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Evaluation of the rapid lateral flow assay (LFA) for detection of five major carbapenemase enzyme families in genotypically characterised bacterial isolates
For correspondence: Dr Shaoli Basu, Department of Laboratory Medicine-Microbiology, P. D. Hinduja hospital and Medical Research Centre, Mumbai 400 016, Maharashtra, India e-mail: dr_shaoli.basu@hindujahospital.com
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Received: ,
Accepted: ,
Abstract
Background & objectives
Antimicrobial resistance has steadily become a grave public health problem worldwide, making carbapenems, often the last choice of antibiotic, for treating infections involving multidrug-resistant Gram-negative organisms. The production of carbapenemases is one of the primary mechanisms responsible for the development of resistance. KPC, NDM, VIM, IMP, and OXA-48-like are the most prevalent carbapenemases. Identification of CROs is based on the resistance phenotype or requires molecular assays, which are not widely available. A lateral flow assay (LFA) has been developed to enable rapid identification of carbapenemase production in cultured bacteria.
Methods
A total of 87 isolates characterised by Whole Genome Sequence (WGS) were tested. Five ATCC strains were used for quality control. LFA was performed according to the kit literature, and the results were compared with those by WGS. The assay was assessed for its sensitivity and specificity for the detection of five major carbapenemases.
Results
A total of 87 isolates of Enterobacterales and Pseudomonas aeruginosa, characterised by WGS, were tested. Of these, 52 were positive for the big five carbapenemases (IMP, VIM, KPC, NDM, and OXA-48-like), and 35 were negative. ATCC strains were run for quality control with each batch of tests. Overall sensitivity of the assay was 98.07 per cent (51/52), with no false positives, having 100 per cent specificity (35/35). The assay correctly detected strains producing KPC, OXA-48-like, VIM, and IMP, being 100 per cent sensitive (n=33/33) when compared with WGS results; whereas, it showed delayed positivity (>15 min) to identify one strain producing NDM-1 (thus considered false negative), accounting to sensitivity of 92.3 per cent (n=12/13) for detection of NDM. It correctly identified six strains simultaneously producing OXA-48-like and NDM, and one strain producing NDM-1 and KPC.
Interpretation & conclusions
The assay, being robust and cost-effective, with a short turn-around-time, will prove to be a great addition to the diagnostic armamentarium, helping in implementing antimicrobial stewardship and preventing AMR.
Keywords
Anti-microbial resistance
big 5 carbapenemase
IMP
KPC
lateral flow assay
NDM
OXA-48-like
VIM
The rapid emergence of AMR worldwide has drastically limited the treatment options, posing a serious public health threat globally. Carbapenems are an important antimicrobial agent frequently used in treating infections involving multidrug-resistant Gram-negative organisms and are prescribed as a last resort antibiotic for critically ill patients1. Development of carbapenem resistance in bacteria is mainly due to three major mechanisms: production of beta-lactamases, hyper-production of efflux pumps, and porin channel mutations, or a combination thereof. Of these, production of carbapenemases remains the most important mechanism in driving carbapenem resistance globally1.
Carbapenemase production leads to resistance to most β-lactam antibiotics, including carbapenems. Most commonly prevalent carbapenemases are classified into three Ambler classes: Class A (Klebsiella pneumoniae carbapenemase; KPC), Class B- metallo β-lactamase (imipenemase; IMP, verona integron metallo β-lactamase; VIM, and New Delhi metallo β-lactamase; NDM), and Class D-oxacillinase (OXA-48-like)1,2. There is significant morbidity and mortality following invasive infections caused by carbapenemase-producing organisms (CPOs). The optimal treatment still remains undefined2. Hence, it becomes crucial to characterise the type of carbapenemase enzyme produced3. Accurate and rapid detection of carbapenemases is of paramount importance to guide antibiotic therapy, and for understanding its epidemiological prevalence and infection control purposes to contain further dissemination.
