Overview of Clostridium Difficile Infections (CDI)

Stephen M. Brecher Ph.D.

VA Boston HealthCare System

BU School of Medicine

The opinions expressed in this presentation are those of the presenter and do not necessarily represent the views of the Veterans Affairs Health- Care System

Overview

• Historical perspective• Organism and key properties• Changing epidemiology• Mild, moderate and severe disease• Laboratory diagnosis• Treatment

Historical Perspective

• In the 1960’s it was noted that patients on antibiotics developed diarrhea1

• “Staphylococcal Colitis”• Originally thought to be caused by S. aureus and treated

with oral bacitracin• Stool cultures routinely ordered for S. aureus

• Early 1970’s, a new explanation• “Clindamycin Colitis”

• Severe diarrhea, pseudomembrane colitis, and occasional deaths documented in patients on clindamycin

1. Gorbach S.L. 1999. NEJM.341: 1689-1691

“Antibiotic Associated Pseudomembranous Colitis Due to Toxin-Producing Bacteria”1

• Bartlett and co-workers demonstrated cytotoxicity in tissue culture and enterocolitis in hamsters from stool isolates from patients with pseudomembranous colitis

• Isolate was Clostridium difficile• Bacillus difficilis (now confirmed as C. difficile) was

cultured from healthy neonates (with difficulty, hence the name) in 19352

1. Bartlett, J.G. et al. 1978. NEJM. 298: 531-534 2. Hall, J.C. and O’Toole E. 1935. Am J Dis Child. 49: 390-402.

Factors That Complicated the Discovery of CDI

• C. difficile is found in healthy infants who appear to be refractile to CDI– Infant intestinal cells do not appear to have

receptors for toxins A and B

• Antibiotics often cause diarrhea unrelated to C. difficile by disrupting intestinal flora which helps digest food

Clostridium difficile

• Gram Positive, anaerobic, spore-forming bacillus

• Vegetative cells die quickly in an aerobic environment

• Spores are a survival form and live for a very long time in the environment

• Grows on selective media in 2 days and smells like horse manure (p-cresol)

Source of Infections

• Spores in the hospital, nursing home or long-term care environment associated with ill patients– Large numbers of spores on beds, bed-rails, chairs, curtains,

medical instruments, ceiling, etc.– Airborne transmission1

• Asymptomatic carriers in those same environments– Low risk compared to patients with active disease

• Unknown in community based infections, but food has been implicated2

1. Best, E.L. et al. 2010. CID. 50. 1450-1457

2. Jhung,M.A. et al. 2008. Emerg Infect Dis. 14: 1039-1045

Risk Factors for Infection

• Antibiotics (some more than others)• Hospitalization or long-term care facility• Increasing age (>65,>>80)• Co-morbidity• Surgery• ? Proton-pump inhibitors• Community Associated Cases

– Peri-partum– Close contact of CDI case– Food

Role of Antibiotics• All antibiotics (including metronidazoloe

and vancomycin) are associated with CDI• High Risk Group

– Clindamycin– Cephalosporins/Penicillins/Beta-Lactams– Fluoroquinolones

• Alteration of normal colonic flora thought to favor growth of C. difficile– Antibiotics do not know they are supposed to

kill/inhibit only the “bad guys”– May not be a factor in community CDI

Incidence in The United States

• CDI is not a reportable disease so exact number of cases and deaths remain unknown

• Based on discharge diagnoses, CDI cases have tripled over the last 5 years

• Deaths in the UK are around 9,000/year

Pathogenesis

• Toxigenic strains produce 2 large protein exotoxins that are associated with virulence– Toxins A and B

– Mutants strains that do not make toxins A and B are not virulent

– Some strains make a third toxin known as Binary Toxin

• By itself, not pathogenic

• May act synergistically with toxins A and B in severe colitis

• More common in animal strains

Asymptomatic C. difficile colonization

Pathogenesis of CDI

C. difficile exposure

Antimicrobial

C. difficile infectionHospitalization

From Johnson S, Gerding DN. Clin Infect Dis. 1998;26:1027-1036; with permission.

PathogenesisChanging Epidemiology

• Increasing morbidity and mortality noted beginning in 2000

• Outbreaks in US and Canada (over 200 deaths)

• Was this due to poor infection control, emergence of antibiotic resistance, or something else?

