- Email: [email protected]
How to: diagnose infection caused by Clostridium difficile
Accepted Manuscript How to: Diagnose infection caused by Clostridium difficile Cécile Gateau, Jeanne Couturier, John Coia, Frédéric Barbut PII:
S1198-743X(17)30678-X
DOI:
10.1016/j.cmi.2017.12.005
Reference:
CMI 1147
To appear in:
Clinical Microbiology and Infection
Received Date: 1 October 2017 Revised Date:
30 November 2017
Accepted Date: 7 December 2017
Please cite this article as: Gateau C, Couturier J, Coia J, Barbut F, How to: Diagnose infection caused by Clostridium difficile, Clinical Microbiology and Infection (2018), doi: 10.1016/j.cmi.2017.12.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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How to: diagnose infection caused by Clostridium difficile
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Cécile Gateau1, Jeanne Couturier1,2, John Coia3,4, Frédéric Barbut1,2,4
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Author’s affiliation:
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Antoine, 34 rue Crozatier, 75012 Paris, France
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EA4065, Université Paris Descartes, Paris, France
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Scottish Microbiology Reference Laboratories, Glasgow, Scotland
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European study group on Clostridium difficile
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National Reference Laboratory for C. difficile, Assistance Publique-Hôpitaux de Paris, Hôpital Saint-
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Contact information / corresponding author:
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Pr Frédéric Barbut
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[email protected],
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National Reference Laboratory for C. difficile,
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Hôpital Saint-Antoine, AP-HP
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34 rue Crozatier,
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75012 Paris, France.
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Tel: 33 1 49 28 30 11
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Fax: 33 1 49 28 30 09
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Statement indicating:
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Length of the abstract: 250 words
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Length of the paper (excluding abstract and references): 3441 words
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ACCEPTED MANUSCRIPT Abstract
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Background: Clostridium difficile is recognized as the major agent responsible for nosocomial
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diarrhoea. In the context of recent increase in the incidence and severity of CDI, an accurate
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diagnosis is essential for optimal treatment and prevention, but continues to be challenging.
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Aims: The present article reviews each key step of CDI diagnosis including stool selection, methods
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and strategies used, and interpretation of the results.
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Sources: The most recent guidelines for CDI diagnosis published by scientific societies were
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reviewed.
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Content: CDI diagnosis is based on clinical presentation and laboratory tests confirming the
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presence of toxigenic strain or toxins in stools. Stool selection is crucial and can be improved by
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implementing rejection criteria and strict policy for appropriate testing. Multiple laboratory tests
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detecting different targets (free toxin or presence of a potentially toxigenic strain) are commercially
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available. However, none of these tests combine high sensitivity and specificity to diagnose CDI, low
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hands-on time and low cost. An optimized diagnosis can be achieved by implementing a 2- or 3-step
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algorithm. Algorithms currently recommended by the ESCMID consist in a screening test with high
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sensitivity followed with a more specific test to detect free toxins. Presence of free toxins in stools
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has been shown to better correlate with severe outcome whereas nucleic acid amplification tests
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(NAAT) may lead to an over-diagnosis by detecting asymptomatic carriers of a toxigenic strain.
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Implication: To date, no single test can accurately diagnose CDI. Guidelines from the ESCMID
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recommend a 2 or 3-step algorithm for an optimal CDI detection.
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Keywords: Clostridium difficile, diagnostic methods, nucleic acid amplification test, glutamate
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dehydrogenase, toxins, diarrhoea, colitis
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Clostridium difficile is an anaerobic Gram-positive spore-forming bacillus responsible for a
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wide spectrum of clinical symptoms ranging from a mild self-limited diarrhea to pseudomembranous
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colitis (PMC), toxic megacolon, septic shock and possible death [1–5]. Risk factors for C. difficile
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infections (CDI) include age above 65 years, previous hospitalization, recent antibiotic therapy (in
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particular
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fluoroquinolones), immunosuppression and proton pump inhibitors [3,6]. Asymptomatic carriage is
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observed in 3% of patients on admission to hospital [7]. This frequency is higher among healthy
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neonates (30%-50%) and hospitalized patients (20 to 30%) [8]. Treatment and isolation of
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asymptomatic carriers are currently not recommended despite some evidence that they could play a
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role in C. difficile transmission [7,9]. In the U.S.A, C. difficile is the leading cause of healthcare-
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associated infections with more than 453 000 CDI per year, leading to 29 600 deaths [10]. In Europe,
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the estimated number of healthcare-associated CDI is 126 000 per year with a 3% attributable
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mortality [11]. C. difficile is also recognized as a major pathogen in the community despite substantial
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underdiagnosis [12]. The incidence of CDI has been increasing worldwide partly due to the
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emergence of an epidemic strain (NAP1/027/BI) responsible for large outbreaks of severe CDI in the
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last few years [13,14]. This hypervirulent clone is now endemic in Europe and the United States,
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despite large variations across countries or states [10,15]. A rapid and accurate diagnosis is essential
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to guide the treatment and to prevent transmission. It has been shown that rapid diagnosis impacts
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positively on the patient’s care by reducing delays in initiation of isolation and treatment for
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confirmed CDI cases. A negative test will also result in rapid discontinuation of empirical therapy and
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isolation [16,17]. Reliable data are also crucial for monitoring CDI incidence over time and
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comparison between different healthcare facilities. The purpose of this article is to review the
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methods and strategies currently available for CDI diagnosis and to highlight essential factors of CDI
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testing optimization.
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cephalosporins,
amoxicillin-clavulanate,
clindamycin,
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How to define a case of C. difficile infection? Only toxigenic strains, producing toxin A and/or B, are pathogenic [18]. According to the
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European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines, a CDI is
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defined as “(i) a clinical picture compatible with CDI and microbiological evidence of toxin A and/or
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toxin B producing C. difficile in stool without evidence of another cause of diarrhea or (ii) patient with
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PMC” [5]. A similar definition is given by the Society for Healthcare Epidemiology of America (SHEA)
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and the Infectious Diseases Society of America (IDSA): “A case of CDI is defined by the presence of
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symptoms (usually diarrhea) and either a stool test positive for C. difficile toxins or toxigenic C.
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difficile, or colonoscopic or histopathologic findings revealing PMC ”[19].
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How to select stool samples?
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Stool selection is essential since currently available tests do not accurately distinguish CDI
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from asymptomatic carriage of toxigenic C. difficile. This selection can be improved by implementing
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rejection criteria and a strict policy for appropriate testing. Continuous education of physicians and
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nurses along with monitoring and feedback are also necessary to reduce inappropriate testing [20].
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Only liquid or unformed stools, i.e. specimens taking the shape of the container, should be
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processed to avoid the identification of asymptomatic carriers. Because of insufficient volume, stool
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swabs cannot be used for toxin tests. However, swabs can be analyzed by culture or nucleic acid
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amplification tests (NAAT) for epidemiological studies or in case of a patient presenting with an ileus.
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For optimal recovery, stool specimens should be cultured within two hours after collection and must
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be preserved in a leak-proof container. Beyond this time, even if some spores can survive at 4°C, the
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number of viable C. difficile vegetative cells will significantly decrease. For toxin assay, stools can be
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stored at 4°C for a maximum of 3 days. If testing is delayed, specimens have to be frozen at -80°C
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[21,22]. Stool samples from children less than 3 years old should only be tested in specific cases (e.g
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neonates with Hirschprung disease or suspicion of an outbreak) because asymptomatic carriage is
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common in this population [23].
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Repeated testing should be discouraged [24] because of a low diagnostic gain (defined as the
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rate of negative samples that convert to positive) [25,26]. In addition, this practice increases the
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likelihood of false positive results due to lack of specificity of the methods [27]. This practice was
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frequent when EIA (Enzyme Immunoassay) for toxins was used as a stand-alone test to overcome the
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lack of sensitivity of these tests. If the new proposed algorithms are used (see section “which
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algorithm to use”), then a negative result with a screening test can reliably rule out the diagnosis of
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CDI due to the high negative predictive value (NPV) at low disease prevalence. However, in cases of
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outbreak situations where CDI prevalence is higher, the NPV of the algorithm will decrease, and a
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repeat sample in cases of ongoing clinical suspicion may be justified.
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Clinical cure is defined by the resolution of the symptoms. A “test-of-cure” is not
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recommended [28], since toxins and/or spores can persist in stools up to 6 weeks, despite symptom
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resolution [29].
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Stool samples should be taken prior to initiation of treatment to avoid false negative results.
