Identification can only be carried out on a good quality blood smear.  

A mediocre blood smear is the cause of the majority of false positives. 

Research is carried out at a magnification of 1,000 (ocular 10 and lens 100), under immersion. The surface area of the smear is chosen in relation to the spread: research is carried out in the area where the red blood cells are contiguous, but not superimposed. The scan in this area will be regular, transversal, each passage set off so that the same field is not read several times.

In the peripheral blood, the parasite is intra-globular. It is a unicellular parasite comprising obligatorily a red-colored nucleus and a blue-colored cytoplasm. The highlighting of the colorless food vacuole and the black-colored malaria pigment is more incidental. 

The pitfalls:

• artifacts of drying or coloring 
• foreign bodies 
• projection of a blood platelet on to a red blood cell  

The trophozoite: stage starting with the penetration of the merozoite into the red blood cell up to the division of the nucleus. The young trophozoite is characterized by a thin cytoplasm encircling the food vacuole. The nucleus is pushed to the edge. The parasite measures up to 1/3 of the diameter of the red blood cell. As it develops, the parasitic modifications are characterized by a deformation process and an increase in volume of the cytoplasm without any division of the nucleus. 

The schizont: stage characterized by a division of the nucleus unaccompanied by a cytoplasmic division. It has an irregular and tormented appearance and the food vacuole is difficult to see. 

The malarial rosette: it represents the ultimate stage of the mature schizont. The repeated division of the nucleus unaccompanied by a cytoplasmic division culminates in the formation of a multi-nucleus parasitic cell. The food vacuole has disappeared. The malaria pigment (hemozoin) is concentrated at the center, the nuclei are spread out on the margins. They vary in number and disposition depending on the plasmodial species. 

The gametocyte: the contours of the gametocytes are rounded or oval. The nucleus is often badly individualized, the hemozoin is often abundant. The tint of the cytoplasm varies according to the sex of the gametocyte, pure blue for the females and lilac for the males. The absence of a food vacuole enables a gametocyte to be differentiated from an aged trophozoite. The banana shape is characteristic of the Plasmodium falciparum species.

General appearance of the blood smear:

• Plasmodium falciparum: an often intense and monotone parasitism with parasites at the same stage of evolution as the trophozoite (ring form tropozoite ). The polyparasitism of the red blood cell is frequent. 
• Plasmodium vivax: fairly non-dense parasitism with mottled coloring (mixture of the different stages of evolution). Polyparasitism is exceptional. 
• Plasmodium malariae: disparate parasitism with mottled coloring and an abundance of malarial pigment. 
• Plasmodium ovale: disparate parasitism, mottled coloring and possibility of polyparasitism.  

Appearance of the parasitized red blood cell:

• Plasmodium falciparum: non-modified size and shape. The Maurer dots, classically, are often badly highlighted by the coloring. The stains are regular in size, limited in number, brown-red in color. They can be compared with finger-nail scratches. 
• Plasmodium vivax: the red blood cell has an increased volume and appears pale (de-hemoglobinized). It is sometimes deformed. At the older trophozoite and schizont stage, very numerous, small, round brown-red granulations may be observed: Schüffner granulations. 
• Plasmodium malariae: the red blood cell is smaller in volume, without modification in shape. It is darker in color. The Zieman pointillé** is very inconsistent. 
• Plasmodium ovale: the red blood cell is moderately larger. It often takes an oval shape with a fringed extremity. It is at times pale in color, but above all, it has Schüffner granulations from a young trophozoite stage.  

 Morphological appearance of the parasite:

1. Plasmodium falciparum: 

• The young trophozoite is small (1/4 of the size of the red blood cell). It is often stuck against the wall of the red blood cell and takes the appearance of a bracelet arabe** (bilobal nucleus and fine cytoplasm). There is no malarial pigment. 
• The mature trophozoite is more voluminous and irregular (shape of a racket, butterfly net). 
• The schizont and the rosette not generally encountered in the peripheral blood outside crises of pernicious malaria with strong parasitemia.  

the mature gametocyte is characteristic: its banana shape deforms the red blood cells whose presence is difficult to distinguish. It is from 8 to 15 microns in size. 