Many tests have been used to detect carbapenemase activity in cultured bacterial isolates, like, the Carba NP tests, the BYG test, and the carbapenem inactivation method. These tests broadly detect only the presence of carbapenemase activity and do not distinctly identify its type. Also, there is a lack of reliable phenotypical tests or chemical inhibitors for detecting OXA-48-like producers4. OXA-48-like enzymes weakly hydrolyse carbapenems. Hence, elevated minimum inhibitory concentrations (MICs) to carbapenems are not always seen with OXA-48-like producers and may easily go undetected with routine laboratory diagnosis2.
Phenotypic assays are laborious, time-consuming, and require separate inhibitor-based confirmatory tests to identify each class of β-lactamases. Molecular techniques like real-time multiplex PCR, such as the Xpert® Carba-R, require special infrastructure, are expensive, and are unavailable in many routine clinical laboratories (particularly in low and middle-income settings)3. Thus, the development of a lateral flow assay (LFA) has the potential to improve both the quality and time of detection of these major five carbapenemases in the routine microbiology laboratory, providing rapid and accurate results at a moderate cost. Currently, there are two LFA: NG-Test Carba 5 (NG Biotech, Guipry, France) and RESIST-5 O.K.N.V.I. assay (CORIS BioConcept, Gembloux, Belgium) are being used in clinical laboratories for the detection of major five carbapenemases from isolates3,4.
In this study, we evaluated a new multiplex LFA, the first of its kind to be developed in India, for the detection of the major five carbapenemases (IMP, VIM, NDM, KPC, OXA-48-like) and assessed its sensitivity and specificity.
Materials & Methods
This study was conducted at P. D. Hinduja Hospital and Medical Research Centre, Mahim, Mumbai, in the Microbiology Laboratory from December 2023 to May 2024, after being approved by the Institute’s Ethics Committee. A total of 87 isolates previously characterised by whole genome sequencing (WGS) were tested. ATCC strains (n=5) (Klebsiella pneumoniae ATCC®BAA-1705, Klebsiella pneumoniae ATCC®BAA-1706, Escherichia coli NCTC®13476, Klebsiella pneumoniae ATCC®BAA-2146, Klebsiella pneumoniae NCTC®13442) were used as quality control. Positive and negative controls were run with every batch of test run and when a new lot of the kits was received.
These strains were sub-cultured on 5 per cent Sheep Blood Agar (5% SBA), incubated at 37⁰C in ambient air for 16-18 h, and were tested by 3B BlackBio- TRURAPID®O.K.N.V.I. RESIST-5, an LFA that directly identifies the presence of carbapenemases from a bacterial colony. The assay consists of two cassettes and includes distinct areas for the detection of VIM and IMP on one cassette and areas for OXA-48-like, KPC, and NDM detection on the other cassette. Both these cassettes include a specific control (C) area (Figure). The assay was performed, and the results were interpreted according to the manufacturer’s instructions. The results were then compared with the genotypic characteristics of the isolates obtained by whole genome sequence (WGS). WGS was performed for all the isolates. The Illumina DNA Prep protocol was used for DNA extraction.
- Lateral flow assay of 3B BlackBio- TRURAPID® O.K.N.V.I. RESIST-5.
Sequencing was performed on Illumina MiSeqDx sequencing systems. FASTQ files we analysed on PHoeNix v2.0.0 bioinformatics platform (CDC, US). The assay was assessed for its sensitivity and specificity.
