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Medical ParasitologyLaboratory

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Introduction

Selection of the proper procedure is paramount in thesuccessful diagnosis of parasite infections

Specimens must be collected properly and transportedwithout delay.

Specimens should be processed immediately orpreserved to maintain the quality of the specimen.

Processing of specimens involves a number ofstandardized procedures used in the routine analysis.

Improperly collected specimens may lead to theinability to identify parasites or incorrect interpretationof results.

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Collection Fecal Specimens

Specimens should be collected in clean, wide-mouthed containers with tight fitting lids and sealed in

plastic bags for transport.

Specimens should always be properly labeled,

including the patient's name, identification number,physician's name and the date and time that thespecimen was collected.

Care should be taken to avoid contamination with

urine or water, which might harm existing organismsor introduce free-living organisms from theenvironment.

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Collection Fecal Specimens

Fresh specimen are mandatory for the recovery of motiletrophozoites.

Liquid specimen should be examined within 30 mins of

passage.

Semi-formed specimens may contain mixture of protozoan

trophozoites and cysts, should be examined within 1 hour.

Acceptable specimens should contain a sufficient amount

of material to perform the examination procedures,approximately 2-5 grams of feces.

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Microscopic Examination of Fecal

Specimen

The microscopic examination consists of three parts :

Direct wet smear

Concentration

Permanent stains

Each part of the exam provides valuable information thataids in the overall diagnosis.

The methods are chosen from many that are reliable andwell proven.

Each laboratory must choose those methods that fit its

individual needs and work flow

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Direct Wet Smear

To assess worm burden of patient To provide quick diagnosis of heavily infected specimen

Trophozoites and cysts of protozoa are lens-like in shapeand clear.

To check organism motility

Helminth eggs and larvae are readily identified withoutstain, but the stain can be used advantageously.

The first technique employed on fresh stool before goingto other techniques

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Direct Wet Smear

Procedure

Place 1 drop of saline and 1 drop of iodine in themiddle of each slide

Take a 2 mg sample and thoroughly emulsify stoolin saline and iodine

Place a coverslip

Systematically scanned with 10x objective. If

something suspicious is seen, the 40x objectivecan be used for more detailed.

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Use cleaned microscope

slidesPlace a drop of saline Take a small amount

of stool with a wooden

stick

Mix stool with saline

Mistake:

Too much stool!

Place coverslip

Avoid air bubbles!

Press cover slip slightly,

remove excess liquid with paper towel

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Preservation of Specimen

To preserve protozoan morphology To prevent continued development of some helminth

eggs and larvae

The stool specimens can placed in the preservatives

either immediately after passage or upon arrival at thelaboratory

The decision to use a preservative may be influencedby the time the specimen may be in transit and theexisting laboratory workload.

Various types of fixatives :

PVA (polyvinyl alcohol)

Formalin

SAF (sodium acetate-acetic acid-formalin)

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Preservation of Specimen

PVA Highly recommended as a means of preserving protozoan cysts and trophozoites

Permits specimens to be shipped

Can prepare permanent stained

Remains stable for long period (month to year) when kept in sealed containers at

room temperature.

FORMALIN FIXATIVE Has been used for helminth eggs and larva, and also protozoan cysts

Formalin should be added to the fecal material in a ratio of 3 parts formalin to 1part feces.

Can be routinely used for direct examination, concentration techniques, and

monoclonal antibody studies.

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Fecal Concentration Methods

The objectives of employing concentration

techniques in stool examination are to :

1. Increase the number of cysts, trophozoites, eggs or

larvae

2. Eliminate most of the fecal debris

3. Present the organisms in an unaltered state so that

they can be identified readily

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Concentration method1. Sedimentation

performed by suspending the fecal material in an aqueoussuspending fluid and through centrifugation, heavier protozoa,

oocysts, eggs and larvae are separated from the lighter fecaldebris.

2. Floatation

enhance the separation of parasitic elements by combining thefecal material with solutions of greater specific gravity, the

lighter protozoa, oocysts, eggs and larvae float to the top and canbe recovered in the surface film.