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Indeed, in a prospective study including 51 patients with CDI, Sunkesula et al. [30] determined the
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conversion time of positive to negative test results after initiation of treatment. They showed that
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14%, 35%, and 45% of PCR positive tests are converted to negative after 1, 2, and 3 days of
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treatment, respectively. Physicians should be aware that any empirical therapy for suspected CDI
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started before stool collection can lead to a false-negative test result.
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What are the different diagnostic methods?
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Several laboratory tests, detecting different targets, are currently available to diagnose CDI
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(Fig 1). These tests detect either free toxins in stools (enzyme immunoassay [EIA], stool cytotoxicity
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assay [CTA]), the presence of C. difficile (EIA for glutamate dehydrogenase, [GDH]), or the presence of
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a toxigenic C. difficile strain (toxigenic culture [TC], nucleic acid amplification tests [NAAT]). Stool CTA
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and TC are considered as the gold-standard methods for detecting toxins or a toxigenic strain,
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respectively. Paradoxically, neither are routinely used because of technical issues and long
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turnaround time.
TC is a two-step method where C. difficile strains are first isolated on a selective medium [31–
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33] and then tested for their ability to produce toxins in vitro. Different selective media are available
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and they usually derive from the cycloserine cefoxitin fructose agar (CCFA) medium initially described
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by George et al. [31]. Subsequently, additive such as sodium taurocholate or lysozyme were added
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to stimulate germination and to enhance recovery. Chromogenic media have also been developed:
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they were shown to be as sensitive as other selective media, yielding identification within 24 h of
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incubation [32]. Plates are incubated in an anaerobic atmosphere for 48h at 36°C + 1°C. Strain
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identification can be performed using gallery strips, gas liquid chromatography, latex agglutination
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for GDH or matrix assisted laser desorption ionization-time-of-flight [MALDI-TOF] mass spectrometry.
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After isolation of a strain, its pathogenic potential is ascertained by testing for its in vitro toxin
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production directly from a suspension of colonies or from the broth supernatant of bacterial growth.
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TC is considered as the reference method to detect toxigenic C. difficile and remains a gold standard
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for evaluating new molecular methods. Although the turnaround time of this method is too long for
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routine diagnosis (2 to 5 days), culture is essential for subsequent typing, molecular analysis and
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determination of antimicrobial susceptibility.
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GDH is a metabolic enzyme expressed by all C. difficile strains. It can be detected by immuno-
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enzymatic (ELISA) or immuno-chromatographic assays. A positive result only indicates the presence
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of C. difficile, without predicting the ability of the strain to produce toxins. Different guidelines now
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proposed GDH EIA tests as a screening method for CDI diagnosis. Because of its high NPV (80.0%-
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100% [reviewed in [28] and [34]]), a negative test for GDH will generally rule out the infection.
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However, a NPV should be interpreted with caution and strongly depends on the prevalence of the
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disease: with a NPV of 99% and a CDI prevalence of 10%, one positive stool out of ten will be
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discarded if GDH is used as a screening test. A positive GDH result has to be confirmed by a second
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more specific test detecting toxins. CTA is considered as the reference method for detecting free toxins (mainly toxin B) in stools.
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This method consists in inoculating a stool filtrate on a cell culture and observing a specific cytopathic
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effect (CPE) (cell rounding) after 1 or 2 days of incubation at 36 + 1°C. Specificity of the CPE is
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assessed by neutralization with antisera directed against C. difficile toxin B or against C. sordellii
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toxins, which share the same antigens. In a large prospective study in the United Kingdom, positivity
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of stool CTA was shown to better correlate with clinical outcome than presence of toxigenic C.
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difficile [14]. Despite good sensitivity and specificity [35] and low cost of CTA, this method is currently
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used by a very limited number of laboratories because of lack of standardization (type of cells used,
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dilution of stool samples, incubation period) and long turn-around time.
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EIA mainly detect both toxins A and B (with or without differentiation) using monoclonal or polyclonal
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embedded
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(ELISA)
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immuno-
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chromatographic/lateral flow membrane devices. Many commercial EIAs are available. They provide
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rapid results and are easy to use. Nevertheless, many studies highlighted their lack of sensitivity
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(ranging from 29% to 86% reviewed in [28]) compared to CTA, precluding their use as stand-alone
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tests for CDI diagnosis.
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NAATs are based on real-time PCR, loop isothermal amplification or microarray technologies.
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Since the first FDA-approved test in 2009, new tests are regularly marketed (Table I). Some platforms
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are designed for low volume laboratories or point-of-care, whereas others are more suitable for high
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throughput testing. NAATs detect a large variety of targets including tcdB, tcdA, ∆117 deletion in tcdC
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(as a surrogate marker of the 027 epidemic strain) or binary toxin (cdt) genes. Some test systems
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combine detection of toxigenic strains of C. difficile with tests for other gastrointestinal pathogens in
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a syndromic approach [36]. Other characteristics like DNA extraction method, use of internal
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controls, manual assays or fully automated systems, must be considered when choosing a molecular
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method. NAATs are very sensitive (average sensitivity of 96% (95% CI = 0.93-0.98) compared to TC
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[37]) and have a high NPV. Like TC, the use of NAAT can lead to overdiagnosis of CDI due to
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asymptomatic carriage of toxigenic strain or inappropriate test ordering (e.g. patient without
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diarrhea). Indeed, these methods do not detect free toxins in the stool but only the genes encoding
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the toxins. In addition, one potential concern is genetic variation in tcdB or tcdA genes [38–40] that
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could lead to false negative results. As a result, the European Society of Clinical Microbiology and
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Infectious Diseases (ESCMID) guidelines do not recommend using NAAT as a stand-alone test for C.
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difficile diagnosis but rather to use NAAT as a screening test given its high negative predictive value
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for CDI. Then, a more specific toxin test will be used to identify patients most likely to have CDI.
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Results of NAATs can be achieved in one hour but they remain more expensive than TC or antigen-
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based assays. This is often a major obstacle to implement this technique as a screening method in a
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laboratory.
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Which strategy to implement?
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The optimal approach for detection of CDI is still matter of debates [41]. According the
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ESCMID, no single test can be recommended as a stand-alone test for diagnosing CDI, given their low
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positive predictive value (PPV) at a low CDI prevalence. Therefore, to optimize CDI diagnosis, two-
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step algorithms are currently recommended [28](fig.2). The first test should have a high NPV (i.e. a
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highly sensitive test) that reliably classifies patients as non-CDI; it can either be a GDH EIA or NAAT.
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The choice between both assays depends on the local organization, financial constraints and stool
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numbers tested per day. In case of a positive result, a second test with a high positive predictive
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value (PPV) (i.e. a highly specific test such as toxin A/B EIA) should be used. Patients with a positive
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second test can be reliably classified as CDI. Patients with a negative second test for toxins should be
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clinically evaluated: they can be either truly infected (with toxin level below the threshold detection
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of the toxin EIA assay) or carriers of a toxigenic strain (Fig. 2) [28]. If GDH was the initial test, then an
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optional third step can be performed by TC or NAAT to discern toxigenic from non-toxigenic strains.
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An alternative algorithm is to simultaneously detect GDH and toxins A and B by EIA. Different
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tests are now commercially available (C. diff Quik Chek Complete, TechLab, Alere; CERTEST
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Clostridium difficile GDH+toxin A+B, Theradiag; C. difficile GDH-toxins A-B, MonlabTest, Orgentec).
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The diagnosis of CDI can be reliably excluded in case of negative results for both GDH and toxins, and
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conversely patients with both positive GDH and toxin results can be classified as CDI. Samples with
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GDH-negative and toxin-positive results are rarely observed and need to be retested. In case of GDH-
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positive samples that are negative for both toxins, NAATs are optionally recommended by the
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ESCMID in order to determine whether a toxigenic C. difficile strain is present.
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How to interpret the results?
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The diagnosis of CDI relies on clinical evidence in association with laboratory tests. There is
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general agreement across international guidelines that EIA for toxins should not be used as a stand-
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alone test, while the debate regarding the clinical interpretation of the presence of a toxigenic strain
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without detectable toxins in stools is ongoing. A large observational study of more than 12 000
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patients with diarrhea was conducted in the UK [14]. Patients with a positive CTA assay had
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increased mortality and higher blood leukocyte counts compared to patients with diarrhea but
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negative for C. difficile, whereas those with detection of toxigenic C. difficile without free toxin (TC
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positive, CTA negative) did not. The authors concluded that detection of free toxin best correlated
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with poor clinical outcome and best defined CDI. These results were confirmed in a prospective
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observational cohort study at a single center in US, showing that patients positive for NAAT and toxin
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had more complications, a higher fecal lactoferrin level and a higher blood leukocyte count than
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patients positive for NAAT but negative for toxins [42]. Patients with positive TC but negative CTA are
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usually classified as potential C. difficile excretors (i.e., asymptomatic carriers with diarrhea not due
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to CDI) [14,42]; the isolation of these patients is recommended by some to prevent cross-
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transmission but this should be assessed case by case. In fact, a negative test for toxin in stool
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sample does not completely rule out the diagnosis of CDI. It has been shown that 11% of diarrheic
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patients harboring toxigenic strains of C. difficile but without detectable toxin in stools have
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pseudomembranes during endoscopic examination, suggesting that some CDI cases may be missed
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by reliance on toxin tests only [43].
