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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 3  |  Page : 406-409
 

Detection of clarithromycin-resistant Helicobacter pylori by polymerase chain reaction using residual samples from rapid urease test


1 Department of Biomedical Laboratory Science, Dankook University College of Health Sciences, Cheonan 31116, Korea
2 Department of Dental Hygiene, Dankook University College of Health Sciences, Cheonan 31116, Korea

Date of Web Publication12-Oct-2017

Correspondence Address:
Ga-Yeon Kim
Department of Dental Hygiene, Dankook University College of Health Sciences, 119, Dandae-Ro, Dongnan-Gu, Cheonan-Si, Chungnam 31116
Korea
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_246

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 ~ Abstract 

Background: Approximately 50% of the world population is infected with Helicobacter pylori, which corresponds to a high infection rate. Furthermore, the incidence of antibiotic-resistant H. pylori has increased with the recent rise in use of antibiotics for H. pylori elimination, suggesting growing treatment failures. Aim: The study was aimed to assess the use of residual samples from rapid urease test (RUT) for biomolecular testing as an effective and accurate method to detect antibiotic-resistant H. pylori. Settings and Design: This study was a retrospective study performed using data obtained from medical records of previously isolated H. pylori strains. Materials and Methods: RUT was conducted for 5440 biopsy samples from individuals who underwent health examination in South Korea. Subsequently, 469 RUT residual samples were randomly selected and subjected to polymerase chain reaction (PCR) to detect antibiotic-resistant H. pylori. Statistical Analysis Used: The Chi-square test was used to analyse categorical data. P < 0.05 was considered statistically significant. Results: The results showed a concordance between the results of PCR and conventional RUT in 450 of 469 samples, suggesting that the H. pylori PCR test is a time- and cost-effective detection method. Conclusions: This study demonstrated that PCR test can aid physicians to prescribe the appropriate antibiotics at the time of diagnosis, thus preventing the reduction in H. pylori eradication due to antibiotic resistance, averting progression to serious diseases and increasing the treatment success rate.


Keywords: Clarithromycin, drug resistance, Helicobacter pylori, polymerase chain reaction, rapid urease test


How to cite this article:
Jeon JS, Kim JK, Kim GY. Detection of clarithromycin-resistant Helicobacter pylori by polymerase chain reaction using residual samples from rapid urease test. Indian J Med Microbiol 2017;35:406-9

How to cite this URL:
Jeon JS, Kim JK, Kim GY. Detection of clarithromycin-resistant Helicobacter pylori by polymerase chain reaction using residual samples from rapid urease test. Indian J Med Microbiol [serial online] 2017 [cited 2018 Jul 18];35:406-9. Available from: http://www.ijmm.org/text.asp?2017/35/3/406/216626



 ~ Introduction Top


The incidence of antibiotic-resistant Helicobacter pylori strains has increased with the recent rise in antibiotic use for H. pylori elimination, suggesting growing treatment failures.[1],[2],[3] In the present study, a biomolecular test was performed using residual samples from rapid urease test (RUT), to evaluate its potential use as an effective and precise method to detect antibiotic-resistant H. pylori strains. This method will allow physicians to select the appropriate treatment regimen upon detecting antibiotic resistance, without subjecting patients to additional invasive procedures.


 ~ Materials and Methods Top


Sample collection

This study was approved by the corresponding Institutional Review Board. This study was a retrospective study using data obtained from medical records of previously isolated H. pylori strains.

The study involved 5440 samples from individuals who underwent endoscopy examination and RUT from November 2011 to November 2015. A total of 469 samples (413 positive samples, 56 negative samples) were randomly selected from the 5440 samples for polymerase chain reaction (PCR) assessment.

Polymerase chain reaction assay using rapid urease test residual samples

PCR test was performed using RUT residual samples that were obtained from endoscopy examination of patients who underwent health examination. The primers and probes for PCR are specific for the 5′-nontranslated region of the H. pylori genome.

Following collection, specimens were subjected to DNA extraction on the day of collection or stored at 4°C for nucleic acid extraction on the next day. For H. pylori PCR specimens, nucleic acid extraction was performed using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, in a QIAcube automated DNA extraction system (Qiagen). H. pylori PCR was performed using 40 μL of eluate that passed through the QIAamp spin column (Qiagen).

