Epidemiology of MRSA

Differences between CA and HA MRSA

M Louie MD FRCPC ProvLab Alberta University of Calgary
March 05 2008

Epidemiology of MRSA: differences between CA and HA MRSA

Objectives 1. Microbiologic differences 2. Best methods for screening / detection of MRSA 3. Methods for surveillance 4. Optimal reporting

Impact of MRSA
Significant morbidity and mortality
(Cosgrove CID 03; Whitby MJA 01)

Prolonged hospital length of stay

(Cosgrove ICHE 05; Engemann CID 03)

Excess costs
(Kim ICHE 01; Goetghebeur CJIDMM 07)

Worldwide Prevalence of MRSA 2006

Grundmann, Lancet 2006

Classical MRSA Epidemic Strains

70.0% 60.0% Percent MRSA Isolates 50.0% 40.0% 30.0% 20.0% 10.0% 0.0%
1995 n=227 1996 n=309 1997 n=638 1998 n=565 1999 n=1083 2000 n=773 2001 n=1461 2002 n=1363 2003 n=1450 2004 n=1623

CMRSA1 CMRSA2 CMRSA3 CMRSA4 CMRSA5 CMRSA6

Year

Canadian Nosocomial Infection Surveillance Program (Mulvey)

NML Type CMRSA1 CMRSA2 CMRSA3 CMRSA4 CMRSA5 CMRSA6 CMRSA7 CMRSA8 CMRSA9 CMRSA10

Global Clones USA600 / Iberian / Archaic USA100 / NYork / Japan USA800 / Pediatric USA700 / Brazilian / Hungarian USA200 / EMRSA16 USA500 / EMRSA15 USA400 / MW2 I

SCC

II I, IV III I I III IV

USA300

IV

NML, Mulvey; JID 2002 Simor

Dissemination of CA-MRSA

Victoria and Vancouver (USA300)

Calgary (USA 300)

USA 400

Toronto (USA 300)

Wylie and Nowicki. JCM. 2005, 43:2830-6.; Mulvey et al. EID 2005, 11:844-850; Gilbert et al. CMAJ. 2006, 175:149-154. (Courtesy of Mulvey)

Alberta Surveillance
In June 2005, MRSA was declared a pathogen under surveillance to determine the epidemiology of MRSA in AB.

Carriage Study Calgary 271 IVDA 5.5 % CMRSA10

Total MRSA Submissions for June 1 2005 - July 31 2007 (n=5674)

400 350 Number of Samples Submitted 300 250 200 150 100 50 0
O ct -0 5 O ct -0 6 A ug -0 5 A ug -0 6 Fe b07 Fe b06 Ap r06 Ju n05 Ju n06 Ap r-0 7 D ec -0 5 D ec -0 6 Ju n07
Region 1 n=293) Region 2 (n=180) Region 3 n=2590) Region 4 (n=612) Region 5 (n=98) Region 6 (n=1370) Region 7 (n=205) Region 8 (n=179) Region 9 (n=147)

Month

PFGE Types for June 05- Oct 06

1400 1300 Number of Samples Submitted 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 Region Region Region Region Region Region Region Region Region 1 2 3 4 5 6 7 8 9 Region of Alberta non assigned CMRSA 10 CMRSA 9 CMRSA 8 CMRSA 7 CMRSA 6 CMRSA 5 CMRSA 4 CMRSA 3 CMRSA 2 CMRSA 1

Collection Sites of Patients with 1st clinical MRSA isolates by CMRSA Type (June 1 2005 - July 31, 2007; n=5721)

80 70 % o f is o la te s c o lle c te d 60 50 40 30 20 10 0 CMRSA10 (n=2762) CMRSA7 (n=372) CMRSA2 (n=1760) CMRSA6 (n=310) CMRSA8 (n=58) Not Assigned (n=448) Inpatient (m=1834) Outpatient (m=2863) LTC (m=1760) Other (m=616)

CMRSA Type

Distribution of CMRSA 2 Cases by Age and Gender (June 1 2005 - July 31 2007) CMRSA2 Male (n=859) 800 700 600 500 400 300 200 100 0 <10 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-89 90-99 100-110 CMRSA2 Female (n=876)

Distribution of CMRSA 10 Cases by Age and Gender (June 1 2005 - July 31 2007)
CMRSA1 Male (n=1 0 754) 800 700 600 500 400 300 200 1 00 0 <1 0 1 9 0-1 20-29 30-39 40-49 50-59 60-69 70-79 80-89 90-99 1 00-1 0 1 CMRSA1 Female (n=963) 0

