Stool examination details

1. STOOL EXAMINATION
PRESENTER – DR. ANGELA SUNA
MODERATOR – DR. REENA BANERJEE

2. INTRODUCTION
• Waste products excreted from the digestive tract are composed of
water (up to 75%), indigestible residue, undigested food, food
which is digested but not absorbed, bile, epithelial cells,
secretions from digestive tract, inorganic material, and bacteria.
• Normal amount of feces in an adult is 100-200 grams per day.

3. INDICATIONS OF STOOL EXAMINATION
• Detection of parasites
• Evaluation of chronic diarrhea
• Evaluation of dysentery
• Bacteriologic examination
• Chemical examination
• Differentiating infection by invasive bacteria (like Salmonella or
Shigella) from that due to toxin producing bacteria (like
Escherichia coli or Vibrio cholerae)
• Identification of Rotavirus

4. Important organisms causing diarrhea and
their associations:
• Broad spectrum antibiotics (clindamycin, ampicillin); acute onset diarrhea
during hospitalization – C. difficile
• Immunocompromised hosts – Isospora, Cryptosporidium, Mycobacterium avium,
CMV
• Pediatric diarrhea – Rotavirus, Norwalk virus, Enterovirus
• Canned food – Clostridium botulinum
• Traveller’s diarrhea (watery) – Enterotoxigenic E.coli
• Acute dysentery – Shigella, Enteroinvasive E.coli, C. jejuni, E. histolytica
• Fried rice (reheated rice syndrome) – Bacillus cereus
• Poultry products - Salmonella

5. • Raw oysters and seafood – Vibrio sp. esp V. vulnificus
• Camping outdoors or in woods (backpacker’s diarrhea) – Giardia
lamblia
• Voluminous rice water diarrhea – Vibrio cholerae
• Undercooked meet (hamburger), bean sprouts, raw leafy vegetables
– Enterohemorrhagic E.coli O157:H7
• Acute bloody diarrhea, pseudoappendicitis – yersinia enterocolitica
(common source – infected pets or animals)

6. COLLECTION OF SPECIMEN
• Type of specimen - A random specimen of stool (at least 4 ml or 4 cm3 )
• Container - Clean, dry, container with a tightly fitting lid
• Transportation - Immediately to the laboratory (this is because
trophozoites of Entamoeba histolytica rapidly degenerate and alter in
morphology)
• Examined as early as possible after receipt in the laboratory (preferably
within 1 hour of collection)
• Storage – Refrigeration or use of a fixative
• 10% formalin (for preservation of eggs, larvae, and cysts)
• polyvinyl alcohol (for preservation of trophozoites and cysts, and for
permanent staining)

7. • One negative report for ova and parasites does not exclude the
possibility of infection.
• Three separate samples, collected at 3-day intervals, have been
recommended to detect all parasite infections.

8. Precautions
• Stool should not be contaminated with urine, water, soil, or
menstrual blood
• Urine and water destroy trophozoites; soil will introduce
extraneous organisms and also hinder proper examination
• Patient should not be receiving oily laxatives, antidiarrheal
medications, bismuth, antibiotics like tetracycline, or antacids for
7 days before stool examination.
• Patient should not have undergone a barium swallow examination.

9. REFERENCE RANGES
Bulk 100-200 grams/day
Color Brown
Water Up to 75%
Ph 7.0-7.5
Red blood cells Absent
White blood cells Few
Epithelial cells Present
Crystals Calcium oxalate, triple phosphate
Fat (Adults) <7 grams/day (gravimetric method),
<6 grams/day (titrimetric method)
Urobilinogen 50-300 mg/24 hours
Parasites Nil
Ova, cysts, trophozoites Nil

10. MACROSCOPIC EXAMINATION
• consistency (watery, loose, soft or formed)
• color,
• Odor
• presence of blood, mucus, adult worms or segments of
tapeworms.

