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. 2. 3.

Proper collection and handling of specimen Amount needed of sample needed for a proper examination 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 10 % Formalin Advantages All purpose fixative Preserves morphology of helminths ova and larvae, protozoan cyst and coccidia Disadvantages Inadequate preservation of morphology of protozoan trophozoite Inadequate preservation of morphology of protozoan trophozoite

Merthiolate Iodine Fixes and stains Formaldehyde microorganisms (MIF)

Polyvinyl Alcohol (PVA)

Schauddins Fixative

Preserves protozoan trophozoites and Cysts

Preserves protozoan trophozoites and Cysts Same with formalin

Inadequate preservation of morphology of helminths ova and larvae and coccidia

Inadequate preservation of morphology of helminths ova and larvae and coccidia Requires additive for adhesion of specimen to slides

Saffranin acetate Acetic acid - Formalin (SAF)

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 Prioritize examination of liquid stools especially those containing mucus and blood as they contain motile amoeba

ALWAYS

Macroscopic Examination

Color

Black, yellow, brown, green Formed Soft Watery

Consistency

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

NSS

iodine

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 No parasite seen

If ova or parasites are NOT SEEN, report as:

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 No parasite seen

If ova or parasites are NOT SEEN, report as:

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 No parasite seen

If ova or parasites are NOT SEEN, report as:

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 No parasite seen

If ova or parasites are NOT SEEN, report as:

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 No microfilariae seen

If no microfilariae is SEEN, report as:

9. Malarial Blood Films

Thick and Thin Gold standard Giemsa!

Blood films for malaria

Thick smear

Thin smear

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

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

Start the count here

CCMOVBD

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

Parasite Counting
ENFI Image

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

The Malaria Parasite

Trophozoites

Three developmental stages seen in blood

films:
CCMOVBD CCMOVBD

Schizont

Gametocyte

1. Trophozoite
2. Schizont 3. Gametocyte

CCMOVBD

CCMOVBD

Plasmodium vivax

(trophozoite stages in thin smear)

Ring form

Developing trophozoite

Mature trophozoite

CCMOVBD

CCMOVBD

CCMOVBD

P. ovale

Microgametocyte (left) Macrogametocyte (right) Fimbriated edges of cell Smaller than P. vivax

Plasmodium knowlesi (schizont stage)

DPDx CDC

DPDx.

DPDx CDC

Interpretation and Recording of Result

species P.Falciparum p.Vivax P.Ovale P.Malariae No Malarial Parasite seen stages Trophozoite stage Gametocyte stage Trophozite and gametocyte stage Parasite/ ul blood Actual Actual actual

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 maam Julie Villar (UST) Notes from Maam Winifreda De Leon (PERC, UP Manila) Notes from Sir Sherwin Galit (RITM)