Prepared By:
Mr. Raed Z. Ahmed
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Blood Examination
• The most commonly used technique for blood examination
is stained blood films.
• Geimsa stain is usually used to stain the films.
• Delafild’s haematoxylin stain is used for microfilariae.
• Either thick or thin films may be used depending on the
circumstances.
• The thick film is more sensitive in detecting parasite and
also saves time in examination.
• The thin film technique cause very little distortion of the
parasite, and permits species identification when it may not
be possible in thick films, but many fields must be
examined to detect parasite when they are few in number.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Continue ………
• Therefore, both thick and thin films must always be prepared
when searching for plasmodia and trypanosomes.
• If a precise identification can not be made from thick film,
the thin film will be available.
• Thick films should be used when searching for microfilariae.
• The most economical use of slides is achieved by making a
combination thick and thin slide.
• However, combination films must dry thoroughly 8-10 hrs.
to overnight before they can be satisfactorily stained.
• Slides for malaria should be stained in the same day.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Continue ………
• The thin films will dry quickly and can be stained as soon as
they are dry, and examine for parasites.
• If parasites are not seen in the thin film, stain the thick film
using Field’s stain, and examine for parasites.
• Direct wet mounts of fresh whole blood (or centrifuged blood)
are usually used for detection of microfilariae and
trypanosomes, this only gives evidence of infection and
stained films are necessary for confirmation of species present.
• In areas where malaria, trypanosomes, and/or microfilariae
may all present, both wet and stained films should be prepared
and examined.
• If neither trypanosomes nor microfilariae occur in region, only
stained smears need to be made for detection of plasmodia.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Examination of Thick & Thin blood smear
• For optimum staining, the thick and thin films should be made
on separate slides and different concentrations used for
staining.
• When it’s done good quality staining of thick film is of
primary importance, best results are obtained if the blood
smear have dried overnight.
• Fixation of thin blood film done by adding 3 drops of
methanol, or dipping it in a container of methanol for few
seconds, with prolonged fixation it may be difficult to
demonstrate Schuffner’s dots and Maurer’s dots.
• To permit dehemoglobinization, thick film should not be
fixed; therefore avoid exposure to methanol or methanol
vapor
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Reading of Thick Film
• Focus on film with 10x objective and search for microfilariae.
They are easily detected with 10x objective.
• If microfilariae are present, switch to oil- immersion objective
and identify the species.
• Also, look for malaria parasites with oil- immersion objective, at
least 100 fields should be examined.
• Microscopy of thick film should reveal the following features:
1. The background should be clean, free from debris, with a
pale mottled- gray color derived from the lysed erythrocytes.
2. Leukocyte nuclei are stained a deep, rich purple.
3. Malaria parasite are well defined with deep- red chromatin
and pale purplish blue cytoplasm.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Reading of Thin Film
• Focus with the 10x objective on the thin terminal end of the film where
the RBCs are in one layer, put oil drop on the slide and switch to the oilimmersion objective.
• When examining fro malaria parasites and trypanosomes, at least 200
fields should be examined.
• Microscopy of thin film should reveal the following features:
1. The background should be clean and free from debris; erythrocytes
are stained a pale greyish pink.
2. Neutrophil leukocytes have deep purple nuclei and well defined
granules.
3. Malaria parasite are well defined with deep- red chromatin and pale
purplish blue cytoplasm.
4. Like plasmodia, the cytoplasm of trypanosomes stain blue, the
nucleus and kinetoplast stain red or purple.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Identification of malarial parasites
• In thin films, look at the appearance of the parasite and the
appearance of the RBCs containing the parasites.
1. The appearance of the parasites
2. The appearance of the RBC containing the parasites:
 Size & Shape: Is the parasitized cell the same size as the
blood cell without parasite or Is it enlarged?
 Stippling: Is the RBC filled with pink or red staining
dots?
• Schuffner’s stippling in the “ghost” of the erythrocyte can
some times be seen at the edges of the film and indicate
infection with Plasmodium vivax or P. ovale,.