The extracted DNA was stored at −80°C until use for a dual priming oligonucleotide-based multiplex PCR using the Seeplex® H. pylori-ClaR ACE Detection kit (Seegene, Seoul, Korea) based on the highly conserved 5′ noncoding region of the H. pylori genome, according to the manufacturer's protocol. A diagnostic kit that can detect H. pylori and mutations at A2142 and A2143, known mutations associated with antibiotic resistance, was used in the PCR (Seegene, Seoul, Korea).

Data are presented as the median and range. The Chi-square test was used to analyse the categorical data. P < 0.05 was considered statistically significant.


 ~ Results Top


The present study involved a total of 5440 samples from 3562 men (65.5%) and 1878 women (34.5%), which were submitted for RUT from November 2011 to November 2015. A total of 2351 samples (1533 men and 818 women) were RUT-positive with a detection rate of 43.2% (43.0% and 43.6% for men and women, respectively) [Table 1]. The samples were also analysed by age of the patients [Table 2]; the mean age of the subjects was 55.5 years, and the number of subjects in their 50s was the highest with 1478 subjects, followed by 1196 subjects in their 60s and 1012 subjects in their 40s. The mean age of RUT-positive patients was 53.1 years, and the detection rate in those in their thirties was the highest at 54.9%, followed by 54.2% and 50.8% in those in their 20s and 40s, respectively [Figure 1]. When both sex and age were factored in, the detection rate was the highest in men in their 30s with 39.1% and women in their 20s with 19.8%.
Table 1: Rapid urease test detection rate by gender

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Table 2: Rapid urease test detection rate by age

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Figure 1: Positive detection rate of rapid urease test by age of the patients

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From the 5440 samples submitted for RUT, 469 (413 RUT-positive and 56 RUT-negative samples) were randomly selected and tested by PCR. The PCR results showed that 418 of 469 samples (89.1%) were positive for H. pylori. Among the positive samples confirmed by PCR, 108 (25.8%) samples had the A2142G and/or A2143G point mutations associated with antibiotic resistance in H. pylori, consisting of 16 (3.8%) samples with an A2142G mutation, 94 (22.5%) samples with an A2143G mutation and 2 (0.5%) samples with both A2142G and A2143G mutations [Table 3] and [Figure 2].
Table 3: Frequency of mutations associated with Helicobacter pylori resistance to clarithromycin

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Figure 2: Helicobacter pylori-specific sequencing analysis for a simultaneous detection of A2142G and A2143G mutations. GTGGAGGTGAAAATTCCTCCTACCCGCGGCAAGACGGRRAGAC CCCGTGGACCT. *R: A + G

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Finally, the correlation between the RUT and PCR results was analysed [Table 3]. Out of the 469 randomly selected samples, 450 had a consistent result for the two tests, with a concordance rate of 95.9%. However, 19 samples showed contradicting results for the two tests; these samples consisted of seven samples with RUT-positive and PCR-negative results, and 12 samples with PCR-positive and RUT-negative results. Comparing the results of the RUT and PCR, the positive detection rate was 88.1% and 89.1% by RUT and PCR, respectively.


 ~ Discussion Top


A triple therapy of a proton pump inhibitor in combination with two antibiotics (amoxicillin and clarithromycin or metronidazole and clarithromycin), depending on the presence or absence of antibiotic resistance and history of antibiotic use, has been used as the first-line treatment for H. pylori eradication in infected patients.[4] Consequently, an increase in antibiotic-resistant H. pylori strains associated with antibiotic use has been reported.[5] To address the emergence of antibiotic resistance, European guidelines recommend bacterial culturing before the initiation of antibiotic treatment, highlighting the importance of early assessment for antibiotic resistance.[6] South Korea has a high rate of H. pylori infection, and the rate of H. pylori reinfection after eradication is higher than that in Western countries.[7] Chronic gastritis caused by H. pylori infection is a major factor in the development of gastric cancer.[8] In addition, the prevalence of H. pylori infection increases with age,[4] and once infected, H. pylori infection is difficult to resolve naturally.[9] Therefore, this study was aimed to assess the utility of a combination of RUT and PCR testing as a rapid and appropriate diagnostic method for H. pylori to address antibiotic resistance.

