The diagnostic performance and accuracy of 3 molecular assays for the detection of Clostridium difficile in stool samples, compared with the Xpert® C. difficile assay

Journal Pre-proof The diagnostic performance and accuracy of 3 molecular assays for the detection of Clostridium difficile in stool samples, compared with the Xpert® C. difficile assay

Maya Azrad, Linda Tkhawkho, Zohar Hamo, Avi Peretz PII:

S0167-7012(19)30530-5

DOI:

https://doi.org/10.1016/j.mimet.2019.105784

Reference:

MIMET 105784

To appear in:

Journal of Microbiological Methods

Received date:

24 June 2019

Revised date:

19 November 2019

Accepted date:

19 November 2019

Please cite this article as: M. Azrad, L. Tkhawkho, Z. Hamo, et al., The diagnostic performance and accuracy of 3 molecular assays for the detection of Clostridium difficile in stool samples, compared with the Xpert® C. difficile assay, Journal of Microbiological Methods (2018), https://doi.org/10.1016/j.mimet.2019.105784

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© 2018 Published by Elsevier.

Journal Pre-proof

The diagnostic performance and accuracy of 3 molecular assays for the detection of Clostridium difficile in stool samples, compared with the Xpert® C. difficile assay Maya Azrada, Linda tkhawkhoa, Zohar Hamoa, Avi Peretza,b a

Clinical Microbiology Laboratory, The Baruch Padeh Medical Center, Poriya, Tiberias, Israel. The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel.

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b

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*Corresponding author:

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Dr. Avi Peretz

Tiberias, Israel

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e-mail: [email protected]

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Phone: +972-4-665-2322 Fax: +972-4-665-2531

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Hanna Senesh 818/2

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Journal Pre-proof Abstract This research compares the performance and diagnostic accuracy of three molecular tests for the detection of Clostridium difficile in stool samples, with the Xpert® C. difficile assay. Fifty-nine positive and twenty-five stool negative samples were analyzed by the BDmax™ Cdiff, the Simplexa C. difficile Direct, and the GenomEra™ C. difficile, and compared with the Xpert® C. difficile routinely used in our laboratory.

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The highest sensitivity was 94.9% for the BDmax™ Cdiff, followed by GenomEra™ C. difficile with 93.2%, and Simplexa™ C. difficile Direct with 89.8%. The

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specificities of all assays were 100%. GenomEra™ C. difficile had the highest

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retesting rate (12%). Simplexa™ C. difficile Direct benefits from both short hands-on

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time and total-turnaround time and 0% retesting.

The differences in performance and accuracy between these three molecular assays

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C. difficile.

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are insignificant and all may be used as part of the routine algorithm for detection of

Key words: Clostridium difficile, Performance, Diagnostic Accuracy, Molecular assays

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Journal Pre-proof Introduction Clostridium difficile is a leading cause of nosocomial diarrhea, the most common nosocomial infection acquired in hospitals and institutions for prolonged hospitalization [1]. This infection is mainly mediated by toxin A and toxin B that are secreted by the bacterium [2]. During the last decade, the global incidence of CDI (C. difficile infection) has increased dramatically due to the appearance of a new virulent strain,

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ribotype 027/NAP1 [3], which is characterized by a relatively severe illness with high antibiotic resistance, mortality, and recurrence rates [4,5].

Thus, it is very important

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to detect CDI early and accurately for better monitoring and treatment.

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Diagnosis of C. difficile can be performed by several methods; the first

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developed assays relied on toxin detection. Although highly sensitive, toxigenic culture, which tests the ability of C. difficile isolates to produce toxins in vitro, is not

cytotoxicity

assay

is

also

laborious

and

time-consuming

[6].

