Keywords: Toxigenic culture, PCR, Inhibitor rate, Turnaround time, Clinical severity CDI: Clostridium difficile infection EIA: Enzyme immunoassay CCNA: Cell culture neutralization assay TC: Toxigenic culture Nucleic acid amplification techniques IBD: Inflammatory bowel disease

Nucleic acid amplification techniques (NAATs) represent a major advance in the diagnosis of C. difficile infection (CDI). This review analyzes the different options available for a molecular diagnosis of CDI, as well as the strengths and weaknesses of NAATs. The performances of seven commercials NAATs are compared (BD GeneOhm Cdiff, Illumigene C. difficile, Xpert C. difficile, BD Max Cdiff, Portrait Toxigenic C. difficile, Progastro Cd, Seeplex Diarrhea ACE). The sensitivity and the rapidity of NAATs are excellent: additional efforts should focus on the discrimination between infection and colonization. Reporting the DNA load of toxigenic C. difficile in the stool sample may represent a solution. Diagnostic algorithms combining immunoassays and NAATs could also improve the specificity and reduce the global cost of this analysis.

INTRODUCTION

Clostridium difficile infection (CDI) is a leading cause of nosocomial diarrhea. CDI is a lifethreatening disease, associated with a 30-day mortality ranging from 3% to 30% [START_REF] Jones | Clostridium difficile: a European perspective[END_REF]. CDI is defined by the presence of compatible symptoms (diarrhea or ileus) and the detection of toxigenic C. difficile (or its toxins) [START_REF] Cohen | 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)[END_REF]. A positive laboratory test is not sufficient by itself, and should always be interpreted in the light of clinical data [START_REF] Wilcox | What is the current role of algorithmic approaches for diagnosis of Clostridium difficile infection?[END_REF].

CURRENT METHODS AVAILABLE FOR CDI DIAGNOSIS

Two types of tests can be used for CDI diagnosis:

-detection of C. difficile toxins A and B: by enzyme immunoassays (EIA) or cell culture neutralization assay (CCNA).

-detection of toxigenic C. difficile strains: by toxigenic culture (TC) or nucleic acids amplification techniques (NAATs).

Enzyme immunoassays (EIA) detecting C. difficile toxins (TcdA and TcdB) are the most frequently employed tests. They are convenient to use, they provide a quick result for a low cost, and their specificity is good. The major drawback of EIA is their lack of sensitivity, ranging from 40 to 60% compared to TC [START_REF] Peterson | Laboratory testing for Clostridium difficile infection: light at the end of the tunnel[END_REF][START_REF] Sharp | Evaluation of the C.Diff Quik Chek Complete Assay, a new glutamate dehydrogenase and A/B toxin combination lateral flow assay for use in rapid, simple diagnosis of Clostridium difficile disease[END_REF]. Therefore, EIA detecting toxins cannot be recommended as a single test. Other immunoenzymatic tests detect the presence of glutamate dehydrogenase (GDH), an enzyme produced by C. difficile. GDH assays have a high negative predictive value, but they also detect non-toxigenic strains. Thus, GDH-positive results must be confirmed by another method. TC has a better sensitivity and specificity than GDH, but takes at least two days to complete. A quick diagnosis is needed to initiate treatment and infection control procedures.

Nucleic acids amplification techniques (NAATs) were developed to address these specific concerns. Sensitivity and specificity of NAATs are as good as the TC reference standard, and they provide a result in a few hours (1-3 hours). In recent published guidelines, NAATs are finally recognized as a superior method for the diagnosis of CDI [START_REF] Surawicz | Guidelines for Diagnosis, Treatment, and Prevention of Clostridium difficile Infections[END_REF]. The present review describes the different options available for a molecular diagnosis of CDI and analyze the strengths and weaknesses of NAATs.

GOLD STANDARD: CCNA OR TC?

New diagnostic assays must be compared against a reference method to evaluate their performances. The reference choice will alter the performances of the technique under evaluation. Two gold standards exist for CDI diagnosis: CCNA and TC. CCNA consist of incubation of the filtered stool sample onto a layer of cells sensitive to C. difficile toxins.

Stools samples producing a specific cytopathic effect are considered positive. TC consists of inoculation of the stool sample into a selective medium, incubated anaerobically for 2 days.

Then, the isolates of C. difficile recovered are tested for their ability to produce toxins.

The sensitivity of CCNA is only 67-86% compared to TC [7][START_REF] Eastwood | Comparison of nine commercially available Clostridium difficile toxin detection assays, a real-time PCR assay for C. difficile tcdB, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods[END_REF][START_REF] Lalande | Evaluation of a loopmediated isothermal amplification assay for diagnosis of Clostridium difficile infections[END_REF]. Thus, more stool samples are considered positive using TC than using CCNA, but that does not necessarily means TC is a better reference method. Indeed, CCNA detects the toxins present in stool samples but TC only evaluates the ability of C. difficile strains to produce toxins in vitro. The presence of free toxins in stool samples assessed by CCNA is thought to be more specific of CDI [START_REF] Planche | Reference assays for Clostridium difficile infection: one or two gold standards?[END_REF]. TC also detects patients colonized by a toxigenic strain of C. difficile, without any symptoms.

Pseudomembranous colitis diagnosed by sigmoidoscopy is pathognomonic of CDI [START_REF] Finegold | Clinical considerations in the diagnosis of antimicrobial agentassociated gastroenteritis[END_REF]. The role of flexible sigmoidoscopy in CDI diagnosis was evaluated in 2003 [START_REF] Johal | Clostridium difficile associated diarrhoea in hospitalised patients: onset in the community and hospital and role of flexible sigmoidoscopy[END_REF]. CCNA was positive in only 52% (29/56) of the CDI cases diagnosed by flexible sigmoidoscopy. These results need to be nuanced: 73 patients were excluded because CCNA was already known to be positive before sigmoidoscopy. Still, if all of these 73 patients had pseudomembranous colitis (best case scenario for CCNA), the sensitivity of CCNA would have been 79%

(102/129). Among the 26 CCNA negative samples from the patients with pseudomembranous colitis, 9 were available for further testing. TC was positive in all of these cases (9/9). Another study evaluated the performances of CCNA and TC against a reference standard of clinically significant diarrhea and detection of toxigenic C. difficile [START_REF] Dubberke | Impact of clinical symptoms on interpretation of diagnostic assays for Clostridium difficile infections[END_REF].

CCNA had a lower sensitivity than TC (62.9% and 100%, respectively) but its specificity was not significantly different (93.9% and 92.2%). Thus, CCNA is not sensitive enough for the diagnosis of CDI: almost all the recent studies use TC as a reference standard. It also makes sense to compare NAATs to TC, because both of these techniques detect toxigenic strains of C. difficile in stool samples, and not free toxins.

Many methods have been described to culture C. difficile from stool samples and to assess its ability to produce toxins. In the first step, the stool can be treated by heat or alcohol to kill vegetative bacteria and select the hardy spores of C. difficile. Then, stool samples are inoculated onto an agar plate (direct plating) or into a selective broth (enriched TC) for enrichment before plating. Recently, 27 culture methods were evaluated for the recovery of low concentrations of C. difficile from stools [START_REF] Hink | A systematic evaluation of methods to optimize culture-based recovery of Clostridium difficile from stool specimens[END_REF]. Enriched TC methods provided the highest recovery rates, especially using cycloserin-cefoxitin mannitol broth after heat-shock or ethanol-shock. After a C. difficile strain is isolated, it is possible to determine if the strain is toxigenic with CCNA or NAATs. EIA should not be used directly on C. difficile strains, because results are not reproducible [START_REF] She | Evaluation of enzyme immunoassays to detect Clostridium difficile toxin from anaerobic stool culture[END_REF]. Studies using different reference methods (TC or CCNA) are not comparable. But even in studies using TC as a unique reference method, comparison is problematic. Indeed, TC is not standardized. The performances of NAATs should be interpreted differently if the gold standard is TC (direct plating) or enriched TC (selective broth). The sensitivity of the evaluated NAATs would appear higher compared to direct plating.

NAATS: IN-HOUSE ASSAYS

In-house NAATs detecting toxigenic C. difficile directly from stool samples were developed 20 years ago [START_REF] Kato | Detection of toxigenic Clostridium difficile in stool specimens by the polymerase chain reaction[END_REF]. One the principal difficulties encountered was the presence of PCR inhibitors in feces. Using a basic DNA extraction procedure (boiling of the stool followed by centrifugation), 30% of the positive samples failed to yield PCR products [START_REF] Boondeekhun | Detection of Clostridium difficile enterotoxin gene in clinical specimens by the polymerase chain reaction[END_REF]. Thus, the extraction procedure has to be optimized specifically for feces. Depending on the DNA extraction method, PCR sensitivity to detect toxigenic C. difficile varies from 63 to 100% [START_REF] Persson | Five commercial DNA extraction systems tested and compared on a stool sample collection[END_REF].

