Observational Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 7, 2016; 22(25): 5822-5830
Published online Jul 7, 2016. doi: 10.3748/wjg.v22.i25.5822
Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori
Xiao-Feng Luo, Jian-Hua Jiao, Wen-Yue Zhang, Han-Ming Pu, Bao-Jin Qu, Bing-Ya Yang, Min Hou, Min-Jun Ji
Xiao-Feng Luo, Wen-Yue Zhang, Bing-Ya Yang, Min Hou, Min-Jun Ji, Department of Pathogen Biology, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China
Jian-Hua Jiao, Bao-Jin Qu, Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Han-Ming Pu, Department of Gastroenterology, Nanjing Qixia District Hospital, Nanjing 210046, Jiangsu Province, China
Author contributions: Luo XF and Jiao JH contributed equally to this work; Luo XF and Zhang WY performed the nested-ASP-PCR experiments and analyzed the data; Pu HM and Qu BJ collected the samples; Hou M contributed new reagents and materials; Yang BY performed the bacterial culturing and DNA extraction; Jiao JH and Ji MJ conceived and designed the research; Luo XF, Jiao JH and Ji MJ drafted the manuscript; all authors read and approved the final version of the paper.
Institutional review board statement: The study was reviewed and approved by the Nanjing Medical University Institutional Review Board (No. (2015)-246).
Informed consent statement: All patients in this study provided a signed informed consent statement.
Conflict-of-interest statement: Each author certifies that he or she has no commercial associations that might pose a conflict of interest in connection with the submitted article.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Min-Jun Ji, Department of Pathogen Biology, Nanjing Medical University, No. 101 Longmian Dadao, Nanjing 211166, Jiangsu Province, China. jiminjun@njmu.edu.cn
Telephone: +86-25-86869400
Received: December 21, 2015
Peer-review started: December 31, 2015
First decision: March 22, 2016
Revised: April 8, 2016
Accepted: April 20, 2016
Article in press: April 20, 2016
Published online: July 7, 2016

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR).

METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion.

RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin.

CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Keywords: Helicobacter pylori, Nested-allele specific primer-polymerase chain reaction, Rapid urease test, Clarithromycin resistance, Drug sensitivity testing

Core tip: In recent years, antibiotic resistance in Helicobacter pylori (H. pylori) has become a global problem, especially the resistances towards metronidazole, clarithromycin and amoxicillin. To combat this growing problem, it is important to determine antimicrobial resistance of a patient’s infection before treatment. Normally, the detection of clarithromycin resistance is based mainly on phenotypic methods performed after culturing. However, bacterial culture has many inherent disadvantages. In the present study, the nested-allele specific primer-polymerase chain reaction was established to detect the different mutations at positions 2142, 2143 and 2144 in the 23SrRNA gene of H. pylori, which can be completed within several hours. This method is expected to contribute towards improving the efficacy of H. pylori eradication therapy.