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Malaria

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[Plasmodium falciparum] [Plasmodium knowlesi] [Plasmodium malariae] [Plasmodium ovale] [Plasmodium vivax]

Causal Agent

Blood parasites of the genus Plasmodium. There are approximately 156 named species of Plasmodium which infect various species of vertebrates. Four species are considered true parasites of humans, as they utilize humans almost exclusively as a natural intermediate host: P. falciparum, P. vivax, P. ovale and P. malariae. However, there are periodic reports of simian malaria parasites being found in humans, most reports implicating P. knowlesi. At the time of this writing, it has not been determined if P. knowlesi is being naturally transmitted from human to human via the mosquito, without the natural intermediate host (macaque monkeys, genus Macaca). Therefore, P. knowlesi is still considered a zoonotic malaria.

Life Cycle

lifecycle

The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host The number 1. Sporozoites infect liver cells The number 2 and mature into schizonts The number 3, which rupture and release merozoites The number 4. (Of note, in P. vivax and P. ovale a dormant stage [hypnozoites] can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later.) After this initial replication in the liver (exo-erythrocytic schizogony The letter A), the parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony The letter B). Merozoites infect red blood cells The number 5. The ring stage trophozoites mature into schizonts, which rupture releasing merozoites The number 6. Some parasites differentiate into sexual erythrocytic stages (gametocytes) The number 7. Blood stage parasites are responsible for the clinical manifestations of the disease.

The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood meal The number 8. The parasites’ multiplication in the mosquito is known as the sporogonic cycle The letter C. While in the mosquito’s stomach, the microgametes penetrate the macrogametes generating zygotes The number 9. The zygotes in turn become motile and elongated (ookinetes) The number 10 which invade the midgut wall of the mosquito where they develop into oocysts The number 11. The oocysts grow, rupture, and release sporozoites The number 12, which make their way to the mosquito’s salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle.

Geographic Distribution

Malaria generally occurs in areas where environmental conditions allow parasite multiplication in the vector.  Malaria today is usually restricted to tropical and subtropical areas and altitudes below 1,500 m., although in the past malaria was endemic in much of North America, Europe and even parts of northern Asia, and today is still present on the Korean peninsula. However, this present distribution could be affected by climatic changes and population movements. Plasmodium falciparum is the predominant species in the world. P. vivax and P. ovale are traditionally thought to occupy complementary niches, with P. ovale predominating in Sub-Saharan Africa and P. vivax in the other areas; but their geographical ranges do overlap. These two species are not always distinguishable on the basis of morphologic characteristics alone, and the use of molecular tools will help clarify their diagnosis and exact distribution. P. malariae has wide global distribution, being found in South America, Asia, and Africa, but it is less frequent than P. falciparum in terms of association with cases of infection. P. knowlesi is found in southeast Asia.

More on: Malaria Risk Information and Prophylaxis by Country

Clinical Presentation

The symptoms of uncomplicated malaria can be rather non-specific and the diagnosis can be missed if health providers are not alert to the possibility of this disease. Since untreated malaria can progress to severe forms that may be rapidly (<24 hours) fatal, malaria should always be considered in patients who have a history of exposure (mostly: past travel or residence in disease-endemic areas). The most frequent symptoms include fever and chills, which can be accompanied by headache, myalgias, arthralgias, weakness, vomiting, and diarrhea. Other clinical features include splenomegaly, anemia, thrombocytopenia, hypoglycemia, pulmonary or renal dysfunction, and neurologic changes. The clinical presentation can vary substantially depending on the infecting species, the level of parasitemia, and the immune status of the patient. Infections caused by P. falciparum are the most likely to progress to severe, potentially fatal forms with central nervous system involvement (cerebral malaria), acute renal failure, severe anemia, or acute respiratory distress syndrome. Other species can also have severe manifestations. Complications of P. vivax malaria include splenomegaly (with, rarely, splenic rupture), and those of P. malariae include nephrotic syndrome.

Plasmodium falciparum
Ring-form trophozoites of P. falciparum in thick and a thin blood smear.

 

Ring-form trophozoites (rings) of Plasmodium falciparum are often thin and delicate, measuring on average 1/5 the diameter of the red blood cell. Rings may possess one or two chromatin dots. They may be found on the periphery of the RBC (accolé, appliqué) and multiply-infected RBCs are not uncommon. Ring forms may become compact or pleomorphic depending on the quality of the blood or if there is a delay in making smears. There is usually no enlargement of infected RBCs.
Figure A
Figure A: Rings of P. falciparum in a thick blood smear.
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Figure D: Rings of P. falciparum in a thick blood smear.
Figure G: Rings of P. falciparum in a thin blood smear.
Figure J: Rings of P. falciparum in a thin blood smear.
Figure B: Rings of P. falciparum in a thick blood smear.
Figure E: Rings of P. falciparum in a thick blood smear.
Figure H: Rings of P. falciparum in a thin blood smear.
Figure K: Rings of P. falciparum in a thin blood smear.
Figure C: Rings of P. falciparum in a thick blood smear.
Figure F: Rings of P. falciparum in a thick blood smear.
Figure I: Rings of P. falciparum in a thin blood smear.
Figure L: Rings of P. falciparum in a thin blood smear. Image courtesy of the Arizona State Public Health Laboratory.
Ring-form trophozoites of P. falciparum in thin blood smears exhibiting Maurer’s clefts.

 

Maurer’s clefts can be seen in P. falciparum infections containing older ring-form trophozoites and asexual stages. Maurer’s clefts resemble the Schüffner’s dots seen in P. vivax and P. ovale, but are usually larger and more coarse. Visualization of these structures is dependent on the quality of the smear preparation and the pH of the Giemsa stain. Like Schüffner’s dots, Maurer’s clefts appear to play a role in the metabolic pathways of the infected RBCs.
Figure A: Ring-form trophozoites of P. falciparum in a thin blood smear, exhibiting Maurer's clefts.
Figure E: Ring-form trophozoites of P. falciparum in a thin blood smear, exhibiting Maurer's clefts.
Figure B: Ring-form trophozoites of P. falciparum in a thin blood smear, exhibiting Maurer's clefts.
Figure F: Ring-form trophozoites of P. falciparum in a thin blood smear, exhibiting Maurer's clefts.
Figure C: Ring-form trophozoites of P. falciparum in a thin blood smear, exhibiting Maurer's clefts.
Figure D: Ring-form trophozoites of P. falciparum in a thin blood smear, exhibiting Maurer's clefts.
Developing and older trophozoites of P. falciparum in thick and a thin blood smear.

