Introduction
Infections caused by carbapenem-resistant organisms (CRO) are emerging as a major challenge to public health [
1,
2]. Over the past couple of years, a marked increase in the prevalence of CRE has been reported worldwide [
3], and infections caused by CRO are associated with a significant mortality [
4]. Of note, the incidence of invasive disease with CRE increases especially in high-risk populations, e.g., in patients treated in intensive care units and hematology wards [
5]. Here, the majority of these infections are associated with increased length of stay and mortality rates ranging from 30 to 70% [
6].
Gram-negative organisms essentially become carbapenem-resistant via two main routes, i.e., carbapenemase production or expression of cephalosporinases (AmpC) and/or extended spectrum β-lactamases (ESBL) in combination with cell wall permeability changes [
7,
8]. Importantly, carbapenemases from molecular classes A, B, C, and D [
9] are characterized by specific β-lactam hydrolytic profiles and susceptibility against inhibition by different β-lactamase inhibitors [
10].
Therapeutic options for treatment of CRO-related infections are limited [
11]. New promising therapy options for CRO are based on new β-lactams and β-lactam/β-lactamase inhibitor combinations (e.g., ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam) [
12]. These substances exhibit specific activity in CRO depending on the respective mechanism underlying carbapenem resistance, and in turn, determination of carbapenemase resistance mechanisms may already allow for informed decision making on optimal treatment [
13].
Approaches to rapidly identify carbapenemase production in CRO include colorimetric assays (e.g., Carba NP), combined disk test (CDT), and the modified carbapenem inactivation method (mCIM). The long turnaround times (up to 24 h) of the two latter are a major limitation [
14]. Faster identification by the available molecular multiplex PCR assays limited to the most frequent carbapenemase-encoding genes remain expensive [
15,
16] and may have reduced sensitivity related to the possible presence of carbapenemases not included in the PCR panel. In that respect, a solution that combines antibiotic susceptibility testing with preliminary identification and classification of carbapenemases may represent an attractive alternative for rapid diagnostics of infections with CRO.
The BD Phoenix CPO detect (PCD) assay is the first automated test strategy that combines antimicrobial susceptibility testing with a built-in carbapenemase detection assay able to identify carbapenemase activity in clinical Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. In addition, the assay provides assignment to the respective four Ambler classes, thus potentially setting basis for rational choice of available antibiotics with dedicated CRO activity. The integration of the PCD assay into routine workflows may thus shorten the time to target antibiotic treatment. Here, we tested the performance of PCD in identifying carbapenemase production and assignment to Ambler classes in comparison to a multiplex carbapenemase PCR assay. Moreover, antibiotic susceptibility testing of meropenem, imipenem, and ceftazidime-avibactam was compared to minimal inhibitory concentrations (MICs) determined by gradient diffusion (Etest, bioMérieux; Liofilchem).
Discussion
Over the last years, a significant increase in CRO infections has been observed in many countries worldwide [
4,
22]. The increasing clinical importance of CRO infections urgently demands methods for rapid identification and classification of carbapenemase in order to provide early optimized therapy. Indeed, early and accurate information on the presence and classification of a carbapenemase enables trained clinicians to make targeted use of new antibiotic substances, i.e., ceftazidime-avibactam or meropenem-vaborbactam [
23]. In this respect, the PCD assay, marketed for testing antibiotic susceptibility and parallel identification of carbapenemase production, addresses a relevant clinical need. While carbapenemase detection and differentiation methods using immunological or molecular approaches provide faster results, the specific strength of an in-build phenotypic carbapenemase detection during routine susceptibility testing lies in the on-the-flight analysis character, providing evidence for carbapenemase production or exclusion of such activity for all isolates under routine investigation.
This study aimed to determine the usefulness of the PCD assay to detect carbapenemase production and to categorize respective enzymes according to the Ambler classification [
24]. As previously described by others [
8,
25,
26], the PCD assay provides accurate results for the identification of carbapenem resistance, with results available in less than 16 h. Moreover, the PCD assay proved able to identify carbapenemase production in our isolate collection with an overall high sensitivity (98.26%), but unexpectedly low specificity (17.72%). Five recent studies examined the analytical performance of the PCD test, using phenotypic carbapenemase detection assays (LDT Carba NP test, β-CARBA test, bioMérieux Rapidec Carba NP assay; [
8,
25,
26]) or a PCR assay (BD MAX™ Check-Points CPO PCR assay; assay, in-house multiplex PCR; [
27,
28]) as a reference. These studies found a comparable high overall sensitivity of 89.4–100%, but in contrast to the present study, a better specificity (68.6–87.1%) for the detection of carbapenemase activity in different collections of clinical
Enterobacterales isolates [
25,
26], partly including a small fraction of non-fermenters (18.03%, 17.09%, and 29.97%, respectively) [
8,
27,
28].
