Malaria Parasites: Species, Life Cycle, and Morphology

Abstract

Plasmodium parasites are parasitized in vertebrates and female Anopheles mosquitoes. Although there are many different species, there are four main species of Plasmodium parasites in humans, namely, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Furthermore, there is a fifth, rarer, human-monkey species, known as Plasmodium knowlesi. This chapter describes the biology, life history, and morphological characteristics of human Plasmodium to provide a comprehensive understanding of Plasmodium in humans.

4.1 Etiology and Classification of Malaria

Malaria is an ancient infectious disease that poses a serious threat to human health. There are written records relating to malaria epidemics in China as far back as 3000 years ago. It was not until 1880 in Algeria that Charles-Louis-Alphonse Laveran, a French army doctor, first discovered Plasmodium in the blood of patients with fever by microscopic examination. In 1897, Ronald Ross, a British army doctor serving in India, confirmed that the Anopheles mosquito was the vector of malaria and elucidated the life cycle of the Anopheles mosquito and the mode of transmission of its bite. It was then that the mystery of malaria was unraveled.

Plasmodium, the causative agent of malaria, is a unicellular eukaryotic protozoan belonging to Sporozoasida, the order Eucoccidiida, the family Plasmodidae, and the genus Plasmodium. There are many different species of Plasmodium, and the hosts can be amphibians, reptiles, birds, mammals, and other vertebrates, but the hosts of the species are highly specific, and there are significant biological differences between species. There are four main species of human malaria parasites, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, which cause falciparum malaria, Vivax malaria, Ovale malaria, and Quartan malaria, respectively. There are currently eight main named species (or subspecies), with P. vivax, P. ovale, and P. malariae belonging to the subgenus Plasmodium and P. falciparum belonging to the subgenus Laverania (Fig. 4.1). In addition, P. ovale contains two subtypes, P. ovale curtisi and P. ovale wallikeri, but it is controversial whether they are two subspecies. P. falciparum, P. vivax, and P. ovale are only parasitic in humans, whereas P. malariae can infect humans and apes in Africa (Xinping and Chuan, 2018; Shaowu et al., 2000; Sutherland et al., 2010).

Fig. 4.1

The classification of main human Plasmodium

Plasmodium knowlesi, a monkey Plasmodium endemic in the virgin forests of Malaysia, has been shown to cause natural infection from monkey to human, human to human, and human to monkey through mosquito vectors and is recognized as the fifth human Plasmodium species. However, P. knowlesi infections are less common, have a limited epidemiological range, and do not develop in humans as hyperparasitemia, hence the mild clinical symptoms of P. knowlesi infection in humans. Other rare cases of human infection with Plasmodium simium, Plasmodium cynomolgi, Plasmodium schwetzi, and Plasmodium inui have also been reported in the literature. The route and mode of infection and the degree of risk to humans have yet to be confirmed (Huaimin et al., 2006).

4.2 Life Cycle

The development and reproduction of Plasmodium require completion by vertebrates and insect vectors, and the hosts of human Plasmodium include humans and Anopheles. Plasmodium parasitizes human liver parenchyma cells and red blood cells (RBCs). Occasionally, Plasmodium may be present outside RBCs in peripheral blood smears, e.g., by released merozoites, or when hemolytic reactions occur (Chap. 6, Fig. 6.14). In mosquitos, it parasitizes the mosquito stomach and finally gathers in salivary glands. The four major human Plasmodium species share a similar life cycle, which includes the exoerythrocytic (liver stage) and erythrocytic phases in humans and the gametogony and sporogony phases in Anopheles mosquitoes. Understanding the life cycle of Plasmodium is of great importance to the morphology of Plasmodium under the microscope (Xinping and Chuan, 2018; Xiaoqiu, 2007).

