Case
 45-yo male veteran
 Good health, but woke with pain on top of left foot
 Looked like a small pimple, or boil
 Redness, some swelling, a little warm to the touch
 Expressed some pus from it
 The next day, more painful
 Expressed serosanguinous fluid from it
 Gram stain
Gram stain
Culture results
MRSA isolate
Sheep blood agar (TSA)
S. aureus, not MRSA
MRSASelect Agar
Rapid Identification of Methicillin Resistant
Staphylococcus aureus (MRSA) Directly From
Clinical Specimens
Steven Mahlen, PhD, D(ABMM)
Medical Director, Microbiology
Madigan Army Medical Center
Tacoma, WA
SAFMLS 2010: ST 8-1
Objectives
 Be able to discuss the importance of MRSA screening
 Be able to summarize the MRSA diagnostics methods
available today
 Be able to describe the validation of multiple specimen types
on culture media
Disclaimer:
Used Bio-Rad MRSASelect Agar for the study
--They did not pay us for the study
--Other chromogenic media may have worked as well
S. aureus: the basics
 Staphylococcus aureus and Staphylococcus epidermidis are
normally found on the skin or mucous membranes of
essentially all people.
 S. aureus may be found in the nose of 1/3 of the US
population
 S. aureus may also colonize other sites including the groin,
axilla, perianal region, and throat
 Breakdown in physical or immunological defenses can lead to
disease and/or death
Kuehnert MJ et al. 2006. Journal of Infectious Diseases. 193:172-9
S. aureus: the basics
 Gram-positive coccus,
clusters
 Key lab ID tests:
 Beta-hemolytic
 Creamy-white to yellow
colonies
 Catalase +
 Coagulase (bound and
free) +
Methicillin-resistant s. aureus (MRSA):
the basics
 Initially, S. aureus strains were sensitive to penicillin
 Penicillin:
Beta-lactam antibiotic
Acts against penicillin-binding proteins (PBPs)
 PBPs responsible for maintaining cell wall, peptidoglycan layer
 Resistance emerged quickly
Penicillinase: cleaved penicillin, rendered useless
1940
MRSA: The basics
 Methicillin was the response
1960
Synthetic antibiotic, related to penicillin
Worked against S. aureus strains that produced penicillinase
 Penicillinase could not cleave
S. aureus quickly became resistant
 “MRSA”; 1961
 Altered PBP
 Result: all beta-lactam antibiotics useless
Methicillin
MRSA
 mecA: gene that codes for methicillin resistance
 On mobile genetic element
Readily moves to other S. aureus strains
 Resistance to methicillin brings other resistances with it:
All beta-lactams
May include resistance to:
 Fluoroquinolones
 Macrolides
 Other antibiotics
 Often also called a multi-drug resistant organism (MDRO)
Definitions
 Colonization: Presence of MRSA in a host without
causing a specific immune response or clinical disease
 “Asymptomatic colonization or carrier state”
 Healthy people can be colonized with MRSA and have no ill
effects
 However, they can pass the bacteria to others
 Most MRSA is spread on the hands, clothing and equipment of
healthcare workers
Definitions
 Invasive Disease: The clinical manifestation of
symptoms caused by the invasion of MRSA into skin
tissue or otherwise sterile sites
 Staphylococci are generally harmless unless they enter the body
through a cut or other wound
 Often cause only minor skin problems in healthy people
 Illness is much more serious in immunocompromised patients
Invasive Disease
 S. aureus is the second most prevalent pathogen isolated in our
laboratory (Madigan Army Medical Center)
 Mostly abscesses, skin diseases (such as boils)
 Surprisingly (perhaps), not many cases of bacteremia
 At the Seattle VA, it is the most prevalent pathogen
 ~40% of bacteremia
 ~50% of all abscesses
Definitions
 HA-MRSA (hospital-associated MRSA):
 Obtained >2 days into hospital stay
 Often resistant to a greater number of antibiotics
(aminoglycosides, macrolides, fluoroquinolones)
 Responsible for great percentage of invasive disease; ~85%
nationwide
Klevens, RM et al. JAMA. 2007;298(15):1763-1771; Diep et al Lancet 2006; Han et al J Clin Micro 2007.