11. Color/Appearance of Fecal Specimens
• Brown: Normal
• Black: Bleeding in upper gastrointestinal tract (proximal to cecum),
Drugs (iron salts, bismuth salts, charcoal)
• Red: Bleeding in large intestine, undigested tomatoes or beets
• Clay-colored (gray-white): Biliary obstruction
• Silvery: Carcinoma of ampulla of Vater
• Watery: Certain strains of Escherichia coli, Rotavirus enteritis,
cryptosporidiosis
• Rice water: Cholera
• Unformed with blood and mucus: Amebiasis, inflammatory bowel disease
• Unformed with blood, mucus, and pus: Bacillary dysentery
• Unformed, frothy, foul smelling, which float on water: Steatorrhea

12. MICROSCOPIC EXAMINATION

13. Preparation of Slides
• A drop of normal saline is placed
near one end of a glass slide and
a drop of Lugol iodine solution is
placed near the other end.
• A small amount of feces (about
the size of a match-head) is
mixed with a drop each of saline
and iodine using a wire loop, and
a cover slip is placed over each
preparation separately.

14. Concentration Procedure
• USE :
• If wet mount examination is negative and there is clinical suspicion of
parasitic infection, fecal concentration is indicated.
• It is used for detection of ova, cysts, and larvae of parasites.
• Two main types:
• Sedimentation techniques : Ova and cysts settle at the bottom. Example:
Formol ethyl acetate sedimentation procedure.
• Floatation techniques: Ova and cysts float on surface. Examples: Saturated
salt floatation technique and zinc sulphate concentration technique.

15. Fecal suspension
(10ml formalin + 1g
stool) passed
through gauze filter
7ml filtered
material + 3ml ethyl
acetate; centrifuge
for 1 min
Eggs,larvae and
cysts sediment at
bottom
Formalin, fecal
debris and ether
above the deposit
Fecal debris lossed
up with applicator
and supernatant
poured out.
Wet mount
preparation

16. Formol-ethyl acetate concentration method
(i) it can detect eggs and larvae of almost all helminths, and cysts
of protozoa,
(ii) it preserves their morphology well,
(iii) it is rapid, and
(iv) risk of infection to the laboratory worker is minimal because
pathogens are killed by formalin.

17. Classification of Intestinal Parasites of
Humans

18. Entamoeba histolytica
• C/F – asymptomatic, amoebic
dysentery, amoebic liver
abscess, weight loss, camping
abd pain
• Amoeboma
1. Identification of trophozoites
and cysts on stool
examination
1. Dx feature of trophozoite –
ingested RBCs
2. Saline wet mount – definite
directional motility
2. Amoebic vs Bacillary
1. Charcot leyden crystals +/-

19. Stool exam negative but intestinal amoebiasis
suspected clinically
3. Detection of antigen of E. histolytica – Enzyme immunoassay
4. Detection of DNA specific to E. histolytica – PCR based assay
5. Serologic tests
Disadvantage – cannot distinguish between recent and past infection
6. Endoscopic biopsy of ulcer in intestine – 50% cases show
trophozoites ; stain- Periodic acid Schiff stain

20. PROTOZOA - Entamoeba histolytica
Trophozite in H&E
stain

21. Giardia intestinalis
• “Backpacker’s diarrhea”,
“beaver fever”
• Asymptomatic, mild diarrhea,
severe disease with diarrhea,
malabsorption, weight loss and
steatorrhea
1. Demonstration of
trophozoites/cysts
1. Trophozoites – fresh liquid
stools, flakes of mucus
2. Cysts – formed/loose stools
2. Detection of antigen of G.
lamblia – Enzyme
immunoassay
3. Direct fluorescent antibody
assay- kit form

23. Coccidia – Isospora belli, Cryptosporidium
parvum, Cyclospora cayetanensis
• C/F – self limited, mild
diarrheal illness
• Immunocompromised patients
– severe and protracted
diarrhea, life threatening
1. Demonstration of oocysts
1. I.belli – unstained direct wet mount, Formalin
ether conc.
Modified ZN stain – Uniform red pink
UV light – Autofluorescence
2. C.parvum – Modified ZN stain- pink red
3. C.cayetenensis –
Direct wet mount – refractile globule (morula like)
Mod. ZN stain – Like parvum but larger
UV light – Intense blue autofluorescence
2. Enzyme immunoassay
3. Direct fluorescent antibody assay –
C.parvum

24. PROTOZOA - Coccidia
Oocyst in Modified ZN
stain
Direct wet mount

25. Microsporidia – Enterocytozoon bieneusi,
Encephaliozoon intestinalis, Encephalitozoon
hellem
• C/F – opportunistic infection in
immunocompromised patients:
persistent diarrhea, weight
loss
• Keratoconjunctivitis, hepatitis,
peritonitis, resp. infection,
kidney disease.
• Cannot be demonstrated on
wet mounts
1. Modified trichome stain of
stool sample – small spores,
stain ed, +/- transverse
bands
2. Small intestine biopsy –
spores within intestinal cells

