Principles of Parasite Diagnosis

The feces is the sample how do we see it

Direct smear · flotation

Sedimentation · Baermann

Egg counts · coproculture

THe sample feces what is the immunoligcal and molecular

• coproantigen test

• Copro-PCR

The sample is blood how do we see it

Thick and thin smears

Modified Knott's

The sample is feces what is immunoligcal and molecular

• heartworm anitgen

• anitbody ELISA IFAT and CFT

• PCR

The sample is skin and hair what does it look like

Direct collection

Scraping · KOH · tape

The sample if skin and hair what is immunoligcal and molecular

• no immunoligcal

• PCR

The sample is tissue and organs what is do we see

Pepsin digestion · histology

Imaging · necropsy

The sample is tissue and organs what is the immunoligcal and molecular

• immunohistochemistry

• PCR

Three questions before you order a test

1 · Where does it live? (Gut · lung · blood skin · tissue)

2 · What stage does it shed? (Egg · oocyst · larva microfilaria · antigen · DNA)

3 · Is it time to look yet? Is the animal past the prepatent period?

Skip the third question why

and a perfectly good test will mislead you. During the prepatent period the animal is genuinely infected — and genuinely negative.

What is the Direct fecal smear test

Feces is the most common sample in practice — cheap, non-invasive, and it samples the gut without opening the animal.

What is the Principle for Direct fecal smear

§ About 2 mg of feces — a match-head — in a drop of saline, coverslip, read at 100–400×.

§ The saline keeps protozoa alive and moving.

• can also add legel’s iodine on the slide if we don’t see any motile

What are the strengths for Direct fecal smear

Quick and simple

A quick first look in urgent cases

The only routine method that shows motile trophozoites — Giardia, Tritrichomonas

What are the limitations for Direct fecal smear

A tiny sample — light infections are missed routinely

Debris obscures the field

A positive is as good as any positive. A negative cannot rule out infection.

Two things decide whether a flotation works:

1 the solution

2 the procedure.

A solution denser than the eggs but lighter than the debris: what will happen

the eggs rise, the debris sinks, and a coverslip on the meniscus collects what floated.

Are fluke eggs heavier ?

are heavier and don’t float so we go through sedimentaition which simply using water/letting the eggs sediment to the bottom

If we choose Sodium nitrate, commercial what its for

The routine choice — common eggs and cysts

If we choose zinc sulfate, commercial what its for

Giardia cysts; some lungworm larvae

If we choose Sheather’s sugar, commercial what its for

Cryptosporidium; least distortion

If we choose Ssaturated sodium chloride , commercial what its for

Cheap — but crystallizes and distorts

What is the trade off for Fecal flotation

trade offs that if you’re using high specific gravity solution the chances of getting the distored morpholgy of the egss is to increase because of the increase osmotic pressure

Passive / standing flotation what are the characteristics

1. Feces + flotation solution in a cup

2. Strain into a tube or fecalizer

3. Coverslip on; stand 10–20 min

4. Lift the coverslip and read it

Simple and fast · commercial kits · lower sensitivity

Centrifugal flotation (Wisconsin) what are the characterisitcs

1. Feces + flotation solution in a cup

2. Strain into a centrifuge tube

3. Coverslip on; spin ~1,500 rpm × 5 min

4. Lift the coverslip and read it

5. Can be used as a quantitative float — eggs per gram of feces

Several times more eggs · higher sensitivity ·

needs a centrifuge

Why is centrifugation important

Centrifugation is the single biggest improvement available to you

Centrifugation increases the sensitivity of the test!

What is the principle for Quantitative fecal float — McMaster, FLOTAC, Wisconsin

Flotation concentrates parasite

eggs/oocysts; count stages within the McMaster

chamber grids and convert the count to EPG/OPG.

What is the key output for Quantitative fecal float — McMaster, FLOTAC, Wisconsin

Eggs or oocysts per gram of feces

(EPG/OPG): an estimate of shedding intensity,

not a direct adult-worm count.

What are the strengths for Quantitative fecal float — McMaster, FLOTAC, Wisconsin

Rapid, inexpensive, standardized, and

useful for comparing animals or tracking group-

level shedding over time.

What are the limitations for Quantitative fecal float — McMaster, FLOTAC, Wisconsin

Lower sensitivity for low egg burdens;

results vary with sample quality, fecal consistency,

parasite biology, and the method’s detection limit.

What are the sensitivity for Quantitative fecal float — McMaster, FLOTAC, Wisconsin

McMaster 25–50 EPG; FLOTAC 1–5

EPG; Wisconsin flotation 1 EPG

What is the key use of Quantitative fecal float — McMaster, FLOTAC, Wisconsin

Herd/flock parasite surveillance, identifying high

shedders, targeted selective treatment, and fecal egg

count reduction testing (FECRT) for anthelmintic efficacy;

What is the McMaster method

What is FLOTAC method

What is the purpose of Sedimentation

§ Heavy eggs settle in water instead of floating

§ For fluke and acanthocephalan eggs

What is the limit for Sedimentation

the debris settles too — slow to

read

Why do we use Baermann

§ Live larvae migrate out of the feces into

water

§ For lungworm L1 and Strongyloides

What is the limit for Baermann

Limit: fresh feces only, and overnight

What is Coproculture

§ Eggs hatch to L3 in vermiculite over 7–14 days

§ Names the genus when all strongyle eggs look alike

What are the limits for Coproculture

Limit: slow; now yielding to sequencing

Blood tests for parasites — the modified Knott's test what is the principle

• Concentrates microfilariae from blood by lysing red blood cells and centrifugation.

