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
• 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
University of Massachusetts
University of Utah
Associated Regional and
Gary Doern, PhD
John Matsen, MD
Focus on Healthcare Associated
• Influenced by the
tremendous impact of
these infections on
• Strong collaborators at
• Emerging technology for
diagnosis of S. aureus,
• Widespread MRSA
infection reported in many
hospitals within the US
• MRSA detection in clinical
• mecA gene had been
• Rapid cycle PCR was
– 1605 Air Thermo-cycler
(Idaho Technology, Idaho
Carl Wittwer, MD, PhD
Carroll KC et al. 1996.
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
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!
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
• 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
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
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
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
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.
• 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
– 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
MRSA ACP assay
Xpert ® SA nasal
LightCycler ® MRSA
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
– 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
• She died on post-op day 15 of ―a diphtheritic
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
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—
• Deletions in tcdC
– 18 bp deletions at nucleotides
• Binary toxin positive
• Fluoroquinolone resistant
McDonald LC, et. al. N Engl J Med
Quebec study-- 12
• 30-d attributable
mortality was 6.9%
• 33 pts. required
• More pts. received
Loo, et. al. 2005. N Engl J Med
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
• 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
• 2,940 specimens (37%
• 640 patients
• 1,101 had 2 nd sample
• 247 pts had 3 samples-
For 0.8% this was the
Glutamate Dehydrogenase Testing
C DIFF CHEK Antigen Test
Zheng et al. JCM
Snell, et al. JCM
Ticehurst, et al
JHH Micro Lab
Fenner L, et al
JCM 2008; 46:328
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
98 89 99.7 Cell culture cytotoxicity
93.4 96.6 99.2 Bacterial culture; PCR
Two step Clostridium difficile
Report as: C difficile antigen not
Reported as : C difficile
toxin assay negative
C difficile antigen detected. The presence
of antigen may not correlate with disease.
Toxin assay will be performed.
C difficile cytotoxicity neutralization
Reported as: Positive
by C difficile
PCR Testing for Clostridium
• 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
404 10 Cytotoxin
272 15.7 Cytotoxin
TOX-B test compared to JHH toxigenic culture
Sens. 63.6%-67.2%; Spec 99.2%
Available Molecular Assays
Assay Target Extraction Internal
Gene Xpert tcdB
tcd B Manual Yes 75-90
tcd B Easy Mag Yes 180 min 77.3-91.9; 99 $25
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
– 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
• 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).
• Methods are not
• JHH procedure
– Spore enrichment using
– Inoculate stool to:
• Pre-reduced CCFA-HB
• CCMB-TAL broth
– Incubate 5 days
– Confirm ID; toxin
production by CCCNA
negative direct toxin
test increased yield
Reller ME et. al. J Clin
Microbiol. 2007; 45:3601.
C. difficile Disease
Where are We Now?
– 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
– Many risk factors identified
– Others require more study (PPIs)
– 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
Larry Reimer, Dave Hillyard, Christine Litwin, Harry Hill,
Lisa Steed, Jeanmarie Mayer, Punam Verma, Tom Novicki,
Musa Hindiyeh, Jim Dunn
Matthew Samore, Carrie Byington,
Andy Pavia, Judy Daly, Susan Mottice
Rebecca Buxton, Joann Cloud
ARUP Microbiology Laboratory Medical Technologists
Thank You Very Much!
Brooks Jackson, Pat Charache, Steve Dumler, Stefan Riedel, Alex
Valsamakis, Megan Reller, Bill Merz, Jim Dick, Sean Zhang
Hasan Bhally, Megan Reller, Victor Flauta, Julie Kingery, Aneela
Trish Perl, Sara Cosgrove, Lisa Maragakis, Aaron Milstone, George
Siberry, Alan Chen, Rich Rothman, Charlotte Gaydos
Mian Cai, Paul Stamper, Tracy Ross
JHH Microbiology Laboratory Technologists/ C difficile team