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How do European countries perform?
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Underdiagnosis of CDI is a major issue in Europe. In a Spanish point-prevalence study, 66% of
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patients with CDI were undiagnosed or misdiagnosed because of lack of clinical suspicion (47.6%) or a
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lack of sensitivity of the diagnostic methods (19%) [44]. The European multicenter point prevalence
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study (EUCLID) conducted in 482 healthcare facilities from 20 countries indicated that 23% of
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samples positive for C. difficile were not diagnosed by participating laboratories due to a lack of
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clinical suspicion and that 1.5% were misdiagnosed due to false negative results. These results
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highlight the substantial burden of undetected CDI cases in Europe, which is likely to hamper control
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measures [15].
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A survey of diagnostic capacity was performed in Europe in 2011 and 2014 under the
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auspices of the European Clostridium difficile Infection Surveillance Network [45]. In 2011, 126
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laboratories from 31 countries completed a survey on local diagnostics. In 2014, a follow-up study
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was carried out by 84 of these 126 laboratories (67%) from 26 countries. The use of an optimal
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strategy (namely a 2-step algorithm as defined by the ESCMID guidelines) increased from 19% to
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46% [45]. The indications for CDI diagnostics reported in 2011 were on all stool samples in 2%, on all
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diarrheal stool specimens in 28%, in case of antibiotic-associated diarrhea in 50%, in cases of
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healthcare-associated diarrhea in 32%, and only upon physician’s request in 33%. In 2014, an
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improvement has been observed in indications for sending samples including an increased use of the
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Bristol stool scale to assess stool consistency for sample selection, an increased awareness from
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physicians for testing patients previously not monitored for CDI (e.g. outpatients, high-risk
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populations) and a better implementation of guidelines for sample selection (e.g. the three-day rule).
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In conclusion, the detection of C. difficile and its toxins should be carried out systematically in
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cases of healthcare-associated diarrhea or in case of any unexplained diarrhea. The implementation
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of an optimal diagnostic strategy based on a two-step algorithm provides a good trade-off between
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sensitivity and specificity for the CDI diagnostic and will allow a better management of the patients.
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ACCEPTED MANUSCRIPT Transparency declaration
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Gateau C. has nothing to disclose.
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Dr. Couturier reports non-financial support from Astellas, outside the submitted work.
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Dr. Coia has nothing to disclose.
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Dr. BARBUT reports grants, personal fees and non-financial support from Astellas, personal fees from
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Pfizer, grants and personal fees from Sanofi Pasteur, grants and non-financial support from Anios,
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grants, personal fees and non-financial support from MSD, grants from Biomérieux, grants from
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Quidel Buhlman, grants from Diasorin, grants from Cubist, grants from Biosynex, grants from
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GenePoc, outside the submitted work.
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Funding
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This study did not receive any external funding
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Acknowledgments
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The authors are grateful to the European Study Group on Clostridium difficile (ESGCD).
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1.
Leffler D, Lamont J. Clostridium difficile Infection. N Engl J Med. 2015;372(16):1539–48.
267 268
2.
Magill SS, Edwards JR, Bamberg W, Beldavs ZG, Dumyati G, Kainer MA, et al. Multistate PointPrevalence Survey of Health Care–Associated Infections. N Engl J Med. 2014;370(13):1198–208.
269 270
3.
Rupnik M, Wilcox MH, Gerding DN. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol. 2009;7(7):526–36.
271
4.
Kelly, MD CP, LaMont, MD JT. Clostridium difficile infection. Annu Rev Med. 1998;49(1):375–90.
272 273 274
5.
Bauer MP, Kuijper EJ, van Dissel JT. European Society of Clinical Microbiology and Infectious Diseases (ESCMID): treatment guidance document for Clostridium difficile infection (CDI). Clin Microbiol Infect. 2009;15(12):1067–79.
275 276 277
6.
Loo VG, Bourgault A-M, Poirier L, Lamothe F, Michaud S, Turgeon N, et al. Host and Pathogen Factors for Clostridium difficile Infection and Colonization. N Engl J Med. 2011;365(18):1693– 703.
278 279
7.
Longtin Y, Gilca R, Loo VG. Effect Of Detecting and Isolating Asymptomatic Clostridium difficile Carriers—Reply. JAMA Intern Med. 2016;176(10):1573.
280 281
8.
Sunenshine RH, McDonald LC. Clostridium difficile-associated disease: new challenges from an established pathogen. Cleve Clin J Med. 2006;73(2):187–97.
282
9.
Curry SR. Clostridium difficile. Clin Lab Med. 2017;37(2):341–69.
283 284
10.
Lessa FC, Winston LG, McDonald LC. Burden of Clostridium difficile Infection in the United States. N Engl J Med. 2015;372(24):2368–70.
285 286 287 288
11.
European Centre for Disease Prevention and Control. Point prevalence survey of healthcare associated infections and antimicrobial use in European acute care hospitals. https://ecdc.europa.eu/sites/portal/files/media/en/publications /Publications/healthcareassociated-infections-antimicrobial-use-PPS.pdf; 2011.
289 290 291
12.
Khanna S, Shin A, Kelly CP. Management of Clostridium difficile Infection in Inflammatory Bowel Disease: Expert Review from the Clinical Practice Updates Committee of the AGA Institute. Clin Gastroenterol Hepatol. 2017;15(2):166–74.
292 293
13.
Gerding DN. Clostridium difficile 30 years on: what has, or has not, changed and why? Int J Antimicrob Agents. 2009;33:S2–8.
294 295 296
14.
Planche TD, Davies KA, Coen PG, Finney JM, Monahan IM, Morris KA, et al. Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection. Lancet Infect Dis. 2013;13(11):936–45.
297 298 299 300
15.
Davies KA, Longshaw CM, Davis GL, Bouza E, Barbut F, Barna Z, et al. Underdiagnosis of Clostridium difficile across Europe: the European, multicentre, prospective, biannual, pointprevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID). Lancet Infect Dis. 2014;14(12):1208–19.
AC C
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265
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ACCEPTED MANUSCRIPT 16.
Barbut F, Surgers L, Eckert C, Visseaux B, Cuingnet M, Mesquita C, et al. Does a rapid diagnosis of Clostridium difficile infection impact on quality of patient management? Clin Microbiol Infect. 2014;20(2):136–44.
304 305 306
17.
Saade E, Deshpande A, Kundrapu S, Sunkesula VCK, Guerrero DM, Jury LA, et al. Appropriateness of empiric therapy in patients with suspected Clostridium difficile infection. Curr Med Res Opin. 2013;29(8):985–8.
307 308
18.
Savidge TC, Pan W-H, Newman P, O’brien M, Anton PM, Pothoulakis C. Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine. Gastroenterology. 2003;125(2):413–20.
309 310 311 312
19.
Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, et al. Clinical Practice Guidelines for Clostridium difficile Infection in Adults: 2010 Update by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infect Control Hosp Epidemiol. 2010;31(5):431–55.
313 314 315
20.
Jury LA, Tomas M, Kundrapu S, Sitzlar B, Donskey CJ. A Clostridium difficile Infection (CDI) Stewardship Initiative Improves Adherence to Practice Guidelines for Management of CDI. Infect Control Hosp Epidemiol. 2013;34(11):1222–4.
316 317 318
21.
Freeman J, Wilcox MH. The effects of storage conditions on viability of Clostridium difficile vegetative cells and spores and toxin activity in human faeces. J Clin Pathol. 2003 Feb;56(2):126–8.
319 320 321
22.
Barbut F, Beaugerie L, Delas N, Fossati-Marchal S, Aygalenq P, Petit J, et al. Comparative Value of Colonic Biopsy and Intraluminal Fluid Culture for Diagnosis of Bacterial Acute Colitis in Immunocompetent Patients. Clin Infect Dis. 1999 Aug;29(2):356–60.
322 323
23.