The differences observed in the available studies regarding the specificity of PCD may be due to a number of different drivers. The studies differed grossly in overall sample size, with number of included isolates ranging from
n = 95 to
n = 294. In addition, the relative proportion of bacterial species substantially varied between studies, and in particular, the number of non-fermenters included exhibits large heterogeneity (
n = 27 to
n = 53; [
8,
27,
28]), highlighting the potential impact of isolate selection on findings during assay evaluation studies. Especially, inclusion of carbapenem-resistant non-fermenters can potentially have a considerable influence on the test performance, given the lower pre-test probability for carbapenemase detection compared to
Enterobacterales. Inherent problems in detecting carbapenemase production in non-fermenters relate to the expression of a broad spectrum of different β-lactam hydrolyzing enzymes (e.g., AmpC- or ESBL-type enzymes) readily leading to false positives in hydrolysis-based carbapenemase detection assays [
21,
29,
30]. The present study included 108
P. aeruginosa and 21
A. baumannii strains, representing more than two-thirds of all tested isolates, which is in clear contrast to previously published studies. Inclusion of a large subset of challenging species may have made an important contribution to the overall unsatisfactory specificity of the PCD assay in the present study. In fact, compared to the reference methods used here, the PCD produced a high rate of false positive results in non-carbapenemase-producing
P. aeruginosa, which accounted for 75.38% of all false positives
Discrepant false positive PCD results not necessarily must result from low specificity of the PCD, but certainly might also be related to false negative reference assay results. In fact, a limitation of our study is the restriction of the reference method to detect only the most common carbapenemase-encoding genes, including class A (KPC, GES, NMC-A/IMI, BIC, SME), class B (VIM, NDM, IMP), and class D (Oxa-48, Oxa-23, Oxa-24) carbapenemases [
17,
31]. Although being the most prevalent and clinically relevant determinants of carbapenemase-mediated resistance in Germany [
32‐
35], the presence of carbapenemases not covered by the PCR assay employed cannot be excluded. To compensate for the potential sensitivity limitation, all PCR-negative isolates were additionally tested using a phenotypic carbapenemase assay (i.e., CIM; [
21]). While certainly, also this assay does not definitely exclude carbapenemase activity that might still be accessible for detection by the PCD. However, based on the combination of phenotypic and molecular methods applied to detect or exclude the presence of a carbapenemase in this isolate collection, it is plausible to assume that most of false positives are in fact a result of an overall low specificity of the PCD. Nevertheless, conclusive evidence certainly may only be obtained by whole genome sequencing.
Only two isolates tested were false negative for carbapenemase production, and both were found to carry
blaGES. Indeed, GES-type carbapenemases [
26] have previously been described as being difficult to detect by colorimetric methods [
36]. The low number of false negatives is in line with previous studies, highlighting the strength of the PCD to serve as a tool to exclude presence of a carbapenemase in clinical isolates [
27]. In fact, in a prospective study including 368/372
Enterobacterales isolates from various routine specimens were correctly identified as non-CPOs (own unpublished observation). To identify additional carbapenemase type-specific sensitivity problems of the PCD, a systematic analysis of isolates carrying rarer carbapenemase types will be necessary.
Given the clinical availability of novel β-lactamase inhibitors (i.e., avibactam, relebactam, vaborbactam) specific for defined subsets of carbapenemases, reliable information on the carbapenemase class may have immediate therapeutic implications. For example, detection of a class A carbapenemase could potentially allow start of ceftazidime-avibactam for therapy of a CRE, even if no standardized susceptibility testing is available [
37]. To allow for such strategy, reliable, highly sensitive carbapenemase classification is mandatory. In the isolate collection analyzed here, the PCD assay correctly assigned a specific carbapenemase class in 79.17% of
Enterobacterales and only 67.16% of non-fermenter isolates. These findings are in sharp contrast to results published recently, showing that 94.6% of isolates were correctly assigned to an Ambler class [
27]. Others, however, reported similar low overall accuracy of 85.0%, 68.99%, and 78.95% for carbapenemase classification by the PCD [
8,
25,
26].
In conclusion, data shown here demonstrate that the PCD assay is a reliable tool for the detection of CPOs with a high sensitivity, but low specificity. Implementation into workflows for routine analysis of clinical isolates thus demands additional confirmatory test to validate positive PCD results from the PCD analysis, especially for non-fermenter organisms. These should include assays allowing for definitive identification and typing of a carbapenemase. The accuracy of the in-build carbapenemase differentiation tool does not have the necessary accuracy to allow for its use in clinical decision making algorithms. The potential value of the PCD assay at present lies in its ability to exclude presence of carbapenemases, however, in a setting with low incidence of CRO, the usefulness in routine practice appears low.
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