• A new, hypervirulent strain was detected

Epidemic Strain• Strain typed BI/NAP1/0271,2

• Is highly resistant to fluoroquinolones2,4

• Binary toxin genes are present• Produces large quantities of toxins A and B1,3

• Has a tcdC gene deletion1

1. Warny M, et al. Lancet. 2005;366:1079-1084.2. Hubert B, et al. Clin Infect Dis. 2007;44:238-244.3. CDC Fact Sheet. July 2005. 4. McDonald LC, et al. N Engl J Med. 2005;353:2433-2441.

Adapted from McDonald LC, et al. N Engl J Med. 2005;353:2433-2441; with permission.

In Vitro Production of Toxins in Epidemic Strain

From Warny M, et al. Lancet. 2005;366:1079-1084; with permission.

Not So Fast

• 2 recent papers questioned whether this strain is more virulent– NAP-1 strain was detected around 25% of time in

their hospital (BID in Boston) but was not associated with increased severity of disease (non-epidemic setting)1

– 18 and 39 bp deletion containing strains were not associated with increased severity of CDI at the Mayo Clinic2

• Age >65 and prior NH stay implicated1. Cloud, J. et al. 2009. Cl Gastro and Hept. 7:868-873

2. Verdoorn, B. P. et al. Diag Micro and ID. 10.1016/j.diagmicrobio.2009.0815

Should You Treat the Patient or Treat the Strain?

• Routine diagnostics laboratory tests do not provide strain type

• Routine tests not always reliable

• Always treat the patient based on symptoms, history, risk factors and markers of severe disease

Symptoms of CDI

• Asymptomatic colonization• Diarrhea

– mild moderate severe

• Abdominal pain and distension• Fever• Pseudomembranous colitis• Toxic megacolon• Perforated colon sepsis death

Markers of Severe Disease

• Leukocytosis– Prominent feature of severe disease– Rapidly elevating WBC– Up to >100 K

• Albumin (<2.5)• Creatinine (2xbaseline)• Serum lactate (>5.0colectomy)• Hypertension and hypotension• Pseudomembranous colitis• Toxic megacolon• Severe distension and abdominal pain

Laboratory Diagnosis of CDI

Laboratory Laboratory DiagnosisDiagnosis

Enzyme Immunoassay (EIA)Enzyme Immunoassay (EIA)Glutamate Glutamate Dehydrogenase (GDH)Dehydrogenase (GDH)

Cell Culture Cell Culture Neutralization Neutralization Assay (CCNA)Assay (CCNA)

Toxigenic Culture Toxigenic Culture (Culture and CCNA)(Culture and CCNA)

Molecular Based (PCR Or LAMP)Molecular Based (PCR Or LAMP)

Stool CultureStool Culture

Specimen and Testing Guidelines• Only test loose or liquid stool

• Test within 2 hours or keep refrigerated for up to 2 days

• Limit testing to 1 test/patient/week

• Do not perform a test for cure

• Do not perform tests on asymptomatic patients

• Test outpatients with diarrhea

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“Plate Sin With Gold and it Goes Untarnished”

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Cell Culture Neutralization AssayPerformance Revisited

Study Method Sensitivity(%)

Specificity(%)

Eastwood, et al. JCM 2009; 47:3211

In house Vero cell assay*

86.4 99.2

Barbut, et al. JCM 2009;47:1276

In house MRC-5 cells*

75.8 100

Stamper, et al JCM 2009;47:373

TechLab TOX-B test*

67.2 99.1

Peterson, et al. CID 2007;45:1152

In house MRC-5cells+

76.7 97.1

* Compared to toxigenic culture. + Compared to clinical case definition and multiple test methods.

Slide provided by Dr. Karen Carroll

Conflicting Results with EIA

1. Stamper PD, et al. J Clin Microbiol. 2009;47:373-378.2. Musher DM, et al. J Clin Microbiol. 2007;45:2737-2739.3. Sloan LM, et al. J Clin Microbiol. 2008;46:1996-2001.4. Gilligan PH. J Clin Microbiol. 2008;46:1523-1525.5. Ticehurst JR. J Clin Microbiol. 2006;44:1145-1149.6. Nice review by Planche T, et al. 2008. www.thelancet.com/infection

Recently Published EIA Papers(1-6)

