In-house Carba NP - II test to identify and differentiate carbapenemase-producing Gram-negative bacteria among various clinical isolates in comparison with immunochromatography assay

Introduction

Carbapenems are the most effective antibacterial agents available for the treatment of multidrug-resistant bacterial infections. However, the irrational and widespread use of carbapenems in unwarranted and avoidable situations has led to the emergence of certain enzymes (carbapenemase) that have the ability to hydrolyze the carbapenem ring and render the antibiotic useless. Carbapenem resistance in Gram-negative pathogens like Enterobacteriaceae, Pseudomonas species, Acinetobacter species has been on rise in recent years and is an ongoing public-health problem of global concern.1

Carbapenem resistance is mediated by transferable carbapenemase-encoding genes, hence they spread rapidly culminating in life threatening outbreaks and massively narrowing down the treatment options.2 Carbapenem resistant Gram-negative (CR-GNB) bacteria may contain any of the following enzymes such as KPC (Ambler class A), Metallo-b-lactamases (MBLs) of the VIM, IMP, and NDM types (Ambler class B), and OXA-48-type enzymes (Ambler class D).1

The most effective carbapenemases, with respect to carbapenem hydrolysis and spread, are KPC, VIM, IMP, NDM and OXA-48 types.2 KPCs inactivate all beta-lactam antibiotics and are only partially inhibited by beta-lactamase inhibitors like clavulanic acid, tazobactam and boronic acid. MBLs hydrolyze all beta-lactams except aztreonam and are not inhibited by the beta lactamase inhibitors like clavulanic acid, tazobactam and boronic acid. They have zinc in their active center, hence their inhibition is achieved in vitro using metal chelators, such as ethylenediaminetetraacetic acid (EDTA).2, 3 Therefore the identification of type of carbapenemase produced by CR-GNB is important for appropriate selection of antibiotic therapy.4

Many phenotypic tests are available for detection of carbapenem resistance; however, commonly employed methods are -

In resource limited middle and low-income countries, a reproducible, reliable, and cost-effective in-house test to detect and differentiate Carbapenemases belonging to Ambler’s molecular Class A, B, and D can play instrumental role in guiding the treating doctor for appropriate antibiotic therapy, which will subsequently contribute to successful implementation and adoption of antimicrobial stewardship in the times of global antibiotic crisis. 

Aims and Objectives

Materials and Methods

Carba NP - II test

1. The isolates that showed resistance to carbapenems on routine AST were subsequently subjected to Carba NP - II test.

2. First Carba NP test solutions are prepared:

   1. 0.1 N NaOH- by adding 40 mg in 10 ml distilled water,

   2. 10% HCL,

   3. Diluted solution of Phenol Red - prepared by using 0.5% wt/vol phenol red + 16.6ml distilled water (0.5% phenol red can be prepared by adding 50 mg of phenol red powder in 10 ml distilled water).

3. Carba NP - II test requires an additional:

   1. 10 mM ZnSo4.7H2O - prepared by adding 28.8 mg in 10 ml distilled water.

   2. 20 mM Tris HCL - by adding 314 mg in 10 ml distilled water.

   3. Imipenem - Cilastatin 1 gram injectable powder of Lupin Pharmaceuticals.

   4. Piperacillin and Tazobactam injectable powder of Intas Pharmaceuticals.

   5. 30 mM EDTA - by adding 111.7 mg in 10 ml distilled water.

4. Using these four solutions A, B, C, and D are prepared (Figure 1) 4

5. Carba NP - II test is performed using these four solutions (Figure 2).

6. Interpretation of Carba NP - II test results

   1. Colour change from RED to ORANGE or YELLOW indicates positive results (Ambler Class A, or B or D). No colour change indicates negative results. (Figure 3a & b).

   2. Quality Control Strains Used.

      1. Klebsiella pneumoniae ATCC 1705 MHT contain KPC.

      2. Escherichia coli ATCC BAA 2469 contain NDM.

      3. Klebsiella pneumoniae ATCC BAA 2524 contain OXA-48.

Figure 1

Preparation of four solutions using base solutions

Figure 2

Timeline ofCarba NP - II test

Figure 3

A: Interpretation of test results; B: Class B and Class D results

Immunochromatography Assay Test

1. The isolates that were subjected to Carba NP - II test were also evaluated by ICT, following the kit procedural manual (Figure 4). Positive control provided with the kit was used.

2. Interpretation of ICT test results: The presence of any red line in the test area indicates a positive result of its corresponding gene type. If the control line doesn't appear, the result is invalid, regardless of the appearance of any test line. (Figure 5)

Figure 4

Timeline of ICT test

Figure 5

ICT results

Observations and Results

Figure 6

Barchart showing Age and Gender distribution.(X-axis: Denotes Age & Gender, Y-axis: Percentage of CR-GNB)

Figure 7

Pie chart showing sample distribution

Figure 8

Pie chart showing CR-GNB distribution

Discussion

This study aimed to evaluate the performance of Carba NP - II test and immunochromatography test for the characterization and identification of carbapenemase-producing gram-negative bacteria.

In our study, 34.51% were carbapenem drug-resistant on routine AST, whereas a study by V. Mangayarkarasi et al 2018 reported 13.33% carbapenem resistance.3 In our study 30.8% were identified as Acinetobacter baumanii, 28.7% Klebsiella pneumoniae, 21.5% Pseudomonas aeruginosa, 13.8% Escherichia coli and the remaining were Enterobacter and Citrobacter whereas in Mangayarkarasi et al 45% were identified as Escherichia coli, 18% identified as Pseudomonas spp., 16% identified as Klebsiella pneumoniae, 10% identified as Acinetobacter spp., 7% identified as Citrobacter spp. and 4% identified as Proteus spp.3, 4, 5, 6

In-house Carba NP - II Test showed 100% sensitivity and specificity comparable to study of Laurent Dortet et al1 whereas Nitin Kumar et al showed sensitivity and specificity for KPC detection by Modified Carba NP as 91.7% and 100% and for MBL detection as (96.7% and 100 %).7, 8 ICT in our study showed 99% sensitivity and 100 % specificity comparable to atul garg atal showed 92% and 96% sensitivity and specificity9, 10, 11 whereas Eltahlawi RA et al study demonstrated an overall sensitivity of 69.3%. 12, 13, 14, 15

Conclusion

In-house Carba NP - II Test is an affordable and readily implementable method that can be adapted in microbiology laboratories in resource-limited countries for the identification of different classes of carbapenemases which will aid the clinician in providing appropriate antimicrobial therapy to the patients. Commercially available immunochromatographic assays are another good option for differentiation and identification of carbapenemases with similar results. However, these ICT kits are expensive.

Ethical Approval

Letter No. / Reference Code: IEC/NRI/68/2023, dated 08/08/2023

Source of Funding

None

Conflict of Interest

None