Lab Examination Parasitology Unibraw

PARASITOLOGICAL LABORATORY EXAMINATION

DEPARTMENT OF PARASITOLOGY FACULTY OF MEDICINE BRAWIJAYA UNIVERSITY TEGUH WS ‘08

Introduction 

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Parasitic diseases:  Are considered to be “non life threatening”  neglected  Symptoms are mostly non specific or asymptomatic  Only some have specific symptoms  Clinical Diagnosis  difficult  to find confirmed diagnosis needs supportive examination  laboratory examination To get the definitive diagnosis  basic knowledge is needed:  Habitat of the parasite  sample/materials  Taking and sending sample (fresh or preserved)  Examination technique TEGUH WS ‘08

MATERIALS/SAMPLES STOOLS BLOOD URINE OTHER BODY LIQUIDS:

1. 2. 3. 4. • • • •

Sputum, Liquor Cerebrospinalis (LCS) Pleural fluid, Ascites etc.

OTHER MATERIALS:

5. • •

Skin scratch Tissue biopsy etc TEGUH WS ‘08

ROUTINE STOOL EXAMINATION 

WHY STOOLS??  ≈ GI tract/intestine is the most habitat of parasites

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WHAT SHOULD BE EXAMINED?  qualitative examination Macroscopic  consistency: formed / soft / loose / watery  Color: yellow / green / white  Odor : specific  Mucous  Blood  Fat  Food materials TEGUH WS ‘08

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CONSISTENCY FORMED

CYSTS

SOFT

LOOSE

TROPHS

WATERY TEGUH WS ‘08

Microscopic Qualitative : 

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Intestinal parasites or their derivatives : 

Worm eggs : Nematodes, Trematodes, Cestodes

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Protozoa : Cysts / Trophozoites

Artifacts /pseudoparasites 

RBC ≈ intra luminal bleeding/ ulcer

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White cells (PMN) ≈ inflammatory processes

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Charchot-Leyden crystals (breakdown products of eosinophils) ≈ allergic phenomena associated ŵ amebiasis & worm infections

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Macrophages ≈ bacterial & parasitic infection, similar ŵ Amoeba

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Epithelial cells ≈ normal desquamation of intestinal cells

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Eggs of Arthropods, plant nematodes and spurious parasites, yeasts etc TEGUH WS ‘08

Microscopic 

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Specific examination/procedure :  Concentration:  Sedimentation  Floatation  Culture Quantitative :  parasite burden in stools  number of worm eggs in stools (worm burden) TEGUH WS ‘08

Qualitative 

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Wet Mount  Direct smear : Saline,Lugol /KI, Eosin  Thick smear :  Cellophane Covered Thick Smear  Kato’s thick smear Preserved & Stained Film Concentration techniques:  Sedimentation :  

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simple centrifugation

Floatation  

Simple floatation Centrifugal floatation  direct  ZnSo4 TEGUH WS ‘08

DIRECT WET MOUNTS/ UNPRESERVED EXAMINATION 

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NORMAL SALINE SOLUTION 0,85% EOSIN 2% LUGOL/IODINE 1%

1.

Place a drop of solution (Saline, Eosin or Iodine) on a clean slide

2.

Take about 2 grams of stools and mix it to be uniform suspension

3.

Cover with 22 x 22 mm coverglass

4.

Examine under microscope TEGUH WS ‘08

THICK SMEAR Cellophane Covered Thick Smear (Kato & Miura, 1954) 

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Cut wettable cellophane into 22 x 30 mm strips and soak them for 24 hours or longer in a mixture of 100 parts of glycerol, 100 parts of destilled water 1 part of 3% aqueous Malachite green Place ± 50-60 mg (≈ bean) fresh feces on a clean standard slide and cover with one of the cellophane strips Invert the preparation, place it on paper towels, and press to spread the fecal material uniformly to the egdes of the strips Reverse the slide and allow it to stand at 40º C for 30 minutes or at room temperature for 1 hour Examine the slide under a low power objective; higher magnification can be used to confirm identification TEGUH WS ‘08

