C difficile

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C difficile

One Microbiologist’s Story of

Mentorship, Technology and

Diagnostic Problem Solving

Karen C Carroll, M.D.

Professor of Pathology and Medicine

Director, Division of Medical Microbiology

The Johns Hopkins University School of Medicine


Relevant Disclosures

Research Funding

• BD Diagnostics, Inc. Sparks, MD

• Gen-Probe (Prodesse) Inc. San Diego, CA

• Microphage, Inc. Boulder, CO

• Akonni, Inc. Gaithersburg, MD

• Great Basin Scientific, SLC, Utah

• Innovative Biosensors, Frederick, MD


Fellowship Training

University of Massachusetts

Medical Center

University of Utah

Associated Regional and

University Pathologists

Gary Doern, PhD

Larry Reimer,

MD

John Matsen, MD


Focus on Healthcare Associated

Infections

• Influenced by the

tremendous impact of

these infections on

patients

• Strong collaborators at

both institutions

• Emerging technology for

diagnosis of S. aureus,

vancomycin-resistant

enterococci, and

Clostridium difficile


Methicillin-Resistant Staphylococcus

aureus:1990’s

• Widespread MRSA

infection reported in many

hospitals within the US

• MRSA detection in clinical

laboratories was

problematic

• mecA gene had been

sequenced

• Rapid cycle PCR was

invented

– 1605 Air Thermo-cycler

(Idaho Technology, Idaho

Falls, ID)

Carl Wittwer, MD, PhD


Carroll KC et al. 1996.

AJCP 106:600-05.

Rapid Detection of mecA gene

from BACTEC Blood Cultures

Positive blood culture gram pos cocci clusters

Direct tube coagulase positive

Aliquot removed for PCR, centrifuged

Achromopeptidase lysis

Rapid cycle PCR Air Thermocycler 1605

533 bp fragment mecA gene


Rapid Detection of mecA gene

from BACTEC Blood Cultures

• 100% correlation with conventional methods!

• $7.67 per specimen!

• Same day results: 2.5 h!

• No supplemental testing!

WOW!

Carroll KC, Leonard RB, Newcomb-Gayman PL, Hillyard DR. 1996. Rapid Detection of

the Staphylococcal mecA gene from BACTEC Blood Culture Bottles by the Polymerase

Chain Reaction. AJCP 106:600-05.


Community Associated MRSA Clones

• Individuals without known healthcare risk factors (mid

1990’s)

• Spread to a variety of populations:

– Prison/Jail inmates

– Athletic teams (professional, college and high school)

– Day care centers

– IV drug users

– Indigenous populations

• Genetically different from HA-MRSA

• More drug susceptible

CDC1999. Four pediatric deaths from CA-MRSA—Minnesota and N. Dakota 1997-1999.

MMWR 48:707-10. Naimi, et al. 2003. Comparison of CA & HA-MRSA Infection.

JAMA. 290 (22); 2976–2984.


MRSA Epidemiology in Baltimore:

2000 and Beyond

• Among clinical S. aureus isolates in Baltimore

2002-2003 (n=1720), 8% were CA-MRSA

Fridkin, et al. 2005. NEJM 352: 1436-1444.

• Incidence of invasive MRSA infections--Baltimore

– 29.7 cases/100,000 pop. were CA-MRSA

Klevens RM, et al. JAMA 2007;298:1763

Francis JS, et. Al. 2005. Clin. Infect. Dis. 40:100-7.


MRSA: Impact on JHH

Microbiology Laboratory

Nasal surveillance of patients on high

risk units began 2000

Implementation CHROMagar MRSA 2004

Flayhart D, et al. 2005. J Clin Microbiol 43:5536-40.

Increased demand for epidemiology support:

Strain typing, SCCmec typing

PVL testing

Environmental testing

Pressure to implement molecular testing

Trish Perl, M.D.


Farley JE, Stamper PD, Cai M, et.al 2008.

Comparison of the BD GeneOhm MRSA

PCR assay to culture using BBL

CHROMagar MRSA for detection of MRSA

from nasal surveillance cultures in an atrisk

community population.

