Evaluation of the SpeeDx Carba (beta) multiplex real-time PCR assay for detection of NDM, KPC, OXA-48-like, IMP-4-like and VIM carbapenemase genes

Abstract

Background Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM. Methods DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard. The Carba (beta) assay was then directly used to screen 460 DNA extracts of faecal specimens, with positive results subjected to the aforementioned in-house assays plus Sanger sequencing. Results The Carba (beta) assay correctly identified the presence of the respective carbapenemase genes in 154 of 156 isolates and provided negative results for all 24 non-CPO isolates. Two isolates provided positive results for OXA-48-like carbapenemase by the Carba (beta) assay only. The Carba (beta) assay had sensitivities of 100% for all targets, and specificities of 100% for KPC, NDM, IMP-4-like, and VIM targets, and 98.5% for OXA-48-like targets. When applied directly to faecal specimens, eight samples were positive by the Carba (beta) assay, two of which were confirmed by in-house TaqMan probe PCR or DNA sequencing. Conclusions The Carba (beta) assay is highly sensitive and specific for detecting key carbapenemase genes in isolates. Further testing is required to assess this assay’s suitability for direct screening of clinical specimens.

Evaluation of High-Resolution Melting Curve Analysis (HRM) assay for Detection of Pseudomonas aeruginosa PASGNDM699: A dangerous New Delhi metallo-β-lactamase (NDM) strain

“…They con rmed the identi cation of NDM strains at dilution 103 and stated that the HRM method had limitations in the detection of different dilutions. Although identi cation of MBL and NDM strains has been performed in various studies in Sweden [28], USA [29], Australia [30], and Italy [31] in gram-negative Bacteria by HRM method, the novelty of the present study was the use of HRM method to identify P. aeruginosa PASGNDM699 strain. It was also found that the HRM method is highly potent in detecting PASGNDM699 strains that are resistant to colistin and carbapenem.…”

“…DNA concentration was determined using a spectrophotometer Nanodrop-200 (Hangzhou Allsheng Instruments Co., Ltd, China). Primers sequence were initially set up as outlined in Ly et al [16], Bordin et al [6], Monteiro et al [17], Kosykowska et al [18], and Alkasaby et al [19].…”

“…Several mechanisms to attain resistance against β-lactam antibiotics [3,4]. These include: mutations to the active site of penicillin-binding-protein(PBP) to prevent drug binding, modi cation of the cell wall to prevent drug entry and assist active removal of antibiotic compounds, and producing the class of enzyme known as β-lactamase, which includes serine βlactamases and metallo-β-lactamase (MBLs) [5,6]. These strains hydrolyze the β-lactam ring of drug compound, thereby inactivating them.…”

Near-Infrared Spectroscopy Evaluations for the Differentiation of Carbapenem-Resistant from Susceptible Enterobacteriaceae Strains

“…Initial carbapenem resistance was determined by measuring reduced susceptibility to ertapenem by disk diffusion; and DNA extracts of the isolates were PCR tested for the presence of carbapenemase determinants ( bla NDM , bla OXA-48 . bla KPC , bla VIM , and bla IMP types) as previously described [ 27 ].…”

“…All K. pneumoniae samples ( n = 73) were further tested by a multiplex PCR method previously described (SpeeDx Pty Ltd., Australia) [ 27 ]. Briefly, samples were screened for carbapenemase genes bla KPC , bla NDM , bla OXA-48-like , bla IMP-4-like and bla VIM in a single multiplex reaction.…”