cepheid ondemand report vol 4 issue 1 spring 2011(1)

8/10/2019 Cepheid OnDemand Report Vol 4 Issue 1 Spring 2011(1)

1/12

Volume4,Issue1

A Quarterly Publication by Cepheid

Cepheid

report

Clostridium difficileRibotype 027

Why Should We Care?

Wake-Up Call

Proficiency Testing for Today's Technology

The Need for

Better Diagnostic Tests forPediatric TuberculosisThe AERAS-CHC-Cepheid Study

in Cambodia

report


2/12

Executive Editor

David Persing, M.D., Ph.D.

Lead Author

Ellen Jo Baron, Ph.D.

Contributing Author

Fred Tenover, Ph.D.

Cepheids ON-DEMAND Report

is distributed four times a year.

We welcome communication

from users of Cepheid systems

and tests and invite suggestions

for articles in future issues. Send

correspondence to:

Cepheid ON-DEMAND Report

1327 Chesapeake Terrace

Sunnyvale, CA 94089

[email protected]

To sign up for e-mail notification

of new issues of Cepheids

ON-DEMAND Report, visit

www.cepheidondemand.com

Contents are 2011 by Cepheid unlessotherwise indicated. Rights reserved onall guest columns. The contents of this

publication may not be reproduced in anyform without written permission ofthe editor. The mention of trade names,commercial products, or organizations

does not imply endorsement by Cepheid.

I am privileged at Cepheid to work with the some of the best minds in the diagnosticsbusiness. In this month's issue of the On-Demand newsletter, Dr. Ellen Jo Baron

highlights the role of decentralized molecular testing in TB diagnostics, with a speciafocus on the nearly impossible challenge of diagnosing pediatric TB. This is a truly

moving story which begs the question: Is there not a better solution? Cepheid iscommitted to developing less invasive diagnostic tools for TB in kids and in all patients

with HIV where current diagnostic options are, simply put, woefully suboptimal. I amoptimistic that Ellen Jo and her colleagues inside and outside the company will come

up with a better way.

Also highlighted in this issue is 1) the need for suitable external controls for molecula

diagnostic assays, and 2) the impact of the emergence of the 027 strain of C. difficileWe hope you enjoy reading this information as we much as we have in preparing it.

Clostridium difficile

Ribotype 027:

Why Should We Care?

The Need for

Better Diagnostic

Tests for Pediatric

Tuberculosis

COVER STORY 3 INSIDE 8

Wake-Up Call:

Proficiency

Testing for Todays

Technology

INSIDE 10

CONTENTS SPRING 2011

From the EditorDavid PersingM.D., Ph.D.Chief Medical and

Technology Officer, Cepheid


3/12 SPRING 2011 | CEPHEID 3

The Need for Better Diagnostic Tests for Pediatric Tuberculosis

THE AERAS-CHC-CEPHEID STUDY IN

CAMBODIA

Almost unfathomable to contemplate is the fact that one-third of the worlds

population is infected with tuberculosis (TB). Each year approximately 9 million newinfections occur (most people who become infected do not exhibit symptoms o

disease for many years, if ever) but around 1.6 million people die of their disease

approximately one death every 9 seconds. 80% of the worlds cases occur in 22

high burden countries, identified by the World Health Organization (WHO) (Figure

1). Tuberculosis can be cured, especially if drug-susceptible and drug-resistant

strains are detected early and treated appropriately. A keystone of the WHOs

global plan to stop TB (Stop TB Partnership; http://www.stoptb.org/global/plan/)

involves using smear microscopy to identify patients and to treat those patients with

a course of antibiotics, with the patient being observed taking his/her drugs by a

healthcare worker for at least the first two months. This directly observed therapy

short-course (DOTS) regimen includes the oral drugs rifampin, isoniazid (INH)

pyrazinamide, and ethambutol for 2 months, followed by 4 more months of INH

and rifampin alone; and for patients with susceptible strains, >95% are cured. If theorganism is known to be susceptible to rifampin and INH through drug susceptibility

testing (DST) performed on organisms isolated in pure culture, then ethambutol may

be removed from the regimen. An expanding group of patients infected with multi-

drug resistant TB (MDR-TB; i.e., strains resistant to multiple drugs including both

