|Year : 2020 | Volume
| Issue : 2 | Page : 85-87
Lateral flow assay for the rapid detection of carbapenemases in Enterobacterales
Kalyani Borde1, P Swathi2, Dilip Mathai3
1 Department of Microbiology, Apollo Institute of Medical Sciences and Research, Hyderabad, Telangana, India
2 Department of Microbiology, Yashoda Hospitals – Somajiguda, Hyderabad, Telangana, India
3 Department of Medicine and Adult Injectious Diseases, Apollo Institute of Medical Sciences and Research, Hyderabad, Telangana, India, Telangana
|Date of Submission||12-Jan-2021|
|Date of Acceptance||13-Jan-2021|
|Date of Web Publication||5-Apr-2021|
Dr. Kalyani Borde
Department of Microbiology, Apollo Institute of Medical Sciences and Research, Hyderabad, Telangana
Source of Support: None, Conflict of Interest: None
With the development of carbapenemase inhibitors such as avibactam, relebactam and vaborbactam, it has become important to detect the type of carbapenemase produced for guiding antibiotic therapy. Among the several methods available for the same, we chose lateral flow assay (Resist-3 O. K. N., Coris BioConcepts, Gembloux, Belgium) and compared it against a commercially available molecular test (Xpert Carba R assay, version 2, Cepheid, Sunnyvale, CA, USA). Sixteen clinical isolates, which were resistant to carbapenems on phenotypic testing, were selected. Fourteen of these were Klebsiella pneumoniae and two were Escherichia coli a total of 22 carbapenemases. (OXA-48-14, New Delhi metallo-beta-lactamase [NDM]-7 and K. pneumoniae carbapenemase [KPC]-1) were detected by the Carba-R assay. Six isolates (K. pneumoniae-5 and E. coli-1) had co-production of OXA-48 and NDM. Resist-3 assay detected all twenty two enzymes. The distribution of enzymes in these 16 isolates was identical to that shown by the Carba-R (100% agreement). Hence, we conclude that lateral flow immunochromatography assay is a simple, rapid and cost-effective method for the detection of carbapenemases. This would help the clinician select the appropriate antibiotic and support antibiotic stewardship in the Indian settings.
Keywords: Carbapenemase, immunochromatography, lateral flow
|How to cite this article:|
Borde K, Swathi P, Mathai D. Lateral flow assay for the rapid detection of carbapenemases in Enterobacterales. J Acad Clin Microbiol 2020;22:85-7
| Introduction|| |
Carbapenemase production has emerged as a predominant mechanism of resistance among Enterobacterales. According to the ICMR report, 20%–30% Enterobacterales are carbapenem resistant. Klebsiella pneumoniae is resistant to carbapenems up to 50% of the times. Most common carbapenemases in India are New Delhi metallo-beta-lactamase (NDM), K. pneumoniae carbapenemase (KPC) and OXA-48., Antibiotic therapy needs to be individualised according to the mechanism of resistance of the bacteria for the best outcomes. Molecular assays have found limited use due to cost and infrastructure constraints. In comparison, immunochromatography is easier and cheaper. Hence, we decided to compare the immunochromatography test results (Resist-3 O. K. N., Coris BioConcepts, Gembloux, Belgium) with a molecular test (Xpert Carba R assay, version 2, Cepheid, Sunnyvale, CA, USA).
| Materials and Methods|| |
This study was carried out at a tertiary care hospital in southern India as a part of research on multidrug-resistant bacteria. All isolates from blood, respiratory secretions, urine and body fluids during the three-month study period were tested on VITEK-2 (bioMérieux, Marcy-l'Etoile, France) for the identification and susceptibility. CLSI (2020) breakpoints were used for the interpretation of antibiotic-susceptibility results.
Those that were carbapenem resistant were tested on GeneXpert System Cepheid, Sunnyvale, CA, USA and on lateral flow assay (LFA) kits (Coris BioConcept, Belgium) for the presence of carbapenemase enzymes. A 0.5 McFarland suspension was prepared from a single colony for Carba-R testing, and the test was performed as per the manufacturer's instructions on GeneXpert System (Cepheid, Sunnyvale, CA, USA). LFA kits were obtained from Coris BioConcept, Belgium for the research purposes only. For LFA, a turbid suspension was prepared by emulsifying isolated colonies in the lysis buffer provided with the kit. Lysis buffer helped in releasing the carbapenemase enzyme. Three drops of this suspension were transferred to the well on the immunochromatography strip. Appearance of the control band indicated a valid run. Bands corresponding to O (OXA-48), K (KPC) and N (NDM) were indicative of production of those enzymes. Escherichia coli (ATCC 25922) was used as the negative control [Figure 1].
|Figure 1: Lateral flow assay for carbapenemase detection: from left to right - negative control, OXA-48 detected, Klebsiella pneumoniae carbapenemase detected, New Delhi metallo-beta-lactamase detected, OXA-48 plus New Delhi metallo-beta-lactamase detected|
Click here to view
| Results|| |
A total of 8739 samples (blood, respiratory secretions, urine and body fluids) were submitted for culture during the three-month study period. A total of 1948 samples showed significant growth. Sixteen of these were non-repetitive, carbapenemase-resistant Enterobacterales which caused clinically significant infections. They were recovered from blood (n = 6), respiratory secretions (n = 6), urine (n = 2) and body fluids (n = 2).
