High Prevalence of Helicobacter pylori Resistance to Clarithromycin : a Hospital-Based Cross-Sectional Study in Nakhon Ratchasima Province , Northeast of Thailand

Helicobacter pylori is a gram-negative, helical shaped and microaerophilic bacterium that colonizes in the pathogenesis of chronic gastritis, peptic ulcer disease, and gastric malignancy (Komoto et al., 1998). Since the discovery of Helicobacter pylori in 1983, strong evidence has indicated that H. pylori eradication is current standard treatment and can prevent of chronic gastritis, peptic ulcer recurrence and malignancy change (Mihara et al., 1999). Prevalence of H. pylori infection has geographic variation in which there is a higher prevalence in the developing country including Thailand (Tan and Goh, 2008). The


Introduction
Helicobacter pylori is a gram-negative, helical shaped and microaerophilic bacterium that colonizes in the pathogenesis of chronic gastritis, peptic ulcer disease, and gastric malignancy (Komoto et al., 1998).Since the discovery of Helicobacter pylori in 1983, strong evidence has indicated that H. pylori eradication is current standard treatment and can prevent of chronic gastritis, peptic ulcer recurrence and malignancy change (Mihara et al., 1999).Prevalence of H. pylori infection has geographic variation in which there is a higher prevalence in the developing country including Thailand (Tan and Goh, 2008).The

RESEARCH ARTICLE
High Prevalence of Helicobacter pylori Resistance to Clarithromycin: a Hospital-Based Cross-Sectional Study in Nakhon Ratchasima Province, Northeast of Thailand Taweesak Tongtawee 1,2,3 *, Chavaboon Dechsukhum 2,3 , Likit Matrakool 1,3 , Sukij Panpimanmas 1,3 , Ryan A Loyd 3,4 , Soraya J Kaewpitoon 3,4,5 , Natthawut Kaewpitoon 3,5,6   distribution pattern of H. pylori infection ranges from 25 to 50% in developed countries to more than 80% in the developing world (Beswick et al., 2006).Although the bacterium is susceptible to most antimicrobial agents in vitro, but the successful treatment of H. pylori is a challenge (Gerrits et al., 2006).H. pylori treatment has been regimented that clarithromycin common practicum, however, clarithromycin resistant H. pylori strains represent the main cause of treatment failure and eradication.The primary resistance rate of H. pylori to clarithromycin is different among each region of the world.The overall global resistance from a systemic review in 2004 was 9.9% (95% CI 8.3-11.7)(Megraud, 2004), and during 2006-2009 was 17.2% (95% CI 16.5-17.9)(De Francesco et al., 2010).A highest of resistance rate was found in Japan, while, the lowest rate was found in Korea and Thailand (Kobayashi et al., 2002).Nationwide survey of H. pylori antibiotic resistance in Thailand was reported.The result show that antibiotic resistance was presented in 50.3% including metronidazole 36%, ciprofloxacin 7.7%, levofloxacin 7.2%, amoxicillin 5.2%, multi-drugs in 4.2%, clarithromycin 3.7%, and tetracycline 1.7%, respectively (Vilaichone et al., 2013).Therefore, this study aimed to determine the prevalence of H. pylori to clarithromycin resistance associated with 23S ribosomal RNA point mutations, and the effect on clarithromycin base triple therapy for H. pylori eradication in Nakhon Ratchasima province, northeast region of Thailand.

Population and Diagnosis of Helicobacter pylori associated gastritis
A hospital-based study was carried out among 300 patients diagnosed with H. pylori during June 2014 and January 2015.The study was performed in Suranaree University of Technology hospital, Suranree University of Technology, Nakhon Ratchasima, Northeastern Thailand.All patients provided a written informed consent.A diagnosis of H. pylori associated gastritis was made if H. pylori were seen on histopathological examination and the rapid urease test were positive.Finally, we prove bacterial infection by Polymerase chain reaction (PCR) method.Briefly,

Biopsy specimens and histological analysis
Biopsy was done according to the updated Sydney classification system (Dixon et al., 1994), which indicates sampling from 5 biopsy sites, each specimen should be obtained from the lesser curvature of the corpus about 4 cm proximal to the angulus (1), from the lesser curvature (2) and greater curvature of the antrum (3), both within 2 to 3 cm of the pylorus, from the middle portion of the greater curvature of the corpus, approximately 8 cm from the cardia (4), and from the incisura angularis (5).Gastric tissue specimens for histological analysis were sent to pathologist.The hematoxylin and eosin stain and Giemsa stain were used for identification of H. pylori.The pathological analysis made by 5 pathologist of Bangkok Pathological Laboratory outside Suranaree University of technology.

