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Faculty of medicine and health sciences LECTURE 1 Diagnostic Parasitology Introduction Asst. Prof. Dr. LINA M. Q. AL-AREQI  Introduction to Diagnostic Parasitology Equipment and Reagents Equipment a...

Faculty of medicine and health sciences LECTURE 1 Diagnostic Parasitology Introduction Asst. Prof. Dr. LINA M. Q. AL-AREQI  Introduction to Diagnostic Parasitology Equipment and Reagents Equipment and reagents used in diagnostic parasitology are: Instruments Microscope Centrifuge  Stains and reagents Giemsa stain  Alcohol Leishman stain  Ether Ziel Neelsen stain  Dobell’s iodine Eosin  Physiological saline Trichrome stain  Fixatives Iron hematoxylin stain  Anticoagulants Carbol fuchsin negative stain Fluorescent stains Papanicolaou stains Other requirements Syringes and needles Cotton swabs Slides and cover glasses Sterile scalpel Spreaders Tubes and Containers Paster pipettes Racks Scotch cellophane tape Anal swabs Fecal occult blood cassette Applicator sticks  Laboratory safety precautions Personal protection Infections may be caused by microorganisms entering the body through the:  Skin  Eyes  Mouth  Respiratory tract  All fresh specimens should be handled carefully, since each specimen represents a potential source of infectious material (bacteria, viruses, fungi, and parasites).  Since diagnostic parasitology work is most often performed within the microbiology division of a clinical laboratory, all general guidelines for safety would also apply.  Laboratory staff must practice a high standard of personal hygiene that includes:  Wearing lab coat that are fully buttoned and it must be worn at all times for work in the laboratory.  Washing hands and arms with soap and water before attending outpatients, visiting wards, after handling specimens and infected material, when leaving the laboratory, and at the end of the day’s work.  Protective gloves should be worn when taking samples from patients and when handling specimens and it should be removed aseptically and hands must then be washed.  Laboratory safety precautions  Covering any cuts, insect bites, open sores, or wounds on the hands or other exposed parts of the body with a water-proof adhesive dressing.  Wearing closed shoes and not walking barefoot.  Safety glasses, face shields (visors) or other protective devices must be worn when it is necessary to protect the eyes and face from splashes, impacting objects and sources of artificial ultraviolet radiation.  Labeling the fixatives properly.  Specific areas designated for specimen handling (biological safety cabinets may be necessary under certain circumstances).  Using proper containers for centrifugation of the specimens.  Not eating, drinking, chewing gum, smoking, or applying cosmetics in any part of the laboratory and not sitting on laboratory benches and the food or drink should never be stored in a laboratory refrigerator.  Not licking gummed labels or placing pens, pencils or other articles near the mouth, eyes, or in hair.  It is prohibited to wear protective laboratory clothing outside the laboratory, e.g. in canteens, coffee rooms, offices, libraries, staff rooms and toilets.  Laboratory working areas  The laboratory should be kept neat, clean and free of materials that are not pertinent to the work.  Work surfaces must be decontaminated after any spill of potentially dangerous material and at the end of the working day.  All contaminated materials and specimens must be decontaminated before disposal or cleaning for reuse.  Packing and transportation must follow applicable national and/or international regulations.  Type of clinical specimens Stages of parasites can also be identified from soil, water, insects, and samples from animal or vegetable sources. The clinical specimens usually obtained for laboratory identification of organisms parasitic to man are:  Fecal  Cerebrospinal fluid  Skin scraping or (CSF) snips  Blood  Bone marrow  Rectal scraping  Urine  Lymph gland  Duodenal aspirate Aspirate  Vaginal discharge  Liver aspirate  Sputum  Spleen aspirate  Fecal specimens The most common medically important parasites that may be found in fecal specimens are: Type of parasite Parasites Form Entamoeba histolytica Cyst, trophozoite Protozoa Giardia lamblia Cyst, trophozoite Balantidium coli Cyst, trophozoite Taenia solium egg, segment Cestodes Taenia saginata egg, segment Vampirolepis nana egg Fasciola hepatica egg Fasciola gigantica egg Trematodes Fasciolopsis buski egg Schistosoma mansoni egg Paragonimus westermani egg Ascaris lumbricoides egg, worm Enterobius vermicularis egg, worm Nematodes Strongyloides stercoralis Larva, egg Trichuris trichiura egg  Blood specimens The most common medically important parasites that may be found in blood specimens are: Class of parasite Type of parasite Form Protozoa Plasmodium species Ring form, trophozoite, schizont, gametocyte Trypanosoma species Trypomastigote Leishmania donovani Amastigote Nematodes Wuchereria bancrofti Microfilaria Loa loa Microfilaria  Urine specimens The most common medically important parasites that may be found in urine specimens are: Class of parasite Type of parasite Form Protozoa Trichomonas vaginalis Trophozoite Schistosoma mansoni Egg Trematodes Schistosoma haematobium Egg Wuchereria bancrofti Microfilaria Nematodes Onchocerca volvulus Microfilaria Vaginal Discharge The most common medically important parasites that may be found in vaginal discharge is: Class of parasite Type of parasite Form Protozoa Trichomonas Trophozoite vaginalis Sputum specimen The most common medically important parasites that may be found in sputum specimens are: Class of parasite Type of parasite Form Trematodes Paragonimus Egg westermani C.S.F specimen The most common medically important parasites that may be found in C.S.F specimens are: Class of parasite Type of parasite Form Trypanosoma species Trypomastigote Protozoa Naegleria Trophozoite Bone marrow aspiration The most common medically important parasites that may be found in bone marrow aspiration specimens are: Class of parasite Type of parasite Form Protozoa Leishmania donovani Amastigote Lymph gland aspiration The most common medically important parasites that may be found in lymph gland aspiration are: Class of parasite Type of parasite Form Leishmania donovani Amastigote Protozoa Trypanosoma species Trypomastigote Toxoplasma gondii Trophozoite Wuchereria bancrofti Microfilaria Nematodes Onchocerca volvulus Microfilaria Liver aspiration The most common medically important parasites that may be found in liver aspiration are: Class of parasite Type of parasite Form Entamoeba histolytica Trophozoite Protozoa Leishmania donovani Amastigote Spleen aspiration The most common medically important parasites that may be found in spleen aspiration are: Class of parasite Type of parasite Form Protozoa Leishmania donovani Amastigote Skin specimen The most common medically important parasites that may be found in skin scraping or snips are: Class of parasite Type of parasite Form Protozoa Leishmania species Amastigote Onchocerca volvulus Microfilaria Nematodes Dracunculu smedinensis Larva Rectal scraping The most common medically important parasites that may be found in rectal scraping are Class of parasite Type of parasite Form Protozoa Entamoeba histolytica Trophozoite Trematodes Schistosoma species Egg Duodenal aspiration The most common medically important parasites that may be found in duodenal aspiration are: Class of parasite Type of parasite Form Protozoa Giardia lamblia Trophozoite Schistosoma species Egg Trematodes Fasciola hepatica egg Nematodes Strongyloides stercoralis Larva  Faculty of medicine and health sciences LECTURE 2 Diagnostic Parasitology Collection, Preservation and Macroscopic Examination of Stool Asst. Prof. Dr. LINA M. Q. AL-AREQI  Sample collection  Morphologic forms of protozoa and helminthes may be detected from a properly collected and prepared stool specimen.  When present, the protozoan forms known as trophozoites and cysts may be recovered from these samples.  Helminth stages, such as eggs, larvae, segments, and adult worms, may also be found.  Because parasites are often shed (i.e., enter and subsequently passed in the stool intermittently, they may not appear in a stool specimen on a daily basis; therefore, multiple specimens are recommended for adequate detection.  The typical stool collection protocol consists of three specimens, one specimen collected every other day or a total of three collected in 10 days. One exception is in the diagnosis of amebiasis, in which up to six specimens in 14 days is acceptable.  Precautions  The stool sample usually collects by the patient and there are certain important consideration to be borne in mind relative to the collection of stool samples:  When handling all specimens, gloves and a protective coat should be worn at all times.  Certain medications and substances may interfere with the detection of parasites.  Such substances include: antacids, kaolin, mineral oil and other oily materials, non-absorbable antidiarrheal preparations, barium or bismuth (7-10 days needed for clearance of effects and gallbladder dyes (3 weeks).  Collection of specimens from patients who have taken antiprotozoal or antihelminths medications should be delayed for 2 weeks following therapy.  Stool specimens should be collected in a clean, watertight container with a tight-fitting lid.  The acceptable amount of stool required for parasite study is 2 to 5 g, often referred to as the size of a walnut.  Urine should not be allowed to contaminate the stool specimen because it has been known to destroy some parasites.  