What’s New in Poo

What’s New in Poo

SOMC Grand RoundsJanuary 6, 2012

Timothy R. Cassity, Ph. D.Microbiologist

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

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


• 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 toxin-producing 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%)


• Routine Ova and Parasite Exam– Very low yield with immunocompetent patients

with no travel history


• Routine Ova and Parasite Exam– Useful for patients with unexplained anemia

(Strongyloides stercoralis)


• Routine Ova and Parasite Exam– Ascaris lumbricoides found in rare instances.

SOMC Ova and ParasiteOva & Parasite and Trichrome Stain Results from SOMC

November 1, 2006 through October 31, 2011

Organism/ O & P Result Number (patients) Percentage Clinical Setting

No parasites seen 3649 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 0.03% Immigrant (Mexico)

Cryptosporidium species 11 0.30% Diarrhea/abdominal pain

Dientamoeba fragilis 2 0.05% Pediatric

Number of positive patients 72 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


• 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


• 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.


• 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


• 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)


• Present day “state of the art” testing is an amplified DNA probe for a unique genomic region found only in the toxigenic strains.


• 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.


• 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.


• Potential shortcomings of amplified DNA probes:– Toxigenic C. difficile is found in many people who

have no symptoms


• 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%.


• 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)


• 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)


• 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


• 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)


• 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


• 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)


• Re-growth of normal bowel flora (particularly anaerobic flora)– Will re-grow eventually if not assaulted– In recurrences more aggressive replacement is

necessary


• 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


– 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


– 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. 28-Nov. 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.


• 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)


• Fecal DNA tests demonstrate high detection rates of early-stage colon cancer.– Have been shown to be more sensitive than fecal

occult blood tests.


• 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.


• 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.


• 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.


• 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


• 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


• No stool DNA tests for colon cancer screening have been endorsed by the U.S. Preventive Services Task Force


• Medicare does not pay for the test, but some private insurers do

Questions?

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