J Clin Microbiol 46:743-6

Farley JE, Ross T, Stamper P, et. al. 2008.

Methicillin resistant Staphylococcus aureus

nasal colonization among newly arrested males

in Baltimore, Maryland. Am J Infect Control

36:644-50.

Stamper PD, Louie L, Wong H, et. al. 2011. Genotypic and phenotypic

characterization of MSSA isolates misidentified as MRSA by the BD

GeneOhm MRSA assay. J Clin Microbiol 49:1240-4.

vs.


Study Results…….

• Prevalence of MSSA and MRSA nasal colonization

among 602 newly arrested men in Baltimore — 40.4%

and 15.8%; 80% were USA 300 or related subtypes

• BD GeneOhm MRSA PCR assay: Sensitivity and

specificity 89.0%, 91.7%, respectively

• 42 false positives by PCR

• 13/42 contained genotypic MRSA but phenotypic

MSSA:

– 1 USA 300 isolate had intact mecA gene that was not expressed

– 11 USA 400-like isolates lacked mecA; remnants of type IVa cassette

– 1 USA 100 had remnant of type II cassette and lacked mecA

Am J Infect Control 2008; 36:644-50; J Clin Microbiol 2008; 46:743-6;

J Clin Microbiol 2011; 49:1240-4.


Available and Emerging

Technologies

PNA-FISH

(AdvanDx,

Inc.)

BD GeneOhm

MRSA ACP assay

(BD-GeneOhm)

Xpert ® SA nasal

complete (Cepheid)

Phage technology

(Microphage Inc.)

Microarrays

LightCycler ® MRSA

Advanced Test

(Roche Molecular

Systems, Inc.)

IBIS T6000

PCR/ESI-MS

(Abbott

Diagnostics)


Have Emerging Technologies

Impacted MRSA Rates?

Jain R, et al. Veterans affairs initiative to prevent MRSA

infections. NEJM 2011;364;1419

• ―MRSA bundle‖ - Oct 07 to June 10 in 150 hospitals

– Universal nasal surveillance

– Contact precautions

– Hand hygiene

– Institutional ―culture change‖

• Screening performed using CHROMagar or PCR

• Results

– MRSA transmission declined by 17-21%

– HA-MRSA infection declined by 62%


Another Devastating Healthcare

Associated Infection: C difficile

• A 22 yo patient was admitted to JHH with

vomiting and weight loss.

• After a long hospitalization she underwent

surgery for pyloric ulcer with severe stenosis.

• Post-operatively she did well ―….until the

diarrhea developed‖ on post-operative day

10.

• She died on post-op day 15 of ―a diphtheritic

colitis‖……

Finney JMT. 1893. Gastro-enterostomy for cicatrizing ulcer of the

pylorus. Bull Johns Hopkins Hosp. 4:53-55.


Discovery of C difficile as Cause

of Pseudomembranous Colitis


Clostridium difficile

EIA Testing at Johns Hopkins

Manabe Y, et. al. Ann. Intern. Med. 1995;123:835-40.

• Study performed JHH-268 non-oncology pts.

• EIA only

– 72% (31/43)--diagnosed with 1 st specimen

– 84% (36/43)—diagnosed with 2 nd specimen

– 93% (40/43)-diagnosed with 3 rd specimen

• Institutional practice became— ―stools for C diff X 3‖


Emergence of Epidemic Toxin Variant

Strain of C. difficile

US-8 facilities/6 States

reported outbreaks in 2001

• 50% of 187 isolates were

clonal-- PFGE (NAP-1)/

ribotype 027 (baseline < 1%)

• Toxin variant strain—

toxinotype III

• Deletions in tcdC

– 18 bp deletions at nucleotides

330-347

• Binary toxin positive

• Fluoroquinolone resistant

McDonald LC, et. al. N Engl J Med

2005;353:2433

Quebec study-- 12

hospitals

• 30-d attributable

mortality was 6.9%

(1.5% baseline)

• 33 pts. required

colectomy (1.9%)

• More pts. received

quinolone antibiotics

Loo, et. al. 2005. N Engl J Med

353:2442.