• Sediment is examined microscopically for microfilarial detection and identification.

What are the strengths for Blood tests for parasites — the modified Knott's test

• More sensitive than a direct blood smear.

• Helps identify microfilariae based on size and morphology.

What are the limitations for Blood tests for parasites — the modified Knott's test

• Detects only circulatingvmicrofilariae, not adult worms.

• Negative in occult infections (adult worms present, no microfilariae) and when microfilariae are scarce.

When do we use skin - scraping, KOH, and tape

Ticks, lice, nits and fleas you collect and identify — intact, because a crushed mite or a tick without its mouthparts loses the characters you need. Flea dirt on damp paper leaves a red halo. Mites you cannot see, so you go into the skin.

What is superficial scraping

§ Broad and shallow, over a large area and several sites

§ Sarcoptes, Notoedres, Cheyletiella, Otodectes

§ Scrape the crusted places — ear margins, elbows, and hocks

A negative scrape does not rule out what

mange — burrowing mites are few and deep.

What is KOH digest

1 volume of scraping in 10 volumes of 5–10% KOH dissolves hair and crust; mites, mite eggs and lice survive it intact

What is Tape preparation

surface mites and lice; perianal tape can be used for pinworms such as Oxyuris equi, and it collects tapeworm segments in dogs and cats.

What are the characteristics of Deep scraping

§ Squeeze the fold to express mites from the follicle

§ Scrape until there is capillary ooze — that is the endpoint

§ For follicular mites — Demodex

Tissue and organ methods is utilized for

For a parasite that never leaves the tissues, or has no stage in the circulating blood (e.g. Trichinella), no fecal or

blood method will find it. You have to go to where it lives.

What is Pepsin digestion —

muscle in acid-pepsin at 37 °C for 60–90 min, sedimented and examined: the standard recovery of Trichinella larvae

Direct observation and necropsy what do we mean by that

total worm counts and organ washing (e.g. eyeworms)

What are the limitations for Tissue and organ methods

Invasive, slow, and needs a parasitologist or other specialist

What is the thin smear test

What is thick smear

What is wet mount

Antigen = parasite material test are for?

§ Detects parasite antigen/protein — mostly current infection

§ Heartworm antigen test — from adult female Dirofilaria

§ Coproantigen tests — Giardia in a SNAP device

§ SNAP 4Dx Plus: D. immitis antigen + five tick-borne antibodies, 8 minutes

Antibody = host response test for what

§ Detects the host's response — past or present

§ ELISA, IFAT, CFT, agglutination, immunodiffusion

§ Where antigen tests are insensitive — feline heartworm,

Toxoplasma, Neospora

§ A positive does not by itself mean current infection

What are the strengths for Immunological tests

finds infections microscopy cannot — prepatent, intermittently

shedding, or occult. Fast, in-house, and needs no eye for morphology.

What are the limitations for immunological test

cross-reactivity between related species and maternal antibody in the young; antibody

persists long after the infection has gone; and a test exists for only a handful of parasites.

What is Molecular tests — PCR, sequencing

Principle. Extract parasite DNA from feces, blood, tissue or environmental samples → amplify a marker gene → read

the product, or sequence it and name the species.

What are the strengths for Molecular tests — PCR, sequencing

Sensitivity — copro-PCR finds what microscopy cannot

Species and genotype, where every egg looks the same

What are the limitations for Molecular tests — PCR, sequencing

Expensive, and mostly not available in the clinic

Turnaround of days, when you need an answer today

A positive detects DNA — not necessarily a living, clinically

relevant worm

When the result does not fit the clinical picture WHat do we do

Recheck the biology and the specimen before you change the diagnosis.

How do we recheck the sample

§ Check the sample — right site, enough of

it, fresh? A frozen sample kills the larvae a

Baermann needs.

§ Check the target — does this method see

the stage? A flotation cannot find L1 larvae.

§ Check the timing — still prepatent, or

shedding on and off? A two-week-old

puppy sheds nothing yet.

§ Check the meaning — could a positive be

incidental? Giardia DNA in a well dog with

normal feces.

§ Then act — repeat, resample, add another

method, or rethink the differential.

How to approach any parasite diagnosis! Six steps, always in this order.

1 · SUSPECT signs, signalment, exposure

2 · LOCATE where does it live, general lifecycle?

3 · TARGET what stage does it shed?

4 · SAMPLE feces · blood · skin · tissue

5 · TEST the method that sees that stage

6 · INTERPRET does it fit the patient?

Errors arise at three stages : Pre-analytical

wrong patient or sample, poor collection, timing, transport, storage, contamination

Errors arise at three stages : analytical

wrong test, too insensitive, cross-reaction, inhibition, equipment or reader error

Errors arise at three stages : post-analytical

misidentification, wrong interpretation, transcription, host biology ignored

What are examples of False positive — parasite absent, test positive

Pseudoparasite – parasite look alike

Contaminated sample, or a specimen mix-up

Spurious parasitism – Coprophagy, prey

Cross-reaction in an antigen or antibody test

Antibody from past exposure, not current infection

DNA carry-over contaminating a PCR

What are exampls of errors in False negative — parasite present, test negative

Prepatent period – no shedding yet

Wrong sample, wrong site, wrong time

Spurious parasitism – Intermittent, Sporadic shedding or parasitemia

Occult infection, Single sex nematode infection

PCR inhibitors, or poor DNA extraction

Parasite sequestered in tissue, or recently treated