1. Clostridium difficile: Shifting Sands of a Pesky Pathogen
Bob Fader, Ph.D. D (ABMM)
Section Chief, Microbiology
Scott & White Memorial Hospital
Temple, TX

2. Objectives
Identify the reasons for the increase and severity of Clostridium difficile diarrhea
List the methods of lab testing for C. difficile and discuss the pros and cons of each method
Describe treatment modalities for C. difficile disease
Discuss Infection Control processes required for containment of C. difficile in the health care setting

4. Clostridium difficile – The Basics
Anaerobic Gram positive bacillus
Spore-former
Normal bowel flora in 2 - 5% of population
Up to 20% in LTC facilities
Infection most often associated with prior antibiotic therapy
Originally strictly a hospital-acquired infection but now can also be seen in outpatients as well

5. C. difficile – Advanced Info
C. difficile diarrhea is a toxin-mediated disease
Large increase in number and severity of cases seen since 2004
Largely due to strain with deletion in regulatory gene for toxin production
Results in 20-fold increase in toxin production
Increasing number of strains also resistant to the fluoroquinolones

6. APIC C. difficile Survey National Prevalence Study of Clostridium difficile in U.S. Healthcare Facilities
Health care institutions requested to collect data on C. difficile on any one day (May-August 2008)
648 facilities
13 of every 1000 pts either infected or colonized
times higher than previous estimates

7. So what happened?
Appearance of a new strain of C. difficile in hospitals in Quebec
Quickly spread south to the U.S. and over to Europe
Strain is known as NAP1 (toxinotype III)

8. Spread of NAP1 strain of C. difficile

9. Deaths in the UK caused by C. difficile

11. C. difficile Epidemiology
Normal colon
Pseudo membranous colitis

12. C. difficile Toxin Genes

13. Increased Toxin A Production in vitro
In vitro production of toxins A and B by C. difficile isolates. Median concentration and IQRs are shown. C. difficile strains included 25 toxinotype 0 and 15 NAP1/027 strains (toxinotype III) from various locations.
Approximately 16 fold increased toxin A production by the epidemic strain
From Warny M, et al. Lancet. 2005;366:

14. Increased Toxin B Production in vitro
In vitro production of toxins A and B by C. difficile isolates. Median concentration and IQRs are shown. C. difficile strains included 25 toxinotype 0 and 15 NAP1/027 strains (toxinotype III) from various locations.
Approximately 23 fold increase in toxin B production
From Warny M, et al. Lancet. 2005;366:

15. Comparison of Molecular Characteristics of 2 C
Comparison of Molecular Characteristics of 2 C. difficile Isolates with Historical Standard-Type Strains and a Recently Recognized Epidemic Strain, by Selected Characteristics, OH and PA, 2005
Characteristic
Standard Strain
Epidemic Strain
Ohio Strain
Pennsylvania Strain
Toxinotype
PFGE* pattern
Binary toxin
18 bp deletion in tcdC
< 80% related to NAP1† _
III
NAP1 + IX
85% related to NAP1
XIV/XV
64% related to NAP1
*Pulsed-field gel electrophoresis.
† North American pulsed-field type 1.
McDonald LC, Killgore GE, Thompson A, Owens RC Jr, Kazakova SV, Sambol SP, Johnson S, Gerding DN. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med. 2005;353:
CDC. MMWR. 2005;54:

16. Resistance of Current (after 2000) BI/NAP1 Isolates to Clindamycin and Fluoroquinolones Compared with Current Non-BI/NAP1 Isolates and Historic (before 2001) BI/NAP1 Isolates
No. (%)
Intermediate or
Resistant to:
Current
BI/NAP1
Isolates
n=24 (%)
non-
P-Value for
vs. Non-
Historic
n=14 (%)
Current vs.
Clindamycin
19 (79)
1.0
10 (71)
0.7
Levofloxacin
24 (100)
23 (96)
14 (100)
Gatifloxacin
10 (42)
<0.001
Moxifloxacin
From McDonald LC, et al. N Engl J Med. 2005;353:

17. C. difficile-associated diseases (CDAD)
Antibiotic-associated diarrhea
Pseudomembranous colitis
Toxic megacolon
Perforations of the colon
Sepsis
Death (rarely)

19. C. difficile Clinical Symptoms
Watery diarrhea
Fever
Loss of appetite
Nausea
Abdominal pain/tenderness

20. Risk factors for C. difficile
Prior antibiotic exposure
GI surgery/endoscopy
Long LOS in health care setting
Serious underlying illness
Immunocompromising conditions
Advanced age
Proton pump inhibitors?

