Malaria

1. Diagnosis & Blood Stages of
Malaria Parasites
Dr.Ankur kumar.

3. Time for taking blood:
• Blood should be collected-
few hours after the height of the paroxysm of
fever and
before taking antimalarial drugs.
• Parasite density is maximum during this
period

5. Characteristic of thin film
Even and uniform
Consist of a single layer of RBCs
The “feathery tail end” is formed near the
center of the slide
Margins of the film should not extend to the
sides of the slide

6. • Stained with one of the Romanowsky’s stains –
Leishman’s
Giemsa
Field’s
Wright’s or
JSB (Jaswant Singh and Bhattacharya)
stain

7. Advantages-
Peripheral smear is simple, rapid and cheap
• Thick smear is useful in:
Detecting the parasites:
 It is 40 times more sensitive than thin smear
 detect as low as 5–10 parasites per mcl of blood
Quantification of parasitemia
Demonstrating the malaria pigments
• Thin smear is useful in
speciation of malaria parasite. (Speciation is not possible
by thick smear as the RBCs are dehemoglobinized)
Disadvantages-
 Labor intensive and requires experienced microscopist
 Low sensitivity: detection limit of thin smear is more
than 200 parasites per μL of blood.

8. Speciation
• The speciation by thin smear is based on the
detection of the
asexual forms (ring forms, late trophozoites and
schizonts) and
gametocytes.
• In falciparum malaria, only the gametocytes
and ring forms are demonstrated (not
schizonts and late trophozoites) in peripheral
blood

9. DIAGNOSIS
Demonstration of Parasites- Light Microscopy
 The diagnosis of malaria rests on the demonstration of asexual forms of the
parasite in stained peripheral-blood smears. It is the Gold standard for
confirmation of malaria.
 After a negative blood smear, repeat smears should be made if there is a high
degree of suspicion.
 Of the Romanowsky stains, Giemsa at pH 7.2 is preferred; Wright’s,
Field’s, or Leishman’s stain or, JSB stain can also be used.
 Both thin and and thick blood smears should be examined. The thin blood
smear should be rapidly air-dried, fixed in anhydrous methanol, and stained; the
RBCs in the tail of the film should then be examined under oil immersion (×1000
magnification).
 The level of parasitemia is expressed as the number of parasitized erythrocytes
per 1000 RBCs. 9

10. DIAGNOSIS
 The thick blood film should be of uneven thickness. The smear should be
dried thoroughly and stained without fixing. As many layers of
erythrocytes overlie one another and are lysed during the staining
procedure,
 the thick film has the advantage of concentrating the parasites (by 40- to
100-fold compared with a thin blood film) and thus increasing diagnostic
sensitivity.
 Both parasites and white blood cells (WBCs) are counted, and the number
of parasites per unit volume is calculated from the total leukocyte count.
Alternatively, a WBC count of 8000/μL is assumed. A minimum of 200
WBCs should be counted under oil immersion.
 This figure is converted to the number of parasitized erythrocytes per
microliter-> Quantitative test-> Prognostic value.
10

11. DIAGNOSIS
 Before a thick smear is judged to be negative, 100–200 fields should
be examined under oil immersion.
 The presence of only malarial pigments in the absence of Malarial
parasites suggests recent P. falciparum infection.
 Disadvange of thick smear is that Plasmodial spp cant be identified.
 In high-transmission areas, the presence of up to 10,000
parasites/μL of blood may be tolerated without symptoms or signs
in partially immune individuals.
 Thus the detection of malaria parasites is sensitive but only poorly
specific in identifying malaria as the cause of illness.
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16. Fluorescence Microscopy-
 Kawamoto technique is a fluorescent-
staining method for demonstrating the malarial
parasite.
 the blood smears are prepared in a slide and is
stained with Acrydine organge.
 this results in differential staining of Malarial
parasite- Nuclear DNA- Stained GREEN,
Cytoplasmic RNA- Stained RED
16

