Comparative performance study of six commercial molecular assays for rapid detection of toxigenic Clostridium difficile

Accepted Manuscript Comparative performance study of six commercial molecular assays for rapid detection of toxigenic Clostridium difficile Yossi Paitan, Tamar Miller Roll, Amos Adler PII:

S1198-743X(17)30104-0

DOI:

10.1016/j.cmi.2017.02.016

Reference:

CMI 864

To appear in:

Clinical Microbiology and Infection

Received Date: 26 December 2016 Revised Date:

13 February 2017

Accepted Date: 14 February 2017

Please cite this article as: Paitan Y, Miller Roll T, Adler A, Comparative performance study of six commercial molecular assays for rapid detection of toxigenic Clostridium difficile, Clinical Microbiology and Infection (2017), doi: 10.1016/j.cmi.2017.02.016. 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.

ACCEPTED MANUSCRIPT Intended category: Original article E-only

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detection of toxigenic Clostridium difficile

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Yossi Paitana*, Tamar Miller Rollb and Amos Adlerb

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Comparative performance study of six commercial molecular assays for rapid

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Meir Medical Center, Kfar Saba, Israel; bNational Center for Infection Control, Ministry of

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Health, Tel-Aviv, Israel.

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Running Title: Rapid molecular detection of C. difficile

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Laboratory, Meir Medical Center, 59 Tschernihovsky St., Kfar Saba, 44281, Israel

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*Corresponding author: Yossi Paitan, PhD, MHA, Director of Clinical Microbiology

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Phone: +972 (0)9 7472371

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Mobile: +972 (0)54 2478547

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Fax:

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E-mail: [email protected]

+972 (0)9 7472369

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Key Words: Clostridium difficile, Rapid detection, Molecular detection, Toxigenic

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Clostridium difficile, Clostridium difficile infection.

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Objectives: Rapid and accurate detection of C. difficile in stool impacts patient treatment and

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containment efforts. Detection of C. difficile toxin genes using nucleic acid amplification

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techniques (NAAT) is part of a multistep algorithm. Our objective was to directly compare

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the diagnostic accuracy and applicability of 6 commercial C. difficile NAAT assays.

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Methods: In this study, 210 specimens were analysed in parallel by 6 commercial NAAT.

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Toxigenic culture was used as a reference method.

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ABSTRACT

Results: We analysed 98 positive and 112 negative samples. The Xpert C. difficile had 99%

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(95% CI 88.49-98.32), Illumigene C. Difficile, and Quidel AmpliVue C. difficile both 93%

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(95% CI 85.84-97.08) and BDmax Cdiff and GenomEra C. difficile both 92% (95% CI 84.55-

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96.41). All assays had very high specificity (>99%). Invalid results requiring retesting were

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the highest in GenomEra (6.7%; 14/210) and BDmax (4.3%; 9/210), followed by AmpliVue

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(1.4%; 3/210) and Xpert (0.96%; 2/210). No retesting was required with Simplexa and

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sensitivity (95% CI 94.45-99.97), followed by Simplexa C. difficile Universal Direct 95%

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the longest for BDmax, mostly due to the different reaction times of assays. Total hands-on

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time was comparable for all 6 assays.

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Conclusions: All assays had high sensitivity and specificity. The differences in the TAT,

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Illumigene. The turn-around time (TAT) was the shortest for the Illumigene and Xpert and

repeat testing rates and in platform characteristics could help laboratories decide which assay

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would integrate better in their setting and to better select a molecular platform for C. difficile

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detection.