 

Developing trophozoites of P. falciparum tend to remain in ring form, but may become thicker and more compact. The amount of pigment and chromatin may also increase. Compact or amoeboid forms may be seen in smears where there was a delay in processing the blood.
Figure A: Trophozoites of P. falciparum in a thick blood smear.
Figure E: Trophozoites of P. falciparum in a thin blood smear.
Figure B: Trophozoite of P. falciparum in a thin blood smear.
Figure F: Trophozoites of P. falciparum in a thin blood smear.
Figure C: Trophozoite of P. falciparum in a thin blood smear.
Figure D: Trophozoite of P. falciparum in a thin blood smear. In this figure, a gametocyte can also be seen in the upper half of the image.
Gametocytes of P. falciparum in thick and a thin blood smear.

 

Gametocytes of Plasmodium falciparum are crescent- or sausage-shaped, and are usually about 1.5 times the diameter of an RBC in length. The cytoplasm of the macrogametocytes (female) are usually a darker, deeper blue; the cytoplasm of the microgametocytes (male) is usually more pale. The red chromatin and pigment is more coarse and concentrated in the macrogametocytes than the microgametocytes. Sometimes in thin blood smears, the remnants of the host RBC can be seen; this is often referred to as Laveran’s bib.
Figure A: Gametocyte of P. falciparum in a thick blood smear. Note also the presence of many ring-form trophozoites.
Figure E: Gametocyte of P. falciparum in a thin blood smear. Also seen in this image are ring-form trophozoites exhibiting Maurer's clefts.
Figure I: Gametocytes of P. falciparum in a thin blood smear. The gametocyte in the upper right is undergoing exflagellation, a process that normally occurs in the mid-gut of the mosquito host. However, it may be observed in human blood specimens when there is a delay in processing the blood.
Figure B: Gametocytes of P. falciparum in a thick blood smear. Note also the presence of many ring-form trophozoites.
Figure F: Gametocyte of P. falciparum in a thin blood smear. In these specimens, Laveran's bibs can be seen.
Figure C: Gametocytes of P. falciparum in a thick blood smear.
Figure G: Gametocytes of P. falciparum in a thin blood smear. In these specimens, Laveran's bibs can be seen.
Figure D: Gametocyte of P. falciparum in a thin blood smear. Also seen in this image are ring-form trophozoites and an RBC exhibiting basophilic stippling (upper left).
Figure H: Gametocyte of P. falciparum in a thin blood smear, showing Laveran's bib. Also seen in this image are ring-form trophozoites exhibiting Maurer's clefts.
Schizonts of P. falciparum in a thin blood smear.

 

Schizonts are rarely seen in peripheral blood of Plasmodium falciparum infections, except in severe cases. When seen, schizonts contain anywhere from 8-24 merozoites. A mature schizont usually fills about 2/3 of the infected RBC.
Figure A: Schizont of P. falciparum in a thin blood smear.
Figure B: Schizont of P. falciparum in a thin blood smear.
Figure C: Schizont of P. falciparum in a thin blood smear. Trophozoites are also seen in this image.
Plasmodium knowlesi
Ring-form trophozoites of P. knowlesi in a thin blood smear.

 

Early ring-form trophozoites (rings) of P. knowlesi are similar to P. falciparum, as rings may show double chromatin dots. Appliqué forms may appear, as well as rectangular rings harboring one or more accessory chromatin dots. Red blood cells may also be multiply-infected. When full-grown, non-amoeboid rings may occupy half or more of the host RBC.
Figure A: Ring-form trophozoites of P. knowlesi in a Giemsa-stained thin blood smear from a human patient that traveled to the Philippines. Note a multiply-infected RBC in this image. Image courtesy of the Wadsworth Center, New York State Department of Health.
Figure E: Ring-form trophozoites of P. knowlesi in a Giemsa-stained thin blood smear from a human patient that traveled to the Philippines. Image courtesy of the Wadsworth Center, New York State Department of Health.
Figure B: Ring-form trophozoite of P. knowlesi in a Giemsa-stained thin blood smear from a human patient that traveled to the Philippines. Image courtesy of the Wadsworth Center, New York State Department of Health.
Figure F: Ring-form trophozoites of P. knowlesi in a Giemsa-stained thin blood smear from a human patient that traveled to the Philippines. Note a multiply-infected RBC in this image. Image courtesy of the Wadsworth Center, New York State Department of Health.
Figure C: Ring-form trophozoites of P. knowlesi in a Giemsa-stained thin blood smear from a human patient that traveled to the Philippines. Note a multiply-infected RBC in this image. Image courtesy of the Wadsworth Center, New York State Department of Health.
Figure D: Ring-form trophozoites of P. knowlesi in a Giemsa-stained thin blood smear from a human patient that traveled to the Philippines. Image courtesy of the Wadsworth Center, New York State Department of Health.
Older, developing trophozoites of P. knowlesi in a thin blood smear.

 

In developing trophozoites of P. knowlesi, band forms may appear that are similar in appearance to P. malariae. As the vacuole is lost during maturation of the trophozoite stage, the parasite becomes smaller and more compact. The pigment appears as dark grains and the red nucleus increases in size. Stippling appears, often referred to as ‘Sinton and Mulligan’s’ stippling, as it is not of the Schüffner type.
Figure A: Band-form trophozoite of P. knowlesi in a Giemsa-stained thin blood smear from a human patient that traveled to the Philippines. Image courtesy of the Wadsworth Center, New York State Department of Health.
Figure B: Band-form (upper) and ring-form (lower) trophozoites of P. knowlesi, from the same specimen as Figure A.
Gametocytes of P. knowlesi in thin blood smears.