DNA isolation method Real Time PCR methods
The DNA of H. pylori was extracted from gastric biopsy specimen using the QIAamp DNA FFPE tissue kit (Qiagen, USA).The DNA extraction was performed according to manufacturer protocol.Briefly, ten tissue sections of 5 µM thick were collected in 1.5 ml microcentrifuge tubes.The deparaffinization step was accomplished by xylene and 95% ethanol.The protein digestion step was done by incubation the tissue in proteinase K digestion buffer at 56°C and 90° C for 1 hour of each step.Then the tissue solution was transferred to spin QIAamp MinElute column to let the DNA combine to the membrane.The samples were then subjected to centrifugation at 6000 × g.After that the samples were subjected to repeated washing step by using the provided washing buffers.In the final step was DNA samples were eluted using elution buffer at the final volume of 50 µl.The detection of 23S rRNA mutation was performed, by using the real-time PCR technique.The hybridization fluorescent probe was utilized for PCR product detection.The real-time PCR procedure was accomplished by using Light Cycler ® 480 instrument (Roche diagnostics, Neuilly sur Seine, France).The identifications of target PCR products were accomplished by melting curve analyses.The target PCR products were amplified by using the primers HPYS and HPYA as previously reported in the previous literature (Me nard et al., 2002).PCR-RFLP can also detect the point mutation A2142C of the 23S rRNA gene associated with resistance of H. pylori to clarithromycin.The amplified products have a size of 267 bp.The hybridization probes include the one that is in the mutation sites of the 23S rRNA gene of H. pylori, the sensor probe.The sequence is 5-GGCAAGACGGAAAGACC-3; nucleotides 2504 to 2520).This sensor probe is labeled by LC-red 640 at 5' and phosphorylated at 3'.The anchor probe will hybridized to the PCR product at the site 3 bp upstream to the sensor probe.The probe sequence is 5 TGTAGTGGAGGTGAAAATTCCTCCTACCC-3; nucleotides 2473 to 2501, GenBank accession number U27270).The probe is labeled with fluorescein at 3'. 3 µl DNA templates were subjected to PCR reaction in the final volume of 20 µl.The reaction mixture consists of MgCl2 (25 mM), forward and reverse primers (20 M each), sensor and anchor probes (20 M each), and 2 µl of FastStart DNA Master Hybridization Probes (Roche Diagnostics).PCR amplification comprised an initial denaturation cycle at 95°C for 10 min, followed by 50 amplification cycles (with a temperature transition rate of 20°C/s) consisting of 95°C for 0 s, annealing at 60°C for 10 s, and extension at 72°C for 17 s.After amplification a melting step was performed, consisting of 95°C for 0 s, cooling to 45°C for 30 s (with a temperature transition rate of 20°C/s), and finally a slow rise in the temperature to 85°C at a rate of 0.1°C/s with continuous acquisition of fluorescence decline.According to previous report using this real-time PCR protocol, this melting curve analysis can detect the possible three mutant geneotypes along with the wild type according to different Tm.The reported Tm of the wild type, A2121C, A2142G and A2143G were 61.5, 58.0, 53, 53.6 °C respectively.

Statistical analysis
SPSS for Windows (version 16.0; SPSS, Chicago, IL, USA) was used for the statistical analysis; baseline demographic data of the infected patients were analyzed.The correlation of eradication rate and pattern of mutation were analyzed by chi-square test.All results were considered statistically significant when the P-values were less than 0.05.

