Stool-Based Diagnosis of Helicobacter Pylori, Detection of Virulence Genes and Molecular Analysis of Clarithromycin Resistance in Patients With Gastrointestinal Complaints

Öz

Aim: This study aimed to detect Helicobacter pylori DNA, major virulence genes (cagA, vacA s1/s2, iceA1/A2), and clarithromycin resistance-associated mutations directly from stool samples of patients presenting with gastrointestinal complaints using molecular techniques. Material and Method: Stool samples from 60 patients collected between April and July 2023 were included in the study. Following DNA extraction, conventional PCR was performed to detect H.pylori and its virulence genes (cagA, vacA s1/s2, iceA1/A2). Real-time PCR (qPCR) was used to identify mutations associated with clarithromycin resistance. Results: H.pylori DNA was detected in 28.3% (n=17) of the samples. Among positive cases, the cagA gene was identified in 17.6% and the vacA s1 subtype in 23.5%. The vacA s2 and iceA1/ A2 genes were not detected. In qPCR analysis, clarithromycinresistance mutations were found in 68.4% of the H.pylori–positive samples (n=19). Conclusion: Molecular stool-based testing appears to be a relevant non-invasive approach for both diagnosis and antibiotic resistance profiling of H.pylori. However, technical limitations such as low DNA yield, PCR inhibitors, and small sample size may have reduced sensitivity. Non-invasive molecular strategies may serve as practical and effective alternatives for treatment stratification, particularly in settings where endoscopic procedures are not readily accessible.

Anahtar Kelimeler

• Helicobacter pylori
• clarithromycin resistance
• stool
• molecular diagnosis
• cagA
• vacA

Kaynakça

1. 1. Ailloud F, Didelot X, Woltemate S, Pfaffinger G, Overmann J, Bader RC, et al. Niche-specific adaptation, intragastric migrations and selective sweeps shape within-host evolution of Helicobacter pylori. Nat Commun. 2019;10:2273. https://doi.org/10.1038/s41467-019-10050-1
2. 2. Sugano K, Tack J, Kuipers EJ, Graham DY, El-Omar EM, Miura S, et al. Kyoto global consensus report on Helicobacter pylori gastritis. Gut. 2015;64(9):1353–1367. https://doi.org/10.1136/gutjnl-2015-309252
3. 3. McNicholl AG, Bordin DS, Lucendo A, Fadeenko G, Fernandez MC, Voynovan I, et al. Combination of bismuth and standard triple therapy eradicates Helicobacter pylori infection in more than 90% of patients. Clin Gastroenterol Hepatol. 2020;18(1):89–98. https://doi.org/10.1016/j.cgh.2019.03.048
4. 4. Sholeh M, Khoshnood S, Azimi T, Mohamadi J, Kaviar VH, Hashemian M, et al. The prevalence of clarithromycin-resistant Helicobacter pylori isolates: a systematic review and meta-analysis. Peer J. 2023;11:e15121. https://doi.org/10.7717/peerj.15121
5. 5. Keskin M, Yavuz A. A novel rapid and accurate method for detecting Helicobacter pylori: the modified antigen test. Eur Rev Med Pharmacol Sci. 2022;26(4):1148–1155. https://doi.org/10.26355/eurrev_202202_28106
6. 6. Qiu E, Li Z, Han S. Methods for detection of Helicobacter pylori from stool sample: current options and developments. Braz J Microbiol. 2021;52(4):2057–2062. https://doi.org/10.1007/s42770-021-00589-x
7. 7. Van den Poel B, Gils S, Micalessi I, Carton S, Christiaens P, Cuyle PJ, et al. Molecular detection of Helicobacter pylori and clarithromycin resistance in gastric biopsies: a prospective evaluation of RIDA® GENE Helicobacter pylori assay. Acta Clinica Belgica. 2021;76(3):177–183. https://doi.org/10.1080/17843286.2019.1685741
8. 8. Ren X, Shi Y, Suo B, Yao X, Lu H, Li C, et al. Individualized diagnosis and eradication therapy for Helicobacter pylori infection based on gene detection of clarithromycin resistance in stool specimens: a systematic review and meta‐analysis. Helicobacter. 2023;28(3):e12958. https://doi.org/10.1111/hel.12958