INH and rifampin) require longer therapy with more antimicrobial agents, typically

fluoroquinolones, aminoglycosides, p-aminosalicylic acid, ethionamide, and others

some of which must be injected. The sooner a patient harboring a resistant strain

begins taking the drugs that will actually work, the greater the likelihood that the

patient will be cured, even though the therapy must continue for a much longer time

than if the strain were susceptible.

Ellen Jo Baron

Ph.D., D(ABMM)

Prof. Emerita, Stanford University

Director of Medical Affairs,Cepheid

The Need for

Better Diagnostic

Tests for PediatricTuberculosis


4/12


4 VOLUME 4, ISSUE 1 | CEPHEID

produce. If they cough, they usually swallow the sputum. That

is the premise on which the collection of gastric aspirates is

based: the swallowed acid fast bacilli (AFB) can be recovered

from the stomach, especially in the morning before the

stomach contents are emptied into the gastrointestinal tract

At least one study has shown that stool (ultimate destination

of swallowed sputum) is another potentially useful specimen

for detection of TB in children, although others have not had

such positive results.7, 8

But recovering AFB from respiratory secretions regardless of

the site from which they can be collected is more difficult in

children. At most only 15% of children with TB have smear-

positive respiratory samples.9Their organisms tend to stay in

the perihilar lymph nodes and rarely rupture into the bronchia

tree, as they do in adults. Consequently, childrens respiratorysamples may have few or no mycobacteria to detect regardless

of the system used. Children older than 8 years old are more

likely to exhibit adult-like pulmonary TB with coughing and

culture-positive sputum. Thus today, diagnosis of active

tuberculosis in children, particularly those


5/12


SPRING 2011 | CEPHEID 5

algorithms include history of contact with a

TB-positive patient, HIV status, tuberculin

skin-test results, chest radiographs,

coughing or other respiratory symptoms,

fever, weight loss, malaise, and smear and

culture results from different sample types.

Given the lack of a gold standard, the true

endpoint cannot be known for certain. This

makes evaluation of any organism-basedtest, such as molecular tests for genomic

sequences, extremely difficult.

Cambodia is one of the worlds poorest

nations. Bordering Thailand and Vietnam,

Cambodia is still recovering from the

devastating reign of the Khmer Rouge and

their ruthless leader, Pol Pot. Sixty percent

of the population is below 20 years old, and

it is the worlds only country with a growing

mortality rate in children


6/12

for specimen collection are the same or better

than are performed in the best childrens hospitals

throughout the world.

During the gastric aspirate collection procedure, the

childs oxygen saturation is monitored closely with

a transcutaneous oxygen tension monitor attached

to his or her toe. If the child starts breathing too

shallowly and the oxygen tension drops too much,the procedure is stopped. The parent is there at

the head of the bed to hold and

comfort the child (Figure 6). The

child is wrapped tightly in the

sarong and laid on the bed. Nurses

measure the length of tubing to be

used and then carefully insert the

nasogastric tube all the way into

the stomach. Using a syringe,

they withdraw the contents of the

stomach and expel it into a 50 cc

centrifuge tube. The markings on

the tube are used to measure the

volume of material recovered; if

less than 5 ml, the nurse will instill

sterile saline into the stomach

and withdraw the liquid. It may

take several syringe aspirations to

reach the 5 ml required volume.

The aspirate is immediately

buffered to maintain a neutral

pH and the specimen is placed

on ice packs for transport to the

laboratory in Phnom Penh, which

takes place each afternoon.

Later on the second day, the

children come back to the treatment rooms for

collection of an aerosol-induced sputum specimen.