Out of 16 clinical isolates, 14 were K. pneumoniae and two were E. coli. OXA-48 was the most common enzyme detected in eight isolates, followed by OXA-48 and NDM co-production in six isolates, KPC and NDM in one isolate each. In this way, a total of 22 carbapenemases (OXA-48-14, NDM-7 and KPC-1) were detected by both the assays. Six isolates (K. pneumoniae-5 and E. coli-1) had co-production of OXA-48 and NDM. Resist-3 assay detected all twenty two enzymes. The distribution of enzymes in these sixteen isolates was identical to that shown by the Carba-R (100% agreement).
| Discussion|| |
Infections due to carbapenem-resistant bacteria are a cause of great worry in Indian hospitals. Newer beta lactamase inhibitor combinations are effective against KPC-or OXA-producers but not against metallo-beta-lactamases like NDM. Siderophore class of antibiotics (e.g., cefiderocol) seems to be effective against NDM-producers but remains to be evaluated in India. Looking at this scenario, it is evident that the microbiology laboratory needs to identify the type of carbapenemase in addition to the detection of phenotypic resistance.
For the detection of NDM, LFA could be less sensitive. Various authors also observed that picking up the colony from near the carbapenem disc increased the sensitivity for the detection of NDM. However, in our study, all the NDM-producing isolates were correctly identified despite being picked up from the original culture plate. The band for NDM was always less prominent, even a faint line was considered as positive according to the kit literature. The results in non-Enterobacterales might not be as encouraging due to non-carbapenemase mechanisms of carbapenem resistance in these organisms. There could also be the presence of uncommon carbapenemases such as GES, IMI, GIM and SIM in these organisms.
In India, NDM has been shown to be the predominant type of carbapenemase. However, in our study, OXA-48 was the most common carbapenemase (50%), in line with the observation of Shankar et al., indicating a possible shift from NDM to OXA in this region.
Major advantage of this test was a considerably (at least four times) lower cost as compared to the molecular assay and a lower turn-around time of around 15 minutes only. Furthermore, no special training is needed to perform the test and it could be done by any laboratory personnel. A possible drawback is the lower sensitivity for NDM detection and lack of validation for detecting carbapenemases from the direct clinical samples. The authors acknowledge the drawbacks of the study as the small sample size and non-availability of positive control strains for individual carbapenemase enzyme. These findings should be confirmed with larger studies performed on clinical isolates from India.
| Conclusion|| |
LFAs are cost-efficient and user-friendly, for the detection of carbapenemases in Enterobacterales. They should be more extensively studied in India to guide antibiotic therapy. We need to work towards pharmaceutical and diagnostic collaboration for research in this field. Immunochromatography for carbapenemase detection can help improve the algorithm of managing carbapenem-resistant Enterobacterales in India.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae
: Here is the storm! Trends Mol Med 2012;18:263-72.
Govindaswamy A, Bajpai V, Khurana S, Batra P, Mathur P, Malhotra R. Prevalence and characterization of carbapenemase-producing Escherichia coli
from a tertiary care hospital in India. J Glob Infect Dis 2019;11:123-4.
Veeraraghavan B, Shankar C, Karunasree S, Kumari S, Ravi R, Ralph R. Carbapenem resistant Klebsiella pneumoniae
isolated from bloodstream infection: Indian experience. Pathog Glob Health 2017;111:240-6.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: M100-S30. Wayne, PA: Clinical & Laboratory Standards Institute; 2020.
Petty LA, Henig O, Patel TS, Pogue JM, Kaye KS. Overview of meropenem-vaborbactam and newer antimicrobial agents for the treatment of carbapenem-resistant Enterobacteriaceae
. Infect Drug Resist 2018;11:1461-72.
Kazmierczak KM, Tsuji M, Wise MG, Hackel M, Yamano Y, Echols R, et al
. In vitro
activity of cefiderocol, a siderophore cephalosporin, against a recent collection of clinically relevant carbapenem-non-susceptible Gram-negative bacilli, including serine carbapenemase- and metallo-b-lactamase-producing isolates (SIDERO-WT-2014 study). Int J Antimicrob Agents 2019;53:177e84.
Saleh A, Göttig S, Hamprecht AG. Multiplex immunochromatographic detection of OXA-48, KPC, and NDM carbapenemases: Impact of inoculum, antibiotics, and agar. J Clin Microbiol 2018;56:e00050-18.
Verma N, Prahraj AK, Mishra B, Behera B, Gupta K. Detection of carbapenemase-producing Pseudomonas aeruginosa
by phenotypic and genotypic methods in a tertiary care hospital of East India. J Lab Physicians 2019;11:287-91.
] [Full text]
Shankar C, Venkatesan M, Rajan R, Mani D, Lal B, Prakash JAJ, et al
. Molecular characterization of colistin-resistant Klebsiella pneumoniae
its clonal relationship among Indian isolates. Indian J Med Res 2019;149:199-207.
] [Full text]