Schutze GE, Willoughby RE. Clostridium difficile Infection in Infants and Children. Pediatrics. 2013;131(1):196–200.
324 325
24.
Renshaw AA, Stelling JM, Doolittle MH. The lack of value of repeated Clostridium difficile cytotoxicity assays. Arch Pathol Lab Med. 1996;120(1):49–52.
326 327
25.
Rohner P, Pittet D, Pepey B, Nije-Kinge T, Auckenthaler R. Etiological agents of infectious diarrhea: implications for requests for microbial culture. J Clin Microbiol. 1997;35(6):1427–32.
328 329
26.
Manabe YC, Vinetz JM, Moore RD, Merz C, Charache P, Bartlett JG. Clostridium difficile colitis: an efficient clinical approach to diagnosis. Ann Intern Med. 1995;123(11):835–40.
330 331
27.
Peterson LR, Robicsek A. Does my patient have Clostridium difficile infection? Ann Intern Med. 2009;151(3):176–9.
332 333 334
28.
Crobach MJT, Planche T, Eckert C, Barbut F, Terveer EM, Dekkers OM, et al. European Society of Clinical Microbiology and Infectious Diseases: update of the diagnostic guidance document for Clostridium difficile infection. Microbiol Infect Dis. 2016 Aug;22 Suppl 4:S63-81.
335 336 337
29.
Sethi AK, Al-Nassir WN, Nerandzic MM, Bobulsky GS, Donskey CJ. Persistence of Skin Contamination and Environmental Shedding of Clostridium difficile during and after Treatment of C. difficile Infection. Infect Control Hosp Epidemiol. 2010;31(01):21–7.
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Sunkesula VCK, Kundrapu S, Muganda C, Sethi AK, Donskey CJ. Does Empirical Clostridium difficile Infection (CDI) Therapy Result in False-Negative CDI Diagnostic Test Results? Clin Infect Dis. 2013;57(4):494–500.
341 342
31.
George WL, Sutter VL, Citron D, Finegold SM. Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol. 1979;9(2):214–9.
343 344
32.
Eckert C, Burghoffer B, Lalande V, Barbut F. Evaluation of the Chromogenic Agar chromID C. difficile. J Clin Microbiol. 2013;51(3):1002–4.
345 346
33.
Marler LM, Siders JA, Wolters LC, Pettigrew Y, Skitt BL, Allen SD. Comparison of five cultural procedures for isolation of Clostridium difficile from stools. J Clin Microbiol. 1992;30(2):514–6.
347 348
34.
Shetty N, Wren MWD, Coen PG. The role of glutamate dehydrogenase for the detection of Clostridium difficile in faecal samples: a meta-analysis. J Hosp Infect. 2011;77(1):1–6.
349 350
35.
Bartlett JG. How to identify the cause of antibiotic-associated diarrhea. J Crit Illn. 1994;9(12):1063–7.
351 352 353
36.
Spina A, Kerr KG, Cormican M, Barbut F, Eigentler A, Zerva L, et al. Spectrum of enteropathogens detected by the FilmArray GI Panel in a multicentre study of communityacquired gastroenteritis. Clin Microbiol Infect. 2015;21(8):719–28.
354 355 356
37.
O’Horo JC, Jones A, Sternke M, Harper C, Safdar N. Molecular techniques for diagnosis of Clostridium difficile infection: systematic review and meta-analysis. Mayo Clin Proc. 2012;87(7):643–51.
357 358
38.
Squire MM, Carter GP, Mackin KE, Chakravorty A, Norén T, Elliott B, et al. Novel molecular type of Clostridium difficile in neonatal pigs, Western Australia. Emerg Infect Dis. 2013;19(5):790–2.
359 360
39.
Geric Stare B, Rupnik M. Clostridium difficile toxinotype XI (A-B-) exhibits unique arrangement of PaLoc and its upstream region. Anaerobe. 2010;16(4):393–5.
361 362 363
40.
Eckert C, Emirian A, Le Monnier A, Cathala L, De Montclos H, Goret J, et al. Prevalence and pathogenicity of binary toxin-positive Clostridium difficile strains that do not produce toxins A and B. New Microbes New Infect. 2015;3:12–7.
364 365 366
41.
Fang FC, Polage CR, Wilcox MH. Point-Counterpoint: What Is the Optimal Approach for Detection of Clostridium difficile Infection? Gilligan P, editor. J Clin Microbiol. 2017 Mar;55(3):670–80.
367 368
42.
Polage CR, Gyorke CE, Kennedy MA, Leslie JL, Chin DL, Wang S, et al. Overdiagnosis of Clostridium difficile Infection in the Molecular Test Era. JAMA Intern Med. 2015;175(11):1792.
369 370 371
43.
Gerding DN, Olson MM, Peterson LR, Teasley DG, Gebhard RL, Schwartz ML, et al. Clostridium difficile-associated diarrhea and colitis in adults. A prospective case-controlled epidemiologic study. Arch Intern Med. 1986;146(1):95–100.
372 373 374
44.
Alcalá L, Martín A, Marín M, Sánchez-Somolinos M, Catalán P, Peláez T, et al. The undiagnosed cases of Clostridium difficile infection in a whole nation: where is the problem? Microbiol Infect Dis. 2012;18(7):E204-213.
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45.
Van Dorp SM, Notermans DW, Alblas J, Gastmeier P, Mentula S, Nagy E, et al. European Clostridium difficile Infection Surveillance Network (ECDIS-Net) project on behalf of all participants. Survey of diagnostic and typing capacity for Clostridium difficile infection in Europe, 2011 and 2014.Euro Surveill; 2016.21;21(29).
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ACCEPTED MANUSCRIPT Table 1: Classification of commercially available NAAT for C. difficile (not exhaustive).
Detection of a single target Method
Target
®
Illumigene C.difficile (Meridian Bioscience)
tcdA
®
AmpliVue (QUIDELMolecular)
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tcdA
Simplexa™ C.difficile Universal Direct (FOCUS Diagnostics)
tcdB
Portrait Toxigenic C.difficile Assay (Portrait, Great Basin)
COBAS™ Cdiff (Roche) Prodesse ProGastro Cd assay (Gen Probe) ®
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ICEPlex C.difficile Kit (PrimeraDax)
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BD MAX™ Cdiff (BD Diagnostics)
tcdB
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BD GeneOhm™ Cdiff Assay (BD Diagnostics)
tcdB
tcdB
tcdB
tcdB
tcdB
Detection of several targets
Method ®
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GenoType Cdiff (Hain Lifescience)
Target tcdA, tcdB, several deletions in tcdC (including ∆117) cdtA, cdtB, gyrA, tpi
®
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IMDx C.difficile (IntelligentMDx)
tcdA, tcdB
®
Xpert C.difficile (Cepheid) ®
tcdB, cdt, deletion in position 117 in tcdC
Verigene C.difficile Test (Nanosphere)
tcdA, tcdB, binary toxin, deletion in position 117 in tcdC
RIDA GENE Clostridium difficile & Toxin A/B (R-biopharm)
tcdA, tcdB
®
®
Genspeed C.Diff Onestep (Genspeed Biotech)
gdh, tcdA, tcdB, binary toxin
®
Quidel Molecular Direct C.difficile Assay (Quidel Corporation)
tcdA, tdcB
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Target
®
Seeplex Diarrhea ACE Detection (Seegene) ®
FilmArray Gastrointestinal Panel (Biomerieux)
®
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RIDA GENE Gastro Panel (R-biopharm)
9 bacteria (including C. difficile tcdA tcdB genes) 3 viruses 3 parasites 22 bacteria (including C. difficile tcdB gene) 4 viruses 20 bacteria (including C. difficile tcdA tcdB genes) 5 viruses 4 parasites 10 bacteria (including C. difficile tcdA tcdB genes) 5 viruses 4 parasites
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xTAG Gastrointestinal Pathogen Panel (Theradiag)
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Fig.1: Advantages, disadvantages and targets of the different methods used for the diagnosis of CDI
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CTA: Cytotoxicity assay; EIA: Enzyme immunoassay; GDH: Glutamate dehydrogenase; NAAT: Nucleic acid
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amplification test; NPV: Negative predictive value; TAT: Turnaround time; CDI: Clostridium difficile infection.
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Fig. 2: Algorithms for the diagnosis of CDI (adapted from the ESCMID guidelines [28])
EIA: Enzyme immunoassay; GDH: Glutamate dehydrogenase; NAAT: Nucleic acid amplification test; CDI:
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Clostridium difficile infection ; TC: Toxigenic culture.
