laboratory diagnosis of malaria parasite in south sudan

1. Plasmodium species
Mathew Tut Moses
RHSI 2015

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)

6. Malaria life cycle
• Refer to L.C diagram!

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.

18. Recognizing Malaria Parasites
Inside a red blood cell
One or more red chromatin dots
Blue cytoplasm

19. RING TROPHOZOITE
SCHIZONT GAMETOCYTE
Blue
Cytoplasm
Red
Chromatin
Brown
Pigment
Recognizing Erythrocytic Stages:
Schematic Morphology

20. Malaria Parasite Erythrocytic Stages
Ring form
Trophozoite
Schizont
Gametocytes

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

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

36. QBC

37. The QBC TestThe QBC Test

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

41. RDT Test FormatRDT Test Format

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

46. TT
HH
AA
NN
KK
SS

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

51. Questions
• There is no vaccine for malaria?
a. True
b. False

52. Questions
• Name 3 ways to prevent malaria: