9. Blood Parasites

Red Blood cells offer parasites an excellent environment for invasion and survival. Haemosporina are the only protozoan parasites which can invade the red blood corpuscles of vertebrates. Most, if not all, have multiplicative phases in the reticulo-endothelial system.

The red blood cells are thin-walled and constantly moving, with the result that absorption of food materials and elimination of waste products of metabolism are relatively easy to achieve. In addition, they also contain rich supplies of protein and oxygen.

It is now known that malarial parasites do not actually penetrate the red blood cell but, in fact, enter the cell membrane by endocytosis and enclosed in a parasitophorous membrane.

9.1. Malaria

Introduction

Malaria is the most important tropical disease known to mankind, causing many death and much morbidity throughout the world. (Diag. 1) It remains a significant problem in many tropical areas especially in sub-Saharan Africa. In many areas of the world the situations deteriorating as a result of environmental changes, including global warming, civil disturbances, increasing travel and drug resistance. (Greenwood, B.M, 1997) There is approximately 100 million cases of malaria worldwide with about 1 million of these proving fatal.

Diagram 1. Map illustrating the enormous distribution of malaria throughout the world.

Malaria is caused by protozoa of the Plasmodium species. There are 4 species which infect both humans and aniamls; Plasmodium malariae (quartian malaria), Plasmodium vivax (benign tertian malaria), Plasmodium falciparum (malignant tertian malaria, subtertian malaria) and Plasmodium ovale (ovale tertian malaria).

The transmission of the protozoa, Plasmodium requires two hosts, an intermediate invertebrate host (vector), and a definitive vertebrate host (mammals, birds and lizards).

All Plasmodium species undergo the general haemosporina developmental cycle which can be summarised as:

1. initial or continual schizogny in the vertebrate host with initiation of gametogeny;

2. formation of gametes in the arthropod host and subsequent fertilisation and formation of a zygote;

3. formation of sporozoites from the zygote by repeated nuclear division followed by cytoplasmic divisions. (Smyth, J.D, 1994)

There is no requirement for resistant stages since the transfer of the parasites between the vertebrate and invertebrate hosts is made by withdrawal or injection during the bloodsucking act, there is little or no exposure to the hazards of the outside world; thus by blood transfusion or inoculation, via the blood stages of the parasite.

Life Cycle

Malaria is transmitted by the female anopheline mosquito. (Fig. 1) The life cycle of all species of human malaria parasites is essentially the same. It comprises an exogenous sexual phase (sporogony) with multiplication in certain Anopheles mosquitoes and an endogenous asexual phase (schizogony) with multiplication in the vertebrate host. (Diag. 2) The latter phase includes the development cycle in the red cells (erythrocytic schizogony) and the phase taking place in the parenchyma cells in the liver (pre-erythrocytic schizogony).

Figure 1. An Anopheline mosquito, the vector of the prozotoa group Plasmodia, the parasite known to cause malaria in both man and humans. Malaria is transmitted by female Anopheles mosquitoes to the definitive host whilst the mosquito sucks on the victims blood.

When a female Anopheles mosquito bites an infected person, it ingests blood which may contain the mature sexual cells (male and female gametocytes) which undergo a series of developmental stages in the stomach of the mosquito. Exflagellation occurs resulting in the production in a number of male and female gametes. Fertilisation occurs producing a zygote which matures to an ookinete. This penetrates the stomach wall of the mosquito where it grows into an oocyst and it further matures to become a motile sporozoite.

Diagram 2. Diagram of the malaria life cycle. 1) Sporozoites, injected through the skin by female anopheline mosquito; 2) sporozoites infect hepatocytes; 3) some sporozoites develop into hypnozoites (P. vivax and P. ovale): 4) liver stage parasite develops; 5 – 6) tissue schizogony; 7) merozoites are released into the circulation; 8) ring stage trophozoites in red cells; 9) erythrocytic schizogony; 10) merozoites invade other red cells; 11) some parasites develop into female (macro-) or male (micro-) gametocytes, taken up by the mosquito; 12) mature macrogametocyte and exflagellating microgametocytes; 13) ookinete penetrates gut wall; 14) development of oocyst; 15) sporozoites penetrate salivary glands. (Bell, D.R, 1995)

The length of the developmental stage in the mosquito not only depends on the Plasmodium species but also the mosquito host and the ambient temperature. This may range from 8 days in Plasmodium vivax to as long as 30 days in Plasmodium malariae.