PRESERVATION OF STOOL SPECIMENS 

Polyvinyl Alkohol (PVA)

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Schaudinn’s Preservation 

Conventional

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Cupric sulfate modification

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Formalin Preservation

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Merthiolate-Iodine-Formalin (MIF) Preservation

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CONCENTRATION METHODS 

SEDIMENTATION  

For cysts, worm eggs, trophozoite Principle : difference of Specific gravity (SG) 

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FLOATATION  

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SG of solution < SG of the Parasites  Parasite will be placed on lower level

For Nematodes eggs Principle : SG > SG Parasite  Eggs will be floated/ placed on upper level

COMBINATION  

Centrifugal floatation Centrifugal sedimentation TEGUH WS ‘08

Simple Sedimentation  

For operculated worm eggs, larvae, cysts Procedure :  Thoroughly comminute ≥ 10 g feces in 50 – 100 cc tap water in a 250 ml or larger beaker glass using a stick or tongue depressor blade  Strain the suspension through two layers of wet gauze into a sedimentation flask  allow the material to sediment ± 1 hour  Decant the supernatant carefully (so as not to lose any sediment)  Resuspend the sediment by adding more tap water  allow ± 1 hour. This wash procedure can be repeated ≈ clarity of supernatan  Using long capillary pipette, remove small portion of sediment, place on a glass slide examine under microscope. If too thick add a drop of saline, or dilute with iodine

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CENTRIFUGATION  

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For Schistosomes eggs & Protozoon cysts 2-3 grams of stools + 20 mL tap water (± 10 x volume of stools) Strain the suspension through two layers of wet gauze into centrifugation tube Centrifuge with speed 1500-2300 rpm for 1-2 minutes Decant the supernatant. This procedure can be repeated until supernatant is clear (usually 2 times) remove small portion of sediment, place on a glass slide examine under microscope

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FLOATATION  

Principle : SG of solution > SG of Parasites  parasite will float Solution of NaCl /Brine solution: SG 1.200  

Ascaris, Trichuris, Hokworms eggs  Good results Schistosomes eggs, Ss + Hw larvae  shrinking

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Opercululated egs  sediment Sugar solution, Glycerin

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ZnSO4 solution (SG 1.180)  centrifugal floatation

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ZnSO4 CENTRIFUGAL FLOATATION     

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Non operculated worm eggs, Protozoon cysts,larvae 331 grams ZnSO4 crytals + 670 mL water = SG 1,18 1 part of stools + 10 parts of water  mix Strain  pour into 15 mL conical centrifuge tube Centrifuge at 400-500 or 2300 rpm for 1-2 menit  decant the supernatant Add 2 – 3 mL destilled water  recentrifuge (repeat 2-3 x) Add 3-4 mL ZnSO4 sol with SG 1.180 Centrifuge at full speed for 1- 2 minutes Use freshly flamed wire loop (diameter 5-7 mm) to touch the center of the surface film  object glass  examined with or without cover glass TEGUH WS ‘08

Method for using wire loop to remove surface film from ZnSO4 floatation technique

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QUANTITATIVE For Estimating:  Number of worm infecting host/worm burden   degree or severity of infection  Especially for intestinal nematode (Ascaris, Trichuris, Hookworms)  Most frequent method used  Kato-Katz’s Thick Smear  Stoll Egg Count Technique

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Kato Katz’s Thick Smear Procedure : As same as Kato Thick Smear, but:  Use wire strain for straining the stools  Amount of stool can be calculated  use thick paper - 1 mm thick  a hole Φ 9 mm ≈ 50-60 mg of stools, or - ½ mm thick a hole Φ 6,5 mm ≈ 20-25 mg of stools - cover with cellophane soaked with Malachyte green  allow it at 40º C for 30 minutes or at room temperature for 1 hour - examine under the microscope and count the number of eggs