The tcdB gene (coding for TcdB) is often chosen as NAATs' target, because toxin B is produced by all the toxigenic strains of C. difficile. TcdB is also sufficient for the virulence of C. difficile in the hamster model of infection [START_REF] Lyras | Toxin B is essential for virulence of Clostridium difficile[END_REF][START_REF] Kuehne | The role of toxin A and toxin B in Clostridium difficile infection[END_REF]. The tcdA gene (coding for TcdA) is less frequently used because 3.3% of the toxigenic strains from Europe do not produce toxin A [START_REF] Bauer | Clostridium difficile infection in Europe: a hospital-based survey[END_REF]. The prevalence of TcdA -/TcdB + strains is higher (27.0%) in Korea [START_REF] Shin | Multicentre study of the prevalence of toxigenic Clostridium difficile in Korea: results of a retrospective study 2000-2005[END_REF]. Nevertheless, a conserved region of the tcdA gene still exist in almost all the toxigenic strains, even in the strains deficient for toxin A production [START_REF] Kozak | Targeting the tcdA gene: is this appropriate for detection of A and/or B Clostridium difficile toxin-producing strains?[END_REF]. Only one C. difficile strain isolated from piglets present a complete deletion of tcdA [START_REF] Squire | Novel molecular type of Clostridium difficile in neonatal pigs, Western Australia[END_REF].

In-house NAATs present a good diagnostic accuracy. Peterson et al. reported a sensitivity of 93.3% and a specificity of 97.4% for their real-time PCR assay [START_REF] Peterson | Detection of toxigenic Clostridium difficile in stool samples by real-time polymerase chain reaction for the diagnosis of C. difficileassociated diarrhea[END_REF]. Multiplex in-house NAATs were also developed to detect toxigenic C. difficile and report a presumptive identification of PCR-ribotype 027 [START_REF] Barbut | Rapid diagnosis of Clostridium difficile infection by multiplex real-time PCR[END_REF]. Compared to enriched TC, the sensitivity and specificity were 86.6% and 97.4%, respectively. The presumptive identification of PCR-ribotype 027 could not be evaluated, because this PCR-ribotype was not detected during the study.

Even if in-house NAATs display good sensitivity and specificity, they also present some disadvantages. They require expertise in molecular biology and cannot be carried out immediately 7 days a week. In-house assays also require a method validation to comply with clinical laboratory accreditation standards [START_REF] Saunders | Guidance on the development and validation of diagnostic tests that depend on nucleic acid amplification and detection[END_REF]. Commercial molecular assays were developed to resolve these shortcomings.

NAATS: COMMERCIAL ASSAYS

BD GeneOhm Cdiff

The first and most extensively evaluated commercial assay developed is the BD GeneOhm Cdiff (BD Diagnostics, La Jolla, CA). This assay targets the tcdB gene. Extraction of DNA is manual: the specimen is submitted to mechanical lysis, then to thermal lysis. Addition of one freeze-thaw cycle can reduce the effects of some PCR inhibitors. Master mix has to be reconstituted immediately before use (3 hours of stability). To detect PCR inhibiting samples, an internal control is already included in the reaction mix. DNA is amplified using the SmartCycler System (Cepheid, Sunnyvale, CA). Fourteen clinical samples and 2 controls can be tested in each run. The turn-around-time (extraction and PCR) is about 120 min, including 45 min hands-on time. The use of frozen master mix reduces hands-on time by 15%, even if it is not recommended by the manufacturer. Sensitivity, specificity and inhibitor rates are similar with frozen or freshly reconstituted master mix [START_REF] Munson | Modifications of commercial toxigenic Clostridium difficile PCR resulting in improved economy and workflow efficiency[END_REF].

Compared to TC, reported sensitivities and specificities of the BD GeneOhm range from 82.1% to 97.7% (median: 95.0%) and from 90.6% to 99.7% (median: 98.4%), respectively (Table 1). Invalid result rates reported in the same studies range from 0% to 6.4% (median: 0.9%) [START_REF] Peterson | Laboratory testing for Clostridium difficile infection: light at the end of the tunnel[END_REF]7,[START_REF] Eastwood | Comparison of nine commercially available Clostridium difficile toxin detection assays, a real-time PCR assay for C. difficile tcdB, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods[END_REF][START_REF] Selvaraju | Detection of toxigenic Clostridium difficile in pediatric stool samples: an evaluation of Quik Check Complete Antigen assay, BD GeneOhm Cdiff PCR, and ProGastro Cd PCR assays[END_REF][START_REF] Viala | Comparison of commercial molecular assays for toxigenic Clostridium difficile detection in stools: BD GeneOhm Cdiff, XPert C. difficile and illumigene C. difficile[END_REF]. False-positive results due to a defective SmartCycler module have been reported once by Louie et al. [START_REF] Louie | An Unusual Cause of False Positive Results with the BD GeneOhm Cdiff Assay[END_REF]. These authors recommend that users request access to amplification curves. Although the software provides automatic interpretation, verification of the amplification curves by the laboratory staff may add another level of security.

Illumigene C. difficile

The illumigene C. difficile (Meridian Bioscience, Cincinnati, OH) assay uses loop-mediated isothermal DNA amplification (LAMP) to target a conserved region of the tcdA gene. This assay can also detect toxigenic strains deficient for toxin A. Extraction of DNA is manual: the specimen is diluted and subjected to thermal lysis. Diluent contains formalin-treated Staphylococcus aureus, acting as an internal control of amplification. Specimen are then processed through two chambers: one with primers specific to toxigenic C. difficile and the other for S. aureus. Positive and negative controls must be tested for each new kit lot. DNA is amplified using the illumipro-10 (Meridian Bioscience, Cincinnati, OH) under isothermal conditions (65°C). DNA amplification forms magnesium pyrophosphate, which increases the turbidity of the reaction solution. Then, optical detection determines whether a sample is positive. The turn-around-time is about 60 min.

Compared to TC, reported sensitivities and specificities of the illumigene range from 73.0% to 98.1% (median: 93.3%) and from 91.5% to 100% (median: 99.7%), respectively (Table 1). It should be noted that one study reports a significantly lower sensitivity, without a clear explanation [START_REF] Walkty | Laboratory Diagnosis of Clostridium difficile Infection -Evaluation of an Algorithmic Approach in Comparison with the Illumigene(R) Assay[END_REF]. Invalid result rates range from 0.8% to 4.3% (median: 2.6%) [START_REF] Lalande | Evaluation of a loopmediated isothermal amplification assay for diagnosis of Clostridium difficile infections[END_REF][START_REF] Bruins | Evaluation of three enzyme immunoassays and a loop-mediated isothermal amplification test for the laboratory diagnosis of Clostridium difficile infection[END_REF].

Xpert C. difficile

The Xpert C. difficile (Cepheid, Sunnyvale, CA) uses disposable unitary cartridges, which contain all the reagents necessary to perform multiplex real-time PCR. This assay targets tcdB (toxin B), cdt (binary toxin), and tcdC117 (a deletion frequently associated with PCRribotype 027). Hands-on time is minimal, because DNA extraction and amplification are completely automatized. The turn-around time is about 1 hour. Each module can process one sample independently from the others. Cartridges contain an internal control (spores of

Bacillus globigii).

Compared to TC, reported sensitivities and specificities of the Xpert C. difficile range from 93.5% to 100% (median: 100%) and from 91.7% to 99.2% (median: 95.5%), respectively (Table 1). Invalid result rates reported in the same studies range from 0.9% to 13.4% (median: 3.7%) [START_REF] Tenover | Impact of strain type on detection of toxigenic Clostridium difficile: comparison of molecular diagnostic and enzyme immunoassay approaches[END_REF][START_REF] Zidaric | Comparison of two commercial molecular tests for the detection of Clostridium difficile in the routine diagnostic laboratory[END_REF][START_REF] Novak-Weekley | Clostridium difficile testing in the clinical laboratory by use of multiple testing algorithms[END_REF].

Besides detecting toxigenic C. difficile, the Xpert C. difficile assay reports presumptive identification of PCR-ribotype 027 (positive for cdt and tcdC117). PCR-ribotype 027 strains are highly virulent. Notably, the production of binary toxin is associated with an increased mortality [START_REF] Bacci | Binary toxin and death after Clostridium difficile infection[END_REF]. However, false positives have been reported for the presumptive identification of PCR-ribotype 027 using the Xpert assay [START_REF] Kok | Presumptive identification of Clostridium difficile strain 027/NAP1/BI on Cepheid Xpert: interpret with caution[END_REF]. This problem seems to arise with strains that possess cdt and present a point mutation at the position 117 of tcdC instead of the typical single-base deletion.

BD MAX Cdiff

The BD MAX Cdiff (BD Diagnostics, La Jolla, CA) allows the amplification of the tcdB gene by real-time PCR. A stool specimen is diluted in a sample buffer tube and associated with a specific bar code. This assay is entirely automated, resulting in minimal hands-on time. An enzymatic extraction is performed with achromopeptidase, then DNA is captured on magnetic beads. DNA is eluted by heat, and added to a lyophilized reagent mix. PCR reactions are performed in microfluidic chambers, allowing fast temperature variations and short thermocycling times. A plasmid containing a synthetic target DNA sequence is included in each extraction tube, acting as an internal control. Twenty-four samples can be processed for each run. The turn-around-time is about 100 min (70 min extraction, 30 min of DNA amplification).

