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BASIC TERMINOLOGY AND PRINCIPLES Symbiosis: Living together Commensalism: One symbiont benefits, other unaffected Mutualism: Both symbionts benefit Parasitism: One symbiont benefits, other is damaged 12/10/2012 Dr.T.V.Rao MD 2

The Reality of Parasites:

The Reality of Parasites 1.3 billion persons infected with Ascaris (1: 4 persons on earth) 300 million with Schistosomiasis 100 million new malaria cases/ year 12/10/2012 Dr.T.V.Rao MD 3

Laboratory Methods For Parasites In Faeces:

4 Laboratory Methods For Parasites In Faeces No technique is 100% successful in detecting parasites by a 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 found in the faeces. Whilst clinical symptoms or a case history may provide clues as to which parasites may be present, each faecal specimen should be treated as an unknown, as parasite stages unrelated to the clinical picture may be present. 12/10/2012 Dr.T.V.Rao MD

Faecal specimens may contain several stages of Parasites:

5 Faecal specimens may contain several stages of Parasites Faecal specimens are examined for the presence of protozoa and helminthes larvae or eggs. The stages of protozoa found in stools are trophozoites and cysts. The stages of helminthes usually found in stools are eggs and larvae, though whole adult ’ s worms or segments of worms may also be seen. Adult worms and segments of tapeworms are usually visible to the naked eye, but eggs, larvae, trophozoites, and cysts can be seen only with the microscope. 12/10/2012 Dr.T.V.Rao MD

Collection of faecal specimens:

6 Collection of faecal specimens 1. Because of the fragile nature of many intestinal parasites, and the need to maintain their morphology for accurate identification, reliable microscopic diagnosis can ’ t be made unless the stool is collected properly. 2. Approximately 10 grams of fresh faeces uncontaminated by urine, oil, water, dyes or radio-opaque into a clean plastic container. 3. The container should be free from antiseptics and disinfectants. 4. Label all samples clearly with the patient ’ s name, reference number, date, and time of collection. 12/10/2012 Dr.T.V.Rao MD

Collection of faecal specimens:

Collection of faecal specimens 5. All samples should be accompanied by a requisition form from the physician giving relevant clinical details and recent travel history. 6. Samples and forms from patients with a confirmed or suspected diagnosis of certain infectious diseases such as AIDS or hepatitis should be clearly labeled with “ Risk of Infection ” or “ Biohazard ” 7 12/10/2012 Dr.T.V.Rao MD

Collection of faecal specimens:

Collection of faecal specimens 7.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 the faeces to retain the morphology as near to the original as possible. 8. Formed samples can be kept in a refrigerator at + 4c for a short while, but not in incubator. 9. Any whole worms or segments passed should be placed in a separate container 12/10/2012 Dr.T.V.Rao MD 8

Collect the Information of the Patient :

Collect the Information of the Patient History (Age, occupation, residency, previous infection) Complaint Clinical examination Investigations - Laboratory investigations - Radiology - Surgical intervention (Exploratory) Provisional diagnosis Confirm the diagnosis 12/10/2012 Dr.T.V.Rao MD 9

The Microscopy in Parasitology:

10 The Microscopy in Parasitology The Microscope is the parasitologist ’ s main tool. If possible the Microscope - should be binocular; most suitable objectives are the x10, x40, and x100. The Microscope must be covered and immersion oil removed from the lens -with xylene or ether when not in use. Calibration of the Microscope Eyepiece Micrometer: On many occasions measuring the size of suspected parasites in faeces is helpful for identification.(eyepiece micrometer) 12/10/2012 Dr.T.V.Rao MD

Microscopic Examination of Wet Mount:

11 Microscopic Examination of Wet Mount Wet mount is the simplest and easiest technique for the examination of faeces, and this method should be performed in all laboratories at the peripheral level. A wet mount can be prepared directly from faecal material or from concentrated specimens. The basic types of wet mount that should be used for each faecal examination are saline, iodine, and buffered methylene blue. 12/10/2012 Dr.T.V.Rao MD

The saline wet mount:

12 The saline wet mount Is used for the initial microscopic examination of stools. It is employed primarily to demonstrate worm's eggs, larvae, protozoan trophozoites, and cysts. This type of mount can also reveal the presence of red blood cells and white blood cells. 12/10/2012 Dr.T.V.Rao MD

The Iodine wet mount:

13 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. The buffered methylene blue (BMB) wet mount should be prepared each time amoebic trophozoites are seen in a saline wet mount, or when their presence is suspected. 12/10/2012 Dr.T.V.Rao MD

Direct saline and iodine mounts:

Direct saline and iodine mounts 1 . With a wax pencil writes the patient ’ s name or number and the date at the left-hand end of the slide. 14 12/10/2012 Dr.T.V.Rao MD

Preparing a faecal specimen :

Preparing a faecal specimen Place a drop of saline in the centre of the left half of the slide and place a drop of iodine solution in the centre of the right half of the slide. Note: If the presence of amoebic trophozoites is suspected, warm saline (37c) should be used. 12/10/2012 Dr.T.V.Rao MD 15

Preparation of Wet film:

Preparation of Wet film With an applicator stick (match or tooth pick), pick up a small portion of the specimen (size of a match head) and mix the drop of saline. 12/10/2012 Dr.T.V.Rao MD 16

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STOOL EXAMINATION 12/10/2012 Dr.T.V.Rao MD 17

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Acetic acid RBC haemolysis Clear ova Ether Dissolve fat M.f 12/10/2012 Dr.T.V.Rao MD 19


