Loop-Mediated Isothermal Amplification Assay for Identifying Neisseria gonorrhoeae Nonmosaic penA- Targeting Strains Potentially Eradicable by Cefixime

Abstract

Treatment regimens for gonorrhea have limited efficacy worldwide due to the rapid spread of antimicrobial resistance. Cefixime (CFM) is currently not recommended as a first-line treatment for gonorrhea due to the increasing number of resistant strains worldwide. Nonetheless, Neisseria gonorrhoeae strains can be eradicated by CFM at a 400 mg/day dose, provided that the strains are CFM responsive (MIC ≤ 0.064 mg/L). To develop a nonculture test for predicting the CFM responsiveness of N. gonorrhoeae strains, we developed an assay to detect N. gonorrhoeae nonmosaic penA using loop-mediated isothermal amplification (LAMP). To avoid false-positive reactions with commensal Neisseria spp. penA, we amplified specific regions of the N. gonorrhoeae penA (NG-penA-LAMP1) and also the nonmosaic N. gonorrhoeae penA (NG-penA-LAMP3). This assay was validated using isolated N. gonorrhoeae (n = 204) and Neisseria spp. (n = 95) strains. Clinical specimens (n = 95) with confirmed positivity in both culture and real-time PCR were evaluated to validate the system. The combination of the previously described NG-penA-LAMP1 and our new NG-penA-LAMP3 assays had high sensitivity (100%) and specificity (100%) for identifying N. gonorrhoeae carrying the nonmosaic type. To determine whether CFM could be applicable for gonorrhea treatment without culture testing, we developed a LAMP assay that targets penA allele-specific nonmosaic types for use as one of the tools for point-of-care testing of antimicrobial resistance. IMPORTANCE Neisseria gonorrhoeae is among the hot topics of "resistance guided therapy," one of the top 5 urgent antimicrobial threats according to the Centers for Disease Control and Prevention (CDC). There is a need either to develop new agents or to make effective use of existing agents, with the current limited number of therapeutic agents available. Knowing the drug susceptibility information of the target microorganism prior to treating patients is very useful in selecting an effective antibiotic, especially in gonococcal infections where drug resistance is prominent, and is also important in preventing treatment failure. In this study, we developed a new method for obtaining drug susceptibility profiles of Neisseria gonorrhoeae using the loop-mediated isothermal amplification (LAMP) method. The LAMP assay does not require expensive devices. Therefore, this method is expected to be a tool for point-of-care testing of antimicrobial resistance for individualized treatment in the future.

Keywords: Neisseria gonorrhoeae; cefixime; loop-mediated isothermal amplification; penA; sexually transmitted infections.

FIG 1

Relationship between the CFM MIC and penA type of N. gonorrhoeae strains from clinical specimens. The vertical and the horizontal axes show the CFM MIC value and the number of isolates, respectively. The black and gray bars represent nonmosaic penA and mosaic penA, respectively.