This document discusses various laboratory methods for diagnosing malaria, including microscopic diagnosis, fluorescent microscopy, quantitative buffy coat testing, antigen detection tests, serology tests, and PCR. Microscopic examination of blood smears remains the gold standard, allowing identification of parasite species and quantification of parasitemia. Thick and thin blood films are prepared and examined under a microscope after staining. Rapid diagnostic tests can provide a preliminary diagnosis but cannot identify species or quantify parasitemia like microscopy. More sensitive methods include fluorescent microscopy, PCR, and quantitative buffy coat testing, but they require specialized equipment and reagents.
3. Blood smear
• Remains the gold standard for diagnosis
• Prepare smears as soon as possible after
collecting venous blood to avoid
• Changes in parasite morphology
• Staining characteristics
• Take care to avoid fixing the thick smear
• Risk of fixing thick when thin is fixed
with methanol if both smears on
same slide
• Let alcohol on finger dry to avoid
fixing thick
• Be careful if drying with heat 302/19/15 VAIDEGI.D
4. Collection of Blood (Smears)
1.
The second or third
finger is usually
selected and cleaned
2.
Puncture at the side of
the ball of the finger.
3.
Gently squeeze toward
the puncture site.
4.
Slide must
always be
grasped by its
edges.
5.
Touch the drop of blood to
the slide from below.
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5. Preparing thick and thin films
2.
Spread the first
drop to make a 1
cm circle.
3.
Touch a fresh drop
of blood to the edge
of another slide.
6.
Wait for both to
dry before fixing
and staining.
5.
Pull the drop of blood
across the first slide in
one motion.
1.
Touch one drop
of blood to a
clean slide.
4.
Carry the drop of blood to
the first slide and hold at
45 degree angle.
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6. Malaria Blood Smear
Used to determine the species
entire thin film should be examined
about 20-40 minutes for an
experienced observer
Thin films:
•Dry
•Fix
•Stain
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8. Interpreting Thick and Thin
Films
THICK FILM
•lysed RBCs
•larger volume
•0.25 μl blood/100 fields
•more difficult to diagnose species
•good screening test
THIN FILM
•fixed RBCs, single layer
•smaller volume
•0.005 μl blood/100 fields
•good species differentiation
•requires more time to read
•low density infections can be missed
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10. Species Differentiation on Thin
Films
P. falciparum P. vivax P. ovale P. malariae
Rings
Trophozoites
Schizonts
Gametocytes
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11. Plasmodium falciparum
Rings: double chromatin dots; appliqué forms;
multiple infections in same red cell
Gametocytes: mature (M)and
immature (I) forms (I is rarely
seen in peripheral blood)
Trophozoites: compact
(rarely seen in
peripheral blood)
Schizonts: 8-24 merozoites
(rarely seen in peripheral blood)
Infected erythrocytes: normal size
M I
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12. Plasmodium vivax
Trophozoites: ameboid; deforms the erythrocyte
Gametocytes: round-ovalSchizonts: 12-24 merozoites
Rings
Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)
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13. Plasmodium ovale
Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots)
“malariae - like parasite in vivax - like erythrocyte”
Rings
Trophozoites: compact
Schizonts: 6-14 merozoites;
dark pigment; (“rosettes”)
Gametocytes: round-oval
1302/19/15 VAIDEGI.D
14. Infected erythrocytes: size normal to decreased (3/4X)
Plasmodium malariae
Trophozoite:
compact
Trophozoite:
typical
band form
Schizont:
6-12 merozoites;
coarse, dark pigment
Gametocyte:
round; coarse,
dark pigment
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17. Calculating Parasite Density
Count the number of parasitized and
nonparasitized RBCs in the same fields on
thin smear
Count 500-2000 RBCs
% parasitemia =
# parasitized RBCs
total # of RBCs
X 100
Count ≥ 200 WBCs on thick film
Assume WBC is 8000/µl (or count it)
parasites/µl = parasites counted
WBC counted
X WBC count/µl
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18. Estimating Parasite Density
Alternate Method
Count the number of asexual parasites per
high-power field (HPF) on a thick blood film
1-10 parasites per 100 HPF +
11-100 parasites per 100 HPF ++
1-10 parasites per each HPF +++
> 10 parasites per each HPF ++++ 1802/19/15 VAIDEGI.D
19. Fluorescent Microscopy
Modification of light microscopy
Fluorescent dyes detect RNA and DNA that
is contained in parasites
Nucleic material not normally in mature
RBCs
Kawamoto technique
Stain thin film with acridine orange (AO)
Requires special equipment – fluorescent
microscope
Nuclei of malaria parasites floresce
bright green and cytoplasm red.