Enzyme

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by

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routinely used due to its complexity and long turn-around time. In vivo toxin detection

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inmunoassays (EIA) for the detection of free toxins and/or the C. difficile antigen glutamate dehydrogenase (GDH) in stool are much quicker and easier to perform. However, these methods suffer from low diagnostic accuracy [7]. More recently, as molecular assays for the detection of specific C. difficile genes have become simpler to operate and highly available, their use has been increased in clinical laboratories for the detection of C. difficile in stool samples. Most of these assays are based on nucleic acid amplification of genes encoded for toxin A and/or B and some even target the binary toxin and the tcdc deletion for specific identification of the ribotype 027/NAP1 strain.

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Journal Pre-proof Although

costly,

these

molecular

methods

offer

high

sensitivity

and

specificity, ease of performance, and short hands-on time and total turnaround time. At the same time, several studies have reported on over-sensitivity in comparison with the toxigenic culture or cytotoxin assays [8-13]. Since these molecular tests identify only the presence of toxins' genes rather than toxins production, a positive PCR result may represent not only patients with an active disease but also asymptomatic patients with C. difficile colonization, which is highly prevalent in health care facilities [13].

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Despite this over diagnosis and consequently over treatment, more laboratories have chosen to implement such molecular assays, apparently due to public health

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burden and lead to disease outbreaks.

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concern that imprecise diagnosis will result in underestimation of CDI prevalence and

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The current study aimed to compare the performance and the diagnostic accuracy of 3 molecular assays, the BDmax™ Cdiff (BD Diagnostics, Franklin Lakes,

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

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GenomEra™ C. difficile (Abacus Diagnostica, Turku, Finland), with the Xpert® C.

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difficile (Xpert C. difficile; Cepheid, Sunnyvale, CA) – our routine laboratory method for the detection of C. difficile in stool samples.

Methods

Sample collecting Eighty-four stool samples were collected at the Poriya Baruch Padeh Medical Center. All samples were analyzed by the Xpert® C. difficile, according to the manufacturer's instructions. Briefly, a sterile cotton swab was dipped in the stool and transferred into the provided test cartridge, which was then loaded onto the GeneXpert

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Journal Pre-proof system (Cepheid). Samples were stored at 4°C until testing in the other three methods was performed (for no longer than 2 days). C. difficile detection by molecular assays All samples were further analyzed by three molecular-based assays: the BDmax™ Cdiff, Simplexa™ C. difficile Direct, and GenomEra™ C. difficile according to the manufacturers' instructions. Briefly, sample preparation for the BDmax™ Cdiff included sampling of 10 µl stool using a sterile loop and adding it to

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a sample buffer tube, supplied in the kit. Then, the sample tube was vortexed for 1 minute and loaded onto the BDmax™ instrument, with additional provided reagents.

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For Simplexa™ C. difficile Direct, one Reaction Mix vial was thawed for each

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sample. Meanwhile, a sterile cotton swab was dipped into the stool sample and

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transported into a Sample Prep Buffer vial (both swab and buffer were provided by the manufacturer). After swirling, the swab was discarded and 50 µl of sample buffer

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and 50 µl of Reaction Mix were added to their specific wells on a direct amplification

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disk, which was loaded onto the LIAISON ® MDX instrument.

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For GenomEra™ C. difficile, 1 μL sterile loop was used for sampling the stool sample and transferring it into the provided GenomEra Z-tube. After 5-minute vortex, 35 μL of the prepared sample was pipetted onto the Test Chip. Then the Test Chip was loaded onto the GenomEra® CDX instrument. The assays' results are automatically appear at the end of each run, thus there is no need for special interpretation. In case of a result other than negative or positive for C. difficile (such as borderline, failed, etc.), the specimen was retested. Table 1 summarizes the main differences between the four molecular tests.

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Journal Pre-proof TABLE 1. Characteristics of C. difficile molecular assays. Assay

Platform

Targets

Automated extraction

Samples

Hands-on

per run

a

time

Total turnaround Timea,b

Xpert®

GeneXpert (Cepheid)

C. difficile

tcdB,

Yes

1-4

3.0 mins

50 mins

cdtB (binary

toxin),

tcdc deletion BDmax™

BDmax (BD)

tcdB

Yes

1-24

3.0 mins

88 mins

Simplexa™

LIAISON® MDX

tcdB

Yes

1-8

2.5 mins

59 mins

C.

