Changing aspects of Clostridium difficile infection

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Changing aspects of Clostridium difficile
infection

• Elisabeth Nagy MD. PhD. DSc.
• Institute of Clinical Microbiology, Faculty of
  Medicine, University of Szeged, Hungary
• 5th ESCMID School
• Santander, 10-16 June, 2006

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History of the detection of C. difficile

• 1893 Finney first description of PMC
• 1935 Hall first isolation of C. difficile
• 1943 Hamber AAD in guinea pigs (penicillin)
• 1974 Tadesco first epidemie of AAD after
  clindamycin treatment
• 1974 Hafiz isolation of C. difficile from
  environmental samples
• 1974 the supernatant of the faces samples
• had cytotoxic effect (virus infection?)
• 1978 Bartlett the toxin-producing C. difficile
  is an enteric pathogen
• 1980- accepted as nosocomial enteric pathogen
• Today C. difficile associated diarrhea (CDAD)

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Changing aspect of C. difficile infections

• According to recent data
• Antibiotics as predisposing factor for CDAD is
  overestimated (USA 30 of CDAD cases had no
  previous antibiotics)
• Community acquired CDAD is increasingly
  recognized (UK CDAD diagnosed by GPs increased
  from 1 to 22 cases/100,000 between 1994 and
  2004)
• The assumption that C. difficle is not pathogenic
  for neonates and children needs reconsideration
  (because no good evidences support it)

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Biological characteristics of C. difficile

• Gram-positive bacilli
• 0,5-1,9 µm wide, 3-16 µm long
• Motil rods
• Subterminal, terminal spores
• Sacharolytic and proteolytic activity,
• Strict anaerobes they do not produce catalase
  and oxidase
• GLC isocapronic acid, acetic acid isobutyric
  acid, and butyric acid

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Pathogenic factors I.

• Adhesion
• Chemotaxis
• S-layer
• Enzymes hyaluronidase, collagenase
• Toxins
• Toxin A
• Toxin B
• Actin-specific ADP-ribozyl-transferase (binary
  toxin)
• High molecular weight protein?
• Second unstable enterotoxin?

M. Cerquetti et al., (2002) FEMS Immun Med
Microbiol
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Pathogenic factors II.
C. P.

• S-layer glycoprotein
• low-MW SLP 32-38 kDa
• variabile
• high-MW SLP 42-48 kDa
• amidase activity
• virulence factor defense
• against phagocytosis
• heterogenity defense

high-MW SLP
low-MW SLP
S-layer protein polyacrilamide gel-electrophoresis
(Terhes G. et al. 2006))
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Pathogenic factors III. (C. difficile toxins)

• Toxin A and B
• TCDa-308 kDa
• TCDb-270 kDa
• Similar structure with gt45 homology on aminoacid
  level
• Receptor
• TCDa- Gala1-3-Galß1-4-GlcNAc
• TCDb-unknown
• Target Rho protein - desintegration of the
  cytosceleton

• Binary toxin
• (actin specific ADP-ribosyltransferase)
• CDTa-enzimatic domain-48 kDa
• CDTb-binding domain-94 kDa
• Receptor unknown
• Target actin

CDTa
CDTb
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The activity of A and B toxins on the intestinal
epithelial cells
Toxin A
Receptor
Toxin B
Receptor?
Fluid secretion
Cytotoxic effect
Rho glycolisation
Lamina propria
monocyta
TNF
Neutrophil chemotaxis
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C. difficile binary toxin(actin-specific
ADP-ribozyl-transferase)

• Similar to other Clostridium binary toxins
• cdtA, cdtB are chromosomal genes, located
  outside of the Pa-Loc region
• Cytotoxic effect on Vero cell-line
  (disintegration of the actin cytoskeleton)
• Two proteins CDTa and CDTb
• CDTa enzymatic function
• CDTb receptor binding

CDTa
CDTb
Actin cytoskeleton desorganisation
?
translocation
ADP-R
actin
actin
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Pa-Loc (Pathogenecity Locus) of C. difficile
2065 bp 791 bp 2796 bp
1843 bp 846 bp 2511 bp
2583 bp 2160 bp 1765 bp
1ncI 1ncI 1ncI
Pst1 Pst1
Pst1 Pst1
B-u1-b
3pB-d ACU-2