The sporozoites migrate from the body cavity of the mosquito to the salivary glands and the mosquito now becomes infective. Sporozoites enter into the blood stream of a host when the mosquito feeds on blood. Following the inoculation, the sporozoites leave the blood within 40 minutes and enter the parenchymal cells of the liver (hepatocytes). In all 4 species, asexual development occurs in the liver cells, a process known as pre-erythrocytic schizogony, to produce thousands of tiny merozoites which are relaeased into the circulation after about 16 days. However in P. vivax and P. ovale some sporozoites differentiate into hypnozoites which remain dormant in hepatocytes for considerable periods of time. When they are “reactivated” they undergo asexual division and produce a clinical relapse.

In P. falciparum and P. malariae hypnozoites are not formed and the parasite develops directly into pre-erythrocytic schizonts.

Once in the circulation, the merozoites invade the red cells and develop in to trophozoites. In the course of their development they absorb the haemoglobin of the red cells leaving as the product of digestion a pigment called haemozoin, a combination of haematin and protein. This iron-containing pigment is seen in the body of the parasite in the form of dark granules, which are more obvious in the later stages of development.

Diagram 3. Diagram illustrating the various stages of the three common species of malaria which infect man. (adapted and redrawn from Smyth, J.D, 1994)

After a period of growth the trophozoite undergoes an asexual division, erythrocytic schizogony. When the mature trophozoite starts to divide in the red blood cell, separate merozoites are formed resulting in a schizont. When fully developed, the schizont ruptures the red blood cell containing it, liberating the merozoites into the circulation. These merozoites will then infect new red cells and the process of asexual reproduction in the blood tends to proceed. Some of the merozoites entering red blood cells do not form trophozoites then schizonts but develop into gametocytes and this process takes place in deep tissue capillaries. This erythrocytic cycle of schizogony is repeated over and over again in the course of infection, leading to a progressive increase of parasitaemia.

Infection with all four strains of malaria have many clinical features in common. These are related to the liberation of fever-producing substances, especially during schizogony. The common features are:

Fever: Often irregular. The regular pattern of fever does not occur until the illness has continued for a week or more. Where it depends on synchronised schizogony.

Anaemia: The anaemia is haemolytic in type. It is more severe in infections with P. falciparum because in this infection cells of all ages can be invaded. Also, the parasitaemia in this infection can be much higher than in other malarias.

Splenomegaly: The spleen enlarges early in the acute attack of malaria. When a patient has been subjected to many attacks, the spleen may be of an enormous size and lead to secondary hypersplenism.

Jaundice: A mild jaundice due to haemolysis may occur in malaria. Severe jaundice only occurs in P. falciparum infection, and is due to liver involvement.

9.2. Species Specific Characteristics

Plasmodium falciparum

Introduction

Plasmodium falciparum is the most important malaria parasite, found in the tropics and sub-tropics, being responsible for approximately 50% of all malaria cases. The incubation period of P. falciaprum malaria is the shortest, between 8 and 11 days and has a periodicity of 36 – 48 hours. It can be differentiated from the other species by the morphology of the different stages found in the peripheral blood. In infections with Plasmodium falciparum usually only young trophozoites and gametocytes are seen in peripheral blood smears, the schizonts are usually found in capillaries sinuses of internal organs and in the bone marrow. The disease it produces runs an acute course and often terminating fatally. It is a significant cause of abortions and stillborns and even death of non-immune pregnant women.

Life cycle

The aspects of the life cycle which are specific to P. falciparum are as follows:

a) It attacks all ages of erythrocytes so that a high density of parasites can be reached quickly. In extreme cases up to 48% of the red blood cells can be parasitised.

b) Multiple infections resulting in several ring forms in a corpuscle are not uncommon.

c) The latter stages in the asexual cycle do not occur in the peripheral blood as in other forms of malaria, so that only rings and crescents are found in blood films. After 24 hours the ring forms and older trophozoites show a tendency to clump together and adhere to the visceral capillary walls and become caught up in the vessels of the heart, intestine, brain or bone marrow in which the later sexual stages are completed.

d) Sporulation is not as well synchronised as in other malaria forms so that the fever may last longer.

e) Exo-erythrocytic forms do not persist in the tissues and hence relapses do not occur.