26. PROTOZOA - Microsporidia
Microsporidia spores in
Trichome stain

27. HELMINTHS - Ascaris lumbricoides
(Roundworm)
• Most common helminthic infection in humans, commonly children.
• Asymptomatic
• Loeffler’s syn – migration of larvae through lungs
• cough, wheezing, eosinophilia, B/L irregular pulmonary densities
• Local effects – abd. pain, diarrhea, intestinal obstruction d/t large
mass of worms, intestinal perforation
• Worms causing obstruction in :
• Pancreatic duct – Pancreatitis
• Common bile duct – Obstructive jaundice
• liver abscess, appendicitis
• Malabsorption

28. 1. Demonstration of eggs
1. Direct wet mount – moderate to heavy infections
Fertilized (double shelled/decorticated)
Unfertilized (double shelled/decorticate)
Ovum – single, central granular mass
Decorticated eggs – Absent outer uneven shell, resemble hookworm eggs
2. Identification of adult worms – passed spontaneously in stool

30. Ancyclostoma duodenale (old world hookworm),
Necator americanus (new world hookworm)
• “Ground itch” – Inflammation
and marked itching on skin at
the site of larval penetration
• Loeffler’s syndrome
• Iron deficiency anemia – d/t
chronic blood loss
• Abd. pain and diarrhea –
increased worm load
1. Demonstration of eggs
Direct wet mount – moderate to heavy
infection
• Fresh stool – eggs show 4-8 gray, granular
cells
• >12 hrs – eggs show rhabditiform larva
folded upon itself (embryonated egg)
• >24 hrs – free rhabditiform larvae
2. Blood – eosinophilia, Microcytic
hypochromic anemia (chronic blood loss)
Occult blood test - positive

31. HELMINTHS - Hookworms

32. Trichuris trichiura
• C/F – heavy infection can cause
diarhea with blood and mucus in
stools, IDA or rectal prolapse
• Identification of typical eggs on
stool examination
• Quantification of eggs – assess the
severity of infection

33. Strongyloides stercoralis
• C/F-redness and itching at site of
perforation of filariform larva
• Loeffler’s syndrome
• Heavy inf.- abd. pain, diarrhea,
malabsorption
• Chronic inf.- abd. pain, diarrhea,
urticaria
• Immunocompromised patients-
fatal hyperinfection (sec to
autoinfection)
Severe pneumonia, neurologic
complications, abd.pain, shock,
septicemia.
Identification of larvae
Fresh stool – Rhabditiform larvae (shorter
buccal cavity in hookworm)
Duodenal fluid aspiration
Entero-test (string test)
- Gelatin capsule with textured string
Disseminated inf.- larvae in sputum
(-) stool exam (-)duodenal aspirate (-)string
test (+) strong clinical suspicion – Enzyme
immunoassay

34. HELMINTHS - Strongyloides stercoralis

35. Enterobius vermicularis
(Pinworm/Seatworm/oxyuris)
• Common in children
• C/F – intense nocturnal
perineal/perianal itching
• Present in appendix
• Vulvovaginitis
• Formation of pelvic and
peritoneal granuloma
• Usually not found in routine
stool exam
• Demonstration of worm
Adult female worm migrates
from intestine to perianal skin @
night and deposit eggs.
“Cellophane tape test” or anal
swab
Specimen collected late at night
or early morning before pt
passes urine, feces or takes a
bath.

36. HELMINTHS - Enterobius vermicularis

37. Taenia solium
1. Intestinal infection – insignificant, passage of a flat segment of
worm in feces
2. Cysticercosis – Ingestion of food contaminated with T.solium eggs
or autoinfection
• Nodules with cysticercus cellulosae found in :
Skeletal muscle
Subcutaneous tissue
Heart, liver, brain
3. Neurocysticercosis – seizures, inc ICP, psychiatric disturbances,
sudden death.