Stool should not be retrieved from toilet bowl water because free-living protozoa and nematodes may be confused with human parasites.  Toilet paper in the stool specimen may mask parasites or make examination of the sample difficult.  Procedures  The stool sample may be collected as the following: Give the patient a clean, dry leak-proof container without preservatives and instruct him/her to collect approximately 5 g of faeces. Instruct the patient to close the screw-top of container immediately after collection of the sample. After receiving the sample transfer it immediately to the laboratory. The specimen container should be labeled with the patient’s name and identification number, the physician’s name, and the date and time of sample collection.  Preservation of stool specimens  A freshly collected stool sample, which is immediately submitted to the laboratory, is the ideal specimen for parasitic examination.  Fecal specimens that cannot be processed and examined in the recommended time should be placed in an appropriate preservative or combination of preservatives for examination later.  Fixatives  Fixatives are substances that preserve the morphology of protozoa and prevent further development of certain helminths eggs and larvae.  Several preservatives are available commercially and the ratio of fixative to stool is important for the successful recovery of parasites and, whatever fixative is used, the recommended ratio is three parts fixative to one part stool.  The most common fixatives are:  Formalin  Modified Polyvinyl alcohol(PVA) copper or zinc  Sodium acetate acetic acid Formalin (SAF)  Merthiolate Iodine Formalin (MIF)  Schaudinn’s Fixative Formalin Uses  Formalin has been used for many years as an all-purpose fixative for the recovery of helminthes eggs and larvae and protozoan cyst.  It may be routinely used for direct examinations and concentration procedures.  Two concentrations of formalin are commonly used:  Five percent concentration ideally preserves protozoan cysts.  Ten percent concentration preserves helminth eggs and larvae. Preparation  The most common formalin preparation is 10% formalin, prepared as follows:  Formaldehyde………………………………………… 100 ml …….  Saline solution, 0.85% 900 ml NaCl.……….…………………….. Advantages There are different advantages for the use of formalin:  It is easy to prepare.  It preserves specimens for up to several years (long shelf life).  The concentration methods, like formalin- ether concentration can be performed from the preserved stool samples without loss of concentration abilities. Disadvantages  The permanent staining procedures can't be performed from formalin preserved stool samples, because the morphology of the parasite does not preserve adequately.  It cannot preserve the trophozoites usually and morphologic details of cysts and eggs may fade with time.  It can interfere with PCR, especially after extended fixation time.  Modified Polyvinyl alcohol copper or zinc  Modified Polyvinyl alcohol(PVA) copper or zinc is the fixative that prepared of copper sulfate or zinc sulfate instead of the mercury compound. Uses  Modified Polyvinyl alcohol (PVA) copper or zinc is recommended for the preservation of the trophozoite and cyst stages of the intestinal protozoa, and also suitable for helminthes eggs and larvae. Preparation  Modified Polyvinyl alcohol(PVA) copper is prepared as follows:  Copper sulfate solution ……………. 600 ml  Ethyl alcohol, 95% ………………….…………………….. 300 ml  Advantages  The preservation of the two stages of protozoa is excellent.  They can be used for concentration methods and permanent stained smears.  It has a long shelf life ( months to years ), when stored at room temperature.  It is free of mercury compound. Disadvantages  Specimen preserved by PVA can’t be used with immunoassay kits.  The organism morphology may be poor. Sodium acetate-acetic acid-formalin Uses  Sodium acetate-acetic acid-formalin (SAF) is good routine fixative for protozoan cyst and trophozoites, helminthes eggs, and larvae. Preparation  SAF fixative is prepared as follows:  Sodium acetate………………………………………………. 1.5g  Acetic acid, glacial…………………….…………………….. 2.0 ml  Formaldehyde, 37 to 40% solution………………………….. 4.0 ml  Distilled water……………………………………………… 92.0 ml Advantages  SAF is easy to prepare and it has long shelf life ( months to years).  The preserved stool samples permits concentration techniques, most monoclonal detection kits, and permanent staining.  It is free of mercury compound. Disadvantages  The SAF fixative has poor adhesive properties when SAF preserved samples are used to prepare permanent stained smears, therefore the addition of albumin (Mayer’s albumin) to the microscope slide may be necessary to ensure adhesion of the specimen to the slide.  Merthiolate-iodine-formaldehyde Uses Merthiolate-iodine-formaldehyde (MIF) was originally developed as a screening procedure for intestinal parasites. MIF is a good stain preservative for most kinds and stages of parasites found in feces; it is especially useful for field surveys. It is used with all common types of stools and aspirates; protozoa, eggs, and larvae can be diagnosed without further staining in temporary wet mounts. Preparation The MIF preservative is prepared in two stock solutions, stored separately, and mixed immediately before use. Solution I (stored in a brown bottle) Solution II (Lugol’s Solution) (good for several weeks in a tightly stoppered brown bottle)  Distilled water………………………………………. 50 ml  Distilled water……………………………………………….. 100 ml  Formaldehyde…………………….…………………….. 5 ml  Thimerosal (tincture of Merthiolate, 1:1,000)…………….. 40 ml  Potassium iodide crystals (KI)..........................………….. 10 g  Glycerin……………………………………………… 1 ml  Iodine crystals (add after KI dissolves)…………….. 5g Combine 9.4 ml of solution I with 0.6 ml of solution II just before use. Advantages  MIF combines preservation that fixes and stains the most kinds and stages found in faeces.  It is easy to prepare and it has long shelf life ( months to years).  It is very useful for field surveys.  The preserved material permits concentration techniques. Disadvantages  MIF contains mercury compounds provide stain color (thimerosal); disposal a problem.  The morphology of organisms on permanent stained smears generally not as good as that seen with other fixatives.  The iodine interferes with other stains and fluorescence and it may cause distortion of protozoa. Schaudinn’s Fixative Uses  Schaudinn’s fixative is designed to be used with fresh stool specimens or samples from the intestinal mucosal surface.  Schaudinn’s fixative is good for preservation of morphology of protozoan trophozoites and cysts Preparation  Schaudinn’s fixative is prepared as follows:  Mercuric Chloride, Saturated Aqueous Solution……. 600 ml  Ethyl alcohol, 95%…………….…………………….. 300 ml  Immediately before use, add 5 ml of glacial acetic acid per 100 ml of stock solution.  The mercuric chloride, saturated aqueous solution is prepared as follows:  Mercuric chloride (HgCl2)…………………… 110g  Distilled water…………….…………………….. 1000 ml Advantages Schaudinn’s fixative provides excellent preservation of protozoan trophozoites and cysts For many years, it considered the “gold standard” Easy preparation of permanent stained smears. Disadvantages The major disadvantages are less suitable for concentration procedures. It contains mercuric chloride. Inadequate preservation of morphology of helminth eggs and larvae. It is poor adhesion of liquid or mucoid specimens to slides  Macroscopic examination of stool Faecal samples are best described by:  Colour  Consistency  The presence or absence of macroscopic blood, mucus or exudate.  The presence of worms or segments. Colour The colour can be described as:  Brown is normal color, results from the intestinal oxidation of stercobilinogen to urobilinogen.  Black (occult blood)  Brown, pale yellow (fat)  White (obstructive jaundice). Ingestion of various compounds may give a distinctive color to the stool (iron, black; barium, light tan to white). Consistency The consistency can be described as:  Formed  Semiformed (soft)  Liquid (watery) The consistency of stool may give an indication of the organism stages that might be present. Trophozoites (potentially motile forms) of the intestinal protozoa are usually found in liquid specimens; both trophozoites and cysts might be found in a soft specimen; and the cyst forms are usually found in formed specimens. Others  The fecal specimens should also be examined macroscopically to see the presence of the following:  Blood or mucus.  Whether the specimen contains worms, e.g. Ascaris lumbricoides (large roundworm), Enterobius vermicularis (threadworm) or segments (proglottids) of Taenia species (tapeworm).  The presence of blood in or on the specimen may indicate several things and should always be reported.  Dark stools may indicate bleeding high in the gastrointestinal tract, and fresh (bright red) blood most often is the result of bleeding at a lower level.  In certain parasitic infections, blood and mucus may be present and soft or liquid stool accompanied by blood is more suggestive of an amebic infection; these areas of blood and mucus should be carefully examined for the presence of trophic amebae.  Occult blood in the stool may or may not be related to a parasitic infection and could result from a number of different conditions.  Adult pinworms and A. lumbricoides are occasionally found on the surface or in the stool.  Tapeworm proglottids may be found on or beneath the stool on the bottom of the collection container.  