Practical Laboratory Diagnosis of

Parasitic Diseases

JUDE ANTHONY C. TRINIDAD, RMT, MPH (units), MSMT (units)

Outline

Part 1 – Proper Collection, Preparation and Examination

of Specimens

Part 2 – Basic Parasitology procedures

PART 1: PROPER

COLLECTION,PREPARATION AND

EXAMINATION OF SPECIMENS

Specimens..

Stool

Blood

Sputum

Skin scrapings

Tissue specimens

Urine

Others

General Considerations

1. Proper collection and handling of specimen

2. Amount needed of sample needed for a proper

examination

3. Proper labeling of the specimen

General Consideration

Number and types of specimen

Helminth egg are passed on continual basis

Protozoans, passed intermittently

Time factor in examination

Watery, liquid, diarrheic specimens: within 30 minutes

Formed specimen: > 1 hour or within the day

General Preservatives for Stool Specimens

Preservatives Advantages Disadvantages

10 % Formalin All purpose fixative

Preserves morphology of

helminths ova and larvae,

protozoan cyst and coccidia

Inadequate preservation of

morphology of protozoan

trophozoite

Merthiolate – Iodine –

Formaldehyde

(MIF)

Fixes and stains

microorganisms

Inadequate preservation of

morphology of protozoan

trophozoite

Polyvinyl Alcohol

(PVA)

Preserves protozoan

trophozoites and Cysts

Inadequate preservation of

morphology of helminths ova

and larvae and coccidia

Schauddins Fixative Preserves protozoan

trophozoites and Cysts

Inadequate preservation of

morphology of helminths ova

and larvae and coccidia

Saffranin – acetate –

Acetic acid - Formalin

(SAF)

Same with formalin Requires additive for adhesion

of specimen to slides

Collection of specimens

Use clean , dry wide – mouthed container

Opposite side of the diaper (for babies)

Bring the specimen ASAP to the laboratory

Collection Of Specimens

Remember!

NEVER

Leave specimen exposed to the air in container without lids

Accept specimens mixed with urine or water

Examine specimen without putting on gloves

ALWAYS

Prioritize examination of liquid stools especially those containing

mucus and blood as they contain motile amoeba

Macroscopic Examination

Color

Black, yellow, brown, green

Consistency

Formed

Soft

Watery

Macroscopic Examination

Macroscopic Examination

Microscopic Examination

To detect motile Trophozoites

To detect ova, larva and cysts

To detect RCs, PCs, fat globules and Charcot Leyden

Crystal

Procedure for stool microscopy

Examine the ENTIRE COVERSLIP!

Common Fault in Specimen Examination

Pollen Bubbles

Sending specimen to a reference laboratory

Preservatives used

10% formalin

PVA - fixative

Disposal of specimens

Burning

Add 10 % formalin

Slides, funnels, and centrifuge tubes should be put in a pan

of disinfectant

PART 2: Basic Procedures in

Parasitology

1. Direct Fecal Smear

Simplest and most rapid of all fecal examination

techniques

iodineNSS

Interpreting and Reporting

If ova or parasites are SEEN, report as:

POSITIVE FOR species and stage of the parasite

Ex: POSITIVE FOR Ascaris lumbricoides ova

If ova or parasites are NOT SEEN, report as:

No parasite seen

2. Kato Katz Technique

Quantitative method!

2. Kato Katz Technique

EPG = number of eggs x 24

If ova or parasites are SEEN, report as:

POSITIVE FOR species and EPG

Ex: POSITIVE FOR Ascaris lumbricoides ova,72 eggs per gram

stool

If ova or parasites are NOT SEEN, report as:

No parasite seen

3. Formalin Ether/ Ethyl acetate

Concentration technique

Principle: sedimentation

Uses bigger volume of stool

Ethyl acetate

Fecal debris

Formalin

Sediment containing parasite

Interpreting and Reporting

If ova or parasites are SEEN, report as:

POSITIVE FOR species and stage of the parasite

Ex: POSITIVE FOR Ascaris lumbricoides ova

If ova or parasites are NOT SEEN, report as:

