Case

1. 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

2. Gram stain

3. Culture results MRSA isolate S. aureus, not MRSA Sheep blood agar (TSA) MRSASelect Agar

4. 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

5. 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

6. 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

7. S. aureus: the basics • Gram-positive coccus, clusters • Key lab ID tests: • Beta-hemolytic • Creamy-white to yellow colonies • Catalase + • Coagulase (bound and free) +

8. 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

9. 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

10. 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)

11. 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

12. 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

13. 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

14. 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.

15. 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.

16. Data from Abu Ghraib/Camp Cropper: Gram-positive isolates from 2005-2007

17. 2006: Iraq-wide data, Gram-positive isolates (in-patient only)

18. S. aureus antibiogram: Abu Ghraib/Camp Cropper 2006 % Susceptible

19. S. aureus antibiogram: Iraq-wide, 2006 (inpatient data) % Susceptible

20. What’s the Big Deal? • Multi-drug resistant organisms are harder to treat • Worsened clinical outcomes • Longer hospital stays • Increased risk of passing bugs to new patients • Increased costs

21. What’s the Solution?Surveillance.

22. Why Do Surveillance? • Incidence of MRSA in hospitals is steadily increasing

23. 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

24. Why Do Surveillance? • Incidence of MRSA in hospitals is steadily increasing • Cost Savings

25. 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

26. Why Do Surveillance? • Incidence of MRSA in hospitals is steadily increasing • Cost Savings • (Limited) Evidence-based method to reduce incidence

27. Outcomes: Active Surveillance Controls MRSA BSIs Huang et al., CID 2006;43:971-8

28. Why Do Surveillance? • Incidence of MRSA in hospitals is steadily increasing • Cost Savings • (Limited) Evidence-based method to reduce incidence • Because they said so

30. Setting Up a Surveillance Program • What is the overall goal? • Which site should be cultured? • What should the overall strategy look like?

31. MRSA Surveillance • The goal is to decrease hospital acquired infections • Basic hypothesis: • Staph infections are spread from person-to-person • Identifying colonized patients using surveillance cultures at admission along prompt isolation of patients (plus good hand hygiene) will decrease MRSA transmission and infection

32. 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

33. Surveillance Strategies • Passive Surveillance • Targeted Active Surveillance (TAS) + High Risk Surveillance • Universal Surveillance

34. 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:

35. 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:

36. 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.;

37. Testing Methodologies

38. 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

39. Conventional Culture • Recommended by CLSI • Blood Agar + Cefoxitin Disk; confirm with PBP2a latex agglutination • or a plate containing 6 μg/ml of oxacillin in Mueller-Hinton agar supplemented with NaCl (4% w/v; 0.68 mol/L) • Some MRSAs are sensitive to NaCl

40. Chromogenic Media • FDA approved • Screening only—not for diagnosis or guiding treatment • low complexity • $ Spectra MRSA (Remel, Lenexa, KS) 24 hours MRSASelect (Bio-Rad, Woodinville, WA) 24 hours BBL CHROMagar MRSA (Becton Dickinson, San Diego, CA) 48 hours

41. 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 • $$

42. Cepheid GeneXpert • FDA approved • 1-16 site random access • Extraction, amplification, detection • TAT ~ 1 hour • moderate complexity • $$-$$$

43. MRSA testing methods: Let’s Compare

44. How Much Time? * Includes overnight broth enrichment

45. 1. Nares Samples Collected from Patients 3. Incubate Swab Overnight in TSY Enrichment Broth MRSASelect Agar, Bio-Rad 2. Samples Processed and Tested Using the BD GeneOhm MRSA Assay on the SmartCycler 4. Subculture TSY Broth to CHROMagar-MRSA and CNA BD GeneOhm™ MRSA Assay on the SmartCycler® PCR vs. Culture at the Seattle VA

46. PCR vs. Culture All Units totals

47. 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

48. 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

49. 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

50. Types and number of specimens directly plated onto MRSASelect agar (Bio-Rad)