Improvement of the Xpert Carba-R Kit for the Detection of Carbapenemase-Producing Enterobacteriaceae

Abstract

The Xpert Carba-R kit, version 2 (v2), which has been improved for the efficient detection of bla_OXA-181 and bla_OXA-232 genes, was tested on a collection of 150 well-characterized enterobacterial isolates that had a reduced susceptibility to carbapenems. The performance of the Xpert Carba-R v2 was high, as it was able to detect the five major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48). Thus, it is now well adapted to the carbapenemase-producing Enterobacteriaceae epidemiology of many countries worldwide.

TEXT

Carbapenemase-producing Enterobacteriaceae (CPEs) have been increasingly reported worldwide (1). The most clinically relevant carbapenemases encountered in Enterobacteriaceae belong to Ambler class A (KPC type); Ambler class B or metallo-β-lactamases (MBLs), such as IMP, VIM, and NDM types; or Ambler class D (OXA-48-like) (1). In the United States and Europe, a mixture of KPC, NDM, VIM, and OXA-48-like carbapenemases dominates (1), while IMP-producing Enterobacteriaceae are more prevalent in the Far East (2). Carbapenem resistance may be the result of combined mechanisms of outer-membrane permeability defect (e.g., porin defect) and noncarbapenemase β-lactamases (e.g., acquired or overexpressed chromosome-encoded cephalosporinase and extended-spectrum β-lactamases) (1). However, it was recently demonstrated that despite having identical MICs for carbapenems, carbapenemase-producing Klebsiella pneumoniae exhibit a marked inoculum effect and were more resistant to the bactericidal effect of meropenem than were non-carbapenemase-producing K. pneumoniae (3). The finding suggested that MIC measurements alone are not sufficient to predict therapeutic efficacy of carbapenems against CPE and that the rapid detection of carbapenemase production is essential for the guidance of antimicrobial treatment (3). Finally, CPEs are known to be highly proficient in disseminating and causing outbreaks through high-risk lineages (e.g., KPC-producing K. pneumoniae ST258), and successful plasmids (e.g., IncL OXA-48 plasmid) (4, 5). Therefore, rapid confirmation of carbapenemase production is essential not only for effective therapy but also for the prompt implementation of infection control measures capable of preventing CPE dissemination (6).

Molecular diagnostic assays have been increasingly used for the rapid screening of patients for carbapenemase producers directly from rectal swabs (7). Most of them can detect the four main carbapenemases (KPC, NDM, VIM, and OXA-48-like), which represent >95% of the carbapenemases produced by Enterobacteriaceae. The Xpert Carba-R kit (Cepheid, Sunnyvale, CA, USA) is the only commercially available assay that can also detect IMP-1 group producers (7). However, the Xpert Carba-R kit did not detect some OXA-48 variants, e.g., OXA-181 and OXA-232. Although still rare in France, the identification of these two variants rose from 0.8% in 2012 (8) to 3.4% in 2014 and 6.7% by mid-2015 of the total CPEs received at the French National Reference Centre for Antibiotic Resistance (L. Dortet, unpublished data). Accordingly, the use of the Xpert Carba-R kit as a unique screening method for CPE carriage led to the reports of several outbreaks caused by OXA-181-producing Enterobacteriaceae in France (9, 10).

Here, we evaluated the performance of a novel version of the Xpert Carba-R kit, the Xpert Carba-R version 2 (v2), which has been improved for the efficient detection of bla_OXA-181 and bla_OXA-232 genes. The Xpert Carba-R v2 was tested on fresh bacterial colonies of a collection of 150 enterobacterial isolates that were representative of the French epidemiology. This collection comprised 61 non-CPE isolates, including 43 isolates with decreased susceptibility to at least one carbapenem (imipenem, meropenem, or ertapenem) according to EUCAST guidelines (11). True CPEs consisted of 9 KPC, 11 NDM, 9 VIM, 5 IMP, and 41 OXA-48-like (20 OXA-48, 2 OXA-162, 9 OXA-181, 5 OXA-204, 3 OXA-232, and 2 OXA-244) producers and 14 isolates producing multiple carbapenemases (3 NDM-1 + OXA-48, 6 NDM-1 + OXA-181, 2 NDM-1 + OXA-232, 1 NDM-5 + OXA-232, 1 NDM-1 + VIM-2, and 1 VIM-4 + OXA-48) (Table 1). The Xpert Carba-R v2 was able to detect all KPC, NDM, VIM, and OXA-48 variants, including OXA-181 and OXA-232. In addition, both resistance determinants were correctly identified in all of the multiple carbapenemase producers. Concerning IMP-type carbapenemases, all of those in the IMP-1 group (IMP-1 and IMP-11 in our study) were detected. As claimed by the manufacturer, the two IMP-8 producers, which are not IMP-1 group members, were not detected. Although these enzymes are prevalent in Taiwan (2), they are still extremely rare in Europe (8). None of the non-carbapenemase producers gave positive PCR results, except for two OXA-163 and one OXA-405 producers. These two OXA-48 variants efficiently hydrolyze expanded-spectrum cephalosporins but were devoid of any carbapenemase activity due to a 4-amino-acid deletion in the active site (12). Although OXA-405 was reported from a unique Serratia marcescens isolate recovered in France (12), OXA-163 producers that were initially described in Argentina have already spread to Egypt (13), paving the way for their possible dissemination in North African countries and Europe.

TABLE 1.

Results of the Xpert Carba-R v2 on a collection of non-carbapenemase- and carbapenemase-producing Enterobacteriaceae

^a↗↗↗ Case, overexpression of a likely chromosome-encoded cephalosporinase.

^bMICs determined using Etest method (bioMérieux, Marcy-l'Etoile, France) according to the manufacturer's instructions.

^c+ (shading), positive result; −, negative result.

^dCase, cephalosporinase.

^eESBL, extended-spectrum β-lactamase.

Standard PCR/sequencing results were used to calculate the performances of the Xpert Carba-R v2 on colonies and were 97.8% (95% confidence interval [CI], 91.85% to 99.72%) and 95.3% (95% CI, 86.91% to 99.02%) sensitivity and specificity, respectively. Extrapolating these results to the global French CPE epidemiology, the Xpert Carba-R v2 may be able to detect 99.6% (2,018/2,026) of the CPEs identified by the Associated French National Reference Center between 2012 and 2014, missing only seven IMI-1/2 and one FRI-1 producers (8, 14).

Our study demonstrated that the Xpert Carba-R kit v2 is now well adapted to the French epidemiology of CPE, which reflects that of many countries, especially those in northern Europe, and underlines the reactivity of Cepheid to evolve their assay according to the ever-changing epidemiology of carbapenemases. As it may be used directly on rectal swabs, the Xpert Carba-R v2 offers an efficient option for the rapid screening of CPE-colonized patients. Accordingly, the use of the Xpert Carba-R v2 for the investigation of an outbreak may efficiently reduce the impact and cost of patient hospitalization by reducing the time to obtain a result for a CPE from 24 hours (needed time to culture bacteria on selective media and perform complementary testing for detection of CPE) to <1 hour.