(Focus Diagnostics)

tcdB

Yes

6 mins

59 mins

GenomEra

GenomEra® CDX

™

(Abacus Diagnostica)

1-4

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Direct

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difficile

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Cdiff

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C. difficile Per four samples

b

Total turnaround Time includes hands -on time, reaction duration, and time required for results

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a

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interpretation

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Journal Pre-proof Data analysis We used the Xpert® C. difficile as the reference method for calculating sensitivity,

specificity,

and

negative and

positive predictive values.

Therefore,

specimens that were found positive or negative by the Xpert® C. difficile were defined as "True Positive" or "True Negative", respectively. Fisher's exact test was applied for analyzing the differences between assays' sensitivities. Concordance rates were calculated for each pair of molecular assays as

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the percentage of samples that had the same results out of total samples. Kappa coefficient was calculated for analyzing the level of agreement. Statistical significance

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was determined with p Value < 0.05. Data was analyzed using SAS® version 9.3

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(SAS Institute, Cary, NC, US).

Results

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Eighty-four samples were collected; twenty-five were found negative and

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Discordant results

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fifty-nine were found positive by the Xpert® C. difficile.

The results of 76 (90.5%) samples were concordant in all 4 molecular methods. Four positive samples (4.8%) had a negative result in either GenomEra™ C. difficile (2) or Simplexa™ C. difficile Direct (2). Three positive samples (3.6%) were negative by two methods; 1 sample was defined negative by both GenomEra™ C. difficile and Simplexa™ C. difficile Direct, 2 samples were defined negative by both BDmax™ Cdiff and Simplexa™ C. difficile Direct. Only one positive sample (1.2%) was found negative in all 3 tested methods. There were no cases false positive results. Overall, agreement levels between all four methods were very high, with all kappa coefficients above 0.8 (Table 2). 7

Journal Pre-proof TABLE 2. Concordance rates between C. difficile molecular assays The compared assays

Concordance

Kappa

rate (%)

coefficient

BDmax™ Cdiff/ Xpert® C. difficile

4.69 0.92

BDmax™ Cdiff / GenomEra™ C. difficile

49 0.87

BDmax™ Cdiff/ Simplexa™ C. difficile Direct

4.69 0.92

Simplexa™ C. difficile Direct/ Xpert C. difficile

4.69 0.84

Simplexa™ C. difficile Direct/ GenomEra™ C.

4.69 0.84

®

difficile 4.6. 0.89

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GenomEra™ C. difficile / Xpert® C. difficile

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Eighty-one samples had the same result by Xpert® C. difficile and the BDmax™ Cdiff, indicating a concordance rate of 96.4%. The concordance rates of

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Xpert® C. difficile with the Simplexa™ C. difficile Direct or with the GenomEra™ C.

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difficile were 92.8% and 95.2%, respectively. Agreement rates between the three tested molecular assays are presented in Table 2.

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Specimens retesting

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Out of 84 samples, 10 (12%) were retested by GenomEra™ C. difficile due to

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"Failed" (9) or "Borderline" (1) result (Table 3).

TABLE 3. Performance of C. difficile molecular assays. Sensitivitya

Assay

Specificitya

PPVb

NPVc

Retesting rated

BDmax™ Cdiff Simplexa™

C.

difficile

94.9 (85.8-98.9)

100 (86.3-100)

100 (93.6-100)

89.3 (71.8-97.7)

3.6

89.8 (79.2-96.2)

100 (86.3-100)

100 (93.3-100)

80.6 (62.5-92.6)

0

93.2 (83.5-98.1)

100 (86.3-100)

100 (86.3-100)

86.2 (68.3-96.1)

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Direct GenomEra™ C. difficile a

Values are presented in percentages, 95% confidence interval is shown in parentheses.

b

PPV= Positive predictive value

c

NPV= Negative predictive value

d

Retesting rate was calculated as percentage of retested samples out of total samples (84).

