Chapter 9.
Malaria

9.1  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, so absorption of food materials and elimination of waste products of metabolism are relatively easy to achieve. They also contain rich supplies of protein and oxygen.

Malarial parasites do not actually penetrate the red blood cell, but enter the cell membrane by endocytosis and enclose in a parasitophorous membrane.

Introduction

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

Illustration 9-1. Map illustrating the enormous distribution of malaria throughout the world. (SOURCE: CDC)

Malaria is caused by protozoa of the Plasmodium species.  There are four species which infect both humans and animals; 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 summarized as:

·         initial or continual schizogony (reproduction by multiple asexual fission) in the vertebrate host with initiation of gametogony (the formation or production of gametes);

·         formation of gametes in the arthropod host and subsequent fertilization and formation of a zygote;

·         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. 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. 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).

Illustration 9-3.  The malaria parasite life cycle.  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 .   (SOURCE:  PHIL 3405- CDC/Alexander J. da Silva, PhD/Melanie Moser)

 

Illustration 9-2.  Distinguishing characteristics of the Anopheles mosquito.

Image 9-1. An Anopheline mosquito, the vector of the protozoa group Plasmodia, the parasite known to cause malaria in both man and non-human primates. Malaria is transmitted by female Anopheles mosquitoes to the definitive host while the mosquito blood-feeds on its victims.  (SOURCE:  PHIL 2070/6765 - CDC/ James Gathany)

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 (the extrusion of rapidly waving flagellum-like microgametes from microgametocytes) occurs resulting in the production in a number of male and female gametes.  Fertilization 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.

Illustration 9-4. 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. (SOURCE: Unknown)

 
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 eight 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 four species, asexual development occurs in the liver cells, a process known as pre-erythrocytic schizogony, to produce thousands of tiny merozoites which are released 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 into trophozoites. In the course of their development they absorb the hemoglobin of the red cells and leave as the product of digestion a pigment called hemozoin, a combination of hematin 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.

 
Illustration 9-5. Diagram illustrating the various stages of the three common species of malaria which infect man. (SOURCE: Unknown)

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 parasitemia.  

Infections 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 synchronized schizogony.

Anemia: The anemia is hemolytic in type. It is more severe in infections with P. falciparum because in this infection cells of all ages can be invaded. Also, the parasitemia 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 hemolysis 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 exists in the tropics and sub-tropics, and is responsible for approximately 50% of all malaria cases.  The incubation period of P. falciparum malaria is the shortest, between eight 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:

·         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 parasitized.

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

·         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.

·         Sporulation is not as well synchronized as in other malaria forms so that the fever may last longer.

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

Morphology of 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. 

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 recognized.  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 accole 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.     Displaced: 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.

Illustration 9-6. 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.

 
Morphology of Gametocytes

Gametocytes are the sexual stage of the malaria parasite. Plasmodium falciparum  gametocytes appear in the peripheral circulation after 7-12 days of patent parasitemia and by then, they have assumed their typical crescent shapes.  They soon reach their peak density, and then decline in numbers, disappearing in about three months as a rule.   

Image 9-2. 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). (SOURCE:  PHIL 5946 - CDC/ Steven Glenn, Laboratory & Consultation Division)

Image 9-3. Plasmodium falciparum gametocytes appear in the peripheral circulation after 7-12 days of patent parasitemia and by then, they have assumed their typical crescent shapes. (Giemsa stain) (SOURCE:  PHIL 5941 - CDC/ Steven Glenn, Laboratory & Consultation Division)

The female form, or macrogametocyte, is usually more slender and somewhat longer than the male, and the cytoplasm takes up a deeper blue color 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.

Morphology of Schizonts

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

Image 9-4. Plasmodium falciparum schizont. Rarely seen in the peripheral blood, a good indicator of a potentially serious parasitemia. They have 8 – 36 merozoites and a large golden brown pigment. (Giemsa stain) (SOURCE:  PHIL 5854 - CDC/Steven Glenn, Laboratory & Consultation Division)

Clinical Disease

Symptoms include headache, photophobia, muscle aches and pains, anorexia, nausea and vomiting.  Complications include severe anemia cerebral malaria, renal disease, black water fever, dysentery, pulmonary edema 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 characterized by the presence of more than one developmental stage in the peripheral blood film.  The parasites parasitize 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:

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

·         The periodicity of the asexual cycle is closely synchronized.