38. 1. Examination of feces
1. Identification of eggs – similar in T.solium and T.saginata
Egg with embryo – round granular mass with 3 pairs of hooklets, surrounded
by fine membrane.
2. Identification of gravid segments/proglottids (identification of species)
• T.solium :8-13 lateral branches in proglottid
• T.saginata : >13 lateral branches
3. Identification of scolex (head) – rarely seen; 4 suckers with a crown of
hooklets.
2. Diagnosis of cysticercosis
• Indirect hemagglutination assay – >/= 1:64
• Glycoprotein immunoblot assay – Neurocysticercosis
• X-ray – Calcified cysts
• CT – Diagnosis of neurocysticercosis

40. Taenia saginata
• C/F – insignificant, sometimes abd pain and diarrhea
• Lab Dx:
1. Identification of eggs –in feces and perianal skin
2. Identification of gravid segments
3. Identification of scolex – 4 suckers with no hooklets

42. BACTERIAL INFECTIONS
• Gram stain – Staphylococcal enterocolitis and Cholera
• Fecal leucocyte examination – Inflammatory process in colon
• Seen in : Shigella, Salmonella, campylobacter, Yersinia, E.coli(EIEC), C.difficile,
E.histolytica
• Non infectious – Inflammatory Bowel disease
• Drop of liquid stool/fleck of mucus, stained with 2 drops of methylene blue
• Alternative : Fecal lactoferrin or Fecal Calprotectin
• Stool culture : preserved in Carey Blair transport medium if not cultured
within 2 hrs.
• Non-hospitalized pts with diarrhea for > 5 days
• Dysentery
• Severe diarrhea
• Outbreaks of diarrheal illness

43. Helicobacter Pylori Antigen Detection in stool
• Gastritis, peptic ulcer, gastric adenocarcinoma, gastric MALT
lymphoma.
• Dx – 14C-labelled urea breath test, gastric biopsy (Gram (-) curved
rods), Enzyme immunoassay (Initial dx, confirm eradication
following tx)
• Stool antigen tests :
• Enzyme immunoassay (EIA)
• Immunochromatographic test (ICT)

44. VIRAL INFECTIONS
Rotavirus (dsRNA) Gastroenteritis, M/C
cause of diarrhea in
infants and small
children; can be fatal
d/t dehydration
Immunoassay for viral
antigen
Norovirus (ssRNA) Gastroenteritis, M/C
cause of diarrhea in
adults; self-limiting
Not typically done
Enteric adenovirus
(dsDNA)
Gastroenteritis in infants
and children
Antigen detection
Note – Rotavirus vaccine (RVV); given orally at 6 weeks, 10 weeks and 14 weeks of age.

45. CHEMICAL EXAMINATION
• Test for occult blood in stool
• Test for malabsorption of fat
• Test for reducing sugars
• Test for urobilinogen in feces
• Fecal osmotic gap
• Fecal pH
• Fecal Calprotectin
• Fecal Alpha-1 Antitrypsin Clearance
• Fecal Elastase-1

46. Test for Occult Blood in Stools
• Recommended as screening procedure for detection of
asymptomatic colorectal carcinoma
• Tests :
1. Based on peroxide-like activity of hemoglobin – colored recation
product
2. Fecal immunochemical test (FIT) for hemoglobin – more specific
for lower GI bleeding
3. Radioisotope test using 51Cr – amount of blood loss can be
calculated

47. Test for Malabsorption of Fat
• Normal - <7 g/day in adults
• Steatorrhea – increased fat excretion in feces
• Tests :
1. Qualitative – direct microscopic examination
• Random stool specimen sained with fat stain - >/= 60 fat globules (steatorrhea)
2. Quantitative – definite
• Gravimetric method
• Titrimetric method – most widely used
>/= 7g/day – abnormal
>/= 14g/day – disease causing fat malabsorption

48. Test for Reducing Sugars
• If lactase is deficient, lactose is converted to lactic acid with
production of gas.
• In infants - diarrhea, vomiting, and failure to thrive.
• Benedict’s test or ClinitestTM tablet test for reducing sugars is used
to test freshly collected stool sample for lactose.
• Oral lactose tolerance test is abnormal (after oral lactose, blood
glucose fails to rise above 20 mg/dl of basal value) in lactase
deficiency.
• Lactose breath hydrogen testing : Amount of hydrogen is measured
in breath; breath hydrogen more than 20 ppm above baseline
within 4 hours indicates positive test.