 

Mature macrogametocytes of P. knowlesi are usually spherical and fill the host RBC. The cytoplasm stains blue and the eccentric nucleus stains red. Pigment is coarse and black, and is scattered irregularly in the cytoplasm. The microgametocyte is often, but not always, smaller than the macrogametocyte.  The cytoplasm usually stains a pale pink, while the nucleus stains a darker red. The nucleus may make up half the parasite. The coarse, black pigment is scattered irregularly thought the cytoplasm.
Figure A: Gametocyte of P. knowlesi in a Giemsa-stained thin blood smear from a patient that traveled to the Philippines. Image courtesy of the Wadsworth Center, New York State Department of Health.
Figure B: Gametocyte of P. knowlesi in a Giemsa-stained thin blood smear from a patient that traveled to the Philippines. Note also a ring-form trophozoite in the lower left of this image. Image courtesy of the Wadsworth Center, New York State Department of Health.
Schizonts of P. knowlesi in a thin blood smear.

 

In developing schizonts, Sinton and Mulligan’s stippling may be observed. The nucleus continues to divide until there are up to 16 (average 10) merozoites. As the schizont matures, it fills the host RBC and the pigment collects into one or a few masses. In the mature schizont, the merozoites may appear ‘segmented’ and the pigment has collected into a single mass.
Figure A: Mature schizont in a Giemsa-stained thin blood smear from a patient that traveled to the Philippines.Images courtesy of the Wadsworth Center, New York State Department of Health.
Figure E: Developing schizont in a Giemsa-stained thin blood smear from the same patient as Figures A-D.
Figure B: Mature schizont in a Giemsa-stained thin blood smear from a patient that traveled to the Philippines. Note also a ring-form trophozoite to the right of the schizont in this figure. Images courtesy of the Wadsworth Center, New York State Department of Health.
Figure C: Developing schizont in a Giemsa-stained thin blood smear from the same patient seen in Figures A and B.
Figure D: Mature schizont in a Giemsa-stained thin blood smear from the same patient seen in Figures A-C.
Plasmodium malariae
Ring-form trophozoites of P. malariae in thick and think blood smears.

 

Ring-form trophozoites have one (rarely two) chromatin dots and a cytoplasm ring that tends to be thicker than P. falciparum. ‘Bird’s-eye’ forms may appear. There is no enlargement of infected RBCs.
Figure A: Ring-form (lower right) and developing (upper left) trophozoites of P. malariae in a thick blood smear.
Figure B: "Birds-eye" trophozoite of P. malariae in a thin blood smear.
Figure C: Ring-form trophozoite of P. malariae in a thin blood smear.
Figure D: Ring-form trophozoite of P. malariae in a thin blood smear.
Trophozoites of P. malariae in a thick blood smear.

 

In developing trophozoites of P. malariae, chromatin is rounded or streaky and the cytoplasm is usually compact with no vacuole. Pigment may be coarse and peripheral. As the trophozoites mature, the cytoplasm may elongate across the host RBC, forming a ‘band-form’, or may be oval with a vacuole forming a ‘basket-form’. Chromatin is usually in a single mass, less definite in outline. Pigment granules become larger and tend to have a more peripheral arrangement.
Figure A: Trophozoite of P. malariae in a thick blood smear.
Figure B: Trophozoite of P. malariae in a thick blood smear.
Band-form trophozoites of P. malariae in a thin blood smear.

 

In developing trophozoites of P. malariae, chromatin is rounded or streaky and the cytoplasm is usually compact with no vacuole. Pigment may be coarse and peripheral. As the trophozoites mature, the cytoplasm may elongate across the host RBC, forming a ‘band-form’, or may be oval with a vacuole forming a ‘basket-form’. Chromatin is usually in a single mass, less definite in outline. Pigment granules become larger and tend to have a more peripheral arrangement.
Figure A: Band-form trophozoite of P. malariae in a thin blood smear.
Figure E: Band-form trophozoite of P. malariae in a thin blood smear.
Figure B: Band-form trophozoite of P. malariae in a thin blood smear.
Figure C: Band-form trophozoite of P. malariae in a thin blood smear.
Figure D: Band-form trophozoite of P. malariae in a thin blood smear.
Basket-form trophozoites of P. malariae in a thin blood smear.

 

In developing trophozoites of P. malariae, chromatin is rounded or streaky and the cytoplasm is usually compact with no vacuole. Pigment may be coarse and peripheral. As the trophozoites mature, the cytoplasm may elongate across the host RBC, forming a ‘band-form’, or may be oval with a vacuole forming a ‘basket-form’. Chromatin is usually in a single mass, less definite in outline. Pigment granules become larger and tend to have a more peripheral arrangement.
Figure A: Basket-form trophozoite of P. malariae in a thin blood smear.
Figure B: Basket-form trophozoite of P. malariae in a thin blood smear.
Figure C: Basket-form trophozoite of P. malariae in a thin blood smear.
Gametocytes of P. malariae in thick and a thin blood smear.

 

Gametocytes of P. malariae are compact and tend to fill the host RBC. There is no enlargement of the infected RBC and sometimes there is a reduction in size. The cytoplasm stains blue and the chromatin pink to red. Abundant dark pigment may be scattered throughout the cytoplasm.
Figure A: Gametocyte of P. malariae in a thick blood smear.
Figure E: Developing gametocyte of P. malariae in a thin blood smear.
Figure B: Gametocyte of P. malariae in a thick blood smear.
Figure F: Developing gametocyte of P. malariae in a thin blood smear.
Figure C: Gametocyte of P. malariae in a thin blood smear.
Figure D: Gametocyte of P. malariae in a thin blood smear.
Schizonts of P. malariae in thick and a thin blood smear.

 

Schizonts of P. malariae have 6-12 (usually 8-10) merozoites, often arranged in a rosette or irregular cluster. Mature schizonts nearly fill the normal-sized host RBC. Pigment is course and often peripheral. Schizonts can be common in peripheral blood circulation.
Figure A: Schizont of P. malariae in a thick blood smear.
Figure E: Schizonts of P. malariae in a thin blood smear.
Figure I: Schizont of P. malariae in a thin blood smear.
Figure B: Schizont of P. malariae in a thick blood smear.
Figure F: Schizont of P. malariae in a thin blood smear.
Figure J: Schizont of P. malariae in a thin blood smear.
Figure C: Schizonts of P. malariae in a thick blood smear.
Figure G: Schizont of P. malariae in a thin blood smear.
Figure D: Schizont of P. malariae in a thick blood smear.
Figure H: Schizont of P. malariae in a thin blood smear.
Plasmodium ovale
Ring-form trophozoites of P. ovale in thick and a thin blood smear.