Compared to TC, reported sensitivities and specificities of the BD MAX Cdiff range from 90.5% to 97.7% and from 97.9% to 99.7%, respectively (Table 1). Invalid result rates range from 1.8% to 3.8% in the first run, but decrease to 0-0.4% when the test is repeated [START_REF] Dalpke | Evaluation of the fully automated BD MAX Cdiff and Xpert C. difficile assays for the direct detection of Clostridium difficile in stool specimens[END_REF][START_REF] Guern | Evaluation of a new molecular test, the BD Max Cdiff, for detection of toxigenic Clostridium difficile in fecal samples[END_REF].

Portrait Toxigenic C. difficile

The Portrait Toxigenic C. difficile assay (Great Basin, Salt Lake City, UT) uses isothermal helicase-dependent amplification (HDA) of tcdB with blocked primers [START_REF] Hicke | Automated detection of toxigenic Clostridium difficile in clinical samples: isothermal tcdB amplification coupled to array-based detection[END_REF]. Extraction, amplification and detection are automated. DNA is continuously amplified at 65°C, using a helicase for denaturation. Then, biotin-labelled amplified DNA is bound to capture probes on a silicon chip and revealed by horseradish peroxidase. Each test contains an internal control (S. aureus nuc gene), a hybridization control and a detection control.

Buchan et al. reported a sensitivity of 98.2% and a specificity of 92.8% against TC [START_REF] Buchan | Multicenter clinical evaluation of the Portrait toxigenic C. difficile assay for detection of toxigenic Clostridium difficile strains in clinical stool specimens[END_REF].

Invalid result rate was 4.6% in the first run, but dropped to 0.5% after the test was repeated. This assay does not require thermal cycling nor real-time fluorescence detection, thus limiting the cost of instrument and reagents.

ProGastro Cd

The ProGastro Cd assay (Hologic Gen-Probe Inc., San Diego, CA) is a real-time PCR diagnostic test targeting the tcdB gene. Stool samples are diluted in S.T.A.R. buffer (Roche Diagnostics Corporation, Indianapolis, IN), then DNA is extracted using the NucliSENS easyMAG System (bioMérieux Inc., Durham, NC). DNA amplification is performed in a SmartCycler. Internal control is included for each specimen. Positive and negative controls must be tested in each run. The turn-around-time is about 3 hours, which is higher than other molecular assays.

Performances of the ProGastro Cd in comparison to TC are variable. Stamper et al. found a low sensitivity (77.3%) and a high specificity (99.2%) [START_REF] Stamper | Evaluation of a new commercial TaqMan PCR assay for direct detection of the Clostridium difficile toxin B gene in clinical stool specimens[END_REF], whereas Selvaraju et al. reported an excellent sensitivity (100%) and a lower specificity (93.4%) [START_REF] Selvaraju | Detection of toxigenic Clostridium difficile in pediatric stool samples: an evaluation of Quik Check Complete Antigen assay, BD GeneOhm Cdiff PCR, and ProGastro Cd PCR assays[END_REF]. These discordant results may be partly explained because Stamper et al. used enriched TC, a more sensitive reference method. These studies report an invalid result rate between 0 and 1.8% [START_REF] Selvaraju | Detection of toxigenic Clostridium difficile in pediatric stool samples: an evaluation of Quik Check Complete Antigen assay, BD GeneOhm Cdiff PCR, and ProGastro Cd PCR assays[END_REF][START_REF] Stamper | Evaluation of a new commercial TaqMan PCR assay for direct detection of the Clostridium difficile toxin B gene in clinical stool specimens[END_REF].

Seeplex Diarrhea ACE

The Seeplex Diarrhea-B1 ACE (Seegene Diagnostics, Seoul, South Korea) detects simultaneously toxigenic C. difficile (tcdB), along with Salmonella spp., Shigella spp., Vibrio spp., and Campylobacter spp. by multiplex PCR. This assay uses dual priming oligonucleotides (DPO), consisting of two priming regions bound by a linker [START_REF] Chun | Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene[END_REF]. DPO increases the specificity of DNA extension, which is important for multiplex PCR. DNA extraction is not included and must be performed with a kit from another manufacturer.

After DNA amplification, PCR products are submitted to capillary electrophoresis. The turnaround time is about 4 hours.

The Seeplex has a sensitivity of 90% and a specificity of 97.1% against TC [START_REF] Shin | Comparison of BD GeneOhm Cdiff and Seegene Seeplex ACE PCR assays using toxigenic Clostridium difficile culture for direct detection of tcdB from stool specimens[END_REF]. False negative results occurred mainly for the lowest concentrations of C. difficile in stool samples (assessed by semi-quantitative culture).

The Seeplex allows simultaneous detection of C. difficile and other enteropathogenic bacteria. Infections due to Salmonella, Shigella, Vibrio and Campylobacter are usually community-acquired. The recovery rate of these bacteria in nosocomial diarrhea is low, about 1% [START_REF] Bauer | Derivation and validation of guidelines for stool cultures for enteropathogenic bacteria other than Clostridium difficile in hospitalized adults[END_REF]. Nosocomial outbreaks caused by these bacteria are possible but rare in developed countries. However, immunosuppressed patients may develop nosocomial diarrhea due to reactivation of one of these enteropathogenic bacteria, especially under cancer chemotherapy [START_REF] Delaloye | Nosocomial nontyphoidal salmonellosis after antineoplastic chemotherapy: reactivation of asymptomatic colonization?[END_REF]. Therefore, this assay seems mainly interesting to explore either community-acquired diarrhea or nosocomial diarrhea in immunosuppressed patients.

Overview

Performances of the different commercial molecular assays are summarized in Table 1. Only studies where TC or enriched TC was performed as a reference method on all the samples are included.

Pooled sensitivities for BD GeneOhm, illumigene, and Xpert C. difficile assays were calculated using Meta-DiSc (Figure 1), including only studies where TC (without enrichment) was performed as a reference method on all the samples. We did not present estimates of pooled specificity because: (i) the reference method (TC) is not very specific of CDI, (ii) sometimes NAATs can be more sensitive than TC without enrichment.

Pooled sensitivity was 93% for BD GeneOhm (95% confidence interval [CI], 89%-95%), 92% for illumigene (95% CI, 88%-94%) and 99% for Xpert C. difficile (95% CI, 97%-100%). The Xpert C. difficile presented the highest pooled sensitivity, compared to TC without enrichment. However, the additional cases detected by the Xpert C. difficile could also correspond to asymptomatic colonization [START_REF] Gyorke | Evaluation of Clostridium difficile fecal load and limit of detection during a prospective comparison of two molecular tests, the illumigene C. difficile and Xpert C. difficile/Epi tests[END_REF].

ROLE OF DIAGNOSTIC ALGORITHMS

GDH: a first-line test

Glutamate dehydrogenase (GDH) is an enzyme produced in large amounts by C. difficile isolates. Thus, EIA detecting GDH present a higher sensitivity than EIA detecting toxins [START_REF] Gilligan | Is a two-step glutamate dehyrogenase antigen-cytotoxicity neutralization assay algorithm superior to the premier toxin A and B enzyme immunoassay for laboratory detection of Clostridium difficile?[END_REF].

Eastwood et al. estimated GDH sensitivity at 87.6% compared to TC [START_REF] Eastwood | Comparison of nine commercially available Clostridium difficile toxin detection assays, a real-time PCR assay for C. difficile tcdB, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods[END_REF]. The negative predictive value of GDH is high (> 98%) [START_REF] Eastwood | Comparison of nine commercially available Clostridium difficile toxin detection assays, a real-time PCR assay for C. difficile tcdB, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods[END_REF], but positive results must be confirmed by another test. Therefore, GDH is often used as the first step of diagnostic algorithms.

Impact of strain type on GDH

The sensitivity of GDH seems to depend on C. difficile PCR-ribotype. GDH sensitivity was evaluated at 90.9% on PCR-ribotype 027 isolates, but dropped to 69.4% on non-027 isolates, compared to enriched TC [START_REF] Tenover | Impact of strain type on detection of toxigenic Clostridium difficile: comparison of molecular diagnostic and enzyme immunoassay approaches[END_REF]. In the same study, NAATs sensitivity of the Xpert C. difficile kit was similar on 027 isolates (90.9%) and on non-027 isolates (91.7%). However, this part of the study involved only 47 positive stool samples, and the authors combined the results of 2 different GDH algorithms. More recently, an in vitro study reported that the production of GDH was highly conserved among C. difficile ribotypes [START_REF] Carman | Glutamate dehydrogenase is highly conserved among Clostridium difficile ribotypes[END_REF], but the authors worked directly with C. difficile strains and not with clinical stool samples. Depending on the local epidemiology, algorithms using GDH as a first line test may have a lower sensitivity than previously admitted.