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Toxigenic Clostridium difficile (C. difficile ) is a leading cause of hospital-acquired diarrhoea,

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and is associated with significant morbidity, mortality and healthcare costs [1, 2]. Since the

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beginning of the millennium, the epidemiology of C. difficile infection (CDI) has changed, in

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part due to the emergence of an epidemic strain, ribotype 027 [3] and to an increase in

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community-associated CDI cases [4, 5]. Timely and accurate detection of C. difficile in stool

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could impact patient treatment and improve containment efforts [2]. Currently, toxigenic

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culture assay and cell cytotoxicity assay are used as reference methods; however, their

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Introduction

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is questionable. Generally, nucleic acid amplification techniques (NAAT) have better

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sensitivity than enzyme immunoassay (EIA) based assays for detection of C. difficile in stool

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[6]. As recommended by ESCMID guidelines [7], detection of C. difficile toxin genes using

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NAAT is a part of a multistep algorithm, starting with EIA for toxins A/B (with or without

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glutamate dehydrogenase (GDH) EIA, followed by confirmatory molecular assay in case of

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application is not practical for routine diagnosis and their correlation with clinical outcomes

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Despite the wide array of molecular tests currently available, relatively few studies have

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conflicting GDH/Tox A/B EIA results [6, 7, 8].

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compare, the diagnostic accuracy of 6 commercial NAAT for C. difficile detection and the

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directly compared the performance of these assays. The purpose of this study was to directly

applicability of each as a routine test.

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Materials and methods

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Specimens

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In this study, 210 unique specimens were analysed in parallel with all 6 assays. Samples were

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collected from May 2014 through October 2014. All liquid or unformed stool specimens were

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tested according to our routine laboratory protocol, which is a two-step algorithmic approach

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Complete (QCC) (Techlab, Blacksburg, VA) for detecting GDH and toxins A/B [8].

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Specimens positive for GDH and toxins are considered positive, specimens negative for both

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are considered negative and specimens with inconclusive results (GDH-positive and toxin-

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negative) are further tested using a molecular test, the Xpert C. difficile assay (Cepheid,

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Sunnyvale, CA) [9]. An average of 180 valid specimens a month are processed in our

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laboratory, with 9-10 (5%) QCC positive, 158-159 (88%) negative and 12-13 (7%)

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inconclusive (GDH-positive and toxin-negative), of which about 7-8 (60%) are positive by

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following the ESCMID guidelines [7], consisting of an initial EIA test, the C. Diff Quik Chek

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Medical Center is about 9%.

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Xpert. Based on these figures, the estimated prevalence of toxigenic C. difficile in Meir

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and frozen at -80°C. We collected 83 consecutive QCC negative samples, 58 positives and 69

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consecutive QCC inconclusive specimens (of which 40 were Xpert positive). The first tube

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was used for comparison analysis. About 10 to 12 specimens were thawed each day and

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analysed in parallel with all 6 assays. The second tube was used for toxigenic culture.

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Following routine analysis, selected specimens (volume > 1 ml) were divided into two tubes

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Thawed stool samples were inoculated on ChromID C. difficile agar plates (BioMérieux,

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C. difficile culture and molecular analysis of isolates

Marcy l'Etoile, France) and incubated in an anaerobic chamber. Suspicious colonies were

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identified based on morphology and confirmed by the detection of the species-specific gene,

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tpi by PCR [10]. The presence of tcdA and tcdB genes (coding for toxins A and B) was tested

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with PCR [11]. A presumptive diagnosis of the epidemic strain, BI/NAP1/027, was done by

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detecting the 18-bp deletion in the tcdC gene [11] and the binary toxin (cdtB) gene [12].

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C. difficile assays

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NJ), Simplexa C. difficile Universal Direct (Simplexa) (Focus Diagnostics, Cypress, CA) and

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GenomEra C. difficile (GenomEra) (Abacus Diagnostica, Turku, Finland) target the tcdB

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gene using quantitative real-time PCR technology (QR-PCR). The Meridian Illumigene C.

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Difficile (Illumigene) (Meridian Bioscience, Cincinnati, OH) and Quidel AmpliVue C.

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difficile (AmpliVue) (Quidel Molecular, San Diego, CA), target a highly conserved fragment

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of tcdA (toxin gene A) [13], using loop-mediated isothermal DNA amplification (LAMP) and

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isothermal helicase dependent amplification (IHDA) technologies, respectively. The Xpert C.

