Cepheid

report
A Quarterly Publication by Cepheid

The Need for

Better Diagnostic Tests for
Pediatric Tuberculosis
The AERAS-CHC-Cepheid Study
in Cambodia
Clostridium difficile Ribotype 027
Why Should We Care?
Wake-Up Call
Proficiency Testing for Today's Technology

Volume 4, Issue 1

CONTENTS SPRING 2011

Executive Editor
David Persing, M.D., Ph.D.

COVER STORY 3
The Need for
Better Diagnostic
Tests for Pediatric
Tuberculosis

Lead Author
Ellen Jo Baron, Ph.D.
Contributing Author
Fred Tenover, Ph.D.

Cepheids ONDEMAND 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 ONDEMAND Report
1327 Chesapeake Terrace
Sunnyvale, CA 94089

INSIDE 8
Clostridium difficile
Ribotype 027:
Why Should We Care?

INSIDE 10
Wake-Up Call:
Proficiency
Testing for Todays
Technology

editor@cepheidondemand.com
To sign up for e-mail notification
of new issues of Cepheids
ONDEMAND Report, visit
www.cepheidondemand.com

Contents are 2011 by Cepheid unless

otherwise indicated. Rights reserved on
all guest columns. The contents of this
publication may not be reproduced in any
form without written permission of
the editor. The mention of trade names,
commercial products, or organizations
does not imply endorsement by Cepheid.

David Persing
M.D., Ph.D.

From the Editor

Chief Medical and

Technology Officer, Cepheid

I am privileged at Cepheid to work with the some of the best minds in the diagnostics
business. 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 special
focus 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 is
committed 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 am
optimistic 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 molecular
diagnostic assays, and 2) the impact of the emergence of the 027 strain of C. difficile.
We hope you enjoy reading this information as we much as we have in preparing it.

The Need for Better Diagnostic Tests for Pediatric Tuberculosis

The Need for

Better Diagnostic
Tests for Pediatric
Tuberculosis
THE AERAS-CHC-CEPHEID STUDY IN
CAMBODIA

Ellen Jo Baron
Ph.D., D(ABMM)
Prof. Emerita, Stanford University
Director of Medical Affairs,Cepheid

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

population is infected with tuberculosis (TB). Each year approximately 9 million new
infections occur (most people who become infected do not exhibit symptoms of
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 the
organism 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 multidrug 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.

SPRING 2011 | CEPHEID

The Need for Better Diagnostic Tests for Pediatric Tuberculosis

FIGURE 1. Global burden of tuberculosis in 2008 (from www.stoptb.org site)

Although the international DOTS strategy has been widely

implemented, its overall impact on the prevalence and
incidence of tuberculosis, particularly in countries with a
high burden of HIV patients, has not met expectations. One
reason is that even those patients on effective therapy remain
infectious longer than previously realized.1 Another reason is
that the side effects of anti-tuberculosis drugs are not trivial.
The drugs can cause hepatitis, rashes, malaise, nausea,
thrombocytopenia, and neuropathy. In addition, some of
the pills are enormous and hard to swallow (Figure 2). No
wonder patients must be observed taking their medications; if
not being observed they would avoid taking them. The WHO
Tuberculosis Fact Sheet states that although the incidence
of TB was falling in all areas of the world, the population
growth in some areas offsets that small victory to result in a
net increase in the number of infected persons in Africa, the
Eastern Mediterranean, and South-East Asia (www.who.int/
mediacentre/factsheets/fs104/en/).
Recent estimates indicate that infants and children comprise
15-20% of cases of tuberculosis disease in resource-limited
countries.2 In some places this may be as high as 50%.3 In
developing regions, an increasing proportion of these children
are co-infected with HIV, which results in more severe TB
disease and higher mortality.4 Children often acquire their
disease from infected adults in their own family or in the
community.5 In fact, the incidence of TB in children, who
usually present with symptoms within a year of infection,
provides a snapshot of transmission within a community.6
Diagnosis of tuberculosis in children, however, is a major
challenge. Infants and young children may cough, but they
cannot be instructed to expectorate the sputum that they

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
Each year approximately 9 million new infections occur (most
people who become infected do not exhibit symptoms of
disease for many years, if ever) but around 1.6 million people
die of their disease; approximately one death every 9 seconds.