This procedure is at least as uncomfortable as the

gastric procedure. The child is first given a bolus

aerosol puff of albuterol to open the lung airways

(Figure 7). The parent holds the child on his or her

lap for the next 15 minutes while the child breathes

in the saline mist, guaranteed to irritate the lung lining

and provoke coughing. The child is then wrapped

in the sarong again, the oxygen monitor is placed,

and the endotracheal tube is threaded down thepatients trachea about halfway to the bronchial separation. One end

of the tubing is attached to a Lukens trap and the trap is attached to

an electric vacuum-suction device (Figure 8). Sputum provoked by

the saline mist is collected in the trap (Figure 9). Other samples for

future testing, including stool specimens, are collected on this day.

On the third day, the child is again brought to the treatment room

early in the morning for collection of the second gastric aspirate. Will

the patient and his parents ever want to see that sarong again? The

skin test result is interpreted and recorded and the patient is free to

go home.

The procedures followed

at the provincial hospital in

Svay Rieng for specimen

collection are the same or

better than are performed

in the best childrens

hospitals throughout

the world.




FIGURE 6. (top) Inserting nasogas-tric tube into child wrapped in sarong;oxygen monitor cord is visible coming

from the bottom of the sarong.

FIGURE 9. (top) During the collof the aerosol-induced sputum.

FIGURE 10. (center) Samples placed into the insulated cold box

transport.

FIGURE 11. (bottom) TB labortechnologist performing Gen-Prob

say on MGIT-positive sample at P

Institute, Phnom Penh.

FIGURE 7. (center) Child sitting onfathers lap receiving albuterol.

FIGURE 8. (bottom) The patient isprepared for insertion of the endotra-

cheal tube. The suction device is on

the table to the right of the father atthe head of the bed.


7/12


8/12

Clostridium difficileRibotype 027: Why Should We Care?


The first hint that something

new was happening with

regard to Clostridumdifficileinfections (CDI)

was a study presented at

the 2004 annual meeting

of the Infectious Diseases

Society of America by

epidemiologists from the

Centers for Disease Control

and Prevention (CDC)

and other investigatorsdescribing an emerging

epidemic strain that was

typically resistant to

fluoroquinolones.1

C. difficile organisms lacking toxin B

were not pathogenic, even if they

produced toxin A, but toxin B-only

strains were fully pathogenic.5 Newstudies have shown the importance

of both toxins in the hamster model.

Toxin B, the cytotoxin, results in the

destruction of the colonic mucosal cells

that leads directly to production of the

necrotic and eroded mucosal lining

known as pseudomembranous colitis

The single nucleotide deletion found in

strains of the emerging toxinotype III

also referred to by PCR ribotyping as

type 027, by restriction endonuclease

analysis (REA) as BI, and by pulsed-field

gel electrophoresis (PFGE) as NAP1

down-regulated the toxin-limiting

action of the tcdCgene, allowing these

strains to produce considerably highe

amounts of both toxins A and B than wild

type toxinotype 0 strains.7In fact, these

strains produced 20 times more toxins

A and B than most previously circulating

strains.8The 027 strain also had an 18-

base pair deletion within tcdC, although

the effect of that deletion, which keeps

the protein in frame, was unknown

Finally, the epidemic strain produced athird toxin, called binary toxin, which is

not exclusive to this ribotype but may

contribute to increased capacity to

cause disease. Independent studies

have shown binary toxin to have ADP-

ribosyltransferase activity.9

Ribotype 027 isolates had anothe

characteristic that no doubt contributed

to their ability to spread quickly in a

In 2005, a Lancet paper described

an emerging strain associated with

outbreaks of severe disease in North

America and Europe.2 Authors froma hospital in Quebec noticed that

patients with CDI were dying within

30 days of diagnosis at a rate in 2003

that was twice that observed in earlier

periods. After testing 124 strains of C.

