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