Journal article
Proof-of-concept, rapid, instrument-free molecular detection of Neisseria gonorrhoeae and ciprofloxacin susceptibility
Journal of Antimicrobial Chemotherapy, Vol.77(11), pp.2933-2936
2022
PMID: 35880750
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.
Details
- Title
- Proof-of-concept, rapid, instrument-free molecular detection of Neisseria gonorrhoeae and ciprofloxacin susceptibility
- Authors
- Abdulrahman K. S. Ayfan (Author) - University of QueenslandJoanne Macdonald (Author) - University of the Sunshine Coast, Queensland, School of Science, Technology and EngineeringAdam D. Irwin (Author) - University of QueenslandHosam M. Zowawi (Author) - University of QueenslandBrian M. Forde (Author) - University of QueenslandDavid L. Paterson (Author) - University of QueenslandMonica M. Lahra (Author) - Prince of Wales HospitalDavid M. Whiley (Corresponding Author) - University of Queensland
- Publication details
- Journal of Antimicrobial Chemotherapy, Vol.77(11), pp.2933-2936
- Publisher
- Oxford University Press
- DOI
- 10.1093/jac/dkac242
- ISSN
- 1460-2091
- PMID
- 35880750
- Grant note
- Queensland Government APP1197743 / National Health and Medical Research Council (NHMRC) Investigator Grant; National Health and Medical Research Council (NHMRC) of Australia IH190100021 / Australian Research Council Research Hub for Antimicrobial Resistance; Australian Research Council Advance Queensland Industry Research Fellowship from the Queensland Government
- Organisation Unit
- Centre for Bioinnovation; School of Science, Technology and Engineering; University of the Sunshine Coast, Queensland
- Language
- English
- Record Identifier
- 99686198802621
- Output Type
- Journal article
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- Infectious Diseases
- Microbiology
- Pharmacology & Pharmacy
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