Mutational Analysis of Clarithromycin and Levofloxacin Resistance in Helicobacter pylori from Gastric Biopsy Specimens in a Tertiary Care Hospital in Dhaka, Bangladesh (original) (raw)
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Infection and Drug Resistance, 2021
Aims and Objectives: More than half of the world's population is infected with Helicobacter pylori, which can cause chronic gastritis. WHO has regarded clarithromycinresistant H. pylori as a high priority pathogen. Hence, accurate diagnosis and detection of clarithromycin-and levofloxacin-resistant H. pylori strains is essential for proper management of infection. The objective of this study was to develop and optimize multiplex quantitative PCR assay for detection of mutations associated with clarithromycin and levofloxacin resistance in H. pylori directly from the gastric biopsies. Materials and Methods: Specific primers and probes were designed to amplify ureA and mutations in 23S rRNA and gyrA genes. Singleplex and triplex qPCR assays were optimized and the assay's sensitivities and specificities were determined. The optimized multiplex qPCR assay was performed on 571 gastric biopsies. Results: In this study, 14.7% (84/571) of the gastric biopsies were positive for H. pylori by conventional methods and 23.8% (136/571) were positive by the ureA-qPCR with 96.4% sensitivity and 88.5% specificity, while the +LR and −LR were 8.72 and 0.04, respectively. The ureA-positive samples (n=136) were subjected to multiplex qPCR which detected A2142G and A2143G mutations in the 23S rRNA gene (20.6%, 28/136) conferring clarithromycin resistance and gyrA mutations N87K, N87I, D91N, and D91Y (11.8%, 16/136) leading to levofloxacin resistance. The sensitivity and specificity of qPCR of 23S rRNA gene were 100% and 98.7%, respectively, while 100% and 99.8% for qPCR of gyrA, respectively. Conclusion: The effectiveness of this qPCR is that it is sensitive in detecting low bacterial load and will help in timely detection of clarithromycin-and levofloxacin-resistant strains, especially in case of mixed infections. Since it is culture independent, it can inform clinicians about antibiotics to be included in the first-line therapy, thereby improving the management of H. pylori infection at a much greater pace.
African Journal of Biotechnology, 2011
Currently, a seven-day, triple-drug regimen has been recommended as one of the first-line therapies for Helicobacter pylori management in which clarithromycin is a key component. Development of clarithromycin resistance leads to the long term assessment of the efficacy of clarithromycin in the triple-drug regimen. The aim of this study was to rapidly and directly assess clarithromycin resistance point mutations on gastric biopsy specimens by using PCR-RFLP method. Biopsy samples were obtained over a 6-months period of 2009, from 200 dyspeptic patients referred to Shahrekord University of Medical Sciences, Iran. Initially, rapid urease test was performed and then DNA was isolated from each tissue and used for molecular analysis such as PCR (for H. pylori diagnostic) and PCR-RFLP (for Cla resistance determination). RUT and PCR results showed that 164 (82%) of the patients were H. pylori-positive. Resistance was evaluated in 164 samples by using enzymes BsaI and MboII. Thirty nine (39) (23/78%) clarithromycin-resistant strains were detected which were identified as 15 (9.15%) A2143G, 15 (9.15%) A2142G and 9 (5.49%) mix strains. The results showed that PCR-RFLP method had a high accuracy to detect A2142G and A2143G mutations associated with resistance to clarithromycin in the minimum possible time.
Journal of the Medical Association of Thailand = Chotmaihet thangphaet, 2012
Polymerase chain reaction (PCR) for detection of single nucleotide mutations in 23S rRNA gene of clarithromycin resistance Helicobacter pylori were investigated worldwide. In Thailand, the prevalence of these mutations had not been extensively investigated. The authors conducted a 32-months prospective study to estimate the prevalence of clarithromycin resistant Helicobacter pylori and to compare the sensitivity and specificity of hybridization real time PCR for 23S rRNA gene of Helicobacter pylori detection with the combination of rapid urease test, immunohistochemistry straining and Helicobacter pylori culture. A total of 200 patients with endoscopic examination with gastric biopsy were enrolled from January 2006 to September 2008. Eight gastric specimens were biopsied and performed rapid urease test, immunohistochemistry straining for Helicobacter pylori, Helicobacter pylori culture and hybridization real time PCR for 23S rRNA dectection, as well as single nucleotide mutation det...
Molecular detection of Helicobacter pylori resistance to clarithromycin isolated from Sabah
2014
Helicobacter pylori is a pathogenic bacterium that has been associated with peptic ulcer disease and cancers of human gastrointestinal tract. Currently, c1arithromycin has remained as one of the most powerful antibiotics used as first line treatment for H. pylori infection. In Malaysia, 2.14% of c1arithromycin resistance H. pylori cases were reported. The small percentage indicates the low incidence of c1arithromycin resistance H. pylori in this country. This study aims to detect A2142G and A2143G mutations of 235 rRNA gene in H. pylori isolated from 5abah and to determine prevalence of c1arithromycin resistance in 5abah isolates using polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). campylobacter-like organism (CLO) tested gastric biopsies were collected from Queen Elizabeth Hospital and subjected for DNA extraction. PCR amplification of 165 rRNA was performed to confirm the presence of H. pylori in the extracted DNA. Next, 235 rRNA gene was ampl...
International Journal of Molecular Sciences
Point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure, thereby reducing the efficacy of standard antibiotics in the treatment of Helicobacter pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, we performed a molecular genetic analysis of three target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. In addition, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses revealed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-c...
2020
Background: Clarithromycin resistant Helicobacter pylori (H. pylori) strains represent a worldwide health problem. These stains are usually carrying mutations within the 23S rRNA gene associated with clarithromycin resistance. This study aimed to detect H. pylori and clarithromycin resistant associated mutations from Sudanese patients with gastritis symptoms.Materials and Methods: Two hundred and eighty-eight gastric biopsies were collected using gastrointestinal endoscopy from patients with gastritis symptoms in different hospitals in Khartoum state. H. pylori was detected by PCR using primers targeting 16S rRNA and 23S rRNA. Then allele-specific PCR and DNA sequencing were used to screen for the presence of A2142G and A2143G point mutations.Results: Out of 288 samples, H. pylori was detected in 97 (33.7%) sample. Allele-specific PCR detected the variant A2142G in 9/97 (9.3%) sample, while A2143G mutation was not found in any sample. The DNA sequencing revealed the presence of muta...
Arquivos de Gastroenterologia, 2016
Background - Antimicrobial resistance is the major factor leading to eradication failure in H. pylori treatment. Molecular tests are useful to detect genetic mutations predictive of clarithromycin and fluoroquinolones resistance. Knowledge of the local prevalence rate of resistance is important to define the best recommended treatment. Objective - To assess the prevalence of primary resistance of H. pylori to clarithromycin and fluoroquinolones, using a molecular test, in a Southeastern urban Brazilian population. Methods - A total of 72 H. pylori seropositive patients [65% female, mean age 39 (19-73) years] never treated before for this infection were studied. All patients underwent gastroscopy in addition to antrum and corpus biopsies and molecular test GenoType HelicoDR (Hain Life Science, Germany) to detect H. pylori and point mutations in genes responsible for clarithromycin and fluoroquinolone resistance. The molecular procedure was divided into three steps: DNA extraction fro...
Gut Pathogens
Background Eradication of Helicobacter pylori provides the most effective treatment for gastroduodenal diseases caused by H. pylori infection. Clarithromycin, a member of the macrolide family, still remains the most important antibiotic used in H. pylori eradication treatment. But the increasing prevalence of clarithromycin resistant H. pylori strains due to point mutations in the V region of the 23S rRNA, poses a great threat in treating the ailing patients. So, we aimed for PCR-mediated rapid detection of the point mutation at 2143 position of 23S rRNA gene in H. pylori that is relevant to clarithromycin resistance from culture and simultaneously from biopsy specimens to avoid the empirical treatment. Results Newly developed PCR assay using DNA of pure culture detected point mutation in 23S rRNA gene in 21 (8.04%) of 261 clinical strains tested. The agar dilution method showed that all these 21 strains were resistant to clarithromycin indicating the perfect match of the PCR based ...
Journal of Digestive Diseases, 2010
OBJECTIVE: To determine the prevalence of primary clarithromycin resistance amongst Helicobacter pylori (H. pylori) strains in Malaysian patients with gastroduodenal diseases, by using restriction fragment length polymorphism (RFLP) in domain V of 23S rRNA.METHODS: Gastric biopsies were obtained from H. pylori positive patients undergoing gastroscopy. DNA extraction was followed by PCR amplification using the primers Hp23-1 and Hp23-2 flanking a region of 425bp within the bacterial 23S rRNA peptidyltranferase (Hp23S fragment). Analysis of the 23S rRNA gene mutations is based on the generation of restriction sites for two restriction enzymes: BbsI and BsaI, which correspond to the base substitutions characteristic of clarithromycin resistance from A to G at positions 2142 and 2143, respectively.RESULTS: Gastric biopsy samples were obtained from 107 patients. A fragment of size 425bp corresponding to that expected from amplification of domain V of 23S rRNA was PCR-amplified from only 105 samples. The amplicon was subsequently subjected to restriction by BbsI and BsaI. Only 1 sample (0.95%) had the BbsI mutation (base substitution at A2142G) and 2 samples (1.90%) the BsaI mutation (base substitution at A2143G). Thus 3 of 105 (2.9%) samples harbored clarithromycin resistant strains.CONCLUSION: In our experience, PCR-RFLP is a rapid and precise method to detect the resistance of H. pylori to clarithromycin. Using this method, a low prevalence of clarithromycin resistance was detected in our local Malaysian strains. This augurs well for the continued use of clarithromycin as a first line drug in the treatment and eradication of H. pylori infection.