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close this bookBasic Malaria Microscopy (part I and II) (WHO; 1991; 72 pages)
View the documentPreface
View the documentIntroduction
View the documentLearning Unit 1. Malaria, the disease
View the documentLearning Unit 2. Cleaning and storing microscope slides
View the documentLearning Unit 3. Keeping accurate records
View the documentLearning Unit 4. Blood films
View the documentLearning Unit 5. Staining blood films with Giemsa stain
View the documentLearning Unit 6. The microscope
View the documentLearning Unit 7. Examining blood films
View the documentLearning Unit 8. Examining blood films for malaria parasites
View the documentLearning Unit 9. Artefacts in blood films
View the documentLearning Unit 10. Routine examination of blood films for malaria parasites
View the documentLearning Unit 11. Life cycle of the malaria parasite
View the documentLearning Unit 12. Supervisory aspects of malaria microscopy
View the documentBack Cover
 

Learning Unit 8. Examining blood films for malaria parasites

Learning objectives

By the end of this Unit you should be able to:

• name the various parts of the malaria parasite

• distinguish malaria parasites in thin blood films, and recognize and name the three stages of trophozoite, schizont and gametocyte

• distinguish between the malaria species, and recognize and name the stage and species of malaria parasite seen in thin blood films

• distinguish between the malaria species, and recognize and name the stage and species of parasite seen in thick blood films.

Recognition of a malaria parasite

Malaria parasites take up Giemsa stain in a special way in both thick and thin blood films. You must be able to distinguish the various parts of the parasite, as shown in the diagram that follows.


Parts of a malaria parasite inside a red blood cell

Malaria parasites pass through a number of developmental stages. In all stages, however, the same parts of the parasite will stain the same colour:

• Chromatin (part of the parasite nucleus) is usually round in shape and stains a deep red.

• Cytoplasm occurs in a number of forms, from a ring shape to a totally irregular shape. It always stains blue, although the shade of blue may vary between the malaria species.

More will be said about the malaria species later in this Learning Unit.

Stages of the malaria parasite

Stages of the malaria parasite that you will see in blood films are described below. For this exercise, they are shown inside the red blood cell.

During the practical exercises you will learn to recognize the various stages of the parasites in thin blood films. You should ask your tutor or facilitator to confirm the stage of parasite that you identify. You can also use the key on pages 51 and 52 to help you identify whether what you see is a parasite or not and, if it is, what stage it is. (Remember, the key is for thin films only, and it does not show the effect of the parasite on the red blood cell.)

The trophozoite stage

This stage is the most commonly seen; it is often called the ring stage, although it sometimes takes the form of an incomplete ring.


The ring stages of malaria parasites

Because the trophozoite stage is a growing stage, the parasite within the red blood cell may vary in size from small to quite large. Pigment appears as the parasite grows. Malaria pigment is a by-product of the growth or metabolism of the parasite. It does not stain, but has a colour of its own, which may range from pale yellow to dark brown or black.

The schizont stage

At the schizont stage the malaria parasite starts to reproduce. This reproduction is referred to as asexual because the parasite is neither male nor female but reproduces itself by simple division. There are several obvious phases in this stage, ranging from parasites with two chromatin pieces to parasites with a number of chromatin dots and definite cytoplasm. These are clearly seen in the diagram that follows.


Stages of schizont growth

Note: The process of forming schizonts, which takes place in the liver and in blood, is referred to as schizogony.

The gametocyte stage

The gametocyte stage is sexual in that the parasites become either male or female in preparation for the next stage, which takes place in the stomach of the female anopheline mosquito. Gametocytes may be either round or banana-shaped, depending on the species. The way in which the parasite takes up the stain will also help you to identify whether what you see is male (microgametocyte) or female (macrogametocyte).


Male and female gametocytes

Key to identifying malaria parasite stages in thin blood films

This key applies to those features inside red blood cells which appear to be parasites.

