Download The D Test

Clindamycin induction
test in treating patients
infected with methicilin
resistant Staphylococcus
aureus
Presented by Iyad Kaddora
Staphylococcus aureus
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Belongs to Micrococcaceae family.
Gram positive cocci.
Clusters resembling grape.
Part of the humans normal flora.
Exist in air and water.
Number one cause of nosocomial infections.
Approximately 25% to 30% of the population is
colonized.
Staphylococcus aureus images
Health info. 2005
S. aureus Virulence Factors
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Surface proteins
Invasions
Surface factors
Biochemical properties
Exotoxins
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Inherent and acquired resistance to antimicrobial agents
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molbio.princeton. university
40% of nosocomial S. aureus
infections are methicillin resistant
First reported in 1960s
 Causes severe morbidity and mortality worldwide
 Endemic in many European and American hospitals
 Many in-patients are colonized or infected
 25% hospital personnel may be carriers
 Spread by hand, usually of health
care workers
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Risk factors for methicilin resistant
Staphylococcus aureus colonization
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Advanced age
Male gender
Previous hospitalization
Length of hospitalization
Chronic medical illness
Prior and prolonged antibiotic therapy
Presence and size of a wound
Exposure to colonized or infected patient
Proportion of nosocomial MRSA
among the intensive care unit
patients
NNIS System.
Rates of hospital MRSA isolates
from 324 geographically distributed
US health care institutions in year
2003
Pfizer. © 2005 Pfizer Inc.
Antibiotics Resistance
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Antibiotics are used to treat bacterial infections,
but recently they start becoming less effective.
In the past few years S. aureus has began to show
more resistant to commonly used antibiotics.
S. aureus has developed new strains called
methicilin resistant Staphylococcucs aureus
(MRSA).
Methcilin resistance now refers to multiantibiotic resistant group.
Clindamycin antibiotic is drug of choice
for treating patients infected with MRSA
Clindamycin
 β-lactam antibiotic.
 Inhibits the synthesis of protein in bacterial organism.
 Prevents the bacteria from replicating.
The failure of clindamycin treatment to MRSA infected
patients, raises a big concern to health care professional
and attending physicians.
Antibiotics resistance mechanisms
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The resistance to erythromycin and clindamycin
occurs by one of two mechanisms.
Efflux: typically mediated by msrA gene.
Ribosome alteration: occur through methylation
of the ribosomal target site, and this resistance is
mediated by erm gene.
Objective:
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Many studies have been performed regarding MRSA.
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New test has been developed to test the future
resistance of MRSA strains to clindamycin.
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The hypotheses: MRSA strains are becoming more
resistant to clindamycin during 2004/2005 year
compared to 2000/2001.
Materials and methodology
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The testing specimens were collected in year
2000/2001, and year 2004/2005.
The specimens were saved in order to be evaluated for
clindamycin resistance.
D test is performed on MRSA strains from sources
other than urine.
MRSA has to be erythromycin resistant and
clindamycin sensitive.
The D Test
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D test is way to detect inducible clindamycin
resistance.
In order for the resistance to show up inducing
agent is required.
D test output:
1. Positive D test.
2. Negative D test.
D-test principle
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D test is done to allow erythromycin induce production
of methylase.
Erythromycin ribosome methylase is a gene that
encodes enzymes which confer inducible or constitutive
resistance to clindamycin.
Induced clindamycin resistance is detected by forming a
D shape letter on the agar plate.
A flattening of the zone in the area between the two disks
(letter D shape) will indicate the organism’s ability to induce
clindamycin resistance in the future.
Clindamycin disk
“D”
Erythromycin disk
D-test positivist on the original wound culture isolate from the case patient. E, erythromycin; CL, clindamycin.
Clinical Infectious Diseases 2003;37:1257-60
D test procedure:
Make 0.5 McFarland suspensions (turbidity
measurement) in3 ml sterile inoculum’s water.
 Sterile swab is used to inoculate
the organism to blood agar plate.
 Place an erythromycin disk 15 to
26 mm away from the clindamycin disk,
in the blood agar plate.
 Incubate 18-24 hours at 35°C.
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Disk induction testing.
(A)Constitutive clindamycin
resistance.
(B) Negative disk induction test
indicating the absence of
inducible clindamycin resistance.