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Stoll Egg Count Technique 

Equipments:  Long necked “Stoll flask” 60 mL with marker on 56 and 60 mL 

Stick/applicator for taking stools Stoll Pipette (0,075 ml and 0,15 ml)

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Glass beads (diameter 6 mm)

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Materials :  NaOH Solution 0,1 N 

Dissolve 4 g of NaOH in small amount of distilled water Add distilled water to bring total volume to 1000 ml

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Store until needed

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Stoll Egg Count Technique Procedure:  Add 0,1 N NaOH solution to the 56 mL mark of the Stoll flask  Using applicator stick carefully add feces to the flask  the level of fluid raised to the 60 cc mark  Add 5-10 small glass beads to the flask, stopper the flask  shake vigorously with up-and-down motion for a minute or more to obtain a homogenous suspension. If the stool is hard,  allow the mixture to stand for several hours or overnight  Use Stoll pipette to remove sample 0,15 mL from the center of the suspension  Expel the pipette content on to a slide  cover with 22x44 mm cover glass  Count all of the eggs on the slide (p). The number of egg per gram of feces, is the number of eggs obtained x 100. (NEPG = p x 100)  Correction factors according to consistency of stools:  Soft  x 1,5 ; Loose  x 2; Watery  x 3 TEGUH WS ‘08

Worm burden Daily production of eggs  Trichuris trichiura = 5.000/ day  Necator americanus = 9.000/ day  Ascaris lumbricoides = 200.000/day 

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If the total amount of feces be weight and the NEPG had been calculated  the number of worms infecting host can be estimated Single adult Ascaris may be dangerous, due to migrating tendency Hookworms infection with NEPG > 2500  clinical symptoms Trichuris infection with NEPG > 20.000  clinical symptoms

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Culture of feces Aims :  To differentiate the worms which had similar morphology of eggs, as:  Strongyloides stercoralis  

Necator americanus Ancylostoma duodenale

Trychostrogylus spp The larvae of those worm can be differentiated morphologically 

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HARADA MORI METHOD  

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Use conical/screw cap test tube vol. 15 mL Use a 13x120 mm paper strip that tapers at the end inserted into the tube. Write the specimen identification number on it Spread approximately 0.5 – 1.0 of feces on to the middle one-third of the strip,  forms a layer several millimeters thick Insert the strip into conical screw cap test tube  several millimeters of the tube bottom Carefully add distilled water until the water is slightly below the fecal mass  do not wash!! Keep test tube upright in the test-tube rack at room temperature (24º-28ºC) for 7-10 days Larvae in fluid can be heat-killed and removed from the bottom of the tube using a glass pipette  drop on a slide  iodineexamine TEGUH WS ‘08

Polyethylene tube      

Modification of Harada Mori culture by Sasa Using polyethylene sac instead of screw cap tube Filter paper strip or newspaper strip Easier and cheaper 5-10 days (about a week) The aim and procedure are the same as Harada Mori

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Sand culture technique  

For Soil Transmitted Helminths Procedure:  Sterile Petri-dish filled by sterile sand  Add feces on to it and covered  Rhabditiform larva (+) after 1 day  Filariform larva (+) after D 3-7

Filter paper/slant Culture   

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write sample identity Smear feces on to middle 1/3 part Place the strip on to a slide  petri dish so that one end rests on a short piece of glass tubing Add a shallow layer of distilled water Cover dish and allow it to stand at 24º-28ºC TEGUH WS ‘08

The culture can be examined under dissecting microscope for the presence of free living adult or larvae of S. stercoralis. Hookworm and trichostrogylid larva can be found in water

Cellulose Tape Preparation Procedure 

Grahams-Scotch’s Adhesive Tape Swab

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For diagnosing intestinal parasites which migrate to perianal region: 

Enterobius vermicularis (adult and eggs)