Staining itself is cheap
Sensitivities around 90%
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21. Quantitative Buffy Coat(QBC)
Useful for screening large numbers of
samples
Quick, saves time
Requires centrifuge, special stains
Malaria parasite floresce green yellow
against dark red –black background.
3 main disadvantages
Species identification and quantification
difficult
High cost of capillaries and equipment
Can’t store capillaries for later reference
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23. Malaria Serology
Antibody detection
•Immunologic assays to detect host response
•Antibodies to asexual parasites appear some days after
invasion of RBCs and may persist for months
•Positive test indicates past infection
•Not useful for treatment decisions
•Valuable epidemiologic tool
Useful for
Identifying infective donor in transfusion-transmitted malaria
Investigating congenital malaria, esp. if mom’s smear is
negative
Diagnosing, or ruling out, tropical splenomegaly syndrome
Retrospective confirmation of empirically-treated non-immunes
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24. Malaria Antigen Detection
Target antigens for malaria
(rapid detection test) RDT
Card / cassette / dipstick
HRP2
HRP2 & aldolase
pLDH Pf & pan
pLDH Pf & Pv
HRP2, pLDH pan
HRP2, pLDH pan & pLDH
Pv
aldolase
"COMBO" tests
A: HRP-2 (histidine-rich protein 2)
(ICT)
B: pLDH (parasite lactate
dehydrogenase)(Flow)
C: HRP-2 (histidine-rich protein 2)
(PATH)
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30. Disadvantages
The use of the RDT does not eliminate the need for
malaria microscopy
Cannot detect mixed infections
may not be able to detect infections with lower
parasitemia
Cannot detect P. ovale and P. malariae
microscopy is needed to quantify parasitemia
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31. INDIRECT FLUORESCENT
ANTIBODY(IFA)
Indirect fluorescent
antibody (IFA) test.
The fluorescence
indicates that the
patient serum being
tested contains
antibodies that are
reacting with the
antigen preparation
(here, Plasmodium
falciparum parasites).
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32. ELISA
Not practical for routine diagnosis of acute
malaria because:
•Delaied development of antibody
•persistence of antibodies
Serology does not detect current infection
but rather measures past experience
Valuable epidemiologic tool
Useful for
Identifying infective donor in transfusion-
transmitted malaria
Investigating congenital malaria, esp. if
mom’s smear is negative
Retrospective confirmation of empirically-
treated non-immunes 3202/19/15 VAIDEGI.D
33. Polymerase Chain Reaction
(PCR)
•Molecular technique to identify parasite
genetic material
•Uses whole blood collected in
anticoagulated tube or directly onto filter
paper
•Threshold of detection 5 parasites/µl
•Definitive species-specific diagnosis now
possible
•Can identify mutations – try to correlate to
drug resistance
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34. PCR
•Parasitemia not quantifiable
•May have use in epidemiologic studies
•Requires specialized equipment, reagents,
and training
Lane S: Molecular base pair
standard (50-bp ladder). Black
arrows :size of standard bands.
Lane 1: P. vivax (size: 120 bp).
Lane 2: P. malariae (size: 144
bp).
Lane 3: P. falciparum (size: 205
bp).
Lane 4: P. ovale (size: 800 bp).
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35. Comparison of methods for diagnosing
Plasmodium infection in blood
PARAMETER MICROSCOPY PCR FLUORESCENCE Dipstick HRP-2 Dipstick pLDH, ICT-Pf/Pv
Sensitivity
(parasites/micol)
50 5 50 >100 >100
Specificity All species All species
P.f good, others
difficult
P. falciparum
P. falciparum and P.vivax good P.o
and P.m only Pldh
prarasite density
or parasitemia
Yes No No
crude
estimation
crude estimation
time for result 30-60 min 24 hr 30-60 min 20 min 20 min
skill level High High Moderate Low Low
equipment Microcsope
PCR
appratus
QBC apparatus
or direct
fluorescence
microscope
Kit only Kit only
cost /test Low High moderate/low Moderate Moderate
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