Morphology

Trophozoites

Red blood cells in Plasmodium falciparum infections are not enlarged and they may have a spiky outline which is common in cells which have dried slowly. The typical arrangement cytoplasm in young trophozoites is the well-known ring formation which thickens and invariably contains several vacuoles as the trophozoite develops. Chromatin is characteristically found as a single bead, but double beads and small curved rod forms frequently occur. (Figs. 2 & 3)

Maurer’s dots are slow to appear and are first seen as minute purplish dots, 6 or less in number. The points become spots, still few in number and are now characteristic enough to be recognised. Maurer describes them as fine ringlets, loops or streaks. They are seldom absent from the red blood cells containing large rings but the staining of the spots is very sensitive to pH and are seldom seen if the pH falls below 6.8.

Trophozoites of P. falciparum can be found on the edge of the red blood cells. These are known as acole forms and are found as three distinct types:

1. Common: The single chromatin bead lies on the edge of the cell with most of the

cytoplasm extended along the edge on both sides of the bead.

2. Rim: The complete parasite lies in a thickened line along the edge of the cell with no evidence of ring formation.

3. Dispalced: The parasites are displaced beyond the edge of the host cell. All degrees of displacement may occur, from partial to marked displacement with most of the parasite lying beyond the cell margin.

Pigment is not a characteristic finding in the early stages of P. falciparum infections.

Figure 2. Diagrammatic illustration of the morphology of the different stages of the Plasmodium falciparum life cycle in thin blood films. 1) P. falciparum early trophozoites / ring forms. 2) Developing trophozoites (rarely seen in peripheral blood). 3) Immature schizonts (rarely seen in peripheral blood). 4) Mature schizonts, almost fill the red blood cell. 5) Microgametocytes, large numbers appear after 7 – 12 days. 6) Macrogametocytes, large numbers appear after 7 - 12 days. (adapted and redrawn from Jeffrey & Leach)

Gametocytes

Gametocytes are the sexual stage of the malaria parasite. Plasmodium falciparum gametocytes appear in the peripheral circulation after 8 - 11 days of patent parasitaemia and by then, they have assumed their typical crescentic shapes. They soon reach their peak density, and then decline in numbers, disappearing in about 3 months as a rule. (Figs. 2 & 4)

Figure 3. Young trophozoite / ring stage of Plasmodium falciparum. The ring thickens and invariably contains several vacuoles as the trophozoite develops. Maurer’s dots are slow to appear and are first seen as minute purplish dots. (Giemsa stain)

Figure 4. Plasmodium falciparum gametocytes appear in the peripheral circulation after 8 - 11 days of patent parasitaemia and by then, they have assumed their typical crescentic shapes. (Giemsa stain)

The female form, or macrogametocyte, is usually more slender and somewhat longer than the male, and the cytoplasm takes up a deeper blue colour with Giemsa stain. The nucleus is small and compact, staining dark red, while the pigment granules are closely aggregated around it. The male form, or microgametocyte, is broader than the female and is more inclined to be sausage shaped. The cytoplasm is either pale blue or tinted with pink and the nucleus, which stains dark pink, is large and less compact than in the female, while the pigment granules are scattered in the cytoplasm around it.

In humans, gametocytes do not multiply, nor cause symptoms but they are the forms which are infective to the mosquito. When a female Anopheline mosquito takes a blood meal, the male and female gametocytes continue their sexual development.

Schizonts

Schizonts are rarely seen in the peripheral blood and their presence may indicate a potentially serious parasitaemia. Schizonts are have 8 - 36 merozoites and a large mass of golden brown pigment (haemozoin) is seen in the pre-schizont and schizont stage. (Fig. 5)

Figure 5. Plasmodium falciparum schizont. Rarely seen in the peripheral blood, a good indicator of a potentially serious parasitaemia. They have 8 – 36 merozoites and a large golden brown pigment. (Giemsa stain)

Clinical Disease

Symptoms include headache, photophobia, muscle aches and pains, anorexia, nausea and vomiting. Complications include severe anaemia cerebral malaria, renal disease, black water fever, dysentery, pulmonary oedema and tropical splenomegaly syndrome.

