The Clinical Laboratory’s Role in Detecting

and Reporting MRSA, VISA, and VRSA!

Janet Fick Hindler, MCLS MT(ASCP)*
UCLA Medical Center
Los Angeles, CA
*currently working with CDC’s Division of Laboratory
Systems through an Interagency Personnel Agreement
Following review of this presentation,
you will be able to……
♦ Discuss the epidemiology of nosocomial
and community-acquired MRSA infections.
♦ Describe the recommendations in the 2003
NCCLS standards for antimicrobial
susceptibility testing (AST) of S. aureus.
♦ Briefly discuss use of mecA and PBP2a
assays for detecting MRSA.
Following review of this presentation,
you will be able to (con’t) ……
♦ List effective ways to report MRSA, VISA,
and VRSA when isolated from diagnostic
specimens.
♦ Summarize the role of the microbiology
laboratory in contributing to proper
management of MRSA infections.
 MRSA
Epidemiology
Methicillin-resistant Staphylococcus aureus
 Epidemiology of MRSA

♦MRSA in hospitalized patients

♦Community-onset MRSA
 S. aureus
Bloodstream Infections
45
40
35
30
% MRSA

25 1997
20
1998
15
10 1999
5
0
Community onset Nosocomial

Diekema et al. 2001. Clin Infect Dis. 32(Suppl 2):S114.

Patient Risk Factors For Nosocomial
Colonization or Infection with MRSA

♦ Transfer of patient from nursing home

♦ Central venous or arterial catheter
♦ Severity of illness (& GI surgery)
♦ Extended length of hospitalization
(nosocomial transmission)

Safdar and Maki. 2002. Ann Intern Med. 136:834.

Wenzel and Wong. 1999. Clin. Infect. Dis. 28:1126.
 Patient Risk Factors For Nosocomial
Colonization or Infection with MRSA (con’t)

♦ Advanced age > 65 yr

♦ Ward changes; ICU, rehabilitation units
♦ Antimicrobial therapy – selection for resistance
− cephalosporins
− multiple antimicrobial agents

Safdar and Maki. 2002. Ann Intern Med. 136:834.

Wenzel and Wong. 1999. Clin. Infect. Dis. 28:1126.
 MRSA in
Non-Healthcare Settings
♦Correctional facilities
♦Men who have sex with men (MSM)
♦Competitive sports
♦School children

Salgado et al. 2003. Clin Infect Dis. 36(Suppl. 2):S131.

MMWR. 2003; 52:88.
 Some Control Measures for
Colonization or Infection with MRSA

♦ Focused hand-related hygiene

♦ Cohort patients & nurses if colonized/
infected
♦ “Patient profiling” from identified
nursing homes

Safdar and Maki. 2002. Ann Intern Med. 136:834.

 Some Control Measures for
Colonization or Infection with MRSA
(con’t)
♦ Screening cultures on admission - isolation
of carriers (contact precautions)
♦ Formal training in vascular access and care
♦ Micro Lab notification of Infection Control
♦ Closely monitored antimicrobial therapy

Safdar and Maki. 2002. Ann Intern Med. 136:834.

 MRSA
Testing
NCCLS Antimicrobial Susceptibility
Testing (AST) Standards
♦ Instructions for performing test (2003)
– M2-A8 Disk Diffusion
– M7-A6 MIC

♦ M100-S13 (2003)* “The Tables”

– Suggests drugs to test/report
– Interpretive Criteria (breakpoints)
– Quality Control ranges

*Updated annually
 NCCLS AST Standards
♦Describe “reference methods”
♦Clinical labs can use:
– NCCLS methods as written OR
– Method that performs comparably to NCCLS
“reference method” (e.g. FDA-cleared
diagnostic device)

Vitek Etest
MicroScan Other
 Staphylococcus spp. Therapy
Organism 1st Choice Drugs Alternative Drugs

oxacillin-S penicillinase-stable a cephalosporin,

penicillin vancomycin, ß-lac/ß-lac
inhibitor combo, imipenem
or meropenem,
clindamycin, a
fluoroquinolone

oxacillin-R vancomycin +/- linezolid, quin-dalfo, a

gentamicin +/- fluoroquinolone, a
rifampin tetracycline, trim-sulfa

The Medical Letter; 2001; 43:69.

