D-Zone Test: Introduction, Principle, Procedure, Result Interpretation, Limit

D-Zone Test: Introduction, Principle, Procedure, Result Interpretation, Limitations and Keynotes

Introduction of D-Zone Test

Putting clindamycin disk 15 mm away from the edge of an erythromycin disk is on an MHA plate incubating of organisms after 18–24 hours if shows flattening of the clindamycin zone of inhibition adjacent to the erythromycin disk appears as letter D zone and thus the test is called D-Zone test. It is applied for the detection of isolates whether inducible macrolide, lincosamide, streptogramin B resistance (MLSBi), or constitutive macrolide, lincosamide, streptogramin B resistance (MLCBc), or macrolide streptogramin (MS) phenotype or. erythromycin and clindamycin (ERY, CL) sensitive phenotype. It is most commonly used in Staphylococcus aureus.

Principle of D-Zone Test

D-zone test is proceeded by disk diffusion, placing a 15 μg erythromycin disk in proximity to a 2μg clindamycin disk on Muller-Hinton agar (MHA) plate that has been inoculated with Staphylococcus aureus; the plate is then incubated overnight. A flattening of the zone of inhibition around the clindamycin disk adjacent to the erythromycin disk is considered a positive result and indicates that the erythromycin has induced clindamycin resistance (D-zone test Positive). For erythromycin-resistant isolates, induction tests can help laboratories determine whether results for clindamycin should be reported as susceptible (when the induction test is negative) or as resistant (when the induction test is positive).

Requirements for D-zone Test

Test organism
Muller-Hinton agar
Antimicrobial disks -erythromycin (15 μg) and clindamycin (2μg)
Sterile swab stick
Mc-Farland Densitometer
Normal saline
Bunsen burner
Inocoluating wire
Incubator
Forceps/ antimicrobial disk dispenser
Waste bin
Control strain

Procedure for D-zone Test

Prepare 0.5 McFarland standard suspension of erythromycin-resistant Staphylococcus aureus isolates using either direct colony suspension or log-phase method of inoculum preparation.
Make a lawn culture of bacteria in agar plates and allow it to stand for 3 to 15 minutes in MHA.
Put clindamycin (2 μg ) and erythromycin (15 μg ) disks approximately 15 mm apart (measured edge to edge).
Invert plates and incubate the plate for 16 to 18 hours at 35°C in an ambient-air incubator.
Check the zone of inhibition.

Result Interpretation of D-Zone Test

Inducible clindamycin resistance: flattening of the zone of inhibition adjacent to the erythromycin disk (D-zone)
No inducible clindamycin resistance: completely round zone of inhibition around clindamycin disk
Hazy growth within the zone of inhibition around the clindamycin disk indicates clindamycin resistance, even if no D-zone is apparent.

Mechanism of Action of Erythromycin and Clindamycin and Development of Resistance

Erythromycin and clindamycin are two different classes of antimicrobial agents i.e. macrolide and lincosamide respectively that inhibit protein synthesis by binding to the 50S subunits of the ribosome in bacterial cells. Resistance to both of these antimicrobial agents can occur in staphylococci via methylation of their ribosomal target site and such resistance is typically mediated by erm genes.

Clinical significance of Inducible Clindamycin resistance

Macrolide-lincosamide-streptogramin B (MLSB) resistance is mediated by the target side modification mechanism, resulting in resistance to erythromycin, clindamycin, and streptogramin B.

Limitations of D-Zone Test

Even being positive results for inducible clindamycin resistance, clindamycin may still be effective in some patients.
The D-zone test is only standardized to detect inducible clindamycin resistance for some bacteria like Staphylococcus, S.pneumoniae, and beta-hemolytic Streptococcus.
Strict adherence to protocol is required to ensure reliable findings since numerous factors such as inoculum size of the test organisms, rate of growth, pH and formulation of media, incubation condition and duration, disk content can affect the result.

Keynotes

Inducible clindamycin-resistant isolates should be reported as clindamycin-resistant alongside a comment stating that inducible clindamycin resistance is in vitro results and clindamycin may still be effective in some patients
Clindamycin is an attractive agent for empirical therapy for suspected S. aureus infections because of its excellent pharmacokinetic and pharmacodynamic properties. Clinical failures of clindamycin therapy for the treatment of Methicillin-Resistant Staphylococcal aureus (MRSA) infections have been documented for strains that were clindamycin sensitive but erythromycin resistant. The failures were due to inducible resistance to clindamycin.
MHA with 5% sheep blood or TSA supplemented with 5% sheep blood is used for streptococci (beta-hemolytic streptococci and pneumococcus) D-Zone test in which antimicrobial disks are placed 12 mm apart for 20 to 24 hours at 35°C in a 5% CO₂ incubator.

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