Toxin A/B EIAs Revisited

• Series of complaints prompted re-evaluation of C difficile

EIA test in use in the lab

• Sensitivity had fallen to 38%!

Ticehurst JR, et al 2006. J Clin Microbiol 44:1145.

• Literature from others followed:

Planche, et al. 2008. Lancet Infect Dis 8:777.

Eastwood et al. 2009. J Clin Microbiol 47:3211.

• Reviews of 6 Tox A/B tests

– None met acceptability criteria: sensitivity 90%; false positives < 3%

– Sensitivities ranged from 60% to 81%; specificities from 91% to

99.4% compared to toxigenic culture

Cohen SH, et al 2010. SHEA/IDSA guidelines ICHE 31:431-55

• Enzyme immunoassays -- suboptimal


Re-Implementation of Cytotoxin

Testing: Is Repeat Testing Useful?

Renshaw A, et. al. Arch

Pathol Lab Med

1996;120:49.

• 4, 238 specimens

• 2009 pts.

• 36% repeated tests

• New information

provided in only 0.5-

0.8% of cases

Borek A, et. al. J Clin

Microbiol 2005:43;2994.

• 2,940 specimens (37%

repeated tests)

• 640 patients

• 1,101 had 2 nd sample

submitted-100%

concordance

• 247 pts had 3 samples-

For 0.8% this was the

initial positive


Glutamate Dehydrogenase Testing

C DIFF CHEK Antigen Test

Evaluator Sens

Zheng et al. JCM

2004;42:3837

Snell, et al. JCM

2004;42:4863

Ticehurst, et al

JHH Micro Lab

JCM 2006;44:1145

Fenner L, et al

JCM 2008; 46:328

(%)

Spec

(%)

NPV

(%)

Comparator Methods

93 89 99 Cell culture cytotoxicity;

PCR gluD gene; culture

93.5 98 98.5 Bacterial culture plus PCR

for tox genes; TOX A/B

EIA

98 89 99.7 Cell culture cytotoxicity

assay;

Bacterial culture

93.4 96.6 99.2 Bacterial culture; PCR

gluD gene


Two step Clostridium difficile

Testing Algorithm

GDH negative

Report as: C difficile antigen not

detected

Negative

Reported as : C difficile

toxin assay negative

.

GDH positive

C difficile antigen detected. The presence

of antigen may not correlate with disease.

Toxin assay will be performed.

C difficile cytotoxicity neutralization

assay.

Positive

Reported as: Positive

by C difficile

neutralization assay


PCR Testing for Clostridium

difficile

• Early reports appeared in the literature in 1991

– end detection

– cumbersome extractions

• Decade later

– DNA extraction methods from fecal samples improved (e.g.

QIAamp DNA stool MiniKit (Qiagen, Valencia, CA; Infectio Diagnostic Inc.)

– Real-time platforms became available

– Analytical sensitivity compared to CCCNA (10-100 more

sensitive ~ 10 genome copies per PCR)

– Analytical specificity also improved

Kato N, et al. J Clin Microbiol 1191:29;33. Gumerlock et al Rev Infect Dis

1991:13:1053 Belanger SD. et. al. 2003. J Clin Microbiol 41:730; Van den Berg RJ, et

al 2005. J Clin Microbiol 43:5338


NAAT Clinical Trial Work

Reference Assay

Stamper, et

al; JCM

2009;47:373

Stamper, et

al. JCM

2009;47:3846

BD-

GeneOhm

Prodesse

ProGastro

#

Prev

(%)

Comp

Methods

404 10 Cytotoxin

Toxigenic

culture

272 15.7 Cytotoxin

Toxigenic

culture

TOX-B test compared to JHH toxigenic culture

Sens. 63.6%-67.2%; Spec 99.2%

Sens

(%)

90.9

83.6

83.3

77.3

Spec

(%)

95.2

98.2

95.6

99.2

PPV

(%)

70.2

89.5

69.4

94.4

NPV

(%)