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Commercial NAAT such as the BDmax Cdiff (BDmax) (BD Diagnostics, Franklin Lakes,

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Northbrook, IL) detect multiple targets including tcdB (toxin gene B), cdtB (binary toxin

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gene) and ∆117 in the tcdC gene (toxin negative regulatory gene) and can indicate the

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presence of presumptive ribotype 027 strain, using multiplexed QR-PCR technology.

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The basic characteristics of the 6 assays tested are shown in Table 1. All assays were

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performed according to manufacturers' instructions and results were interpreted according to

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difficile (Xpert) (Cepheid, Sunnyvale, CA) and the Verigene C. difficile (CDF; Nanosphere,

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positive or negative for toxigenic C. difficile (i.e. unresolved, indeterminate, borderline,

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failed, system errors, etc.) were retested from the original specimen or diluted and retested, as

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indicated by the manufacturer. All discordant valid results in any assay were retested using

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manufacturer's recommendations. All specimens that produced an invalid result other than

the specific assay.

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Data analysis

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Toxigenic culture results were used as a reference. However, 5 cases (2 positives and 3

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negatives among 210) had insufficient sample for culture analysis, and 1 initial SOP positive

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case (QCC inconclusive, Xpert positive) was culture negative. For these 6 cases, the initial

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SOP analysis and all 6 assays result were concordant and were therefore used as the final

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were considered True Positive (TP). Two samples that were insufficient for culture analysis

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and 1 culture negative sample which were all positive by initial SOP and by all 6 assays, were

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also considered TP. Eighty-six culture negative samples and 23 non- toxigenic cultures were

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considered True Negative (TN). An additional 3 samples insufficient for culture analysis

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which were negative at initial SOP and with all the 6 assays were considered TN. Any assay

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that did not produce a valid result upon retesting was excluded from the overall performance

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analysis of the specific assay. Predictive values were not calculated, as not all samples were

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result regardless of lack of culture results. All specimens found positive by toxigenic culture

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collected consecutively.

Results Assay performance

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(47%) positives and 112 (53%) negatives. Among 98 positive samples, 2 (2%) were

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We analysed 210 specimens following standard routine procedure (Table 2), yielding 98

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95 specimens, 2 were non-toxigenic (tcdA and tcdB negative). Among 93 tcdA and tcdB

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positive isolates, 12 (13%) were identified as presumed ribotype 027 (positive by cdtB PCR

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and 18bp tcdC deletion). Of note, presumptive ribotype 027 was identified in only 10 of the

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insufficient for culture analysis, 1 (1%) did not grow, and 95 (97%) grew C. difficile. Of these

respective samples by the Xpert system. Among 112 negative samples, no growth was

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observed in 86 (76.8%), 3 (2.7%) were insufficient for culture analysis and 23 (20.5%) grew

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non-toxigenic C. difficile (tcdA and tcdB negative).

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Among 210 specimens analysed, all 6 assays reported valid results upon initial testing in 186

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samples and in 207 upon retesting. In 3 samples (2 in BDmax and 1 in Xpert), results

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remained invalid. The sensitivity and specificity of the assays evaluated in this study are

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shown in Table 3. The Xpert assay had 99% sensitivity (95% CI 94.45-99.97) and 100%

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98.32) and 100% specificity (95% CI 96.76-100), Illumigene and AmpliVue both with 93%

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sensitivity (95% CI 85.84-97.08) and 100% specificity (95% CI 96.76-100), BDmax with

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92% sensitivity (95% CI 84.55-96.41), 100% specificity (95% CI 96.76-100) and GenomEra

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with 92% sensitivity (95% CI 84.55-96.41), 99.11% specificity (95%CI 95.13-99.98).