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Journal Pre-proof Additional 3 (3.6%) samples were re-analyzed by BDmax™ Cdiff due to "Invalid" result. Upon retesting, all these 13 samples had a positive or negative result. No retesting was required using the Simplexa™ C. difficile Direct. Assay performance We observed 3 false negative results with the BDmax™ Cdiff, which had the highest sensitivity (94.9%) and negative predictive value (NPV) (89.3%) (Table 3). Four samples had a false negative result with the GenomEra™ C. difficile, which had

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a sensitivity of 93.2% and NPV of 86.2%. The Simplexa™ C. difficile Direct, which detected 6 samples as false negative, had the lowest sensitivity (89.8%) and NPV

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(80.7%). All assays had a specificity and a positive predictive value (PPV) of 100%.

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The sensitivities of all assays were not significantly different (p > 0.5 for all

Discussion

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comparisons).

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The goal of this study was to evaluate the diagnostic performance and

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accuracy of 3 molecular assays for the detection of C. difficile, compared with the Xpert® C. difficile, which has been implemented in many laboratories. Overall, the sensitivities and specificities of all three tested methods were high; BDmax™ Cdiff had the highest sensitivity (94.9%), followed by GenomEra™ C. difficile (93.2%) and Simplexa™ C. difficile Direct (89.8%). These differences that were not statistically significant (p > 0.5), may be attributed to the different primers used in each assay. Although we compared the assays to different reference methods used in previous studies, BDmax™ Cdiff had higher sensitivity and specificity in the current study (94.9% and 100%, versus 87-93.7% and 97.9-98.8%, respectively) [7,14,15] Also, the retesting rate (3.6%) was lower compared to a few studies (4.4-5.8%) [7, 14]

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Journal Pre-proof but higher than what was found in one study (1.8%) [15]. These differences might result from changes in the assay protocol that may have been introduced over the years. The specificity of GenomEra™ C. difficile in our study was comparable to that found in previous reports (99.6-99.7%) [7, 16] However, the sensitivity in the current study was lower than in a previous report (97.5%) [7] and higher than in another study (87.2%) [16] Retesting rate was lower in both previous studies (5.3-5.8%), compared to what we found (12%). Regarding Simplexa™ C. difficile Direct, the specificity was

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found to be 100% in the current study as well as in other studies [14, 17]. Both lower sensitivity (87%) [14] and a higher sensitivity (98%) [17] compared to our study

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(89.8%) were reported. However, both studies have used different protocol for sample

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processing than our protocol. Retesting rate (0%) was not different from previous

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reports (0-1.3%) [14, 18].

In light of the high performance of all tested assays, the decision of which

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molecular assay to implement into a clinical laboratory may rely on the ease of

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operation, hands-on time, total turnaround time, and ease of results interpretation.

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Regarding both operation and hands-on time, Simplexa™ C. difficile Direct was the easiest to perform and had the shortest hands-on time per 4 samples. Additionally, it offers simple and clear result interpretation. The reaction lasts 55 minutes; thus the time to result is adequate (59 mins). BDmax™ Cdiff also had a short sample processing time (3 mins). However, it requires more preparation steps such as sample vortex and reagents loading. Additionally, its reaction time for 4 samples was the longest – 84 minutes, resulting in the high total turnaround time (88 mins). Results interpretation was uncomplicated. GenomEra™ C. difficile had the longest sample preparation time due to a 5-minute vortex step. However, as the reaction lasts only 52 minutes, the overall time to result is still satisfactory (59 minutes). Also, interpretation

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Journal Pre-proof of results is straightforward. Xpert® C. difficile is still the most advantageous in matter of total turnaround time (50 mins). Also, no multiple preparatory steps are required before sample loading to the instrument. Furthermore, it has the advantage of identifying the hyper virulent C. difficile strain NAP1/Ribotype 027. In view of the quite expensive costs of the molecular assays, high retesting rate must be taken into consideration. With regard to this aspect, Simplexa™ C. difficile Direct should be praised for having no invalid results at all, minimizing the

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need for retesting samples. In contrast, both BDmax™ Cdiff and GenomEra™ C. difficile had several instances of invalidity, with a retesting rate of 3.6% and 12%,

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respectively, which increase both the time and financial expenditure.