The results of RUT conducted between 2011 and 2015 showed that the detection rate of H. pylori was 43.2% in 5,440 samples analysed in this study; this result was in agreement with a previous report that >50% of the world's population is infected with H. pylori.[10] The prevalence of H. pylori infection is higher in developing countries than in developed countries,[11],[12] and transmissions mainly occur through person-to-person contact (e.g., mother to child, spouse to spouse), sanitation facilities or contaminated water and food.[13] Geographical location, ethnicity, age and socioeconomic factors also contribute to H. pylori epidemic.[4] Therefore, appropriate countermeasures against H. pylori infection are needed.

Results of this study showed that among the RUT-positive samples, 1,533 and 818 samples were from men and women, respectively, with a gender ratio of 1.87:1. This finding indicated that men were more likely to be RUT-positive than women although the difference was not statistically significant (P = 0.367). However, when the results were analysed by age, the positive detection rate was the highest in those in their thirties (54.9%). Results of previous studies indicated that the detection rate increases with age;[9],[13],[14] however, the present study showed that the detection rate decreased after the age of thirties. Because these results were based on data obtained from a single hospital in Cheonan area, South Korea, further studies are needed to investigate the association between the detection rate and geographical characteristics or other factors.

Mutations in the 23S rRNA gene of H. pylori associated with resistance to clarithromycin were detected in 108 patients by H. pylori PCR. The A2143G point mutation was detected in 94 patients, while the A2142G point mutation was detected in 16 patients and concurrent A2142G/A2143G mutations were detected in two patients. According to the genotype, clarithromycin-resistant H. pylori strains were detected in 23% of the 469 subjects. A recent study reported that the rate of clarithromycin resistance in South Korea has increased to 37%.[15] Although result of this study showed a lower clarithromycin-resistance rate than that reported in a previous study, the result should not be extrapolated because it was solely derived from the genotype and the analysis was performed at a single institution in Cheonan. Because the present study was performed retrospectively, the subject selection was unplanned, and there may be variations in test and experimental procedures. However, the finding that one in five individuals had antibiotic-resistant H. pylori should not be overlooked. Interestingly, A2143G and A2142G mutations were concurrently detected in two patients in the present study. A previous study postulated that such cases may represent infections with hetero-resistance strains, in which the wild-type and mutant strains may coexist in an individual.[9] A previous study reported that the majority of H. pylori-infected patients were colonised by a mixed population of different H. pylori strains, such as the wild-type and mutant strains or susceptible and resistant strains.[16] Because mixed H. pylori infections with different strains may lower the eradication rate,[9] antibiotics should be used with caution in such cases.

Comparing the conventional RUT and H. pylori PCR results, 450 of 469 samples showed the same result, with a concordance rate of 95.4%. The detection rate was 88.1% and 89.1% by RUT and H. pylori PCR, respectively, suggesting that RUT residual samples can be used for PCR with sufficient sensitivity. In fact, RUT result is dependent on the bacterial load[17] although RUT has a relatively high sensitivity of >95%.[4],[18] RUT can only yield a reliable result if the number of bacteria present is at least 1 × 105 copies.[19] Thus, in cases of samples with a low bacterial load, more sensitive test methods are needed to detect H. pylori infection[11] and at least two tests should be performed to increase the reliability of the detection.[20] Therefore, RUT alone is insufficient to detect H. pylori; however, a combination of RUT and H. pylori PCR using RUT residual samples can produce reliable results, in addition to permitting the detection of antibiotic resistance or mixed infections. Results of this study indicated that the combination of the two tests can increase the chance for H. pylori eradication.


 ~ Conclusions Top


H. pylori PCR is a highly useful test method with a high detection rate that is not inferior to RUT. In addition, H. pylori PCR permits the detection of clarithromycin-resistant bacteria. This test method has the advantage of minimising inconvenient invasive procedures and cost associated with follow-up endoscopy for detecting resistant bacteria when antibiotics are ineffective or marginally effective to eradicate H. pylori. Therefore, a combination of RUT and PCR is a valuable diagnostic method. A faster identification for antibiotic resistance at the genetic level than that achieved with the conventional test method described above will enable physicians to determine the most effective antibiotics for individual patient, thereby increasing the treatment success rate through a more rapid administration of such antibiotics, leading to an overall improvement of the patient health.