PL-d
5 tcdD
tcdB tcdE
tcdA
tcdC 3
Deletion (1.6-6kb)
Toxin A detection by using toxin A ELISA
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B2
A2


2,0 kb
2,0 kb

B1
B3
A1
A3

3,1 kb
2,0 kb
3,1 kb
3,1 kb
R tcd D tcdB
tcdE tcdA
tcdC
PaLoc
To day 24 different toxinotypes
M. Rupnik JCM 1998
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?
Schroeder et al. AFP (2005) 71 921-928
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S. M. Poutanen et al. CMAJ (2004) 171 51-58
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C. difficile asymptomatic carriage

• Asymptomatic carriage in human1
• Healthy adults 3-8 (0.3 - Europe, 15 - Japan)
• Infants 15-63 (colonization starts during the
  1st-2nd week)
• During hospital stay 20 (30) and it is about
  2-5x higher than the number of patients with
  symptoms
• After antibiotic usage 46
• Transient colonization or component of the
  stable flora? (Kato et al, 2001, JMM)
• Extra-intestinal carriages not known (in
  pregnant women the vaginal carriage rate is
  lt18)2
• Asymptomatic carriage in domestic animals
  20-40 (zoonozis???)3

1 Spencer (1998) JAC 41, Suppl. C, 5 2 McFarland
et al., (1999) AJIC 27 301 3 Arroyo et al.,
(2005) JMM 54 163
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Clinical syndromes caused by C. difficile

• Pseudomembranous colitis (PMC) 90-95
• Antibiotic associated colitis (AAC) 60-75
• Antibiotic-associated diarrhoea (AAD) 11-33
• Extraintestinal infections
• abscesses, peritonitis polymicrobial pelvic
  anaerobic infections, reactive arthritis, sepsis
  etc.

Colonoscopic picture of differet forms of
C. difficile associated disease (CDAD)
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Antibiotics and anti-cancer substances frequently
associated with C. difficile infection
Frequently Less frequently Cephalosporine
s Tricarcillin-clavulanic acid Ampicillin,
amoxicillin Chloramphenicole Other
beta-lactams Amphotericin B Clindamycin Metro
nidazole Erythromycin other macrolides Rifampici
n Tetracycline 5-Fluorouracil Trimethoprime-
sulfamethoxasole Methotrexate Doxorubicin
Aminoglycosids
Wilcox et al., (2003) Best Pract Res Clin
Gastroenter 17 475-493.
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Predisposing factors of C. difficile caused
infections

• Advanced age
• Prolonged hospital stay
• Immunsuppression, underlying sever diseases
• Antibiotic or anti-cancer treatment
• Intra-abdominal surgery
• Others
• Healthcare workers
• Cross-transmission between neonates and mothers

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C. difficile infection in health care workers
(Arfons et al., (2005) CID 40 1384-1385)
.
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Cross-transmission between neonates and mothers
recurrent C. difficile infections
(McFarland et al., (1999) AJIC 27 301-303.)
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Emergence C. difficile PCR ribotype 027 (NAP1) in
Canada/USA and Europe

• Since March 2003, increased rates of CDAD
  reported in Canada and USA
• Special features of these cases
• More sever course
• Higher mortality
• More complications
• Increased virulence is due to
• Higher amount of toxin production
• Fluoroquinolon resistance
• Typing methods showed
• ribotype 027
• Toxinotype III
• PFGE NAP1

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Emergence C. difficile PCR ribotype 027 (NAP1) in
Canada/USA and Europe

• 2004 Québec (Canada) in 12 hospitals
• 1703 patients with 1719 episodes of CDAD was
  observed.
• 22.5 cases/1,000 admissions
• 6.9 mortality
• incidence and mortality increased with the age
• 2005 January, 30 hospitals report showed a
• prevalence of CDAD was five fold higher than the
  historical average
• 2000 and 2003 USA
• Increased number of CDAD cases in 11 states
• Reports on sever CA cases in low prevalence
  population

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Emergence C. difficile PCR ribotype 027 (NAP1) in
Canada/USA and Europe

