An overview of Malaria Microscopy

&
Quantifying malaria parasites

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 Malaria microscopy
• Diagnosis can be based on clinical criteria
and/or parasite demonstration.
• The condition is considered when
– a person who has a febrile illness and
• come from the area endemic for malaria
• received blood transfusion
• used intra venous drugs
• Microscopy-by peripheral blood films
• Serology-for epidemiological studies
• Molecular techniques-species identification 2
Malaria differential diagnosis in Ethiopia
• Non complicated
– Relapsing fever, Typhus & Typhoid fever,
Influenza.VL
• Complicated Malaria
– Decreased Level of Consciousness
• Viral encephalitis, Bacterial
meningoencephalitis, Cerebral typhoid
,Complicated typhus, Sepsis
– Renal failure
• Glomerulonephritis, Acute tubular necrosis due
to hypovolemia or hypotension
– Jaundice associated with fever 3
 Diagnosis
Laboratory findings
Severe anemia (hemoglobin <5g/dl,
haemocrit < 15%):normal 11-19.5g/dl,34-
54% .
Hypoglycemia (<40 mg/dL)
Acidosis (bicarbonate <15 mmol/L)
Hyperlactatemia (>5 mmol/L)
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 Malaria microscopy
Microscopic detection
• Microscopic identification is the method most
frequently used to demonstrate an active
infection.
1) Collection of blood: Peripheral blood
should be collected before starting treatment
with antimalarial and during fever.
• Smears should be examined at least twice
daily until parasites are detected.
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 Malaria microscopy
2) Examination:
• Both thick and thin smears are prepared from
the peripheral blood.
• Stained with Giemsa or Field’s stain.
• Thick smear is used for detecting parasites,
quantitating parasitaemia and demonstrating
malaria pigments.
• If thick smear is positive, thin blood smear are
examined for marking a species diagnosis.
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 Malaria microscopy

• Threshold of detection

– thin film: 100 parasites/μl

– thick film: 5 -20 parasites/μl

• Simple, inexpensive yet labor-intensive

• Accuracy depends on laboratory skill

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 Thick vs Thin Films
• Thick film:
– 30 times more sensitive (~20
parasites/µL)
– Rapid detection(low parasitemia)
• E.g. P.malariae
– Blood is not fixed
• Red cells Lyse during staining
–Parasites and white cells seen in a
much larger volume of blood
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 Thick vs Thin Films
• Thin blood film:
– Identification/confirmation of plasmodium
species
– Blood cells are fixed
– Parasites seen in the red cells
– Change in morphology of parasitized red cells
– Enables to investigate anemia and white cell
abnormalities.
– If no malaria parasites, suggest an alternative
diagnosis
• e.g. sickle cell disease 9
 Thick vs Thin Films
 THICK FILM THIN FILM
– Lysed RBCs – Fixed RBCs, single
layer
– Larger volume (3 drops)
– Smaller volume /1
– 0.25 μl blood/100 fields drop
– Good screening test – 0.005 μl blood/100
– Positive or negative fields
– Parasite density – Good species
– More difficult to differentiation
diagnose species
– Requires more time to
read 10
 Malaria microscopy

• Components

1) Blood film preparation

2) Drying & Fixation
3) Staining

4) Reporting

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 Blood film preparation
1.The second or third
4.Slide must always
finger is usually
be grasped by its
selected and cleaned.
edges.
2.Puncture at the 5.Touch the drop of
side of the ball of blood to the slide
the finger. from below.

3.Gently squeeze
toward the puncture
site.
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 Preparation cont’d…
1.Touch one 4.Carry the drop
drop of blood of blood to the
to a clean first slide and hold
slide. at 45degree angle.
read newsprint”

2.Spread the
first drop to 5.Pull the drop of
“see but not

make a blood across the

15x15mm first slide in one
circle. motion.
3.Touch a fresh 6.Wait for both to
drop of blood dry before fixing
to the edge of and staining.
another slide. 13
 Drying thick blood films
– keep a separate box or deep tray for drying
– Cover it with a lid made from netting to
protect the films from insects and dust
– If the box or tray is placed in a warm sunny
place, the thick film will dry quickly
• do not allow the blood to remain in the
sun after it has dried
– In humid climates
• use a hand dryer or an incubator to dry
thick blood films 14
 Fixing thin blood films
– Use absolute methanol (methyl alcohol)
– The alcohol must be free from water
• Procedure
– Place the slide horizontally on a staining
rack
– Apply a small drop (3drops) of absolute
methanol or
– ethanol to the thin film the alcohol
• The alcohol shouldn’t touch the thick film
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 Staining
• Staining media

