2. Related Tasks:
By the end of this session, students are expected to be
able to:
Describe Morphological Characteristics of Plasmodium
species.
Explain the mode of transmission of Plasmodium species
Explain the effects of Plasmodium species on the host
Perform the laboratory diagnosis of plasmodium species
3. Introduction to Malaria
A serious and sometimes fatal disease caused
by a parasite transmitted by a mosquito
Patients with malaria are typically very sick
with high fever, shaking chills, and flu-like illness
Four kinds of malaria parasites can infect
humans
Plasmodium Falciparum (deadly)
Plasmodium Vivax
Plasmodium Ovale
Plasmodium Malarie
4. Malaria
• Malaria is typically found in warmer regions of the
world. In tropical and subtropical climates.
• Malaria parasites which grow and develop inside the
mosquito need warmth to complete their growth
before they are mature enough to be transmitted to
humans.
5. Transmission
People get bitten by an infected female anopheles mosquito (only
anopheles mosquitoes can transmit malaria)
When the mosquito bites, a small amount of blood is taken which
contains a small amount of microscopic parasites
The parasite grows and matures in the mosquito’s gut for about 7 days
and then travels to the mosquito’s salivary glands
When the mosquito takes its next blood meal, these parasites are mixed
with the saliva and injected into the bite
Once in the blood of the human the parasites travel to the liver and
multiply
After 8 days or more the parasites leave the liver and enter red blood
cells where they continue to multiply
Malaria can be transmitted through blood transfusions, organ
transplants, or the shared use of needles or syringes, and to a mother to
her fetus before or during delivery (MTCT)
7. Who is at risk for malaria?
• Anyone can get malaria
• Most cases occur in residents of countries with
malaria transmission and travelers to those countries
• In non-endemic countries, cases can occur in non-
travelers as congenital malaria, introduced malaria,
or transfusional malaria
8. Symptoms of Malaria
• Fever
• Flu-like illness including: shaking, chills. Headache,
muscle ache, and tiredness
• Nausea, vomiting and diarrhea may also occur.
• Anemia and jaundice may occur due to the loss of
red blood cells
• Plasmodium falciparum may cause kidney failure,
seizures, mental confusion, coma and death
9. Symptoms (cont.)
Symptoms begin 10 days to 4 weeks after
infection although a person may feel ill as early as
7 days or as late as 1 year later
Plasmodium vivax and plasmodium ovale can
relapse
In plasmodium vivax and plasmodium ovale
infections some parasites can remain dormant in
the liver for several months for up to 4 years after a
person has been bitten by an infected mosquito
If an individual has symptoms after traveling in an
malaria risk area they should seek medical help
immediately
10. Lab. Diagnosis of malaria
• Clinical Diagnosis
• Malaria Blood Smear
• Fluorescent microscopy
• Quantitative Buffy coat
• Antigen Detection
• Serology
• Other tests
11. Clinical diagnosis of Malaria
• 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
12. Cont. …
• Clinical case description: Fever case with any of the following:
Chills, Sweating, Jaundice, Splenomegaly Convulsions, Coma,
Shock, Pulmonary edema & Death (in severe cases)
• Case classification:
SUSPECT : Any case of fever
PROBABLE : Case that meets the clinical
case definition
CONFIRMED: A suspected/probable case that is laboratory
confirmed
13. 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/Field
• thick film: 5 -20 parasites/Field
• Requirements: equipment, training, reagents,
supervision
• Simple, inexpensive yet labor-intensive
• Accuracy depends on laboratorian skill
14. Interpreting Thick and Thin Films
THICK FILM
lysed RBCs
larger volume
0.25 μl blood/100 fields
blood elements more
concentrated
good screening test
positive or negative
parasite density
more difficult to diagnose
species
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
15. Malaria blood smear Cont.
• Prepare smears as soon as possible after collecting capillary
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
16. Collection of Blood Smears
5.
Touch the drop of
blood to the slide
from below.
4.
Slide must always be
grasped by its edges.
2.
Puncture at the side
of the ball of the
finger.
3.
Gently squeeze
toward the puncture
site.
1.
The second or third
finger is usually
selected and cleaned.
17. Preparing thick and thin films
1.
Touch one drop of
blood to a clean
slide.
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.
4.
Carry the drop of blood
to the first slide and hold
at 45 degree angle.
21. Diagnostic Points for Plasmodium
falciparum• Red Cells are not enlarged.