Discussion
H. pylori is remains a major public health problem in Thailand.Recently, the results show that 84% and 15% of them were the non-ulcer gastritis/duodinitis, and peptic ulcer disease.Unfortunately, 1% of them was the gastric cancer, this figure indicates that H. pylori infection was a serious problem in the northeastern Thailand where has the high incidence of cholangiocarcinoma; bile ducat cancer.In addition, the new discovery has been a strong, positive correlation between opisthorchiasis-associated cholangiocarcinoma and infection with Helicobacter.The findings indicate that the liver fluke Opisthorchis viverrini in the biliary tree of the hamsters harbors H. pylori and Helicobacter-like bacteria.Accordingly, the association between O. viverrini and H. pylori may be an obligatory mutualism (Deenonpoe et al., 2015).Recent results showed the majority of them had a history with alcoholic drinking, smoking, and spicy food consumption.Previous studied had shown that smoking has effects in H. pylori related gastric carcinogenesis.The smoking behavior contributed to the increased risk of gastric carcinogenesis from gastric atrophy, and had little influence on H. pylori infection or gastric atrophy development (Hishida et al., 2010).
Alcohol consumption has a close relationship with peptic ulcer diseases.Chronic active gastritis is reportedly associated with chronic alcohol ingestion.Nonetheless, the inflammatory changes are likely to be related to concurrent H. pylori infection that is common among alcoholics.Moreover, chronic alcoholism is also correlated with the presence of gastric metaplasia.Both clinically and experimentally, alcohol had been shown to affect the mucosal barrier and histology.These ulcerogenic effects play a crucial role in altering gastric mucosal defense mechanisms.Concurrent consumption of alcohol and cigarette smoking significantly increases the risk of gastric ulcers.In animal experiments, cigarette smoking potentiated ethanol-induced gastric mucosal damage.The reduction of mucus secretion, increase in leukotriene B4 level, increased activities of inducible nitric oxide synthase, xanthine oxidase and myeloperoxidase, and the expression of adhesion molecules in the gastric mucosa accompanied such potentiating effects (Ko and Cho, 2000).
Among clarithromycin-resistant strains tested, all were due to A2144G point mutation in 23S rRNA gene.The most of them was wild type + mutation (susceptible + resistance), followed by mutation, A2143/2142CG (resistance), and wild type.This result is similar to Wang et al (2000), H. pylori strain resistant to metronidazole and clarithromycin in Hong Kong.Dual resistant strains reduced the eradication rate to 66.7%.Among clarithromycin-resistant strains tested, all were due to A2144G point mutation in 23S rRNA gene.A meta-analysis of 93 studies with 10,178 subjects has been reported (Fischbach et al., 2007).Clarithromycin resistance affects the efficacy of first line triple therapy regimen as well.In standard, clarithromycin base triple therapy regimen, the efficacy decreased by an estimated 66 % (95% CI 58.2-74.2) and the eradication rate ranged 0%-50%.A standard clarithromycin base triple therapy recommended by American College of Gastroenterology guideline (Chey et al., 2007) and the second Asia-Pacific consensus for H. pylori eradication (Fock et al., 2009), however, antibiotic-resistant H. pylori strain increasing, the efficacy of this regimen can be reduced.Nationwide survey of antibiotic resistance to H. pylori in Thailand has been reported and found that the antibiotic resistance was 50% and these bacteria resistant to 3.7% clarithromycin (Vilaichone et al., 2013).Recent result, the prevalence of H. pylori strains resistant to clarithromycin was 76.2%.The clarithromycin-based triple therapy regimen, the efficacy decreased by 70% for H. pylori eradication.According to this result, the majority of histologicallyproven H. pylori infected cases (76.2%) have mutant genotypes, which confer the resistant property to clarithromycin, the clinical data indicated that most of the cases poor response to the treatment for standard regimen.This observation indicated that even in the cases who have resistant strain, this treatment protocol (clarithromycin base triple therapy) an ineffective to eradicate the bacteria in our area and answer clinical practice outcome.The underlying reasons could be explained these hypotheses, the possible reason that underlies the mixed geneotypes are multiple infections of the same patient by two strains.The other is the occurring of mutation after one infection.The further genotypic analyses have to pursue to confirm these possible mechanisms.
In conclusion, prevalence of clarithromycin resistant H. pylori is geographically different among regions of Thailand.Physician should be concern of local resistance prevalence in each area and choose the most appropriate regimen for H. pylori eradication.Larger multi-center studies about mutation pattern are needed to test this hypothesis.