difficileand finding 58 hospital-acquired

and 14 community-acquired strains of

toxinotype III (a previously uncommon

toxinotype), they realized that a new

strain was emerging. Along with CDC

and experts from the United Kingdom,

they fully characterized 15 of the new

toxinotype III isolates, comparing them

with historical strains of the previous

prevailing toxinotype 0.3

Several reports were published in the

medical literature describing more

severe disease, more rapid and more

common relapses, and higher mortality

rates associated with isolates of the

new strain.4 Scientists studying this

strain, now being called hypervirulent

and epidemic, found some interesting

characteristics. The most strikingfinding, thought to contribute to its

enhanced virulence, was a missing base

at nucleotide 117 of the pathogenicity

locus (PaLoc) regulatory gene tcdC.

This regulator gene controls the

expression of both the toxin A gene

(tcdA) and toxin B gene (tcdB) located

in the pathogenicity locus. A landmark

paper by Lyras and colleagues from

Australia showed in animal models that

Clostridium difficileRibotype 027


9/12

Clostridium difficile Ribotype 027: Why Should We Care?


healthcare environment, they produced

very high levels of spores compared

to other C. difficile strains.7,8 Some

infection preventionists even fumigatedpatient rooms with aerosolized

hydrogen peroxide, the same sporicidal

compound used to clear the Hart Senate

Office Building after the anthrax scare

in 2001,10in an effort to control spread

of the disease. Higher levels of spores

in the environment may lead to more

efficient spread from patient to patient,

but may also lead to higher loading

doses per patient who is exposed.

Most models of infection show that

there is a relationship between the

size of the inoculating dose and the

severity of disease another potential

explanation for poor patient outcomes

in the hospital outbreak setting.

Some clinicians have expressed the

opinion that the 027 strain deserves

more aggressive treatment measures,

such as using vancomycin instead

of metronidazole, even for the initial

episode, but the current prevailing

view is that treatment should be based

on disease severity and not on straintype, since there are reports of mild

disease in sporadic (as opposed to

epidemic) cases of 027 infection.11This

observation could be consistent with

the spore loading dose hypothesis;

in a hospital outbreak setting,

environmental spore counts are likely to

be higher, especially so for an organism

like the 027 strain that produces more

than its fair share of spores. Sporadic or

Why Should We Care?

community exposure levels are not as

likely to be concentrated.

Hospital outbreaks of C. difficile strain027 seem to represent a special

challenge; only extensive bundles

of interventions seemed to work in

eradicating the organism from a patient

care unit once it gained a foothold,

as noted by Muto and colleagues.12

Many authorities have agreed that

knowing that 027 was present in their

hospital would prompt them to scale

up their infection control activities

earlier and impose more controls,

including limitations of fluoroquinolone

use, as quickly as possible.13Knowing

that the patient is infected with the

027 may also be useful for predicting

the likelihood of relapse after therapy,

either as an inpatient or after discharge.

The clearest evidence for a strain-

associated difference in relapse rate

was presented as part of a recent

study of the newest antimicrobial agent

for CDI, i.e., fidaxomicin. The study,

published in the New England Journal

of Medicine, showed that in a cohort of

patients being treated with vancomycinand/or fidaxomicin, a greater number

of recurrences of disease occurred in

patients who harbored the 027 strain

compared with those who carried a

variety of other C. difficilestrains.14

So lets ask the question again: of

what value is the rapid identification

of patients infected with ribotype 027

strains versus other strain types? First,

the identification of multiple patients i

a healthcare setting with ribotype 02

strains (especially from the same ward

indicates a possible outbreak muc

more rapidly than epidemiologic dat

would alert infection preventionists t

a problem. This could be an indicatio

that the infection control bundle

described by Muto et al12 should b

considered; including switching from

alcohol-based hand gels to soap an

water for hand disinfection, longe

isolation of patients, possible pharmac

controls including general restriction o

fluoroquinolones at an institution, an

more aggressive tracking of cases. Thi

is also an indication that the individua

patient should be monitored closely fopossible relapse. Indication of infectio

caused by ribotype 027 should not b

used to guide therapy, since therap

should be based on severity of diseas

(treat the patient, not the microbe).

