Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

Evaluation of EasyScreenTM  ESBL/CPO Detection Kit using direct-PCR from patient culture and broth samples (#201)

Dilshan M Abeysekera 1 , Doug S Millar 1 , John R Melki 1 , Elaine McGrath 2 , Martin Cormican 2
  1. Genetic Signatures, Newtown, NSW, Australia
  2. Division of Microbiology, University Hospital Galway, County Galway , Ireland


Beta-lactam and carbapenem antibiotics are the most commonly used worldwide in the treatment of bacterial infections. The recent emergence of ESBL and CPO is a significant global concern in healthcare settings as standard treatments may be rendered ineffective. Thus accurate and rapid detection of these resistant organisms will have a significant impact on patient management. We evaluated a EasyScreenTM  ESBL/CPO Detection Kit to detect most significant and commonly encountered bacterial resistance genes TEM, CTX-M, SME, GES, IMP, NDM, OXA-23 like, OXA-48 like, OXA-51 like, MCR-1, DHA, SHV, VIM, IMI, CMY, KPC and their subtypes in patient culture and broth samples.



To test the EasyScreenTM  ESBL/CPO Detection Kit’s sensitivity/specificity synthetic DNA constructs, validation organisms and panels from Vircell, Zeptometrix and QCMD were used. The assay was performed on 30 known clinical isolates from University Hospital Galway. 9 isolates were re-cultured in BHI-broth (with Ceftazidime). Inoculated samples were lysed/converted at 95°C for 15 minutes and directly amplified on the CFX 96/384 PCR-machines and compared with data from conventional DNA extracted and amplified samples.



Results from the validation panels yielded 100% concordance with the expected resistance patterns. The agreement with known clinical samples was 98%. Mixed infections could be easily detected using the different channels of the PCR instrument.



The EasyScreenTM  ESBL/CPO Detection Kit can be used to detect resistant genes directly from cultures and broths without the need for a DNA extraction/purification step. The PCR protocol can be significantly manipulated to reduce run-time given the high copy number. Turn-around-time is approximately 2hr for cultures and 1hr for broths. 

The 3baseTM technique expands the detection capacity of multiplex-PCR for some target genes (CTX-M, IMP-14) to detect various subtypes within the target by affecting the DNA sequence homology. Also, novel variants or new resistant markers can readily be incorporated into existing assays easily given the properties of the 3baseTM converted DNA, thus improving the coverage of such assays. This assay provides a rapid, sensitive, and cost-effective alternative for the detection of ESBL and CPO’s.