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1654. Evaluation of a rapid detection method of clarithromycin resistance genes in Mycobacterium avium using the Amplification Refractory Mutation System-Loop-Mediated Isothermal Amplification method


 
 
 Clarithromycin (CLR) is the key drug in multidrug therapy for Mycobacterium avium complex (MAC) diseases and the only drug for which drug susceptibility is correlated with a clinical response in these diseases. In the case of CLR-resistant MAC, a point mutation is present at either position 2058 or 2059 of the peptidyl transferase active center in the domain V region of 23S rRNA at the macrolide binding site. Using conventional investigation, we clarified the correlation between drug susceptibility testing and mutation of drug resistance genes. In this study, we adapted a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene in M. avium isolates (Figure 1). Furthermore, we evaluated the usefulness as point-of-care testing (POCT) technology using clinical isolates.
 Figure 1. The designs of CLR resistance A2058G mutant-type mismatch primers used for the ARMS-LAMP assay. a) A strand-displacing DNA polymerase extends the DNA from FIP while separating from the DNA chain. The primer F3 binds to its complementary region on the DNA to displace the newly synthesized DNA. An analogous reaction is performed by BIP and B3. α (α = A, wild type; G, A2058G) and β (β = A, wild type; C, A2058G) are indicated by the point mutation at position 2058 of the 23S rRNA gene. The bold area indicates the mismatched base C (cytosine). b) The synthesized DNA self-anneals because of the complementary region at both ends and forms ‘dumbbell’ structures. c) After repeated rounds, a complementary region on the same chain is amplified.
 
 
 
 
 Primers for ARMS-LAMP were designed using PrimerExplorerV5 software based on the nucleotide sequence data for 23S rRNA in M. avium strain 104 (Figure 2). Using the minimum inhibitory concentration of CLR, drug susceptibility was determined for 18 clinical M. avium isolates. Of these, eight CLR-susceptible and 10 CLR-resistant strains were analyzed by sequencing the 23S rRNA gene and ARMS-LAMP.
 Figure 2. Alignment of the nucleotide sequences including the domain V region of 23S rRNA at the macrolide binding site. The constructed LAMP primer sets are shown in solid boxes (forward primers, F1-3) and dashed boxes (backward primers, B1-3). The bold area indicates the point mutation at position 2058 or 2059 of the 23S rRNA gene.
 
 
 
 
 Sequence analysis revealed that all eight CLR-sensitive strains tested were wild type, whereas all 10 CLR-resistant strains were mutants. Using ARMS-LAMP, no amplification with the mutant-type mismatch primer sets (MTPS) was observed in the eight wild-type strains, but amplification was observed with MTPS in the 10 mutant strains (Table 1).
 Table 1. MICs of CLR and results of ARMS–LAMP using Mycobacterium avium isolates.
 
 
 
 
 The developed rapid detection method for the CLR resistance gene using ARMS-LAMP can determine drug resistance in a few hours without the need for special equipment. ARMS-LAMP may be a new clinically beneficial POCT technology for examination that is novel and extremely practical.
 
 
 
 All Authors: No reported disclosures