Faculty of medicine and health sciences LECTURE 3 Diagnostic Parasitology Fecal concentration techniques Asst. Prof. Dr. LINA M. Q. AL-AREQI  Fecal concentration techniques  Several diagnostic methods can be used in the microscopic examination of a fecal specimen:  Direct wet mount examination (stained and unstained) of fresh stool specimens.  Concentration procedures with wet mount examination of the concentrate.  Preparation of permanently stained smears  In general, concentration and permanent staining procedures should be performed on all specimens  Wet mount preparation Purposes  The microscopic examination of a direct smear has several purposes to:  detects protozoan cysts, trophozoites, helminths eggs, and helminth larvae.  provide a quick diagnosis of a heavily infected specimen.  Diagnose parasites that may be lost in concentration techniques. Principle  The wet mount procedure uses a glass slide on which a drop of physiologic saline (0.85%) has been placed at one end and a drop of iodine (Dobell or 1 : 5 dilution of Lugol solution) at the other end.  A small amount (2 mg) of feces is added to each drop and mixed well and each preparation should be covered square coverslip.  The preparation should be thin enough so that newsprint can be read through it and should not overflow beyond the edges of the coverslip.  The iodine stains nuclei and glycogen of the cyst but not chromatoid bodies.  A drop of eosin reagent may also be mixed with a small amount of the specimen and cover with a cover glass.  The eosin does not stain living trophozoites but provides a pink background which can make them easier to see.  Wet mounts are also made from the fecal specimen following a concentration procedure.  Concentration techniques Purposes  The purpose of concentration is to aggregate parasites present into a small volume of the sample and to remove as much debris as possible that might hinder the laboratory technician’s ability to see any parasites present clearly.  It provides the ability to detect small numbers of parasites that might not be detected using direct wet preparations.  They are used to detect protozoan cysts, oocysts, helminth eggs, and larvae, whereas the trophozoites get destroyed during the concentration procedures.  Types of concentration techniques  There are two types of concentration methods: sedimentation and flotation. 1-Sedimentation techniques  In sedimentation method, the feces is suspended in a solution with low specific gravity, so that the eggs and cysts get sedimented at the bottom, either spontaneously or by centrifugation. Formalin–ethyl acetate sedimentation Principle The principle of this technique is based on specific gravity, in which the ethyl acetate is added to a saline-washed formalin-fixed sample and the tube is then centrifuged. Parasites are heavier than the solution and settle in the sediment of the tube, whereas fecal debris is usually lighter and rises to the upper layers of the test tube Advantages Rapid test in which the one sample can be prepared in five minutes. The fecal odour is removed. It will recover most of the cyst, ova and larvae in the specimens. The morphology of the most parasites is retained for easy identification. There is less risk of infection from bacteria and viruses. It is inexpensive, easy to performed and can be done at any level of the health infrastructure. Disadvantages  It requires several pieces of apparatus which does not make it an easy filed method.  The preparation contains some debris.  The trophozoites form are not detected in this method.  Hymenolepis nana and Fasciola species do not concentrated well.  The ether is flammable and the formalin can cause irritation.  2-Flotation techniques  The flotation method uses solutions that have higher specific gravity than the organisms to be floated so that certain parasitic forms rise to the top, while fecal debris sinks to the bottom.  Zinc Sulfate Flotation technique Principle  The zinc sulfate flotation technique is based on differences in specific gravity between the sample debris, which in this case is heavy and sinks to the bottom of the test tube, and potential parasites, which are lighter and float toward the top of the tube.  In this procedure, zinc sulfate, with a specific gravity of 1.18 to 1.20, is used as the concentrating solution. When the zinc sulfate is added to the specimen and centrifuged, the parasites float to the surface and can be skimmed from the top of the tube.  In comparison, the specific gravity of protozoa and many of the helminth eggs is lower.  For example, the specific gravity of a hookworm egg is 1.055, an Ascaris egg 1.110, a Trichiura egg 1.150, and Giardia cysts 1.060. To ensure detection of all possible organisms, both the surface film and the sediment must be examined. Advantages The advantage of this technique is that more fecal debris is removed and it yields a cleaner preparation, making it easier for microscopic examination. Disadvantages The major disadvantages are that the walls of eggs and cysts will often collapse, thus hindering identification; also, some parasite eggs do not float. The operculated trematode and cestode eggs may not be detected because the high concentration of the zinc sulfate suspension causes the opercula to open and the egg to fill with fluid and sink to the bottom of the tube. Some helminths eggs are very dense and will not float; therefore, some parasites will be missed. Delay in examination can produce some distortion , particularly cysts. The possibility of risk of infection from unfixed material. The trophozoites form are not detected in this method.  Saturated sodium chloride floatation technique Principle A simple and popular method is salt-floatation using a saturated solution of sodium chloride, having a specific gravity of 1.2. Advantages The saturated sodium chloride technique is a useful and inexpensive method of concentrating hookworm or Ascaris eggs, e.g. in field surveys.. It concentrates nematode ova well. Disadvantages It is not applicable for eggs of tapeworms, unfertilized egg of Ascaris lumbricoides, eggs of trematodes, and protozoan cysts.  Sheather’s sugar flotation technique Principle Sheather’s flotation is sugar flotation, using a saturated solution of sugar, having a specific gravity of 1.20 to float many of the common parasite ova. Advantages Sheather’s sugar floatation technique is recommended for the detection of cryptosporidia infection. It reveals most nematode eggs and protozoan cyst. Disadvantages Flukes eggs and tape worm eggs are not demonstrate well. The most nematode larvae are not demonstrate well.  Faculty of medicine and health sciences LECTURE 4 Diagnostic Parasitology Microscopic Examination of Stool Asst. Prof. Dr. LINA M. Q. AL-AREQI  Microscopic examination of stool Systematic microscopic examination of stool specimens  The stool is emulsified in normal saline and a large drop is placed on a glass slide and is then covered with a cover slip.  Iodine preparation leads to better visualization of morphological details of ova and cysts as it stains the glycogen in them.  It however has the disadvantage that the live trophozoites of Entamoeba histolytica cannot be seen as the iodine kills it.  The eosin does not stain living parasites but provides a pink background which can make them easier to see.  Buffered methylene blue stain is used for the nuclear stain in E. histolytica. Systematic microscopic examination of stool specimens  In sedimentation technique; transfer the sediment to a slide, and cover with a cover glass.  The stool is examined under a light microscope and the entire 22- by 22-mm coverslip should be systematically (zigzag pattern) examined with the low power objective (10x) and low light intensity; any suspicious objects may then be examined with the high dry objective (40x). Use of an oil immersion objective (100x) on wet mounts is not routinely recommended, but it may be used in the permanent stained smear. Criteria for identification of parasite stages Identification of trophozoites  The trophozoites of protozoa are motile either because of slow movements of the cell (amoebae) or because they have rapidly moving flagella (long whip-like threads) or cilia (numerous short hairs).  Trophozoites are chiefly found in: Watery stools Stools containing mucus Soft or semi formed stools  Identification of trophozoites  The following features are useful for the identification of motile forms of intestinal protozoa:  Size  Shape  Cytoplasm  Pseudopodia or flagella or cilia  Nuclei (number and site)  Vacuoles  Inclusion bodies containing RBCs, bacteria, yeast cells, debris, etc.  Nuclear membrane (chromatin)  Nuclear karyosome Identification of cysts  Cysts are the resistant forms of certain intestinal amoebae, flagellates and ciliates.  They are small, round or oval in shape and non-motile and they may have one or several nuclei.  The presence of chromatoid bodies, the number of the nuclei, size and shape of cysts is useful for the correct identification of species. Identification of eggs  The identification of eggs should be approached in a systematic manner, taking into account the size and shape of the egg, the thickness of the shell.  The presence or absence of specialized structures such as polar plugs, knobs, spines, opercula, hooklets or mammilated outer coats can also be aids to identification.  The presence and characteristics of larvae within the eggs may also be useful.  