20 Examination 1. Put the slide with the mounts on the microscope stage and focus on the mount with the x10 or low-power objectiv e. 2. Regulate the light in the microscope field with the sub stage diaphragm. You should be able to see objects in the field distinctly. Too much or too little light is not good. 3. Examine the entire coverslip area with the x10 objective; focus the objective on the top left-hand corner and move the slide systematically backwards and forwards, or up and down. 12/10/2012 Dr.T.V.Rao MD

Examination Cont.:

21 Examination Cont . 4. When organisms or suspicious material are seen, switch to the high-dry objective, and increase the light by opening the sub stage diaphragm to observe the detailed morphology. -This is a systematic examination. If mounts are examined in this way, any parasites present will usually be found. If the mount is not examined systematically, parasites may be missed. Examine each microscope field carefully, focusing up and down, before moving to the next field. 12/10/2012 Dr.T.V.Rao MD

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STOOL EXAMINATION A Rapid Methods Saline smear Iodine smear saline Iodine 1% Huge number of: Eggs Protozoal troph. Motility (Amoeb, flagellates) Huge number of: Cyst morphological details 12/10/2012 Dr.T.V.Rao MD 23

Need for Concentration Methods for Faecal examination:

Need for Concentration Methods for Faecal examination A concentration procedure is performed mainly to separate the parasites from faecal debris. The concentration procedure not only increases the numbers of parasites in the sediment but it also unmasks them, making them more visible by removing organic and inorganic debris 12/10/2012 Dr.T.V.Rao MD 24

STOOL EXAMINATION Scanty infection Concentration techniques:

STOOL EXAMINATION Scanty infection Concentration techniques Sedimentation Floatation Heavy eggs (Ascaris egg) Operculated eggs (Trematodes) Larvae (Strong sterc.) Cysts Non Operculated eggs Trematodes ( S. m.) Cestode Nematode( Hookworms,Trichostong) Cysts 12/10/2012 Dr.T.V.Rao MD 25

STOOL EXAMINATION Saline sedimentation:

STOOL EXAMINATION Saline sedimentation 10 g stool Saline Mesh wire gauze Conical flask Sediment Emulsify 12/10/2012 Dr.T.V.Rao MD 26


STOOL EXAMINATION Kato technique Mesh screen Template Hole Remove the template Cellophane soaked by glycerin (clears faeces ( Egg count/ g stool Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni 12/10/2012 Dr.T.V.Rao MD 27

Stool examination other Texhniques:

Stool examination other Texhniques Stoll’s technique for eggs of Ascaris, T. trichiura., Hookworms, S. mansoni Baermann’s technique Detec. Of Nematode L. /stool, soil Cultures for Nematode larvae using Filter paper culture for Larvae of: St. stercoralis (A,L) and Hookworms 12/10/2012 Dr.T.V.Rao MD 28


29 Artifacts Artifacts other things, living or artificial, present in the stool that are not parasites and could mislead the laboratory worker. Note: “ Artifacts not to be mistaken for cysts ” . 12/10/2012 Dr.T.V.Rao MD

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31 Plant cells Air bubble Plant hairs Plant fibre Pollen grains Non-human coccidial oocysts Fat droplets Soapy plaques Starch cell Charcot leyden crystals Muscle fibers Fatty acids Macrophage Epithelial cells 12/10/2012 Dr.T.V.Rao MD

Examining other Specimens:

Examining other Specimens 12/10/2012 Dr.T.V.Rao MD 32

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NaOH Sputum Centrifuge 12/10/2012 Dr.T.V.Rao MD 33

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Lumbar puncutre Centrifuge Examine sed. 12/10/2012 Dr.T.V.Rao MD 35

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BLOOD EXAMINATION BLOOD FILMS Thin Thick Bld drop spread Air dry methyl alcohol Geimsa Air dry Geimsa Circular motion Malaria, Babesia, Filaria, Tryp. 12/10/2012 Dr.T.V.Rao MD 37

Observe the Thin and Thick Smear:

Observe the Thin and Thick Smear 12/10/2012 Dr.T.V.Rao MD 38

QBC Method is used in ….:

QBC Method is used in …. The QBC Malaria method is the simplest and most sensitive method for diagnosing the following diseases. Malaria Babesiosis Trypanosomiasis (Chagas disease, Sleeping Sickness) Filariasis (Elephantiasis, Loa-Loa) Relapsing Fever (Borreliosis) 12/10/2012 Dr.T.V.Rao MD 39



How to read the QBC results ….:

How to read the QBC results …. When the operator looks through the wall of the tube, the nucleus of the parasite fluoresces bright green, and the cytoplasm shows up as yellow-orange. The shape and colours are quite characteristic, and since the parasites are concentrated up to 1000X, there are usually a large number of them in any field of view in this area of the tube. 12/10/2012 Dr.T.V.Rao MD 41


URINE EXAMINATION SEDIMENTATION CONCENTRATION 15-20 min Centrifuge (2 min) Clean conical glass receptacle 12/10/2012 Dr.T.V.Rao MD 42

URINE EXAMINATION Membrane filtration technique:

URINE EXAMINATION Membrane filtration technique air 10 ml urine Nucleopore filter Eggs of Schistosoma + Saline 12/10/2012 Dr.T.V.Rao MD 43

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Indirect immunological diagnosis:

Indirect immunological diagnosis Serology – All tests available IHA ELISA More useful in Amoebiasis Leishmaniasis Malaria Toxoplasmosis Trichinosis Filariasis Echinococcosis Skin Tests – Specificity low, cross reactions common Casoni’s test Leishmanin test 12/10/2012 Dr.T.V.Rao MD 45

PowerPoint Presentation:

Programme Created by Dr.T.V.Rao MD for Basic learning in Human Parasitology for Undergraduate Medical Students Email 12/10/2012 Dr.T.V.Rao MD 46

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