D.M. Wolk

Arizona Health Science Center -Tucson • University Medical Center • 380 beds • 100,000 patients/year

Clinical Laboratory Response to Sepsis ASMCUE, San Diego, CA 5/21/2010

University of Arizona Donna M. Wolk, MLS(ASCP), MHA, Ph.D., D(ABMM) Division Chief, Clinical and Molecular Microbiology, Arizona Health Science Center Principle Investigator, Infectious Disease Research Core, BIO5 Institute Associate Professor, Pathology

for Collaborative Bioresearch

Objectives • Identify key clinical symptoms and the microbial and immunological conditions that characterize sepsis

What is Clinical Microbiology?

• Describe microbiology’s role to deliver sepsis-related laboratory results and support Surviving Sepsis Campaigns • Describe Medical Laboratory Scientists’ (MLS) training requirements for careers in medical microbiology laboratories • Define current standards and limitations of blood culture methods and the premises for new diagnostic method development to support rapid de-escalation of antibiotics

The study of pathogenic microorganisms such as bacteria, fungi, parasites and viruses.

D.M. Wolk

Clinical Microbiologists • We work in hospitals and in research laboratories. • You may not see us at a patient’s bedside - but we are behind the scenes, working to support patient care through diagnostic laboratory testing.

What is a clinical microbiologist? • Doctoral level scientists (MD, Ph.Ds) who direct and oversee the operations, practices, services, and technical personnel in diagnostic clinical microbiology laboratories in hospitals, public health, or other medical settings and provides consultation to physicians regarding infections

• We are saving lives; …one laboratory test at a time.

Advancing diagnostics…saving lives.

Clinical microbiologists oversee … • Operations to isolate and identify infectious organisms (bacteria, viruses, parasites, or fungi) from blood, urine, body fluid, sputum, wounds, or other specimens • Practices to provide crucial information that guides the selection of antimicrobial therapy for patients , as well as the proper specimen selection, collection, and culture of pathogenic microorganisms and normal flora.

Clinical microbiologists oversee … • Services that are critical to the well-being of sick patients by enabling the correct diagnosis to be made • MLS Personnel, highly trained professionals (board certified medical laboratory scientists) who make countless decisions each day that save lives and benefit the sick and at-risk members of our society

D.M. Wolk

Clinical Microbiology Laboratories • CML are the centerpiece of infectious disease diagnosis and the cornerstone of infection control /prevention. – Use laboratory techniques including culture, microscopic examination, biochemical, molecular and immunological tests. – Identify the best course of antibiotic treatment by establishing which antibiotics will be most effective in combating the organism causing an infection.

Clinical Microbiology Subspecialties • • • • • • • •

Bacteriology Virology Mycobacteriology Mycology Parasitology Serology Antimicrobial Testing Molecular Microbiology

Medical Laboratory Scientosts • • • •

4 year degree plus board certification Medical Technology/Medical Laboratory Science Microbiology Molecular Biology

• • • •

ASCP – American Society for Clinical Pathology MT(ASCP) M(ASCP) MP(ASCP)

• Others

Ph.D and Post-doctoral Programs

Microbiolgy

D.M. Wolk

The American College of Microbiology • Within the College, the Committee on Postgraduate Educational Programs (CPEP) has developed standards for the training of postdoctoral-level (Ph.D., M.D., Sc.D., O.D., Dr. P.H., etc.) medical microbiologists and immunologists in laboratory medicine. • CPEP-training (1-2 years) prepares scientists or clinicians for employment in clinical and medical microbiology laboratories, clinical and medical immunology laboratories, public health laboratories, university and industrial research programs, and college and university educational settings. http://www.asm.org/Academy/index.asp?bid=2108

Molecular Diagnostics: Subspecialty • A subspecialty of Laboratory Medicine in which molecular biologic methods are used to test nucleic acids (DNA/RNA) • Molecular Diagnostics (Clinical Pathology) • Molecular Microbiology, most of testing and revenue • Histocompatibility (HLA) • Molecular Hematopathology and Cytogenetics • Molecular Genetics, Inherited Disease • Molecular Pathology • Oncology • Cytology • Forensics and Identity Testing

Clinical Microbiologists in Health Care

Specialized Practice in Clinical Microbiology

• The health of individuals and communities hinges on the services provided by clinical microbiology laboratories and the expertise of the professionals who provide these services

D.M. Wolk

Clinical Microbiologists are Sentinels – Serve as the sentinels for anomalies, natural outbreaks of infection and bioterrorism events which threaten the public health. – The first to recognize the emergence of resistance to frequently used antimicrobial agents.