S1198-743X(17)30678-X
DOI:
10.1016/j.cmi.2017.12.005
Reference:
CMI 1147
To appear in:
Clinical Microbiology and Infection
Received Date: 1 October 2017 Revised Date:
30 November 2017
Accepted Date: 7 December 2017
Please cite this article as: Gateau C, Couturier J, Coia J, Barbut F, How to: Diagnose infection caused by Clostridium difficile, Clinical Microbiology and Infection (2018), doi: 10.1016/j.cmi.2017.12.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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How to: diagnose infection caused by Clostridium difficile
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Cécile Gateau1, Jeanne Couturier1,2, John Coia3,4, Frédéric Barbut1,2,4
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Author’s affiliation:
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1
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Antoine, 34 rue Crozatier, 75012 Paris, France
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2
EA4065, Université Paris Descartes, Paris, France
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3
Scottish Microbiology Reference Laboratories, Glasgow, Scotland
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European study group on Clostridium difficile
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National Reference Laboratory for C. difficile, Assistance Publique-Hôpitaux de Paris, Hôpital Saint-
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Contact information / corresponding author:
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Pr Frédéric Barbut
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[email protected],
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National Reference Laboratory for C. difficile,
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Hôpital Saint-Antoine, AP-HP
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34 rue Crozatier,
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75012 Paris, France.
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Tel: 33 1 49 28 30 11
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Fax: 33 1 49 28 30 09
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Statement indicating:
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Length of the abstract: 250 words
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Length of the paper (excluding abstract and references): 3441 words
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ACCEPTED MANUSCRIPT Abstract
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Background: Clostridium difficile is recognized as the major agent responsible for nosocomial
27
diarrhoea. In the context of recent increase in the incidence and severity of CDI, an accurate
28
diagnosis is essential for optimal treatment and prevention, but continues to be challenging.
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Aims: The present article reviews each key step of CDI diagnosis including stool selection, methods
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and strategies used, and interpretation of the results.
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Sources: The most recent guidelines for CDI diagnosis published by scientific societies were
32
reviewed.
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Content: CDI diagnosis is based on clinical presentation and laboratory tests confirming the
34
presence of toxigenic strain or toxins in stools. Stool selection is crucial and can be improved by
35
implementing rejection criteria and strict policy for appropriate testing. Multiple laboratory tests
36
detecting different targets (free toxin or presence of a potentially toxigenic strain) are commercially
37
available. However, none of these tests combine high sensitivity and specificity to diagnose CDI, low
38
hands-on time and low cost. An optimized diagnosis can be achieved by implementing a 2- or 3-step
39
algorithm. Algorithms currently recommended by the ESCMID consist in a screening test with high
40
sensitivity followed with a more specific test to detect free toxins. Presence of free toxins in stools
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has been shown to better correlate with severe outcome whereas nucleic acid amplification tests
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(NAAT) may lead to an over-diagnosis by detecting asymptomatic carriers of a toxigenic strain.
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Implication: To date, no single test can accurately diagnose CDI. Guidelines from the ESCMID
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recommend a 2 or 3-step algorithm for an optimal CDI detection.
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Keywords: Clostridium difficile, diagnostic methods, nucleic acid amplification test, glutamate
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dehydrogenase, toxins, diarrhoea, colitis
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Clostridium difficile is an anaerobic Gram-positive spore-forming bacillus responsible for a
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wide spectrum of clinical symptoms ranging from a mild self-limited diarrhea to pseudomembranous
51
colitis (PMC), toxic megacolon, septic shock and possible death [1–5]. Risk factors for C. difficile
52
infections (CDI) include age above 65 years, previous hospitalization, recent antibiotic therapy (in
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particular
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fluoroquinolones), immunosuppression and proton pump inhibitors [3,6]. Asymptomatic carriage is
55
observed in 3% of patients on admission to hospital [7]. This frequency is higher among healthy
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neonates (30%-50%) and hospitalized patients (20 to 30%) [8]. Treatment and isolation of
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asymptomatic carriers are currently not recommended despite some evidence that they could play a
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role in C. difficile transmission [7,9]. In the U.S.A, C. difficile is the leading cause of healthcare-
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associated infections with more than 453 000 CDI per year, leading to 29 600 deaths [10]. In Europe,
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the estimated number of healthcare-associated CDI is 126 000 per year with a 3% attributable
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mortality [11]. C. difficile is also recognized as a major pathogen in the community despite substantial
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underdiagnosis [12]. The incidence of CDI has been increasing worldwide partly due to the
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emergence of an epidemic strain (NAP1/027/BI) responsible for large outbreaks of severe CDI in the
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last few years [13,14]. This hypervirulent clone is now endemic in Europe and the United States,
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despite large variations across countries or states [10,15]. A rapid and accurate diagnosis is essential
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to guide the treatment and to prevent transmission. It has been shown that rapid diagnosis impacts
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positively on the patient’s care by reducing delays in initiation of isolation and treatment for
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confirmed CDI cases. A negative test will also result in rapid discontinuation of empirical therapy and
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isolation [16,17]. Reliable data are also crucial for monitoring CDI incidence over time and
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comparison between different healthcare facilities. The purpose of this article is to review the
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methods and strategies currently available for CDI diagnosis and to highlight essential factors of CDI
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testing optimization.
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cephalosporins,
amoxicillin-clavulanate,
clindamycin,
and
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How to define a case of C. difficile infection? Only toxigenic strains, producing toxin A and/or B, are pathogenic [18]. According to the
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European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines, a CDI is
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defined as “(i) a clinical picture compatible with CDI and microbiological evidence of toxin A and/or
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toxin B producing C. difficile in stool without evidence of another cause of diarrhea or (ii) patient with
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PMC” [5]. A similar definition is given by the Society for Healthcare Epidemiology of America (SHEA)
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and the Infectious Diseases Society of America (IDSA): “A case of CDI is defined by the presence of
81
symptoms (usually diarrhea) and either a stool test positive for C. difficile toxins or toxigenic C.
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difficile, or colonoscopic or histopathologic findings revealing PMC ”[19].
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How to select stool samples?
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Stool selection is essential since currently available tests do not accurately distinguish CDI
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from asymptomatic carriage of toxigenic C. difficile. This selection can be improved by implementing
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rejection criteria and a strict policy for appropriate testing. Continuous education of physicians and
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nurses along with monitoring and feedback are also necessary to reduce inappropriate testing [20].
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Only liquid or unformed stools, i.e. specimens taking the shape of the container, should be
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processed to avoid the identification of asymptomatic carriers. Because of insufficient volume, stool
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swabs cannot be used for toxin tests. However, swabs can be analyzed by culture or nucleic acid
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amplification tests (NAAT) for epidemiological studies or in case of a patient presenting with an ileus.
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For optimal recovery, stool specimens should be cultured within two hours after collection and must
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be preserved in a leak-proof container. Beyond this time, even if some spores can survive at 4°C, the
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number of viable C. difficile vegetative cells will significantly decrease. For toxin assay, stools can be
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stored at 4°C for a maximum of 3 days. If testing is delayed, specimens have to be frozen at -80°C
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[21,22]. Stool samples from children less than 3 years old should only be tested in specific cases (e.g
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neonates with Hirschprung disease or suspicion of an outbreak) because asymptomatic carriage is
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common in this population [23].
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Repeated testing should be discouraged [24] because of a low diagnostic gain (defined as the
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rate of negative samples that convert to positive) [25,26]. In addition, this practice increases the
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likelihood of false positive results due to lack of specificity of the methods [27]. This practice was
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frequent when EIA (Enzyme Immunoassay) for toxins was used as a stand-alone test to overcome the
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lack of sensitivity of these tests. If the new proposed algorithms are used (see section “which
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algorithm to use”), then a negative result with a screening test can reliably rule out the diagnosis of
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CDI due to the high negative predictive value (NPV) at low disease prevalence. However, in cases of
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outbreak situations where CDI prevalence is higher, the NPV of the algorithm will decrease, and a
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repeat sample in cases of ongoing clinical suspicion may be justified.
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Clinical cure is defined by the resolution of the symptoms. A “test-of-cure” is not
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recommended [28], since toxins and/or spores can persist in stools up to 6 weeks, despite symptom
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resolution [29].
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Stool samples should be taken prior to initiation of treatment to avoid false negative results.
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Indeed, in a prospective study including 51 patients with CDI, Sunkesula et al. [30] determined the
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conversion time of positive to negative test results after initiation of treatment. They showed that
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14%, 35%, and 45% of PCR positive tests are converted to negative after 1, 2, and 3 days of
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treatment, respectively. Physicians should be aware that any empirical therapy for suspected CDI
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started before stool collection can lead to a false-negative test result.
