PRACTICAL AND ACCURATE DIAGNOSIS OF CLINICALLY IMPORTANT PARASITES

Transcription

1 PRACTICAL AND ACCURATE DIAGNOSIS OF CLINICALLY IMPORTANT PARASITES Michael W. Dryden, DVM, PhD Professor of Veterinary Parasitology Department of Diagnostic Medicine/Pathobiology College of Veterinary Medicine Kansas State University Manhattan KS 66506

2 Techniques: 1. Direct smear Essentially non-diagnostic for most intestinal parasites May be useful for the diagnosis of protozoal parasites that have motile trophozoite stages Limitation: small sample size

3 2. Fecal Flotation: Techniques Useful for concentration of parasite material in feces Based on differential specific gravity of parasite eggs/cysts, fecal debris and flotation solution. Flotation solution must have a higher specific gravity than parasite egg or oocysts. Specific gravity refers to weight of object compared to equal volume of water. Specific gravity of water is g/ml and most parasite eggs are g/ml Flotation solutions should be > 1.24 g/ml

4 Specific Gravity of Common Parasite Eggs - dogs and cats Physaloptera sp Taenia Trichuris vulpis Toxocara cati Toxocara canis Ancylostoma sp

5 Flotation Solutions Sugar: 454g / 355ml water 1.27 sp. gr. (add 2ml 37% formaldehyde) Sodium Chloride: 400g / 1000ml water 1.2 sp. gr. Magnesium sulfate: 400g / 1000ml water 1.2 sp. gr. Zinc sulfate: 371g / 1000ml water 1.18 sp. gr. Sodium nitrate: 400g / 1000ml water 1.18 sp. gr.

6 Fecal Techniques Passive Standing fecal Commercial kit Centrifugation swinging head fixed head

7 Simple vs. Centrifugation

8 Simple: Standing Fecal Exam Mix feces with 3 to 5 ml flotation solution Pour into shell vial or centrifuge tube Place in a supporting rack Fill with solution and place coverslip Remove and examine for parasite eggs or oocysts 5 to 20 minutes later.

9 Fecal Exam Preparation 1. Put two to five grams (approximate) feces into a small cup. 2. Add a few ml of flotation solution to break up the fecal mass.

10 Fecal Exam Preparation 3. Strain contents through a tea strainer and then pour into a 12 15ml centrifuge tube.

11 Fecal Exam Preparation 4. Add flotation solution forming a slight positive meniscus.

12 Fecal Exam Preparation - 5. Leave undisturbed in a test tube rack for 15 minutes. 6. Then lift coverslip directly off tube and put onto microscope slide. 7.Examine entire coverslip at 10X. Use 40X to identify. 8. Record results Standing Fecal

13 Ovassaay

14 Centrifugation Technique - Swinging Head 2 to 5-gm sample placed into cup or beaker and 10 ml flotation solution added & solution was mixed thoroughly Mixture poured through a tea strainer or cheese cloth into a 15-ml centrifuge tube Four to 5 ml of flotation solution (sp. gr. > 1.2) added to fill the tube forming a slight slight positive meniscus. A square 18 mm coverslip placed on tube Tube placed in centrifuge and balanced Centrifuge in a swinging bucket centrifuge for 5 minutes at 280xG; then let stand for 10 additional minutes. Coverslip carefully removed, placed on a microscope slide and examined systematically under 100x magnification.

15 Centrifuge

16 Centrifugation Technique Centrifuge in a swinging bucket centrifuge for 5 minutes at 280xG; then let stand for 10 additional minutes.

17 Fixed Head Centrifuge Technique Mix feces with 3 to 5 ml flotation solution in a beaker Pour into centrifuge tube straining through a tea strainer Add flotation solution, fill tube to within to 1cm of top. Place in fixed head centrifuge and centrifuge at 280xg for 5 min. Remove tube and place in a rack, then fill with flotation solution forming slight positive meniscus and cover with cover slip. Let sit for 10 minutes Remove and examine for parasite eggs or oocysts

18 Fecal Techniques - Study Centrifugation swinging head (280g for 5 min.) Commercial kit (passive) fecal exam Ovassay (15 min.) 1.1 vs. 1.2 specific gravity solutions

19 ovassay Comparison of Fecal Techniques Ovassay - Centrifugation - Hooks average of 3-2 gram samples centrifuge Av. eggs recovered

20 200 ovassay Comparison of Fecal Techniques Ovassay - Centrifugation - Rounds average of 3-2 gram samples centrifuge Av. eggs recovered zinc 1.1 zinc 1.2 sugar 1.2