Plasmodium vivax

Introduction

Plasmodium vivax is found almost everywhere malaria is endemic and is the most predominant of the malaria parasites. Causing 43% of all cases of malaria in the world, it also has the widest geographical distribution. Although the disease itself is not usually life threatening, it can cause severe acute illness.

Plasmodium vivax does not infect West Africans due to the fact that West Africans do not possess the Duffy Antigen on the red blood cells which the parasite requires to enter the red blood cell. It has an incubation period of between 10 and 17 days which is sometimes prolonged to months or years due to the formation of hypnozoites. It has a periodicity of 48 hours. Plasmodium vivax infections are usually characterised by the presence of more than one developmental stage in the peripheral blood film. The parasites parasitise young enlarged erythrocytes and Schüffner’s dots develop on the erythrocyte membrane.

Life cycle

The aspects of the life cycle which are specific to P. vivax are as follows:

a) The degree of infectivity is low, only the young immature corpuscles are infected; about 2% of erythrocytes are parasitised.

b) The periodicity of the asexual cycle is closely synchronised.

c) Hypnozoites develop in the liver, so that relapses may occur.

Morphology

Trophozoites

Most trophozoites of P. vivax are already several hours old when they appear in peripheral blood and by that time the Schüffner’s dots are already visible. The trophozoites are actively amoeboid and contain single or sometimes double chromatin dots that are either circular or ovoid. As the trophozoites mature, the Schüffner’s dots increase in number and size and the parasite changes from large irregular rings to rounded or ovoid forms in mature trophozoites. (Fig. 6 & 7)

Figure 6. Trophozoites of Plasmodium vivax are already several hours old when they appear in the peripheral blood and therefore, you can already see the Schüffners dots. They contain single or sometimes double chromatin dots. (Giemsa stain)

Figure 7. Diagrammatic illustration of the morphology of the different stages of the Plasmodium vivax life cycle in thin blood films.

1) Early trophozoites / ring forms (accole forms, not shown here, are occasionally seen). 2) Developing trophozoites are large and irregular with a prominent vacuole. 3) Immature schizonts, are amoeboid and almost fill the red blood cell. 4) Mature schizonts, almost fill the red blood cell. 5) Microgametocytes , large numbers appear after 3 – 5 days. 6) Macrogametocytes, large numbers appear after 3 – 5 days. (Adapted and redrawn from Jeffrey & Leach)

Gametocytes

Mature female gametocytes are large rounded parasites which fill or nearly fill the host cell. The cytoplasm is blue and fairly homogenous. The nuclear chromatin is a single, well-defined purplish mass, varied in form and usually peripheral in distribution. (Fig 7 & 8) Mature male gametocytes can be distinguished from females by the large, loose and ill-defined mass of chromatin and by their paler colour and smaller mass.

Figure 8. Mature female Plasmodium vivax gametocytes are large rounded parasites which fill or nearly fill the host cell. The cytoplasm is blue and fairly homogenous. The nuclear chromatin is a single, well-defined purplish mass, varied in form and usually peripheral in distribution. (Giemsa stain)

Schizonts

The parasitised red cells are much enlarged containing Schüffner’s dots. The parasites are large, filling the enlarged red cell. There are between 12-24 merozoites in the schizonts (usually16). The pigment is a golden brown central loose mass. (Fig. 9)

Figure 9. A schizont of Plasmodium vivax. The parasites are large, filling the enlarged red cell. There are between 12 - 24 merozoites in the schizonts (usually16). The pigment is a golden brown central loose mass. (Giemsa stain)

Clinical Disease

Symptoms include headache, photophobia, muscle aches and pains, anorexia, nausea and vomiting. Complications due to P. vivax are relatively rare and arise due do a previous debility or pre-existing disease.

Plasmodium ovale

Introduction

Plasmodium ovale is widely distributed in tropical Africa especially the west coast, despite that it is a species that is rarely encountered. It has also been reported in South America and Asia. It has an incubation period of 10 – 17 days which is sometimes prolonged to months or years due to the formation of hypnozoites. It has a periodicity of 48 hours, the fever it produces is milder than the benign tertian P. falciparum.