• Rings appear fine and delicate and there may be several in one
cell.
• Some rings may have two chromatin dots.
• Presence of marginal or applique forms.
• It is unusual to see developing forms in peripheral blood films.
• Gametocytes have a characteristic crescent shape appearance.
However, they do not usually appear in the blood for the first
four weeks of infection.
• Maurer's dots may be present.
22. 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
23. Diagnostic Points for P. vivax
• Red cells containing parasites are usually enlarged.
• Schuffner's dots are frequently present in the red cells
• The mature ring forms tend to be large and coarse.
• Developing forms are frequently present.
24. Diagnostic Points for P. malariae
• Ring forms may have a squarish appearance.
• Band forms are a characteristic of this species.
• Mature schizonts may have a typical daisy head appearance
with up to ten merozoites.
• Red cells are not enlarged.
• Chromatin dot may be on the inner surface of the ring.
25. Plasmodium vivax
Trophozoites: ameboid; deforms the erythrocyte
Gametocytes: round-ovalSchizonts: 12-24 merozoites
Rings
Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)
26. Diagnostic Points for P. ovale
• Red cells enlarged.
• Comet forms common .
• Rings large and coarse.
• Schuffner's dots, when present, may be prominent.
• Mature schizonts similar to those of P. malariae but
larger and more coarse.
• Quantifying parasites:
% parasitemia = (parasitized RBCs/total RBCs) × 100
27. Species Differentiation on Thin Films
Feature P. falciparum P. vivax P. ovale P. malariae
Enlarged infected RBC + +
Infected RBC shape round round,
distorted
oval,
fimbriated
round
Stippling infected RBC Mauer clefts Schuffner
spots
Schuffner
spots
none
Trophozoite shape small ring,
appliqu
large ring,
amoeboid
large ring,
compact
small ring,
compact
Chromatin dot often double single large single
Mature schizont rare, 12-30
merozoites
12-24
merozoites
4-12
merozoites
6-12
merzoites
Gametocyte crescent shape large,
round
large,
round
compact,
round
28.
29. Species Differentiation on Thin Films
P. falciparum P. vivax P. ovale P. malariae
Rings
Trophozoites
Schizonts
Gametocytes
30. 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.
31. Parasitemia and clinical correlates
Parasitemia Parasites/µl Remarks
2-5% 100,000-
250,00
Hyperparasitemia/severe
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).
32. 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
33. 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%
34. 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%
35. Quantitative Buffy Coat Cont. …
Useful for screening large numbers of samples
Quick, saves time
Requires centrifuge, special stains
Three main disadvantages
Species identification and quantification difficult
High cost of capillaries and equipment
Can’t store capillaries for later reference
38. 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
39. 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
40. 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
42. 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)
43. Reporting result of Malaria parasite
• Blood film for malaria/(BFFM) or Blood smear for malaria
(BSFM):
• Malaria parasite seen (+ve)
• No malaria parasite seen (-ve)
• For RDTs or ICT report:
• RDTs/ICT for malaria is Negative or,
• RDTs/ICT for malaria is Positive.
44. OTHER TESTS
Polymerase chain reaction (PCR)
Detection of Anti-malarial antibodies
Intraleucocytic malaria pigment
Flowcytometry
Mass spectrometry
45. Preventing Malaria
Keep mosquitoes from biting you especially at
night
Take anti-malaria drugs to kill the parasites
Eliminate places around your home where
mosquitoes breed
Spray insecticides on your home’s walls to kill adult
mosquitoes that come inside
Sleep under mosquito nets – especially effective if
they have been treated with insecticides
Wear insect repellant and long sleeve clothing
when outdoors at night
Currently there is no vaccine for malaria
47. Recap Questions
• Which kind of malaria parasite is most deadly?
a. Plasmodium falciparum
b. Plasmodium vivax
c. Plasmodium ovale
d. Plasmodium malariae
48. Questions
• Malaria is typically found in which climate?
a. Subtropical
b. Tundra
c. Tropical
d. A and C
49. Questions
• Which of the following is not a symptom of malaria?
a. Fever
b. Rash
c. Nausea
d. Headache
50. Questions
• Which two kinds of malaria can relapse?
a. Plasmodium falciparum and plasmodium ovale
b. Plasmodium ovale and plasmodium malariae
c. Plasmodium malariae and plasmodium vivax
d. Plasmodium vivax and plasmodium ovale