21 ovassay Comparison of Fecal Techniques Ovassay - Centrifugation - Whips centrifuge 20 average of 3-2 gram samples Av. eggs recovered NA zinc 1.1 zinc 1.2 sugar 1.2

22 Specific Gravity Hydrometers

23 Specific Gravity Hydrometers Fisher ( Hydrometer sp. gr. Cat# B $18.56 Cole Parmer ( General Purpose Hydrometer sp. gr. Cat# P $18.80 Ward s Biology ( Specific Gravity Hydrometer sp. gr. Cat# 15 W 0887 $10.50

24 Comparison of Fecal Flotation Techniques Sugar vs. salt Time to examination Centrifugation vs. standing fecal Centrifugation vs. standing fecal vs. commercial kit Swinging vs. fixed head centrifuge Dip method

25 Toxocara Comparison of Fecal Techniques Sugar 5 Min.Centrifugation vs. Float Results are mean of 3-2 gram samples 293 Specific Gravity = 1.2 Av. eggs recovered Centrifuge Float 5min Float 10min Float 15min Float 20min

26 Comparison of Flotation Techniques NaNo 3 5 Min.Centrifugation vs. Float 300 Toxocara Results are mean of 3-2 gram samples Specific Gravity = Av. eggs recovered Centrifuge Float 5min Float 10min Float 15min Float 20min

27 Comparison of Flotation Techniques Sugar 5 Min.Centrifugation vs. Float Ancylostoma Trichuris Av. eggs recovered Results are mean of 3-2 gram samples Specific Gravity = a = 2 of 3 samples negative for Trichuris a a a Centrifuge Float 5min Float 10min Float 15min Float 20min

28 40 Comparison of Flotation Techniques NaNo 3 5 Min.Centrifugation vs. Float Ancylostoma Trichuris Results are mean of 3-2 gram samples Specific Gravity = 1.2 a = 2 of 3 samples negative for Trichuris 30 Av. eggs recovered a a Centrifuge Float 5min Float 10min Float 15min Float 20min

29 Comparison of Fecal Flotation Techniques Sugar vs. salt Time to examination Centrifugation vs. standing fecal Centrifugation vs. standing fecal vs. commercial kit Swinging vs. fixed head centrifuge Dip method

30 Fixed vs. Swinging Head Centrifuge Mix feces with 3 to 5 ml flotation solution in a beaker Pour into centrifuge tube straining through a tea strainer Add flotation solution, fill tube to within to 1cm of top. Place in fixed head centrifuge and centrifuge at 280xg for 5 min. Remove tube and place in a rack, then fill with flotation solution forming slight positive meniscus and cover with cover slip. Let sit for 10 minutes Remove and examine for parasite eggs or oocysts

31 300 Toxocara Comparison of Fecal Techniques Fixed vs Swinging Head Centrifuges Ancylostoma Results are mean of 3-2 gram samples Specific Gravity = min. Cent. - Tubes removed & let stand 10 min. 210 Av. eggs recovered Swinging Fixed Swinging Fixed Sugar Sodium Nitrate

32 Comparison of Fecal Flotation Techniques Sugar vs. salt Time to examination Centrifugation vs. standing fecal Centrifugation vs. standing fecal vs. commercial kit Swinging vs. fixed head centrifuge Dip method

33 Comparison of Flotation Techniques 5 Min. Cent. 280xG - Fixed Head Toxocara Ancylostoma 300 Results are mean of 3-2 gram samples Specific Gravity = 1.2 Av. eggs recovered min. stand 3 rod dips 10 min. stand 3 rod dips Sugar Sodium Nitrate

34 Summary Sp. gravity < 1.2 will not recover Toxocara and Trichuris eggs Centrifugation is more efficient than Fecalizer, Ovassay or standing fecal. After centrifugation leaving coverslips on for an additional 10 minutes increased egg recovery Centrifugation in sugar recovered more eggs than sodium nitrate

35 Summary Sodium nitrate recovered more eggs than sugar in the standing fecal flotation. Ovassay failed to recover Trichuris in 5 of 12 samples even after 15 minutes.

36 Centrifuges - Used IEC Clinical Centrifuge Model CL, with 6 place swinging bucket rotor. $250 - $950 (Older model: not in production, excellent general purpose centrifuge: make sure it has rotor and shields) search under Clinical centrifuge or Table/Bench top centrifuge or IEC clinical centrifuge model CL