17. Quantitative Buffy Coat (QBC)-
 This is a sensitive microscopic test based on the ability of
Acridin Orange to stain the nucleic acid contained parasites.
 In this method, blood is collected in a capillary tube coated
with fluorescence dye and subjected to microheamatocrit
centrifugation in a special centrifugation apparatus.
 After centrifugation, the buffy coat in the centrifused
capillary tube is examined directly under a fluorescence
microscope.
 Dye stained malarial parasites appear bright green. It can
detect parasite count as low as 3-4 parasites/ µL.
 The disadvantage is expensive, less specific & inability to
identify parasite spp. 17

19. Immunochromatographic tests-
They are used for Malarial Antigen detection
from blood or urine aka-
 Malarial rapid diagnostic tests, Antigen capture assay
or Dipstics.
The ICTs are monoclonal antibody based assays
to detect
– Plasmodium falciparum histidine-rich protein-2,
– Parasite lactose dehydrogenase or
– Plasmodium aldolase.
PfHRP-2 persists in blood for few months so
can’t be used to predict t/t failure.
100 parasites/ µL density is required for the test . 19

21. Comparison of peripheral smear, quantitative
buffy coat and rapid diagnostic tests

22. SERODIAGNOSIS
 Serological tests are used for-
• Screening of blood bank to identify the infected donors.
• Confirm past malaria in patients.
• Epidemiological survey
 Indirect heamaglutination (IHA), Indirect
Fluorescence antibody (IFA) & ELISA are most
frequently used test to detect malarial antibody in
serum.
 ELISA –Inhibition Test is a recent method to detect
malarial antigen in serum.
22

23. DIAGNOSIS
Molecular diagnosis
 DNA and RNA probes are highly sensitive and specific molecular
methods for d/g of falciparum malaria.
 DNA probes can detect <10 parasites/ µL of blood.
 PCR including real time assay like QT-NASBA (quantitative
nucleic acid sequence-based amplification) can detect parasite as
low as 1 parasite in 20 µL of blood using PBRK1 primer> 100 times
more sensitive than Thick smear.
 PCR can detect parasites in dry blood spots and drug resistance
malaria.
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24. 24

25. Plasmodium Falciparum
Ring Stage (Immature Trophozoites)-
 RBC- Rbcs that are infected are not enlarged;
multiple infection of rbcs is more common in P.
falciparum than in other species.
 Parasite-
 P. falciparum rings have delicate cytoplasm and
one or two small chromatin dots.
Occasional appliqué forms (rings appearing on
the periphery of the rbc) can be present.

28. Plasmodium Falciparum
 Trophozoites
 RBC- normal; rarely, Maurer’s clefts (under certain staining
conditions)
 Parasite-P. falciparum trophozoites are rarely seen in
peripheral blood smears.
 Older, ring stage parasites are re-ferred to as trophozoites.
 The cytoplasm of mature trophozoites tends to be more
dense than in younger rings.
 As P. falciparum trophozoites grow and mature, they tend to
retain their ring-like shape and some-times trace amounts
of yellow pigment can be seen within the cytoplasm.
 Growing trophozoites in P. fal-ciparum can appear slightly
amoeboid in shape.

31. Plasmodium Falciparum
Schizont
RBC- normal; rarely, Maurer’s clefts (under
certain staining conditions)
Parasite- seldom seen in peripheral blood;
mature = 8-24 small merozoites; dark pigment,
clumped in one mass.

34. Plasmodium Falciparum
Gametocyte-
RBC-distorted by parasite
Parasite- crescent or sausage shape; chromatin
in a single mass (macrogametocyte) or diffuse
(microgametocyte); dark pigment mass.

37. Plasmodium Vivax
Rings-
RBC- normal to 1-1/4 X, round; occasionally
fine Schüffner’s dots;multiple infection of
RBC not uncommon.
Parasite-P. vivax rings have large chromatin
dots and cytoplasm can become ameboid as
they develop.

41. Plasmodium Vivax
Trophozoites-
RBC- enlarged 1-1/2–2 X; may be distorted;
fine Schüffner’s dots.
Parasite-P. vivax trophozoites show amoeboid
cytoplasm, large chromatin dots, and have
fine, yellowish-brown pigment. Schüffner's
dots may appear more fine in comparison to
those seen in P. ovale.

46. Plasmodium Vivax
Schizont-
RBC- enlarged 1-1/2–2 X; may be distorted;
fine Schüffner’s dots.
Parasite- large,may almost fill RBC; mature =
12-24 merozoites; yellowish-brown, coalesced
pigment.

