A comparative study between real-time PCR and loop-mediated isothermal amplification to detect carbapenemase and/or ESBL genes in Enterobacteriaceae directly from bronchoalveolar lavage fluid samples

Abstract. Objectives: To evaluate and compare the efficacy of real-time PCR (Xpert Carba-R) and loop-mediated isothermal amplification (Eazyplex^® SuperBug CRE) for detecting carbapenemase carriage in Enterobacteriaceae directly from bronchoalveolar lavage (BAL).

Methods

Negative BAL samples were spiked with 21 well-characterized carbapenemase-producing Enterobacteriaceae strains to a final concentration of 10²–10⁴ cfu/mL. Xpert Carba-R (Cepheid, Sunnyvale, CA, USA), which detects five targets (bla_KPC, bla_NDM, bla_VIM, bla_OXA-48 and bla_IMP-1), and the Eazyplex^® SuperBug CRE system (Amplex-Diagnostics GmbH, Germany), which detects seven genes (bla_KPC, bla_NDM, bla_VIM, bla_OXA-48, bla_OXA-181, bla_CTXM-1 and bla_CTXM-9), were evaluated for the detection of these genes directly from BAL samples.

Results

Xpert Carba-R showed 100% agreement with carbapenemase characterization by PCR and sequencing for all final bacteria concentrations. Eazyplex^® SuperBug CRE showed 100%, 80% and 27% agreement with PCR and sequencing when testing 10⁴, 10³ and 10² cfu/mL, respectively. False negative results for Eazyplex^® SuperBug CRE matched the highest cycle threshold values for Xpert Carba-R. Hands-on time for both assays was about 15 min, but Eazyplex^® SuperBug CRE results were available within 30 min, whereas Xpert Carba-R took around 50 min.

Conclusions

We here describe the successful use of two commercial diagnostic tests, Xpert Carba-R and Eazyplex^® SuperBug CRE, to detect bacterial carbapenem resistance genes directly in lower respiratory tract samples. Our results could be used as proof-of-concept data for validation of these tests for this indication.