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 smearpositive respiratory samples.9 Their organisms tend to stay in
the perihilar lymph nodes and rarely rupture into the bronchial
tree, as they do in adults. Consequently, childrens respiratory
samples 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 <8 years old, is
based on a combination of clinical, radiological, and laboratory
criteria. A large analysis looking at nine different algorithms for
diagnosis of childhood TB, however, found major differences
in the number of cases defined by each algorithm, from a
low of 7% to a high of 89%!10 Criteria evaluated in various

The Need for Better Diagnostic Tests for Pediatric Tuberculosis

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-based
test, 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 <5 years old. It is
this setting where Dr. Anne Goldfeld has
chosen to try to make things better.
Dr. Anne Goldfeld, Professor in the
Department of Immunology and Infectious
Diseases at Harvard University, has been
actively working in the field of pediatric
infectious diseases for many years
(Figure 3). As a principle investigator
on a number of pediatric tuberculosis
diagnosis and treatment projects around
the resource-limited world, she works
with the Cambodian Health Committee
(http://www.globalhealthcommittee.org), a
subsidiary of the Global Health Committee,
whose mission is to improve the health and
well-being of patients, primarily children,
with tuberculosis and AIDS. The work
started in Cambodia. Central to these
efforts is the Maddox Chivan Childrens
Center in Phnom Penh, Cambodia, which
was built in part with grants from Angelina
Jolie and Brad Pitt. Dr. Goldfelds work
was recently featured on ABC News.
(See link: http://abcnews.go.com/Health/
global-tuberculosis-war-fighting-spread-tbcambodia-india/story?id=12395019)
AERAS (from the Greek word for air) is
another non-profit organization dedicated
to ridding the world of the scourge of
tuberculosis by developing an effective
vaccine that is affordable and available
throughout the world. Since 1921, the only
vaccine for TB has been BCG (Bacillus
Calmette-Gurin), named after the original
investigators who developed it in Lille,

France, i.e., Calmette and Gurin. In the

Pasteur Institute museum in Lille one can
see test tubes containing some of the actual
dried-out cultures of Mycobacterium bovis
on Lowenstein-Jensen agar worked on by
Calmette and Gurin. Dr. Anne Goldfeld
and Dr. Rinn Song, a Fellow in Infectious
Diseases from Children's Hospital in
Boston, are spearheading a project in the
far south-eastern Cambodian province of
Svay Rieng, bordering Vietnam, funded
partially by AERAS. The objectives are to
characterize the nature of tuberculosis
disease in the local children and to determine
which method of diagnosis would be best
for monitoring the effectiveness of the new
vaccine. As many as 1000 children will be
studied in the vaccine pilot project. Several
diagnostic tests are being evaluated,
including an interferon-gamma releasing
assay, acid fast smears, cultures in liquid
and on solid media, and the Xpert MTB/
RIF (CE-IVD), which was recently endorsed
by the World Health Organization. The
GeneXpert Test was developed with
support from the Foundation for Innovative
New Diagnostics (FIND) and the National
Institute for Allergy and Infectious Diseases
(NIAID); the first large multi-center trial of
the system was published recently in the
New England Journal of Medicine.11
Children with symptoms of respiratory
disease are brought to the provincial hospital
in Svay Rieng (Figure 4) by their parents.
They have been promised free diagnostic
tests, for which they must stay overnight
for 2 nights. All of their food and bedding is
provided during the stay, and each parent
receives a few personal hygiene products
and a new sarong for the child. The
children may not appreciate these sarongs,
as they are used to wrap their arms and
legs tightly to prevent them from squirming
during the specimen-collection procedures
that they must endure. At the hospital,
they are first evaluated by a physician for
symptoms. Radiographs are obtained and
blood is drawn for laboratory tests. Urine is
obtained for additional laboratory tests. A
tuberculin skin test is placed, and the child
and parent stay overnight in a nice room
with colorful, child-size hammocks (Figure
5). The first gastric aspirate is obtained the
next morning. The doctors and nurses at
the district hospital are highly skilled and
well-trained. The procedures they follow