Plasmodium falciparum (after G. Villain)

2. Plasmodium vivax : 

• The young trophozoïte occupies 1/3 of the red blood cell. The cytoplasm is thicker than that of Plasmodium falciparum. There is no malarial pigment. 
• The mature trophozoïte is characteristic (amoeboid body). The cytoplasm is drawn out in all directions, deformed, very polymorphous. The food vacuole is badly delimited and the malarial pigment fine. 
• The schizont comprises nuclei which are irregular in size and in distribution in the parasite. 
• The rosette is voluminous: it occupies the whole of the red blood cell and comprises 12 to 18 irregularly distributed merozoites. The pigment is grouped in the center. 
• The gametocyte is rounded or oval in shape and occupies almost all the red blood cell. The nucleus is eccentric and the pigment abundant.  

Plasmodium vivax (after G. Villain)

3. Plasmodium malariae : 

• The young trophozoite is comparable in size and shape to that of Plasmodium vivax, but at this stage it has a grain of pigment. 
• The older trophozoite is more voluminous. The pigment is very abundant. Two classic aspects may be observed: the equatorial strip, the rigid and transversal form of the parasite in the red blood cell, and the hemogregarine form where the parasite is coiled like a worm, with abundant pigment.  

• The rosette is characteristic with its regular daisy-like appearance, at its center a very abundant malarial pigment and on the periphery, 8 to 12 merozoites regularly disposed.  

The gametocyte is globular and has an abundance of malarial pigment. It occupies almost all the red blood cell.

Plasmodium malariae (after G. Villain)

4. Plasmodium ovale: 

• The young trophozoite is comparable to that of Plasmodium vivax, but it often has a fine grain of pigment. 
• The older trophozoite is pigmented. The parasite grows and becomes deformed. At this stage, the cytoplasm may have a stringy section, but it never forms an amoeboid body. 
• The schizont can only be differentiated from the older trophozoite by the division of the nucleus.
• The rosette comprises 8 merozoites, sometimes more, distributed more or less regularly. The pigment is dense, but finer than the Plasmodium malariae.  

The gametocyte resembles that of Plasmodium vivax, but it does not occupy the whole of the red blood cell.

Plasmodium ovale (after G. Villain)

Plasmodium falciparum:

• monotone appearance of the blood smear with pluriparasitism 
• parasitic density sometimes very high 
• non-deformed infected red blood cells 
• parasites often young and small in size 
• absence of rosettes 
• gametocytes in banana form  

Plasmodium  vivax:

• mottled blood smear 
• parasitized red blood cells increased in volume and de-hemoglobinized 
• presence of Schüffner granulations at the trophozoite stage 
• irregular rosette with 12 to 18 merozoites 
• gametocyte occupying the whole of the red blood cell 
• amoeboid body  

Plasmodium malariae:

• mottled blood smear 
• infected red blood cell diminished in size 
• very abundant malarial pigment 
• trophozoite in equatorial strip 
• very regular rosette with 8 to 12 merozoites 
• gametocyte occupying the whole of the red blood cell  

Plasmodium ovale:

• mottled blood smear with frequent polyparasitism 
• parasitized red blood cells oval in shape with fringed extremity 
• Schüffner granulations from the young trophozoite stage 
• irregular rosette with 8 merozoites, sometimes more 
• gametocyte not occupying the whole of the red blood cell  

ATTENTION

Beyond the discovery of an element of pathognomonic morphology (gametocytes of Plasmodium falciparum, amoeboid body of Plasmodium vivax equatorial strip of Plasmodium malariae), the identification of the plasmodial species can only take place based on a number of coherent arguments.