Transcript CLOSTRIDIUM DIFFICILE

CLOSTRIDIUM DIFFICILE
A Disease of the Antibiotic Era
Resident Lecture
2010
Objectives
Learn the Microbiology of C. difficile
Understand the Pathogenesis of Disease
Introduction to the Epidemiology of
Infection by this organism
Overview of Advances in Diagnostic
Testing
A 75-year-old man with peripheral vascular disease, chronic obstructive pulmonary disease, and
diabetes mellitus presented with gangrene of the right fourth toe and underwent tarsalmetatarsal amputation to manage the progression of gangrene, followed by amputation of the
right leg below the knee
Wolf, P. L. et al. N Engl J Med 2005;353:2491
C. difficile
Strict anaerobe
Gram variable rod
Subterminal elongated spores
Irregular, flat, rough colonies
Fluoresce chartreuse
Isolation Media
CCFA
–
–
–
–
–
2.5% egg yolk
Fructose
Neutral red
8-16 ugm/L cefoxitin
250-500 mgm/L cycloserine
BA with lysed horse blood
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–
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Opaque grey-white colonies
Larger than when egg yolk is used
Fluoresce a green-yellow colour
TCCFA
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–
0.1% Na taurocholate
Enhances sporulation
CDMN Selective Agar
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Cysteine hydrochloride
Norfloxacin
Moxalactam
C. difficile grows better
Fecal Flora more inhibited
May eliminate the need for a selective broth
Buchanen’s broth
–
–
–
CMC
Cefoxitin
Cycloserine
C. difficile Laboratory Identification
GLC
Acetic Acid
Butyric Acid
Isobutyric Acid
Isovaleric Acid
Isocaproic Acid
C. difficile Laboratory Identification
Biochemicals
C. difficile
C. sporogenes
Glucose
+W
V
Mannitol
WA
-
Milk
-
D (digested)
Neya
Lecithinase
Lipase
-
-+
-
+
Fructose
AW
-W
C. Difficile –Rapid ID
RapID ANA
– 4 hour enzyme detection system
– 10 reaction cavities
– 18 tests
Latex Agglutination
Toxins Produced by C. difficile
Toxin A
Toxin B
Molecular Wt.
550,000
360,000
Stability (-200C. –
370C.)
4 weeks
4 weeks
Stability (560C)
<6 minutes
<6 minutes
Cytotoxicity (CHO
cells)
3.8 X 106 cu/mgm.
5.3 X 109 cu/
mgm.
Fluid accumulation in
infant mice
> 0.25 ugm.
> 2.5 ugm.
Death of infant mice
None at 5 ugm.
50% at 1-5 ugm.
Rabbit ileal loop
Viscous hemorrhagic
fluid response
Variable fluid
response
C. difficile Pathogenicity Locus
Clinical Microbiology
Reviews 18: 247-263
Clinical Microbiology Reviews 18: 247-263
TcdA and TcdB
Glucosyltransferases
Inactivate Rho, Rac and Cdc42
Actin condensation
Rounding of cells
Membrane blebbing
Apoptosis
Virulence I
Bind to specific receptors on the colonic epithelium
Transported into the cytoplasm
Both toxins inactivate Rho proteins
– GTP-binding proteins important in actin polymerization,
cytoskeletal architecture and cell movement
Disruption of the actin cytoskeleton
Disruption of epithelial cell tight-junction proteins
Defective pathogenicity locus may cause disease
– 22 Toxinotypes to date
Virluence II
Production of inflammatory mediators
– Macrophage inflammatory protein-2 (MIP-2)
– Activate sensory nerves
– Sensory nerves release pro-inflammatory
neuropeptides
Substance P and CGRP
Stimulate inflammatory cells to release proinflammatory cytokines
Neutrophil recruitment
Clinical Microbiology Reviews 18: 247-263
Binary Toxin
Encoded by CDTa and CDTb
2 component ADP-ribosyltransferase activity
>80% homology with C. perfringens iota toxin
In 6.4% of isolates from the UK
Due to genetic exchanges among several
clostridial species
Appears to enhance pathogenicity of NAP 1
C. Difficile Toxin Assay
W. I. 38 cells
Stool in MEM with antibiotics
12,800 g for 10 minutes
0.2 ml. on tissue culture
C. difficile antitoxin
Epidemiology of C. difficile
Normal fecal flora of newborns
2% of healthy adults
10-20% of elderly individuals colonized
20-40% of hospitalized patients colonized
Nosocomial clusters
Isolated from toilets, bedpans, floors
Spores isolated for up to 6 months
Consequences of Ingestion of C.