49. Test for Urobilinogen in Feces
• Normal amount of urobilinogen excreted in feces is 50-300 mg per
day.
• Increased fecal excretion of urobilinogen is seen in hemolytic
anemia.
• Urobilinogen is deceased in biliary tract obstruction, severe liver
disease, oral antibiotic therapy (disturbance of intestinal bacterial
flora), and aplastic anemia (low hemoglobin turnover).
• Stools become pale or clay-colored if urobilinogen is reduced or
absent.
• Fecal urobilinogen is determined by Ehrlich’s aldehyde test

50. FECAL OSMOTIC GAP FECAL pH
• Stool pH below 5.6 is
characteristic of carbohydrate
malabsorption
• Fecal osmotic gap is calculated
from concentration of
electrolytes in stool water by
formula : 290-2([Na+] + [K+]).
(290 is the assumed plasma
osmolality).
• In osmotic diarrheas, osmotic
gap is >150 mOsm/kg, while in
secretory diarrhea, it is
typically below 50 mOsm/kg.

51. Fecal calprotectin
• Calprotectin – protein present in neutrophil granules; released
during inflammation. Uses are :
• Differentiation b/w Irritable bowel syndrome (IBS) and inflammatory bowel
disease
• Assessing response to tx in IBD
• Monitoring of IBD and prediction of relapse
• <50µg/g of feces – IBS
• 50 – 150 – Bacterial infections, chronic NSAIDs use*
• >150 – IBD, Colorectal carcinoma*
* Advice- Colonoscopy if levels are persistently high.

52. Fecal Alpha-1 Antitrypsin Clearance
• Protein losing enteropathy (PLE) – excessive loss of plasma
proteins through GI mucosa leading to Hypoalbuminemia, edema
• Used to differentiate PLE from other caused of hypoalbuminemia
• Protein loss from GI mucosa – measured via α1-antitrypsin (AAT)
clearance, estimation of fecal fat, amount of AAT in stool
collected over a definite time period.

53. Fecal Elastase-1 (FE-1) : Isoenzyme of
elastase (specific for pancreas)
• Very stable in GIT; no proteolytic degradation
• Measurement of FE-1:
• Indirect assessment of pancreatic output, amylase, lipase and trypsin
• Done via Enzyme immunoassay
• Sensitive indicator of exocrine pancreatic insufficiency (EPI)
• Differentiate b/w pancreatic and non-pancreatic cause of
steatorrhea
• Low FE-1 – EPI
• Screening test; when positive 72 hr fecal fat estimation shoul be done (gold
standard)

54. FECAL MOLECULAR TESTING
1. GI molecular panel- Multiplexed nucleic acid test
• Simultaneous identification of multiple bacterial, viral and
parasitic nucleic acids in fecal samples of persons with C/F of
infectious colitis/ gastroenteritis.
• Advantage – avoids collection of multiple samples, result within a
short time
• Disadvantage – expensive test, cannot be specified for individual
patient requirements.

55. 2. Fecal genetic testing for Colon cancer
• American Cancer Society recommends regular screening of all adults
>/= 45 yrs with an average risk of Colorectal cancer.
1. Annual fecal immunochemical test (FIT)
2. Annual guaiac-based fecal occult blood test (gFOBT)
3. Multitarget stool DNA (mtDNA) every 3 years; if positive –
Colonoscopy
4. Colonoscopy every 10 years
5. CT colonscopy every 5 years
6. Flexible sigmoidoscopy every 5 years

56. ARTIFACTS
Fungal spore in wet
mount
Macrophages in trichome
stained stool
WBCs in trichome
stained stool
E.Histolytica trophozoite and cyst

57. Yeast in iodine stained wet
mount
Fungal spore in wet
mount
Plant hair in wet mount
G.lamblia cyst and trophozoite

58. Spore of mushroom Plant material
Hookworm eggs

59. Pollen grain in wet
mount
A.Lumbricoides egg
in wet mount
Pollen grain
Egg of T.solium
Fungal element in
ZN stain
Oocyst of
Cryptosporidium
spp.

60. REFERENCES
▪ Essentials of Clinical Pathology Third Edition by Shirish M.
Kawthalkar
▪ Textbook of Medical Laboratory Technology Third Edition
by Praful B. Godkar, Darshan P
. Godkar
• CDC DPDx – Laboratory Identification of Parasites of Public Health
Concern