Sources of MRSA Isolates by CMRSA Type

1400 N ber of Isolates S um ubm itted 1200 1000 800 600 400 200 0
S of tT is su e R es pi ra to ry U rin ar y S ite s B lo od O th er

CMRSA 2 n=1149 CMRSA 7 n=284 CMRSA 10 n=1403

S ki n

an d

Source

S te ril e

Epidemiological Differences: HA and CA MRSA

HA MRSA Risk factors Carriage Carriage-Infection Clinical disease Recovery Antibiotics
Health care related Chronic illness Nares, groin, rectal Pre-dates infection Respiratory, urinary Older Mean LOS ~21d More resistant

CA MRSA
Specific risk groups (IVDA, homeless, etc) None known Uncertain Throat, vaginal, stool? Unknown SSTI Younger Mean LOS ~3d More susceptible

Popovich 08 CID; Huang 06 JCM; Naimi 03 JAMA

Epidemiology of MRSA: differences between CA and HA MRSA Are there microbiologic differences ?

Staphyloccus aureus
Methicillin-susceptible SA MSSA 90-95 % penicillin-R -lactamase production Plasmid mediated Methicillin-resistant SA MRSA Mediated by mecA gene Encodes for new PBP 2 Decreased affinity for lactam antibiotics Chromosomal mediated S MIC < 2 g/mL R MIC > 4 g/mL

S MIC < 0.12 g/mL R MIC > 0.25 g/mL

Staphylococcus aureus ? is history repeating itself ?

Penicillin resistance
100 90 80 70 60 50 40 30 20 10 0 1940 1945 1950 1955 1960 1965 1970 1975 Years

Methicillin resistance
70 60 % R e s is t a n c e 50 40 30 20 10 0 1980 1990 2000 Years 2010 HA CA

% R e s is ta nc e

HA CA

Methicillin Resistance
mecA located on chromosome
Staphylococcal Cassette Chromosome SCC
antibiotic resistance island may or may not carry other resistance genes several different types of SCC

Mobile genetic element (recombinase enzymes) integration

intM

MSSA chromosome

SCC I IA IV V II III IIIA

Fig. 1. SCCmec elements type IV observed in staphylococci. Based on Ito et al. (2001), Oliveira et al. (2001), Okuma et al. (2002) and Ito et al. (2004)pls, plasmin sensitive surface protein; kdp, kdp operon involved in ATP-dependent potassium transport across the bacterial cell membrane. HVR, hypervariable region; dcs, downstream constant sequence and conserved region in SCCmec types I, II and IV between the IS431 copy and orfX; ips, region between the IS431 right flanking copy of pT181 and the left flanking copy of pI258 in SCCmec III; hsd, type I restriction-modification

Hanssen FEMS Immunol Med Microbiol 2006

SCC TYPES
SCC I Size (kb) 34 + 0 No UK (archaic) 1960s SCC II 52 + Yes Yes Japan HAMRSA 1980s SCC III 66 + Yes Yes New Zealand HAMRSA 1980 1990s SCC IV 20-24 + 0 No CAMRSA SCC V 28 + 0 No CAMRSA

mec A
Trnsps Plasmids R to other ABs

1990s

2004

Resistance Profiles HA vs CA MRSA Isolates Case-Control CIHR Study n=123

100 90 80 70 % Resistance 60 50 40 30 20 10 0 CL
p < 0.009

HA MRSA CA MRSA

E
p < 0.10

Cp
p < 0.02

F
p < 0.34

M
p < 0.84

Ln

TS

Antibiotic

S. aureus Chromosome
SCC

Kuroda Lancet 2001 357:1225-40

Accessory gene regulator agr controls virulence gene expression during growth phase 4 types

Lowy 98 NEJM

CA-MRSA Virulence
MW2 strain (USA400)(CMRSA7) - 19 toxin genes identified including: Panton Valentine Leukocidin Enterotoxins cna (collagen-adhesin protein) USA 300/400 more virulent than other strains of MSSA / MRSA in a mouse bactermia model more resistant to killing by human PMNs isogenic PVL-negative (knockout) USA300/400 strains were as lethal as wild-type strains in mouse sepsis and abscess models similar lysis of human PMNs with wild-type and mutant (knockout) strains novel ACME (arginine catabolic mobile element) arcA gene, similar in structure to SCC found in USA300 SCC IV strains enhance survival and virulence on skin
Baba Lancet 2002 Voyich J Immunol 2005 Voyich JID 2006 Ellington JAC 2008

