Laboratory diagnosis of malaria

Plasmodium falciparum
Basic guidelines
A. Capillary blood should be obtained by fingerstick, or venous blood should be obtained by
venipuncture.
B. Blood smears, at least two thick and two thin, should be prepared as soon as possible after col-
lection. Delay in preparation of the smears can result in changes in parasite morphology
and staining characteristics.
C. Schüffner’s dots can be demonstrated in Giemsa stain, which is preferred to Wright or Wright-
Giemsa stains.

In P. falciparum infections, red blood cells (rbcs) are normal in size. Typically only rings and gametocytes are
seen unless the blood sat before the smears were prepared.
1. Rings
P. falciparum rings have delicate cytoplasm and one or two small chromatin dots. Rbcs that are infected
are not enlarged; multiple infection of rbcs is more common in P. falciparum than in other species. Occa-
sional appliqué forms (rings appearing on the periphery of the rbc) can be present.

Rings in a thick blood smear. Images from a thick blood smear showing more rings.

Rings in a thick blood smear. Images from a thick blood smear showing more rings. Note the classic “headphones”
appearance of many of the rings.
Laboratory diagnosis of malaria

Plasmodium falciparum

Thin, delicate rings in a thin blood smear. Thin, delicate rings in a thin blood smear. Rings in a blood smear. Note the multiply-
Note the double chromatin dot in the in- infected rbcs
fected rbc at top, and the appliqué form in
the infected rbc at bottom.

Rings in thin blood smears. Note the multiply-infected rbcs, some appliqué forms and some classic “head phone” form of several of the in-
fected red blood cells.

Rings and some developing trophozoites seen in thin smears, Note also the presence of Maurer’s clefts, which are often seen in older
ring forms. Maurer’s clefts stain best with an alkaline pH of 7.2—7.6.
Laboratory diagnosis of malaria

Plasmodium falciparum
2. Gametocytes
P. falciparum gametocytes are crescent or sausage shaped. The chromatin is in a single mass
(macrogamete) or diffuse (microgamete).

Gametocytes in a thick blood smear. Gametocytes in a thick smear. Note also the presence of several rings.

Gametocyte in a thin smear with rings and Two gametocytes in a thin smear. Gametocyte in a thin smear showing the
Maurer’s clefts. membrane of the rbc.

Gametocytes; the one on the right is un- Gametocytes in thin blood smears. Note the presence of “Laveran’s bib” (black arrow),
dergoing exflagellation which is not always visible.
Laboratory diagnosis of malaria

Plasmodium falciparum
3. Trophozoites
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.

Trophozoites in a thick blood smear. Mature, compact trophozoites in a thin Compact trophozoites in a thin blood
blood smear. smear.

4. Schizonts
P. falciparum schizonts are seldom seen in peripheral blood. Mature schizonts have 8 to 24 small
merozoites; dark pigment, clumped in one mass.

Ruptured schizont in a thin blood Another schizont in a thin blood

Mature schizont in a thin blood smear.
smear. smear.
Laboratoy diagnosis of malaria

Plasmodium vivax
Basic guidelines
A. Capillary blood should be obtained by fingerstick, or venous blood should be obtained by
venipuncture.
B. Blood smears, at least two thick and two thin, should be prepared as soon as possible after col-
lection. Delay in preparation of smears can result in changes in parasite morphology and
staining characteristics.
C. Schüffner’s dots can be demonstrated in Giemsa stain, which is preferred to Wright or Wright-
Giemsa stains.

In P. vivax infections, red blood cells (rbcs) can be normal to enlarged (up to 1 1/2× to 2×) in size
and may be distorted. Under optimal conditions, Schüffner's dots may be seen in Giemsa-stained
slides.
1. Rings
P. vivax rings have large chromatin dots and cytoplasm can become ameboid as they develop.

Ring in a thick blood smear. The “halo” is Ameboid ring in an enlarged and dis- Ameboid ring in an enlarged and distorted
suggestive of Schüffner’s dots. torted infected rbc. infected rbc. Schüffner’s dots are visible.

More ring forms in a thin blood smear. Note the slight enlargement of the infected Rings and a trophozoites in a thin blood
rbcs. smear.
Laboratoy diagnosis of malaria

Plasmodium vivax
2. Trophozoites
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.

Trophozoites in thick blood smears.

Large, ameboid trophozoites in thin blood smears. Note the presence of Schüffner's dots, which are best seen when the blood is
stained with Giemsa, and not Wright’s stain.

Thin smears showing mature trophozoites. Note the resemblance to the band forms of P. malariae. The enlarged size of the in-
fected rbcs helps distinguish the two species.
Laboratoy diagnosis of malaria

Plasmodium vivax
3. Gametocytes
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.

Gametocyte in a thick blood smear. Gametocytes in thin blood smears.