4.2.1 Development of Plasmodium in Humans

The exoerythrocytic phase is also known as the liver stage. When female Anopheles mosquitoes carrying Plasmodium sporozoites in salivary glands take a blood meal from humans, the sporozoites with saliva invade human peripheral blood, and they can remain under the skin for several hours. The majority of sporozoites then enter the capillaries, and a very small proportion invade the capillary lymphatics. The sporozoites follow the blood flow into the hepatic sinusoids, cross the Kupffer cell or sinusoidal endothelial cell space, and eventually invade the hepatic parenchymal cells. It takes approximately 30 minutes from the time the sporozoites enter the blood vessels to the time they invade the hepatocytes. Development and asexual schizogenesis of Plasmodium sporozoites are completed in parenchymal cells of the liver, and the development of Plasmodium to schizonts is termed schizonts of the exoerythrocytic phase or liver stage. The mature schizonts of the exoerythrocytic phase are 45–60 μm in diameter, which give birth to 10–30,000 merozoites, and escape from the hepatocytes in the form of merosomes, which bud out. After entering the peripheral blood, merosomes release merozoites, some of which are engulfed by macrophages and some of which successfully invade RBCs and begin the development of the erythrocytic phase. The duration of the exoerythrocytic phase varies in Plasmodium species, ranging from 6 to 12 days, with 5 to 6 days for P. falciparum, 8 days for P. vivax, 9 days for P. ovale, and 11 to 12 days for P. malariae. There are currently two genetically distinct types of sporozoites of P. vivax and P. ovale, namely, tachysporozoites and bradysporozoites. Tachysporozoites invade hepatocytes and continue to proliferate in the exoerythrocytic phase, releasing liver-stage merozoites that invade RBCs and cause clinical episodes through schizosomal proliferation. Bradysporozoites do not continue developing after invading hepatocytes temporarily but remain dormant (latent period). After a period of dormancy ranging from a few months to a few years (usually longer than 3 months), bradysporozoites develop into mature exoerythrocytic schizonts and release merozoites to invade RBCs to cause clinical symptoms, which is also known as “relapse.” In Wuhan city, an overseas imported case infected with P. ovale that had a latent period of more than 14 months was observed, and a case with a latent period of more than 2 years was reported in China. Since no bradysporozoites are observed in either P. falciparum or P. malariae, there is no relapse of P. falciparum or P. malariae. However, in Wuhan city, several imported cases infected with P. malariae were found to present clinical symptoms after more than 4 months of returning from overseas countries, which showed a similar latent period with P. vivax and P. ovale. In fact, the parasitemia density of P. malariae is generally lower than that of other species. The extremely low density causes long-term asymptomatic erythrocyte parasitism in P. malariae; however, clinical symptoms may occur due to the subsequently increased density.

The erythrocytic phase is also known as the blood stage or RBC stage. Following release to peripheral blood, some merozoites invade RBCs within a few seconds or minutes and develop in RBCs for reproduction by fission, while other merozoites are engulfed by phagocytic cells. The process of merozoite invasion into RBCs includes three consecutive stages. First, merozoites recognize and attach to receptors on the surface of the RBC membrane through specific sites. Second, RBCs are deformed, and the cell membrane is concave around merozoites to form Plasmodium vacuoles. Finally, the vacuoles are sealed after merozoite invasion. The merozoites develop into small trophozoites (ring trophozoites or trophozoites of the former forms) in RBCs. After swallowing hemoglobin and other nutrients, the nucleus is enlarged, the cytoplasm is increased, and iron ion-containing pigments are produced by breaking down hemoglobin. The color and shape of the malaria pigments vary somewhat between species of Plasmodium. Then, the small trophozoites develop gradually into large trophozoites (late trophozoites or mature trophozoites), also known as an amoeba-like shape, because of their amoeboid movements. The nucleus and cytoplasm of mature trophozoites begin to divide and develop into schizonts. Each nucleus of a mature schizont is surrounded by a piece of cytoplasm, giving it a grainy and clear state. The mature schizonts have 8–32 nuclei (merozoites), and the number of nuclei varies according to the species. When the schizonts are mature, the RBCs are exhausted and then broken. RBC fragments, merozoites, and malarial pigments are released into peripheral blood and cause the onset of clinical symptoms of malaria. Some merozoites are consumed by macrophages, while others reinvade new RBCs and begin a new erythrocytic phase. This cycle is known as the fission proliferation cycle. The duration of the erythrocytic phase varies with Plasmodium species, with 36–48 hours for P. falciparum, 48 hours for P. vivax and P. ovale, and 72 hours for P. malariae, producing a corresponding fever cycle. After more than ten hours of development in RBCs, the early trophozoites of P. falciparum gradually hide in microvessels, blood sinuses, and places with slow blood flow and continue to develop into late trophozoites and schizonts. Therefore, the late trophozoites and schizonts of P. falciparum are generally not easily found in peripheral blood, except for severe cases of falciparum malaria. After several fission proliferations of Plasmodium in RBCs, merozoites that have invaded RBCs no longer undergo asexual divisions but develop into female (macrogametocytes) and male gametocytes (microgametocytes). At this time, if female Anopheles bites and feeds on human blood, the mature female and male gametophytes are sucked into the mosquito's stomach, and sexual reproduction begins. If they remain in humans, they will age and die out spontaneously, being cleared out or simply engulfed by WBCs within 30–60 days. The immature gametophytes of P. falciparum are mainly in the microvessels and blood sinuses of the liver, spleen, bone marrow, and other organs. In general, they appear in the peripheral blood after maturity, and the time is approximately 7–10 days after the emergence of asexual bodies in the peripheral blood. P. falciparum can parasitize all types of RBCs, P. vivax and P. ovale mainly parasitize reticulocytes, and P. malariae mostly parasitizes aged RBCs. In addition to new infections in malaria patients, the most common reason leading to the recurrence of clinical symptoms is the massive proliferation of residual Plasmodium in blood, which is called “resurgence.” Therefore, no matter what kind of malaria parasite is infected, resurgence is occurring as long as the anti-malaria treatment fails.