Parameter Range

Sensitivity 32 – 98.7%

Specificity 92 – 100%

PPV 76.4 – 96%

NPV 88 – 100%

My Opinion

• EIA Testing is too insensitive to use as in a disease as important as CDI

• It is time to move on

• Increased cost of other tests are warranted because the failure to diagnose CDI is more costly– mural dyslexia is all too common in

hospitals

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C. Diff Quik Chek Complete• Lateral flow GDH and EIA on one test card• 2 recent studies

– Quinn et al reported that if• Both + = +• Both - = -• 13.2% discrepant, re-test. Use PCR

– Sharp et al reported that 88% of specimens were both positive or both negative

• Used random access PCR to resolve remaining 12%

1. Quinn, C. D. 2010. J Clin Microbiol. 48: 603-605

2. Sharp, SE et al. 2010. J Clin Microbiol. 48: 2082-2086

Commercially Available Molecular Assays

AssayAssay TargetTarget InstrumentInstrument TATTAT

BD BD GeneOhmGeneOhm™ ™ Cdiff AssayCdiff Assay

ToxinBToxinB SmartCycler SmartCycler AmplificationAmplification 75-120 75-120

minmin

Prodesse Prodesse proGASTRproGASTROO™™

Toxin BToxin B easyMag easyMag extractionextraction

SmartCycler SmartCycler AmplificationAmplification

~ 3 hrs~ 3 hrs

Cepheid Cepheid XpertXpert™ ™

C. difficileC. difficile

Toxin BToxin B

(RUO IDs (RUO IDs 027/BIstrai027/BIstrains)ns)

GeneXpertGeneXpert

45 min45 min

Meridian Illumigene

conserved conserved 5’ sequence 5’ sequence of the of the tcdAtcdA gene gene

illumigene Incubator/reader

45 min45 min

Slide provided by Dr. Susan Novak-WeekleyakSli

BD GeneOhm™ Cdiff Assay Procedure Overview

SpecimenPreparation

Lysis - DNAExtraction

ReconstitutionOf Reagents

Real-time PCRAnalysis on theSmartCycler®

DefinitiveOn-screenResults

Results in <2 Hours

Stool Specimen

Slide courtesy BD GeneOhmlideS provide

proproGASTROGASTRO™™ Cd Cd WorkflowWorkflow

Specimens Clarified Stool

110 µl of nucleic acid for analysis

Dilute Internal Control. Combine 20 µl clarified stool and 10 µl Internal

Control

Run Lysis, Add Beads

Perform extraction and elute in 110 µl

Add buffer, Vortex & spin

Slide courtesy Gen-Probe

proproGASTROGASTRO™™ Cd Cd WorkflowWorkflow

Hands-on Time = 57 min Hands-off Time = 137 min TOTAL TIME = 194 min

Extracted nucleic acid

Prepare controls Combine 5 µl of nucleic acid &20 µl of Mastermix in reaction tube

Insert each reaction tube in SmartCycler

Program instrument and run assay

Mastermix

Slide courtesy Gen-Probe

Xpert® C. difficile PCR Test for Clostridium difficile Toxin B Only

Results in an early as 30 minutes; assay complete in 45 minutesIf negative

• 11 minute sample extraction with ~1 minute of hands on time.

• No centrifugation required.

• All required material included in kit.

• Identical simple process for single or batched samples.

Assay Protocol

Slide Courtesy of Meridian

• illumipro-10™ provides walk-away amplification and detection

• No precision pipetting (3 x 50μl pipetting steps)

• Amplicons contained in sealed & locked illumigene™ device

• Total assay:~ 2 min hands on time< 60 minutes to resultNo centrifugation or precision pipetting

required

Assay Protocol

Slide Courtesy of Meridian

Summary of Summary of C. difficileC. difficile PCR PCR Published DataPublished Data

Publication Publication PCR AssayPCR Assay Sens/SpecSens/SpecTerhes, 2009Terhes, 2009 BD vs. Tox Culture*BD vs. Tox Culture* 96%/99%96%/99% Not all Not all

negatives specimens tested negatives specimens tested by culture*. by culture*.