21. Changes in age-specific C. difficile associated disease 2000-2005
Zilberberg, M.D. et al. Increase in Adult Clostridium difficile-related hospitalizations and case-fatality rate, United States, Emerg Infect Dis 2008 Vol 14 No 6

22. Stomach Acid-Suppressing Medications and Community-Acquired CDAD, England
From Dial S, et al. JAMA. 2005;294:

23. Severe CDAD in Populations Previously at Low Risk—Four States, 2005
Recent reports to the Pennsylvania Department of Health and CDC
Young patients without serious underlying disease
C. difficile toxin-positive by routine diagnostic testing
Responded to CDAD-specific therapy
Peripartum
Within 4 weeks of delivery
Reports from PA, NJ, OH, and NH
Community-associated
No hospital exposure in prior 3 months
Reports from Philadelphia and 4 surrounding counties
CDC. MMWR. 2005;54:

24. Severe CDAD in Populations Previously at Low Risk—Four States, 2005 (2)
Characteristic,
No. (%)
Community
(N=23)
Peripartum
(N=10)
Total
(N=33)
Aged < 18 years
11 (48)
0 (0)
11 (33)
Female
15 (65)
10 (100)
25 (76)
Antimicrobial exposure
9 (90)
24 (73)
Bloody diarrhea
6 (26)
2 (20)
8 (24)
Hospitalization necessary
4 (40)
10 (24)
ER visit necessary
3 (13)
5 (15)
Relapse
8 (35)
5 (50)
13 (39)
CDC. MMWR. 2005;54:

25. Severe CDAD in Populations Previously at Low Risk—Four States, 2005
Transmission to close contacts in 4 cases
8 cases without antimicrobial exposure
5 children; 3 required hospitalization
3 had close contact with diarrheal illness
Another 3 cases with < 3 doses of antimicrobials
Clindamycin most common exposure (10 cases)
Estimated minimum annual incidence of community-associated disease
7.6 cases per 100,000 population
1 case per 5,000 outpatient antimicrobial prescriptions
CDC. MMWR. 2005;54:

26. Laboratory Diagnosis of C
Laboratory Diagnosis of C. difficile “A disease in search of a good diagnostic test”

27. C. difficile - Lab Testing General Caveats
Lab testing should only be performed on diarrheal stools (conform to the shape of the container)
One or two specimens collected on consecutive days are sufficient for diagnosis
No more than one specimen per day
Swab specimens are insufficient
Once pt positive, no further testing for at least 2 weeks **No “Test of Cure”**

28. C. difficile - Lab testing Options
Culture on CCFA agar (Cycloserine Cefoxitin Fructose agar)
Most sensitive and specific
If isolated still need to confirm toxin production
72-96 hour turn-around-time
Requires anaerobic culture

29. C. difficile – Lab Testing continued
Latex agglutination –
originally marketed as toxin assay
now known to detect glutamate dehydrogenase enzyme
present in C. difficile but also in other organisms (C. sordellii)
Same day or next day results
Need to confirm positives with toxin assay

30. C. difficile – Lab Testing continued
Enzyme immunoassays
Detection of Toxin A only
Will not detect ToxA negative/ToxB positive strains
Detection of Toxins A&B
Better sensitivity than toxin A alone
Microwell, lateral flow assay formats
Same day or next day results
Most popular (>90% of labs)
Sensitivities – 65-85%