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

Morphology of 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.

Image 9-5. Trophozoites of Plasmodium vivax are already several hours old when they appear in the peripheral blood and therefore, you can already see the Schüffner's dots. They contain single or sometimes double chromatin dots. (Giemsa stain) (SOURCE:  PHIL 5928 - CDC/ Steven Glenn, Laboratory & Consultation Division)

Illustration 9-7. 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) Microgametocyte, large numbers appear after 3–5 days. 6) Macrogametocyte, large numbers appear after 3–5 days.  (SOURCE: Unknown)

Morphology of 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. Mature male gametocytes can be distinguished from females by the large, loose and ill-defined mass of chromatin and by their paler color and smaller mass.

Image 9-6. 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) (SOURCE:  PHIL 5138 - CDC/ Dr. Mae Melvin)

 
Morphology of Schizonts

The parasitized 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.

Image 9-7.  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) (SOURCE:  PHIL 5925 - CDC/ Steven Glenn, Laboratory Training & Consultation Division)

Clinical Disease

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

Plasmodium ovale

Introduction

Plasmodium ovale is widely distributed in tropical Africa especially the west coast, but it is not often 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 that caused by the benign tertian P. falciparum.

Life Cycle

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

·         It morphologically resembles P. malariae in most of its stages.

·         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.

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

·         Hypnozoites develop in the liver so that relapses may occur.

Morphology of Trophozoites

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.  

Illustration 9-8. 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.

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.

Image 9-8. 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. (SOURCE:  PHIL 5935 - CDC/ Steven Glenn, Laboratory & Consultation Division)

Morphology of 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.

Image 9-9. Gametocyte of Plasmodium ovale. The mature gametocytes are round, filling two thirds of the red cell. (Giemsa stain) (SOURCE:  PHIL 3474 – CDC/Dr. Mae Melvin)  

Morphology of 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.  

Image 9-10. 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) (SOURCE:  PHIL 5846 - CDC/ Steven Glenn, Laboratory & Consultation Division)

 
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.  It is 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 specific to P. malariae are as follows:

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

·         Mature erythrocytes are attacked, but reticulocytes generally are not

·         The density of parasites is very low; about 0.2% of erythrocytes are parasitized.

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

Morphology of Trophozoites

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

Illustration 9-9. 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) Micro-gametocytes, low numbers appear after 7–14 days.  6) Macrogametocytes, low numbers appear after 7-14 days.

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.  

Image 9-11. Trophozoite of Plasmodium malariae. These can be very distinct and distorted by taking the form of a band across the cell. (Giemsa stain) (SOURCE:  PHIL 639 - CDC/ Dr. Mae Melvin, Steve Glenn)

Morphology of 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.

Image 9-12. 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) (SOURCE:  PHIL 5837 - CDC/ Steven Glenn, Laboratory & Consultation Division)

Morphology of 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.

Image 9-13. 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) (SOURCE:  PHIL 4826 - CDC/Dr. Mae Melvin)

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. Laboratory 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 parasitized 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 parasitemia.  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 (destroyed), 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 two 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 three seconds.

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

5.   The slide is dipped into Field’s stain B for three 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 leukocyte 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 recognizable.

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

·     As a result of hemolysis of the red blood cells due to staining of an unfixed film, the only elements seen are leukocytes 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 artifacts 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 one 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 one 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 visualize 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 parasitized cells and the parasites can be seen.

4.     On a well stained film the chromatin stains red/purple and the cytoplasm blue.  Leukocytes 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?

Table 9-1. Stages and appearance of Plasmodium species in blood. (SOURCE: CDC) 

Table 9-2.  Characteristics of Plasmodium parasites (SOURCE: CDC)

Rings of the four main species of malaria may look alike. If you see rings, look for older stages.  Patients 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 Parasitemia of Plasmodium falciparum

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

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

A red blood cell infected with multiple parasites counts as one parasitized red cell.  The percentage parasitemia should be calculated by counting the number of parasitized red blood cells in 1000 cells in a thin blood film.

Method 2

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

The parasitemia 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 % parasitemia 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.   Accole 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, hemozoin, 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.

Table 9-3. Differential diagnostic features of human Plasmodia species - Giemsa stained thin film of peripheral blood.