Two-steps algorithms

To lessen the cost of NAATs, Swindells et al. proposed a two-steps diagnostic algorithm [START_REF] Swindells | Evaluation of diagnostic tests for Clostridium difficile infection[END_REF].

First, both GDH and toxins are detected by EIA with the C. Diff Quik Chek Complete (TechLab, Blacksburg, VA). Three possibilities may occur: (i) the sample is negative for GDH: CDI is ruled out; (ii) the sample is positive for both GDH and toxin production: CDI is likely;

(iii) the sample is positive for GDH only: NAAT must be performed. If NAAT is negative, CDI is ruled out. If NAAT is positive, CDI is probable.

The value of this algorithm was investigated by Culbreath et al. [START_REF] Culbreath | Evolution of Testing Algorithms at a University Hospital for Detection of Clostridium difficile Infections[END_REF]. In their institution, this algorithm was 56% cheaper than applying the Xpert C. difficile systematically. This algorithm also presents the advantage to individualize a subgroup of patients with free toxins in the stools. For some authors, the presence of free toxin assessed by EIA is more specific of the disease than the presence of toxigenic C. difficile assessed by NAATs [START_REF] Planche | Reference assays for Clostridium difficile infection: one or two gold standards?[END_REF].

The GDH sensitivity is lower than NAATs, and some positive cases will be missed by the diagnostic algorithms. This is well illustrated by the application of Bayes' theorem to diagnostic tests [START_REF] Akobeng | Understanding diagnostic tests 2: likelihood ratios, pre-and post-test probabilities and their use in clinical practice[END_REF]. The post-test probability represents the probability that a particular patient has the disease after the result of a diagnostic test is known. It depends on the diagnostic test performances and on the pre-test probability. The pre-test probability is estimated from the patient clinical data and the local prevalence of the disease. Peterson et al. evaluated an EIA detecting toxins and GDH (C. Diff Quik Chek Complete) as well as a commercial NAATs (BD GeneOhm) on 1000 samples, compared to enriched TC [START_REF] Peterson | Laboratory testing for Clostridium difficile infection: light at the end of the tunnel[END_REF]. The posttest probability of CDI after a negative EIA, GDH, or NAATs was estimated using the results from their study (Figure 2). NAATs are the best assays to rule out CDI. Thus, some laboratories routinely use diagnostic algorithms, but they also allow physicians to directly request NAATs in case of a high suspicion of CDI [START_REF] Culbreath | Evolution of Testing Algorithms at a University Hospital for Detection of Clostridium difficile Infections[END_REF].

Globally, two-steps algorithms constitute an excellent alternative to the exclusive use of NAATs. Indeed, these algorithms provide additional information (EIA is performed on all the stool samples to detect free toxins), associated with a relatively low cost.

Other considerations: biosafety level

C. difficile is a biosafety level 2 (BSL-2) pathogen. Laboratory-acquired infections have been described, especially due to PCR-ribotype 027 strains [START_REF] Bouza | Laboratory-acquired Clostridium difficile polymerase chain reaction ribotype 027: a new risk for laboratory workers?[END_REF]. Laboratory personnel working on stool samples should use class II biosafety cabinets. Multi-step algorithms may expose laboratory workers to a greater risk of infection, even though this risk is probably low.

TARGET POPULATION

The asymptomatic colonization rate is about 1.9% in healthy adults [START_REF] Aronsson | Antimicrobial agents and Clostridium difficile in acute enteric disease: epidemiological data from Sweden, 1980-1982[END_REF]. However, this rate can be as high as 51% for residents of long term care facilities [START_REF] Riggs | Asymptomatic carriers are a potential source for transmission of epidemic and nonepidemic Clostridium difficile strains among long-term care facility residents[END_REF]. Therefore, it is important to test only diarrheal stool samples, especially using a sensitive test like NAATs.

CDI testing rate / CDI incidence

CDI is an underdiagnosed disease. In a Spanish study, 118 laboratories sent all the diarrheal stool samples they had received on a single day to a reference laboratory [START_REF] Alcala | The undiagnosed cases of Clostridium difficile infection in a whole nation: where is the problem?[END_REF]. TC was performed by the reference laboratory on all the samples, regardless of the diagnosis requested by clinicians. Toxigenic C. difficile was recovered by the reference laboratory for 45 samples:

-31% (14/45) were successfully diagnosed by the participating laboratories.

-20% (9/45) were diagnosed as false-negative by the participating laboratories. In fact, 83% of the participating laboratories used EIA, which has a low sensitivity.

-49% (22/45) were not tested at all for C. difficile presence by the participating laboratories because it was not requested by clinicians.

CDI rates are principally influenced by the frequency of the laboratory tests carried out to detect toxigenic C. difficile. Across European countries, the testing rate for CDI varies from 3 to 141 per 10,000 patients-days [START_REF] Bauer | Clostridium difficile infection in Europe: a hospital-based survey[END_REF]. Countries from Northern Europe have a higher incidence rate of CDI, but they also have a higher testing rate. To optimize the detection of CDI, all the cases of unexplained diarrhea should be tested.

Changing epidemiology of CDI

The three major risk factors for CDI are exposure to antibiotics, exposure to healthcare facilities and older age [START_REF] Carroll | Biology of Clostridium difficile: implications for epidemiology and diagnosis[END_REF]. Recently, increased rates of infection were observed in patients previously thought to be at low risk for CDI [START_REF] Freeman | The changing epidemiology of Clostridium difficile infections[END_REF]. Community-acquired CDI has become more prevalent the last 10 years, and represent now 20-59% of all the CDI cases [START_REF] Khanna | Community-acquired Clostridium difficile infection: an emerging entity[END_REF][START_REF] Dumyati | Community-associated Clostridium difficile infections, Monroe County, New York, USA[END_REF][START_REF] Leffler | Editorial: not so nosocomial anymore: the growing threat of community-acquired Clostridium difficile[END_REF][START_REF] Shin | Characterization of cases of Clostridium difficile infection (CDI) presenting at an emergency room: molecular and clinical features differentiate community-onset hospital-associated and communityassociated CDI in a tertiary care hospital[END_REF].

Moreover, peripartum women may be at risk for severe CDI, even if they are younger than typical patients suffering from CDI [START_REF] Venugopal | Clostridium difficile infection rates and spectrum of disease among peripartum women at one hospital from 2003 to 2007 with molecular typing analysis of recovered Clostridium difficile isolates[END_REF]. Caesarean section seems to be an additional risk factor, probably due to antimicrobial prophylaxis [START_REF] Kuntz | Trends in Clostridium difficile infection among peripartum women[END_REF].

The epidemiology of the PCR-ribotypes responsible for CDI is changing in European hospitals.

Between 2005 and 2008, the prevalence of the PCR-ribotype 078 increased from to 3% to 8%, whereas the prevalence of the PCR-ribotype 027 slightly dropped from 6% to 5% [START_REF] Bauer | Clostridium difficile infection in Europe: a hospital-based survey[END_REF][START_REF] Barbut | Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates[END_REF]. This is important because the genotype of C. difficile strains seems to be correlated with the severity of the disease. In a large study including 2222 adults suffering from CDI, Walker et al. explored the relationship between strain-type and mortality [START_REF] Walker | Relationship Between Bacterial Strain Type, Host Biomarkers, and Mortality in Clostridium difficile Infection[END_REF]. The 14-day mortality was highest for PCR-ribotypes 078 (25%) and 027 (20%), compared to an overall mortality of 13% (p<0.0001). Inclusion of a large number of CDI cases was necessary to detect a difference between genotypes with sufficient power [START_REF] Walker | Regarding "Clostridium Difficile Ribotype Does not Predict Severe Infection[END_REF].

Repeated NAATs testing

After a positive result, NAATs should not be repeated. NAATs can remain positive for weeks after an episode of CDI. The only "test of cure" is the resolution of the diarrhea. Moreover, infection control precautions should only be maintained for the duration of diarrhea, irrespective of laboratory results [START_REF] Cohen | 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)[END_REF].

After a negative result, the post-test probability of CDI is low because NAATs possess a high negative predictive value. When NAATs are repeated within 14 days, negative results are confirmed in 97.5% of the cases [START_REF] Luo | Is repeat PCR needed for diagnosis of Clostridium difficile infection?[END_REF]. Repeated testing generates additional costs, which are not justified in light of this low diagnostic yield.

Caution to Interpret test in children and IBD patients

The rate of colonization by toxigenic C. difficile is high in children under the age of 2 years, dropping progressively from 38% (1 to 12 months) to 28% (12 to 24 months) [START_REF] Rousseau | Prevalence and diversity of Clostridium difficile strains in infants[END_REF]. Several hypotheses have been proposed to explain why C. difficile does not cause disease in infants.