Potential Limitations in differentiating between HA and CA MRSA

Defining CA-MRSA
Community acquired Community onset Community associated
Buck 05 EID Folden 05 JHospInfect Salgado 03 CID

Potential Limitations in differentiating between HA and CA MRSA

Transmission of CA MRSA in hospital settings
USA400 post-partum infections(mastitis, cellulitis, abscesses), New York (Saiman CID 2003) USA400 outbreak in a neonatal and maternity unit, New York (Bratu EID 2005) USA300 prosthetic joint infections, Atlanta (Kourbatova
AJIC 2005)

USA300 in 28% healthcare-associated bacteremias, 20% nosocomial MRSA BSIs, Atlanta (Seybold,CID 2006)
Gonzalez 2006 ICHE Boyce 2008 CID

Potential Limitations in differentiating between HA and CA MRSA

Host specific factors

??

Microbial specific factors virulence

??

Constant evolution
Hanssen 06 FEMS Immunol Med Microbiol

Epidemiology of MRSA: differences between CA and HA MRSA

What are the best methods for acreening / detecting MRSA?

Laboratory Detection of MRSA

Approach Different

Screening Specimens

Clinical Specimens
DFJ Brown 2005 JAC

Screening Specimens for MRSA

Nasal Peri-anal Rectal Groin Axilla HA MRSA

Vagina Glaborous skin Throat CA MRSA ? Stool

Clinical Specimens for MRSA

http://www.metrowestcleangear.com/MRSA.htm

www.mja.com

Approach Different
Clinical Specimens
staphylococci grows in pure or mixed culture

Screening Specimens
staphylococci competes and grows with normal flora

Screening Cultures for MRSA Carriage

Solid media with or without prior broth enrichment of screening samples Enrichment media used to enhance detection of MRSA by overnight incubation before plating on solid media

Enrichment Broth Cultures

Allows small numbers of MRSA to grow during overnight or shorter incubation prior to subculture on solid agar media Increased length of time to detection & cost of processing (unless pooled swabs in one broth from same patient) Improved sensitivity in high-risk groups and in screening for clearance of MRSA

MRSA AGAR SCREEN Solid Agar Selective Media for Screening Swabs
Mannitol Salt Agar and ORSA media with/without oxacillin/cefoxitin .
Sensitivity 55-80%; Specificity 85-95%

MRSA-Select BioRad
Sensitivity > 90%; Specificity <24h >89%,>24h <50%

CHROMagars
Sensitivity >95%; Specificity >97%

MRSA Select vs MSA-8Fox

Simor et al JCM 2006 (#specimens) (# MRSA)
Overall (6199)(181) Catheter Exits (647)(14) Nares Swabs (2483)(57) Peri/Rectal (2312)(64) Sputum (58)(4) SSTI (632)(42)

Media
MRSA Select MSA-8Fox MRSA Select MSA-8Fox MRSA Select MSA-8Fox MRSA Select MSA-8Fox MRSA Select MSA-8Fox MRSA Select MSA-8Fox

Sensitivity % @ 24 h
93* 69* 100 50 90 70 97 66 100 50 91 81

Specificity % @ 24 h
99.5 92 99.8 98 99.6 97 99.3 85 100 100 99.5 96
p <0.0001

MRSA AGAR SCREEN

No single medium will recover ALL MRSA Complex formulations are more expensive Laboratory preference based on:
Ease to read Ability to differentiate from MSSA/CoNS Cost effectiveness

Clinical Isolates Screened for MRSA

Clinical S. aureus isolates are purified and screened for methicillin resistance

Approach Different
Clinical Specimens
Staphylococci grows in pure or mixed culture

Screening Specimens
Staphylococci competes and grows with normal flora

Agar SCREEN Presumptive MRSA

MRSA CONFIRMATION

MRSA Confirmation
Culture methods
Methicillin susceptibility testing

Non-Culture methods
Detect mec A gene or product
Phenotypic Genotypic

Methicillin-Resistant Staphylococcus aureus Detection

Testing Conditions as per CLSI

Medium: CA-Mueller-Hinton w NaCl Inoculum: direct colony suspension = 0.5 McFarland standard Incubation: 35C, ambient air, 24h