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specificity (95% CI 96.73-100), followed by Simplexa with 95% sensitivity (95% CI 88.49-

Discordant specimens

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Among 210 specimens, we observed discordant valid results for 11 positive and 1 negative

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upon retesting. A test failure was observed in the analysis of 5 of the 12 specimens (3 in

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BDmax and 2 in GenomEra), which had to be repeated again to obtain a valid result.

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Toxigenic C. difficile was isolated in all 11 positive specimens. Non-toxigenic C. difficile was

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isolated in the negative discordant specimen, which tested positive only with GenomEra. It

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should be noted that it retested twice as positive once from the specimen and once from the

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first extraction sample.

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Repeat testing rates

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specimens in one or more assays (Table 2). In all 12 cases, the discordant result repeated

As indicated in Table 3, Simplexa and Illumigene did not require repeat testing. Two

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specimens (0.96%; 2/210) had to be re-tested using Xpert, 1 was resolved and 1 remained

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invalid. Three AmpliVue specimens (1.4%; 3/210) had to be retested, all of which were

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resolved. Nine BDmax specimens (4.3%; 9/210) had to be retested; 7 were resolved and 2

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remained invalid. With GenomEra, 14 specimens (6.7%; 14/210) had to be retested, all of

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which were resolved. All 3 unresolved upon repeat specimens (2 BDmax and 1 Xpert) were

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retested with the initial sample suspension and with a second sample prepared from the

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clinical specimen.

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The 6 assays were compared in terms of throughput, total hands on time and total turnaround-

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time (TAT) — a timed run by one operator processing 10-12 samples (Table 1). The

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Simplexa, Illumigene, GenomEra and BDmax assays are run on batched platforms. The

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Simplexa assay has the highest capacity (1-96), followed by BDmax (1-24), Illumigene (1-

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10) and GenomEra (1-4). The Xpert and AmpliVue are random access platforms. Capacity of

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the Xpert depends on the specific instrument, which can accommodate 1- 80 cells in the

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Method comparison

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accommodate 1-24 samples. Xpert had the shortest total TAT for processing 10-12 specimens

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(70 min), followed by Illumigene (77 min), and GenomEra (52 min for 4 specimens in a 1

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instrument setup or extrapolated to 80 min for 12 specimens in a 3 instrument setup). These

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were followed by Simplexa (90 min), AmpliVue (105 min) and BDmax (142 min). The total

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hands-on time per batch of 10-12 samples was measured, including all manual steps from

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Infinity version. The AmpliVue test uses a disposable cell and dry baths and can

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aliquoting, platform preparation, loading, computer setup, reading result and samples and

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reagents disposal. We did not observe a difference in the total hands-on time (excluding

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incubation time), which was the shortest for the Xpert and Simplexa (25 minutes) and the

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specimen arrival to disposal: vortexing samples, marking, incubation, mix preparation,

longest for AmpliVue (35 minutes).

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Discussion

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Due to increases in the incidence and severity of C. difficile infection [14], accurate, readily

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available, rapid detection has become of extremely important. In recent years, the number of

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FDA-cleared or CE-marked commercial molecular C. difficile detection assays has increased

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and accordingly, the number of clinical laboratories using these assays. However, attempting

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has been compared to the reference method, cell culture cytotoxicity assays or toxigenic

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culture and not directly to other assays, resulting in variations in assay performance among

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studies.

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to choose one assay by comparing published studies is very problematic. Often, performance

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cleared and 1 CE-marked molecular C. difficile detection assay: Xpert, Simplexa, Illumigene,

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GenomEra, BDmax, and AmpliVue. To our knowledge, this is the first and most

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comprehensive study to directly compare these 6 assays simultaneously using the same

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samples.