Definitions
 CA-MRSA (community-acquired MRSA):
 No history of recent hospitalization, medical procedure, or
indwelling device
 Typically skin and soft tissue infections (SSTIs)
 Athletes, prisoners, military recruits
 ~14% nationwide
 Military deployments
 Harder to tell CA and HA apart now
 Clusters of isolates with multiple resistance to erythromycin,
clindamycin, tetracycline, ciprofloxacin, and mupirocin
Klevens, RM et al. JAMA. 2007;298(15):1763-1771; Diep et al Lancet 2006; Han et al J Clin Micro 2007.
Data from Abu Ghraib/Camp Cropper: Grampositive isolates from 2005-2007
180
160
140
120
100
S aureus
Enterococci
80
60
40
20
0
2005
2006
2007
2006: Iraq-wide data, Grampositive isolates (in-patient only)
250
200
150
Saureus
Enteroc
100
50
0
Camp
Cropper
Ibn Sina
Balad
Total
S. aureus antibiogram: Abu
Ghraib/Camp Cropper 2006
Oxacillin
Cipro
Clinda
Erythro
Tetra
Bactrim
Outpatient
60
83
92
56
91
96
Inpatient
52
52
97
50
61
76
% Susceptible
S. aureus antibiogram: Iraq-wide,
2006 (inpatient data)
Oxacillin
Cipro
50-57%
52-70%
Clinda
Erythro
71-97%
43-54%
% Susceptible
Tetra
61-88%
Bactrim
67-89%
What’s the Big Deal?
 Multi-drug resistant organisms are harder to treat
 Worsened clinical outcomes
 Longer hospital stays
 Increased risk of passing
 Increased costs
bugs to new patients
What’s the Solution?
Surveillance.
Why Do Surveillance?
1.
Incidence of MRSA in hospitals is steadily increasing
Why Do Surveillance?
 10-30% of patients of colonized patients will go on to develop
invasive disease—rates are higher in severely ill patients (ICU)
 ~60% of soft tissue infections from EDs were MRSA
 2% in 1974; 22% in 1995; 64% in 2004
Moran GJ, et al. N Engl J Med 2006;355:666-74; Klevens RM et al. CID 2006;42:389-91
Why Do Surveillance?
1.
2.
Incidence of MRSA in hospitals is steadily increasing
Cost Savings
MRSA Cost for Hospitals
 Duke: Median attributable cost of MRSA blood stream
infection: $27 K
 Duke: MSSA vs. MRSA-- no increased hospital days; $19K
increase in cost
 BI Deaconess: MRSA vs. MSSA-- 1.36-fold increase in
hospital charges for MRSA vs. MSSA
 ENH: $2.25 million increased costs per year; 5 extra hospital
days
Cosgrove et al. ICHE 2005;26:166-174 Abramson and Sexton ICHE 1999;20:408-11; Kaye et al.
EID 2004:10:1125-8“Designing a Program to Eliminate MRSA Transmission Part 2: Making the
Business Case” Dr. Robicsek – APIC, 2007
Why Do Surveillance?
1.
2.
3.
Incidence of MRSA in hospitals is steadily increasing
Cost Savings
(Limited) Evidence-based method to reduce incidence
Outcomes: Active Surveillance
Controls MRSA BSIs
Huang et al., CID 2006;43:971-8
Why Do Surveillance?
1.
2.
3.
4.
Incidence of MRSA in hospitals is steadily increasing
Cost Savings
(Limited) Evidence-based method to reduce incidence
Because they said so
Setting Up a Surveillance Program
 What is the overall goal?
 Which site should be cultured?
 What should the overall strategy look like?
MRSA Surveillance

The goal is to decrease hospital acquired infections

Basic hypothesis:
1. Staph infections are spread from person-to-person
2. Identifying colonized patients using surveillance cultures at
admission along prompt isolation of patients (plus good hand
hygiene) will decrease MRSA transmission and infection
Which Site to Sample? Nares!