No parasite seen

4. Zinc Sulfate Centrifugal Floatation

technique

Principle: Floatation

Reagent: 33% Zinc Sulfate

Specific gravity of 1.18, 1.20

Interpreting and Reporting

If ova or parasites are SEEN, report as:

POSITIVE FOR species and stage of the parasite

Ex: POSITIVE FOR Ascaris lumbricoides ova

If ova or parasites are NOT SEEN, report as:

No parasite seen

5. Tape Peri – Anal Swab Technique

diagnosis of Enterobius vermicularis infection

Collection is done by pressing the adhesive portion of the

cellulose tape to the peri anal folds or region

The procedure..

Interpreting and Reporting

Results are reported as POSITIVE or NEGATIVE for

Enterobius vermicularis

6. Harada Mori Technique

Uses POSITIVE stool

Uses filter paper strips and test tubes

7. Stoll Egg Count

A method for determining the number of nematode eggs

per gram of feces in order to estimate the worm burden

0.1 NaOH, Stool displacement flask

Saponifies fat and frees eggs from fecal debris

8. Filarial Blood Films (FBFs)

For the Identification of microfilariae

Dehemoglobinized before staining

Microfilariae

Recording and Reporting

If microfilariae is SEEN, report as:

POSITIVE FOR species microfilariae

Ex: POSITIVE FOR Wuchereria bancrofti microfilariae

If no microfilariae is SEEN, report as:

No microfilariae seen

9. Malarial Blood Films

Thick and Thin

Gold standard

Giemsa!

Blood films for malaria

Layer of red blood cells 10-20 times thicker than in a thin film

Used to detect parasites and estimate parasite density

Gives sensitivity to diagnosis

sir jude semi pogi lang

Thin smearThick smear

Single layer of red blood cells

Used to identify parasite species, after they have been seen in the thick film

Gives specificity to diagnosis

Used as label to identify patient

Reading of Malarial Blood film and

Quantification of Parasite Density

Read the thick smear first!

For Quantification, count 200 WBC or 500 WBC

Parasite ul blood

= no.of parasite counted x actualWBC Count

no. of WBC counted

Parasite counting in thick film

1. Cross-sectional method(recommended technique in parasite counting)

CCMOVBD

Start the count here

Parasite Counting

Why establish a parasite count?

1) To determine the parasitological severity of malaria infection

2) To determine/monitor the parasitological response to

antimalarial treatment given (therapeutic assessment)

3) To know the severity of infections in an area

ENFI Image

3. Gametocyte

The Malaria Parasite

Three developmental

stages seen in blood

films:

1. Trophozoite

2. Schizont

CCMOVBD CCMOVBD

Trophozoites

CCMOVBD

GametocyteSchizont

CCMOVBD

Plasmodium vivax(trophozoite stages in thin smear)

CCMOVBD

Ring form Mature trophozoite

CCMOVBDCCMOVBD

Developing trophozoite

CCMOVBD

P. ovale

Microgametocyte (left) Macrogametocyte (right)

Fimbriated edges of cell

Smaller than P. vivax

Plasmodium knowlesi

(schizont stage)

DPDx.

DPDx CDC

DPDx CDC

Interpretation and Recording of Result

species stages Parasite/ ul blood

P.Falciparum Trophozoite stage

Gametocyte stage

Actual

Actual

p.Vivax Trophozite and

gametocyte stage

actual

P.Ovale

P.Malariae

No Malarial Parasite

seen

Example

Plasmodium falciparum, trophozoite stage – 2876

parasite/ul blood

Plasmodium vivax, trophozoite and gametocyte stages –

7635 parasite/ ul blood

Questions???

THANK YOU!

Sources..

Lecture from Department of Parasitology, RITM

Notes from Sir Greg Martin and ma’am Julie Villar (UST)

Notes from Ma’am Winifreda De Leon (PERC, UP Manila)

Notes from Sir Sherwin Galit (RITM)