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Another aspect that should be mentioned is the number of samples that can be

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simultaneously loaded onto the instrument. BDmax™ has a maximum capacity of 24 samples per run. Moreover, although fewer samples can be analyzed per run, reagent

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tubes, which are supplied in in sealed pouches per 24 samples, are stable for up to 7

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days at 2–25°C after initial opening. These characteristics indicate BDmax™ The LIAISON® MDX and

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compatibility for large-scale clinical laboratories.

GenomEra® CDX enable runs of maximum 8 and 4 samples at a time, respectively. For Simplexa™ C. difficile Direct, each Sample Prep Buffer and Reaction Mix are provided in 2 separate tubes per one sample, thus there is no minimum samples per run requirement or short assay expiration. Additionally, the direct amplification disk into which samples are loaded can be kept after partial use (for fewer than 8 samples) for one year at room temperature. Similarly, the GenomEra Z-tubes of the GenomEra™ C. difficile assay are provided separately per one sample and fewer than 4 samples can be loaded per run. Therefore, both Simplexa™ C. difficile Direct and GenomEra™ C. difficile are suitable for small clinical laboratories. The Xpert® C.

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Journal Pre-proof difficile assay is also more relevant for small laboratories as it processes 1-4 samples simultaneously. The main difference from Simplexa™ C. difficile Direct and GenomEra™ C. difficile is that the samples are loaded into 4 different modules, enabling independent starting time for each of the 4 samples. Several limitations exist in the current study; first, we did not perform a toxigenic culture, which may represent the "gold standard" for detecting C. difficile. Second, we did not collect any data regarding the patient's clinical symptoms; that

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may have thrown light on specific cases with discordant results.

Despite previous concerns that molecular tests for detection of C. difficile are

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over-sensitive and lead to over-diagnosis, their use in the clinical laboratory has been

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markedly increased in recent years. The main reason for this is apparently the reduced

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costs and improved patient management that were noted since their implementation as part of CDI diagnosis [19, 20]

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In summary, all three molecular assays that have been tested in the current

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study were found to have good performance and accuracy. While choosing one

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molecular platform, differences regarding the ease of operation, time to result, retesting rate, maximal capacity, and other technical aspects should be taken into account.

Acknowledgments Reagents used in the current study were provided and were partially funded by the

Israeli representatives

of Focus

Diagnostics

(DiaSorin),

BD Diagnostics

(BACTLAB diagnostics) and Abacus Diagnostica (Gamidor Diagnostics).

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Journal Pre-proof Funding Reagents used in the current study were provided and were partially funded by the

Israeli representatives

of Focus

Diagnostics

(DiaSorin),

BD Diagnostics

(BACTLAB diagnostics) and Abacus Diagnostica (Gamidor Diagnostics). No additional funding was obtained.

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Conflicts of interest

Israeli representatives

of Focus

Diagnostics

(DiaSorin),

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the

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Reagents used in the current study were provided and were partially funded by BD Diagnostics

(BACTLAB diagnostics) and Abacus Diagnostica (Gamidor Diagnostics).

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There are no other potential conflicts of interest to report.

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Journal Pre-proof References 1. Khan HA. Nosocomial infections and their control strategies. Asian Pac J Trop Biomed 2015;5(7):509-14. 2. Jank T, Giesemann T, Aktories K. Rho-glucosylating Clostridium difficile toxins A and

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027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol 2009;10(9):1.

