Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Rapid Detection of OXA-48-like Carbapenemase Genes in Enterobacterales

Abstract

Carbapenem-resistant Enterobacterales (CRE) possessing various carbapenemases, particularly the OXA-48 group, are now rapidly spreading and becoming a major public health concern worldwide. Phenotypic detection of OXA-48-like carbapenemases is still suboptimal due to their weak carbapenemase activity, whereas highly sensitive and specific polymerase chain reaction (PCR)-based methods take at least 3–4 h. We, therefore, developed a recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip assay for the rapid detection of blaOXA-48-like in Enterobacterales. A total of 131 clinical isolates including 61 blaOXA-48-like-carrying Enterobacterales isolates and 70 Gram-negative bacilli isolates containing other bla genes were subjected to the RPA method performed under isothermal conditions at 37 °C within 10 min and visually inspected by LF strip within 5 min. The RPA-LF assay provided 100% sensitivity (95% confidence interval, 92.6–100%) and 100% specificity (93.5–100%) for detecting blaOXA-48-like genes from bacterial colonies. Its detection limit was 100 times less than that of the PCR method. This assay is rapid, easy to perform, and provides excellent performance without any special equipment. It may be applied for directly identifying the blaOXA-48-like genes in Enterobacterales obtained from blood culture. Rapid identification of carbapenemase types is essential for selecting appropriate antimicrobial options, particularly the β-lactams combined with novel β-lactamase inhibitors.

Simple and quick detection of extended-spectrum <i>β</i>-lactamase and carbapenemase-encoding genes using isothermal nucleic acid amplification techniques

“…The RPA-LF assay showed 100% sensitivity and 100% specificity (93.5-100%) for detecting bla OXA-48-like genes in bacterial colonies. Its detection limit was 1/100 that of the PCR method (Hemwaranon et al 2022) . Wang et al (2021) focused on four representative carbapenemase-encoding genes, bla KPC , bla NDM , bla OXA-48-like , and bla IMP , to develop detection methods.…”

“…Ayfan et al (2021) reported a rapid and simple molecular test for detecting bla NDM and bla VIM carbapenemase-encoding genes, with results obtained in approximately 15 min using DNA extracts, with detection limits of 9.2 copies/µL for the bla NDM type and 7.5 copies/µL for the bla VIM type Hemwaranon et al (2022). focused on…”

Replacement of the Double Meropenem Disc Test with a Lateral Flow Assay for the Detection of Carbapenemase-Producing Enterobacterales and Pseudomonas aeruginosa in Clinical Laboratory Practice

“…In recent years, rapid diagnostic testing using LFAs for the detection of enzymes associated with antimicrobial resistance have been widely developed as they demonstrate comparable performance with gold standard PCR-based methods and can be easily applied in clinical microbiology laboratories without requiring specialized personnel and excessive cost [ 21 , 22 , 23 , 24 , 25 ].…”

Unique combination of Recombinase Polymerase Amplification with label-free citrate-stabilised silver nanoparticle assay offers a highly sensitive, fast, accurate and visual molecular detection ofKlebsiella pneumoniae

“…In comparison, RPA provides further advantage of rapid (15-30 min) isothermal amplification at lower temperatures (∼37-42°C) permitting quicker detection 23 . RPA when combined with Lateral Flow Strip (LFS) system is capable of detecting as low as 100-1000 bacteria, highlighting scope for improving the sensitivity of detection post RPA-based DNA amplification 18, 19 . A recent study highlights utility of tailor-made plasmonic aptamer-gold nanoparticle (AuNP) assay for detection of Klebsiella without the need of amplification, however, the sensitivity of this methods is ∼3400 bacteria 24 .…”

“…However, the cost of detection is extremely high due to requirement of fluorescent probes and quantitative PCR (qPCR) equipment. Subsequent developments introduced isothermal amplification methods, including loop-mediated isothermal amplification (LAMP) 16,17 and recombinase polymerase amplification (RPA) [18][19][20][21] and multiple cross displacement amplification (MCDA) 22 , for rapid K. pneumoniae detection without the requirement of expensive thermal cyclers or qPCR. LAMP alone or in combination with clustered regularly interspaced short palindromic repeats (CRISPR) and MCDA combined with lateral flow strip (LFS) has demonstrated comparable sensitivity (1-20 bacteria) compared to qPCR.…”