98.8

97.1

98

95.9


Available Molecular Assays

Summary

Assay Target Extraction Internal

Control

BD

GeneOhm ®

Prodesse

ProGastro

Gene Xpert tcdB

Cdt

nt 117

del

tcdC

tcd B Manual Yes 75-90

min

TAT Performance

Sens. Spec.*

(%)

83.6-96.4;

94- 100

Cost/

test

$25-

$49

tcd B Easy Mag Yes 180 min 77.3-91.9; 99 $25

Automated

(Infinity

platform)

Yes 45 min 94-100; 93-99.6 $45

illumigene tcdA Manual Yes 70 min 98 98 N/A

* As per published literature


“Three Step Algorithms”

C diff Quik CHEK Complete

• Combines GDH testing and toxin

testing (A&B) into one device

• 2 published studies compared to

toxigenic culture:

GDH Toxin

– Sensitivity 100 61.1-78.3

– Specificity 97 100

– PPV 81.8 100

– NPV 100 95

Quinn CD et al J Clin Microbiol 2010; 48:603.

Swindells J et al J Clin Microbiol. 2010;

48:606; Sharp S

J Clin Microbiol 2010;48:2082


Potential Disadvantages to Multi-Step

Algorithms

• Time to detection—Impact on patient care?

Sydnor ERM, et al. 2011. Antimicrobial prescribing practices associated with Clostridium

difficile testing. ICHE (In review)

• Maintenance of multiple test methods

• Cost/re-imbursement issues

• Variability in reported GDH assay performance

Tenover FC, et al. 2010. Impact of strain type on detection of C. difficile: Comparison of

molecular diagnostic and EIA approaches. J. Clin. Microbiol. 48:3719-24.

Carroll KC and Loeffelholz M. Conventional vs. Molecular Methods for the Detection of

Clostridium difficile. J. Clin. Microbiol (In press).


Toxigenic Culture

• Methods are not

standardized

• JHH procedure

– Spore enrichment using

heat

– Inoculate stool to:

• Pre-reduced CCFA-HB

• CCMB-TAL broth

– Incubate 5 days

– Confirm ID; toxin

production by CCCNA

Toxigenic culture

performed after

negative direct toxin

test increased yield

by 23%

Reller ME et. al. J Clin

Microbiol. 2007; 45:3601.

CCFA-HB

(Remel)

CCMB-TAL broth

(Anaerobe Systems)


C. difficile Disease

Where are We Now?

• Biology

– Full genome sequencing of C. difficile isolate 630 and partial

sequencing of 4 others

– Insights into pathogenesis of hypervirulent strains

– Physiology of gut flora and its impact on C difficile partially

elucidated

• Epidemiology

– Many risk factors identified

– Others require more study (PPIs)

• Diagnosis

– EIAs are ―out‖; molecular methods, multi-step algorithms are ―in‖

– Cost-effectiveness models, performance of NAATs in special

groups, impact on epidemiology are needed


Thank You Very Much!

University of Utah / ARUP Labs

Faculty

Larry Reimer, Dave Hillyard, Christine Litwin, Harry Hill,

John Matsen

Fellows

Lisa Steed, Jeanmarie Mayer, Punam Verma, Tom Novicki,

Musa Hindiyeh, Jim Dunn

Research Collaborators

Matthew Samore, Carrie Byington,

Andy Pavia, Judy Daly, Susan Mottice

Research associates

Rebecca Buxton, Joann Cloud

ARUP Microbiology Laboratory Medical Technologists


Thank You Very Much!

Johns Hopkins

Faculty

Brooks Jackson, Pat Charache, Steve Dumler, Stefan Riedel, Alex

Valsamakis, Megan Reller, Bill Merz, Jim Dick, Sean Zhang

Fellows

Hasan Bhally, Megan Reller, Victor Flauta, Julie Kingery, Aneela

Mahmud

Research Collaborators

Trish Perl, Sara Cosgrove, Lisa Maragakis, Aaron Milstone, George

Siberry, Alan Chen, Rich Rothman, Charlotte Gaydos

Research coordinators

Mian Cai, Paul Stamper, Tracy Ross

JHH Microbiology Laboratory Technologists/ C difficile team

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