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What are the different diagnostic methods?
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Several laboratory tests, detecting different targets, are currently available to diagnose CDI
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(Fig 1). These tests detect either free toxins in stools (enzyme immunoassay [EIA], stool cytotoxicity
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assay [CTA]), the presence of C. difficile (EIA for glutamate dehydrogenase, [GDH]), or the presence of
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a toxigenic C. difficile strain (toxigenic culture [TC], nucleic acid amplification tests [NAAT]). Stool CTA
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and TC are considered as the gold-standard methods for detecting toxins or a toxigenic strain,
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respectively. Paradoxically, neither are routinely used because of technical issues and long
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turnaround time.
TC is a two-step method where C. difficile strains are first isolated on a selective medium [31–
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33] and then tested for their ability to produce toxins in vitro. Different selective media are available
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and they usually derive from the cycloserine cefoxitin fructose agar (CCFA) medium initially described
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by George et al. [31]. Subsequently, additive such as sodium taurocholate or lysozyme were added
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to stimulate germination and to enhance recovery. Chromogenic media have also been developed:
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they were shown to be as sensitive as other selective media, yielding identification within 24 h of
131
incubation [32]. Plates are incubated in an anaerobic atmosphere for 48h at 36°C + 1°C. Strain
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identification can be performed using gallery strips, gas liquid chromatography, latex agglutination
133
for GDH or matrix assisted laser desorption ionization-time-of-flight [MALDI-TOF] mass spectrometry.
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After isolation of a strain, its pathogenic potential is ascertained by testing for its in vitro toxin
135
production directly from a suspension of colonies or from the broth supernatant of bacterial growth.
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TC is considered as the reference method to detect toxigenic C. difficile and remains a gold standard
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for evaluating new molecular methods. Although the turnaround time of this method is too long for
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routine diagnosis (2 to 5 days), culture is essential for subsequent typing, molecular analysis and
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determination of antimicrobial susceptibility.
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GDH is a metabolic enzyme expressed by all C. difficile strains. It can be detected by immuno-
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enzymatic (ELISA) or immuno-chromatographic assays. A positive result only indicates the presence
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of C. difficile, without predicting the ability of the strain to produce toxins. Different guidelines now
143
proposed GDH EIA tests as a screening method for CDI diagnosis. Because of its high NPV (80.0%-
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100% [reviewed in [28] and [34]]), a negative test for GDH will generally rule out the infection.
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However, a NPV should be interpreted with caution and strongly depends on the prevalence of the
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disease: with a NPV of 99% and a CDI prevalence of 10%, one positive stool out of ten will be
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discarded if GDH is used as a screening test. A positive GDH result has to be confirmed by a second
148
more specific test detecting toxins. CTA is considered as the reference method for detecting free toxins (mainly toxin B) in stools.
150
This method consists in inoculating a stool filtrate on a cell culture and observing a specific cytopathic
151
effect (CPE) (cell rounding) after 1 or 2 days of incubation at 36 + 1°C. Specificity of the CPE is
152
assessed by neutralization with antisera directed against C. difficile toxin B or against C. sordellii
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toxins, which share the same antigens. In a large prospective study in the United Kingdom, positivity
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of stool CTA was shown to better correlate with clinical outcome than presence of toxigenic C.
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difficile [14]. Despite good sensitivity and specificity [35] and low cost of CTA, this method is currently
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used by a very limited number of laboratories because of lack of standardization (type of cells used,
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dilution of stool samples, incubation period) and long turn-around time.
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EIA mainly detect both toxins A and B (with or without differentiation) using monoclonal or polyclonal
antibodies
embedded
in
micro-well immuno-enzymatic
(ELISA)
or
immuno-
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chromatographic/lateral flow membrane devices. Many commercial EIAs are available. They provide
161
rapid results and are easy to use. Nevertheless, many studies highlighted their lack of sensitivity
162
(ranging from 29% to 86% reviewed in [28]) compared to CTA, precluding their use as stand-alone
163
tests for CDI diagnosis.
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NAATs are based on real-time PCR, loop isothermal amplification or microarray technologies.
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Since the first FDA-approved test in 2009, new tests are regularly marketed (Table I). Some platforms
166
are designed for low volume laboratories or point-of-care, whereas others are more suitable for high
167
throughput testing. NAATs detect a large variety of targets including tcdB, tcdA, ∆117 deletion in tcdC
168
(as a surrogate marker of the 027 epidemic strain) or binary toxin (cdt) genes. Some test systems
169
combine detection of toxigenic strains of C. difficile with tests for other gastrointestinal pathogens in
170
a syndromic approach [36]. Other characteristics like DNA extraction method, use of internal
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controls, manual assays or fully automated systems, must be considered when choosing a molecular
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method. NAATs are very sensitive (average sensitivity of 96% (95% CI = 0.93-0.98) compared to TC
173
[37]) and have a high NPV. Like TC, the use of NAAT can lead to overdiagnosis of CDI due to
174
asymptomatic carriage of toxigenic strain or inappropriate test ordering (e.g. patient without
175
diarrhea). Indeed, these methods do not detect free toxins in the stool but only the genes encoding
176
the toxins. In addition, one potential concern is genetic variation in tcdB or tcdA genes [38–40] that
177
could lead to false negative results. As a result, the European Society of Clinical Microbiology and
178
Infectious Diseases (ESCMID) guidelines do not recommend using NAAT as a stand-alone test for C.
179
difficile diagnosis but rather to use NAAT as a screening test given its high negative predictive value
180
for CDI. Then, a more specific toxin test will be used to identify patients most likely to have CDI.
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Results of NAATs can be achieved in one hour but they remain more expensive than TC or antigen-
182
based assays. This is often a major obstacle to implement this technique as a screening method in a
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laboratory.
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Which strategy to implement?
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The optimal approach for detection of CDI is still matter of debates [41]. According the
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ESCMID, no single test can be recommended as a stand-alone test for diagnosing CDI, given their low
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positive predictive value (PPV) at a low CDI prevalence. Therefore, to optimize CDI diagnosis, two-
188
step algorithms are currently recommended [28](fig.2). The first test should have a high NPV (i.e. a
189
highly sensitive test) that reliably classifies patients as non-CDI; it can either be a GDH EIA or NAAT.
190
The choice between both assays depends on the local organization, financial constraints and stool
191
numbers tested per day. In case of a positive result, a second test with a high positive predictive
192
value (PPV) (i.e. a highly specific test such as toxin A/B EIA) should be used. Patients with a positive
193
second test can be reliably classified as CDI. Patients with a negative second test for toxins should be
194
clinically evaluated: they can be either truly infected (with toxin level below the threshold detection
195
of the toxin EIA assay) or carriers of a toxigenic strain (Fig. 2) [28]. If GDH was the initial test, then an
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optional third step can be performed by TC or NAAT to discern toxigenic from non-toxigenic strains.
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An alternative algorithm is to simultaneously detect GDH and toxins A and B by EIA. Different
198
tests are now commercially available (C. diff Quik Chek Complete, TechLab, Alere; CERTEST
199
Clostridium difficile GDH+toxin A+B, Theradiag; C. difficile GDH-toxins A-B, MonlabTest, Orgentec).
200
The diagnosis of CDI can be reliably excluded in case of negative results for both GDH and toxins, and
201
conversely patients with both positive GDH and toxin results can be classified as CDI. Samples with
202
GDH-negative and toxin-positive results are rarely observed and need to be retested. In case of GDH-
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positive samples that are negative for both toxins, NAATs are optionally recommended by the
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ESCMID in order to determine whether a toxigenic C. difficile strain is present.
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How to interpret the results?
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The diagnosis of CDI relies on clinical evidence in association with laboratory tests. There is
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general agreement across international guidelines that EIA for toxins should not be used as a stand-
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alone test, while the debate regarding the clinical interpretation of the presence of a toxigenic strain
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without detectable toxins in stools is ongoing. A large observational study of more than 12 000
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patients with diarrhea was conducted in the UK [14]. Patients with a positive CTA assay had
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increased mortality and higher blood leukocyte counts compared to patients with diarrhea but
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negative for C. difficile, whereas those with detection of toxigenic C. difficile without free toxin (TC
213
positive, CTA negative) did not. The authors concluded that detection of free toxin best correlated
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with poor clinical outcome and best defined CDI. These results were confirmed in a prospective
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observational cohort study at a single center in US, showing that patients positive for NAAT and toxin
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had more complications, a higher fecal lactoferrin level and a higher blood leukocyte count than
217
patients positive for NAAT but negative for toxins [42]. Patients with positive TC but negative CTA are
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usually classified as potential C. difficile excretors (i.e., asymptomatic carriers with diarrhea not due
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to CDI) [14,42]; the isolation of these patients is recommended by some to prevent cross-
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transmission but this should be assessed case by case. In fact, a negative test for toxin in stool
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sample does not completely rule out the diagnosis of CDI. It has been shown that 11% of diarrheic
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patients harboring toxigenic strains of C. difficile but without detectable toxin in stools have
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pseudomembranes during endoscopic examination, suggesting that some CDI cases may be missed
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by reliance on toxin tests only [43].
