introduction Medical parasitology lab. Islam F. Hassona Faculty of health sciences Medical Laboratory Sciences Department Medical parasitology lab. introduction Islam F. Hassona

Topics Introduction Artifacts Concentration Techniques Sample Collection and Preservation Methods Wet mount Preparation Normal saline 0.85% Iodine BMB Artifacts Concentration Techniques Modified Formal- Ether Sedimentation technique Acid- Ether Sedimentation technique Flotation Techniques By using Sheather’s solution By using Sodium Chloride solution By using Zinc Sulphate

Topics (cont.) Staining of parasites Detecting of Blood Parasites Thick and thin Blood smear Counting of Helminthes Eggs in Feces Chemical Tests Fecal PH test Testing feces for Occult Blood Fecal fat test Stool reducing sugar test Medical Entomology

Grades Attendance: ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ10% Unknowns: ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــ15% Assignment & Participation: ــــــــــــــــــــــــــــ5% Midterm examination:ـــــــــــــــــــــــــــــــــــــ 20% Final examination: ــــــــــــــــــــــــــــــــــــــــــــ50% Practical exam: ـــــــــــــــــــــــــــــــــــ30% Written exam: ـــــــــــــــــــــــــــــــــــــ20%

General Lab Objectives To familiarizes the student with the most widely used techniques for detection of parasites. To be able to identify the parasite stages. To learn the student, how to deal with risk samples.

What is the stool or feces? Waste residue of indigestible material (cellulose during the previous 4 days) Bile pigments and electrolyte. Intestinal secretions, including mucus. Leukocytes that migrate from the bloodstream Epithelial cells that have been shade. Bacteria and Inorganic material(10-20%) chiefly calcium and phosphates. Undigested and unabsorbed food

Fecal Specimen Fecal specimen are examined for protozoa, helminthes larvae or eggs. The stages of protozoa found in stool samples are trophozoites and cysts or oocytes. The stages of helminthes usually found in the stool samples are eggs and larvae, through whole adult worms or segment of worms may also be seen. Adult worms and segment of tape worms are usually visible to naked eye, but eggs, larvae, cyst, oocyst and trophozoites can be seen only with the microscope. In order to see these structure, the fecal material must be properly collected and examined.

Number of Specimens and Collection Time No technique is 100% successful in detecting parasites by single stool examination, and at least three serial stools must be examined before a patient can be considered free from infections in which stages of parasites would be expected to be free in the faeces. Because of the intermittent passage of certain parasites, the possibility of finding organisms is increased by examining multiple specimens. It is suggested that 3 specimens, collected at 2 to 3 day intervals, should be examined both pretreatment and post treatment (to ensure eradication of documented pathogenic protozoa).

Collection of Fecal Specimen Because of fragile nature of many intestinal parasites, and the need to maintain their morphology for accurate identification. Reliable microscopic diagnosis can not made unless the stool is collected properly. The stool specimen must be enough for satisfactory examination of fresh feces uncontaminated by urine, dirt*, water or other body secretion such as menstrual blood. If the sample is too small or contaminated with urine, it should not be accepted. Ask the patient to pass another specimen. * Urine will destroy the amoebic trophozoites and dirt will interfere with examination

Collection of Fecal Specimen (cont.) Collect the specimen in a clean dry screw-capped top container Collect the stool with a clean tongue blade or similar object. The container should be free from antiseptics and disinfectant.

Collection of Fecal Specimen (cont.) The container with the specimen should be clearly labeled with the following: Patient’s name or number. Date and time of collection. All samples should be accompanied by a requisition form from the physician giving relevant clinical details and recent travel history. Samples and forms from patient with a confirmed or suspected diagnosis of certain infectious diseases such as AIDS or hepatitis should be clearly labeled with “Biohazard”

Collection of Fecal Specimen (cont.) Most viable parasites are susceptible to desiccation or temperature variation. If time lapse between collection and observation is considerable, i.e. more than 4 days, it may be necessary to add some form of preservative to feces specimen to retain morphology. Formed samples can be kept in a refrigerator at 4 C° for a short time, but not in incubator. Any whole worms or segments passed should be placed in a separate container

Preservation methods for fecal specimens Preservation allows fecal samples to be examined after a delay in delivery or postage or testing. Many methods for the preservation of stool samples and permanent staining procedures. The most common fixatives are: Polyvinyl Alcohol, PVA Merthiolate Iodine Formalin, MIF Sodium acetate Acetic acid Formalin, SAF Formalin. Bayer’s solution* The preservatives used have different effect on the various stages of the parasites.

Formalin Formalin 4% has been used for many years as an all purpose fixative that is appropriate for helminthes eggs and larvae and for protozoan cyst. The fixative has a long shelf life. Concentration methods, like formalin- ether concentration can be performed from the preserved stool samples without loss of concentration abilities. The major disadvantage of formalin is that permanent staining procedures can't be performed from formalin preserved stool samples.