Ribotype 027 strains have now sprea

worldwide and will likely continue t

cause both epidemic and sporadi

disease. They will likely be a challeng

for years to come.


10/12

Wake-Up Cal l: Proficiency Testing for Todays Technology

Wake-Up Call:Proficiency Testing for Todays Technolog

Last year, a number of Cepheids Xpert

GBS assay users failed some of the

College of American Pathologists

group B streptococcal proficiency

testing (P.T.) samples distributed in

shipment D8. The users reported

positive for group B Streptococcus

(GBS) when the CAP had intended

those samples to be negative and

the provider of the samples had

certified that they did not contain

GBS. Laboratories using other nucleic

acid amplification methods, such as

BD GeneOhm, and those using the

Cepheid SmartCycler GBS product

did not experience any positive results,

and CAPs initial assessment was that

the Xpert GBS assay was producing

false positive results. CAP initially

suspected that there was a cross-

reaction with other organisms presentin the challenge specimens. Although

the early discussions focused on

whether CAP should notify the FDA

about the faulty performance of the

assay, a conference call with several

representatives from Cepheid and CAP

resulted in an agreement to investigate

the situation further. CAPs decision

may have been influenced partially by

a letter received from one participating

laboratory documenting that the

laboratory had recovered viable GBS

from at least one of the supposedlynegative P.T. samples after extended

broth incubation.

At CAPs direction, additional P.T.

samples from the contested lots

were sent to a consultant on the CAP

Microbiology Resource Committee as

well as to Cepheid. Numerous samples

were placed into enrichment broth for

extended culture analysis, and others

HOW CEPHEIDS GBS ASSAY STIMULATED NEW AWARENESS AND HOW YOUR LABORATORY

SHOULD RESPOND TO AVOID SIMILAR PROBLEMS

were retested in additional molecular

platforms, as well as being run on

GeneXpert Systems at Cepheid.

Again, a few of the negative samples

yielded positive results for GBS in the

GeneXpert, but not in the other assays.

This time, the amplified products from

the GeneXpert assay were sent to

an independent laboratory for DNA

sequence analysis, and sure enough,

contained amplified sequences of GBS

DNA. Scientists familiar with the issue

had experienced a previous incident

in which P.T. materials that should

have been negative were inadvertently

contaminated during specimen

preparation with genetic material from

the putative agent, and a number of

corrective action interventions that

had been successful in the past were

suggested.

CAP then convened a conference call

with the P.T. vendor, who acknowledged

that the samples had been prepared

primarily for culture analysis (for which

the presence of dead organisms or

genomic DNA posed no problem) and

that not all processes were in place

to avoid genetic, i.e., nucleic acid,

contamination. Through this process,

it became clear that because of their

high levels of sensitivity (for MRSA as

well as GBS) the GeneXpert assayswere more prone to genomic DNA

carryover than other methods used

by participants (whether culture or

nucleic acid amplification) and that a

new standard for P.T. samples must be

implemented. Fortunately the vendor

had already identified potential sources

of carryover and had begun to alter its

production methods to accommodate

the required stringency for nucleic

acid amplification test methods. CAP

issued a letter to its participants

noting that the results from Cepheid

GeneXpert users would not be graded

In fact, the false positives were

actually true positives.

This incident should also serve as a

wake-up call to GeneXpert users

The high sensitivity of PCR protocols

can readily lead to problems if stringent

specimen handling protocols are not

followed. Batched testing is especially

prone to target contamination, and

in the age of closed system real-

time PCR, target contamination

probably accounts for the majority o

contamination events leading to false

positive results.1For laboratorians, this

is a modern version of the same age-old

problem that causes occasional false-positive mycobacterial cultures and

viral cultures. Sample contamination

appears to be resurfacing in the age o

molecular methods. C. difficilespores

are also notorious for their ability to

contaminate work environments so

the potential for sample contamination

is high.2 Good sample handling

technique is critical to getting good

results.