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The goal of this study was to directly compare the performance and applicability of 5 FDA-

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to the reference method, EIA and other NAAT have been published [15-26]. To date, of the 6

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assays evaluated in our study the Xpert has been described the most, followed by Illumigene,

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BDmax, Simplexa, AmpliVue and GenomEra. Most published studies directly compared 2

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Several reviews and reports regarding evaluation of each of the studied assays in comparison

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comparison of these assays (in different studies) we found that Xpert was compared to

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Illumigene, BDmax, GenomEra and Simplexa. Illumigene was compared to Xpert,

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assays [20-22, 24, 26], and few compared 3 or 4 assays directly [19, 23, 25]. In view of direct

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Simplexa was compared to Xpert, Illumigene, AmpliVue and BDmax; AmpliVue was

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compared to Illumigene, and Simplexa; and GenomEra was compared to Xpert and BDmax.

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In addition, several reviews summarized results of different studies in an attempt to compare

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their relative performance [15-17]. This emphasizes a clear need for direct, simultaneous

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comparison of these assays as performed in the current study.

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All 6 assays resulted in high diagnostic values with greater than 90% sensitivity and close to

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100% Specificity. The Xpert displayed 99% sensitivity (95% CI 94.45-99.97), followed by

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AmpliVue, and Simplexa; BDmax was compared to Xpert, GenomEra, and Simplexa;

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CI 85.84-97.08) and BDmax and GenomEra both with 92% (95% CI 84.55-96.41). All assays

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displayed very high specificity; GenomEra had 99.11% (95%CI 95.13-99.98) and the rest had

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100% (95% CI 96.76-100). The relative performance of the assays observed in our study is

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generally consistent with those of other studies that directly compared 2-4 assays [19-26].

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However, it should be noted that the Xpert assay was compared to all other assays, except for

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AmpliVue. It constantly displayed higher sensitivity than other assays did and varying results

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regarding specificity.

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Simplexa with 95% (95% CI 88.49-98.32), Illumigene, and AmpliVue both with 93% (95%

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Commercial NAAT are associated with significant costs. Therefore, we assessed the rates of

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occurred more often with the GenomEra (6.7%; 14/210) and BDmax (4.3%; 9/210), followed

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by AmpliVue (1.4%; 3/210) and Xpert (0.96%; 2/210). No retesting was required with

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Simplexa and Illumigene. Comparative information regarding invalid results and retesting

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rates is limited and often unavailable [15]. Our results agree with those of other studies;

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retesting rates of 5.3% and 5.8% were observed for GenomEra [22, 24], 4.4% and 5.8% for

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BDmax [22, 23], 0.5%, 2.9% and 1.1% for Xpert [23-25], and 0% to 1.3% for Simplexa [23,

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invalid results requiring specimen retesting in each assay. Invalid results requiring retesting

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in the package insert. The higher retesting rate observed for GenomEra compared to previous

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26]. There are no reports on the retesting rate of AmpliVue; however, it is described as 0.5%

studies, might be due to a protocol change by the manufacturer, eliminating a specimen

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dilution step before reaction, resulting in shorter TAT, but possibly higher PCR inhibition

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rate. A 0.8%-4.4% retesting rate was reported for Illumigene [15]. This is in contrast to our

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finding of no retesting, which could be due to sample conditions (in our study samples were

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frozen before analysis), different pre-analytical exclusion criteria for samples and testing

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timelines. Given the relatively limited number of studies reporting retesting rate data,

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additional studies are needed to address the overall reliability of these tests.

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respectively), and the longest for BDmax (142 min). The differences in TAT were mostly due

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to the reaction times and incubation time of assays, as the total hands- on time did not differ

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significantly, ranging from 25 to 35 min.

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TAT for 10-12 samples was the shortest for the Xpert and Illumigene (70 and 77 min

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distinguish between true C. difficile infections and low-level colonization [27] emphasizes the

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need for strict pre-analytical inclusion/exclusion criteria of samples [18] and interpretation in

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conjunction with the clinical assessment. In addition, it has been suggested that detecting C.

