C difficile infection and the Laboratory:What, How and When

1
C difficile infection and the LaboratoryWhat,
How and When?

• Mike Wren, Clinical Microbiology, UCL Hospital,
  London

July 09 Lewisham
2
Mostly based on ELISA tests How many severe
needing targeted therapy? How many mild possibly
not needing therapy? 20 recurrence rate ? How
many are true recurrence ? How many re-infections
3
Spectrum of C difficile infection Self-limiting
severe
diarrhoea colitis PMC-toxic MgC
Mild diarrhoea
Antibiotic therapy targeted against C difficile
no
yes yes
yes

surgery
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CDI A diagnostic dilemma

• Patient RL 63 yr old M
• Develops D whilst on ward virus PCR neg day1
• C difficile toxin neg day2
• Day 5 D worsening
• C difficile toxin virus PCR neg Day6
• D worsens colitis suspected
• Day7 CT scan confirms colitis
• Bacterial pathogen culture neg Day9
• C difficile toxin pos Day11

• Faecal GDH pos Day2
• Faecal lactoferrin pos Day2
• Culture pos Day4 (tox isolate)
• GDH positive day5
• C difficile ribotype 027

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Clinical cases of CDI but with a negative faecal
toxin test by 7 ELISA kits
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Faecal cytotoxin said to be Gold Standard BUT No
standard method for cell culture assay
(CCA) Depending on study and method CCA
can Miss up to 30 of CDAD cases In PMC
patients Faecal Cytotoxin only detected 27/56
cases with PMC Johal et al, Gut, 2004 Of the 29
faeces with a negative CCA 9 were culture
positive with toxigenic isolate Still the most
sensitive test for detection of toxin in faeces
Sens Spec PPV
NPV Cytotoxin assay 86 99
86 99 (CEP Evaluation 2009)
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Average figures from literature for commercial
kit performance vs toxigenic culture
Sens Spec PPV
NPV Cytotoxin assay 86 99
86 99 Toxin kits 1 80
97 67 98 2
68 91 33 97
3 60 95 46
97 600 samples 4
80 96 56 98
CEP, Wilcox 5
81 93 43 98
Eastwood 6 74
98 81 98 7
68 93 38 97 GDH
97.3 96.2 73.2
99.7 1500 samples, Wren et al The poor PPVs
of toxin detection kits, especially in the
context of widespread testing, raises doubts
about their appropriateness when used as single
tests for the laboratory detection of C difficile
toxins. (NHS CEP Evaluation Report, 2009)
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Faecal Lactoferrin Does it have a
role? Lactoferrin (not specific for
CD) Indicator of intestinal inflammation (key
role in how quickly the disease progresses to
colitis) High levels in patients with
severe/advanced CDAD compared to mild or no
disease Lactoferrin levels in faeces rise
significantly in patients with advanced CDI
compared to those with mild disease association
is statistically significant
LF level
Normal threshold
Asymptomatic Mild
Severe colitis
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Severity score applied to diarrhoeal patients
suspected of CDI
Patients with gt2 points considered to have severe
CDI Zar, FA et al. CID 2007 45
302-307 Patients with scores of 3 or more have a
positive lactoferrin (value of quantifying
lactoferrin vs mortality?)
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• Diagnostic challenges
• Patient with diarrhoea but only colonised with a
  tox isolate (eg. Patient on naso-gastric feeding
  or on laxatives) but may have high faecal toxin
  levels RISK OVER TREATMENT Increases
  susceptibility to CDI
• Colonisation rates can be high
• 2. Patients with clinically severe CDI but
  negative faecal toxin tests.
• RISK NO / UNDER TREATMENT may go on
  to develop colitis
• Development of colitis can be rapid
• 3. Patients with recurrence of symptoms
• a) Relapse of
  original infection?
• b) Re-infection
  with new strain?