Fecal Concentration Methods

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Permanent Stained Smears

To provide contrasting colors for the backgrounddebris and parasites present

To allow recovery and examination and recognitionof detailed organism morphology under oil

immersion examination

Types of permanent stains :

Iron Hematoxylin (All protozoa, helminths egg)

Trichrome stain (All protozoa)

Gram Chromotrope (Microsporidia spp)

DMSO modified Acid Fast (Cryptosporidium spp)

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Cellophane Tape / Scotch Tape

The most commonly used procedures for the

recovery ofEnterobius vermicularis (pinworm) eggs.

Specimens should be collected in the morning, before

the patient bathes or defecates. Cellophane tape around peri-anal region

Use a tongue depressor to pat the scotch tape (sticky

side down) onto the anal area.

Stick the tape onto a glass slide. This identical procedure has also proven helpful in

the recovery ofTaenia eggs.

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Egg Counting Techniques

To estimate the daily output per female worm

To obtain clinical calculation of worm infection

To determine the efficacy of anti helminth

medication, pre and post treatment

Quantitative methods :

Kato-Katz thick smear Stool dilution method

Beavers direct smear

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Egg Counting Techniques

Place faecal on absorbent paper

Press sample with a wire net

Withdraw faeces passed through the net and conveyto the central hole of the card which must be lyingon a glass slide

After filling the hole, carefully withdraw the card

Place a piece of cellophane soaked in malachite-green solution over the sample

Using a rubber stopper or invert slide on a flatsurface to spread sample evently

Count ova in entire smear

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Modified Harada-Mori TechniqueCulture Methods for Larval Stage Nematodes

Smear faeces on one side of the filter paper at the pointedend and at the top end (for handling and labeling)

Place the paper into the plastic bag with unsmeared end

towards the bottom Add water into the bag, water level must be below the

sample level

Seal and hang upright and incubate at room temperature

After 5-6 days cut the pointed tip and drain water into thepetri dish

Examine the fluid under dissecting microscope

Pick up larvae and transfer to a slide

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Aspirates

Material aspirated from lung or liver abscesses Such material should be examined in direct wet mount

and on permanent stained slides.

Culture techniques, using bovine serum based medium,

are also used routinely by some labs Duodenal aspirates may require concentration by

centrifugation prior to direct examination for motileorganisms and permanent staining.

Bronchoscopy procedures yield fluid specimens, suchas bronchial washings and bronchoalveolar lavages

Needle aspirates from many tissues or other materialcan be smeared on a microscope slide.

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Cerebrospinal Fluid

Cerebrospinal fluid is collected in a sterile, tight

sealing container.

The sample should be concentrated by centrifugation,

and the sediment examined in wet mount for thepresence of motile trophozoites.

Permanent stained smears should also be prepared.

Some of the concentrated sediment canbe culturedon non-nutrient agar, incubated and examined for

parasite cysts.

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Sputum

Sputum specimens should be collected in the early morning,

keep in a tight sealing, sterile container.

A proper specimen from the lower respiratory passages.

Can be examined in direct wet mount or concentrated.

Urine/Genital Specimens

Parasites are often recovered in the urinary sediment, in

vaginal and prostatic secretions. Urine is collected into a wide-mouth, sterile container with

a tight fitting lid and forwarded to the laboratory

immediately.

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Corneal and Mouth Scrapings,

Nasal Discharge

Corneal scrapings, should be placed in a sterile air-tightcontainer.

Scrapings can also be examined directly, or processed

as a histologic specimen or can be directly inoculatedonto non-nutrient agar plates.

Mouth scrapings recovered particularly from around thegumline or pyorrheal pockets.

Specimens should be obtained in a sterile, air-tightcontainer or swab and examined by direct wet mount.

Permanent stained smears may also have diagnosticvalue.

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Tissue/Biopsy Material/Skin Snips

Tissue biopsy specimens are often submitted onpatients suspected of cutaneous parasitic infections.

Specimens should be surgically removed and submittedto the laboratory in sterile saline.

Biopsy material can be cultured onto Nicolle-Novy-McNeal (NNN) medium and examined weekly.

