A Single Center’s Experience with a Novel Nucleic Acid Amplification Test for the Detection of the Clostridium difficile

Toxin B Gene from Clinical Samples C. Dang1, S. Lewis2, M. Olson1, A. Romesburg1, J. Liffers1, B. C. Ellis1, K. C. Carroll2

1Johns Hopkins Hospital, Baltimore, MD, 2Johns Hopkins University School of Medicine, Baltimore, MD

Background: Clostridium (now Clostridioides) difficile remains a formidable pathogen.

While controversial, many laboratories use a molecular test for the diagnosis of C. difficile

infection (CDI). We report a single center’s experience from a multi-center study for FDA

510K approval of a new qualitative real-time PCR assay, i.e. the GenePOC™ CDiff assay

(GenePOC Inc., Québec, Canada).

Methods: The Johns Hopkins Hospital (JHH) Microbiology Laboratory tested 200 unformed

stool specimens. To run the GenePOC CDiff test, approximately 5 μl of the stool sample was

tested according to the manufacturer’s package insert. 500 µl of the stool sample was

transferred to an Anaerobe Transport Medium and shipped to an off-site reference lab for

direct culture on CCFA agar and enriched culture using CCMB-TAL broth in case direct

culture was negative. Recovered isolates were determined to be cytotoxin positive by a cell

culture cytotoxin neutralization assay. In addition, since the standard-of-care in our clinical

laboratory is the BD MAX™ Cdiff test (BD Diagnostics, Inc., USA), the GenePOC CDiff

assay was also compared with the BD MAX results.

Results: Two hundred samples were tested at JHH. Eleven samples were eliminated due to

an exceeded transit time to the reference lab for culture. Thirteen samples were excluded

because SBT and PIEs were mixed with different lot numbers. Therefore, a total of 176 stool

specimens had valid test results. Twelve samples were positive for toxigenic C. difficile by

direct culture. All direct culture positive samples were positive by GenePOC CDiff

(sensitivity, 100%). An additional nine specimens were direct culture negative, but positive

by the GenePOC CDiff assay (specificity, 94.5%). Twenty-three samples were positive by

combined culture, sixteen of which were positive by the GenePOC assay (69.6% sensitivity).

There were five GenePOC positive, culture negative samples for a specificity of 96.7%.

Compared to the BD MAX (n = 189), the sensitivity and specificity of the GenePOC test

were 95.5% and 100%, respectively.

Conclusion: The GenePOC CDiff test compares favorably to direct culture on CCFA and to

another FDA-cleared NAAT that detects the toxin B gene in unformed fecal samples.

Clostridium (now Clostridioides) difficile continues to cause significant morbidity and

mortality. Accurate and rapid diagnosis is essential in the appropriate treatment of patients

and in preventing transmission of this infection in the hospital environment. While the

optimum diagnostic method is still a matter of debate, the updated guidelines from the

Infectious Diseases Society of America and the Society for Healthcare Epidemiology (1)

recommend either a two-step algorithm based upon testing for glutamate dehydrogenase or

nucleic acid test (NAT) followed by a toxin test or, in institutions where when there are pre-

agreed institutional criteria for patient stool submission, a NAT alone may be acceptable.

NATs are currently the most sensitive, but least specific tests available for the diagnosis of C.

difficile. Studies have shown a reduction in transmission over time when NATs are used

appropriately in hospitalized patients who meet clinical criteria for C. difficile disease

combined with optimum infection control measures (2).

The GenePOC™ CDiff assay is a qualitative in vitro diagnostic test that utilizes real-time

polymerase chain reaction (PCR) to detect tcdB of toxigenic C. difficile. The goal of this

project was to compare the GenePOC CDiff assay to a reference method (toxigenic culture)

as well as to the standard of care molecular test (BD MAX Cdiff assay) currently in use in

the Johns Hopkins Hospital Microbiology Laboratory.

Test Method: GenePOC CDiff Assay

GenePOC Instrument

Comparative Methods

ABSTRACT (AMENDED) MATERIALS AND METHODS

• The GenePOC™ CDiff assay is comparable to direct toxigenic culture for C.

difficile detection, but less sensitive than enriched culture (CCMB-TAL).

• The GenePOC™ CDiff assay is comparable to the FDA-cleared BD MAX™

Cdiff test, a NAAT that detects the toxin B gene found in C. difficile.