1. Are there one or more red-stained chromatin dots and blue cytoplasm?
Yes: go to 2
No: what you see is not a parasite

2. Are the size and shape right for a malaria parasite?
Yes: what you see is probably a malaria parasite; go to 3
No: what you see is not a parasite

3. Is there malaria pigment in the cell?
Yes: go to 7
No: go to 4

4. Does the parasite have one chromatin dot attached to blue cytoplasm in the form of a regular ring with a vacuole?
Yes: this is a trophozoite stage
No: go to 5

5. Does the parasite have one chromatin dot attached to blue cytoplasm in the form of a small solid or regular ring or with a vacuole?
Yes: this is a trophozoite stage
No: go to 6

6. Is the parasite with one chromatin dot irregular or fragmented?
Yes: this is a trophozoite stage
No: go to 8

7. Does the parasite with malaria pigment have one chromatin dot?
Yes: go to 8
No: go to 9

8. Does the parasite have a vacuole or is it still fragmented in some way?
Yes: this is probably a late trophozoite stage
No: go to 11

9. Does the parasite have two chromatin dots attached to a ring and also have a vacuole?
Yes: this is a trophozoite stage
No: go to 10

10. Does the parasite have between 2 and 32 chromatin dots and pigment?
Yes: this is a schizont stage

11. Is the parasite rounded or banana-shaped?
Rounded: go to 12
Banana-shaped: go to 14

12. Does the rounded parasite have clearly stained chromatin and deep blue cytoplasm?
Yes: this is a female gametocyte
No: go to 13

13. Does the rounded parasite have a reddish overall colour to the staining, so that the chromatin cannot be clearly seen?
Yes: this is a male gametocyte

14. Does the banana-shaped parasite have densely stained blue cytoplasm and bright red chromatin?
Yes: this is a female gametocyte
No: go to 15

15. Does the banana-shaped parasite have a reddish overall colour to the staining, so that the chromatin is indistinct?
Yes: This is a male gametocyte

Species of malaria parasite

You have been learning how to recognize malaria parasites and their stages in thin films and have so far concentrated only on their appearance. However, the effect the parasite has on red blood cells is also important because it will help you to identify the malaria species.

The four species of malaria

There are four species of malaria that affect humans:

Plasmodium falciparum: the commonest species in the hotter parts of the world and responsible for much sickness and even death.

P. vivax: the commonest species in the cooler parts of the tropics, the largest of the malaria parasites found in humans, and the cause of much illness.

P. malariae: a less common species but one that occurs throughout much of the world.

P. ovale: a relatively rare species but reported from time to time in many countries, especially in Africa; sometimes confused with P. vivax.

Appearance of parasite species in thin blood films

The simplest guide to distinguishing between the four species of malaria is the effect the parasite has on infected red blood cells. Features to concentrate on include the size of the red blood cell (whether or not it is enlarged) and whether or not staining reveals Schüffner’s dots or Maurer’s dots (also known as Maurer’s clefts) within the cell.

The diagnostic features outlined in Fig. 3 will help you to decide which species of malaria parasite you have found. If you have any problem using this key, your tutor or facilitator will help you. You should also consult the colour plates (Plates 4-7) in this Guide. The left-hand side of each plate shows the various stages of the different species of malaria parasite as they appear in thin blood films, and the right-hand side shows their appearance in thick blood films.

You will be given plenty of time to practise identification of species in thin films. Once your tutor considers that you are able to identify accurately the stage and species of malaria parasite, you will move on to the examination of parasites in thick blood films.

Appearance of parasite species in thick blood films

Just as the appearance of both red and white blood cells differs in thin and thick blood films, so too there are differences in the appearance of malaria parasites.

The first difference is obvious as soon as you look at a thick film with the x 100 oil immersion objective and the x 7 ocular: there are no red blood cells. The malaria parasites, however, can be seen, although, like the white blood cells, they appear to be smaller than in thin blood films. You may need to look quite carefully before you see them. You will need to refocus, using the fine adjustment, each time you move the microscope field: this will allow you to examine the thick film at different depths.

The fine rings of cytoplasm of the trophozoites may appear incomplete or broken. This is the normal appearance of trophozoites in thick blood films.