A pair of gametocytes in a thin blood Gametocytes in thin blood smears. Note the enlargement of the infected rbc and the
smear. scattered pigment.

Ookinetes in thin blood smears. Ookinetes may form if blood is allowed to sit too long Ookinete in a thick smear.
before processing.
Laboratoy diagnosis of malaria

Plasmodium vivax
4. Schizonts
P. vivax schizonts are large, have 12 to 24 merozoites, yellowish-brown, coalesced pigment, and may
fill the rbc.

Schizont in a thick blood smear. Schizonts in thick blood smears.

Immature schizont in a thin blood Mature schizonts in thin blood smears.

smear.
Laboratory diagnosis of malaria

Plasmodium malariae
Basic guidelines
A. Capillary blood should be obtained by fingerstick, or venous blood should be obtained by
venipuncture.
B. Blood smears, at least two thick and two thin, should be prepared as soon as possible after
collection. Delay in preparation of the smears can result in changes in parasite morphol-
ogy and staining characteristics.
C. Schüffner’s dots can be demonstrated in Giemsa stain, which is preferred to Wright or Wright-
Giemsa stains.

In P. malariae infections, red blood cells (rbcs) are normal or smaller than normal (3/4×) in size.

1. Rings
P. malariae rings have sturdy cytoplasm and a large chromatin dot.

Ring in a thick blood smear. Rings in thin blood smears.

2. Trophozoites
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.

Trophozoite in a thick blood smear.

Band-form trophozoites in thin blood smears.
Laboratory diagnosis of malaria

Plasmodium malariae

Trophozoite in a thick smear. Band-form trophozoites in thin blood smears.

“Basket-form” trophozoites in a thin smear.

“Basket-form” trophozoite in a thin

Trophozoites in thin blood smears. These images show variations on the “basket-form.”
Laboratory diagnosis of malaria

Plasmodium malariae
3. Schizonts
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.

Schizont in a thick blood smear. Schizont in thick blood smears. Note the classic “rosette” appearance of the merozoites.

Schizonts in thin blood smears. The schizont on the left has the appearance of a Schizont in a thin blood smear; note the
rosette pattern of the merozoites.
rosette pattern.

Schizonts in thin blood smears Schizont in a thin blood smear; another

resembling a rosette.
Laboratory diagnosis of malaria

Plasmodium malariae

4. Gametocytes
P. malariae gametocytes are round to oval with scattered brown pigment; they may almost fill the infected
red blood cell.

Gametocytes in thick blood smears. Gametocyte in a thin blood smear.

Gametocytes in thin blood smears.

Gametocyte in thin blood smears.
Laboratory diagnosis of malaria

Plasmodium ovale
Basic guidelines
A. Capillary blood should be obtained by fingerstick, or venous blood should be obtained by
venipuncture.
B. Blood smears, at least two thick and two thin, should be prepared as soon as possible after col-
lection. Delay in preparation of the smears can result in changes in parasite morphology
and staining characteristics.
C. Schüffner’s dots can be demonstrated in Giemsa stain, which is preferred to Wright or Wright-
Giemsa stains.

In P. ovale infections, red blood cells (rbcs) can be normal or slightly enlarged (up to 1 1/4×) in size,
may be round to oval, and are sometimes fimbriated. Under optimal conditions, Schüffner's dots
may be seen in Giemsa stained slides.
1. Rings
P. ovale rings have sturdy cytoplasm and large chromatin dots.

Ring in a thick blood smear. Rings in fimbriated rbcs in thin blood smears.

Rings in thin blood smears. Note the multiply-infected rbcs. Rings in a thin blood smear.
Laboratory diagnosis of malaria

Plasmodium ovale
2. Trophozoites
P. ovale trophozoites have sturdy cytoplasm, large chromatin dots, and can be compact to slightly irregu-
lar.

Trophozoite in a thick blood smear. Compact trophozoites in fimbriated rbcs in thin blood smears. Schüffner’s dots are
also visible.

Ring forms and developing trophozoites in thin blood smears. Compact trophozoite in a fimbriated rbc
in a thin blood smear.
Schüffner’s dots are also visible.

Compact trophozoites showing Schüffner’s dots. The image on the left also shows Ring forms, developing and compact
prominent fimbriation. trophozoites in a thin blood smear.
Laboratory diagnosis of malaria

Plasmodium ovale
3. Gametocytes
P. ovale gametocytes are round to oval and may almost fill the red blood cells. Pigment is brown and
more coarse in comparison to P. vivax.

Gametocyte in a thick blood smear. Gametocyte in a thin blood smear. Gametocyte in a thin blood smear. The
infected rbc shows some fimbriation.

Macrogametocytes in thin blood smears. Notice how they nearly fill the infected rbcs. Course pigment, a discrete red nucleus and
Schüffner’s dots can be seen.

Microgametocyte in thin blood smear. Gametocytes in thin blood smears.