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difficile with NAAT may be less specific than with EIA methods. Yet, molecular C. difficile

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The concern that the high sensitivity of C. difficile NAAT detection may impact the ability to

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despite the possibility of suboptimal clinical specificity [28, 29]. It should be noted that

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NAATs are not recommended as a stand-alone test due to inadequate positive predicted value

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(PPV) [7]. ESCMID guidelines for CDI diagnosis emphasize that diagnosis should be based

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on clinical signs and symptoms in combination with laboratory tests. They recommend a 2-

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step algorithm [7]. Based on ESCMID guidelines, we screen samples for both GDH and toxin

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A/B EIA. Samples with concordant positive or negative results are reported as such. Samples

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detection has been associated with lower hospitalization costs and improved outcomes

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represent samples with CDI or C. difficile carriage [7].

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with a negative GDH result but positive toxin result are retested by NAAT as they may

The current study had several limitations. The use of frozen samples could affect culture and

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molecular results. In addition, the direct application of samples on ChromID agar only, may

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also affect culture results, as some C. difficile strains form colourless colonies on ChromID

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C. difficile agar which could have been missed. The lack of broth enrichment or alcohol

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shock in the culture process may also result in lower sensitivity of the culture method.

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However among 210 study samples, this might have affected only 5 positive cases (3

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insufficient sample for culture, 2 non-toxigenic cultures and 1 no growth) and 3 negative

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and assays result were concordant (indicating a minimal effect of freezing on the results).

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Toxigenic culture might not be sensitive enough to detect very low levels of C. difficile that

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could be detected by PCR [27], as was seen in 3 cases (culture/toxin-PCR negative, but

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cases (insufficient sample for culture). Nevertheless, in these 8 cases, the initial SOP analysis

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cases were positive in all 6 assays. An additional limitation was the lack of clinical data

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regarding the patients, their syndromes (apart from diarrhoea) and outcomes.

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molecular assay positive). However, this potential affect was minimal in our study, as all 3

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Differences found in test performance, reliability, hands-on time, TAT and platform

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In summary, we evaluated the performance of 6 molecular C. difficile detection assays.

characteristics could help improve selection of platforms and determine which assay would

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integrate best into the workflow of an existing laboratory, in a multistep C. difficile detection

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analysis.

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Reagents and instrumentation were provided by Cepheid, Focus Diagnostics, Meridian

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Bioscience, Quidel Molecular, BD Diagnostics and Abacus Diagnostica and their

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representatives in Israel: Medison, Gamidor Diagnostics, DiaSorin, Ilex medical, BACTLAB

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diagnostics and Gamidor Diagnostics, respectively.

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The instruments were returned upon completion of the study.

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Acknowledgments

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Funding: Reagents and instrumentation were provided by Cepheid, Focus Diagnostics,

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representatives in Israel: Medison, Gamidor Diagnostics, DiaSorin, Ilex medical, BACTLAB

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diagnostics and Gamidor Diagnostics, respectively.

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No additional funding was obtained.

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Meridian Bioscience, Quidel Molecular, BD Diagnostics and Abacus Diagnostica and their

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Conflicts of interest: Reagents and instrumentation were provided by Cepheid, Focus

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Diagnostica and their representatives in Israel: Medison, Gamidor Diagnostics, DiaSorin, Ilex

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medical, BACTLAB diagnostics and Gamidor Diagnostics, respectively. The instruments

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were returned upon completion of the study.

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Diagnostics, Meridian Bioscience, Quidel Molecular, BD Diagnostics and Abacus

There are no other potential conflicts of interest to report.