• Maurer’s dots show as stippling in erythrocytes containing
the larger ring forms of Plasmodium falciparum.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Comparison
Thick smear
Thin smear
Lysed RBCs, many layer
Fixed RBCs, single layer
( large volume )
0.25 μl blood/100 fields
( small volume )
0.005 μl blood/100 fields
Good screening test
( positive or negative )
Good species differentiation
Save time in examination
Requires more time to read
Low density infection can be detected as
blood elements more concentrate
Low density infections can be missed
(more sensitive)
(less sensitive)
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Blood Protozoa
Microfilariae
Trypanosoma
Blood Parasite
Plasmodium falciparum
Leishmania
Plasmodium vivax
Plasmodium
Plasmodium ovale
Plasmodium malariae
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Life cycle
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Trypanosoma spp.
• Trypanosoma cruzi (Americans) cause Chaga’s disease.
• Trypanosoma bruzi (Africans) cause sleeping sickness disease.
• Trypanosoma have many stages:
– Amastigote, Promastigote, Epimastigote and Trypomastigote.
• Reservoir host: mammalian animal.
• Intermediate host:
– Tse tse fly (Glossina spp.) for Trypanosoma bruzi
– Reduviidae bug for Trypanosoma cruzi
• Definitive host: Human.
• Infective stage: Metacyclic trypomastigote.
• Diagnostic stage: Trypomastigote.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Continue ……
• Diagnosis:
o Detection of Trypanosoma chancer after bite
o Blood smear within 21 days from the bite, it will show the
parasites.
o Lymph node aspiration (most reliable).
o Lumber puncture if brain affected.
Undulating membrane
kinetoblast
Nucleus
Flagellum
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Trypanosoma Trypomastigotes
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Life cycle
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Leishmania spp.
• There is many species affect man:
– Leishmania tropica : cause skin lesion ( cutaneous )
– Leishmania braziliense : cause muco-cutaneous lesion.
– Leishmania donovani : cause visceral lesion.
• Leishmania have two stages:
– Amastigote (Leishmania stage), in man (reticuloendothelial
cell).
– Promastigote (Leptomonas stage), the infective stage and
present in the lumen gut of the sand fly.
• Reservoir host: dogs and rodents.
• Intermediate host: Sand fly (Phlebotomus).
• Definitive host: Human.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Continue ……
• Diagnosis:
– Thick and thin blood film
– Skin scraping
– Blood culture on N.N.N media*
– Serological tests
Nucleus
Flagellum
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Leishmania promastigotes
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Life cycle
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Plasmodium spp.
• Four species of Plasmodium are the causative agent of malaria, these
are:
– P. vivax, P. malariae, P. falciparum, and P. ovale.
• Intermediate host: Human.
• Definitive host: Anopheles mosquitoes.
• Plasmodium spp. have 4 stages:
– Ring form (young trophozoite.)
– Late ( old ) trophozoite
– Schizonts
– Gametocyte.
• Infective stage: Sporozoites.
• Diagnosis:
– Thick and stained thin blood film to detect parasites.
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Ring form
P. vivax
P. ovale
P. malariae
P. falciparum
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Trophozoite form
P. vivax
P. malariae
P. ovale
P. falciparum
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Schizonts form
P. vivax
P. ovale
P. malariae
P. falciparum
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Gametocyte form
P. vivax
P. malariae
P. ovale
P. falciparum
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
Species Differentiation On Thin Films
Feature
P. falciparum
P. vivax
P. ovale
P. malariae
-
+
+
-
round
round,
distorted
oval,
fimbriated
round
Stippling infected
RBC
Maurer’s
clefts
Schuffner's
spots
Schuffner's
dots
none
Trophozoite shape
small ring,
applique
large ring,
amoeboid
large ring,
compact
small ring,
compact
often double
single
rare, 12-30
merozoites
12-24
merozoites
large
4-12
merozoites
( scattered )
single
6-12
merozoites
Enlarged infected
RBC
Infected RBC shape
Chromatin dot
Mature schizont
Gametocyte
crescent shape
large, round
large, round
Raed Z. Ahmed, Medical Parasitology Lab.,2012-2013
( rosette )
compact,
round