Hasyanee Binmaeil1, Alfizah Hanafiah1,2, Isa Mohamed Rose3, Raja Affendi Raja Ali2,4. 1. Department of Medical Microbiology and Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, Malaysia. 2. GUT Research Group, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, Malaysia. 3. Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, Malaysia. 4. Department of Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, Malaysia.
Abstract
AIMS AND OBJECTIVES: More than half of the world's population is infected with Helicobacter pylori, which can cause chronic gastritis. WHO has regarded clarithromycin-resistant 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.
AIMS AND OBJECTIVES: More than half of the world's population is infected with Helicobacter pylori, which can cause chronic gastritis. WHO has regarded clarithromycin-resistant 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.
Authors: J F Tomb; O White; A R Kerlavage; R A Clayton; G G Sutton; R D Fleischmann; K A Ketchum; H P Klenk; S Gill; B A Dougherty; K Nelson; J Quackenbush; L Zhou; E F Kirkness; S Peterson; B Loftus; D Richardson; R Dodson; H G Khalak; A Glodek; K McKenney; L M Fitzegerald; N Lee; M D Adams; E K Hickey; D E Berg; J D Gocayne; T R Utterback; J D Peterson; J M Kelley; M D Cotton; J M Weidman; C Fujii; C Bowman; L Watthey; E Wallin; W S Hayes; M Borodovsky; P D Karp; H O Smith; C M Fraser; J C Venter Journal: Nature Date: 1997-08-07 Impact factor: 49.962
Authors: Antonio Cuadrado-Lavín; J Ramón Salcines-Caviedes; Miguel F Carrascosa; Purificación Mellado; Idoia Monteagudo; Javier Llorca; Marta Cobo; M Rosario Campos; Blanca Ayestarán; Antonio Fernández-Pousa; Elena González-Colominas Journal: J Antimicrob Chemother Date: 2011-09-29 Impact factor: 5.790
Authors: V de Francesco; M Margiotta; A Zullo; C Hassan; N D Valle; O Burattini; U Cea; G Stoppino; A Amoruso; F Stella; S Morini; C Panella; E Ierardi Journal: Aliment Pharmacol Ther Date: 2006-02-01 Impact factor: 8.171