What’s New in Poo
A lighthearted review of the use of human feces
for the diagnosis and treatment of human
gastrointestinal illnesses from the tainted
viewpoint of a warped Microbiologist
SOMC Grand Rounds
January 6, 2012
Timothy R. Cassity, Ph. D.
Microbiologist
Sorry Winnie, this presentation is
about poo, not Pooh
Diagnostic Use of Stool
• Symptomatic patients – GI complaints
– Most of related to an infectious etiology
• Asymptomatic patients
– Most of screening for potential colon cancers
Symptomatic
• Most common symptom is diarrhea
– Lower GI tract
• Dyspepsia, pain, etc
– Upper GI tract
What’s not very new
• Routine stool culture
– Useful for Salmonella, Shigella, Campylobacter,
and Escherichia coli O157 infections
What’s not very new
• Routine stool culture
– Misses some strains of enteropathogenic or
enterotoxigenic E. coli.
Campylobacter EIA
• An enzyme immunoassay is being evaluated at
SOMC for the detection of Campylobacter
species.
– Can be done in < 30 minutes (culture takes 2 – 3
days)
– Only need one per patient per illness
– Slightly more sensitive than culture
Shiga-Toxin EIA
• An enzyme immunoassay is
being evaluated for the
detection of shiga toxinproducing strains of E. coli
– Requires 16 – 40 hours (culture
takes 2 – 3 days)
– Only need one per patient per
illness
Shiga-Toxin EIA
• An enzyme immunoassay is being evaluated
for the detection of shiga toxin-producing
strains of E. coli
– Significantly more sensitive than culture
– Detects enterotoxigenic E. coli other than O157
(increases sensitivity about 50%)
What’s not very new
• Routine Ova and Parasite Exam
– Very low yield with immunocompetent patients
with no travel history
What’s not very new
• Routine Ova and Parasite Exam
– Useful for patients with unexplained anemia
(Strongyloides stercoralis)
What’s not very new
• Routine Ova and Parasite Exam
– Ascaris lumbricoides found in rare instances.
SOMC Ova and Parasite
Ova & Parasite and Trichrome Stain Results from SOMC
November 1, 2006 through October 31, 2011
Organism/ O & P Result
No parasites seen
Number (patients)
Percentage
3649
Clinical Setting
98.09% Diarrhea/abdominal pain
Blastocystis hominis
31
0.83% Not significant or immunosuppressed?
Giardia lamblia
20
0.54% Diarrhea/abdominal pain
Strongyloides stercoralis
7
0.19% All anemic
Ascaris lumbricoides
0
0% (Pediatric)
Trichuris trichiura
1
Cryptosporidium species
Dientamoeba fragilis
Number of positive patients
11
2
72
0.03% Immigrant (Mexico)
0.30% Diarrhea/abdominal pain
0.05% Pediatric
1.94%
Cryptosporidium/Giardia lamblia DFA
• Direct fluorescent antibody for Giardia lamblia
and Cryptosporidium parvum.
– Can be done in less than 1 hour
– Much more sensitive for finding these organisms,
particularly Cryptosporidium, than traditional ova
and parasite exam.
Parasites – Recommended Algorithm
• Order only Giardia lamblia/Cryptosporidium
DFA on a single stool specimen on patients
over 12 years old unless the patient is anemic,
immunosuppressed or has a significant travel
history.
Clostridium difficile
Clostridium difficile
• An anaerobic, spore-forming gram positive
bacillus, found mostly in the GI tract of
mammals.
• Both toxin-producing and non-toxin-producing
strains exist
– C. difficile culture alone not useful -detects both
types
Clostridium difficile
• Both toxin-producing and non-toxin-producing
strains exist
– GDH (Glutamate dehydrogenase) detects C.
difficile, but detects both types
– GDH can be used to rule out C. difficile, but not
definitively diagnose it.
Clostridium difficile
• Toxigenic culture (Reference Method)
– Very sensitive and specific
– Culture C. difficile, then test isolate that is
cultured for toxin production or toxin genes
– Good academically, not useful clinically
– Requires 2 or more days to a negative result, 3
days to positive result
Clostridium difficile
• Up until the last 2 years, most clinical labs
used (and >90% still do) C. difficile toxin tests
(either toxin A, or toxin A & B combined)
Clostridium difficile
• Present day “state of the art” testing is an
amplified DNA probe for a unique genomic
region found only in the toxigenic strains.