Clinical Microbiologists Collaborate • Optimize both service and cost savings for the health care system. • Perform strategic planning and evidence-based interventions • Educate and collaborate with healthcare team • Support community educational outreach

Clinical Microbiologists Assure Quality

Clinical Microbiologist Mine Data

• Assure quality and compliance with federal state and local regulations, including safety

– Generate reports regarding test positivity rates and antibiotic resistance trends.

• Ensure competency of staff and accuracy of lab results

– Integrate with Laboratory information systems to support communication between laboratory and physicians and other laboratories (including local, state, and national public health laboratories)

D.M. Wolk

Clinical Microbiologists in Public Health

Clinical Microbiologists in Epidemiology

• Deal with live microorganisms that can disrupt the public health through devastating local, regional, national, and worldwide outbreaks.

• The study of factors affecting the health and illness of population; it serves as the foundation of interventions made in the interest of public health and prevention

• Manage outbreaks of infectious disease by identifying and curbing pathogens that could potentially infect dozens, hundreds, or even thousands of people. • Recognize, track, and control outbreaks at the community level. • Characterize both phenotypic and molecular traits of microorganisms .

Clinical Microbiologists in Research • Investigating and understanding the virulence of pathogens and pathogenesis or human response to disease – Many organisms isolated in the laboratory are clinically unimportant under some circumstances and clinically significant in others. Virulence factors are studied. – Search for new or forgotten infectious diseases. – Developing new diagnostic capabilities. – Evidence-based interventions

Clinical Microbiology and Translational Research – Collaborate with industry in development of new diagnostics assays and clinical trials and evidence-based research – Support Experimental Design and Technology Transfer Clinical Lab State of the Law Proof of Utility Medical Principles

1. Standard Practices

2. Quality Systems

Requires FDA Review, In vitro Device

Research Lab State of the Art “Proof of Concept” Scientific Principle GLP

Diagnostic Laboratory Operations

3. Proven outcomes & Indicators

D.M. Wolk

Translational Research and Clinical Trial Unit Method Development, Consulting, and Testing FDA methods GOAL: Bench to Bedside

Clinical Laboratory Response to Sepsis

Bacterial Cells Trigger Immune Response

Bloodstream infections can lead to SEPSIS which is often fatal.

Gram Negative Sepsis • Endotoxin Lipopolysacharride (LPS) from cell wall initiates inflammatory response

Gram Positive Sepsis • Lipotechoic acid • Peptidoglycan • Extracellular toxins

Other Antigens, common to gram neg, pos, etc. Carbohydrate and Lipid

D.M. Wolk

Incidence of Sepsis, According to Organism (Population-Adjusted); HAIs play a role

Blood Culture Collection

Collect Blood Volume Based on Patient Weight Weight, (kg)

ALWAYS: Use HAND HYGIENE & Gloves Include EMPLOYEE ID on lab requisition SCRUB with ChloraPrep® Swab

NO “Quick-Draws”; AIM for 0% Blood Culture Contaminants ?s to 694-2601

< 1.5 – 2 2- 4 4 - 13

~ Weight (lb) 25

> 55 - Adult

20

Divide Total Volume Equally Between Bottles FAN FAN BacT Alert BacT Alert (Aerobic) (Anaerobic) X X X X X (if needed)* X X (if needed)*

* If no anaerobic bottle is required, place full amount into 2 aerobic bottles

PERIPHERAL DRAWS (SKIN)

1. REMOVE Swab

2. SCRUB 30sec 3. DRY SKIN, 30 sec 4. Collect with FRICTION NO blotting/fanning, etc. Blood

5. Inoculate bottles; 10ml ea

LINE DRAWS

1. REMOVE Swab

2. SCRUB 30sec 3. Remove End Cap; 4. Place New 5. Inoculate with FRICTION Collect blood; Adult 20ml End Cap bottles; 10ml ea

Martin, G. et al. N Engl J Med 2003;348:1546-1554 AVOID Contamination Events that Affect Your Patient and Waste Valuable Healthcare Resources

Blood Culture Method and Phenotype

Immune Response = Inflammation • Human’s mount protective host responses (vascular, cellular, chemical) and typical inflammatory functions aim to – remove bacteria and cell debris by phagocytosis. – stop injury with edema which dilutes toxins.

• Sepsis results from body’s out of control response to pathogens, toxins, etc.

D.M. Wolk

Inflammation: Response Options

Gradient of Disease 1. Bacteremia/Bloodstream Infection (BSI): Presence of viable bacteria in blood (Hematopathogen or skin contaminant?) 2. SIRS Systemic Inflammatory Response Syndrome 3. Sepsis: SIRS plus a culture-documented infection 4. Severe Sepsis: Sepsis plus >1 organ dysfunction 5. Septic Shock

Sepsis/ Organ Dysfunction

Overwhelming Infection (Death) Out of control immune response (Death)

– –

Hypotension (despite fluid resuscitation) plus Hypoperfusion (perfusion abnormalities which may include, but are not limited to lactic acidosis, oliguria, or an acute alteration in mental status.)