49. Plasmodium Vivax
Gametocyte-
RBC- enlarged 1-1/2–2 X; may be distorted; fine
Schüffner’s dots.
Parasite- P. vivax gametocytes are round to oval
with scattered brown pigment and may almost fill
the rbc. Schüff-ner's dots may appear more fine in
comparison to those seen in P. ovale. Chromatin
compact, eccentric (macrogametocyte) or
diffuse(microgametocyte); scattered brown
pigment.

53. Plasmodium Ovale
Rings-
RBC- normal to 1-1/4 X, round to oval;
occasionally Schüffner’s dots; occasionally
fimbriated; multiple infection of RBC not
uncommon.
Parasite- sturdy cytoplasm; large chromatin.

56. Plasmodium Ovale
Trophozoite-
RBC- normal to 1-1/4 X; round to oval; some
fimbriated; Schüffner’s dots.
 Parasite-P. ovale trophozoites have sturdy
cytoplasm, large chromatin dots, and can be
compact to slightly irregular, dark brown
pigment.

58. Plasmodium Ovale
Schizonts-
RBC- normal to 1-1/4 X; round to oval; some
fimbriated; Schüffner’s dots.
Parasite- mature = 6-14 merozoites with large
nuclei, clustered around mass of dark-brown
pigment.

61. Plasmodium Ovale
Gametocytes-
RBC- normal to 1-1/4 X; round to oval; some
fimbriated; Schüffner’s dots.
Parasite- round to oval; compact; may almost
fill RBC; chromatin compact, eccentric
(macrogametocyte) or more diffuse
(microgametocyte); scattered brown pigment,
more coarse than P. vivax.

62. Plasmodium Malariae
Rings-
RBC- normal to 3/4 X.
Parasite- sturdy cytoplasm; large chromatin.

63. Plasmodium Malariae
Trophozoite-
RBC- normal to 3/4 X; rarely, Ziemann’s
stippling(under certain staining conditions)
Parasite- P. malariae trophozoites have
compact cytoplasm and a large chromatin dot.
Occasional band forms and/or “basket” forms
with coarse, dark-brown pigment can be seen.

66. Plasmodium Malariae
Schizont-
RBC- normal to 3/4 X; rarely, Ziemann’s
stippling(under certain staining conditions)
Parasite-P. malariae schizonts have 6 to 12
merozoites with large nuclei, clustered around
a mass of coarse, dark-brown pigment.
Merozoites can occasionally be arranged as a
rosette pattern.

69. Plasmodium Malariae
Gametocytes-
RBC- normal to 3/4 X; rarely, Ziemann’s
stippling (under certain staining conditions).
Parasite- round to oval; compact; may almost
fill RBC; chromatin compact, eccentric
(macrogametocyte) or more diffuse
(microgametocyte); scattered brown pigment.

72. 1= ?
2=?
Thick
Smear
SLIDE-1

73. 1=?
3=?
7=?
Thick
Smear
SLIDE-2

74. 1=?
4=?
Thick
Smear
SLIDE-3

75. 1=?
3=?
Thick
Smear
SLIDE-4

76. ANSWERS
 SLIDE-1:
 1=Small trophozoites. (P. falciparum)
 2=Gametocytes (P. falciparum)
 SLIDE-2:
 1=Small trophozoites (P. malariea)
 3=Mature trophozoites (P. malariea)
 7= Mature schizonts. (P. malariea)
 SLIDE-3:
 1= Small trophozoites (P. ovale)
 4= Schizonts. (P. ovale)
 SLIDE-4:
 1= Ameboid trophozoites. (P. vivax)
 3= Mature schizont. (P. vivax)

77. Case Discussion-1
 An 11-year-old boy from the northern part of Thailand
near the Thai-Burmese border was admitted to the
Chiang Mai University Hospital (CMUH) due to
sustained fever for 10 days prior to admission.
 He presented with fever and chills, but had no
symptoms of cough, running nose or other respiratory
tract symptoms.
 His appetite was normal and he also had no jaundice,
vomiting or diarrhoea.
 The boy had no history of underlying diseases and his
birth history was unremarkable. He had no known drug
allergies and his vaccination history was complete.