Methicillin Susceptibility Testing Culture Methods

Oxacillin disk diffusion Cefoxitin disk diffusion S/I/R

Oxacillin E-test MICs

Methicillin Susceptibility Testing Culture Methods

Oxacillin broth microdilution (MICs)

Automated Methods VITEK, MicroScan, Phoenix

Non-Culture Methods Phenotypic Assays

MRSA-Screen (Denka Seiken) Latex-coated monoclonal Abs to detect PBP 2 by latex agglutination directly from colonies

Non-Culture Methods Phenotypic Assays

BBL Crystal MRSA ID (Becton Dickinson) Contains 4ug/mL oxacillin broth Uses O2 sensitive fluorescent indicator, which in the presence of O2 is quenched and colorless Oxacillin-resistant bugs will grow and use O2 in the suspension broth, allowing fluorescence

Non-Culture Methods Genotypic Assays

Velogene Rapid MRSA ID (ID Biomedical) Genotypic assay without need to do PCR Isothermal CyclingProbe technology Uses fluorscein-labelled, biotinylated mecA probe
Negative mecA blue color Positive mecA colorless

Comparison (2001 Arbique et al DMID)

Sens % Spec % TAT Total $ (Kit $/test) 19.22 (15.55) 13.72 (10.00) 34.27 (29.75) 51.50 Number Steps 9 9 21 >20

BBL Crystal Denka Velogene PCR mecA

99 99 96 100

100 100 100 100

4 hr 15 min 2 hr 4-5 hr

Biosensor Assays ATP Bioluminescence method to detect viable MRSA

BacLite Rapid MRSA (Acolyte Biomedica) Swab is enriched in oxacillin broth Immunomagnetic capture with specific anti-S aureus monoclonal ABs Detection using ATP bioluminescence

Molecular Assays Nucleic acid amplification PCR

mecA, nuc, fem genes
In-house and commercial Sensitivity 93 100% Specificity 90 100% TAT
1 hr real-time amplification formats with molecular beacon detection can see fluorsecence during amplification eg. Smart Cycler 5 hr traditional PCR amplification, and gel electrophresis detection

IDI-MRSA
Assay directly on nasal specimens amplifies part of SCCmec Potential for deletion of mecA and parts of SCCmec Real-Time assay on Smart Cycler - TAT < 2 hrs Sensitivity 91.7 98.7% Specificity 93.5 98.4%

Where can we use Direct Molecular Detection of MRSA in Specimens?

Screening specimens Blood cultures positive with Gram Positive Cocci

IDI-MRSA use on admission to ICU

(Cunningham 2007 JHospInfect)

Overall carriage on admission 7% Relative risk reduction 0.65, 95% CI 0.28-1.07)

Are we ready for real time MRSA detection?

Rapid TAT to results from 2 hrs vs 3-4 days Faster time to patient isolation Reduced nosocomial transmission Rapid targeted treatment Assessment of cost-effectiveness High volume testing / workflow Sensitivity/Specificity/ PPV/NPV False negatives and False positives

Methods for Surveillance Molecular Typing

Antibiograms Phage typing Genotyping Methods
Index chromosomal variation Typeability Discrimination Reproducibility Applicability to broad range of organisms Ease of use Portability data sharing Low costs

Methods for Surveillance Molecular Typing

Pulsed field gel electrophoresis
SmaI digestion of gDNA & electrophoresis Current gold standard for benchmarking other methods Standardized protocols Labour intensive Interprative expertise Subjective analysis of complex bands and patterns - profiles Short term epidemiology

Canadian Epidemic MRSA

M 1 2 3 4 M 5 6 7 8 9 M

Methods for Surveillance Molecular Typing

Multi-locus sequence typing (MLST) Sequencing internal fragments of 7 conserved housekeeping genes Requires no interpretation Long term epidemiology / evolution Expensive Sequence types (ST) assigned Web accessible database linking epi data with isolate data May be less discriminatory than PFGE

Methods for Surveillance Molecular Typing

Spa typing
DNA sequencing of a polymorphic 24-bp variable-number tandem repeat (VNTR) within a single gene protein A Code the repeats eg. WGKAQQ Less costly than MLST More rapid and easy to perform Can detect micro and macro variations

Methods for Surveillance Molecular Typing

SCC Typing PCR multiplex amplification of SCC types Potential for same day Currently 5 SCC types with subtypes Novel types not yet characterized Not standardized