MEDICAL PARASITOLOGY LAB .

Fecal Chemical Tests

Testing For Fecal Occult blood

FECAL OCCULT BLOOD TEST “ FOBT”

Hematemesis: bleeding into the gastrointestinal tract may be rapid with the vomiting of blood.

Melaena: the passage of blood through the rectum. When the bleeding is chronic with only small amounts of

blood being passed in the feces. If the blood or it’s products is not recognized in the feces, it

is referred to Occult blood (hidden Blood).

PRINCIPLE OF THE TEST Chemical tests to detect occult blood are based on the

principle that hemoglobin and it’s derivatives react in a similar way to peroxidase enzymes (pseudo-peroxidase activity).

Chromogen such as guaiac, O- toluidine, 4-aminophenazone or benzidine will be used as indicator for oxidation reaction.

Hemoglobin and its derivatives catalyze the transfer of oxygen from hydrogen peroxide to Guaiac, oxidation of the colorless chromogen produces a blue color.

Hemoglobin + H2O2 + Guaiac Oxidized guaiac+ H2O

O2

Pseudo-peroxidase

Blue color

REAGENTS AND METHOD Reagents:

Acetic acid 10% v/v Alcohol 95% v/v 4-aminophenazone (4-aminoantipyrine) Hydrogen Peroxide (H2O2) 10 vol. solution*

Working 4-aminophenazone reagent: The amounts given are sufficient for 1 test with positive and negative

controls. Prepare fresh as follows:

Alcohol 95% v/v ------------------------------------------------- 15 mlAcetic acid 10% v/v ------------------------------------------------ 1ml 4-aminophenazone ------------------------------------------------- 0.4gDissolve the 4-aminophenazone in alcohol solution and

immediately before use add the acetic acid. Mix well

PROCEDURES 1. Dispense about 7ml of distilled water into a test tube

2. Add a sample of feces about 1gm., use a glass or plastic rod to emulsify the feces.

3. Allow the fecal particles to settle or centrifuge the emulsified specimen.

4. Take 3 completely clean tubes and label them as : T: Patient's test. Neg.: Negative control. Pos.: Positive control.

5. Add into each tube as follow: T ------ 5ml supernatant fluid from emulsified feces. Neg. ------ 5ml distilled water. Pos.: ------5ml distilled water in which 5ul of whole blood has

been mixed.

CONTINUE………… 6. Add 5ml of working 4-aminophenazone reagent on top of

the fluid in each tube .Do Not Mix

7. Add 10 drops of the 10 vols. Hydrogen peroxide solution. Do Not Mix, allow to stand for 1 minute.

8. Look for the appearance of a blue color where 4-aminophenazone reagents meets the sample or control solutions.

Neg. control: this should show no color change. Pos. control: this should show a positive reaction.

Color Result

No color change Negative

Pale blue Positive +

Dark blue Positive + +

Blue – black Positive + + +

FALSE REACTIONS False positive: 1

1. Aspirin and anti-inflammatory medications.

2. Red meat (contain Myoglobin)and fish.

3. Green vegetables (Melons), and Horseradish.

4. Menstrual and hemorrhoid contamination.

5. Some intestinal bacteria that produce peroxidase enzymes. False negative: 2

1. Vitamin C greater than 250 mg/dl

2. Iron supplements containing vitamin C

INTERPRETATION The commonest cause of positive occult blood tests in

tropical and other developing countries are Hookworm infections, peptic ulcer, and bleeding from esophagus or liver cirrhosis.

Other causes include carcinoma in gastrointestinal tract, erosive gastritis duo to alcohol or drugs, or swallowed blood from nosebleeds.

If the test is negative but there is high clinical suspicion, a further two specimen should be tested to detect bleeding which be intermittent.

COMMERCIAL METHODS Therefore, to prevent false-positive reactions, the

sensitivity of the test must be increased. Many commercial testing kits are available for

occult blood testing with guaiac reagent. The kits contain guaiac impregnated filter paper, to

which the fecal specimen and hydrogen peroxide are added.

Two or three filter paper areas are provided for application of material taken from different areas of the stool, and positive and negative controls are also included.

CONTINUE...…

Additional, more sensitive and specific methods, for the detection of occult blood have been developed.

Hemoquant, provides a fluorometric test for hemoglobin and porphyrin.

As hemoglobin progresses through the intestinal tract, bacterial actions degrade it to porphyrin that the guaiac test cannot detect, this can result in some false-negative results from upper gastrointestinal bleeding when using the guaiac test.

iFOBT: The immunochemical fecal occult blood test, Hemoccult ICT, is specific for the globin portion of human hemoglobin and uses anti-human hemoglobin antibodies.

Because Hemoccult ICT is specific for human blood in feces, it does not require dietary or drug restrictions.

It is more sensitive to lower GI bleeding that could be an indicator of colon cancer or other gastrointestinal disease.

Can be used for patients who are taking aspirin and other anti-inflammatory medications.

CONTINUE...…

Intestinal Nematodes

Strongyloides stercoralis

STRONGYLOIDES STERCORALIS Adults lives in the small intestine (duodenum and jejunum),

fertilized females are deeply embedded in the mucosa, where they also oviposit.

Male have a pointed curved tail associated with two spicules. Female have straight tail without spicules. In contrast to the Anclystoma spp., both sexes have short

buccal cavity. Infective stage: Filariform larvae. Diagnosis:• Based on recovery of the rhabditiform larvae passed in

stool.• If diarrhea is present, eggs may also be recovered.

Intestinal Nematodes

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Strongyloides stercoralis

S. STERCORALIS ADULT MALE

spicules

spicules

S. STERCORALIS ADULT FEMALE

Intestinal Nematodes

Ancylostoma duodenale

Intestinal Nematodes

Raed Z. Ahmed, Medical Parasitology Lab.,2012

Anclystoma duodenale

ANCYLOSTOMA DUODENALAE (HOOKWORM)

Inhabit human intestine ( jejunum, ileum, and rarely duodenum ) and cause Anclystomiasis.

Male shorter than female and have copulatory bursa and two spicules.

Female is long and has pointed end. Both sexes have long buccal cavity with two pairs of teeth. Infective stage: Filariform larvae. Diagnosis:

Based on finding ova in fresh stool sample. In old sample, larvae present and must be differentiated

from larvae of Strongyloides stercoralis.

HOOKWORM EGGS Hook worm eggs like insects or mites egg, therefore must be differentiated

Mites egg Hook worm eggPlant material

HOOKWORM ADULT

MaleFemale

ANCYLOSTOMA DUODENALAE COPULATRY BURASA

ANCYLOSTOMA DUODENALAE BUCCAL CAPSULE

COMPARISON BETWEEN S. STERCORALIS AND HOOKWORM

Aspect Anclystoma spp. (Hook worm) Strongyloides stercoralis

Rhabditiform larvae

Buccal cavity Long Short

Esophagus One – third of the body One – third of the body

Genital permordium Absence Prominent

Filariform larvae

Esophagus One – third of the body Half of the body

Tail Pointed Notched

larvae Striated sheath Unsheathed

STRONGYLOIDES STERCORALIS LARVAE

Rhabditiform

Filariform

HOOKWORM LARVAE

Filariform Rhabditiform