Note the diffuse pigment.
Laboratory diagnosis of malaria

Plasmodium ovale
4. Schizonts
P. ovale schizonts have 6 to 14 merozoites with large nuclei, clustered around a mass of dark-brown
pigment.

Schizonts in thick blood smears. Schizont in a thin blood smear.

Schizont in a thin blood smear with a

Schizonts in thin blood smears. Note the infected rbcs are oval.
developing trophozoite.
Plasmodium falciparum Blood Stage Parasites,
Thin Blood Smears

Fig. 1: Normal red cell Figs. 19-26: Schizonts (Fig. 26 is a ruptured schizont)
Figs. 2-18: Trophozoites (among these, Figs. 2-10 correspond to Figs. 27, 28: Mature macrogametocytes (female)
ring-stage trophozoites) Figs. 29, 30: Mature microgametocytes (male).

Illustrations from: Coatney GR, Collins WE,Warren M, Contacos PG. The Primate Malarias. U.S. Department of Health, Education and Welfare, Bethesda, 1971.
Plasmodium malariae Blood Stage Parasites,
Thin Blood Smears

Fig. 1: Normal red cell Fig. 23: Developing gametocyte

Figs. 2-5: Young trophozoites (rings) Fig. 24: Macrogametocyte (female)
Figs. 6-13: Trophozoites Fig. 25: Microgametocyte (male)
Figs. 14-22: Schizonts
Illustrations from: Coatney GR, Collins WE,Warren M, Contacos PG. The Primate Malarias. U.S. Department of Health, Education and Welfare, Bethesda, 1971.
Plasmodium ovale Blood Stage Parasites,
Thin Blood Smears

Fig. 1: Normal red cell Figs. 16-23: Schizonts

Figs. 2-5: Young trophozoites (Rings) Fig. 24: Macrogametocytes (female)
Figs. 6-15: Trophozoites Fig. 25: Microgametocyte (male)

Illustrations from: Coatney GR, Collins WE,Warren M, Contacos PG. The Primate Malarias. U.S. Department of Health, Education and Welfare, Bethesda, 1971.
Plasmodium vivax Blood Stage Parasites,
Thin Blood Smears

Fig. 1: Normal red cell Figs. 19-27: Schizonts

Figs. 2-6: Young trophozoites (ring stage parasites) Figs. 28 and 29: Macrogametocytes (female)
Figs. 7-18: Trophozoites Fig. 30: Microgametocyte (male)
Illustrations from: Coatney GR, Collins WE,Warren M, Contacos PG. The Primate Malarias. U.S. Department of Health, Education and Welfare, Bethesda, 1971.
Plasmodium falciparum
Blood Stage Parasites, 7.

Thick Blood Smears

3.

1: Small trophozoites.
2: Gametocytes — normal.
3: Slightly distorted gametocyte. 4.
4: “Rounded-up” gametocyte. 1. 6.
5: Disintegrated gametocyte.
6: Nucleus of leucocyte.
7: Blood platelets. 2. 8.
8: Cellular remains of young erythrocyte.

1.

5. 1.
6.

Illustration from: Wilcox A. Manual for the Microscopical Diagnosis of Malaria in Man.
U.S. Department of Health, Education and Welfare,Washington, 1960.

Plasmodium malariae
Blood Stage Parasites,
Thick Blood Smears 8.

5.
10.
1: Small trophozoites. 6.
2: Growing trophozoites.
3: Mature trophozoites.
4, 5, 6: Immature schizonts with varying numbers
of divisions of the chromatin. 4.
7: Mature schizonts. 2.
8: Nucleus of leucocyte. 7.
9: Blood platelets.
10: Cellular remains of young erythrocytes.

10. 9.
7.

1.
3.

8.
Illustration from: Wilcox A. Manual for the Microscopical Diagnosis of Malaria in Man.
U.S. Department of Health, Education and Welfare,Washington, 1960.
Plasmodium ovale
Blood Stage Parasites,
Thick Blood Smears 3.

7. 4.
6.
1: Small trophozoites.
2: Growing trophozoites. 5.
3: Mature trophozoites. 4.
4: Schizonts.
5: Gametocytes.
6: Nucleus of leucocyte. 2.
7: Blood platelets.

1.
5.
7.

4.
6.

Plasmodium vivax
Blood Stage Parasites, 6.

Thick Blood Smears

1.
8.
1: Ameboid trophozoites.
2: Schizont — 2 divisions of chromatin.
3: Mature schizont.
4: Microgametocyte. 5.
5: Blood Platelets. 7.
6: Nucleus of neutrophil.
7: Eosinophil. 1.
8: Blood platelet associated with cellular 5.
remains of young erythrocytes.

3.

1.

4.
2.

Illustration from: Wilcox A. Manual for the Microscopical Diagnosis of Malaria in Man.
U.S. Department of Health, Education and Welfare,Washington, 1960.