In particular, this could be due to a diminished number of toxin receptors on the intestinal mucosal surface, or to their immaturity [START_REF] Sammons | Clostridium difficile Infection in Children[END_REF]. Although uncommon, it is possible for children to suffer from CDI. That is why the American Academy of Pediatrics published recommendations for the diagnosis of CDI in children [START_REF] Schutze | Clostridium difficile infection in infants and children[END_REF]. Children younger than 1 year old should not be tested for CDI, except if they have Hirschsprung disease or during an outbreak situation. Between 1 and 2-year of life, positive C. difficile tests are difficult to interpret ("possible CDI"). After 3-year of life, positive C. difficile tests indicate probable CDI.

Carriage of toxigenic C. difficile is also significantly higher in inflammatory bowel diseases (IBD) patients (8.2%, n=122) compared to healthy adults (1.0%, n=99) (p=0.02) [START_REF] Clayton | The vexed relationship between Clostridium difficile and inflammatory bowel disease: an assessment of carriage in an outpatient setting among patients in remission[END_REF].

Ulcerative colitis patients present a higher carriage rate of toxigenic C. difficile (9.4%) than Crohn's disease patients (6.9%). PCR-ribotyping suggests that these strains are probably community-acquired. None of these patients presented a clinical episode of CDI during the 6 months following the study [START_REF] Clayton | The vexed relationship between Clostridium difficile and inflammatory bowel disease: an assessment of carriage in an outpatient setting among patients in remission[END_REF]. Thus, clinical interpretation of positive NAATs for IBD patients is particularly difficult. Since the mortality of CDI is higher in IBD patients [START_REF] Ananthakrishnan | Excess hospitalisation burden associated with Clostridium difficile in patients with inflammatory bowel disease[END_REF], recent guidelines recommend that all the IBD patients who are hospitalized for a disease flare should be tested for CDI [START_REF] Surawicz | Guidelines for Diagnosis, Treatment, and Prevention of Clostridium difficile Infections[END_REF].

DISCREPANCIES TOXIN / NAAT

Clinical severity

The higher sensitivity of NAATs compared to EIA has been criticized because the additional cases detected only by NAATs could also correspond to asymptomatic colonization.

Humphries et al. recently evaluated the sensitivity of EIA and NAATs stratified by the severity of CDI, compared to TC [START_REF] Humphries | Performance of Clostridium difficile Toxin Enzyme Immunoassay and Nucleic Acid Amplification Tests Stratified by Patient Disease Severity[END_REF]. The severity of CDI was evaluated based on age, albumin level, white blood cell count, admission to the intensive care unit, and presence of pseudomembranous colitis. EIA was more sensitive in patients with severe CDI (58%) than in patients with mild disease (49%), even though this difference was not statistically significant (p=0.31). NAATs (Illumigene) presented the same sensitivity in patients with mild or severe CDI (98%). In other words, 42% of the severe CDI cases would have been missed for EIA against only 2% for NAATs. Thus, the additional cases detected only by NAATs also involve severe disease.

On the other hand, complication rates of nosocomial CDI were evaluated by Longtin et al. EIA is probably more specific of complicated CDI than NAATs, but the cases detected by NAATs only can also correspond to a severe or complicated disease.

Quantitation / load: useful parameter?

The concentration of toxigenic C. difficile can be estimated by NAATs using standard curves.

Leslie et al. reported the tcdB DNA load of samples positive with EIA or NAAT (Xpert C. difficile) [START_REF] Leslie | Role of fecal Clostridium difficile load in discrepancies between toxin tests and PCR: is quantitation the next step in C. difficile testing?[END_REF]. The tcdB DNA load was 10 to 10,000 times higher for EIA-positive samples compared to EIA-negative NAAT-positive samples. Nevertheless, 48% of the additional cases detected only by NAATs also involve high tcdB DNA load (from 10 5 to 10 8 copies/mL). Further Whole genome sequencing seems particularly promising to investigate epidemiological links between bacterial isolates [START_REF] Loman | High-throughput bacterial genome sequencing: an embarrassment of choice, a world of opportunity[END_REF]. It provides in-depth information about the spread of C. difficile in hospitals and communities, and lead to a better understanding of C. difficile transmission [START_REF] Didelot | Microevolutionary analysis of Clostridium difficile genomes to investigate transmission[END_REF]. Since turnaround-time and costs decrease steadily, whole genome sequencing will probably become affordable for clinical laboratories within a few years.

Title: 2 ,Frédéric Wallet 1 1:

 21 Molecular methods in the diagnosis of Clostridium difficile infections: an update Authors names & affiliations: Rémi Le Guern 1 , Stéphanie Herwegh 1 , René Courcol 1,Institut de Microbiologie, CHU Lille, Lille, France 2 : Université Lille Nord de France, Lille, France

  Further studies are needed to assess the clinical value of a positive NAAT result in case of a low concentration of C. difficile. Cost per test, hands-on time and turnaround time are also important characteristics for the comparison of diagnostic assays (Table 2). Cost per test is minimal ($25) for the Portrait Toxigenic C. difficile and the Progastro Cd. Hands-on time is minimal (10 min) for the three fully automated assays (Xpert C. difficile, BD MAX Cdiff, and Portrait C. difficile) and for the illumigene C. difficile. Turn-around time is minimal (<60 min) for the Xpert C. difficile and the illumigene C. difficile. Two assays also use multiplex PCR to provide additional information (Xpert C. difficile and Seeplex Diarrhea ACE).

  depending on the type of diagnostic test (NAATs versus EIA/CCNA) [76]. Complications were defined as all-cause mortality during the first 30 days, colectomy, admission to the intensive care unit or recurrence of CDI. As expected, more nosocomial cases of CDI were detected with NAATs (Illumigene) than with EIA/CCNA (85 versus 56, respectively). NAATs detected all the 23 cases who presented a complicated course of CDI, and EIA/CCNA detected almost all of these cases (22/23). The prevalence of complicated CDI was 39% for the CDI cases detected by NAATs and EIA/CCNA (22/56), and only 3% for the CDI cases detected by NAATs only (1/29). However, in this study the physicians had only access to the results of NAATs. It is possible that some of the cases detected by NAATs only would have developed complications if they had not received treatment for CDI.

  studies are needed to determine if the DNA load may separate asymptomatic colonization from infection. Reporting the concentration of toxigenic C. difficile to the clinicians may solve the problem of asymptomatic colonization detection by NAATs.EXPERT COMMENTARYClostridium difficile is responsible for 10 to 25% of antibiotic associated diarrhea and for virtually all cases of pseudomembranous colitis. Rapid detection of toxigenic C. difficile is critical to begin optimal therapy of the patient and to interrupt transmission of C. difficile to other patients by applying infection control measures. Although toxigenic culture is the gold standard test to diagnose the infection, it is slow, laborious and requires 72-96 hours to give a reply to the clinician. Commercial EIA assays detecting C. difficile toxins show a low sensitivity ranging from 40 to 60% compared to toxigenic culture and cannot be recommended as single test.NAATs represent a major advance in the diagnosis of CDI. Commercial assays are often easy to use and reduce hands-on time for technical staff, compared to in-house methods.However, the prescription of these tests must be strictly limited to diarrheal stool specimen (Bristol Stool Chart 5 to 7) in patients without laxatives, to avoid the detection of asymptomatic colonization. Moreover, these molecular tests are only useful for the initial diagnosis and do not permit to follow the evolution under treatment. Toxigenic C. difficile DNA (tcdB) may remain positive for several weeks after the resolution of clinical symptoms.NAATs remain expensive but they can be integrated in a well thought algorithm, depending on the local epidemiology. Notably, the use of GDH as a first line test decreases the global cost. NAATs provide a quick and accurate diagnosis, allowing to limit the widespread diffusion of toxigenic C. difficile in our hospitals. The higher cost of NAATs may be counterbalanced by a decrease in healthcare-associated infections costs[78].FIVE-YEAR VIEWNAATs will probably be considered as the standard diagnostic test for CDI, either as a standalone test or included in an algorithm. The sensitivity and rapidity of NAATs are already excellent: additional efforts should focus on the prediction of the disease severity. NAATs results should always be interpreted in light of clinical data, but reporting the DNA load of the toxigenic C. difficile gene target may help to discriminate between colonization and infection. Moreover, reporting the PCR-ribotype strain type and presence of virulence factors could help to predict the severity of the disease. One assay (Xpert C. difficile) already reports the presumptive identification of PCR-ribotype 027 from stool samples, but other PCR-ribotypes seem to emerge as responsible for severe disease. Keeping up with the continuously evolving epidemiology of CDI represents a challenge for clinical laboratories.



  NAATs must only be performed on diarrheal stools (Bristol stool chart 5 to 7)  NAATs combine excellent positive and negative predictive values, in contrast to EIA and GDH assays  Qualitative NAATs cannot discriminate colonization from infection, but reporting the DNA load of toxigenic C. difficile using quantitative NAATs may add diagnostic value  The only "test of cure" is the resolution of the diarrhea  Positive C. difficile NAATs are difficult to interpret in children before 2-year of age and in inflammatory bowel disease patients  Diagnostic algorithms reduce the cost of NAATs and individualize a subgroup of patients with free toxins in stools, which is more specific of the disease  Even if NAATs are more expensive, this may be counterbalanced by a shorter technical working time and a decrease in costs due to healthcare-associated infections

Figure 1 .Figure 2 .