 Penicillinase-Stable Penicillins
♦ Oxacillin = group representative
♦ Oxacillin performs best in in vitro test
systems
♦ Oxacillin results used to predict results of
other penicillinase-stable penicillins:
– Methicillin
– Nafcillin
– Dicloxacillin
♦ MRSA = ORSA
 Staphylococcus spp.
NCCLS Testing Methods
Inoculum: McFarland 0.5 suspension
from fresh colonies
Incubation: 35°C; 24h
Disk diffusion (DD): MHA*
oxacillin
transmitted light
MIC testing: CAMHB** + 2% NaCl
Agar screen: MHA + 4% NaCl +
6 μg/ml oxacillin
*Mueller-Hinton agar
**Cation-adjusted Mueller-Hinton broth NCCLS M100-S13
 Measure Zones for Oxacillin and
Vancomycin
USE Transmitted Reflected
Light Light
 Positive
Oxacillin-salt agar patient
screen for S. aureus
(MHA + 4% NaCl + 6 ug/ml
oxacillin) + control

- control

+ control – S. aureus ATCC 43300

- control – S. aureus ATCC 29213
Negative
patient
 Classic MRSA - Expression

♦ mecA = genetic determinant of MRSA

♦ Heterogeneous expression - in MRSA
(mecA +) population, some cells appear
“S” and others appear “R” under standard
test conditions
♦ Homogeneous expression - in MRSA
(mecA +) population, all cells appear “R”
Heterogeneous
expression

Haze within zone due

to oxacillin-R cells
Homogeneous
expression

Confluent growth
up to disk
 Other MRSA Test Methods

♦Commercial methods – follow

manufacturer’s instructions
precisely
♦mecA assay (e.g., by PCR)
♦PBP2a assay
 Staphylococcus spp.

“Isolates of staphylococci that are

shown to carry the mecA gene, or
that produce PBP2a, the gene
product, should be reported as
oxacillin resistant.”

NCCLS M100-S13; Table 2C

 PBP2a rapid latex agglutination
assay
- Perform on isolated colonies
- Extract PBP2a
- React with latex antibody to PBP2a
 MRSA
Reporting
NCCLS Table 1
Staphylococcus spp. Oxacillin
Group A
(Test & Report) Penicillin
 Staphylococcus spp.
Use Pen and Ox results to deduce results for other ß-lactams

Pen Ox Comments
S S S to penicillins, cephems,
carbapenems
R S R to ß-lactamase-labile pens;
S to ß-lactamase-stable pens;
S to ß-lac / ß-lac inhibitor
combos, cephems, carbapenems
R R R to all ß-lactams

NCCLS M100-S13; Table 1

 azithromycin or
clarithromycin or
erythromycin
Staphylococcus spp.
clindamycin
Group B
(Test but linezolid
Report Selectively)*
trimeth-sulfa

*based on your vancomycin

institution’s policies
 Staphylococcus aureus
clindamycin S
erythromycin S
oxacillin S
penicillin R
vancomycin S
“Cefazolin and other ß-lactams (except amoxicillin,
ampicillin, and penicillins) are active against
oxacillin-S and penicillin-R staphylococci.”

*Consider adding comment to report

to further explain results
Staphylococcus aureus
cefazolin S R*
clindamycin R
erythromycin R
oxacillin R
penicillin R
vancomycin S

*If any β-lactam is tested and tests “S”,

do not report or change to “R” for MRSA
 Staphylococcus aureus
clindamycin S
erythromycin S
oxacillin R
penicillin R
vancomycin S
Historically, MRSA has been multiply resistant to
other anti-staphylococcal agents. However,
some MRSA, particularly community-onset strains
are not multiply resistant.
 VISA / VRSA
Vancomycin-intermediate Staphylococcus aureus
Vancomycin-resistant Staphylococcus aureus
 Staphylococcus spp.
Vancomycin
MIC (µg/ml)
Susceptible ≤ 4
Intermediate 8 - 16
Resistant ≥ 32

VISA = 4 - 16 µg/ml
VRSA = ≥ 32 µg/ml
NCCLS M100-S13; Table 2C
 Detection of VISA / VRSA
♦ Disk diffusion
– VISA – not detected; MIC of 8 µg/ml are “S” by DD
(even at 24 h)
– VRSA - “R”; use transmitted light
♦ MIC
– VISA, VRSA - generally, overnight broth
microdilution systems will detect both
• Vitek, MicroScan may not detect VISA or VRSA
– Check S. aureus with vanco MIC ≥4 µg/ml
 Detection of VISA / VRSA
(con’t)
♦Brain heart infusion (BHI) agar with 6
µg/ml vancomycin*
– VISA and VRSA grow on this
– Incubate 24 h
– QC
• S. aureus ATCC 29213
• E. faecalis ATCC 51299
– Consider use if routine test method has been
shown to miss VISA / VRSA
*medium used for VRE screening
 Detection of VISA
(results obtained from various systems)
MIC (µg/ml)
♦ MicroScan Overnight 8
♦ Etest 6 -8
♦ Sensititre 4,8
♦ Vitek 4
♦ BHI-Van (6 µg/ml) growth
♦ MicroScan rapid ≤2, ≥ 16
♦ Disk diffusion inadequate