Results
A total of 87 isolates of Enterobacterales and Pseudomonas aeruginosa (Table I), characterised by WGS, were tested, of which 52 were positive for the big five carbapenemases (IMP, VIM, KPC, NDM, OXA-48-like), and 35 were negative for the same.
| Organism | Total no. | KPC | OXA-48 like | IMP | VIM | NDM | NDM-5 + OXA 48 like | NDM-1 + KPC | Total positive | Total negative |
|---|---|---|---|---|---|---|---|---|---|---|
| Klebsiella pneumoniae complex | 26 | 3 | 4 | 2 | 4 | 1 | 3 | 1 | 18 | 8 |
| Escherichia coli | 20 | 2 | 4 | 0 | 0 | 6 | 1 | 0 | 13 | 7 |
| Enterobacter spp | 14 | 3 | 0 | 0 | 1 | 2 | 0 | 0 | 6 | 8 |
| Pseudomonas aeruginosa | 12 | 0 | 0 | 2 | 3 | 1 | 1 | 0 | 7 | 5 |
| Klebsiella spp | 7 | 1 | 2 | 1 | 0 | 0 | 0 | 0 | 4 | 3 |
| Serratia marcescens | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 |
| Proteus mirabilis | 2 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 1 |
| Providencia rettgeri | 2 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 2 | 0 |
| Morganella morganii | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
| 87 | 10 | 10 | 5 | 8 | 13 | 5 | 1 | 52 | 35 | |
| Overall | KPC (n=10) | OXA-48 like (n=10) | IMP (n=5) | VIM (n=8) | NDM (n=13) | More than one carbapenemase (NDM+OXA-48 Like, NDM+KPC) (n=6) | ||||
| Sensitivity % | 98.07% | 100 | 100 | 100 | 100 | 92.3 | 100 | |||
| Specificity % | 100% | 100 | 100 | 100 | 100 | 100 | 100 | |||
KPC, Klebsiella pneumonia carbapenemase; IMP, Imipenemase; VIM, Verona integron metallo β-lactamase, NDM, New Delhi metallo β-lactamase; OXA-48-like, Oxacillinase
The assay correctly detected all the strains producing KPC (blaKPC-2, blaKPC-3)(n= 10), OXA-48-like (blaOXA-48, blaOXA-48-like, blaOXA-181, and blaOXA-232)(n=10), VIM (blaVIM-2, blaVIM-27, and blaVIM-1) (n=8) and IMP (blaIMP-4, blaIMP-1) (n=5), being 100 per cent sensitive for detection of these carbapenemases when compared to the whole genome sequence results.
For the detection of NDM (blaNDM-1, blaNDM-5, blaNDM-6, blaNDM-7)(n=13), the assay detected 12 strains as positive, whereas it showed delayed positivity (NDM-line appearing beyond 15 min the recommended time-frame as per the manufacturer) to identify one strain producing blaNDM-1, hence, was considered as false-negative- having a sensitivity of 92.3 per cent for detection of NDM.
Also, it correctly identified six strains that simultaneously produced OXA-48-like and NDM (blaNDM-1 + blaOXA-232, blaNDM-5 + blaOXA-48-like) as well as one strain co-producing blaNDM-1 and blaKPC.
Therefore, the overall sensitivity of the assay was 98.07 per cent (51/52), as seen in table I. All 35 non-producers of these five carbapenemases were detected as negative. All isolates that produced a beta-lactamase other than those targeted by the assay gave a negative result, namely blaOXA-1,4,9,10,50,56,396,488; SME-3,4; SPM-1; NMC-A; as expected, and hence, no false positive results nor cross-reactivity were observed, thus having 100 per cent specificity (35/35), as depicted in table II.
| LFA | |||
|---|---|---|---|
| Positive | Negative | ||
| WGS | Positive | 51 | 1 |
| Negative | 0 | 35 | |
| Overall sensitivity % | 98.07 | ||
| Overall specificity % | 100 | ||
However, inter-lot variation in sensitivity for the detection of NDM was observed, which needs to be addressed in the product monograph under problem-solving points. Hence, it is recommended to run ATCC control strains with every new batch/lot of the assay. Also, the manufacturing company should formulate a stringent internal QC protocol to keep in check any deviation from the expected results before any batch/lot of the assay is marketed.