 12 Figure 1. Forest plot estimates of pooled sensitivities for three popular commercial NAATs compared to toxigenic culture (without enrichment).

Table 1 .

 1 Performances of commercial NAATs against toxigenic culture (TC)

	Assay	Authors	Year	Gold Standard	Number	Sensitivity (%)	Specificity (%)
	BD GeneOhm
		Dubberke [13]	2011	TC	150	97.7	90.6
		Eastwood [8]	2009	TC	552	88.5	95.4
		Le Guern [40]	2012	TC	360	95.5	99.7
		Selvaraju [29]	2011	TC	200	89.6	96.7
		Shin [45]	2012	TC	243	95.7	96.5
		Swindells [51]	2010	TC	150	94.4	99.2
		Viala [30]	2012	TC	94	95.5	97.9
		Zidaric [35]	2011	TC	194	82.1	98.2
		Buchan [42]	2012 Enriched TC	169	97.4	98.5
		Stamper [7]	2008 Enriched TC	401	83.6	98.2
		Peterson [4]	2011 Enriched TC 1000	94.5	96.5
	illumigene
		Bruins [33]	2012	TC	986	93.2	99.7
		Dubberke [13]	2011	TC	150	95.5	91.5
		Viala [30]	2012	TC	94	86.7	100.0
		Walkty [32]	2013	TC	427	73.0	99.7
		Ylisiurua [81]	2013	TC	430	97.7	99.6
		Buchan [42]	2012 Enriched TC	96	93.3	95.1
		Lalande [9]	2011 Enriched TC	472	91.8	99.1
	Xpert
	C. difficile
		Dalpke [39]	2013	TC	448	97.3	97.9
		Dubberke [13]	2011	TC	150	100.0	93.4
		Goldenberg [82]	2010	TC	224	100.0	96.7
		Shin [83]	2012	TC	253	100.0	94.6
		Swindells [51]	2010	TC	150	100.0	99.2
		Tenover [34]	2010	TC	2296	98.8	90.8
		Viala [30]	2012	TC	94	97.8	97.9
		Zidaric [35]	2011	TC	178	96.4	97.3
		Buchan [42]	2012 Enriched TC	275	100.0	91.7
		Novak-Weekley [36]	2010 Enriched TC	428	94.4	96.3
		Tenover [34]	2010 Enriched TC 2296	93.5	94.0
	BD Max
		Dalpke [39]	2013	TC	448	90.5	97.9
		Le Guern [40]	2012	TC	360	97.7	99.7
	Portrait
		Buchan [42]	2012	Enriched	540	98.2	92.8
	ProGastro
		Selvaraju [29]	2011	TC	200	100.0	93.4
		Stamper [43]	2009	Enriched	280	77.3	99.2
	SeeGene
		Shin [45]	2012	TC	243	90.0	97.1

Table 2 .

 2 Characteristics of commercial NAATs

	Assay	Method	Target	DNA Extraction HOT (min) a TAT (min) a	Cost per test
	BD GeneOhm Cdiff	RT-PCR	tcdB	Manual	45	120	$47 b
	Illumigene C. difficile	LAMP	tcdA	Manual	10	60	$33 b
	Xpert C. difficile	Multiplex RT-PCR	tcdB, cdt, tcdC117	Automated	10	60	$52 b
	BD MAX Cdiff	RT-PCR	tcdB	Automated	10	100	$43 b
	Portrait C. difficile	HDA	tcdB	Automated	10	100	$25
	Progastro Cd	RT-PCR	tcdB	Automated (easyMAG)	45	180	$25
	Seeplex Diarrhea ACE	Multiplex PCR	tcdB	Manual	N/A	240	$41 b


Rémi Le Guern

Caroline Loïez

Bruno Grandbastien

René Courcol

Frédéric Wallet
email: frederic.wallet@chru-lille.fr

Performance of stool cultures before and after a 3-day hospitalization: fewer cultures, better for patients and for money

Keywords: rejection criteria, 3-day rule, nosocomial outbreak, bacterial diarrhea, length of hospitalization, cost savings

We evaluated retrospectively the yield of stool culture (SC) depending on the length of hospitalization, and we characterized the patients missed by the 3-day rejection rule. SC detects bacterial enteric pathogens (Campylobacter spp., Salmonella enterica, Yersinia spp., Shigella spp.). During this 5-year study period, 13,039 SCs were requested, and 376 were positive (2.9%). The yield of SC dropped from 11.7% before 3 days of hospitalization to 0.7% after 3 days in children and 4.3% to 0.3% in adults. Finally, only 13 clinically relevant cases (0.2% of SC prescribed after 3 days) were undiagnosed by strict application of the 3-day rule.

In conclusion, rejection of SC prescribed after 3 days of hospitalization allows to reduce workload by 37.8% for children and 65.7% for adults, representing a cost of €12,500 ($16,250) per year in our hospital.

Introduction

Stool culture (SC) are prescribed to detect bacterial enteric pathogens (Campylobacter spp., Salmonella enterica, Yersinia enterocolitica, Shigella spp.), which are frequently community-acquired. Therefore, the yield of SC is low in hospitalized patients (2.6-6.1%) [START_REF] Fan | Application of rejection criteria for stool cultures for bacterial enteric pathogens[END_REF] , [START_REF] Rohner | Etiological agents of infectious diarrhea: implications for requests for microbial culture[END_REF], especially after 3 days of hospitalization (0.5%) [START_REF] Bauer | Derivation and validation of guidelines for stool cultures for enteropathogenic bacteria other than Clostridium difficile in hospitalized adults[END_REF]. Thus, some laboratories reject SC submitted after 3 days of hospitalisation ("3-day rule"), considering Clostridium difficile is the major microorganism responsible for nosocomial bacterial diarrhoea [START_REF] Versalovic | Manual of Clinical Microbiology[END_REF].

In 2001, a European study group [START_REF] Bauer | Derivation and validation of guidelines for stool cultures for enteropathogenic bacteria other than Clostridium difficile in hospitalized adults[END_REF] modified this 3-day rejection rule to propose exceptions for high risk patients in adult population (neutropenic, HIV-positive, or age > 65 years with comorbidities). These guidelines were validated in Freiburg Hospital (Germany): the global yield of stool culture was 1.0% on 3416 samples, and 82.5% of the SC were sent after 3 days of hospitalization. This particular population may not be representative of all European laboratories. In Lisbon (Portugal) the global yield of SC was 9% on 853 samples and 64.6% of the stool cultures were sent after 3 days of hospitalisation [START_REF] Hanscheid | Feasibility of introducing rejection criteria for stool cultures in a teaching hospital in Portugal[END_REF]. The guidelines validated for adults by Bauer et al. were later extended to children in 2007 [START_REF] Seyler | Safety and cost savings of an improved three-day rule for stool culture in hospitalised children and adults[END_REF]. In this paediatric population from Oxford, the global yield of SC was 1.9% on 5262 samples, and 29% of the stool cultures were sent after 3 days of hospitalization.

The first objective of this study is to evaluate retrospectively in Lille University Hospital (France) the yield of SC depending on the length of hospitalization, and to characterize the patients missed by the 3-day rejection rule. It seems necessary to evaluate the application of rejection criteria separately on children (age < 16 years old), because the epidemiology of infectious diarrhoea differs markedly between adults and children [START_REF] Pfeiffer | The patient presenting with acute dysentery-a systematic review[END_REF]. The second aim is to evaluate the potential savings for the laboratory.

Material and Methods

Stool cultures requested from January 2007 to December 2011 in Lille University

Hospital were included retrospectively in this study. Follow-up cultures after a positive result were excluded. For Campylobacter spp. detection, all specimens were streaked onto Karmali medium (bioMérieux, Marcy l'Etoile, France) without microfiltration and incubated at 37°C in a microaerophilic atmosphere (85% nitrogen, 10% carbon dioxide, 5% oxygen) for 48 hours. To detect Salmonella enterica and Shigella spp., all specimen were streaked onto Hektoen medium (bioMérieux, Marcy l'Etoile, France) and incubated at 37°C in air for 24 hours. To enhance the recovery of Salmonella enterica, we used a Selenite F broth (bioMérieux, Marcy l'Etoile, France), which was subcultured onto Hektoen after 18 hours of incubation at 37°C. Yersinia enterocolitica were detected using Schiemann-CIN medium (bioMérieux, Marcy l'Etoile, France) after an incubation of 24 hours at 28°C in air.

To identify bacterial enteric pathogens, we used conventional biochemical techniques from January 2007 to September 2009. For Salmonella enterica and Shigella spp., suspicious colonies on Hektoen were screened for urease activity and streaked onto Kligler agar.

