Evaluation of the Immunochromatographic NG-Test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT for Rapid Detection of KPC-, NDM-, IMP-, VIM-type, and OXA-48-like Carbapenemase Among Enterobacterales

Background Enterobacterales are the most common pathogens for nosocomial infections. The emergence and spread of KPC, NDM, and OXA-48-like carbapenemase-producing Enterobacterales with their extensively drug-resistant characteristics have posed great threats to public health. This study aimed to evaluate the performance of NG-test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT for rapid detection of five carbapenemases (KPC, NDM, VIM, IMP, and OXA-48-like) among Enterobacterales. Methods A total of 186 carbapenem-resistant Enterobacterales clinical isolates and 29 reference strains were used in this study. Carbapenemase genes were confirmed by PCR and DNA sequencing. The sensitivities and specificities of these assays were calculated utilizing the VassarStats software. Results For clinical isolates, the NG-test Carba 5 detected KPC, NDM, OXA-48-like, IMP, and VIM in less than 15 min with the sensitivity and specificity of 100% and 100%, respectively. The RESIST-5 O.O.K.N.V. detected KPC, NDM, OXA-48-like, and VIM with the sensitivity and specificity of 99.4 and 100%. The IMP K-SeT detected all of the IMP producers (6/6). For reference strains, the sensitivity and specificity of NG-test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT were all 100 and 100%, respectively. Conclusion As efficient, rapid, and convenient diagnostic methods, NG-test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT could help to simplify the complex routine workflow for detecting carbapenemases. Rapid and accurate identification of carbapenemase is of significance for both epidemiological and infection control purposes.


INTRODUCTION
The emergence and dissemination of carbapenemase-producing Enterobacterales (CPE) pose a global health threat (Nordmann et al., 2012a). As the ability of carbapenemases to hydrolyze all β-lactam antibiotics leads to few antibiotics retaining activity against CPE, infections caused by CPE are usually burdened by high mortality and poor prognoses (Falagas et al., 2014;Feil, 2016). Tigecycline, colistin, and ceftazidime-avibactam are the only available antimicrobial agents for the treatment of infections caused by CPE in China. However, unlike tigecycline and colistin, the activity of ceftazidime-avibactam is varied to different CPE. The in vitro studies show that ceftazidimeavibactam has excellent in vitro activity against ESBL-, AmpC-,  Enterobacterales, but has no activity against metallo-beta-lactamases Enterobacterales (Shirley, 2018;Yin et al., 2019). As reported, for the treatment of carbapenem-resistant Klebsiella pneumoniae bloodstream infections, initial adequate antibiotic therapy resulted in the only independent factor able to protect against death (Micozzi et al., 2017). Therefore, rapid and accurate identification of carbapenemases is critical for both epidemiological and infection control purposes.
Recently, rapid diagnostic tests, NG-test Carba 5 immunochromatographic assay (NG Biotech, Guipry, France), and BioConcept,Gembloux,Belgium) have been developed to detect the five main carbapenemases, namely, KPC, NDM, OXA-48-like, IMP, and VIM. To date, several studies have evaluated the performance of NG-test Carba 5 and demonstrated that it performed well on bacterial colonies and positive blood cultures with overall sensitivity and specificity that ranged from 97.3 to 100% and 95.3 to 100%, respectively (Boutal et al., 2018;Hopkins et al., 2018;Bodendoerfer et al., 2019;Giordano et al., 2019). The RESIST-5 O.O.K.N.V. detected KPC-type and OXA-48-like carbapenemases from blood cultures with a sensitivity of 100% for both, but 50.0 and 52.2% for NDM-and VIM-type carbapenemases, respectively (Bianco et al., 2020). Comparative studies evaluating the performance of different lateral flow chromatographic assays to detect carbapenemase from bacterial colonies are lacking in China. In this study, we investigated the performance of NG-test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT assay to detect carbapenemases among CPE.

Immunochromatographic Assays
The NG-Test Carba 5 assay consists of an independent cassette (targets KPC-, NDM-, VIM-, and IMP-type and OXA-48-like five main carbapenemases). RESIST-5 O.O.K.N.V. consists of two independent K-SeTs (one for the detection of OXA-163, OXA-48-like, and KPC; another for the detection of VIM and NDM). Both cassettes are provided in a single package and are to be used in parallel on the same bacterial lysis preparation. IMP K-SeT consists of a K-SeT for the detection of IMP metalloβ-lactamase, which was performed as a complementary test of RESIST-5 O.O.K.N.V.
These tests were performed according to the manufacturer's instructions in parallel. Firstly, one single isolated colony of overnight growth was harvested from the plate to an Eppendorf tube or tube with extraction buffer and suspended thoroughly to perform the lysis step. Subsequently, approximately 100 µl of the mixture was loaded on the sample region of the cassette and allowed to migrate for 15 min. Finally, the results were read until the control line turned red in the control region and then recorded whether the lines turned red in the test region of the cassette 15 min later.

Statistical Analysis
The sensitivity and specificity of the assay and upper and lower limits of the 95% confidence intervals (CIs) were calculated utilizing the VassarStats software 3 .  Table 2).