In the PCR world, genetic

targets can be inadvertentl

carried over from positive

patient samples as well as

from organisms, alive or

dead, that may contaminat

the workspace.


11/12

Wake-Up Cal l: Proficiency Testing for Todays Technology

The GeneXpert cartridge

is a closed system, which

is an important attribute

for controlling cross-

contamination during the

sample processing and

amplification steps.

However, paying careful attention to

the first steps of the process, that

of adding the specimen carefully to

the cartridge without carryover of

material from previously processed

specimens, is still important. This is

especially true when batch testing

is performed; tests performed in

random order will not be as subject

to this problem since the target-

specific detection reagents are

unique to each cartridge. Although

some testing of small batches maybe unavoidable, processing of large

batches of identical tests does not

take full advantage of the diagnostic

horsepower under the hood of your

GeneXpert, since batches take time

to accumulate and this occurs at

the expense of turnaround time.

Moreover, it puts your laboratory

at increased risk of experiencing a

false-positive result due to inadvertent

introduction of nucleic acid targets

into a negative patients sample or

test cartridge. Remember to followrecommended procedures no matter

how testing is being done; wash

hands and change gloves before and

after each new sample, clean the

bench and surrounding workspace

frequently with 10% bleach or DNA-

destroying cleaner, and discard

the used cartridges in a hard-sided

container to avoid leakage of contents

into the environment.


Call for Case StudiesSharing your interesting patient cases where GeneXpertmade a difference can be a rewarding experience. David

Persing, Fred Tenover, and Ellen Jo Baron invite you to

send us your case for publication in On-Demand.

The best case of the quarter will win

a copy of the new definitive reference

on Molecular Microbiology from ASM

Press, signed by Dr. David Persingand Dr. Fred Tenover.

Your case and a photo of your lab

will be published in On-Demand,

Cepheids quarterly newsletter:

http://www.cepheidondemand.com/

Write up the case including history,

symptoms, initial findings, sample

type received, assay(s) performed

(including routine laboratory tests), time to results, and

outcomes.

Describe how the GeneXpertSystem made a difference.

Photos or other images are especially welcome.

Send it to [email protected].


12/12

A better way.

REFERENCES


1. Fitzwater, S. P., et al. Prolonged infectiousness of tuberculosis patients in a directly observed

therapy short-course program with standardized therapy. Clin Infect Dis.51: 371-378.

2. Hesseling, A. C., et al. 2002. A critical review of diagnostic approaches used in the diagnosis of

childhood tuberculosis. Int J Tuberc Lung Dis.6: 1038-1045.

3. Marais, B. J. & H. S. Schaaf. Childhood tuberculosis: an emerging and previously neglected

problem. Infect Dis Clin North Am. 24: 727-749.

4. Mwinga, A. 2005. Challenges and hope for the diagnosis of tuberculosis in infants and young

children. Lancet. 365: 97-98.

5. Wood, R., et al. Tuberculosis transmission to young children in a South African community:

modeling household and community infection risks. Clin Infect Dis.51: 401-408.

6. Stefan, D. C., et al. Interferon-gamma release assays for the detection of Mycobacterium

tuberculosis infection in children with cancer. Int J Tuberc Lung Dis.14: 689-694.

7. Donald, P. R., et al. 1996. Stool microscopy and culture to assist the diagnosis of pulmonarytuberculosis in childhood.J Trop Pediatr.42: 311-312.

8. Oberhelman, R. A., et al. Diagnostic approaches for paediatric tuberculosis by use of different

specimen types, culture methods, and PCR: a prospective case-control study. Lancet Infect Dis.

10: 612-620.