 

Ring-form trophozoites usually contain a single chromatin dot, but may contain double-chromatin dots. Multiply-infected RBCs may be seen, making the rings difficult to differentiate from P. falciparum. The sing rings may be difficult to differentiate from P. vivax, as the cytoplasm is usually thick with a large chromatin dot. As the trophozoites mature, they are less amoeboid than P. vivax and may exhibit fimbriation and Schüffner’s dots. Infected RBCs are not usually enlarged as in P. vivax infections.
Figure A: Ring-form trophozoite of P. ovale in a thick blood smear.
Figure B: Ring-form trophozoite of P. ovale in a thick blood smear.
Figure C: Ring-form trophozoites of P. ovale in a thin blood smear. Note the multiply-infected RBC in this image.
Figure D: Ring-form trophozoites of P. ovale in a thin blood smear.
Figure E: Ring-form trophozoites of P. ovale in a thin blood smear. Note the multiply-infected RBC in this image.
Trophozoites of P. ovale in thick and thin blood smears.

 

Developing trophozoites of P. ovale are compact with little vacuolation. Infected RBCs are often slightly enlarged and may exhibit fimbriation and Schüffner’s dots. Pigment is less-coarse and diffuse.
Figure A: Trophozoite of P. ovale in a thick blood smear.
Figure B: Trophozoite of P. ovale in a thin blood smear. Note the fimbriation.
Figure C: Trophozoite of P. ovale in a thin blood smear. Note the fimbriation and Schüffner's dots.
Figure D: Trophozoite of P. ovale in a thin blood smear. Note the fimbriation and Schüffner's dots.
Figure E: Trophozoites of P. ovale in a thin blood smear.
Figure F: Infected RBCs showing developing (lower) and ring-form (upper two) trophozoites of in a thin blood smear.
Figure G: Trophozoites of P. ovale in a thin blood smear.
Gametocytes of P. ovale in thick and thin blood smears.

 

Gametocytes of P. ovale can be difficult to distinguish from those of P. vivax, although there is generally less enlargement of the infected RBC. The mature macrogametocyte fills the host RBC; the microgametocyte is smaller. Schüffner’s dots may be seen with proper staining and fimbriation may occur.
Figure A: Gametocyte of P. ovale in a thick blood smear.
Figure B: Gametocyte of P. ovale (red arrow) nestled between two white blood cells in a thick blood smear.
Figure C: Microgametocyte of P. ovale in a thin blood smear. Note the elongated, oval shape and the Schüffner's dots.
Figure D: Macrogametocyte of P. ovale in a thin blood smear. Note the fimbriation.
Figure E: Macrogametocyte of P. ovale in a thin blood smear. Note the fimbriation.
Figure F: Macrogametocyte of P. ovale in a thin blood smear, showing Schüffner's dots.
Figure G: Macrogametocyte of P. ovale in a thin blood smear.
Figure H: Macrogametocyte of P. ovale in a thin blood smear.
Schizonts of P. ovale in thick and thin blood smears.

 

Schizonts of P. ovale can be similar to P. vivax, although tend to be smaller and contain fewer merozoites (4-16, on average 8). Elongation to an oval shape and fimbriation are common. Schüffner’s dots can be observed with proper staining. Pigment is lighter and less coarse, similar to P. vivax.
Figure A: Schizont of P. ovale in a thick blood smear.
Figure B: Schizonts of P. ovale in a thick blood smear.
Figure C: Schizont of P. ovale in a thick blood smear.
Figure D: Schizont of P. ovale in a thin blood smear. Notice the fimbriation.
Figure E: Schizont of P. ovale in a thin blood smear. Notice the fimbriation.
Figure F: Schizont of P. ovale in a thin blood smear.
Figure G: Schizont (upper right) and ring-form trophozoite (lower left) of P. ovale in a thin blood smear.
Plasmodium vivax
Ring-form trophozoites of P. vivax in thick and thin blood smears.

 

Ring-form trophozoites of P. vivax usually have a thick cytoplasm with a single, large chromatin dot. Rings may be difficult to distinguish from those of P. ovale. The cytoplasm becomes amoeboid and Schüffner’s dots may appear as the trophozoites mature. Infected RBCs are often larger than uninfected RBCs. Multiply-infected RBCs are not uncommon.
Figure A: Ring-form trophozoites of P. vivax in a thick blood smear.
Figure B: Ring-form trophozoites of P. vivax in a thick blood smear.
Figure C: Ring-form trophozoite of P. vivax in a thin blood smear.
Figure D: Ring-form trophozoites of P. vivax in a thin blood smear.
Figure E: Ring-form trophozoites of P. vivax in a thin blood smear.
Figure F: Ring-form trophozoites of P. vivax in a thin blood smear.
Figure G: Ring-form trophozoites of P. vivax in a thin blood smear.
Trophozoites of P. vivax in thick and thin blood smears.

 

Developing trophozoites of P. vivax become increasingly amoeboid, with tenuous pseudopodial processes and large vacuoles.  Schüffner’s dots are visible with proper staining. Pigment tends to be fine and brown. Infected RBCs are usually noticeably larger than uninfected RBCs.
Figure A: Trophozoite of P. vivax in a thick blood smear.
Figure E: Trophozoite of P. vivax in a thin blood smear. Note the amoeboid appearance, Schüffner's dots and enlarged infected RBCs.
Figure B: Trophozoite of P. vivax in a thin blood smear. Note the amoeboid appearance, Schüffner's dots and enlarged infected RBCs.
Figure F: Trophozoite of P. vivax in a thin blood smear. The infected RBCs are also noticeably larger than the uninfected RBCs.
Figure C: Trophozoites of P. vivax in a thin blood smear. Note the amoeboid appearance, Schüffner's dots and enlarged infected RBCs.
Figure G: Trophozoite of P. vivax in a thin blood smear. Note the band-like appearance of the trophozoite in this figure that may be mistaken for a band-form trophozoite of P. malariae. Note, however, the fine, light brown pigment that is distributed throughout the cytoplasm (pigment in P. malariae is usually darker and coarser and distributed on the periphery of the cytoplasm). The infected RBCs are also noticeably larger than the uninfected RBCs.
Figure D: Trophozoite of P. vivax in a thin blood smear. Note the amoeboid appearance, Schüffner's dots and enlarged infected RBCs.
Gametocytes of P. vivax in thick and thin blood smears.