• 027 ribotype was recognized in
• 44 hospitals in England
• 8 hospitals in the Netherlands
• 6 hospitals in Belgium
• Outbreaks were difficult to control
• Correlation with the use of fluoroquinolons and
  cephalosporines in the hospitals
• According to these information and surveillance
  data ECDC start to deal with the situation as
  emerging threat

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Characteristics of the newly emerged epidemic C.
difficile strain

• Different typing methods confirmed the clonality
  PCR ribotype 027, PFGE type NAP1 and toxinotype
  III.
• A and B toxin positive (over production of the
  toxin)
• Binary toxin positive
• By molecular methods a 18 bp deletion in tcdC
  region was shown. (TcdC is considered as a
  negative regulator of the production of toxin A
  and B).
• Sporulation of the epidemic strain is more
  intensive than other C. difficile strains (ICCAC
  2005) and it was increased by non-chlorine-based
  cleaning agents

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Problems in diagnosing C. difficile infection

• What kind of diagnostic methods should be used?
• Isolation on CCFA agar (Oxoid) toxin detection
  from the supernatant of the isolate

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Colonies of C. difficile on anaerobic Brucella
agar, incubation time 48 hours
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Colonies of C. difficile on anaerobic Brucella
agar, incubation time 48 hours
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Problems in diagnosing C. difficile infection

• What kind of diagnostic methods should be used?
• Direct toxin detection from the faces (ELISA, IC,
  cell culture)

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Survey in Europe(F. Barbut and ESGCD CMI 2003)
control

• Eight countries
• 212 questionnaires from different laboratories
  (87.7 perform C. difficile diagnosis routinely)
• Methods used Percentage
• Toxin detection 93
• Cytotoxin assay 17
• Different EIAs 79
• Culture 55
• GDH detection 5.9

positive
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Percentage of laboratories performing culturing
or direct toxin detection in the different
countries
No accepted strategies in Europe
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Clinicians do not think on C. difficile diarrhoea
(Urbán E., et al. 2002)

• 1200-bed university hospital
• Study period 7 months in 2000
• The study was based on direct detection of toxin
  A by EIA and toxin A/B by cytotoxin assay
• No. of samples / positive
• All stool samples /
• cultures positive for other bacteria
  1804 / 49 (2.7)
• CD toxin test requested by the clinicians 150
  / 35 (23)
• CD toxin tested according to specific criteria
  318 / 71 (22)

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Laboratory reports of culture and toxin detection
of C. difficile in stool specimens in
England and Wales increased considerably between
1984 and 1997
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Prevalence of nosocomial diarrhea caused by
C. difficile in Eastern Europe

• Few studies !!
• Poland
• Martirosia G. et al 1993 Less than 10 in a
  surgical department.
• Pituch H. 2002 1200-bed hospital 32 of stool
  specimens positive.
• Hungary
• Urbán et al. 2002 In a 1200-bed university
  hospital 22 of specially selected stool
  specimens positive
• Belorus
• Titov L. et al. 2000 1.8 of stool specimens
  of 509 patients with colitis positive
• From ESGCD data
• Median incidence of CDAD 1.1 for every 1 000
  patients

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Nosocomial epidemices caused by toxin A-/toxin
B C. difficile strains

• Canada (1998 between July and September) 16
  cases, 4 wards, relapse rate 18.75.
• Netherlands (11 month) 24 cases (19 mild, 7
  sever, 1 lethal cases), surgery, relapse rate
  13
• Japan (2001 between February and June) 15 cases,
  oncology ward.
• Argentina (2000-2003) During a 4 year period
  A-/B strains complately replaced A/B strains
  in CDAD nosocomial cases

Toxin A-/B strains cause diagostic problems To
culture the strains is needed also for typing
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Phenotypic methods to type C. difficile

• Antibiotic resistance pattern analysis (Burdon)
• Plasmid analysis
• PAGE of soluble proteins (Wüst)
• Phage and bacteriocin typing (Sell)
• Serotyping (Nakamura, Delmée) (now up to 19
  serogroups)
• Analysis of 35S-methionine-incorpoated cellular
  proteins (Tabaqchali)
• or combined with immunoblotting (Heard)

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Molecular typing methods for C. difficile

• Whole cell DNA REA (HindIII) (Kuijper)
• RFLP Southern blotting
• AP-PCR
• RAPD (Barbut)
• PFGE (SmalI, KspI, SacII or NruI) (Talon)
• PCR ribotyping (Gurtler, ARU, Cardiff) (more
  than 150 different types in the data base)
• Toxinotyping (Rupnik) O XXIV different
  toxinotypes