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Taenia solium & T. saginata eggs (less frequency)

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In the morning, prior to using the toilet or bathing

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Cellulose tape preparations should be taken at least 3-4 consecutive days before a patient is considered to be free from infection

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Adult male of Enterobius vermicularis 

can demonstrated by cellulose tape preparation also

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Can be seen with naked eye, crawling around the anus, but the male TEGUH WS ‘08

Materials and methods Procedures a. Place a strip of cellulose tape on a microscopic slide, starting 1.2 cm from one end, and running toward the same end. Wrap the tape over the edge of the slide. b. Peel back the tape, and, with adhesive side outward over a wooden tongue depressor. c. Press the tape firmly against the right and left perianal fold d. Spread the tape back over the slide, adhesive side down e. Press the tape down on the slide, examine directly under low power of microscope.

TEGUH WS ‘08

ANAL SWAB METHOD    

For diagnosing of Enterobius vermicularis eggs Is an alternative procedure for diagnosing pinworm infection This procedure is more complex than the cellulose tape method Offers no advantages over it

Procedure:  Immerse cotton swab in paraffin and petroleum jelly mixture, place swabs into tubes, store in refrigerator until needed  Spread the buttock and rub the swab over the perianal surface very gently. Insert the swab a short distance into the anal opening. Return the swab to the tube and label  Fill the tube ± half full ŵ xylene to cover the swab  allow for 5 min.  Remove the swab, centrifuge the suspension 500 g/ for 1 min., remove supernatant  examine under microscope

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Examination of Blood     

MALARIA FILARIASIS TOXOPLASMOSIS TRYPANOSOMIASIS LEISHMANIASIS

Examination of Blood Taking of peripheral blood samples  Capillary blood : finger tip, heel, auricle/ear lobe  Circulatory blood : v. mediana cubiti Preparation blood film  Thin blood film/smear : malaria, trypanosomiasis, babesiosis 

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Because of the small amount of blood used in preparation of thin film, they are not as sensitive for detecting parasites as one would like

Thick blood film/smear : malaria, filaria 

The amount of blood used in preparation of thick film is more  more sensitive, but less specific than thin film,

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Capillary blood administration

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Examination of Microfilaria 

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Direct examination (wet blood)  Directly examining the motility of microfilaria  Punction the finger tip with lancet  suck the blood with capillary tube drop blood on to a clean and dry slide  spread with other slide edge  directly examine under the microscope Knott Concentration Technique  Venous blood + anticoagulant  into a tube containing formaline solution 2% (10 cc)  centrifuge for 2 minutes  Decant supernatant, directly examine the sediment, or stained with Giemsa staining Straining using nucleophore filter  Venous blood + anticoagulant were strained using nucleophore filter  microfilaria will strained  stain with Giemsa examine

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Capillary tube

Wet preparation 20 l

Air dried

Stained with Giemsa 1 : 15 for 15 min.

Dehaemoglobin with water

Fixed with methanol Air dried

washed with running water Examined under compound microscope

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Examination of urine (Urinalysis)  

Urinalysis  centrifugation urine sediment Urogenital tract parasites  

Trichomonas vaginalis Schistosoma haematobium

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Others : Polychaete annelids, Ren worm Chyluria  microfilaria of Wuchereria bancrofti, Onchocerca volvulus Trophozoite of Entamoeba histolytica  fistel?

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Strongyloides stercoralis Larvae

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Examination of sputum  

DIRECT SMEAR METHOD For Pulmonary parasites  Paragonimus  Amoeba  Hydatid cyst  Strongyloides stercoralis Larvae

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OTHER SPECIMENS 

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Other body fluids  LCS  Ascites, pleural fluid Scratching /rub down of Skin or mucosal Muscle biopsy Duodenal fluid aspiration Lymph-node biopsy Soil Finger nails

TEGUH WS ‘08

Thankyou

TEGUH WS ‘08