difficile
Excretion
Asymptomatic colonization of gut
Diarrhoea
Colitis
Pseudomembranous colitis
Death
Antibiotic Associated Colitis
3-5 loose stools per day
No alternative cause
Onset during or after antimicrobial
administration
– Erythromycin stimulates gut peristalsis
Wide specturm
Pseudomembranous colitis
Loose watery stools
8-11 days
Prolonged if
– Onset after antibiotics stopped
– Antibiotics continued after onset
C. Difficile Infection
CDI
Diagnosis must be confirmed by laboratory
tests
Spectrum the same as AAD
Not all AAD is CDI
Clinical Presentation
Diarrhea within a few days of antibiotic therapy
Faeces have a distinctive foul odour
Abdominal pain +/- pyrexia
Blood if pseudomembranous colitis
Electrolyte disturbances
Hyoalbuminaemia
Paralytic ileus
Toxic Megacolon/perforation/shock
Increased WBC
PMC Microscopic Appearance
PMC Low Power Microscopic
Other Organisms Implicated in AAD
Enterotoxigenic Clostridium perfringens
– 8% of cases in one study (Wilcox et al)
Staphylococcus aureus
– 1/6 cases (Graves et al)
– 92% MRSA
– 9 bacteraemic
– Fluroquinolones involved
– Leukotoxins and Staphylococcal enterotoxin A
Antibiotic-related risk of C. difficile
Infection
High Risk
Medium Risk
Low Risk
Cephalosporins
Macrolides
Aminoglycosides
Clindamycin
Co-trimoxazole
Metronidazole
Amipicillin/
amoxycillin
Fluoroquinolones
Tetracyclines
Anti-pseudomonal
penicillins
Rifampicin
Vancomycin
Antibiotic Risk Determinants
Frequency of use
Age
Environmental contamination
Association with an outbreak
Combination of antimicrobials
Duration of therapy
– <3 days
– Surgical prophylaxis
ANTIBIOTIC THERAPY
↓
ALTERATION OF COLONIC MICROFLORA
↓
C. DIFFICILE EXPOSURE AND COLONIZATION
↓
RELEASE OF TOXINS A AND B
↓
BINDING TO ENTEROCYTE RECEPTORS
↓
COLONIC MUCOSAL INJURY AND ACUTE INFLAMMATION
↓
DIARRHEA AND COLITIS
Microorganisms Which Inhibit the
Growth of C. difficile
Lactobacillus species
Group D Streptococci
Staphylococcus aureus
Bacteroides species
Bifidobacteria
Antineoplastic Agents
Direct inflammatory effects of cytotoxic agents
Neutropenia
Agents implicated
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Adriamycin
Cyclophosphamide
5-Fluorouricil
Bleomycin
Melphalan
Dichloromethotrexate
Immune Response
70% of people have antibody
Patients who are colonized on admission are
less likely to develop symptomatic infection
Asymptomatic carriers had greater increases in
serum IgG antitoxin A than those with diarrhoea
Higher levels of serum IgM and IgG antitoxin A in
those with one episode, vs. multiple episodes
Epidemiological Update
Changes in antimicrobial drug use
– Fluroquinolones
Alcohol-based waterless, hand sanitizers
New more virulent strains
– Hyperproduction of toxins A and B
Cut-backs in environmental hygeine
JID 2004:189 (1585-1589)
EID 12: 409-415.2006
EID 12: 409-415.2006
Role of Aging
Increased exposure to healthcare facilities
Increased use of antimicrobials
Use of histamine-2 receptor blockers or
proton-pump inhibitors
Decreased host defenses
– Achlorhydria
– Decreased immune responsiveness
N-CDAD in Canada: The CNISP
Study
19 Health Care Facilities in 8 provinces
Toxin assay or Toxin + culture
Case: Acute onset of loose stools in a
patient that was positive for C. difficile
culture/toxin that persisted for at least 2
days, without an alternative explanation for
diarrhea.
Six continuous weeks/200 consecutive
diarrhea specimens Jan. to April, 1997
CNISP Results
371 Potential Cases
269 met case definition of N-CDAD
13.0% of patients with diarrhea had NCDAD
250 cases occurred >3 days after
admission
19 patients readmitted for diarrhea within 1
month of discharge
Can J Infect Dis 12: 82-88
CNISP Results II
Incidence density was 66.3 cases/100,000
patient days and 5.9 cases/1000 patient
admissions
Mean # of N-CDAD per institution in 6 weeks
was 13.7.
Patients not treated with antibiotics were more
likely to die than those that were treated (OR
2.69)
41 patients with CDAD died (15%)
4 died because of N-CDAD directly or indirectly
Can J Infect Dis 12: 82-88
Fig. 1: Annual incidence (per 100 000 population) of Clostridium difficile-associated
diarrhea (CDAD) in Sherbrooke, Que., 1991-2003
Pepin, J. et al. CMAJ 2004;171:466-472
Copyright ©2004 CMA Media Inc. or its licensors
Table 1.