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6. Planche TD, Davies KA, Coen PG, et al. Differences in outcome according to Clostridium difficile testing method: a prospective multicenter diagnostic validation study of C difficile infection. Lancet Infect Dis 2013;13(11):936-45. 7. Hirvonen JJ, Kaukoranta SS. Comparison of BD Max Cdiff and GenomEra C. difficile molecular assays for detection of toxigenic Clostridium difficile from stools in conventional sample containers and in fecal swabs. Eur J Clin Microbiol Infect Dis 2015;34:1005-9. 8. Bruins MJ, Verbeek E, Wallinga JA, et al. Evaluation of three enzyme immunoassays and a loop-mediated isothermal amplification test for the

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Journal Pre-proof laboratory diagnosis of Clostridium difficile infection. Eur J Clin Microbiol Infect Dis 2012;31:3035–9. 9. Eastwood K, Else P, Charlett A, Wilcox M. 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.

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10. Chapin KC, Dickenson RA, Wu F, Andrea SB. Comparison of five assays for detection of Clostridium difficile toxin. J Mol Diagn 2011;13:395–400.

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11. Buchan BW, Mackey TL, Daly JA, et al. Multicenter clinical evaluation of the

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portrait toxigenic C. difficile assay for detection of toxigenic Clostridium

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difficile strains in clinical stool specimens. J Clin Microbiol 2012;50:3932–6. 12. LeGuern R, Herwegh S, Grandbastien B, et al. Evaluation of a new molecular

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test, the BD Max Cdiff, for detection of toxigenic Clostridium difficile in fecal

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samples. J Clin Microbiol 2012;50:3089–90.

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13. Polage CR, Gyorke CE, Kennedy MA, et al. Overdiagnosis of Clostridium difficile

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2015;175:1792-801.

14. Gilbreath JJ, Verma P, Abbott AN, Butler-Wu SM. Comparison of the Verigene Clostridium difficile, Simplexa C. difficile Universal Direct, BD MAX Cdiff, and Xpert C. difficile assays for the detection of toxigenic C. difficile. Diagn Microbiol Infect Dis 2014;80:13-8. 15. Dalpke AH, Hofko M, Zorn M, Zimmermann S. Evaluation of the fully automated BD MAX Cdiff and Xpert C. difficile assays for direct detection of Clostridium difficile in stool specimens. J Clin Microbiol 2013;51:1906-8.

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Journal Pre-proof 16. Alcalá L, Reigadas E, Marín M, et al. Comparison of GenomEra C. difficile and Xpert C. difficile as confirmatory tests in a multistep algorithm for diagnosis of Clostridium difficile infection. J Clin Microbiol 2015;53:332-5. 17. Deak E, Miller SA, Humphries RM. Comparison of the Illumigene, Simplexa, and AmpliVue Clostridium difficile molecular assays for diagnosis of C. difficile infection. J Clin Microbiol 2014;52:960–3. 18. Nolte FS, Ribeiro-Nesbitt DG. Clinical comparison of Simplexa universal direct

samples. J Clin Microbiol 2014;52:281-2.

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19. Catanzaro M, Cirone J. Real-time polymerase chain reaction testing for

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Clostridium difficile reduces isolation time and improves patient management

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in a small community hospital. Am J Infect Control 2012;40:663-6. 20. Sydnor ER, Lenhart A, Trollinger B, et al. Antimicrobial prescribing practices in

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Journal Pre-proof

Author Statement MA, LT,ZH and AP designed the study, analyzed, interpreted the data, and wrote the final manuscript. ZH and LT performed the test . All authors read and approved the

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final manuscript.

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Journal Pre-proof Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Journal Pre-proof 

The current study aimed to compare the performance and the diagnostic accuracy of 3 molecular assays, the BDmax™ Cdiff, Simplexa™ C. difficile Direct), and GenomEra™ C. difficile ,with the Xpert® C. difficile for the detection of C. difficile in stool samples.



The highest sensitivity was 94.9% for the BDmax™ Cdiff, followed by GenomEra™ C. difficile with 93.2%, and Simplexa™ C. difficile Direct with

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The differences in performance and accuracy between these three molecular

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assays are insignificant and all may be used as part of the routine algorithm for

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detection of C. difficile

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

f

89.8%. The specificities of all assays were 100%.

19