Common artifacts in the stool sediments There are a number of structures that closely resemble parasites but in reality are not. These structures, termed artifacts and confusers, are found primarily in stool and blood samples. Such stool artifacts and confusers may be the result of disease processes, medications, and/or dietary habits. Gross and microscopic examination of stool may be complicated by the presence of artifacts resembling parasitic trophozoites, cysts, eggs, larvae, and adult worms. Many such artifacts arise from the large array of vegetable and meat products ingested every day by humans. Cells of human enteric origin may also mimic pathogenic or commensal protozoa in their appearance. Spurious infections with human or nonhuman parasites are known to occur following ingestion of contaminated or infected meats. The use of improper collection techniques offers another mechanism by which specimens are contaminated with extraneous organisms. The presence of free-living organisms in stool caused by specimen contact with water, sewage, or soil may often cause confusion.  Summary of artifacts resembling parasites in stool specimens Type of artifact Resemblance Cells of host origin  PMNs Entamoeba histolytica/E. dispar cysts  Macrophages E. histolytica/E. dispar trophozoites  Columnar epithelial cells Amebic trophozoites  Squamous epithelial cells Amebic trophozoites Free-living protozoa)  Amebae Amebic trophozoites or cysts  Flagellates Any of the intestinal flagellates  Ciliates Balantidium coli trophozoites Protozoan cysts (particularly Endolimax Yeasts nana) Fungal conidia Helminth eggs Plant  Plant cells Protozoan cysts, helminth eggs  Plant hairs Nematode larvae  Vegetable spiral Helminth eggs Pollen grains Nematode larvae Helminth eggs (particularly Ascaris or Taenia eggs) Diatoms Helminth eggs Starch granules, fat globules, air Protozoan cysts bubbles, mucus  Faculty of medicine and health sciences LECTURE 5 Diagnostic Parasitology Egg counting techniques Asst. Prof. Dr. LINA M. Q. AL-AREQI  Egg counting techniques  The are several techniques may be used for used for quantitative estimation of worm burden, such as:  Direct-smear egg count  Kato-Katz technique  Stoll’s technique  Formol detergent field technique  Direct-smear egg count Principle  In the direct-smear egg count, two mg of faeces is mixed in a small drop of saline on a slide and a coverslip is applied avoiding formation of air bubbles. The entire preparation is examined under low power of the microscope and the number of eggs (in 2mg faeces) is counted and then the number of eggs per gram of faeces is calculated. Advantages  The direct-smear egg count is rapid, inexpensive technique and suitable for fluid or hard specimens. Disadvantages  The disadvantages of direct-smear egg count are only small pieces of faces may be examined, the microscope-slide cannot be stored and the risk of infection from bacteria and viruses.  Kato-Katz technique Principle  In the Kato-Katz technique faeces are pressed through a mesh screen to remove large particles. A portion of sieved sample is then transferred to the hole of a template on a slide. After filling the hole, the template is removed and the remaining sample (about 10 mg, 20 mg, or 50 mg depending on size of template) is covered with a piece of cellophane soaked in glycerol. The glycerol clears the faecal material from around the eggs. The eggs are then counted and the number calculated per gram of faeces and the number of eggs found in the fecal smear should be multiplied by the constant 24 to obtain the number of eggs per gram of faeces. Video 1: Advantages  The Kato thick smear is a simple quantitation technique that has been used successfully in the field.  The slides can be prepared in the field, stored in microscope-slide boxes, and shipped great distances, for examination at a central laboratory if required. Disadvantages  The disadvantages of Kato-Katz technique are:  Less sensitive, particularly in light and moderate infections  Unsuitable for fluid or hard specimens  It can alter the morphological appearances of eggs  Less safe and hygienic  Unsuitable for the diagnosis of helminth larvae and is difficult to perform on hard and/or liquid stools.  Stoll’s technique Principle  In Stoll's technique, the fresh fecal material is added to calibrated Stoll flask that contains sodium hydroxide. A few small glass beads are also added to make a uniform suspension of fecal material and sodium hydroxide by vigorously shaking. The eggs are then counted by multiply the number of counted eggs by 100 to give the number of eggs per gram of faeces.  When the faeces is not a formed specimen, the following additional calculation is necessary to give the number of eggs per gram of faeces:  Fluid specimen.................. multiply by 5  Unformed watery specimen....... multiply by 4  Unformed soft specimen.......... multiply by 3  Semiformed specimen........... multiply by 2 Video 2: Advantages  The Stoll's technique is simple quantitation technique, more sensitive and suitable for fluid or hard specimens. Disadvantages  The disadvantages of Stoll's technique is the microscope-slide cannot be stored.  Formol detergent field technique Principle  In this technique, a spoonful of faeces is transferred to formol detergent solution. The suspension is sieved in a beaker, then transferred to the conical based container to be left upright for at least one hour without centrifugation.  The supernatant fluid is then removed and a suitable amount of 10% of formal detergent solution is added and mixed well for a minimum of 30 sec to be left for another hour. The sediment then transferred to a slide and covered with coverslip. The eggs are then counted by multiply the number of counted eggs by 3 to give the approximate number per gram (g) of faeces. Advantages  This technique is reproducible, inexpensive, simple, safe and hygienic to perform (formalin kills faecal pathogens) and it gives good preservation of schistosoma eggs.  It is more sensitive than the Kato-Katz technique because more faeces is used and with simple modification, the technique can also be used to detect other faecal parasites. Disadvantages  The disadvantages of formol detergent field technique include:  The schistosome eggs are fixed and will not overclear or become distorted.  The process is time consuming (several hours).  Faculty of medicine and health sciences LECTURE 6 Diagnostic Parasitology Special Diagnostic Techniques Asst. Prof. Dr. LINA M. Q. AL-AREQI  Special diagnostic techniques  There are several special techniques that may be used in diagnostic parasitology such as:  Scotch cellophane tape  Anal swabs  Miracidial hatching test  Fecal occult blood test  Scotch cellophane tape Uses  Scotch cellophane tape (adhesive cellophane tape) is the most widely used diagnostic procedure for eggs or adult female of Enterobius vermicularis (pinworm) infection.  Therefore, it is important that the specimen be obtained in the morning before the patient bathes or goes to the bathroom (washes or defecates) and at least four to six consecutive negative slides should be observed before the patient is considered free of infection. Principle  In this test; an adhesive transparent cellophane tape held sticky side out, on a wooden tongue depressor. The mounted tape is firmly pressed against the anal margin, covering all sides. The tape is transferred to a glass slide, sticky side down, with a drop of toluene for clearing and examined under the microscope.  Laboratory technicians must be knowledgeable regarding collection of this specimen because they may need to explain the procedure to patients, their families, and/or other health care professionals.  This is particularly important because, in many cases, parents may need to collect these samples from their children in a home setting.  When instructing others, it is also critical to emphasize the importance of exercising proper hygiene and preventive measures during specimen collection to avoid spreading infectious eggs into the environment.  Anal swabs Uses  The anal swab is also available for the detection of pinworm infections and the NIH swab (named after National Institutes of Health) has been widely used for collection of specimens.  At least four to six consecutive negative swabs should be obtained before the patient is considered free of infection. Principle  This consists of a glass rod at one end of which a piece of transparent cellophane is attached with a rubber band. The glass rod is fixed on a rubber stopper and kept in a wide test tube. The cellophane part is used for swabbing by rolling over the perianal area. It is returned to the test tube and sent to the laboratory, where the cellophane piece is detached, spread over a glass side and examined microscopically.  Miracidial hatching test Uses  Miracidial hatching test is helpful in demonstrating the egg viability of the egg of the Schistosome species.  When schistosome eggs are recovered from either urine or stool, they should be carefully examined to determine viability.  The presence of living miracidia within the eggs indicates an active infection that may require therapy.  The viability of the miracidia can be determined in two ways:  The cilia of the flame cells (primitive excretory cells) may be seen on a wet smear by using high power and are usually actively moving.  The miracidia may be released from the eggs by a hatching procedure. Principle  When the urine is dilute or has been left to stand for several hours in the light, the miracidia will hatch from the eggs and the ciliated miracidia are motile.  The eggs usually hatch within several hours when placed in 10 volumes of dechlorinated or spring water (hatching may begin soon after contact with the water).  The eggs that are recovered in the urine (24-h specimen collected with no preservatives) are easily obtained from the sediment and can be examined under the microscope to determine viability.  