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Clinical cases of CDI but with a negative faecal
toxin test by 7 ELISA kits
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• Delayed toxin positivity ?
• Slow production of toxins responses to
  environmental factors?
• Initial absorption of toxin by colonic receptors?
• Infection with a different strain to that
  initially acquired?
• Poor sensitivity of testing methods
• Upregulation of toxins following commencement of
  specific antibiotic therapy? (definitely happens
  in vitro)

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The laboratory diagnosis of CDI The future
direction ? Current tests available
Associated Pros
Cons Cytotoxin detection
Most sens spec
test for

toxin So-called Gold Std

Cell line maintenance

Technical
knowhow / cost

Time to positivity ELISA tests for toxin
Quick
/ convenient /

relatively cheap Now Std

in most laboratories

Insensitive / False ves C
difficile antigen (GDH)
Good sensitivity/ very high

NPV / closely
IIs culture

Independent of toxigenicity Toxigenic culture

Excellent sensitivity

Time to positivity / need to

check toxigenicity of
isolate PCR
Excellent
sensitivty / quick

High NPV / Cost
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What test and when? GDH? Cytotoxin by cell
line? Toxin by ELISA?
And in what
combination? Presence of toxin genes by
PCR? Toxigenic culture? 1. Where lies the
usefulness of PCR? 2. Role of a marker for
intestinal inflammation (FLF)? 3. How does FLF
relate to predicted severity?
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Screen with GDH
(1500) (Se 97.3, Sp 96.2, PPV 73.2, NPV
99.7) Negative (1306)
Positive (194) 8 cult
2t Report No C difficile
Toxin AB Test
Negative (117)
Positive (77)
Tox Culture Report C
difficile
Lactoferrin (69 lactoferrin
ve) Both Neg(50) Both
Pos(23) TC/LF-(42) TC-/LF(2)
Report No CDI CDI
CDI Other IBD (Crohns Campy)
? Colonised All had severe diarrhoea
?colonised
60 with colitis

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Summary of recent GDH studies
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Utility of PCR?
GDH screen
Toxin AB (92) C diff complete
Both ve(15) GDH ve
GDH ve Both -ve
Toxin AB ve(5)
Toxin AB ve(0) (72)

Rarely occurs Report
PCR PCR
Report C difficile Pos
Tox culture Tox culture No C
difficile Culture for epidemiology

OR save
stool at -20C
Report accordingly
PCR15 PCR2

PCR1 tCult15
tCult2
tCult1
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Swindells et al, ECCMID 2009 Cairns and Wren,
2009
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Swindells et al, ECCMID 2009
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• Clostridium difficile the future
• 1. Need a robust laboratory testing algorithm
• What tests and when to do them.
• 
  GDH as screening test
• 
  What next? CTA EIA Tox cult PCR
• 2. Commercial CTA soon will undergo testing
  in near future
• 3. Does Lactoferrin have a role?
• 
  In predicting severity of outcome?
• 
  How does it relate to severity scoring?
• 4. Why do some patients (especially with
  severe disease) give negative
• toxin results?
• Just test
  sensitivity issues? Patient gut related?
• 5. High severity not just associated with Rt
  027.

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Human foreskin fibroblasts
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The Future? Screen with GDH
Positive (All studies
show 10-12 ve) Negative
Second line confirmation
?? No further action Cytotoxin
assay PCR Toxigenic culture
Technically
demanding V. expensive
Easy, but takes up to 4-5days
but will
possibly be
available
commercially The poor PPVs of toxin detection
kits, especially in the context of widespread
testing, raises doubts about their
appropriateness when used as single tests for the
laboratory detection of C difficile toxins. (NHS
CEP Evaluation Report, 2009)
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Alternative algorithms GDH
GDH
GDH -
- -
No CDI CTA
No CDI Tox Cult No CDI
PCR -
-
- CDI
CDI
CDI Tox Cult
TCBroth
Positive takes 24hr
Positive takes 48-96hr Positive
takes 2-3hr or 72-120hr
Easiest to do
Most expensive to do commercial OR
2hr if ELISA ve Most
sensitive of the Could be longest to do OR
routine options shortest to do but least
sensitive