1. Evaluation of Various Laboratory Methods for the Diagnosis of Clostridium difficile Infection
Yousun Chung1, Ji Yeon Sung1, Ho Eun Chang2, Soon Hee Choi2, Yun Ji Hong1, Taek Soo Kim1,2, Kyoung Un Park1,2, Junghan Song1,2, Eui Chong Kim1
1Department of Laboratory Medicine, Seoul National University College of Medicine; 2Department of Laboratory Medicine, Seoul National University Bundang Hospital

2. Clostridium difficile Infection (CDI)
BACKGROUND
Clostridium difficile Infection (CDI)
Leading cause of health care-associated diarrhea
Clinical features overlapping with other antibiotic-associated diarrhea
Increase in the number of cases and in the severity
Emergence of hypervirulent toxigenic strain, BI/NAP1/027
Prompt CDI identification to prevent disease progression and
to reduce additional nosocomial cases
Lack of rapid, accurate, and inexpensive diagnostic test

3. Laboratory Diagnosis of CDI
BACKGROUND
Laboratory Diagnosis of CDI
Culture
Labor-intensive, long TAT
Enzyme immunoassay (EIA)
Easy, rapid TAT, variable performance
Cell cytotoxicity neutralization assay (CCNA)
Labor-intensive, long TAT, Expensive cell lines, Interpretation
Molecular-based assays
High sensitivity and specificity, short TAT, Expensive

4. Aims
Compare the performance of different laboratory methods for the diagnosis of CDI
Evaluate real-time PCR assays in diagnosis of CDI

5. Materials and Methods Materials Laboratory diagnostic methods
188 stool specimens from Seoul National University Bundang Hospital
52 C. difficile colonies: randomly selected
Laboratory diagnostic methods
chromID C. difficile (bioMérieux)
Cell cytotoxicity neutralization assay (CCNA)
VIDAS C. difficile Toxin A & B (bioMérieux)
RIDA QUICK C. difficile Toxin A/B (R-Biopharm)
RIDA SCREEN C. difficile Toxin A/B (R-Biopharm)
RIDA GENE C. difficile & Toxin A/B (R-Biopharm)
IMDx C. difficile (Abbott)

6. chromID C. difficile (bioMérieux)
MATERIALS and METHODS
chromID C. difficile (bioMérieux)
Detection of β-glucosidase producing C. difficile strains
Typical grey to black color of colonies
VIDAS C. difficile Toxin A & B (bioMérieux)
Enzyme linked fluorescent immunoassay
Qualitative detection of C. difficile toxin A/B

7. Cell cytotoxicity neutralization assay (CCNA)
MATERIALS and METHODS
Cell cytotoxicity neutralization assay (CCNA)
Inoculation of stool sample onto cultured human fibroblasts
Reading in 2 hrs, 6 hrs, 24hrs, and 48hrs
Cytopathic effect of toxin causes the fibroblasts to round up
Absence of cytopathic effect with the anti-toxin
Considered as a gold standard for C. difficile toxin
Labor-intensive, long TAT, Expensive cell lines, Interpretation

8. RIDASCREEN® C. difficile Toxin A/B (R-Biopharm)
MATERIALS and METHODS
RIDA®QUICK C. difficile Toxin A/B (R-Biopharm)
Immunochromatographic rapid assay
Determination of Toxin A and Toxin B with anti-Toxin A and anti-Toxin B antibodies
RIDASCREEN® C. difficile Toxin A/B (R-Biopharm)
Enzyme immunoassay
Detection of both toxin A/B with monoclonal antibodies

9. RIDA®GENE C. difficile & Toxin A/B (R-Biopharm)
MATERIALS and METHODS
RIDA®GENE C. difficile & Toxin A/B (R-Biopharm)
Real-time PCR
Detection of C. difficile (16S rRNA) and toxin (tcdA/tcdB genes)
IMDx C. difficile (Abbott)
Real-time PCR
Detection of conserved regions of tcdA and tcdB genes

10. CCNA as the gold standard
RESULTS
CCNA as the gold standard
chromID
VIDAS
RIDA
QUICK
SCREEN
GENE
IMDx
Sensitivity
Stool
79.1
74.6
10.4
47.8
89.6
96.9
Colony 75
12.5
59.4
Specificity
23.8 69
100
95.2
76.2
76.9
57.9
94.7
89.5
68.4
63.2
PPV
62.4
79.4
94.1
85.7
87.5 80
90.5
83.8
81.6
NPV
41.7 63
41.2
53.3
82.1
93.8
39.1
56.7
92.9
92.3

11. Real-time PCR assays as the gold standard
RESULTS
Real-time PCR assays as the gold standard
chromID
VIDAS
RIDA
QUICK
SCREEN
CCNA
Sensitivity
Stool
74.3
65.7
10.0
45.7
89.1
Culture
68.4
13.2
55.3
Specificity
15.9
56.8
100.0
95.5
84.2
50.0
92.9
PPV
58.4
70.7
94.1
90.5
78.8
NPV
28.0
51.0
41.1
52.5
82.1
36.8
29.8
43.3

12. Conclusions Enzyme immunoassay methods showed variable performance
VIDAS showed relatively high sensitivity
Real-time PCR methods showed high sensitivity and specificity
Do not detect the presence of toxin itself
Difficult choices to balance test performance and cost
chromID + VIDAS → Molecular assay