FIGURE 2. Example of huge size of

some DOTS pills

FIGURE 3. Dr. Anne Goldfeld with a

Cambodian TB patient

FIGURE 4. District hospital, Svay Rieng,

Cambodia

FIGURE 5. Patient room at Svay Rieng

provincial hospital

SPRING 2011 | CEPHEID

The Need for Better Diagnostic Tests for Pediatric Tuberculosis

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
theworld.

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 7. (center) Child sitting on
fathers lap receiving albuterol.

FIGURE 8. (bottom) The patient is

prepared for insertion of the endotra

cheal tube. The suction device is on
the table to the right of the father at
the head of the bed.

VOLUME 4, ISSUE 1 | CEPHEID

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 the
patients 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.

FIGURE 9. (top) During the collection

of the aerosol-induced sputum.

FIGURE 10. (center) Samples being

placed into the insulated cold box for
transport.
FIGURE 11. (bottom) TB laboratory

technologist performing Gen-Probe as

say on MGIT-positive sample at Pasteur
Institute, Phnom Penh.

The Need for Better Diagnostic Tests for Pediatric Tuberculosis

What happens with those precious samples?

Each specimen is labeled with at least two

identifiers, and secured in a cold box (Figure 10),
after which the samples are transported by car
to the Pasteur Institute laboratory (Laboratoire de
Biologie Mdicale, Institut Pasteur du Cambodge)
in Phnom Penh, about 2.5 hours away.
This occurs each day; transport includes a
ferry ride across the Mekong River. Stools
are frozen at -80C for processing later, and
the gastric aspirates and sputum samples are
handled immediately. The Pasteur Laboratory
uses state-of-the-art culture and identification
methods, including MGIT liquid culture, GenProbe nucleic acid amplification assay, and
the HAIN line-probe system for species
identification and drug susceptibility testing
(Figure 11). A portion of each sample is
refrigerated for transport to the National Center
for Tuberculosis and Leprosy Control (CENAT)
Laboratory, the government-run TB laboratory
of Cambodia, where the other testing arm of
the study is performed, which includes testing
with the Xpert MTB/RIF (CE-IVD). Cepheid
loaned CHC a 4-module GeneXpert System,
sent experts in to set up the instrument and
conduct training (Figure 12), and is providing
all of the cartridges needed to test both gastric
aspirates and sputum samples. A flowchart for
specimen handling also was provided (Figure
13). The GeneXpert System fit easily into
their existing space, is easy to use, and the
results are beginning to accumulate (Figure
14). According to the previously cited ABC
news report, the system is already making a
difference.

FIGURE 12. Training technicians at the

FIGURE 14. GeneXpert in place on a table

CENAT laboratory in Phom

Penh.

opposite the MGIT in CENAT

laboratory.

1
3
2
6
4
7

Cepheid is proud to be contributing to this

major pediatric tuberculosis diagnosis and
treatment trial. Perhaps better diagnostic
methods for this difficult-to-diagnose disease
will come out of the study. In any case, many
children are receiving high quality care, their
parents are receiving health education, and
improved wellbeing for many Cambodians is a
direct benefit.

FIGURE 13. Flowchart of specimen handling for GeneXpert assay in Khmer.

SPRING 2011 | CEPHEID

Clostridium difficile Ribotype 027: Why Should We Care?