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Distribution of C. difficile ribotypes among
isolates from our hospital
M 018 012 150 018 002 014 M
002 018 161 018 018 001 M
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Therapy

• Stop antibiotic usage, if possible
• Metronidazole 250-500 mg 4x/day p.o. (7-14 days)
• Vancomycin 125-500 mg 4x/day p.o. (5-14 days)
• Saccharomyces boulardii, Lactobacillius GG
• Adsorbents cholestyramine, colestipol, tolevamer
• Immune WPC-40/MucoMilk (Holstein-Frisian cows are
  immunized by inactivated C. difficile toxin and
  killed bacteria. The protein component of the
  milk contains toxin A/B antibodies
• Recurrences
• 20 (out of this multiple recurrences
  50-65)

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Case definition for CDAD

• No real consensus exists on case definition to
  day.
• This is a suggestion of ESGCD and ECDC
• Criterion 1
• Diarrheal stools or toxic megacolon AND
• A positive assay for C. difficile toxin (direct
  EIA for A or B toxin or neutralized cytotoxic
  test, OR a positive stool culture with toxigenic
  strain
• Criterion 2
• PMC observed by endoscope
• Criterion 3
• colonic histopathology characteristics for C.
  difficile infection on a specimen taken during
  endoscopy, colectomy or autopsy

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Case definition for CDAD (cont.)

• Excluded from the above definition
• Asymptomatic patients with a positive stool
  culture for C. difficile
• Asymptomatic patients with a positive assay for
  C. difficile toxin
• Neonates
• Diarrhea with an other cause
• Recurrences

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Case definition for CDAD (cont.)

• Definition of CDAD recurrence
• Two episodes of CDAD in the same patient gt2 month
  apart is distinct event
• An episode that occurs within 2 months is
  considered to be a recurrence of the initial one.
• Relapse or re-infection is difficult to
  distinguish even in the case of typing as more
  ribotypes can be harbored in the same time.

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Case definition for CDAD (cont.)

• The origin of CDAD can be defined
• Healthcare-associated, nosocomial the onset of
  the symptoms occurs at least 48 hours after
  admission or within 4 weeks after discharge.
• Healthcare-associated, imported case CDAD in
  hospitalized patient within 48 hours after
  admission or in outpatient AND a history of
  previous hospitalization or ambulatory care (e.g.
  dialysis etc.) within 4 weeks
• Community-acquired CDAD in outpatient or in
  hospitalized patient within 48 hours after
  admission AND no history of previous
  hospitalization or ambulatory care (e.g. dialysis
  etc.) within 4 weeks

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Guideline to diagnosis of C. difficile
diarrhea(American Collage of Gastroenterology
1997)

• CDAD should be suspected
• in patients received antibiotics within 2 month,
• whose diarrhea began 72 hours or more after
  hospitalization
• stool specimen should be tested for the presence
  of C. difficile and/or its toxins
• Asymptomatic patient
• Investigation should be carried out only for
  epidemiological purposes
• lt1 year
• Diarrhea usually does not correlate with the
  presence of C. difficile or its toxins

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Guideline to diagnosis of C. difficile diarrhea
(American Collage of Gastroenterology 1997)

• When symptomes of CDAD is suspected
• One stool sample should be sent to the laboratory
  (if the result negative and the symptoms persist
  1-2 further sample should be tested)
• Stool culture is the most sensitive method
• Detection of toxin B in the stool specimen is the
  most specific test (toxin A-negative, toxin
  B-positive strains!)
• EIAs are rapid, but less sensitive and less
  specific

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Practical guidelines for the prevention and
control of C. difficile infection
(American Collage of Gastroenterology 1997)

• Limit the use of antimicrobial drugs
• Wash hands between contact with all patients
• In the case of C. difficile carrier prevent
  spread of stool specimen
• Wear gloves when in contact with patients who
  have C. difficile diarrhea
• Disinfect objects contaminated with C. difficile
  with sodium hypochlorite, alkaline glutaraldehyde
  or ethylene oxide
• Educate medical staff about the disease and its
  epidemiology