• The GenePOC™ CDiff assay requires little sample preparation for testing and

will provide accurate results within 70 minutes, allowing for rapid diagnosis of

C. difficile infection.

Controls:

• Each PIE contains reagents for the detection of tcdB and process controls to monitor

processing, amplification, and the absence of reaction inhibitors.

• Positive external controls, provided by GenePOC, were tested each day.

• 150 µL of SBT was used as a negative external control.

Testing Algorithm:

• The stool samples were placed in the SBTs for testing. Afterwards, the SBTs were placed

at 2-8°C in the event repeat testing is required. If the sample did not require repeat testing,

the SBT was frozen at -70±5°C or colder.

• 500 µl of stool sample was transferred into an Anaerobe Transport Medium kept at room

temperature for shipment to a reference lab (Microbiology Specialists, Inc.) for toxigenic

culture (reference method).

• The remaining (minimum of 250 µL) of the homogenized stool specimen was frozen at -

70±5°C (or colder) for further testing (if needed).

GenePOC Specimen Processing:

• 5µl of unformed stool was transferred into

the appropriately labeled Sample Buffer

Tube (SBT) using a disposable inoculating

loop.

• The SBT was closed and vortexed as

described in the GenePOC CDiff test

package insert.

• Mixed SBT was transferred into the PIE

using the disposable transfer pipette.

• The PIE was loaded onto the GenePOC

instrument and run.

• The specimen identifier, SBT barcode, and

PIE barcode were scanned prior to insertion

of the PIE.

• The PIE was inserted into the instrument

carousel.

• A maximum of eight samples can be

processed in a single run including external

controls.

• For runs with less than eight patient

samples, the empty places were filled with

PIEs containing external controls.

• Results were available in 70 min.

Toxigenic Culture (Reference Method)

• Specimens were inoculated onto a CCFA

plate for direct culture and a CCMB-TAL

broth for enriched culture.

• After incubation, the broth was sub-

cultured to another CCFA plate.

• The plates were examined for colonies

characteristic of C. difficile.

• Suspicious isolates were confirmed as C.

difficile by gas liquid chromatography after

incubation in chopped meat carbohydrate

broth for 48 h.

• Toxigenicity of isolates was determined by

cell culture cytotoxicity neutralization

assay.

BD MAX (Standard of care)

• Specimens were inoculated into SBT

tube, vortexed, and run on BD MAX

instrument.

• Real-time PCR for the amplification of

C. difficile toxin B gene.

• Detection of the amplified DNA uses

fluorogenic target specific hybridization

probes.

• Results are available in 100 minutes.

Table 3: GenePOC™ results compared to BD MAX™ results

Specimen Inclusion Criteria:

• At least 1.25ml of unformed stool from patients suspected of having CDI

• Fresh specimens were tested with the GenePOC and the reference method within 48 h

• Specimens were residual, de-identified, and kept at 2-8 C prior to testing

Table 1: GenePOC™ CDiff results compared to direct culture results.

References

1. McDonald LC, Gerding DN, Johnson S, et al. 2018. Clinical practice guidelines for

Clostridium difficile infection in adults and children: 2017 update by the Infectious

Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of

America (SHEA). Clin Infect Dis 66:987-94.

2. Napierala M, Munson E, Skonieczny P, et al. 2013. Impact of toxigenic Clostridium

difficile polymerase chain reaction testing on the clinical microbiology laboratory and

inpatient epidemiology. Diagn Microbiol Infect Dis. 76:534-38.

DIRECT CULTURE

POS NEG

GENEPOC™

CDIFF

ASSAY

POS 12 9

NEG 0 155

Table 4: Analysis of GenePOC™ results versus comparative methods.

GenePOC™ Cdiff vs

Direct Culture

GenePOC™ Cdiff vs

Combined Culture

GenePOC™ CDiff vs

BD MAX™ Cdiff

Sensitivity 100% Sensitivity 69.6% Sensitivity 95.5%

Specificity 94.5% Specificity 96.7% Specificity 100%

Saturday - 288 Johns Hopkins University

410-955-5077

kcarrol7@jhmi.edu

Table 2: GenePOC™ results compared to BD MAX™ results

COMBINED CULTURE

POS NEG

GENEPOC™

CDIFF

ASSAY

POS 16 5

NEG 7 148

BD MAX™ CDIFF ASSAY

POS NEG

GENEPOC™

CDIFF

ASSAY

POS 21 0

NEG 1 152