Diagnostic Techniques Flashcards
Why test for parasites?
- Tailor therapy/ prophylaxis for individuals.
- Detect developing resistance
- Reduce unnecessary use of medications in low risk population’s.
- Detect emergence of new parasites especially with warming climate.
Who needs parasitology testing?
- Clinically ill ( Diarrhea, vomiting, bloody stool, anemia, eosinophilic enteritis, coughing, once bacteria/ virus r/o)
- High risk animals ( young, high exposure, susceptible such as immunocompromised, pregnant, old, young, nursing)
What is the definition of the word Prevalence?
Proportion of animals (%) infected with a parasite
What is the definition of Intensity of infection?
Number of parasite/parasitic stage in an individual animal
• Usually expressed in eggs/oocyst/larva per gram of feces • When expressed as mean/median of a herd/population only positive
animals are considered
What is the definition of sensitivity?
The ability of the test to correctly detect individuals infected with a parasite
(true positives)
What is the definition of specificity?
The ability of the test to correctly detect individuals without a parasite (true
negatives?
What is the definition of a pseudoparasite?
an object or organism that resembles or is mistaken for a parasite
What kind of tests can be run on fecal samples?
- Fecal smear
- Fecal flotation
- Fecal sedimentation
- Baermann method
- McMaster method/ FLOTAC
- Coproculture (Fecal culture)
What are the most important things to remember when doing a fecal examination?
• Important to have fresh samples, preferably directly collected from the animal
• At least 10 g of feces should be collected
• Feces collected from yard, pen or litter box may be old and egg may embryonate, oocyst
may sporulate
• Refrigerate (4 degree C) to prevent development
• Freezing is inefficient – freeze thaw destroys eggs/larvae
What is a direct fecal smear and what are the pros and cons?
- Qualitative fecal exam
- Simple quick and easy
- Good for moving protozoal trophozoites (giardia), mobile amoeba
- Can be fixed with a special stain (e.g for Cryptosporidium)
- Negative result inconclusive, positive result equally valid!
What is a fecal flotation and what is the most important thing to remember when preforming the test?
- Based on differential specific gravity of parasite eggs/cysts, fecal debris and flotation solution.
- Flotation solution must have higher specific gravity than parasite eggs or oocysts.
- Specific gravity of most parasite eggs are 1.1 – 1.2 g/ml
- Flotation solutions should be > 1.2
What parasite would a fecal float be best used for?
- Best for most nematodes and cestode eggs, some protozoal (oo)cysts
What happens if you use a solution for fecal float that is too low in specific gravity? To high?
- Some fluke eggs won’t float especially if solutions of lower specific gravity are
used - Higher specific gravity solution – distorts protozoal trophozoite, cysts and
some helminths eggs, larvae – Proper choice of flotation solution
What is the ideal specific gravity of a fecal float solution?
> 1.2
What are the steps to complete a passive/ standing fecal float?
- ) Feces + flotation solution in beaker / cup.
- ) Strain and transfer into test tube/ fecalizer
- ) Add coverslip and let it stand for some time
- ) Examine coverslip under microscope
What are the steps to complete a centrifugal fecal flotation?
- ) Feces + flotation solution in a beaker/ cup
- ) Strain and transfer to test tube
- ) Add coverslip and centrifuge ~ 1500 rpm for 5 minutes
- ) Examine coverslip under microscope
What are the pros and cons of using a passive/ standing fecal flotation?
Pro:
- Simple and Fast
- Commercial kits available
Cons:
- Lower sensitivity
- Qualitative or semiqualitative.
What are the pros and cons of using centrifugal fecal flotation?
Pro:
- Recovers more eggs/ ova
- Higher sensitivity
- Can be quantitative
Con:
- More labor intensive
- More expensive
What is most important to remember when deciding between a passive/ standing fecal floatation or a centrifugal fecal flotation?
Centrifugation increases the sensitivity of your tests. Centrifugation recovers 3-5 times as many eggs over other passive floatation techniques
What is the McMaster Method?
- Commonly used quantitate technique for helminth eggs in
livestock - It uses a counting chamber that enables a known volume of fecal
suspension to be examined microscopically - Based on the principle of passive flotation (no centrifugation
required) - Quantitative
What is the steps to preforming the McMaster method?
- ) The fecal slurry is made with a known amount of feces and a known amount of flotation solution
- ) The slurry is sieved through a strainer
- ) Filtrate is used to fill the counting slide chamber
- ) Calculation (according to the protocol)
What is the Wisconsin double centrifugation?
Sensitive test that has 2 centrifugation steps, it is quantitative.
Most sensitive flotation technique in low infection intensities (1 epg or less)
What are the two centrifugation steps in the wisconsin double centrifugation?
Two centrifugation steps
- 1st – Fecal slurry made up of feces and water
- 2nd – Sediment of 1st step homogenized with flotation solution
Why is the wisconsin double centrifugation test challenging in clinics?
- Requires larger space (centrifuges)
- More time
- Expensive
What is fecal sedimentation and when would it be used?
- For eggs with higher specific gravity (e.g trematodes and acanthocephalan eggs) that do not float easily or eggs that are easily distorted by flotation solution
- More sensitive than direct smear
What are the steps to the traditional sedimentation method?
Mix 10 g of feces with 100 ml of water
- ) Mix and strain, collect filtrate in a beaker
- ) Allow to sediment for 5 minutes
- ) Allow to sediment for 5 minutes
- ) Siphon off supernatant
- ) Examine the sediment
What is the Baermann method? What would be a parasite this would be an ideal test for?