Fecal occult blood test Uses  The main use for the fecal occult blood test (FOBT) is as a screen for early colon cancer.  Blood in the stool may be the only symptom of early cancer; thus, if detected early, treatment can begin immediately, improving the chance of a cure.  For 1 day before the examination, the patient should not:  eat any meat;  take any drugs containing iron compounds;  brush his or her teeth vigorously  There are different methods in clinical use for testing for occult blood in feces, as:  Chemical methods using guaiac based reagents prepared in the laboratory, e.g. aminophenazone test.  Immunochemical methods using a haemoglobin specific cassette or strip test. Chemical methods Principle  The principle of chemical tests to detect occult blood is based on the fact that hemoglobin and its derivatives comes into contact with peroxidase enzymes, by catalyzing the transfer of an oxygen atom from the hydrogen peroxide to a chromogen aminopyrine (aminophenazone) and oxidation of the chromogen is indicated by the production of a blue color. Immunochemical methods  Modern fecal occult blood testing is moving to an immunochemical test which is specific for human hemoglobin.  Faculty of medicine and health sciences LECTURE 7 Diagnostic Parasitology Staining methods for stool examination Asst. Prof. Dr. LINA M. Q. AL-AREQI  Staining methods for stool examination Permanent stains  The detection and correct identification of many intestinal protozoa frequently depend on the examination of the permanent stained smear with the oil immersion lens (100×objective).  The smaller protozoan organisms are frequently seen on the stained smear when they are easily missed with only the direct smear and concentration methods.  For these reasons, the permanent stain is recommended for every stool sample submitted for a routine parasite examination.  A permanent stained smear, defined as a microscope slide that contains a fixed sample that has been allowed to dry and subsequently stained.  The slides are considered permanent because after staining, they are typically cover- slipped and sealed, thus allowing them to remain intact long term.  A permanent stained smear is designed to confirm the presence of protozoa cysts and/or trophozoites, due to this procedure allows laboratory technicians to observe detailed features of protozoa by staining intracellular organelles.  Although some protozoa may be recognized in the direct or concentrated wet preparation, the identification is considered tentative until confirmed with the permanent stained smear.  In addition, there are some protozoa that only possess a trophozoite stage and will not be detected in the concentrated wet mount preparation.  Dientamoeba fragilis is one example and, if a permanent stained smear is not performed, this parasite will likely be missed.  The permanent stained smear is not the method of choice for the identification of helminths eggs or larvae because these parasites often stain too dark or appear distorted.  Helminth eggs or larvae are best detected and identified using a concentration technique.  On completion of staining, the slides can be sealed with a permanent mounting sealant and can be kept for years, serving as an effective teaching tool.  The slides are reviewed under oil immersion (100×); 300 fields are reviewed before the slide can be considered negative.  Two common stains used for routine testing include; trichrome stain and iron hematoxylin.  Trichrome stain The most widely used permanent stain is the trichrome stain and the laboratory technicians choose this stain because it uses reagents with a relatively long shelf life and the procedure is easy to perform. There are distinct color differences among the cytoplasmic and nuclear structures of select parasitic forms, as well as background material. Appearance of select protozoan structures and background material on trichrome Stain Structure or Material Appearance Cytoplasm of Entamoeba histolytica Light pink or blue-green trophozoites and cysts Cytoplasm of Entamoeba coli cysts Purple tint Nuclear karyosomes Bright red to red-purple Degenerated parasites Light green Background Green  Iron Hematoxylin stain  The iron hematoxylin stain is one of a number of stains that allow one to make a permanent stained slide for detecting and quantitating parasitic organisms.  The iron hematoxylin stain may be used instead of the trichrome technique, but this procedure was considered to be time-consuming.  The iron hematoxylin stain reveals excellent morphology of the intestinal protozoa and in some cases, the nuclear detail of these organisms is considered to be stained clearer and sharper than when stained with trichrome. Appearance of select protozoan structures and background material on iron hematoxylin stain Structure or Material Appearance Protozoa cytoplasm Blue to purple Protozoa nuclear material Dark blue to dark purple Debris and background Light blue, sometimes with pink tint material  Specialized Stains  Specialized stains are also available for specific groups of parasites and these stains include; the modified trichrome stain, modified Ziehl- Neelsen acid- fast stain, Modified Kinyoun’s acid-fast stain, carbol fuchsin negative stain and fluorescent stains.  Modified trichrome staining  The microsporidial spores are also acid fast, their size (1 to 2 μm) makes identification very difficult without special modified trichrome stains or the use of immunoassay reagents.  Appearance of Microsporidia on modified trichrome stain Structure or Material Appearance Spores of microsporidia Pink to red with clear interior Polar tubule Red horizontal or diagonal bar Bacteria, yeast, debris Pink to red Background Green  Modified Ziehl-Neelsen Acid-Fast Stain (Hot Method)  The modified acid-fast stain, has become an important permanent stain procedure for the detection of the oocysts of Cryptosporidium, as well as those of Isospora and Cyclospora  Oocysts in clinical specimens may be difficult to detect without special staining, so the modified acid-fast stains are recommended to demonstrate these organisms.  Application of heat to the carbol fuchsin assists in the staining, and the use of a milder decolorizer allows the organisms to retain their pink-red color.  Although the microsporidial spores are also acid fast, their size (1 to 2 μm) makes identification very difficult without special stains or the use of monoclonal antibody reagents. Appearance of select protozoan structures, yeast and background material on modified acid-fast stain Structure or Material Appearance Oocysts of Cryptosporidium Pink to red and Isospora Oocysts of Cyclospora Variable; clear to pink to red Yeast Blue Background material Blue or light red  Modified Kinyoun’s Acid-Fast Stain (Cold Method)  Unlike the Ziehl-Neelsen modified acid-fast stain, the modified Kinyoun’s stain does not require the heating of reagents for staining.  With additional reports of diarrheal outbreaks due to Cyclospora, it is also important to remember that these organisms are modified acid-fast variable and can be identified by this staining approach.  Carbol Fuchsin Negative Stain  Carbol Fuchsin Negative Stain is used to stain the oocyst of the Cryptosporidium species.  The immersion oil is added to the stained smear directly and then the coverslip is applied.  The stained smear is observed under bright-field microscopy (×400).  Everything but the oocysts stains darkly due to the oocysts are bright and refractile because they contain water and everything else is oil soluble.  Quick-Hot Gram-Chromotrope stain  This is a fast, reliable, and simple method of staining smears to demonstrate microsporidian spores in fecal and other clinical specimens.  Fluorescent stains Auramine O stain & Calcofluor white stain  Coccidia are acid-fast organisms and also stain well with auramine O (phenolized auramine O).  The size and typical appearance of Cryptosporidium, Cyclospora, and Cystoisospora oocysts enable auramine O-stained slides to be examined at low power using the 10× objective.  The smear is examined with a fluorescence microscope with a 10× objective and fluorescein isothiocyanate optical filters (auramine O: excitation max, ~435 nm in water; emission max, ~510 nm).  Screen the whole sample area for the presence of fluorescent oocysts and the suspicious objects can be reexamined using a 20× or 100× objective.  Smears can be restained by any of the carbol fuchsin (modified acid-fast) staining procedures to allow examination with light microscopy.  Calcofluor white stain is used to determine the cysts.  Faculty of medicine and health sciences LECTURE 8 Diagnostic Parasitology Examination Of Urogenital Specimens For Parasites Asst. Prof. Dr. LINA M. Q. AL-AREQI  Examination of urogenital specimens for parasites Collection of urine sample The urine sample usually collects by the patient and there are certain important considerations to be borne in mind relative to the collection of urine samples for examination as:  A random sample is usually sufficient for the performance of most urinary screening tests; but, since the first sample voided in the morning (first-morning) is more concentrated it is usually the sample of choice.  Containers for routine urinalysis should have a wide mouth to facilitate collection from female patients and a wide, flat bottom to prevent overturning. Conical containers are less likely to tip over.  The container should also be a clean, dry, leak-proof, 100 to 200 ml with lid.  The container should be made of a clear plastic material to allow for determination of color and clarity.  