Modified Hodge test (MHT) is a simple phenotypic test for the detection of the presence of carbapenemase enzyme in bacteria. A positive MHT test is observed in Klebsiella pneumoniae carbapenemase (KPC), Metallo-β-lactamase (MBL), and the SME-1 in Serratia marcescens. Modified Hodge test (MHT) has been suggested as a screening test for carbapenemases.
It is based on the inactivation of a carbapenem by carbapenemase-producing strains (test isolate) that enable a carbapenem-susceptible indicator strain (E. coli ATCC® 25922) to extend growth towards a carbapenem-containing disc along with the streak of inoculum of the test strain. Positive test result gives cloverleaf-like indentation.
Culture Medium: Mueller-Hinton broth (MHB), Mueller-Hinton agar (MHA)
- Escherichia coli ATCC 25922 (indicator organism)
- Klebsiella pneumoniae ATCC® BAA-1705™ (positive control)
- Klebsiella pneumoniae ATCC® BAA-1706™ (negative control)
- Test strains (strains showing reduced susceptibility to carbapenems)
Others: Saline, swabs, forceps, inoculating loop, 0.5 McFarland standard
Procedure of Modified Hodge Test (MHT)
- Prepare a 0.5 McFarland dilution of the E.coli ATCC 25922 (indicator organism) in 5 ml of broth or saline.
- Dilute 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of MHB or saline.
- Streak a lawn of the 1:10 dilution of E.coli ATCC 25922 to a Mueller Hinton agar (MHA) plate and allow to dry for 3–5 minutes.
- Place a 10 µg meropenem or ertapenem susceptibility disk in the center of the test area.
- In a straight line, streak test organism from the edge of the disk to the edge of the plate. Repeat the same with the QC strain in another direction. (Up to four organisms can be tested on the same plate with one drug.)
- Incubate overnight at 35°C ± 2°C in ambient air for 16–24 hours.
After 16–24 hours of incubation, examine the plate for a clover leaf-type indentation at the intersection of the test organism and the E. coli 25922, within the zone of inhibition of the carbapenem susceptibility disk.
A. MHT Positive test has a clover leaf-like indentation of the E.coli 25922 growing along the test organism growth streak within the disk diffusion zone.
A positive test indicates carbapenemase production by the test microorganism. By producing carbapenemase, the test microorganism is able to inactivate the carbapenem that diffuses from the disk after the disk has been placed on the MHA. This allows carbapenem susceptible E. coli ATCC® 25922™ to grow toward the disk.
B. MHT Negative test has no growth of the E.coli 25922 along the test organism growth streak within the disc diffusion zone.
Limitations of MHT
- MHT demonstrates acceptable sensitivity for most carbapenemases, particularly KPC enzymes, but low sensitivity for MBLs.
- Isolates producing extended-spectrum -lactamases (ESBLs) or AmpC cephalosporinases in conjunction with porin mutations often yield false-positive results the MHT has limited specificity (around 91%)
- It is occasionally challenging to interpret and time-consuming, as it requires an additional 24-h growth step after AST results become available.
- Get details about Modified Hodge Test for Carpenamase Detection in Enterobacteriaceace in CDC Website
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Hello sir I really like your posts. Can you please explain immonodiffusion- single and double