(C) Positive disk induction test indicating
inducible clindamycin resistance.
Journal of Clinical Microbiology, April 2005, p. 1716-1721, Vol. 43, No. 4
Results of the D test
- All clinical S. aureus Isolates at the Johns Hopkins Hospital
- Nov 1, 2002 through Dec 17, 2002
- 512 Total S. aureus Isolates
Clinical Infectious Diseases 2003;37:1257-60
Clindamycin Susceptibility Testing for Adult & Pediatric
S. aureus Isolates
All Adult S. Aureus
Adult
NonDiscordant
Adult Neg DTest
17%
3%
16%
64%
Adult Dtest
Missing
Adult iMLS
Discordant Susceptibility
•Adult: 36%
•17% iMLS
All Pediatric S. Aureus
•Pediatric: 32%
Ped
NonDiscordant
Ped Neg Dtest
20%
•20% iMLS
4%
8%
68%
Ped Dtest
Missing
Ped iMLS
Clinical Infectious Diseases 2003;37:1257-60
Clindamycin Susceptibility Testing
for MRSA
All MRSA
20%
1%
20%
59%
MRSA
NonDiscordant
MRSA Neg
Dtest
MRSA Dtest
Missing
MRSA iMLS
Clinical Infectious Diseases 2003;37:1257-60
Reported Frequency of In Vitro Inducible
Resistance to Clindamycin in MRSA
Report
Location
(Pediatric vs.
Adult)
Subject for D-test Positive
D-test
Como-Sabetti
Minnesota
(Not specified)
64% of all MRSA
84%
Chicago
(Pediatric)
38% of all MRSA
94%
Houston
(Pediatric)
90% of all MRSA
8%
Baltimore
(combined)
Ped:25%
Ad: 44%
43%
51%
IDSA 2002 #92
Frank
PIDJ2002;21:530
Sattler
PIDJ2002;21:910
Current Study
Clinical Infectious Diseases 2003;37:1257-60
Clindamycin treatment of MRSA infections
 31/33 were D test positive
 9/31 treated with clindamycin
 1 could not be evaluated
 3 received multiple antibiotics
 5 treated with clindamycin only
3/5: minor skin infections resolved on
clindamycin
2/5: clinical failures on clindamycin
Frank PIDJ 2002;21:530.
Lab Protocol for S. aureus
S. aureus
cultured
MRSA
MSSA
Clinda R or
Clinda S and
Eryth S/Clinda S Erythro R
Routine D-test
Report Clinda MIC
+
Clinda S and
Erythro R
Clinda R or
ErythS /Clinda S
D-test at request of Physician
+
Report Clinda=R
Report Clinda MIC
Clindamycin Treatment of S.
aureus Infections
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MRSA
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Important therapeutic option if
Erythromycin and clindamycin sensitive
 Erythromycin-resistant, clindamycin sensitive IF D-test
negative
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MSSA
Beta lactam more widely used
 Concern for treatment failure same as for MRSA, if
clindamycin S, erythromycin R, D test positive
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Clinical Significance of In Vitro Inducible
Resistance to Clindamycin
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Increasing use of clindamycin for MRSA
Importance of awareness of potential for
increased risk of treatment failure with
positive D test:
 MRSA & MSSA
 Rates of positive D test in S. aureus may
varies by age, region, time
 D-test erythromycin R/ clindamycin S MRSA &
MSSA before reporting clindamycin susceptibility
Conclusion
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The high frequency of positive D test for MSSA
isolates raises concern that clindamycin may fail to treat
MSSA.
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Clindamycin should be avoided as a treatment for
patients infected by S. aureus exhibiting inducible
resistance
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The proportion of S. aureus with in vitro inducible
clindamycin resistance may vary by region, age group,
and methicillin sensitivity
Conclusion
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There has been a marked increase in the number of
patients with infections that are unreceptive to
clindamycin treatment.
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The large number of positive D test may explain the
failure of clindamycin in treating the infected patients.
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This study has demonstrated the importance of
performing D test on MRSA Isolates.
Future studies
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Is it gender specific?
Is it age specific?
Repeated infections?
Environment effects?
Molecular mechanism of antibiotics resistance?
Improving the D test.
Acknowledgment
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Dr. Harrison
BSC 661
Questions?