3. Clinical Diagnosis
• Hyperendemic and holoendemic areas
• Laboratory resources not needed
• Fever or history of fever
• Sensitivity ranges from poor to high
• Often has poor specificity and predictive
values
• Overlap with other syndromes
4. Malaria Blood Smear
• Remains the gold standard for diagnosis
• Giemsa stain
• distinguishes between species and life cycle stages
• parasitemia is quantifiable
• Threshold of detection
• thin film: 100 parasites/l
• thick film: 5 -20 parasites/l
• Requirements: equipment, training, reagents,
supervision
• Simple, inexpensive yet labor-intensive
• Accuracy depends on laboratorian skill
5. Interpreting Thick and Thin Films
• THICK FILM • THIN FILM
– lysed RBCs – fixed RBCs, single layer
– larger volume – smaller volume
– 0.25 μl blood/100 fields – 0.005 μl blood/100 fields
– blood elements more
– good species
concentrated
differentiation
– good screening test
– – requires more time to read
positive or negative
– parasite density – low density infections can
– more difficult to diagnose be missed
species
6. Malaria Blood Smear
• 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
7. Collection of Blood Smears
1. 4.
The second or third Slide must always be
finger is usually grasped by its edges.
selected and cleaned.
2. 5.
Puncture at the side Touch the drop of
of the ball of the blood to the slide
finger. from below.
3.
Gently squeeze
toward the puncture
site.
8. Preparing thick and thin films
1. 4.
Touch one drop of Carry the drop of blood
blood to a clean to the first slide and hold
slide. at 45 degree angle.
2. 5.
Spread the first Pull the drop of blood
drop to make a 1 across the first slide in
cm circle. one motion.
3. 6.
Touch a fresh drop Wait for both to
of blood to the edge dry before fixing
of another slide. and staining.
12. Plasmodium falciparum
Infected erythrocytes: normal size
M I
Gametocytes: mature (M)and
immature (I) forms (I is rarely
Rings: double chromatin dots; appliqué forms;
seen in peripheral blood)
multiple infections in same red cell
Schizonts: 8-24 merozoites
(rarely seen in peripheral blood)
Trophozoites: compact
(rarely seen in
peripheral blood)
13. Plasmodium vivax
Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)
Rings Trophozoites: ameboid; deforms the erythrocyte
Schizonts: 12-24 merozoites Gametocytes: round-oval
14. Plasmodium ovale
Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots)
“malariae - like parasite in vivax - like erythrocyte”
Trophozoites: compact
Rings
Schizonts: 6-14 merozoites; Gametocytes: round-oval
dark pigment; (“rosettes”)
15. Plasmodium malariae
Infected erythrocytes: size normal to decreased (3/4X)
Trophozoite: Trophozoite: Schizont: Gametocyte:
compact typical 6-12 merozoites; round; coarse,
band form coarse, dark pigment dark pigment
16. Species Differentiation on Thin Films
Feature P. falciparum P. vivax P. ovale P. malariae
Enlarged infected RBC + +
Infected RBC shape round round, oval, round
distorted fimbriated
Stippling infected RBC Mauer clefts Schuffner Schuffner none
spots spots
Trophozoite shape small ring, large ring, large ring, small ring,
appliqu amoeboid compact compact
Chromatin dot often double single large single
Mature schizont rare, 12-30 12-24 4-12 6-12
merozoites merozoites merozoites merzoites
Gametocyte crescent shape large, large, compact,
round round round
17. Species Differentiation on Thin Films
P. falciparum P. vivax P. ovale P. malariae
Rings
Trophozoites
Schizonts
Gametocytes
18. Species Differentiation on Thick Films
Feature P. falciparum P. vivax P. ovale P. malariae
Uniform trophozoites +
Fragmented trophozoites ++ +
Compact trophozoites + +
Pigmented trophozoites +
Irregular cytoplasm + +
Stippling (“RBC ghosts”) + +
Schizonts visible very rarely often often often
Gametocytes visible occasionally usually usually usually
19. Calculating Parasite Density - 1
• Using 100X oil immersion lens, select
area with 10-20 WBCs/field
• Count the number of asexual parasites and
white blood cells in the same fields on
thick smear
• Count ≥ 200 WBCs
• Assume WBC is 8000/µl (or count it)
parasites/µl = parasites counted X WBC count/µl
WBC counted
20. Calculating Parasite Density - 2
• Count the number of parasitized and
nonparasitized red blood cells (RBCs) in
the same fields on thin smear
• Count asexual stages separately from
gametocytes
• Count 500-2000 RBCs (fewer RBCs if
parasitemia is high)
% parasitemia = # parasitized RBCs X 100
total # of RBCs
21. Calculating Parasite Density
• Can interconvert results in % parasitized
RBCs and parasites /µl if you know the
RBC or WBC counts
• If unknown, can assume
4,000,000 RBCs /µl or 8000 WBCs /µl
22. Parasitemia and clinical correlates
Parasitemia Parasites /µl Remarks
0.0001-0.0004% 5-20 Sensitivity of thick blood
film
0.002% 100 Patients may have
symptoms below this
level, where malaria is
seasonal
0.2% 10,000 Level above which
immunes show symptoms
2% 100,000 Maximum parasitemia of
P.v. and P.o.