Depending on the results of these tests, we continued with VITEK 2 GN (bioMérieux, Marcy l'Etoile, France) or with Shigella antisera (Bio-Rad, Marnes la Coquette, France).

Campylobacter spp. were identified by API Campy (bioMérieux, Marcy l'Etoile, France), and Yersinia enterocolitica using the VITEK 2 GN. From October 2009 to December 2011, we used MALDI-TOF mass spectrometry (Microflex; Bruker Daltonics, Wissembourg, France).

Shigella strains were still identified by seroagglutination since MALDI-TOF cannot identify this specific bacterial genus.

We estimated the average stool culture would cost 7.98€ for our laboratory. This global cost can be decomposed into culture media (1.53€), automated specimen inoculation (1.04€), bacterial identification (0.41€) and technical personnel (5.00€).

Statistical analysis was performed with SPSS 20.0. Mann-Whitney test was used to compare the length of hospitalization for children versus adults. Chi-squared test was used to compare the yield of SC, and this analysis was also stratified by hospitalization length. Pvalues of less than 0.05 were considered to indicate a statistically significant difference.

Results

During this 5-year study period, 13,039 SC were requested, corresponding to 10,635 patients (including 2,238 children). 376 were positive (2.9%): 239 SC for Campylobacter spp.

(63.6% of the positive stool cultures), 111 for Salmonella enterica (29.5%), 18 for Shigella spp. (4.8%), and 8 for Yersinia enterocolitica (2.1%).

The global yield of SC was higher for children (7.6%) than for adults (1.7%) (p<0.001) (Table 1). Only 37.8% of SC were prescribed after 3 days of hospitalization for children compared to 65.7% for adults (p<0.001). As the yield of SC falls sharply when the length of hospitalization increases, we performed a stratified analysis based on 4 categories of hospitalization length (Figure 1). At Day 0, the yield of SC was 18.8% for children and 7.9% for adults (p<0.001). After Day 3, no significant difference was found between the yield of SC in children (0.7%) versus adults (0.3%) (p=0.09).

Strict application of the 3 day-rule rejection criteria would have led to miss 7 cases of bacterial enteric pathogens for children (3.5% of all positive SC) and 23 cases for adults (13.0% of all positive SC) (Table 2). Among these 30 missed cases, 4 were asymptomatic carriers, 4 had non-diarrheal manifestations of enteric infection (SC prescribed after bacteraemia or abscess due to enteric pathogens), and 9 were hospitalized patients with a community acquired diarrhoea at Day 0 but the first SC was prescribed after more than 3 days of hospitalization. Only 13 of the 30 positive SC prescribed after 3 days of hospitalization would have been relevant (4 children and 9 adults), representing 0.2% of all the SC prescribed for nosocomial diarrhoea.

Patients suffering from nosocomial diarrhoea often received chemotherapy during the preceding weeks (7 of the 13 clinically relevant cases) but only 2 of them had severe neutropenia (absolute neutrophil count < 0.5 x 10 9 /L). Four patients had haematological malignancies, 2 had solid tumours and 1 was treated with methotrexate for Crohn's disease. The yield of SC was higher during warmer months: 4.0% from May to September (with a maximum of 5.0% in July) against 2.0% from October to April (with a minimum of 1.7% in February). The number of SC prescribed per month remained stable, regardless of the season (mean = 217, standard deviation =18).

Using the 3-day rule, 7,830 SC would have been rejected (60.0% of all the SC received). The potential savings for the laboratory would have been about 12,500€ per year (culture media 2,400€, automated specimen inoculation 1,630€, bacterial identification 640€ and technical personnel 7,830€).

Discussion

Even if the yield of SC prescribed after 3 days of hospitalization was low (0.7% for children and 0.3% for adults), 13 clinically relevant cases would have been missed by the strict application of the 3-day rule (4 children and 9 adults). Among these 9 adult cases of nosocomial diarrhoea undiagnosed, only 2 would have been detected by the application of Bauer guidelines (1 had age > 65 years associated with comorbidities, and 1 had severe neutropenia).

Interestingly, we found that 7 patients out the 13 clinically relevant cases of nosocomial diarrhoea received chemotherapy. Asymptomatic colonization with enteropathogenic bacteria can lead to invasive disease triggered by cancer chemotherapy, as previously reported for Salmonella [START_REF] Delaloye | Nosocomial nontyphoidal salmonellosis after antineoplastic chemotherapy: reactivation of asymptomatic colonization?[END_REF], probably due to mucositis and immunosuppression. In our study, 4 patients developed nosocomial Campylobacter diarrhoea (2 C. coli, 2 C. jejuni) and 3 developed Salmonella enterica diarrhoea after receiving chemotherapy.

After 3 days of hospitalization, there was no significant difference between the yield of SC in children compared to adults. Although children seem particularly susceptible to develop community-acquired diarrhoea, that does not justify the research of enteropathogenic bacteria after 3 days of hospitalization. Globally, SC did not add much value to the diagnosis of nosocomial diarrhoea, except for patients under cancer chemotherapy.

The higher number of positive SC cases detected in warm months (May to September) can be explained by the epidemiology of Campylobacter and Salmonella infections (representing 93% of our positive cases). Indeed, the increase of ambient temperature is known to be associated with Campylobacter [START_REF] Nichols | Campylobacter epidemiology: a descriptive study reviewing 1 million cases in England and Wales between 1989 and 2011[END_REF] or Salmonella [START_REF] Ravel | Seasonality in human salmonellosis: assessment of human activities and chicken contamination as driving factors[END_REF] infections. However, the number of SC prescribed per month remained stable during the year. This may be explained by the winter seasonality of viral gastroenteritis [START_REF] Mounts | Cold weather seasonality of gastroenteritis associated with Norwalk-like viruses[END_REF], contrasting with the summer seasonality of most bacterial enteric infections.

Physicians prescribe SC in case of diarrhoea, not knowing if the aetiological agent is a bacteria, a parasite or a virus.

The introduction of rejection criteria for SC can potentially delay the diagnosis of nosocomial outbreaks due to bacterial enteric pathogens [START_REF] Bruins | Detection of a nosocomial outbreak of salmonellosis may be delayed by application of a protocol for rejection of stool cultures[END_REF]. These outbreaks are very rare nowadays, thanks to higher standards of hygiene. Actually, no cluster of nosocomial cases has been identified during the 5 years covered by this study.

In conclusion, rejection of SC prescribed after 3 days of hospitalization allows to reduce workload by 37.8% for children and 65.7% for adults, representing cost-savings of 12,500€ (16,250 $) per year. Few cases of clinically relevant enteric infections are missed, but even the exception criteria proposed by Bauer et al. do not identify all of these cases. Patients under cancer chemotherapy seem particularly at risk to develop nosocomial diarrhoea caused by enteropathogenic bacteria. Overall, this study highlights the importance of communication between clinical microbiologists and physicians before the prescription of SC for patients suffering from nosocomial diarrhoea.

Tables and Figures legends

Table 1. Characteristics of the study population (children versus adults)

Table 2 .

 2 Epidemiology of the bacterial enteric pathogens detected after 3 days of Stool culture yield stratified on the length of hospitalization (children versus adults)

	hospitalization
	C.: Campylobacter, S.: Salmonella, Y.: Yersinia
	Figure 1. *: p<0.001, NS: non significant

Table 1 .

 1 Characteristics of the study population (children versus adults)

		Children	Adults
	Stool Culture (SC)	Positive	Negative Positive	Negative	P-value
	Total Number of SC (%)	199 (7.6%)	2,431 (92.4%)	177 (1.7%)	10,232 (98.3%)	<0.001
	SC sent before 3 days (%)	192 (11.7%)	1,443 (88.3%)	154 (4.3%)	3,420 (95.7%)	<0.001
	SC sent after 3 days (%)	7 (0.7%)	988 (99.3%)	23 (0.3%)	6,812 (99.7%)	0.09
	Median length of hospitalization (days)	0	2	1	8	<0.001
	Age (mean years ±	4.84	3.44	43.89	60.07
	standard deviation)	(± 4.62)	(± 4.86)	(±20.49)	(± 18.95)
	Sex Ratio	1,26	1,21	1,36	1,14	0.22

Table 2 .

 2 Epidemiology of the bacterial enteric pathogens detected after 3 days of

	hospitalization
	Epidemiology	n (%)	Bacterial species
	Children
	Community acquired diarrhoea	1 (14%)	1 C. jejuni
	Nosocomial diarrhoea (with chemotherapy)	2 (29%)	1 C. coli, 1 S. enterica
	Nosocomial diarrhoea (without chemotherapy)	2 (29%)	1 C. coli, 1 S. enterica
	Non-diarrheal manifestation of enteric infection	1 (14%)	1 S. enterica
	Asymptomatic carrier	1 (14%)	1 C. jejuni
	Total	7 (100%)	2 C. jejuni, 2 C. coli, 3 S. enterica
	Adults
	Community acquired diarrhoea	8 (35%)	1 C. jejuni, 5 C. coli, 2 S. enterica
	Nosocomial diarrhoea (with chemotherapy)	5 (22%)	2 C. jejuni, 1 C. coli, 2 S. enterica
	Nosocomial diarrhoea (without chemotherapy)	4 (17%)	2 C. jejuni, 1 S. enterica, 1 Y. enterocolitica
	Non-diarrheal manifestation of enteric infection	3 (13%)	3 S. enterica
	Asymptomatic carrier	3 (13%)	1 C. jejuni, 1 C. coli, 1 S. enterica
	Total	23 (100%)	6 C. jejuni, 7 C. coli, 9 S. enterica, 1 Y. enterocolitica
	C.: Campylobacter, S.: Salmonella, Y.: Yersinia

Funding sources

None.