Life cycle

The features of the life cycle which are specific to P. ovale are as follows:

a) It morphologically resembles P. malariae in most of its stages.

b) The changes produced in the erythrocytes in general are similar to those produced by P. vivax, but Schüffner’s dots appear considerably earlier (in the ring stage) and are coarser and more numerous.

c) In the oocyst the pigment granules are (usually) characteristically arranged in two rows crossing each other at right angles.

d) Hypnozoites develop in the liver so that relapses may occur.

Morphology

Parasites of P. ovale are usually found in enlarged and stippled red blood cells (James’s dots), similar to those found in P. vivax infections. Host cells show an oval shape, particularly those containing younger stages of the parasites and the host cell may also show “spiking” or fimbriation. (Fig. 10)

Figure 10. Diagrammatic illustration of the morphology of the different stages of the Plasmodium ovale life cycle in thin blood films. 1) Early trophozoites / ring forms, are dense rings with well- defined masses of chromatin. 2) Developing trophozoites, small and compact with an inconspicuous vacuole. 3) Immature schizonts, compact and almost fill the red blood cell. 4) Mature schizonts, fill ¾ of the red blood cell. 5) Microgametocytes, low numbers appear after 12 - 14 days. 6) Macrogametocytes, low numbers appear after 12 – 14 days. (Adapted and redrawn from Jeffrey & Leach)

Trophozoites

Young trophozoites are found as compact rings in cells containing Schüffner’s dots. The trophozoite rings remain compact as they develop and show little of the amoeboid features common in P. vivax. Small, scattered pigment granules can be seen in developing trophozoites which disperse as the trophozoite matures. Late trophozoites are round and consolidated with an increase in cytoplasm, they are very similar to P. vivax at this stage. (Figs. 10 & 11)

Figure 11. Trophozoite of Plasmodium ovale. Young trophozoites are found as compact rings in cells containing Schüffner’s dots. The trophozoite rings remain compact as they develop. Late trophozoites are round and consolidated with an increase in cytoplasm, they are very similar to P. vivax at this stage.

Gametocytes

The mature gametocytes are round, filling two thirds of the red cell. The red blood cell is slightly enlarged and stippled and contains pigment which has a distinct arrangement of concentric rods, mostly at the periphery. (Figs. 10 & 12)

Figure 12. Gametocyte of Plasmodium ovale. The mature gametocytes are round, filling two thirds of the red cell. (Giemsa stain)

Schizonts

The parasite is smaller than red blood cells and contains 6-12 merozoites, usually 8 in a single ring. The pigment is a brown / greenish central clump. The red cell slightly enlarged, stippled, frequently oval and fimbriated. (Fig. 13)

Figure 13. Schizont of Plasmodium ovale. The parasite is smaller than the red blood cell and contains 6 – 12 merozoites. The red cell is slightly enlarged, stippled, frequently oval and fimbriated. (Giemsa stain)

Clinical Disease

Symptoms, like those of P. vivax, include headache, photophobia, muscle aches and pains, anorexia, nausea and vomiting. Complications due to P. ovale are relatively rare and arise due do a previous debility or pre-existing disease.

Plasmodium malariae

Introduction

Plasmodium malariae occurs mainly in the subtropical and temperate areas where P. falciparum and P. vivax occur. However it is less frequently seen, responsible for approximately 7% of all malaria in the world. It has an incubation period of 18 – 40 days and a periodicity of 72 hours.

Life cycle

The features of the life cycle which are specific to P. malariae are as follows:

a) Infected erythrocytes are not larger than uninfected ones and sometimes even smaller.

b) Mature erythrocytes are attacked and rarely reticulocytes, so that the density of parasites is very low; about 0.2% of erythrocytes are parasitised.

c) It is often difficult to distinguish between a large trophozoite and an immature gametocyte.

Morphology

Parasites of P. malariae are typically compact heavily pigmented parasites which are usually smaller and more deeply stained than normal. They tend to parasitise small, old red blood cells, they do not contain any inclusion dots and the parasitaemia is usually low.

Figure 14. Diagrammatic illustration of the morphology of the different stages of the Plasmodium malariae life cycle in thin blood films.