The following instructions should be given to the patient before collection of urine sample: Instruct the patient to wash his/her hands before and after collection of urine. Instruct the patient to collect the urine sample without mixing with stool, water or soup or other contaminants. When midstream urine sample is required ask the patient to pass small amount of urine out and pass the remaining of urine to the container. When terminal urine sample is required ask the patient to urinate the last portion of urine into the container. Instruct the patient to collects about 20 ml of urine. Instruct the patient to place the lid, secure tightly and rapidly transport of urine sample to the laboratory.  Collection of urine sample for parasites Filariasis can be diagnosed from urine samples, and Trichomonas vaginalis and Schistosoma haematobium eggs can be detected in the urine. Collection of a midday urine specimen or a 24-h collection in a container without preservatives is recommendedwhen the S. haematobium is suspected. In patient with hematuria ,instruct the his/her to run slowly for at least half an hour before collection of urine sample, where the peak egg excretion of S. haematobium occurs between noon and 3p.m. and the eggs may be found trapped in the blood and mucus in the terminal portion (last-voided portion) of the urine specimen.  Microfilariae may be detected in urine of patients with chyluria, of patients with very heavy filarial infections, and of patients treated with diethylcarbamazine and the specimens may be collected as first-voided or as a 24-h collection in a container without preservatives. Collection of first-voided urine, particularly after prostatic massage in male patients, is useful for the diagnosis of T. vaginalis infection. All specimens and request forms must be labeled properly with the patient’s name and identification number, the date and time of collection. Labels must be attached to the container, not to the lid, and should not become detached if the container is refrigerated or frozen. Improperly labeled and collected specimens should be rejected by the laboratory, and appropriate personnel should be notified to collect a new specimen.  Urine sample preservation The most routinely used method of preservation is refrigeration at 2 C° to 8 C°, which decreases bacterial growth and metabolism. Preservatives that can be used to preserve random screening samples include formalin, toluene , thymol, chloroform, boric acid, and chlorhexidine.  Macroscopic examination of urine  The urine specimens may be examined physically and chemically.  The physical examination of urine includes; appearance, color and specific gravity.  Urine is normally clear yellow in color and the more concentrated urine may appear dark yellow.  The specific gravity is the ratio of the weight of a volume of urine to the weight of the same volume of distilled water at a constant temperature.  It is an indicator of the concentration of dissolved material in the urine; however, it is dependent not only upon the number of particles but also upon the weight of the particles in the solution.  The chemical examination of urine includes pH, protein, glucose, ketones, occult blood, bilirubin, urobilinogen, nitrite, leukocyte esterase, and strip test method for specific gravity.  There are many methods for chemical examination of urine but the rapid method is a reagent strip.  Microscopic examination of urine  The purpose of microscopic examination of urine sediments is to detect and identify insoluble materials present in the urine specimen.  Helminthic larval stages and eggs and some protozoa infecting humans may be found in the urine whether or not they cause pathologic sequelae in the urinary tract.  Microfilariae may be detected in the urine of heavily infected patients or of patients recently treated with diethylcarbamazine.  The microfilariae that may be found in urine are Wuchereria bancrofti and Onchocerca volvulus.  Microfilariae and S. haematobium eggs can be easily concentrated by passing the specimen through a membrane filter and the filter can then be observed through a microscope.  Collection of genital specimens  The genital specimen from both males and females should be collected by a physician or specialist nurse. Microscopic examination of genital specimens  T. vaginalis infections are primarily diagnosed from direct saline (wet) mounts by detecting live motile flagellates.  Permanently stained smears can be made from patient specimens for specific identification of the organism.  Although a number of stains can be used, Giemsa and Papanicolaou stains are the ones most frequently used to diagnose T. vaginalis infections.  Faculty of medicine and health sciences LECTURE 9 Diagnostic Parasitology Collection and examination of blood specimens Asst. Prof. Dr. LINA M. Q. AL-AREQI  Collection of blood for malaria parasites  The blood and bone marrow specimens may be used for detection of malaria parasites.  The blood smears are best prepared directly from capillary blood, but the EDTA anticoagulated venous blood can also be used.  The capillary blood can be collected from a finger, heel or (rarely) an ear lobe may be performed on patients of any age.  Both thin and thick blood films must be prepared on the same slide for diagnosis of malaria parasites.  Collection of blood for malaria parasites  Before collection of blood sample, it is necessary to keep in mind the following considerations:  Wear protective gloves when taking blood samples or handling blood.  Blood to be examined for malaria parasites is usually collected at a health centre.  The most suitable time for collection is at the height of an episode of fever, when the parasites are most numerous in the blood.  Blood specimens should always be collected before antimalarial drugs are given.  Staining of blood smears  The specimen may consist of a thin blood film that has been fixed in absolute methanol and allowed to dry, a thick blood film that has been allowed to dry thoroughly and is not fixed, or a combination of a fixed thin film and an adequately dried thick film (not fixed) on the same slide.  Staining of blood smears  There are different stains that may be used for staining the of malaria parasites such as Giemsa stain, Leishman stain and Wright stain, but Giemsa-stained peripheral blood smears are the specimens of choice for the laboratory diagnosis of malaria and for staining of blood smears with Giemsa stain; one of the following concentrations may be used: Three percent (3%) concentration Five percent (5%) concentration Ten percent (10%) concentration  Microscopic examination  Diagnosis rests on microscopic examination of blood, using both thick and thin Giemsa-stained smears.  The thick smear is used to screen for the presence of organisms and reporting the degree of malaria parasites, and the thin smear is used for species identification.  It is important to identify the species because the treatment of different species can differ.  Concentrating parasites in venous blood by centrifugation can be also used when the malaria parasites cannot be found in blood films.  Differentiation between Plasmodium species  The comparison between the four species of Plasmodia in thin blood film as the following: Plasmodium falciparum Ring form  The ring form of P. falciparum has delicate cytoplasm; 1-2 small chromatin dots; occasional appliqué (accollé) forms and double chromatin dot.  The normal appearance of infected red blood cell (RBC) and multiple infection is more common than in other species. About 5% of red blood cells or more are seen parasitized in the blood smear, on the other hand the presence of large number of ring forms.  Trophozoite  The trophozoite of P. falciparum is seldom seen in peripheral blood; compact cytoplasm and dark pigment may be seen.  The normal appearance or slightly crenated infected RBC with and multiple infection and Maurer’s clefts may also be seen. Schizont  The schizont of P. falciparum is seldom seen in peripheral blood and mature schizont contains 8-32 small merozoites.  The normal appearance infected RBC with and multiple infection and Maurer’s clefts may also be seen. Gametocyte  The gametocyte of P. falciparum has crescent or sausage shape with a single mass chromatin in macrogametocyte or diffuse in microgametocyte.  The infected RBC may be distorted by parasite. Plasmodium vivax Ring form  The ring form of P. vivax has large cytoplasm with occasional pseudopods and large chromatin dot.  The normal or round appearance of infected RBC and occasionally appears with fine Schüffner’s dots. Trophozoite  The trophozoite of P. vivax has large ameboid cytoplasm and large chromatin dot.  The enlarged appearance of infected RBC with Schüffner’s dots. Schizont  The mature schizont of P. vivax contains 12-24 small merozoites.  The enlarged appearance of infected RBC with Schüffner’s dots. Gametocyte  The gametocyte of P. vivax has round to oval shape with eccentric chromatin mass in macrogametocyte or diffuse microgametocyte.  The infected RBC may be enlarged or distorted by parasite. Plasmodium malariae Ring form The ring form of P. malariae has sturdy cytoplasm and large chromatin dot and occasional ‘bird’s-eye’ ring form may be seen. The normal appearance of infected RBC. Trophozoite  The trophozoite of P. malariae has compact cytoplasm and large chromatin and occasional band forms may be seen.  The normal appearance of infected RBC. Schizont  The mature schizont of P. malariae contains (6-12) merozoites and occasional rosettes form may be seen.  The normal appearance of infected RBC. Gametocyte  The gametocyte of P. malariae has round to oval shape with eccentric chromatin mass in macrogametocyte or diffuse microgametocyte.  