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Clostridium difficile two-step

algorithmic testing

Adam Caulfield, PhD

Spectrum Health Regional

Laboratory

SCACM

September 29, 2017

Outline

• C. difficile epidemiology

• Clinical criteria for testing

• Lab criteria for testing

• Test methods

• Standalone nucleic acid amplification tests (NAAT)

• Algorithm: enzyme immunoassay (EIA) + NAAT

• Comparison data

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Clostridium difficile epidemiology

• C. difficile is a spore-forming,

Gram-positive anaerobic bacillus

• Leading cause of infectious

diarrhea in the US (15-25%)

• Approx 450,000 infections,

29,000 deaths annually in the US

• During colonization, intestinal

inflammation is caused by

interaction between bacterial

exotoxin and colonic epithelial

receptors.

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Clostridium difficile epidemiology

• 30-50% of antibiotics prescribed

in hospitals are unnecessary or

incorrect.

• C. difficile accounts for 15-25%

of antibiotic associated diarrhea.

• $9,000-13,000 per case

• US annual cost: $500 million –

$1.5 billion (Seo et al, 2016, J

Clin Lab Anal).

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Guidelines

• IDSA/SHEA: Cohen S.H. et al., 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). Infect Control Hosp Epidemiol, 2010. 31(5): p431-55.

• ACG: Surawicz C.M. et al., Guidelines for diagnosis, treatment, and prevention of Clostridium difficile

infections. Am J Gastroenterol, 2013. 108(4): p478-98.

• ESCMID: European Guidelines: Crobach et al, European Society of Clinical Microbiology and Infectious

Diseases: update of the diagnostic guidance document for Clostridium difficile infection. Clin Microbiol

Infection. 2016. 22: S63-S81

• ASM: Spring 2018?

• 1.) Only patients with significant diarrheal stool are tested (3+ in 24 hr).

• 2.) Do not test for cure.

• 3.) Do not perform repeat testing. Within 7 days is generally considered to be

unnecessary (positive results can persist after effective treatment).

• 4.) Asymptomatic patients should not be tested as a positive result represents

colonization and should not be treated.

• 5.) Method: either NAAT or two-step (EIA composed of GDH/toxin plus NAAT)

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Clinical diagnosis

• C. difficile infection (CDI) –

no formal clinical

definition.

• Limit testing in low risk

populations (influences

predictive value of testing).

• Avoid testing of patients

with other explanations for

diarrhea: laxatives, IBD,

recent abdominal surgery,

viral illness, etc. 6

C. difficile test clinical ordering criteria

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C. difficile test lab ordering criteria

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• Consistency – some labs

use Bristol scoring system,

Test if 5-7 score.

• Must conform to the shape of

the container.

• Frequency – repeat ordering

within 7 days is restricted

electronically by EMR.

• Age – ≤ 1 year of age,

restricted testing.

(Heaton et al, 1992, Gut)

A role for stewardship teams – reduce fluoroquinolone usage

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Clindamycin, cephalosporins, and fluoroquinolones are associated

with the highest risk of CDI (Dingle et al, 2017, Lancet Infect Dis).

• The incidence of C. difficile incidence in England declined by 80% after 2006, following the

implementation of national control policies.

• Hypotheses for role of stewardship in CDI reduction:

• (1) If C. difficile infection declines were driven by reductions in use of particular antibiotics, then

incidence of C. difficile infections caused by resistant isolates should decline faster than that caused

by susceptible isolates across multiple genotypes.

• (2) If C. difficile infection declines were driven by improvements in hospital infection control, then

transmitted (secondary) cases should decline regardless of susceptibility.

WGS of 4,000+ isolates for fluoroquinolone resistance.

Quinolone usage – High Risk for C. difficile

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“Restricting fluoroquinolone prescribing appears to explain the decline in

incidence of C. difficile infections, above other measures” (Dingle et al, 2017,

Lancet Infect Dis).

Healthcare-onset C. difficile rates

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C. difficile toxins and associated genes

• Toxin A (tcdA) – Enterotoxin: causes fluid accumulation in bowel.

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(McDonald et al, 2005, New Engl J Med)

• Toxin B (tcdB) – Cytotoxin: cytopathic to lab-cultured cells.

• tcdC – regulatory gene of pathogenicity locus.

• Binary toxin (cdtA and cdtB) – associated with hypervirulent strain

NAP1/027/BI. Predictor of severe disease (illeus, toxic megacolon,

pseudomembranous colitis), ICU admission, colectomy, and death

(See et al, 2014, Clin Infect Dis).

C. difficile testing methods

• Diagnostic methods target either production of toxin protein or the presence of toxin genes.

• Culture + cell cytotoxicity assay • Historical gold standard

• Identification from selective media, then cytotoxicity assay

• Strong performance, yet labor intensive and slow

• Glutamate Dehydrogenase (GDH) antigen + Toxin A/B EIAs

• Rapid, GDH has high sensitivity, toxin component has high

specificity, less expensive

• NAAT (not all are PCR)

• High sensitivity, expensive, lower clinical specificity

• Combination?! EIA as initial screen (not recommended as

standalone test), with PCR arbitration.

