PERFORMANCE COMPARISON AND EVALUATION OF THE SIMPLEXA™ C. DIFFICILE DIRECT TEST FOR THE LABORATORY DIAGNOSIS OF C. DIFFICILE IN LIQUID STOOL SPECIMENS

ABSTRACT METHODS

text Background: Clostridium difficile infection (CDI) is a leading

cause of infectious diarrhea in the U. S. healthcare environment.

Although the organism is not invasive, disease is caused by

toxigenic strains that produce toxin A and B, and less frequently,

a binary toxin, 027(BI/NAP1). Several commercial tests

encompassing enzyme immunoassays (EIA) that detect

Glutamate Dehydrogenase (GDH) and toxin, and PCR targeting

the genes that code for tcdA, tcdB or the combination of

tcdB/NAP1/027/BI are available for the laboratory diagnosis of

CDI. The objective of this study was to compare the Simplexa™

C. difficile Direct PCR assay to our in-house two-step/three-step

algorithm (GDH, EIA, PCR) for the detection of the gene that

codes for tcdB and NAP. Methods: A total of 196 liquid stool

specimens were tested by our in-house GDH, toxin A and B

enzyme immunoassay method (C. Diff Quik Chek Complete,

Alere, TechLab, Blacksburg, VA) and three molecular assays:

Great Basin (Salt Lake City, UT); Simplexa™ C. difficile Direct

(DiaSorin Molecular LLC, Cypress, CA), and the Xpert™ C.

difficile/Epi performed on the GeneXpert Infinity (Cepheid,

Sunnyvale, CA). All samples were stored at 4° and

prospectively tested within 24 hrs. Results were analyzed by

comparing both methods (Simplexa™ and Infinity) to the two-

step/three-step algorithm in which the Great Basin PCR assay

was used as a confirmatory test if the EIA toxin result was

negative. Results: There was complete agreement (100%)

between the Simplexa™ and Infinity with our in-house two-

step/three-step testing method when the GDH and toxin assay

(EIA were positive. Twenty five samples were GDH positive and

EIA negative; of these, the Great Basin and Simplexa™

demonstrated 100% agreement; the Infinity detected one

additional positive and negative, respectively. The two-step

method 159 negatives (GDH and EIA negative). Of these, the

Simplexa™, 157 and the Infinity, 156 which resulted in two false-

positives by the Simplexa™ versus 3 false-positives by the

Infinity. The overall agreement between the Simplexa™ and

Infinity was 98.5%. Conclusion: The Simplexa™ compared

favorably to the Infinity and our in-house two-step/three-step

testing algorithm supporting its utility as a reliable method for

confirmatory testing in laboratories using algorithmic testing as

routine diagnostic platform.

Clostridium difficile accounts for the majority of infectious

diarrhea in the healthcare environment in the developed world.

In the United States alone, healthcare costs associated with CDI

are estimated to range from $500 million to $1.5 billion annually

(1). In an effort to reduce healthcare costs including reduced

length of hospital stays related to CDI, clinical microbiology

laboratories are challenged and pressured to provide rapid,

accurate, and cost-effective diagnostic test results. Current

diagnostic tests detect toxigenic C. difficile or its toxins. Many

institutions have adopted molecular testing technologies for C.

difficile that are more sensitive compared to conventional

methods, namely immunoassays that detect the glutamate

dehydrogenase antigen (GDH) and toxin (2). Both molecular and

conventional methods are replacing the traditional culture-based

methods, toxigenic culture and cell cytotoxicity neutralization.

In this study, we compared the performance of two PCR assays

with our current in-house testing algorithm using PCR as a

confirmatory test.

1. There was complete agreement between the Simplexa™ C. difficile Direct and

Xpert Infinity with our in-house two-step/three-step algorithm.

2. Overall agreement of Simplexa™ C. difficile Direct with Xpert Infinity was 98.5%.

3. A limitation to our study was the small sample size (196 samples).

4. We demonstrated that the Simplexa™ C. difficile Direct assay is a rapid and

reliable method for confirmatory testing in laboratories that follow a two-step

testing algorithm.

1. Crobach, M. J. T, T. Plance, C. Eckert, F. Barbut, E. M. Terveer, O. M. Dekkers, M. H. Wilcox, and E. J.

Kuijper. 2016. Clin. Microbiol. Infect. 22: S61 - S81.