225
How do European countries perform?
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Underdiagnosis of CDI is a major issue in Europe. In a Spanish point-prevalence study, 66% of
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patients with CDI were undiagnosed or misdiagnosed because of lack of clinical suspicion (47.6%) or a
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lack of sensitivity of the diagnostic methods (19%) [44]. The European multicenter point prevalence
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study (EUCLID) conducted in 482 healthcare facilities from 20 countries indicated that 23% of
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samples positive for C. difficile were not diagnosed by participating laboratories due to a lack of
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clinical suspicion and that 1.5% were misdiagnosed due to false negative results. These results
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highlight the substantial burden of undetected CDI cases in Europe, which is likely to hamper control
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measures [15].
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A survey of diagnostic capacity was performed in Europe in 2011 and 2014 under the
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auspices of the European Clostridium difficile Infection Surveillance Network [45]. In 2011, 126
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laboratories from 31 countries completed a survey on local diagnostics. In 2014, a follow-up study
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was carried out by 84 of these 126 laboratories (67%) from 26 countries. The use of an optimal
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strategy (namely a 2-step algorithm as defined by the ESCMID guidelines) increased from 19% to
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46% [45]. The indications for CDI diagnostics reported in 2011 were on all stool samples in 2%, on all
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diarrheal stool specimens in 28%, in case of antibiotic-associated diarrhea in 50%, in cases of
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healthcare-associated diarrhea in 32%, and only upon physician’s request in 33%. In 2014, an
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improvement has been observed in indications for sending samples including an increased use of the
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Bristol stool scale to assess stool consistency for sample selection, an increased awareness from
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physicians for testing patients previously not monitored for CDI (e.g. outpatients, high-risk
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populations) and a better implementation of guidelines for sample selection (e.g. the three-day rule).
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In conclusion, the detection of C. difficile and its toxins should be carried out systematically in
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cases of healthcare-associated diarrhea or in case of any unexplained diarrhea. The implementation
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of an optimal diagnostic strategy based on a two-step algorithm provides a good trade-off between
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sensitivity and specificity for the CDI diagnostic and will allow a better management of the patients.
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ACCEPTED MANUSCRIPT Transparency declaration
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Gateau C. has nothing to disclose.
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Dr. Couturier reports non-financial support from Astellas, outside the submitted work.
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Dr. Coia has nothing to disclose.
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Dr. BARBUT reports grants, personal fees and non-financial support from Astellas, personal fees from
256
Pfizer, grants and personal fees from Sanofi Pasteur, grants and non-financial support from Anios,
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grants, personal fees and non-financial support from MSD, grants from Biomérieux, grants from
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Quidel Buhlman, grants from Diasorin, grants from Cubist, grants from Biosynex, grants from
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GenePoc, outside the submitted work.
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Funding
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This study did not receive any external funding
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Acknowledgments
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The authors are grateful to the European Study Group on Clostridium difficile (ESGCD).
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266
1.
Leffler D, Lamont J. Clostridium difficile Infection. N Engl J Med. 2015;372(16):1539–48.
267 268
2.
Magill SS, Edwards JR, Bamberg W, Beldavs ZG, Dumyati G, Kainer MA, et al. Multistate PointPrevalence Survey of Health Care–Associated Infections. N Engl J Med. 2014;370(13):1198–208.
269 270
3.
Rupnik M, Wilcox MH, Gerding DN. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol. 2009;7(7):526–36.
271
4.
Kelly, MD CP, LaMont, MD JT. Clostridium difficile infection. Annu Rev Med. 1998;49(1):375–90.
272 273 274
5.
Bauer MP, Kuijper EJ, van Dissel JT. European Society of Clinical Microbiology and Infectious Diseases (ESCMID): treatment guidance document for Clostridium difficile infection (CDI). Clin Microbiol Infect. 2009;15(12):1067–79.
275 276 277
6.
Loo VG, Bourgault A-M, Poirier L, Lamothe F, Michaud S, Turgeon N, et al. Host and Pathogen Factors for Clostridium difficile Infection and Colonization. N Engl J Med. 2011;365(18):1693– 703.
278 279
7.
Longtin Y, Gilca R, Loo VG. Effect Of Detecting and Isolating Asymptomatic Clostridium difficile Carriers—Reply. JAMA Intern Med. 2016;176(10):1573.
280 281
8.
Sunenshine RH, McDonald LC. Clostridium difficile-associated disease: new challenges from an established pathogen. Cleve Clin J Med. 2006;73(2):187–97.
282
9.
Curry SR. Clostridium difficile. Clin Lab Med. 2017;37(2):341–69.
283 284
10.
Lessa FC, Winston LG, McDonald LC. Burden of Clostridium difficile Infection in the United States. N Engl J Med. 2015;372(24):2368–70.
285 286 287 288
11.
European Centre for Disease Prevention and Control. Point prevalence survey of healthcare associated infections and antimicrobial use in European acute care hospitals. https://ecdc.europa.eu/sites/portal/files/media/en/publications /Publications/healthcareassociated-infections-antimicrobial-use-PPS.pdf; 2011.
289 290 291
12.
Khanna S, Shin A, Kelly CP. Management of Clostridium difficile Infection in Inflammatory Bowel Disease: Expert Review from the Clinical Practice Updates Committee of the AGA Institute. Clin Gastroenterol Hepatol. 2017;15(2):166–74.
292 293
13.
Gerding DN. Clostridium difficile 30 years on: what has, or has not, changed and why? Int J Antimicrob Agents. 2009;33:S2–8.
294 295 296
14.
Planche TD, Davies KA, Coen PG, Finney JM, Monahan IM, Morris KA, et al. Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection. Lancet Infect Dis. 2013;13(11):936–45.
297 298 299 300
15.
Davies KA, Longshaw CM, Davis GL, Bouza E, Barbut F, Barna Z, et al. Underdiagnosis of Clostridium difficile across Europe: the European, multicentre, prospective, biannual, pointprevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID). Lancet Infect Dis. 2014;14(12):1208–19.
AC C
EP
TE D
M AN U
SC
RI PT
265
14
ACCEPTED MANUSCRIPT 16.
Barbut F, Surgers L, Eckert C, Visseaux B, Cuingnet M, Mesquita C, et al. Does a rapid diagnosis of Clostridium difficile infection impact on quality of patient management? Clin Microbiol Infect. 2014;20(2):136–44.
304 305 306
17.
Saade E, Deshpande A, Kundrapu S, Sunkesula VCK, Guerrero DM, Jury LA, et al. Appropriateness of empiric therapy in patients with suspected Clostridium difficile infection. Curr Med Res Opin. 2013;29(8):985–8.
307 308
18.
Savidge TC, Pan W-H, Newman P, O’brien M, Anton PM, Pothoulakis C. Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine. Gastroenterology. 2003;125(2):413–20.
309 310 311 312
19.
Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, et al. Clinical Practice Guidelines for Clostridium difficile Infection in Adults: 2010 Update by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infect Control Hosp Epidemiol. 2010;31(5):431–55.
313 314 315
20.
Jury LA, Tomas M, Kundrapu S, Sitzlar B, Donskey CJ. A Clostridium difficile Infection (CDI) Stewardship Initiative Improves Adherence to Practice Guidelines for Management of CDI. Infect Control Hosp Epidemiol. 2013;34(11):1222–4.
316 317 318
21.
Freeman J, Wilcox MH. The effects of storage conditions on viability of Clostridium difficile vegetative cells and spores and toxin activity in human faeces. J Clin Pathol. 2003 Feb;56(2):126–8.
319 320 321
22.
Barbut F, Beaugerie L, Delas N, Fossati-Marchal S, Aygalenq P, Petit J, et al. Comparative Value of Colonic Biopsy and Intraluminal Fluid Culture for Diagnosis of Bacterial Acute Colitis in Immunocompetent Patients. Clin Infect Dis. 1999 Aug;29(2):356–60.
322 323
23.
Schutze GE, Willoughby RE. Clostridium difficile Infection in Infants and Children. Pediatrics. 2013;131(1):196–200.