Pepin, J. et al. CMAJ 2004;171:466-472
Copyright ©2004 CMA Media Inc. or its licensors
Fig. 2: Proportions of patients with CDAD by class of antibiotic received in the 2 months
preceding the diagnosis of CDAD, 1991-2003
Pepin, J. et al. CMAJ 2004;171:466-472
Copyright ©2004 CMA Media Inc. or its licensors
Age-Specific Incidence and Mortality Attributed to Clostridium difficile-Associated Diarrhea
Loo, V. G. et al. N Engl J Med 2005;353:2442-2449
Characteristics of the Highly
Virulent Strain
1400 deaths in Quebec since 2003
PFGE: NAP 1 strain
Toxinotype III
Ribotype 027
Partial deletion of the tcdC gene
Levels of Toxins A and B are 16 to 23 times
higher than previous toxinotypes
Produced Binary toxin
Resistant to fluoroquinolones
CNISP Study 2004-5
Incidence 6 per 1000 hospital admissions
NAP 1 strain preset in 8 provinces
Mortality rate rose from 1.5% to 5.8%
since 1997
NAP 1 strain 2.3 times more likely to have
a serious outcome
Fig. 1: Case-fatality rates among patients with Clostridium difficile-associated disease
(CDAD) from Nov
Eggertson, L. CMAJ 2006;174:607-a-608-a
Copyright ©2006 CMA Media Inc. or its licensors
Diagnosis of C. difficile Associated
Diarrhea
Tissue Culture Cytotoxin Assay the gold
standard
Toxigenic Culture
– 4-6 isolates to CMC
– 24 hr. filtrate for toxin detection
Immunoassay for toxin detection
Glutamate dehrydrogenase detection
PCR
Controversies in Diagnosis
Sensitivity of a single cytotoxin assay 79%
Toxigenic culture may increase sensitivity
to 96%
Toxin positive, culture negative patients
may progress to death
Toxin A negative/B positive patients
Repeat Testing: Is it necessary
Immunoassay Performance vs
Cytotoxicity Assay
Test
Sensitivity
Specificity
Alexon-Trend Prospect II CD Toxin A
82-85
90-100
Bartel Toxin A, Prima
87
98
bioMerieux VIDAS Toxin A II
65-85
99-100
Biosite Triage C. difficile Panel
(antigen or toxin A)
68-100
83-90
Meridian ImmunoCard Toxin A
65-92
95-100
Meridian Premier Cytoclone CD Toxin 77-83
A/B
99-100
Meridian Toxin A
87-94
97-99
Meridian Premier Toxins A and B
84-95
97-00
TechLab/Wampole CD Tox A/B
80-94
99-100
Immunoassay Performance vs
Cytotoxic Culture
Authour
Test
Sensitivity Specificity
Alcala et al
Tox A/B (Remel)
49%
95.8%
Tox A/B (Quick
Chek)
54.9%
95.5%
ImmunoCard A&B
66.7%
95.1%
Ticehurst et al
Tox A/B II EIA 38%
100%
Fenner et al
Tox A/B II EIA 50.7%
99.4%
Commercial PCR Assays
Authour
Test
Sensitivity
Specificity
Stamper
BD GeneOhm
83.6%
98.2%
Kvach
BD GeneOhm
91.4%
100%
Novak-Weekly
Xpert C. difficile
assay
94.4%
96.3%
Swindells et al
BD GeneOhm
100%
97%
Xpert C. difficile
Assay
100%
97%
ProGastro Cd
assay
77.3
94.4
Stamper
Testing Frequency
Aichinger: Patients
with >2 tests in 7 days
– Repeat EIA: Increase
yield by 1.9%
– Repeat PCR:
Increase yield by 1.7%
Cardona: 8,256 tests
from 3, 112 patients
– 49% of tests repeated
Day
% Positive
0
0.9
1
1.8
2
3.8
3
2.6
4-6
5.4
7-10
10.6
Methods to Subtype C. difficile
Restriction Endonuclease Analysis
– Many subtypes
– Outbreaks divided 50/50 between the
outbreak strain and others
– Requires expertise to read the gels
– Time consuming
Methods to Subtype C. difficile
Pulse Field Gel electrophoresis
– Some strains autodigest and not typable using this method
– Thiourea decreases the amount of autodigestion of DNA
AP-PCR
– Non-specific amplifications of segments of the bacterial
chromosome by PCR using short primers under low-stringency
conditions
– Difficulties with reproducibility
PCR-ribotyping
– Specific primer complimentary to the 3’ end of the 16S rRNA
gene and to the 5’ end of the 23 S rRNA gene
– Variable-length intergenic spacer regions of up to 20 16-23 S
rRNA gene pairs
Treatment of C. difficile Associated
Colitis
Discontinue preciptitating antibiotics
Oral Vancomycin 125 mgm qds for 7-10 d
Oral Metronidazole 400-500 mg tds for 710d
IV give both antibiotics together
Recent reports of resistance to
metronidazole
Treatment of C. difficile Associated
Colitis
Relapses common
¼ of symptom-free patients continue to
excrete C. difficile in their faeces
Relapses are often re-infections with
different strains
Treatment of C. difficile Associated
Colitis
S. boulardii
– Evidence mixed
– Cases of fungaemia in immunocompromised
patients
– Virulence differs with the source
Other agents
– Lactobacillus acidophilus
– Lactobacillus GG
– Non-toxigenic C. difficile
Prevention of Infection
Role of restrictive antibiotic policies
Transmission via contaminated hands
– Gloves
– Antiseptic soaps
– Alcohol-based hand rinse
Transmission via the environment
– Role of Na hypochlorite vs. Quaternary
ammonium compounds