 

Macrogametocytes of P. vivax are round to oval and usually fill the host cell. The infected RBC is usually noticeably larger than uninfected RBCs. The cytoplasm is usually a darker blue and contains fine brown pigment throughout. Schüffner’s dots may be seen with proper staining. Microgametocytes are usually the size of an uninfected RBC and have a paler blue, pink or gray cytoplasm.
Figure A: Gametocyte (upper) and trophozoite (lower) of P. vivax in a thick blood smear.
Figure B: Gametocyte of P. vivax in a thick blood smear.
Figure C: Macrogametocytes of P. vivax in a thin blood smear. Note the enlargement of the gametocytes compared to uninfected RBCs.
Figure D: Macrogametocyte of P. vivax in a thin blood smear. Note the enlargement of the gametocytes compared to uninfected RBCs.
Figure E: Macrogametocyte of P. vivax in a thin blood smear. Note the enlargement of the gametocytes compared to uninfected RBCs.
Figure F: Macrogametocyte of P. vivax in a thin blood smear. Note the enlargement of the gametocytes compared to uninfected RBCs.
Figure G: Macrogametocytes of P. vivax in a thin blood smear.
Figure H: Macrogametocyte of P. vivax in a thin blood smear.
Ookinetes of P. vivax in thick and thin blood smears.

 

Ookinetes are motile zygotes formed by the combination of macrogametocytes and exflagellated microgametocytes in the mid-gut of the mosquito host. Ookinetes invade epithelial cells of the mosquito’s mid-gut where an oocyst is formed. Ookinetes are not found in peripheral blood in the human host and are very rarely found on blood smears. Their presence on smears usually indicates a substantial delay occurred between the time the blood was collected and the time the slide was prepared. The following ookinetes were observed on a specimen courtesy of the Florida State Department of Health. The patient had traveled to India.
Figure A: Ookinete of P. vivax in a thick blood smear.
Figure E: Ookinete of P. vivax in a thin blood smear.
Figure B: Ookinete of P. vivax in a thick blood smear.
Figure C: Ookinete of P. vivax in a thick blood smear.
Figure D: Ookinete of P. vivax in a thin blood smear.
Schizonts of P. vivax in thick and thin blood smears.

 

Developing schizonts of P. vivax are large and amoeboid. Chromatin is arranged in two or more masses; pigment is also usually arranged in more than one mass. Mature schizonts contain 12-24 merozoites, each of which contains a dot of chromatin and a mass of cytoplasm. Pigment is usually organized in one or two clumps. Like other stages, infected RBCs are usually larger than uninfected RBCs.
Figure A: Schizont of P. vivax in a thick blood smear.
Figure B: Schizont of P. vivax in a thick blood smear.
Figure C: Schizont of P. vivax in a thick blood smear.
Figure D: Schizont of P. vivax in a thick blood smear.
Figure E: Schizont of P. vivax in a thick blood smear.
Figure I: Ruptured schizont of P. vivax in a thin blood smear, showing free merozoites and pigment.
Figure F: Schizont of P. vivax in a thin blood smear.
Figure G: Schizont of P. vivax in a thin blood smear.
Figure H: Schizont of P. vivax in a thin blood smear.

Diagnostic Findings

Microscopy

Microscopy (morphologic analysis) continues to be the “gold standard” for malaria diagnosis. Parasites may be visualized on both thick and thin blood smears stained with Giemsa, Wright, or Wright-Giemsa stains. Giemsa is the preferred stain, as it allows for detection of certain morphologic features (e.g. Schüffner’s dots, Maurer’s clefts, etc.) that may not be seen with the other two. Ideally, the thick smears are used to detect the presence of parasites while the thin smears are used for species-level identification. Quantification may be done on both thick and thin smears.

