Transcript Malaria Blood Smear • Remains the gold standard for diagnosis Threshold of detection

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
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
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
Collection of Blood Smears
1.
The second or third
finger is usually
selected and cleaned.
4.
Slide must always be
grasped by its edges.
2.
5.
Puncture at the side
of the ball of the
finger.
Touch the drop of
blood to the slide
from below.
3.
Gently squeeze
toward the puncture
site.
Preparing thick and thin films
1.
4.
Touch one drop of
blood to a clean
slide.
Carry the drop of blood
to the first slide and hold
at 45 degree angle.
2.
5.
Spread the first
drop to make a 1
cm circle.
Pull the drop of blood
across the first slide in
one motion.
3.
Touch a fresh drop
of blood to the edge
of another slide.
6.
Wait for both to dry
before fixing and
staining.
Recognizing Malaria Parasites
Inside a red blood cell
One or more red chromatin dots
Blue cytoplasm
Recognizing Erythrocytic Stages:
Schematic Morphology
Blue
Cytoplasm
RING
Red
Chromatin
TROPHOZOITE
Brown
Pigment
SCHIZONT
GAMETOCYTE
Malaria Parasite Erythrocytic Stages
Ring form
Schizont
Trophozoite
Gametocytes
Plasmodium falciparum
Infected erythrocytes: normal size
M
Rings: double chromatin dots; appliqué forms;
multiple infections in same red cell
I
Gametocytes: mature (M)and
immature (I) forms (I is rarely
seen in peripheral blood)
Schizonts: 8-24 merozoites
(rarely seen in peripheral blood)
Trophozoites: compact
(rarely seen in
peripheral blood)
Plasmodium vivax
Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)
Rings
Schizonts: 12-24 merozoites
Trophozoites: ameboid; deforms the erythrocyte
Gametocytes: round-oval
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;
dark pigment; (“rosettes”)
Gametocytes: round-oval
Plasmodium malariae
Infected erythrocytes: size normal to decreased (3/4X)
Trophozoite:
compact
Trophozoite:
typical
band form
Schizont:
6-12 merozoites;
coarse, dark pigment
Gametocyte:
round; coarse,
dark pigment
Species Differentiation on Thin Films
Feature
P. falciparum
P. vivax
P. ovale
+
+
round
round,
distorted
oval,
fimbriated
round
Mauer clefts
Schuffner
spots
Schuffner
spots
none
Enlarged infected RBC
Infected RBC shape
Stippling infected RBC
Trophozoite shape
Chromatin dot
Mature schizont
Gametocyte
small ring,
appliqu
often double
rare, 12-30
merozoites
crescent shape
large ring, large ring,
amoeboid compact
single
large
12-24
4-12
merozoites merozoites
large,
round
large,
round
P. malariae
small ring,
compact
single
6-12
merzoites
compact,
round
Species Differentiation on Thin Films
P. falciparum
Rings
Trophozoites
Schizonts
Gametocytes
P. vivax
P. ovale
P. malariae
Species Differentiation on Thick Films
Feature
Uniform trophozoites
P. falciparum
P. vivax
P. ovale
++
+
P. malariae
+
Fragmented trophozoites
Compact trophozoites
+
Pigmented trophozoites
+
+
Irregular cytoplasm
+
+
Stippling (“RBC ghosts”)
+
+
very rarely
often
often
often
occasionally
usually
usually
usually
Schizonts visible
Gametocytes visible