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Practical Laboratory Diagnosis of Parasitic Diseases
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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)