Comparison of Plasmodium Species Which Cause Malaria in Humans
Plasmodium species Stages found in blood Appearance of Erythrocyte (RBC) Appearance of Parasite
P. falciparum Ring normal; multiple infection of RBC more common than in other species; Maurer’s clefts (under certain staining conditions) delicate cytoplasm; 1 to 2 small chromatin dots; occasional appliqué (accolé) forms
Trophozoite normal; rarely, Maurer’s clefts (under certain staining conditions) seldom seen in peripheral blood; compact cytoplasm; dark pigment
Schizont normal; rarely, Maurer’s clefts (under certain staining conditions) seldom seen in peripheral blood; mature = 8 to 24 small merozoites; dark pigment, clumped in one mass
Gametocyte distorted by parasite crescent or sausage shape; chromatin in a single mass (macrogametocyte) or diffuse (microgametocyte); dark pigment mass
P. vivax Ring normal to 1.25x, round; occasionally fine Schüffner’s dots; multiple infection of RBC not uncommon large cytoplasm with occasional pseudopods; large chromatin dot
Trophozoite enlarged 1.5 to 2x; may be distorted; fine Schüffner’s dots large amoeboid cytoplasm; large chromatin; fine, yellowish-brown pigment
Schizont enlarged 1.5 to
2x; may be distorted; fine Schüffner’s dots		 large, may almost fill RBC; mature = 12 to 24 merozoites; yellowish-brown, coalesced pigment
Gametocyte enlarged 1.5 to 2x; may be distorted; fine Schüffner’s dots round to oval; compact; may almost fill RBC; chromatin compact, eccentric (macrogametocyte) or diffuse (microgametocyte); scattered brown pigment
P. ovale Ring normal to 1.25x, round to oval; occasionally Schüffner’s dots; occasionally fimbriated; multiple infection of RBC not uncommon sturdy cytoplasm; large chromatin
Trophozoite normal to 1.25x; round to oval; some fimbriated; Schüffner’s dots compact with large chromatin; dark-brown pigment
Schizont normal to 1.25x, round to oval, some fimbriated, Schüffner’s dots mature = 6 to 14 merozoites with large nuclei, clustered around mass of dark-brown pigment
Gametocyte normal to 1.25x; round to oval, some fimbriated; Schüffner’s dots round to oval; compact; may almost fill RBC; chromatin compact, eccentric (macrogametocyte) or more diffuse (microgametocyte); scattered brown pigment
P. malariae Ring normal to 0.75x sturdy cytoplasm; large chromatin
Trophozoite normal to 0.75x; rarely, Ziemann’s stippling (under certain staining conditions) compact cytoplasm; large chromatin; occasional band forms; coarse, dark-brown pigment
Schizont normal to 0.75x; rarely, Ziemann’s stippling (under certain staining conditions) mature = 6 to 12 merozoites with large nuclei, clustered around mass of coarse, dark-brown pigment; occasional rosettes
Gametocyte normal to 0.75x; rarely, Ziemann’s stippling (under certain staining conditions) round to oval; compact; may almost fill RBC; chromatin compact, eccentric (macrogametocyte) or more diffuse (microgametocyte); scattered brown pigment
P. knowlesi Ring normal to 0.75x; multiple infection not uncommon. delicate cytoplasm; 1 to 2 prominent chromatin dots; occasional appliqué (accolé) forms
Trophozoite normal to 0.75x; rarely, Sinton and Mulligan’s stippling (under certain staining conditions) compact cytoplasm; large chromatin; occasional band forms; coarse, dark-brown pigment
Schizont normal to 0.75x; rarely, Sinton and Mulligan’s stippling (under certain staining conditions) mature = up to 16 merozoites with large nuclei, clustered around mass of coarse, dark-brown pigment; occasional rosettes; mature merozoites appear segmented
Gametocyte normal to 0.75x; rarely, Sinton and Mulligan’s stippling (under certain staining conditions) round to oval; compact; may almost fill RBC; chromatin compact, eccentric (macrogametocyte) or more diffuse (microgametocyte); scattered brown pigment

Molecular Diagnosis

Agarose gel (2%) analysis of a PCR diagnostic test for species-specific detection of Plasmodium DNA.

Morphologic characteristics of malaria parasites can determine a parasite species, however, microscopists may occasionally fail to differentiate between species in cases where morphologic characteristics overlap (especially Plasmodium vivax and P. ovale), as well as in cases where parasite morphology has been altered by drug treatment or improper storage of the sample. In such cases, the Plasmodium species can be determined by using confirmatory molecular diagnostic tests. In addition, molecular tests such as PCR can detect parasites in specimens where the parasitemia may be below the detectable level of blood film examination. The methods currently used at CDC are described below.

Species-specific PCR diagnosis of malaria

Plasmodium genomic DNA is extracted from 200 µl whole blood using the QIAamp Blood Kit (Cat. No. 29106; Qiagen Inc., Chatsworth, CA.) or a similar product that can yield the comparable concentration of genomic DNA from the same volume of blood.

Detection and identification of Plasmodium to the species level is done with a  real-time PCR assay as described by Rougemont et al 2004. This is a dual duplex assay that detects P. falciparum and P. vivax in one reaction, and P. malariae and P. ovale in a parallel reaction, using species-specific TaqMan probes.  In cases where infection by more than one Plasmodium species  is suspected, there is an option to use a conventional nested PCR assay (Snounou el al, 1993) that has an improved resolution of mixed infection compared to the real-time PCR assay.

Agarose gel (2%) analysis of a PCR diagnostic test for species-specific detection of Plasmodium DNA. PCR was performed using nested primers of Snounou et al.1

  • Lane S: Molecular base pair standard (50-bp ladder). Black arrows show the size of standard bands.
  • Lane 1: The red arrow shows the diagnostic band for P. vivax (size: 120 bp).
  • Lane 2: The red arrow shows the diagnostic band for P. malariae (size: 144 bp).
  • Lane 3: The red arrow shows the diagnostic band for P. falciparum (size: 205 bp).
  • Lane 4: The red arrow shows the diagnostic band for P. ovale (size: 800 bp).
Reference:

Mathieu Rougemont, Madeleine Van Saanen, Roland Sahli, Hans Peter Hinrikson, Jacques Bille and Katia Jaton. Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays. J. Clin. Microbiol. 2004, 42(12):5636.

Snounou G, Viriyakosol S, Zhu XP, et al. High sensitivity detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol 1993;61:315-320.

Antibody Detection

Agarose gel (2%) analysis of a PCR diagnostic test for species-specific detection of Plasmodium DNA.
Positive IFA result with P. malariae schizont antigen.

Malaria antibody detection for clinical diagnosis is performed using the indirect fluorescent antibody (IFA) test. The IFA procedure can be used as a diagnostic tool to determine if a patient has been infected with Plasmodium. Because of the time required for development of antibody and also the persistence of antibodies, serologic testing is not practical for routine diagnosis of acute malaria. However, antibody detection may be useful for:

  • screening blood donors involved in cases of transfusion-induced malaria when the donor’s parasitemia may be below the detectable level of blood film examination
  • testing a patient who has been recently treated for malaria but in whom the diagnosis is questioned

Species-specific testing is available for the four human species: P. falciparum, P. vivax, P. malariae, and P. ovale. Cross reactions often occur between Plasmodium species and Babesia species. Blood stage Plasmodium species schizonts (meronts) are used as antigen. The patient’s serum is exposed to the organisms; homologous antibody, if present, attaches to the antigen, forming an antigen-antibody (Ag-Ab) complex. Fluorescein-labeled antihuman antibody is then added, which attaches to the patient’s malaria-specific antibodies. When examined with a fluorescence microscope, a positive reaction is when the parasites fluoresce an apple green color.

Reference:

Sulzer AJ, and Wilson M. The fluorescent antibody test for malaria. Crit Rev Clin Lab Sci 1971;2:601-609.