 The nose is the most consistent site for MRSA colonization—
1/3rd of US population
 MRSA carriage also in throat, axilla, groin, perianal
 Some evidence for increased sensitivity with collection of
samples from additional sites
 Some evidence that additional sites have marginal to no yield
Wertheim et al. 2005. Lancet Infectious Disease. 5: 751-762
Surveillance Strategies
 Passive Surveillance
 Targeted Active Surveillance (TAS) + High Risk
Surveillance
 Universal Surveillance
Surveillance Strategies
 Passive Surveillance: isolate patients when MRSA is
found on clinical culture requests— only identify ~15%
of MRSA colonized patients
 Targeted Active Surveillance (TAS) + High Risk
Surveillance:
 Universal Surveillance:
Surveillance Strategies
 Passive Surveillance—isolate patients when MRSA is
found on clinical culture requests— only identify ~15%
of MRSA colonized patients
 Targeted Active Surveillance (TAS) + High Risk
Surveillance: screen all ICU patients for MRSA, include
patients age>90, on dialysis, transplant, or living in long
term care—most recommended based on the available
evidence
 Universal Surveillance:
Surveillance Strategies
 Passive Surveillance—isolate patients when MRSA is
found on clinical culture requests— only identify ~15%
of MRSA colonized patients
 Targeted Active Surveillance (TAS) + High Risk
Surveillance: screen all ICU patients for MRSA, include
patients age>90, on dialysis, transplant, or living in long
term care—most recommended based on the available
evidence
 Universal Surveillance: screen everybody—limited
evidence; what intervals?
Siegel JD, et. al. Management of multidrug-resistant organisms in healthcare settings, 2006.
http://www.cdc.gov/ncidod/dhqp/; Salgado CD, Farr BM. Infect Control Hosp Epidemiol 2006;
27:116-121.;
Testing Methodologies
Broth Enrichment (BE)
 Overnight Incubation
 Brain-Heart Infusion (BHI); Trypticase Soy Yeast Extract
(TSY); Tryptone Soy Broth (TSB); many other
formulations
increase in sensitivity of chromogenic
media 10-25%
decrease in turn-around-time by 1 day
Safdar, N et al. : J Clin Microbiol. 2003 Jul;41(7):3163-6; Nahimana, I et al. Clin Microbiol Infect.
2006 Dec;12(12):1168-74; Lee, S et al. Ann Clin Lab Sci. 2007 Summer;37(3):248-50
Conventional Culture
 Recommended by CLSI
 Blood Agar + Cefoxitin Disk; confirm with PBP2a latex
agglutination
 or a plate containing 6 μg/ml of oxacillin in MuellerHinton agar supplemented with NaCl (4% w/v; 0.68
mol/L)
 Some MRSAs are sensitive to NaCl
Chromogenic Media
Spectra MRSA (Remel,
Lenexa, KS)
 FDA approved
24 hours
not for diagnosis or
guiding treatment
 low complexity
$
MRSASelect
(Bio-Rad, Woodinville, WA)
24 hours
BBL CHROMagar MRSA
(Becton Dickinson,
San Diego, CA)
48 hours
 Screening only—
BD GeneOhm MRSA Assay
 FDA approved
 14 samples (plus 2 controls)
per batched run
 Amplification and detection
(separate extraction
procedure)
 TAT ~ 1 hour
 high complexity
 $$
Cepheid GeneXpert
 FDA approved
 1-16 site random access
 Extraction, amplification,
detection
 TAT ~ 1 hour
 moderate complexity
 $$-$$$
MRSA testing methods: Let’s Compare
Method
Cost
Hands-On/
Complexity
TAT
Best Choice For…
Conventional
Culture (with
o/n BE)
Not recommended—
there are better
methods
Culture w/
Chromagar
Labs with limited staff
and resources
GeneOhm
Lab staff with adequate
staffing requiring quick
TAT
GeneXpert
Labs requiring quick
TAT with limited staffing
but financial resources
How Much Time?