Clostridium difficile
• Our experience with amplified probe:
– Increased sensitivity, 29% better than C. difficile
toxin testing
– Correlates well (>95% agreement) with toxigenic
culture, the recognized reference method.
Clostridium difficile
• Potential shortcomings of amplified DNA probes:
– Excellent sensitivity – can detect as few as 16 C.
difficile toxin gene copies per 50 microliters of stool
– Must correlate with clinical information!
• Presence of C. difficile does not confirm a diagnosis of C.
difficile-associated disease.
Clostridium difficile
• Potential shortcomings of amplified DNA
probes:
– Toxigenic C. difficile is found in many people who
have no symptoms
Clostridium difficile
• Potential shortcomings of amplified DNA
probes:
– Only method FDA approved for pediatrics, but
remember children under 2 years of age have a
high carriage rate, up to 50%.
Clostridium difficile
• Specimen requirements
– Specimens only accepted on symptomatic patients
(i.e. diarrhea stool)
– Rejected for testing:
• Rattler’s
• Formed stool
• Stick Test negative (applicator stick stands)
Clostridium difficile
• Specimen requirements
– Specimens only accepted on symptomatic patients
(i.e. diarrhea stool)
– Acceptable specimens:
• Liquid stool (takes shape of container)
• Stick Test Positive (applicator stick falls)
Clostridium difficile
• Specimen requirements
– Because sensitivity is >95%, no more than 1
specimen needed
• One specimen per patient per week, unless approved
by the Microbiologist or Pathologist
Clostridium difficile
• Specimen requirements
– No lab test is indicated for “test of cure.”
Resolution of diarrhea is sufficient.
Clostridium difficile - Treatment
• Adequate treatment involves two dimensions
– Eradication of toxin-producing C. difficile
– Re-growth of normal bowel flora (particularly
anaerobic flora)
Clostridium difficile - Treatment
• Eradication of toxin-producing C. difficile
– Metronidazole not effective for moderate to
severe cases
• Kills C. difficile OK, but kills anaerobic GI flora better
• Treatment effective initially, but recurrence common
Clostridium difficile - Treatment
• Eradication of toxin-producing C. difficile
– Oral vancomycin is the agent of choice
– Less toxic to normal GI anaerobes
– A taper regimen is preferable – antibiotics kill only
vegetative cells.
• Longer coverage is necessary to control germinating
spores
• Longer coverage is necessary to allow re-growth of
normal anaerobic GNRs.
Clostridium difficile - Treatment
• Fidaxomycin
– Less toxic to normal bowel flora
than metronidazole or vancomycin
– Compared to vancomycin 125 mg
qid x 10 to 14 days, fidaxomycin
had a lower relapse rate.
– Expensive – standard course of
therapy approximately $2,800
(wholesale price)
Clostridium difficile - Treatment
• Re-growth of normal bowel flora (particularly
anaerobic flora)
– Will re-grow eventually if not assaulted
– In recurrences more aggressive replacement is
necessary
Clostridium difficile - Treatment
• Re-growth of normal bowel flora (particularly
anaerobic flora)
– Stool transplant is effective in >90% of cases
– Stool transplant involves a lot of testing of donor
stool (which takes several days to complete), and
is not aesthetically pleasing
– “Synthetic” stool developed for this purpose
Therapeutic Use of Human Feces
Fecal Transplant
• Used primarily to treat Cl. Difficile associated
disease
Fecal Transplant
• How they are performed:
Fecal Transplant
• How they are performed:
– Obtain approximately 200 – 300 grams of feces
from a close, healthy donor
Fecal Transplant
• How they are performed:
– Donor stool is screened for:
• Typical and atypical enteric pathogens (stool culture, Y.
enterocolitica, A. hydrophila, and others)
• Ova and parasites
– The donor is screened serologically for:
• Hepatitis A, B, and C
• HIV-1 and HIV-2
• Syphilis
Fecal Transplant
• How they are performed:
– The recipient is screened for:
• Hepatitis A, B, and C
• HIV-1 and HIV-2
Fecal Transplant
• How they are performed:
– Recipient is treated with oral vancomycin 500 mg
bid for 3 – 4 days (to kill vegetative C. difficile
cells)
Fecal Transplant
• How they are performed:
– If donor stool is suitable, 200 – 300 grams of
donor feces is blended with 200 – 300 mL of saline
to make a slurry
– Donor stool slurry is filtered through a coffee filter
to remove particulate matter
Fecal Transplant
• How they are performed:
– Donor stool is “implanted” in the recipient via as
nasogastric tube, colonoscope, or enema.
