Rapid detection ofEnterococcusand vancomycin resistance using recombinase polymerase amplification

Abstract

Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium, have been a global concern, often causing serious healthcare-associated infections. We established a rapid approach for detecting E. faecium and vancomycin-resistance genes (vanA and vanB) in clinical samples using isothermal recombinase polymerase amplification (RPA) combined with a lateral-flow (LF) strip. Specific RPA primer sets and probes for ddl (to identify the presence of E. faecium) vanA and vanB genes were designed. The RPA reaction was performed under isothermal condition at 37 °C within 20 min and read using the LF strip within a further 5 min. A total of 141 positive blood-cultures and 136 stool/rectal swab samples were tested using RPA-LF method compared to the conventional PCR method. The RPA-LF method exhibited 100% sensitivity in both blood-culture (60 E. faecium; 35 vanA type and two vanB type) and stool/rectal-swab samples (63 E. faecium and 36 vanA type) without cross-reaction (100% specificity). The lower detection limit of the RPA-LF was approximately 10 times better than that of the conventional PCR method. The RPA-LF method is an alternative rapid method with excellent sensitivity and specificity for detecting E. faecium, vanA, and vanB, and it has the potential to be used as a point-of-care device for VRE therapy and prevention.

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

“…However, preparation times for each step were not included. The RPA-LF assay also provided the limit of detection lower than those of the RPA-AGE and the conventional PCR-AGE method, similar to previous reports (10–100 times) [ 27 , 28 , 29 , 30 , 31 ]. To reduce the cost of the RPA-LF assay, the RPA reaction using the RPA reagents of TwistAmp™ Kit was performed in the small final volume of 12.5 µL as described previously [ 28 , 41 ].…”

“…The RPA-LF assay is now widely applied to detect and identify antimicrobial resistance genes, such as mobilized colistin resistance-1 ( mcr-1 ) [ 26 ], macrolide efflux A ( mefA ) [ 27 ], methicillin resistance ( mecA ) [ 28 ], and extended-spectrum β-lactamase (ESBL) genes [ 29 ]. It was also successful to apply for rapid and direct detection of Staphylococcus aureus plus methicillin resistance as well as Enterococcus plus vancomycin resistance in positive blood cultures, suggesting the use of RPA-LF assay as a POC device [ 30 , 31 ]. The international patent for RPA primers of bla OXA-48-like by Sutton et al [ 36 ] was reported in 2017.…”

“…The presence of a colored band generated by a specific RPA product can be observed by naked eyes within 5 min. The RPA method in combination with the LF strip (RPA-LF) is simple, and now widely applied to detect and identify various antimicrobial resistance genes [ 26 , 27 , 28 , 29 , 30 , 31 ]. We, therefore, developed the RPA-LF assay for the rapid detection of bla OXA-48-like genes, which could be used as a POC diagnostic tool for patients with bloodstream infections, or for infection control purposes in low-resource laboratories.…”

Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications

“…Srirattakarn et al established RPA-LFS to monitor MRS, the sensitivity and specificity of RPA-LFS were 92.1% and 100% respectively (Srisrattakarn et al, 2020). RPA-LFS could also be used to detect vancomycinresistant enterococci (Panpru et al, 2021). Singpanomchai et al established an allele-specific RPA-SYBR amplification system to detect multidrug-resistant tuberculosis, this method designed specific primers for the alleles of four major mutations rpoB516, rpoB526, rpoB531, and katG315.…”

“…established RPA-LFS to monitor MRS, the sensitivity and specificity of RPA-LFS were 92.1% and 100% respectively ( Srisrattakarn et al., 2020 ). RPA-LFS could also be used to detect vancomycin-resistant enterococci ( Panpru et al., 2021 ). Singpanomchai et al.…”