Tenover, et al. 1998. JCM. 36:1020

 Confirmation of VISA / VRSA

If you suspect a VISA / VRSA:

1. Repeat ID and susceptibility tests
2. Contact your institution’s infection
control department
3. Contact CDC at SEARCH@cdc.gov
4. Contact your local health department
5. SAVE ISOLATE!!
 VISA
♦ 8 cases to date in USA
♦ Pts. Previously had MRSA
♦ Pt. previously treated with vancomycin
♦ Most are MRSA

Fridkin et. al. 2001. Clin. Infect. Dis. 32:108.

Fridkin et. al. 2003. Clin. Infect. Dis. 36:429.
VISA colonies may be
smaller and slower growing
than typical S. aureus as
shown on this 48h BAP

VISA

Marlowe, et al. 2001. JCM. 39:2637.

MRSA (not VISA)
 VRSA – 1st Case
Michigan July 2002
♦ 40 y female, diabetic, hemodialysis
♦ Pt. previously had MRSA and VRE (vanA)
♦ Pt. previously treated with vancomycin
♦ VRSA isolated from catheter exit site and
foot wound
♦ Wounds healed at 3 months
♦ VRSA had mecA and vanA
MMWR. 2002; 51:565-7.
 VRSA – 1st Case
Michigan July 2002 (con’t)
♦ Routine vancomycin tests (NCCLS methods)
– MIC = 1024 µg/ml
– DD = no zone
– Screen plate (BHI + 6 µg/ml vancomycin) = growth

♦ “S” to chloramphenicol, linezolid,

minocycline, quin-dalfo, TMP-SMZ

MMWR 2002; 51:565-7.

 VRSA
st
1 Case VANCOMYCON

LINEZOLID

From Michigan
Health Dept. Lab
 VRSA – 2nd Case
Pennsylvania September 2002
♦ Chronic foot ulcer grew VRSA
♦ Routine vancomycin tests (NCCLS methods)
– MIC = 32 µg/ml
– DD = 12 mm
– Screen plate (BHI + 6 µg/ml vancomycin) = growth
♦ Etest vancomycin = 64 µg/ml
♦ “S” to chloramphenicol, linezolid, minocycline,
quin-dalfo, rifampin, TMP-SMZ
MMWR 2002; 51:902.
 QC of AST for S. aureus
NCCLS Recommendations
♦Disk diffusion
– S. aureus ATCC 25923
– QC daily or weekly
♦MIC
– S. aureus ATCC 29213
– QC daily or weekly
♦Oxacillin-salt agar screen
– S. aureus ATCC 43300
– S. aureus ATCC 29213
– Test each day of use NCCLS M100-S13
 Supplemental QA / QC of AST
for S. aureus
NCCLS Recommendations

♦Verification of AST results on patient’s

isolates
♦NCCLS M100-S13 (2003) - New
Verification Tables!

NCCLS M100-S13
 “Verify” Results
Patient’s Isolates - HOW?
♦ Check transcription
♦ Reexamine plate/tray, purity plate, etc.
♦ Check previous isolates on patient
THEN………
♦ Confirm ID and/or
♦ Repeat AST (alternate method?)
♦ Get assistance from reference lab
NCCLS M100-S13
 Verifying MRSA
♦NCCLS suggests verification if you
feel this is appropriate for your
institution
♦Consider verifying MRSA when
isolated from new patient (first time
MRSA for that patient)
Verifying VISA / VRSA
♦ Confirm identification
♦ Confirm AST results
♦ SAVE isolate in your lab
♦ Send isolate to referral lab that uses
NCCLS dilution reference method
Expanded guidelines coming to you SOON for
clinical laboratories on MASTER website and
from your Public Health Laboratory!
Visit this site and order “FREE”: CD ROM
www.phppo.cdc.gov/dls/master/default.asp