– pH 7.1–7.2

• Two options

– Field’s stain

– Giemsa stain

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 Staining cont’d
• Field’s stain
– water-based Romanowsky stain
– composed of Field’s stain A and Field’s stain
B.
– Don’t requires dilution when staining thick
films.
– “B” requires dilution when staining thin BF.
– Is more stable
– stains fresh blood films well, particularly
thick films. 17
 Staining cont’d
• Giemsa stain
– alcohol-based Romanowsky stain
– Requires dilution in pH 7.1–7.2 buffered water.
– It gives the best staining of thin films
– It also stains thick films well
• overnight drying
• the concentration of stain is low
• the staining time is sufficiently long
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 Staining cont’d
• Thin film Field’s staining technique
1) Cover the methanol-fixed thin film with ~0.5
ml of diluted Field’s stain B.
2) Add an equal volume of Field’s stain A & mix
3) Leave to stain for 1 minute
4) Wash off the stain with clean water
5) Wipe the back of the slide clean and place it
in a draining rack for the film to air-dry

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 Staining cont’d
• Thick film Field’s staining technique
1) dried thick film facing downwards, dip the slide into
Field’s stain A for 5 seconds
– Drain off the excess stain
2) Wash gently for about 5 seconds in clean water.
– Drain off the excess water.
3) Dip the slide into Field’s stain B for 3 seconds.
– Drain off the excess stain.
4) Wash gently in clean water
– Wipe the back of the slide clean and air-dry.

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 Staining cont’d
Giemsa staining technique
– Dilution
– 3% for 30 minutes

• 1ml stock/97 ml distilled water

– 10% for 10 minutes

• 10ml stock/90 ml distilled water

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 Giemsa staining cont’d
• Method
1) Pour 3% or 10% giemsa in a jar
2) Put slides in a rack inside the jar__Slides
should be fully covered with the stain
3) Stain for 30 or 10 minutes
4) Pour clean water gently and rinse
5) Air dry

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 Reporting malaria films
• apply a drop of immersion oil to an area of the
film which appears mauve coloured
– usually around the edges
• scan with the 10 and 40 objectives
• Select an area that is well stained and not too
thick & and change to 100 objective
• Examine for malaria parasites and malaria
pigment.
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 Reporting malaria films cont’d
• Reporting thick films
– Examine at least 100 high power (100
objective) microscope fields for parasites.
– In areas where P. malariae exists, examine
approximately 200 fields
– If thick film is positive examine thin films
for species identification.

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 Reporting thick films con’d…
• Report the approximate numbers of parasites
(trophozoites, schizonts, and gametocytes)
• also whether malaria pigment is present in
white cells (always mention when the pigment
is in neutrophils).
Parasites
1–10 per 100 high power fields . . . . . . . .+
11–100 per 100 high power fields . . . . . . .++
1–10 in every high power field . . . . . .+++
More than 10 in every high power field . .++ 25
 Malaria pigment in white cells

• This appears as brown-black granules in

monocytes and occasionally in neutrophils.
• It should always be reported.
• Pigment in white cells, particularly in
neutrophils is associated with severe falciparum
malaria.
• It can give an indication of whole body parasite
density, especially when the peripheral blood film
shows no parasites or very few.
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 Reporting malaria films cont’d
Reporting thin blood films
• Significance
– To detect changes which take place in
parasitized RBCs
• to identify Plasmodium species
• To detect mixed infections
– to count the percentage of parasitized red cells
in severe falciparum infections
– to perform white cell differential count
– To examine the morphology RBCs in severe 27
 Parasitized Red Cell Changes
– Enlargement and irregular shape- P. vivax.
– Oval cells with ragged ends-P. ovale.
– Red stippling (Schuffner’s dots) -P. vivax
and P. ovale
– less distinct irregular stippling (Maurer’s
clefts) - P. falciparum.
– Parasites forming a band across red cells-
feature of P. malariae.
– Several parasites in a single red cell- P.
falciparum
– Peripheral rings (Acole forms) & double 28
 Quantifying malaria parasites
• Two methods
– Quantitative
– Qualitative
• Greenwood and Armstrong method
– Estimating parasite numbers/µl of blood from the
thick film
– This is carried out by multiplying the average
number of parasites per high power field by 500.
– Between 10–50 should be examined to
determine the average number of trophozoites
per (HPF). 29
Greenwood and Armstrong method
cont’d
• The factor of 500 (Greenwood and Armstrong)
– 5–8µl is the volume of blood required to make a
satisfactory thick film
– The volume of blood in one HPF is about 0.002µl
– the number of parasites per HPF multiplied by
500 gives the estimated number of parasites/µl of
blood
• Greenwood and Armstrong found to be more
accurate and quicker method.

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Quantifying malaria parasites
con’d…
• Estimating parasite numbers/µl of blood by
counting parasites against white cells
1) Select a part of the thick film where the white
cells are evenly distributed and the parasites are
well stained.
2) Systematically count 100 WBCs, estimating at the
same time the numbers of parasites in each field
covered.
3) Repeat this in two other areas of the film and take
an average of the three counts.
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Counting parasites against WBCs cont’d…

WBC count x Parasites counted against

100WBC 100

NB: Patients total Leukocyte count is needed

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 Quantifying malaria parasites
con’d…
• WHO (Falciparum parasitemia)

– 250, 000 parasites/µl

• heavy infection and poor prognosis

– 500 000 parasites/µl

• very high risk of mortality

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