1) Early trophozoites / ring forms, compact rings containing one mass of chromatin. 2) Developing trophozoites, small and compact (often band forms) with an inconspicuous vacuole. 3) Immature schizonts, compact and almost fill the red blood cell which contains scattered pigment. 4) Mature schizonts, almost fill the red blood cell. 5) Microgametocytes, low numbers appear after 7 – 14 days. 6) Macrogametocytes, low numbers appear after 7 - 14 days. (Adapted and redrawn from Jeffrey & Leach)

Trophozoites

Trophozoites are found as fairy large fleshy rings with a single chromatin dot. These can be very distorted and can often take the form of bands across the cell. All trophozoites have a single chromatin dot and contain pigment. (Fig. 14 & 15)

Figure 15. Trophozoite of Plasmodium malariae. These can be very distinct and distorted by taking the form of a band across the cell. (Giemsa stain)

Gametocytes

Gametocytes contain large amounts of black pigment, with chromatin present as a compact mass in females and diffuse in males. They occupy less than two thirds of the red blood cell. (Figs. 14 & 16)

Figure 16. Plasmodium malariae gametocyte. They contain large amounts of black pigment, with chromatin present as a compact mass in females (macrogametocyte) and diffuse in males (microgametocyte). (Giemsa stain)

Schizonts

Schizonts are usually few in numbers with 6 - 12 large merozoites in a single ring. Pigment is usually present as a central black mass. The parasites present are generally only found at one stage of schizogony development. (Fig 17)

Figure 17. Schizont of Plasmodium malariae. They are usually few in numbers with 6 – 12 large merozoites in a single ring. Pigment is usually present as a central black mass. (Giemsa stain)

Clinical Disease

Symptoms include headache, photophobia, muscle aches and pains, anorexia, nausea and vomiting. Plasmodium malariae, like P. vivax and P. ovale are relatively benign. However, chronic infections in children may lead to nephrotic syndrome due to immune complexes depositing on the glomerular wall.

9.3. Diagnosis of malaria parasites

Introduction

The definitive diagnosis of malaria infection is still based on finding malaria parasites in blood films. In thin films the red blood cells are fixed so the morphology of the parasitised cells can be seen. Species identification can be made, based upon the size and shape of the various stages of the parasite and the presence of stippling (i.e. bright red dots) and fimbriation (i.e. ragged ends). However, malaria parasites may be missed on a thin blood film when there is a low parasitaemia. Therefore, examination of a thick blood film is recommended. With a thick blood film, the red cells are approximately 6 - 20 layers thick which results in a larger volume of blood being examined.

Thick Blood Films

In examining stained thick blood films, the red blood cells are lysed, so diagnosis is based on the appearance of the parasite. In thick films, organisms tend to be more compact and denser than in thin films.

Field’s stain method for Thick blood films

The method recommended for staining thick blood is Field’s Stain which is made from 2 components. Field’s A is a buffered solution of azure dye and Field’s B is a buffered solution of eosin. Both Field’s A and B are supplied ready for use by the manufacturer.

Method

1. Place a drop of blood on a microscope slide and spread to make an area of approximately 1 cm². It should just be possible to read small print through a thick film.

2. The film is air dried and NOT fixed in methanol.

3. The slide is dipped into Field’s stain A for 3 seconds.

4. The slide is then dipped into tap water for 3 seconds and gently agitated.

5. The slide is dipped into Field’s stain B for 3 seconds and washed gently in tap water for a few seconds until the excess stain is removed.

6. The slide is drained vertically and left to dry.

Microscopic Features of the Field’s stained thick blood film

· The end of the film at the top of the slide when it was draining should be looked at. The edges of the film will also be better than the centre, where the film may be too thick or cracked.

· In a well-stained film the malaria parasites show deep red chromatin and pale blue cytoplasm.

· White cells, platelets and malaria pigment can also be seen on a thick film. The leucocyte nuclei stain purple and the background is pale blue. The red cells are lysed and only background stroma remains. The occasional red cell may fail to lyse.

· Schizonts and gametocytes, if present, are also easily recognisable.

· A thick film should be examined for at least 10 minutes, which corresponds to approximately 200 oil immersion fields, before declaring the slide negative.

N.B.

· As a result of haemolysis of the red blood cells due to staining of an unfixed film, the only elements seen are leucocytes and parasites, the appearance of the latter being altered. Consequently:

1. The young trophozoites appear as incomplete rings or spots of blue cytoplasm with

detached chromatin dots.