The normal appearance of infected RBC. Plasmodium ovale Ring form The ring form of P. ovale has sturdy cytoplasm and large chromatin dot. The enlarged appearance of infected RBC with Schüffner’s dots and occasionally fimbriated shape. Trophozoite  The trophozoite of P. ovale has compact cytoplasm and large chromatin.  The enlarged appearance of infected RBC with Schüffner’s dots and occasionally fimbriated shape. Schizont  The mature schizont of P. ovale contains 6-12 merozoites.  The enlarged appearance of infected RBC with Schüffner’s dots and occasionally fimbriated shape. Gametocyte  The gametocyte of P. ovale has round to oval shape with eccentric chromatin mass in macrogametocyte or diffuse microgametocyte.  The enlarged infected RBC with Schüffner’s dots and occasionally fimbriated shape.  Types of staining techniques The Romanowsky stains that may be used for staining the blood parasites are alcohol- based such as Giemsa, Leishman and Wright stains and aqueous-based such as Field’stain. The most commonly used are Giemsa stain and Leishman stain.  Advantages  The Giemsa stain can stain both thick and thin smears and it uses for estimation degree of malaria parasites.  The leishman stain takes less time for preparation. Disadvantages  The Giemsa stain takes more time for preparation and staining procedures and thin smear requires fixation before staining.  The leishman stain can stain the thin smear more better than thick smear.  Malaria parasite density estimation There are different method that may be used for estimation the density of parasite and one of them is estimation the number of parasites per microliter of blood. In this method, the numbers parasites are estimated per microliter of blood against white cells as the following: Bring two tally counters, one for counting the white blood cells and other for counting the malaria parasites. When there are 100 or more parasites have been seen against 200 white blood cells have been counted stop the count.  Malaria parasite density estimation When there are less than 100 parasites have been seen against 200 white blood cells have been counted, continue the counting until the number of white blood cells become 500. In the procedure in (2 and 3) , calculate the number of parasites per microliter of blood by multiplying the number of parasites by 8000 and then dividing by the number of white blood cells (200 or 500).  Blood for other parasites examination Trypanosoma  The specimens that may be used for demonstration of Trypanosoma cruzi are: blood specimen, and lymph nodes aspiration.  The specimens that may be used for demonstration of T. brucei are: blood specimen, lymph nodes aspiration and CSF specimen.  During the early stages, microscopic examination of the blood or an aspirate of the chancre or enlarged lymph node reveals (either wet films or thick or thin smears) trypomastigotes.  Both stained and wet preparations should be examined, the latter for motile organisms.  Examination of cerebrospinal fluid (c.s.f.) for trypanosomes, cells (including Morula "Mott"cells), coupled with an elevated protein level, indicates that the patient has entered the late, encephalitic stage.  Microfilaria  The primary method of filarial diagnosis is microscopic examination of the microfilariae in a Giemsa-stained smear of blood or a tissue scraping of an infected nodule.  The microfilaria can be differentiated based on the following:  The site of infection and the type of sample (skin or blood)  The presence or the absence of the sheath  The extending of the nuclei in the tail of microfilaria.  Whether the collection of sample is nocturnal or diurnal  There are many species of filariae, but most are parasites of animals and rarely affect humans.  The most important types are  Wuchereria bancrofti  Mansonella perstans  Brugia malayi  Mansonella ozzardi  Brugia timori  Mansonella streptocerca  Loa loa  Onchocerca volvulus  Examination of skin specimens Skin snips  Multiple Giemsa-stained slides of tissue biopsies, known as skin snips, collected from suspected infected areas are the specimens of choice for the recovery of O. volvulus microfilariae.  The skin snips should be obtained with as little blood as possible to avoid contamination of the sample with other species of microfilariae that may be present in the blood.  They can be collected using a sterile needle and razor blade (or scalpel).  Sites for collection of skin snip specimens from patients with nodules:  on the chest (over the ribs)  on the hips  on the legs (tibia)  on the back (shoulder-blades  Sites for collection of slit skin specimens from patients without nodules  Top of the buttocks  Calves (upper outer part)  Back (shoulder blades)  The collected skin snips is immersed in a conical centrifuge tube containing about 1 ml of fresh physiological saline and centrifuged.  The skin snip and sediment are examined microscopically for microfilariae using the 10X objective.  Skin scraping  The skin scraping should be collected from the margin of the ulcer or lesion by using sterile scalpel.  Skin scraping is used for demonstrating the presence of amastigotes microscopically, in a smear taken from the skin lesion, after staining with Giemsa or leishman stain.  Faculty of medicine and health sciences LECTURE 10&11 Diagnostic Parasitology Examination of aspirates, biopsy and other materials for parasites Asst. Prof. Dr. LINA M. Q. AL-AREQI Examination of aspirates, biopsy and other materials for parasites Aspirates  The examination of aspirated material for the diagnosis of parasitic infections may be extremely valuable, particularly when routine testing methods have failed to demonstrate the organisms.  Aspirates most commonly processed in the parasitology laboratory include fine-needle aspirates.  Examination of lymph node aspirate  Examination of lymph node aspirate may be used for the detection of motile trophozoites of Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense.  These protozoa may be seen in swollen lymph node in early stages of African trypanosomiasis.  Impression smears from tissues should be prepared and stained with Giemsa stain.  Aspiration of cyst material  Aspiration of cyst material for the diagnosis of hydatid disease is a dangerous procedure and is normally performed only when open surgical techniques are used for cyst removal.  The entire structure of the larval form of E. granulosus is called hydatid cyst and within the cyst, miniaturizations of the entire hydatid cyst may occur; these are referred to as daughter cysts.  Both types of cysts are surrounded by a protective cyst wall and laminated layers of germinal tissue.  Aspiration of cyst material  The components found in the fluid of older E. granulosus cysts that typically include daughter cysts, free scolices, hooklets, and miscellaneous nondescript material may evolve are defined as hydatid sand.  The protoscolex of E. granulosus may be invaginated or evaginated and the hooks of the scolex can be easily seen with focusing.  The viability protoscoleces may be determinated by staining with eosin where the viable protoscoleces do not stain and non-viable protoscoleces stain red.  Biopsy material  Biopsy specimens are recommended for the diagnosis of tissue parasites and success in detection of parasites in tissue depends in part on specimen collection and on the presence of sufficient material to perform the recommended diagnostic procedures.  The larva of T. solium (cysticercus cellulosae) which is spherical (or slightly oval), yellowish white, can be detected in the biopsy specimens from different site of human body.  Biopsy material It contains two chambers: outer one is a bladder like sac filled with 0.5 mL of vesicular fluid and the inner chamber contains the growing scolex with hooklets and a spiral canal. Examination of the affected skeletal muscle biopsy is the method of choice for recovery of the encysted larvae of Trichinella spiralis  Corneal scrapings  Corneal scrapings are the specimen of choice for recovery of Acanthamoeba infections of the eye.  A rapid method for the diagnosis of Acanthamoeba keratitis involves the use of calcofluor white, which is a chemofluorescent dye with an affinity for the polysaccharide polymers of amebic cysts.  Buffy coat and bone marrow preparations  A buffy coat is a layer of white blood cells between the plasma and red blood cells that results from centrifuging whole blood.  Buffy coat preparations can be made from centrifuging blood in capillary tubes and then breaking the tubes to obtain the buffy coat layer and red cells below it.  The motile trypanosome can be seen in the plasma above the buffy coat and the amastigote forms of visceral leishmaniasis are frequently found in blood monocytes and neutrophils in buffy coat preparation.  Buffy coat and bone marrow preparations Buffy coat cells may be extracted from blood specimens, stained with Giemsa stain, Material obtained by needle aspiration from bone marrow can be used to demonstrate amastigote stages of Leishmania donovani. Smears can then be prepared by fixing in methanol and staining with Giemsa stain.  Faculty of medicine and health sciences LECTURE 12 Diagnostic Parasitology Immunodiagnosis & Intradermal tests & Cultivation of parasite Asst. Prof. Dr. LINA M. Q. AL-AREQI  Immunodiagnosis Several serological tests have been developed for detection of antibodies to parasites using antigens from cultured parasites or from natural or experimental infections in animals or humans. In some instances, diagnosis is attempted by serological demonstration of parasitic antigens in blood, tissues or secretions of suspected patients  Advantages  The advantages of the serological tests include:  They are rapid tests in which the more than one sample can be performed at the same time.  