Antigen Detection

In addition to microscopy and molecular methods, there are methods for detecting malaria parasites on the basis of antigens or enzymatic activities associated with the parasites. These methods are often packaged as individual test kits called rapid diagnostic tests or RDTs.

These methods include, among others:

  • detection of an antigen (histidine rich protein-2, HRP-2) associated with malaria parasites (P. falciparum)
  • detection of a Plasmodium specific aldolase
  • detection of a Plasmodium associated lactate dehydrogenase (pLDH) either through its enzymatic activity or by immunoassay

There is currently only one RDT licensed for use in the United States. For additional information visit https://www.cdc.gov/malaria/hcp/diagnosis-testing/malaria-diagnostic-tests.html

Additional Guidance

Patients suspected of having an orthoebolavirus or orthomarburgvirus after travel to an area experiencing an Ebola or Marburg outbreak should be evaluated urgently for malaria infection as the symptoms of malaria infection are very similar to those of Ebola and Marburg. CDC provides guidance for malaria diagnosis in patients with suspected orthoebolavirus or orthomarburgvirus infection in the United States. 

Bench Aids

Treatment Information

Information about treatment of malaria in the United States is available at https://www.cdc.gov/malaria/treatment/index.html.

Anopheles Mosquitoes 

Malaria is transmitted to humans by female mosquitoes of the genus Anopheles. Female mosquitoes take blood meals for egg production, and these blood meals are the link between the human and the mosquito hosts in the parasite life cycle. The successful development of the malaria parasite in the mosquito (from the “gametocyte” stage to the “sporozoite” stage) depends on several factors. The most important is ambient temperature and humidity (higher temperatures accelerate the parasite growth in the mosquito) and whether the Anophelessurvives long enough to allow the parasite to complete its cycle in the mosquito host (“sporogonic” or “extrinsic” cycle, duration 9 to 18 days). In contrast to the human host, the mosquito host does not suffer noticeably from the presence of the parasites.

Diagram of Adult Female Mosquito
Diagram of Adult Female Mosquito
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Map showing which malaria vectors populate various parts of the world
Map of the world showing the distribution of predominant malaria vectors
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Anopheles freeborni mosquito pumping blood
Anopheles freeborni mosquito pumping blood
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Sequential images of the mosquito taking its blood meal

General Information

There are approximately 3,500 species of mosquitoes grouped into 41 genera. Human malaria is transmitted only by females of the genus Anopheles. Of the approximately 430 Anopheles species, only 30-40 transmit malaria (i.e., are “vectors”) in nature. The rest either bite humans infrequently or cannot sustain development of malaria parasites.

Geographic Distribution

Anophelines are found worldwide except Antarctica. Malaria is transmitted by different Anopheles species in different geographic regions. Within geographic regions, different environments support a different species.

Anophelines that can transmit malaria are found not only in malaria-endemic areas, but also in areas where malaria has been eliminated. These areas are thus at risk of re-introduction of the disease.

Life Stages

Like all mosquitoes, anopheles mosquitoes go through four stages in their life cycle: egg, larva, pupa, and adult. The first three stages are aquatic and last 7-14 days, depending on the species and the ambient temperature. The biting female Anopheles mosquito may carry malaria. Male mosquitoes do not bite so cannot transmit malaria or other diseases. The adult females are generally short-lived, with only a small proportion living long enough (more than 10 days in tropical regions) to transmit malaria.

Eggs

Adult females lay 50-200 eggs per oviposition. Eggs are laid singly directly on water and are unique in having floats on either side. Eggs are not resistant to drying and hatch within 2-3 days, although hatching may take up to 2-3 weeks in colder climates.

Larvae

Mosquito larvae have a well-developed head with mouth brushes used for feeding, a large thorax, and a segmented abdomen. They have no legs. In contrast to other mosquitoes, Anopheles larvae lack a respiratory siphon and for this reason position themselves so that their body is parallel to the surface of the water.

Anopheles Egg
Top: Anopheles Egg; note the lateral floats.
Bottom: Anopheles eggs are laid singly.

Larvae breathe through spiracles located on the 8th abdominal segment and therefore must come to the surface frequently.

The larvae spend most of their time feeding on algae, bacteria, and other microorganisms in the surface microlayer. They do so by rotating their head 180 degrees and feeding from below the microlayer. Larvae dive below the surface only when disturbed. Larvae swim either by jerky movements of the entire body or through propulsion with the mouth brushes.

Larvae develop through 4 stages, or instars, after which they metamorphose into pupae. At the end of each instar, the larvae molt, shedding their exoskeleton, or skin, to allow for further growth.

Anopheles Larva
Anopheles Larva. Note the position, parallel to the water surface.
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The larvae occur in a wide range of habitats but most species prefer clean, unpolluted water. Larvae of Anopheles mosquitoes have been found in fresh- or salt-water marshes, mangrove swamps, rice fields, grassy ditches, the edges of streams and rivers, and small, temporary rain pools. Many species prefer habitats with vegetation. Others prefer habitats that have none. Some breed in open, sun-lit pools while others are found only in shaded breeding sites in forests. A few species breed in tree holes or the leaf axils of some plants.

Pupae

The pupa is comma-shaped when viewed from the side. This is a transitional stage between larva and adult. The pupae does not feed, but undergoes radical metamorphosis. The head and thorax are merged into a cephalothorax with the abdomen curving around underneath. As with the larvae, pupae must come to the surface frequently to breathe, which they do through a pair of respiratory trumpets on the cephalothorax. After a few days as a pupa, the dorsal surface of the cephalothorax splits and the adult mosquito emerges onto the surface of the water.

The duration from egg to adult varies considerably among species and is strongly influenced by ambient temperature. Mosquitoes can develop from egg to adult in as little as 7 days but usually take 10-14 days in tropical conditions.

Anopheles Pupa
Anopheles Pupa
Anopheles Adults
Anopheles Adults. Note (bottom row) the typical resting position.

 

 

 

 

 

 

 

 

 

 

 

 

 

Adults

Like all mosquitoes, adult anopheles have slender bodies with 3 sections: head, thorax and abdomen.