Clostridium difficile
Ribotype 027:
The first hint that something
new was happening with
regard to Clostridum
difficile infections (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 investigators
describing an emerging
epidemic strain that was
typically resistant to
fluoroquinolones.1

VOLUME 4, ISSUE 1 | CEPHEID

In 2005, a Lancet paper described

an emerging strain associated with
outbreaks of severe disease in North
America and Europe.2 Authors from
a 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.
difficile and 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 striking
finding, 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

C. difficile organisms lacking toxinB

were not pathogenic, even if they
produced toxin A, but toxin B-only
strains were fully pathogenic.5 New
studies have shown the importance
of both toxins in the hamster model.6
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 tcdC gene, allowing these
strains to produce considerably higher
amounts of both toxins A and B than wild
type toxinotype 0 strains.7 In fact, these
strains produced 20 times more toxins
A and B than most previously circulating
strains.8 The 027 strain also had an 18base pair deletion within tcdC, although
the effect of that deletion, which keeps
the protein in frame, was unknown.
Finally, the epidemic strain produced a
third 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 ADPribosyltransferase activity.9
Ribotype 027 isolates had another
characteristic that no doubt contributed
to their ability to spread quickly in a

Clostridium difficile Ribotype 027: Why Should We Care?

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 fumigated
patient
rooms
with
aerosolized
hydrogen peroxide, the same sporicidal
compound used to clear the Hart Senate
Office Building after the anthrax scare
in 2001,10 in 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 strain
type, since there are reports of mild
disease in sporadic (as opposed to
epidemic) cases of 027 infection.11 This
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

community exposure levels are not as

likely to be concentrated.
Hospital outbreaks of C. difficile strain
027 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.13 Knowing
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 strainassociated 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 vancomycin
and/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. difficile strains.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 in

a healthcare setting with ribotype 027
strains (especially from the same ward)
indicates a possible outbreak much
more rapidly than epidemiologic data
would alert infection preventionists to
a problem. This could be an indication
that the infection control bundles
described by Muto et al12 should be
considered; including switching from
alcohol-based hand gels to soap and
water for hand disinfection, longer
isolation of patients, possible pharmacy
controls including general restriction of
fluoroquinolones at an institution, and
more aggressive tracking of cases. This
is also an indication that the individual
patient should be monitored closely for
possible relapse. Indication of infection
caused by ribotype 027 should not be
used to guide therapy, since therapy
should be based on severity of disease
(treat the patient, not the microbe).15
Ribotype 027 strains have now spread
worldwide and will likely continue to
cause both epidemic and sporadic
disease. They will likely be a challenge
for years to come.

SPRING 2011 | CEPHEID

Wake-Up Call: Proficiency Testing for Todays Technology

Wake-Up Call:

Proficiency Testing for Todays Technology

HOW CEPHEIDS GBS ASSAY STIMULATED NEW AWARENESS AND HOW YOUR LABORATORY
SHOULD RESPOND TO AVOID SIMILAR PROBLEMS
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 crossreaction with other organisms present
in 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 supposedly
negative 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

10

VOLUME 4, ISSUE 1 | CEPHEID

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.

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.

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 assays
were 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

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 realtime PCR, target contamination
probably accounts for the majority of
contamination events leading to false
positive results.1 For laboratorians, this
is a modern version of the same age-old
problem that causes occasional falsepositive mycobacterial cultures and
viral cultures. Sample contamination
appears to be resurfacing in the age of
molecular methods. C. difficile spores
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.

This incident should also serve as a

wake-up call to GeneXpert users.

In the PCR world, genetic

targets can be inadvertently
carried over from positive
patient samples as well as
from organisms, alive or
dead, that may contaminate
the workspace.

Wake-Up Call: Proficiency Testing for Todays Technology

Call for Case Studies

The GeneXpert cartridge
is a closed system, which
is an important attribute
for controlling crosscontamination 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 targetspecific detection reagents are
unique to each cartridge. Although
some testing of small batches may
be 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 follow
recommended 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 DNAdestroying cleaner, and discard
the used cartridges in a hard-sided
container to avoid leakage of contents
into the environment.

Sharing your interesting patient cases where GeneXpert

made 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 Persing
and 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 GeneXpert System made a difference.
Photos or other images are especially welcome.
Send it to editor@cepheidondemand.com.

SPRING 2011 | CEPHEID

11

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The Need for Better Diagnostic Tests for Pediatric Tuberculosis

Clostridium difficile Ribotype 027: Why Should We Care?

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