PVA This fixative is recommended for the preservation of the trophozoite and cyst stages of the intestinal protozoa, and also suitable for helminthes eggs and larvae. The preservation of the two stages of protozoa is excellent. The PVA is a plastic resin that serves as adhesive for the stool material. Has a long shelf life. ( months to years ). Concentration methods can’t performed from the specimen preserved by PVA.

PVA (cont.) The greatest advantage of this fixative is that a permanent stain can be prepared from stool specimen preserved by PVA, giving excellent result with trichrome staining. Specimen preserved by PVA can’t be used with immunoassay kits. Toxic, because contain mercury compound.

SAF Good routine fixative for protozoan cyst and trophozoites, helminthes eggs, and larvae. Has long shelf life. ( months to years). The preserved stool samples permits concentration techniques, most monoclonal detection kits, and permanent staining. Unlike the PVA, the SAF fixative has poor adhesive properties when SAF preserved samples are used to prepare permanent stained smears. ( Mayer’s albumin has been recommended as an adhesive. The combination of SAF preserved material and CB, IHK, and mod. Ziel Neelsen provides excellent staining of protozoan where staining of SAF preserved material with Trichrome gives poor results.

SAF (cont.) Specific advantages of the use of SAF are: SAF preserved material can be used for concentration techniques and permentant stained smears (CB, IHK). SAF preserved material can be used for some immunoassay methods. SAF is easy to prepare and has a long shelf life. Unlike the PVA, the SAF fixative contain no mercury compounds. It is therefore much less toxic than PVA

MIF This fixative was originally developed as a screening procedure for intestinal parasites. MIF combines preservation and staining for most kinds and stages found in faeces. It’s contains Merthiolate, Iodine, and Formalin. The preserved material permits concentration techniques. The major disadvantages are the short shelf life ( duo to iodine) and permanent stained smears can’t be prepared from MIF preserved material.

Fixative used for the preservation of stool samples: an overview of the advantages and disadvantages: Formalin 4% PVA SAF MIF Toxicity +/- +++ ( duo to Hg ) Shelf life Long (months) (months/years) Limited Preparation Easy Difficult Quality of fixation Egg: ++ Cyst: ++ Cyst: +++ Troph’s: +/- Troph’s: +++ Formalin ether concentration Possible Not possible Permanent stained smear Only Trichrome IHK, CB, mod. Ziel Neelsen

Preservation of worms Cestodes Wash in water to remove the mucus. Large tapeworms such as Taenia can be washed for several hours to relax the musculature, and can then be fixed in 10% formol saline b/w two glass slides to give flatter specimens. Trematodes These should be treated in a similar manner to cestodes, and mounted with the ventral sucker uppermost Nematodes Adult are washed in saline to remove mucus. Worms up to about 7 cm in length are fixed in hot(60-70˚C) 70% alcohol, which straightens out living worms, except those which have natural curvatures at the head or the tail. Alternatively, they can be fixed in hot 5% formalin. Large worms such Ascaris lumbricoides can be fixed and preserved in cold 5% formalin

Stool Analysis A stool analysis is a series of tests done on a stool (feces) sample to help diagnose certain conditions affecting the digestive tract . These conditions can include infection (such as from parasites, viruses, or bacteria), poor nutrient absorption, or cancer.

Stool Analysis (cont.) Laboratory analysis includes macroscopic, microscopic examination, chemical tests, and microbiologic tests. The stool will be checked for color, consistency, weight (volume), shape, odor, and the presence of mucus and parasites stages. The stool may be examined for hidden (occult) blood, fat, meat fibers, bile, white blood cells, and sugars called reducing substances. The pH of the stool also may be measured. A stool culture is done to find out if bacteria may be causing an infection.

Fecal Sample Examination Macroscopic Examination Color Consistency abnormal features adult worm or segment Microscopic Examination WBC/ HPF RBC/ HPF Mucus Yeast Cyst, trophozoite, or both Larvae, egg, or both Chemical Examination Fecal PH test Fecal fatty acid testing Testing feces for Occult Blood Stool reducing substances testing

Macroscopic Examination Color: Brown is normal color, results from the intestinal oxidation of stercobilinogen to urobilin. Bright red to dark red to black stools occur when iron or bismuth is taken or when there is intestinal hemorrhage. Pale yellow stools indicate the biliary obstruction, steatorrea, and also associated with diagnostic procedures that use barium sulfate. White stools occur when there is obstructive jaundice. Green stool may observed in patient taking oral antibiotic, because of oxidation of bilirubin to biliverdin.