Fig. 3. Species differentiation of malaria parasites by host-cell change in Giemsa-stained thin blood films


Plate 4. Appearance of Plasmodium falciparum stages in Giemsa-stained thin and thick blood films


Plate 5. Appearance of Plasmodium vivax stages in Giemsa-stained thin and thick blood films


Plate 6 Appearance of Plasmodium ovale stages in Giemsa-stained thin and thick blood films


Plate 7. Appearance of Plasmodium malariae stages in Giemsa-stained thin and thick blood films


Fig 4. Species differentiation of malaria parasites by cytoplasmic pattern of trophozoites in Giemsa-stained thick blood films

Plate 8. Species identification of malaria parasites in Giemsa-stained thick blood films

 

Stage of parasite in peripheral blood

Species

Trophozoite

Schizont

Gametocyte

Plasmodium falciparum

Young, growing trophozoites and/or mature gametocytes usually seen.

   

Size; small to medium; number: often numerous; shape: ring and comma forms common; chromatin: often two dots; cytoplasm: regular, fine to fleshy; mature forms: sometimes present in severe malaria, compact with pigment as few coarse grains or a mass.

Usually associated with many young ring forms. Size: small, compact; number: few, uncommon, usually in severe malaria; mature forms: 12-30 or more merozoites in compact cluster; pigment: single dark mass,

Immature pointed-end forms uncommon. Mature forms: banana-shaped or rounded; chromatin: single, well defined; pigment: scattered, coarse, rice-grain-like; pink extrusion body sometimes present. Eroded forms with only chromatin and pigment often seen.

Plasmodium Vivax

All stages seen; Schüffner’s stippling in “ghost” of host red cells, especially at film edge.

   

Size small to large; number: few to moderate; shape: broken ring to irregular forms common; chromatin: single, occasionally two; cytoplasm: irregular or fragmented; mature forms: compact, dense; pigment: scattered, fine.

Size: large; number: few to moderate; mature forms: 12-24 merozoites, usually 16, in irregular cluster; pigment: loose mass.

Immature forms difficult to distinguish from mature trophozoites. Mature forms: round, large; chromatin: single, well defined; pigment, scattered, fine. Eroded forms with scanty or no cytoplasm and only chromatin and pigment present

Plasmodium ovale

All stages seen; prominent Schüffner’s stippling in “ghost” of host red cells, especially at film edge.

   

Size: may be smaller than P. Vivax; number: usually few; shape: ring to rounded, compact forms; chromatin: single, prominent; cytoplasm: fairly regular. Fleshy; pigment: scattered, coarse.

Size: rather like P. malariae; number: few; mature forms: 4-12 merozoites, usually 8, in loose cluster; pigment: concentrated mass.

Immature forms difficult to distinguish from mature trophozoites. Mature forms: round, may be smaller than P. Vivax: chromatin: single, well defined; pigment: scattered, coarse. Eroded forms with only chromatin and pigment present.

Plasmodium malariae

All stages seen.

   

Size: small; number: usually few; shape: ring to rounded, compact forms; chromatin: single, large; cytoplasm: regular, dense; pigment: scattered, abundant, with yellow tinge in older forms.

Size: small, compact; number: usually few; mature forms: 6-12 merozoites, usually 8, in loose cluster, some apparently without cytoplasm; pigment: concentrated.

Immature and certain mature forms difficult to distinguish from mature trophozoites. Mature forms: round: compact; chromatin: single, well defined; pigment: scattered, coarse, may be peripherally distributed. Eroded forms with only chromatin and pigment present.

Similarly, the absence of red blood cells may make the Schüffner’s dots difficult to see; in fact, in the thicker parts of the film, it may not be possible to see the stippling at all. However, the “ghosts” of red cells can usually be seen surrounding parasites in the thinner parts of the films, often towards the edge, and this will help you make your diagnosis.

Note: The Maurer’s dots of P. falciparum cannot be seen in thick films.

Fig. 4 provides a key for species identification in thick films; you can also get help from Plates 4-7 (right-hand side) and from Plate 8. With practice, you will soon become competent at identification of species.

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