9. Marais, B. J. 2007. Childhood tuberculosis--risk assessment and diagnosis. S Afr Med J. 97:

978-982.

10. Hatherill, M., et al. Structured approaches for the screening and diagnosis of childhood

tuberculosis in a high prevalence region of South Africa. Bull World Health Organ.88: 312-320.

11. Boehme, C. C., et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J

Med.363: 1005-1015.

Wake-Up Call: Proficiency Testing for Todays Technology

1. Sloan, L. M. 2007. Real-Time PCR in Clinical Microbiology: Verification, Validation, and

Contamination Control. Clinical Microbiology Newsletter. 29: 87-95.

2. Dumford, D. M., 3rd, et al. 2009. What is on that keyboard? Detecting hidden environmental

reservoirs of Clostridium difficileduring an outbreak associated with North American pulsed-field

gel electrophoresis type 1 strains.Am J Infect Control.37: 15-19.

Clostridium difficileRibotype 027: Why Should We Care?

1. McDonald, L. C., et al. 2004. Emergence of an epidemic strain of Clostridium difficilein the

United States 20014: potential role for virulence factors and antimicrobial resistance traits -

Abstract LB-2. Presented at 42nd Annual Meeting of the Infectious Diseases Society of America

Boston, MA, 2004.

2. Warny, M., et al. 2005. Toxin production by an emerging strain of Clostridium difficileassociated

with outbreaks of severe disease in North America and Europe. Lancet. 366: 1079-1084.

3. Kuijper, E. J., et al. 2006. Emergence of Clostridium difficile-associated disease in North America

and Europe. Clin Microbiol Infect.12 Suppl 6: 2-18.

4. O'Connor, J. R., et al. 2009. Clostridium difficileinfection caused by the epidemic BI/NAP1/027

strain. Gastroenterology. 136: 1913-1924.

5. Lyras, D., et al. 2009. Toxin B is essential for virulence of Clostridium difficile. Nature. 458: 1176-

1179.

6. Kuehne, S. A., et al. The role of toxin A and toxin B in Clostridium difficileinfection. Nature. 467:711-713.

7. Merrigan, M., et al. Human hypervirulent Clostridium difficilestrains exhibit increased sporulation

as well as robust toxin production.J Bacteriol.192: 4904-4911.

8. Vohra, P. & I. Poxton. Comparison of toxin and spore production in clinically relevant strains of

Clostridium difficile. Microbiology.

9. Carter, G. P., et al. 2007. Binary toxin production in Clostridium difficileis regulated by CdtR, a

LytTR family response regulator.J Bacteriol.189: 7290-7301.

10. Boyce, J. M., et al. 2008. Impact of hydrogen peroxide vapor room decontamination on

Clostridium difficileenvironmental contamination and transmission in a healthcare setting. Infect

Control Hosp Epidemiol.29: 723-729.

11. Voelker, R. Increased Clostridium difficilevirulence demands new treatment approach.JAMA.

303: 2017-2019.

12. Muto, C. A., et al. 2007. Control of an outbreak of infection with the hypervirulent Clostridium

difficileBI strain in a university hospital using a comprehensive "bundle" approach. Clin Infect Dis

45: 1266-1273.

13. Owens, R. C., Jr., et al. 2008. Antimicrobial-associated risk factors for Clostridium difficile

infection. Clin Infect Dis.46 Suppl 1: S19-31.

14. Louie, T. J., et al. Fidaxomicin versus vancomycin for Clostridium difficileinfection. N Engl J Med.

364: 422-431.

15. Cohen, S. H., et al. Clinical Practice Guidelines for Clostridium difficileInfection in Adults: 2010

Update by the Society for Healthcare Epidemiology of America (SHEA) and the InfectiousDiseases Society of America (IDSA). Infect Control Hosp Epidemiol.31: 431-455.

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cepheid ondemand report vol 4 issue 1 spring 2011(1)

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