9. 9. Graham DY, Moss SF. Antimicrobial susceptibility testing for Helicobacter pylori is now widely available: when, how, why. Am J Gastroenterol. 2022;117(4):524–528. https://doi.org/10.14309/ajg.0000000000001659
10. 10. Rimbara E, Fischbach LA, Graham DY. Optimal therapy for Helicobacter pylori infections. Nat Rev Gastroenterol Hepatol. 2011;8(2):79–88. https://doi.org/10.1038/nrgastro.2010.210
11. 11. Marrero Rolon R, Cunningham SA, Mandrekar JN, Polo ET, Patel R. Clinical evaluation of a real-time PCR assay for simultaneous detection of Helicobacter pylori and genotypic markers of clarithromycin resistance directly from stool. J Clin Microbiol. 2021;59(5):10–1128. https://doi.org/10.1128/JCM.03040-20
12. 12. Beckman E, Saracino I, Fiorini G, Clark C, Slepnev V, Patel D, et al. A novel stool PCR test for Helicobacter pylori may predict clarithromycin resistance and eradication of infection at a high rate. J Clin Microbiol. 2017;55(8):2400–2405. https://doi.org/10.1128/JCM.00506-17
13. 13. Kakiuchi T, Okuda M, Matsuo M, Fujimoto K. Smart gene™ as an effective non‐invasive point‐of‐care test to detect Helicobacter pylori clarithromycin‐resistant mutation. J Gastroenterol Hepatol. 2022;37(9):1719–1725. https://doi.org/10.1111/jgh.15887
14. 14. Oktem-Okullu S, Cekic-Kipritci Z, Kilic E, Seymen N, Mansur-Ozen N, Sezerman U, et al. Analysis of correlation between the seven important Helicobacter pylori (H.pylori) virulence factors and drug resistance in patients with gastritis. Gastroenterol Res Pract. 2020;2020:3956838. https://doi.org/10.1155/2020/3956838
15. 15. Lin TF, Hsu PI. Second-line rescue treatment of Helicobacter pylori infection: where are we now?. World J Gastoentrol. 2018;24(40):4548. https://doi.org/10.3748/wjg.v24.i40.4548
16. 16. Thung I, Aramin H, Vavinskaya V, Gupta S, Park JY, Crowe SE, et al. The global emergence of Helicobacter pylori antibiotic resistance. Alimentary pharmacology & therapeutics. 2016;43(4):514–533. https://doi.org/10.1111/apt.13497
17. 17. Şen N, Yılmaz Ö, Şimşek İ, Küpelioğlu AA, Ellidokuz, H. Detection of Helicobacter pylori DNA by a simple stool PCR method in adult dyspeptic patients. Helicobacter. 2005;10(4):353–359. https://doi.org/10.1111/j.1523-5378.2005.00326.x
18. 18. Smith SI, Fowora MA, Lesi OA, Agbebaku E, Odeigah P, Abdulkareem FB, et al. Application of stool-PCR for the diagnosis of Helicobacter pylori from stool in Nigeria-a pilot study. Springerplus. 2012;1:1–5. https://doi.org/10.1186/2193-1801-1-78
19. 19. Fan C-J, Li Z, Zhai L-L, Wang H, Zhao X-L, Xie D-L, et al. Diagnostic accuracy of a real-time PCR assay for detection of Helicobacter pylori and resistance to clarithromycin and levofloxacin directly from stool. Eur Rev Med Pharmacol Sci. 2024;28(12):3836–3840. https://doi.org/10.26355/eurrev_202406_36460
20. 20. Moss SF, Dang LP, Chua D, Sobrado J, Zhou Y, Graham, DY. Comparable results of Helicobacter pylori antibiotic resistance testing of stools vs gastric biopsies using next-generation sequencing. Gastroenterology. 2022;162(7):2095–2097. https://doi.org/10.1053/j.gastro.2022.02.027
21. 21. Pichon M, Pichard B, Barrioz T, Plouzeau C, Croquet V, Fotsing G, et al. Diagnostic accuracy of a noninvasive test for detection of Helicobacter pylori and resistance to clarithromycin in stool by the Amplidiag H.pylori+ ClariR real-time PCR assay. J Clin Microbiol. 2020;58(4):10–1128. https://doi.org/10.1128/JCM.01787-19
22. 22. Ren X, Shi Y, Suo B, Yao X, Lu H, Li C, et al. Individualized diagnosis and eradication therapy for Helicobacter pylori infection based on gene detection of clarithromycin resistance in stool specimens: a systematic review and meta‐analysis. Helicobacter. 2023;28(3):e12958. https://doi.org/10.1111/hel.12958
23. 23. Compaore TR, Traore K, Compaore NI, Traore L, Zida S, Soubeiga ST, et al. Helicobacter pylori virulence genes cagA, babA2, and vacA detection in dyspeptic patients from Burkina Faso. Am J Mol Biol. 2023;13(3):141–155. https://doi.org/10.4236/ajmb.2023.133010
24. 24. Sicinschi LA, Correa P, Bravo LE, Schneider BG. Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive, one-step polymerase chain reaction in stool samples from children. J Microbiol Methods. 2003;52(2):197–207. https://doi.org/10.1016/S0167-7012(02)00158-6