Discussion
The prevalence of CRE in India varies from as high as 40 per cent in E. coli to as high as 72 per cent in Klebsiella pneumoniae5. The most potent carbapenemases, in terms of resistance to carbapenems and their global presence, are the ‘Big 5’ carbapenemases, namely, KPC, VIM, IMP, NDM, and OXA-48-like.They have been detected in many Gram-negative organisms like Enterobacterales, as well as in glucose-nonfermenting bacteria, e.g., P. aeruginosa. Even though carbapenemases have been in existence for more than two decades; however, their physiological role and antimicrobial resistance profile are still being studied to understand them fully. The first carbapenemase and the first MBL, Imipenemase (IMP-1), was detected in 1991 in Japan6, whereas VIM-1 was discovered in 1997 in Verona, Italy7. KPCs were first detected in 1996 in the northeastern United States, and the first documented case of a bacterial infection caused by New Delhi metallo-β-lactamase-1 (NDM-1) was reported from India in 20088. OXA-48-like variants having carbapenemase activity were discovered in 2001 from a carbapenem-resistant K. pneumoniae isolated in Istanbul, Turkey9. Since their discovery, these carbapenemases have successfully spread to all continents, and different variants of these are reported from different parts of the world.
Till date, a total of 88 variants of IMP-type metallo-β-lactamase have been discovered with IMP-1-like variants (IMP-1, IMP-2, IMP-3, IMP-5, IMP-6, IMP-10, IMP-11, IMP-15, IMP-25, IMP-29, IMP-30, and IMP-40) most prevalent world-wide. IMP-2 is most prevalent in Italy, and IMP-5 in Portugal, IMP-4 in the USA, and both IMP-1 and 2 in the Middle East10,11.
VIM-type MBLs have been mostly identified in Pseudomonas spp. as well as in Enterobacterales and Acinetobacter spp.; at least 71 VIM-variants have been identified in several countries, with VIM-1 having global distribution10,12. Twenty four variants of NDM have been identified so far, with NDM-1 widely prevalent in Acinetobacter species, Enterobacter species, and K. pneumoniae isolates, while NDM-5 is most common in E. coli10,13. OXA-48-like carbapenemases are one of the important causes of non-susceptibility to the carbapenems among the Enterobacterales and include the following enzymes: OXA-48, OXA-162, OXA-181, OXA-204, OXA-232, OXA-244, OXA-245, OXA-247, OXA-436, OXA-484, and OXA-519. OXA-48, OXA-181, OXA-232, OXA-204, OXA-162, and OXA-244, in that order, are the most common enzymes identified among the OXA-48-like carbapenemases. More than 150 KPC-variants have been reported worldwide. KPC-2 and KPC-3 persist as the most common ones11. Currently, most clinical microbiology laboratories do not undertake the characterisation of the mechanism of carbapenem resistance for therapeutic decision-making.
The type of carbapenemase(s) produced by an organism has a significant impact on treatment, as antibiotics like ceftazidime-avibactam or meropenem-vaborbactam, ceftazidime-avibactam plus aztreonam have activity against specific types of carbapenemases. Meropenem-vaborbactam and imipenem-cilastatin-relebactam are active only against KPC producing Enterobacterales. Neither ceftazidime-avibactam, meropenem-vaborbactam, nor imipenem-cilastatin-relebactam has activity against metallo-β-lactamase (e.g., NDM) producing Enterobacterales. While Ceftazidime-avibactam is currently the preferred treatment option for OXA-48-like-producing organisms, the combination of Ceftazidime-avibactam with aztreonam is the preferred treatment option for NDM and other metallo-β-lactamase-producing infections14.
Phenotypic assays currently recommended by CLSI are culture-based assays which detect resistance of the cultured isolate in the presence of an antibiotic (e.g., modified carbapenem inactivation method (mCIM), and hydrolysis methods, i.e., Carba NP test.
Modified Hodge test (MHT) has acceptable sensitivity for most carbapenemases, especially KPC enzymes, but low sensitivity for MBLs. In one of the studies, MHT was reported to have only 50 per cent sensitivity in detecting NDM-producing isolates15 with low specificity and being time-consuming.