Conflict of interest statement

None declared.


Rémi Le Guern
email: remi.leguern@gmail.com

Frédéric Wallet
email: frederic.wallet@chru-lille.fr

Emmanuel Vega
email: emmanuel.vega@chru-lille.fr

René J Courcol
email: rene.courcol@chru-lille.fr

Caroline Loïez
email: caroline.loiez@chru-lille.fr

Postneurosurgical meningitis due to Psychrobacter sanguinis

ABSTRACT

We report the first case of a post-neurosurgical meningitis due to Psychrobacter sanguinis. Psychrobacter spp. have been observed in marine environments and in a wide range of food products. Despite a strict questioning of the patient and the medical staff, we did not find the source of this bacterium.

CASE REPORT

A 64-year-old woman living near the English Channel suddenly experienced severe headaches and vomiting. Clinical examination revealed nuchal rigidity but no focal neurologic signs. There was no hyperthermia, nor altered consciousness (Glasgow Coma Scale evaluated at 14). A computed tomography angiography (CTA) showed a subarachnoid hemorrhage with enlargement of ventricles, due to a basilar artery aneurysm. The patient was immediately transferred to a neurosurgery unit. An external ventricular drain was set on to relieve the elevated intracranial pressure, under local anesthesia. She did not receive any prophylactic antibiotic therapy. Endovascular coiling was performed on day 1 after admission to seal off the basilar aneurysm.

A leak of cerebro-spinal fluid (CSF) was observed at the drain insertion site, on day 3. The patient kept touching the drain insertion site with her bare hands, despite repeated instructions. On day 8, the external ventricular drain was removed due to the persistence of the CSF leak. There was no local sign of inflammation or infection. However, the patient presented hyperthermia (38.5°C) the same day and a lumbar puncture was performed. The CSF was hemorrhagic, with increased proteins concentration (1.64 g/L) and decreased glucose (0.19 g/L). A mild hypoglycorrhachia occurs frequently after subarachnoidal hemorrhage, therefore it was not considered as indicative of meningitis. No bacteria was shown on Gram-stain, and the cultures remained sterile.

On day 15, the patient presented hyperthermia at 39°C, with altered sensorium and drowsiness. A new lumbar puncture allowed a definitive diagnosis of post-neurosurgical meningitis. CSF analysis showed a hyperleukocytosis (5900 WBC/mm 3 ), a profound hypoglycorrhachia (0.07 g/L) and elevated protein levels. Direct examination revealed few Gram-negative coccobacilli. An empiric antimicrobial therapy was initiated immediately using a combination of meropenem and fosfomycin at 2g x 3 per day and 4g x 4 per day, respectively. A single dose of amikacine (25 mg/kg) was also administered. No bacterial growth was observed at 48h after incubation. On day 4, the brain heart infusion (BHI) broth became turbid due to the growth of Gram-negative coccobacilli. The BHI was subcultured onto blood agar incubated at 37°C, chocolate agar incubated at 37°C and 5% CO 2 , and onto blood agar incubated at 37°C in an anaerobic atmosphere. The Gram-negative coccobacilli only grew on the blood and chocolate agar under aerobic conditions after 48 hours. These colonies were catalase, oxidase and urease positive. Unfortunately, MALDI-TOF (Bruker Daltonik S. A., Wissembourg, France) using FlexControl software (version 3.0) gave no reliable identification : the Biotyper database contains the spectra of 5,627 species and is regularly updated by the Bruker Company. To identify of this agent, sequencing of the 16S rRNA gene was performed and a 1,500-bp DNA fragment was amplified using the universal primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1525R

(5'-AAAGGAGGTGATCCAGCC-3'). DNA sequence analysis was performed with leBIBI and BLAST. There was a 100% identity on 1400 bp with the Psychrobacter sanguinis type strain (Genbank accession no. HM212668). Partial 16S sequences of 19 Psychrobacter species type strains and our isolate were analyzed using the ARB software environment [START_REF] Ludwig | ARB: a software environment for sequence data[END_REF]. A phylogenetic tree was created using the ARB neighbor-joining algorithm (Figure 1).

In vitro antimicrobial susceptibility was determined using agar disc diffusion, using the critical zone diameters recommended by CA-SFM 2013 (Comité de l'Antibiogramme de la Société Française de Microbiologie; http://www.sfm.asso.fr). Our isolate of P. sanguinis was susceptible to amoxicillin, ticarcillin, cefepim, ceftazidim, imipenem, meropenem, amikacin, ciprofloxacin and trimethoprim/sulfamethoxazole. Therefore, meropenem and fosfomycin were switched to amoxicillin (4g x 3 per day), as part of antimicrobial therapy de-escalation strategy. Amoxicillin was continued until the patient had received 15 days of antimicrobial therapy.

The patient experienced a rapid recovery under treatment. The fever subsided 4 days after the initiation of meropenem and fosfomycin. A lumbar puncture performed the same day revealed only 12 WBC/mm 3 , and a normal level of glucose (0.54 g/L). Proteins levels were still slightly elevated (0.92 g/L). No other complications were observed during the hospitalization.

We report the first case of a post-neurosurgical meningitis due to P. sanguinis. Members of the Psychrobacter genus are small Gram-negative coccobacillus. Psychrobacter spp. have been observed in marine environments like deep-sea [START_REF] Maruyama | Phylogenetic analysis of psychrophilic bacteria isolated from the Japan Trench, including a description of the deep-sea species Psychrobacter pacificensis sp[END_REF] or sea ice [START_REF] Bowman | Diversity and association of psychrophilic bacteria in Antarctic sea ice[END_REF], and in a wide range of food products including seafood (2) and cheese (4). P. sanguinis is a new species described in 2012: the type strain was isolated from the blood of an 84-year male in the USA (8).

Because P. sanguinis is a recently described species, there is a lack of data available concerning its pathogenicity. P. sanguinis was first isolated from the blood cultures of 4 patients in New York [START_REF] Wirth | Psychrobacter sanguinis sp. nov., recovered from four clinical specimens over a 4-year period[END_REF]. However, P. sanguinis is probably an environmental bacteria, and was recovered from seaweeds in India (Genbank accession no. JX501674 and JX501676). P.

sanguinis is able to grow on marine agar, which contains 3.5% NaCl to simulate sea water, and its temperature range is 4-37°C [START_REF] Wirth | Psychrobacter sanguinis sp. nov., recovered from four clinical specimens over a 4-year period[END_REF]. Other Psychrobacter species have been found in various environmental sites, and are only considered as rare opportunistic human pathogens.

P. arenosus is a species closely related with P. sanguinis based on their 16S rRNA sequence, and was described in 2004 [START_REF] Yarza | The All-Species Living Tree project: a 16S rRNA-based phylogenetic tree of all sequenced type strains[END_REF]. P. arenosus was isolated from costal sea ice in Japan [START_REF] Romanenko | Psychrobacter maritimus sp. nov. and Psychrobacter arenosus sp. nov., isolated from coastal sea ice and sediments of the Sea of Japan[END_REF], and associated with one published case of bacteremia after blood transfusion in France (3).

In our case report, investigations were performed to identify the source of P. sanguinis but it is difficult to understand how P. sanguinis was able to cause a post-neurosurgical meningitis.

One hypothesis is that the bacteria was present in the hospital environment and entered the central nervous system by colonization of the external ventricular drain. Indeed, the patient frequently touched the drain with her bare hands despite repeated instruction from the hospital staff. It is also possible that the mode of entry is a transient bacteremia, but it is less likely.

Four set of blood cultures were drawn during the hospitalization and remained sterile.

This first report of P. sanguinis meningitis emphasizes the need to incubate culture medium for more than 48h in post-neurosurgical patients. Neurosurgery can lead to infection with rare bacteria of low pathogenicity levels, even one week after an external ventricular drain was removed.

Nucleotide sequence accession numbers:

The 16S rDNA sequence data of this P. sanguinis strain was deposited to Genbank under accession number KJ439038. Psychrobacter type strains, the Psychrobacter sanguinis isolate KJ439038 from our study, and an Acinetobacter calcoaceticus type strain as a root. Bootstrap values (> 50%) are indicated at branch nodes.

Figure 1 .

 1 Figure 1. Neighbor-joining phylogenetic tree based on 16S rRNA sequences from 19