Malaria

• The definitive diagnosis of malaria infection is still based on finding

malaria parasites in blood films.

• In thin films the red blood cells are fixed so the morphology of the
parasitized cells can be seen. Species identification can be made
based upon:
– The size and shape of the various stages of the parasite
– Presence of stippling (i.e. bright red dots)
– Fimbriation (i.e. ragged ends).

• Malaria parasites may be missed on a thin blood film when there is

a low parasitaemia. Therefore, examination of a thick blood film is
recommended. With a thick blood film, the red cells are
approximately 6 - 20 layers thick which results in a larger volume of
blood being examined.
Plasmodium falciparum life cycle in
thin blood films
• 1) P. falciparum early
trophozoites / ring forms.
• 2) Developing trophozoites
(rarely seen in peripheral
blood).
• 3) Immature schizonts (rarely
seen in peripheral blood).
• 4) Mature schizonts, almost fill
the red blood cell.
• 5) Macrogametocytes, large
numbers appear after 7 – 12
days.
• 6) Microgametocytes, large
numbers appear after 7 - 12
days.)
Plasmodium falciparum
• Crescent shaped Plasmodium
falciparum gametocytes appear in the
peripheral circulation at 8 - 11 days of
parasitaemia (Giemsa stain)

• Young trophozoite (ring stage)

of Plasmodium falciparum. The
ring thickens and invariably
contains several vacuoles as
the trophozoite develops.
Maurer’s dots are slow to
appear and are first seen as
minute purplish dots. (Giemsa
stain)
Plasmodium falciparum
• Plasmodium falciparum
schizont. Rarely seen in
the peripheral blood, a
good indicator of a
potentially serious
parasitaemia. They have
8 – 36 merozoites and a
large golden brown
pigment. (Giemsa stain)
P.vivax
• Early trophozoites / ring forms
(accole forms, not shown here,
are occasionally seen).
• 2) Developing trophozoites are
large and irregular with a
prominent vacuole.
• 3) Immature schizonts, are
amoeboid and almost fill the red
blood cell.
• 4) Mature schizonts, almost fill the
red blood cell.
• 5) Microgametocytes , large
numbers appear after 3 – 5 days.
• 6) Macrogametocytes, large
numbers appear after 3 – 5 days.
(Adapted and redrawn from
Jeffrey & Leach)
P.vivax
• Trophozoites of
Plasmodium vivax
already several hours old
when they appear in the
peripheral blood. You
can already see the
Schüffners dots. They
contain single or
sometimes double
chromatin dots. (Giemsa
stain)
P.vivax
• Mature female
Plasmodium vivax
gametocytes are large
rounded parasites which
fill or nearly fill the host
cell.
• The cytoplasm is blue
and fairly homogenous.
• The nuclear chromatin is
a single, well-defined
purplish mass, varied in
form and usually
peripheral in distribution.
(Giemsa stain)
P.vivax
• A schizont of Plasmodium
vivax. The parasites are
large, filling the enlarged
red cell. There are
between 12 - 24
merozoites in the
schizonts (usually16).
The pigment is a golden
brown central loose
mass. (Giemsa stain)
P.ovale
• 1) Early trophozoites / ring forms,
are dense rings with well- defined
