[Plasmodium falciparum] [Plasmodium knowlesi] [Plasmodium malariae] [Plasmodium ovale] [Plasmodium vivax]
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.
The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host . Sporozoites infect liver cells and mature into schizonts , which rupture and release merozoites . (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 parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony ). Merozoites infect red blood cells . The ring stage trophozoites mature into schizonts, which rupture releasing merozoites . Some parasites differentiate into sexual erythrocytic stages (gametocytes) . 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 parasites’ multiplication in the mosquito is known as the sporogonic cycle . While in the mosquito’s stomach, the microgametes penetrate the macrogametes generating zygotes . The zygotes in turn become motile and elongated (ookinetes) which invade the midgut wall of the mosquito where they develop into oocysts . The oocysts grow, rupture, and release sporozoites , which make their way to the mosquito’s salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle .
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.
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.
Ring-form trophozoites of P. falciparum in thick and a thin blood smear.
Ring-form trophozoites of P. falciparum in thin blood smears exhibiting Maurer’s clefts.
Developing and older trophozoites of P. falciparum in thick and a thin blood smear.
Gametocytes of P. falciparum in thick and a thin blood smear.
Schizonts of P. falciparum in a thin blood smear.
Ring-form trophozoites of P. knowlesi in a thin blood smear.
Older, developing trophozoites of P. knowlesi in a thin blood smear.
Gametocytes of P. knowlesi in thin blood smears.
Schizonts of P. knowlesi in a thin blood smear.
Ring-form trophozoites of P. malariae in thick and think blood smears.
Trophozoites of P. malariae in a thick blood smear.
Band-form trophozoites of P. malariae in a thin blood smear.
Basket-form trophozoites of P. malariae in a thin blood smear.
Gametocytes of P. malariae in thick and a thin blood smear.
Schizonts of P. malariae in thick and a thin blood smear.
Ring-form trophozoites of P. ovale in thick and a thin blood smear.
Trophozoites of P. ovale in thick and thin blood smears.
Gametocytes of P. ovale in thick and thin blood smears.
Schizonts of P. ovale in thick and thin blood smears.
Ring-form trophozoites of P. vivax in thick and thin blood smears.
Trophozoites of P. vivax in thick and thin blood smears.
Gametocytes of P. vivax in thick and thin blood smears.
Ookinetes of P. vivax in thick and thin blood smears.
Schizonts of P. vivax in thick and thin blood smears.
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.
|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|
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).
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.
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.
Sulzer AJ, and Wilson M. The fluorescent antibody test for malaria. Crit Rev Clin Lab Sci 1971;2:601-609.
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/diagnosis_treatment/rdt.html
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/.