Microscopic Findings

The four Plasmodium species that cause human malaria can be distinguished most of the time (but not always) based on the morphology of their blood stages. The Table describes their most salient morphologic characteristics, in each of 4 morphologically distinguishable stages: Ring: early developmental stage of the asexual erythrocytic parasite; the term is derived from the morphologic appearance of this stage, which includes chromatin (red), cytoplasm (blue), often arranged in a ring shape around a central vacuole; biologically, the ring is a young trophozoite. Trophozoite: next developmental stage of the asexual erythrocytic parasite; it has lost its "ring" appearance, and has begun to accumulate pigment (colored yellow to black). Schizont: late developmental stage of the asexual erythrocytic parasite; it has begun its division into merozoites, and thus is characterized by the presence of multiple contiguous chromatin dots (to be distinguished from multiple chromatin dots from multiple infections, which tend not to be contiguous). Gametocyte: sexual erythrocytic stage. Other Parasite Stages Seen Rarely in Blood: There are some other malaria stages that can be seen, but are rare and/or do not assist in diagnosis. They are the merozoites, which are released when the schizont ruptures, and which will re-invade new erythrocytes to continue the asexual erythrocytic cycle; and on exceptional occasions, when blood samples are left at room temperature, the gametocytes can exflagellate and the blood smears can have exflagellating gametocytes, microgametes, or even ookinetes (the product of the fusion of a male and female gametes, usually found only in the mosquito).

Comparison of Plasmodium Species Which Cause Human Malaria

Plasmodium species

Stages found in blood Ring Images Trophozoite Images

Appearance of Erythrocyte (RBC) normal; multiple infection of RBC more common than in other species normal; rarely, Maurer's clefts (under certain staining conditions) normal; rarely, Maurer's clefts (under certain staining conditions)

Appearance of Parasite delicate cytoplasm; 1 to 2 small chromatin dots; occasional appliqu (accoll) forms seldom seen in peripheral blood; compact cytoplasm; dark pigment seldom seen in peripheral blood; mature = 8 to 24 small merozoites; dark pigment, clumped in one mass crescent or sausage shape; chromatin in a single mass (macrogametocyte) or diffuse (microgametocyte); dark pigment mass

P. falciparum Images Schizont Images

Gametocyte distorted by parasite Images

Ring Images

normal to 11/4 , round; occasionally fine Schffner's dots; multiple infection of RBC not uncommon enlarged 11/2 to 2 ; may be distorted; fine Schffner's dots enlarged 11/2 to 2 ; may be distorted; fine Schffner's dots

large cytoplasm with occasional pseudopods; large chromatin dot

Trophozoite Images P. vivax Images Schizont Images

large ameboid cytoplasm; large chromatin; fine, yellowishbrown pigment large, may almost fill RBC; mature = 12 to 24 merozoites; yellowish-brown, coalesced pigment round to oval; compact; may almost fill RBC; chromatin compact, eccentric (macrogametocyte) or diffuse (microgametocyte); scattered brown pigment

Gametocyte Images

enlarged 11/2 to 2 ; may be distorted; fine Schffner's dots

Ring Images

normal to 11/4 , round to oval; occasionally Schffner's dots; occasionally fimbriated; multiple infection of RBC not uncommon normal to 11/4 ; round to oval; some fimbriated; Schffner's dots normal to 11/4 , round to oval, some fimbriated, Schffner's dots

sturdy cytoplasm; large chromatin

Trophozoite P. ovale Images Images

compact with large chromatin; dark-brown pigment

Schizont Images

mature = 6 to 14 merozoites with large nuclei, clustered around mass of dark-brown pigment round to oval; compact; may almost fill RBC; chromatin compact, eccentric (macrogametocyte) or more diffuse (microgametocyte); scattered brown pigment sturdy cytoplasm; large chromatin compact cytoplasm; large chromatin; occasional band

Gametocyte Images

normal to 11/4 ; round to oval, some fimbriated; Schffner's dots

P. malariae Images

Ring normal to 3/4 Images Trophozoite normal to 3/4 ; rarely, Ziemann's stippling

Images

(under certain staining conditions) normal to 3/4 ; rarely, Ziemann's stippling (under certain staining conditions)

forms; coarse, dark-brown pigment mature = 6 to 12 merozoites with large nuclei, clustered around mass of coarse, darkbrown pigment; occasional rosettes round to oval; compact; may almost fill RBC; chromatin compact, eccentric (macrogametocyte) or more diffuse (microgametocyte); scattered brown pigment

Schizont Images

Gametocyte Images

normal to 3/4 ; rarely, Ziemann's stippling (under certain staining conditions)

While morphologic characteristics provide valuable criteria for determination of malaria parasite species, they occasionally fail to differentiate between species that share morphologic characteristics (especially Plasmodium vivax and P. ovale) or in cases where parasite morphology has been altered by drug treatment or improper storage of the sample. In such cases, molecular diagnostic tests can provide useful complementary information. In addition, molecular tests can be more sensitive and their interpretation is less open to subjectivity than microscopy. The method currently used at CDC is described below.

Species-specific PCR diagnosis of malaria

Plasmodium genomic DNA is extracted from 200 l 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 speciation of Plasmodium is done with a two step nested PCR using the primers of Snounou et al. In the first step (PCR1), 1 l of extracted DNA is amplified using genus specific primers; in the second step (PCR2), 1 l of PCR1 amplification product is further amplified using species specific primers. Ten l of each PCR2 amplified DNA product is separated by 2% agarose gel electrophoresis, stained for 15 min with ethidium bromide and visualized by UV illumination.

A A: 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: 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.

Malaria antibody detection can be performed using various techniques. For the clinical laboratory, the most practical approach is the indirect fluorescent antibody (IFA) test. This test, with malaria parasites as antigens, detects most sensitively antibody responses to a wide range of plasmodial antigens. The IFA procedure can be used 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 malaria. However, serology 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 with a febrile illness who is suspected of having malaria and from whom repeated blood smears are negative

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 anti-human

antibody is then added, which attaches to the patient's malaria-specific antibody. When examined with a fluorescence microscope, a positive reaction is when the parasites appear fluorescent yellow.

A A: Positive malaria IFA showing a fluorescent schizont.

Reference: Sulzer AJ,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 include, among others: detection of an antigen (histidine rich protein-2, HRP-2) associated with malaria parasites (especially P. falciparum and P. vivax) detection of a Plasmodium associated lactate dehydrogenase (pLDH) either through its enzymatic activity or by immunoassay

The detection of HRP-2 and of pLDH forms the basis for diagnostic kits that have been, and continue to be evaluated. Consensus information, when available, will be reviewed.

Bench Aids
The bench aids are in PDF format, and in order to access these files you must have Adobe Acrobat Reader. If you do not have Acrobat Reader, please click here for more information. If you experience problems downloading these files due to file size, email us at dpdx@cdc.gov. Bench aid: Comparison of the Plasmodium Species Which Cause Human Malaria (PDF file: 28 kb) Bench aid: Plasmodium spp. Blood Stage Parasites, Thick Blood Smears (PDF file: 938 kb) Bench aid: Plasmodium spp. Blood Stage Parasites, Thin Blood Smears (PDF file: 512 kb)

The following bench aid was developed for the Democratic Republic of Congo by the National Malaria Control Program of that country, with assistance from the Centers for Disease Control and Prevention and United States Agency for International Development. Bench aid: Planches pour le Diagnostic microscopique du paludisme (PDF file: 885 kb)