masses of chromatin.
• 2) Developing trophozoites, small
and compact with an
inconspicuous vacuole.
• 3) Immature schizonts, compact
and almost fill the red blood cell.
• 4) Mature schizonts, fill ¾ of the
red blood cell.
• 5) Microgametocytes, low
numbers appear after 12 - 14
days.
• 6) Macrogametocytes, low
numbers appear after 12 – 14
days. (Adapted and redrawn from
Jeffrey & Leach)
Trophozoite of Plasmodium ovale
• Young trophozoites are
found as compact rings in
cells containing
Schüffner’s dots. The
trophozoite rings remain
compact as they develop.
Late trophozoites are
round and consolidated
with an increase in
cytoplasm, they are very
similar to P. vivax at this
stage.
Gametocyte of Plasmodium ovale
• mature gametocytes
are round, filling two
thirds of the red cell.
(Giemsa stain)
Schizont of Plasmodium ovale.
• The parasite is
smaller than the red
blood cell and
contains 6 – 12
merozoites. The red
cell is slightly
enlarged, stippled,
frequently oval and
fimbriated. (Giemsa
stain)
P. malariae
• 1) Early trophozoites / ring forms,
compact rings containing one
mass of chromatin.
• 2) Developing trophozoites, small
and compact (often band forms)
with an inconspicuous vacuole.
• 3) Immature schizonts, compact
and almost fill the red blood cell
which contains scattered pigment.
• 4) Mature schizonts, almost fill the
red blood cell.
• 5) Microgametocytes, low
numbers appear after 7 – 14 days.
6) Macrogametocytes, low
numbers appear after 7 - 14 days.
(Adapted and redrawn from
Jeffrey & Leach)
Trophozoite of Plasmodium
malariae.
• These can be very
distinct and distorted
by taking the form of
a band across the
cell. (Giemsa stain)
•
Plasmodium malariae gametocyte.
• They contain large
amounts of black
pigment, with
chromatin present as
a compact mass in
females
(macrogametocyte)
and diffuse in males
(microgametocyte).
(Giemsa stain)
Plasmodium malariae
• They are usually few
in numbers with 6 –
12 large merozoites in
a single ring. Pigment
is usually present as
a central black mass.
(Giemsa stain)
 Babesiosis
• Babesia are tickborne sporozoan parasites
• May also be transmitted via Blood
transfusion or organ transplantation
• Infect RBC & appear as ring-like structures
which may be confused with Plasmodium
in stained blood films
• In Babesia 4 or 5 may be seen in one RBC
• Individual rings are smaller than those of
malaria
Babesia
Leishmaniasis
• Geimsa stain of
splenic aspirate
showing amastigotes
within macrophage
• Geimsa stain of bone
marrow aspirate
showing amastigotes
within macrophage
Muco-cut leishmaniasis
Cutaneous Leishmaniasis
Visceral Leishmaniasis
Trypanosoma brucei
Trypanosoma brucei
Wuchereria and Brugia
Brugia malayi & Wuchereria
bancrofti
Life Cycle of Loa loa
Microfilaria of Loa loa
Onchocerca volvulus
Microfilaria of Onchocerca volvulus
 Collection of blood
• Blood for parasites is collected by finger
stick
• If collected in a container it is collected in a
EDTA bottle(Purple top)
EDTA container
 How to make a thick blood
smear/film