2. The stippling of P. vivax and P. ovale may be less obvious although occasionally ghost

stippling may be seen.

3. The cytoplasm of late trophozoites of P. vivax and P. ovale may be fragmented.

· Caution should be exercised when examining thick blood films as artefacts and blood platelets may be confused with malaria parasites.

Thin Blood Films

When examining thin blood films for malaria you must look at the infected red blood cells and the parasites inside the cells.

1. Rapid Field’s stain for thin films

This is a modification of the original Field’s stain to enable rapid staining of fixed thin films. This method is suitable for malaria parasites, Babesia sp., Borrelia sp. and Leishmania sp.

Method.

1. Air dry the film

2. Fix in methanol for 1 minute.

3. Flood the slide with 1 ml of Field’s stain B, diluted 1 in 4 with distilled water.

4. Immediately, add an equal volume of undiluted Field’s stain A, mix well and allow to stain for 1 minute.

5. Rinse well in tap water and drain dry.

Uses.

This is a useful method for rapid presumptive species identification of malarial parasites. It shows adequate staining of all stages including stippling (mainly Maurer’s dots). However, staining with Giemsa is always the method of choice for definitive species differentiation.

2. Giemsa stain for thin films.

Method.

1. Air dry thin films

2. Fix in methanol for 1 minute

3. Wash in tap water and flood the slide with Giemsa diluted 1 in 10 with buffered distilled water pH 7.2. The diluted stain must be freshly prepared each time.

4. Stain for 25 - 30 minutes.

5. Run tap water on to the slide to float off the stain and to prevent deposition of precipitate on to the film. Dry vertically.

6. Examine the film using the x100 objective.

Microscopic features of the thin blood film

1. Examine the tail end of the slide where the red cells are separated into a one-cell-layer

thick.

2. An alkaline buffer pH 7.2 is vital for clear differentiation of nuclear and cytoplasmic material and to visualise inclusions such as Schüffner’s / James’s dots in the red cells. Acidic buffer is unsuitable.

3. The red cells are fixed in the thin film so the morphology of the parasitised cells and the parasites can be seen.

4. On a well stained film the chromatin stains red/purple and the cytoplasm blue. Leucocytes have purple nuclei, the red stippling, if present should be clearly visible.

Infected Red Blood Cells

1. Look at the size of the infected red blood cells.

2. Are there any Schüffner’s dots present or not?

(Adapted and redrawn from WHO, 1991)

Rings of the four main species of malaria may look alike. If you see rings, look for older stages. Patient’s with a P. falciparum infection only, rings are usually seen; older stages are present only in severe infections.

In poorly stained slides, Schüffner’s dots may not be visible, so it is essential that correct staining methods are used. Also Schüffner’s dots may not be seen in the earlier rings of P. vivax or P. ovale.

Estimation of Percentage Parasitaemia of Plasmodium falciparum

Counting of red blood cells infected with parasites of P. falciparum is essential and the percentage parasitaemia should always be reported as this has implications for prognosis and the pattern of treatment employed.

The recommended procedure for estimating the percentage parasitaemia in a thin blood film is by expressing the number of infected cells as a percentage of the red blood cells e.g. 3 parasitised red cells / 100 red blood cells or 3% parasitaemia.

A red blood cell infected with multiple parasites counts as one parasitised red cell.

The percentage parasitaemia should be calculated by counting the number of parasitised red bloodcells in 1000 cells in a thin blood film.

Method 2

Alternatively the World Health Organisation recommend a method which compares the number of parasites in a thick blood film with the white blood cell count.

The parasitaemia is estimated by first counting the number of parasites per 200 white blood cells in a thick blood film and then calculating the parasite count / ml from the total white blood cell count / ml.

Knowledge of either % parasitaemia or total parasite count is essential for the correct clinical management of P. falciparum malaria.