Stamper, 2009Stamper, 2009 BD vs. Tox Culture BD vs. Tox Culture 83.6%/98.2%83.6%/98.2%

Eastwood, 2009Eastwood, 2009 BD vs. CCCNBD vs. CCCN

BD vs. Tox CultureBD vs. Tox Culture92.2%/94%92.2%/94%

88.5%/95.4%88.5%/95.4%

Kvach, 2010Kvach, 2010 BD vs. Tox Culture*BD vs. Tox Culture* 91.4%/100% 91.4%/100% Not all Not all negatives specimens tested negatives specimens tested by culture*. by culture*.

Stamper, 2009Stamper, 2009 Prodesse vs. Tox Prodesse vs. Tox CultureCulture

77.3%/99.2%77.3%/99.2%

Huang, 2009Huang, 2009 Cepheid Xpert vs. Cepheid Xpert vs. CCCNCCCN

97.1%/93.9%97.1%/93.9%

Novak-Weekley, Novak-Weekley, 20102010

Cepheid Xpert vs. Tox Cepheid Xpert vs. Tox CultureCulture

94.4%/96.3%94.4%/96.3%

Swindells, 2010Swindells, 2010 Cepheid Xpert vs Tox Cepheid Xpert vs Tox CultureCulture

BD vs Tox cultureBD vs Tox culture

100%/99.2%100%/99.2%

94.4%/99.2%, 94.4%/99.2%, Small “N”Small “N”

Slide courtesy of Dr. Susan Novak-Weekley

Illumigene• No peer reviewed full studies in literature yet as test

is new. There are a few abstracts– Elagin et al. (from Meridian) reported 95.2%

sensitivity and 95.3% specificity as compared to toxigenic cx and another form of PCR1

– Lalande et al. reported 96.1% and 98.7% but N was small2

– Rhees et al reported 91.3% and 94.3%3

1. Elagin, S et al. 26th CVS. 2010

2. Lalande, V at. Al. ICCAC. D-131. 2010

3. Rhees, J.R. et al. ICAAC. D-128. 2010

Consequences of Bad Tests

• Repeat testing

• Low sensitivity– False negative patients don’t get treated

and spread the organism

• Low specificity– False positive patients get costly

treatments and IC protocols

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My Recommendations

• EIA alone is not accurate and should be discontinued (sensitivity unacceptable)

• 2 step tests with GDH/EIA have shown increased accuracy over EIA alone

• PCR works best but is significantly more expensive– Cost of test is a small part of total cost

• Some do 2-step and confirm with PCR

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Treatments

• Metronidazole (oral or IV)• Oral vancomycin • IVIG• Fidaxomicin (OPT-80)• Nitazoximide• Rifaximin chasers• Probiotics• C. difficile non-toxigenic strains (phase 2)• Monoclonal Antibodies for recurrent Disease1

1. Lowry, I. et al. N Engl J Med 2010; 362:197-205

BacteriotherapyFecal Transplantation

• In recurrent disease, Aas et al have reported success using fresh stool– Select healthy family member– Mix 30-50 mg in 50-90 ml of normal saline– Homogenize in a food blender– Filter x 2 in coffee filter paper– Administer via NG tube

• 1/16 patients relapsed

Aas, J. et al. CID.2003. 36:580-585

Infection Control (short course)

• Multi-step interdisciplinary program essential

• Ingredients– Hand-washing with soap/warm-water – Cleaning protocols aimed at killing spores (– Precautions– Antibiotic stewardship– Accurate testing

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Summary

• CDI is increasing in incidence, severity and poor outcomes

• No reasonable explanation for treatment failures

• Community based infections are not well understood

• Extremely important to accurately detect all disease and aggressively treat severe disease

• Cost alone should not be the decision maker

11.5

10.7

10.5

10.3

12.0

11.1

11.0

11.8

q

p

Y Chromosome

Testis Determining Factor (TDF)

Gadgetry (MAC- locus)

Channel Flipping (FLP)

Catching and Throwing (BLZ-1)

Self-confidence (BLZ-2)

(note: unlinked to ability)

Ability to remember and tell jokes (GOT-1)

Sports Page (BUD-E)

Addiction to death & destruction

movies (MOV-E)

Air Guitar (RIF)

Ability to identify aircraft (DC10)

Pre-adolescent fascination with Arachnid

and Reptilia (MOM-4U)

Spitting (P2E)

Sitting on the john reading (SIT)

Inability to express emotion over the

phone (ME-2)

Selective hearing loss (HUH?)

Total lack of recall for dates (OOPS)

Gitschier, J., Science, 1993 (261) p. 679