Carba NP test and variants
It has good sensitivity, ranging from 73 to 100 per cent for detecting most carbapenemases16,17. However, it was reported to have as low as six per cent in detecting OXA-48-like carbapenemases18. Also, this test requires reagent preparation, which has a short shelf life. False-negative results can occur with OXA-48-like enzymes, and the interpretation of results can be subjective due to minor colour changes.
mCIM can identify the presence of carbapenemases; however, it cannot distinguish between serine and metallo-β-lactamases. A further modification to this assay with the addition of EDTA has been proposed, which can differentiate between serine and MBLs. Of note, if a single isolate coproduces both serine and metallo-carbapenemases, false-negative results may occur. This is important, as there are a significant number of isolates co-producing both OXA-48-like and NDM enzymes17. A significant drawback of these tests is low sensitivity and the long turnaround time required, as this is important in clinical decision-making and in-hospital infection control purposes, with results available on the same day being ideal.
In the present study, we evaluated the 3B BlackBio-TRURAPID® O.K.N.V.I. RESIST-5assay, an LFA designed to detect OXA-48-like carbapenemase, which possess weak carbapenem-hydrolysing activities, VIM, NDM, KPC, and IMP carbapenemase families. There are very few assays that detect all of these major five carbapenemases2,4,19. Previous studies evaluating similar LFA detecting (4 carbapenemases) OXA-48-like, KPC, VIM, and NDM found sensitivity ranging from 84.1 per cent to 100 per cent and a specificity varying between 98 per cent to 100 per cent20-22. Studies evaluating assays detecting the five major carbapenemases reported a sensitivity of 82.6-98.4 per cent and a specificity of 98 per cent -100 per cent23-25. In all the above studies, the LFA assay was compared to either a phenotypic method for carbapenemase detection or NG-Test CARBA-5®, or a molecular method such as Xpert Carba-R assay, or in-house PCR.
This study had the advantage of evaluating the LFA against strains characterised by WGS, an important technology for mapping the complete genome of bacteria. It is the gold standard method to detect the resistance genes present in an organism for various antibiotics. Also, clinical isolates from India and the US have been used to assess this assay. Hence, these isolates had the most prevalent sub-variants of the ‘Big 5’ carbapenemases.
We found excellent concordance in detecting carbapenemase genes between WGS and the results of 3B BlackBio-TRURAPID® O.K.N.V.I. RESIST-5 assay, for all but one isolate, which was delayed positive (false-negative) for NDM. There was no cross-reactivity or false-positive detection of carbapenemases other than the targeted ones.
Overall, understanding the type of carbapenemase produced is important not only for epidemiological prevalence and infection control practices but also plays a significant role in deciding the use of appropriate antibiotics, especially the newly available and more potent BL/BLI combinations.
The availability of this assay will prove to be a powerful tool to detect and characterise even dual carbapenemase producers, even in a non-specialised clinical laboratory setting, and a cost-effective alternative to molecular amplification methods, which are more costly and less widely available. The speed of this assay and being user-friendly represent a major technical advance and make it particularly attractive to improve patient management, and a reduction in the steadily growing antibiotic resistance through targeted treatment and better infection control.
Acknowledgment
This work was supported by US CDC cooperative agreements NU2GGH001869 (‘Capacity Building and Strengthening of Hospital Infection Control to Detect and Prevent Antimicrobial Resistance in India’ with All India Institute of Medical Sciences (AIIMS-New Delhi) by providing isolates from CDC AR Bank (n=36) and NU3HCK000001 (Global Healthcare Detection and Response with Johns Hopkins University) by providing JHU clinical isolates (n=29).
Financial support & sponsorship
The study received funding support from GAIHN-AR India project, funded by the US CDC JHU PTE/prime (award No. 5NU3HCK000001-03-00; subaward no: 2005869446) for WGS of 22 Indian isolates.
Conflicts of Interest
None.
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.
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