Hands-On Time Complexity
per Sample
TAT
Conventional Culture *
10 min
low
4 days
BBL-CHROMAgar *
5 seconds
low
3 days
(positives-2 days)
MRSASelect *
5 seconds
low
1 day
MRSA-Spectra *
5 seconds
low
2 days
BD-GeneOhm
4.5 min
high
1 hour/14 samples
Gene-Xpert
2 min
moderate
1 hour
* Includes overnight broth enrichment
PCR vs. Culture at the Seattle VA
1. Nares Samples Collected from Patients
3. Incubate Swab
Overnight in TSY
Enrichment Broth
MRSASelect Agar, BioRad
BD GeneOhm™ MRSA Assay on the SmartCycler®
2. Samples Processed and Tested Using the BD
GeneOhm MRSA Assay on the SmartCycler
4. Subculture TSY Broth to CHROMagarMRSA and CNA
PCR vs. Culture
PCR
All Units totals
Culture
pos
neg
pos
94
6
neg
8
229
Prevalence
30%
Sensitivity
94%
Specificity
97%
PPV
92%
NPV
97%
PCR vs. Culture-Sensitivity
Is PCR more sensitive than culture??
 Depends on the study
 Overall consensus in literature is YES
 Increase in sensitivity is marginal in many studies
 Highest sensitivities for culture were with broth enrichment
(BE) step (60-70% sensitivity without BE)
 Results from our investigations found that BE + CHROMagar
had comparable sensitivity to PCR
 Most reported specificities are in the 85-95% range for both
methods
Next step
• We wanted to see if CHROMagar could be used directly on
clinical specimens
• Most studies:
– As surveillance (nares, etc.)
• Only two studies used directly as primary media:
– MRSASelect agar (BioRad) and CHROMagar MRSA
(BioConnections) primarily from surveillance sites with some
wounds (Davies et al., 2008, Br. J. Biomed. Sci. 65:13-17)
• 96% sensitivity for both types of media
– CHROMagar MRSA (BBL) from blood culture specimens
• 100% sensitive and specific
Initial study
• 333 consecutive clinical specimens
– Inpatients and outpatients
– Non-surveillance
– Direct plating onto MRSASelect agar (Bio-Rad)
• Not FDA-approved at time of study
– Compared to standard culture
• Sheep blood agar
• CNA
• Chocolate agar
• MacConkey agar
Types and number of specimens directly
plated onto MRSASelect agar (Bio-Rad)
Specimen Type (Number Tested)
Number [%] Positive for MRSA
Superficial Wounds/Ulcers (106)
28 [26]
Abscesses and aspirates (45)
11 [24]
Endotracheal aspirates (42)
6 [14]
Sputum (expectorated) (33)
5 [15]
Joint fluids (28)
2 [7]
Peritoneal fluids (26)
0 [0]
Tissues (various anatomic sites) (17)
2 [12]
Pleural fluid (14)
0 [0]
Bronchial washes (6)
0 [0]
Bone (5)
0 [0]
Bronchoalveolar lavage (5)
0 [0]
Cath tips and drains (5)
0 [0]
Cerebrospinal fluid (1)
0 [0]
Total specimens (333)
54 [16]
Initial study: results
• Overall: 54 positive specimens for MRSA (16%)
• Complete agreement of conventional culture with MRSASelect
agar for normally sterile sites
– Deep wound specimens, fluids, tissues
• MRSASelect agar was more sensitive for more complex specimens
– Sputum
– Endotracheal aspirates
– Superficial wounds and ulcers
– 10/54 positives
• 19% of samples, then, picked up only by MRSASelect
• CHROMagar not necessary for normally sterile specimens?
Based on this: phase 2
 305 consecutive clinical specimens
 Normally polymicrobial
 Sputa, endotracheal aspirates, superficial wounds/ulcers
 Inpatients and outpatients
 Direct plating, again, onto MRSASelect agar
 Compared to conventional culture
Phase 2: results
 Overall, MRSA from 88 specimens (29%)
 Overall, MRSASelect agar 18% more detection than
conventional culture
 One isolate found on conventional culture alone
Data for normally polymicrobial sites
Total
Superficial wounds
and ulcers
Endotracheal
aspirates and sputa
Number of cultures evaluated
486
240
246
MRSA isolated on any plate
127 (26%)
73 (30%)
54 (22%)
11 (15%)
15 (28%)
MRSA isolated from MRSASelect
26 (20%)
agar only
Conclusions
 Addition of MRSASelect Agar as a primary plate increased the
sensitivity of culturing MRSA from normally polymicrobial
sites
 Better for finding colonization?
 Increased turn-around-time by 1 full day
 May impact therapeutic decisions
 Definitely impacts infection control