Fecal Transplant
• Stool transplants are effective in >90% of
cases
– Used only for patients with recurrences
• average duration of C. difficile in study was 11 months
– 77 patients studied
– 70 (91%) resolved within 6 days of treatment
Reference:
Shefchik, C. 2011. Clostridium Difficile Treated By Fecal Microbiota Transplant. Presented
at the American College of Gastroenterology's 76th Annual Scientific Meeting, Oct. 28Nov. 2 in Washington, D.C
Stools and Asymptomatic Patients
Asymptomatic Patients
• Most tests are methods of screening for GI
bleeding or potential colon cancer
– Tests that detect GI bleeding
– Tests that detect genetic changes associated with
cancer cells
Hemocult (guaiac) Testing
• Traditional test for occult blood
– Easy to do
– Inexpensive
– Sensitivity
– Many interfering substances
– Dietary changes may be necessary before
collecting stool specimen
Immunochromatographic Testing for
Human Hemoglobin (FOBT)
• Traditional test for occult blood
– Easy to do, but more expensive than Hemocult
– Sensitivity much better (approximately 300 X)
than Hemocult
– Few or interfering substances
– Dietary changes not necessary before collecting
stool specimen
Comparison of iFOBT and Hemocult
Hemocult (Guaiac Test)
Immunochromatographic
Test Method
Chemical
Immunological
Interpretation
Look for blue color after
addition of developer
Look for a line – similar to a
pregnancy test
Specificity
Depends on patients diet
>96% for human hemoglobin
Sensitivity
≈50% - Detects 90,000 ng/mL
or higher of hemoglobin
>87% - Detects as low as 50
ng/mL of human hemoglobin
Overall Accuracy
>97%
< 86%
Dietary Restrictions
None
Several
Samples required
One (or two)
Three
Cancer Cell DNA Screening
• Cells continually shed from the lining of the
colon, including cells from precancerous
polyps and cancerous tumors.
• Adenomas and tumors have DNA segments
that are different from normal human colonic
cells.
Cancer Cell DNA Screening
• DNA “markers” are shed with the cells from
abnormal cells
– Shedding is continuous
– But, multiple markers are needed to achieve high
detection rates
– Only 0.01% of DNA found in stool is of human
origin, so a sensitive method is needed to detect
the markers (amplified DNA probes)
Cancer Cell DNA Screening
• Fecal DNA tests demonstrate high detection
rates of early-stage colon cancer.
– Have been shown to be more sensitive than fecal
occult blood tests.
Cancer Cell DNA Screening
• Currently, only 1 test is available
– It is not FDA approved
• A number of second generation tests have
been developed and undergoing final clinical
validation in multicenter studies by the FDA.
Cancer Cell DNA Screening
• Interpretation of results:
– Negative result – NO DNA markers common to
colon cancer or precancerous polyps are found.
– Positive result - DNA markers common to colon
cancer or precancerous polyps were found
Additional evaluation — usually colonoscopy —
would be recommended.
Cancer Cell DNA Screening
• Interpretation of results:
– False Negative result – These may occur if colon
cancer or polyps do not harbor DNA markers
targeted by the stool DNA test, or if markers are
present in extremely low amounts.
Cancer Cell DNA Screening
• Interpretation of results:
– False Positive result – (Marker +, negative
colonoscopy) - These may occur if colon cancer or
polyps do not harbor DNA markers targeted by
the stool DNA test, or if markers are present in
extremely low amounts.
– Algorithm being developed that may include an
upper GI and re-evaluation of DNA markers
Cancer Cell DNA Screening
• Stool DNA tests for colon cancer screening are
endorsed by:
– American Cancer Society
– U.S. Multi-Society Task Force on Colorectal Cancer
– American College of Radiology
Cancer Cell DNA Screening
• No stool DNA tests for colon cancer screening
have been endorsed by the U.S. Preventive
Services Task Force
Cancer Cell DNA Screening
• Medicare does not pay for the test, but some
private insurers do
Questions?