4.2.2 Development of Plasmodium in Anopheles Mosquito

Gametogenesis: After Plasmodium at each stage enters mosquito stomachs by sucking the blood of malaria patients and Plasmodium carriers, mature female and male gametocytes continue to develop in the mosquito stomachs, while another asexual Plasmodium is digested, including small and large trophozoites, schizonts, and immature gametocytes. Female gametocytes form circular and inactive female gametes following nucleus meiosis. The nucleus of male gametocytes first divides into 4–8 pieces, and the cytoplasm extends 4–8 flagellate filaments, known as the filament phenomenon. Then, each nucleus enters a filament, which detaches from the mother and forms a flagellated male gamete. The male gamete may swim close to female gametes directionally. When in contact with the female gamete, it can burrow into the female gamete within seconds, forming a rounded zygote. The zygote develops into motile and elongated ookinetes. The ookinete passes through the epithelial cells on the mosquito gastric wall and stays between the epithelial cells and the outer elastic fibrous membrane, developing into an oocyst. If the patient's peripheral blood has a long interval between collection and production, then female and male gametes, the filament phenomenon (Figs. 6.25 and 6.57), ookinetes (Fig. 4.2), and other morphologies can also be observed in the peripheral blood smear.

Fig. 4.2

A P. vivax ookinete (Giemsa staining, ×1000)

P. vivax ookinetes may be present in human peripheral blood if the time interval from blood collection to the preparation of blood smears is long enough.

Sporozoite reproduction: The oocysts grow and protrude into the wall of the mosquito’s stomach in the form of a tumor, and there can be several to dozens, or even more, of oocysts on the wall of the mosquito's stomach. The nuclei and cytoplasm in the oocysts are divided repeatedly and undergo sporozoite reproduction. Mature oocysts are approximately 40–60 μm in diameter and contain approximately 1000–10,000 spindle-shaped sporozoites, which are 10–15 μm long and approximately 1 μm wide, with a nucleus pointed at both ends and a curved body. The sporozoites can be released from the rupture or burrowed out of the oocyst and concentrated in the salivary glands of the mosquito via the hemolymph, developing into mature sporozoites. The duration of Plasmodium development in mosquitoes is related to temperature and humidity. The most suitable condition for Plasmodium sporozoites reproduction in mosquitoes is at a temperature of 24–26 °C and relative humidity of 75%–80%. A temperature lower than 16 °C or higher than 30 °C will delay development and may cause degeneration until death. Mature sporozoites escape through the crevice of the oocysts or diffuse into the blood and eventually accumulate in the salivary gland adenocytes. If a female mosquito with sporozoites bites another person, sporozoites inject humans with saliva and initiate development in humans. Under the most suitable conditions, the development time of Plasmodium in Anopheles mosquitoes is 10–12 days for P. falciparum, 9–10 days for P. vivax, approximately 16 days for P. ovale, and 25–28 days for P. malariae.

The biological characteristics and life cycles of the four Plasmodium species are shown in Table 4.1 and Fig. 4.3.

Table 4.1 Biological characteristics of four human Plasmodium species

Fig. 4.3

The life cycle of Plasmodium species

4.3 Morphology of Human Plasmodium in the Erythrocytic Phase

The basic structure of Plasmodium includes the nucleus, cytoplasm and cell membrane. After ingestion of hemoglobin, malaria pigments, a product of digestion and breakdown of hemoglobin, appear in the pRBCs (Fig. 4.4). Blood smears with stainings, such as Giemsa or Wright's staining, show red or purplish red nuclei, blue or dark blue cytoplasm, and brown or black-brown malaria pigments. The four human Plasmodium species have the same basic structure, but the morphology of each stage of development varies. In addition to the morphological characteristics of the Plasmodium itself, the pRBCs can also change in morphology (Xinping and Chuan, 2018; Xiaoqiu, 2007).