- For parasites that shed larvae in the feces (e.g. Dictyocaulus, lungworms)
- Feces suspended in water overnight -> Migration of live larvae out of the fecesinto water
- Should only be used in fresh feces (live larvae)
What is a coproculture (fecal culture)? What is it used for?
- Eggs of strongyles/trichostrongyles are virtually impossible to
diagnose morphologically so eggs are grown in a culture
medium (vermiculite) at room temperature for several days to
the third stage larvae (L3) - L3s are morphologically distinguishable
- Usually done in pooled samples
What are the steps to a coproculture?
- ) 20-30 grams of feces + tap water vermiculate
- ) Incubate at room temperature, maintain moisture
- ) After 7-14 days Larvae recovered (Baermann technique)
- ) Microscopy
What are the limitations of fecal based diagnosis?
- ) Chances of false negatives
- ) Chances of false positives
Why may there be false negatives on fecal tests?
- Sporadic shedding
- Prepatent period – Parasites not yet mature to shed eggs/larvae
- Single-sex nematode infections
- Senescent infections
- Old feces (hatched eggs)
What is a false negative test?
Negative test result but the host has parasites
What is a false positive test?
Positive test result but no parasitism
Why may there be a false positive on fecal tests?
- Coprophagia/Predation – Spurious
parasites - Pseudoparasites
- Many parasite species shed identical eggs – Diagnostic challenge
What is a direct blood smear? What kind are there?
S implest blood parasite detection procedure is by direct microscopic examination of whole blood.
- Thick smear
- Thin smear
What is a thick bloodsmear and what is a parasite it is good to use for?
o Examining a drop of blood on a slide
o Mainly for motile microfilariae of D. immitis
o Quick and easy method but less reliabl
What is a thin bloodsmear and what is a parasite it is good to use for?
o A drop of blood spread on the slide
o Not suitable for microfilaria larvae
o Suitable for Trypanosomes, Babesia, Thelaria, Anaplasma
What is the steps for thin blood smears?
- ) Make slide ( Make blood smear, you know how to do this)
- ) Fix in methanol for 2 minutes
- ) Stains ( Giemsa, fields stain)
What is the Modified Knotts test used for?
Rapid detection and identification of microfilariae (larvae) of filarial nematodes in
blood
- Concentrates microfilaria from 1 ml of blood à more sensitive
What can be used to diagnose ectoparasites?
- Skin scrapping KOH digestion
• Collection and examination
What is superficial skin scraping? What wouls you use it for? Where should you scrape?
- On animal with pruritic or scaly skin ( Scabis, Cheyletiella, Otodectes etc.)
- The best areas to scrape are typically crusty ear margins and crusty areas on the elbows or hocks.
What is deep skin scrapping? What is a parasite this is used for, and what is the process to take a deep skin scrape?
For mites that reside deep in the hair follicle (E.g. Demodex )
- Squeeze the skin
- Scrape unitl there is light capillary bleeding – multiple sites
What is KOH digestion? What/ when is it used for?
If the scrapping contains much debris § If lice/mites are suspected and not found by inspection
What are the steps to KOH digestion?
- ) 1 volume of skin scrappings + 10 volumes of KOH in large beaker
- ) Heat until hair disolves
- ) Centrifuge and transfer the sediment to a petridish and observe under microscope at 10x ( you can see mite eggs, mites, and lice with this)
What is tissue digestion used for? What are the general instrictions? What is an example of a parasite you can use this for?
- For parasites that have their larval or adult stages in host tissues and organs
- Tissue is digested in digest solution (mostly acid pepsin) at a certain temp for certain time -> centrifugation -> digest examined
(e. g – Trichinella spp., some arthropods)
What is the peri-anal tape method? What are its pros and its cons?
- Preferred method to detect eggs of pinworms in large animals
- Eggs often attach to perianal region and may not been present in the feces
- In dogs/cats- used to detect segments and eggs of cyclophyllid cestodes (e.g Taenia spp.)
- Risky technique in regions where Echinococcus spp. is endemic.
What are immunologic tests against parasites? What is an example of one?
Detecting antibodies (against the parasites) and antigens (of the parasites) • ELISA – testing serum antibodies against parasites, parasite antigens
What are the strengths of Immunologic testing for parasites?
- Can identify presence of parasites when eggs are not recovered by flotation (e.g prepatent infection, intermittent egg shedding)
- Relatively simple, fast (commercial kits)
What are the limitations of immunological testing of parasites?
- Measures immune response to exposure – not necessarily active infection
- Cross-reactivity
- Not universally available
What are coproantigen tests? What is an example?
Coproantigen tests: Parasite antigens in feces Increasingly common for in-house detections (e.g cyst wall antigen for Giardia in dog/cat feces)
Example: Blood antigen test – E.g. 4dx snap test (IDEXX)
Coproantigen ELISA exists for Ascaris, hookworms, giardia.
What immunological tests are available for protozoal infections?
Antibody tests – Protozoal infections
- Complement fixation test
- Immunodiffusion
- Haemagglutination test
- Florescent antibody test
What are PCR tests for parasites?
- Extraction of parasitic DNA from eggs/larvae in feces (Copro PCR); blood or tissue stages, or adult stage
- Amplification of a specific genetic marker/gene or genes
- Analyzing the DNA sequence to ID the species
What is the Advantages of PCR parasite testing?
- Higher Sensitivity ( copro pcr than egg based method)
- Accurate
- Sequences for species level ID, genotyping, phylogenetics
What are limitations of using PCR testing for parasite identification?
- Not possible in clinics
- Expensive, Highly technical