78. Case Discussion-1
He had a history of unknown partial treatments
from a rural Burmese hospital 1 week prior to
moving to Thailand.
His father remembered that the boy had been
given some artesunate tablets without
improvement of his condition. Three days before
admission to the CMUH, he developed stomach-
ache and had several episodes of vomiting.
He also complained about myalgia, but did not
have any petechiaes or haemorrhage.

79. Case Discussion-1
 On admission, his physical examination revealed a high-grade
fever (39.1°C), heart rate 140 bpm and respiratory rate 30
breaths/minute.
 He looked sick and had a flushed face. He had no pallor, but
mildly injected conjunctivae and pharynx, mildly enlarged tonsils
and left submandibular lymphadenopathy.
 His breath sound was normal, but he had tachycardia. He had mild
tenderness on the upper right side of the abdomen and epigastrium,
without abdominal distension, guarding or rebound tenderness.
 His liver was mildly enlarged with firm and sharp margins
without tenderness. His spleen was normal.
 There were no petechiae or haemorrhages on his skin. He had
drowsiness but good orientations to time, place and person. No
neurological deficits were found during admission.

80. Case Discussion-1
INVESTIGATIONS-
 Laboratory studies showed a
 haemoglobin concentration of 12.2 g/dL,
 a haematocrit 38.0% and
 a white cell count 11810 cells/mm3 with 70%
neutrophils, 11.2% lymphocytes and 13.7% monocytes.
 The platelet count was 383000 cells/mm3 .
 His peripheral blood smear showed normochromic red
blood cells with several target cells and few basophilic
stipplings,

81. Case Discussion-1
 A liver function test revealed a total protein of 8.1 g/dL,
albumin 4.4 g/dL, globulin 3.6 g/dL, alkaline
phosphatase 229 U/ L,
 cholesterol 118 mg/dL, aspartate aminotransferase 35
U/L, alanine transaminase 32 U/L, total bilirubin 0.53
mg/dL and direct bilirubin 0.09 mg/dL.
 His blood glucose was 99 mg/dL. His blood chemistry
levels and urinary analysis were normal.
 Anti-HIV was negative. His erythrocyte sedimentation
rate was 25 mm/h and C reactive protein level was
below 3.3 mg/L.

82. Case Discussion-2
 A 20-year-old woman, who immigrated 2 weeks ago from
Niger, Africa, presented to the emergency department of an
urban teaching hospital with fever, hypotension, and malaise.
 She was resuscitated with 5.5 L of normal saline solution and
norepinephrine.
 Thin blood smear demonstrated Plasmodium falciparum with
parasitemia of 10% to 15%.
 She had rapid reversal of circulatory shock, cleared her
parasitemia in less than 48 hours with antimalarial therapy,
and was discharged home on hospital day 6 in good condition.

83. Case Discussion-3
 Patient is 61 years old, with hypertension, residing in
France. She developed fever after an 11-day stay in Africa.
Rapidly, her neurological status declined to Glasgow 4.
 Blood smears showed 40% P. falciparumparasitemia. No
other cause for encephalopathy was found.
 The patient arrived in the rehabilitation unit after the anti-
malaria treatment and two month in the recovery unit.
 MRI showed hypersignals from the white matter of the
brain in the occiput, putamen, and corpus callosum,
suggestive of cerebral vasculitis without hemorrhage.

84. Case Discussion-4
 A 23-year old male returned to Singapore from a 3 months study trip to
rural Ghana.
 Te patient was an Asian student at a tertiary institution in Singapore who
had never taken treatment or prophylaxis for malaria despite spending his
childhood and early adulthood living and travelling in malaria endemic
areas of South East Asia.
 Te patient was empirically treated with artemether–lumefantrine (AL)
obtained from a village chemist on the third day of an illness typical for
malaria, though no confrmatory laboratory tests were performed.
 With ongoing symptoms on the 9th day of illness, P. falciparum infection was
documented at a private medical clinic in Accra, with a written report
suggesting that 0.8% of red cells were parasitized (original slides unavailable
for review).
 A further treatment course of oral AL was prescribed and completed.
 From the 5th day of illness until presentation at NUH hospital on day 14,
5 days after the second course of AL, the patient complained of intermittent
passage of dark red urine.