6. Death Infection Controlled = REGULATION = (Survival)

Clinical Symptoms • Systemic Inflammatory Response Syndrome (SIRS) • Two or more of the following: – Temperature of >38oC or 90 bpm – Respiratory rate of >20 or PaCO2 12 x 109/L or

0

•

Prolongs length of stay in hospital, 4-8 days Angus DC, et al. Crit Care Med. 2001;29(7):1303-1310.

Age (y)

Angus DC, et al., Crit. Care Med. 2001. Walston et al. Am J Respir. 2003 Williams, et al Critical Care 2004, 8:R291-R298

2004 through 2005 MEDPAR Hospital Discharge Databases

D.M. Wolk

Primary Point of Contact is often the Emergency Dept.

“Until a cure for sepsis is found, early detection is the surest hope for survival” The Sepsis Alliance

Bacteremia Fungemia, Parasitemia, Viremia, Other

Goal: Rapid Administration of “Targeted” Antibiotics

Infection

Burns, Trauma, Pancreatits, Other

SEPSIS

SIRS Severe Inflammatory Response Syndrome

Death can occur quickly, in ED, needs results in 4-6 hrs for maximum impact

Duration of hypotension before initiation of effective antimicrobial therapy is the most critical determinant of survival in human septic shock

• “There is strong evidence showing that the rapid administration of an effective antibiotic saves more lives than virtually any other medical intervention”

• Appropriate Antibiotics For every hour sooner, survival increases by ~ 7-10%

• • • • •

• Appropriate antibiotic therapy increases survival by ~ 25-45%

Dellinger RP, et al. 2008. Crit Care Med 2008;36:296-327. Jaeschke RZ, et al Pol Arch Med Wewn 2008;118:92-95. Kollef MH, et al Chest 1999;115:462-474. Garnacho-Montero J, et al, Crit Care Med 2003;31:2742-2751. Valles J, et al, J Infect 2008;56:27-34.

1

Fraction of total patients

Rapid Targeted Antibiotic Therapy Saves Lives

0.8

Survival Fraction Cumulative Effective Antimicrobial Initiation

0.6

0.4

0.2

0

Time from hypotension onset (hrs)

Kumar A. et al., Crit Care Med 2006, 34:1286

D.M. Wolk

Rapid De-escalation of Rx Saves Lives

Rapid Intervention?

• Speed of de-escalation is critical • Discontinue IV broad-spectrum antibiotics within 3 - 5 days – Re-evaluate daily to focus therapy, prevent resistance, avoid toxicity, and minimize costs. Kumar et. al, 2006. Crit. Care Med. (34) Sharma, S. and Kumar, A. 2008 29(4)

Rapid Diagnostic Methods Urgently Needed to Support Diagnosis and Appropriate Antibiotic Therapy

Translational Research Infectious Disease Research Core Vision: To improve infectious disease diagnosis and prevention by the development of new diagnostic laboratory methods http://idrc.bio5.org

Translational Research in Clinical Laboratories Diagnostic Method Development

Advancing Diagnostics…Saving Lives

D.M. Wolk

Microbiology Lab Drives Antibiotic De-escalation

Collaboration with Emergency Dept. and “Surviving Sepsis Campaign” 1. “Sepsis Bundles” - aimed at reducing mortality with criteria for patient assessment, early recognition of symptoms, and aggressive intervention and therapies 2. “Code Sepsis” was initiated in UMC Emergency Dept., Laboratory, Nursing, etc.

1. Results phoned to Pharmacy 2. Assess patient for current therapy, co-morbidities, previous antibiotics 3. Pharmacy contacts MD to intervene 4. If warranted, antibiotic intervention occurs

Our Goals for Rapid Laboratory Testing • Provide rapid laboratory results to target antibiotic therapy and maximize effectiveness – Decrease mortality and morbidity – Shorten antibiotic treatment duration, and avoid development of resistance and side effects – Decrease length of stay – Decrease overall hospital costs

Methods for Rapid Identification of Pathogens in Blood Culture Bottles

D.M. Wolk

Traditional vs. Rapid Identification Day 0

Day 1 Day 2 Call to physician Positive BC

Day 3

Day 4

Traditional Species ID

Antibiotic

AdvanDx (bioMérieux) PNA FISHTM Testing

Gram Stain Empiric Rx

BC collection

Broad-spectrum

Targeted Rx

Molecular Species ID and Antibiotics

Empiric Rx Targeted Rx; No more bottleneck

Fluorescence from Probe binding to rRNA in Bacteria Fix, Hybridize, Wash Mount, Read w/ Fluorescent Scope 97-100% sensitivity and specificity per insert

PNA FISH Targets rRNA in Microbe Chromosomes 16S rRNA

• Advantages of small subunit rRNA Target – Sequences are known and unique between species

1.5

T.A.T.= T.A.T.= 1.5 1.5 Hr Hr H.O.T. H.O.T. == 12 12 min min..

