Modified Hodge Test (MHT): Procedure, and Why CLSI No Longer Recommends It

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.

Current status: CLSI removed MHT from its M100 performance standards in 2018, citing limited specificity (false positives in ESBL/AmpC producers with porin mutations) and poor sensitivity for metallo-beta-lactamases. CLSI now recommends the modified carbapenem inactivation method (mCIM) or the Carba NP test instead. MHT is covered here because it remains widely used in laboratories with limited resources and because it's the conceptual basis the newer tests build on, not because it's still the current recommended standard.

Requirements

Culture Medium:  Mueller-Hinton broth (MHB), Mueller-Hinton agar (MHA)

Organisms:

1. Escherichia coli ATCC 25922 (indicator organism)
2. Klebsiella pneumoniae ATCC® BAA-1705™ (positive control)
3. Klebsiella pneumoniae ATCC® BAA-1706™ (negative control)
4. Test strains (strains showing reduced susceptibility to carbapenems)

Others: Saline, swabs, forceps, inoculating loop, 0.5 McFarland standard

Procedure of Modified Hodge Test (MHT)

1. Prepare a 0.5 McFarland dilution of the E.coli ATCC 25922 (indicator organism) in 5 ml of broth or saline.
2. Dilute 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of MHB or saline.
3. 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.
4. Place a 10 µg meropenem or ertapenem susceptibility disk in the center of the test area.
5. 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.)
6. Incubate overnight at 35°C ± 2°C in ambient air for 16–24 hours.

Interpretation/Results

Figure: Figure 1. The MHT performed on a 100 mm MHA plate. (1) K. pneumoniae ATCC BAA 1705, positive result (2) K. pneumoniae ATCC BAA 1706, negative result; and (3) a clinical isolate, positive result

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.coli25922 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.

Learning and Remembering

Quick hook: A positive MHT looks like a clover leaf because the indicator E. coli literally grows toward the disk wherever the carbapenem next to it has been chewed up by the test organism's enzyme, growth follows the path the antibiotic no longer blocks.

Clinical story: MHT was introduced by CLSI in 2009 as the first widely available phenotypic screen for carbapenemase production, at a time when KPC-producing Klebsiella was just beginning its global spread and labs needed something they could run without specialized equipment. It did its job for nearly a decade, but the same simplicity that made it accessible also made it unreliable, by 2018, enough false positives from ESBL and AmpC producers, and enough missed MBLs, had accumulated that CLSI pulled it from the guidelines entirely. The test isn't gone from real-world labs, especially where budgets are tight, but its story is a useful one: a test doesn't have to be wrong to be replaced, it just has to be reliably less right than what came after it.

One sentence that captures it: MHT answers "can this organism inactivate a carbapenem," which sounds like the same question as "does this organism produce a carbapenemase," but the gap between those two questions is exactly why CLSI moved on to mCIM and Carba NP.

Exam facts

Further Readings

1. CLSI. M100—Performance Standards for Antimicrobial Susceptibility Testing. Clinical and Laboratory Standards Institute; current annual edition.
2. CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 28th informational supplement, M100-S28. Clinical and Laboratory Standards Institute; 2018. (MHT removed from recommended methods in this edition.)
3. CDC. Modified Hodge Test for Carbapenemase Detection in Enterobacteriaceae. Modified Hodge Test for Carpenamase Detection in Enterobacteriaceace