Fig. 4.4

Yellowish- or dark-brown malarial pigments (Giemsa staining, ×1000)

Accumulation of malarial pigments is seen in leukocyte phagocytosis after medical treatment (red arrow), which is often observed in cases with high parasitemias or severe cases.

4.3.1 Morphological Characteristics of the Stages of Plasmodium Development in RBCs

The morphological characteristics of Plasmodium are grouped into three main developmental stages, namely, the trophozoite, schizont, and gametophyte stages, based on the morphological characteristics of each stage in the pRBCs. The trophozoites and schizonts belong to the asexual stage, and the gametophytes belong to the sexual stage.

• I. Trophozoites

  Based on morphological characteristics, they can be subdivided into the small trophozoite stage and the large trophozoite stage. Small trophozoites, also known as ring-form and early trophozoites, have a small nucleus, little cytoplasm, a vacuole in the middle and a ring-shaped body. As Plasmodium feeds, grows, and develops, the nucleus and cytoplasm increase in size, and pseudopods and malaria pigments appear. Maurer’s clefts appear inside pRBCs parasitized by P. falciparum, Schüffner’s dots appear inside pRBCs parasitized by P. vivax and P. ovale, and Ziemann’s dots appear inside pRBCs parasitized by P. malariae. At this point, Plasmodium develops into large trophozoites, which can also be called late trophozoites or mature trophozoites. The morphology and size of the large trophozoites vary considerably between Plasmodium parasites and are key to morphological differentiation.

• II. Schizonts

  As the trophozoites mature, the nucleus and cytoplasm begin to divide in a dichotomy called schizonts. The nucleus undergoes repeated divisions, and the cytoplasm divides as well, with each nucleus surrounded by cytoplasm, called a merozoite. The schizonts can be subdivided into immature (early) schizonts and mature (late) schizonts. The nuclei of immature schizonts are few and tightly packed in undivided cytoplasm, whereas when there are more nuclei, the respective cytoplasm surrounds the nuclei and becomes grainy and clear, and the malaria pigment tends to concentrate; then, the schizonts are mature. The morphology of the schizonts and the number of schizonts vary considerably between Plasmodium parasites, which is the key to morphological differentiation.

• III. Gametocytes

  After Plasmodium parasites have undergone several fission proliferations and some merozoites invade RBCs, the nucleus and cytoplasm develop into round, oval or crescent-shaped, sausage-shaped gametophytes. Depending on morphological characteristics, gametophytes can be divided into female and male and can also be subdivided into immature gametophytes and mature gametophytes. The mature gametophyte of P. falciparum is crescentic and sausage shaped, which differs markedly from the round, oval-shaped gametophytes of P. vivax, P. ovale, and P. malariae.

4.3.2 Morphological Characteristics of Four Human Plasmodium Species in Thin Blood Smears

The morphological characteristics of Plasmodium in thin blood smears after Giemsa staining are shown in Table 4.2. The morphology of large (late or mature) trophozoites is sometimes similar to that of nearly mature female gametocytes. The identification is described in Table 4.3.

Table 4.2 Morphological identification of four Plasmodium species on a thin blood smear (Giemsa staining)

Table 4.3 Morphological identification between large (late or mature) trophozoites and nearly mature female gametocytes

4.3.3 Morphological Characteristics of Four Human Plasmodium Species in Thick Blood Smears

The RBCs of the thick blood smears stacked cascade, Plasmodium shrank fold or part of Plasmodium was missing, the lysis of RBCs could not be used as a reference, and the hemolysis process also formed more impurities than thin blood smears, resulting in morphological identification being more difficult than thin blood smears. However, thick blood smears have more blood volume and a smaller area, and when RBCs are concentrated, the detection rate of Plasmodium is significantly higher than that of thin blood smears and less likely to be missed. The morphological characteristics of Plasmodium in thin blood smears after Giemsa staining are shown in Table 4.4.

Table 4.4 Morphology of human malaria parasites on a thick blood smear

Figures 4.5, 4.6, 4.7, and 4.8 describe the morphological characteristics of Plasmodium in thick and thin blood smears.

Fig. 4.5

Morphology of P. falciparum at the erythrocytic stage

Fig. 4.6

Morphology of P. vivax at the erythrocytic stage

Fig. 4.7

Morphology of P. ovale at the erythrocytic stage

Fig. 4.8

Morphology of P. malariae at the erythrocytic stage