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associated diarrhea in adult and pediatric patients. Diagn Microbiol Infect Dis

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17

ACCEPTED MANUSCRIPT TABLE 1. C. difficile assay characteristics Assay

Target(s)

Automated

1 Hands Reaction TATc Assay

extraction on timea

timeb

Platform

type

tcdB, cdt, ∆117 in tcdC

Yes

25

45

70 QR-PCR GeneXpert (Cepheid)d

Simplexa

tcdB

No

25(+10)

55

90 QR-PCR Integrated cycler (3M)e

Illumigene tcdA conserved fragment

No

27(+10)

40

77

GenomEra tcdB

No

28

52

80 QR-PCR GenomEra CDX (Abacus Diagnostica)g

BDmax

Yes

32

110

142 QR-PCR BDmax (BD)e

No

35(+10)

60

105 IHDA

AmpliVue tcdA conserved fragment

LAMP

IllumiPro-10 (Meridian Bioscience)f

AmpliVue (Quidel Molecular)h

SC

tcdB

RI PT

Xpert

2 3

IHDA: isothermal helicase dependent amplification.

4

a

5

M AN U

QR-PCR: quantitative real-time PCR; LAMP: loop-mediated isothermal DNA amplification;

Hands-on time (incubation time), in minutes, to process 10-12 specimens according to

manufacturers' instructions.

6

b

Indicates the reaction time in minutes.

c

Total turnaround time in minutes for results from 10-12 specimens including hands-on time,

TE D

7 8 9

d

Random access platform: The number of specimens per run depends on platform size .

10

e

Batched platform: The BDmax and Simplexa assays can be batched for up to 24 and 96

11

AC C

EP

incubation and reaction time.

specimens, respectively. In the BDmax, after first batch lysis completed, an additional 24

12

lysis reaction may be loaded.

13

f

Batched platform, a 2*5 cell instrument, enabling up to 10 specimens in each run.

14

g

Batched platform, a 4 cell instrument, multiple instruments can be used in parallel.

15

h

Disposable cell test assay, 16 tests per kit.