2-step algorithm

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Patient with diarrhea and risk for

C. difficile infection

Stool tested by EIA for GDH antigen and Toxin A/B

GDH positive

Toxin negative

GDH negative

Toxin positive

Indeterminate result

NAAT for C. difficile toxin

GDH positive

Toxin positive

Results consistent with

C. difficile infection

GDH negative

Toxin negative

Results not consistent with

C. difficile infection

NAAT positive NAAT negative

Mayo Clinic study

• 268 beds, 6000 staff

• 500 prospective stool

specimens submitted

for C. difficile testing

• GDH/toxin EIA

• NAAT (x4)

• EIANAAT

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Phoenix, AZ

Methods

• Specimens tested by methods individually and as part of

2-step algorithm

• Tested 4 different NAAT methods; all detect one or

multiple toxin-associated genes.

• Combined Reference Standard: for comparison, a

specimen was considered positive if all 4 molecular tests

were positive, and negative if all 4 negative. • If a discrepancy (only 1-3 molecular tests positive), toxigenic

culture was performed as definitive gold standard method.

• 18/500 = 3.6% discrepancy rate. 7/18 = 39% positive by toxigenic

culture.

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Molecular platforms used for testing

Cepheid Xpert (PCR) tcdB, the binary toxin gene

(CDT), and tcdCΔ117

Focus Simplexa (PCR) tcdB

Meridian illumigene (LAMP) tcdA

Quidel AmpliVue (HDA) tcdA

NAAT assay characteristics

Assay

Target

Specimen

workflow

Hands-

on steps

Time, mina Invalid

Result

Rate, %

Capital

equipment

requirement Name Type

Hands-

on Total Alere

Quik Chek

EIA GDH antigen;

toxins A & B

Single or batch 4 9 min 35 min 0.0 No

Cepheid

Xpert

RT-PCR Toxin B gene

(tcdB);

binary toxin gene

(CDT); tcdCΔ117

Single 2 10 min 60 min 1.4 Yes

Focus

Simplexa

RT-PCR Toxin B gene

(tcdB)

Batch (1-94

patients)

5 35 min 100 min 0.4 Yes

Meridian

Illumigene

LAMP Conserved tcdA

fragment

Batch (1-5

patients)

5 25 min 75 min 0.8 No

Quidel

AmpliVue

HDA Conserved tcdA

fragment

Batch 5 30 min 110 min 3.4 No

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a Time for laboratory staff to test a batch of 10 samples.

Results: GDH/toxin EIA performance

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Results: NAAT performance

Results: performance of 2-step algorithm

• 88.8% of specimens

covered by EIA

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Spectrum Health data

Cepheid PCR

Positive Negative Total

Ale

re E

IA

GDH+ Toxin+ 11 2 13

GDH+ Toxin- 12 6 18

GDH- Toxin- 3 91 94 Total 26 99 125

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• Limitations:

• Smaller sample set (125)

• Not comparing to toxigenic culture as gold standard method.

Sensitivity Specificity PPV NPV

GDH 88.5% 91.9% 74.2% 96.8%

Toxin 42.3% 98.0% 84.6% 86.6%

What do positive/negative results really mean?

• Outcomes of 6500+ patients with diarrheal disease. (Planche et al., 2013, Lancet Infect Dis)

• Group 1: Toxin EIA (+), PCR (+)

• Group 2: Toxin EIA (-), PCR (+)

• Group 3: Toxin EIA (-), PCR (-)

• European Society of Clinical Microbiology and Infectious Disease

(ESCMID) guidelines recommend that NAAT methods are not to be used

as stand-alone tests, but rather as part of an algorithm including toxin EIA

testing. (Crobach et al., Clin Microbiol Infect, 2016)

• Due to low sensitivity of toxin EIA, it may miss some patients with CDI, so 2

step algorithm is a compromise using NAAT to detect toxin gene in patients

with high enough bacterial burden to be GDH positive.

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More severe disease, longer duration of

diarrhea, more infection-related complications,

increased mortality.

No statistical difference

Impact of 2-step algorithm

• Clinical: • Avoid overdiagnosis/overtreatment of C. difficile carriers or at least

those with less severe disease.

• Financial: • Cheaper for lab, cheaper for patients

• With 90% of patients covered by the cheaper assay and only 10%

of patients requiring both tests, the average test charge per patient

would drop from $119 to $45. (SHGR does ~5,000 tests/yr)

• Reduced CMS penalty?

• Lab workflow • Slightly more complicated, batched testing required

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C. difficile test distribution

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Fre

quency

Hour of Day

C. difficile PCR

Two weeks (2/27/17 - 3/12/17)

Frequency Distribution of "Received in Lab" Times

Cerner result display

• Test change: March, 2017

• We chose not to display GDH antigen result in an attempt

to avoid confusion with result interpretation.

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GDH/toxin

+/+

-/-

+/- - See PCR result

SH Regional C. difficile testing

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Elsewhere: NAAT

Cepheid

illumigene

Great Basin

3 sites have incorporated

EIA molecular testing

12 hospitals in 9 cities

Conclusions

• CDI is a clinical diagnosis, aided by lab results.

• “Treat the patient, not the test.”

• Pre-analytical patient selection is more important than test method.

• Study: GDH performed well as a screening target (high NPV) and the

high specificity of toxin EIA obviates need for molecular confirmatory

testing. However, labs must maintain NAAT assay for specimens

with discrepant EIA results.

• Algorithm adds complexity, reduces overall cost.

• The decision to use either standalone NAAT or 2-step algorithm must

be made at the institutional level. Both approaches have pros/cons

and are supported by current guidelines.

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