324 325
24.
Renshaw AA, Stelling JM, Doolittle MH. The lack of value of repeated Clostridium difficile cytotoxicity assays. Arch Pathol Lab Med. 1996;120(1):49–52.
326 327
25.
Rohner P, Pittet D, Pepey B, Nije-Kinge T, Auckenthaler R. Etiological agents of infectious diarrhea: implications for requests for microbial culture. J Clin Microbiol. 1997;35(6):1427–32.
328 329
26.
Manabe YC, Vinetz JM, Moore RD, Merz C, Charache P, Bartlett JG. Clostridium difficile colitis: an efficient clinical approach to diagnosis. Ann Intern Med. 1995;123(11):835–40.
330 331
27.
Peterson LR, Robicsek A. Does my patient have Clostridium difficile infection? Ann Intern Med. 2009;151(3):176–9.
332 333 334
28.
Crobach MJT, Planche T, Eckert C, Barbut F, Terveer EM, Dekkers OM, et al. European Society of Clinical Microbiology and Infectious Diseases: update of the diagnostic guidance document for Clostridium difficile infection. Microbiol Infect Dis. 2016 Aug;22 Suppl 4:S63-81.
335 336 337
29.
Sethi AK, Al-Nassir WN, Nerandzic MM, Bobulsky GS, Donskey CJ. Persistence of Skin Contamination and Environmental Shedding of Clostridium difficile during and after Treatment of C. difficile Infection. Infect Control Hosp Epidemiol. 2010;31(01):21–7.
AC C
EP
TE D
M AN U
SC
RI PT
301 302 303
15
ACCEPTED MANUSCRIPT 30.
Sunkesula VCK, Kundrapu S, Muganda C, Sethi AK, Donskey CJ. Does Empirical Clostridium difficile Infection (CDI) Therapy Result in False-Negative CDI Diagnostic Test Results? Clin Infect Dis. 2013;57(4):494–500.
341 342
31.
George WL, Sutter VL, Citron D, Finegold SM. Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol. 1979;9(2):214–9.
343 344
32.
Eckert C, Burghoffer B, Lalande V, Barbut F. Evaluation of the Chromogenic Agar chromID C. difficile. J Clin Microbiol. 2013;51(3):1002–4.
345 346
33.
Marler LM, Siders JA, Wolters LC, Pettigrew Y, Skitt BL, Allen SD. Comparison of five cultural procedures for isolation of Clostridium difficile from stools. J Clin Microbiol. 1992;30(2):514–6.
347 348
34.
Shetty N, Wren MWD, Coen PG. The role of glutamate dehydrogenase for the detection of Clostridium difficile in faecal samples: a meta-analysis. J Hosp Infect. 2011;77(1):1–6.
349 350
35.
Bartlett JG. How to identify the cause of antibiotic-associated diarrhea. J Crit Illn. 1994;9(12):1063–7.
351 352 353
36.
Spina A, Kerr KG, Cormican M, Barbut F, Eigentler A, Zerva L, et al. Spectrum of enteropathogens detected by the FilmArray GI Panel in a multicentre study of communityacquired gastroenteritis. Clin Microbiol Infect. 2015;21(8):719–28.
354 355 356
37.
O’Horo JC, Jones A, Sternke M, Harper C, Safdar N. Molecular techniques for diagnosis of Clostridium difficile infection: systematic review and meta-analysis. Mayo Clin Proc. 2012;87(7):643–51.
357 358
38.
Squire MM, Carter GP, Mackin KE, Chakravorty A, Norén T, Elliott B, et al. Novel molecular type of Clostridium difficile in neonatal pigs, Western Australia. Emerg Infect Dis. 2013;19(5):790–2.
359 360
39.
Geric Stare B, Rupnik M. Clostridium difficile toxinotype XI (A-B-) exhibits unique arrangement of PaLoc and its upstream region. Anaerobe. 2010;16(4):393–5.
361 362 363
40.
Eckert C, Emirian A, Le Monnier A, Cathala L, De Montclos H, Goret J, et al. Prevalence and pathogenicity of binary toxin-positive Clostridium difficile strains that do not produce toxins A and B. New Microbes New Infect. 2015;3:12–7.
364 365 366
41.
Fang FC, Polage CR, Wilcox MH. Point-Counterpoint: What Is the Optimal Approach for Detection of Clostridium difficile Infection? Gilligan P, editor. J Clin Microbiol. 2017 Mar;55(3):670–80.
367 368
42.
Polage CR, Gyorke CE, Kennedy MA, Leslie JL, Chin DL, Wang S, et al. Overdiagnosis of Clostridium difficile Infection in the Molecular Test Era. JAMA Intern Med. 2015;175(11):1792.
369 370 371
43.
Gerding DN, Olson MM, Peterson LR, Teasley DG, Gebhard RL, Schwartz ML, et al. Clostridium difficile-associated diarrhea and colitis in adults. A prospective case-controlled epidemiologic study. Arch Intern Med. 1986;146(1):95–100.
372 373 374
44.
Alcalá L, Martín A, Marín M, Sánchez-Somolinos M, Catalán P, Peláez T, et al. The undiagnosed cases of Clostridium difficile infection in a whole nation: where is the problem? Microbiol Infect Dis. 2012;18(7):E204-213.
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45.
Van Dorp SM, Notermans DW, Alblas J, Gastmeier P, Mentula S, Nagy E, et al. European Clostridium difficile Infection Surveillance Network (ECDIS-Net) project on behalf of all participants. Survey of diagnostic and typing capacity for Clostridium difficile infection in Europe, 2011 and 2014.Euro Surveill; 2016.21;21(29).
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ACCEPTED MANUSCRIPT Table 1: Classification of commercially available NAAT for C. difficile (not exhaustive).
Detection of a single target Method
Target
®
Illumigene C.difficile (Meridian Bioscience)
tcdA
®
AmpliVue (QUIDELMolecular)
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tcdA
Simplexa™ C.difficile Universal Direct (FOCUS Diagnostics)
tcdB
Portrait Toxigenic C.difficile Assay (Portrait, Great Basin)
COBAS™ Cdiff (Roche) Prodesse ProGastro Cd assay (Gen Probe) ®
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ICEPlex C.difficile Kit (PrimeraDax)
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BD MAX™ Cdiff (BD Diagnostics)
tcdB
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BD GeneOhm™ Cdiff Assay (BD Diagnostics)
tcdB
tcdB
tcdB
tcdB
tcdB
Detection of several targets
Method ®
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GenoType Cdiff (Hain Lifescience)
Target tcdA, tcdB, several deletions in tcdC (including ∆117) cdtA, cdtB, gyrA, tpi
®
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IMDx C.difficile (IntelligentMDx)
tcdA, tcdB
®
Xpert C.difficile (Cepheid) ®
tcdB, cdt, deletion in position 117 in tcdC
Verigene C.difficile Test (Nanosphere)
tcdA, tcdB, binary toxin, deletion in position 117 in tcdC
RIDA GENE Clostridium difficile & Toxin A/B (R-biopharm)
tcdA, tcdB
®
®
Genspeed C.Diff Onestep (Genspeed Biotech)
gdh, tcdA, tcdB, binary toxin
®
Quidel Molecular Direct C.difficile Assay (Quidel Corporation)
tcdA, tdcB
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Target
®
Seeplex Diarrhea ACE Detection (Seegene) ®
FilmArray Gastrointestinal Panel (Biomerieux)
®
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RIDA GENE Gastro Panel (R-biopharm)
9 bacteria (including C. difficile tcdA tcdB genes) 3 viruses 3 parasites 22 bacteria (including C. difficile tcdB gene) 4 viruses 20 bacteria (including C. difficile tcdA tcdB genes) 5 viruses 4 parasites 10 bacteria (including C. difficile tcdA tcdB genes) 5 viruses 4 parasites
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®
xTAG Gastrointestinal Pathogen Panel (Theradiag)
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Fig.1: Advantages, disadvantages and targets of the different methods used for the diagnosis of CDI
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CTA: Cytotoxicity assay; EIA: Enzyme immunoassay; GDH: Glutamate dehydrogenase; NAAT: Nucleic acid
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amplification test; NPV: Negative predictive value; TAT: Turnaround time; CDI: Clostridium difficile infection.
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Fig. 2: Algorithms for the diagnosis of CDI (adapted from the ESCMID guidelines [28])
EIA: Enzyme immunoassay; GDH: Glutamate dehydrogenase; NAAT: Nucleic acid amplification test; CDI:
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Clostridium difficile infection ; TC: Toxigenic culture.