Genotypic and Phenotypic Detection of Clarithromycin Resistance in Clinical Isolates of Helicobacter pylori with Point Mutations in23S rRNA: Molecular and Clinical Study

Abstract

Clarithromycin is the cornerstone of Helicobacter pylori (H. pylori) treatment until the emergence of clarithromycin resistant strains in high ratio. The study aimed to detect the presence of H. pylori in study patients using noninvasive test which include the conventional techniques and advanced molecular techniques using gastric tissue during oral endoscopy in patients with gastrointestinal diseases. Further to determine the phenotypic technique for detection of clarithromycin resistance H. pylori, to detect the point mutations in 23S rRNA which associate with clarithromycin-resistant H. pylori isolates in A2143G, A2144G genetic loci using restriction fragment length polymorphism technique and to evaluate the distribution of clarithromycin-resistant H. pylori strains and their association with genotypic markers, such as vacA gene and allelic variants of the vacA gene using conventional PCR. In total, 115 Al-Anbar Governorate patients strongly suspected of H. pylori infection were participated in the current study. They were referred to Ramadi Teaching Hospitals and Private Clinics in Al-Anbar Governorate for upper gastrointestinal endoscopy, were involved in the current study from January 2020 until February 2021. Invasive techniques including rapid urease testing (RUT) and gastric tissue culture in addition to non-invasive techniques including 14C-Urea breath test (14C-UBT), stool antigen test (SAT), CagA-IgG serology, and RT-PCR were performed to detect the H. pylori infection. The study result revealed the prevalence rate of H. pylori in study patients was ranged from 47.8 to 70.4 % using different methods. The positive results for each test were as follows: RT-PCR 81, (70.4 %) UBT 79 (68.7 %), SAT 77 (67 %), RUT 76 (66.1%), CagA-IgG 61 (53 %), and culture 55 (47.8 %). The 14CUBT showed the highest overall performance with 97.5 % sensitivity, 97 % specificity, and total accuracy of 97.3 % followed by SAT, RUT, Cag-IgG, and culture method. In total, 55 (47.8%) H. pylori isolates were cultured from the 115 biopsy specimens, among which 16 (29.1%), 38 (69.1%), 20 (36.4%), and 40 (72.7%) showed some degree of resistance to levofloxacin, clarithromycin, ciprofloxacin, and metronidazole, respectively. The frequency of A2144G and A2143G point mutations were 23 (60.5%) and 19 (50%), respectively. Also, it is required inantibiotic resistance surveillance and for identification and evaluation of effective H. pylori therapies. The vacA gene was detected in 55 (67.9%) of the 81 H. pylori isolates examind. The vacA s-region and m-region were found in all 55 vacA positive strains. Out of 55 isolates, 25/55 (45.5%) had the (s1) allele, 30/55 (54.5%) had (s2) allele, 21/55 (38.2%) had (m1) allele, and 34/55(61.8%) had (m2) allele. Allele combination: four vacA genotypes were detected, with s2m2 allele combination 29/55 (52.7%) being the most prevalent, followed by s1m1 20/55 (36.4%), s1m2 5/55 (9.1%), and s2m1 genotype found in only one strain. In terms of endoscopic aspects of H. pylori infected patients' mucosa, 17/18 (94.4%) patients with combined gastric and duodenal ulcers and 33/47 (70.2%) patients with gastritis tested positive for the vacA genotype). Patients with gastritis were more likely to have the vacA m2 allele (75.8%), followed by s2 (66.7%) and s1 (33.3%) while patients with combined gastric and duodenal ulcers were more likely to have the vacA s1, m1, and m2 alleles (70.6%), (64.7%), (35.3%), respectively. Thus, in H. pylori-infected patients with gastritis and combined gastric and duodenal ulcers (66.7 % and 64.7 %, (p = 0.01 and p=0.000), respectively), vacA s2m2 and s1m1 were significantly found. The s2 and s2m2 genotypes were detected more usually in patients aged <= 37 years (P value 0.000). Additionally the relationship between genotype s1 and genotype s1m1 and this age group was insignificant. In the current investigation, more virulent H. pylori strains (s1 and s1m1) were found to have a significant correlation with older patient’s ≥ 54. H. pylori strains that showing vacA positive were more resistance to clarithromycin (60% percent versus 19.2% for vacA-negative bacteria; P = 0.001), according to the distribution of H. pylori genotypes in relation to clarithromycin resistance. Furthermore, as expected, clarithromycin resistance was significantly linked to vacA s2m2 and s1m1 in H. pylori isolates (75.9% and 50%; p= 0.000 and p= 0.021, respectively). The study concluded the prevalence rate of H. pylori in Al-Anbar Governorate patients was ranged from 47.8 to 70.4 % using different invasive and non-invasive detection methods. Based on the accuracy of the studied methods for H. pylori detection, they can be arranged in order as follows: RT-PCR > UBT > SAT > RUT> CagA IgG > culture. The UBT may be recommended as first choice due to its higher performance compared to other methods. This study confirmed markedly the role of real-time PCR as a more sensitive, reliable and accurate than another diagnostic study method. High level of resistance to clarithromycin by study isolates is highly observed. PCR-RFLP technique was assumed the clarithromycin resistance due to point mutations efficiently. Other mechanisms of clarithromycin resistance due to presence of non-restricted DNA of study isolates are highly suggestive. In the study patients, the most common vacA genotype combinations were s2m2 and s1m1, which were mainly related with gastritis and combined gastric and duodenal ulcers. Further, s2m2 bacteria are more resistant to clarithromycin than s1m1 and s1m2 mosaic combinations. The study concludes that there is a strong correlation between smoking, alcohol consumption, and H. pylori infection that was considerably more prevalent in study males. Also, patients who drank alcohol and smoked had a greater risk of H. pylori infection than those who did not.