DISCUSSION
Of note, the rapid detection and identification of carbapenemase can help to prevent the spread and infection control of carbapenemase-producing isolates in health facilities (Boutal et al., 2018;Takissian et al., 2019). A nationwide survey indicated that bla KPC-2 (57% and 627/1,105) and bla NDM (31% and 343/1,105) were the most common carbapenemase genes among carbapenem-resistant Enterobacterales clinical isolates in China (Zhang et al., 2017;Han et al., 2020). Rapid identification of the carbapenemase type can help to guide therapy for the treatment of infection caused by carbapenemase-producing isolates. As reported, most KPC-2 or OXA-48-like carbapenemase-producing isolates are susceptible to ceftazidime-avibactam (Falcone and Paterson, 2016;Bassetti et al., 2018;Stewart et al., 2018;Zou et al., 2019). On the contrary, ceftazidime-avibactam has no activities against metallo-β-lactamase (NDM-, VIM-, or IMP-type) producers; thus other therapeutic options have to be considered, such as tigecycline, colistin, or other available antimicrobial agents (Davido et al., 2017;Zusman et al., 2017; Jayol et al., 2018;Emeraud et al., 2019;Yin et al., 2019;Zou et al., 2019). Currently, several phenotypic methods have been developed for the detection and identification of carbapenemases. Modified carbapenem inactivation methods (mCIMs) had sensitivities ranging from 93 to 100% and specificities from 97 to 100% for the detection of CPE, and excellent reproducibility was shown across laboratories (Pierce et al., 2017;Tsai et al., 2020). The mCIM and EDTA-CIM detected metallo-carbapenemases with a sensitivity of 89.3% and a specificity of 98.7% (Tsai et al., 2020). The mCIM was easy to perform and interpret for Enterobacterales, but timeconsuming (overnight incubation). Besides, invalid or uncertain results may occur with some isolates, and certain carbapenemase types are not detected consistently.
Carba NP test detected most carbapenemases with high sensitivities from 73 to 100% among Enterobacterales, but with low sensitivity in detecting OXA-48-like carbapenemase producers (Nordmann et al., 2012b;Tijet et al., 2013;Vasoo et al., 2013;Yusuf et al., 2014;Papagiannitsis et al., 2015;Tamma et al., 2017). The modified Carba NP test had a sensitivity of 99% (Tamma et al., 2017). One limitation of the manual versions of the Carba NP test and its variants was frequent reagent preparation due to the short shelf life of the imipenem-containing solution. Other commercially available assays, including the Rapidec Carba NP test, the Neo-Rapid Carb screen, and the Rapid Carb Blue screen have sensitivities ranging from 89 to 98% and specificities approaching 100% (Tamma et al., 2017). Similar to the Carba NP test, the limitations of these commercial assays are as follows: false-negative results occurred with OXA-48-like carbapenemase and the interpretation of results can be subjective due to slight color changes (Tamma and Simner, 2018). The sensitivites of the modified Hodge test (MHT) have been reported to be between 93 and 98% in KPC producers, but low sensitivites for metallo-beta-lactamases (Girlich et al., 2012;Mathers et al., 2013;Vasoo et al., 2013;Tsai et al., 2020). Reported sensitivities and specificities of MALDI-TOF MS ranged from 77 to 100% and 94 to 100%, respectively, with a turnaround time of within 4 h (Knox et al., 2014;Papagiannitsis et al., 2015;Oho et al., 2020). Carbapenemase inhibition tests with boronic acid derivatives (BA) and dipicolinic acid (DPA)/EDTA were tested in Enterobacterales. The sensitivity for identification of class A, B, and OXA-48 carbapenemases was 95, 90, and 100%, with 96 to 100% specificity (van Dijk et al., 2014).
Compared with the phenotypic method, the multiplex immunochromatographic assay for the detection of KPC-, NDM-, VIM-, IMP-, and OXA-48-like carbapenemases was easy to perform and only relatively little hands-on time (no more than 5 min) was required. Several studies assessing the immunochromatographic for the detection of common carbapenemases showed high sensitivity and specificity results in bacteria (Boutal et al., 2018;Hopkins et al., 2018;Bodendoerfer et al., 2019). The NG-test Carba 5 has evaluated the detection of CPE from spiked blood cultures with a sensitivity and specificity of 97.7 to 98.3% and 96.1 to 100%, respectively (Giordano et al., 2019;Takissian et al., 2019). The RESIST-5 O.O.K.N.V. detected KPC-type and OXA-48-like carbapenemases from blood cultures with a high sensitivity of 100%, but with low sensitivities of 50.0 and 52.2% in detecting NDM-and VIM-type carbapenemases, respectively, and the results were quite different from our research of NDM metallo-beta-lactamases (97.1% sensitivity) (Bianco et al., 2020).
Our study had three limitations. The first limitation is including only six bla IMP -positive strains and one bla VIMpositive strain. Second, there is a lack of other KPC-and OXA-48-like variant carbapenemases such KPC-3 and OXA-163 in this study; these variants are rare in China. Third, limited non-CPE isolates were used to assess the specificity of the assays, and we need to sufficiently investigate this isolates to evaluate the performance of these assays in this study. Recently, we collected one K. pneumoniae harboring a new variant, rare carbapenemase bla KPC-33 , and we found that immunochromatographic assays cannot detect this new variant. Although other bla KPC -positive strains including bla KPC-3 -, bla KPC-33 -, or bla VIM -type metalloβ-lactamase positive strains are rare in China, we still need to collect these strains to make a more comprehensive assessment on the performance of immunochromatographic testing for detection of carbapenemases.

CONCLUSION
In conclusion, NG-test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT assays could be efficient, rapid, and convenient diagnostic tools for detecting the most common carbapenemases in China. These might help to control the spread of carbapenemaseproducing isolates in health facilities.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The study protocol was approved by the Institutional Review Board of Huashan Hospital, Fudan University (Number: 2018-408).

AUTHOR CONTRIBUTIONS
FH designed the study. RH, YG, MP, QS, and SW performed the experimental work. RH, YG, YY, and DY collected the data. FH and RH analyzed the data. All authors read and approved the final manuscript.