– Highly abundant target, multi-copy

Fuchs et al. 1998. AEM 64:4973-4982

– PNA probe can bind in highly conserved regions that are not accessible to DNA probes

D.M. Wolk

Advantages over conventional nucleic acid probes

Treatment Algorithm – Streptococci1

• The strength of the FISH signal, is > a DNA probe

Positive Blood Culture

Gram-Positive Cocci in Pairs and Chains

Gram Stain Performed – PNA Chemistry creates very small dye-labeled probes ~ 12 - 20 mers, so easy to hybridize to target and they bind tightly

42%2

E. faecalis

– Neutral charge: No natural repulsion with charged backbones – Exquisite base-discrimination – More specific binding to target DNA or RNA – Very low background, therefore greater sensitivity

27%2

31%2 Nonenterococci (Mainly streptococci)

Other enterococci (Mainly E. faecium)

0% Amp R 5% VRE

100% AmpR 65% VRE

N/A

Rx = Ampicillin

Rx = Linezolid

Rx = Ampicillin/Ceftriaxone

1. Toombs et al. IDSA 2006, Toronto, Canada. Oral Presentation 131. and Forrest, UMMC 2. Johnson et al. Poster # C-147. ASM 2006. Orlando, Florida.

Reduction in Mortality, Streptococci Forrest, G. N. et. al Antimicrob Agents Chemother 52

Treatment Algorithm – Yeast1

• A quasi-experimental study: PNA FISH was added to a treatment algorithm for HA-enterococcal bacteremia

Positive Blood Culture

Yeast

Gram Stain Performed

• Primary outcome assessed = Time from blood culture draw to implementation of effective antimicrobial therapy

53% 1

C. albicans

16 %

C. glabrata

• n =129 in the pre-intervention, n= 95 w/ PNA FISH • Results – – – –

E. faecalis identified (1.1 vs. 4.1 days; p < 0.001) -3d E. faecium identiied (1.1 vs. 3.4 days; p < 0.001) -2.3d Decreased 30-d mortality (26% vs. 45%; p = 0.04) Overall hospital savings $20,000/year

1% FluR

>28% FluR

Fluconazole w/o prior azole exposure2

Aggressive therapy. Consider non-azole

1. 2. 3. 4.

Pfaller et al. J Clinical Microbiology. 2002 Mar;40(3):852-856 Forrest et al. J. Clinical Microbiology. 2006 Sep;44(9):3381-3 Pappas et al. IDSA Guidelines. CID. 2004 Jan;38: 161-189 Spellberg et al. CID. 2006 Jan; 42:244-51

3,4

31%

Non albican, Non-glabrata

9% FluR

Treat based on species distribution until culture identification is confirmed

D.M. Wolk

CLINICAL APPLICATION Forrest et al. J Clin Microbiol. 2006 Sep;44(9)

Ongoing studies - UMC Sepsis/Antimicrobial Team • Enterococci:

– Reporting of C. albicans by PNA FISH led to early switch to generic fluconazole without compromising patient safety

– 42% patients received AMT intervention by PNA FISH

• Candida sp. – $1800 cost savings per candidemia patient

– 46% intervention with PNA FISH

• MRSA/SA, in progress PCR – Conventional methods misidentified 6 C. dubliniensis as C. albicans (Germ tube only ~ 95-98% accurate)

Xpert™ MRSA/SA on Cephied GeneXpert ® system • Detection of Staphylococcus aureus and MRSA directly from positive blood culture bottles • mecA, spa and orf targets - to avoid false pos. from SCCmec variants • TAT ~ < 1 hr

– Based on retrospective analysis >85% of UMC patients with S. aureus BSI from would have a rapid change in therapy with direct PCR

Cost and LOS analysis pending to remove bottlenecks

Xpert MRSA/SA Assay: Pre-clinical Performance: Xpert MRSA/SA Performance, Wolk, et al. 2009. J. Clin. Microbiol., 47

D.M. Wolk

Disclosures THANK YOU

• Abbott, AdvanDX, Bio-Rad, Cepheid, Ibis, Microphage, Qiagen, for research support • Abbott, bioMerieux, BioRad, Cepheid, Qiagen, Thermo-Fisher for CME - speaking engagements

Dr. Vicki Wysocki, Erin Johnson, Desiree Johnson, Lorraine Dominguez, Andrew Clark, Dee Gamage, Joe Marano, Elizabeth Ingram, Erica Corcoran

Advancing Diagnostics… Saving Lives©