Financial support and sponsorship

This work was supported by the Dankook University in 2017 under Grant R201700373.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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Malfertheiner P, Megraud F, O'Morain CA, Atherton J, Axon AT, Bazzoli F, et al. Management of Helicobacter pylori infection – The Maastricht IV/florence consensus report. Gut 2012;61:646-64.  Back to cited text no. 1
    
2.
Gatta L, Vakil N, Vaira D, Scarpignato C. Global eradication rates for Helicobacter pylori infection: Systematic review and meta-analysis of sequential therapy. BMJ 2013;347:f4587.  Back to cited text no. 2
    
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Hartgrink HH, Jansen EP, van Grieken NC, van de Velde CJ. Gastric cancer. Lancet 2009;374:477-90.  Back to cited text no. 3
    
4.
Hunt RH, Xiao SD, Megraud F, Leon-Barua R, Bazzoli F, van der Merwe S, et al. Helicobacter pylori in developing countries. World Gastroenterology Organisation Global Guideline. J Gastrointestin Liver Dis 2011;20:299-304.  Back to cited text no. 4
    
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Ubhayawardana NL, Weerasekera MM, Weerasekera D, Samarasinghe K, Gunasekera CP, Fernando N, et al. Detection of clarithromycin-resistant Helicobacter pylori strains in a dyspeptic patient population in Sri Lanka by polymerase chain reaction-restriction fragment length polymorphism. Indian J Med Microbiol 2015;33:374-7.  Back to cited text no. 5
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Suzuki H, Nishizawa T, Hibi T. Helicobacter pylori eradication therapy. Future Microbiol 2010;5:639-48.  Back to cited text no. 6
    
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Kim MS, Kim N, Kim SE, Jo HJ, Shin CM, Lee SH, et al. Long-term follow-up Helicobacter pylori reinfection rate and its associated factors in Korea. Helicobacter 2013;18:135-42.  Back to cited text no. 7
    
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Kim N, Park RY, Cho SI, Lim SH, Lee KH, Lee W, et al. Helicobacter pylori infection and development of gastric cancer in Korea: Long-term follow-up. J Clin Gastroenterol 2008;42:448-54.  Back to cited text no. 8
    
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Cho AR, Lee MK. A comparison analysis on the diagnosis of Helicobacter pylori infection and the detection of clarithromycin resistance according to biopsy sites. Korean J Lab Med 2010;30:381-7.  Back to cited text no. 9
    
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Lacy BE, Rosemore J. Helicobacter pylori: Ulcers and more: The beginning of an era. J Nutr 2001;131:2789S-93S.  Back to cited text no. 10
    
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Go MF. Review article: Natural history and epidemiology of Helicobacter pylori infection. Aliment Pharmacol Ther 2002;16 Suppl 1:3-15.  Back to cited text no. 11
    
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Brown LM. Helicobacter pylori: Epidemiology and routes of transmission. Epidemiol Rev 2000;22:283-97.  Back to cited text no. 12
    
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Salih BA. Helicobacter pylori infection in developing countries: The burden for how long? Saudi J Gastroenterol 2009;15:201-7.  Back to cited text no. 13
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Wu HC, Tuo BG, Wu WM, Gao Y, Xu QQ, Zhao K, et al. Prevalence of peptic ulcer in dyspeptic patients and the influence of age, sex, and Helicobacter pylori infection. Dig Dis Sci 2008;53:2650-6.  Back to cited text no. 14
    
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Lee JW, Kim N, Kim JM, Nam RH, Chang H, Kim JY, et al. Prevalence of primary and secondary antimicrobial resistance of Helicobacter pylori in Korea from 2003 through 2012. Helicobacter 2013;18:206-14.  Back to cited text no. 15
    
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Kim JW, Kim JG, Chae SL, Cha YJ, Park SM. High prevalence of multiple strain colonization of Helicobacter pylori in Korean patients: DNA diversity among clinical isolates from the gastric corpus, antrum and duodenum. Korean J Intern Med 2004;19:1-9.  Back to cited text no. 16
    
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Zhou L, Zhao F, Hu B, Fang Y, Miao Y, Huang Y, et al. Acreative Helicobacter pylori diagnosis scheme based on multiple genetic analysis system: Qualification and quantitation. Helicobacter 2015;20:343-52.  Back to cited text no. 17
    
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Ou Z, Xiong L, Li DY, Geng L, Li L, Chen P, et al. Evaluation of a new fluorescence quantitative PCR test for diagnosing Helicobacter pylori infection in children. BMC Gastroenterol 2013;13:7.  Back to cited text no. 20
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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