31. Microwell enzyme immunoassay
Lateral flow EIA

32. C. difficile – Lab Testing continued
Cell culture Cytotoxicity Assay –
Detects Toxin B (Cytotoxin) only
Will not detect ToxA positive/ToxB negative strains
Considered the “gold standard” for toxin testing
Requires tissue culture capability
Costly, slow, and time consuming
24-48 hour turn-around-time

33. C. difficile – Lab Testing continued
Rapid enzyme EIA for glutamate dehydrogenase enzyme
Used as initial screen
If negative you can report out as negative
Positive rapid test requires confirmation by cell culture cytotoxicity assay, EIA or PCR
High specificity for negatives but still cannot provide a rapid result for a positive patient

34. C. difficile – Lab Testing continued
Polymerase chain reaction
BD GeneOhm Cdiff PCR assay has recently been FDA approved
Targets the Toxin B gene
Fresh stool: Sensitivity 93.8%/Specificity 95.5%
Frozen stool: Sensitivity 100%/Specificity 97.7%
Evidence suggests a single negative is enough to rule out infection

35. C. difficile – Lab Testing continued
EIA
PCR
Sens
84%
100%
Spec
97.7%
PPV
94.9%
NPV
92.2%
Polymerase chain reaction for toxin A and toxin B (S&W)
Primers and probes for Toxin A and B
146 specimens
PCR picked up 7 additional positives
(37 vs 44)
Smith, D, Hocker, K, Fader, B, Luna, RA, Versalovic, J, Rao, A. Rapid PCR screening strategy for hospital acquired infections including vancomycin-resistant Enterococci and Clostridium difficile in adult patients. (ASM Annual meeting, 2008)

37. C. difficile Treatment Discontinue current antibiotics
23% cure rate
Antibiotics – metronidazole
oral vancomycin
concern about selection of VRE
20% relapse rate
Continue with metronidazole
2nd relapse – switch to vancomycin

38. C. difficile Treatment continued
“Severe disease”
Zar and CDC criteria
Vancomycin shown superior to metronidazole
Toxic megacolon – direct installation of vancomycin + IV metronidazole

40. C. difficile Treatment continued
Other antibiotics? (rifamixin, nitazoxinide)
Toxin binding compound was in development
Recently stopped study
Monoclonal antibodies
Vaccine in development
Probiotics – Saccharomyces boulardii and Lactobacilli - questionable results
usually in combination with antibiotics

41. Treatment – Stool Transplant
Reserved for severe pseudomembranous colitis and toxic megacolon
Stool donated by healthy volunteer – usually family member
Fresh stool is homogenized, filtered through coffee filter X2
Administered by nasogastric tube or by enema
Usually 5 treatments to help restore normal flora
Recurrent Clostridium difficile Colitis: Case Series Involving 18 Patients Treated with Donor Stool Administered via a Nasogastric Tube. Johannes Aas, Charles E. Gessert, and Johan S. Bakken; Clin. Infect. Dis :

42. Infection Control Recommendations
Conduct surveillance. Track positive numbers and outcomes
Emphasize early diagnosis and treatment to physicians
Enforce strict Infection Control policies
Contact precautions
Hand washing with soap and water as opposed to alcohol-based hand rinse when caring for C. diff positive pts

44. Infection Control Recommendations
Environmental cleaning and disinfection strategy (need sporicidal agent)
Terminal cleaning - Routine cleaning (Sani-Master 4) vs (Dispatch)
SaniMaster 4 – ammonium chloride
Dispatch – sodium hypochlorite
Sani-wipes for equipment and other items
Glo-Germ® to evaluate cleaning

45. The rails are considered a “high touch” area in the patient environment.

46. We have reusable medical equipment that is usually shared between patients. I would have to make the assumption their stethoscope and bp cuff were not cleaned after the last patient.

47. Surveillance - Should we Screen?
Is C. difficile the next organism for active surveillance on inpatient admission?
If so, should you also screen for Vancomycin- Resistant Enterococcus (VRE) with the same specimen?
If nothing else, all positive C. difficile tests from the laboratory should be reported to Infection Control for tracking