Carba NP Test (CNPt): Principle, Procedure, Results

Last updated on June 21st, 2021

The Carba NP test (CNPt) is a biochemical test for the rapid detection (≤2 h) of carbapenemase production in Gram-negative bacilli. Clinical and Laboratory Standards Institute (CLSI) recommends Carba NP test for ensuring the production of carbapenemases in EnterobacteriaceaePseudomonas aeruginosa, and Acinetobacter spp with elevated carbapenem MICs or reduced disk diffusion inhibition zones, for epidemiological or infection control purposes.

CNPt is highly sensitive for the detection of Klebsiella pneumoniae carbapenemase (KPC) and metallo-beta-lactamase (MBL) producers but less sensitive for detection of OXA-48-like carbapenemase.

Principle

When bacterial lysate hydrolyse imipenem, a carboxylic derivative is formed which decreases the pH of the medium. This change in pH is detected by a pH indicator, by changing the color of the medium from red to yellow.

Test Requirements

The test requires the separate preparation of few solutions.

Solution A: Concentrated solution of 0.5% w/v phenol red with 0.1 mmol/liter ZnSO4

  1. Prepare a concentrated solution of 0.5% w/v phenol red (0.5gm in 100ml distilled water)
  2. Mix 2ml of concentrated phenol red solution (vortex strongly before pipetting to resuspend the solution) with 16.6 mL of distilled water.
  3. Adjust the pH to 7.8 using drops of 1N sodium hydroxide (NaOH) solution.
  4. Add 180 µl of 10 mM zinc sulphate (ZnSo4) to obtain a final concentration of 0.1mM.

Note: This solution can be prepared and used at room temperature for 1 week and at -20ºC for months.

Solution B: Solution A + 6 mg/ml imipenem

Batches of imipenem powder solution can prepared and kept in advance at 4 ºC for 2 weeks if not mixed with solution A.

  1. 20mM Tris-HCl lysis buffer

Test Procedure

  1. Add 100μl of 20 mM Tris-HCl lysis buffer (available commercially) in each of two 1.5-ml Eppendorf tubes.
  2. Suspend 10μl of bacterial suspension in each of those 2 Eppendorf tubes. Ensure that the bacterial colonies have been resuspended or mix up and down using a pipette.

OR,

Alternatively, bacterial colony grown overnight on MHA can be scraped off using a loop and suspended in 100μl of 20 mM Tris-HCl lysis buffer mixed using a vortex device for 5 seconds.

  1. Add 100μl of solution A in the first and 100μl of solution B (solution A+6mg/ml of imipenem) in the second Eppendorf tube respectively.
  2. Incubate both tubes at 37°C for a maximum of 2 hours
  3. Observe the color change in both tubes and interpret accordingly.

Test Interpretation

The Carba NP test is interpreted with the change in color from the original red solution in both tubes.

Color in Tube A (without antibiotic)Color in tube B (with imipenem)Interpretation
RedRedNon- carbapenemase producer
RedOrange/yellowCarbapenemase producer
YellowYellowNon-interpretable

Usually, the time required for obtaining positive result varies for various type of carbapenemase

Type of carbapenemaseTime period for positive result
KPC producers2 to 30min
OXA-48 like producers20 min to 1hour
Metallo beta-lactamases (NDM, VIM, IMP)15 min to 1 hour

Modification of Carba NP

Many variants of the Carba NP test have since been described with changes in the inoculum size, extraction reagents, starting pH, pH indicators, and reading times.

  1. Blue Carba test utilizes the pH indicator bromothymol blue.
  2. The modified Carba NP test is another variant that uses 0.02% cetyltrimethylammonium bromide lysis buffer and a starting pH of 7.5 instead of 7.8, enabling the improved identification of carbapenemase producer. This also detects OXA 48 type carbapenemases to some extent.
  3. The Carba NP test II is another modification that identifies both carbapenemase production and also discriminates which class of carbapenemase is produced. It relies on pH changes due to imipenem hydrolysis in conjunction with specific enzyme inhibition through the use of tazobactam for KPC detection and EDTA for MBL detection.
  4. The Carb Acineto NP test is another variation designed to overcome difficulties with detecting carbapenemases produced by A. baumannii. The test requires modified lysis conditions and an increased bacterial inoculum compared to the original Carba NP test.

Carba NP limitations

  1. Although rapid results are obtained, the test requires fresh preparation of reagents (imipenem-containing solution) that have a maximum shelf-life of 72 h and appropriate storage.
  2. Fails to detect OXA-48 type producing strains.

References and further readings

  1. Pragasam A.K, Veeraraghavan B, Bakthavatchalam Y.D, Gopi R, Aslam R.F “ Strengths and limitations of various screening methods for carbapenem-resistant  Enterobacteriaceae including new method recommended by clinical and laboratory standards institute, 2017: A tertiary care experience”, Indian journal of medical microbiology, 2017; Volume: 35  |  Issue : 1  |  Page : 116-119
  2. Rudresh S.M, Ravi G. S, Sunitha L, Hajira S N, Kalaiarasan E , Harish B N “Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among Gram-negative bacteria” Journal of Laboratory Physicians. 2017 Oct-Dec; 9(4): 303–307
About Nisha Rijal 46 Articles
I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.