The head is specialized for acquiring sensory information and for feeding. The head contains the eyes and a pair of long, many-segmented antennae. The antennae are important for detecting host odors as well as odors of aquatic larval habitats where females lay eggs. The head also has an elongate, forward-projecting proboscis used for feeding, and two sensory palps.

The thorax is specialized for locomotion. Three pairs of legs and a single pair of wings are attached to the thorax.

The abdomen is specialized for food digestion and egg development. This segmented body part expands considerably when a female takes a blood meal. The blood is digested over time serving as a source of protein for the production of eggs, which gradually fill the abdomen.

Anopheles mosquitoes can be distinguished from other mosquitoes by the palps, which are as long as the proboscis, and by the presence of discrete blocks of black and white scales on the wings. Adult Anopheles can also be identified by their typical resting position: males and females rest with their abdomens sticking up in the air rather than parallel to the surface on which they are resting.

Adult mosquitoes usually mate within a few days after emerging from the pupal stage. In some species, the males form large swarms, usually around dusk, and the females fly into the swarms to mate. The mating habitats of many species remain unknown.

Males live for about a week, feeding on nectar and other sources of sugar. Females will also feed on sugar sources for energy but usually require a blood meal for the development of eggs. After obtaining a full blood meal, the female will rest for a few days while the blood is digested and eggs are developed. This process depends on the temperature but usually takes 2-3 days in tropical conditions. Once the eggs are fully developed, the female lays them then seeks blood to sustain another batch of eggs.

The cycle repeats itself until the female dies. Females can survive up to a month (or longer in captivity) but most do not live longer than 1-2 weeks in nature. Their chances of survival depend on temperature and humidity, but also upon their ability to successfully obtain a blood meal while avoiding host defenses.

Female Anopheles dirus feeding
Female Anopheles dirus feeding

 

 

 

 

 

 

Factors Involved in Malaria Transmission and Malaria Control

Understanding the biology and behavior of Anopheles mosquitoes can aid in designing appropriate control strategies. Factors that affect a mosquito’s ability to transmit malaria include its innate susceptibility to Plasmodium, its host choice, and its longevity. Long-lived species that prefer human blood and support parasite development are the most dangerous. Factors that should be taken into consideration when designing a control program include the susceptibility of malaria mosquitoes to insecticides and the preferred feeding and resting location of adult mosquitoes.

More on: How to Reduce Malaria’s Impact

Preferred Sources for Blood Meals

One important behavioral factor is the degree to which an Anopheles species prefers to feed on humans (anthropophily) or animals such as cattle (zoophily). Anthrophilic Anopheles are more likely to transmit the malaria parasites from one person to another. Most Anopheles mosquitoes are not exclusively anthropophilic or zoophilic; many are opportunistic and feed upon whatever host is available. However, the primary malaria vectors in Africa, An. gambiae and An. funestus, are strongly anthropophilic and, consequently, are two of the most efficient malaria vectors in the world.

Life Span

Once ingested by a mosquito, malaria parasites must undergo development within the mosquito before they are infectious to humans. The time required for development in the mosquito (the extrinsic incubation period) takes 9 days or longer, depending on the parasite species and the temperature. If a mosquito does not survive longer than the extrinsic incubation period, then she will not be able to transmit any malaria parasites.

It is not possible to measure directly the life span of mosquitoes in nature, but many studies have indirectly measured longevity by examination of their reproductive status or via marking, releasing, and recapturing adult mosquitoes. The majority of mosquitoes do not live long enough to transmit malaria, but some may live as long as three weeks in nature. Though evidence suggests that mortality rate increases with age, most workers estimate longevity in terms of the probability that a mosquito will live one day. Usually these estimates range from a low of 0.7 to a high of 0.9. If survivorship is 90% daily, then a substantial proportion of the population would live longer than 2 weeks and would be capable of transmitting malaria. Any control measure that reduces the average lifespan of the mosquito population will reduce transmission potential. Insecticides thus need not kill the mosquitoes outright, but may be effective by limiting their lifespan.

Patterns of Feeding and Resting

Most Anopheles mosquitoes are crepuscular (active at dusk or dawn) or nocturnal (active at night). Some Anopheles mosquitoes feed indoors (endophagic) while others feed outdoors (exophagic). After blood feeding, some Anopheles mosquitoes prefer to rest indoors (endophilic) while others prefer to rest outdoors (exophilic). Biting by nocturnal, endophagic Anopheles mosquitoes can be markedly reduced through the use of insecticide-treated bed nets (ITNs) or through improved housing construction to prevent mosquito entry (e.g., window screens). Endophilic mosquitoes are readily controlled by indoor spraying of residual insecticides. In contrast, exophagic/exophilic vectors are best controlled through source reduction (destruction of larval habitats).

Insecticide Resistance

Insecticide-based control measures (e.g., indoor spraying with insecticides, ITNs) are the principal way to kill mosquitoes that bite indoors. However, after prolonged exposure to an insecticide over several generations, mosquitoes, like other insects, may develop resistance, a capacity to survive contact with an insecticide. Since mosquitoes can have many generations per year, high levels of resistance can arise very quickly. Resistance of mosquitoes to some insecticides has been documented within a few years after the insecticides were introduced. There are over 125 mosquito species with documented resistance to one or more insecticides. The development of resistance to insecticides used for indoor residual spraying was a major impediment during the Global Malaria Eradication Campaign. Judicious use of insecticides for mosquito control can limit the development and spread of resistance, particularly via rotation of different classes of insecticides used for control. Monitoring of resistance is essential to alert control programs to switch to more effective insecticides.

Susceptibility/Refractoriness

Some Anopheles species are poor vectors of malaria, as the parasites do not develop well (or at all) within them. There is also variation within species. In the laboratory, it has been possible to select for strains of An. gambiae that are refractory to infection by malaria parasites. These refractory strains have an immune response that encapsulates and kills the parasites after they have invaded the mosquito’s stomach wall. Scientists are studying the genetic mechanism for this response. It is hoped that some day, genetically modified mosquitoes that are refractory to malaria can replace wild mosquitoes, thereby limiting or eliminating malaria transmission.

DPDx is an educational resource designed for health professionals and laboratory scientists. For an overview including prevention, control, and treatment visit www.cdc.gov/parasites/.