23. Parasitemia and clinical correlates
Parasitemia Parasites/µl Remarks
2-5% 100,000- Hyperparasitemia/severe
250,00 malaria*, increased
mortality
10% 500,000 Exchange transfusion may
be considered/ high
mortality
*WHO criteria for severe malaria are parasitemia > 10,000 /µl and
severe anaemia (haemaglobin < 5 g/l).
Prognosis is poor if > 20% parasites are pigment containing
trophozoites and schizonts (more mature forms) and/or if > 5% of
neutrophils contain visible pigment.
Hänscheid T. (1999) Diagnosis of malaria: a review of alternatives to conventional
microscopy. Clin Lab. Haem. 21, 235-245.
24. 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
25. 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
– Staining itself is cheap
– Sensitivities around 90%
26. Quantitative Buffy Coat (QBC ®)
• Fluorescent microscopy after centrifugation
• AO-coated capillary is filled with 50-100 µl
blood
• Parasites concentrate below the granulocyte
layer in tube
• May be slightly more sensitive than light
microscopy but some reports of 55-84%
27. Quantitative Buffy Coat (QBC ®)
• Useful for screening large numbers of
samples
• Quick, saves time
• Requires centrifuge, special stains
• 3 main disadvantages
– Species identification and quantification
difficult
– High cost of capillaries and equipment
– Can’t store capillaries for later reference
28. 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
29. Malaria Serology – antibody detection
• Valuable epidemiologic tool in some settings
• 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
30. Malaria Antigen Detection
• Immunologic assays to detect specific antigens
• Commercial kits now available as
immunochromatographic rapid diagnostic tests
(RDTs), used with blood
• P. falciparum histidine-rich protein 2 (PfHRP-2)
• parasite LDH (pLDH)
• Monoclonal and polyclonal antibodies used in
antigen (Ag) capture test
• Species- and pan-specific Ab
• Cannot detect mixed infections
• Cross reactivity with rheumatoid factor
reportedly corrected
32. Detection of Plasmodium antigens
A: HRP-2 (histidine-rich protein 2) (ICT)
B: pLDH (parasite lactate dehydrogenase)(Flow)
C: HRP-2 (histidine-rich protein 2) (PATH)
33. Malaria Antigen Detection - RDTs
Feature PfHRP-2 tests pLDH tests
Test Use of monoclonal (Ab) Use of monoclonal and
principle polyclonal Ab
Detects a histidine rich protein Detects a parasite enzyme,
of P.f. lactate dehydrogenase
Water-soluble protein is pLDH is found in sexual and
released from parasitized RBCs asexual forms
Not present in mature Differentiation between
gametocytes malarial species is based on
antigenic differences between
pLDH isoforms
34. Malaria Antigen Detection - RDTs
Feature PfHRP-2 tests pLDH tests
Advantages Threshold for parasite Threshold for parasite detection
detection as low as 10 ≥ 100 parasites/µl
parasites/µl (but sensitivity
drops at < 100 parasites /µl) Can detect all species which
infect humans
Does not cross react with other Can differentiate between P.f.