Macroscopic Examination (cont.) Consistency: degree of moisture, will be a guide as to whether the trophozoite stage or the cyst stage of protozoa is likely to present. Formed, write “F” Soft , write “S” Loose , write “L” Watery , write “W”

Macroscopic Examination (cont.) Abnormal features: If mucus is present writ “M”, and “B” if blood is present. The presence of mucus coated stool is indicative for intestinal inflammation or irritation. Adult worm or segments: The feces may have adult helminthes or segments present such as Ascaris lumbricoides, Entrobius vermicularis, or Taenia spp. gravid segment, these can be seen by naked eye. And frequently motile for several days and may migrate to the top of the container.

Notice If several specimens are received at the same time; those containing blood and mucus should be examined first, followed by liquid specimens. These specimens are the most likely to contain amoebic trophozoites ( which die soon after being passed), and must be examined within 1 hour after passage. Formed specimens may be examined at any time during the first day, but should not be left overnight ( cyst may disintegrate). Excessive bulky stools may indicate conditions such as giardiasis.

Microscopic Examination of wet mount Wet mount is the simplest and easiest technique for the examination of feces, and this method should be performed in all laboratories at peripheral level. A wet mount can be prepared directly from fecal material or from concentrated specimens. The basic types of wet mount that should be used for each fecal examination are normal saline (0.85% NaCl), iodine, and buffered methylene blue.

Microscopic Examination of wet mount (cont.) The Saline Wet Mount Is used for the initial microscopic examination of stool specimens. It is employed primarily to demonstrate worms eggs, larvae. Protozoan trophozoites, and cysts. This type of mount can also reveal the presence of red blood cells and white blood cells. If the presence of amoebic trophozoites is suspected, warm saline (37˚C) should be used.

Microscopic Examination of wet mount (cont.) The Iodine Wet Mount Is used mainly to stain glycogen and the nuclei of cysts, if present. Cysts can usually be specifically identified in this mount. Trophozoite can not be revealed by this type of wet mount, because iodine kill trophozoite.

Microscopic Examination of wet mount (cont.) The Buffered Methylene Blue Wet Mount Should be prepared each time amoebic trophozoites are seen in a saline wet mount, or when their presence is suspected. BMB stains amoebic trophozoites, but not stain amoebic cysts, flagellate trophozoites or flagellate cysts. BMB stain is appropriate only for fresh unpreserved specimens. BMB stain live organism only, it isn’t used on preserved samples in which the organism have been killed Wait for five minutes to allow the stain to penetrate the trophozoites. It will overstrain the trophozoites in 30 minutes.

Notice Formed stool: take the portion of stool from an area to include inside and outside parts of the specimen. Stool with mucus: if mucus is present, label a second slide with the patient’s name or number. Put a drop of saline on the slide, pick up a small portion of mucus and mix with the saline. Trophozoites, if present, are sometimes more readily found in mucus than in the solid parts of the stool. Loose watery stool: if mucus is not present, pick up a small portion of the stool (any part) and mix with the saline.

Making Direct smear Microscopy Materials and reagents: Microscopic slides. Cover slips. Applicator sticks. Marker or pen for labeling. Reagents: Saline solution(isotonic) Lugols iodine(1% solution) BMB

Wet mount procedures Examine the slide on microscope: 10X 40X Make sure you have the condenser iris diaphragm open so that there is just enough light to work with (the higher the aperture, the lower the contrast). When using the 4X and 10X objectives, the diaphragm should be almost closed; open it a little for use with the 40X objective and further for use with the oil lens. 3. The condenser should be moved to almost its top position (you should not be able to see the lamp filament). Do not use the condenser to adjust the light level, use the diaphragm.

When examining a wet-mount for cysts and ova, start in one corner of the coverslip using your 10X objective and cover the slide in overlapping fields (see diagram #1). Use your 40X lens to examine any suspicious objects, and after you have completed the examination, repeat about 1/4 of it using the 40X objective to find the smaller cysts. Note that the addition of a drop of iodine to the sample will stain many eggs and cysts increasing their contrast.

Disposable of microscopic slides In diluted detergent Great care is needed in handling all laboratory specimens and rubber gloves should always be worn

DIRECT SMEAR A small amount of feces is transferred to a microscope slide and mixed with saline to produce a layer thin enough to read newsprint through. A coverslip is placed on the slide for viewing under the microscope. Rapid screening of fecal material with the ability to observe motile trophozoites that may be distorted and killed in hypertonic flotation solutions Reliability of Results Dependent on experience and skill of person examining the slide and the presence and viability of the trophozoites in the small-sample volume.

Result of Examination If no parasites are found: “No ova or parasites seen”, and specify whether this result was obtained by direct examination or by a concentration method (name method used). Never state categorically: “No parasites” If any parasites are seen, write the scientific name of the parasite with stages Example: Giardia lambilia cyst