They are used for detection of asymptomatic infections or chronic carriers.  They are helpful in monitoring the response to treatment.  They are useful for those parasites that require invasive technique for microscopic diagnosis.  Some techniques are performed at anywhere and not required further requirements.  Disadvantages  The immunodiagnosis in parasitic infections has only limited value due to various factors.  Parasites are complex antigenically and exhibit wide ranging cross-reactions so that serological tests are not sufficiently specific.  The distinguishing between past and current infection is difficult.  The species of parasites cannot be differentiated by these methods.  They cannot detect the stages of the parasites e.g : malaria parasites.  They cannot detect the sites of infection for the systemic parasites.  These techniques are not available for all types of parasites and they have limited values to detect the intestinal parasites.  Types of serological tests  The following serological test may be used to detect the parasitic infections:  Enzyme-linked immunosorbent assay (ELISA)  Indirect hemagglutination (IHA)  Indirect immunofluorescence antibody (IFA)  Complement fixation test (CFT)  Enzyme-linked immunosorbent assay  There are different types of ELISAs available for the detection and quantitation of either the antigen or antibodies in serum and other body fluids.  The direct and indirect ELISA techniques are the most commonly used techniques where the direct (sandwich) ELISA is used for the detection of antigen and the indirect ELISA is used for the quantitative estimation of antibodies in the serum and other body fluids. Principle Principle  Indirect hemagglutination  IHA is used to detect the antibodies in the serum. Principle  Indirect immunofluorescence antibody  The indirect immunofluorescence test is used for detection of specific antibodies in the serum and other body fluids.  Principle:  Complement fixation test  CFT is to detect either antigen or antibody in the patient serum. Principle  Applications of immunodiagnosis  There are some of the applications of immunodiagnosis: Amoebiasis  Serology is of no value in the diagnosis of acute amoebic dysentery or luminal amoebiasis, but in invasive amoebiasis, particularly in liver abscess, serology is very useful by using IHA technique.  Cellulose acetate precipitation (CAP) is also used for invasive amoebiasis and tech Lab E. histolytica test was able to detect galactose lectin (galnac) antigen in almost all patients of amoebic liver abscess.  Giardiasis  ELISA have been developed for detection of Giardia and commercially available ELISA Kit to detect Giardia-specific antigen 65 (GAS 65).  The sensitivity of the test is 95% and specificity is 100%, when compared to conventional microscopy. Trypanosomiasis  Serological tests used to detect trypanosomiasis are IHA, IFA, and ELISA.  Specific antibodies are detected by these tests in the serum within 2–3 weeks infection.  Specific antibodies can be demonstrated by IFA and ELISA in CSF.  Leishmaniasis  IHA, IFA and CF with leishmania antigen are usually positive in kala-azar.  IFA test is positive very early in the disease, even before the appearance of symptoms and becomes negative within 6 months of cure. Malaria  IFA, ELISA and IHA are sensitive and specific, but are not useful for diagnosis of acute malaria because antibodies persist for some years after cure and a negative test may however help to exclude malaria.  Rapid immuno-chromatographic tests are available for diagnosis of the malaria parasites.  Toxoplasmosis  Serological tests offer the most useful diagnostic method in toxoplasmosis and IFA, IHA and CF were other useful tests.  At present ELISA is routinely used in toxoplasma serology and it is very informative as it provides titres of IgM and IgG antibody separately for better interpretation of the results. Cryptosporidiosis  IFA and ELISA using purified oocysts as antigens have been used to detect circulating antibodies specific to Cryptosporidium parvum.  Intestinal helminths  Antibodies can be demonstrated in most intestinal helminthiases, but extensive cross-reactions limit their use in diagnosis. Filariasis  IHA and bentonite flocculation test (BFT) with antigen from Dirofilaria immitis gives positive reaction in patients, and high titres in tropical pulmonary eosinophilia, but cross-reactions are frequent.  Immunochromatographic filariasis card test (ICT) is a new and rapid filarial antigen test that detects soluble Wuchereria bancrofti antigens in the serum of infected humans.  Trichinellosis  BFT is useful for detection of antibodies against trichinellosis. Echinococcosis  Several serological tests have been developed using hydatid fluid or scolex antigens from hydatid cysts in sheep.  IHA, IFA and ELISA are very sensitive and cross-reactions occur with cysticercosis.  Intradermal tests Intradermal tests have been used in many parasitic infections and they are sensitive and persist for many years, sometimes even for life, but specificity is relatively low.  Casoni’s test  Casoni’s test had been used widely in the diagnosis of hydatid disease since its original description in 1911.  The antigen is sterile hydatid fluid drawn from hydatid cysts from cattle, sheep, pig or humans, filtered and tested for sterility.  Intradermal injection of 0.2 ml of the antigen induces a wheal and flare reaction within 20 minutes in positive cases and a saline control is used.  False-positive tests are seen in schistosomiasis and some other conditions, therefore Casoni’s test is now largely replaced by serological tests.  Leishmanin test  Leishmanin (Montenegro) test is sensitive and relatively specific and the antigen is obtained from cultured leishmania and consists of killed promastigotes in phenol saline.  Intradermal injection of 0.1 ml induces a papule 5 mm or more in diameter in 48-72 hours.  This delayed hypersensitivity test is positive in cutaneous leishmaniasis and negative in diffuse cutaneous and visceral leishmaniases.  Fairley’s test  This skin test is group-specific and gives positive results in all schistosomiases.  The intradermal allergic test uses antigen infected snails, cercariae, eggs, and adult schistosomes from experimentally infected laboratory animals. Skin test in bancroftian fi lariasis  Intradermal injection of filarial antigens (extracts of microfilariae, adult worms and third-stage larvae of Brugia malayi, or the dog filaria, Dirofilaria immitis induce an immediate hypersensitivity reaction, but the diagnostic value of the skin test is very limited due to the high rate of false-positive and negative reactions.  Cultivation of parasite  Many parasites can now be grown in culture, but this has not become a routine diagnostic method in parasitic infections.  It is sometimes employed for accurate identification of the parasite species and it is more often employed for obtaining large yields of the parasite as a source of antigen, for animal inoculation, drug sensitivity testing, for experimental or physiological studies and for teaching purposes. The protozoa which can be cultured in the laboratory are: Entamoeba histolytica. Giardia lamblia, Trichomonas vaginalis, Leishmania species, Acanthamoeba species, Naegleria fowleri, Balantidium coli and Plasmodium species  The larval-stage intestinal nematodes such as Strongyloides stercoralis and Hookworm can be also cultured.  E. histolytica and other intestinal amoebae can be grown in diphasic or monophasic media, in media containing other microorganisms or in axenic cultures.  Boeck and Drbohlav’s diphasic medium, the classical culture medium for amoeba has been modified by various workers.  Cultures can be obtained from feces containing cysts or trophozoites. Balamuth’s monophasic liquid medium is also used commonly for cultivation of amoebae and other intestinal protozoa.  Both protozoa and bacteria present in stools grow in the above media and the bacterial growth can be reduced by addition of penicillin or other antibiotics that do not inhibit protozoa.  Axenic cultures (pure cultures without bacteria or other microorganisms) were first developed by Diamond in 1961.  Balantidium coli grows well in Balamuth’s’ medium and Giardia lamblia had been established in association with candida.  Trichomonas vaginalis grows very well in several commercially available media such as trypticase serum media.  Naegleria and Acanthamoeba from CSF can be grown on agar plates heavily seeded with Escherichia coli.  The classical Novy-MCNeal-Nicolle (NNN) medium first described in 1904 for cultivation of leishmania is equally satisfactory for trypanosomes also.  Cultivation of malaria parasites was first obtained by Bass and Jones in 1912 , in which about 10-12 ml of defibrinated or heparinised blood rich in ring forms of malaria parasite, mixed with 0.2 ml of 50% dextrose solution are incubated at 37° C in a sterile test tube in an upright position.  The blood separates into the erythrocytes below, plasma above and the buffy coat in between.  Malaria parasites grow in the erythrocyte layer immediately below the buffy coat and smears are collected from this layer at intervals, without tilting the tube.  Segmented schizonts are usually observed after incubation for 24 to 36 hours.  The intestinal nematode may culture by using of the following methods:  Harada-Mori strip method  Petri dish method  charcoal method  Koga agar method  Baermann method