2. Fang, F. C., C. R. Polage, and M. H. Wilcox. 2017. Point-Countepoint: What is the optimal approach for

detection of Clostridium difficile infection? J. Clin Microbiol. 55:670 – 680.

Acknowledgements:

Kaisha Gonzalez, DiaSorin Molecular LLC, for her technical support

Test kits were provided by DiaSorin Molecular LLC

REFERENCES

RESULTS SUMMARY

INTRODUCTION

J. Snyder1 and G. Thomson2, 1University of Louisville, 2University of Louisville Hospital, Louisville, KY

1. Both PCR methods demonstrated complete agreement with our in-house testing algorithm in which specimens

were GDH positive and Toxin positive.

2. Of the 25 specimens that were GDH positive, 15

were toxin positive and 10 were toxin negative.

There was 100% agreement between the Great

Basin and Simplexa™ ; Of these the Xpert Infinity

detected 16 positives and 9 negatives resulting in

one false-positive.

3. Of the 159 GDH negative, Toxin negative

specimens, 3 false positives were recorded, 2 for

Simplexa™ and 1 for the Xpert Infinity.

4. In the single incident involving a negative GDH

and positive Toxin, all three PCR methods agreed

with the EIA toxin results.

5. The overall agreement between the Simplexa™

and Xpert Infinity was 98.5% (Table 2).

2. In-House Testing Algorithm

a) Glutamate Dehyrdogenase Antigen plus Toxin EIA (C. Diff Quik Check Complete; Alere,

TechLab, Blacksburg, VA)

b) Confirmatory Molecular Assays:

1) Great Basin (Salt Lake City, UT)

2) Xpert™ C. difficile/Epi (GeneXpert Infinity; Cepheid, Sunnyvale, CA)™

3) Simplexa™ C. difficile Direct (DiaSorin Molecular, LLC; Cypress, CA)™ performed on

the Integrated Cycler (Figure 2)

*All specimens tested (Direct PCR) independent of two-step algorithm

1. Samples: Total of 196 liquid stools (conform to shape of container) from hospitalized

patients

a) Tested prospectively within 24 hours

b) Stored at 4°C

A. Study Design (Figure 1)

Figure 2: Integrated Cycler Figure 1: In-house Algorithmic Testing Figure 3: Stepwise procedure for specimen testing

152

Table 2:

Simplexa™ Positives Negatives

Positives 28 2 30

Negatives 1 164 165

29 166 195

Kappa Accuracy /Overall Percent Agreement (OPA) 98.5%

Upper 95% CI 100.0%

Lower 95%CI 96.7%

For Non-Reference Positive Percent Agreement (PPA) 96.6%

Upper 95% CI 100.0%

Lower 95%CI 89.9%

Negative Percent Agreement (NPA) 98.8%

Upper 95% CI 100.0%

Lower 95%CI 97.1%

Population Distribution

Percent Positive 15.4%

Upper 95% CI 20.4%

Lower 95%CI 10.3%

Percent Negative 84.6%

Upper 95% CI 89.7%

Lower 95%CI 79.6%

3 Simplexa™ samples showed discordant results with Infinity (36, 74, and 142)

36-NEG GDH/NEG Toxin/Neg Simplexa™/POS Infinity--- False Positive Infinity

74-NEG GDH/NEG Toxin/Neg Simplexa™/ POS Infinity--- False Positive Infinity

142-NEG GDH/NEG Toxin/POS Simplexa™/ Neg Infinity--- False Positive Simplexa™

Agreement of Simplexa™ versus Infinity

Infinity

N*=number tested

**GDH (+), Toxin (+) and GDH (-), Toxin (-), did not meet protocol to be tested on the Great Basin assay

Table 1: Results of Two-Step/Three-Step Testing Algorithm and PCRs for

Detection of C. difficile

Results *N Positive Negative Positive Negative Positive Negative

GDH (+), Toxin (+) 11 11 0 11 0

GDH (+), Toxin (-) 25 15 10 15 10 16 9

GDH (-), Toxin (-) 159 2 157 3 156

GDH (-), Toxin (+) 1 1 0 1 0 1 0

Total = 196

Simplexa™ Infinity

Not tested**

Not tested**

Great Basin