This preparation can then be fixed and stained as forthin blood films.

Other types of tissue biopsies, skin snips and bonemarrow and lymph node specimens should be

processed by histology and reviewed by the pathologist.

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Blood Parasites

Blood specimensFingerstick and venipuncture are acceptable

specimens for diagnosis; fingerstick preferred for

malaria; make smears within 1 hour

Time of collection is also important for the recovery andidentification of the microfilariae

The success of blood film preparation depends on the

use of clean, unscratched, grease-free slides.

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Blood Parasites:

Permanent Stain Smears

Giemsa stain Recommended all purpose stain for blood and tissue parasites

May not stain the sheath of some Microfilariae

Cytoplasm stains blue and nuclear material red (except formicrofilarial nuclei which will stain blue)

Buffer pH critical for proper staining

Permanent stained smears

Giemsa

Wrights (Wright-Giemsa)

Hematoxylin and Eosin (H&E)

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Blood Parasites:

Permanent Stain Smears

Wrights Stain Commonly used hematology stain

Not recommended for use as a confirmatory parasitology stain

May not see stippling (Schffners dots) with Wrights staining

Recommended that follow-up Giemsa stain is performed on allpositive smears for tissue parasites

Hematoxylin and Eosin (H&E) Stain Commonly used histology stain

May be used for the detection of tissue parasites May see sheath of many Microfilariae

Recommended that follow-up Giemsa stain is performed on all

positive smears for tissue parasites

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Thin Blood Smear

Often preferred for routine estimation of parasitesbecause organisms are easier to see and count

Inadequate for detecting low parasite density

Species identification. Procedure :

A small drop of blood is placed at one end of a clean microscopeslide.

The end of another slide, held at a 30 degree angle is placed inthe middle of the blood drop.

The blood is allowed to spread along the width of the spreaderslide and then in a rapid even motion, the spreader slide is

pushed across the length of the original slide.

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Thick Blood Smear

Using about 2-3 times more blood than the thin film

Better than the thin film in detecting low levels ofparasites

Procedure : Place 2-3 small drops of whole blood close together at one end of an alcohol-cleaned,

dry slide.

With one corner of another clean slide, mix the drops together in a circular motion over

an area of 2 cm in diameter (about the size of a nickel).

Mixing should continue for at least 30 seconds to prevent formation of fibrin strands. (Ifanti-coagulated blood is used, this step should be eliminated.)

Slides should be allowed to air dry thoroughly at room temperature. Do not heat.

Dry slides should be laked to remove hemo-globin. This can be accomplished by placing

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Malaria diagnosisMicroscopic

Thickbloodfilm:10l

blood(3drops)

Bloodsmear:2lblood(1drop)

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A diagnosis problem ?Traditionally diagnosis infection based on finding parasite.

Problems: Some parasites morphologically indistinguishable.

Parasites hidden in host tissue.

Low sensitivity.

Three types ofmolecular tests.

1. Biochemical (first generation) : Isoenzymes

2. Immunological (Antibodies).

3. Nucleic acid : PCR (Polymerase Chain Reaction)

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Biochemical molecular tests:Enzyme patterns.

Advantages:

Simple technique.

Large number of typing enzymes available.

Many samples typed at same time.

Power to distinguish morphologically similar parasites.

Disadvantages:

Significant tissue needed for analysis

visceral leishmaniasis requires spleen, liver. Technique not rapid can take days.

Sometimes incorrect diagnosis enzyme labile.

Technique simple but equipment expensive.

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Antibody based diagnosis.

Rely on identification of specific antibodies.

Advantages:

Rapid easy field-based tests.

Both individual & mass population screening.

Ig subclasses to improve specificity/sensitivity.

Disadvantages: Cannot distinguish past / present infections.

Expensive to develop significant research prior to

commercialization.

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Nucleic acid based diagnosisPCR

Advantages:

Highly sensitive and specific

Able to detect parasite at very low levels (below

microscopic detection levels) Able to detect mixed infections

Disadvantages:

Expensive - especially PCR .

PCR can fail: - Contamination & false positives.

DNA probes do not distinguish between dead & livingparasites

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Thank You