16 17 18

1

ACCEPTED MANUSCRIPT TABLE 2. Detailed results of study specimens

19 20

QCC

Xpert

Culture results tif positive tcdA tcdB

cdtB

culture

Xpert

Simplexa Illumigene GenomEra

BDmax

AmpliVue

∆117 in tcdC

ND

+

+

+

-

-

Pos

Pos

Pos

2

Positive

ND

+

+

+

-

18bp

Pos

Pos

Pos

9

Positive

ND

+

+

+

+

18bp

Pos

Pos

Pos

2

Positive

ND

+

+

+

+

36bp

Pos

Pos

Pos

1

Positive

ND

+

+

+

+

54bpl

Pos

Pos

Pos

2

Positive

ND

Insufficientb

NA

NA

NA

NA

Pos

Pos

26 GDH + onlyc Positive

+

+

+

-

-

Pos

Pos

2

GDH + only

Positive

+

+

+

+

18bp

Pos

Pos

2

GDH + only

Positive

+

-

-

-

1

GDH + only

Positive

No growth

NA

NA

NA

22 GDH + only

Negative

+

-

-

-

4

GDH + only

Negative

-

-

-

-

3

GDH + only

Negative

Insufficientb

NA

NA

NA

-

-

-

-

2

Negative

ND

-

-

1

Negative

ND

-

-

1

Positive

ND

+

+

1

Positive

ND

+

+

1

GDH + only

Positive

+

1

GDH + only

Positive

+

1

GDH + only

1

GDH + only

1

GDH + only

1

GDH + only

1

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos

M AN U

Pos

-

Pos

Pos

Pos

Pos

Pos

Pos

NA

Pos

Pos

Pos

Pos

Pos

Pos

-

Neg

Neg

Neg

Neg

Neg

Neg

-

Neg

Neg

Neg

Neg

Neg

Neg

NA

Neg

Neg

Neg

Neg

Neg

Neg

-

Neg

Neg

Neg

Neg

Neg

Neg

TE D

ND

-

-

-

Neg

Neg

Neg

Neg

Invalid

Neg

-

-

-

Invalid

Neg

Neg

Neg

Neg

Neg

+

+

-

Pos

Pos

Pos

Neg/Neg

Pos

Pos

+

-

-

Pos

Neg/Neg

Neg/Neg

Neg/Bor/Negd Neg/Neg

Pos

Pos

Pos

Pos

UNR/Neg/Neg Neg/Neg

Nege/Neg Neg/Neg

Neg/Neg

Neg/Neg

Neg/IND/Neg

Neg/Neg

EP

79 Negative

Pos

SC

40 Positive

RI PT

Noa Initial SOP results

+

-

-

+

+

+

54bp

AC C

+

Neg/Neg

Positive

+

+

+

-

-

Pos

Pos

Neg/Neg

Neg/Bor/Negd Neg/IND/Neg

Neg/Neg

Positive

+

+

+

+

18bp

Pos

Neg/Neg

Neg/Neg

Neg/Neg

Neg/Neg

Neg/Neg

Positive

+

+

+

-

-

Pos

Pos

Pos

Neg/Neg

Pos

Pos

Positive

+

+

+

-

-

Pos

Pos

Neg/Neg

Neg/Neg

Neg/Neg

Pos

GDH + only

Positive

+

+

+

-

-

Pos

Pos

Pos

Pos

Neg/Neg

Pos

1

GDH + only

Positive

+

+

+

-

-

Pos

Neg/Neg

Neg/Neg

Neg/Neg

Neg/Neg

Neg/Neg

1

GDH + only

Positive

+

+

+

-

-

Pos

Neg/Neg

Neg/Neg

Pos

Pos

Neg/Neg

1

Negative

ND

+

-

-

-

-

Neg

Neg

Neg

Pos/Pos

Neg

Neg

21

2

ACCEPTED MANUSCRIPT 22

negative; Pos: positive; NA: not analyzed; UNR: unresolved; Bor: borderline; IND:

23

Indeterminate.

24

a

Number of specimens.

25

b

Insufficient sample for culture analysis.

26

c

Inconclusive C. Diff Quik Chek Complete test: GDH-positive and toxin-negative

27

d

The retest in the GenomEra assay was borderline, suggesting re-analysis which was negative.

28

e

The first sample was cdtB positive, retesting was negative for all.

29

SC

RI PT

QCC: C. Diff Quik Chek Complete (Techlab, Blacksburg, VA); ND: not determined; Neg:

30

AC C

EP

TE D

M AN U

31

3

ACCEPTED MANUSCRIPT TABLE 3. Assay performance

32 33

No. of

Specimens

valid

No. of

No. of

No. of

No. of

Sensitivityb

Specificityb

requiring repeat

Resolved

resultsa

TP

TN

FP

FN

(95% CIc)

(95% CIc)

testingd

upon repeate

Xpert

209

97

111

0

1

99 (94.45-99.97)

100 (96.73-100)

Simplexa

210

93

112

0

5

95 (88.49-98.32)

100 (96.76-100)

0 (0%)

NA

Illumigene

210

91

112

0

7

93 (85.84-97.08)

100 (96.76-100)

0 (0%)

NA

AmpliVue

210

91

112

0

7

93 (85.84-97.08)

100 (96.76-100)

3 (1.4%)

3

GenomEra

210

90

111

1

8

92 (84.55-96.41)

99.11 (95.13-99.98)

14 (6.7%)

14 (100%)

BDmax

208

90

110

0

8

92 (84.55-96.41)

100 (96.70-100)

9 (4.3%)

7

1 (50%)

RI PT

2 (0.96 %)

(100%)

(78%)

SC

Assay

34

TP: True positive; TN: True negative; FP: False positive; FN: False negative; NA: not

M AN U

applicable.

35 36

a

Among 210 study specimens collected.

37

b

All values shown are percentage.

38

c

95% confidence interval (CI)

d

Percentage of the 210 samples is indicated in parentheses.

40

e

Among 28 repeated tests, 20 tests resolved as concordant, 5 discordant and 3 could not be

41

TE D

39

resolved, percentage of the resolved samples is indicated in parentheses.

EP

42 43

AC C

44

4