Effects of anticoagulant on the microscopic diagnosis of malarial parasites

Thin blood films for malaria diagnosis are best prepared from venous or capillary blood taken directly from the patient, without the addition of anticoagulant. However, this is not usually possible in a clinical laboratory, as many samples are received from general practices and other hospitals. All anticoagulants have some effect on the morphology of malaria parasites and the red blood cell they inhabit. This effect depends on the stage of the parasite, the time taken for the blood to reach the laboratory and the type of anticoagulant used. If it is necessary to use an anticoagulant, the films should be prepared as soon as possible after the blood has been taken. If the films cannot be made immediately, potassium EDTA is the anticoagulant of choice. However if the blood is left for several hours in EDTA, the following effects may be seen.

1. Sexual stages may continue to develop and male gametocytes can exflagellate, liberating gametes into the plasma. These can be mistaken for organisms such as Borrelia. Gametocytes of P. falciparum which have a characteristic crescent shape, may round up and then resemble those of P. malariae.

2. Acole forms, which are characteristic of P. falciparum, may be seen in P. vivax because of attempted re - invasion of the red blood cell by merozoites.

3. Mature trophozoites of P. vivax may condense when exposure becomes prolonged and in cases of extreme exposure, red blood cells containing gametocytes and mature schizonts may be totally destroyed along with the contained parasites. The malaria pigment, haemozoin, always remains and can provide a clue to the presence and, to an expert eye identity of the parasite.

4. The morphology of the red blood cell may be altered by shrinkage or crenation.

References

Greenwood, B.M (1997): What’s new in Malaria control? London School of Hygiene and Tropical Medicine, Keppel Street, London

Murray, PR, Drew, WL, Koyayashi, GS & Thomson, JH: Medical Microbiology. Mosby Books Inc., New York (1990)

Peters, W & Gilles, HM: Tropical Medicine & Parasitology. Wolfe Medical Publications Ltd.

Jeffrey & Leach: Atlas of Medical Helminthology and Protozoology. E & S Livingstone Ltd.

Garcia, LS & Bruckner, DA: Diagnostic Medical Parasitology. Elsevior Science Publishing Co. Inc.

Smyth, J.D: Introduction to Animal Parasitology. Cambridge University Press (1994)

Snell, JJS, Farrell, ID & Roberts, C: Quality Control, Principles and Practice in the Microbiology Laboratory. Public Health Laboratory Service. ISBN 0 901 144 312.

Malaria species	*P. falciparum*	*P. vivax*	*P. ovale*	*P. malariae*
Red Cell Changes	Maurer’s dots	Schüffner’s dots	James’s Dots / Fimbriation	Ziemann’s dots
Trophozoite – Ring form	Cytoplasm very fine in young rings; thick and irregular in old rings. Acole forms, multiple infections common	Cytoplasm fine in young rings. Red cell unaltered in size.	Cytoplasm thicker than that found in P. vivax. Red cell unaltered in size.	Cytoplasm noticeably thicker. Red cell unaltered in size.
Trophozoite – Growing form	Red cell unaltered in size, sometimes stippled with Maurer’s dots. Parasite is compact; pigment is dense brown or black mass.	Red cell enlarged, stippled. Parasite - amoeboid, vacuolated; pigment fine and scattered, golden brown.	Red cell unaltered in size, or slightly enlarged. Stippled; may be oval and fimbriated. Parasite – compact, rounded; pigment fine brown grains.	Red cell unaltered in size. Parasite – compact, ugly, rounded or band- shaped; dark brown / black pigment often concentrates in a line along one edge of the band.
Mature schizonts	Red cell unaltered in size. Parasite – merozoites 8 – 36; pigment clumped, black. Rare in peripheral blood	Red cell much enlarged, stippled. Parasite – large, filling enlarged red cell. Merozoites 12 - 24, usually 16; pigment golden brown central loose mass.	Red cell slightly enlarged, stippled, frequently oval and fimbriated. Parasite – smaller than red cell. Merozoites 6 – 12, usually 8 in a single ring; pigment, brown / greenish central clump.	Red cell unaltered in size. Parasite – fills red cell. Merozoites 6 - 12, usually 8, sometimes forming a rosette; pigment, brown / black central clump.
Gametocytes	Red cell distorted. Parasite – crescentric. Rare in early cases < 10 days.	Red cell enlarged, stippled. Parasite - large, fills red cell.	Red cell slightly enlarged, stippled. Parasite – round, filling 2/3 of the red cell.	Red cell unaltered in size. Parasite – small, round, fills the red cell.