species – P.v., P.o., P.m. and non-falciparum malaria
Does not cross react with human
LDH
Positive only in viable parasites,
potentially useful for monitoring
success of treatment
35. Malaria Antigen Detection - RDTs
Feature PfHRP-2 tests pLDH tests
D Some tests only detect P.f. Cannot differentiate between
non-falciparum species
Cannot detect mixed infections Cannot detect mixed infections
Sensitivity and specificity Sensitivity and specificity
decreases < 100 parasites/µl decreases < 100 parasites/µl
Can remain positive up to 14
days post treatment, in spite of
asexual and sexual parasite
clearance, due to circulating
antigens
36. Malaria Antigen Detection - RDTs
Feature PfHRP-2 tests pLDH tests
Sensitivity/ Sensitivity 92-100% Sensitivity P.f. 88-98%
Specificity* Specificity 85- 100% P.v. 89-94%
Specificity P.f. 93-99%
P.v. 99-100%
Commercial Approximately US$ 0.60 –1.00 Approximately US$ 2.50
cost/test**
Commercial 1) PATH falciparum Malaria 1) OptiMAL® - Flow, Inc.
products IC Strip test – Program for 2) Binax NOW ®ICT
Appropriate Technology in Malaria - Binax, Inc.
Health
2) MAKROmed™
3) Orchid ®
* Compared to microscopy, results from multiple studies
** Varies by size of order and vendor
37. Polymerase Chain Reaction (PCR)
• Molecular technique to identify parasite
genetic material
• Uses whole blood collected in
anticoagulated tube (200 µl) or directly
onto filter paper (5 µl)
– 100% DNA is extracted
– 10% blood volume used in PCR reaction
38. Polymerase Chain Reaction (PCR)
• Threshold of detection at CDC
– 0.1 parasite/µl if whole blood in tube
– 2 parasites/µl if using filter paper
• Definitive species-specific diagnosis now
possible
• Can identify mutations – try to correlate to drug
resistance
• Parasitemia not quantifiable
• May have use in epidemiologic studies
• Requires specialized equipment, reagents, and
training
39. Real-Time PCR
New technique based on
fluorescence
Promising because it has
potential to quantify
parasitemia, decreases
contamination, may detect
multiple wavelengths in
same tube identifying
multiple species in one run,
saves hands-on time
Needs further research and
validation for malaria
44. Preventing TTM:
Detection of Parasites/Parasite Products
100 parasites/unit PCR (0.05 to 0.1 parasites/l)
(25 X 10-5/l)
Microscopy (5 parasites/l)
10 parasites/unit Antigen detection
(2.5 X 10-5/l) (10 to 100 parasites/ l)
10-5 10-3 10-1 10 103
Parasite densities (parasites/l)
Detection of 10 parasites/unit requires a sensitivity:
-4,000 times better than PCR
-200,000 times better than microscopy
45. Mass Screening for Malaria
in Populations for Resettlement
• Blood smear examinations to detect
asymptomatic parasitemia
• Not useful for predicting individual risks
• undetectable parasitemias
• dormant liver phase parasites
• Can be used to make a decision about the
need for mass treatment of the entire
group
46. Issues in application of
diagnostics
• Roll Back Malaria objective – At least 60% of those
suffering from malaria have prompt access to and are
able to use correct, affordable and appropriate
treatment within 24 hours of the onset of symptoms
• Cost should not focus on unit cost alone
• Must put in context of case management
– Amount of drugs being inappropriately dispensed
– Increasing drug resistance
– Increasingly costly, complex, and toxic alternative drugs
– Epidemiology of malaria, populations served
– Provider and patient acceptability, esp. of negative results
47. Issues in application of
diagnostics
• Rapid diagnostic tests have potential to complement
conventional microscopy or provide a diagnostic
modality when none is available
• Operational research is needed to evaluate best uses
and cost effectiveness
• Potential uses
– Epidemics and emergencies
– Inadequate or absent lab services, unskilled staff
– Mobile clinics
– Low transmission areas; areas with high levels of drug
resistance
– Epidemiologic surveys, seroprevalence data
