Thai adults in other studies (23.2%) (4). Because there have been few studies that focused on the rate of clarithromycin-resistant H. pylori in Thai children, the results of this study will be useful for estimating the rate of clarithromycin-resistant H. pylori infection in adults in Thailand in the future.The cagA gene was present in 59 (49.2%) of the H. pylori-positive samples. Of these samples, 20 (33.9%) samples contained the Western CagA type (containing the EPIYA-C segments) and 4 (6.8%) contained the East Asian CagA type (containing the EPIYA-D segments). The remaining 35 samples, which lacked any EPIYA-C or EPIYA-D motifs, were considered to contain the Western CagA type (1). To confirm the absence of the EPIYA-C and EPIYA-D segments in these 35 samples, DNA sequencing was performed on 14 cagA genes by using the second PCR product, and none of the cagA genes analyzed had an EPIYA-C or EPIYA-D segment. In summary, H. pylori isolates containing the East Asian CagA type (6.8%) were significantly less prevalent than H. pylori isolates containing the Western CagA type (93.2%). Most of the H. pylori strains isolated in Asian countries with high incidences of deaths from gastric cancer, such as Japan and China, have been reported to contain the East Asian CagA type (10). Since differences in the prevalences of the East Asian CagA type in Asian countries have been suggested to be one of the reasons underlying the differential mortality rates associated with gastric cancer (2), further investigation is needed to confirm this possibility.In this study, we detected a high proportion (59.3%) of CagA that contained neither the EPIYA-C nor the EPIYA-D segment. The low incidence of Western CagA containing the EPIYA-C segment in Thailand may be one of the reasons for the low mortality rate associated with gastric cancer in Thailand.In conclusion, we show that the prevalence of the CagA types of H. pylori in Thai children differs from that reported for other Asian countries. Furthermore, our study demonstrates the usefulness of this approach for detecting and typing CagA in H. pylori by using feces. This method may prove useful in further investigations of the prevalences of the CagA types in H. pylori isolates from infected individuals.