Proof-of-concept, rapid, instrument-free molecular detection of Neisseria gonorrhoeae and ciprofloxacin susceptibility

Abstract

Objectives To develop instrument-free point-of-care methods using recombinase polymerase amplification (RPA) technology coupled with a simple lateral flow detection system to detect Neisseria gonorrhoeae and susceptibility to ciprofloxacin. Methods For identification of gonococcal infection, an RPA-based method was developed targeting the gonococcal porA pseudogene (NG-porA-RPA). For ciprofloxacin susceptibility, predictive WT sequences at codons 91 and 95 of the gonococcal gyrA DNase gene were targeted. Given the known complexities of SNP detection using RPA (e.g. the ability to accommodate mismatches) we trialled several different assays incorporating various additional non-template mismatches in the oligonucleotide sequences to reduce affinity for the mutant (resistant) gyrA sequences. Assays were evaluated using a bank of N. gonorrhoeae (n = 10) and non-gonococcal (n = 5) isolates and a panel of N. gonorrhoeae nucleic acid amplification test (NAAT)-positive clinical sample extracts (n = 40). Results The NG-porA-RPA assay was specific to N. gonorrhoeae and provided a positive percentage agreement (PPA) of 87.5% (35/40) compared with a commercial N. gonorrhoeae NAAT when applied to the 40 clinical sample extracts. For gyrA, the non-template bases successfully reduced banding intensity for double-mutant strains (mutations at both 91 and 95), but not for rarer single-mutant (91 only) strains. The most promising gyrA assay, NG-gyrA-RPA08, correctly detected 83% (25/30) of infections from NAAT-positive clinical samples confirmed to have WT gyrA sequences based on Sanger sequencing. Conclusions These proof-of-concept data show that RPA technology has considerable promise for detecting N. gonorrhoeae and associated antibiotic susceptibility and would offer a diagnostic-based stewardship strategy identified as urgently needed by the WHO.

Democratizing nucleic acid-based molecular diagnostic tests for infectious diseases at resource-limited settings – from point of care to extreme point of care

“…[86][87][88][89][90][91][92] Ayfan et al reported the rapid and compact molecular detection assay of Neisseria gonorrhoeae and ciprofloxacin susceptibility cases by combining an RPA assay with LFA-mediated detection. 93 Abdou Mohamed et al were among the early researchers 94 to develop a multiplex nucleic acid enzyme-gold nanoparticle-based diagnostic platform for identifying the target pathogens alongside the respective antibiotic resistant genes within a span of 2 hours. Their test deployed an enzyme comprising two nucleic acid strands instead of amino acids, having the capability of selective DNA cleaving.…”