• Place a small drop of blood in the center of

the pre-cleaned, labeled slide.
• Using the corner of another slide spread
the drop in a circular pattern until it is the
size of a dime (1.5 cm2
 Making thick smear
• A thick smear of proper density is one
which, if placed (wet) over newsprint,
allows you to barely read the words.
• Lay the slides flat and allow the smears to
dry thoroughly (protect from dust and
insects!).
 How to make a thin smear

• Place a small drop of blood on the pre-

cleaned, labeled slide, near its frosted end.
• Bring another slide at a 30-45° angle up to
the drop, allowing the drop to spread along
the contact line of the 2 slides.
• Quickly push the upper (spreader) slide
toward the unfrosted end of the lower slide.
 Making a thin film
• Make sure that the smears have a good
feathered edge. This is achieved by using
the correct amount of blood and spreading
technique.
• Allow the thin smears to dry
• You can fix the thin smears by dipping
them in absolute methanol.
Thick and thin films
 Staining
• Giemsa stain
• Fields stain
• Leishman stain
 Field stain
• Sold as 2 powders
• Fields stain A ( methylene blue)
• Field satin B ( Eosin )

Each of those is dissolved in Distilled water

separately
The stain solutions
Field Stain technique Thick film
• Dip the smear in Field’s stain A for 5
seconds
• Dip it in water for 5 seconds
• Dip in Field’s stain B for 3 seconds
• Dip in water for five seconds
Allow to dry and examine
Staining thin film with Field stain
• Immerse the thin film in absolute Methanol
for 2 seconds to fix the RBC and other
cells
• Dip in Field stain B for 5 seconds
• Dip in water for 5 seconds
• Dip in FielD stain A for 6 seconds
• Dip in water for 5 seconds
• Allow to dry and examine
 Giemsa stain
• Sold as one powder
Preparation of Stock solution:
-Dissolve 3.8 g of giemsa powder in 250ml
of Methanol and heat up to about 60oC
- Add 250 ml of Glycerol to the solution ,
filter and keep for I month before use
Giemsa stock solution
 Working Giemsa solution
• Add 1.5 of stock solution to 48.5ml of
distilled water to make 3% working
solution
• Alternatively take 5 ml of stock solution
and add to 45 ml of distilled water to make
10% solution
 Staining thick film with Giemsa

• Dip the thick blood smear into

diluted( working) Giemsa stain and leave it
there for 10 minutes if 10% solution is used

• Leave it for 30 minutes 3 % solution is used

• Dip in water to rinse, allow to dry and
examine
Staining a thin film with Giemsa
• Dip the thin film in absolute methanol for 5
seconds to fix. Allow the methanol to
evaporate
• Dip in working Giemsa solution for 10
minutes if 10% working solutionis used and
30 minutes if 3 % working solution is used
• Dip in water to rinse , allow to dry and
examine
 Special Procedures for Detecting
Microfilariae

• Blood microfilariae:
• Capillary (fingerstick) blood
Since microfilariae concentrate in the
peripheral capillaries, thick and thin
smears prepared from fingerstick blood
are recommended.
 Leishman stain
• Sold as one powder
• Contains Methylene blue, Eosin and Azure
Preparation
Dissolve 1.5 g in 1 liter of Abs Methanol
Leishman stain solution
 Staining a thin film with
Leishman stain
• Fix the smear by putting about 1ml of
Leishman’s stain (undiluted) using Pasteur
pipette and leave for around 45seconds.
• Add 3ml of buffered water to dilute the
stain on the slide and leave to stain for 8-
10 minutes.
• Wash the stain off with buffered
water,wipe the back of the slides sing a
cotton wool ,and allow to dry and examine
 Staining thick film with
Leishman
• Dilute the stain with buffer water by mixing
1part of stain to 3 parts of buffered water
• Drop the diluted stain on the thick smear
and leav to stain for 8 -10 minutes
• Wash with buffered water and wipe the
back of the slide
• Allow to dry and examine using the X 100
objective
Knott’s technique for microfilaria

• Prepare 2% formaldehyde (2 ml of 37%

formaldehyde + 98 ml H2O).
• Mix 9 ml of this 2% formaldehyde with 1 ml of
patient’s venous blood.
• Centrifuge at 3000 rpm for 10 minutes; discard
supernatant. Sediment is composed of WBCs and
microfilariae (if present).
 Knott’s technique
• Examine as temporary wet mounts.

• Prepare thick and thin smears; allow to dry; dip in

absolute methanol before Giemsa staining to
enhance staining of microfilariae.

• Examine the stained smears

 Videos to watch on youtube
• Microscopic staining for blood parasites-
Multi-lingual captions
• Malaria Microscopy- A step by step guide
Babesia
Babesia
Brugia malayi
Loa loa
Loa loa unstained
Loa loa
Loa loa
Onchocerca volvulus
Thick film with Malaria parasites
No RBCs in film
Thin Film with P. falciparum
Plasmodium malariae band
form
P.malariae
P ovale oval and fimbriated rbc
not enlarged
P. Vivax ( enlarge rbc with
Schuffners dots)
P. falciparum
P falciparum
T. brucei gambiense
T.cruzi
T .cruzi
Triatomid bug
Ixodes scapularis
Glossina( tsets fly)
Anopheles mosquito
Anopheles after a meal
Rapid Diagnostic Test
End of Presentation