Improving the processing time for the detection of carbapenemase-producing enterobacterales using an evolving algorithm

Carbapenemase-producing enterobacterales (CPE) have disseminated worldwide, with the enzyme OXA-48 (so-named by its hydrolysis of the antibiotic oxacillin) emerging as the most prevalent carbapenemase, whilst there is also a place for Klebsiella pneumoniae carbapenemase [1,2]. Recognition of CPE in the routine laboratory is challenging: CPE are not universally resistant to carbapenems and not all carbapenem-resistant enterobacterales produce carbapenemases [3]. Recovery of CPE from surveillance specimens is dependent on utilizing screening media. Detecting CPE among clinical isolates is initially reliant on minimum inhibitory concentration values from automated antimicrobial susceptibility testing. Expert rules are activated where carbapenem minimum inhibitory concentrations meet alert criteria to prompt subsequent confirmation or exclusion of CPE. Recognition of OXA-48 enzymes is difficult, as these enzymes weakly hydrolyse carbapenems. High level temocillin resistance (>128 mg/L) is recommended as a phenotypic marker for OXA-48 production, but lacks specificity, so that the use of confirmatory tests for CPE has been recommended [3–5]. However, as a standardized detection algorithm has only recently been defined [6], individual laboratories have been compelled to develop inhouse strategies for CPE recognition and confirmation, either via a molecular genetic approach or externally by national reference facilities, with associated delays. In 2016, when chromogenic screening media and phenotypic and molecular genetic detection methodologies became available, we developed a local algorithm to enhance recognition and reduce reporting turnaround times.Wepresent the evolution of methods to detect and confirm CPE over eight years and the development of an algorithm to improve processing times. Our accredited microbiology laboratory processes approximately 140,000 patient specimens annually, utilizing the latest European Committee for Antimicrobial Susceptibility Testing (EUCAST) recommendations [4]. Processing of rectal swabs for CPE was introduced in February 2011. Between February 2011 and October 2016, CPE detection methodology from rectal swabs initially employed pre-enrichment in tryptone soya broth, which was sub-cultured onto MacConkey agar with a carbapenem disc. This method later evolved to direct culture onto MacConkey agar with an ertapenem disc (MacE). Enterobacterales isolates recovered from within a defined zone diameter of the carbapenem disc on the MacConkey screening agar were considered suspect CPE. For suspect CPE from clinical specimens, alert criteria of ≥0.5 mg/L for meropenem and ertapenem were employed for Enterobacterales other than Enterobacter species on the BD PhoenixTM Antimicrobial Susceptibility Testing analysis software system (BD Diagnostics Systems, Sparks, MD, USA); Enterobacter species were subjected to an alert criterion of ≥8 mg/L for ertapenem. The modified Hodge test was performed on suspect isolates as an additional phenotypic test. Presumptive CPEs were referred to the national CPE reference laboratory service for confirmation. In 2011, a further phenotypic-based assay, the Klebsiella pneumoniae carbapenemase, MBL and OXA-Confirm Kit (Rosco Diagnostica, Taastrup, Denmark) was incorporated. As technologies evolved [7], the algorithmwas revised. In October 2016 (Figure 1), the chromID® Carba Smart (bioMerieux, Marcy L’Etoile, France) screening agar replaced the MacE. The RESIST-3 O.K.N K-SeT (Coris BioConcept, Gembloux, Belgium) was introduced and positive results allowed preliminary reporting of suspect CPE. An assay based on molecular genetics, the Xpert® Carba-R (Cepheid, Sunnyvale, CA, USA), was also introduced. Alert criteria of ≥0.5 mg/L for ertapenem and ≥0.125 mg/L for meropenem were employed for antimicrobial susceptibility testing analysis. A final confirmatory test, the carbapenemase inactivation method [8], was introduced to further substantiate a CPE negative result. In March 2017, reporting procedures were amended to allow final reporting of CPE based on the Xpert® Carba-R findings with antimicrobial susceptibility testing confirmation. Due to the reliability of the Xpert® Carba-R positive result, referral to the reference laboratory was no longer deemed essential for confirmation, but continued for national epidemiological purposes.