title: PCR-Based Rapid Identification System Using Bridged Nucleic Acids for Detection of Clarithromycin-Resistant Mycobacterium avium-M. intracellulare Complex Isolates.
abstract: The nontuberculous mycobacteria (NTM) cause miscellaneous disorders in humans, especially in the lungs, which present with a variety of radiological features. To date, knowledge of the pathogenic role of the Mycobacterium avium-intracellulare complex (MAC) in the human lung and the definitive criteria for initiating multidrug therapy are still lacking. However, there is little doubt that clarithromycin is the most efficacious drug among the various treatment regimens for lung NTM. In this study, with the use of a bridged nucleic acid (BNA) probe a detection system based on a real-time PCR (BNA-PCR) for the identification of the point mutations at position 2058 or 2059 in domain V of the 23S rRNA gene responsible for clarithromycin resistance was developed and has been assessed using MAC isolates from clinical samples. Out of 199 respiratory specimens, the drug susceptibility test demonstrated 12 strains resistant to clarithromycin, while the BNA-PCR showed 8 strains carrying the point mutation at position 2058 or 2059 of the 23S rRNA gene. This system revealed that there were mycobacterial strains resistant to clarithromycin which do not carry previously identified resistance genes. This paper documents a novel system for detecting clarithromycin-resistant strains and demonstrates that although these mutations are tacitly assumed to account for >90% of the reported resistant mutants, there is a significant fraction of resistant mutants that do not harbor these mutations. Therefore, unknown mechanisms affecting clarithromycin resistance remain to be elucidated.
authors.affiliation: Department of Respiratory Medicine, Saitama Medical University, Moroyama, Saitama, Japan Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada mycobacteriumtuberculosis@hotmail.com.
authors.forename: Takashi
authors.initials: T
authors.lastname: Hirama
authors.affiliation: Department of Respiratory Medicine, Saitama Medical University, Moroyama, Saitama, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Shiono
authors.affiliation: Department of Respiratory Medicine, Saitama Medical University, Moroyama, Saitama, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Egashira
authors.affiliation: The Microbiology Laboratory of Central Laboratory, Saitama Medical University, Moroyama, Saitama, Japan.
authors.forename: Etsuko
authors.initials: E
authors.lastname: Kishi
authors.affiliation: Department of Respiratory Medicine, Saitama Medical University, Moroyama, Saitama, Japan Clinical Laboratories for Cardiovascular Diseases, Jichi Medical University, Oomiya, Saitama, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Hagiwara
authors.affiliation: Department of Respiratory Medicine, Saitama Medical University, Moroyama, Saitama, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Nakamura
authors.affiliation: Department of Respiratory Medicine, Saitama Medical University, Moroyama, Saitama, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Kanazawa
authors.affiliation: Department of Respiratory Medicine, Saitama Medical University, Moroyama, Saitama, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Nagata
mesh_terms: D000328:Adult; D000368:Aged; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017291:Clarithromycin / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D005260:Female; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D009154:Mutation; D015269:Mycobacterium avium Complex / Q000145:classification / Q000187:drug effects* / Q000235:genetics*; D015270:Mycobacterium avium-intracellulare Infection / Q000175:diagnosis* / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology
chemical_list: D000900:Anti-Bacterial Agents; D012338:RNA, Ribosomal, 23S; D017291:Clarithromycin